Comparative analysis of nitrifying bacteria in full-scale oxidation ditch and aerated nitrification biofilter by using fluorescent in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE).

PubMed

Mertoglu, Bulent; Calli, Baris; Girgin, Emine; Inanc, Bulent; Ozturk, Izzet

2005-01-01

In this study, nitrification performances and composition of nitrifying populations in a full-scale oxidation ditch and a high-rate submerged media nitrification biofilter were comparatively analyzed. In addition to different reactor configurations, effects of differing operational conditions on the nitrification efficiency and bacterial diversity were also explored and evaluated thoroughly. In microbial analysis of sludge samples fluorescent in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) techniques were used complementary to each other. The extended aeration oxidation ditch subjected to the study is operated as a nitrogen and phosphorus removal system consisting of anaerobic, anoxic, and aerobic zones. The high-rate submerged media aerated filter is operated as nitrification step following the conventional activated sludge unit and the nitrified wastewater is discharged to the sea without complete nitrogen removal. In situ hybridization results have indicated that Nitrosomonas-like ammonia oxidizing and Nitrospira-related nitrite oxidizing bacteria were intensively present in vigorous flocs in nitrification biofilter while carbonaceous bacteria belong to beta subclass of Proteobacteria were considerably dominant in oxidation ditch. Low quantities of nitrifiers in oxidation ditch were also confirmed by the dissimilarity in intensive bands between two systems obtained with DGGE analysis.

Dynamics of Vaginal Bacterial Communities in Women Developing Bacterial Vaginosis, Candidiasis, or No Infection, Analyzed by PCR-Denaturing Gradient Gel Electrophoresis and Real-Time PCR▿

PubMed Central

Vitali, Beatrice; Pugliese, Ciro; Biagi, Elena; Candela, Marco; Turroni, Silvia; Bellen, Gert; Donders, Gilbert G. G.; Brigidi, Patrizia

2007-01-01

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA. PMID:17644631

Dynamics of vaginal bacterial communities in women developing bacterial vaginosis, candidiasis, or no infection, analyzed by PCR-denaturing gradient gel electrophoresis and real-time PCR.

PubMed

Vitali, Beatrice; Pugliese, Ciro; Biagi, Elena; Candela, Marco; Turroni, Silvia; Bellen, Gert; Donders, Gilbert G G; Brigidi, Patrizia

2007-09-01

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.

Monitoring of the microbial communities involved in the soy sauce manufacturing process by PCR-denaturing gradient gel electrophoresis.

PubMed

Tanaka, Yasushi; Watanabe, Jun; Mogi, Yoshinobu

2012-08-01

Soy sauce is a traditional seasoning produced through the fermentation of soybeans and wheat using microbes. In this study, the microbial communities involved in the soy sauce manufacturing process were analyzed by PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE). The bacterial DGGE profile indicated that the bacterial microbes in the koji were Weissella cibaria (Weissella confusa, Weissella kimchii, Weissella salipiscis, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus iners, or Streptococcus thermophilus), Staphylococcus gallinarum (or Staphylococcus xylosus), and Staphylococcus kloosii. In addition to these bacteria, Tetragenococcus halophilus was also detected in the mash during lactic acid fermentation. The fungal DGGE profile indicated that the fungal microbes in the koji were not only Aspergillus oryzae but also several yeasts. In the mash, Zygosaccharomyces rouxii appeared in the early fermentation stage, Candida etchellsii (or Candida nodaensis) and Candida versatilis were detected at the middle fermentation stage, and Candida etchellsii was detected at the mature fermentation stage. These results suggest that the microbial communities present during the soy sauce manufacturing process change drastically throughout its production. This is the first report to reveal the microbial communities involved in the soy sauce manufacturing process using a culture-independent method. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

Microbial Diversity during Fermentation of Sweet Paste, a Chinese Traditional Seasoning, Using PCR-Denaturing Gradient Gel Electrophoresis.

PubMed

Mao, Ping; Hu, Yuanliang; Liao, Tingting; Wang, Zhaoting; Zhao, Shumiao; Liang, Yunxiang; Hu, Yongmei

2017-04-28

The aim of this study was to elucidate the changes in the microbial community and biochemical properties of a traditional sweet paste during fermentation. PCR-denaturing gradient gel electrophoresis (DGGE) analysis showed that Aspergillus oryzae was the predominant species in the koji (the fungal mixture), and the majority of the fungi isolated belonged to two Zygosaccharomyces species in the mash. The bacterial DGGE profiles revealed the presence of Bacillus subtilis during fermentation, and Lactobacillus acidipiscis, Lactobacillus pubuzihii, Lactobacillus sp., Staphylococcus kloosi, and several uncultured bacteria were also detected in the mash after 14 days of main fermentation. Additionally, during main fermentation, amino-type nitrogen and total acid increased gradually to a maximum of 6.77 ± 0.25 g/kg and 19.10 ± 0.58 g/kg (30 days) respectively, and the concentration of reducing sugar increased to 337.41 ± 3.99 g/kg (7 days). The 180-day fermented sweet paste contained 261.46 ± 19.49 g/kg reducing sugar and its pH value remained at around 4.65. This study has used the PCR-DGGE technique to demonstrate the microbial community (including bacteria and fungi) in sweet paste and provides useful information (biochemical properties) about the assessment of the quality of sweet paste throughout fermentation.

Bacterial analysis of combined periodontal-endodontic lesions by polymerase chain reaction-denaturing gradient gel electrophoresis.

PubMed

Xia, Minghui; Qi, Qingguo

2013-01-01

We used denaturing gradient gel electrophoresis (DGGE) to compare bacterial profiles in periodontium and root canals of teeth with combined periodontal-endodontic lesions. Samples of dental plaque and necrotic pulp were collected from thirteen extracted teeth with advanced periodontitis. Genomic DNA was extracted for polymerase chain reaction (PCR) analysis using universal bacterial primers. The PCR products were then loaded onto DGGE gels to obtain fractionated bands. Characteristic DGGE bands were excised and DNA was cloned and sequenced. The number of bands, which indicates the number of bacterial species, was compared between dental plaques and necrotic pulp tissues from the same tooth. Although the difference was statistically significant (P < 0.01), there was no positive correlation; similarity (Dice coefficient) was 13.1% to 62.5%. Some bacteria species were present in both the periodontal pockets and root canals of the same tooth; however, periodontal bacteria did not always invade the root canals, and some bacteria in root canals were not present in periodontal pockets of the same tooth. In some teeth, unique bacteria in root canals had not passed from periodontal pockets. A basic local alignment search tool (BLAST) sequence search in Genbank indicated that new bacteria species were present in periodontal pockets and root canals. Their characteristics must thus be further analyzed.

Use of denaturing gradient gel electrophoresis to detect Actinobacteria associated with the human faecal microbiota.

PubMed

Hoyles, Lesley; Clear, Jessica A; McCartney, Anne L

2013-08-01

With the exceptions of the bifidobacteria, propionibacteria and coriobacteria, the Actinobacteria associated with the human gastrointestinal tract have received little attention. This has been due to the seeming absence of these bacteria from most clone libraries. In addition, many of these bacteria have fastidious growth and atmospheric requirements. A recent cultivation-based study has shown that the Actinobacteria of the human gut may be more diverse than previously thought. The aim of this study was to develop a denaturing gradient gel electrophoresis (DGGE) approach for characterizing Actinobacteria present in faecal samples. Amount of DNA added to the Actinobacteria-specific PCR used to generate strong PCR products of equal intensity from faecal samples of five infants, nine adults and eight elderly adults was anti-correlated with counts of bacteria obtained using fluorescence in situ hybridization probe HGC69A. A nested PCR using Actinobacteria-specific and universal PCR-DGGE primers was used to generate profiles for the Actinobacteria. Cloning of sequences from the DGGE bands confirmed the specificity of the Actinobacteria-specific primers. In addition to members of the genus Bifidobacterium, species belonging to the genera Propionibacterium, Microbacterium, Brevibacterium, Actinomyces and Corynebacterium were found to be part of the faecal microbiota of healthy humans. Copyright © 2013 Elsevier Ltd. All rights reserved.

Population structure and abundance of arsenite-oxidizing bacteria along an arsenic pollution gradient in waters of the upper isle River Basin, France.

PubMed

Quéméneur, Marianne; Cébron, Aurélie; Billard, Patrick; Battaglia-Brunet, Fabienne; Garrido, Francis; Leyval, Corinne; Joulian, Catherine

2010-07-01

Denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR (qPCR) were successfully developed to monitor functional aoxB genes as markers of aerobic arsenite oxidizers. DGGE profiles showed a shift in the structure of the aoxB-carrying bacterial population, composed of members of the Alpha-, Beta- and Gammaproteobacteria, depending on arsenic (As) and E(h) levels in Upper Isle River Basin waters. The highest aoxB gene densities were found in the most As-polluted oxic surface waters but without any significant correlation with environmental factors. Arsenite oxidizers seem to play a key role in As mobility in As-impacted waters.

SIMILARITY OF PARTICLE-ASSOCIATED AND FREE-LIVING BACTERIAL COMMUNITIES IN NORTHERN SAN FRANCISCO BAY, CALIFORNIA

EPA Science Inventory

We used denaturing gradient gel electrophoresis (DGGE) of 16S rDNA PCR amplicons to analyze the composition of Bacteria communities in samples collected during the summer, low flow season from northern San Francisco Bay, California. There were clear compositional differences in ...

Application of denaturing high-performance liquid chromatography for monitoring sulfate-reducing bacteria in oil fields.

PubMed

Priha, Outi; Nyyssönen, Mari; Bomberg, Malin; Laitila, Arja; Simell, Jaakko; Kapanen, Anu; Juvonen, Riikka

2013-09-01

Sulfate-reducing bacteria (SRB) participate in microbially induced corrosion (MIC) of equipment and H2S-driven reservoir souring in oil field sites. Successful management of industrial processes requires methods that allow robust monitoring of microbial communities. This study investigated the applicability of denaturing high-performance liquid chromatography (DHPLC) targeting the dissimilatory sulfite reductase ß-subunit (dsrB) gene for monitoring SRB communities in oil field samples from the North Sea, the United States, and Brazil. Fifteen of the 28 screened samples gave a positive result in real-time PCR assays, containing 9 × 10(1) to 6 × 10(5) dsrB gene copies ml(-1). DHPLC and denaturing gradient gel electrophoresis (DGGE) community profiles of the PCR-positive samples shared an overall similarity; both methods revealed the same samples to have the lowest and highest diversity. The SRB communities were diverse, and different dsrB compositions were detected at different geographical locations. The identified dsrB gene sequences belonged to several phylogenetic groups, such as Desulfovibrio, Desulfococcus, Desulfomicrobium, Desulfobulbus, Desulfotignum, Desulfonatronovibrio, and Desulfonauticus. DHPLC showed an advantage over DGGE in that the community profiles were very reproducible from run to run, and the resolved gene fragments could be collected using an automated fraction collector and sequenced without a further purification step. DGGE, on the other hand, included casting of gradient gels, and several rounds of rerunning, excising, and reamplification of bands were needed for successful sequencing. In summary, DHPLC proved to be a suitable tool for routine monitoring of the diversity of SRB communities in oil field samples.

Effects of a Campylobacter jejuni infection on the development of the intestinal microflora of broiler chickens.

PubMed

Johansen, C H; Bjerrum, L; Finster, K; Pedersen, K

2006-04-01

The effect of a Campylobacter jejuni colonization on the development of the microflora of the cecum and the ileum of broiler chickens was studied using molecular methods. The infection did affect the development and complexity of the microbial communities of the ceca, but we found no permanent effect of a C. jejuni infection on the ileal microflora of the broilers. In addition, denaturant gradient gel electrophoresis (DGGE) profiles generated from cecal and ileal contents revealed several DGGE bands that were present in the control chickens, but not in the chickens colonized with C. jejuni. Some of these DGGE bands could be affiliated with Lactobacillus reuteri, Clostridium perfringens, and the genus Klebsiella.

Comparison of automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) techniques for analysing the influence of diet on ruminal bacterial diversity.

PubMed

Saro, Cristina; Molina-Alcaide, Eduarda; Abecia, Leticia; Ranilla, María José; Carro, María Dolores

2018-04-01

The objective of this study was to compare the automated ribosomal intergenic spacer analysis (ARISA) and the denaturing gradient gel electrophoresis (DGGE) techniques for analysing the effects of diet on diversity in bacterial pellets isolated from the liquid (liquid-associated bacteria (LAB)) and solid (solid-associated bacteria (SAB)) phase of the rumen. The four experimental diets contained forage to concentrate ratios of 70:30 or 30:70 and had either alfalfa hay or grass hay as forage. Four rumen-fistulated animals (two sheep and two goats) received the diets in a Latin square design. Bacterial pellets (LAB and SAB) were isolated at 2 h post-feeding for DNA extraction and analysed by ARISA and DGGE. The number of peaks in individual samples ranged from 48 to 99 for LAB and from 41 to 95 for SAB with ARISA, and values of DGGE-bands ranged from 27 to 50 for LAB and from 18 to 45 for SAB. The LAB samples from high concentrate-fed animals tended (p < 0.10) to show greater peak numbers and Shannon index values than those isolated from high forage-fed animals with ARISA, but no differences were identified with DGGE. The SAB samples from high concentrate-fed animals had lower (p < 0.05) peak numbers and Shannon index values than those from animals fed high-forage diets with ARISA, but only a trend was noticed for these parameters with DGGE (p < 0.10). The ARISA detected that animals fed alfalfa hay diets showed lower (p < 0.05) SAB diversity than those fed grass hay diets, but no differences were observed with DGGE. No effect of forage type on LAB diversity was detected by any technique. In this study, ARISA detected some changes in ruminal bacterial communities that were not detected by DGGE, and therefore ARISA was considered more appropriate for assessing bacterial diversity of ruminal bacterial pellets. The results highlight the impact of the fingerprinting technique used to draw conclusions on dietary factors affecting bacterial diversity in ruminal bacterial pellets.

Population Structure and Abundance of Arsenite-Oxidizing Bacteria along an Arsenic Pollution Gradient in Waters of the Upper Isle River Basin, France▿ †

PubMed Central

Quéméneur, Marianne; Cébron, Aurélie; Billard, Patrick; Battaglia-Brunet, Fabienne; Garrido, Francis; Leyval, Corinne; Joulian, Catherine

2010-01-01

Denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR (qPCR) were successfully developed to monitor functional aoxB genes as markers of aerobic arsenite oxidizers. DGGE profiles showed a shift in the structure of the aoxB-carrying bacterial population, composed of members of the Alpha-, Beta- and Gammaproteobacteria, depending on arsenic (As) and Eh levels in Upper Isle River Basin waters. The highest aoxB gene densities were found in the most As-polluted oxic surface waters but without any significant correlation with environmental factors. Arsenite oxidizers seem to play a key role in As mobility in As-impacted waters. PMID:20453153

Comparison of benthic bacterial community composition in nine streams

Treesearch

Xueqing Gao; Ola A. Olapade; Laura G. Leff

2005-01-01

In this study, the abundance of major bacterial taxa (based on fluorescent in situ hybridization, FISH) and the structure of the bacterial community (based on denaturing gradient gel electrophoresis, DGGE) were determined in the benthos of 9 streams in the southeastern and midwestern United States and related to differences in environmental...

Camparison of benthic bacterial community composition in nine streams

Treesearch

Xuqing Gao; Ola A. Olapade; Laura G. Leff

2005-01-01

In this study, the abundance of major bacterial taxa (based on fluorescent in situ hybridization, FISH) and the structure of the bacterial community (based on denaturing gradient gel electrophoresis, DGGE) were determined in the benthos of 9 streams in the southeastern and midwestern United States and related to differences in environmental conditions. Taxa examined...

Culture-Independent Analysis of Probiotic Products by Denaturing Gradient Gel Electrophoresis

PubMed Central

Temmerman, R.; Scheirlinck, I.; Huys, G.; Swings, J.

2003-01-01

In order to obtain functional and safe probiotic products for human consumption, fast and reliable quality control of these products is crucial. Currently, analysis of most probiotics is still based on culture-dependent methods involving the use of specific isolation media and identification of a limited number of isolates, which makes this approach relatively insensitive, laborious, and time-consuming. In this study, a collection of 10 probiotic products, including four dairy products, one fruit drink, and five freeze-dried products, were subjected to microbial analysis by using a culture-independent approach, and the results were compared with the results of a conventional culture-dependent analysis. The culture-independent approach involved extraction of total bacterial DNA directly from the product, PCR amplification of the V3 region of the 16S ribosomal DNA, and separation of the amplicons on a denaturing gradient gel. Digital capturing and processing of denaturing gradient gel electrophoresis (DGGE) band patterns allowed direct identification of the amplicons at the species level. This whole culture-independent approach can be performed in less than 30 h. Compared with culture-dependent analysis, the DGGE approach was found to have a much higher sensitivity for detection of microbial strains in probiotic products in a fast, reliable, and reproducible manner. Unfortunately, as reported in previous studies in which the culture-dependent approach was used, a rather high percentage of probiotic products suffered from incorrect labeling and yielded low bacterial counts, which may decrease their probiotic potential. PMID:12513998

Compositions of maple sap microflora and collection system biofilms evaluated by scanning electron microscopy and denaturing gradient gel electrophoresis.

PubMed

Lagacé, L; Jacques, M; Mafu, A A; Roy, D

2006-05-25

The bacterial microflora of maple sap and biofilms in collection system tubing were studied through the use of bacterial counts, scanning electron microscopy (SEM) of surfaces and the analysis of 16S rRNA gene by denaturing gradient gel electrophoresis (DGGE). Samples were taken at five times during the 2002 and 2003 seasons in order to follow the changes in the microflora of this complex ecosystem. Bacterial counts showed the growth of bacterial populations as the season advanced. These populations were mainly composed of psychrotrophic bacteria and Pseudomonas spp. SEM results confirmed the suspected presence of biofilms on the inner surfaces of tubing samples. Bacterial colonization and biofilm formation progressively increased during the season for both lateral and main line surfaces, and biofilms were mainly composed of rod shape bacteria. The bacterial microflora profiles obtained for sap and corresponding biofilm by DGGE showed up to 12 major bands. The Shannon-Weaver index of diversity (H) calculated from DGGE bands were statistically higher for sap samples compared to biofilm. The diversity index was relatively stable or increasing for lateral line sap and biofilm samples during the season while the diversity index for sap and biofilm samples of the main line showed a decreasing profile as the season progressed. Sequence analysis of major DGGE bands revealed the predominance of bacteria from the genera Pseudomonas, Rahnella and another, unidentified genus. The results describe the composition of sap collection system microflora as well as the formation of biofilms and will be useful for further studies on factors affecting maple product quality.

16S rRNA PCR-Denaturing Gradient Gel Electrophoresis of Oral Lactobacillus casei Group and Their Phenotypic Appearances.

PubMed

Piwat, S; Teanpaisan, R

2013-01-01

This study aimed to develop a 16S rRNA PCR-denaturing gradient gel electrophoresis (DGGE) to identify the species level of Lactobacillus casei group and to investigate their characteristics of acid production and inhibitory effect. PCR-DGGE has been developed based on the 16S rRNA gene, and a set of HDA-1-GC and HDA-2, designed at V2-V3 region, and another set of CARP-1-GC and CARP-2, designed at V1 region, have been used. The bacterial strains included L. casei ATCC 393, L. paracasei CCUG 32212, L. rhamnosus ATCC 7469, L. zeae CCUG 35515, and 46 clinical strains of L. casei/paracasei/rhamnosus. Inhibitory effect against Streptococcus mutans and acid production were examined. Results revealed that each type species strain and identified clinical isolate showed its own unique DGGE pattern using CARP1-GC and CARP2 primers. HDA1-GC and HDA2 primers could distinguish the strains of L. paracasei from L. casei. It was found that inhibitory effect of L. paracasei was stronger than L. casei and L. rhamnosus. The acid production of L. paracasei was lower than L. casei and L. rhamnosus. In conclusion, the technique has been proven to be able to differentiate between closely related species in L. casei group and thus provide reliable information of their phenotypic appearances.

16S rRNA PCR-Denaturing Gradient Gel Electrophoresis of Oral Lactobacillus casei Group and Their Phenotypic Appearances

PubMed Central

Piwat, S.; Teanpaisan, R.

2013-01-01

This study aimed to develop a 16S rRNA PCR-denaturing gradient gel electrophoresis (DGGE) to identify the species level of Lactobacillus casei group and to investigate their characteristics of acid production and inhibitory effect. PCR-DGGE has been developed based on the 16S rRNA gene, and a set of HDA-1-GC and HDA-2, designed at V2-V3 region, and another set of CARP-1-GC and CARP-2, designed at V1 region, have been used. The bacterial strains included L. casei ATCC 393, L. paracasei CCUG 32212, L. rhamnosus ATCC 7469, L. zeae CCUG 35515, and 46 clinical strains of L. casei/paracasei/rhamnosus. Inhibitory effect against Streptococcus mutans and acid production were examined. Results revealed that each type species strain and identified clinical isolate showed its own unique DGGE pattern using CARP1-GC and CARP2 primers. HDA1-GC and HDA2 primers could distinguish the strains of L. paracasei from L. casei. It was found that inhibitory effect of L. paracasei was stronger than L. casei and L. rhamnosus. The acid production of L. paracasei was lower than L. casei and L. rhamnosus. In conclusion, the technique has been proven to be able to differentiate between closely related species in L. casei group and thus provide reliable information of their phenotypic appearances. PMID:24191230

Application of the denaturing gradient gel electrophoresis (DGGE) technique as an efficient diagnostic tool for ciliate communities in soil.

PubMed

Jousset, Alexandre; Lara, Enrique; Nikolausz, Marcell; Harms, Hauke; Chatzinotas, Antonis

2010-02-01

Ciliates (or Ciliophora) are ubiquitous organisms which can be widely used as bioindicators in ecosystems exposed to anthropogenic and industrial influences. The evaluation of the environmental impact on soil ciliate communities with methods relying on morphology-based identification may be hampered by the large number of samples usually required for a statistically supported, reliable conclusion. Cultivation-independent molecular-biological diagnostic tools are a promising alternative to greatly simplify and accelerate such studies. In this present work a ciliate-specific fingerprint method based on the amplification of a phylogenetic marker gene (i.e. the 18S ribosomal RNA gene) with subsequent analysis by denaturing gradient gel electrophoresis (DGGE) was developed and used to monitor community shifts in a polycyclic aromatic hydrocarbon (PAH) polluted soil. The semi-nested approach generated ciliate-specific amplification products from all soil samples and allowed to distinguish community profiles from a PAH-polluted and a non-polluted control soil. Subsequent sequence analysis of excised bands provided evidence that polluted soil samples are dominated by organisms belonging to the class Colpodea. The general DGGE approach presented in this study might thus in principle serve as a fast and reproducible diagnostic tool, complementing and facilitating future ecological and ecotoxicological monitoring of ciliates in polluted habitats. Copyright 2009 Elsevier B.V. All rights reserved.

Case Study of the Distribution of Mucosa-Associated Bifidobacterium Species, Lactobacillus Species, and Other Lactic Acid Bacteria in the Human Colon

PubMed Central

Nielsen, D. S.; Møller, P. L.; Rosenfeldt, V.; Pærregaard, A.; Michaelsen, K. F.; Jakobsen, M.

2003-01-01

The distribution of mucosa-associated bacteria, bifidobacteria and lactobacilli and closely related lactic acid bacteria, in biopsy samples from the ascending, transverse, and descending parts of the colon from four individuals was investigated by denaturing gradient gel electrophoresis (DGGE). Bifidobacterial genus-specific, Lactobacillus group-specific, and universal bacterial primers were used in a nested PCR approach to amplify a fragment of the 16S rRNA gene. DGGE profiles of the bifidobacterial community were relatively simple, with one or two amplicons detected at most sampling sites in the colon. DGGE profiles obtained with Lactobacillus group-specific primers were complex and varied with host and sampling site in the colon. The overall bacterial community varied with host but not sampling site. PMID:14660412

Association of Streptomyces community composition determined by PCR-denaturing gradient gel electrophoresis with indoor mold status

PubMed Central

Johansson, Elisabet; Reponen, Tiina; Meller, Jarek; Vesper, Stephen; Yadav, Jagjit

2014-01-01

Both Streptomyces species and mold species have previously been isolated from moisture-damaged building materials; however, an association between these two groups of microorganisms in indoor environments is not clear. In this study we used a culture-independent method, PCR denaturing gradient gel electrophoresis (PCR-DGGE) to investigate the composition of the Streptomyces community in house dust. Twenty-three dust samples each from two sets of homes categorized as high-mold and low-mold based on mold specific quantitative PCR-analysis were used in the study. Taxonomic identification of prominent bands was performed by cloning and sequencing. Associations between DGGE amplicon band intensities and home mold status were assessed using univariate analyses, as well as multivariate recursive partitioning (decision trees) to test the predictive value of combinations of bands intensities. In the final classification tree, a combination of two bands was significantly associated with mold status of the home (p = 0.001). The sequence corresponding to one of the bands in the final decision tree matched a group of Streptomyces species that included S. coelicolor and S. sampsonii, both of which have been isolated from moisture-damaged buildings previously. The closest match for the majority of sequences corresponding to a second band consisted of a group of Streptomyces species that included S. hygroscopicus, an important producer of antibiotics and immunosuppressors. Taken together, the study showed that DGGE can be a useful tool for identifying bacterial species that may be more prevalent in mold-damaged buildings. PMID:25331035

Bacterial community structure in the hyperarid core of the Atacama Desert, Chile

USGS Publications Warehouse

Drees, Kevin P.; Neilson, Julia W.; Betancourt, Julio L.; Quade, Jay; Henderson, David A.; Pryor, Barry M.; Maier, Raina M.

2006-01-01

Soils from the hyperarid Atacama Desert of northern Chile were sampled along an east-west elevational transect (23.75 to 24.70 degrees S) through the driest sector to compare the relative structure of bacterial communities. Analysis of denaturing gradient gel electrophoresis (DGGE) profiles from each of the samples revealed that microbial communities from the extreme hyperarid core of the desert clustered separately from all of the remaining communities. Bands sequenced from DGGE profiles of two samples taken at a 22-month interval from this core region revealed the presence of similar populations dominated by bacteria from the Gemmatimonadetes and Planctomycetes phyla.

Evaluation of different PCR primers for denaturing gradient gel electrophoresis (DGGE) analysis of fungal community structure in traditional fermentation starters used for Hong Qu glutinous rice wine.

PubMed

Lv, Xu-Cong; Jiang, Ya-Jun; Liu, Jie; Guo, Wei-Ling; Liu, Zhi-Bin; Zhang, Wen; Rao, Ping-Fan; Ni, Li

2017-08-16

Denaturing gradient gel electrophoresis (DGGE) has become a widely used tool to examine microbial community structure. However, when DGGE is applied to evaluate the fungal community of traditional fermentation starters, the choice of hypervariable ribosomal RNA gene regions is still controversial. In the current study, several previously published fungal PCR primer sets were compared and evaluated using PCR-DGGE, with the purpose of screening a suitable primer set to study the fungal community of traditional fermentation starters for Hong Qu glutinous rice wine. Firstly, different primer sets were used to amplify different hypervariable regions from pure fungal cultures. Except NS1/FR1+ and ITS1fGC/ITS4, other primer sets (NL1+/LS2R, NL3A/NL4GC, FF390/FR1+, NS1/GCFung, NS3+/YM951r and ITS1fGC/ITS2r) amplified the target DNA sequences successfully. Secondly, the selected primer sets were further evaluated based on their resolution to distinguish different fungal cultures through DGGE fingerprints. Three primer sets (NL1+/LS2R, NS1/GCFung and ITS1fGC/ITS2r) were finally selected for investigating the fungal community structure of different traditional fermentation starters for Hong Qu glutinous rice wine. The internal transcribed spacer (ITS) region amplified by ITS1fGC/ITS2r, which is more hypervariable than the 18S rRNA gene and 26S rRNA gene, provides an excellent tool to separate amplification products of different fungal species. Results indicated that PCR-DGGE profile using ITS1fGC/ITS2r showed more abundant fungal species than that using NL1+/LS2R and NS1/GCFung. Therefore, ITS1fGC/ITS2r is the most suitable primer set for PCR-DGGE analysis of fungal community structure in traditional fermentation starters for Hong Qu glutinous rice wine. DGGE profiles based on ITS1fGC/ITS2r revealed the presence of twenty-four fungal species in traditional fermentation starter. A significant difference of fungal community can be observed directly from DGGE fingerprints and principal component analysis. The statistical analysis results based on the band intensities of fungal DGGE profile showed that Saccharomyces cerevisiae, Saccharomycopsis fibuligera, Rhizopus oryzae, Monascus purpureus and Aspergillus niger were the dominant fungal species. In conclusion, the comparison of several primer sets for fungal PCR-DGGE would be useful to enrich our knowledge of the fungal community structures associated with traditional fermentation starters, which may facilitate the development of better starter cultures for manufacturing Chinese Hong Qu glutinous rice wine. Copyright © 2017 Elsevier B.V. All rights reserved.

Prescreening of microbial populations for the assessment of sequencing potential.

PubMed

Hanning, Irene B; Ricke, Steven C

2011-01-01

Next-generation sequencing (NGS) is a powerful tool that can be utilized to profile and compare microbial populations. By amplifying a target gene present in all bacteria and subsequently sequencing amplicons, the bacteria genera present in the populations can be identified and compared. In some scenarios, little to no difference may exist among microbial populations being compared in which case a prescreening method would be practical to determine which microbial populations would be suitable for further analysis by NGS. Denaturing density-gradient electrophoresis (DGGE) is relatively cheaper than NGS and the data comparing microbial populations are ready to be viewed immediately after electrophoresis. DGGE follows essentially the same initial methodology as NGS by targeting and amplifying the 16S rRNA gene. However, as opposed to sequencing amplicons, DGGE amplicons are analyzed by electrophoresis. By prescreening microbial populations with DGGE, more efficient use of NGS methods can be accomplished. In this chapter, we outline the protocol for DGGE targeting the same gene (16S rRNA) that would be targeted for NGS to compare and determine differences in microbial populations from a wide range of ecosystems.

Successive changes in community structure of an ethylbenzene-degrading sulfate-reducing consortium.

PubMed

Nakagawa, Tatsunori; Sato, Shinya; Yamamoto, Yoko; Fukui, Manabu

2002-06-01

The microbial community structure and successive changes in a mesophilic ethylbenzene-degrading sulfate-reducing consortium were for the first time clarified by the denaturing gradient gel electrophoresis (DGGE) analysis of the PCR amplified 16S rRNA gene fragments. At least ten bands on the DGGE gel were detected in the stationary phase. Phylogenetic analysis of the DGGE bands revealed that the consortium consisted of different eubacterial phyla including the delta subgroup of Proteobacteria, the order Sphingobacteriales, the order Spirochaetales, and the unknown bacterium. The most abundant band C was closely related to strain mXyS1, an m-xylene-degrading sulfate-reducing bacterium (SRB), and occurred as a sole band on DGGE gels in the logarithmic growth phase that 40% ethylbenzene was consumed accompanied by sulfide production. During further prolonged incubation, the dominancy of band C did not change. These results suggest that SRB corresponds to the most abundant band C and contributes mainly to the degradation of ethylbenzene coupled with sulfate reduction.

Geobacteraceae Community Composition Is Related to Hydrochemistry and Biodegradation in an Iron-Reducing Aquifer Polluted by a Neighboring Landfill†

PubMed Central

Lin, Bin; Braster, Martin; van Breukelen, Boris M.; van Verseveld, Henk W.; Westerhoff, Hans V.; Röling, Wilfred F. M.

2005-01-01

Relationships between community composition of the iron-reducing Geobacteraceae, pollution levels, and the occurrence of biodegradation were established for an iron-reducing aquifer polluted with landfill leachate by using cultivation-independent Geobacteraceae 16S rRNA gene-targeting techniques. Numerical analysis of denaturing gradient gel electrophoresis (DGGE) profiles and sequencing revealed a high Geobacteraceae diversity and showed that community composition within the leachate plume differed considerably from that of the unpolluted aquifer. This suggests that pollution has selected for specific species out of a large pool of Geobacteraceae. DGGE profiles of polluted groundwater taken near the landfill (6- to 39-m distance) clustered together. DGGE profiles from less-polluted groundwater taken further downstream did not fall in the same cluster. Several individual DGGE bands were indicative of either the redox process or the level of pollution. This included a pollution-indicative band that dominated the DGGE profiles from groundwater samples taken close to the landfill (6 to 39 m distance). The clustering of these profiles and the dominance by a single DGGE band corresponded to the part of the aquifer where organic micropollutants and reactive dissolved organic matter were attenuated at relatively high rates. PMID:16204512

Bulk and Rhizosphere Soil Bacterial Communities Studied by Denaturing Gradient Gel Electrophoresis: Plant-Dependent Enrichment and Seasonal Shifts Revealed

PubMed Central

Smalla, K.; Wieland, G.; Buchner, A.; Zock, A.; Parzy, J.; Kaiser, S.; Roskot, N.; Heuer, H.; Berg, G.

2001-01-01

The bacterial rhizosphere communities of three host plants of the pathogenic fungus Verticillium dahliae, field-grown strawberry (Fragaria ananassa Duch.), oilseed rape (Brassica napus L.), and potato (Solanum tuberosum L.), were analyzed. We aimed to determine the degree to which the rhizosphere effect is plant dependent and whether this effect would be increased by growing the same crops in two consecutive years. Rhizosphere or soil samples were taken five times over the vegetation periods. To allow a cultivation-independent analysis, total community DNA was extracted from the microbial pellet recovered from root or soil samples. 16S rDNA fragments amplified by PCR from soil or rhizosphere bacterium DNA were analyzed by denaturing gradient gel electrophoresis (DGGE). The DGGE fingerprints showed plant-dependent shifts in the relative abundance of bacterial populations in the rhizosphere which became more pronounced in the second year. DGGE patterns of oilseed rape and potato rhizosphere communities were more similar to each other than to the strawberry patterns. In both years seasonal shifts in the abundance and composition of the bacterial rhizosphere populations were observed. Independent of the plant species, the patterns of the first sampling times for both years were characterized by the absence of some of the bands which became dominant at the following sampling times. Bacillus megaterium and Arthrobacter sp. were found as predominant populations in bulk soils. Sequencing of dominant bands excised from the rhizosphere patterns revealed that 6 out of 10 bands resembled gram-positive bacteria. Nocardia populations were identified as strawberry-specific bands. PMID:11571180

Bacterial Population Changes in a Membrane Bioreactor for Graywater Treatment Monitored by Denaturing Gradient Gel Electrophoretic Analysis of 16S rRNA Gene Fragments

PubMed Central

Stamper, David M.; Walch, Marianne; Jacobs, Rachel N.

2003-01-01

The bacterial population of a graywater treatment system was monitored over the course of 100 days, along with several wastewater biochemical parameters. The graywater treatment system employed an 1,800-liter membrane bioreactor (MBR) to process the waste, with essentially 100% recycling of the biomass. Graywater feed consisting of 10% galley water and 90% laundry water, selected to approximate the graywater composition on board U.S. Navy ships, was collected offsite. Five-day biological oxygen demand (BOD5), oils and greases (O/G), nitrogen, and phosphorus were monitored in the feed and were found to vary greatly day to day. Changes in the bacterial population were monitored by PCR amplification of region 332 to 518 (Escherichia coli numbering) of the 16S rRNA gene and denaturing gradient gel electrophoresis (DGGE) analysis of the resultant PCR products. DGGE analysis indicated a diverse and unstable bacterial population throughout the 100-day period, with spikes in feed strength causing significant changes in community structure. Long-term similarity between the communities was 0 to 25%, depending on the method of analysis. In spite of the unstable bacterial population, the MBR system was able to meet effluent quality parameters approximately 90% of the time. PMID:12571004

Bacterial population changes in a membrane bioreactor for graywater treatment monitored by denaturing gradient gel electrophoretic analysis of 16S rRNA gene fragments.

PubMed

Stamper, David M; Walch, Marianne; Jacobs, Rachel N

2003-02-01

The bacterial population of a graywater treatment system was monitored over the course of 100 days, along with several wastewater biochemical parameters. The graywater treatment system employed an 1,800-liter membrane bioreactor (MBR) to process the waste, with essentially 100% recycling of the biomass. Graywater feed consisting of 10% galley water and 90% laundry water, selected to approximate the graywater composition on board U.S. Navy ships, was collected offsite. Five-day biological oxygen demand (BOD(5)), oils and greases (O/G), nitrogen, and phosphorus were monitored in the feed and were found to vary greatly day to day. Changes in the bacterial population were monitored by PCR amplification of region 332 to 518 (Escherichia coli numbering) of the 16S rRNA gene and denaturing gradient gel electrophoresis (DGGE) analysis of the resultant PCR products. DGGE analysis indicated a diverse and unstable bacterial population throughout the 100-day period, with spikes in feed strength causing significant changes in community structure. Long-term similarity between the communities was 0 to 25%, depending on the method of analysis. In spite of the unstable bacterial population, the MBR system was able to meet effluent quality parameters approximately 90% of the time.

Detection and Identification of Lactobacillus Species in Crops of Broilers of Different Ages by Using PCR-Denaturing Gradient Gel Electrophoresis and Amplified Ribosomal DNA Restriction Analysis

PubMed Central

Guan, Le Luo; Hagen, Karen E.; Tannock, Gerald W.; Korver, Doug R.; Fasenko, Gaylene M.; Allison, Gwen E.

2003-01-01

The microflora of the crop was investigated throughout the broiler production period (0 to 42 days) using PCR combined with denaturing gradient gel electrophoresis (PCR-DGGE) and selective bacteriological culture of lactobacilli followed by amplified ribosomal DNA restriction analysis (ARDRA). The birds were raised under conditions similar to those used in commercial broiler production. Lactobacilli predominated and attained populations of 108 to 109 CFU per gram of crop contents. Many of the lactobacilli present in the crop (61.9% of isolates) belonged to species of the Lactobacillus acidophilus group and could not be differentiated by PCR-DGGE. A rapid and simple ARDRA method was developed to distinguish between the members of the L. acidophilus group. HaeIII-ARDRA was used for preliminary identification of isolates in the L. acidophilus group and to identify Lactobacillus reuteri and Lactobacillus salivarius. MseI-ARDRA generated unique patterns for all species of the L. acidophilus group, identifying Lactobacillus crispatus, Lactobacillus johnsonii, and Lactobacillus gallinarum among crop isolates. The results of our study provide comprehensive knowledge of the Lactobacillus microflora in the crops of birds of different ages using nucleic acid-based methods of detection and identification based on current taxonomic criteria. PMID:14602636

Denaturing gradient gel electrophoresis and barcoded pyrosequencing reveal unprecedented archaeal diversity in mangrove sediment and rhizosphere samples.

PubMed

Pires, Ana C C; Cleary, Daniel F R; Almeida, Adelaide; Cunha, Angela; Dealtry, Simone; Mendonça-Hagler, Leda C S; Smalla, Kornelia; Gomes, Newton C M

2012-08-01

Mangroves are complex ecosystems that regulate nutrient and sediment fluxes to the open sea. The importance of bacteria and fungi in regulating nutrient cycles has led to an interest in their diversity and composition in mangroves. However, very few studies have assessed Archaea in mangroves, and virtually nothing is known about whether mangrove rhizospheres affect archaeal diversity and composition. Here, we studied the diversity and composition of Archaea in mangrove bulk sediment and the rhizospheres of two mangrove trees, Rhizophora mangle and Laguncularia racemosa, using denaturing gradient gel electrophoresis (DGGE) and pyrosequencing of archaeal 16S rRNA genes with a nested-amplification approach. DGGE profiles revealed significant structural differences between bulk sediment and rhizosphere samples, suggesting that roots of both mangrove species influence the sediment archaeal community. Nearly all of the detected sequences obtained with pyrosequencing were identified as Archaea, but most were unclassified at the level of phylum or below. Archaeal richness was, furthermore, the highest in the L. racemosa rhizosphere, intermediate in bulk sediment, and the lowest in the R. mangle rhizosphere. This study shows that rhizosphere microhabitats of R. mangle and L. racemosa, common plants in subtropical mangroves located in Rio de Janeiro, Brazil, hosted distinct archaeal assemblages.

Denaturing Gradient Gel Electrophoresis and Barcoded Pyrosequencing Reveal Unprecedented Archaeal Diversity in Mangrove Sediment and Rhizosphere Samples

PubMed Central

Pires, Ana C. C.; Cleary, Daniel F. R.; Almeida, Adelaide; Cunha, Ângela; Dealtry, Simone; Mendonça-Hagler, Leda C. S.; Smalla, Kornelia

2012-01-01

Mangroves are complex ecosystems that regulate nutrient and sediment fluxes to the open sea. The importance of bacteria and fungi in regulating nutrient cycles has led to an interest in their diversity and composition in mangroves. However, very few studies have assessed Archaea in mangroves, and virtually nothing is known about whether mangrove rhizospheres affect archaeal diversity and composition. Here, we studied the diversity and composition of Archaea in mangrove bulk sediment and the rhizospheres of two mangrove trees, Rhizophora mangle and Laguncularia racemosa, using denaturing gradient gel electrophoresis (DGGE) and pyrosequencing of archaeal 16S rRNA genes with a nested-amplification approach. DGGE profiles revealed significant structural differences between bulk sediment and rhizosphere samples, suggesting that roots of both mangrove species influence the sediment archaeal community. Nearly all of the detected sequences obtained with pyrosequencing were identified as Archaea, but most were unclassified at the level of phylum or below. Archaeal richness was, furthermore, the highest in the L. racemosa rhizosphere, intermediate in bulk sediment, and the lowest in the R. mangle rhizosphere. This study shows that rhizosphere microhabitats of R. mangle and L. racemosa, common plants in subtropical mangroves located in Rio de Janeiro, Brazil, hosted distinct archaeal assemblages. PMID:22660713

Analysis of microbial diversity in Shenqu with different fermentation times by PCR-DGGE.

PubMed

Liu, Tengfei; Jia, Tianzhu; Chen, Jiangning; Liu, Xiaoyu; Zhao, Minjie; Liu, Pengpeng

Shenqu is a fermented product that is widely used in traditional Chinese medicine (TCM) to treat indigestion; however, the microbial strains in the fermentation process are still unknown. The aim of this study was to investigate microbial diversity in Shenqu using different fermentation time periods. DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) profiles indicated that a strain of Pediococcus acidilactici (band 9) is the predominant bacteria during fermentation and that the predominant fungi were uncultured Rhizopus, Aspergillus oryzae, and Rhizopus oryzae. In addition, pathogenic bacteria, such as Enterobacter cloacae, Klebsiella oxytoca, Erwinia billingiae, and Pantoea vagan were detected in Shenqu. DGGE analysis showed that bacterial and fungal diversity declined over the course of fermentation. This determination of the predominant bacterial and fungal strains responsible for fermentation may contribute to further Shenqu research, such as optimization of the fermentation process. Copyright © 2017. Published by Elsevier Editora Ltda.

Association of Atopobium vaginae, a recently described metronidazole resistant anaerobe, with bacterial vaginosis

PubMed Central

Ferris, Michael J; Masztal, Alicia; Aldridge, Kenneth E; Fortenberry, J Dennis; Fidel, Paul L; Martin, David H

2004-01-01

Background Bacterial vaginosis (BV) is a polymicrobial syndrome characterized by a change in vaginal flora away from predominantly Lactobacillus species. The cause of BV is unknown, but the condition has been implicated in diverse medical outcomes. The bacterium Atopobium vaginae has been recognized only recently. It is not readily identified by commercial diagnostic kits. Its clinical significance is unknown but it has recently been isolated from a tuboovarian abcess. Methods Nucleotide sequencing of PCR amplified 16S rRNA gene segments, that were separated into bands within lanes on polyacrylamide gels by denaturing gradient gel electrophoresis (DGGE), was used to examine bacterial vaginal flora in 46 patients clinically described as having normal (Lactobacillus spp. predominant; Nugent score ≤ 3) and abnormal flora (Nugent score ≥ 4). These women ranged in age from 14 to 48 and 82% were African American. Results The DGGE banding patterns of normal and BV-positive patients were recognizably distinct. Those of normal patients contained 1 to 4 bands that were focused in the centre region of the gel lane, while those of BV positive patients contained bands that were not all focused in the center region of the gel lane. More detailed analysis of patterns revealed that bands identified as Atopobium vaginae were present in a majority (12/22) of BV positive patients, while corresponding bands were rare (2/24) in normal patients. (P < 0.001) Two A. vaginae isolates were cultivated from two patients whose DGGE analyses indicated the presence of this organism. Two A. vaginae 16S rRNA gene sequences were identified among the clinical isolates. The same two sequences were obtained from DGGE bands of the corresponding vaginal flora. The sequences differed by one nucleotide over the short (~300 bp) segment used for DGGE analysis and migrated to slightly different points in denaturing gradient gels. Both isolates were strict anaerobes and highly metronidazole resistant. Conclusion The results suggest that A. vaginae may be an important component of the complex bacterial ecology that constitutes abnormal vaginal flora. This organism could play a role in treatment failure if further studies confirm it is consistently metronidozole resistant. PMID:15018635

Denaturing Gradient Gel Electrophoresis-Polymerase Chain Reaction Comparison of Chitosan Effects on Anaerobic Cultures of Broiler Cecal Bacteria and Salmonella Typhimurium.

PubMed

Hume, Michael; Sohail, Muhammad Umar

2018-04-01

Enteropathogen colonization and product contamination are major poultry industry problems. The emergence of antibiotic resistance, and associated risks to human health, is limiting the use of antibiotics as first-line defense against enteropathogens in poultry. The chitin derivative, chitosan, has drawn substantial attention for its bactericidal properties. Different molecular weight (MW) chitosans can have varied effects against different bacteria in monoculture. In the current study, cecal contents from each of three market-age broilers and Salmonella Typhimurium, as indicator enteropathogen, were exposed to in vitro anaerobic culture to three chitosan preparations (0.08%, wt/vol), low (LMW), medium (MMW), and coarse (CMW). Effects of chitosan and the carrier solvent acetic acid, on cecal bacteria and Salmonella, were examined by denaturing gradient gel electrophoresis (DGGE) and Salmonella enumeration. Bacterial profiles for the three cecal contents were shown by DGGE to be very different. Each of the three cecal contents grown in the presence of 0.08% acetic acid was very different from the same contents grown without the chitosan solvent. Culturing cecal contents in the presence of chitosan altered the bacterial DGGE profiles from the control and acetic acid-only cultures. The DGGE chitosan-treated profiles for all three cecal sources were identical to each other regardless of the MW chitosan in the culture medium. Compared with Salmonella in monoculture, Salmonella decreased (p < 0.05) by about 1.5 log CFU/mL when grown in mixed culture with cecal contents. Salmonella monocultures in the presence of 0.08% of the chitosan solvent acetic acid decreased (p < 0.05) counts by almost 3.5 log CFU/mL. Combining acetic acid and cecal contents reduced (p < 0.05) Salmonella by 7 log CFU/mL. Adding the chitosan preparations to the mixtures reduced (p < 0.05) Salmonella by 8 log CFU/mL.

Different substrates and starter inocula govern microbial community structures in biogas reactors.

PubMed

Satpathy, Preseela; Steinigeweg, Sven; Cypionka, Heribert; Engelen, Bert

2016-01-01

The influence of different starter inocula on the microbial communities in biogas batch reactors fed with fresh maize and maize silage as substrates was investigated. Molecular biological analysis by Denaturing Gradient Gel Electrophoresis (DGGE) of 16S rRNA gene fragments showed that each inoculum bore specific microbial communities with varying predominant phylotypes. Both, bacterial and archaeal DGGE profiles displayed three distinct communities that developed depending on the type of inoculum. Although maize and silage are similar substrates, different communities dominated the lactate-rich silage compared to lactate-free fresh maize. Cluster analysis of DGGE gels showed the communities of the same substrates to be stable with their respective inoculum. Bacteria-specific DGGE analysis revealed a rich diversity with Firmicutes being predominant. The other abundant phylotypes were Bacteroidetes and Synergistetes. Archaea-specific DGGE analysis displayed less diverse community structures, identifying members of the Methanosarcinales as the dominant methanogens present in all the three biogas digesters. In general, the source of inoculum played a significant role in shaping microbial communities. Adaptability of the inoculum to the substrates fed also influenced community compositions which further impacted the rates of biogas production.

Diversity and dynamics of antibiotic-resistant bacteria in cheese as determined by PCR denaturing gradient gel electrophoresis.

PubMed

Flórez, Ana Belén; Mayo, Baltasar

2015-12-02

This work reports the composition and succession of tetracycline- and erythromycin-resistant bacterial communities in a model cheese, monitored by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Bacterial 16S rRNA genes were examined using this technique to detect structural changes in the cheese microbiota over manufacturing and ripening. Total bacterial genomic DNA, used as a template, was extracted from cultivable bacteria grown without and with tetracycline or erythromycin (both at 25 μg ml(-1)) on a non-selective medium used for enumeration of total and viable cells (Plate Count agar with Milk; PCA-M), and from those grown on selective and/or differential agar media used for counting various bacterial groups; i.e., lactic acid bacteria (de Man, Rogosa and Sharpe agar; MRSA), micrococci and staphylococci (Baird-Parker agar; BPA), and enterobacteria (Violet Red Bile Glucose agar; VRBGA). Large numbers of tetracycline- and erythromycin-resistant bacteria were detected in cheese samples at all stages of ripening. Counts of antibiotic-resistant bacteria varied widely depending on the microbial group and the point of sampling. In general, resistant bacteria were 0.5-1.0 Log10 units fewer in number than the corresponding susceptible bacteria. The PCR-DGGE profiles obtained with DNA isolated from the plates for total bacteria and the different bacterial groups suggested Escherichia coli, Lactococcus lactis, Enterococcus faecalis and Staphylococcus spp. as the microbial types resistant to both antibiotics tested. This study shows the suitability of the PCR-DGGE technique for rapidly identifying and tracking antibiotic resistant populations in cheese and, by extension, in other foods. Copyright © 2015 Elsevier B.V. All rights reserved.

Association of diverse bacterial communities in human bile samples with biliary tract disorders: a survey using culture and polymerase chain reaction-denaturing gradient gel electrophoresis methods.

PubMed

Tajeddin, E; Sherafat, S J; Majidi, M R S; Alebouyeh, M; Alizadeh, A H M; Zali, M R

2016-08-01

Bacterial infection is considered a predisposing factor for disorders of the biliary tract. This study aimed to determine the diversity of bacterial communities in bile samples and their involvement in the occurrence of biliary tract diseases. A total of 102 bile samples were collected during endoscopic retrograde cholangiopancreatography (ERCP). Characterization of bacteria was done using culture and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) methods. Antimicrobial susceptibility of the isolates was determined based on the Clinical and Laboratory Standards Institute (CLSI) guidelines and identity of the nucleotide sequences of differentiated bands from the DGGE gels was determined based on GenBank data. In total, 41.2 % (42/102) of the patients showed bacterial infection in their bile samples. This infection was detected in 21 % (4/19), 45.4 % (5/11), 53.5 % (15/28), and 54.5 % (24/44) of patients with common bile duct stone, microlithiasis, malignancy, and gallbladder stone, respectively. Escherichia coli showed a significant association with gallstones. Polymicrobial infection was detected in 48 % of the patients. While results of the culture method established coexistence of biofilm-forming bacteria (Pseudomonas aeruginosa, E. coli, Klebsiella pneumoniae, Enterococcus spp., and Acinetobacter spp.) in different combinations, the presence of Capnocytophaga spp., Lactococcus spp., Bacillus spp., Staphylococcus haemolyticus, Enterobacter or Citrobacter spp., Morganella spp., Salmonella spp., and Helicobacter pylori was also characterized in these samples by the PCR-DGGE method. Multidrug resistance phenotypes (87.5 %) and resistance to third- and fourth-generation cephalosporins and quinolones were common in these strains, which could evolve through their selection by bile components. Ability for biofilm formation seems to be a need for polymicrobial infection in this organ.

The use of PCR-DGGE to determine bacterial fingerprints for poultry and red meat abattoir effluent.

PubMed

de Smidt, O

2016-01-01

Strict legislation and chemical composition monitoring of effluent may be useful, but the data generated do not allow for source tracking, and enforcing legislation remains problematic in the South African setting. These difficulties emphasize the necessity for effluent source traceability. Denaturing gradient gel electrophoresis (DGGE) targeting the V3 region of the 16S rRNA gene was considered as fingerprinting technique for effluent originating from abattoirs slaughtering different animal species. The influence of treatment to remove excess fat from effluent prior to molecular analyses and different PCR approaches on the detection of bacterial diversity were considered. Use of a treatment option to remove fat and a nested PCR approach resulted in up to 51% difference in inter-sample diversity similarity. A robust approach with no pre-treatment to remove PCR inhibitors, such as fat, and direct amplification from genomic DNA yielded optimal/maximal bacterial diversity fingerprints. Repeatable fingerprints were obtained for poultry abattoir effluent over a 4-month period, but profiles for the red meat abattoir varied with maximum similarity detected only 33·2%. Genetic material from faecal indicators Aeromona spp and Clostridium spp were detected. Genera unique to each effluent were present; Anoxybacillus, Patulibacter and Oleispira in poultry abattoir effluent and Porphyromonas and Peptostreptococcus in red meat abattoir effluent. This study was the first to demonstrate the application of denaturing gradient gel electrophoresis (DGGE) to construct bacterial diversity fingerprints for high-throughput abattoir effluents. Proved redundancy of fat removal as PCR inhibitor and change in diversity similarity introduced by nested PCR approach. The importance of limiting excessive handling/processing which could lead to misrepresented diversity profiles was emphasized. © 2015 The Society for Applied Microbiology.

Investigating Freshwater Periphyton Community Response to Uranium with Phospholipid Fatty Acid and Denaturing Gradient Gel Electrophoresis Analyses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Small, Jack A.; Bunn, Amoret L.; McKinstry, Craig A.

2008-04-01

Periphyton communities can be used as monitors of ecosystem health and as indicators of contamination in lotic systems. Measures of biomass, community structure and genetic diversity were used to investigate impacts of uranium exposure on periphyton. Laboratory exposures of periphyton in river water amended with uranium were performed for 5 days, followed by 2 days of uranium depuration in unamended river water. Productivity as measured by biomass was not affected by concentrations up to 100 µg L-1 uranium. Phospholipid fatty acid (PLFA) profiles and denaturing gradient gel electrophoresis (DGGE) banding patterns found no changes in community or genetic structure relatedmore » to uranium exposure. We suggest that the periphyton community as a whole is not impacted by exposures of uranium up to a dose of 100 µg L-1. These findings have significance for the assessment and prediction of uranium impacts on aquatic ecosystems.« less

Microbiota during fermentation of chum salmon (Oncorhynchus keta) sauce mash inoculated with halotolerant microbial starters: analyses using the plate count method and PCR-denaturing gradient gel electrophoresis (DGGE).

PubMed

Yoshikawa, Shuji; Yasokawa, Daisuke; Nagashima, Koji; Yamazaki, Koji; Kurihara, Hideyuki; Ohta, Tomoki; Kawai, Yuji

2010-06-01

Nine different combinations of mugi koji (barley steamed and molded with Aspergillus oryzae) and halotolerant microorganisms (HTMs), Zygosaccharomyces rouxii, Candida versatilis, and Tetragenococcus halophilus, were inoculated into chum salmon sauce mash under a non-aseptic condition used in industrial fish sauce production and fermented at 35 +/- 2.5 degrees C for 84 days to elucidate the microbial dynamics (i.e., microbial count and microbiota) during fermentation. The viable count of halotolerant yeast (HTY) in fermented chum salmon sauce (FCSS) mash showed various time courses dependent on the combination of the starter microorganisms. Halotolerant lactic acid bacteria (HTL) were detected morphologically and physiologically only from FCSS mash inoculated with T. halophilus alone or with T. halophilus and C. versatilis during the first 28 days of fermentation. Only four fungal species, Z. rouxii, C. versatilis, Pichia guilliermondii, and A. oryzae, were detected throughout the fermentation by PCR-denaturing gradient gel electrophoresis (PCR-DGGE). In FCSS mash, dominant HTMs, especially eumycetes, were nonexistent. However, under the non-aseptic conditions, undesirable wild yeast such as P. guilliermondii grew fortuitously. Therefore, HTY inoculation into FCSS mash at the beginning of fermentation is effective in preventing the growth of wild yeast and the resultant unfavorable flavor. 2009 Elsevier Ltd. All rights reserved.

Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

NASA Technical Reports Server (NTRS)

Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Said; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

2002-01-01

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

Parallel Characterization of Anaerobic Toluene- and Ethylbenzene-Degrading Microbial Consortia by PCR-Denaturing Gradient Gel Electrophoresis, RNA-DNA Membrane Hybridization, and DNA Microarray Technology

PubMed Central

Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Saïd; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

2002-01-01

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis. PMID:12088997

Monitoring the Bacterial Population Dynamics in Sourdough Fermentation Processes by Using PCR-Denaturing Gradient Gel Electrophoresis

PubMed Central

Meroth, Christiane B.; Walter, Jens; Hertel, Christian; Brandt, Markus J.; Hammes, Walter P.

2003-01-01

Four sourdoughs (A to D) were produced under practical conditions by using a starter mixture of three commercially available sourdough starters and a baker's yeast constitutively containing various species of lactic acid bacteria (LAB). The sourdoughs were continuously propagated until the composition of the LAB flora remained stable. Two LAB-specific PCR-denaturing gradient gel electrophoresis (DGGE) systems were established and used to monitor the development of the microflora. Depending on the prevailing ecological conditions in the different sourdough fermentations, only a few Lactobacillus species were found to be competitive and became dominant. In sourdough A (traditional process with rye flour), Lactobacillus sanfranciscensis and a new species, L. mindensis, were detected. In rye flour sourdoughs B and C, which differed in the process temperature, exclusively L. crispatus and L. pontis became the predominant species in sourdough B and L. crispatus, L. panis, and L. frumenti became the predominant species in sourdough C. On the other hand, in sourdough D (corresponding to sourdough C but produced with rye bran), L. johnsonii and L. reuteri were found. The results of PCR-DGGE were consistent with those obtained by culturing, except for sourdough B, in which L. fermentum was also detected. Isolates of the species L. sanfranciscensis and L. fermentum were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively. PMID:12514030

Clinical signs and bacterial communities of deciduous necrotic root canals detected by PCR-DGGE analysis: research association.

PubMed

de Paula, Viviane Andrade Cancio; de Carvalho Ferreira, Dennis; Cavalcante, Fernanda Sampaio; do Carmo, Flávia Lima; Rosado, Alexandre Soares; Primo, Laura Guimarães; dos Santos, Kátia Regina Netto

2014-08-01

This study sought to investigate the possible association between clinical and radiographic data of the patients with the bacterial community profiles involved in cases of necrosis in primary root canals. Microbial community profiles for 25 samples from necrotic deciduous root canals were analyzed using the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprinting approach. These results were related to the clinical and radiographic data of these patients. The analysis showed a large diversity of microbial communities in necrotic deciduous root canals. The statistical results pointed out that posterior and anterior teeth were associated with <20 bands and >20 bands in PCR-DGGE method, respectively. A relationship was verified between ages >4 years old and posterior teeth and, ages ≤4 years old and anterior teeth. The data showed a polymicrobial community and pointed out the association of age with necrosis in anterior and posterior teeth. Copyright © 2014 Elsevier Ltd. All rights reserved.

PCR-DGGE analysis of the microbial communities in three different Chinese "Baiyunbian" liquor fermentation starters.

PubMed

Xiong, Xiaomao; Hu, Yuanliang; Yan, Nanfeng; Huang, Yingna; Peng, Nan; Liang, Yunxiang; Zhao, Shumiao

2014-08-01

A systematic investigation was performed on the bacterial, Bacillus, fungal, and yeast communities of the three types of Daqu (mechanically prepared, manually prepared, and mixed prepared) used in Baiyunbian Company by reconditioning PCR-denaturing gradient gel electrophoresis (PCR-DGGE). The DGGE results showed that the microbes in the three types of Daqu were mainly thermotolerant and thermophilic microbes, and the most dominant bacterial species were Bacillus and Virgibacillus, followed by Lactobacillus and Trichococcus. Furthermore, the dominant fungi were found to be molds, such as Rasamsonia, Penicillium, Aspergillus, and Monascus, and the dominant yeasts were Saccharomyces cerevisiae, Saccharomycopsis fibuligera, Pichia anomala, and Debaryomyces hansenii. In general, the three types of Daqu showed slight differences in microbial communities, and the Shannon indexes (H') of the manually prepared and mechanically prepared Daqu were similar. The results suggest that mechanically prepared Daqu can replace manually prepared Daqu in liquor production, and this research provides useful information for liquor production and process improvement.

Application of rDNA-PCR amplification and DGGE fingerprinting for detection of microbial diversity in a Malaysian crude oil.

PubMed

Liew, Pauline Woanying; Jong, Bor Chyan

2008-05-01

Two culture-independent methods, namely ribosomal DNA libraries and denaturing gradient gel electrophoresis (DGGE), were adopted to examine the microbial community of a Malaysian light crude oil. In this study, both 16S and 18S rDNAs were PCR-amplified from bulk DNA of crude oil samples, cloned, and sequenced. Analyses of restriction fragment length polymorphism (RFLP) and phylogenetics clustered the 16S and 18S rDNA sequences into seven and six groups, respectively. The ribosomal DNA sequences obtained showed sequence similarity between 90 to 100% to those available in the GenBank database. The closest relatives documented for the 16S rDNAs include member species of Thermoincola and Rhodopseudomonas, whereas the closest fungal relatives include Acremonium, Ceriporiopsis, Xeromyces, Lecythophora, and Candida. Others were affiliated to uncultured bacteria and uncultured ascomycete. The 16S rDNA library demonstrated predomination by a single uncultured bacterial type by >80% relative abundance. The predomination was confirmed by DGGE analysis.

Analysis of a diverse assemblage of diazotrophic bacteria from Spartina alterniflora using DGGE and clone library screening.

PubMed

Lovell, Charles R; Decker, Peter V; Bagwell, Christopher E; Thompson, Shelly; Matsui, George Y

2008-05-01

Methods to assess the diversity of the diazotroph assemblage in the rhizosphere of the salt marsh cordgrass, Spartina alterniflora were examined. The effectiveness of nifH PCR-denaturing gradient gel electrophoresis (DGGE) was compared to that of nifH clone library analysis. Seventeen DGGE gel bands were sequenced and yielded 58 nonidentical nifH sequences from a total of 67 sequences determined. A clone library constructed using the GC-clamp nifH primers that were employed in the PCR-DGGE (designated the GC-Library) yielded 83 nonidentical sequences from a total of 257 nifH sequences. A second library constructed using an alternate set of nifH primers (N-Library) yielded 83 nonidentical sequences from a total of 138 nifH sequences. Rarefaction curves for the libraries did not reach saturation, although the GC-Library curve was substantially dampened and appeared to be closer to saturation than the N-Library curve. Phylogenetic analyses showed that DGGE gel band sequencing recovered nifH sequences that were frequently sampled in the GC-Library, as well as sequences that were infrequently sampled, and provided a species composition assessment that was robust, efficient, and relatively inexpensive to obtain. Further, the DGGE method permits a large number of samples to be examined for differences in banding patterns, after which bands of interest can be sampled for sequence determination.

Study of the bacterial diversity of foods: PCR-DGGE versus LH-PCR.

PubMed

Garofalo, Cristiana; Bancalari, Elena; Milanović, Vesna; Cardinali, Federica; Osimani, Andrea; Sardaro, Maria Luisa Savo; Bottari, Benedetta; Bernini, Valentina; Aquilanti, Lucia; Clementi, Francesca; Neviani, Erasmo; Gatti, Monica

2017-02-02

The present study compared two culture-independent methods, polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and length-heterogeneity polymerase chain reaction (LH-PCR), for their ability to reveal food bacterial microbiota. Total microbial DNA and RNA were extracted directly from fourteen fermented and unfermented foods, and domain A of the variable regions V1 and V2 of the 16S rRNA gene was analyzed through LH-PCR and PCR-DGGE. Finally, the outline of these analyses was compared with bacterial viable counts obtained after bacterial growth on suitable selective media. For the majority of the samples, RNA-based PCR-DGGE revealed species that the DNA-based PCR-DGGE was not able to highlight. When analyzing either DNA or RNA, LH-PCR identified several lactic acid bacteria (LAB) and coagulase negative cocci (CCN) species that were not identified by PCR-DGGE. This phenomenon was particularly evident in food samples with viable loads<5.0 Logcfug -1 . Furthermore, LH-PCR was able to detect a higher number of peaks in the analyzed food matrices relative to species identified by PCR-DGGE. In light of these findings, it may be suggested that LH-PCR shows greater sensitivity than PCR-DGGE. However, PCR-DGGE detected some other species (LAB included) that were not detected by LH-PCR. Therefore, certain LH-PCR peaks not attributed to known species within the LH-PCR database could be solved by comparing them with species identified by PCR-DGGE. Overall, this study also showed that LH-PCR is a promising method for use in the food microbiology field, indicating the necessity to expand the LH-PCR database, which is based, up to now, mainly on LAB isolates from dairy products. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of different primers for PCR-DGGE analysis of cheese-associated enterococci.

PubMed

Lorbeg, Petra Mohar; Majhenic, Andreja Canzek; Rogelj, Irena

2009-08-01

Enterococci represent an important part of bacterial microbiota in different types of artisanal cheeses, made from either raw or pasteurized milk. Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) of ribosomal DNA is currently one of the most frequently used fingerprinting method to study diversity and dynamics of microbial communities and also a tool for microbial identification. Among several primer pairs for DGGE analysis published so far, six primer pairs amplifying different variable regions of 16S rDNA were selected and applied in our DGGE analysis of 12 species belonging to genus Enterococcus and eight other bacterial species often found in cheeses (seven lactobacilli and one Lactoccocus lactis). When DGGE procedures were optimized, the same set of primers was used for DGGE analysis of five cheese samples. Our study demonstrates that the use of different primer pairs generate significant differences in DGGE analysis of enterococcal population, consequently, appropriate primers regarding the purpose of analysis can be selected. For differentiation and identification of pure enterococcal isolates, primer pair P1V1/P2V1 showed the most promising results since all 12 enterococcal isolates gave distinctive DGGE fingerprints, but with multiple bands patterns; therefore, these primers do not seem to be appropriate for identification of enterococcal species in mixed cultures. Use of primer pairs HDA1/HDA2 and V3f/V3r amplifying V3 region showed better potential for detection and identification of enterococci in mixed communities, but since some bacterial species showed the same fingerprint, for clear identification combination of DGGE and some other method (e.g. species specific PCR) or combined DGGE analysis using two primer pairs generating distinctive results should be used.

Bacterial population in traditional sourdough evaluated by molecular methods.

PubMed

Randazzo, C L; Heilig, H; Restuccia, C; Giudici, P; Caggia, C

2005-01-01

To study the microbial communities in artisanal sourdoughs, manufactured by traditional procedure in different areas of Sicily, and to evaluate the lactic acid bacteria (LAB) population by classical and culture-independent approaches. Forty-five LAB isolates were identified both by phenotypic and molecular methods. The restriction fragment length polymorphism and 16S ribosomal DNA gene sequencing gave evidence of a variety of species with the dominance of Lactobacillus sanfranciscensis and Lactobacillus pentosus, in all sourdoughs tested. Culture-independent method, such as denaturing gradient gel electrophoresis (DGGE) of the V6-V8 regions of the 16S rDNA, was applied for microbial community fingerprint. The DGGE profiles revealed the dominance of L. sanfranciscensis species. In addition, Lactobacillus-specific primers were used to amplify the V1-V3 regions of the 16S rDNA. DGGE profiles flourished the dominance of L. sanfranciscensis and Lactobacillus fermentum in the traditional sourdoughs, and revealed that the closely related species Lactobacillus kimchii and Lactobacillus alimentarius were not discriminated. Lactobacillus-specific PCR-DGGE analysis is a rapid tool for rapid detection of Lactobacillus species in artisanal sourdough. This study reports a characterization of Lactobacillus isolates from artisanal sourdoughs and highlights the value of DGGE approach to detect uncultivable Lactobacillus species.

Assessment of the microbial diversity of Brazilian kefir grains by PCR-DGGE and pyrosequencing analysis.

PubMed

Leite, A M O; Mayo, B; Rachid, C T C C; Peixoto, R S; Silva, J T; Paschoalin, V M F; Delgado, S

2012-09-01

The microbial diversity and community structure of three different kefir grains from different parts of Brazil were examined via the combination of two culture-independent methods: PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing. PCR-DGGE showed Lactobacillus kefiranofaciens and Lactobacillus kefiri to be the major bacterial populations in all three grains. The yeast community was dominated by Saccharomyces cerevisiae. Pyrosequencing produced a total of 14,314 partial 16S rDNA sequence reads from the three grains. Sequence analysis grouped the reads into three phyla, of which Firmicutes was dominant. Members of the genus Lactobacillus were the most abundant operational taxonomic units (OTUs) in all samples, accounting for up to 96% of the sequences. OTUs belonging to other lactic and acetic acid bacteria genera, such as Lactococcus, Leuconostoc, Streptococcus and Acetobacter, were also identified at low levels. Two of the grains showed identical DGGE profiles and a similar number of OTUs, while the third sample showed the highest diversity by both techniques. Pyrosequencing allowed the identification of bacteria that were present in small numbers and rarely associated with the microbial community of this complex ecosystem. Copyright © 2012 Elsevier Ltd. All rights reserved.

Soil Microbial Community Structure across a Thermal Gradient following a Geothermal Heating Event

PubMed Central

Norris, Tracy B.; Wraith, Jon M.; Castenholz, Richard W.; McDermott, Timothy R.

2002-01-01

In this study microbial species diversity was assessed across a landscape in Yellowstone National Park, where an abrupt increase in soil temperature had occurred due to recent geothermal activity. Soil temperatures were measured, and samples were taken across a temperature gradient (35 to 65°C at a 15-cm depth) that spanned geothermally disturbed and unimpacted soils; thermally perturbed soils were visually apparent by the occurrence of dead or dying lodgepole pine trees. Changes in soil microbial diversity across the temperature gradient were qualitatively assessed based on 16S rRNA sequence variation as detected by denaturing gradient gel electrophoresis (DGGE) using both ribosomal DNA (rDNA) and rRNA as PCR templates and primers specific for the Bacteria or Archaea domain. The impact of the major heating disturbance was apparent in that DGGE profiles from heated soils appeared less complex than those from the unaffected soils. Phylogenetic analysis of a bacterial 16S rDNA PCR clone library from a recently heated soil showed that a majority of the clones belonged to the Acidobacterium (51%) and Planctomyces (18%) divisions. Agar plate counts of soil suspensions cultured on dilute yeast extract and R2A agar media incubated at 25 or 50°C revealed that thermophile populations were two to three orders of magnitude greater in the recently heated soil. A soil microcosm laboratory experiment simulated the geothermal heating event. As determined by both RNA- and DNA-based PCR coupled with DGGE, changes in community structure (marked change in the DGGE profile) of soils incubated at 50°C occurred within 1 week and appeared to stabilize after 3 weeks. The results of our molecular and culture data suggest that thermophiles or thermotolerant species are randomly distributed in this area within Yellowstone National Park and that localized thermal activity selects for them. PMID:12450855

Soil microbial community structure across a thermal gradient following a geothermal heating event.

PubMed

Norris, Tracy B; Wraith, Jon M; Castenholz, Richard W; McDermott, Timothy R

2002-12-01

In this study microbial species diversity was assessed across a landscape in Yellowstone National Park, where an abrupt increase in soil temperature had occurred due to recent geothermal activity. Soil temperatures were measured, and samples were taken across a temperature gradient (35 to 65 degrees C at a 15-cm depth) that spanned geothermally disturbed and unimpacted soils; thermally perturbed soils were visually apparent by the occurrence of dead or dying lodgepole pine trees. Changes in soil microbial diversity across the temperature gradient were qualitatively assessed based on 16S rRNA sequence variation as detected by denaturing gradient gel electrophoresis (DGGE) using both ribosomal DNA (rDNA) and rRNA as PCR templates and primers specific for the Bacteria or Archaea domain. The impact of the major heating disturbance was apparent in that DGGE profiles from heated soils appeared less complex than those from the unaffected soils. Phylogenetic analysis of a bacterial 16S rDNA PCR clone library from a recently heated soil showed that a majority of the clones belonged to the Acidobacterium (51%) and Planctomyces (18%) divisions. Agar plate counts of soil suspensions cultured on dilute yeast extract and R2A agar media incubated at 25 or 50 degrees C revealed that thermophile populations were two to three orders of magnitude greater in the recently heated soil. A soil microcosm laboratory experiment simulated the geothermal heating event. As determined by both RNA- and DNA-based PCR coupled with DGGE, changes in community structure (marked change in the DGGE profile) of soils incubated at 50 degrees C occurred within 1 week and appeared to stabilize after 3 weeks. The results of our molecular and culture data suggest that thermophiles or thermotolerant species are randomly distributed in this area within Yellowstone National Park and that localized thermal activity selects for them.

Detritus-Dependent Development of the Microbial Community in an Experimental System: Qualitative Analysis by Denaturing Gradient Gel Electrophoresis†

PubMed Central

van Hannen, Erik J.; Mooij, Wolf; van Agterveld, Miranda P.; Gons, Herman J.; Laanbroek, Hendrikus J.

1999-01-01

Correlations between the biomass of phytoplankton and the biomass of bacteria and between the biomass of bacteria and the biomass of protozoans suggest that there is coupling between these compartments of the “microbial loop.” To investigate this coupling on the species level, bacteria and protozoans from untreated lake water inocula were allowed to grow on detritus of the green alga Ankistrodesmus falcatus or the cyanobacterium Oscillatoria limnetica in continuous-flow systems for 1 month. Denaturing gradient gel electrophoresis (DGGE) of the 16S and 18S rRNA genes was used to monitor the development of the bacterial community structure and the eukaryotic community structure, respectively. Nonmetric multidimensional scaling of the DGGE profiles revealed the changes in the microbial community structure. This analysis showed that significantly different bacterial communities developed on the green algal detritus and on the cyanobacterial detritus. Although similar results were obtained for the eukaryotic communities, the differences were not significant. Hence, our findings indicate that the origin of detritus can affect the structure of at least the bacterial community. A phylogenetic analysis of 20 18S ribosomal DNA clones that were isolated from the continuous cultures revealed that many sequences were related to the sequences of bacterivorous protozoans (members of the Ciliophora, Rhizopoda, Amoeba, and Kinetoplastida). One clone grouped in a recently established clade whose previously described members are all parasites. The affiliations of about 20% of the clones could not be determined. PMID:10347030

Actively Growing Bacteria in the Inland Sea of Japan, Identified by Combined Bromodeoxyuridine Immunocapture and Denaturing Gradient Gel Electrophoresis▿ †

PubMed Central

Hamasaki, Koji; Taniguchi, Akito; Tada, Yuya; Long, Richard A.; Azam, Farooq

2007-01-01

A fundamental question in microbial oceanography concerns the relationship between prokaryote diversity and biogeochemical function in an ecosystem context. We combined bromodeoxyuridine (BrdU) magnetic bead immunocapture and PCR-denaturing gradient gel electrophoresis (BUMP-DGGE) to examine phylotype-specific growth in natural marine assemblages. We also examined a broad range of marine bacterial isolates to determine their abilities to incorporate BrdU in order to test the validity of the method for application to diverse marine assemblages. We found that 27 of 29 isolates belonging to different taxa could incorporate BrdU. BUMP-DGGE analysis revealed phylogenetic affiliations of DNA-synthesizing, presumably actively growing bacteria across a eutrophic to mesotrophic transect in the Inland Sea of Japan. We found that the BrdU-incorporating (growing) communities were substantially different from the total communities. The majority (34/56) of phylotypes incorporated BrdU and were presumably growing, and these phylotypes comprised 10 alphaproteobacteria, 1 betaproteobacterium, 11 gammaproteobacteria, 11 Cytophaga-Flavobacterium-Bacteroides group bacteria, and 1 unclassified bacterium. All BrdU-responsive alphaproteobacteria were members of the Rhodobacterales, suggesting that such bacteria were dominant in the growing alphaproteobacterial populations in our samples. The BrdU-responsive gammaproteobacteria belonged to the Oceanospirillales, the SAR86 cluster, the Pseudomonadales, the Alteromonadales, and the Vibrionales. Thus, contemporaneous cooccurrence of diverse actively growing bacterial taxa was a consistent pattern in our biogeochemically varied study area. PMID:17337555

Bacteria community study of combined periodontal-endodontic lesions using denaturing gradient gel electrophoresis and sequencing analysis.

PubMed

Li, Hong; Guan, Rui; Sun, Jinghua; Hou, Benxiang

2014-10-01

The entire microbial population and predominant microflora of root canals (RCs) and adjacent periodontal pockets (PPs) from teeth with combined periodontal-endodontic lesions were determined and compared. Pooled RC and PP samples were collected from the molars of 20 patients diagnosed with combined periodontal-endodontic lesions. DNA was extracted for polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-DGGE), cloning, and sequence analysis. A coefficient of similarity (Cs) was used to determine the similarity of the bacterial profiles from RCs and PPs. Significantly fewer bands were produced by PCR-DGGE from RCs (5.9 ± 1.7) than from PPs (8.0 ± 1.8) (P <0.001). The average Cs of the RC and PP samples was 93.81% ± 10.26%. Overall, 60 genera/species were identified by sequencing. Of these, the predominant genera in RCs were Porphyromonas sp. (13.9%), Filifactor sp. (12.5%), and Parvimonas sp. (11.1%), similar to the genera obtained from PP samples. In total, 43 genera/species were common to the RC and PP samples. The most prevalent bacteria in both the RC and PP samples were (in descending order) Filifactor alocis, Parvimonas micra, Porphyromonas gingivalis, and Tannerella forsythia. The high similarity in the sets of organisms present in both RC and PP samples in this study suggests that the pocket could be a source of RC infection. The data also demonstrate that combined periodontal-endodontic lesions consist of a diverse and complex microbial community.

Comparison among amoA Primers Suited for Quantification and Diversity Analyses of Ammonia-Oxidizing Bacteria in Soil

PubMed Central

Shimomura, Yumi; Morimoto, Sho; Hoshino, Yuko Takada; Uchida, Yoshitaka; Akiyama, Hiroko; Hayatsu, Masahito

2012-01-01

Ammonia monooxygenase subunit A gene (amoA) is frequently used as a functional gene marker for diversity analysis of ammonia-oxidizing bacteria (AOB). To select a suitable amoA primer for real-time PCR and PCR-denaturing gradient gel electrophoresis (DGGE), three reverse primers (degenerate primer amoA-2R; non-degenerate primers amoA-2R-GG and amoA-2IR) were examined. No significant differences were observed among the three primers in terms of quantitative values of amoA from environmental samples using real-time PCR. We found that PCR-DGGE analysis with the amoA-2IR primer gave the best results in this studied soil. These results indicate that amoA-2IR is a suitable primer for community analysis of AOB in the environment. PMID:22075625

Analysis of DGGE profiles to explore the relationship between prokaryotic community composition and biogeochemical processes in deep subseafloor sediments from the Peru Margin.

PubMed

Fry, John C; Webster, Gordon; Cragg, Barry A; Weightman, Andrew J; Parkes, R John

2006-10-01

The aim of this work was to relate depth profiles of prokaryotic community composition with geochemical processes in the deep subseafloor biosphere at two shallow-water sites on the Peru Margin in the Pacific Ocean (ODP Leg 201, sites 1228 and 1229). Principal component analysis of denaturing gradient gel electrophoresis banding patterns of deep-sediment Bacteria, Archaea, Euryarchaeota and the novel candidate division JS1, followed by multiple regression, showed strong relationships with prokaryotic activity and geochemistry (R(2)=55-100%). Further correlation analysis, at one site, between the principal components from the community composition profiles for Bacteria and 12 other variables quantitatively confirmed their relationship with activity and geochemistry, which had previously only been implied. Comparison with previously published cell counts enumerated by fluorescent in situ hybridization with rRNA-targeted probes confirmed that these denaturing gradient gel electrophoresis profiles described an active prokaryotic community.

Oral administration of kefiran exerts a bifidogenic effect on BALB/c mice intestinal microbiota.

PubMed

Hamet, M F; Medrano, M; Pérez, P F; Abraham, A G

2016-01-01

The activity of kefiran, the exopolysaccharide present in kefir grains, was evaluated on intestinal bacterial populations in BALB/c mice. Animals were orally administered with kefiran and Eubacteria, lactobacilli and bifidobacteria populations were monitored in faeces of mice at days 0, 2, 7, 14 and 21. Profiles obtained by Denaturing Gradient Gel Electrophoresis (DGGE) with primers for Eubacteria were compared by principal component analysis and clearly defined clusters, correlating with the time of kefiran consumption, were obtained. Furthermore, profile analysis of PCR products amplified with specific oligonucleotides for bifidobacteria showed an increment in the number of DGGE bands in the groups administered with kefiran. Fluorescent In Situ Hybridisation (FISH) with specific probes for bifidobacteria showed an increment of this population in faeces, in accordance to DGGE results. The bifidobacteria population was also studied on distal colon content after 0, 2 and 7 days of kefiran administration. Analysis of PCR products by DGGE with Eubacteria primers showed an increment in the number and intensity of bands with high GC content of mice administered with kefiran. Sequencing of DGGE bands confirmed that bifidobacteria were one of the bacterial populations modified by kefiran administration. DGGE profiles of PCR amplicons obtained by using Bifidobacterium or Lactobacillus specific primers confirmed that kefiran administration enhances bifidobacteria, however no changes were observed in Lactobacillus populations. The results of the analysis of bifidobacteria populations assessed on different sampling sites in a murine model support the use of this exopolysaccharide as a bifidogenic functional ingredient.

Investigation of bacterial and archaeal communities: novel protocols using modern sequencing by Illumina MiSeq and traditional DGGE-cloning.

PubMed

Kraková, Lucia; Šoltys, Katarína; Budiš, Jaroslav; Grivalský, Tomáš; Ďuriš, František; Pangallo, Domenico; Szemes, Tomáš

2016-09-01

Different protocols based on Illumina high-throughput DNA sequencing and denaturing gradient gel electrophoresis (DGGE)-cloning were developed and applied for investigating hot spring related samples. The study was focused on three target genes: archaeal and bacterial 16S rRNA and mcrA of methanogenic microflora. Shorter read lengths of the currently most popular technology of sequencing by Illumina do not allow analysis of the complete 16S rRNA region, or of longer gene fragments, as was the case of Sanger sequencing. Here, we demonstrate that there is no need for special indexed or tailed primer sets dedicated to short variable regions of 16S rRNA since the presented approach allows the analysis of complete bacterial 16S rRNA amplicons (V1-V9) and longer archaeal 16S rRNA and mcrA sequences. Sample augmented with transposon is represented by a set of approximately 300 bp long fragments that can be easily sequenced by Illumina MiSeq. Furthermore, a low proportion of chimeric sequences was observed. DGGE-cloning based strategies were performed combining semi-nested PCR, DGGE and clone library construction. Comparing both investigation methods, a certain degree of complementarity was observed confirming that the DGGE-cloning approach is not obsolete. Novel protocols were created for several types of laboratories, utilizing the traditional DGGE technique or using the most modern Illumina sequencing.

Use of pyrosequencing and denaturing gradient gel electrophoresis to examine the effects of probiotics and essential oil blends on digestive microflora in broilers under mixed Eimeria infection.

PubMed

Hume, Michael E; Barbosa, Nei A; Dowd, Scot E; Sakomura, Nilva K; Nalian, Armen G; Martynova-Van Kley, Alexandra; Oviedo-Rondón, Edgar O

2011-11-01

A protective digestive microflora helps prevent and reduce broiler infection and colonization by enteropathogens. In the current experiment, broilers fed diets supplemented with probiotics and essential oil (EO) blends were infected with a standard mixed Eimeria spp. to determine effects of performance enhancers on ileal and cecal microbial communities (MCs). Eight treatment groups included four controls (uninfected-unmedicated [UU], unmedicated-infected, the antibiotic BMD plus the ionophore Coban as positive control, and the ionophore as negative control), and four treatments (probiotics BC-30 and Calsporin; and EO, Crina Poultry Plus, and Crina PoultryAF). Day-old broilers were raised to 14 days in floor pens on used litter and then were moved to Petersime batteries and inoculated at 15 days with mixed Eimeria spp. Ileal and cecal samples were collected at 14 days and 7 days postinfection. Digesta DNA was subjected to pyrosequencing for sequencing of individual cecal bacteria and denaturing gradient gel electrophoresis (DGGE) for determination of changes in ileal and cecal MC according to percentage similarity coefficient (%SC). Pyrosequencing is very sensitive detecting shifts in individual bacterial sequences, whereas DGGE is able to detect gross shifts in entire MC. These combined techniques offer versatility toward identifying feed additive and mild Eimeria infection modulation of broiler MC. Pyrosequencing detected 147 bacterial species sequences. Additionally, pyrosequencing revealed the presence of relatively low levels of the potential human enteropathogens Campylobacter sp. and four Shigella spp. as well as the potential poultry pathogen Clostridiun perfringens. Pre- and postinfection changes in ileal (56%SC) and cecal (78.5%SC) DGGE profiles resulted from the coccidia infection and with increased broiler age. Probiotics and EO changed MC from those seen in UU ilea and ceca. Results potentially reflect the performance enhancement above expectations in comparison to broilers not given the probiotics or the specific EO blends as feed supplements.

Identification of yeasts and evaluation of their distribution in Taiwanese Kefir and Viili starters.

PubMed

Wang, S Y; Chen, H C; Liu, J R; Lin, Y C; Chen, M J

2008-10-01

The objective of the present study was to investigate yeast communities in kefir grains and viili starters in Taiwan through conventional microbiological cultivation and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The DNA sequencing was used as a validity technique to ensure that all isolates within each group belonged to just one species, and to confirm the identified results of PCR-DGGE. Results indicated that a combination of conventional microbiological cultivation with PCR-DGGE and sequencing could successfully identify 4 yeast species from both types of cultures in Taiwan. Kluyveromyces marxianus, Saccharomyces turicensis, and Pichia fermentans were found in Taiwanese kefir grains with a distribution of 76, 22, and 2%, respectively, whereas Klu. marxianus, Saccharomyces unisporus and P. fermentans were identified in viili starters corresponding to 58, 11, and 31% of the total cell counts, respectively. Furthermore, the culture-independent method was applied to identify the yeast species using DGGE. Only 2 yeast species, Klu. marxianus and S. turicensis, were found in kefir grains and 2, Klu. marxianus and P. fermentans, in viili starters. These results suggest that in samples containing multiple species, PCR-DGGE may fail to detect some species. Sequences of yeast isolates reported in this study have been deposited in the GenBank database under accession nos. DQ139802, AF398485, DQ377652, and AY007920.

Changes in Microbial Communities, Including both Uncultured and Culturable Bacteria, with Mid-Ocean Ballast-Water Exchange during a Voyage from Japan to Australia

PubMed Central

Tomaru, Akiko; Kawachi, Masanobu; Demura, Mikihide; Fukuyo, Yasuwo

2014-01-01

We assessed changes in the microbial communities in ballast water during a trans-Pacific voyage from Japan to Australia that included a mid-ocean ballast-water exchange. Uncultured (i.e., total) and culturable bacteria were counted and were characterized by using denaturing gradient gel electrophoresis (DGGE). There was a clear decrease over time in numbers of uncultured microorganisms, except for heterotrophic nanoflagellates, whereas the abundance of culturable bacteria initially decreased after the ballast-water exchange but then increased. The increase, however, was only up to 5.34% of the total number of uncultured bacteria. Cluster analysis showed that the DGGE profiles of uncultured bacteria clearly changed after the exchange. In contrast, there was no clear change in the DGGE profiles of culturable bacteria after the exchange. Multidimensional scaling analysis showed changes in microbial communities over the course of the voyage. Although indicator microbes as defined by the International Convention for the Control and Management of Ships' Ballast Water and Sediments were occasionally detected, no coliform bacteria were detected after the exchange. PMID:24817212

Senior Thai fecal microbiota comparison between vegetarians and non-vegetarians using PCR-DGGE and real-time PCR.

PubMed

Ruengsomwong, Supatjaree; Korenori, Yuki; Sakamoto, Naoshige; Wannissorn, Bhusita; Nakayama, Jiro; Nitisinprasert, Sunee

2014-08-01

The fecal microbiotas were investigated in 13 healthy Thai subjects using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Among the 186 DNA bands detected on the polyacrylamide gel, 37 bands were identified as representing 11 species: Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides uniformis, Bacteroides vulgatus, Clostridium colicanis, Eubacterium eligenes, E. rectale, Faecalibacterium prausnitzii, Megamonas funiformis, Prevotella copri, and Roseburia intestinalis, belonging mainly to the groups of Bacteroides, Prevotella, Clostridium, and F. prausnitzii. A dendrogram of the PCR-DGGE divided the subjects; vegetarians and non-vegetarians. The fecal microbiotas were also analyzed using a quantitative real-time PCR focused on Bacteroides, Bifidobacterium, Enterobacteriaceae, Clostrium coccoides-Eubacterium rectale, C. leptum, Lactobacillus, and Prevotella. The nonvegetarian and vegetarian subjects were found to have significant differences in the high abundance of the Bacteroides and Prevotella genera, respectively. No significant differences were found in the counts of Bifidabacterium, Enterobacteriaceae, C. coccoides-E. rectale group, C. leptum group, and Lactobacillus. Therefore, these findings on the microbiota of healthy Thais consuming different diets could provide helpful data for predicting the health of South East Asians with similar diets.

Bulk soil and rhizosphere bacterial community PCR-DGGE profiles and beta-galactosidase activity as indicators of biological quality in soils contaminated by heavy metals and cultivated with Silene vulgaris (Moench) Garcke.

PubMed

Martínez-Iñigo, M J; Pérez-Sanz, A; Ortiz, I; Alonso, J; Alarcón, R; García, P; Lobo, M C

2009-06-01

The biological quality of two heavy metal contaminated soils (soil C: Typic Calcixerept, pH 8.3 and soil H: Typic Haploxeraf, pH 7.3) was investigated after growing the metal-tolerant plant Silene vulgaris (Moench) Garcke for two vegetative periods. The activity of the enzyme beta-galactosidase, which is sensitive to the presence of contaminants in soil, and the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) profiles of 16S rRNA gene fragments of culturable bacteria from bulk soil and rhizosphere were determined. The microbial enzymatic activity was higher in planted soils than in bare soils at the contamination level of 600 mg of total heavy metals kg(-1) soil. After growing S. vulgaris, beta-galactosidase activity was almost recovered in the calcareous soil. In this soil new bands appeared in the PCR-DGGE profiles of the rhizosphere bacterial community as a response to the exposure to heavy metals.

Dynamics of oral microbial community profiling during severe early childhood caries development monitored by PCR-DGGE.

PubMed

Tao, Ye; Zhou, Yan; Ouyang, Yong; Lin, HuanCai

2013-09-01

To monitor the longitudinal changes in oral microbial diversity of children with severe early childhood caries (S-ECC) compared to caries free (CF) controls. Dental plaque samples of 12 children in each group at 8, 14, 20, 26 and 32 months of age were analysed. Total microbial genomic DNA was isolated from each sample, and PCR-denaturing gradient gel electrophoresis (DGGE) analyses were carried out. The number of bands was significantly higher in the CF group (18.17±4.91 bands) than in the S-ECC group (14.54±5.56 bands) at 32 months of age (P<0.05). A total of 21 genera were identified in all subjects, and there were no significant differences between the two groups at genus level. DGGE profiles showed that most of the clusters were constructed from one individual over time in the both groups. The onset of S-ECC is accompanied by a decrease in microbial diversity. The overall composition of the microbiota is highly similar within an individual over time. Copyright © 2013 Elsevier Ltd. All rights reserved.

Estimation of contamination sources of human enteroviruses in a wastewater treatment and reclamation system by PCR-DGGE.

PubMed

Ji, Zheng; Wang, Xiaochang C; Xu, Limei; Zhang, Chongmiao; Funamizu, Naoyuki; Okabe, Satoshi; Sano, Daisuke

2014-06-01

A polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method was employed to estimate the contamination sources of human enteroviruses and understand how their dominant strains vary in a wastewater treatment and reclamation system consisting of sewage collection, wastewater treatment with membrane bioreactor and open lakes for reclaimed water storage and reuse. After PCR-DGGE using a selected primer set targeting enteroviruses, phylogenetic analysis of acquired enterovirus gene sequences was performed. Enteroviruses identified from the septic tank were much more diverse than those from grey water and kitchen wastewater. Several unique types of enterovirus different from those in wastewater samples were dominant in a biological wastewater treatment unit. Membrane filtration followed by chlorination was proved effective for physically eliminating enteroviruses; however, secondary contamination likely occurred as the reclaimed water was stored in artificial lakes. Enterovirus 71 (EV71), a hand-foot-and-mouth disease (HFMD) viral pathogen, was detected mainly from the artificial lakes, implying that wastewater effluent was not the contamination source of EV71 and that there were unidentified non-point sources of the contamination with the HFMD viral pathogen in the reclaimed water stored in the artificial lakes. The PCR-DGGE targeting enteroviruses provided robust evidence about viral contamination sources in the wastewater treatment and reclamation system.

Assessing the impact of fungicide enostroburin application on bacterial community in wheat phyllosphere.

PubMed

Gu, Likun; Bai, Zhihui; Jin, Bo; Hu, Qing; Wang, Huili; Zhuang, Guoqiang; Zhang, Hongxun

2010-01-01

Fungicides have been used extensively for controlling fungal pathogens of plants. However, little is known regarding the effects that fungicides upon the indigenous bacterial communities within the plant phyllosphere. The aims of this study were to assess the impact of fungicide enostroburin upon bacterial communities in wheat phyllosphere. Culture-independent methodologies of 16S rDNA clone library and 16S rDNA directed polymerase chain reaction with denaturing gradient gel electrophoresis (PCR-DGGE) were used for monitoring the change of bacterial community. The 16S rDNA clone library and PCR-DGGE analysis both confirmed the microbial community of wheat plant phyllosphere were predominantly of the gamma-Proteobacteria phyla. Results from PCR-DGGE analysis indicated a significant change in bacterial community structure within the phyllosphere following fungicide enostroburin application. Bands sequenced within control cultures were predominantly of Pseudomonas genus, but those bands sequenced in the treated samples were predominantly strains of Pantoea genus and Pseudomonas genus. Of interest was the appearance of two DGGE bands following fungicide treatment, one of which had sequence similarities (98%) to Pantoea sp. which might be a competitor of plant pathogens. This study revealed the wheat phyllosphere bacterial community composition and a shift in the bacterial community following fungicide enostroburin application.

Molecular and Microscopical Investigation of the Microflora Inhabiting a Deteriorated Italian Manuscript Dated from the Thirteenth Century

PubMed Central

Michaelsen, Astrid; Piñar, Guadalupe

2010-01-01

This case study shows the application of nontraditional diagnostic methods to investigate the microbial consortia inhabiting an ancient manuscript. The manuscript was suspected to be biologically deteriorated and SEM observations showed the presence of fungal spores attached to fibers, but classic culturing methods did not succeed in isolating microbial contaminants. Therefore, molecular methods, including PCR, denaturing gradient gel electrophoresis (DGGE), and clone libraries, were used as a sensitive alternative to conventional cultivation techniques. DGGE fingerprints revealed a high biodiversity of both bacteria and fungi inhabiting the manuscript. DNA sequence analysis confirmed the existence of fungi and bacteria in manuscript samples. A number of fungal clones identified on the manuscript showed similarity to fungal species inhabiting dry or saline environments, suggesting that the manuscript environment selects for osmophilic or xerophilic fungal species. Most of the bacterial sequences retrieved from the manuscript belong to phylotypes with cellulolytic activities. PMID:20449583

Multiple approaches to characterize the microbial community in a thermophilic anaerobic digester running on swine manure: a case study.

PubMed

Tuan, Nguyen Ngoc; Chang, Yi-Chia; Yu, Chang-Ping; Huang, Shir-Ly

2014-01-01

In this study, the first survey of microbial community in thermophilic anaerobic digester using swine manure as sole feedstock was performed by multiple approaches including denaturing gradient gel electrophoresis (DGGE), clone library and pyrosequencing techniques. The integrated analysis of 21 DGGE bands, 126 clones and 8506 pyrosequencing read sequences revealed that Clostridia from the phylum Firmicutes account for the most dominant Bacteria. In addition, our analysis also identified additional taxa that were missed by the previous researches, including members of the bacterial phyla Synergistetes, Planctomycetes, Armatimonadetes, Chloroflexi and Nitrospira which might also play a role in thermophilic anaerobic digester. Most archaeal 16S rRNA sequences could be assigned to the order Methanobacteriales instead of Methanomicrobiales comparing to previous studies. In addition, this study reported that the member of Methanothermobacter genus was firstly found in thermophilic anaerobic digester. Copyright © 2014 Elsevier GmbH. All rights reserved.

PCR-denaturing gradient gel electrophoresis analysis of microbial community in soy-daddawa, a Nigerian fermented soybean (Glycine max (L.) Merr.) condiment.

PubMed

Ezeokoli, Obinna T; Gupta, Arvind K; Mienie, Charlotte; Popoola, Temitope O S; Bezuidenhout, Cornelius C

2016-03-02

Soy-daddawa, a fermented soybean (Glycine max (L.) Merr.) condiment, plays a significant role in the culinary practice of West Africa. It is essential to understand the microbial community of soy-daddawa for a successful starter culture application. This study investigated the microbial community structure of soy-daddawa samples collected from Nigerian markets, by PCR-denaturing gradient gel electrophoresis (DGGE) targeting the V3-V5 region of the 16S rRNA gene of bacteria and internal transcribed spacer 2 (ITS2) region of fungi. Six bacterial and 16 fungal (nine yeasts and seven molds) operational taxonomic units (OTUs)/species were obtained at 97% sequence similarity. Taxonomic assignments revealed that bacterial OTUs belonged to the phyla Firmicutes and Actinobacteria, and included species from the genera Atopostipes, Bacillus, Brevibacterium and Nosocomiicoccus. Densitometric analysis of DGGE image/bands revealed that Bacillus spp. were the dominant OTU/species in terms of population numbers. Fungal OTUs belonged to the phyla Ascomycota and Zygomycota, and included species from the genera, Alternaria, Aspergillus, Candida, Cladosporium, Dokmaia, Issatchenkia, Kodamaea, Lecythophora, Phoma, Pichia, Rhizopus, Saccharomyces and Starmerella. The majority of fungal species have not been previously reported in soy-daddawa. Potential opportunistic human pathogens such as Atopostipes suicloacalis, Candida rugosa, Candida tropicalis, and Kodamaea ohmeri were detected. Variation in soy-daddawa microbial communities amongst samples and presence of potential opportunistic pathogens emphasises the need for starter culture employment and good handling practices in soy-daddawa processing. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of Bacteria Potentially Responsible for Oxic and Anoxic Sulfide Oxidation in Biofilters of a Recirculating Mariculture System

PubMed Central

Cytryn, Eddie; van Rijn, Jaap; Schramm, Andreas; Gieseke, Armin; de Beer, Dirk; Minz, Dror

2005-01-01

Bacteria presumably involved in oxygen- or nitrate-dependent sulfide oxidation in the biofilters of a recirculating marine aquaculture system were identified using a new application of reverse transcription-PCR denaturing gradient gel electrophoresis (DGGE) analysis termed differential-transcription (DT)-DGGE. Biofilter samples were incubated in various concentrations of sulfide or thiosulfate (0 to 5 mM) with either oxygen or nitrate as the sole electron acceptor. Before and after short-term incubations (10 to 20 h), total DNA and RNA were extracted, and a 550-bp fragment of the 16S rRNA genes was PCR amplified either directly or after reverse transcription. DGGE analysis of DNA showed no significant change of the original microbial consortia upon incubation. In contrast, DGGE of cDNA revealed several phylotypes whose relative band intensities markedly increased or decreased in response to certain incubation conditions, indicating enhanced or suppressed rRNA transcription and thus implying metabolic activity under these conditions. Specifically, species of the gammaproteobacterial genus Thiomicrospira and phylotypes related to symbiotic sulfide oxidizers could be linked to oxygen-dependent sulfide oxidation, while members of the Rhodobacteraceae (genera Roseobacter, Rhodobacter, and Rhodobium) were putatively active in anoxic, nitrate-dependent sulfide oxidation. For all these organisms, the physiology of their closest cultured relatives matches their DT-DGGE-inferred function. In addition, higher band intensities following exposure to 5 mM sulfide and nitrate were observed for Thauera-, Hydrogenophaga-, and Dethiosulfovibrio-like phylotypes. For these genera, nitrate-dependent sulfide oxidation has not been documented previously and therefore DT-DGGE might indicate a higher relative tolerance to high sulfide concentrations than that of other community members. We anticipate that DT-DGGE will be of general use in tracing functionally equivalent yet phylogenetically diverse microbial populations in nature. PMID:16204531

Improvement of DGGE analysis by modifications of PCR protocols for analysis of microbial community members with low abundance.

PubMed

Wang, Yong-Feng; Zhang, Fang-Qiu; Gu, Ji-Dong

2014-06-01

Denaturing gradient gel electrophoresis (DGGE) is a powerful technique to reveal the community structures and composition of microorganisms in complex natural environments and samples. However, positive and reproducible polymerase chain reaction (PCR) products, which are difficult to acquire for some specific samples due to low abundance of the target microorganisms, significantly impair the effective applications of DGGE. Thus, nested PCR is often introduced to generate positive PCR products from the complex samples, but one problem is also introduced: The total number of thermocycling in nested PCR is usually unacceptably high, which results in skewed community structures by generation of random or mismatched PCR products on the DGGE gel, and this was demonstrated in this study. Furthermore, nested PCR could not resolve the uneven representative issue with PCR products of complex samples with unequal richness of microbial population. In order to solve the two problems in nested PCR, the general protocol was modified and improved in this study. Firstly, a general PCR procedure was used to amplify the target genes with the PCR primers without any guanine cytosine (GC) clamp, and then, the resultant PCR products were purified and diluted to 0.01 μg ml(-1). Subsequently, the diluted PCR products were utilized as templates to amplify again with the same PCR primers with the GC clamp for 17 cycles, and the products were finally subjected to DGGE analysis. We demonstrated that this is a much more reliable approach to obtain a high quality DGGE profile with high reproducibility. Thus, we recommend the adoption of this improved protocol in analyzing microorganisms of low abundance in complex samples when applying the DGGE fingerprinting technique to avoid biased results.

Analysis of artifacts suggests DGGE should not be used for quantitative diversity analysis.

PubMed

Neilson, Julia W; Jordan, Fiona L; Maier, Raina M

2013-03-01

PCR-denaturing gradient gel electrophoresis (PCR-DGGE) is widely used in microbial ecology for the analysis of comparative community structure. However, artifacts generated during PCR-DGGE of mixed template communities impede the application of this technique to quantitative analysis of community diversity. The objective of the current study was to employ an artificial bacterial community to document and analyze artifacts associated with multiband signatures and preferential template amplification and to highlight their impacts on the use of this technique for quantitative diversity analysis. Six bacterial species (three Betaproteobacteria, two Alphaproteobacteria, and one Firmicutes) were amplified individually and in combinations with primers targeting the V7/V8 region of the 16S rRNA gene. Two of the six isolates produced multiband profiles demonstrating that band number does not correlate directly with α-diversity. Analysis of the multiple bands from one of these isolates confirmed that both bands had identical sequences which lead to the hypothesis that the multiband pattern resulted from two distinct structural conformations of the same amplicon. In addition, consistent preferential amplification was demonstrated following pairwise amplifications of the six isolates. DGGE and real time PCR analysis identified primer mismatch and PCR inhibition due to 16S rDNA secondary structure as the most probable causes of preferential amplification patterns. Reproducible DGGE community profiles generated in this study confirm that PCR-DGGE provides an excellent high-throughput tool for comparative community structure analysis, but that method-specific artifacts preclude its use for accurate comparative diversity analysis. Copyright © 2013 Elsevier B.V. All rights reserved.

Molecular approaches to analysing the microbial composition of raw milk and raw milk cheese.

PubMed

Quigley, Lisa; O'Sullivan, Orla; Beresford, Tom P; Ross, R Paul; Fitzgerald, Gerald F; Cotter, Paul D

2011-11-01

The availability and application of culture-independent tools that enable a detailed investigation of the microbiota and microbial biodiversity of food systems has had a major impact on food microbiology. This review focuses on the application of DNA-based technologies, such as denaturing gradient gel electrophoresis (DGGE), temporal temperature gradient gel electrophoresis (TTGE), single stranded conformation polymorphisms (SSCP), the polymerase chain reaction (PCR) and others, to investigate the diversity, dynamics and identity of microbes in dairy products from raw milk. Here, we will highlight the benefits associated with culture-independent methods which include enhanced sensitivity, rapidity and the detection of microorganisms not previously associated with such products. Copyright © 2011 Elsevier B.V. All rights reserved.

Simultaneous construction of PCR-DGGE-based predictive models of Listeria monocytogenes and Vibrio parahaemolyticus on cooked shrimps.

PubMed

Liao, C; Peng, Z Y; Li, J B; Cui, X W; Zhang, Z H; Malakar, P K; Zhang, W J; Pan, Y J; Zhao, Y

2015-03-01

The aim of this study was to simultaneously construct PCR-DGGE-based predictive models of Listeria monocytogenes and Vibrio parahaemolyticus on cooked shrimps at 4 and 10°C. Calibration curves were established to correlate peak density of DGGE bands with microbial counts. Microbial counts derived from PCR-DGGE and plate methods were fitted by Baranyi model to obtain molecular and traditional predictive models. For L. monocytogenes, growing at 4 and 10°C, molecular predictive models were constructed. It showed good evaluations of correlation coefficients (R(2) > 0.92), bias factors (Bf ) and accuracy factors (Af ) (1.0 ≤ Bf ≤ Af ≤ 1.1). Moreover, no significant difference was found between molecular and traditional predictive models when analysed on lag phase (λ), maximum growth rate (μmax ) and growth data (P > 0.05). But for V. parahaemolyticus, inactivated at 4 and 10°C, molecular models show significant difference when compared with traditional models. Taken together, these results suggest that PCR-DGGE based on DNA can be used to construct growth models, but it is inappropriate for inactivation models yet. This is the first report of developing PCR-DGGE to simultaneously construct multiple molecular models. It has been known for a long time that microbial predictive models based on traditional plate methods are time-consuming and labour-intensive. Denaturing gradient gel electrophoresis (DGGE) has been widely used as a semiquantitative method to describe complex microbial community. In our study, we developed DGGE to quantify bacterial counts and simultaneously established two molecular predictive models to describe the growth and survival of two bacteria (Listeria monocytogenes and Vibrio parahaemolyticus) at 4 and 10°C. We demonstrated that PCR-DGGE could be used to construct growth models. This work provides a new approach to construct molecular predictive models and thereby facilitates predictive microbiology and QMRA (Quantitative Microbial Risk Assessment). © 2014 The Society for Applied Microbiology.

[Microbial diversity of sediments from the coasts of Dalian Changshan Islands].

PubMed

Li, Jialin; Wang, Zhonghua; Qin, Song; Wang, Guangyi

2011-05-01

To understand the impacts of anthropogenic activities on structure and composition of bacterial communities and to evaluate how bacterial communities respond to environmental gradients at coastal sediments. The diversity of bacterial communities in sediments from tourist and mariculture zones at coastal area of Dalian Changshan Islands was assessed using terminal restriction fragment length polymorphism (t-RFLP) and denaturing gradient gel electrophoresis (DGGE) approaches. Meanwhile, 16S rRNA clone library was constructed to reveal the composition and structure of bacterial communities in the most seriously polluted site (D4). There were much higher values of richness, Shannon-wiener and evenness index at D4 site by the analysis of terminal restriction fragments (t-RFs). The clustering result on the t-RFs areas and DGGE patterns showed that the bacterial diversity of tourist zone were more similar, while the distinction was increased with pollution levels among the tourist and mariculture zones. The 16S rRNA clone of D4 revealed that the Proteobacteria were the dominant phylum, and gamma-proteobacteria was the main class within Proteobacteria. The study documented changes in bacterial community structure by human impacts of mariculture than geographical location.

Genetic difference but functional similarity among fish gut bacterial communities through molecular and biochemical fingerprints.

PubMed

Mouchet, Maud A; Bouvier, Corinne; Bouvier, Thierry; Troussellier, Marc; Escalas, Arthur; Mouillot, David

2012-03-01

Considering the major involvement of gut microflora in the digestive function of various macro-organisms, bacterial communities inhabiting fish guts may be the main actors of organic matter degradation by fish. Nevertheless, the extent and the sources of variability in the degradation potential of gut bacterial communities are largely overlooked. Using Biolog Ecoplate™ and denaturing gradient gel electrophoresis (DGGE), we explored functional (i.e. the ability to degrade organic matter) and genetic (i.e. identification of DGGE banding patterns) diversity of fish gut bacterial communities, respectively. Gut bacterial communities were extracted from fish species characterized by different diets sampled along a salinity gradient in the Patos-Mirim lagoons complex (Brazil). We found that functional diversity was surprisingly unrelated to genetic diversity of gut bacterial communities. Functional diversity was not affected by the sampling site but by fish species and diet, whereas genetic diversity was significantly influenced by all three factors. Overall, the functional diversity was consistently high across fish individuals and species, suggesting a wide functional niche breadth and a high potential of organic matter degradation. We conclude that fish gut bacterial communities may strongly contribute to nutrient cycling regardless of their genetic diversity and environment. © European Union 2011.

Effects of lead and zinc mining contamination on bacterial community diversity and enzyme activities of vicinal cropland.

PubMed

Qu, Juanjuan; Ren, Guangming; Chen, Bao; Fan, Jinghua; E, Yong

2011-11-01

In the process of mining activity, many kinds of heavy metals enter into soils with dust, causing serious contamination to the environment. In this study, six soils were sampled from cropland at different distances from a lead/zinc mine in Heilongjiang Province, China. The total contents of lead and zinc in the vicinal cropland exceeded the third level of environmental quality standard for soil in China, which indicated that soils in this area were moderately contaminated. Bacterial community diversity and population were greatly decreased when the concentrations of lead and zinc were beyond 1,500 and 995 mg kg(-1), respectively, as analyzed by plate counting and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The bands of DGGE patterns varied with the degree of contamination. The activities of soil urease, phosphatase, and dehydrogenase were negatively correlated with the concentrations of lead and zinc. The highest inhibitory effect of heavy metals on soil enzyme activities was observed in urease. It was noted that PCR-DGGE patterns combined with soil enzyme activity analysis can be indices for the soil quality assessment by heavy metal contamination.

Suppression of Listeria monocytogenes by the Native Micro-Flora in Teewurst Sausage.

PubMed

Austin-Watson, Clytrice; Grant, Ar'Quette; Brice, Michline

2013-10-21

Modern consumers are interested in the use of non-chemical methods to control pathogens when heat sterilization is not an option. Such is the case with teewurst sausage, a raw spreadable sausage and a popular German commodity. Although Listeria was not found in teewurst, the optimal microbial growing conditions of teewurst coupled with the ubiquity of L. monocytogenes in nature, makes the possibility of contamination of products very possible. This pilot study was conducted to examine teewurst's native micro-flora's ability to suppress the outgrowth of L. monocytogenes at 10 °C using standard plate counts and PCR-DGGE. Traditional plating methods showed L. monocytogenes growth significantly decreased when in competition with the teewurst's native micro-flora ( p < 0.05). The native micro-flora of the teewurst suppressed the overall growth of L. monocytogenes by an average of two logs, under these conditions. Denaturing Gradient Gel Electrophoresis (DGGE) amplicons with unique banding patterns were extracted from DGGE gel for identification. Brochothrix thermosphacta and Lactobacillus curvatus were identified as a part of the teewurst's native micro-flora. Although the native micro-flora did not decrease L. monocytogenes to below limits of detection, it was enough of a decrease to warrant further investigation.

Evaluation of bacterial flora during the ripening of Kedong sufu, a typical Chinese traditional bacteria-fermented soybean product.

PubMed

Feng, Zhen; Gao, Wei; Ren, Dan; Chen, Xi; Li, Juan-juan

2013-04-01

Kedong sufu is a typical bacteria-fermented sufu in China. Isolation and identification of the autochthonous bacteria involved would allow the design of specific starters for this speciality. The purpose of the present study was to evaluate the bacterial flora during the ripening of Kedong sufu using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and culturing. In terms of bacterial diversity, 22 strains were isolated and identified and 27 strains were detected by DGGE. Regarding bacterial dynamics, the results of culturing and PCR-DGGE exhibited a similar trend towards dominant strains. Throughout the fermentation of sufu, Enterococcus avium, Enterococcus faecalis and Staphylococcus carnosus were the dominant microflora, while the secondary microflora comprised Leuconostoc mesenteroides, Staphylococcus saprophyticus, Streptococcus lutetiensis, Kocuria rosea, Kocuria kristinae, Bacillus pumilus, Bacillus cereus and Bacillus subtilis. This study is the first to reveal the bacterial flora during the ripening of Kedong sufu using both culture-dependent and culture-independent methods. This information will help in the design of autochthonous starter cultures for the production of Kedong sufu with desirable characteristic sensory profiles and shorter ripening times. © 2012 Society of Chemical Industry.

Characterisation of microbial communities in Chinese liquor fermentation starters Daqu using nested PCR-DGGE.

PubMed

Zhang, Liqiang; Wu, Chongde; Ding, Xiaofei; Zheng, Jia; Zhou, Rongqing

2014-12-01

In this study, characterises of the microbial community structures of three typical Chinese liquor Daqu, as well as different kinds of light flavour Daqu were investigated using nested PCR-denaturing gradient gel electrophoresis (DGGE). The results showed that microbial diversity was considerably different, and the microfloral compositions were highly variable among various Daqu. Lactic acid bacteria, which accounted for 30.95 % of all identified bacteria, were dominant in all Daqu samples, whereas Bacillus species were also predominant in the Luzhou (14.8 %) and Langjiu Daqu (18.2 %). Citrobacter and Burkholderia were first identified in light flavour Daqu. Aspergillus was the dominant moulds, and the non-Saccharomyces yeast species, Saccharomycopsis fibuligera, Wallemia sebi, Wallemia muriae, and Pichia subpelliculosa, were the dominant yeasts. Rasamsonia, Galactomyces, Geotrichum and Wallemia were first identified using nested PCR-DGGE. Cluster analysis indicated that the microbial community structures of different Daqu samples exhibited some differences. These may be ascribed to the different peak production temperatures, raw material constituents and microhabitats around the liquor enterprises. The current study provides insights into the microbial community structures of three typical Daqu samples, and may facilitate the development of starter cultures for manufacturing Chinese liquor.

Water quality and microbial diversity in cisterns from semiarid areas in Brazil.

PubMed

Alves, Fellipe; Köchling, Thorsten; Luz, Julio; Santos, Sylvana Melo; Gavazza, Savia

2014-09-01

Harvesting rainwater is a common practice worldwide, particularly in areas with no access to a public water supply or insufficient groundwater reserves. More than two million people living in semiarid regions of Brazil consume rainwater stored in cisterns, and little information is available regarding the water quality. Despite the initial good quality of the rainwater, its harvest and storage can introduce contaminants that must be eliminated before consumption. To evaluate the influence of handling, cistern age and precipitation on the quality of harvested rainwater, we monitored seven cisterns in the semiarid Brazilian Northeast over 4 years. Microbial and physicochemical parameters were monitored once a month, and denaturant gradient gel electrophoresis (DGGE) was performed at the end of the monitoring period. Coliform bacteria were detected in 100% of samples, while Escherichia coli were observed in 73.8%. The alkalinity and conductivity were the highest for the recently built cisterns due to the dissolution of construction materials. The DGGE of the 16S r DNA did not reveal the presence of E. coli. Instead, DGGE bands sequencing indicated that species primarily affiliated with Alphaproteobacteria were present in all cisterns, indicating the presence of microbial ecosystems capable of purifying and stabilizing the stored rainwater.

DGGE and multivariate analysis of a yeast community in spontaneous cocoa fermentation process.

PubMed

Ferreira, A C R; Marques, E L S; Dias, J C T; Rezende, R P

2015-12-28

Cocoa bean is the main raw material used in the production of chocolate. In southern Bahia, Brazil, cocoa farming and processing is an important economic activity. The fermentation of cocoa is the processing stage that yields important chocolate flavor precursors and complex microbial involvement is essential for this process. In this study, PCR-denaturing gradient gel electrophoreses (DGGE) was used to investigate the diversity of yeasts present during the spontaneous fermentation of cocoa in southern Bahia. The DGGE analysis revealed a richness of 8 to 13 distinct bands of varied intensities among the samples; and samples taken at 24, 36, and 48 h into the fermentation process were found to group with 70% similarity and showed the greatest diversity of bands. Hierarchical clustering showed that all samples had common operational taxonomic units (OTUs) and the highest number of OTUs was found in the 48 h sample. Variations in pH and temperature observed within the fermenting mass over time possibly had direct effects on the composition of the existing microbial community. The findings reported here indicate that a heterogeneous yeast community is involved in the complex cocoa fermentation process, which is known to involve a succession of specialized microorganisms.

Comparison of PCR-DGGE and PCR-SSCP analysis for bacterial flora of Japanese traditional fermented fish products, aji-narezushi and iwashi-nukazuke.

PubMed

An, Choa; Takahashi, Hajime; Kimura, Bon; Kuda, Takashi

2010-08-30

The bacterial flora of two Japanese traditional fermented fish products, aji-narezushi (salted and long-fermented horse mackerel (Trachurus japonicas) with rice) and iwashi-nukazuke (salted and long-fermented sardine (Sardinops melanostica) with rice bran), was analysed using non-culture-based polymerase chain reaction (PCR) denaturing gradient gel electrophoresis (DGGE) and culture-based PCR single-strand conformation polymorphism (SSCP) methods. Viable plate counts in aji-narezushi and iwashi-nukazuke were about 6.3-6.6 and 5.7-6.9 log colony-forming units g(-1) respectively. In the PCR-DGGE analysis, Lactobacillus acidipiscis was detected as the predominant bacterium in two of three aji-narezushi samples, while Lactobacillus versmoldensis was predominant in the third sample. By the PCR-SSCP method, Lb. acidipiscis and Lactobacillus plantarum were isolated as the predominant bacteria, while Lb. versmoldensis was not detected. The predominant bacterium in two of three iwashi-nukazuke samples was Tetragenococcus muriaticus, while Tetragenococcus halophilus was predominant in the third sample. The results suggest that the detection of some predominant lactic acid bacteria species in fermented fish by cultivation methods is difficult. Copyright (c) 2010 Society of Chemical Industry.

The effect of heavy metal contamination on the bacterial community structure at Jiaozhou Bay, China.

PubMed

Yao, Xie-Feng; Zhang, Jiu-Ming; Tian, Li; Guo, Jian-Hua

In this study, determination of heavy metal parameters and microbiological characterization of marine sediments obtained from two heavily polluted sites and one low-grade contaminated reference station at Jiaozhou Bay in China were carried out. The microbial communities found in the sampled marine sediments were studied using PCR-DGGE (denaturing gradient gel electrophoresis) fingerprinting profiles in combination with multivariate analysis. Clustering analysis of DGGE and matrix of heavy metals displayed similar occurrence patterns. On this basis, 17 samples were classified into two clusters depending on the presence or absence of the high level contamination. Moreover, the cluster of highly contaminated samples was further classified into two sub-groups based on the stations of their origin. These results showed that the composition of the bacterial community is strongly influenced by heavy metal variables present in the sediments found in the Jiaozhou Bay. This study also suggested that metagenomic techniques such as PCR-DGGE fingerprinting in combination with multivariate analysis is an efficient method to examine the effect of metal contamination on the bacterial community structure. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

[Ciliate diversity and spatiotemporal variation in surface sediments of Yangtze River estuary hypoxic zone].

PubMed

Feng, Zhao; Kui-Dong, Xu; Zhao-Cui, Meng

2012-12-01

By using denaturing gradient gel electrophoresis (DGGE) and sequencing as well as Ludox-QPS method, an investigation was made on the ciliate diversity and its spatiotemporal variation in the surface sediments at three sites of Yangtze River estuary hypoxic zone in April and August 2011. The ANOSIM analysis indicated that the ciliate diversity had significant difference among the sites (R = 0.896, P = 0.0001), but less difference among seasons (R = 0.043, P = 0.207). The sequencing of 18S rDNA DGGE bands revealed that the most predominant groups were planktonic Choreotrichia and Oligotrichia. The detection by Ludox-QPS method showed that the species number and abundance of active ciliates were maintained at a higher level, and increased by 2-5 times in summer, as compared with those in spring. Both the Ludox-QPS method and the DGGE technique detected that the ciliate diversity at the three sites had the similar variation trend, and the Ludox-QPS method detected that there was a significant variation in the ciliate species number and abundance between different seasons. The species number detected by Ludox-QPS method was higher than that detected by DGGE bands. Our study indicated that the ciliates in Yangtze River estuary hypoxic zone had higher diversity and abundance, with the potential to supply food for the polyps of jellyfish.

Comparing the effects of three pre-treatment disintegration techniques on aerobic sludge digestion: biodegradability enhancement and microbial community monitoring by PCR-DGGE.

PubMed

Jaziri, Kais; Casellas, Magali; Dagot, Christophe

2012-06-01

The objectives of this work were to compare and investigate the effect of three activated sludge disintegration processes before aerobic sludge digestion on 1) aerobic biodegradability enhancement and 2) microbial community evolution using the polymerase chain reaction-denaturant gel gradient electrophoresis (PCR-DGGE) technique. The comparison of three disintegration processes: thermal treatment (95 degrees C, 2h), sonication (100,000 kJ/kgTS) and ozonation (0.108 g O3/gTS) showed that the disintegration processes acted differently according to the composition of the soluble phase and to the DNA damage. Thermal treatment led to significant protein solubilization and to DNA modification. Sonication and ozonation resulted in similar soluble phase compositions and did not lead to any DNA modifications. During activated sludge aerobic digestion, intrinsic biodegradability enhancement was observed for thermal and ozone activated sludge pre-treatments. The analysis of the DGGE patterns at the end of aerobic digestion showed that population diversity was affected by both the aerobic digestion and the pre-treatment. The dissimilarity percentages measured at the end of aerobic digestion in the control sample and in the treated sludge were equal to 22, 25 and 20% for thermal treatment, sonication and ozonation respectively. This study indicated that PCR-DGGE could be a useful tool for the comparison of disintegration processes before and after aerobic digestion.

Investigation of bacterial community in activated sludge with an anaerobic side-stream reactor (ASSR) to decrease the generation of excess sludge.

PubMed

Kim, Young Mo; Chon, Dong-Hyun; Kim, Hee-Sik; Park, Chul

2012-09-01

The goal of this study was to investigate the bacterial community in activated sludge with an anaerobic side-stream reactor (ASSR), a process permitting significant decrease in sludge production during wastewater treatment. The study operated five activated sludge systems with different sludge treatment schemes serving as various controls for the activated sludge with ASSR. Bacterial communities were analyzed by denaturing gradient gel electrophoresis (DGGE), sequencing and construction of phylogenetic relationships of the identified bacteria. The DGGE data showed that activated sludge incorporating ASSR contained higher diversity of bacteria, resulting from long solids retention time and recirculation of sludge under aerobic and anaerobic conditions. The similarity of DGGE profiles between ASSR and separate anaerobic digester (control) was high indicating that ASSR is primarily related to conventional anaerobic digesters. Nevertheless, there was also unique bacteria community appearing in ASSR. Interestingly, sludge in the main system and in ASSR showed considerably different bacterial composition indicating that ASSR allowed enriching its own bacterial community different than that from the aeration basin, although two reactors were connected via sludge recirculation. In activated sludge with ASSR, sequences represented by predominant DGGE bands were affiliated with Proteobacteria. The remaining groups were composed of Spirochaetes, Clostridiales, Chloroflexi, and Actinobacteria. Their putative role in the activated sludge with ASSR is also discussed in this study. Copyright © 2012 Elsevier Ltd. All rights reserved.

Influence of pasteurization, brining conditions and production environment on the microbiota of artisan Gouda-type cheeses.

PubMed

Van Hoorde, Koenraad; Heyndrickx, Marc; Vandamme, Peter; Huys, Geert

2010-05-01

To monitor the effect of the indigenous milk microbiota and of technological and environmental parameters on the microbiota established in ripened cheese, the diversity and dynamics of the predominant microbial communities in artisan Gouda-type cheeses produced under different conditions was studied. A total of 22 cheese types differing in milk source, milk treatment, production environment and brining conditions were analyzed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) using total DNA extracts as well as DNA extracted from culturable fractions. Through band position analysis and band sequencing, the majority of DGGE bands could be attributed to lactic acid bacteria (LAB), although a few bands also belonged to staphylococci and gamma-Proteobacteria. Aided by principal component analysis (PCA) and multivariate analysis of variance (MANOVA), cheeses produced at different locations could clearly be differentiated. The same approach also allowed to distinguish raw and pasteurized milk cheeses, the former showing a more diverse microbiota in terms of a higher species richness and number of DGGE bands. No substantial differences were found between cheeses brined at two different locations. In conclusion, the combined PCR-DGGE approach relying on both total DNA extracts and culturable fractions proved its value for analyzing the effect of technological and environmental parameters on the diversity and dynamics of the microbiota in Gouda-type cheeses. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Genetic diversity pattern of microeukaryotic communities and its relationship with the environment based on PCR-DGGE and T-RFLP techniques in Dongshan Bay, southeast China

NASA Astrophysics Data System (ADS)

Zhang, Wenjing; Mo, Yuanyuan; Yang, Jun; Zhou, Jing; Lin, Yuanshao; Isabwe, Alain; Zhang, Jian; Gao, Xiu; Yu, Zheng

2018-07-01

Microeukaryotes play important roles in aquatic ecosystems, and could act as drivers of the biological nutrient cycling processes. However, compared with prokaryotic ones, little is known about the genetic diversity pattern of their community, and the environmental factors affecting their ecological pattern, especially in marine ecosystems. In this study, we used denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) to explore the genetic diversity structure of microeukaryotic communities in Dongshan Bay, southeast China. Our results revealed that microeukaryotic diversity ranged from 31 to 48 phylotypes (on average, 42) using the DGGE approach, while from 22 to 38 phylotypes (on average, 30) based on T-RFLP method, and the Shannon-Wiener diversity (DGGE-based) was relatively higher, suggesting that DGGE displayed a slightly higher resolution than T-RFLP. Surprisingly, the DGGE showed significant horizontal difference among microeukaryotic communities, but was similar with T-RFLP analysis that had no significant influence on microeukaryotic diversity at vertical scale. Further, redundancy analysis (RDA) indicated that the DGGE-based microeukaryotic communities distribution was significantly correlated with three environmental factors (Chl-a, TP and salinity), whereas microeukaryotic community revealed by T-RFLP was affected by four environmental factors namely salinity, temperature, depth and NOX-N. Compared with RDA, BIO-ENV analysis showed that heterotrophic bacteria and NOX-N were important environmental variable influencing microeukaryotic communities in both methods. These differences may be attributed to the noisy effects caused by the relatively large number of environmental variables. Generally speaking, despite differences in beta-diversity ordination for both DGGE and T-RFLP methods, there exists some consistency in the results of both techniques in terms of microeukaryotes responses to the environmental variables. These results suggested that environmental parameters had a great effect on spatial distribution of microeukaryotic community and contributed to marine ecosystem health to be further evaluated.

Effect of the length of the cycle on biodegradable polymer production and microbial community selection in a sequencing batch reactor.

PubMed

Dionisi, Davide; Majone, Mauro; Vallini, Giovanni; Gregorio, Simona Di; Beccari, Mario

2007-01-01

The effect of the length of the cycle on the enrichment and selection of mixed cultures in sequencing batch reactors (SBRs) has been studied, with the aim of biodegradable polymers (namely, polyhydroxyalkanoates (PHAs)) production from organic wastes. At a fixed feed concentration (20 gCOD/L) and organic loading rate (20 gCOD/L/day), the SBR was operated at different lengths of the cycle, in the range 1-8 h. Process performance was measured by considering the rates and yields of polymer storage and of the competing phenomenon of growth. The selected biomass was enriched with microorganisms that were able to store PHAs at high rates and yields only when the length of the cycle was 2 or 4 h, even though in these conditions the process was unstable. On the other hand, when the length of the cycle was 1 or 8 h, the dynamic response of the selected microorganisms was dominated by growth. The best process performance was characterized by storage rates in the range 500-600 mgCOD/gCOD/h and storage yields of 0.45-0.55 COD/COD. The corresponding productivity of the process was in the range 0.25-0.30 gPHA/L/h, the highest values obtained until now for mixed cultures. The microbial composition of the selected biomasses was analyzed through denaturing gradient gel electrophoresis (DGGE) and reverse-transcriptase denaturing gradient gel electrophoresis (RT-DGGE). The instability of the runs characterized by high storage rate was associated with a higher microbial heterogeneity compared to the runs with a stable growth response.

Community Analysis and Recovery of Phenol-degrading Bacteria from Drinking Water Biofilters

PubMed Central

Gu, Qihui; Wu, Qingping; Zhang, Jumei; Guo, Weipeng; Wu, Huiqing; Sun, Ming

2016-01-01

Phenol is a ubiquitous organic contaminant in drinking water. Biodegradation plays an important role in the elimination of phenol pollution in the environment, but the information about phenol removal by drinking water biofilters is still lacking. Herein, we study an acclimated bacterial community that can degrade over 80% of 300 mg/L phenol within 3 days. PCR detection of genotypes involved in bacterial phenol degradation revealed that the degradation pathways contained the initial oxidative attack by phenol hydroxylase, and subsequent ring fission by catechol 1,2-dioxygenase. Based on the PCR denatured gradient gel electrophoresis (PCR-DGGE) profiles of bacteria from biological activated carbon (BAC), the predominant bacteria in drinking water biofilters including Delftia sp., Achromobacter sp., and Agrobacterium sp., which together comprised up to 50% of the total microorganisms. In addition, a shift in bacterial community structure was observed during phenol biodegradation. Furthermore, the most effective phenol-degrading strain DW-1 that correspond to the main band in denaturing gradient gel electrophoresis (DGGE) profile was isolated and identified as Acinetobacter sp., according to phylogenetic analyses of the 16S ribosomal ribonucleic acid (rRNA) gene sequences. The strain DW-1 also produced the most important enzyme, phenol hydroxylase, and it also exhibited a good ability to degrade phenol when immobilized on granular active carbon (GAC). This study indicates that the enrichment culture has great potential application for treatment of phenol-polluted drinking water sources, and the indigenous phenol-degrading microorganism could recover from drinking water biofilters as an efficient resource for phenol removal. Therefore, the aim of this study is to draw attention to recover native phenol-degrading bacteria from drinking water biofilters, and use these native microorganisms as phenolic water remediation in drinking water sources. PMID:27148185

Community Analysis and Recovery of Phenol-degrading Bacteria from Drinking Water Biofilters.

PubMed

Gu, Qihui; Wu, Qingping; Zhang, Jumei; Guo, Weipeng; Wu, Huiqing; Sun, Ming

2016-01-01

Phenol is a ubiquitous organic contaminant in drinking water. Biodegradation plays an important role in the elimination of phenol pollution in the environment, but the information about phenol removal by drinking water biofilters is still lacking. Herein, we study an acclimated bacterial community that can degrade over 80% of 300 mg/L phenol within 3 days. PCR detection of genotypes involved in bacterial phenol degradation revealed that the degradation pathways contained the initial oxidative attack by phenol hydroxylase, and subsequent ring fission by catechol 1,2-dioxygenase. Based on the PCR denatured gradient gel electrophoresis (PCR-DGGE) profiles of bacteria from biological activated carbon (BAC), the predominant bacteria in drinking water biofilters including Delftia sp., Achromobacter sp., and Agrobacterium sp., which together comprised up to 50% of the total microorganisms. In addition, a shift in bacterial community structure was observed during phenol biodegradation. Furthermore, the most effective phenol-degrading strain DW-1 that correspond to the main band in denaturing gradient gel electrophoresis (DGGE) profile was isolated and identified as Acinetobacter sp., according to phylogenetic analyses of the 16S ribosomal ribonucleic acid (rRNA) gene sequences. The strain DW-1 also produced the most important enzyme, phenol hydroxylase, and it also exhibited a good ability to degrade phenol when immobilized on granular active carbon (GAC). This study indicates that the enrichment culture has great potential application for treatment of phenol-polluted drinking water sources, and the indigenous phenol-degrading microorganism could recover from drinking water biofilters as an efficient resource for phenol removal. Therefore, the aim of this study is to draw attention to recover native phenol-degrading bacteria from drinking water biofilters, and use these native microorganisms as phenolic water remediation in drinking water sources.

Molecular characterization of microbial population dynamics during sildenafil citrate degradation.

PubMed

De Felice, Bruna; Argenziano, Carolina; Guida, Marco; Trifuoggi, Marco; Russo, Francesca; Condorelli, Valerio; Inglese, Mafalda

2009-02-01

Little is known about pharmaceutical and personal care products pollutants (PPCPs), but there is a growing interest in how they might impact the environment and microbial communities. The widespread use of Viagra (sildenafil citrate) has attracted great attention because of the high usage rate, the unpredictable disposal and the unknown potential effects on wildlife and the environment. Until now information regarding the impact of Viagra on microbial community in water environment has not been reported. In this research, for the first time, the genetic profile of the microbial community, developing in a Viagra polluted water environment, was evaluated by means of the 16S and 18S rRNA genes, for bacteria and fungi, respectively, amplified by polymerase chain reaction (PCR) and separated using the denaturing gradient gel electrophoresis (DGGE) technique. The DGGE results revealed a complex microbial community structure with most of the population persisting throughout the experimental period. DNA sequences from bands observed in the different denaturing gradient gel electrophoresis profiles exhibited the highest degree of identity to uncultured bacteria and fungi found previously mainly in polluted environmental and treating bioreactors. Biotransformation ability of sildenafil citrate by the microbial pool was studied and the capability of these microorganisms to detoxify a polluted water ecosystem was assessed. The bacterial and fungal population was able to degrade sildenafil citrate entirely. Additionally, assays conducted on Daphnia magna, algal growth inhibition assay and cell viability determination on HepG2 human cells showed that biotransformation products obtained from the bacterial growth was not toxic. The higher removal efficiency for sildenafil citrate and the lack of toxicity by the biotransformation products obtained showed that the microbial community identified here represented a composite population that might have biotechnological relevance to retrieve sildenafil citrate contaminated sites.

Molecular and Culture-Based Assessment of the Microbial Diversity of Diabetic Chronic Foot Wounds and Contralateral Skin Sites

PubMed Central

Oates, Angela; Bowling, Frank L.; Boulton, Andrew J. M.

2012-01-01

Wound debridement samples and contralateral (healthy) skin swabs acquired from 26 patients attending a specialist foot clinic were analyzed by differential isolation and eubacterium-specific PCR-denaturing gradient gel electrophoresis (DGGE) in conjunction with DNA sequencing. Thirteen of 26 wounds harbored pathogens according to culture analyses, with Staphylococcus aureus being the most common (13/13). Candida (1/13), pseudomonas (1/13), and streptococcus (7/13) were less prevalent. Contralateral skin was associated with comparatively low densities of bacteria, and overt pathogens were not detected. According to DGGE analyses, all wounds contained significantly greater eubacterial diversity than contralateral skin (P < 0.05), although no significant difference in total eubacterial diversity was detected between wounds from which known pathogens had been isolated and those that were putatively uninfected. DGGE amplicons with homology to Staphylococcus sp. (8/13) and S. aureus (2/13) were detected in putatively infected wound samples, while Staphylococcus sp. amplicons were detected in 11/13 noninfected wounds; S. aureus was not detected in these samples. While a majority of skin-derived DGGE consortial fingerprints could be differentiated from wound profiles through principal component analysis (PCA), a large minority could not. Furthermore, wounds from which pathogens had been isolated could not be distinguished from putatively uninfected wounds on this basis. In conclusion, while chronic wounds generally harbored greater eubacterial diversity than healthy skin, the isolation of known pathogens was not associated with qualitatively distinct consortial profiles or otherwise altered diversity. The data generated support the utility of both culture and DGGE for the microbial characterization of chronic wounds. PMID:22553231

[Analysis of the oral microbiota in twin children].

PubMed

Du, Qin; Wang, Yan; Xu, Xin; Li, Yuqing; Li, Mingyun; Zou, Jing; Zhou, Xuedong

2014-04-01

To analyze the differences between the oral microbiota of monozygotic and dizygotic twins by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). A total of 20 pairs of twin children were included in this study, in which 10 pairs were monozygotic (MZ) twins, and 10 pairs were dizygotic (DZ) twins. Of the 20 pairs, 10 pairs of twins had primary dentition, and 10 pairs had mixed dentition; 17 children had caries, and 23 children had no caries. Genomic DNA was extracted from saliva samples. The 16s rRNA was amplified and analyzed by PCR-DGGE. The PCR-DGGE band number and Shannon index were calculated. Cluster analysis showed high similarity in the oral bacterial community seen in co-twins. However, no significant difference was seen between MZ and DZ twins. In the primary dentition, the PCR-DGGE band number and Shannon index of children with caries (11.00 +/- 1.56, 1.05 +/- 0.36) were lower than those of children without caries (14.00 +/- 2.74, 1.44 +/- 0.37) (P < 0.05). In mixed dentition, the PCR-DGGE band number and Shannon index of children with caries (11.88 +/- 4.05, 1.18 +/- 0.36) were lower than those of children without caries (14.31 +/- 5.71, 1.28 +/- 0.47), but the differences were not statistically significant (P > 0.05). Environmental factors may have a stronger effect on the constitution of oral microbiota in children compared with genetic factors. Children without caries may have a richer microbial diversity compared with children with caries.

Efficiency of temporary storage of geothermal waters in a lake system: Monitoring the changes of water quality and bacterial community structures.

PubMed

Szirányi, Barbara; Krett, Gergely; Kosáros, Tünde; Janurik, Endre; Pekár, Ferenc; Márialigeti, Károly; Borsodi, Andrea K

2017-12-01

Disposal of used geothermal waters in Hungary often means temporary storage in reservoir lakes to reduce temperature and improve water quality. In this study, the physical and chemical properties and changes in the bacterial community structure of a reservoir lake system in southeast region of Hungary were monitored and compared through 2 years, respectively. The values of biological oxygen demand, concentrations of ammonium ion, total inorganic nitrogen, total phosphorous, and total phenol decreased, whereas oxygen saturation, total organic nitrogen, pH, and conductivity increased during the storage period. Bacterial community structure of water and sediment samples was compared by denaturing gradient gel electrophoresis (DGGE) following the amplification of the 16S rRNA gene. According to the DGGE patterns, greater seasonal than spatial differences of bacterial communities were revealed in both water and sediment of the lakes. Representatives of the genera Arthrospira and Anabaenopsis (cyanobacteria) were identified as permanent and dominant members of the bacterial communities.

Effects of Hypoxia on the Phylogenetic Composition and Species Distribution of Protists in a Subtropical Harbor.

PubMed

Rocke, Emma; Jing, Hongmei; Xia, Xiaomin; Liu, Hongbin

2016-07-01

Tolo Harbor, a subtropical semi-enclosed coastal water body, is surrounded by an expanding urban community, which contributes to large concentrations of nutrient runoff, leading to algal blooms and localized hypoxic episodes. Present knowledge of protist distributions in subtropical waters during hypoxic conditions is very limited. In this study, therefore, we combined parallel 454 pyrosequencing technology and denaturing gradient gel electrophoresis (DGGE) fingerprint analyses to reveal the protist community shifts before, during, and after a 2-week hypoxic episode during the summer of 2011. Hierarchical clustering for DGGE demonstrated similar grouping of hypoxic samples separately from oxic samples. Dissolved oxygen (DO) concentration and dissolved inorganic nitrogen:phosphate (DIN:PO4) concentrations significantly affected OTU distribution in 454 sequenced samples, and a shift toward a ciliate and marine alveolate clade II (MALV II) species composition occurred as waters shifted from oxic to hypoxic. These results suggest that protist community shifts toward heterotrophic and parasitic tendencies as well as decreased diversity and richness in response to hypoxic outbreaks.

[Fungal community structure in phase II composting of Volvariella volvacea].

PubMed

Chen, Changqing; Li, Tong; Jiang, Yun; Li, Yu

2014-12-04

To understand the fungal community succession during the phase II of Volvariella volvacea compost and clarify the predominant fungi in different fermentation stages, to monitor the dynamic compost at the molecular level accurately and quickly, and reveal the mechanism. The 18S rDNA-denaturing gradient gel electrophoresis (DGGE) and sequencing methods were used to analyze the fungal community structure during the course of compost. The DGGE profile shows that there were differences in the diversity of fungal community with the fermentation progress. The diversity was higher in the stages of high temperature. And the dynamic changes of predominant community and relative intensity was observed. Among the 20 predominant clone strains, 9 were unknown eukaryote and fungi, the others were Eurotiales, Aspergillus sp., Melanocarpus albomyces, Colletotrichum sp., Rhizomucor sp., Verticillium sp., Penicillium commune, Microascus trigonosporus and Trichosporon lactis. The 14 clone strains were detected in the stages of high and durative temperature. The fungal community structure and predominant community have taken dynamic succession during the phase II of Volvariella volvacea compost.

Effects of organic loading rates on reactor performance and microbial community changes during thermophilic aerobic digestion process of high-strength food wastewater.

PubMed

Jang, Hyun Min; Lee, Jae Won; Ha, Jeong Hyub; Park, Jong Moon

2013-11-01

To evaluate the applicability of single-stage thermophilic aerobic digestion (TAD) process treating high-strength food wastewater (FWW), TAD process was operated at four organic loading rates (OLRs) from 9.2 to 37.2 kg COD/m(3)d. The effects of OLRs on microbial community changes were also examined. The highest volumetric removal rate (13.3 kg COD/m(3)d) and the highest thermo-stable protease activity (0.95 unit/mL) were detected at OLR=18.6 kg COD/m(3)d. Denaturing gradient gel electrophoresis (DGGE) profiles and quantitative PCR (qPCR) results showed significant microbial community shifts in response to changes in OLR. In particular, DGGE and phylogenetic analysis demonstrate that the presence of Bacillus sp. (phylum of Firmicutes) was strongly correlated with efficient removal of organic particulates from high-strength food wastewater. Copyright © 2013 Elsevier Ltd. All rights reserved.

Raw Cow Milk Bacterial Population Shifts Attributable to Refrigeration

PubMed Central

Lafarge, Véronique; Ogier, Jean-Claude; Girard, Victoria; Maladen, Véronique; Leveau, Jean-Yves; Gruss, Alexandra; Delacroix-Buchet, Agnès

2004-01-01

We monitored the dynamic changes in the bacterial population in milk associated with refrigeration. Direct analyses of DNA by using temporal temperature gel electrophoresis (TTGE) and denaturing gradient gel electrophoresis (DGGE) allowed us to make accurate species assignments for bacteria with low-GC-content (low-GC%) (<55%) and medium- or high-GC% (>55%) genomes, respectively. We examined raw milk samples before and after 24-h conservation at 4°C. Bacterial identification was facilitated by comparison with an extensive bacterial reference database (∼150 species) that we established with DNA fragments of pure bacterial strains. Cloning and sequencing of fragments missing from the database were used to achieve complete species identification. Considerable evolution of bacterial populations occurred during conservation at 4°C. TTGE and DGGE are shown to be a powerful tool for identifying the main bacterial species of the raw milk samples and for monitoring changes in bacterial populations during conservation at 4°C. The emergence of psychrotrophic bacteria such as Listeria spp. or Aeromonas hydrophila is demonstrated. PMID:15345453

Atrazine, chlorpyrifos, and iprodione effect on the biodiversity of bacteria, actinomycetes, and fungi in a pilot biopurification system with a green cover.

PubMed

Elgueta, Sebastian; Correa, Arturo; Campo, Marco; Gallardo, Felipe; Karpouzas, Dimitrios; Diez, Maria Cristina

2017-09-02

The use of biopurification systems can mitigate the effects of pesticide contamination on farms. The primary aim of this study was to evaluate the effect of pesticide dissipation on microbial communities in a pilot biopurification system. The pesticide dissipation of atrazine, chlorpyrifos and iprodione (35 mg kg -1 active ingredient [a.i.]) and biological activity were determined for 40 days. The microbial communities (bacteria, actinomycetes and fungi) were analyzed using denaturing gradient gel electrophoresis (DGGE). In general, pesticide dissipation was the highest by day 5 and reached 95%. The pesticides did not affect biological activity during the experiment. The structure of the actinomycete and bacterial communities in the rhizosphere was more stable during the evaluation than that in the communities in the control without pesticides. The rhizosphere fungal communities, detected using DGGE, showed small and transitory shifts with time. To conclude, rhizosphere microbial communities were not affected during pesticide dissipation in a pilot biopurification system.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosatelli, M.C.; Faa, V.; Sardu, R.

This study reports the molecular characterization of [beta]-thalassemia in the Sardinian population. Three thousand [beta]-thalassemia chromosomes from prospective parents presenting at the genetic service were initially analyzed by dot blot analysis with oligonucleotide probes complementary to the most common [beta]-thalassemia mutations in the Mediterranean at-risk populations. The mutation which remained uncharacterized by this approach were defined by denaturing gradient gel electrophoresis (DGGE) followed by direct sequence analysis on amplified DNA. The authors reconfirmed that the predominant mutation in the Sardinian population is the codon 39 nonsense mutation, which accounts for 95.7% of the [beta]-thalassemia chromosomes. The other two relatively commonmore » mutations are frameshifts at codon 6 (2.1%) and at codon 76 (0.7%), relatively uncommon in other Mediterranean-origin populations. In this study they have detected a novel [beta]-thalassemia mutation, i.e., a frameshift at codon 1, in three [beta]-thalassemia chromosomes. The DGGE procedure followed by direct sequencing on amplified DNA is a powerful approach for the characterization of unknown mutations in this genetic system.« less

Effects of oxytetracycline on the abundance and community structure of nitrogen-fixing bacteria during cattle manure composting.

PubMed

Sun, Jiajun; Qian, Xun; Gu, Jie; Wang, Xiaojuan; Gao, Hua

2016-09-01

The effects of oxytetracycline (OTC) on nitrogen-fixing bacterial communities were investigated during cattle manure composting. The abundance and community structure of nitrogen-fixing bacteria were determined by qPCR and denaturing gradient gel electrophoresis (DGGE), respectively. The matrix was spiked with OTC at four levels: no OTC, 10mg/kg dry weight (DW) OTC (L), 60mg/kg DW OTC (M), and 200mg/kg DW OTC (H). The high temperature period of composting was shorter with M and H, and the decline in temperature during the cooling stage was accelerated by OTC. OTC had a concentration-dependent inhibitory effect on the nitrogenase activity during early composting, and the nifH gene abundance declined significantly during the later composting stage. The DGGE profile and statistical analysis showed that OTC changed the nitrogen-fixing bacterial community succession and reduced the community richness and dominance. The nitrogen-fixing bacterial community structure was affected greatly by the high level of OTC. Copyright © 2016. Published by Elsevier Ltd.

Denaturing gradient gel electrophoresis fingerprinting of soil bacteria in the vicinity of the Chinese Great Wall Station, King George Island, Antarctica.

PubMed

Pan, Qi; Wang, Feng; Zhang, Yang; Cai, Minghong; He, Jianfeng; Yang, Haizhen

2013-08-01

Bacterial diversity was investigated in soil samples collected from 13 sites around the Great Wall Station, Fildes Peninsula, King George Island, Antarctica, using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes. The classes alpha-, beta-, and gamma-Proteobacteria, as well as the phylum Actinobacteria, were found to be the dominant bacteria in the soils around the Great Wall Station. Although the selected samples were not contaminated by oil, a relationship between soil parameters, microbial biodiversity, and human impact was still seen. Sample sites in human impacted areas showed lower bacterial biodiversity (average H' = 2.65) when compared to non-impacted sites (average H' = 3.05). There was no statistically significant correlation between soil bacterial diversity and total organic carbon (TOC), total nitrogen, or total phosphorus contents of the soil. Canonical correlation analysis showed that TOC content was the most important factor determining bacterial community profiles among the measured soil parameters. In conclusion, microbial biodiversity and community characteristics within relatively small scales (1.5 km) were determined as a function of local environment parameters and anthropogenic impact.

Detection and quantification of major toxigenic Microcystis genotypes in Moo-Tan reservoir and associated water treatment plant.

PubMed

Yen, Hung-Kai; Lin, Tsair-Fuh; Tseng, I-Cheng

2012-02-01

Two molecular methods, denaturing gradient gel electrophoresis (DGGE) and quantitative real-time polymerase chain reaction (qPCR) with the Universal ProbeLibrary (UPL) probe, were developed and used for the characterization and quantification of several microcystin producers in Moo-Tan Reservoir (MTR), Taiwan and its associated water treatment plant (Shih-Men Water Treatment Plant, SMWTP). Internal transcribed spacer (ITS) sequence, a highly diversified region between the 16S rRNA and 23S rRNA genes, was used to further identify the isolated strains from MTR and also used in DGGE for the detection of the specific DNA fragments and biomarkers for 11 strains observed in MTR. These ITS-DGGE biomarkers were successfully applied to monitor the community changes of potential toxigenic Microcystis sp. over a period of five years. Two highly specific primers were combined with UPL probes to measure microcystins synthesis gene (mcyB) and phycocyanin intergenic spacer region (cpcB) concentrations in water samples. The copy concentrations of UPL-mcyB and UPL-cpcB correlated well with MC-RR concentrations/water temperature and Microcystis sp. cell numbers in the water samples, respectively. For SMWTP, toxin concentrations were low, but the DGGE bands clearly demonstrated the presence of potential microcystin producers in both water treatment plants and finished water samples. It was demonstrated that toxigenic Microcystis sp. may penetrate through the treatment processes and pose a potential risk to human health in the drinking water systems.

Fecal Microbiota in Healthy Subjects Following Omnivore, Vegetarian and Vegan Diets: Culturable Populations and rRNA DGGE Profiling.

PubMed

Ferrocino, Ilario; Di Cagno, Raffaella; De Angelis, Maria; Turroni, Silvia; Vannini, Lucia; Bancalari, Elena; Rantsiou, Kalliopi; Cardinali, Gianluigi; Neviani, Erasmo; Cocolin, Luca

2015-01-01

In this study, the fecal microbiota of 153 healthy volunteers, recruited from four different locations in Italy, has been studied by coupling viable counts, on different microbiological media, with ribosomal RNA Denaturing Gradient Gel Electrophoresis (rRNA-DGGE). The volunteers followed three different diets, namely omnivore, ovo-lacto-vegetarian and vegan. The results obtained from culture-dependent and -independent methods have underlined a high level of similarity of the viable fecal microbiota for the three investigated diets. The rRNA DGGE profiles were very complex and comprised a total number of bands that varied from 67 to 64 for the V3 and V9 regions of the 16S rRNA gene, respectively. Only a few bands were specific in/of all three diets, and the presence of common taxa associated with the dietary habits was found. As far as the viable counts are concerned, the high similarity of the fecal microbiota was once again confirmed, with only a few of the investigated groups showing significant differences. Interestingly, the samples grouped differently, according to the recruitment site, thus highlighting a higher impact of the food consumed by the volunteers in the specific geographical locations than that of the type of diet. Lastly, it should be mentioned that the fecal microbiota DGGE profiles obtained from the DNA were clearly separated from those produced using RNA, thus underlining a difference between the total and viable populations in the fecal samples.

Habitual dietary intake is associated with stool microbiota composition in monozygotic twins.

PubMed

Simões, Catarina D; Maukonen, Johanna; Kaprio, Jaakko; Rissanen, Aila; Pietiläinen, Kirsi H; Saarela, Maria

2013-04-01

The impact of diet on the gut microbiota has usually been assessed by subjecting people to the same controlled diet and thereafter following the shifts in the microbiota. In the present study, we used habitual dietary intake, clinical data, quantitative polymerase chain reaction, and denaturing gradient gel electrophoresis (DGGE) to characterize the stool microbiota of Finnish monozygotic twins. The effect of diet on the numbers of bacteria was described through a hierarchical linear mixed model that included the twin individuals, stratified by body mass index, and their families as random effects. The abundance and diversity of the bacterial groups studied did not differ between normal-weight, overweight, and obese individuals with the techniques used. Intakes of energy, monounsaturated fatty acids, n3 polyunsaturated fatty acids (PUFAs), n6 PUFAs, and soluble fiber had significant associations with the stool bacterial numbers (e.g., increased energy intake was associated with reduced numbers of Bacteroides spp.). In addition, co-twins with identical energy intake had more similar numbers and DGGE-profile diversities of Bacteroides spp. than did the co-twins with different intake. Moreover, the co-twins who ingested the same amounts of saturated fatty acids had very similar DGGE profiles of Bacteroides spp., whereas the co-twins with similar consumption of fiber had a very low bifidobacterial DGGE-profile similarity. In conclusion, our findings confirm that the diet plays an important role in the modulation of the stool microbiota, in particular Bacteroides spp. and bifidobacteria.

Fecal Microbiota in Healthy Subjects Following Omnivore, Vegetarian and Vegan Diets: Culturable Populations and rRNA DGGE Profiling

PubMed Central

Ferrocino, Ilario; Di Cagno, Raffaella; De Angelis, Maria; Turroni, Silvia; Vannini, Lucia; Bancalari, Elena; Rantsiou, Kalliopi; Cardinali, Gianluigi; Neviani, Erasmo; Cocolin, Luca

2015-01-01

In this study, the fecal microbiota of 153 healthy volunteers, recruited from four different locations in Italy, has been studied by coupling viable counts, on different microbiological media, with ribosomal RNA Denaturing Gradient Gel Electrophoresis (rRNA-DGGE). The volunteers followed three different diets, namely omnivore, ovo-lacto-vegetarian and vegan. The results obtained from culture-dependent and -independent methods have underlined a high level of similarity of the viable fecal microbiota for the three investigated diets. The rRNA DGGE profiles were very complex and comprised a total number of bands that varied from 67 to 64 for the V3 and V9 regions of the 16S rRNA gene, respectively. Only a few bands were specific in/of all three diets, and the presence of common taxa associated with the dietary habits was found. As far as the viable counts are concerned, the high similarity of the fecal microbiota was once again confirmed, with only a few of the investigated groups showing significant differences. Interestingly, the samples grouped differently, according to the recruitment site, thus highlighting a higher impact of the food consumed by the volunteers in the specific geographical locations than that of the type of diet. Lastly, it should be mentioned that the fecal microbiota DGGE profiles obtained from the DNA were clearly separated from those produced using RNA, thus underlining a difference between the total and viable populations in the fecal samples. PMID:26035837

Natural attenuation is enhanced in previously contaminated and coniferous forest soils.

PubMed

Kauppi, Sari; Romantschuk, Martin; Strömmer, Rauni; Sinkkonen, Aki

2012-01-01

Prevalence of organic pollutants or their natural analogs in soil is often assumed to lead to adaptation in the bacterial community, which results in enhanced bioremediation if the soil is later contaminated. In this study, the effects of soil type and contamination history on diesel oil degradation and bacterial adaptation were studied. Mesocosms of mineral and organic forest soil (humus) were artificially treated with diesel oil, and oil hydrocarbon concentrations (GC-FID), bacterial community composition (denaturing gradient gel electrophoresis, DGGE), and oil hydrocarbon degraders (DGGE + sequencing of 16S rRNA genes) were monitored for 20 weeks at 16°C. Degradation was advanced in previously contaminated soils as compared with pristine soils and in coniferous organic forest soil as compared with mineral soil. Contamination affected bacterial community composition especially in the pristine mineral soil, where diesel addition increased the number of strong bands in the DGGE gel. Sequencing of cloned 16S rRNA gene fragments and DGGE bands showed that potential oil-degrading bacteria were found in mineral and organic soils and in both pristine and previously contaminated mesocosms. Fast oil degradation was not associated with the presence of any particular bacterial strain in soil. We demonstrate at the mesocosm scale that previously contaminated and coniferous organic soils are superior environments for fast oil degradation as compared with pristine and mineral soil environments. These results may be utilized in preventing soil pollution and planning soil remediation.

Characterization of eubacterial and archaeal community diversity in the pit mud of Chinese Luzhou-flavor liquor by nested PCR-DGGE.

PubMed

Ding, Xiao-Fei; Wu, Chong-De; Zhang, Li-Qiang; Zheng, Jia; Zhou, Rong-Qing

2014-02-01

The aim of this study was to investigate and compare the microbial community structures of eubacteria and archaea in the pit mud of Chinese Luzhou-flavor liquor from the wall (C(w)) and bottom (C(b)) of cellar through nested PCR-denaturing gradient gel electrophoresis (DGGE). The Shannon-Wiener index (H) calculated from the DGGE profiles showed that the community diversities of eubacteria and archaea in samples from C(b) were almost higher than that from C(w). In addition, cluster analysis of the DGGE profiles revealed that some differences were found in the microbial community structure in samples from different locations. The closely relative microorganisms of all eubacterial 16S rRNA gene sequences fell into four phyla (Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria), including 12 genera and 2 uncultured eubacteria. Moreover, 37.1% eubacteria were affiliated with Clostridium. Particularly, genus Acinetobacter was absent in all samples from C(b) but present in all samples from C(w). The closely relative microorganisms of all archaeal 16S rRNA gene sequences fell into four genera, which included Methanobrevibacter, Methanoculleus, Methanobacterium and Methanosaeta, while the dominant archaea in samples from C(w) and C(b) were similar. Results presented in this study provide further understanding of the spatial differences in microbial community structure in the pit mud, and is of great importance for the production and quality improvement of Luzhou-flavor liquor.

Changes to the structure of Sphingomonas spp. communities associated with biodegradation of the herbicide isoproturon in soil.

PubMed

Shi, Shengjing; Bending, Gary D

2007-04-01

The phenyl-urea herbicide isoproturon is a major contaminant of surface and ground-water in agricultural catchments. Earlier work suggested that within-field spatial variation of isoproturon degradation rate resulted from interactions between catabolizing Sphingomonas spp. and pH. In the current study, changes to the structure of Sphingomonas communities during isoproturon catabolism were investigated using Sphingomonas-specific 16S rRNA gene primers. Growth-linked catabolism at high-pH (>7.5) sites was associated with the appearance of multiple new denaturing gradient gel electrophoresis (DGGE) bands. At low-pH sites, there was no change in DGGE banding at sites in which there was cometabolism, but at sites in which there was growth-linked catabolism, degradation was associated with the appearance of a new band not present at high pH sites. Sequencing of DGGE bands indicated that a strain related to Sphingomonas mali proliferated at low pH sites, while strain Sphingomonas sp. SRS2, a catabolic strain identified in earlier work, together with several further Sphingomonas spp., proliferated at high-pH sites. The data indicate that degradation was associated with complex changes to the structure of Sphingomonas spp. communities, the precise nature of which was spatially variable.

Distribution of oil-degrading bacteria in coastal seawater, Toyama Bay, Japan.

PubMed

Tanaka, Daisuke; Tanaka, Shunsuke; Yamashiro, Yoko; Nakamura, Shogo

2008-10-01

Oil-degrading bacteria are considered to play an important role in the biodegradation of spilled or released oil in the sea. The distribution of indigenous oil-degrading bacteria in the coastal seawater of Toyama Bay, Japan, was examined. Surface seawater samples with or without oil film in fishing port were analyzed by denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V3 region of bacterial 16S rDNA. Sequence analysis revealed that several DGGE bands clearly detected only in samples with oil film corresponded to Cyanobacteria. Moreover, we cultured surface seawater samples with oil film in two different liquid culture media, a marine broth and an NSW medium; each culture contained 0.5% (w/v) C-heavy oil. Emulsification of the oil was observed at day 6 in the marine broth and day 9 in the NSW medium. Time-dependent changes of bacterial communities in those culture media were analyzed by DGGE. Interestingly, we found that Alcanivorax sp. became one of the dominant bacteria in each culture medium when emulsification of the oil began. Alcanivorax sp. is one of the well-known oil-degrading bacteria in seawater and is associated with the production of biosurfactants. These results suggest that Cyanobacteria and Alcanivorax play important roles in the bioremediation of oil-contaminated areas in Toyama Bay.

Microbial community dynamics in anaerobic bioreactors and algal tanks treating piggery wastewater.

PubMed

Patil, Sayali S; Kumar, Martin S; Ball, Andrew S

2010-06-01

Integrated biosystem is becoming a major aspect of wastewater management practice. Microbial communities in piggery wastewater sampled from anaerobic (thermophilic and mesophilic) and aerobic digesters (algal tanks) during waste remediation were analyzed by culture-independent techniques based on polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The use of Muyzer's 314F-GC, 518R bacterial primers, and archaeal A934F, 1309R primers followed by partial 16s rDNA sequence analysis of the main bands from DGGE revealed the presence of unknown and as yet uncultured microorganisms but also showed functional and ecologically significant denitrifying, acetogenic bacteria along with autotrophic, hydrogenotrophic, and acetoclastic methanogen archaea. Thermophilic digesters were dominated by gamma-Proteobacteria, Methanothermobacter sp., while mesophilic digesters showed dominance by Firmicutes, uncultured bacteria, Methanosarcina, and Methanoculleus genera. Under aerobic conditions within algal tanks, pH rose from 7.17 to 9.32, with a significant decrease in total ammonia nitrogen, chemical oxygen demand, and soluble phosphorus levels. PCR-DGGE proved a useful tool for investigating the dynamics of microbial community in the bio-processing of piggery wastewater. Knowledge of the microbial communities involved in digestion of piggery wastewater will allow optimization of integrated biosystem by removing the main pollutants like inorganic ammonium-nitrogen, phosphorus, and pathogens from intensive farming system.

Suppression of Listeria monocytogenes by the Native Micro-Flora in Teewurst Sausage

PubMed Central

Austin-Watson, Clytrice; Grant, Ar’Quette; Brice, Michline

2013-01-01

Modern consumers are interested in the use of non-chemical methods to control pathogens when heat sterilization is not an option. Such is the case with teewurst sausage, a raw spreadable sausage and a popular German commodity. Although Listeria was not found in teewurst, the optimal microbial growing conditions of teewurst coupled with the ubiquity of L. monocytogenes in nature, makes the possibility of contamination of products very possible. This pilot study was conducted to examine teewurst’s native micro-flora’s ability to suppress the outgrowth of L. monocytogenes at 10 °C using standard plate counts and PCR-DGGE. Traditional plating methods showed L. monocytogenes growth significantly decreased when in competition with the teewurst’s native micro-flora (p < 0.05). The native micro-flora of the teewurst suppressed the overall growth of L. monocytogenes by an average of two logs, under these conditions. Denaturing Gradient Gel Electrophoresis (DGGE) amplicons with unique banding patterns were extracted from DGGE gel for identification. Brochothrix thermosphacta and Lactobacillus curvatus were identified as a part of the teewurst’s native micro-flora. Although the native micro-flora did not decrease L. monocytogenes to below limits of detection, it was enough of a decrease to warrant further investigation. PMID:28239131

Molecular microbiological characterization of preterm neonates at risk of bronchopulmonary dysplasia.

PubMed

Payne, Matthew S; Goss, Kevin C W; Connett, Gary J; Kollamparambil, Tanoj; Legg, Julian P; Thwaites, Richard; Ashton, Mark; Puddy, Victoria; Peacock, Janet L; Bruce, Kenneth D

2010-04-01

The role of infection in bronchopulmonary dysplasia (BPD) is unknown. We present an observational study of 55 premature infants born weighing less than 1.3 kg within two level III neonatal intensive care units. Endotracheal aspirates (ETA) and nasogastric aspirates (NGA) were studied with denaturing gradient gel electrophoresis (DGGE) profiling to elucidate the total bacterial community, and species-specific PCR was used to detect the presence of Mycoplasma hominis, Ureaplasma urealyticum, and Ureaplasma parvum. DGGE identified bacterial species in 59% of NGA and ETA samples combined. A diverse range of species were identified including several implicated in preterm labor. Species-specific PCR identified M. hominis in 25% of NGA and 11% of ETA samples. Among the 48 infants surviving up to 36 wk-postconceptual age, ordinal logistic regression showed the odds ratio for BPD or death where Ureaplasma was present/absent as 4.80 (95% CI 1.15-20.13). After adjusting for number of days ventilated, this was reduced to 2.04 (0.41-10.25). These data demonstrate how the combined use of DGGE and species-specific PCR identifies a high exposure in utero and around the time of birth to bacteria that might be causally related to preterm delivery and subsequent lung injury.

Analysis of the bacterial community in aged and aging pit mud of Chinese Luzhou-flavour liquor by combined PCR-DGGE and quantitative PCR assay.

PubMed

Liang, Huipeng; Li, Wenfang; Luo, Qingchun; Liu, Chaolan; Wu, Zhengyun; Zhang, Wenxue

2015-10-01

The community structure of bacteria in aged and aging pit mud, which was judged according to their sensory and physicochemical characteristics, was analysed using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative real-time PCR (qPCR). The phyla Firmicutes, Actinobacteria, Proteobacteria, Synergistetes and Unclassified Bacteria were detected and the fermentative Firmicutes was predominant in both types of pit mud in the PCR-DGGE analysis. Among Firmicutes, Clostridiales was dominant in aged pit mud while Bacillales and Lactobacillales were dominant in aging pit mud. The diversity of bacterial communities in aged pit mud was higher than that in aging pit mud. In the qPCR analysis the abundance of Clostridium IV in aged pit mud was higher than that in aging pit mud and there were significant differences in the quantity of Clostridium IV between aged and aging pit mud of the same cellar (P < 0.05). There were some significant differences in the microbial community structure between aged and aging pit mud. The differences in the quantity of Clostridium IV might be involved in the distinction that the aged pit mud has a strong aroma while the aging pit mud does not. © 2014 Society of Chemical Industry.

Microbiological study of lactic acid bacteria in kefir grains by culture-dependent and culture-independent methods.

PubMed

Chen, Hsi-Chia; Wang, Sheng-Yao; Chen, Ming-Ju

2008-05-01

Lactic acid bacteria (LAB) in different original kefir grains were first assessed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) by a culture-dependent way, and were further confirmed by DNA sequencing techniques. Results indicated that a combined method of cultivation with PCR-DGGE and subsequent DNA sequencing could successfully identify four LAB strains from three kefir grains from Taiwan (named Hsinchu, Mongolia and Ilan). Lactobacillus kefiri accounted, in the three kefir grains, for at least half of the isolated colonies while Lb. kefiranofaciens was the second most frequently isolated species. Leuconostoc mesenteroides was less frequently found but still in the three kefir grains conversely to Lactococcus lactis which based on culture-dependent isolation was only found in two of the kefir grains. It was interesting to find that all three kefir grains contain similar LAB species. Furthermore, the DGGE as a culture-independent method was also applied to detect the LAB strains. Results indicated that Lb. kefiranofaciens was found in all three kefir grains, whereas Lb. kefiri was only observed in Hsinchu kefir grain and Lc. lactis was found in both Mongolia and Ilan samples. Two additional strains, Pseudomonas spp. and E. coli, were also detected in kefir grains.

PCR-DGGE analysis of bacterial community dynamics in kava beverages during refrigeration.

PubMed

Dong, J; Kandukuru, P; Huang, A S; Li, Y

2011-07-01

Kava beverages are highly perishable even under refrigerated conditions. This study aimed to investigate the bacterial community dynamics in kava beverages during refrigeration.  Four freshly made kava beverages were obtained from kava bars and stored at 4°C. On days 0, 3 and 6, the aerobic plate count (APC), lactic acid bacteria (LAB) count and yeast and mould count (YMC) of the samples were determined. Meanwhile, bacterial DNA was extracted from each sample and subjected to the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Moreover, species-specific PCR assays were employed to identify predominant Pseudomonas spp. involved in kava spoilage. Over the storage period, the APC, LAB count and YMC of the four kava beverages all increased, whereas their pH values decreased. The DGGE profile revealed diverse bacterial populations in the samples. LAB, such as Weissella soli, Lactobacillus spp. and Lactococcus lactis, were found in the kava beverages. Species-specific PCR assays detected Pseudomonas putida and Pseudomonas fluorescens in the samples; Ps. fluorescens became dominant during refrigeration. LAB and Pseudomonas may play a significant role in the spoilage of kava beverages. This study provides important information that may be used to extend the shelf life of kava beverages. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

PCR-DGGE analysis of lactic acid bacteria and yeast dynamics during the production processes of three varieties of Panettone.

PubMed

Garofalo, C; Silvestri, G; Aquilanti, L; Clementi, F

2008-07-01

To study lactic acid bacteria (LAB) and yeast dynamics during the production processes of sweet-leavened goods manufactured with type I sourdoughs. Fourteen sourdough and dough samples were taken from a baking company in central Italy during the production lines of three varieties of Panettone. The samples underwent pH measurements and plating analysis on three solid media. The microbial DNA was extracted from both the (sour)doughs and the viable LAB and yeast cells collected in bulk, and subjected to PCR-denaturing gradient gel electrophoresis (DGGE) analysis. The molecular fingerprinting of the cultivable plus noncultivable microbial populations provide evidence of the dominance of Lactobacillus sanfranciscensis, Lactobacillus brevis and Candida humilis in the three fermentation processes. The DGGE profiles of the cultivable communities reveal a bacterial shift in the final stages of two of the production processes, suggesting an effect of technological parameters on the selection of the dough microflora. Our findings confirm the importance of using a combined analytical approach to explore microbial communities that develop during the leavening process of sweet-leavened goods. In-depth studies of sourdough biodiversity and population dynamics occurring during sourdough fermentation are fundamental for the control of the leavening process and the manufacture of standardized, high-quality products.

Long-term impact of farm management and crops on soil microorganisms assessed by combined DGGE and PLFA analyses

PubMed Central

Stagnari, Fabio; Perpetuini, Giorgia; Tofalo, Rosanna; Campanelli, Gabriele; Leteo, Fabrizio; Della Vella, Umberto; Schirone, Maria; Suzzi, Giovanna; Pisante, Michele

2014-01-01

In the present study, long-term organic and conventional managements were compared at the experimental field of Monsampolo del Tronto (Marche region, Italy) with the aim of investigating soil chemical fertility and microbial community structure. A polyphasic approach, combining soil fertility indicators with microbiological analyses (plate counts, PCR-denaturing gradient gel electrophoresis [DGGE] and phospholipid fatty acid analysis [PLFA]) was applied. Organic matter, N as well as some important macro and micronutrients (K, P, Mg, Mn, Cu, and Zn) for crop growth, were more available under organic management. Bacterial counts were higher in organic management. A significant influence of management system and management x crop interaction was observed for total mesophilic bacteria, nitrogen fixing bacteria and actinobacteria. Interestingly, cultivable fungi were not detected in all analyzed samples. PLFA biomass was higher in the organic and Gram positive bacteria dominated the microbial community in both systems. Even if fungal biomass was higher in organic management, fungal PCR-DGGE fingerprinting revealed that the two systems were very similar in terms of fungal species suggesting that 10 years were not enough to establish a new dynamic equilibrium among ecosystem components. A better knowledge of soil biota and in particular of fungal community structure will be useful for the development of sustainable management strategies. PMID:25540640

A methodology for a quantitative interpretation of DGGE with the help of mathematical modelling: application in biohydrogen production.

PubMed

Tapia, Estela; Donoso-Bravo, Andres; Cabrol, Léa; Alves, Madalena; Pereira, Alcina; Rapaport, Alain; Ruiz-Filippi, Gonzalo

2014-01-01

Molecular biology techniques provide valuable insights in the investigation of microbial dynamics and evolution. Denaturing gradient gel electrophoresis (DGGE) analysis is one of the most popular methods which have been used in bioprocess assessment. Most of the anaerobic digestion models consider several microbial populations as state variables. However, the difficulty of measuring individual species concentrations may cause inaccurate model predictions. The integration of microbial data and ecosystem modelling is currently a challenging issue for improved system control. A novel procedure that combines common experimental measurements, DGGE, and image analysis is presented in this study in order to provide a preliminary estimation of the actual concentration of the dominant bacterial ribotypes in a bioreactor, for further use as a variable in mathematical modelling of the bioprocess. This approach was applied during the start-up of a continuous anaerobic bioreactor for hydrogen production. The experimental concentration data were used for determining the kinetic parameters of each species, by using a multi-species chemostat-model. The model was able to reproduce the global trend of substrate and biomass concentrations during the reactor start-up, and predicted in an acceptable way the evolution of each ribotype concentration, depicting properly specific ribotype selection and extinction.

Effect of nitrate injection on the bacterial community in a water-oil tank system analyzed by PCR-DGGE.

PubMed

Jurelevicius, Diogo; von der Weid, Irene; Korenblum, Elisa; Valoni, Erika; Penna, Mônica; Seldin, Lucy

2008-04-01

Sulfide production by sulfate-reducing bacteria (SRB) is a major concern for the petroleum industry since it is toxic and corrosive, and causes plugging due to the formation of insoluble iron sulfides (reservoir souring). In this study, PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) using two sets of primers based on the 16S rRNA gene and on the aps gene (adenosine-5-phosphosulfate reductase) was used to track changes in the total bacterial and SRB communities, respectively, present in the water-oil tank system on an offshore platform in Brazil in which nitrate treatment was applied for 2 months (15 nitrate injections). PCR-DGGE analysis of the total bacterial community showed the existence of a dominant population in the water-oil tank, and that the appearance and/or the increase of intensity of some bands in the gels were not permanently affected by the introduction of nitrate. On the other hand, the SRB community was stimulated following nitrate treatment. Moreover, sulfide production did not exceed the permissible exposure limit in the water-oil separation tank studied treated with nitrate. Therefore, controlling sulfide production by treating the produced water tank with nitrate could reduce the quantity of chemical biocides required to control microbial activities.

Occurrence and Phylogenetic Diversity of Sphingomonas Strains in Soils Contaminated with Polycyclic Aromatic Hydrocarbons

PubMed Central

Leys, Natalie M. E. J.; Ryngaert, Annemie; Bastiaens, Leen; Verstraete, Willy; Top, Eva M.; Springael, Dirk

2004-01-01

Bacterial strains of the genus Sphingomonas are often isolated from contaminated soils for their ability to use polycyclic aromatic hydrocarbons (PAH) as the sole source of carbon and energy. The direct detection of Sphingomonas strains in contaminated soils, either indigenous or inoculated, is, as such, of interest for bioremediation purposes. In this study, a culture-independent PCR-based detection method using specific primers targeting the Sphingomonas 16S rRNA gene combined with denaturing gradient gel electrophoresis (DGGE) was developed to assess Sphingomonas diversity in PAH-contaminated soils. PCR using the new primer pair on a set of template DNAs of different bacterial genera showed that the method was selective for bacteria belonging to the family Sphingomonadaceae. Single-band DGGE profiles were obtained for most Sphingomonas strains tested. Strains belonging to the same species had identical DGGE fingerprints, and in most cases, these fingerprints were typical for one species. Inoculated strains could be detected at a cell concentration of 104 CFU g of soil−1. The analysis of Sphingomonas population structures of several PAH-contaminated soils by the new PCR-DGGE method revealed that soils containing the highest phenanthrene concentrations showed the lowest Sphingomonas diversity. Sequence analysis of cloned PCR products amplified from soil DNA revealed new 16S rRNA gene Sphingomonas sequences significantly different from sequences from known cultivated isolates (i.e., sequences from environmental clones grouped phylogenetically with other environmental clone sequences available on the web and that possibly originated from several potential new species). In conclusion, the newly designed Sphingomonas-specific PCR-DGGE detection technique successfully analyzed the Sphingomonas communities from polluted soils at the species level and revealed different Sphingomonas members not previously detected by culture-dependent detection techniques. PMID:15066784

Do Patterns of Bacterial Diversity along Salinity Gradients Differ from Those Observed for Macroorganisms?

PubMed Central

Zhang, Yong; Shen, Ji; van der Gast, Christopher; Hahn, Martin W.; Wu, Qinglong

2011-01-01

It is widely accepted that biodiversity is lower in more extreme environments. In this study, we sought to determine whether this trend, well documented for macroorganisms, also holds at the microbial level for bacteria. We used denaturing gradient gel electrophoresis (DGGE) with phylum-specific primers to quantify the taxon richness (i.e., the DGGE band numbers) of the bacterioplankton communities of 32 pristine Tibetan lakes that represent a broad salinity range (freshwater to hypersaline). For the lakes investigated, salinity was found to be the environmental variable with the strongest influence on the bacterial community composition. We found that the bacterial taxon richness in freshwater habitats increased with increasing salinity up to a value of 1‰. In saline systems (systems with >1‰ salinity), the expected decrease of taxon richness along a gradient of further increasing salinity was not observed. These patterns were consistently observed for two sets of samples taken in two different years. A comparison of 16S rRNA gene clone libraries revealed that the bacterial community of the lake with the highest salinity was characterized by a higher recent accelerated diversification than the community of a freshwater lake, whereas the phylogenetic diversity in the hypersaline lake was lower than that in the freshwater lake. These results suggest that different evolutionary forces may act on bacterial populations in freshwater and hypersaline lakes on the Tibetan Plateau, potentially resulting in different community structures and diversity patterns. PMID:22125616

Spatial and temporal changes in microbial community structure associated with recharge-influenced chemical gradients in a contaminated aquifer

USGS Publications Warehouse

Haack, S.K.; Fogarty, L.R.; West, T.G.; Alm, E.W.; McGuire, J.T.; Long, D.T.; Hyndman, D.W.; Forney, L.J.

2004-01-01

In a contaminated water-table aquifer, we related microbial community structure on aquifer sediments to gradients in 24 geochemical and contaminant variables at five depths, under three recharge conditions. Community amplified ribsosomal DNA restriction analysis (ARDRA) using universal 16S rDNA primers and denaturing gradient gel electrophoresis (DGGE) using bacterial 16S rDNA primers indicated: (i) communities in the anoxic, contaminated central zone were similar regardless of recharge; (ii) after recharge, communities at greatest depth were similar to those in uncontaminated zones; and (iii) after extended lack of recharge, communities at upper and lower aquifer margins differed from communities at the same depths on other dates. General aquifer geochemistry was as important as contaminant or terminal electron accepting process (TEAP) chemistry in discriminant analysis of community groups. The Shannon index of diversity (H) and the evenness index (E), based on DGGE operational taxonomic units (OTUs), were statistically different across community groups and aquifer depths. Archaea or sulphate-reducing bacteria 16S rRNA abundance was not clearly correlated with TEAP chemistry indicative of methanogenesis or sulphate reduction. Eukarya rRNA abundance varied by depth and date from 0 to 13% of the microbial community. This contaminated aquifer is a dynamic ecosystem, with complex interactions between physical, chemical and biotic components, which should be considered in the interpretation of aquifer geochemistry and in the development of conceptual or predictive models for natural attenuation or remediation.

Analysis of β-Subgroup Proteobacterial Ammonia Oxidizer Populations in Soil by Denaturing Gradient Gel Electrophoresis Analysis and Hierarchical Phylogenetic Probing

PubMed Central

Stephen, John R.; Kowalchuk, George A.; Bruns, Mary-Ann V.; McCaig, Allison E.; Phillips, Carol J.; Embley, T. Martin; Prosser, James I.

1998-01-01

A combination of denaturing gradient gel electrophoresis (DGGE) and oligonucleotide probing was used to investigate the influence of soil pH on the compositions of natural populations of autotrophic β-subgroup proteobacterial ammonia oxidizers. PCR primers specific to this group were used to amplify 16S ribosomal DNA (rDNA) from soils maintained for 36 years at a range of pH values, and PCR products were analyzed by DGGE. Genus- and cluster-specific probes were designed to bind to sequences within the region amplified by these primers. A sequence specific to all β-subgroup ammonia oxidizers could not be identified, but probes specific for Nitrosospira clusters 1 to 4 and Nitrosomonas clusters 6 and 7 (J. R. Stephen, A. E. McCaig, Z. Smith, J. I. Prosser, and T. M. Embley, Appl. Environ. Microbiol. 62:4147–4154, 1996) were designed. Elution profiles of probes against target sequences and closely related nontarget sequences indicated a requirement for high-stringency hybridization conditions to distinguish between different clusters. DGGE banding patterns suggested the presence of Nitrosomonas cluster 6a and Nitrosospira clusters 2, 3, and 4 in all soil plots, but results were ambiguous because of overlapping banding patterns. Unambiguous band identification of the same clusters was achieved by combined DGGE and probing of blots with the cluster-specific radiolabelled probes. The relative intensities of hybridization signals provided information on the apparent selection of different Nitrosospira genotypes in samples of soil of different pHs. The signal from the Nitrosospira cluster 3 probe decreased significantly, relative to an internal control probe, with decreasing soil pH in the range of 6.6 to 3.9, while Nitrosospira cluster 2 hybridization signals increased with increasing soil acidity. Signals from Nitrosospira cluster 4 were greatest at pH 5.5, decreasing at lower and higher values, while Nitrosomonas cluster 6a signals did not vary significantly with pH. These findings are in agreement with a previous molecular study (J. R. Stephen, A. E. McCaig, Z. Smith, J. I. Prosser, and T. M. Embley, Appl. Environ. Microbiol 62:4147–4154, 1996) of the same sites, which demonstrated the presence of the same four clusters of ammonia oxidizers and indicated that selection might be occurring for clusters 2 and 3 at acid and neutral pHs, respectively. The two studies used different sets of PCR primers for amplification of 16S rDNA sequences from soil, and the similar findings suggest that PCR bias was unlikely to be a significant factor. The present study demonstrates the value of DGGE and probing for rapid analysis of natural soil communities of β-subgroup proteobacterial ammonia oxidizers, indicates significant pH-associated differences in Nitrosospira populations, and suggests that Nitrosospira cluster 2 may be of significance for ammonia-oxidizing activity in acid soils. PMID:9687457

Effects of copper on the abundance and diversity of ammonia oxidizers during dairy cattle manure composting.

PubMed

Yin, Yanan; Song, Wen; Gu, Jie; Zhang, Kaiyu; Qian, Xun; Zhang, Xin; Zhang, Yajun; Li, Yang; Wang, Xiaojuan

2016-12-01

This study investigated the effects of adding Cu(II) at two exposure levels (50 and 500mgkg -1 , i.e., Cu50 and Cu500 treatments, respectively) on the activity of ammonia-oxidizing microorganisms during dairy cattle manure composting. The results showed that the pH, NH 4 + -N, NO 3 - -N, and potential ammonia oxidation values were inhibited significantly by the addition of Cu(II). Furthermore, the abundances of the ammonia-oxidizing archaea (AOA) amoA gene and ammonia-oxidizing bacteria (AOB) amoA gene were determined by quantitative PCR, and their compositions were evaluated by denaturing gradient gel electrophoresis (DGGE). AOA was the dominant ammonia oxidizing microorganism, of which the abundance was much higher than AOB during composting. Cu50 and Cu500 had significant inhibitory effects on the abundance of the amoA gene. The DGGE profile and statistical analysis showed that Cu(II) changed the AOA and AOB community structure and diversity, where Nitrosomonas and Crenarchaeota dominated throughout the composting process. Copyright © 2016. Published by Elsevier Ltd.

Application of Sequence-Dependent Electrophoresis Fingerprinting in Exploring Biodiversity and Population Dynamics of Human Intestinal Microbiota: What Can Be Revealed?

PubMed Central

Huys, Geert; Vanhoutte, Tom; Vandamme, Peter

2008-01-01

Sequence-dependent electrophoresis (SDE) fingerprinting techniques such as denaturing gradient gel electrophoresis (DGGE) have become commonplace in the field of molecular microbial ecology. The success of the SDE technology lays in the fact that it allows visualization of the predominant members of complex microbial ecosystems independent of their culturability and without prior knowledge on the complexity and diversity of the ecosystem. Mainly using the prokaryotic 16S rRNA gene as PCR amplification target, SDE-based community fingerprinting turned into one of the leading molecular tools to unravel the diversity and population dynamics of human intestinal microbiota. The first part of this review covers the methodological concept of SDE fingerprinting and the technical hurdles for analyzing intestinal samples. Subsequently, the current state-of-the-art of DGGE and related techniques to analyze human intestinal microbiota from healthy individuals and from patients with intestinal disorders is surveyed. In addition, the applicability of SDE analysis to monitor intestinal population changes upon nutritional or therapeutic interventions is critically evaluated. PMID:19277102

Microbiological study on bioremediation of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) contaminated soil by agricultural waste composting.

PubMed

Chen, Yaoning; Ma, Shuang; Li, Yuanping; Yan, Ming; Zeng, Guangming; Zhang, Jiachao; Zhang, Jie; Tan, Xuebin

2016-11-01

This paper studied the degradation of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) in contaminated soil under composting and natural conditions, respectively. BDE-47 residue in agricultural waste-composting pile was determined during 45-day composting. The microbial communities were determined by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), and the relationships between the DGGE results and physico-chemical parameters were evaluated by redundancy analysis (RDA) and heatmap-clustering analysis. The results showed that the degradation rate of BDE-47 was significantly higher in agricultural waste-composting pile compared with control group, which was enhanced up to almost 15 % at the end of composting. There were different environmental factors which affected the distribution of composting bacterial and fungal communities. The bacterial community composition was more significantly affected by the addition of BDE-47 compared with other physico-chemical parameters, and BDE-47 had stronger influences on bacterial community than fungal community during the composting. Meanwhile, the most variation in distribution of fungal community was explained by pile temperature.

[Effects and Biological Response on Bioremediation of Petroleum Contaminated Soil].

PubMed

Yang, Qian; Wu, Man-li; Nie, Mai-qian; Wang, Ting-ting; Zhang, Ming-hui

2015-05-01

Bioaugmentation and biostimulation were used to remediate petroleum-contaminated soil which were collected from Zichang city in North of Shaanxi. The optimal bioremediation method was obtained by determining the total petroleum hydrocarbon(TPH) using the infrared spectroscopy. During the bioremediation, number of degrading strains, TPH catabolic genes, and soil microbial community diversity were determined by Most Probable Number (MPN), polymerase chain reaction (PCR) combined agarose electrophoresis, and PCR-denaturing gradient electrophoresis (DGGE). The results in different treatments showed different biodegradation effects towards total petroleum hydrocarbon (TPH). Biostimulation by adding N and P to soils achieved the best degradation effects towards TPH, and the bioaugmentation was achieved by inoculating strain SZ-1 to soils. Further analysis indicated the positive correlation between catabolic genes and TPH removal efficiency. During the bioremediation, the number of TPH and alkanes degrading strains was higher than the number of aromatic degrading strains. The results of PCR-DGGE showed microbial inoculums could enhance microbial community functional diversity. These results contribute to understand the ecologically microbial effects during the bioremediation of petroleum-polluted soil.

Cyanobacterial Community Structure In Lithifying Mats of A Yellowstone Hotspring-Implications for Precambrian Stromatolite Biocomplexity

NASA Technical Reports Server (NTRS)

Lau, Evan; Nash, C. Z.; Vogler, D. R.; Cullings, K.; DeVincenzi, Donald (Technical Monitor)

2002-01-01

Denaturing Gradient Gel Electrophoresis (DGGE) of partial 16S rRNA gene sequences was used to investigate the molecular biodiversity of cyanobacterial communities inhabiting various lithified morpho-structures in two hotsprings of Yellowstone National Park. These morpho-structures - flat-topped columns, columnar cones, and ridged cones - resemble ancient stromatolites, which are possibly biogenic in origin. The top, middle and bottom sections of these lithified morpho-structures, as well as surrounding non-lithified mats were analyzed to determine the vertical and spatial distribution of cyanobacterial communities. Results from DGGE indicate that the cyanobacterial community composition of lithified morpho-structures (flat-topped columns, columnar cones, and ridged cones) were largely similar in vertical distribution as well as among the morpho-structures being studied. Preliminary results indicate that the cyanobacterial communities in these lithified morpho-structures were significantly different from communities in surrounding non-lithified mats. These results provide additional support to the theory that certain Phormidium/Leptolyngbya species are involved in the morphogenesis of lithifying morpho-structures in hotsprings and may have played a role in the formation of ancient stromatolites.

Symbiodinium diversity in the sea anemone Entacmaea quadricolor on the east Australian coast

NASA Astrophysics Data System (ADS)

Pontasch, S.; Scott, A.; Hill, R.; Bridge, T.; Fisher, P. L.; Davy, S. K.

2014-06-01

The diversity of Symbiodinium spp. in Entacmaea quadricolor was analysed from five locations along ~2,100 km on the east coast of Australia using denaturing gradient gel electrophoresis (DGGE) of the internal transcribed spacer 2 region (ITS2) combined with bacterial cloning. DGGE revealed that E. quadricolor predominantly associated with six types of clade C (four of which are novel) and that most anemones harboured multiple types simultaneously. Anemones from southern locations associated with a mixed assemblage of C25 and a variant of C3. This assemblage also dominated the central location, but was absent at the northern location. At central and northern sites, two novel variants of C42(type2) and C1 were found. Anemones hosting C42(type2) also showed a low abundance of variants of C3 and C1, and E1 was found in one sample, as revealed by bacterial cloning. The occurrence of geographically distinct ITS2 types or a consortium of types might reflect a need to optimise physiological performance of the symbiosis at different latitudes.

Genetic diversity patterns of microeukaryotic plankton communities in Shenhu Bay, southeast China

NASA Astrophysics Data System (ADS)

Zhang, Wenjing; Pan, Yongbo; Yu, Lingyu; Liu, Lemian

2017-06-01

Microeukaryotic plankton is an abundant and diverse component of marine environments and plays an important role in microbial food webs. However, few studies have been conducted on the genetic diversity of microeukaryotes in the subtropical bays of China. In the present study, we investigated the microeukaryotic plankton in the Shenhu Bay by using denaturing gradient gel electrophoresis (DGGE) and sequencing of prominent bands. Our results indicated that Copepoda and Dinophyceae were the most diverse groups, and that the microeukaryotic communities varied significantly between summer and autumn, with the autumn communities exhibited a higher diversity than summer communities. Furthermore, the community composition and diversity from both surface and bottom waters showed more significant differences in summer than in autumn. Environmental parameters also displayed obvious seasonal patterns. Redundancy analysis (RDA) showed that temperature was the most significant environmental factor shaping the seasonal patterns of the microplanktonic members in the Shenhu Bay. Community-level molecular techniques such as DGGE appear as useful tools to detect the presence of red tide causing species and to guide the management of coastal water mariculture.

Seasonal variation of plankton communities influenced by environmental factors in an artificial lake

NASA Astrophysics Data System (ADS)

Li, Xuemei; Yu, Yuhe; Zhang, Tanglin; Feng, Weisong; Ao, Hongyi; Yan, Qingyun

2012-05-01

We evaluated the seasonal variation in plankton community composition in an artificial lake. We conducted microscopic analysis and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified partial 16S rRNA and 18S rRNA genes to characterize the plankton community. The clustering of unweighted pair group method with arithmetic mean (UPGMA) was then used to investigate the similarity of these plankton communities. DGGE fingerprinting revealed that samples collected at the different sites within a season shared high similarity and were generally grouped together. In contrast, we did not observe any seasonal variation based on microscopic analysis. Redundancy analysis (RDA) of the plankton operational taxonomic units (OTUs) in relation to environmental factors revealed that transparency was negatively correlated with the first axis ( R=-0.931), and temperature and total phosphorus (TP) were positively correlated with the first axis ( R=0.736 and R=0.660, respectively). In conclusion, plankton communities in the artificial lake exhibited significant seasonal variation. Transparency, phosphorus and temperature appear to be the major factors driving the differences in plankton composition.

Community analysis of hydrogen-producing extreme thermophilic anaerobic microflora enriched from cow manure with five substrates.

PubMed

Yokoyama, Hiroshi; Moriya, Naoko; Ohmori, Hideyuki; Waki, Miyoko; Ogino, Akifumi; Tanaka, Yasuo

2007-11-01

The present study analyzed the community structures of anaerobic microflora producing hydrogen under extreme thermophilic conditions by two culture-independent methods: denaturing gradient gel electrophoresis (DGGE) and clone library analyses. Extreme thermophilic microflora (ETM) was enriched from cow manure by repeated batch cultures at 75 degrees C, using a substrate of xylose, glucose, lactose, cellobiose, or soluble starch, and produced hydrogen at yields of 0.56, 2.65, 2.17, 2.68, and 1.73 mol/mol-monosaccharide degraded, respectively. The results from the DGGE and clone library analyses were consistent and demonstrated that the community structures of ETM enriched with the four hexose-based substrates (glucose, lactose, cellobiose, and soluble starch) consisted of a single species, closely related to a hydrogen-producing extreme thermophile, Caldoanaerobacter subterraneus, with diversity at subspecies levels. The ETM enriched with xylose was more diverse than those enriched with the other substrates, and contained the bacterium related to C. subterraneus and an unclassified bacterium, distantly related to a xylan-degrading and hydrogen-producing extreme thermophile, Caloramator fervidus.

Effects of imidacloprid on soil microbial communities in different saline soils.

PubMed

Zhang, Qingming; Xue, Changhui; Wang, Caixia

2015-12-01

The effects of imidacloprid in the soil environment are a worldwide concern. However, the impact of imidacloprid on soil microorganisms under salt stress is almost unknown. Therefore, an indoor incubation test was performed, and the denaturing gradient gel electrophoresis (DGGE) approach was used to determine the response of different saline soil bacterial and fungal community structures to the presence of imidacloprid (0.4, 2, 10 mg kg(-1)). The results showed that the soil bacterial diversity slightly declined with increasing imidacloprid concentration in soils with low salinity. In moderately saline soils, a new band in the DGGE profile suggested that imidacloprid could improve the soil bacterial diversity to some degree. An analysis of variance indicated that the measured soil bacterial diversity parameters were significantly affected by dose and incubation time. Compared with the control, the soil fungal community structure showed no obvious changes in low and moderately saline soils treated with imidacloprid. The results of these observations provide a basic understanding of the potential ecological effects of imidacloprid on different microorganisms in saline soils.

Microbial ecology overview during anaerobic codigestion of dairy wastewater and cattle manure and use in agriculture of obtained bio-fertilisers.

PubMed

Toumi, Jihen; Miladi, Baligh; Farhat, Amel; Nouira, Said; Hamdi, Moktar; Gtari, Maher; Bouallagui, Hassib

2015-12-01

The anaerobic co-digestion of dairy wastewater (DW) and cattle manure (CM) was examined and associated with microbial community's structures using Denaturing Gradient Gel Electrophoresis (DGGE). The highest volatile solids (VS) reduction yield of 88.6% and biogas production of 0.87 L/g VS removed were obtained for the C/N ratio of 24.7 at hydraulic retention time (HRT) of 20 days. The bacterial DGGE profile showed significant abundance of Uncultured Bacteroidetes, Firmicutes and Synergistetes bacterium. The Syntrophomonas strains were discovered in dependent association to H2-using bacteria such as Methanospirillum sp., Methanosphaera sp. and Methanobacterium formicicum. These syntrophic associations are essential in anaerobic digesters allow them to keep low hydrogen partial pressure. However, high concentrations of VFA produced from dairy wastes acidification allow the growth of Methanosarcina species. The application of the stabilised anaerobic effluent on the agriculture soil showed significant beneficial effects on the forage corn and tomato plants growth and crops. Copyright © 2015 Elsevier Ltd. All rights reserved.

Responses of soil microbial activity to cadmium pollution and elevated CO2.

PubMed

Chen, Yi Ping; Liu, Qiang; Liu, Yong Jun; Jia, Feng An; He, Xin Hua

2014-03-06

To address the combined effects of cadmium (Cd) and elevated CO2 on soil microbial communities, DGGE (denaturing gradient gel electrophoresis) profiles, respiration, carbon (C) and nitrogen (N) concentrations, loessial soils were exposed to four levels of Cd, i.e., 0 (Cd0), 1.5 (Cd1.5), 3.0 (Cd3.0) and 6.0 (Cd6.0) mg Cd kg(-1) soil, and two levels of CO2, i.e., 360 (aCO2) and 480 (eCO2) ppm. Compared to Cd0, Cd1.5 increased fungal abundance but decreased bacterial abundance under both CO2 levels, whilst Cd3.0 and Cd6.0 decreased both fungal and bacterial abundance. Profiles of DGGE revealed alteration of soil microbial communities under eCO2. Soil respiration decreased with Cd concentrations and was greater under eCO2 than under aCO2. Soil total C and N were greater under higher Cd. These results suggest eCO2 could stimulate, while Cd pollution could restrain microbial reproduction and C decomposition with the restraint effect alleviated by eCO2.

Influence of long-term repeated prescribed burning on mycelial communities of ectomycorrhizal fungi.

PubMed

Bastias, Brigitte A; Xu, Zhihong; Cairney, John W G

2006-01-01

To demonstrate the efficacy of direct DNA extraction from hyphal ingrowth bags for community profiling of ectomycorrhizal (ECM) mycelia in soil, we applied the method to investigate the influence of long-term repeated prescribed burning on an ECM fungal community. DNA was extracted from hyphal ingrowth bags buried in forest plots that received different prescribed burning treatments for 30 yr, and denaturing gradient gel electrophoresis (DGGE) profiles of partial fungal rDNA internal transcribed spacer (ITS) regions were compared. Restriction fragment length polymorphism (RFLP) and sequence analyses were also used to compare clone assemblages between the treatments. The majority of sequences derived from the ingrowth bags were apparently those of ECM fungi. DGGE profiles for biennially burned plots were significantly different from those of quadrennially burned and unburned control plots. Analysis of clone assemblages indicated that this reflected altered ECM fungal community composition. The results indicate that hyphal ingrowth bags represent a useful method for investigation of ECM mycelial communities, and that frequent long-term prescribed burning can influence below-ground ECM fungal communities.

Enhanced biodegradation of antibiotic combinations via the sequential treatment of the sludge resulting from pharmaceutical wastewater treatment using white-rot fungi Trametes versicolor and Bjerkandera adusta.

PubMed

Aydin, Sevcan

2016-07-01

While anaerobic treatment is capable of treating pharmaceutical wastewater and removing antibiotics in liquid phases, solid phases may still contain significant amounts of antibiotics following this treatment. The main goal of this study was to evaluate the use of white-rot fungi to remove erythromycin, sulfamethoxazole, and tetracycline combinations from biosolids. The degradation potential of Trametes versicolor and Bjerkandera adusta was evaluated via the sequential treatment of anaerobic sludge. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analyses were used to identify competition between the autochthonous microbial communities and white-rot fungi. Solid-phase treatment using white-rot fungi substantially reduced antibiotic concentrations and toxicity in sludge. According to PCR-DGGE results, there is an association between species of fungus and antibiotic type as a result of the different transformation pathways of fungal strains. Fungal post-treatment of sludge represents a promising method of removing antibiotic combinations, therefore holding a significant promise as an environmentally friendly means of degrading the antibiotics present in sludge.

Analysis of dissimilatory sulfite reductase and 16S rRNA gene fragments from deep-sea hydrothermal sites of the Suiyo Seamount, Izu-Bonin Arc, Western Pacific.

PubMed

Nakagawa, Tatsunori; Ishibashi, Jun-Ichiro; Maruyama, Akihiko; Yamanaka, Toshiro; Morimoto, Yusuke; Kimura, Hiroyuki; Urabe, Tetsuro; Fukui, Manabu

2004-01-01

This study describes the occurrence of unique dissimilatory sulfite reductase (DSR) genes at a depth of 1,380 m from the deep-sea hydrothermal vent field at the Suiyo Seamount, Izu-Bonin Arc, Western Pacific, Japan. The DSR genes were obtained from microbes that grew in a catheter-type in situ growth chamber deployed for 3 days on a vent and from the effluent water of drilled holes at 5 degrees C and natural vent fluids at 7 degrees C. DSR clones SUIYOdsr-A and SUIYOdsr-B were not closely related to cultivated species or environmental clones. Moreover, samples of microbial communities were examined by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA gene. The sequence analysis of 16S rRNA gene fragments obtained from the vent catheter after a 3-day incubation revealed the occurrence of bacterial DGGE bands affiliated with the Aquificae and gamma- and epsilon-Proteobacteria as well as the occurrence of archaeal phylotypes affiliated with the Thermococcales and of a unique archaeon sequence that clustered with "Nanoarchaeota." The DGGE bands obtained from drilled holes and natural vent fluids from 7 to 300 degrees C were affiliated with the delta-Proteobacteria, genus Thiomicrospira, and Pelodictyon. The dominant DGGE bands retrieved from the effluent water of casing pipes at 3 and 4 degrees C were closely related to phylotypes obtained from the Arctic Ocean. Our results suggest the presence of microorganisms corresponding to a unique DSR lineage not detected previously from other geothermal environments.

Microbiological and meteorological analysis of two Australian dust storms in April 2009.

PubMed

Lim, Natalie; Munday, Chris I; Allison, Gwen E; O'Loingsigh, Tadhg; De Deckker, Patrick; Tapper, Nigel J

2011-12-15

Dust is an important source of bioaerosols including bacteria. In this study, the microbiology and meteorology of specific dust storms in Australia were investigated. The samples were collected from two dust events in April 2009 that were characterised by intense cold fronts that entrained dust from the highly erodible and drought-stricken Mallee and Riverina regions of Victoria and central NSW. In the first storm, the dust travelled eastward over Canberra and Sydney, and in the second storm, the dust travelled east/southeastward over Canberra and Melbourne. Rain fell on both cities during the second dust storm. Dust and rain samples were collected, cultured, and the composition compared using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Multiple bands were evident on DGGE indicative of a diverse microflora, and identification of several bands confirmed the presence of multiple genera and species representing three phyla. Numerous bands represented Bacillus species, and these were present in multiple dust samples collected from both Canberra and Melbourne. Interestingly, the microflora present in rain samples collected in Canberra during the second dust storm was quite different and the DGGE banding patterns from these samples clustered separately to most dust samples collected at the same time. Identification of several DGGE bands and PCR products from these rain samples indicated the presence of Pseudomonas species. These results indicate that Australian dust and rain have a diverse microflora and highlights the contribution of dust events to the distribution of microbes in the environment. Copyright © 2011 Elsevier B.V. All rights reserved.

Temporal analysis of the effect of extruded flaxseed on the swine gut microbiota.

PubMed

Holman, Devin B; Baurhoo, Bushansingh; Chénier, Martin R

2014-10-01

Flaxseed is a rich source of α-linolenic acid, an essential ω-3 fatty acid reported to have beneficial health effects in humans. Feeding swine a diet supplemented with flaxseed has been found to enrich pork products with ω-3 fatty acids. However, the effect of flaxseed supplementation on the swine gut microbiota has not been assessed to date. The purpose of this study was to investigate if extruded flaxseed has any impact on the bacterial and archaeal microbiota in the feces of growing-finishing pigs over a 51-day period, using denaturing gradient gel electrophoresis (DGGE) and real-time PCR. Bacterial DGGE profile analysis revealed major temporal shifts in the bacterial microbiota with only minor ones related to diet. The archaeal microbiota was significantly less diverse than that of Bacteria. The majority of bacterial DGGE bands sequenced belonged to the Firmicutes phylum while the archaeal DGGE bands were found to consist of only 2 species, Methanobrevibacter smithii and Methanosphaera stadtmanae. The abundance of Bacteroidetes decreased significantly from day 0 to day 21 in all diet groups while the abundance of Firmicutes was relatively stable across all diet cohorts and sampling times. There was also no significant correlation between pig mass and the ratio of Firmicutes to Bacteroidetes. While the addition of extruded flaxseed to the feed of growing-finishing pigs was beneficial for improving ω-3 fatty acid content of pork, it had no detectable impact on the fecal bacterial and archaeal microbiota, suggesting that extruded flaxseed may be used to improve meat quality without adverse effect on the swine gut microbiota or animal performance.

Dynamic changes of yak (Bos grunniens) gut microbiota during growth revealed by polymerase chain reaction-denaturing gradient gel electrophoresis and metagenomics

PubMed Central

Nie, Yuanyang; Zhou, Zhiwei; Guan, Jiuqiang; Xia, Baixue; Luo, Xiaolin; Yang, Yang; Fu, Yu; Sun, Qun

2017-01-01

Objective To understand the dynamic structure, function, and influence on nutrient metabolism in hosts, it was crucial to assess the genetic potential of gut microbial community in yaks of different ages. Methods The denaturing gradient gel electrophoresis (DGGE) profiles and Illumina-based metagenomic sequencing on colon contents of 15 semi-domestic yaks were investigated. Unweighted pairwise grouping method with mathematical averages (UPGMA) clustering and principal component analysis (PCA) were used to analyze the DGGE fingerprint. The Illumina sequences were assembled, predicted to genes and functionally annotated, and then classified by querying protein sequences of the genes against the Kyoto encyclopedia of genes and genomes (KEGG) database. Results Metagenomic sequencing showed that more than 85% of ribosomal RNA (rRNA) gene sequences belonged to the phylum Firmicutes and Bacteroidetes, indicating that the family Ruminococcaceae (46.5%), Rikenellaceae (11.3%), Lachnospiraceae (10.0%), and Bacteroidaceae (6.3%) were dominant gut microbes. Over 50% of non-rRNA gene sequences represented the metabolic pathways of amino acids (14.4%), proteins (12.3%), sugars (11.9%), nucleotides (6.8%), lipids (1.7%), xenobiotics (1.4%), coenzymes, and vitamins (3.6%). Gene functional classification showed that most of enzyme-coding genes were related to cellulose digestion and amino acids metabolic pathways. Conclusion Yaks’ age had a substantial effect on gut microbial composition. Comparative metagenomics of gut microbiota in 0.5-, 1.5-, and 2.5-year-old yaks revealed that the abundance of the class Clostridia, Bacteroidia, and Lentisphaeria, as well as the phylum Firmicutes, Bacteroidetes, Lentisphaerae, Tenericutes, and Cyanobacteria, varied more greatly during yaks’ growth, especially in young animals (0.5 and 1.5 years old). Gut microbes, including Bacteroides, Clostridium, and Lentisphaeria, make a contribution to the energy metabolism and synthesis of amino acid, which are essential to the normal growth of yaks. PMID:28183172

Fate of a metal-resistant inoculum in contaminated and pristine soils assessed by denaturing gradient gel electrophoresis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stephen, J.R.; Chang, Y.J.; MacNaughton, S.J.

Cesium, cadmium, cobalt, and strontium are four contaminants frequently found in soils at biotoxic levels. Introduction of certain nongenetically modified bacteria has been frequently suggested as a method for the immobilization of heavy metal contaminants in soil, thereby reducing runoff and bioavailability. In this study, the authors have used the polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) to track the survival of the five bacterial species added to soil microcosms with and without the addition of a mixture of these metals. The PCR primers targeted conserved regions of the 165 rDNA molecular present in all bacteria. Themore » reaction products were shown to reflect the relative abundance of the bacteria both in mixtures of pure cultures and against a background of all the eubacterial species present in the soil following inoculation. Three of the species (Pseudomonas aeruginosa FRD-1, Shewanella putrifaciens 200, and Desulfovibrio vulgaris Hildenborough) decreased rapidly following inoculation into both soils. The proportion of Alcaligenes eutrophus CH34 remained at a constant level throughout the 8-week experiment in both soil treatments. Sphingomonas aromaticivorans B0695 showed toxic metal-dependent survival in that its relative abundance dropped rapidly in pristine soil but remained at approximately inoculation levels throughout the experiment in contaminated microcosms.« less

Biodiversity in Oscypek, a Traditional Polish Cheese, Determined by Culture-Dependent and -Independent Approaches

PubMed Central

Alegría, Ángel; Szczesny, Pawel; Mayo, Baltasar; Bardowski, Jacek

2012-01-01

Oscypek is a traditional Polish scalded-smoked cheese, with a protected-designation-of-origin (PDO) status, manufactured from raw sheep's milk without starter cultures in the Tatra Mountains region of Poland. This study was undertaken in order to gain insight into the microbiota that develops and evolves during the manufacture and ripening stages of Oscypek. To this end, we made use of both culturing and the culture-independent methods of PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing of 16S rRNA gene amplicons. The culture-dependent technique and PCR-DGGE fingerprinting detected the predominant microorganisms in traditional Oscypek, whereas the next-generation sequencing technique (454 pyrosequencing) revealed greater bacterial diversity. Besides members of the most abundant bacterial genera in dairy products, e.g., Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, and Enterococcus, identified by all three methods, other, subdominant bacteria belonging to the families Bifidobacteriaceae and Moraxellaceae (mostly Enhydrobacter), as well as various minor bacteria, were identified by pyrosequencing. The presence of bifidobacterial sequences in a cheese system is reported for the first time. In addition to bacteria, a great diversity of yeast species was demonstrated in Oscypek by the PCR-DGGE method. Culturing methods enabled the determination of a number of viable microorganisms from different microbial groups and their isolation for potential future applications in specific cheese starter cultures. PMID:22247135

Combined Analyses of Bacterial, Fungal and Nematode Communities in Andosolic Agricultural Soils in Japan

PubMed Central

Bao, Zhihua; Ikunaga, Yoko; Matsushita, Yuko; Morimoto, Sho; Takada-Hoshino, Yuko; Okada, Hiroaki; Oba, Hirosuke; Takemoto, Shuhei; Niwa, Shigeru; Ohigashi, Kentaro; Suzuki, Chika; Nagaoka, Kazunari; Takenaka, Makoto; Urashima, Yasufumi; Sekiguchi, Hiroyuki; Kushida, Atsuhiko; Toyota, Koki; Saito, Masanori; Tsushima, Seiya

2012-01-01

We simultaneously examined the bacteria, fungi and nematode communities in Andosols from four agro-geographical sites in Japan using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and statistical analyses to test the effects of environmental factors including soil properties on these communities depending on geographical sites. Statistical analyses such as Principal component analysis (PCA) and Redundancy analysis (RDA) revealed that the compositions of the three soil biota communities were strongly affected by geographical sites, which were in turn strongly associated with soil characteristics such as total C (TC), total N (TN), C/N ratio and annual mean soil temperature (ST). In particular, the TC, TN and C/N ratio had stronger effects on bacterial and fungal communities than on the nematode community. Additionally, two-way cluster analysis using the combined DGGE profile also indicated that all soil samples were classified into four clusters corresponding to the four sites, showing high site specificity of soil samples, and all DNA bands were classified into four clusters, showing the coexistence of specific DGGE bands of bacteria, fungi and nematodes in Andosol fields. The results of this study suggest that geography relative to soil properties has a simultaneous impact on soil microbial and nematode community compositions. This is the first combined profile analysis of bacteria, fungi and nematodes at different sites with agricultural Andosols. PMID:22223474

Combined analyses of bacterial, fungal and nematode communities in andosolic agricultural soils in Japan.

PubMed

Bao, Zhihua; Ikunaga, Yoko; Matsushita, Yuko; Morimoto, Sho; Takada-Hoshino, Yuko; Okada, Hiroaki; Oba, Hirosuke; Takemoto, Shuhei; Niwa, Shigeru; Ohigashi, Kentaro; Suzuki, Chika; Nagaoka, Kazunari; Takenaka, Makoto; Urashima, Yasufumi; Sekiguchi, Hiroyuki; Kushida, Atsuhiko; Toyota, Koki; Saito, Masanori; Tsushima, Seiya

2012-01-01

We simultaneously examined the bacteria, fungi and nematode communities in Andosols from four agro-geographical sites in Japan using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and statistical analyses to test the effects of environmental factors including soil properties on these communities depending on geographical sites. Statistical analyses such as Principal component analysis (PCA) and Redundancy analysis (RDA) revealed that the compositions of the three soil biota communities were strongly affected by geographical sites, which were in turn strongly associated with soil characteristics such as total C (TC), total N (TN), C/N ratio and annual mean soil temperature (ST). In particular, the TC, TN and C/N ratio had stronger effects on bacterial and fungal communities than on the nematode community. Additionally, two-way cluster analysis using the combined DGGE profile also indicated that all soil samples were classified into four clusters corresponding to the four sites, showing high site specificity of soil samples, and all DNA bands were classified into four clusters, showing the coexistence of specific DGGE bands of bacteria, fungi and nematodes in Andosol fields. The results of this study suggest that geography relative to soil properties has a simultaneous impact on soil microbial and nematode community compositions. This is the first combined profile analysis of bacteria, fungi and nematodes at different sites with agricultural Andosols.

Comparison of bacterial community structures of terrestrial cyanobacterium Nostoc flagelliforme in three different regions of China using PCR-DGGE analysis.

PubMed

Han, Pei-pei; Shen, Shi-gang; Jia, Shi-ru; Wang, Hui-yan; Zhong, Cheng; Tan, Zhi-lei; Lv, He-xin

2015-07-01

Filamentous Nostoc flagelliforme form colloidal complex, with beaded cells interacting with other bacteria embedded in the complex multilayer sheath. However, the species of bacteria in the sheath and the interaction between N. flagelliforme and associated bacteria remain unclear. In this study, PCR-denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial communities of N. flagelliforme from three regions of China. DGGE patterns showed variations in all samples, exhibiting 25 discrete bands with various intensities. The diversity index analysis of bands profiles suggested the high similarity of bacterial communities to each other but also the dependence of microbial composition on each location. Phylogenetic affiliation indicated that the majority of the sequences obtained were affiliated with Actinobacteria, Cyanobacteria, Proteobacteria, Acidobacteria, Bacteroidetes, of which Cyanobacteria was dominant, followed the Proteobacteria. Members of the genus Nostoc were the most abundant in all samples. Rhizobiales and Actinobacteria were identified, whereas, Craurococcus, Caulobacter, Pseudomonas, Terriglobus and Mucilaginibacter were also identified at low levels. Through comparing the bacterial composition of N. flagelliforme from different regions, it was revealed that N. flagelliforme could facilitate the growth of other microorganisms including both autotrophic bacteria and heterotrophic ones and positively contributed to their harsh ecosystems. The results indicated N. flagelliforme played an important role in diversifying the microbial community composition and had potential application in soil desertification.

Temporal changes in community composition of heterotrophic bacteria during in situ iron enrichment in the western subarctic Pacific (SEEDS-II)

NASA Astrophysics Data System (ADS)

Kataoka, Takafumi; Suzuki, Koji; Hayakawa, Maki; Kudo, Isao; Higashi, Seigo; Tsuda, Atsushi

2009-12-01

Little is known about the effects of iron enrichment in high-nitrate low-chlorophyll (HNLC) waters on the community composition of heterotrophic bacteria, which are crucial to nutrient recycling and microbial food webs. Using denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments, we investigated the heterotrophic eubacterial community composition in surface waters during an in situ iron-enrichment experiment (SEEDS-II) in the western subarctic Pacific in the summer of 2004. DGGE fingerprints representing the community composition of eubacteria differed inside and outside the iron-enriched patch. Sequencing of DGGE bands revealed that at least five phylotypes of α-proteobacteria including Roseobacter, Cytophaga-Flavobacteria- Bacteroides (CFB), γ-proteobacteria, and Actinobacteria occurred in almost all samples from the iron-enriched patch. Diatoms did not bloom during SEEDS-II, but the eubacterial composition in the iron-enriched patch was similar to that in diatom blooms observed previously. Although dissolved organic carbon (DOC) accumulation was not detected in surface waters during SEEDS-II, growth of the Roseobacter clade might have been particularly stimulated after iron additions. Two identified phylotypes of CFB were closely related to the genus Saprospira, whose algicidal activity might degrade the phytoplankton assemblages increased by iron enrichment. These results suggest that the responses of heterotrophic bacteria to iron enrichment could differ among phylotypes during SEEDS-II.

Diversity of ndo Genes in Mangrove Sediments Exposed to Different Sources of Polycyclic Aromatic Hydrocarbon Pollution▿

PubMed Central

Gomes, Newton C. Marcial; Borges, Ludmila R.; Paranhos, Rodolfo; Pinto, Fernando N.; Krögerrecklenfort, Ellen; Mendonça-Hagler, Leda C. S.; Smalla, Kornelia

2007-01-01

Polycyclic aromatic hydrocarbon (PAH) pollutants originating from oil spills and wood and fuel combustion are pollutants which are among the major threats to mangrove ecosystems. In this study, the composition and relative abundance in the sediment bacterial communities of naphthalene dioxygenase (ndo) genes which are important for bacterial adaptation to environmental PAH contamination were investigated. Three urban mangrove sites which had characteristic compositions and levels of PAH compounds in the sediments were selected. The diversity and relative abundance of ndo genes in total community DNA were assessed by a newly developed ndo denaturing gradient gel electrophoresis (DGGE) approach and by PCR amplification with primers targeting ndo genes with subsequent Southern blot hybridization analyses. Bacterial populations inhabiting sediments of urban mangroves under the impact of different sources of PAH contamination harbor distinct ndo genotypes. Sequencing of cloned ndo amplicons comigrating with dominant DGGE bands revealed new ndo genotypes. PCR-Southern blot analysis and ndo DGGE showed that the frequently studied nah and phn genotypes were not detected as dominant ndo types in the mangrove sediments. However, ndo genotypes related to nagAc-like genes were detected, but only in oil-contaminated mangrove sediments. The long-term impact of PAH contamination, together with the specific environmental conditions at each site, may have affected the abundance and diversity of ndo genes in sediments of urban mangroves. PMID:17905873

Effect of alginate and inulin on intestinal microbial ecology of weanling pigs reared under different husbandry conditions.

PubMed

Janczyk, Pawel; Pieper, Robert; Smidt, Hauke; Souffrant, Wolfgang B

2010-04-01

The effects of inulin and alginate on intestinal microbial ecophysiology were investigated in piglets fed a diet (C) with 0.1% alginate (C+A) or 1.5% inulin (C+I) from weaning at day 28. The experiment was performed at an experimental farm (EF) and a commercial farm (CF). Digesta was collected from the ileum, caecum and colon of four piglets from each group on days 29, 30, 33 and 39. The metabolite concentrations changed with age. Colonic and caecal metabolites were affected by prebiotic treatment. Changes in microbiota composition were assessed by cultivation and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Enterococci increased in C+A at EF and decreased in C+I at both farms. Lactobacilli decreased in all segments in the experimental groups on days 30 and 33. Yeasts in C+I were five times lower at CF than at EF on day 39. The richness and diversity of DGGE profiles increased in the experimental groups. The evenness of colon digesta-derived DGGE profiles was higher in the experimental groups than in C and this situation was reversed in the distal small intestine. Multivariate redundancy analysis confirmed the recorded effects. In summary, both prebiotics affected the intestinal microbiota, and the changes were more pronounced at the CF.

Characterisation of the spoilage bacterial microbiota in oyster gills during storage at different temperatures.

PubMed

Chen, Huibin; Liu, Zhiyu; Wang, Meiying; Chen, Shaojun; Chen, Tuanwei

2013-12-01

The spoilage bacterial community in oyster gill was investigated during storage at 4, 10 and 20 °C. Aerobic plate counts and pH values were determined. Total bacterial DNA was extracted from oyster gill and bulk cells of plate count media. The major bacterial species during fresh or different temperatures storage were determined by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The initial aerobic plate count in oyster gill reached 6.70 log CFU g(-1). PCR-DGGE fingerprinting analysis of the 16S rRNA gene V3 region revealed that most of the strains in fresh oyster gill belonged to the genera Lactococcus and Enterobacter. The major spoilage bacteria at a storage temperature of 20 °C were Leuconostoc pseudomesenteroides, an uncultured bacterium, Cytophaga fermentans, Lactococcus lactis, Pseudoalteromonas sp., Enterococcus mundtii, Clostridium difficile and an uncultured Fusobacteria; those at 10 °C were Lactococcus spp., Lactobacillus curvatus, Weissella confusa and C. difficile; those at 4 °C were Lactococcus, Weissella, Enterobacter and Aeromonas. The other minor species were L. curvatus, Pseudomonas sp. and E. mundtii. Lactococcus spp. was the most common main spoilage bacteria in oyster gill during chilled storage. PCR-DGGE revealed the complexity of the bacterial microbiota and the major bacteria species in oyster gill for fresh and storage. © 2013 Society of Chemical Industry.

Structural and metabolic responses of microbial community to sewage-borne chlorpyrifos in constructed wetlands.

PubMed

Zhang, Dan; Wang, Chuan; Zhang, Liping; Xu, Dong; Liu, Biyun; Zhou, Qiaohong; Wu, Zhenbin

2016-06-01

Long-term use of chlorpyrifos poses a potential threat to the environment that cannot be ignored, yet little is known about the succession of substrate microbial communities in constructed wetlands (CWs) under chlorpyrifos stress. Six pilot-scale CW systems receiving artificial wastewater containing 1mg/L chlorpyrifos were established to investigate the effects of chlorpyrifos and wetland vegetation on the microbial metabolism pattern of carbon sources and community structure, using BIOLOG and denaturing gradient gel electrophoresis (DGGE) approaches. Based on our samples, BIOLOG showed that Shannon diversity (H') and richness (S) values distinctly increased after 30days when chlorpyrifos was added. At the same time, differences between the vegetated and the non-vegetated systems disappeared. DGGE profiles indicated that H' and S had no significant differences among four different treatments. The effect of chlorpyrifos on the microbial community was mainly reflected at the physiological level. Principal component analysis (PCA) of both BIOLOG and DGGE showed that added chlorpyrifos made a difference on test results. Meanwhile, there was no difference between the vegetation and no-vegetation treatments after addition of chlorpyrifos at the physiological level. Moreover, the vegetation had no significant effect on the microbial community at the genetic level. Comparisons were made between bacteria in this experiment and other known chlorpyrifos-degrading bacteria. The potential chlorpyrifos-degrading ability of bacteria in situ may be considerable. Copyright © 2016. Published by Elsevier B.V.

Simazine application inhibits nitrification and changes the ammonia-oxidizing bacterial communities in a fertilized agricultural soil.

PubMed

Hernández, Marcela; Jia, Zhongjun; Conrad, Ralf; Seeger, Michael

2011-12-01

s-Triazine herbicides are widely used for weed control, and are persistent in soils. Nitrification is an essential process in the global nitrogen cycle in soil, and involves ammonia-oxidizing Bacteria (AOB) and ammonia-oxidizing Archaea (AOA). In this study, we evaluated the effect of the s-triazine herbicide simazine on the nitrification and on the structure of ammonia-oxidizing microbial communities in a fertilized agricultural soil. The effect of simazine on AOB and AOA were studied by PCR-amplification of amoA genes of nitrifying Bacteria and Archaea in soil microcosms and denaturing gradient gel electrophoresis (DGGE) analyses. Simazine [50 μg g(-1) dry weight soil (d.w.s)] completely inhibited the nitrification processes in the fertilized agricultural soil. The inhibition by simazine of ammonia oxidation observed was similar to the reduction of ammonia oxidation by the nitrification inhibitor acetylene. The application of simazine-affected AOB community DGGE patterns in the agricultural soil amended with ammonium, whereas no significant changes in the AOA community were observed. The DGGE analyses strongly suggest that simazine inhibited Nitrosobacteria and specifically Nitrosospira species. In conclusion, our results suggest that the s-triazine herbicide not only inhibits the target susceptible plants but also inhibits the ammonia oxidation and the AOB in fertilized soils. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Do neighboring lakes share common taxa of bacterioplankton? Comparison of 16S rDNA fingerprints and sequences from three geographic regions.

PubMed

Lindström, E S; Leskinen, E

2002-07-01

Bacterioplankton community composition was studied in 12 lakes in three different geographic regions in Scandinavia using denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S rDNA. Area-specific abundant taxa were found in the lakes in two of the regions. In the region of Uppland the lakes had an alpha-proteobacterium, belonging to the subgroup Alpha V in common. The Alpha V bacteria appeared to be favored by neutral or higher pH values. The lakes in Lappland were found to harbor Actinobacteria, which appeared to be favored in bog lakes. No abundant taxon was found to be in common for the lakes in Svalbard, the third region studied.

Distinctive archaebacterial species associated with anaerobic rumen protozoan Entodinium caudatum.

PubMed

Tóthová, T; Piknová, M; Kisidayová, S; Javorský, P; Pristas, P

2008-01-01

The diversity of archaebacteria associated with anaerobic rumen protozoan Entodinium caudatum in long term in vitro culture was investigated by denaturing gradient gel electrophoresis (DGGE) analysis of hypervariable V3 region of archaebacterial 16S rRNA gene. PCR was accomplished directly from DNA extracted from a single protozoal cell and from total community genomic DNA and the obtained fingerprints were compared. The analysis indicated the presence of a solitary intensive band present in Entodinium caudatum single cell DNA, which had no counterparts in the profile from total DNA. The identity of archaebacterium represented by this band was determined by sequence analysis which showed that the sequence fell to the cluster of ciliate symbiotic methanogens identified recently by 16S gene library approach.

Acclimatization of a mixed-animal manure inoculum to the anaerobic digestion of Axonopus compressus reveals the putative importance of Mesotoga infera and Methanosaeta concilii as elucidated by DGGE and Illumina MiSeq.

PubMed

Lee, Jonathan T E; He, Jianzhong; Tong, Yen Wah

2017-12-01

In this study, a multifarious microbial mix from different sources is acclimatized over a period of three months to digesting cowgrass, and the changes in the community structure are examined with both a traditional denaturing gradient gel electrophoresis method as well as a next generation sequencing MiSeq method. It is shown that the much more in depth analysis by Illumina gives more information about the relative abundance and thus putative importance of the role of various microbes, in particular the bacterium Mesotoga infera and the archaeon Methanosaeta concilii. Copyright © 2017 Elsevier Ltd. All rights reserved.

Analysis of Dissimilatory Sulfite Reductase and 16S rRNA Gene Fragments from Deep-Sea Hydrothermal Sites of the Suiyo Seamount, Izu-Bonin Arc, Western Pacific

PubMed Central

Nakagawa, Tatsunori; Ishibashi, Jun-Ichiro; Maruyama, Akihiko; Yamanaka, Toshiro; Morimoto, Yusuke; Kimura, Hiroyuki; Urabe, Tetsuro; Fukui, Manabu

2004-01-01

This study describes the occurrence of unique dissimilatory sulfite reductase (DSR) genes at a depth of 1,380 m from the deep-sea hydrothermal vent field at the Suiyo Seamount, Izu-Bonin Arc, Western Pacific, Japan. The DSR genes were obtained from microbes that grew in a catheter-type in situ growth chamber deployed for 3 days on a vent and from the effluent water of drilled holes at 5°C and natural vent fluids at 7°C. DSR clones SUIYOdsr-A and SUIYOdsr-B were not closely related to cultivated species or environmental clones. Moreover, samples of microbial communities were examined by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA gene. The sequence analysis of 16S rRNA gene fragments obtained from the vent catheter after a 3-day incubation revealed the occurrence of bacterial DGGE bands affiliated with the Aquificae and γ- and ɛ-Proteobacteria as well as the occurrence of archaeal phylotypes affiliated with the Thermococcales and of a unique archaeon sequence that clustered with “Nanoarchaeota.” The DGGE bands obtained from drilled holes and natural vent fluids from 7 to 300°C were affiliated with the δ-Proteobacteria, genus Thiomicrospira, and Pelodictyon. The dominant DGGE bands retrieved from the effluent water of casing pipes at 3 and 4°C were closely related to phylotypes obtained from the Arctic Ocean. Our results suggest the presence of microorganisms corresponding to a unique DSR lineage not detected previously from other geothermal environments. PMID:14711668

Analysis of Microbial Communities in Biofilms from CSTR-Type Hollow Fiber Membrane Biofilm Reactors for Autotrophic Nitrification and Hydrogenotrophic Denitrification.

PubMed

Shin, Jung-Hun; Kim, Byung-Chun; Choi, Okkyoung; Kim, Hyunook; Sang, Byoung-In

2015-10-01

Two hollow fiber membrane biofilm reactors (HF-MBfRs) were operated for autotrophic nitrification and hydrogenotrophic denitrification for over 300 days. Oxygen and hydrogen were supplied through the hollow fiber membrane for nitrification and denitrification, respectively. During the period, the nitrogen was removed with the efficiency of 82-97% for ammonium and 87-97% for nitrate and with the nitrogen removal load of 0.09-0.26 kg NH4(+)-N/m(3)/d and 0.10-0.21 kg NO3(-)-N/m(3)/d, depending on hydraulic retention time variation by the two HF-MBfRs for autotrophic nitrification and hydrogenotrophic denitrification, respectively. Biofilms were collected from diverse topological positions in the reactors, each at different nitrogen loading rates, and the microbial communities were analyzed with partial 16S rRNA gene sequences in denaturing gradient gel electrophoresis (DGGE). Detected DGGE band sequences in the reactors were correlated with nitrification or denitrification. The profile of the DGGE bands depended on the NH4(+) or NO3(-) loading rate, but it was hard to find a major strain affecting the nitrogen removal efficiency. Nitrospira-related phylum was detected in all biofilm samples from the nitrification reactors. Paracoccus sp. and Aquaspirillum sp., which are an autohydrogenotrophic bacterium and an oligotrophic denitrifier, respectively, were observed in the denitrification reactors. The distribution of microbial communities was relatively stable at different nitrogen loading rates, and DGGE analysis based on 16S rRNA (341f /534r) could successfully detect nitrate-oxidizing and hydrogen-oxidizing bacteria but not ammonium-oxidizing bacteria in the HF-MBfRs.

Using DGGE and 16S rRNA gene sequence analysis to evaluate changes in oral bacterial composition.

PubMed

Chen, Zhou; Trivedi, Harsh M; Chhun, Nok; Barnes, Virginia M; Saxena, Deepak; Xu, Tao; Li, Yihong

2011-01-01

To investigate whether a standard dental prophylaxis followed by tooth brushing with an antibacterial dentifrice will affect the oral bacterial community, as determined by denaturing gradient gel electrophoresis (DGGE) combined with 16S rRNA gene sequence analysis. Twenty-four healthy adults were instructed to brush their teeth using commercial dentifrice for 1 week during a washout period. An initial set of pooled supragingival plaque samples was collected from each participant at baseline (0 h) before prophylaxis treatment. The subjects were given a clinical examination and dental prophylaxis and asked to brush for 1 min with a dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride (Colgate Total). On the following day, a second set of pooled supragingival plaque samples (24 h) was collected. Total bacterial genomic DNA was isolated from the samples. Differences in the microbial composition before and after the prophylactic procedure and tooth brushing were assessed by comparing the DGGE profiles and 16S rRNA gene segments sequence analysis. Two distinct clusters of DGGE profiles were found, suggesting that a shift in the microbial composition had occurred 24 h after the prophylaxis and brushing. A detailed sequencing analysis of 16S rRNA gene segments further identified 6 phyla and 29 genera, including known and unknown bacterial species. Importantly, an increase in bacterial diversity was observed after 24 h, including members of the Streptococcaceae family, Prevotella, Corynebacterium, TM7 and other commensal bacteria. The results suggest that the use of a standard prophylaxis followed by the use of the dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride may promote a healthier composition within the oral bacterial community.

Effects of root inoculation with bacteria on the growth, Cd uptake and bacterial communities associated with rape grown in Cd-contaminated soil.

PubMed

Chen, Zhao-jin; Sheng, Xia-fang; He, Lin-yan; Huang, Zhi; Zhang, Wen-hui

2013-01-15

Two metal-resistant and plant growth-promoting bacteria (Burkholderia sp. J62 and Pseudomonas thivervalensis Y-1-3-9) were evaluated for their impacts on plant growth promotion, Cd availability in soil, and Cd uptake in rape (Brassica napus) grown in different level (0, 50, and 100 mg kg(-1)) of Cd-contaminated soils. The impacts of the bacteria on the rape-associated bacterial community structures were also evaluated using denaturing gradient gel electrophoresis (DGGE) analysis of bacterial DNA extracted from the root interior and rhizosphere and bulk soil samples collected at day 60 after inoculation. Canonical correspondence analysis (CCA) was used to have a comparative analysis of DGGE profiles. Inoculation with live bacteria not only significantly increased root (ranging from 38% to 86%), stem (ranging from 27% to 65%) and leaf (ranging from 23% to 55%) dry weights and water-extractive Cd contents (ranging from 59% to 237%) in the rhizosphere soils of the rape but also significantly increased root (ranging from 10% to 61%), stem (ranging from 41% to 57%) and leaf (ranging from 46% to 68%) total Cd uptake of rape compared to the dead bacterial-inoculated controls. DGGE and sequence analyses showed that the bacteria could colonize the rhizosphere soils and root interiors of rape plants. DGGE-CCA also showed that root interior and rhizosphere and bulk soil community profiles from the live bacteria-inoculated rape were significantly different from those from the dead bacteria-inoculated rape respectively. These results suggested that the bacteria had the potential to promote the growth and Cd uptake of rape and to influence the development of the rape-associated bacterial community structures. Copyright © 2012 Elsevier B.V. All rights reserved.

Stable-Isotope Probing of Bacteria Capable of Degrading Salicylate, Naphthalene, or Phenanthrene in a Bioreactor Treating Contaminated Soil

PubMed Central

Singleton, David R.; Powell, Sabrina N.; Sangaiah, Ramiah; Gold, Avram; Ball, Louise M.; Aitken, Michael D.

2005-01-01

[13C6]salicylate, [U-13C]naphthalene, and [U-13C]phenanthrene were synthesized and separately added to slurry from a bench-scale, aerobic bioreactor used to treat soil contaminated with polycyclic aromatic hydrocarbons. Incubations were performed for either 2 days (salicylate, naphthalene) or 7 days (naphthalene, phenanthrene). Total DNA was extracted from the incubations, the “heavy” and “light” DNA were separated, and the bacterial populations associated with the heavy fractions were examined by denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Unlabeled DNA from Escherichia coli K-12 was added to each sample as an internal indicator of separation efficiency. While E. coli was not detected in most analyses of heavy DNA, a low number of E. coli sequences was recovered in the clone libraries associated with the heavy DNA fraction of [13C]phenanthrene incubations. The number of E. coli clones recovered proved useful in determining the relative amount of light DNA contamination of the heavy fraction in that sample. Salicylate- and naphthalene-degrading communities displayed similar DGGE profiles and their clone libraries were composed primarily of sequences belonging to the Pseudomonas and Ralstonia genera. In contrast, heavy DNA from the phenanthrene incubations displayed a markedly different DGGE profile and was composed primarily of sequences related to the Acidovorax genus. There was little difference in the DGGE profiles and types of sequences recovered from 2- and 7-day incubations with naphthalene, so secondary utilization of the 13C during the incubation did not appear to be an issue in this experiment. PMID:15746319

Bacterial community dynamics and product distribution during pH-adjusted fermentation of vegetable wastes.

PubMed

Ye, N-F; Lü, F; Shao, L-M; Godon, J-J; He, P-J

2007-10-01

To estimate the effect of pH on the structures of bacterial community during fermentation of vegetable wastes and to investigate the relationship between bacterial community dynamics and product distribution. The bacterial communities in five batch tests controlled at different pH values [uncontrolled (about pH 4), 5, 6, 7 and 8] were monitored by denaturing gradient gel electrophoresis (DGGE) and single-strand conformation polymorphism (SSCP). The two fingerprinting methods provided consistent results and principal component analysis indicated a close similarity of bacterial community at pH 7 and 8 in addition to those at pH 4-6. This clustering also corresponded to dominant metabolic pathway. Thus, pH 7-8 shifted from alcohol-forming to acid-forming, especially butyric acid, whereas both alcohol-forming and acid-forming dominated at pH 5-6, and at pH 4, fermentation was inhibited. Shannon-weaver index was calculated to analyse the DGGE profiles, which revealed that the bacterial diversities at pH 7 and 8 were the highest while those at pH 5 and 4 (uncontrolled) were the lowest. According to sequencing results of the bands excised from DGGE gels, lactic acid bacteria and Clostridium sp. were predominant at all pH values, but varieties in species were observed as pH changed and time prolonged. The bacterial community during fermentation was materially influenced by pH and the diverse product distribution was related to the shift of different bacterial population. The study reveals that the impact of pH on fermentation product distribution is implemented primarily by changes of bacterial community. It also provides information about the comparison of two fingerprinting methods, DGGE and SSCP.

Molecular typing of Brucella melitensis endemic strains and differentiation from the vaccine strain Rev-1.

PubMed

Noutsios, Georgios T; Papi, Rigini M; Ekateriniadou, Loukia V; Minas, Anastasios; Kyriakidis, Dimitrios A

2012-03-01

In the present study forty-four Greek endemic strains of Br. melitensis and three reference strains were genotyped by Multi locus Variable Number Tandem Repeat (ML-VNTR) analysis based on an eight-base pair tandem repeat sequence that was revealed in eight loci of Br. melitensis genome. The forty-four strains were discriminated from the vaccine strain Rev-1 by Restriction Fragment Length Polymorphism (RFLP) and Denaturant Gradient Gel Electrophoresis (DGGE). The ML-VNTR analysis revealed that endemic, reference and vaccine strains are genetically closely related, while most of the loci tested (1, 2, 4, 5 and 7) are highly polymorphic with Hunter-Gaston Genetic Diversity Index (HGDI) values in the range of 0.939 to 0.775. Analysis of ML-VNTRs loci stability through in vitro passages proved that loci 1 and 5 are non stable. Therefore, vaccine strain can be discriminated from endemic strains by allele's clusters of loci 2, 4, 6 and 7. RFLP and DGGE were also employed to analyse omp2 gene and reveled different patterns among Rev-1 and endemic strains. In RFLP, Rev-1 revealed three fragments (282, 238 and 44 bp), while endemic strains two fragments (238 and 44 bp). As for DGGE, the electrophoretic mobility of Rev-1 is different from the endemic strains due to heterologous binding of DNA chains of omp2a and omp2b gene. Overall, our data show clearly that it is feasible to genotype endemic strains of Br. melitensis and differentiate them from vaccine strain Rev-1 with ML-VNTR, RFLP and DGGE techniques. These tools can be used for conventional investigations in brucellosis outbreaks.

The Primary Results of Analyses on The Archaeal and Bacterial Diversity of Active Cave Environments Settled in Limestones at Southern Turkey

NASA Astrophysics Data System (ADS)

Tok, Ezgi; Kurt, Halil; Tunga Akarsubasi, A.

2016-04-01

The microbial diversity of cave sediments which are obtained from three different caves named Insuyu, Balatini and Altınbeşik located at Southern Turkey has been investigated using molecular methods for biomineralization . The total number of 22 samples were taken in duplicates from the critical zones of the caves at where the water activity is observed all year round. Microbial communities were monitored by 16S rRNA gene based PCR-DGGE (Polymerase Chain Reaction - Denaturating Gradient Gel Electrophoresis) methodology. DNA were extracted from the samples by The PowerSoil® DNA Isolation Kit (MO BIO Laboratories inc., CA) with the modifications on the producer's protocol. The synthetic DNA molecule poly-dIdC was used to increase the yield of PCR amplification via blocking the reaction between CaCO3 and DNA molecules. Thereafter samples were amplified by using both Archaeal and Bacterial universal primers (ref). Subsequently, archaeal and bacterial diversities in cave sediments, were investigated to be able to compare with respect to their similarities by using DGGE. DGGE patterns were analysed with BioNumerics software 5.1. Similarity matrix and dendograms of the DGGE profiles were generated based on the Dice correlation coefficient (band-based) and unweighted pair-group method with arithmetic mean (UPGMA). The structural diversity of the microbial community was examined by the Shannon index of general diversity (H). Similtaneously, geochemical analyses of the sediment samples were performed within the scope of this study. Total organic carbon (TOC), x-ray diffraction spectroscopy (XRD) and x-ray fluorescence spectroscopy (XRF) analysis of sediments were also implemented. The extensive results will be obtained at the next stages of the study currently carried on.

[Dynamics of bacterial community during the bloom caused by Skeletonema costatum and Akashiwo sanguinea in Xiamen sea area].

PubMed

Li, Yi; Yang, Caiyun; Li, Dong; Tian, Yun; Zheng, Tianling

2012-10-04

To investigate the dynamics of bacterial community in Xiamen sea during the bloom mainly caused by Skeletonema costatum and Akashiwo sanguine in August 2011. Bacterial community structures of samples from two bloom sites and one non-bloom site were evaluated by PCR-DGGE (Denaturing gradient gel electrophoresis, DGGE). The genetic diversity of bacterial community was analyzed based on the DGGE fingerprint. The correlation between bacterial community and environmental parameters was studied by Canoco. The bacterial community was largely related to pH and N/P during the start-up stage of the bloom; while in the demise stage, it was mostly correlated to salinity and temperature. According to the results of sequence analysis of DGGE dominant bands, Gammaproteobacteria accounted for 47.7% during the bloom and Pseudoalteromonas, Pseudomonas, Alteromonas, Hydrogenophaga, Actibacter and Oleibacter were dominant genus in bacterial community. The Shannon-Weaver diversity index showed that the diversity of bacterial community in bloom site increased firstly and then decreased during this bloom. Hydrogenophaga was dominant in the start-up stage of bloom, while Pseudomonas and Pseudoalteromonas were dominant in the demise stage of bloom. The diversity of attached bacteria and free-living bacteria in bloom sites reached maximum in the same day (the concentration of algae was high) , both of them changed greatly during the bloom while the environment factors which correlated with the two communities were different. It is the first report about dynamics of bacterial community during the bloom caused by several algae together. This work is helpful to understand the dynamics of bacterial community during the bloom, and provides a theoretical basis for bloom's control in the future.

Influence of uranium on bacterial communities: a comparison of natural uranium-rich soils with controls.

PubMed

Mondani, Laure; Benzerara, Karim; Carrière, Marie; Christen, Richard; Mamindy-Pajany, Yannick; Février, Laureline; Marmier, Nicolas; Achouak, Wafa; Nardoux, Pascal; Berthomieu, Catherine; Chapon, Virginie

2011-01-01

This study investigated the influence of uranium on the indigenous bacterial community structure in natural soils with high uranium content. Radioactive soil samples exhibiting 0.26% - 25.5% U in mass were analyzed and compared with nearby control soils containing trace uranium. EXAFS and XRD analyses of soils revealed the presence of U(VI) and uranium-phosphate mineral phases, identified as sabugalite and meta-autunite. A comparative analysis of bacterial community fingerprints using denaturing gradient gel electrophoresis (DGGE) revealed the presence of a complex population in both control and uranium-rich samples. However, bacterial communities inhabiting uraniferous soils exhibited specific fingerprints that were remarkably stable over time, in contrast to populations from nearby control samples. Representatives of Acidobacteria, Proteobacteria, and seven others phyla were detected in DGGE bands specific to uraniferous samples. In particular, sequences related to iron-reducing bacteria such as Geobacter and Geothrix were identified concomitantly with iron-oxidizing species such as Gallionella and Sideroxydans. All together, our results demonstrate that uranium exerts a permanent high pressure on soil bacterial communities and suggest the existence of a uranium redox cycle mediated by bacteria in the soil.

Continous application of bioorganic fertilizer induced resilient culturable bacteria community associated with banana Fusarium wilt suppression

PubMed Central

Fu, Lin; Ruan, Yunze; Tao, Chengyuan; Li, Rong; Shen, Qirong

2016-01-01

Fusarium wilt of banana always drives farmers to find new land for banana cultivation due to the comeback of the disease after a few cropping years. A novel idea for solving this problem is the continuous application of bioorganic fertilizer (BIO), which should be practiced from the beginning of banana planting. In this study, BIO was applied in newly reclaimed fields to pre-control banana Fusarium wilt and the culturable rhizobacteria community were evaluated using Biolog Ecoplates and culture-dependent denaturing gradient gel electrophoresis (CD-DGGE). The results showed that BIO application significantly reduced disease incidences and increased crop yields, respectivly. And the stabilized general bacterial metabolic potential, especially for the utilization of carbohydrates, carboxylic acids and phenolic compounds, was induced by BIO application. DGGE profiles demonstrated that resilient community structure of culturable rhizobacteria with higher richness and diversity were observed in BIO treated soils. Morever, enriched culturable bacteria affiliated with Firmicutes, Gammaproteobacteria and Actinobacteria were also detected. In total, continuous application of BIO effectively suppressed Fusarium wilt disease by stabilizing culturable bacterial metabolic potential and community structure. This study revealed a new method to control Fusarium wilt of banana for long term banana cultivation. PMID:27306096

Salecan Enhances the Activities of β-1,3-Glucanase and Decreases the Biomass of Soil-Borne Fungi

PubMed Central

Chen, Yunmei; Xu, Haiyang; Zhou, Mengyi; Wang, Yang; Wang, Shiming; Zhang, Jianfa

2015-01-01

Salecan, a linear extracellular polysaccharide consisting of β-1,3-D-glucan, has potential applications in the food, pharmaceutical and cosmetic industries. The objective of this study was to evaluate the effects of salecan on soil microbial communities in a vegetable patch. Compositional shifts in the genetic structure of indigenous soil bacterial and fungal communities were monitored using culture-dependent dilution plating, culture-independent PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative PCR. After 60 days, soil microorganism counts showed no significant variation in bacterial density and a marked decrease in the numbers of fungi. The DGGE profiles revealed that salecan changed the composition of the microbial community in soil by increasing the amount of Bacillus strains and decreasing the amount of Fusarium strains. Quantitative PCR confirmed that the populations of the soil-borne fungi Fusarium oxysporum and Trichoderma spp. were decreased approximately 6- and 2-fold, respectively, in soil containing salecan. This decrease in the amount of fungi can be explained by salecan inducing an increase in the activities of β-1,3-glucanase in the soil. These results suggest the promising application of salecan for biological control of pathogens of soil-borne fungi. PMID:26247592

Impact of non-ionic surfactant on the long-term development of lab-scale-activated sludge bacterial communities.

PubMed

Lozada, Mariana; Basile, Laura; Erijman, Leonardo

2007-01-01

The development of bacterial communities in replicate lab-scale-activated sludge reactors degrading a non-ionic surfactant was evaluated by statistical analysis of denaturing gradient gel electrophoresis (DGGE) fingerprints. Four sequential batch reactors were fed with synthetic sewage, two of which received, in addition, 0.01% of nonylphenol ethoxylates (NPE). The dynamic character of bacterial community structure was confirmed by the differences in species composition among replicate reactors. Measurement of similarities between reactors was obtained by pairwise similarity analysis using the Bray Curtis coefficient. The group of NPE-amended reactors exhibited the highest similarity values (Sjk=0.53+/-0.03), indicating that the bacterial community structure of NPE-amended reactors was better replicated than control reactors (Sjk=0.36+/-0.04). Replicate NPE-amended reactors taken at different times of operation clustered together, whereas analogous relations within the control reactor cluster were not observed. The DGGE pattern of isolates grown in conditioned media prepared with media taken at the end of the aeration cycle grouped separately from other conditioned and synthetic media regardless of the carbon source amendment, suggesting that NPE degradation residuals could have a role in the shaping of the community structure.

Development of a molecular approach to describe the composition of Trichoderma communities.

PubMed

Meincke, Remo; Weinert, Nicole; Radl, Viviane; Schloter, Michael; Smalla, Kornelia; Berg, Gabriele

2010-01-01

Trichoderma and its teleomorphic stage Hypocrea play a key role for ecosystem functioning in terrestrial habitats. However, little is known about the ecology of the fungus. In this study we developed a novel Trichoderma-specific primer pair for diversity analysis. Based on a broad range master alignment, specific Trichoderma primers (ITSTrF/ITSTrR) were designed that comprise an approximate 650bp fragment of the internal transcribed spacer region from all taxonomic clades of the genus Trichoderma. This amplicon is suitable for identification with TrichoKey and TrichoBLAST. Moreover, this primer system was successfully applied to study the Trichoderma communities in the rhizosphere of different potato genotypes grown at two field sites in Germany. Cloning and sequencing confirmed the specificity of the primer and revealed a site-dependent Trichoderma composition. Based on the new primer system a semi-nested approach was used to generate amplicons suitable for denaturing gradient gel electrophoresis (DGGE) analysis and applied to analyse Trichoderma communities in the rhizosphere of potatoes. High field heterogeneity of Trichoderma communities was revealed by both DGGE. Furthermore, qPCR showed significantly different Trichoderma copy numbers between the sites. Copyright 2009 Elsevier B.V. All rights reserved.

Efficacy of rifaximin vaginal tablets in treatment of bacterial vaginosis: a molecular characterization of the vaginal microbiota.

PubMed

Cruciani, Federica; Brigidi, Patrizia; Calanni, Fiorella; Lauro, Vittoria; Tacchi, Raffaella; Donders, Gilbert; Peters, Klaus; Guaschino, Secondo; Vitali, Beatrice

2012-08-01

Bacterial vaginosis (BV) is a common vaginal disorder characterized by an alteration of the vaginal bacterial morphotypes, associated with sexually transmitted infections and adverse pregnancy outcomes. The purpose of the present study was to evaluate the impact of different doses of rifaximin vaginal tablets (100 mg/day for 5 days, 25 mg/day for 5 days, and 100 mg/day for 2 days) on the vaginal microbiota of 102 European patients with BV enrolled in a multicenter, double-blind, randomized, placebo-controlled study. An integrated molecular approach based on quantitative PCR (qPCR) and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) was used to investigate the effects of vaginal tablets containing the antibiotic. An increase in members of the genus Lactobacillus and a decrease in the BV-related bacterial groups after the antibiotic treatment were demonstrated by qPCR. PCR-DGGE profiles confirmed the capability of rifaximin to modulate the composition of the vaginal microbial communities and to reduce their complexity. This molecular analysis supported the clinical observation that rifaximin at 25 mg/day for 5 days represents an effective treatment to be used in future pivotal studies for the treatment of BV.

Efficacy of Rifaximin Vaginal Tablets in Treatment of Bacterial Vaginosis: a Molecular Characterization of the Vaginal Microbiota

PubMed Central

Cruciani, Federica; Brigidi, Patrizia; Calanni, Fiorella; Lauro, Vittoria; Tacchi, Raffaella; Donders, Gilbert; Peters, Klaus; Guaschino, Secondo

2012-01-01

Bacterial vaginosis (BV) is a common vaginal disorder characterized by an alteration of the vaginal bacterial morphotypes, associated with sexually transmitted infections and adverse pregnancy outcomes. The purpose of the present study was to evaluate the impact of different doses of rifaximin vaginal tablets (100 mg/day for 5 days, 25 mg/day for 5 days, and 100 mg/day for 2 days) on the vaginal microbiota of 102 European patients with BV enrolled in a multicenter, double-blind, randomized, placebo-controlled study. An integrated molecular approach based on quantitative PCR (qPCR) and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) was used to investigate the effects of vaginal tablets containing the antibiotic. An increase in members of the genus Lactobacillus and a decrease in the BV-related bacterial groups after the antibiotic treatment were demonstrated by qPCR. PCR-DGGE profiles confirmed the capability of rifaximin to modulate the composition of the vaginal microbial communities and to reduce their complexity. This molecular analysis supported the clinical observation that rifaximin at 25 mg/day for 5 days represents an effective treatment to be used in future pivotal studies for the treatment of BV. PMID:22585228

Decreased microbial diversity and Lactobacillus group in the intestine of geriatric giant pandas (Ailuropoda melanoleuca).

PubMed

Peng, Zhirong; Zeng, Dong; Wang, Qiang; Niu, Lili; Ni, Xueqin; Zou, Fuqin; Yang, Mingyue; Sun, Hao; Zhou, Yi; Liu, Qian; Yin, Zhongqiong; Pan, Kangcheng; Jing, Bo

2016-05-01

It has been established beyond doubt that giant panda genome lacks lignin-degrading related enzyme, gastrointestinal microbes may play a vital role in digestion of highly fibrous bamboo diet. However, there is not much information available about the intestinal bacteria composition in captive giant pandas with different ages. In this study, we compared the intestinal bacterial community of 12 captive giant pandas from three different age groups (subadults, adults, and geriatrics) through PCR-denaturing gradient gel electrophoresis (DGGE) and real-time PCR analysis. Results indicated that microbial diversity in the intestine of adults was significantly higher than that of the geriatrics (p < 0.05), but not significant compared to the subadults (p > 0.05). The predominant bands in DGGE patterns shared by the twelve pandas were related to Firmicutes and Proteobacteria. Additionally, in comparison to healthy individuals, antibiotic-treated animals showed partial microbial dysbiosis. Real-time PCR analyses confirmed a significantly higher abundance of the Lactobacillus in the fecal microbiota of adults (p < 0.05), while other bacterial groups and species detected did not significantly differ among the three age groups (p > 0.05). This study revealed that captive giant pandas with different ages showed different intestinal bacteria composition.

Analysis of the microbial communities on corroded concrete sewer pipes--a case study.

PubMed

Vincke, E; Boon, N; Verstraete, W

2001-12-01

Conventional as well as molecular techniques have been used to determine the microbial communities present on the concrete walls of sewer pipes. The genetic fingerprint of the microbiota on corroded concrete sewer pipes was obtained by means of denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. The DGGE profiles of the bacterial communities present on the concrete surface changed as observed by shifts occurring at the level of the dominance of bands from non-corroded places to the most severely corroded places. By means of statistical tools, it was possible to distinguish two different groups, corresponding to the microbial communities on corroded and non-corroded surfaces, respectively. Characterization of the microbial communities indicated that the sequences of typical bands showed the highest level of identity to sequences from the bacterial strains Thiobacillus thiooxidans, Acidithiobacillus sp., Mycobacterium sp. and different heterotrophs belonging to the alpha-, beta- and gamma-Proteobacteria, Acidobacteria and Actinobacteria. In addition, the presence of N-acyl-homoserine lactone signal molecules was shown by two bio-assays of the biofilm on the concrete under the water level and at the most severely corroded places on the concrete surface of the sewer pipe.

Molecular detection of Lactobacillus species in the neovagina of male-to-female transsexual women

PubMed Central

Petricevic, Ljubomir; Kaufmann, Ulrike; Domig, Konrad J.; Kraler, Manuel; Marschalek, Julian; Kneifel, Wolfgang; Kiss, Herbert

2014-01-01

There is a general opinion that penile skin lined neovagina of transsexual women is not able to support the growth of lactobacilli. This study was undertaken to prove if lactobacilli strains could survive in neovagina and to characterise the most dominant Lactobacillus species. Sixty three male-to-female transsexual women without abnormal vaginal discharge, clinical signs of infection were recruited on an ongoing basis from among transsexual outpatients in an academic research institution and tertiary care centre. Neovaginal smears were taken for molecular Lactobacillus spp. profiling by denaturing gradient gel electrophoresis (PCR–DGGE). Lactobacillus species were detected from 47/63 transsexual women (75%). The 279 Lactobacillus signals detected by PCR-DGGE technique belonged to 13 different species. Lactobacilli of the L. delbrueckii group (L. gasseri, L. crispatus, L. johnsonii, L. iners, L. jensenii) were predominant. More than 90% of women harboured a combination of two or more neovaginal Lactobacillus species. In this study we report the frequent occurrence of lactobacilli from neovagina of transsexual women. Both, frequency and composition were similar to the normal lactic acid bacterial microflora in both women of reproductive age and postmenopausal women. PMID:24434849

Microbial community of cyanobacteria mats in the intertidal zone of oil-polluted coast of Saudi Arabia.

PubMed

Al-Thukair, A A; Abed, R M M; Mohamed, L

2007-02-01

Cyanobacterial mats are found at various locations along the coast of the Eastern Province of Saudi Arabia. Those mats were affected by severe oil pollution following 1991 oil spill. In this study, samples from Abu Ali Island were collected at three selected sampling sites across the intertidal zone (Lower, Middle, and Upper) in order to understand the effect of extreme environmental conditions of high salinity, temperature and desiccation on distribution of cyanobacteria along the oil polluted intertidal zone. Our investigation of composition of cyanobacteria and diatoms was carried out using light microscopy, and Denaturant Gradient Gel Electrophoresis (DGGE) technique. Light microscopy identification revealed dominant cyanobacteria to be affiliated with genera Phormidium, Microcoleus, and Schizothrix, and to a lesser extent with Oscillatoria, Halothece, and various diatom species. The analysis of DGGE of PCR-amplified 16S rRNA fragments showed that the diversity of cyanobacteria decreases as we proceed from the lower to the upper intertidal zone. Accordingly, the tidal regime, salinity, elevated ambient air temperature, and desiccation periods have a great influence on the distribution of cyanobacterial community in the oil polluted intertidal zone of Abu Ali Island.

Bacterial community structure and activity of sulfate reducing bacteria in a membrane aerated biofilm analyzed by microsensor and molecular techniques.

PubMed

Liu, Hong; Tan, Shuying; Sheng, Zhiya; Liu, Yang; Yu, Tong

2014-11-01

The activities and vertical spatial distribution of sulfate reducing bacteria (SRB) in an oxygen (O2 )-based membrane aerated biofilm (MAB) were investigated using microsensor (O2 and H2 S) measurements and molecular techniques (polymerase chain reaction-denaturing gradient gel electrophoresis [PCR-DGGE] and fluorescence in situ hybridization [FISH]). The O2 concentration profile revealed that O2 penetrated from the bottom (substratum) of the gas permeable membrane, and was gradually consumed within the biofilm until it was completely depleted near the biofilm/bulk liquid interface, indicating oxic and anoxic zone in the MAB. The H2 S concentration profile showed that H2 S production was found in the upper 285 µm of the biofilm, indicating a high activity of SRB in this region. The results from DGGE of the PCR-amplified dissimilatory sulfite reductase subunit B (dsrB) gene and FISH showed an uneven spatial distribution of SRB. The maximum SRB biomass was located in the upper biofilm. The information from the molecular analysis can be supplemented with that from microsensor measurements to better understand the microbial community and activity of SRB in the MAB. © 2014 Wiley Periodicals, Inc.

Bacterial microbiota profile in gills of modified atmosphere-packaged oysters stored at 4 °C.

PubMed

Chen, Huibin; Wang, Meiying; Lin, Xiangzhi; Shi, Caihua; Liu, Zhiyu

2017-02-01

As filter-feeding bivalves, oysters can accumulate microorganisms into their gills, causing spoilage and potential safety issues. This study aims to investigate the changes in the gill microbiota of oysters packed under air and modified atmospheres (MAs, 50% CO 2 : 50% N 2 , 70% CO 2 : 30% O 2 , and 50% CO 2 : 50% O 2 ) during storage at 4 °C. The diversity of bacterial microbiota in oyster gills was profiled through polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis on the 16S rRNA gene V3 region to describe the variation during the entire storage period. The DGGE profile revealed high bacterial diversity in the air- and MA-packaged oyster gills, and the spoilage bacterial microbiota varied in the MA-packaged oyster gills. Results indicated that CO 2 :O 2 (70%:30%) was suitable for oyster MA packaging and that high bacterial loads in oyster gills need to be considered during storage. In addition, Lactobacillus and Lactococcus species were found to grow dominantly in fresh oyster gills under MA packaging, which supports the potential application of MA packaging for oyster storage. Copyright © 2016 Elsevier Ltd. All rights reserved.

Analysis on the Spatial Difference of Bacterial Community Structure in Micro-pressure Air-lift Loop Reactor

NASA Astrophysics Data System (ADS)

Wan, L. G.; Lin, Q.; Bian, D. J.; Ren, Q. K.; Xiao, Y. B.; Lu, W. X.

2018-02-01

In order to reveal the spatial difference of the bacterial community structure in the Micro-pressure Air-lift Loop Reactor, the activated sludge bacterial at five different representative sites in the reactor were studied by denaturing gradient gel electrophoresis (DGGE). The results of DGGE showed that the difference of environmental conditions (such as substrate concentration, dissolved oxygen and PH, etc.) resulted in different diversity and similarity of microbial flora in different spatial locations. The Shannon-Wiener diversity index of the total bacterial samples from five sludge samples varied from 0.92 to 1.28, the biodiversity index was the smallest at point 5, and the biodiversity index was the highest at point 2. The similarity of the flora between the point 2, 3 and 4 was 80% or more, respectively. The similarity of the flora between the point 5 and the other samples was below 70%, and the similarity of point 2 was only 59.2%. Due to the different contribution of different strains to the removal of pollutants, it can give full play to the synergistic effect of bacterial degradation of pollutants, and further improve the efficiency of sewage treatment.

Dietary supplementation with sorbitol results in selective enrichment of lactobacilli in rat intestine.

PubMed

Sarmiento-Rubiano, Luz Adriana; Zúñiga, Manuel; Pérez-Martínez, Gaspar; Yebra, María Jesús

2007-01-01

A potential prebiotic action has been ascribed to sorbitol, but in vivo evidence of this remains scarce. In the present work, the effect of sorbitol was compared to that of fructo-oligosaccharides (FOS) in a rat model. Microbiota changes, particularly in lactobacilli, were analyzed on fecal, colonic and cecal samples. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene amplicons using universal primers showed that FOS and sorbitol diets exerted a strong influence upon gut microbiota patterns. When Lactobacillus group-specific primers were used, DGGE profiles revealed five DNA bands that belonged to Lactobacillus johnsonii, Lactobacillus sp. AD102, Lactobacillus intestinalis, Lactobacillus murinus and Lactobacillus reuteri. Although these species are present in all dietary groups, quantification by real-time PCR showed that sorbitol and FOS intake increased L. reuteri cell numbers, and sorbitol also contributed to maintaining the levels of Lactobacillus sp. AD102. Analysis of organic acid concentrations showed that sorbitol intake significantly increased colonic and cecal butyrate levels. Hence, sorbitol, which is widely used as a low-calorie sweetener, has the capacity, in our animal model, to modify gut microbiota activity in such a way as to possibly contribute to healthy colonic mucosa.

Competing factors of compost concentration and proximity to root affect the distribution of streptomycetes.

PubMed

Inbar, Ehud; Green, Stefan J; Hadar, Yitzhak; Minz, Dror

2005-07-01

Streptomycetes are important members of soil microbial communities and are particularly active in the degradation of recalcitrant macromolecules and have been implicated in biological control of plant disease. Using a streptomycetes-specific polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (PCR-DGGE) methodology coupled with band excision and sequence analysis, we examined the effect of grape marc compost amendment to soil on cucumber plant-associated streptomycetes community composition. We observed that both compost amendment and proximity to the root surface influenced the streptomycetes community composition. A strong root selection for a soil-derived Streptomycete, most closely related to Streptomyces thermotolerans, S. iakyrus, and S. thermocarboxydus, was independent of compost amendment rate. However, while the impact of compost amendment was mitigated with increasing proximity to the root, high levels of compost amendment resulted in the detection of compost-derived species on the root surface. Conversely, in rhizosphere and non-rhizosphere soils, the community composition of streptomycetes was affected strongly even by modest compost amendment. The application of a streptomycetes-specific PCR primer set combined with DGGE analysis provided a rapid means of examining the distribution and ecology of streptomycetes in soils and plant-associated environments.

Continous application of bioorganic fertilizer induced resilient culturable bacteria community associated with banana Fusarium wilt suppression

NASA Astrophysics Data System (ADS)

Fu, Lin; Ruan, Yunze; Tao, Chengyuan; Li, Rong; Shen, Qirong

2016-06-01

Fusarium wilt of banana always drives farmers to find new land for banana cultivation due to the comeback of the disease after a few cropping years. A novel idea for solving this problem is the continuous application of bioorganic fertilizer (BIO), which should be practiced from the beginning of banana planting. In this study, BIO was applied in newly reclaimed fields to pre-control banana Fusarium wilt and the culturable rhizobacteria community were evaluated using Biolog Ecoplates and culture-dependent denaturing gradient gel electrophoresis (CD-DGGE). The results showed that BIO application significantly reduced disease incidences and increased crop yields, respectivly. And the stabilized general bacterial metabolic potential, especially for the utilization of carbohydrates, carboxylic acids and phenolic compounds, was induced by BIO application. DGGE profiles demonstrated that resilient community structure of culturable rhizobacteria with higher richness and diversity were observed in BIO treated soils. Morever, enriched culturable bacteria affiliated with Firmicutes, Gammaproteobacteria and Actinobacteria were also detected. In total, continuous application of BIO effectively suppressed Fusarium wilt disease by stabilizing culturable bacterial metabolic potential and community structure. This study revealed a new method to control Fusarium wilt of banana for long term banana cultivation.

Influence of Uranium on Bacterial Communities: A Comparison of Natural Uranium-Rich Soils with Controls

PubMed Central

Mondani, Laure; Benzerara, Karim; Carrière, Marie; Christen, Richard; Mamindy-Pajany, Yannick; Février, Laureline; Marmier, Nicolas; Achouak, Wafa; Nardoux, Pascal; Berthomieu, Catherine; Chapon, Virginie

2011-01-01

This study investigated the influence of uranium on the indigenous bacterial community structure in natural soils with high uranium content. Radioactive soil samples exhibiting 0.26% - 25.5% U in mass were analyzed and compared with nearby control soils containing trace uranium. EXAFS and XRD analyses of soils revealed the presence of U(VI) and uranium-phosphate mineral phases, identified as sabugalite and meta-autunite. A comparative analysis of bacterial community fingerprints using denaturing gradient gel electrophoresis (DGGE) revealed the presence of a complex population in both control and uranium-rich samples. However, bacterial communities inhabiting uraniferous soils exhibited specific fingerprints that were remarkably stable over time, in contrast to populations from nearby control samples. Representatives of Acidobacteria, Proteobacteria, and seven others phyla were detected in DGGE bands specific to uraniferous samples. In particular, sequences related to iron-reducing bacteria such as Geobacter and Geothrix were identified concomitantly with iron-oxidizing species such as Gallionella and Sideroxydans. All together, our results demonstrate that uranium exerts a permanent high pressure on soil bacterial communities and suggest the existence of a uranium redox cycle mediated by bacteria in the soil. PMID:21998695

Influence of Effluent Irrigation on Community Composition and Function of Ammonia-Oxidizing Bacteria in Soil

PubMed Central

Oved, Tamar; Shaviv, Avi; Goldrath, Tal; Mandelbaum, Raphi T.; Minz, Dror

2001-01-01

The effect of effluent irrigation on community composition and function of ammonia-oxidizing bacteria (AOB) in soil was evaluated, using techniques of molecular biology and analytical soil chemistry. Analyses were conducted on soil sampled from lysimeters and from a grapefruit orchard which had been irrigated with wastewater effluent or fertilizer-amended water (FAW). Specifically, comparisons of AOB community composition were conducted using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified fragments of the gene encoding the α-subunit of the ammonia monooxygenase gene (amoA) recovered from soil samples and subsequent sequencing of relevant bands. A significant and consistent shift in the population composition of AOB was detected in soil irrigated with effluent. This shift was absent in soils irrigated with FAW, despite the fact that the ammonium concentration in the FAW was similar. At the end of the irrigation period, Nitrosospira-like populations were dominant in soils irrigated with FAW, while Nitrosomonas-like populations were dominant in effluent-irrigated soils. Furthermore, DGGE analysis of the amoA gene proved to be a powerful tool in evaluating the soil AOB community population and population shifts therein. PMID:11472914

Diversity of Bacterial Communities in Container Habitats of Mosquitoes

PubMed Central

Ponnusamy, Loganathan; Xu, Ning; Stav, Gil; Wesson, Dawn M.; Schal, Coby

2010-01-01

We investigated the bacterial diversity of microbial communities in water-filled, human-made and natural container habitats of the mosquitoes Aedes aegypti and Aedes albopictus in suburban landscapes of New Orleans, Louisiana in 2003. We collected water samples from three classes of containers, including tires (n=12), cemetery urns (n=23), and miscellaneous containers that included two tree holes (n=19). Total genomic DNA was extracted from water samples, and 16S ribosomal DNA fragments (operational taxonomic units, OTUs) were amplified by PCR and separated by denaturing gradient gel electrophoresis (DGGE). The bacterial communities in containers represented diverse DGGE-DNA banding patterns that were not related to the class of container or to the local spatial distribution of containers. Mean richness and evenness of OTUs were highest in water samples from tires. Bacterial phylotypes were identified by comparative sequence analysis of 90 16S rDNA DGGE band amplicons. The majority of sequences were placed in five major taxa: Alpha-, Beta- and Gammaproteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, and an unclassified group; Proteobacteria and Bacteroidetes were the predominant heterotrophic bacteria in containers. The bacterial communities in human-made containers consisted mainly of undescribed species, and a phylogenetic analysis based on 16S rRNA sequences suggested that species composition was independent of both container type and the spatial distribution of containers. Comparative PCR-based, cultivation-independent rRNA surveys of microbial communities associated with mosquito habitats can provide significant insight into community organization and dynamics of bacterial species. PMID:18373113

Bioremediation of oil-based drill cuttings by a halophilic consortium isolated from oil-contaminated saline soil.

PubMed

Rezaei Somee, Maryam; Shavandi, Mahmoud; Dastgheib, Seyed Mohammad Mehdi; Amoozegar, Mohammad Ali

2018-05-01

Oil-based drill cuttings are hazardous wastes containing complex hydrocarbons, heavy metals, and brine. Their remediation is a crucial step before release to the environment. In this work, we enriched a halophilic consortium, from oil-polluted saline soil, which is capable of degrading diesel as the main pollutant of oil-based drill cuttings. The degradation ability of the consortium was evaluated in microcosms using two different diluting agents (fine sand and biologically active soil). During the bioremediation process, the bacterial community dynamics of the microcosms was surveyed using PCR amplification of a fragment of 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE). The diesel degradation rates were monitored by total petroleum hydrocarbon (TPH) measurement and the total count of heterotrophic and diesel-degrading bacteria. After 3 months, the microcosm containing fine sand and drill cuttings with the ratio of 1:1 (initial TPH of 36,000 mg/kg) showed the highest TPH removal (40%) and its dominant bacterial isolates belonged to the genera Dietzia, Arthrobacter , and Halomonas . DGGE results also confirmed the role of these genera in drill cuttings remediation. DGGE analysis of the bacterial diversity showed that Propionibacterium, Salinimicrobium, Marinobacter , and Dietzia are dominant in active soil microcosm; whereas Bacillus, Salinibacillus , and Marinobacter are abundant in sand microcosm. Our results suggest that the bioaugmentation strategy would be more successful if the diluting agent does not contain a complex microbial community.

Comparison of characterization and microbial communities in rice straw- and wheat straw-based compost for Agaricus bisporus production.

PubMed

Wang, Lin; Mao, Jiugeng; Zhao, Hejuan; Li, Min; Wei, Qishun; Zhou, Ying; Shao, Heping

2016-09-01

Rice straw (RS) is an important raw material for the preparation of Agaricus bisporus compost in China. In this study, the characterization of composting process from RS and wheat straw (WS) was compared for mushroom production. The results showed that the temperature in RS compost increased rapidly compared with WS compost, and the carbon (C)/nitrogen (N) ratio decreased quickly. The microbial changes during the Phase I and Phase II composting process were monitored using denaturing gradient gel electrophoresis (DGGE) and phospholipid fatty acid (PLFA) analysis. Bacteria were the dominant species during the process of composting and the bacterial community structure dramatically changed during heap composting according to the DGGE results. The bacterial community diversity of RS compost was abundant compared with WS compost at stages 4-5, but no distinct difference was observed after the controlled tunnel Phase II process. The total amount of PLFAs of RS compost, as an indicator of microbial biomass, was higher than that of WS. Clustering by DGGE and principal component analysis of the PLFA compositions revealed that there were differences in both the microbial population and community structure between RS- and WS-based composts. Our data indicated that composting of RS resulted in improved degradation and assimilation of breakdown products by A. bisporus, and suggested that the RS compost was effective for sustaining A. bisporus mushroom growth as well as conventional WS compost.

Selection of Sphingomonadaceae at the base of Laccaria proxima and Russula exalbicans fruiting bodies.

PubMed

Boersma, F G Hidde; Warmink, Jan A; Andreote, Fernando A; van Elsas, Jan Dirk

2009-04-01

The dense hyphal network directly underneath the fruiting bodies of ectomycorrhizal fungi might exert strong influences on the bacterial community of soil. Such fruiting bodies might serve as hot spots for bacterial activity, for instance by providing nutrients and colonization sites in soil. Here, we assessed the putative selection of specific members of the Sphingomonadaceae family at the bases of the fruiting bodies of the ectomycorrhizal fungi Laccaria proxima and Russula exalbicans in comparison to the adjacent bulk soil. To do so, we used a previously designed Sphingomonadaceae-specific PCR-denaturing gradient gel electrophoresis (DGGE) system and complemented this with analyses of sequences from a Sphingomonadaceae-specific clone library. The analyses showed clear selective effects of the fruiting bodies of both fungi on the Sphingomonadaceae community structures. The effect was especially prevalent with R. exalbicans. Strikingly, similar fungi sampled approximately 100 m apart showed similar DGGE patterns, while corresponding bulk soil-derived patterns differed from each other. However, the mycospheres of L. proxima and R. exalbicans still revealed divergent community structures, indicating that different fungi select for different members of the Sphingomonadaceae family. Excision of specific bands from the DGGE patterns, as well as analyses of the clone libraries generated from both habitats, revealed fruiting body-specific Sphingomonadaceae types. It further showed that major groups from the mycospheres of R. exalbicans and L. proxima did not cluster with known bacteria from the database, indicating new groups within the family of Sphingomonadaceae present in these environments.

[Bacterial diversity in the oral cavity of adolescents with different caries susceptibilities].

PubMed

Yangyang, Zhang; Jinzhi, He; Xin, Xu; Xuedong, Zhou

2015-12-01

To analyze the differences between the bacterial diversities in the saliva of caries-free and caries-susceptible adolescents through polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE). Twenty adolescent subjects aged 12-18 years were recruited and subdivided into two groups: caries-free adolescents (n = 10) and caries-susceptible adolescents (n = 10). Saliva samples were collected. Total DNA was isolated directly from each sample. A portion of the 16S rRNA gene locus was PCR-amplified by using universal primers. Microbial diversity was analyzed through PCR-DGGE. Analyzing the DGGE profile, we found that the composition of the saliva microbiome exhibited great intra-individual differences; the average band numbers of the caries-free adolescent group and the caries-susceptible adolescent group were 32.5 ± 3.7 and 27.3 ± 3.4, respectively. The differences between the groups were statistically significant (P = 0.008). Shannon-Wiener's indexes of the caries-susceptible adolescent group and the caries-free adolescent group were 2.5 ± 0.2 and 2.6 ± 0.2, respectively, but the differences between the groups were not significant (P = 0.405). Clustering analysis results suggested that most of the samples in the same group clustered together; this observation showed a high community structure similarity. The microbial diversity and complexity of bacteria in saliva are significantly higher in caries-free adolescents than in caries-susceptible adolescents. During caries development, bacterial diversity in the saliva likely decreases.

Sulphur-oxidizing and sulphate-reducing communities in Brazilian mangrove sediments.

PubMed

Varon-Lopez, Maryeimy; Dias, Armando Cavalcante Franco; Fasanella, Cristiane Cipolla; Durrer, Ademir; Melo, Itamar Soares; Kuramae, Eiko Eurya; Andreote, Fernando Dini

2014-03-01

Mangrove soils are anaerobic environments rich in sulphate and organic matter. Although the sulphur cycle is one of the major actors in this ecosystem, little is known regarding the sulphur bacteria communities in mangrove soils. We investigated the abundance, composition and diversity of sulphur-oxidizing (SOB) and sulphate-reducing (SRB) bacteria in sediments from three Brazilian mangrove communities: two contaminated, one with oil (OilMgv) and one with urban waste and sludge (AntMgv), and one pristine (PrsMgv). The community structures were assessed using quantitative real-time polymerase chain reaction (qPCR), polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and clone libraries, using genes for the enzymes adenosine-5'-phosphosulphate reductase (aprA) and sulphite reductase (Dsr) (dsrB). The abundance for qPCR showed the ratio dsrB/aprA to be variable among mangroves and higher according to the gradient observed for oil contamination in the OilMgv. The PCR-DGGE patterns analysed by Nonmetric Multidimensional Scaling revealed differences among the structures of the three mangrove communities. The clone libraries showed that Betaproteobacteria, Gammaproteobacteria and Deltaproteobacteria were the most abundant groups associated with sulphur cycling in mangrove sediments. We conclude that the microbial SOB and SRB communities in mangrove soils are different in each mangrove forest and that such microbial communities could possibly be used as a proxy for contamination in mangrove forests. © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.

Diversity and composition of sulfate- and sulfite-reducing prokaryotes as affected by marine-freshwater gradient and sulfate availability.

PubMed

Fan, Lan-Feng; Tang, Sen-Lin; Chen, Chang-Po; Hsieh, Hwey-Lian

2012-01-01

Sulfate- and sulfite-reducing prokaryotes (SSRP) communities play a key role in both sulfur and carbon cycles. In estuarine ecosystems, sulfate concentrations change with tides and could be limited in tidal freshwater reach or deep sediments. In a subtropical estuary of northern Taiwan in December 2007, we examined the compositional changes of SSRP communities. We examined three sites: from the lower estuarine brackish-water reach (site GR and mangrove vegetation site, GM) to the upper estuarine tidal freshwater reach (site HR), as well as from surface to a 50-cm depth. The partial sequence of sulfite reductase (dsrB) genes was used as a molecular marker of SSRP, linked to polymerase chain reaction and denaturing gradient gel electrophoresis (DGGE) techniques. SSRP communities of the DGGE profiles varied with sites according to one-way analyses of similarities (Global R = 0.69, P = 0.001). Using cluster analysis, the DGGE profile was found to show site-specific clusters and a distinct depth zonation (five, six, and two SSRP communities at the GM, GR, and HR sites, respectively). SSRP composition was highly correlated to the combination of salinity, reduced sulfur, and total organic carbon contents (BIO-ENV analysis, r ( s ) = 0.56). After analyzing a total of 35 dsrB sequences in the DGGE gel, six groups with 15 phylotypes were found, which were closely related to marine-freshwater gradient. Moreover, sequences neighboring sulfite-reducing prokaryotes were observed, in addition to those affiliated to sulfate-reducing prokaryotes. Four phylotypes harvested in HR resembled the genus Desulfitobacterium, a sulfite-reducing prokaryote, which failed to use sulfate as an electron acceptor and were active in freshwater and sulfate-limited habitat. The other five phylotypes in the HR reach belonged to the sulfate-reducing prokaryotes of the genera Desulfatiferula, Desulfosarcina, Desulfovibrio, and Desulfotomaculum, which appeared to tolerate low salinity and low sulfate supply. SSRP phylotypes at the mangrove-vegetated GM site (five phylotypes in two groups) were phylogenetically less diverse, when compared with those at the non-mangrove-vegetated GR site (three phylotypes in three groups) and the tidally influenced freshwater HR site (nine phylotypes in five groups). Phylotypes found at GR and GM were all affiliated to marine sulfate-reducing prokaryote strains of the genera Desulfofaba, Desulfobotulus, Desulfatiferula, Desulfosarcina, and Desulfotomaculum. Notably, a phylotype recorded in the surface sediment at GR resembled the genus Desulfobulbus, which was recorded from freshwater environment consisting of the freshwater input at GR during ebb tides.

Growth of Quailbush in Acidic, Metalliferous Desert Mine Tailings: Effect of Azospirillum brasilense Sp6 on Biomass Production and Rhizosphere Community Structure

PubMed Central

de-Bashan, Luz E.; Hernandez, Juan-Pablo; Nelson, Karis N.; Bashan, Yoav

2010-01-01

Mine tailing deposits in semiarid and arid environments frequently remain devoid of vegetation due to the toxicity of the substrate and the absence of a diverse soil microbial community capable of supporting seed germination and plant growth. The contribution of the plant growth promoting bacterium (PGPB) Azospirillum brasilense Sp6 to the growth of quailbush in compost-amended, moderately acidic, high-metal content mine tailings using an irrigation-based reclamation strategy was examined along with its influence on the rhizosphere bacterial community. Sp6 inoculation resulted in a significant (2.2-fold) increase in plant biomass production. The data suggest that the inoculum successfully colonized the root surface and persisted throughout the 60-day experiment in both the rhizosphere, as demonstrated by excision and sequencing of the appropriate denaturing gradient gel electrophoresis (DGGE) band, and the rhizoplane, as indicated by fluorescent in situ hybridization of root surfaces. Changes in rhizosphere community structure in response to Sp6 inoculation were evaluated after 15, 30, and 60 days using DGGE analysis of 16S rRNA polymerase chain reaction amplicons. A comparison of DGGE profiles using canonical correspondence analysis revealed a significant treatment effect (Sp6-inoculated vs. uninoculated plants vs. unplanted) on bacterial community structure at 15, 30, and 60 days (p<0.05). These data indicate that in an extremely stressed environment such as acid mine tailings, an inoculated plant growth promoting bacterium not only can persist and stimulate plant growth but also can directly or indirectly influence rhizobacterial community development. PMID:20632001

Genetic profiling of the oral microbiota associated with severe early-childhood caries.

PubMed

Li, Y; Ge, Y; Saxena, D; Caufield, P W

2007-01-01

The determination of the composition of the microbial community in the oral cavity is usually based on cultivation methods; however, nearly half of the bacteria in the saliva and the dental plaque are not cultivable. In this study, we evaluated the difference in oral microbial diversity between children with severe early-childhood caries (S-ECC) and caries-free (CF) controls by means of a cultivation-independent approach called denaturing gradient gel electrophoresis (DGGE). Pooled dental plaque samples were collected from 20 children aged 2 to 8 years. Total microbial genomic DNA was isolated from those subjects, and a portion of the 16S rRNA gene locus was PCR amplified by using universal primers. We observed that the mean species richness of the bacterial population was greater in the CF children (n = 12) (42 +/- 3.7) than in the S-ECC children (n = 8) (35 +/- 4.3); the difference was statistically significant (P = 0.005). The overall diversity of plaque samples as measured by the Shannon index was 3.5 for the S-ECC group and 3.7 for the CF group (P = 0.004). Differences in DGGE profiles were distinguished on the basis of a cluster analysis. Sequence analysis of excised DGGE bands consisted of 2.7 phylotypes, on average. After adjusting for the number of observed bands, we estimated that the S-ECC group exhibited 94.5 total phylotypes and that the CF group exhibited 113.4. These results suggest that the microbial diversity and complexity of the microbial biota in dental plaque are significantly less in S-ECC children than in CF children.

Analysis of Inter-Individual Bacterial Variation in Gut of Cicada Meimuna mongolica (Hemiptera: Cicadidae)

PubMed Central

Zhou, Wenting; Nan, Xiaoning; Zheng, Zhou; Wei, Cong; He, Hong

2015-01-01

Intestinal bacterial community plays a crucial role in the nutrition, development, survival, and reproduction of insects. When compared with other insects with piercing-sucking mouthparts, the habitats of cicada nymphs and adults are totally different. However, little is known about the differences in the gut bacterial communities in the nymphs and adults within any cicada species. The diversity of bacteria in the gut of nymphs and adults of both genders of Meimuna mongolica (Distant) was studied using the denaturing gradient gel electrophoresis (DGGE) method. Few inter-individual variations among gut microbiota were observed, suggesting that M. mongolica typically harbors a limited and consistent suite of bacterial species. Bacteria in the genera Pseudomonas and Enterobacter were the predominant components of the gut microflora of M. mongolica at all life stages. Bacteria of Pantoea, Streptococcus, and Uruburuella were also widespread in the cicada samples but at relatively lower concentrations. The relative stability and similarity of the PCR-DGGE patterns indicate that all individuals of this cicada species harbor a characteristic bacterial community which is independent from developmental stages and genders. Related endosymbionts that could be harbored in bacteromes of cicadas were not detected in any gut samples, which could be related to the cicada species and the distribution of these endosymbionts in the cicada cavity, or due to some of the possible limitations of PCR-DGGE community profiling. It is worthwhile to further address if related cicada endosymbiont clades distribute in the alimentary canals and other internal organs through diagnostic PCR using group-specific primer sets. PMID:26411784

The biogeography of fungal communities in wetland sediments along the Changjiang River and other sites in China

PubMed Central

Wu, Bing; Tian, Jianqing; Bai, Chunming; Xiang, Meichun; Sun, Jingzu; Liu, Xingzhong

2013-01-01

Whether fungal community structure depends more on historical factors or on contemporary factors is controversial. This study used culture-dependent and -independent (polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE)) methods to assess the influence of historical and contemporary factors on the distributions of fungi in the wetland sediments at 10 locations along the Changjiang River and at 10 other locations in China. The culture-dependent approach detected greater species diversity (177 operational taxonomic units (OTUs)) than PCR-DGGE analysis (145 OTUs), and the species in the genera of Penicillium (relative frequency=16.8%), Fusarium (15.4%), Aspergillus (7.6%), Trichoderma (5.8%) and Talaromyces (4.2%) were dominant. On the basis of DGGE data, fungal diversity along the Changjiang River increased from upstream to downstream; altitude explained 44.8% of this variation in diversity. And based on the data from all 20 locations, the fungal communities were geographically clustered into three groups: Southern China, Northern China and the Qinghai-Tibetan Plateau. Multivariate regression tree analysis for data from the 20 locations indicated that the fungal community was influenced primarily by location (which explained 61.8% of the variation at a large scale), followed by total potassium (9.4%) and total nitrogen (3.5%) at a local scale. These results are consistent with the concept that geographic distance is the dominant factor driving variation in fungal diversity at a regional scale (1000–4000 km), whereas environmental factors (total potassium and total nitrogen) explain variation in fungal diversity at a local scale (<1000 km). PMID:23446835

Litter Breakdown and Microbial Succession on Two Submerged Leaf Species in a Small Forested Stream

PubMed Central

Newman, Molli M.; Liles, Mark R.; Feminella, Jack W.

2015-01-01

Microbial succession during leaf breakdown was investigated in a small forested stream in west-central Georgia, USA, using multiple culture-independent techniques. Red maple (Acer rubrum) and water oak (Quercus nigra) leaf litter were incubated in situ for 128 days, and litter breakdown was quantified by ash-free dry mass (AFDM) method and microbial assemblage composition using phospholipid fatty acid analysis (PLFA), ribosomal intergenic spacer analysis (RISA), denaturing gradient gel electrophoresis (DGGE), and bar-coded next-generation sequencing of 16S rRNA gene amplicons. Leaf breakdown was faster for red maple than water oak. PLFA revealed a significant time effect on microbial lipid profiles for both leaf species. Microbial assemblages on maple contained a higher relative abundance of bacterial lipids than oak, and oak microbial assemblages contained higher relative abundance of fungal lipids than maple. RISA showed that incubation time was more important in structuring bacterial assemblages than leaf physicochemistry. DGGE profiles revealed high variability in bacterial assemblages over time, and sequencing of DGGE-resolved amplicons indicated several taxa present on degrading litter. Next-generation sequencing revealed temporal shifts in dominant taxa within the phylum Proteobacteria, whereas γ-Proteobacteria dominated pre-immersion and α- and β-Proteobacteria dominated after 1 month of instream incubation; the latter groups contain taxa that are predicted to be capable of using organic material to fuel further breakdown. Our results suggest that incubation time is more important than leaf species physicochemistry in influencing leaf litter microbial assemblage composition, and indicate the need for investigation into seasonal and temporal dynamics of leaf litter microbial assemblage succession. PMID:26098687

Analyzing the relation between the microbial diversity of DaQu and the turbidity spoilage of traditional Chinese vinegar.

PubMed

Li, Pan; Li, Sha; Cheng, Lili; Luo, Lixin

2014-07-01

Vinegar is a traditional fermented condiment, and the microbial diversity of DaQu makes the quality of vinegar products. Recently, turbidity spoilage of vinegar sharply tampered with the quality of vinegar. In this study, the relation between the microbial diversity of DaQu and turbidity spoilage of vinegar was analyzed by plating technique, PCR-denaturing gradient gel electrophoresis (DGGE), and high-performance liquid chromatography (HPLC). The 16S rRNA sequencing and DGGE analysis indicated that Bacillus (Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus thuringiensis) and Lactobacillus (including Lactobacillus acidipiscis and Lactobacillus pobuzihii) species were the dominant contaminants in vinegar products. Meanwhile, DGGE analysis showed that the dominant bacteria in DaQu belonged to genera Bacillus, Lactobacillus, Pseudomonas, Weissella, Saccharopolyspora, Enterobacter, and Pantoea. However, only two yeast species (Pichia kudriavzevii and Saccharomycopsis fibuligera) and seven mold species including Aspergillus oryzae, Aspergillus niger, Aspergillus candidus, Rhizopus microspores, Eurotium herbariorum, Absidia corymbifera, and Eupenicillium javanicum were detected in the DaQu. The population level of fungi was below 5 log CFU/g in DaQu. The chemical and physical properties of vinegar and sediments were also determined. On the basis of a combined microbial diversity-chemical analysis, we demonstrated that turbidity spoilage of vinegar was a result of cooperation among the low population level and abundance of fungal species in DaQu, the suitable climate conditions, and the contaminants in vinegar. This is the first report to analyze the relation between the microbial diversity of DaQu and turbidity spoilage of vinegar.

Bio-Augmentation of Cupriavidus sp. CY-1 into 2,4-D Contaminated Soil: Microbial Community Analysis by Culture Dependent and Independent Techniques

PubMed Central

Chang, Young-Cheol; Reddy, M. Venkateswar; Umemoto, Honoka; Sato, Yuki; Kang, Mi-Hye; Yajima, Yuka; Kikuchi, Shintaro

2015-01-01

In the present study, a 2,4-dichlorophenoxyacetic acid (2,4-D) degrading bacterial strain CY-1 was isolated from the forest soil. Based on physiological, biochemical and 16S rRNA gene sequence analysis it was identified as Cupriavidus sp. CY-1. Further 2,4-D degradation experiments at different concentrations (200 to 800 mg l-1) were carried out using CY-1. Effect of NaCl and KNO3 on 2,4-D degradation was also evaluated. Degradation of 2,4-D and the metabolites produced during degradation process were analyzed using high pressure liquid chromatography (HPLC) and GC-MS respectively. The amount of chloride ions produced during the 2,4-D degradation were analyzed by Ion chromatography (IC) and it is stoichiometric with 2,4-D dechlorination. Furthermore two different types of soils collected from two different sources were used for 2,4-D degradation studies. The isolated strain CY-1 was bio-augmented into 2,4-D contaminated soils to analyze its degradation ability. Culture independent methods like denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP), and culture dependent methods like colony forming units (CFU) and most probable number (MPN) were used to analyze the survivability of strain CY-1 in contaminated soil. Results of T-RFLP were coincident with the DGGE analysis. From the DGGE, T-RFLP, MPN and HPLC results it was concluded that strain CY-1 effectively degraded 2,4-D without disturbing the ecosystem of soil indigenous microorganisms. PMID:26710231

Community structure of ammonia-oxidizing bacteria under long-term application of mineral fertilizer and organic manure in a sandy loam soil.

PubMed

Chu, Haiyan; Fujii, Takeshi; Morimoto, Sho; Lin, Xiangui; Yagi, Kazuyuki; Hu, Junli; Zhang, Jiabao

2007-01-01

The effects of mineral fertilizer (NPK) and organic manure on the community structure of soil ammonia-oxidizing bacteria (AOB) was investigated in a long-term (16-year) fertilizer experiment. The experiment included seven treatments: organic manure, half organic manure N plus half fertilizer N, fertilizer NPK, fertilizer NP, fertilizer NK, fertilizer PK, and the control (without fertilization). N fertilization greatly increased soil nitrification potential, and mineral N fertilizer had a greater impact than organic manure, while N deficiency treatment (PK) had no significant effect. AOB community structure was analyzed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of the amoA gene, which encodes the alpha subunit of ammonia monooxygenase. DGGE profiles showed that the AOB community was more diverse in N-fertilized treatments than in the PK-fertilized treatment or the control, while one dominant band observed in the control could not be detected in any of the fertilized treatments. Phylogenetic analysis showed that the DGGE bands derived from N-fertilized treatments belonged to Nitrosospira cluster 3, indicating that N fertilization resulted in the dominance of Nitrosospira cluster 3 in soil. These results demonstrate that long-term application of N fertilizers could result in increased soil nitrification potential and the AOB community shifts in soil. Our results also showed the different effects of mineral fertilizer N versus organic manure N; the effects of P and K on the soil AOB community; and the importance of balanced fertilization with N, P, and K in promoting nitrification functions in arable soils.

Community Structure of Ammonia-Oxidizing Bacteria under Long-Term Application of Mineral Fertilizer and Organic Manure in a Sandy Loam Soil▿

PubMed Central

Chu, Haiyan; Fujii, Takeshi; Morimoto, Sho; Lin, Xiangui; Yagi, Kazuyuki; Hu, Junli; Zhang, Jiabao

2007-01-01

The effects of mineral fertilizer (NPK) and organic manure on the community structure of soil ammonia-oxidizing bacteria (AOB) was investigated in a long-term (16-year) fertilizer experiment. The experiment included seven treatments: organic manure, half organic manure N plus half fertilizer N, fertilizer NPK, fertilizer NP, fertilizer NK, fertilizer PK, and the control (without fertilization). N fertilization greatly increased soil nitrification potential, and mineral N fertilizer had a greater impact than organic manure, while N deficiency treatment (PK) had no significant effect. AOB community structure was analyzed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of the amoA gene, which encodes the α subunit of ammonia monooxygenase. DGGE profiles showed that the AOB community was more diverse in N-fertilized treatments than in the PK-fertilized treatment or the control, while one dominant band observed in the control could not be detected in any of the fertilized treatments. Phylogenetic analysis showed that the DGGE bands derived from N-fertilized treatments belonged to Nitrosospira cluster 3, indicating that N fertilization resulted in the dominance of Nitrosospira cluster 3 in soil. These results demonstrate that long-term application of N fertilizers could result in increased soil nitrification potential and the AOB community shifts in soil. Our results also showed the different effects of mineral fertilizer N versus organic manure N; the effects of P and K on the soil AOB community; and the importance of balanced fertilization with N, P, and K in promoting nitrification functions in arable soils. PMID:17098920

Performance of a pilot-scale submerged membrane bioreactor (MBR) in treating bathing wastewater.

PubMed

Xia, Siqing; Guo, Jifeng; Wang, Rongchang

2008-10-01

Bathing wastewater was treated by a pilot-scale submerged membrane bioreactor (MBR) for more than 60 days. The results showed that the removal rates of main pollutants of wastewater such as COD(Cr), LAS, NH(4)(+)-N and total nitrogen (TN) were above 93%, 99%, 99%, and 90%, respectively. The results of denaturing gel gradient electrophoresis (DGGE) and fluorescent in situ hybridization (FISH) indicated that the bacteria were stable. The abundant nitrobacteria intercepted by the membrane led to the high removal rate of ammonia and TN. FISH and 16S rDNA gene sequence analysis revealed that some specific phylogenetic group of bacteria, the Pseudomonas sp. Ochrobactrum anthropi sp. and Enterobacter sp. probably played a major role in the development of the mature biofilms, which led to the severe irreversible membrane biofouling.

Synergistic in vitro and in vivo antimicrobial effect of a mixture of ZnO nanoparticles and Lactobacillus fermentation liquor.

PubMed

Kuang, Huijuan; Yang, Lin; Shah, Nagendra P; Aguilar, Zoraida P; Wang, Lijun; Xu, Hengyi; Wei, Hua

2016-04-01

In this study, we investigated the antibacterial activity of ZnO nanoparticles (NPs) and Lactobacillus-fermentation liquor (LFL) against two pathogenic bacteria in vitro and in vivo. Bactericidal tests were performed on solid agar plates and quantitative real-time PCR (qPCR), and denaturing gradient gel electrophoresis (DGGE) techniques were used to examine the antibacterial activity of the mixture of ZnO NPs and LFL in vivo. The results showed that the mixture exhibited higher antibacterial activity against Salmonella typhimurium in vitro in comparison with ZnO NPs alone. The results showed that ZnO NPs and LFL significantly enhanced microbial diversity in mouse intestine which suggested a synergistic antibacterial activity against the tested pathogenic bacteria that could be used for the control of the spread and persistence of bacterial infections.

[Observation of genetic diversity in dental plaque of elder people with root caries].

PubMed

Ma, Shan-fen; Liang, Jing-ping; Jiang, Yun-tao; Zhu, Cai-lian

2011-08-01

Bacterial community in dental plaque of elder people was analyzed to learn about the microhabitat composition and diversity. Dental plaque samples were collected from 25 elders. PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) was used to evaluate the microbial diversity by displaying PCR-generated 16SrDNA fragments that migrate at different distances, reflecting the different sequence of fragment. SPSS12.0 software was used to analyze the variance of genotypes between different groups of bacteria. Genotypes of bacteria in dental plaques in the root caries group was significantly more than the other two groups. Crown caries group and caries-free group had no significant difference. The genetic diversity of the dental plaque microflora in the root caries group is significantly higher than coronal caries group and caries-free group.

Spatial and seasonal prokaryotic community dynamics in ponds of increasing salinity of Sfax solar saltern in Tunisia.

PubMed

Boujelben, Ines; Gomariz, María; Martínez-García, Manuel; Santos, Fernando; Peña, Arantxa; López, Cristina; Antón, Josefa; Maalej, Sami

2012-05-01

The spatial and seasonal dynamics of the halophilic prokaryotic community was investigated in five ponds from Sfax solar saltern (Tunisia), covering a salinity gradient ranging from 20 to 36%. Fluorescence in situ hybridization indicated that, above 24% salinity, the prokaryotic community shifted from bacterial to archaeal dominance with a remarkable increase in the proportion of detected cells. Denaturing gradient gel electrophoresis (DGGE) profiles were rather similar in all the samples analyzed, except in the lowest salinity pond (around 20% salt) where several specific archaeal and bacterial phylotypes were detected. In spite of previous studies on these salterns, DGGE analysis unveiled the presence of microorganisms not previously described in these ponds, such as Archaea related to Natronomonas or bacteria related to Alkalimnicola, as well as many new sequences of Bacteroidetes. Some phylotypes, such as those related to Haloquadratum or to some Bacteroidetes, displayed a strong dependence of salinity and/or magnesium concentrations, which in the case of Haloquadratum could be related to the presence of ecotypes. Seasonal variability in the prokaryotic community composition was focused on two ponds with the lowest (20%) and the highest salinity (36%). In contrast to the crystallized pond, where comparable profiles between autumn 2007 and summer 2008 were obtained, the non-crystallized pond showed pronounced seasonal changes and a sharp succession of "species" during the year. Canonical correspondence analysis of biological and physicochemical parameters indicated that temperature was a strong factor structuring the prokaryotic community in the non-crystallizer pond, that had salinities ranging from 20 to 23.8% during the year.

Exploring Microbial Iron Oxidation in Wetland Soils

NASA Astrophysics Data System (ADS)

Wang, J.; Muyzer, G.; Bodelier, P. L. E.; den Oudsten, F.; Laanbroek, H. J.

2009-04-01

Iron is one of the most abundant elements on earth and is essential for life. Because of its importance, iron cycling and its interaction with other chemical and microbial processes has been the focus of many studies. Iron-oxidizing bacteria (FeOB) have been detected in a wide variety of environments. Among those is the rhizosphere of wetland plants roots which release oxygen into the soil creating suboxic conditions required by these organisms. It has been reported that in these rhizosphere microbial iron oxidation proceeds up to four orders of magnitude faster than strictly abiotic oxidation. On the roots of these wetland plants iron plaques are formed by microbial iron oxidation which are involved in the sequestering of heavy metals as well organic pollutants, which of great environmental significance.Despite their important role being catalysts of iron-cycling in wetland environments, little is known about the diversity and distribution of iron-oxidizing bacteria in various environments. This study aimed at developing a PCR-DGGE assay enabling the detection of iron oxidizers in wetland habitats. Gradient tubes were used to enrich iron-oxidizing bacteria. From these enrichments, a clone library was established based on the almost complete 16s rRNA gene using the universal bacterial primers 27f and 1492r. This clone library consisted of mainly α- and β-Proteobacteria, among which two major clusters were closely related to Gallionella spp. Specific probes and primers were developed on the basis of this 16S rRNA gene clone library. The newly designed Gallionella-specific 16S rRNA gene primer set 122f/998r was applied to community DNA obtained from three contrasting wetland environments, and the PCR products were used in denaturing gradient gel electrophoresis (DGGE) analysis. A second 16S rRNA gene clone library was constructed using the PCR products from one of our sampling sites amplified with the newly developed primer set 122f/998r. The cloned 16S rRNA gene sequences all represented novel culturable iron oxidizers most closely related to Gallionella spp. Based on their nucleotide sequences four groups could be identified, which were comparable to the DGGE banding pattern obtained before with the gradient tubes enrichments. The above mentioned nested PCR-DGGE method was used to study the distribution and community composition of Gallionella-like iron-oxidizing bacteria under the influence of plants species, soil depth, as well as season. Soil samples from Appels, Belgium, an intertidal, freshwater marsh known to hold intensive iron cycling, were taken from 5 different vegetation types in April, July and October 2007. Soil cores were sliced at 1-cm intervals and subjected to chemical and molecular analyses. The DGGE patterns showed that the community of iron-oxidizing bacteria differed with vegetation type, and sediment depth. Samples taken in autumn held lower diversity in Gallionella-related iron oxidizers than those sampled in spring and summer.

Radiation-induced impacts on the degradation of 2,4-D and the microbial population in soil microcosms.

PubMed

Niedrée, Bastian; Vereecken, Harry; Burauel, Peter

2013-01-01

In a soil microcosm experiment, the influence of low-level (137)Cs and (90)Sr contamination on the degradation of (14)C-ring-labeled 2,4-dichlorophenoxyacetic acid (2,4-D) was studied. Two differently treated soils (one native soil and one soil sterilized and reinoculated with a biotic soil aliquot) were artificially contaminated with various concentrations of (137)Cs and (90)Sr as nitrate salts. The cumulative doses increased up to 4 Gy for 30 days of incubation in soil microcosms. Changes in microbial community structure were observed with help of the denaturing gradient gel electrophoresis (DGGE). A radiation-induced impact appeared only in the microcosms treated with 30 times the maximum contamination appearing in the exclusion zone around reactor 4 in Chernobyl. In contrast to the less contaminated soils, the mineralization of 2,4-D was delayed for 4 days before it recovered. Slight shifts in the microbial communities could be traced to radiation effects. However, other parameters had a major impact on mineralization and community structure. Thus the sterilization and reinoculation and, of course, application of the 2,4-D were predominantly reflected in the (14)CO(2) emissions and the DGGE gel patterns. Copyright © 2012 Elsevier Ltd. All rights reserved.

Monitoring the bacterial community dynamics in a petroleum refinery wastewater membrane bioreactor fed with a high phenolic load.

PubMed

Silva, Cynthia C; Viero, Aline F; Dias, Ana Carolina F; Andreote, Fernando D; Jesus, Ederson C; De Paula, Sergio O; Torres, Ana Paula R; Santiago, Vania M J; Oliveira, Valeria M

2010-01-01

The phenolic compounds are a major contaminant class often found in industrial wastewaters and the biological treatment is an alternative tool commonly employed for their removal. In this sense, monitoring microbial community dynamics is crucial for a successful wastewater treatment. This work aimed to monitor the structure and activity of the bacterial community during the operation of a laboratory-scale continuous submerged membrane bioreactor (SMBR), using PCR and RT-PCR followed by Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA libraries. Multivariate analyses carried out using DGGE profiles showed significant changes in the total and metabolically active dominant community members during the 4-week treatment period, explained mainly by phenol and ammonium input. Gene libraries were assembled using 16S rDNA and 16S rRNA PCR products from the fourth week of treatment. Sequencing and phylogenetic analyses of clones from 16S rDNA library revealed a high diversity of taxa for the total bacterial community, with predominance of Thauera genus (ca. 50%). On the other hand, a lower diversity was found for metabolically active bacteria, which were mostly represented by members of Betaproteobacteria (Thauera and Comamonas), suggesting that these groups have a relevant role in the phenol degradation during the final phase of the SMBR operation.

Dominant genera of cyanobacteria in Lake Taihu and their relationships with environmental factors.

PubMed

Feng, Lijun; Liu, Shiyou; Wu, Wenxian; Ma, Jiawen; Li, Pei; Xu, Hailing; Li, Na; Feng, Yaoyu

2016-07-01

Cyanobacterial blooms in freshwaters have become one of the most widespread of environmental problems and threaten water resources worldwide. Previous studies on cyanobacteria in Lake Taihu often collected samples from one site (like Meiliang Bay or Zhushan Bay) and focused on the variation in patterns or abundance of Microcystis during the blooming season. However, the distribution of cyanobacteria in Lake Taihu shows differing pattern in various seasons. In this study, water samples were collected monthly for one year at five sites in Lake Taihu with different trophic status and a physicochemical analysis and denaturing gradient gel electrophoresis (DGGE) were conducted. DGGE fingerprint analysis showed that Microcystis (7/35 bands) and Synechococcus (12/35 bands) were the two most dominant genera present during the study period at all five sites. Cyanobium (3/35 bands) was the third most common genus which has seldom been previously reported in Lake Taihu. Redundancy analysis (RDA) indicated that the cyanobacterial community structure was significantly correlated with NO3 (-)-N, CODMn, and NH4 (+)-N in the winter and spring, whereas it was correlated with water temperature in the summer and autumn. Limiting the nutrient input (especially of N and C loading) in Lake Taihu would be a key factor in controlling the growth of different genera of cyanobacteria.

Occurrence and activity of Archaea in aerated activated sludge wastewater treatment plants.

PubMed

Gray, Neil D; Miskin, Ian P; Kornilova, Oksana; Curtis, Thomas P; Head, Ian M

2002-03-01

The occurrence, distribution and activity of archaeal populations within two aerated, activated sludge wastewater treatment systems, one treating domestic waste and the second treating mixed domestic and industrial wastewater, were investigated by denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction (PCR)-amplified ribosomal RNA gene fragments and process measurements. In the plant receiving mixed industrial and domestic waste the archaeal populations found in the mixed liquor were very similar to those in the influent sewage, though a small number of DGGE bands specific to the mixed liquor were identified. In contrast, the activated sludge treating principally domestic waste harboured distinct archaeal populations associated with the mixed liquor that were not prevalent in the influent sewage. We deduce that the Archaea in the plant treating mixed wastewater were derived principally from the influent, whereas those in the plant treating solely domestic waste were actively growing in the treatment plant. Archaeal 16S rRNA gene sequences related to the Methanosarcinales, Methanomicrobiales and the Methanobacteriales were detected. Methanogenesis was measured in activated sludge samples incubated under oxic and anoxic conditions, demonstrating that the methanogens present in both activated sludge plants were active only in anoxic incubations. The relatively low rates of methanogenesis measured indicated that, although active, the methanogens play a minor role in carbon turnover in activated sludge.

[Diversity of oil-degrading bacteria isolated form the Indian Ocean sea surface].

PubMed

Wu, Changliang; Wang, Xin; Shao, Zongze

2010-09-01

In order to investigate the diversity of oil-degrading bacteria in the surface seawater across the India Ocean, and to obtain new oil-degrading bacteria. Potential oil-degrading bacteria were selected out via 1:1 mixture of diesel and crude oil as sole carbon source. Meanwhile, the community structure of 13 enrichments was analyzed by polymerase chain reaction with denaturing gradient gel electrophoresis (PCR-DGGE). We obtained 51 unique strains of 29 genera after screening via morphological, physiological, biochemical and 16S rRNA analyses. They mainly belonged to a and gamma-Proteobacteria. The four genera Alcanivorax (accounting for 18%), Novosphingobium (10%), Marinobacter (6%) and Thalassospira (6%) were the most predominant bacteria. Ecological analyses showed that the bacteria had high diversity with Shannon-Winner index (H) of 4.57968, and distributed even with Evenness index (E) as 0.8664771. Then Further experiments revealed oil-degrading capability of 49 strains. In addition, our investigation revealed oil-degrading ability of genera Sinomonas, Knoellia and Mesoflavibacter for the first time. DGGE fingerprint patterns indicated that the genus Alcanivorax was an important oil-degrading bacteria in the surface seawater across the India Ocean. This study demonstrated a high diversity of the oil-degradation bacteria in the surface seawater of Indian Ocean, these bacteria are of potential in bioremediation of marine oil pollution.

Biodiversity of Bacterial Ecosystems in Traditional Egyptian Domiati Cheese▿

PubMed Central

El-Baradei, Gaber; Delacroix-Buchet, Agnès; Ogier, Jean-Claude

2007-01-01

Bacterial biodiversity occurring in traditional Egyptian soft Domiati cheese was studied by PCR-temporal temperature gel electrophoresis (TTGE) and PCR-denaturing gradient gel electrophoresis (DGGE). Bands were identified using a reference species database (J.-C. Ogier et al., Appl. Environ. Microbiol. 70:5628-5643, 2004); de novo bands having nonidentified migration patterns were identified by DNA sequencing. Results reveal a novel bacterial profile and extensive bacterial biodiversity in Domiati cheeses, as reflected by the numerous bands present in TTGE and DGGE patterns. The dominant lactic acid bacteria (LAB) identified were as follows: Leuconostoc mesenteroides, Lactococcus garvieae, Aerococcus viridans, Lactobacillus versmoldensis, Pediococcus inopinatus, and Lactococcus lactis. Frequent non-LAB species included numerous coagulase-negative staphylococci, Vibrio spp., Kocuria rhizophila, Kocuria kristinae, Kocuria halotolerans, Arthrobacter spp./Brachybacterium tyrofermentans. This is the first time that the majority of these species has been identified in Domiati cheese. Nearly all the dominant and frequent bacterial species are salt tolerant, and several correspond to known marine bacteria. As Domiati cheese contains 5.4 to 9.5% NaCl, we suggest that these bacteria are likely to have an important role in the ripening process. This first systematic study of the microbial composition of Domiati cheeses reveals great biodiversity and evokes a role for marine bacteria in determining cheese type. PMID:17189434

Biodiversity of bacterial ecosystems in traditional Egyptian Domiati cheese.

PubMed

El-Baradei, Gaber; Delacroix-Buchet, Agnès; Ogier, Jean-Claude

2007-02-01

Bacterial biodiversity occurring in traditional Egyptian soft Domiati cheese was studied by PCR-temporal temperature gel electrophoresis (TTGE) and PCR-denaturing gradient gel electrophoresis (DGGE). Bands were identified using a reference species database (J.-C. Ogier et al., Appl. Environ. Microbiol. 70:5628-5643, 2004); de novo bands having nonidentified migration patterns were identified by DNA sequencing. Results reveal a novel bacterial profile and extensive bacterial biodiversity in Domiati cheeses, as reflected by the numerous bands present in TTGE and DGGE patterns. The dominant lactic acid bacteria (LAB) identified were as follows: Leuconostoc mesenteroides, Lactococcus garvieae, Aerococcus viridans, Lactobacillus versmoldensis, Pediococcus inopinatus, and Lactococcus lactis. Frequent non-LAB species included numerous coagulase-negative staphylococci, Vibrio spp., Kocuria rhizophila, Kocuria kristinae, Kocuria halotolerans, Arthrobacter spp./Brachybacterium tyrofermentans. This is the first time that the majority of these species has been identified in Domiati cheese. Nearly all the dominant and frequent bacterial species are salt tolerant, and several correspond to known marine bacteria. As Domiati cheese contains 5.4 to 9.5% NaCl, we suggest that these bacteria are likely to have an important role in the ripening process. This first systematic study of the microbial composition of Domiati cheeses reveals great biodiversity and evokes a role for marine bacteria in determining cheese type.

Comparison of bacterial community structure and dynamics during the thermophilic composting of different types of solid wastes: anaerobic digestion residue, pig manure and chicken manure

PubMed Central

Song, Caihong; Li, Mingxiao; Jia, Xuan; Wei, Zimin; Zhao, Yue; Xi, Beidou; Zhu, Chaowei; Liu, Dongming

2014-01-01

This study investigated the impact of composting substrate types on the bacterial community structure and dynamics during composting processes. To this end, pig manure (PM), chicken manure (CM), a mixture of PM and CM (PM + CM), and a mixture of PM, CM and anaerobic digestion residue (ADR) (PM + CM + ADR) were selected for thermophilic composting. The bacterial community structure and dynamics during the composting process were detected and analysed by polymerase chain reaction–denaturing gradient gel electrophoresis (DGGE) coupled with a statistic analysis. The physical-chemical analyses indicated that compared to single-material composting (PM, CM), co-composting (PM + CM, PM + CM + ADR) could promote the degradation of organic matter and strengthen the ability of conserving nitrogen. A DGGE profile and statistical analysis demonstrated that co-composting, especially PM + CM + ADR, could improve the bacterial community structure and functional diversity, even in the thermophilic stage. Therefore, co-composting could weaken the screening effect of high temperature on bacterial communities. Dominant sequencing analyses indicated a dramatic shift in the dominant bacterial communities from single-material composting to co-composting. Notably, compared with PM, PM + CM increased the quantity of xylan-degrading bacteria and reduced the quantity of human pathogens. PMID:24963997

Effects of poly-β-hydroxybutyrate (PHB) on Siberian sturgeon (Acipenser baerii) fingerlings performance and its gastrointestinal tract microbial community.

PubMed

Najdegerami, Ebrahim H; Tran, Tiet Ngoc; Defoirdt, Tom; Marzorati, Massimo; Sorgeloos, Patrick; Boon, Nico; Bossier, Peter

2012-01-01

Poly-β-hydroxybutyrate (PHB) is a natural polymer that can be depolymerized into water-soluble short-chain fatty acid monomers. These monomers can act as microbial control agents. In this study, the effects of partially replacing the diet of Siberian sturgeon fingerlings with 2% and 5% PHB were investigated. Replacing 2% of the diet with PHB improved weight gain, specific growth rate (SGR) and survival in the sturgeon fingerlings during the 10-week experimental period. Community-level physiological profiling and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) were used to analyze the microbial community diversity and community organization in the sturgeon gastrointestinal tract. DGGE analysis revealed that PHB affected the intestinal microbial species richness and diversity. The highest species richness was observed with 2% PHB. DNA sequencing of the dominant bands in 2% and 5% PHB treatments revealed that PHB stimulated bacteria belonging to the genera Bacillus and Ruminococcaceae. Principal component analysis, Lorenz curves and the Shannon index of Biolog Ecoplate data revealed that aerobic metabolic potential of the bacterial community was different in the PHB-treated fishes as compared with the control situation. Overall, our results indicate that PHB act as microbial control agents and replacement of 2% of Siberian sturgeon fingerling diet with PHB has beneficial effects.

Culture-independent analysis of lactic acid bacteria diversity associated with mezcal fermentation.

PubMed

Narváez-Zapata, J A; Rojas-Herrera, R A; Rodríguez-Luna, I C; Larralde-Corona, C P

2010-11-01

Mezcal is an alcoholic beverage obtained from the distillation of fermented juices of cooked Agave spp. plant stalks (agave must), and each region in Mexico with denomination of origin uses defined Agave species to prepare mezcal with unique organoleptic characteristics. During fermentation to produce mezcal in the state of Tamaulipas, not only alcohol-producing yeasts are involved, but also a lactic acid bacterial community that has not been characterized yet. In order to address this lack of knowledge on this traditional Mexican beverage, we performed a DGGE-16S rRNA analysis of the lactic acid bacterial diversity and metabolite accumulation during the fermentation of a typical agave must that is rustically produced in San Carlos County (Tamaulipas, Mexico). The analysis of metabolite production indicated a short but important malolactic fermentation stage not previously described for mezcal. The denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA genes showed a distinctive lactic acid bacterial community composed mainly of Pediococcus parvulus, Lactobacillus brevis, Lactobacillus composti, Lactobacillus parabuchneri, and Lactobacillus plantarum. Some atypical genera such as Weissella and Bacillus were also found in the residual must. Our results suggest that the lactic acid bacteria could strongly be implicated in the organoleptic attributes of this traditional Mexican distilled beverage.

Microbial communities and chemical changes during fermentation of sugary Brazilian kefir.

PubMed

Magalhães, Karina Teixeira; de M Pereira, G V; Dias, Disney Ribeiro; Schwan, Rosane Freitas

2010-07-01

The microorganisms associated with sugary Brazilian kefir beverage were investigated using a combination of culture-dependent and -independent methods. A total of 289 bacteria and 129 yeasts were identified via phenotypic and genotypic methods. Lb. paracasei (23.8%) was the major bacterial isolate identified, followed by Acetobacter lovaniensis (16.31%), Lactobacillus parabuchneri (11.71%), Lactobacillus kefir (10.03%) and Lactococcus lactis (10.03%). Saccharomyces cerevisiae (54.26%) and Kluyveromyces lactis (20.15%) were the most common yeast species isolated. Scanning electron microscopy showed that the microbiota was dominated by lemon-shaped yeast cells growing in close association with Lactobacillus (long and curved). Some lactic acid bacteria detected by sequence analysis of DGGE (denaturing gradient gel electrophoresis) bands were not recovered at any time through fermentation by plating. Conversely, DGGE fingerprints did not reveal bands corresponding to some of the species isolated by culturing methods. The bacteria Acetobacter lovaniensis and the yeast Kazachstania aerobia are described for the first time in sugary kefir. During the 24 h of fermentation, the concentration of lactic acid ranged from 0.2 to 1.80 mg/ml, and that of acetic acid increased from 0.08 to 1.12 mg/ml. The production of ethanol was limited, reaching a final mean value of 1.24 mg/ml.

Rice root-associated bacteria – insights in community structures across ten cultivars

PubMed Central

Hardoim, Pablo Rodrigo; Andreote, Fernando Dini; Reinhold-Hurek, Barbara; Sessitsch, Angela; van Overbeek, Leonard Simon; van Elsas, Jan Dirk

2015-01-01

In this study, the effect of plant genotype, soil type and nutrient use efficiency on the composition of different bacterial communities associated with rice roots were investigated. Thus, total bacteria, Alpha- and Beta-proteobacteria, Pseudomonas and Actinobacteria were studied using PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE). Rice genotype determined to a large extent the composition of the different bacterial communities across cultivars. Several cultivars belonging to Oryza sativa subspecies indica tended to select similar bacterial communities, whereas those belonging to subspecies japonica and aromatica selected ones with divergent community structures. An effect of soil type was pronounced for the Actinobacteria communities, while a small effect of ‘improved’ and ‘traditional’ plants was noted for all communities analysed. A few dominant bands in PCR-DGGE, affiliated with Rhizobium radiobacter, Dickeya zeae, Mycobacterium bolletii and with members of the Rhizobiales, Rhodospirillaceae and Paenibacillaceae were spread across cultivars. In contrast, a majority of bands (e.g. affiliated with Enterobacter cloacae or Burkholderia kururiensis) was only present in particular cultivars or was erratically distributed amongst rice replicates. The data suggested that both bacterial adaptation and plant genotype contribute to the shaping of the dynamic bacterial communities associated with roots of rice plants. PMID:21426364

Inoculation of starter cultures in a semi-dry coffee (Coffea arabica) fermentation process.

PubMed

Evangelista, Suzana Reis; Miguel, Maria Gabriela da Cruz Pedrozo; Cordeiro, Cecília de Souza; Silva, Cristina Ferreira; Pinheiro, Ana Carla Marques; Schwan, Rosane Freitas

2014-12-01

The aim of this study was to evaluate the use of yeasts as starter cultures in coffee semi-dry processing. Arabica coffee was inoculated with one of the following starter cultures: Saccharomyces cerevisiae UFLA YCN727, S. cerevisiae UFLA YCN724, Candida parapsilosis UFLA YCN448 and Pichia guilliermondii UFLA YCN731. The control was not inoculated with a starter culture. Denaturing gradient gel electrophoresis (DGGE) was used to assess the microbial population, and organic acids and volatile compounds were quantified by HPLC and HS-SPME/GC, respectively. Sensory analyses were evaluated using the Temporal Dominance of Sensations (TDS). DGGE analysis showed that the inoculated yeasts were present throughout the fermentation. Other yeast species were also detected, including Debaryomyces hansenii, Cystofilobasidium ferigula and Trichosporon cavernicola. The bacterial population was diverse and was composed of the following genera: Weissella, Leuconostoc, Gluconobacter, Pseudomonas, Pantoea, Erwinia and Klebsiella. Butyric and propionic acids, were not detected in any treatment A total of 47 different volatiles compounds have been identified. The coffee inoculated with yeast had a caramel flavor that was not detected in the control, as assessed by TDS. The use of starter cultures during coffee fermentation is an interesting alternative for obtaining a beverage quality with distinctive flavor. Copyright © 2014 Elsevier Ltd. All rights reserved.

Microbial shifts of the silkworm larval gut in response to lettuce leaf feeding.

PubMed

Liang, Xue; Fu, Yuming; Tong, Ling; Liu, Hong

2014-04-01

Silkworm (Bombyx mori L.) larvae were used as an ideal animal protein source for astronauts in the bioregenerative life support system (BLSS). Here, we compared the difference in bacterial communities of the silkworm larval gut between the BLSS rearing way (BRW) and the traditional rearing way (TRW) through culture-dependent approach, 16S rRNA gene analysis, and denaturing gradient gel electrophoresis (DGGE). The culture-dependent approach revealed that the numbers of gut bacteria of silkworm in the BRW significantly decreased compared with that of the TRW. The analysis of clone libraries showed that the gut microbiota in the BRW was significantly less diverse than that in the TRW. Acinetobacter and Bacteroides were dominant populations in the BRW, and Bacillus and Arcobacter dominated in the TRW. DGGE profiles confirmed the difference of silkworm gut bacterial community between two rearing ways. These results demonstrate that gut bacteria change from the BRW contributes to the decrease of silkworm physiological activity. This study increases our understanding of the change of silkworm gut microbiota in response to lettuce leaf feeding in the BRW. We could use the dominant populations to make probiotic products for nutrient absorption and disease prevention in the BLSS to improve gut microecology, as well as the yield and quality of animal protein.

Occurrence of fungi in combs of fungus-growing termites (Isoptera: Termitidae, Macrotermitinae).

PubMed

Guedegbe, Herbert J; Miambi, Edouard; Pando, Anne; Roman, Jocelyne; Houngnandan, Pascal; Rouland-Lefevre, Corinne

2009-10-01

Fungus-growing termites cultivate their mutualistic basidiomycete Termitomyces species on a substrate called a fungal comb. Here, the Suicide Polymerase Endonuclease Restriction (SuPER) method was adapted for the first time to a fungal study to determine the entire fungal community of fungal combs and to test whether fungi other than the symbiotic cultivar interact with termite hosts. Our molecular analyses show that although active combs are dominated by Termitomyces fungi isolated with direct Polymerase Endonuclease Restriction - Denaturing Gradient Gel Electrophoresis (PCR-DGGE), they can also harbor some filamentous fungi and yeasts only revealed by SuPER PCR-DGGE. This is the first molecular evidence of the presence of non-Termitomyces species in active combs. However, because there is no evidence for a species-specific relationship between these fungi and termites, they are mere transient guests with no specialization in the symbiosis. It is however surprising to notice that termite-associated Xylaria strains were not isolated from active combs even though they are frequently retrieved when nests are abandoned by termites. This finding highlights the implication of fungus-growing termites in the regulation of fungi occurring within the combs and also suggests that they might not have any particular evolutionary-based association with Xylaria species.

Gut content microbiota of introduced bigheaded carps (Hypophthalmichthys spp.) inhabiting the largest shallow lake in Central Europe.

PubMed

Borsodi, Andrea K; Szabó, Attila; Krett, Gergely; Felföldi, Tamás; Specziár, András; Boros, Gergely

2017-01-01

Studying the microbiota in the alimentary tract of bigheaded carps (Hypophthalmichthys spp.) gained special interest recently, as these types of investigations on non-native fish species may lead to a better understanding of their ecological role and feeding habits in an invaded habitat. For microbiological examinations, bigheaded carp gut contents and water column samples from Lake Balaton (Hungary) were collected from spring to autumn in 2013. Denaturing Gradient Gel Electrophoresis (DGGE) and pyrosequencing of the 16S rRNA gene were performed to reveal the composition. According to the DGGE patterns, bacterial communities of water samples separated clearly from that of the intestines. Moreover, the bacterial communities in the foreguts and hindguts were also strikingly dissimilar. Based on pyrosequencing, both foregut and hindgut samples were predominated by the fermentative genus Cetobacterium (Fusobacteria). The presence of some phytoplankton taxa and the high relative abundance of cellulose-degrading bacteria in the guts suggest that intestinal microbes may have an important role in digesting algae and making them utilizable for bigheaded carps that lack cellulase enzyme. In turn, the complete absence of typical heterotrophic freshwater bacteria in all studied sections of the intestines indicated that bacterioplankton probably has a negligible role in the nutrition of bigheaded carps. Copyright © 2016 Elsevier GmbH. All rights reserved.

Seasonal changes in bacterial communities associated with healthy and diseased Porites coral in southern Taiwan.

PubMed

Lin, Chorng-Horng; Chuang, Chih-Hsiang; Twan, Wen-Hung; Chiou, Shu-Fen; Wong, Tit-Yee; Liu, Jong-Kang; Kao, Chyuan-Yao; Kuo, Jimmy

2016-12-01

We compared the bacterial communities associated with healthy scleractinian coral Porites sp. with those associated with coral infected with pink spot syndrome harvested during summer and winter from waters off the coast of southern Taiwan. Members of the bacterial community associated with the coral were characterized by means of denaturing gradient gel electrophoresis (DGGE) of a short region of the 16S rRNA gene and clone library analysis. Of 5 different areas of the 16S rRNA gene, we demonstrated that the V3 hypervariable region is most suited to represent the coral-associated bacterial community. The DNA sequences of 26 distinct bands extracted from DGGE gels and 269 sequences of the 16S rRNA gene from clone libraries were determined. We found that the communities present in diseased coral were more heterogeneous than the bacterial communities of uninfected coral. In addition, bacterial communities associated with coral harvested in the summer were more diverse than those associated with coral collected in winter, regardless of the health status of the coral. Our study suggested that the compositions of coral-associated bacteria communities are complex, and the population of bacteria varies greatly between seasons and in coral of differing health status.

Effect of Activated Carbon Amendment on Bacterial Community Structure and Functions in a PAH Impacted Urban Soil

PubMed Central

2012-01-01

We collected urban soil samples impacted by polycyclic aromatic hydrocarbons (PAHs) from a sorbent-based remediation field trial to address concerns about unwanted side-effects of 2% powdered (PAC) or granular (GAC) activated carbon amendment on soil microbiology and pollutant biodegradation. After three years, total microbial cell counts and respiration rates were highest in the GAC amended soil. The predominant bacterial community structure derived from denaturing gradient gel electrophoresis (DGGE) shifted more strongly with time than in response to AC amendment. DGGE band sequencing revealed the presence of taxa with closest affiliations either to known PAH degraders, e.g. Rhodococcus jostii RHA-1, or taxa known to harbor PAH degraders, e.g. Rhodococcus erythropolis, in all soils. Quantification by real-time polymerase chain reaction yielded similar dioxygenases gene copy numbers in unamended, PAC-, or GAC-amended soil. PAH availability assessments in batch tests showed the greatest difference of 75% with and without biocide addition for unamended soil, while the lowest PAH availability overall was measured in PAC-amended, live soil. We conclude that AC had no detrimental effects on soil microbiology, AC-amended soils retained the potential to biodegrade PAHs, but the removal of available pollutants by biodegradation was most notable in unamended soil. PMID:22455603

Nutrients removal in hybrid fluidised bed bioreactors operated with aeration cycles.

PubMed

Martin, Martin; Enríquez, L López; Fernández-Polanco, M; Villaverde, S; Garcia-Encina, P A

2007-01-01

Abstract Two hybrid fluidised bed reactors filled with sepiolite and granular activated carbon (GAC) were operated with short cycled aeration for removing organic matter, total nitrogen and phosphorous, respectively. Both reactors were continuously operated with synthetic and/or industrial wastewater containing 350-500 mg COD/L, 110-130 mg NKT/L, 90-100 mg NH3-N/L and 12-15 mg P/L for 8 months. The reactor filled with sepiolite, treating only synthetic wastewater, removed COD, ammonia, total nitrogen and phosphorous up to 88, 91, 55 and 80% with a hydraulic retention time (HRT) of 10 h, respectively. These efficiencies correspond to removal rates of 0.95 kgCODm(-3)d(-1) and 0.16 kg total N m(-3)d(-1). The reactor filled with GAC was operated for 4 months with synthetic wastewater and 4 months with industrial wastewater, removing 98% of COD, 96% of ammonia, and 66% of total nitrogen, with an HRT of 13.6 h. No significant phosphorous removing activity was observed in this reactor. Microbial communities growing with both reactors were followed using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) techniques. The microbial fingerprints, i.e. DGGE profiles, indicated that biological communities in both reactors were stable along the operational period even when the operating conditions were changed.

[Bacterial diversity in sequencing batch biofilm reactor (SBBR) for landfill leachate treatment using PCR-DGGE].

PubMed

Xiao, Yong; Yang, Zhao-hui; Zeng, Guang-ming; Ma, Yan-he; Liu, You-sheng; Wang, Rong-juan; Xu, Zheng-yong

2007-05-01

For studying the bacterial diversity and the mechanism of denitrification in sequencing bath biofilm reactor (SBBR) treating landfill leachate to provide microbial evidence for technique improvements, total microbial DNA was extracted from samples which were collected from natural landfill leachate and biofilm of a SBBR that could efficiently remove NH4+ -N and COD of high concentration. 16S rDNA fragments were amplified from the total DNA successfully using a pair of universal bacterial 16S rDNA primer, GC341F and 907R, and then were used for denaturing gradient gel electrophoresis (DGGE) analysis. The bands in the gel were analyzed by statistical methods and excided from the gel for sequencing, and the sequences were used for homology analysis and then two phylogenetic trees were constructed using DNAStar software. Results indicated that the bacterial diversity of the biofilm in SBBR and the landfill leachate was abundant, and no obvious change of community structure happened during running in the biofilm, in which most bacteria came from the landfill leachate. There may be three different modes of denitrification in the reactor because several different nitrifying bacteria, denitrifying bacteria and anaerobic ammonia oxidation bacteria coexisted in it. The results provided some valuable references for studying microbiological mechanism of denitrification in SBBR.

Effect of seed sludge on characteristics and microbial community of aerobic granular sludge.

PubMed

Song, Zhiwei; Pan, Yuejun; Zhang, Kun; Ren, Nanqi; Wang, Aijie

2010-01-01

Aerobic granular sludge was cultivated by using different kinds of seed sludge in sequencing batch airlift reactor. The influence of seed sludge on physical and chemical properties of granular sludge was studied; the microbial community structure was probed by using scanning electron microscope and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The results showed that seed sludge played an important role on the formation of aerobic granules. Seed sludge taken from beer wastewater treatment plant (inoculum A) was more suitable for cultivating aerobic granules than that of sludge from municipal wastewater treatment plant (inoculum B). Cultivated with inoculum A, large amount of mature granules formed after 35 days operation, its SVI reached 32.75 mL/g, and SOUR of granular sludge was beyond 1.10 mg/(g x min). By contrast, it needed 56 days obtaining mature granules using inoculum B. DGGE profiles indicated that the dominant microbial species in mature granules were 18 and 11 OTU when inoculum A and B were respectively employed as seed sludge. The sequencing results suggested that dominant species in mature granules cultivated by inoculum A were Paracoccus sp., Devosia hwasunensi, Pseudoxanthomonas sp., while the dominant species were Lactococcus raffinolactis and Pseudomonas sp. in granules developed from inoculum B.

Effect of long-term industrial waste effluent pollution on soil enzyme activities and bacterial community composition.

PubMed

Subrahmanyam, Gangavarapu; Shen, Ju-Pei; Liu, Yu-Rong; Archana, Gattupalli; Zhang, Li-Mei

2016-02-01

Although numerous studies have addressed the influence of exogenous pollutants on microorganisms, the effect of long-term industrial waste effluent (IWE) pollution on the activity and diversity of soil bacteria was still unclear. Three soil samples characterized as uncontaminated (R1), moderately contaminated (R2), and highly contaminated (R3) receiving mixed organic and heavy metal pollutants for more than 20 years through IWE were collected along the Mahi River basin, Gujarat, western India. Basal soil respiration and in situ enzyme activities indicated an apparent deleterious effect of IWE on microbial activity and soil function. Community composition profiling of soil bacteria using 16S rRNA gene amplification and denaturing gradient gel electrophoresis (DGGE) method indicated an apparent bacterial community shift in the IWE-affected soils. Cloning and sequencing of DGGE bands revealed that the dominated bacterial phyla in polluted soil were affiliated with Firmicutes, Acidobacteria, and Actinobacteria, indicating that these bacterial phyla may have a high tolerance to pollutants. We suggested that specific bacterial phyla along with soil enzyme activities could be used as relevant biological indicators for long-term pollution assessment on soil quality. Graphical Abstract Bacterial community profiling and soil enzyme activities in long-term industrial waste effluent polluted soils.

Occurrence and diversity of naphthalene dioxygenase genes in soil microbial communities from the Maritime Antarctic.

PubMed

Flocco, Cecilia G; Gomes, Newton C M; Mac Cormack, Walter; Smalla, Kornelia

2009-03-01

The diversity of naphthalene dioxygenase genes (ndo) in soil environments from the Maritime Antarctic was assessed, dissecting as well the influence of the two vascular plants that grow in the Antarctic: Deschampsia antarctica and Colobanthus quitensis. Total community DNA was extracted from bulk and rhizosphere soil samples from Jubany station and Potter Peninsula, South Shetland Islands. ndo genes were amplified by a nested PCR and analysed by denaturant gradient gel electrophoresis approach (PCR-DGGE) and cloning and sequencing. The ndo-DGGE fingerprints of oil-contaminated soil samples showed even and reproducible patterns, composed of four dominant bands. The presence of vascular plants did not change the relative abundance of ndo genotypes compared with bulk soil. For non-contaminated sites, amplicons were not obtained for all replicates and the variability among the fingerprints was comparatively higher, likely reflecting a lower abundance of ndo genes. The phylogenetic analyses showed that all sequences were affiliated to the nahAc genes closely related to those described for Pseudomonas species and related mobile genetic elements. This study revealed that a microdiversity of nahAc-like genes exists in microbial communities of Antarctic soils and quantitative PCR indicated that their relative abundance was increased in response to anthropogenic sources of pollution.

Kinetics of nitrification in a fixed biofilm reactor using dewatered sludge-fly ash composite ceramic particle as a supporting medium.

PubMed

Lee, Mong-Chuan; Lin, Yen-Hui; Yu, Huang-Wei

2014-11-01

A mathematical model system was derived to describe the kinetics of ammonium nitrification in a fixed biofilm reactor using dewatered sludge-fly ash composite ceramic particle as a supporting medium. The model incorporates diffusive mass transport and Monod kinetics. The model was solved using a combination of the orthogonal collocation method and Gear's method. A batch test was conducted to observe the nitrification of ammonium-nitrogen ([Formula: see text]-N) and the growth of nitrifying biomass. The compositions of nitrifying bacterial community in the batch kinetic test were analyzed using PCR-DGGE method. The experimental results show that the most staining intensity abundance of bands occurred on day 2.75 with the highest biomass concentration of 46.5 mg/L. Chemostat kinetic tests were performed independently to evaluate the biokinetic parameters used in the model prediction. In the column test, the removal efficiency of [Formula: see text]-N was approximately 96 % while the concentration of suspended nitrifying biomass was approximately 16 mg VSS/L and model-predicted biofilm thickness reached up to 0.21 cm in the steady state. The profiles of denaturing gradient gel electrophoresis (DGGE) of different microbial communities demonstrated that indigenous nitrifying bacteria (Nitrospira and Nitrobacter) existed and were the dominant species in the fixed biofilm process.

Diversity of pufM genes, involved in aerobic anoxygenic photosynthesis, in the bacterial communities associated with colonial ascidians.

PubMed

Martínez-García, Manuel; Díaz-Valdés, Marta; Antón, Josefa

2010-03-01

Ascidians are invertebrate filter feeders widely distributed in benthic marine environments. A total of 14 different ascidian species were collected from the Western Mediterranean and their bacterial communities were analyzed by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene. Results showed that ascidian tissues harbored Bacteria belonging to Gamma- and Alphaproteobacteria classes, some of them phylogenetically related to known aerobic anoxygenic phototrophs (AAPs), such as Roseobacter sp. In addition, hierarchical cluster analysis of DGGE patterns showed a large variability in the bacterial diversity among the different ascidians analyzed, which indicates that they would harbor different bacterial communities. Furthermore, pufM genes, involved in aerobic anoxygenic photosynthesis in marine and freshwater systems, were widely detected within the ascidians analyzed, because nine out of 14 species had pufM genes inside their tissues. The pufM gene was only detected in those specimens that inhabited shallow waters (<77 m of depth). Most pufM gene sequences were very closely related to that of uncultured marine bacteria. Thus, our results suggest that the association of ascidians with bacteria related to AAPs could be a general phenomenon and that ascidian-associated microbiota could use the light that penetrates through the tunic tissue as an energy source.

Effect of activated carbon amendment on bacterial community structure and functions in a PAH impacted urban soil.

PubMed

Meynet, Paola; Hale, Sarah E; Davenport, Russell J; Cornelissen, Gerard; Breedveld, Gijs D; Werner, David

2012-05-01

We collected urban soil samples impacted by polycyclic aromatic hydrocarbons (PAHs) from a sorbent-based remediation field trial to address concerns about unwanted side-effects of 2% powdered (PAC) or granular (GAC) activated carbon amendment on soil microbiology and pollutant biodegradation. After three years, total microbial cell counts and respiration rates were highest in the GAC amended soil. The predominant bacterial community structure derived from denaturing gradient gel electrophoresis (DGGE) shifted more strongly with time than in response to AC amendment. DGGE band sequencing revealed the presence of taxa with closest affiliations either to known PAH degraders, e.g. Rhodococcus jostii RHA-1, or taxa known to harbor PAH degraders, e.g. Rhodococcus erythropolis, in all soils. Quantification by real-time polymerase chain reaction yielded similar dioxygenases gene copy numbers in unamended, PAC-, or GAC-amended soil. PAH availability assessments in batch tests showed the greatest difference of 75% with and without biocide addition for unamended soil, while the lowest PAH availability overall was measured in PAC-amended, live soil. We conclude that AC had no detrimental effects on soil microbiology, AC-amended soils retained the potential to biodegrade PAHs, but the removal of available pollutants by biodegradation was most notable in unamended soil. © 2012 American Chemical Society

Impact of fungicides on the diversity and function of non-target ammonia-oxidizing microorganisms residing in a litter soil cover.

PubMed

Puglisi, Edoardo; Vasileiadis, Sotirios; Demiris, Konstantinos; Bassi, Daniela; Karpouzas, Dimitrios G; Capri, Ettore; Cocconcelli, Pier S; Trevisan, Marco

2012-10-01

Litter soil cover constitutes an important micro-ecosystem in sustainable viticulture having a key role in nutrient cycling and serving as a habitat of complex microbial communities. Ammonia-oxidizing bacteria (AOB) and archaea (AOA) are known to regulate nitrification in soil while little is known regarding their function and diversity in litter. We investigated the effects of two fungicides, penconazole and cyprodinil, commonly used in vineyards, on the function and diversity of total and active AOB and AOA in a microcosm study. Functional changes measured via potential nitrification and structural changes assessed via denaturating gradient gel electrophoresis (DGGE) at the DNA and RNA levels were contrasted with pesticide dissipation in the litter layer. The latter was inversely correlated with potential nitrification, which was temporarily inhibited at the initial sampling dates (0 to 21 days) when nearly 100 % of the applied pesticide amounts was still present in the litter. Fungicides induced changes in AOB and AOA communities with RNA-DGGE analysis showing a higher sensitivity. AOA were more responsive to pesticide application compared to AOB. Potential nitrification was less sensitive to the fungicides and was restored faster than structural changes, which persisted. These results support the theory of microbial redundancy for nitrification in a stressed litter environment.

Growth of Chlorella vulgaris and associated bacteria in photobioreactors

PubMed Central

Lakaniemi, Aino‐Maija; Intihar, Veera M.; Tuovinen, Olli H.; Puhakka, Jaakko A.

2012-01-01

Summary The aim of this study was to test three flat plate photobioreactor configurations for growth of Chlorella vulgaris under non‐axenic conditions and to characterize and quantify associated bacterial communities. The photobioreactor cultivations were conducted using tap water‐based media to introduce background bacterial population. Growth of algae was monitored over time with three independent methods. Additionally, the quantity and quality of eukaryotes and bacteria were analysed using culture‐independent molecular tools based on denaturing gradient gel electrophoresis (PCR‐DGGE) and quantitative polymerase chain reaction (QPCR). Static mixers used in the flat plate photobioreactors did not generally enhance the growth at the low light intensities used. The maximum biomass concentration and maximum specific growth rate were 1.0 g l−1 and 2.0 day−1 respectively. Bacterial growth as determined by QPCR was associated with the growth of C. vulgaris. Based on PCR‐DGGE, bacteria in the cultures mainly originated from the tap water. Bacterial community profiles were diverse but reproducible in all flat plate cultures. Most prominent bacteria in the C. vulgaris cultures belonged to the class Alphaproteobacteria and especially to the genus Sphingomonas. Analysis of the diversity of non‐photosynthetic microorganisms in algal mass cultures can provide useful information on the public health aspects and unravel community interactions. PMID:21936882

Effects of Different Organic Manures on the Biochemical and Microbial Characteristics of Albic Paddy Soil in a Short-Term Experiment

PubMed Central

Zhang, Qian; Zhou, Wei; Liang, Guoqing; Wang, Xiubin; Sun, Jingwen; He, Ping; Li, Lujiu

2015-01-01

This study aimed to evaluate the effects of chemical fertilizer (NPK), NPK with livestock manure (NPK+M), NPK with straw (NPK+S), and NPK with green manure (NPK+G) on soil enzyme activities and microbial characteristics of albic paddy soil, which is a typical soil with low productivity in China. The responses of extracellular enzyme activities and the microbial community diversity (determined by phospholipid fatty acid analysis [PLFA] and denaturing gradient gel electrophoresis [DGGE]) were measured. The results showed that NPK+M and NPK+S significantly increased rice yield, with NPK+M being approximately 24% greater than NPK. The NPK+M significantly increased soil organic carbon (SOC) and available phosphate (P) and enhanced phosphatase, β-cellobiosidase, L-leucine aminopeptidase and urease activities. The NPK+S significantly increased SOC and available potassium (K) and significantly enhanced N-acetyl-glucosamidase, β-xylosidase, urease, and phenol oxidase activities. The NPK+G significantly improved total nitrogen (N), ammonium N, available P, and N-acetyl-glucosamidase activity. The PLFA biomass was highest under NPK+S, followed by NPK+M and NPK+G treatments. Principal component analysis (PCA) of the PLFA indicated that soils with NPK+M and NPK+S contained higher proportions of unsaturated and cyclopropane fatty acids (biomarkers of fungi and gram-negative bacteria) and soil under NPK+G contained more straight chain saturated fatty acids (representing gram-positive bacteria). PCA of the DGGE patterns showed that organic amendments had a greater influence on fungal community. Cluster analysis of fungal DGGE patterns revealed that NPK+G was clearly separated. Meanwhile, the bacterial community of NPK+M treatment was the most distinct. RDA analysis revealed changes of microbial community composition mostly depended on β-xylosidase, β-cellobiosidase activities, total N and available K contents. The abundances of gram-negative bacterial and fungal PLFAs probably effective in improving fertility of low-yield albic paddy soil because of their significant influence on DGGE profile. PMID:25879759

Effects of different organic manures on the biochemical and microbial characteristics of albic paddy soil in a short-term experiment.

PubMed

Zhang, Qian; Zhou, Wei; Liang, Guoqing; Wang, Xiubin; Sun, Jingwen; He, Ping; Li, Lujiu

2015-01-01

This study aimed to evaluate the effects of chemical fertilizer (NPK), NPK with livestock manure (NPK+M), NPK with straw (NPK+S), and NPK with green manure (NPK+G) on soil enzyme activities and microbial characteristics of albic paddy soil, which is a typical soil with low productivity in China. The responses of extracellular enzyme activities and the microbial community diversity (determined by phospholipid fatty acid analysis [PLFA] and denaturing gradient gel electrophoresis [DGGE]) were measured. The results showed that NPK+M and NPK+S significantly increased rice yield, with NPK+M being approximately 24% greater than NPK. The NPK+M significantly increased soil organic carbon (SOC) and available phosphate (P) and enhanced phosphatase, β-cellobiosidase, L-leucine aminopeptidase and urease activities. The NPK+S significantly increased SOC and available potassium (K) and significantly enhanced N-acetyl-glucosamidase, β-xylosidase, urease, and phenol oxidase activities. The NPK+G significantly improved total nitrogen (N), ammonium N, available P, and N-acetyl-glucosamidase activity. The PLFA biomass was highest under NPK+S, followed by NPK+M and NPK+G treatments. Principal component analysis (PCA) of the PLFA indicated that soils with NPK+M and NPK+S contained higher proportions of unsaturated and cyclopropane fatty acids (biomarkers of fungi and gram-negative bacteria) and soil under NPK+G contained more straight chain saturated fatty acids (representing gram-positive bacteria). PCA of the DGGE patterns showed that organic amendments had a greater influence on fungal community. Cluster analysis of fungal DGGE patterns revealed that NPK+G was clearly separated. Meanwhile, the bacterial community of NPK+M treatment was the most distinct. RDA analysis revealed changes of microbial community composition mostly depended on β-xylosidase, β-cellobiosidase activities, total N and available K contents. The abundances of gram-negative bacterial and fungal PLFAs probably effective in improving fertility of low-yield albic paddy soil because of their significant influence on DGGE profile.

Culture-Independent Techniques for Rapid Detection of Bacteria Associated with Loss of Chloramine Residual in a Drinking Water System

PubMed Central

Hoefel, Daniel; Monis, Paul T.; Grooby, Warwick L.; Andrews, Stuart; Saint, Christopher P.

2005-01-01

Chloramination is often the disinfection regimen of choice for extended drinking water systems. However, this process is prone to instability due to the growth of nitrifying bacteria. This is the first study to use alternative approaches for rapid investigation of chloraminated drinking water system instability in which flow cytometric cell sorting of bacteria with intact membranes (membrane-intact fraction) (BacLight kit) or with active esterases (esterase-active fraction) (carboxyfluorescein diacetate) was combined with 16S rRNA gene-directed PCR and denaturing gradient gel electrophoresis (DGGE). No active bacteria were detected when water left the water treatment plant (WTP), but 12 km downstream the chloramine residual had diminished and the level of active bacteria in the bulk water had increased to more than 1 × 105 bacteria ml−1. The bacterial diversity in the system was represented by six major DGGE bands for the membrane-intact fraction and 10 major DGGE bands for the esterase-active fraction. PCR targeting of the 16S rRNA gene of chemolithotrophic ammonia-oxidizing bacteria (AOB) and subsequent DGGE and DNA sequence analysis revealed the presence of an active Nitrosospira-related species and Nitrosomonas cryotolerans in the system, but no AOB were detected in the associated WTP. The abundance of active AOB was then determined by quantitative real-time PCR (qPCR) targeting the amoA gene; 3.43 × 103 active AOB ml−1 were detected in the membrane-intact fraction, and 1.40 × 104 active AOB ml−1 were detected in the esterase-active fraction. These values were several orders of magnitude greater than the 2.5 AOB ml−1 detected using a routine liquid most-probable-number assay. Culture-independent techniques described here, in combination with existing chemical indicators, should allow the water industry to obtain more comprehensive data with which to make informed decisions regarding remedial action that may be required either prior to or during an instability event. PMID:16269672

Spatial variations of bacterial community and its relationship with water chemistry in Sanya Bay, South China Sea as determined by DGGE fingerprinting and multivariate analysis.

PubMed

Ling, Juan; Zhang, Yan-Ying; Dong, Jun-De; Wang, You-Shao; Feng, Jing-Bing; Zhou, Wei-Hua

2015-10-01

Bacteria play important roles in the structure and function of marine food webs by utilizing nutrients and degrading the pollutants, and their distribution are determined by surrounding water chemistry to a certain extent. It is vital to investigate the bacterial community's structure and identifying the significant factors by controlling the bacterial distribution in the paper. Flow cytometry showed that the total bacterial abundance ranged from 5.27 × 10(5) to 3.77 × 10(6) cells/mL. Molecular fingerprinting technique, denaturing gradient gel electrophoresis (DGGE) followed by DNA sequencing has been employed to investigate the bacterial community composition. The results were then interpreted through multivariate statistical analysis and tended to explain its relationship to the environmental factors. A total of 270 bands at 83 different positions were detected in DGGE profiles and 29 distinct DGGE bands were sequenced. The predominant bacteria were related to Phyla Protebacteria species (31 %, nine sequences), Cyanobacteria (37.9 %, eleven sequences) and Actinobacteria (17.2 %, five sequences). Other phylogenetic groups identified including Firmicutes (6.9 %, two sequences), Bacteroidetes (3.5 %, one sequences) and Verrucomicrobia (3.5 %, one sequences). Conical correspondence analysis was used to elucidate the relationships between the bacterial community compositions and environmental factors. The results showed that the spatial variations in the bacterial community composition was significantly related to phosphate (P = 0.002, P < 0.01), dissolved organic carbon (P = 0.004, P < 0.01), chemical oxygen demand (P = 0.010, P < 0.05) and nitrite (P = 0.016, P < 0.05). This study revealed the spatial variations of bacterial community and significant environmental factors driving the bacterial composition shift. These results may be valuable for further investigation on the functional microbial structure and expression quantitatively under the polluted environments in the world.

Analysis of oral microbiota in children with dental caries by PCR-DGGE and barcoded pyrosequencing.

PubMed

Ling, Zongxin; Kong, Jianming; Jia, Peng; Wei, Chaochun; Wang, Yuezhu; Pan, Zhiwen; Huang, Wujing; Li, Lanjuan; Chen, Hui; Xiang, Charlie

2010-10-01

Oral microbiota plays a vital role in maintaining the homeostasis of oral cavity. Dental caries are among the most common oral diseases in children and pathogenic bacteria contribute to the development of the disease. However, the overall structure of bacterial communities in the oral cavity from children with dental caries has not been explored deeply heretofore. We used high-throughput barcoded pyrosequencing and PCR-denaturing gradient gel electrophoresis (DGGE) to examine bacterial diversity of oral microbiota in saliva and supragingival plaques from 60 children aged 3 to 6 years old with and without dental caries from China. The multiplex barcoded pyrosequencing was performed in a single run, with multiple samples tagged uniquely by multiplex identifiers. As PCR-DGGE analysis is a conventional molecular ecological approach, this analysis was also performed on the same samples and the results of both approaches were compared. A total of 186,787 high-quality sequences were obtained for evaluating bacterial diversity and 41,905 unique sequences represented all phylotypes. We found that the oral microbiota in children was far more diverse than previous studies reported, and more than 200 genera belonging to ten phyla were found in the oral cavity. The phylotypes in saliva and supragingival plaques were significantly different and could be divided into two distinct clusters (p < 0.05). The bacterial diversity in oral microbiome analyzed by PCR-DGGE and barcoded pyrosequencing was employed to cross validate the data sets. The genera of Streptococcus, Veillonella, Actinomyces, Granulicatella, Leptotrichia, and Thiomonas in plaques were significantly associated with dental caries (p < 0.05). The results showed that there was no one specific pathogen but rather pathogenic populations in plaque that significantly correlated with dental caries. The enormous diversity of oral microbiota allowed for a better understanding of oral microecosystem, and these pathogenic populations in plaque provide new insights into the etiology of dental caries and suggest new targets for interventions of the disease.

Molecular diagnostics of periodontitis.

PubMed

Korona-Głowniak, Izabela; Siwiec, Radosław; Berger, Marcin; Malm, Anna; Szymańska, Jolanta

2017-01-28

The microorganisms that form dental plaque are the main cause of periodontitis. Their identification and the understanding of the complex relationships and interactions that involve these microorganisms, environmental factors and the host's health status enable improvement in diagnostics and targeted therapy in patients with periodontitis. To this end, molecular diagnostics techniques (both techniques based on the polymerase chain reaction and those involving nucleic acid analysis via hybridization) come increasingly into use. On the basis of a literature review, the following methods are presented: polymerase chain reaction (PCR), real-time polymerase chain reaction (real-time PCR), 16S rRNA-encoding gene sequencing, checkerboard and reverse-capture checkerboard hybridization, microarrays, denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), as well as terminal restriction fragment length polymorphism (TRFLP) and next generation sequencing (NGS). The advantages and drawbacks of each method in the examination of periopathogens are indicated. The techniques listed above allow fast detection of even small quantities of pathogen present in diagnostic material and prove particularly useful to detect microorganisms that are difficult or impossible to grow in a laboratory.

Application of anaerobic ammonium-oxidizing consortium to achieve completely autotrophic ammonium and sulfate removal.

PubMed

Liu, Sitong; Yang, Fenglin; Gong, Zheng; Meng, Fangang; Chen, Huihui; Xue, Yuan; Furukawa, Kenji

2008-10-01

The simultaneous ammonium and sulfate removal was detected in an anammox reactor, consisted of ammonium oxidization with sulfate deoxidization, and subsequently traditional anammox process, in via of middle medium nitrite with solid sulfur and N2 as the terminal products. The pure anammox bacteria offered a great biotechnological potential for the completely autotrophic reaction indicated by batch tests. Denaturing gradient gel electrophoresis (DGGE) analysis further revealed that a new organism belonging to Planctomycetales was strongly enriched in the defined niche: the redox of ammonium and sulfate. The new species "Anammoxoglobussulfate" was so considered as holding a critical role in the ammonium oxidization with sulfate deoxidization to nitrite. Afterwards, the Planctomyces existing in the bacteria community performed the anammox process together to achieve the complete nitrogen and sulfate removal. The potential use of sulfate as electron acceptor for ammonium oxidizing widens the usage of anammox bacteria.

[Effect of the initial anode potential on electricity generation in microbial fuel cell].

PubMed

Fan, Ming-Zhi; Liang, Peng; Cao, Xiao-Xin; Huang, Xia

2008-01-01

The initial anode potential of the microbial fuel cell (MFC) was changed by additional circuit in the anode chamber, and the influence of the initial anode potential on the electricigens was studied. When the initial anode potential was 350 mV (vs Hg/Hg2 Cl2), the growth of microorganisms was much slower than that of the microorganisms which grew on the anode with an initial potential of -200 mV or 200 mV (vs Hg/Hg2 Cl2). After stable electricity generation, the anode resistances of the three MFCs, which had initial anode potentials of 350 mV, 200 mV and -200 mV respectively, were 71 Omega, 43 Omega and 80 Omega. The community structures in MFCs, before and after the electricity generation, were also studied by denaturing gradient gel electrophoresis (DGGE). Clostridium sticklandii, Pseudomonas mendocina and Paenibacillus taejonensis were the three most enriched strains on the anode.

Monitoring Biological Activity at Geothermal Power Plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peter Pryfogle

2005-09-01

The economic impact of microbial growth in geothermal power plants has been estimated to be as high as $500,000 annually for a 100 MWe plant. Many methods are available to monitor biological activity at these facilities; however, very few plants have any on-line monitoring program in place. Metal coupon, selective culturing (MPN), total organic carbon (TOC), adenosine triphosphate (ATP), respirometry, phospholipid fatty acid (PLFA), and denaturing gradient gel electrophoresis (DGGE) characterizations have been conducted using water samples collected from geothermal plants located in California and Utah. In addition, the on-line performance of a commercial electrochemical monitor, the BIoGEORGE?, has beenmore » evaluated during extended deployments at geothermal facilities. This report provides a review of these techniques, presents data on their application from laboratory and field studies, and discusses their value in characterizing and monitoring biological activities at geothermal power plants.« less

Microbial biofilms on facial prostheses.

PubMed

Ariani, Nina; Vissink, Arjan; van Oort, Robert P; Kusdhany, Lindawati; Djais, Ariadna; Rahardjo, Tri Budi W; van der Mei, Henny C; Krom, Bastiaan P

2012-01-01

The composition of microbial biofilms on silicone rubber facial prostheses was investigated and compared with the microbial flora on healthy and prosthesis-covered skin. Scanning electron microscopy showed the presence of mixed bacterial and yeast biofilms on and deterioration of the surface of the prostheses. Microbial culturing confirmed the presence of yeasts and bacteria. Microbial colonization was significantly increased on prosthesis-covered skin compared to healthy skin. Candida spp. were exclusively isolated from prosthesis-covered skin and from prostheses. Biofilms from prostheses showed the least diverse band-profile in denaturing gradient gel electrophoresis (DGGE) whereas prosthesis-covered skin showed the most diverse band-profile. Bacterial diversity exceeded yeast diversity in all samples. It is concluded that occlusion of the skin by prostheses creates a favorable niche for opportunistic pathogens such as Candida spp. and Staphylococcus aureus. Biofilms on healthy skin, skin underneath the prosthesis and on the prosthesis had a comparable composition, but the numbers present differed according to the microorganism.

The effectiveness of the biodegradation of raw and processed polystyrene by mealworms

NASA Astrophysics Data System (ADS)

Leluk, Karol; Hanus-Lorenz, Beata; Rybak, Justyna; Bożek, Magdalena

2017-11-01

In our studies biodegradation of four variants of polystyrene was performed. We tested: raw material (PS), processed polystyrene (PSr), building insulation material (EPS) and food packaging boxes (PSp). Materials were characterized by means melt flow ratio (MFR), shore hardness and gloss. The biochemical assessment of macromolecules (proteins, lipids and sugars) in the mealworms organisms fed with tested forms of polystyrene allowed us to set how efficient and beneficial the biodegradation of types of polystyrene is. We also evaluated the variability of bacterial community in larval guts by the use of denaturing gradient gel electrophoresis (DGGE) on the bacterial DNA of 16S rRNA genes amplified in polymerase chain reaction (PCR). The results suggest that EPS and PSp polystyrene are the most digestible for T. molitor larvae. The metabolic degradation of polystyrene is probably strictly connected with the changes in biodiversity of gut bacteria.

Microbial diversity of a Camembert-type cheese using freeze-dried Tibetan kefir coculture as starter culture by culture-dependent and culture-independent methods.

PubMed

Mei, Jun; Guo, Qizhen; Wu, Yan; Li, Yunfei

2014-01-01

The biochemical changes occurring during cheese ripening are directly and indirectly dependent on the microbial associations of starter cultures. Freeze-dried Tibetan kefir coculture was used as a starter culture in the Camembert-type cheese production for the first time. Therefore, it's necessary to elucidate the stability, organization and identification of the dominant microbiota presented in the cheese. Bacteria and yeasts were subjected to culture-dependent on selective media and culture-independent polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of dominant bands to assess the microbial structure and dynamics through ripening. In further studies, kefir grains were observed using scanning electron microscopy (SEM) methods. A total of 147 bacteria and 129 yeasts were obtained from the cheese during ripening. Lactobacillus paracasei represents the most commonly identified lactic acid bacteria isolates, with 59 of a total of 147 isolates, followed by Lactococcus lactis (29 isolates). Meanwhile, Kazachstania servazzii (51 isolates) represented the mainly identified yeast isolate, followed by Saccharomyces cerevisiae (40 isolates). However, some lactic acid bacteria detected by sequence analysis of DGGE bands were not recovered by plating. The yeast S. cerevisiae and K. servazzii are described for the first time with kefir starter culture. SEM showed that the microbiota were dominated by a variety of lactobacilli (long and curved) cells growing in close association with a few yeasts in the inner portion of the grain and the short lactobacilli were observed along with yeast cells on the exterior portion. Results indicated that conventional culture method and PCR-DGGE should be combined to describe in maximal detail the microbiological composition in the cheese during ripening. The data could help in the selection of appropriate commercial starters for Camembert-type cheese.

Effects of Agronomic Treatments on Structure and Function of Ammonia-Oxidizing Communities

PubMed Central

Phillips, Carol J.; Harris, Dave; Dollhopf, Sherry L.; Gross, Katherine L.; Prosser, James I.; Paul, Eldor A.

2000-01-01

The aim of this study was to determine the effects of different agricultural treatments and plant communities on the diversity of ammonia oxidizer populations in soil. Denaturing gradient gel electrophoresis (DGGE), coupled with specific oligonucleotide probing, was used to analyze 16S rRNA genes of ammonia oxidizers belonging to the β subgroup of the division Proteobacteria by use of DNA extracted from cultivated, successional, and native deciduous forest soils. Community profiles of the different soil types were compared with nitrification rates and most-probable-number (MPN) counts. Despite significant variation in measured nitrification rates among communities, there were no differences in the DGGE banding profiles of DNAs extracted from these soils. DGGE profiles of DNA extracted from samples of MPN incubations, cultivated at a range of ammonia concentrations, showed the presence of bands not amplified from directly extracted DNA. Nitrosomonas-like bands were seen in the MPN DNA but were not detected in the DNA extracted directly from soils. These bands were detected in some samples taken from MPN incubations carried out with medium containing 1,000 μg of NH4+-N ml−1, to the exclusion of bands detected in the native DNA. Cell concentrations of ammonia oxidizers determined by MPN counts were between 10- and 100-fold lower than those determined by competitive PCR (cPCR). Although no differences were seen in ammonia oxidizer MPN counts from the different soil treatments, cPCR revealed higher numbers in fertilized soils. The use of a combination of traditional and molecular methods to investigate the activities and compositions of ammonia oxidizers in soil demonstrates differences in fine-scale compositions among treatments that may be associated with changes in population size and function. PMID:11097922

Diversity and Functional Analysis of Bacterial Communities Associated with Natural Hydrocarbon Seeps in Acidic Soils at Rainbow Springs, Yellowstone National Park

PubMed Central

Hamamura, Natsuko; Olson, Sarah H.; Ward, David M.; Inskeep, William P.

2005-01-01

In this paper we describe the bacterial communities associated with natural hydrocarbon seeps in nonthermal soils at Rainbow Springs, Yellowstone National Park. Soil chemical analysis revealed high sulfate concentrations and low pH values (pH 2.8 to 3.8), which are characteristic of acid-sulfate geothermal activity. The hydrocarbon composition of the seep soils consisted almost entirely of saturated, acyclic alkanes (e.g., n-alkanes with chain lengths of C15 to C30, as well as branched alkanes, predominately pristane and phytane). Bacterial populations present in the seep soils were phylogenetically characterized by 16S rRNA gene clone library analysis. The majority of the sequences recovered (>75%) were related to sequences of heterotrophic acidophilic bacteria, including Acidisphaera spp. and Acidiphilium spp. of the α-Proteobacteria. Clones related to the iron- and sulfur-oxidizing chemolithotroph Acidithiobacillus spp. were also recovered from one of the seep soils. Hydrocarbon-amended soil-sand mixtures were established to examine [14C]hexadecane mineralization and corresponding changes in the bacterial populations using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Approximately 50% of the [14C]hexadecane added was recovered as 14CO2 during an 80-day incubation, and this was accompanied by detection of heterotrophic acidophile-related sequences as dominant DGGE bands. An alkane-degrading isolate was cultivated, whose 16S rRNA gene sequence was identical to the sequence of a dominant DGGE band in the soil-sand mixture, as well as the clone sequence recovered most frequently from the original soil. This and the presence of an alkB gene homolog in this isolate confirmed the alkane degradation capability of one population indigenous to acidic hydrocarbon seep soils. PMID:16204508

Does Wheat Genetically Modified for Disease Resistance Affect Root-Colonizing Pseudomonads and Arbuscular Mycorrhizal Fungi?

PubMed Central

Foetzki, Andrea; Luginbühl, Carolin; Winzeler, Michael; Kneubühler, Yvan; Matasci, Caterina; Mascher-Frutschi, Fabio; Kalinina, Olena; Boller, Thomas; Keel, Christoph; Maurhofer, Monika

2013-01-01

This study aimed to evaluate the impact of genetically modified (GM) wheat with introduced pm3b mildew resistance transgene, on two types of root-colonizing microorganisms, namely pseudomonads and arbuscular mycorrhizal fungi (AMF). Our investigations were carried out in field trials over three field seasons and at two locations. Serial dilution in selective King's B medium and microscopy were used to assess the abundance of cultivable pseudomonads and AMF, respectively. We developed a denaturing gradient gel electrophoresis (DGGE) method to characterize the diversity of the pqqC gene, which is involved in Pseudomonas phosphate solubilization. A major result was that in the first field season Pseudomonas abundances and diversity on roots of GM pm3b lines, but also on non-GM sister lines were different from those of the parental lines and conventional wheat cultivars. This indicates a strong effect of the procedures by which these plants were created, as GM and sister lines were generated via tissue cultures and propagated in the greenhouse. Moreover, Pseudomonas population sizes and DGGE profiles varied considerably between individual GM lines with different genomic locations of the pm3b transgene. At individual time points, differences in Pseudomonas and AMF accumulation between GM and control lines were detected, but they were not consistent and much less pronounced than differences detected between young and old plants, different conventional wheat cultivars or at different locations and field seasons. Thus, we conclude that impacts of GM wheat on plant-beneficial root-colonizing microorganisms are minor and not of ecological importance. The cultivation-independent pqqC-DGGE approach proved to be a useful tool for monitoring the dynamics of Pseudomonas populations in a wheat field and even sensitive enough for detecting population responses to altered plant physiology. PMID:23372672

In-Field Spatial Variability in the Degradation of the Phenyl-Urea Herbicide Isoproturon Is the Result of Interactions between Degradative Sphingomonas spp. and Soil pH

PubMed Central

Bending, Gary D.; Lincoln, Suzanne D.; Sørensen, Sebastian R.; Morgan, J. Alun W.; Aamand, Jens; Walker, Allan

2003-01-01

Substantial spatial variability in the degradation rate of the phenyl-urea herbicide isoproturon (IPU) [3-(4-isopropylphenyl)-1,1-dimethylurea] has been shown to occur within agricultural fields, with implications for the longevity of the compound in the soil, and its movement to ground- and surface water. The microbial mechanisms underlying such spatial variability in degradation rate were investigated at Deep Slade field in Warwickshire, United Kingdom. Most-probable-number analysis showed that rapid degradation of IPU was associated with proliferation of IPU-degrading organisms. Slow degradation of IPU was linked to either a delay in the proliferation of IPU-degrading organisms or apparent cometabolic degradation. Using enrichment techniques, an IPU-degrading bacterial culture (designated strain F35) was isolated from fast-degrading soil, and partial 16S rRNA sequencing placed it within the Sphingomonas group. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified bacterial community 16S rRNA revealed two bands that increased in intensity in soil during growth-linked metabolism of IPU, and sequencing of the excised bands showed high sequence homology to the Sphingomonas group. However, while F35 was not closely related to either DGGE band, one of the DGGE bands showed 100% partial 16S rRNA sequence homology to an IPU-degrading Sphingomonas sp. (strain SRS2) isolated from Deep Slade field in an earlier study. Experiments with strains SRS2 and F35 in soil and liquid culture showed that the isolates had a narrow pH optimum (7 to 7.5) for metabolism of IPU. The pH requirements of IPU-degrading strains of Sphingomonas spp. could largely account for the spatial variation of IPU degradation rates across the field. PMID:12571001

Dynamics of indigenous bacterial communities associated with crude oil degradation in soil microcosms during nutrient-enhanced bioremediation.

PubMed

Chikere, Chioma B; Surridge, Karen; Okpokwasili, Gideon C; Cloete, Thomas E

2012-03-01

Bacterial population dynamics were examined during bioremediation of an African soil contaminated with Arabian light crude oil and nutrient enrichment (biostimulation). Polymerase chain reaction followed by denaturing gradient gel electrophoresis (DGGE) were used to generate bacterial community fingerprints of the different treatments employing the 16S ribosomal ribonucleic acid (rRNA) gene as molecular marker. The DGGE patterns of the nutrient-amended soils indicated the presence of distinguishable bands corresponding to the oil-contaminated-nutrient-enriched soils, which were not present in the oil-contaminated and pristine control soils. Further characterization of the dominant DGGE bands after excision, reamplification and sequencing revealed that Corynebacterium spp., Dietzia spp., Rhodococcus erythropolis sp., Nocardioides sp., Low G+C (guanine plus cytosine) Gram positive bacterial clones and several uncultured bacterial clones were the dominant bacterial groups after biostimulation. Prominent Corynebacterium sp. IC10 sequence was detected across all nutrient-amended soils but not in oil-contaminated control soil. Total heterotrophic and hydrocarbon utilizing bacterial counts increased significantly in the nutrient-amended soils 2 weeks post contamination whereas oil-contaminated and pristine control soils remained fairly stable throughout the experimental period. Gas chromatographic analysis of residual hydrocarbons in biostimulated soils showed marked attenuation of contaminants starting from the second to the sixth week after contamination whereas no significant reduction in hydrocarbon peaks were seen in the oil-contaminated control soil throughout the 6-week experimental period. Results obtained indicated that nutrient amendment of oil-contaminated soil selected and enriched the bacterial communities mainly of the Actinobacteria phylogenetic group capable of surviving in toxic contamination with concomitant biodegradation of the hydrocarbons. The present study therefore demonstrated that the soil investigated harbours hydrocarbon-degrading bacterial populations which can be biostimulated to achieve effective bioremediation of oil-contaminated soil.

Effects of artificial defoliation of pines on the structure and physiology of the soil fungal community of a mixed pine-spruce forest

NASA Technical Reports Server (NTRS)

Cullings, Ken; Raleigh, Christopher; New, Michael H.; Henson, Joan

2005-01-01

Loss of photosynthetic area can affect soil microbial communities by altering the availability of fixed carbon. We used denaturing gradient gel electrophoresis (DGGE) and Biolog filamentous-fungus plates to determine the effects of artificial defoliation of pines in a mixed pine-spruce forest on the composition of the fungal community in a forest soil. As measured by DGGE, two fungal species were affected significantly by the defoliation of pines (P < 0.001); the frequency of members of the ectomycorrhizal fungus genus Cenococcum decreased significantly, while the frequency of organisms of an unidentified soil fungus increased. The decrease in the amount of Cenococcum organisms may have occurred because of the formation of extensive hyphal networks by species of this genus, which require more of the carbon fixed by their host, or because this fungus is dependent upon quantitative differences in spruce root exudates. The defoliation of pines did not affect the overall composition of the soil fungal community or fungal-species richness (number of species per core). Biolog filamentous-fungus plate assays indicated a significant increase (P < 0.001) in the number of carbon substrates utilized by the soil fungi and the rate at which these substrates were used, which could indicate an increase in fungal-species richness. Thus, either small changes in the soil fungal community give rise to significant increases in physiological capabilities or PCR bias limits the reliability of the DGGE results. These data indicate that combined genetic and physiological assessments of the soil fungal community are needed to accurately assess the effect of disturbance on indigenous microbial systems.

Culture-Independent Analysis of Bacterial Fuel Contamination Provides Insight into the Level of Concordance with the Standard Industry Practice of Aerobic Cultivation ▿ †

PubMed Central

White, Judith; Gilbert, Jack; Hill, Graham; Hill, Edward; Huse, Susan M.; Weightman, Andrew J.; Mahenthiralingam, Eshwar

2011-01-01

Bacterial diversity in contaminated fuels has not been systematically investigated using cultivation-independent methods. The fuel industry relies on phenotypic cultivation-based contaminant identification, which may lack accuracy and neglect difficult-to-culture taxa. By the use of industry practice aerobic cultivation, 16S rRNA gene sequencing, and strain genotyping, a collection of 152 unique contaminant isolates from 54 fuel samples was assembled, and a dominance of Pseudomonas (21%), Burkholderia (7%), and Bacillus (7%) was demonstrated. Denaturing gradient gel electrophoresis (DGGE) of 15 samples revealed Proteobacteria and Firmicutes to be the most abundant phyla. When 16S rRNA V6 gene pyrosequencing of four selected fuel samples (indicated by “JW”) was performed, Betaproteobacteria (42.8%) and Gammaproteobacteria (30.6%) formed the largest proportion of reads; the most abundant genera were Marinobacter (15.4%; JW57), Achromobacter (41.6%; JW63), Burkholderia (80.7%; JW76), and Halomonas (66.2%; JW78), all of which were also observed by DGGE. However, the Clostridia (38.5%) and Deltaproteobacteria (11.1%) identified by pyrosequencing in sample JW57 were not observed by DGGE or aerobic culture. Genotyping revealed three instances where identical strains were found: (i) a Pseudomonas sp. strain recovered from 2 different diesel fuel tanks at a single industrial site; (ii) a Mangroveibacter sp. strain isolated from 3 biodiesel tanks at a single refinery site; and (iii) a Burkholderia vietnamiensis strain present in two unrelated automotive diesel samples. Overall, aerobic cultivation of fuel contaminants recovered isolates broadly representative of the phyla and classes present but lacked accuracy by overrepresenting members of certain groups such as Pseudomonas. PMID:21602386

Diversity, dynamics, and activity of bacterial communities during production of an artisanal Sicilian cheese as evaluated by 16S rRNA analysis.

PubMed

Randazzo, Cinzia L; Torriani, Sandra; Akkermans, Antoon D L; de Vos, Willem M; Vaughan, Elaine E

2002-04-01

The diversity and dynamics of the microbial communities during the manufacturing of Ragusano cheese, an artisanal cheese produced in Sicily (Italy), were investigated by a combination of classical and culture-independent approaches. The latter included PCR, reverse transcriptase-PCR (RT-PCR), and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes (rDNA). Bacterial and Lactobacillus group-specific primers were used to amplify the V6 to V8 and V1 to V3 regions of the 16S rRNA gene, respectively. DGGE profiles from samples taken during cheese production indicated dramatic shifts in the microbial community structure. Cloning and sequencing of rDNA amplicons revealed that mesophilic lactic acid bacteria (LAB), including species of Leuconostoc, Lactococcus lactis, and Macrococcus caseolyticus were dominant in the raw milk, while Streptococcus thermophilus prevailed during lactic fermentation. Other thermophilic LAB, especially Lactobacillus delbrueckii and Lactobacillus fermentum, also flourished during ripening. Comparison of the rRNA-derived patterns obtained by RT-PCR to the rDNA DGGE patterns indicated a substantially different degree of metabolic activity for the microbial groups detected. Identification of cultivated LAB isolates by phenotypic characterization and 16S rDNA analysis indicated a variety of species, reflecting to a large extent the results obtained from the 16S rDNA clone libraries, with the significant exception of the Lactobacillus delbrueckii species, which dominated in the ripening cheese but was not detected by cultivation. The present molecular approaches combined with culture can effectively describe the complex ecosystem of natural fermented dairy products, giving useful information for starter culture design and preservation of artisanal fermented food technology.

Microbial Diversity of a Camembert-Type Cheese Using Freeze-Dried Tibetan Kefir Coculture as Starter Culture by Culture-Dependent and Culture-Independent Methods

PubMed Central

Mei, Jun; Guo, Qizhen; Wu, Yan; Li, Yunfei

2014-01-01

The biochemical changes occurring during cheese ripening are directly and indirectly dependent on the microbial associations of starter cultures. Freeze-dried Tibetan kefir coculture was used as a starter culture in the Camembert-type cheese production for the first time. Therefore, it's necessary to elucidate the stability, organization and identification of the dominant microbiota presented in the cheese. Bacteria and yeasts were subjected to culture-dependent on selective media and culture-independent polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of dominant bands to assess the microbial structure and dynamics through ripening. In further studies, kefir grains were observed using scanning electron microscopy (SEM) methods. A total of 147 bacteria and 129 yeasts were obtained from the cheese during ripening. Lactobacillus paracasei represents the most commonly identified lactic acid bacteria isolates, with 59 of a total of 147 isolates, followed by Lactococcus lactis (29 isolates). Meanwhile, Kazachstania servazzii (51 isolates) represented the mainly identified yeast isolate, followed by Saccharomyces cerevisiae (40 isolates). However, some lactic acid bacteria detected by sequence analysis of DGGE bands were not recovered by plating. The yeast S. cerevisiae and K. servazzii are described for the first time with kefir starter culture. SEM showed that the microbiota were dominated by a variety of lactobacilli (long and curved) cells growing in close association with a few yeasts in the inner portion of the grain and the short lactobacilli were observed along with yeast cells on the exterior portion. Results indicated that conventional culture method and PCR-DGGE should be combined to describe in maximal detail the microbiological composition in the cheese during ripening. The data could help in the selection of appropriate commercial starters for Camembert-type cheese. PMID:25360757

Analysis of Inter-Individual Bacterial Variation in Gut of Cicada Meimuna mongolica (Hemiptera: Cicadidae).

PubMed

Zhou, Wenting; Nan, Xiaoning; Zheng, Zhou; Wei, Cong; He, Hong

2015-01-01

Intestinal bacterial community plays a crucial role in the nutrition, development, survival, and reproduction of insects. When compared with other insects with piercing-sucking mouthparts, the habitats of cicada nymphs and adults are totally different. However, little is known about the differences in the gut bacterial communities in the nymphs and adults within any cicada species. The diversity of bacteria in the gut of nymphs and adults of both genders of Meimuna mongolica (Distant) was studied using the denaturing gradient gel electrophoresis (DGGE) method. Few inter-individual variations among gut microbiota were observed, suggesting that M. mongolica typically harbors a limited and consistent suite of bacterial species. Bacteria in the genera Pseudomonas and Enterobacter were the predominant components of the gut microflora of M. mongolica at all life stages. Bacteria of Pantoea, Streptococcus, and Uruburuella were also widespread in the cicada samples but at relatively lower concentrations. The relative stability and similarity of the PCR-DGGE patterns indicate that all individuals of this cicada species harbor a characteristic bacterial community which is independent from developmental stages and genders. Related endosymbionts that could be harbored in bacteromes of cicadas were not detected in any gut samples, which could be related to the cicada species and the distribution of these endosymbionts in the cicada cavity, or due to some of the possible limitations of PCR-DGGE community profiling. It is worthwhile to further address if related cicada endosymbiont clades distribute in the alimentary canals and other internal organs through diagnostic PCR using group-specific primer sets. © The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.

Culture-independent analysis of bacterial fuel contamination provides insight into the level of concordance with the standard industry practice of aerobis cultivation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

White, J.; Gilbert, J. A.; Hill, G.

2011-07-01

Bacterial diversity in contaminated fuels has not been systematically investigated using cultivation-independent methods. The fuel industry relies on phenotypic cultivation-based contaminant identification, which may lack accuracy and neglect difficult-to-culture taxa. By the use of industry practice aerobic cultivation, 16S rRNA gene sequencing, and strain genotyping, a collection of 152 unique contaminant isolates from 54 fuel samples was assembled, and a dominance of Pseudomonas (21%), Burkholderia (7%), and Bacillus (7%) was demonstrated. Denaturing gradient gel electrophoresis (DGGE) of 15 samples revealed Proteobacteria and Firmicutes to be the most abundant phyla. When 16S rRNA V6 gene pyrosequencing of four selected fuel samplesmore » (indicated by 'JW') was performed, Betaproteobacteria (42.8%) and Gammaproteobacteria (30.6%) formed the largest proportion of reads; the most abundant genera were Marinobacter (15.4%; JW57), Achromobacter (41.6%; JW63), Burkholderia (80.7%; JW76), and Halomonas (66.2%; JW78), all of which were also observed by DGGE. However, the Clostridia (38.5%) and Deltaproteobacteria (11.1%) identified by pyrosequencing in sample JW57 were not observed by DGGE or aerobic culture. Genotyping revealed three instances where identical strains were found: (i) a Pseudomonas sp. strain recovered from 2 different diesel fuel tanks at a single industrial site; (ii) a Mangroveibacter sp. strain isolated from 3 biodiesel tanks at a single refinery site; and (iii) a Burkholderia vietnamiensis strain present in two unrelated automotive diesel samples. Overall, aerobic cultivation of fuel contaminants recovered isolates broadly representative of the phyla and classes present but lacked accuracy by overrepresenting members of certain groups such as Pseudomonas.« less

Dynamics and Biodiversity of Populations of Lactic Acid Bacteria and Acetic Acid Bacteria Involved in Spontaneous Heap Fermentation of Cocoa Beans in Ghana▿

PubMed Central

Camu, Nicholas; De Winter, Tom; Verbrugghe, Kristof; Cleenwerck, Ilse; Vandamme, Peter; Takrama, Jemmy S.; Vancanneyt, Marc; De Vuyst, Luc

2007-01-01

The Ghanaian cocoa bean heap fermentation process was studied through a multiphasic approach, encompassing both microbiological and metabolite target analyses. A culture-dependent (plating and incubation, followed by repetitive-sequence-based PCR analyses of picked-up colonies) and culture-independent (denaturing gradient gel electrophoresis [DGGE] of 16S rRNA gene amplicons, PCR-DGGE) approach revealed a limited biodiversity and targeted population dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation. Four main clusters were identified among the LAB isolated: Lactobacillus plantarum, Lactobacillus fermentum, Leuconostoc pseudomesenteroides, and Enterococcus casseliflavus. Other taxa encompassed, for instance, Weissella. Only four clusters were found among the AAB identified: Acetobacter pasteurianus, Acetobacter syzygii-like bacteria, and two small clusters of Acetobacter tropicalis-like bacteria. Particular strains of L. plantarum, L. fermentum, and A. pasteurianus, originating from the environment, were well adapted to the environmental conditions prevailing during Ghanaian cocoa bean heap fermentation and apparently played a significant role in the cocoa bean fermentation process. Yeasts produced ethanol from sugars, and LAB produced lactic acid, acetic acid, ethanol, and mannitol from sugars and/or citrate. Whereas L. plantarum strains were abundant in the beginning of the fermentation, L. fermentum strains converted fructose into mannitol upon prolonged fermentation. A. pasteurianus grew on ethanol, mannitol, and lactate and converted ethanol into acetic acid. A newly proposed Weissella sp., referred to as “Weissella ghanaensis,” was detected through PCR-DGGE analysis in some of the fermentations and was only occasionally picked up through culture-based isolation. Two new species of Acetobacter were found as well, namely, the species tentatively named “Acetobacter senegalensis” (A. tropicalis-like) and “Acetobacter ghanaensis” (A. syzygii-like). PMID:17277227

Optimization of Plant Growth-Promoting Bacteria-Assisted Phytostabilization of Mine Tailings

PubMed Central

Grandlic, Christopher J.; Palmer, Michael W.; Maier, Raina M.

2009-01-01

Recent studies have indicated that plant growth-promoting bacteria (PGPB) can improve revegetation of arid mine tailings as measured by increased biomass production. The goals of the present study were first to evaluate how mode of application of known PGPB affects plant growth, and second to evaluate the effect of this inoculation on rhizosphere microbial community structure. PGPB application strategies investigated include preliminary surface sterilization of seeds (a common practice in phytoremediation trials) followed by a comparison of two application methods; immersion and alginate encapsulation. Results with two native desert plant species, Atriplex lentiformis and Buchloe dactyloides, suggest that seed surface sterilization prior to inoculation is not necessary to achieve beneficial effects of introduced PGPB. Both PGPB application techniques generally enhanced plant growth although results were both plant and PGPB specific. These results demonstrate that alginate encapsulation, which allows for long-term storage and easier application to seeds, is an effective way to inoculate PGPB. In addition, the influence of PGPB application on B. dactyloides rhizosphere community structure was evaluated using PCR-DGGE (denaturing gradient gel electrophoresis) analysis of bacterial DNA extracted from rhizosphere samples collected 75 d following planting. A comparative analysis of DGGE profiles was performed using canonical correspondence analysis (CCA). DGGE-CCA showed that rhizosphere community profiles from PGPB-inoculated treatments are significantly different from both uninoculated tailings rhizosphere profiles and profiles from the compost used to amend the tailings. Further, community profiles from B. dactyloides inoculated with the best performing PGPB (Arthro mix) were significantly different from two other PGPB tested. These results suggest that introduced PGPB have the potential to influence the development of the rhizosphere community structure found in plants grown in mine tailings. PMID:20161141

Molecular Diversity of Lactobacillus spp. and Other Lactic Acid Bacteria in the Human Intestine as Determined by Specific Amplification of 16S Ribosomal DNA

PubMed Central

Heilig, Hans G.H.J.; Zoetendal, Erwin G.; Vaughan, Elaine E.; Marteau, Philippe; Akkermans, Antoon D.L.; de Vos, Willem M.

2002-01-01

A Lactobacillus group-specific PCR primer, S-G-Lab-0677-a-A-17, was developed to selectively amplify 16S ribosomal DNA (rDNA) from lactobacilli and related lactic acid bacteria, including members of the genera Leuconostoc, Pediococcus, and Weissella. Amplicons generated by PCR from a variety of gastrointestinal (GI) tract samples, including those originating from feces and cecum, resulted predominantly in Lactobacillus-like sequences, of which ca. 28% were most similar to the 16S rDNA of Lactobacillus ruminis. Moreover, four sequences of Leuconostoc species were retrieved that, so far, have only been detected in environments other than the GI tract, such as fermented food products. The validity of the primer was further demonstrated by using Lactobacillus-specific PCR and denaturing gradient gel electrophoresis (DGGE) of the 16S rDNA amplicons of fecal and cecal origin from different age groups. The stability of the GI-tract bacterial community in different age groups over various time periods was studied. The Lactobacillus community in three adults over a 2-year period showed variation in composition and stability depending on the individual, while successional change of the Lactobacillus community was observed during the first 5 months of an infant’s life. Furthermore, the specific PCR and DGGE approach was tested to study the retention in fecal samples of a Lactobacillus strain administered during a clinical trial. In conclusion, the combination of specific PCR and DGGE analysis of 16S rDNA amplicons allows the diversity of important groups of bacteria that are present in low numbers in specific ecosystems to be characterized, such as the lactobacilli in the human GI tract. PMID:11772617

Diversity and functional analysis of bacterial communities associated with natural hydrocarbon seeps in acidic soils at Rainbow Springs, Yellowstone National Park.

PubMed

Hamamura, Natsuko; Olson, Sarah H; Ward, David M; Inskeep, William P

2005-10-01

In this paper we describe the bacterial communities associated with natural hydrocarbon seeps in nonthermal soils at Rainbow Springs, Yellowstone National Park. Soil chemical analysis revealed high sulfate concentrations and low pH values (pH 2.8 to 3.8), which are characteristic of acid-sulfate geothermal activity. The hydrocarbon composition of the seep soils consisted almost entirely of saturated, acyclic alkanes (e.g., n-alkanes with chain lengths of C15 to C30, as well as branched alkanes, predominately pristane and phytane). Bacterial populations present in the seep soils were phylogenetically characterized by 16S rRNA gene clone library analysis. The majority of the sequences recovered (>75%) were related to sequences of heterotrophic acidophilic bacteria, including Acidisphaera spp. and Acidiphilium spp. of the alpha-Proteobacteria. Clones related to the iron- and sulfur-oxidizing chemolithotroph Acidithiobacillus spp. were also recovered from one of the seep soils. Hydrocarbon-amended soil-sand mixtures were established to examine [14C]hexadecane mineralization and corresponding changes in the bacterial populations using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Approximately 50% of the [14C]hexadecane added was recovered as 14CO2 during an 80-day incubation, and this was accompanied by detection of heterotrophic acidophile-related sequences as dominant DGGE bands. An alkane-degrading isolate was cultivated, whose 16S rRNA gene sequence was identical to the sequence of a dominant DGGE band in the soil-sand mixture, as well as the clone sequence recovered most frequently from the original soil. This and the presence of an alkB gene homolog in this isolate confirmed the alkane degradation capability of one population indigenous to acidic hydrocarbon seep soils.

Association study of Demodex bacteria and facial dermatoses based on DGGE technique.

PubMed

Zhao, YaE; Yang, Fan; Wang, RuiLing; Niu, DongLing; Mu, Xin; Yang, Rui; Hu, Li

2017-03-01

The role of bacteria is unclear in the facial skin lesions caused by Demodex. To shed some light on this issue, we conducted a case-control study comparing cases with facial dermatoses with controls with healthy skin using denaturing gradient gel electrophoresis (DGGE) technique. The bacterial diversity, composition, and principal component were analyzed for Demodex bacteria and the matched facial skin bacteria. The result of mite examination showed that all 33 cases were infected with Demodex folliculorum (D. f), whereas 16 out of the 30 controls were infected with D. f, and the remaining 14 controls were infected with Demodex brevis (D. b). The diversity analysis showed that only evenness index presented statistical difference between mite bacteria and matched skin bacteria in the cases. The composition analysis showed that the DGGE bands of cases and controls were assigned to 12 taxa of 4 phyla, including Proteobacteria (39.37-52.78%), Firmicutes (2.7-26.77%), Actinobacteria (0-5.71%), and Bacteroidetes (0-2.08%). In cases, the proportion of Staphylococcus in Firmicutes was significantly higher than that in D. f controls and D. b controls, while the proportion of Sphingomonas in Proteobacteria was significantly lower than that in D. f controls. The between-group analysis (BGA) showed that all the banding patterns clustered into three groups, namely, D. f cases, D. f controls, and D. b controls. Our study suggests that the bacteria in Demodex should come from the matched facial skin bacteria. Proteobacteria and Firmicutes are the two main taxa. The increase of Staphylococcus and decrease of Sphingomonas might be associated with the development of facial dermatoses.

Microbial Dynamics during Aerobic Exposure of Corn Silage Stored under Oxygen Barrier or Polyethylene Films▿

PubMed Central

Dolci, Paola; Tabacco, Ernesto; Cocolin, Luca; Borreani, Giorgio

2011-01-01

The aims of this study were to compare the effects of sealing forage corn with a new oxygen barrier film with those obtained by using a conventional polyethylene film. This comparison was made during both ensilage and subsequent exposure of silage to air and included chemical, microbiological, and molecular (DNA and RNA) assessments. The forage was inoculated with a mixture of Lactobacillus buchneri, Lactobacillus plantarum, and Enterococcus faecium and ensiled in polyethylene (PE) and oxygen barrier (OB) plastic bags. The oxygen permeability of the PE and OB films was 1,480 and 70 cm3 m−2 per 24 h at 23°C, respectively. The silages were sampled after 110 days of ensilage and after 2, 5, 7, 9, and 14 days of air exposure and analyzed for fermentation characteristics, conventional microbial enumeration, and bacterial and fungal community fingerprinting via PCR-denaturing gradient gel electrophoresis (DGGE) and reverse transcription (RT)-PCR-DGGE. The yeast counts in the PE and OB silages were 3.12 and 1.17 log10 CFU g−1, respectively, with corresponding aerobic stabilities of 65 and 152 h. Acetobacter pasteurianus was present at both the DNA and RNA levels in the PE silage samples after 2 days of air exposure, whereas it was found only after 7 days in the OB silages. RT-PCR-DGGE revealed the activity of Aspergillus fumigatus in the PE samples from the day 7 of air exposure, whereas it appeared only after 14 days in the OB silages. It has been shown that the use of an oxygen barrier film can ensure a longer shelf life of silage after aerobic exposure. PMID:21821764

Does wheat genetically modified for disease resistance affect root-colonizing pseudomonads and arbuscular mycorrhizal fungi?

PubMed

Meyer, Joana Beatrice; Song-Wilson, Yi; Foetzki, Andrea; Luginbühl, Carolin; Winzeler, Michael; Kneubühler, Yvan; Matasci, Caterina; Mascher-Frutschi, Fabio; Kalinina, Olena; Boller, Thomas; Keel, Christoph; Maurhofer, Monika

2013-01-01

This study aimed to evaluate the impact of genetically modified (GM) wheat with introduced pm3b mildew resistance transgene, on two types of root-colonizing microorganisms, namely pseudomonads and arbuscular mycorrhizal fungi (AMF). Our investigations were carried out in field trials over three field seasons and at two locations. Serial dilution in selective King's B medium and microscopy were used to assess the abundance of cultivable pseudomonads and AMF, respectively. We developed a denaturing gradient gel electrophoresis (DGGE) method to characterize the diversity of the pqqC gene, which is involved in Pseudomonas phosphate solubilization. A major result was that in the first field season Pseudomonas abundances and diversity on roots of GM pm3b lines, but also on non-GM sister lines were different from those of the parental lines and conventional wheat cultivars. This indicates a strong effect of the procedures by which these plants were created, as GM and sister lines were generated via tissue cultures and propagated in the greenhouse. Moreover, Pseudomonas population sizes and DGGE profiles varied considerably between individual GM lines with different genomic locations of the pm3b transgene. At individual time points, differences in Pseudomonas and AMF accumulation between GM and control lines were detected, but they were not consistent and much less pronounced than differences detected between young and old plants, different conventional wheat cultivars or at different locations and field seasons. Thus, we conclude that impacts of GM wheat on plant-beneficial root-colonizing microorganisms are minor and not of ecological importance. The cultivation-independent pqqC-DGGE approach proved to be a useful tool for monitoring the dynamics of Pseudomonas populations in a wheat field and even sensitive enough for detecting population responses to altered plant physiology.

Variations in bacterial communities during foliar litter decomposition in the winter and growing seasons in an alpine forest of the eastern Tibetan Plateau.

PubMed

Zhao, Yeyi; Wu, Fuzhong; Yang, Wanqin; Tan, Bo; He, Wei

2016-01-01

Bacterial communities are the primary engineers during litter decomposition and related material cycling, and they can be strongly controlled by seasonal changes in temperature and other environmental factors. However, limited information is available on changes in the bacterial community from winter to the growing season as litter decomposition proceeds in cold climates. Here, we investigated the abundance and structure of bacterial communities using real-time quantitative PCR and denaturing gradient gel electrophoresis (DGGE) during a 2-year field study of the decomposition of litter of 4 species in the winter and growing seasons of an alpine forest of the eastern Tibetan Plateau. The abundance of the bacterial 16S rRNA gene was relatively high during decomposition of cypress and birch litter in the first winter, but for the other litters 16S rRNA abundance during both winters was significantly lower than during the following growing season. A large number of bands were observed on the DGGE gels, and their intensities and number from the winter samples were lower than those from the growing season during the 2-year decomposition experiment. Eighty-nine sequences from the bands of bacteria that had been cut from the DGGE gels were affiliated with 10 distinct classes of bacteria and an unknown group. A redundancy analysis indicated that the moisture, mass loss, and elemental content (e.g., C, N, and P) of the litter significantly affected the bacterial communities. Collectively, the results suggest that uneven seasonal changes in climate regulate bacterial communities and other decomposers, thus affecting their contribution to litter decomposition processes in the alpine forest.

Evaluation of soil bioremediation techniques in an aged diesel spill at the Antarctic Peninsula.

PubMed

de Jesus, Hugo E; Peixoto, Raquel S; Cury, Juliano C; van Elsas, Jan D; Rosado, Alexandre S

2015-12-01

Many areas on the Antarctic continent already suffer from the direct and indirect influences of human activities. The main cause of contamination is petroleum hydrocarbons because this compound is used as a source of energy at the many research stations around the continent. Thus, the current study aims to evaluate treatments for bioremediation (biostimulation, bioaugmentation, and bioaugmentation + biostimulation) using soils from around the Brazilian Antarctic Station "Comandante Ferraz" (EACF), King George Island, Antarctic Peninsula. The experiment lasted for 45 days, and at the end of this period, chemical and molecular analyses were performed. Those analyses included the quantification of carbon and nitrogen, denaturing gradient gel electrophoresis (DGGE) analysis (with gradient denaturation), real-time PCR, and quantification of total hydrocarbons and polyaromatics. Molecular tests evaluated changes in the profile and quantity of the rrs genes of archaea and bacteria and also the alkB gene. The influence of the treatments tested was directly related to the type of soil used. The work confirmed that despite the extreme conditions found in Antarctic soils, the bacterial strains degraded hydrocarbons and bioremediation treatments directly influenced the microbial communities present in these soils even in short periods. Although the majority of the previous studies demonstrate that the addition of fertilizer seems to be most effective at promoting bioremediation, our results show that for some conditions, autochthonous bioaugmentation (ABA) treatment is indicated. This work highlights the importance of understanding the processes of recovery of contaminated environments in polar regions because time is crucial to the soil recovery and to choosing the appropriate treatment.

Stability of a biogas-producing bacterial, archaeal and fungal community degrading food residues.

PubMed

Bengelsdorf, Frank R; Gerischer, Ulrike; Langer, Susanne; Zak, Manuel; Kazda, Marian

2013-04-01

The resident microbiota was analyzed in a mesophilic, continuously operating biogas plant predominantly utilizing food residues, stale bread, and other waste cosubstrates together with pig manure and maize silage. The dominating bacterial, archaeal, and eukaryotic community members were characterized by two different 16S/18S rRNA gene culture-independent approaches. Prokaryotic 16S rRNA gene and eukaryotic 18S rRNA gene clone libraries were constructed and further analyzed by restriction fragment length polymorphism (RFLP), 16S/18S rRNA gene sequencing, and phylogenetic tree reconstruction. The most dominant bacteria belonged to the phyla Bacteriodetes, Chloroflexus, and Firmicutes. On the family level, the bacterial composition confirmed high differences among biogas plants studied so fare. In contrast, the methanogenic archaeal community was similar to that of other studied biogas plants. Furthermore, it was possible to identify fungi at the genus level, namely Saccharomyces and Mucor. Both genera, which are important for microbial degradation of complex compounds, were up to now not found in biogas plants. The results revealed their long-term presence as indicated by denaturating gradient gel electrophoresis (DGGE). The DGGE method confirmed that the main members of the microbial community were constantly present over more than one-year period. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Bacterial community composition associated with freshwater algae: species specificity vs. dependency on environmental conditions and source community.

PubMed

Eigemann, Falk; Hilt, Sabine; Salka, Ivette; Grossart, Hans-Peter

2013-03-01

We studied bacterial associations with the green alga Desmodesmus armatus and the diatom Stephanodiscus minutulus under changing environmental conditions and bacterial source communities, to evaluate whether bacteria-algae associations are species-specific or more generalized and determined by external factors. Axenic and xenic algae were incubated in situ with and without allelopathically active macrophytes, and in the laboratory with sterile and nonsterile lake water and an allelochemical, tannic acid (TA). Bacterial community composition (BCC) of algae-associated bacteria was analyzed by denaturing gradient gel electrophoresis (DGGE), nonmetric multidimensional scaling, cluster analyses, and sequencing of DGGE bands. BCC of xenic algal cultures of both species were not significantly affected by changes in their environment or bacterial source community, except in the case of TA additions. Species-specific interactions therefore appear to overrule the effects of environmental conditions and source communities. The BCC of xenic and axenic D. armatus cultures subjected to in situ bacterial colonization, however, had lower similarities (ca. 55%), indicating that bacterial precolonization is a strong factor for bacteria-algae associations irrespective of environmental conditions and source community. Our findings emphasize the ecological importance of species-specific bacteria-algae associations with important repercussions for other processes, such as the remineralization of nutrients, and organic matter dynamics. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

The influence of nickel on the bioremediation of multi-component contaminated tropical soil: microcosm and batch bioreactor studies.

PubMed

Taketani, Natália Franco; Taketani, Rodrigo Gouvêa; Leite, Selma Gomes Ferreira; Rizzo, Andrea Camardella de Lima; Tsai, Siu Mui; da Cunha, Cláudia Duarte

2015-07-01

Large petrochemical discharges are responsible for organic and inorganic pollutants in the environment. The purpose of this study was to evaluate the influence of nickel, one of the most abundant inorganic element in crude oil and the main component of hydrogen catalysts for oil refining, on the microbial community structure in artificially petroleum-contaminated microcosms and in solid phase bioreactor studies. In the presence of metals, the oil biodegradation in microcosms was significantly delayed during the first 7 days of operation. Also, increasing amounts of moisture generated a positive influence on the biodegradation processes. The oil concentration, exhibiting the most negative influence at the end of the treatment period. Molecular fingerprinting analyses (denaturing gradient gel electrophoresis--DGGE) indicated that the inclusion of nickel into the contaminated soil promoted direct changes to the microbial community structure. By the end of the experiments, the results of the total petroleum hydrocarbons removal in the bioreactor and the microcosm were similar, but reductions in the treatment times were observed with the bioreactor experiments. An analysis of the microbial community structure by DGGE using various markers showed distinct behaviors between two treatments containing high nickel concentrations. The main conclusion of this study was that Nickel promotes a significant delay in oil biodegradation, despite having only a minor effect over the microbial community.

Metagenomic analyses of the dominant bacterial community in the Fildes Peninsula, King George Island (South Shetland Islands)

NASA Astrophysics Data System (ADS)

Foong, Choon Pin; Wong Vui Ling, Clemente Michael; González, Marcelo

2010-08-01

There is little information on the bacterial diversity of the Fildes Peninsula, King George Island. Hence, this study was conducted to determine the bacterial population of sediments and soils from the lakes, river, glacier and an abandoned oil tank area in the Fildes Peninsula, using a metagenomic approach. DNA was extracted from the sediment and soil samples, and analyzed using the 16S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). A total of 299 DNA fragments resolved using the DGGE were sequenced. The results of the analysis provided an overview of the predominant groups of bacteria and the diversity of the bacterial communities. The most abundant phyla of bacteria in Fildes Peninsula were Bacteroidetes, Proteobacteria, Acidobacteria, Gemmatimonadetes, Nitrospira, Firmicutes, Actinobacteria, Chloroflexi, Cyanobacteria, Spirochaetes, Deinococcus-Thermus, WS3 and BRC1. All of the sediment samples from the lakes had different representatives of dominant bacterial species. Interestingly, 15% of the operational taxonomic units (OTUs) did not group into any of the existing phyla in the Ribosomal Database Project (RDP). One of the OTUs had a similarity of <0.90 when compared to the GenBank sequences and probably was a novel bacterium specific to that location. The majority of the bacterial 16S rDNA sequences were found to be closely related to those found elsewhere.

Bacterial diversity of field-caught mosquitoes, Aedes albopictus and Aedes aegypti, from different geographic regions of Madagascar.

PubMed

Zouache, Karima; Raharimalala, Fara Nantenaina; Raquin, Vincent; Tran-Van, Van; Raveloson, Lala Harivelo Ravaomanarivo; Ravelonandro, Pierre; Mavingui, Patrick

2011-03-01

Symbiotic bacteria are known to play important roles in the biology of insects, but the current knowledge of bacterial communities associated with mosquitoes is very limited and consequently their contribution to host behaviors is mostly unknown. In this study, we explored the composition and diversity of mosquito-associated bacteria in relation with mosquitoes' habitats. Wild Aedes albopictus and Aedes aegypti were collected in three different geographic regions of Madagascar. Culturing methods and denaturing gradient gel electrophoresis (DGGE) and sequencing of the rrs amplicons revealed that Proteobacteria and Firmicutes were the major phyla. Isolated bacterial genera were dominated by Bacillus, followed by Acinetobacter, Agrobacterium and Enterobacter. Common DGGE bands belonged to Acinetobacter, Asaia, Delftia, Pseudomonas, Enterobacteriaceae and an uncultured Gammaproteobacterium. Double infection by maternally inherited Wolbachia pipientis prevailed in 98% of males (n=272) and 99% of females (n=413); few individuals were found to be monoinfected with Wolbachia wAlbB strain. Bacterial diversity (Shannon-Weaver and Simpson indices) differed significantly per habitat whereas evenness (Pielou index) was similar. Overall, the bacterial composition and diversity were influenced both by the sex of individuals and by the environment inhabited by the mosquitoes; the latter might be related to both the vegetation and the animal host populations that Aedes used as food sources. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

PCR-DGGE Analysis on Microbial Community Structure of Rural Household Biogas Digesters in Qinghai Plateau.

PubMed

Han, Rui; Yuan, Yongze; Cao, Qianwen; Li, Quanhui; Chen, Laisheng; Zhu, Derui; Liu, Deli

2018-05-01

To investigate contribution of environmental factor(s) to microbial community structure(s) involved in rural household biogas fermentation at Qinghai Plateau, we collected slurry samples from 15 digesters, with low-temperature working conditions (11.1-15.7 °C) and evenly distributed at three counties (Datong, Huangyuan, and Ledu) with cold plateau climate, to perform polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and further sequencing. The bacterial communities in the total 15 digesters were classified into 38 genera with Mangroviflexus (12.1%) as the first dominant, and the archaeal communities into ten genera with Methanogenium (38.5%) as the most dominant. For each county, the digesters with higher biogas production, designated as HP digesters, exclusively had 1.6-3.1 °C higher fermentation temperature and the unique bacterial structure composition related, i.e., unclassified Clostridiales for all the HP digesters and unclassified Marinilabiliaceae and Proteiniclasticum for Ledu HP digesters. Regarding archaeal structure composition, Methanogenium exhibited significantly higher abundances at all the HP digesters and Thermogymnomonas was the unique species only identified at Ledu HP digesters with higher-temperature conditions. Redundancy analysis also confirmed the most important contribution of temperature to the microbial community structures investigated. This report emphasized the correlation between temperature and specific microbial community structure(s) that would benefit biogas production of rural household digesters at Qinghai Plateau.

Comparison of bacterial community structure and dynamics during the thermophilic composting of different types of solid wastes: anaerobic digestion residue, pig manure and chicken manure.

PubMed

Song, Caihong; Li, Mingxiao; Jia, Xuan; Wei, Zimin; Zhao, Yue; Xi, Beidou; Zhu, Chaowei; Liu, Dongming

2014-09-01

This study investigated the impact of composting substrate types on the bacterial community structure and dynamics during composting processes. To this end, pig manure (PM), chicken manure (CM), a mixture of PM and CM (PM + CM), and a mixture of PM, CM and anaerobic digestion residue (ADR) (PM + CM + ADR) were selected for thermophilic composting. The bacterial community structure and dynamics during the composting process were detected and analysed by polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) coupled with a statistic analysis. The physical-chemical analyses indicated that compared to single-material composting (PM, CM), co-composting (PM + CM, PM + CM + ADR) could promote the degradation of organic matter and strengthen the ability of conserving nitrogen. A DGGE profile and statistical analysis demonstrated that co-composting, especially PM + CM + ADR, could improve the bacterial community structure and functional diversity, even in the thermophilic stage. Therefore, co-composting could weaken the screening effect of high temperature on bacterial communities. Dominant sequencing analyses indicated a dramatic shift in the dominant bacterial communities from single-material composting to co-composting. Notably, compared with PM, PM + CM increased the quantity of xylan-degrading bacteria and reduced the quantity of human pathogens. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Establishment of intestinal microbiota with focus on yeasts of unweaned and weaned piglets kept under different farm conditions.

PubMed

Urubschurov, Vladimir; Janczyk, Pawel; Souffrant, Wolfgang-Bernhard; Freyer, Gertraude; Zeyner, Annette

2011-09-01

This study aimed to characterize the intestinal yeasts in weaning piglets and to establish their possible relationships with main bacterial groups. German Landrace piglets were weaned (WP, n=32) at 28 days of age or kept with the dams until day 39 without creep feed (UP, n=32). The experiment was performed at an experimental and a commercial farm (CF). Faeces were collected from the piglets, sows and pen floors on days 28, 33 and 39 for isolation of DNA and cultivation for enumeration of yeasts, enterobacteria, enterococci and lactobacilli. Fragments of the D1 domain of 26S rRNA gene were amplified and separated by denaturing gradient gel electrophoresis (DGGE). No yeasts could be cultured from water and feed samples. No or only low numbers of yeasts were detected among all UP. In WP at CF, yeasts correlated with lactobacilli (r=0.456; P=0.009) and enterobacteria (r=-0.407; P=0.021). Kazachstania slooffiae dominated among the cultured yeasts. It was the only yeast species detected by PCR-DGGE. Yeasts, especially K. slooffiae, established in the porcine gastrointestinal tract after consumption of grain-based feed and may interrelate with the intestinal microbiota. The study provides data indicating importance of K. slooffiae for the development of balanced porcine gut microbiota. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Microbial diversity associated with odor modification for production of fertilizers from chicken litter.

PubMed

Enticknap, Julie J; Nonogaki, Hirofumi; Place, Allen R; Hill, Russell T

2006-06-01

Litter from the chicken industry can present several environmental challenges, including offensive odors and runoff into waterways leading to eutrophication. An economically viable solution to the disposal of waste from chicken houses is treatment to produce a natural, granulated fertilizer that can be commercially marketed for garden and commercial use. Odor of the final product is important in consumer acceptance, and an earthy odor is desirable. By understanding and manipulating the microbial processes occurring during this process, it may be possible to modify the odors produced. Geosmin and related volatiles produced by soil actinomycetes are responsible for earthy odors, and actinomycetes are likely to be present in the composting manure. Bacterial communities at each stage of the process were analyzed by culturing studies and denaturing gradient gel electrophoresis (DGGE). The processing steps changed the culturable bacterial community, but the total community was shown by DGGE to be stable throughout the process. A local agricultural soil was analyzed in parallel as a potential source of geosmin-producing actinomycetes. This agricultural soil had higher microbial diversity than the compost at both the culturable and the molecular levels. Actinomycete bacteria were isolated and analyzed by AromaTrax, a gas chromatography-olfactometry system. This system enables the odor production of individual isolates to be monitored, allowing for rational selection of strains for augmentation experiments to improve the odor of the final fertilizer product.

Association of marine viral and bacterial communities with reference black carbon particles under experimental conditions: an analysis with scanning electron, epifluorescence and confocal laser scanning microscopy.

PubMed

Cattaneo, Raffaela; Rouviere, Christian; Rassoulzadegan, Fereidoun; Weinbauer, Markus G

2010-11-01

Black carbon (BC), the product of incomplete combustion of fossil fuels and biomass, constitutes a significant fraction of the marine organic carbon pool. However, little is known about the possible interactions of BC and marine microorganisms. Here, we report the results of experiments using a standard reference BC material in high concentrations to investigate basic principles of the dynamics of natural bacterial and viral communities with BC particles. We assessed the attachment of viral and bacterial communities using scanning electron, epifluorescence and confocal laser scanning microscopy and shifts in bacterial community composition using 16S rRNA gene denaturing gradient gel electrophoresis (DGGE). In 24-h time-course experiments, BC particles showed a strong potential for absorbing viruses and bacteria. Total viral abundance was reduced, whereas total bacterial abundance was stimulated in the BC treatments. Viral and bacterial abundance on BC particles increased with particle size, whereas the abundances of BC-associated viruses and bacteria per square micrometer surface area decreased significantly with BC particle size. DGGE results suggested that BC has the potential to change bacterial community structure and favour phylotypes related to Glaciecola sp. Our study indicates that BC could influence processes mediated by bacteria and viruses in marine ecosystems. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Comparative study of rhizobacterial community structure of plant species in oil-contaminated soil.

PubMed

Lee, Eun-Hee; Cho, Kyong-Suk; Kim, Jaisoo

2010-09-01

In this study, the identity and distribution of plants and the structure of their associated rhizobacterial communities were examined in an oil-contaminated site. The number of plant species that formed a community or were scattered was 24. The species living in soil highly contaminated with total petroleum hydrocarbon (TPH) (9,000-4,5000 mg/g-soil) were Cynodon dactylon, Persicaria lapathifolia, and Calystegia soldanella (a halophytic species). Among the 24 plant species, the following have been known to be effective for oil removal: C. dactylon, Digitaria sanguinalis, and Cyperus orthostachyus. Denaturing gradient gel electrophoresis (DGGE) profile analysis showed that the following pairs of plant species had highly similar (above 70%) rhizobacterial community structures: Artemisia princeps and Hemistepta lyrata; C. dactylon and P. lapathifolia; Carex kobomugi and Cardamine flexuosa; and Equisetum arvense and D. sanguinalis. The major groups of rhizobacteria were Betaproteobacteria, Gamma-proteobacteria, Chloroflexi, Actinobacteria, and unknown. Based on DGGE analysis, P. lapathifolia, found for the first time in this study growing in the presence of high TPH, may be a good species for phytoremediation of oil-contaminated soils and in particular, C. soldanella may be useful for soils with high TPH and salt concentrations. Overall, this study suggests that the plant roots, regardless of plant species, may have a similar influence on the bacterial community structure in oil-contaminated soil.

Dynamics of Bacterial Communities in Two Unpolluted Soils after Spiking with Phenanthrene: Soil Type Specific and Common Responders

PubMed Central

Ding, Guo-Chun; Heuer, Holger; Smalla, Kornelia

2012-01-01

Considering their key role for ecosystem processes, it is important to understand the response of microbial communities in unpolluted soils to pollution with polycyclic aromatic hydrocarbons (PAH). Phenanthrene, a model compound for PAH, was spiked to a Cambisol and a Luvisol soil. Total community DNA from phenanthrene-spiked and control soils collected on days 0, 21, and 63 were analyzed based on PCR-amplified 16S rRNA gene fragments. Denaturing gradient gel electrophoresis (DGGE) fingerprints of bacterial communities increasingly deviated with time between spiked and control soils. In taxon specific DGGE, significant responses of Alphaproteobacteria and Actinobacteria became only detectable after 63 days, while significant effects on Betaproteobacteria were detectable in both soils after 21 days. Comparison of the taxonomic distribution of bacteria in spiked and control soils on day 63 as revealed by pyrosequencing indicated soil type specific negative effects of phenanthrene on several taxa, many of them belonging to the Gamma-, Beta-, or Deltaproteobacteria. Bacterial richness and evenness decreased in spiked soils. Despite the significant differences in the bacterial community structure between both soils on day 0, similar genera increased in relative abundance after PAH spiking, especially Sphingomonas and Polaromonas. However, this did not result in an increased overall similarity of the bacterial communities in both soils. PMID:22934091

Airborne myxomycete spores: detection using molecular techniques

NASA Astrophysics Data System (ADS)

Kamono, Akiko; Kojima, Hisaya; Matsumoto, Jun; Kawamura, Kimitaka; Fukui, Manabu

2009-01-01

Myxomycetes are organisms characterized by a life cycle that includes a fruiting body stage. Myxomycete fruiting bodies contain spores, and wind dispersal of the spores is considered important for this organism to colonize new areas. In this study, the presence of airborne myxomycetes and the temporal changes in the myxomycete composition of atmospheric particles (aerosols) were investigated with a polymerase chain reaction (PCR)-based method for Didymiaceae and Physaraceae. Twenty-one aerosol samples were collected on the roof of a three-story building located in Sapporo, Hokkaido Island, northern Japan. PCR analysis of DNA extracts from the aerosol samples indicated the presence of airborne myxomycetes in all the samples, except for the one collected during the snowfall season. Denaturing gradient gel electrophoresis (DGGE) analysis of the PCR products showed seasonally varying banding patterns. The detected DGGE bands were subjected to sequence analyses, and four out of nine obtained sequences were identical to those of fruiting body samples collected in Hokkaido Island. It appears that the difference in the fruiting period of each species was correlated with the seasonal changes in the myxomycete composition of the aerosols. Molecular evidence shows that newly formed spores are released and dispersed in the air, suggesting that wind-driven dispersal of spores is an important process in the life history of myxomycetes. This study is the first to detect airborne myxomycetes with the use of molecular ecological analyses and to characterize their seasonal distribution.

Effects of C/N ratio on nitrous oxide production from nitrification in a laboratory-scale biological aerated filter reactor.

PubMed

He, Qiang; Zhu, Yinying; Fan, Leilei; Ai, Hainan; Huangfu, Xiaoliu; Chen, Mei

2017-03-01

Emission of nitrous oxide (N 2 O) during biological wastewater treatment is of growing concern. This paper reports findings of the effects of carbon/nitrogen (C/N) ratio on N 2 O production rates in a laboratory-scale biological aerated filter (BAF) reactor, focusing on the biofilm during nitrification. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and microelectrode technology were utilized to evaluate the mechanisms associated with N 2 O production during wastewater treatment using BAF. Results indicated that the ability of N 2 O emission in biofilm at C/N ratio of 2 was much stronger than at C/N ratios of 5 and 8. PCR-DGGE analysis showed that the microbial community structures differed completely after the acclimatization at tested C/N ratios (i.e., 2, 5, and 8). Measurements of critical parameters including dissolved oxygen, oxidation reduction potential, NH 4 + -N, NO 3 - -N, and NO 2 - -N also demonstrated that the internal micro-environment of the biofilm benefit N 2 O production. DNA analysis showed that Proteobacteria comprised the majority of the bacteria, which might mainly result in N 2 O emission. Based on these results, C/N ratio is one of the parameters that play an important role in the N 2 O emission from the BAF reactors during nitrification.

Comparative analysis of fecal microbiota and intestinal microbial metabolic activity in captive polar bears.

PubMed

Schwab, Clarissa; Gänzle, Michael

2011-03-01

The composition of the intestinal microbiota depends on gut physiology and diet. Ursidae possess a simple gastrointestinal system composed of a stomach, small intestine, and indistinct hindgut. This study determined the composition and stability of fecal microbiota of 3 captive polar bears by group-specific quantitative PCR and PCR-DGGE (denaturing gradient gel electrophoresis) using the 16S rRNA gene as target. Intestinal metabolic activity was determined by analysis of short-chain fatty acids in feces. For comparison, other Carnivora and mammals were included in this study. Total bacterial abundance was approximately log 8.5 DNA gene copies·(g feces)-1 in all 3 polar bears. Fecal polar bear microbiota was dominated by the facultative anaerobes Enterobacteriaceae and enterococci, and the Clostridium cluster I. The detection of the Clostridium perfringens α-toxin gene verified the presence of C. perfringens. Composition of the fecal bacterial population was stable on a genus level; according to results obtained by PCR-DGGE, dominant bacterial species fluctuated. The total short-chain fatty acid content of Carnivora and other mammals analysed was comparable; lactate was detected in feces of all carnivora but present only in trace amounts in other mammals. In comparison, the fecal microbiota and metabolic activity of captive polar bears mostly resembled the closely related grizzly and black bears.

Combination of culture-independent and culture-dependent molecular methods for the determination of bacterial community of iru, a fermented Parkia biglobosa seeds.

PubMed

Adewumi, Gbenga A; Oguntoyinbo, Folarin A; Keisam, Santosh; Romi, Wahengbam; Jeyaram, Kumaraswamy

2012-01-01

In this study, bacterial composition of iru produced by natural, uncontrolled fermentation of Parkia biglobosa seeds was assessed using culture-independent method in combination with culture-based genotypic typing techniques. PCR-denaturing gradient gel electrophoresis (DGGE) revealed similarity in DNA fragments with the two DNA extraction methods used and confirmed bacterial diversity in the 16 iru samples from different production regions. DNA sequencing of the highly variable V3 region of the 16S rRNA genes obtained from PCR-DGGE identified species related to Bacillus subtilis as consistent bacterial species in the fermented samples, while other major bands were identified as close relatives of Staphylococcus vitulinus, Morganella morganii, B. thuringiensis, S. saprophyticus, Tetragenococcus halophilus, Ureibacillus thermosphaericus, Brevibacillus parabrevis, Salinicoccus jeotgali, Brevibacterium sp. and uncultured bacteria clones. Bacillus species were cultured as potential starter cultures and clonal relationship of different isolates determined using amplified ribosomal DNA restriction analysis (ARDRA) combined with 16S-23S rRNA gene internal transcribed spacer (ITS) PCR amplification, restriction analysis (ITS-PCR-RFLP), and randomly amplified polymorphic DNA (RAPD-PCR). This further discriminated B. subtilis and its variants from food-borne pathogens such as B. cereus and suggested the need for development of controlled fermentation processes and good manufacturing practices (GMP) for iru production to achieve product consistency, safety quality, and improved shelf life.

Biodiversity of thermophilic prokaryotes with hydrolytic activities in hot springs of Uzon Caldera, Kamchatka (Russia).

PubMed

Kublanov, Ilya V; Perevalova, Anna A; Slobodkina, Galina B; Lebedinsky, Aleksander V; Bidzhieva, Salima K; Kolganova, Tatyana V; Kaliberda, Elena N; Rumsh, Lev D; Haertlé, Thomas; Bonch-Osmolovskaya, Elizaveta A

2009-01-01

Samples of water from the hot springs of Uzon Caldera with temperatures from 68 to 87 degrees C and pHs of 4.1 to 7.0, supplemented with proteinaceous (albumin, casein, or alpha- or beta-keratin) or carbohydrate (cellulose, carboxymethyl cellulose, chitin, or agarose) biological polymers, were filled with thermal water and incubated at the same sites, with the contents of the tubes freely accessible to the hydrothermal fluid. As a result, several enrichment cultures growing in situ on different polymeric substrates were obtained. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments obtained after PCR with Bacteria-specific primers showed that the bacterial communities developing on carbohydrates included the genera Caldicellulosiruptor and Dictyoglomus and that those developing on proteins contained members of the Thermotogales order. DGGE analysis performed after PCR with Archaea- and Crenarchaeota-specific primers showed that archaea related to uncultured environmental clones, particularly those of the Crenarchaeota phylum, were present in both carbohydrate- and protein-degrading communities. Five isolates obtained from in situ enrichments or corresponding natural samples of water and sediments represented the bacterial genera Dictyoglomus and Caldanaerobacter as well as new archaea of the Crenarchaeota phylum. Thus, in situ enrichment and consequent isolation showed the diversity of thermophilic prokaryotes competing for biopolymers in microbial communities of terrestrial hot springs.

[Composition diversity of lactic acid bacteria (LAB) community Al2 used for alfalfa silage].

PubMed

Wang, Xiao-Fen; Gao, Li-Juan; Yang, Hong-Yan; Wang, Wei-Dong; Cui, Zong-Jun

2006-10-01

Alfalfa is the most important forage grass that is difficult to ensile for good quality. Using silage inoculants are the important way for preservation of alfalfa silage. Through continuous restricted subcultivation, a lactic acid bacteria (LAB) community Al2 was selected from well-fermented alfalfa silage. Plate isolation and Denaturing Gradient Gel Electrophoresis (DGGE), construction of 16S rDNA clone library were used to identify the composition diversity of Al2 community, with 7 strains detected, and they were all belonged to Lactobacillus. The composition ratios of the 7 strains were 55.21%, 19.79%, 14.58%, 3.13%, 3.13%, 3.13%, 1.03% according to 16S rDNA clone library. Al2-1i, Al2-2i, Al2-3i, corresponding to L. plantarum (99.9%), L. kimchii (99.4%), L. farciminis (100%) were detected by plate isolation. Among 3 isolates, Al2-1i had the highest ability of dropping pH and producing lactic acid, and the amount of lactic acid was reach to 18g/L at 24h cultivated in MRS media. The ability of dropping pH and producing lactic acid of Al2-3i was the lowest. From DGGE profiles, the dominant strains in Al2 community were L. plantarum and L. kimchii. L. plantarum was detected during the whole process, and L. kimchii was detected in the later phase.

Characterisation of the microbiota of rice sourdoughs and description of Lactobacillus spicheri sp. nov.

PubMed

Meroth, Christiane B; Hammes, Walter P; Hertel, Christian

2004-03-01

The microbiota of two industrially processed rice sourdoughs was characterised by bacteriological culture in combination with PCR-denaturing gradient gel electrophoresis (DGGE) and 16S/28S rDNA sequence analysis. Rice sourdough I was continuously propagated for several years by back-slopping every week, whereas sourdough II was processed by using a commercial starter culture and back-slopping daily for three days. In rice sourdough II Candida krusei and Saccharomyces cerevisiae as well as Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus kimchii, Lactobacillus plantarum, and Lactobacillus pontis dominated at the first day of fermentation. RAPD analysis of lactobacilli revealed identical profiles for each of the species except for L. fermentum and L. pontis indicating the presence of different strains. Fluctuations within the LAB community during fermentation were monitored by PCR-DGGE. L. pontis decreased in numbers over time and L. curvatus became dominant after 3 days of fermentation. Rice sourdough I contained S. cerevisiae, Lactobacillus paracasei (present with three different RAPD types), Lactobacillus paralimentarius, and a Lactobacillus strain which could not be allotted to any valid species. Phylogenetic analysis based on 16S rDNA sequences revealed Lactobacillus brevis as the closest relative (97.3% sequence similarity). Differences in some phenotypic characteristics and DNA-DNA relatedness indicated that the strain represents a new Lactobacillus species, for which the name Lactobacillus spicheri is proposed.

Microbial Diversity Associated with Odor Modification for Production of Fertilizers from Chicken Litter†

PubMed Central

Enticknap, Julie J.; Nonogaki, Hirofumi; Place, Allen R.; Hill, Russell T.

2006-01-01

Litter from the chicken industry can present several environmental challenges, including offensive odors and runoff into waterways leading to eutrophication. An economically viable solution to the disposal of waste from chicken houses is treatment to produce a natural, granulated fertilizer that can be commercially marketed for garden and commercial use. Odor of the final product is important in consumer acceptance, and an earthy odor is desirable. By understanding and manipulating the microbial processes occurring during this process, it may be possible to modify the odors produced. Geosmin and related volatiles produced by soil actinomycetes are responsible for earthy odors, and actinomycetes are likely to be present in the composting manure. Bacterial communities at each stage of the process were analyzed by culturing studies and denaturing gradient gel electrophoresis (DGGE). The processing steps changed the culturable bacterial community, but the total community was shown by DGGE to be stable throughout the process. A local agricultural soil was analyzed in parallel as a potential source of geosmin-producing actinomycetes. This agricultural soil had higher microbial diversity than the compost at both the culturable and the molecular levels. Actinomycete bacteria were isolated and analyzed by AromaTrax, a gas chromatography-olfactometry system. This system enables the odor production of individual isolates to be monitored, allowing for rational selection of strains for augmentation experiments to improve the odor of the final fertilizer product. PMID:16751521

Rapid intrinsic biodegradation of benzene, toluene, and xylenes at the boundary of a gasoline-contaminated plume under natural attenuation.

PubMed

Takahata, Yoh; Kasai, Yuki; Hoaki, Toshihiro; Watanabe, Kazuya

2006-12-01

A groundwater plume contaminated with gasoline constituents [mainly benzene, toluene, and xylenes (BTX)] had been treated by pumping and aeration for approximately 10 years, and the treatment strategy was recently changed to monitored natural attenuation (MNA). To gain information on the feasibility of using MNA to control the spread of BTX, chemical and microbiological parameters in groundwater samples obtained inside and outside the contaminated plume were measured over the course of 73 weeks. The depletion of electron acceptors (i.e., dissolved oxygen, nitrate, and sulfate) and increase of soluble iron were observed in the contaminated zone. Laboratory incubation tests revealed that groundwater obtained immediately outside the contaminated zone (the boundary zone) exhibited much higher potential for BTX degradation than those in the contaminated zone and in uncontaminated background zones. The boundary zone was a former contaminated area where BTX were no longer detected. Denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction (PCR)-amplified bacterial 16S rRNA gene fragments revealed that DGGE profiles for groundwater samples obtained from the contaminated zone were clustered together and distinct from those from uncontaminated zones. In addition, unique bacterial rRNA types were observed in the boundary zone. These results indicate that the boundary zone in the contaminant plumes served as a natural barrier for preventing the BTX contamination from spreading out.

Growth of ammonia-oxidizing archaea in soil microcosms is inhibited by acetylene.

PubMed

Offre, Pierre; Prosser, James I; Nicol, Graeme W

2009-10-01

Autotrophic ammonia-oxidizing bacteria were considered to be responsible for the majority of ammonia oxidation in soil until the recent discovery of the autotrophic ammonia-oxidizing archaea. To assess the relative contributions of bacterial and archaeal ammonia oxidizers to soil ammonia oxidation, their growth was analysed during active nitrification in soil microcosms incubated for 30 days at 30 degrees C, and the effect of an inhibitor of ammonia oxidation (acetylene) on their growth and soil nitrification kinetics was determined. Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial ammonia oxidizer 16S rRNA genes did not detect any change in their community composition during incubation, and quantitative PCR (qPCR) analysis of bacterial amoA genes indicated a small decrease in abundance in control and acetylene-containing microcosms. DGGE fingerprints of archaeal amoA and 16S rRNA genes demonstrated changes in the relative abundance of specific crenarchaeal phylotypes during active nitrification. Growth was also indicated by increases in crenarchaeal amoA gene copy number, determined by qPCR. In microcosms containing acetylene, nitrification and growth of the crenarchaeal phylotypes were suppressed, suggesting that these crenarchaea are ammonia oxidizers. Growth of only archaeal but not bacterial ammonia oxidizers occurred in microcosms with active nitrification, indicating that ammonia oxidation was mostly due to archaea in the conditions of the present study.

Abundance and Genetic Diversity of nifH Gene Sequences in Anthropogenically Affected Brazilian Mangrove Sediments

PubMed Central

Dias, Armando Cavalcante Franco; Pereira e Silva, Michele de Cassia; Cotta, Simone Raposo; Dini-Andreote, Francisco; Soares, Fábio Lino; Salles, Joana Falcão; Azevedo, João Lúcio; van Elsas, Jan Dirk

2012-01-01

Although mangroves represent ecosystems of global importance, the genetic diversity and abundance of functional genes that are key to their functioning scarcely have been explored. Here, we present a survey based on the nifH gene across transects of sediments of two mangrove systems located along the coast line of São Paulo state (Brazil) which differed by degree of disturbance, i.e., an oil-spill-affected and an unaffected mangrove. The diazotrophic communities were assessed by denaturing gradient gel electrophoresis (DGGE), quantitative PCR (qPCR), and clone libraries. The nifH gene abundance was similar across the two mangrove sediment systems, as evidenced by qPCR. However, the nifH-based PCR-DGGE profiles revealed clear differences between the mangroves. Moreover, shifts in the nifH gene diversities were noted along the land-sea transect within the previously oiled mangrove. The nifH gene diversity depicted the presence of nitrogen-fixing bacteria affiliated with a wide range of taxa, encompassing members of the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Firmicutes, and also a group of anaerobic sulfate-reducing bacteria. We also detected a unique mangrove-specific cluster of sequences denoted Mgv-nifH. Our results indicate that nitrogen-fixing bacterial guilds can be partially endemic to mangroves, and these communities are modulated by oil contamination, which has important implications for conservation strategies. PMID:22941088

Abundance and genetic diversity of nifH gene sequences in anthropogenically affected Brazilian mangrove sediments.

PubMed

Dias, Armando Cavalcante Franco; Pereira e Silva, Michele de Cassia; Cotta, Simone Raposo; Dini-Andreote, Francisco; Soares, Fábio Lino; Salles, Joana Falcão; Azevedo, João Lúcio; van Elsas, Jan Dirk; Andreote, Fernando Dini

2012-11-01

Although mangroves represent ecosystems of global importance, the genetic diversity and abundance of functional genes that are key to their functioning scarcely have been explored. Here, we present a survey based on the nifH gene across transects of sediments of two mangrove systems located along the coast line of São Paulo state (Brazil) which differed by degree of disturbance, i.e., an oil-spill-affected and an unaffected mangrove. The diazotrophic communities were assessed by denaturing gradient gel electrophoresis (DGGE), quantitative PCR (qPCR), and clone libraries. The nifH gene abundance was similar across the two mangrove sediment systems, as evidenced by qPCR. However, the nifH-based PCR-DGGE profiles revealed clear differences between the mangroves. Moreover, shifts in the nifH gene diversities were noted along the land-sea transect within the previously oiled mangrove. The nifH gene diversity depicted the presence of nitrogen-fixing bacteria affiliated with a wide range of taxa, encompassing members of the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Firmicutes, and also a group of anaerobic sulfate-reducing bacteria. We also detected a unique mangrove-specific cluster of sequences denoted Mgv-nifH. Our results indicate that nitrogen-fixing bacterial guilds can be partially endemic to mangroves, and these communities are modulated by oil contamination, which has important implications for conservation strategies.

Characterisation of prototype Nurmi cultures using culture-based microbiological techniques and PCR-DGGE.

PubMed

Waters, Sinéad M; Murphy, Richard A; Power, Ronan F G

2006-08-01

Undefined Nurmi-type cultures (NTCs) have been used successfully to prevent salmonella colonisation in poultry for decades. Such cultures are derived from the caecal contents of specific-pathogen-free birds and are administered via drinking water or spray application onto eggs in the hatchery. These cultures consist of many non-culturable and obligately anaerobic bacteria. Due to their undefined nature it is difficult to obtain approval from regulatory agencies to use these preparations as direct fed microbials for poultry. In this study, 10 batches of prototype NTCs were produced using an identical protocol over a period of 2 years. Traditional microbiological techniques and a molecular culture-independent methodology, polymerase chain reaction combined with denaturing gradient gel electrophoresis (PCR-DGGE), were applied to characterise these cultures and also to examine if the constituents of the NTCs were identical. Culture-dependent analysis of these cultures included plating on a variety of selective and semi-selective agars, examination of colony morphology, Gram-staining and a series of biochemical tests (API, BioMerieux, France). Two sets of PCR-DGGE studies were performed. These involved the amplification of universal and subsequently lactic acid bacteria (LAB)-specific hypervariable regions of a 16S rRNA gene by PCR. Resultant amplicons were subjected to DGGE. Sequence analysis was performed on subsequent bands present in resultant DGGE profiles using the Basic Local Alignment Search Tool (BLAST). Microbiological culturing techniques tended to isolate common probiotic bacterial species from the genera Lactobacillus, Lactococcus, Bifidobacterium, Enterococcus, Clostridium, Escherichia, Pediococcus and Enterobacterium as well as members of the genera, Actinomyces, Bacteroides, Propionibacterium, Capnocytophaga, Proteus, and Klebsiella. Bacteroides, Enterococcus, Escherichia, Brevibacterium, Klebsiella, Lactobacillus, Clostridium, Bacillus, Eubacterium, Serratia, Citrobacter, Enterobacter, Pectobacterium and Pantoea spp. in addition to unculturable bacteria were identified as constituents of the NTCs using universal PCR-DGGE analysis. A number of the sequences detected by LAB-specific PCR-DGGE were homologous to those of a number of Lactobacillus spp., including L. fermentum, L. pontis, L. crispatus, L. salivarius, L. casei, L. suntoryeus, L. vaginalis, L. gasseri, L. aviaries, L. johnsonii, L. acidophilus, and L. mucosae in addition to a range of unculturable lactobacilli. While NTCs are successful due to their complexity, the presence of members of Lactobacillus spp. amongst other probiotic genera, in these samples possibly lends to the success of the NTC cultures as probiotics or competitive exclusion products in poultry over the decades. PCR-DGGE proved to be an effective tool in detecting non-culturable organisms present in these complex undefined cultures. In conclusion, while the culture-dependent identification methods or PCR-DGGE alone cannot comprehensively elucidate the bacterial species present in such complex cultures, their complementarity provides useful information on the identity of the constituents of NTCs and will aid in future development of defined probiotics. Moreover, for the purpose of analysing prototype NTCs during their development, PCR-DGGE overcomes the limitations associated with conventional culturing methods including their low sensitivities, inability to detect unculturable bacteria and unknown species, very slow turnabout time and poor reproducibility. This study demonstrated that PCR-DGGE is indeed more valuable in detecting predominant microbial populations between various NTCs than as an identification methodology, being more applicable as a quality control method used to analyse for batch-to-batch variation during NTC production.

Archaeal communities in boreal forest tree rhizospheres respond to changing soil temperatures.

PubMed

Bomberg, Malin; Münster, Uwe; Pumpanen, Jukka; Ilvesniemi, Hannu; Heinonsalo, Jussi

2011-07-01

Temperature has generally great effects on both the activity and composition of microbial communities in different soils. We tested the impact of soil temperature and three different boreal forest tree species on the archaeal populations in the bulk soil, rhizosphere, and mycorrhizosphere. Scots pine, silver birch, and Norway spruce seedlings were grown in forest humus microcosms at three different temperatures, 7-11.5°C (night-day temperature), 12-16°C, and 16-22°C, of which 12-16°C represents the typical mid-summer soil temperature in Finnish forests. RNA and DNA were extracted from indigenous ectomycorrhiza, non-mycorrhizal long roots, and boreal forest humus and tested for the presence of archaea by nested PCR of the archaeal 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) profiling and sequencing. Methanogenic Euryarchaeota belonging to Methanolobus sp. and Methanosaeta sp. were detected on the roots and mycorrhiza. The most commonly detected archaeal 16S rRNA gene sequences belonged to group I.1c Crenarchaeota, which are typically found in boreal and alpine forest soils. Interestingly, also one sequence belonging to group I.1b Crenarchaeota was detected from Scots pine mycorrhiza although sequences of this group are usually found in agricultural and forest soils in temperate areas. Tree- and temperature-related shifts in the archaeal population structure were observed. A clear decrease in crenarchaeotal DGGE band number was seen with increasing temperature, and correspondingly, the number of euryarchaeotal DGGE bands, mostly methanogens, increased. The greatest diversity of archaeal DGGE bands was detected in Scots pine roots and mycorrhizas. No archaea were detected from humus samples from microcosms without tree seedling, indicating that the archaea found in the mycorrhizosphere and root systems were dependent on the plant host. The detection of archaeal 16S rRNA gene sequences from both RNA and DNA extractions show that the archaeal populations were living and that they may have significant contribution to the methane cycle in boreal forest soil, especially when soil temperatures rise.

Molecular Monitoring of the Fecal Microbiota of Healthy Human Subjects during Administration of Lactulose and Saccharomyces boulardii

PubMed Central

Vanhoutte, Tom; De Preter, Vicky; De Brandt, Evie; Verbeke, Kristin; Swings, Jean; Huys, Geert

2006-01-01

Diet is a major factor in maintaining a healthy human gastrointestinal tract, and this has triggered the development of functional foods containing a probiotic and/or prebiotic component intended to improve the host's health via modulation of the intestinal microbiota. In this study, a long-term placebo-controlled crossover feeding study in which each subject received several treatments was performed to monitor the effect of a prebiotic substrate (i.e., lactulose), a probiotic organism (i.e., Saccharomyces boulardii), and their synbiotic combination on the fecal microbiota of three groups of 10 healthy human subjects differing in prebiotic dose and/or intake of placebo versus synbiotic. For this purpose, denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene amplicons was used to detect possible changes in the overall bacterial composition using the universal V3 primer and to detect possible changes at the subpopulation level using group-specific primers targeting the Bacteroides fragilis subgroup, the genus Bifidobacterium, the Clostridium lituseburense group (cluster XI), and the Clostridium coccoides-Eubacterium rectale group (cluster XIVa). Although these populations remained fairly stable based on DGGE profiling, one pronounced change was observed in the universal fingerprint profiles after lactulose ingestion. Band position analysis and band sequencing revealed that a band appearing or intensifying following lactulose administration could be assigned to the species Bifidobacterium adolescentis. Subsequent analysis with real-time PCR (RT-PCR) indicated a statistically significant increase (P < 0.05) in total bifidobacteria in one of the three subject groups after lactulose administration, whereas a similar but nonsignificant trend was observed in the other two groups. Combined RT-PCR results from two subject groups indicated a borderline significant increase (P = 0.074) of B. adolescentis following lactulose intake. The probiotic yeast S. boulardii did not display any detectable universal changes in the DGGE profiles, nor did it influence the bifidobacterial levels. This study highlighted the capacity of an integrated approach consisting of DGGE analysis and RT-PCR to monitor and quantify pronounced changes in the fecal microbiota of healthy subjects upon functional food administration. PMID:16957220

Effects of stimulation of copper bioleaching on microbial community in vineyard soil and copper mining waste.

PubMed

Andreazza, Robson; Okeke, Benedict C; Pieniz, Simone; Bortolon, Leandro; Lambais, Márcio R; Camargo, Flávio A O

2012-04-01

Long-term copper application in vineyards and copper mining activities cause heavy metal pollution sites. Such sites need remediation to protect soil and water quality. Bioremediation of contaminated areas through bioleaching can help to remove copper ions from the contaminated soils. Thus, the aim of this work was to evaluate the effects of different treatments for copper bioleaching in two diverse copper-contaminated soils (a 40-year-old vineyard and a copper mining waste) and to evaluate the effect on microbial community by applying denaturing gradient gel electrophoresis (DGGE) of 16S ribosomal DNA amplicons and DNA sequence analysis. Several treatments with HCl, H(2)SO(4), and FeSO(4) were evaluated by stimulation of bioleaching of copper in the soils. Treatments and extractions using FeSO(4) and H(2)SO(4) mixture at 30°C displayed more copper leaching than extractions with deionized water at room temperature. Treatment with H(2)SO(4) supported bioleaching of as much as 120 mg kg(-1) of copper from vineyard soil after 115 days of incubation. DGGE analysis of the treatments revealed that some treatments caused greater diversity of microorganisms in the vineyard soil compared to the copper mining waste. Nucleotide Blast of PCR-amplified fragments of 16S rRNA gene bands from DGGE indicated the presence of Rhodobacter sp., Silicibacter sp., Bacillus sp., Paracoccus sp., Pediococcus sp., a Myxococcales, Clostridium sp., Thiomonas sp., a firmicute, Caulobacter vibrioides, Serratia sp., and an actinomycetales in vineyard soil. Contrarily, Sphingomonas was the predominant genus in copper mining waste in most treatments. Paracoccus sp. and Enterobacter sp. were also identified from DGGE bands of the copper mining waste. Paracoccus species is involved in the copper bioleaching by sulfur oxidation system, liberating the copper bounded in the soils and hence promoting copper bioremediation. Results indicate that stimulation of bioleaching with a combination of FeSO(4) and H(2)SO(4) promoted bioleaching in the soils and can be employed ex situ to remediate copper-impacted soils.

Identification of bacteria in a biodegraded wall painting by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.

PubMed Central

Rölleke, S; Muyzer, G; Wawer, C; Wanner, G; Lubitz, W

1996-01-01

Medieval wall paintings are often affected by biodecay. An inventory of the existing microorganisms associated with the damage to the paintings is not yet an integral part of the restoration process. This stems from the lack of effective means for such a stocktaking. Nevertheless, fungi and bacteria cause severe damage through mechanical processes from growth into the painting and its grounding and through their metabolism. Detailed information on the bacterial colonization of ancient wall paintings is essential for the protection of the paintings. We used a molecular approach based on the detection and identification of DNA sequences encoding rRNA (rDNA) to identify bacteria present on an ancient wall painting without prior cultivation of the organisms, since it has been shown that most of these bacteria cannot be cultivated under laboratory conditions. To trace the noncultivated fraction of bacteria, total DNA from a biodegraded wall painting sample from a 13th century fresco was extracted and 194-bp fragments of the 16S rDNA were amplified with eubacterial primers. The 16S rDNA fragments of uniform length obtained from the different bacterial species were separated according to their sequence differences by denaturing gradient gel electrophoresis (DGGE). By sequencing excised and reamplified individual DNA bands, we characterized the phylogenetic affiliation of the corresponding bacteria. Using this approach, we identified members or close relatives of the genera Halomonas, Clostridium, and Frankia. To our knowledge, these groups of bacteria have not yet been isolated and implicated by conventional microbiological techniques as contributing to the biodegradation of wall paintings. PMID:8787403

Application of Reverse Transcriptase-PCR-DGGE as a rapid method for routine determination of Vibrio spp. in foods.

PubMed

Chahorm, Kanchana; Prakitchaiwattana, Cheunjit

2018-01-02

The aim of this research was to evaluate the feasibility of PCR-DGGE and Reverse Transcriptase-PCR-DGGE techniques for rapid detection of Vibrio species in foods. Primers GC567F and 680R were initially evaluated for amplifying DNA and cDNA of ten references Vibrio species by PCR method. The GC-clamp PCR amplicons were separated according to their sequences by the DGGE using 10% (w/v) polyacrylamide gel containing 45-70% urea and formamide denaturants. Two pair of Vibrio species, which could not be differentiated on the gel, was Vibrio fluvialis - Vibrio furnissii and Vibrio parahaemolyticus - Vibrio harveyi. To determine the detection limit, in the community of 10 reference strains containing the same viable population, distinct DNA bands of 3 species; Vibrio cholerae, Vibrio mimicus and Vibrio alginolyticus were consistently observed by PCR-DGGE technique. In fact, 5 species; Vibrio cholerae, Vibrio mimicus, Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio fluvialis consistently observed by Reverse Transcriptase-PCR-DGGE. In the community containing different viable population increasing from 10 2 to 10 5 CFU/mL, PCR-DGGE analysis only detected the two most prevalent species, while RT-PCR-DGGE detected the five most prevalent species. Therefore, Reverse Transcriptase-PCR-DGGE was also selected for detection of various Vibrio cell conditions, including viable cell (VC), injured cells from frozen cultures (IVC) and injured cells from frozen cultures with pre-enrichment (PIVC). It was found that cDNA band of all cell conditions gave the same migratory patterns, except that multiple cDNA bands of Plesiomonas shigelloides under IVC and PIVC conditions were found. When Reverse Transcriptase-PCR-DGGE was used for detecting Vibrio parahaemolyticus in the pathogen-spiked food samples, Vibrio parahaemolyticus could be detected in the spiked samples containing at least 10 2 CFU/g of this pathogen. The results obtained also corresponded to standard method (USFDA, 2004). In comparison with the detection of the Vibrio profiles in fourteen food samples using standard method, Reverse Transcriptase-PCR-DGGE resulted in 100%, 75% and 50% similarity in 3, 1 and 6 food samples, respectively. Copyright © 2017 Elsevier B.V. All rights reserved.

Fungicides degradation in an organic biomixture: impact on microbial diversity.

PubMed

Coppola, Laura; Comitini, Francesca; Casucci, Cristiano; Milanovic, Vesna; Monaci, Elga; Marinozzi, Maria; Taccari, Manuela; Ciani, Maurizio; Vischetti, Costantino

2011-12-15

Biological systems are being developed all over EU countries to protect water-bodies from pesticide contamination at farm level. A laboratory experiment was carried out to test the efficiency of a mixture of compost and straw in bio-degrading different mixtures of fungicides usually applied in vineyards. At the same time the effects of fungicide applications on microbial community of biomixture were also evaluated. Results showed that the biomixture had a good capability of degrading pesticides. Indeed, at the end of the experiment (112 days), the concentration of most of the pesticides was close to complete degradation. Denaturing gradient gel electrophoresis (DGGE) analysis showed an evident modification of microbial diversity after the addition of fungicides. However, at the end of degradation process, no significant changes in the composition of microbial community were seen. In this specific substrate used in the biomixture, yeast flora and ascomycete filamentous fungi seemed to be involved in the degradation activity. Copyright © 2011 Elsevier B.V. All rights reserved.

Microbial Diversity of Acidic Hot Spring (Kawah Hujan B) in Geothermal Field of Kamojang Area, West Java-Indonesia

PubMed Central

Aditiawati, Pingkan; Yohandini, Heni; Madayanti, Fida; Akhmaloka

2009-01-01

Microbial communities in an acidic hot spring, namely Kawah Hujan B, at Kamojang geothermal field, West Java-Indonesia was examined using culture dependent and culture independent strategies. Chemical analysis of the hot spring water showed a characteristic of acidic-sulfate geothermal activity that contained high sulfate concentrations and low pH values (pH 1.8 to 1.9). Microbial community present in the spring was characterized by 16S rRNA gene combined with denaturing gradient gel electrophoresis (DGGE) analysis. The majority of the sequences recovered from culture-independent method were closely related to Crenarchaeota and Proteobacteria phyla. However, detail comparison among the member of Crenarchaeota showing some sequences variation compared to that the published data especially on the hypervariable and variable regions. In addition, the sequences did not belong to certain genus. Meanwhile, the 16S Rdna sequences from culture-dependent samples revealed mostly close to Firmicute and gamma Proteobacteria. PMID:19440252

Comparative evaluation of microbial diversity and metabolite profiles in doenjang, a fermented soybean paste, during the two different industrial manufacturing processes.

PubMed

Lee, Sunmin; Lee, Sarah; Singh, Digar; Oh, Ji Young; Jeon, Eun Jung; Ryu, Hyung SeoK; Lee, Dong Wan; Kim, Beom Seok; Lee, Choong Hwan

2017-04-15

Two different doenjang manufacturing processes, the industrial process (IP) and the modified industrial process (mIP) with specific microbial assortments, were subjected to metabolite profiling using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). The multivariate analyses indicated that both primary and secondary metabolites exhibited distinct patterns according to the fermentation processes (IP and mIP). Microbial community analysis for doenjang using denaturing gradient gel electrophoresis (DGGE), exhibited that both bacteria and fungi contributed proportionally for each step in the process viz., soybean, steaming, drying, meju fermentation, cooling, brining, and aging. Further, correlation analysis indicated that Aspergillus population was linked to sugar metabolism, Bacillus spp. with that of fatty acids, whereas Tetragenococcus and Zygosaccharomyces were found associated with amino acids. These results suggest that the components and quality of doenjang are critically influenced by the microbial assortments in each process. Copyright © 2016 Elsevier Ltd. All rights reserved.

Simultaneous treatment of raw palm oil mill effluent and biodegradation of palm fiber in a high-rate CSTR.

PubMed

Khemkhao, Maneerat; Techkarnjanaruk, Somkiet; Phalakornkule, Chantaraporn

2015-02-01

A high-rate continuous stirred tank reactor (CSTR) was used to produce biogas from raw palm oil mill effluent (POME) at 55°C at a highest organic loading rate (OLR) of 19 g COD/ld. Physical and chemical pretreatments were not performed on the raw POME. In order to promote retention of suspended solids, the CSTR was installed with a deflector at its upper section. The average methane yield was 0.27 l/g COD, and the biogas production rate per reactor volume was 6.23 l/l d, and the tCOD removal efficiency was 82%. The hydrolysis rate of cellulose, hemicelluloses and lignin was 6.7, 3.0 and 1.9 g/d, respectively. The results of denaturing gradient gel electrophoresis (DGGE) suggested that the dominant hydrolytic bacteria responsible for the biodegradation of the palm fiber and residual oil were Clostridium sp., while the dominant methanogens were Methanothermobacter sp. Copyright © 2014 Elsevier Ltd. All rights reserved.

Autoclave method for rapid preparation of bacterial PCR-template DNA.

PubMed

Simmon, Keith E; Steadman, Dewey D; Durkin, Sarah; Baldwin, Amy; Jeffrey, Wade H; Sheridan, Peter; Horton, Rene; Shields, Malcolm S

2004-02-01

An autoclave method for preparing bacterial DNA for PCR template is presented, it eliminates the use of detergents, organic solvents, and mechanical cellular disruption approaches, thereby significantly reducing processing time and costs while increasing reproducibility. Bacteria are lysed by rapid heating and depressurization in an autoclave. The lysate, cleared by microcentrifugation, was either used directly in the PCR reaction, or concentrated by ultrafiltration. This approach was compared with seven established methods of DNA template preparation from four bacterial sources which included boiling Triton X-100 and SDS, bead beating, lysozyme/proteinase K, and CTAB lysis method components. Bacteria examined were Enterococcus and Escherichia coli, a natural marine bacterial community and an Antarctic cyanobacterial-mat. DNAs were tested for their suitability as PCR templates by repetitive element random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE) analysis. The autoclave method produced PCR amplifiable template comparable or superior to the other methods, with greater reproducibility, much shorter processing time, and at a significantly lower cost.

Evidence of active methanogen communities in shallow sediments of the sonora margin cold seeps.

PubMed

Vigneron, Adrien; L'Haridon, Stéphane; Godfroy, Anne; Roussel, Erwan G; Cragg, Barry A; Parkes, R John; Toffin, Laurent

2015-05-15

In the Sonora Margin cold seep ecosystems (Gulf of California), sediments underlying microbial mats harbor high biogenic methane concentrations, fueling various microbial communities, such as abundant lineages of anaerobic methanotrophs (ANME). However, the biodiversity, distribution, and metabolism of the microorganisms producing this methane remain poorly understood. In this study, measurements of methanogenesis using radiolabeled dimethylamine, bicarbonate, and acetate showed that biogenic methane production in these sediments was mainly dominated by methylotrophic methanogenesis, while the proportion of autotrophic methanogenesis increased with depth. Congruently, methane production and methanogenic Archaea were detected in culture enrichments amended with trimethylamine and bicarbonate. Analyses of denaturing gradient gel electrophoresis (DGGE) fingerprinting and reverse-transcribed PCR-amplified 16S rRNA sequences retrieved from these enrichments revealed the presence of active methylotrophic Methanococcoides burtonii relatives and several new autotrophic Methanogenium lineages, confirming the cooccurrence of Methanosarcinales and Methanomicrobiales methanogens with abundant ANME populations in the sediments of the Sonora Margin cold seeps. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Novel Rickettsiella Bacterium in the Leafhopper Orosius albicinctus (Hemiptera: Cicadellidae)

PubMed Central

Iasur-Kruh, Lilach; Weintraub, Phyllis G.; Mozes-Daube, Netta; Robinson, Wyatt E.; Perlman, Steve J.

2013-01-01

Bacteria in the genus Rickettsiella (Coxiellaceae), which are mainly known as arthropod pathogens, are emerging as excellent models to study transitions between mutualism and pathogenicity. The current report characterizes a novel Rickettsiella found in the leafhopper Orosius albicinctus (Hemiptera: Cicadellidae), a major vector of phytoplasma diseases in Europe and Asia. Denaturing gradient gel electrophoresis (DGGE) and pyrosequencing were used to survey the main symbionts of O. albicinctus, revealing the obligate symbionts Sulcia and Nasuia, and the facultative symbionts Arsenophonus and Wolbachia, in addition to Rickettsiella. The leafhopper Rickettsiella is allied with bacteria found in ticks. Screening O. albicinctus from the field showed that Rickettsiella is highly prevalent, with over 60% of individuals infected. A stable Rickettsiella infection was maintained in a leafhopper laboratory colony for at least 10 generations, and fluorescence microscopy localized bacteria to accessory glands of the female reproductive tract, suggesting that the bacterium is vertically transmitted. Future studies will be needed to examine how Rickettsiella affects host fitess and its ability to vector phytopathogens. PMID:23645190

Phylogenetic characterization of a corrosive consortium isolated from a sour gas pipeline.

PubMed

Jan-Roblero, J; Romero, J M; Amaya, M; Le Borgne, S

2004-06-01

Biocorrosion is a common problem in oil and gas industry facilities. Characterization of the microbial populations responsible for biocorrosion and the interactions between different microorganisms with metallic surfaces is required in order to implement efficient monitoring and control strategies. Denaturing gradient gel electrophoresis (DGGE) analysis was used to separate PCR products and sequence analysis revealed the bacterial composition of a consortium obtained from a sour gas pipeline in the Gulf of Mexico. Only one species of sulfate-reducing bacteria (SRB) was detected in this consortium. The rest of the population consisted of enteric bacteria with different characteristics and metabolic capabilities potentially related to biocorrosion. Therefore, several types of bacteria may be involved in biocorrosion arising from natural biofilms that develop in industrial facilities. The low abundance of the detected SRB was evidenced by environmental scanning electron microscopy (ESEM). In addition, the localized corrosion of pipeline steel in the presence of the consortium was clearly observed by ESEM after removing the adhered bacteria.

Biotechnological potential of microbial consortia and future perspectives.

PubMed

Bhatia, Shashi Kant; Bhatia, Ravi Kant; Choi, Yong-Keun; Kan, Eunsung; Kim, Yun-Gon; Yang, Yung-Hun

2018-05-15

Design of a microbial consortium is a newly emerging field that enables researchers to extend the frontiers of biotechnology from a pure culture to mixed cultures. A microbial consortium enables microbes to use a broad range of carbon sources. It provides microbes with robustness in response to environmental stress factors. Microbes in a consortium can perform complex functions that are impossible for a single organism. With advancement of technology, it is now possible to understand microbial interaction mechanism and construct consortia. Microbial consortia can be classified in terms of their construction, modes of interaction, and functions. Here we discuss different trends in the study of microbial functions and interactions, including single-cell genomics (SCG), microfluidics, fluorescent imaging, and membrane separation. Community profile studies using polymerase chain-reaction denaturing gradient gel electrophoresis (PCR-DGGE), amplified ribosomal DNA restriction analysis (ARDRA), and terminal restriction fragment-length polymorphism (T-RFLP) are also reviewed. We also provide a few examples of their possible applications in areas of biopolymers, bioenergy, biochemicals, and bioremediation.

Microbial community structure in a thermophilic aerobic digester used as a sludge pretreatment process for the mesophilic anaerobic digestion and the enhancement of methane production.

PubMed

Jang, Hyun Min; Park, Sang Kyu; Ha, Jeong Hyub; Park, Jong Moon

2013-10-01

An effective two-stage sewage sludge digestion process, consisting of thermophilic aerobic digestion (TAD) followed by mesophilic anaerobic digestion (MAD), was developed for efficient sludge reduction and methane production. Using TAD as a biological pretreatment, the total volatile suspended solid reduction (VSSR) and methane production rate (MPR) in the MAD reactor were significantly improved. According to denaturing gradient gel electrophoresis (DGGE) analysis, the results indicated that the dominant bacteria species such as Ureibacillus thermophiles and Bacterium thermus in TAD were major routes for enhancing soluble organic matter. TAD pretreatment using a relatively short SRT of 1 day showed highly increased soluble organic products and positively affected an increment of bacteria populations which performed interrelated microbial metabolisms with methanogenic species in the MAD; consequently, a quantitative real-time PCR indicated greatly increased Methanosarcinales (acetate-utilizing methanogens) in the MAD, resulting in enhanced methane production. Copyright © 2013 Elsevier Ltd. All rights reserved.

Biofuel components change the ecology of bacterial volatile petroleum hydrocarbon degradation in aerobic sandy soil.

PubMed

Elazhari-Ali, Abdulmagid; Singh, Arvind K; Davenport, Russell J; Head, Ian M; Werner, David

2013-02-01

We tested the hypothesis that the biodegradation of volatile petroleum hydrocarbons (VPHs) in aerobic sandy soil is affected by the blending with 10 percent ethanol (E10) or 20 percent biodiesel (B20). When inorganic nutrients were scarce, competition between biofuel and VPH degraders temporarily slowed monoaromatic hydrocarbon degradation. Ethanol had a bigger impact than biodiesel, reflecting the relative ease of ethanol compared to methyl ester biodegradation. Denaturing gradient gel electrophoresis (DGGE) of bacterial 16S rRNA genes revealed that each fuel mixture selected for a distinct bacterial community, each dominated by Pseudomonas spp. Despite lasting impacts on soil bacterial ecology, the overall effects on VHP biodegradation were minor, and average biomass yields were comparable between fuel types, ranging from 0.40 ± 0.16 to 0.51 ± 0.22 g of biomass carbon per gram of fuel carbon degraded. Inorganic nutrient availability had a greater impact on petroleum hydrocarbon biodegradation than fuel composition. Copyright © 2012 Elsevier Ltd. All rights reserved.

[Effect of temperature on performance of microbial fuel cell using beer wastewater].

PubMed

Wang, Xin; Feng, Yu-Jie; Qu, You-Peng; Li, Dong-Mei; Li, He; Ren, Nan-Qi

2008-11-01

The effects of temperature on performance and biological community structure were investigated in air-cathode microbial fuel cells (MFCs) using beer wastewater amended with 50 mmol/L phosphate buffer solution (PBS). The maximum power density decreased from 483 mW/m2 to 435 mW/m2 when the temperature varied from 30 degrees C to 20 degrees C, meanwhile just a little decreasing on coulombic efficiency and the COD removal rate were observed. Decreasing of temperature resulted in effects both on cathode potential and anode potential, but cathode potential behaved much more sensitive to temperature. The half-saturation constants (Ks) obtained from the fit of Monod-type equation were 228 mg/L (30 degrees C) and 293 mg/L (20 degrees C) respectively. Denaturing gradient gel electrophoresis (DGGE) analysis indicated that operating temperature not only affected the predominant population of the anodic bacterial community, but also had a great impact on the diversity of the cathodic microbial population.

Treatment of high ethanol concentration wastewater by biological sand filters: enhanced COD removal and bacterial community dynamics.

PubMed

Rodriguez-Caballero, A; Ramond, J-B; Welz, P J; Cowan, D A; Odlare, M; Burton, S G

2012-10-30

Winery wastewater is characterized by its high chemical oxygen demand (COD), seasonal occurrence and variable composition, including periodic high ethanol concentrations. In addition, winery wastewater may contain insufficient inorganic nutrients for optimal biodegradation of organic constituents. Two pilot-scale biological sand filters (BSFs) were used to treat artificial wastewater: the first was amended with ethanol and the second with ethanol, inorganic nitrogen (N) and phosphorus (P). A number of biochemical parameters involved in the removal of pollutants through BSF systems were monitored, including effluent chemistry and bacterial community structures. The nutrient supplemented BSF showed efficient COD, N and P removal. Comparison of the COD removal efficiencies of the two BSFs showed that N and P addition enhanced COD removal efficiency by up to 16%. Molecular fingerprinting of BSF sediment samples using denaturing gradient gel electrophoresis (DGGE) showed that amendment with high concentrations of ethanol destabilized the microbial community structure, but that nutrient supplementation countered this effect. Copyright © 2012 Elsevier Ltd. All rights reserved.

Microbial diversity of acidic hot spring (kawah hujan B) in geothermal field of kamojang area, west java-indonesia.

PubMed

Aditiawati, Pingkan; Yohandini, Heni; Madayanti, Fida; Akhmaloka

2009-01-01

Microbial communities in an acidic hot spring, namely Kawah Hujan B, at Kamojang geothermal field, West Java-Indonesia was examined using culture dependent and culture independent strategies. Chemical analysis of the hot spring water showed a characteristic of acidic-sulfate geothermal activity that contained high sulfate concentrations and low pH values (pH 1.8 to 1.9). Microbial community present in the spring was characterized by 16S rRNA gene combined with denaturing gradient gel electrophoresis (DGGE) analysis. The majority of the sequences recovered from culture-independent method were closely related to Crenarchaeota and Proteobacteria phyla. However, detail comparison among the member of Crenarchaeota showing some sequences variation compared to that the published data especially on the hypervariable and variable regions. In addition, the sequences did not belong to certain genus. Meanwhile, the 16S Rdna sequences from culture-dependent samples revealed mostly close to Firmicute and gamma Proteobacteria.

The assessment of the coke wastewater treatment efficacy in rotating biological contractor.

PubMed

Cema, G; Żabczyński, S; Ziembińska-Buczyńska, A

2016-01-01

Coke wastewater is known to be relatively difficult for biological treatment. Nonetheless, biofilm-based systems seem to be promising tool for such treatment. That is why a rotating biological contactor (RBC) system focused on the Anammox process was used in this study. The experiment was divided into two parts with synthetic and then real wastewater. It was proven that it is possible to treat coke wastewater with RBC but such a procedure requires a very long start-up period for the nitritation (190 days), as well as for the Anammox process, where stable nitrogen removal over 70% was achieved after 400 days of experiment. Interestingly, it was possible at a relatively low (20.2 ± 2.2 °C) temperature. The polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) based monitoring of the bacterial community showed that its biodiversity decreased when the real wastewater was treated and it was composed mainly of GC-rich genotypes, probably because of the modeling influence of this wastewater and the genotypes specialization.

Ammonia concentration determines differential growth of ammonia-oxidising archaea and bacteria in soil microcosms.

PubMed

Verhamme, Daniel T; Prosser, James I; Nicol, Graeme W

2011-06-01

The first step of nitrification, oxidation of ammonia to nitrite, is performed by both ammonia-oxidising archaea (AOA) and ammonia-oxidising bacteria (AOB) in soil, but their relative contributions to ammonia oxidation and existence in distinct ecological niches remain to be determined. To determine whether available ammonia concentration has a differential effect on AOA and AOB growth, soil microcosms were incubated for 28 days with ammonium at three concentrations: native (control), intermediate (20 μg NH(4)(+)-N per gram of soil) and high (200 μg NH(4)(+)-N per gram of soil). Quantitative PCR demonstrated growth of AOA at all concentrations, whereas AOB growth was prominent only at the highest concentration. Similarly, denaturing gradient gel electrophoresis (DGGE) analysis revealed changes in AOA communities at all ammonium concentrations, whereas AOB communities changed significantly only at the highest ammonium concentration. These results provide evidence that ammonia concentration contributes to the definition of distinct ecological niches of AOA and AOB in soil.

Ammonia concentration determines differential growth of ammonia-oxidising archaea and bacteria in soil microcosms

PubMed Central

Verhamme, Daniel T; Prosser, James I; Nicol, Graeme W

2011-01-01

The first step of nitrification, oxidation of ammonia to nitrite, is performed by both ammonia-oxidising archaea (AOA) and ammonia-oxidising bacteria (AOB) in soil, but their relative contributions to ammonia oxidation and existence in distinct ecological niches remain to be determined. To determine whether available ammonia concentration has a differential effect on AOA and AOB growth, soil microcosms were incubated for 28 days with ammonium at three concentrations: native (control), intermediate (20 μg NH4+-N per gram of soil) and high (200 μg NH4+-N per gram of soil). Quantitative PCR demonstrated growth of AOA at all concentrations, whereas AOB growth was prominent only at the highest concentration. Similarly, denaturing gradient gel electrophoresis (DGGE) analysis revealed changes in AOA communities at all ammonium concentrations, whereas AOB communities changed significantly only at the highest ammonium concentration. These results provide evidence that ammonia concentration contributes to the definition of distinct ecological niches of AOA and AOB in soil. PMID:21228892

The impact of shrimp farming effluent on bacterial communities in mangrove waters, Ceará, Brazil.

PubMed

Sousa, O V; Macrae, A; Menezes, F G R; Gomes, N C M; Vieira, R H S F; Mendonça-Hagler, L C S

2006-12-01

The effects of shrimp farm effluents on bacterial communities in mangroves have been infrequently reported. Classic and molecular biology methods were used to survey bacterial communities from four mangroves systems. Water temperature, salinity, pH, total heterotrophic bacteria and maximum probable numbers of Vibrio spp. were investigated. Genetic profiles of bacterial communities were also characterized by polymerase chain reaction (PCR) amplification of eubacterial and Vibrio 16S rDNA using denaturing gradient gel electrophoresis (DGGE). Highest heterotrophic counts were registered in the mangrove not directly polluted by shrimp farming. The Enterobacteriaceae and Chryseomonas luteola dominated the heterotrophic isolates. Vibrio spp. pathogenic to humans and shrimps were identified. Eubacterial genetic profiles suggest a shared community structure independent of mangrove system. Vibrio genetic profiles were mangrove specific. Neither microbial counts nor genetic profiling revealed a significant decrease in species richness associated with shrimp farm effluent. The complex nature of mangrove ecosystems and their microbial communities is discussed.

Pretreatment of piggery wastewater by a stable constructed microbial consortium for improving the methane production.

PubMed

Cai, Jian; Mo, Xiwei; Cheng, Guojun; Du, Dongyun

2015-01-01

A stable aerobic microbial consortium, established by successive subcultivation, was employed to solubilize the solid organic fraction in swine wastewater. In the 30 days' successive biological pretreatments, 30-38% of volatile solids and 19-28% total solids in raw slurry were solubilized after 10 hours at 37 °C. Meanwhile, soluble chemical oxygen demand (COD) and volatile fatty acid increased by 48%-56% and 600%-750%, respectively. Furthermore, the molecular microbial profile of the consortium in successive pretreatment was conducted by denaturing gradient gel electrophoresis (DGGE). The results indicated that bacterial species of the consortium rapidly overgrew the indigenous microbial community of raw water, and showed a stable predominance at the long-term treatment. As a consequence of biological pretreatment, pretreatment shortened digestion time by 50% and increased biogas production by 45% compared to raw water in the anaerobic process. The microbial consortium constructed herein is a potential candidate consortium for biological pretreatment of swine wastewater to enhance biogas production.

Analysis of the coral associated bacterial community structures in healthy and diseased corals from off-shore of southern Taiwan.

PubMed

Chiou, Shu-Fen; Kuo, Jimmy; Wong, Tit-Yee; Fan, Tung-Yung; Tew, Kwee Siong; Liu, Jong-Kang

2010-07-01

The methods of denaturing gradient gel electrophoresis (DGGE) and DNA sequencing were used to analyze the ribotypes of microbial communities associated with corals. Both healthy and diseased coral of different species were collected at three locations off the southern coast of Taiwan. Ribotyping results suggested that the microbial communities were diverse. The microbial community profiles, even among the same species of corals from different geographical locations, differ significantly. The coral-associated bacterial communities contain many bacteria common to the habitants of various invertebrates. However, some bacteria were unexpected. The presence of some unusual species, such as Staphylococcus, Clostridium and Legionella, associated with corals that were likely the results of human activities. Human activities, such as thermal pollution from the nearby nuclear plant, active fishing and tourism industries in the region might have all contributed to the change in bacterial communities and the death of coral colonies around the region.

Characterization of the bacterial communities associated with the bald sea urchin disease of the echinoid Paracentrotus lividus.

PubMed

Becker, Pierre T; Egea, Emilie; Eeckhaut, Igor

2008-06-01

The microbial communities involved in the bald sea urchin disease of the echinoid Paracentrotus lividus are investigated using culture-independent techniques. Lesions of diseased specimens from two locations in France, La Ciotat (Mediterranean Sea) and Morgat (Atlantic Ocean), are examined by Scanning Electron Microscopy (SEM) and the diversity of their microbiota is analysed by Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA gene clones libraries construction. Microscopic observations demonstrated that only the central area of the lesions is invaded by bacteria but not the peripheral zone and the surrounding healthy tissues. Molecular analysis identified at least 24 bacterial genomospecies in bald sea urchin lesions: 5 are Alphaproteobacteria, 10 are Gammaproteobacteria, 8 are CFB bacteria and 1 is a Fusobacteria. Out of them, 4 are observed in both locations while 10 occur only in the Atlantic Ocean and 10 only in the Mediterranean Sea. Gammaproteobacteria are the most represented in clones libraries from both locations, with respectively 65% and 43% of the total clones. CFB and Alphaproteobacteria accounted for the majority of the remaining clones and were detected by DGGE in virtually all samples from both stations. Our results demonstrate that bacterial communities observed on diseased individuals of the same echinoid species but originating from distinct locations are not similar and thus support the hypothesis that bacteria involved in the worldwide echinoid disease commonly called the bald sea urchin disease are opportunistic and not specific.

Assessment of Anaerobic Metabolic Activity and Microbial Diversity in a Petroleum-Contaminated Aquifer Using Push-Pull Tests in Combination With Molecular Tools and Stable Isotopes

NASA Astrophysics Data System (ADS)

Schroth, M. H.; Kleikemper, J.; Pombo, S. A.; Zeyer, J.

2002-12-01

In the past, studies on microbial communities in natural environments have typically focused on either their structure or on their metabolic function. However, linking structure and function is important for understanding microbial community dynamics, in particular in contaminated environments. We will present results of a novel combination of a hydrogeological field method (push-pull tests) with molecular tools and stable isotope analysis, which was employed to quantify anaerobic activities and associated microbial diversity in a petroleum-contaminated aquifer in Studen, Switzerland. Push-pull tests consisted of the injection of test solution containing a conservative tracer and reactants (electron acceptors, 13C-labeled carbon sources) into the aquifer anoxic zone. Following an incubation period, the test solution/groundwater mixture was extracted from the same location. Metabolic activities were computed from solute concentrations measured during extraction. Simultaneously, microbial diversity in sediment and groundwater was characterized by using fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE), as well as phospholipids fatty acid (PLFA) analysis in combination with 13C isotopic measurements. Results from DGGE analyses provided information on the general community structure before, during and after the tests, while FISH yielded information on active populations. Moreover, using 13C-labeling of microbial PLFA we were able to directly link carbon source assimilation in an aquifer to indigenous microorganisms while providing quantitative information on respective carbon source consumption.

Prebiotic effect of fructooligosaccharide in the simulator of the human intestinal microbial ecosystem (SHIME® model).

PubMed

Sivieri, Katia; Morales, Martha L Villarreal; Saad, Susana M I; Adorno, Maria A Tallarico; Sakamoto, Isabel Kimiko; Rossi, Elizeu A

2014-08-01

Maintaining "gut health" is a goal for scientists throughout the world. Therefore, microbiota management models for testing probiotics, prebiotics, and synbiotics have been developed. The SHIME(®) model was used to study the effect of fructooligosaccharide (FOS) on the fermentation pattern of the colon microbiota. Initially, an inoculum prepared from human feces was introduced into the reactor vessels and stabilized over 2 weeks using a culture medium. This stabilization period was followed by a 2-week control period during which the microbiota was monitored. The microbiota was then subjected to a 4-week treatment period by adding 5 g/day-1 FOS to vessel one (the "stomach" compartment). Plate counts, Denaturing Gradient Gel Electrophoresis (DGGE), short-chain fatty acid (SCFA), and ammonium analyses were used to observe the influence of FOS treatment in simulated colon compartments. A significant increase (P<.01) in the Lactobacillus spp. and Bifidobacterium spp. populations was observed during the treatment period. The DGGE obtained showed the overall microbial community was changed in the ascending colon compartment of the SHIME reactor. FOS induced increase of the SCFA concentration (P<.05) during the treatment period, mainly due to significant increased levels of acetic and butyric acids. However, ammonium concentrations increased during the same period (P<.01). This study indicates the usefulness of in vitro methods that simulate the colon region as part of research towards the improvement of human health.

Microbial Ecology of an Extreme Acidic Environment, the Tinto River

PubMed Central

González-Toril, E.; Llobet-Brossa, E.; Casamayor, E. O.; Amann, R.; Amils, R.

2003-01-01

The Tinto River (Huelva, southwestern Spain) is an extreme environment with a rather constant acidic pH along the entire river and a high concentration of heavy metals. The extreme conditions of the Tinto ecosystem are generated by the metabolic activity of chemolithotrophic microorganisms thriving in the rich complex sulfides of the Iberian Pyrite Belt. Molecular ecology techniques were used to analyze the diversity of this microbial community. The community's composition was studied by denaturing gradient gel electrophoresis (DGGE) using 16S rRNA and by 16S rRNA gene amplification. A good correlation between the two approaches was found. Comparative sequence analysis of DGGE bands showed the presence of organisms related to Leptospirillum spp., Acidithiobacillus ferrooxidans, Acidiphilium spp., “Ferrimicrobium acidiphilum,” Ferroplasma acidiphilum, and Thermoplasma acidophilum. The different phylogenetic groups were quantified by fluorescent in situ hybridization with a set of rRNA-targeted oligonucleotide probes. More than 80% of the cells were affiliated with the domain Bacteria, with only a minor fraction corresponding to Archaea. Members of Leptospirillum ferrooxidans, Acidithiobacillus ferrooxidans, and Acidiphilium spp., all related to the iron cycle, accounted for most of the prokaryotic microorganisms detected. Different isolates of these microorganisms were obtained from the Tinto ecosystem, and their physiological properties were determined. Given the physicochemical characteristics of the habitat and the physiological properties and relative concentrations of the different prokaryotes found in the river, a model for the Tinto ecosystem based on the iron cycle is suggested. PMID:12902280

Reactor performance and microbial community of an EGSB reactor operated at 20 and 15 degrees C.

PubMed

Xing, W; Zuo, J-E; Dai, N; Cheng, J; Li, J

2009-09-01

To investigate the effects of low temperatures on the performance and microbial community of anaerobic wastewater treatment. An expanded granular sludge bed (EGSB) reactor was employed to treat synthetic brewery wastewater at 20 and 15 degrees C. Reactor performance was represented by chemical oxygen demand (COD) removal efficiency, while the microbial community was analysed using denaturing gradient gel electrophoresis (DGGE) and clone technology. When the hydraulic retention time (HRT) was maintained at 18 h, COD removal efficiencies above 85% were obtained at both 20 and 15 degrees C, with influent COD concentrations up to 7300 and 4100 mg l(-1), respectively. At 15 degrees C, the COD removal efficiency was more easily manipulated by increasing the influent COD concentration. DGGE and clone results for both temperatures revealed that Methanosaeta and Methanobacterium were two dominant methanogens, and that the majority of the eubacterial clones were represented by Firmicutes. When the temperature decreased from 20 to 15 degrees C, both archaeal and eubacterial communities had higher diversity, and the proportion of Methanosaeta (acetate-utilizing methanogens) decreased markedly from 60.0% to 49.3%, together with an increase in proportions of hydrogen-utilizing methanogens (especially Methanospirillum). The feasibility of psychrophilic anaerobic treatment of low and medium strength organic wastewaters was demonstrated, although lower temperature could significantly affect both reactor performance and the anaerobic microbial community. The findings enrich the theory involving the microbial community and the application of anaerobic treatment in a psychrophilic environment.

Characterizing the microbial colonization of a dolostone quarry: implications for stone biodeterioration and response to biocide treatments.

PubMed

Cámara, Beatriz; De los Ríos, Asuncion; Urizal, Marta; de Buergo, Mónica Alvarez; Varas, Maria Jose; Fort, Rafael; Ascaso, Carmen

2011-08-01

This study examines the microbial colonization of three fronts of an abandoned dolostone quarry (Redueña, Madrid, Spain) exposed to atmospheric conditions for different time periods since Roman times to the present. Through scanning electron microscopy in backscattered electron mode (SEM-BSE), endolithic colonization was predominantly detected in the most recently exposed front, while in the longer exposed quarry fronts, epilithic forms of growth were most often observed. These observations were confirmed by denaturing gradient gel electrophoresis (DGGE) analysis. Based on the distribution pattern of microbial colonization in the different quarry fronts, we then established a sequence of colonization events that took place over this long time frame. Bioalteration processes related to this sequential colonization were also identified. Characterizing these sequential processes can be useful for interpreting biodeterioration processes in historic dolostone monuments, especially those affecting constructions in the area of the Redueña stone quarry. In a second experimental stage, different biocide treatments were tested on this quarry rock to find the best way to avoid the microbial colonization effects identified. Through combined SEM-BSE/DGGE analysis, the efficacy of several biocides against the microorganisms inhabiting the dolostones was assessed after 4 and 16 months treatment. In general, all treatments were effective at reducing around 80% of the lichen cover, although effects on endolithic lithobiontic communities were dependent on how well the rock surface had been mechanically cleaned prior to treatment and gradually disappeared over time.

Application of aerobic composting system for space agriculture

NASA Astrophysics Data System (ADS)

Oshima, Tairo; Yoshii, Takahiro; Moriya, Toshiyuki; Yamashita, Masamichi

Composting is a classical technique to decompose organic wastes such as animal bodies, straw, paper, raw sludge, and so on. Compared with burning of wastes, the composting method has many advantages. It is an inexpensive and safer method because of its self-heating without spending extra energy resources. It does not emit toxic pollutants such as dioxin, NOx , and SOx . The composting products can be used as organic fertilizers for agricultural production. Composting is a promising way for digesting organic wastes safely on spaceships or manned exploration on extraterrestrial planets. We have developed a small scale high-temperature composter in order to examine its feasobility to operate food waste disposing facility and fertilizer production in space. This composter has a heated reaction vessel containing compost soil (seed bacteria) provided by a compost factory. To determine the optimal condition for its operation, we analyzed the effect of temperature on metabolic activity (CO2 production rate), and water content. The dynamics of microbial community was studied by polymerase chain reaction - denaturing gradient gel electrophoresis (PCR-DGGE). Water content was maintained to a range between 27% and 40% by continuously adding water. The highest CO2 emission was observed at around 70° C. PCR-DGGE analysis shows that the bacterial community of the compost soil is dramatically changed by changing reaction temperature. We will discuss the application of the composter in space in order to establish the closed recycling loop of bio-elements in space agriculture.

Investigation of Arsenotrophic Microbiome in Arsenic-Affected Bangladesh Groundwater.

PubMed

Sultana, Munawar; Mou, Taslin Jahan; Sanyal, Santonu Kumar; Diba, Farzana; Mahmud, Zahid Hayat; Parvez, Anowar Khasru; Hossain, M Anwar

2017-09-01

Arsenotrophic bacteria contribute to the nutrient cycling in arsenic (As) affected groundwater. This study employed a culture-independent and -dependent investigation of arsenotrophic microbiomes in As affected groundwater samples collected from Madhabpur, Sonatengra, and Union Porishod in Singair Upazila, Manikganj, Bangladesh. Total As contents, detected by Atomic Absorption Spectrophotometry (AAS) of the samples, were 47 µg/L (Madhabpur, SNGW-1), 53 µg/L (Sonatengra, SNGW-2), and 12 µg/L (Union porishod, SNGW-3), whereas the control well (SNGW-4; depths >150 m) showed As content of 6 µg/L. Denaturing Gradient Gel Electrophoresis (DGGE) analysis of the amplified 16S rRNA gene from As-affected groundwater samples revealed the dominance of aerobic bacteria Pseudomonas within heterogeneous bacterial populations. DGGE of heterotrophic enrichments supplemented with arsenite [As (III)] for 4 weeks showed the dominance of Chryseobacterium, Flavobacterium, and Aquabacterium, whereas the dominant genera in that of autotrophic enrichments were Aeromonas, Acinetobacter, and Pseudomonas. Cultured bacteria retrieved from both autotrophic and heterotrophic enrichments were distinguished into nine genotypes belonging to Chryseobacterium, Acinetobacter, Escherichia, Pseudomonas, Stenotrophomonas, Janibacter, Staphylococcus, and Bacillus. They exhibited varying range of As(III) tolerance from 4 to 27 mM. As(III) transformation potential was confirmed within the isolates with oxidation rate as high as 0.143 mM/h for Pseudomonas sp. Sn 28. The arsenotrophic microbiome specifies their potential role in groundwater As-cycling and their genetic information provide the scientific basis for As-bioremediation. © 2017, National Ground Water Association.

Evaluation of the specificity and effectiveness of selected oral hygiene actives in salivary biofilm microcosms.

PubMed

Ledder, Ruth G; Sreenivasan, Prem K; DeVizio, William; McBain, Andrew J

2010-12-01

The microbiological effects of biocidal products used for the enhancement of oral hygiene relate to the active compound(s) as well as other formulation components. Here, we test the specificities of selected actives in the absence of multiple excipients. Salivary ecosystems were maintained in tissue culture plate-based hydroxyapatite disc models (HDMs) and modified drip-flow biofilm reactors (MDFRs). Test compounds stannous fluoride (SF), SDS, triclosan (TCS), zinc lactate (ZL) and ZL with SF in combination (ZLSF) were delivered to the HDMs once and four times daily for 6 days to MDFRs. Plaques were characterized by differential viable counting and PCR-denaturing gradient gel electrophoresis (DGGE). TCS and SDS were the most effective compounds against HDM plaques, significantly reducing total viable counts (P<0.05), whilst SF, ZL and ZLSF were comparatively ineffective. TCS exhibited specificity for streptococci (P<0.01) and Gram-negative anaerobes (P<0.01) following a single dosing and also on repeated dosing in MDFRs. In contrast to single exposures, multiple dosing with ZLSF also significantly reduced all bacterial groups, whilst SF and ZL caused significant but transient reductions. According to PCR-DGGE analyses, significant (P<0.05) reductions in eubacterial diversity occurred following 6 day dosing with both TCS and ZLSF. Concordance of MDFR eubacterial profiles with salivary inocula ranged between 58 and 97%. TCS and ZL(SF) exhibited similar specificities to those reported for formulations. TCS was the most potent antibacterial, after single and multiple dosage regimens.

Characterization of bacterial community associated to biofilms of corroded oil pipelines from the southeast of Mexico.

PubMed

Neria-González, Isabel; Wang, En Tao; Ramírez, Florina; Romero, Juan M; Hernández-Rodríguez, César

2006-06-01

Microbial communities associated to biofilms promote corrosion of oil pipelines. The community structure of bacteria in the biofilm formed in oil pipelines is the basic knowledge to understand the complexity and mechanisms of metal corrosion. To assess bacterial diversity, biofilm samples were obtained from X52 steel coupons corroded after 40 days of exposure to normal operation and flow conditions. The biofilm samples were directly used to extract metagenomic DNA, which was used as template to amplify 16S ribosomal gene by PCR. The PCR products of 16S ribosomal gene were also employed as template for sulfate-reducing bacteria (SRB) specific nested-PCR and both PCR products were utilized for the construction of gene libraries. The V3 region of the 16S rRNA gene was also amplified to analyse the bacterial diversity by analysis of denaturing gradient gel electrophoresis (DGGE). Ribosomal library and DGGE profiles exhibited limited bacterial diversity, basically including Citrobacter spp., Enterobacter spp. and Halanaerobium spp. while Desulfovibrio alaskensis and a novel clade within the genus Desulfonatronovibrio were detected from the nested PCR library. The biofilm samples were also taken for the isolation of SRB. Desulfovibrio alaskensis and Desulfovibrio capillatus, as well as some strains related to Citrobacter were isolated. SRB consists in a very small proportion of the community and Desulfovibrio spp. were the relatively abundant groups among the SRB. This is the first study directly exploring bacterial diversity in corrosive biofilms associated to steel pipelines subjected to normal operation conditions.

Microbial fouling community analysis of the cooling water system of a nuclear test reactor with emphasis on sulphate reducing bacteria.

PubMed

Balamurugan, P; Joshi, M Hiren; Rao, T S

2011-10-01

Culture and molecular-based techniques were used to characterize bacterial diversity in the cooling water system of a fast breeder test reactor (FBTR). Techniques were selected for special emphasis on sulphate-reducing bacteria (SRB). Water samples from different locations of the FBTR cooling water system, in addition to biofilm scrapings from carbon steel coupons and a control SRB sample were characterized. Whole genome extraction of the water samples and SRB diversity by group specific primers were analysed using nested PCR and denaturing gradient gel electrophoresis (DGGE). The results of the bacterial assay in the cooling water showed that the total culturable bacteria (TCB) ranged from 10(3) to 10(5) cfu ml(-1); iron-reducing bacteria, 10(3) to 10(5) cfu ml(-1); iron oxidizing bacteria, 10(2) to 10(3) cfu ml(-1) and SRB, 2-29 cfu ml(-1). However, the counts of the various bacterial types in the biofilm sample were 2-3 orders of magnitude higher. SRB diversity by the nested PCR-DGGE approach showed the presence of groups 1, 5 and 6 in the FBTR cooling water system; however, groups 2, 3 and 4 were not detected. The study demonstrated that the PCR protocol influenced the results of the diversity analysis. The paper further discusses the microbiota of the cooling water system and its relevance in biofouling.

Microscopic and molecular studies of the diversity of free-living protozoa in meat-cutting plants.

PubMed

Vaerewijck, Mario J M; Sabbe, Koen; Baré, Julie; Houf, Kurt

2008-09-01

The diversity of free-living protozoa in five meat-cutting plants was determined. Light microscopy after enrichment culturing was combined with sequencing of PCR-amplified, denaturing gradient gel electrophoresis (DGGE)-separated 18S rRNA gene fragments, which was used as a fast screening method. The general results of the survey showed that a protozoan community of amoebae, ciliates, and flagellates was present in all of the plants. Protozoa were detected mainly in floor drains, in standing water on the floor, on soiled bars of cutting tables, on plastic pallets, and in out-of-use hot water knife sanitizers, but they were also detected on surfaces which come into direct contact with meat, such as conveyer belts, working surfaces of cutting tables, and needles of a meat tenderizer. After 7 days of incubation at refrigerator temperature, protozoa were detected in about one-half of the enrichment cultures. Based on microscopic observations, 61 morphospecies were found, and Bodo saltans, Bodo spp., Epistylis spp., Glaucoma scintillans, Petalomonas spp., Prodiscophrya collini, and Vannella sp. were the most frequently encountered identified organisms. Sequencing of DGGE bands resulted in identification of a total of 49 phylotypes, including representatives of the Amoebozoa, Chromalveolata, Excavata, Opisthokonta, and Rhizaria. Sequences of small heterotrophic flagellates were affiliated mainly with the Alveolata (Apicomplexa), Stramenopiles (Chrysophyceae), and Rhizaria (Cercozoa). This survey showed that there is high protozoan species richness in meat-cutting plants and that the species included species related to known hosts of food-borne pathogens.

Revealing the microbiota of marketed edible insects through PCR-DGGE, metagenomic sequencing and real-time PCR.

PubMed

Osimani, Andrea; Milanović, Vesna; Garofalo, Cristiana; Cardinali, Federica; Roncolini, Andrea; Sabbatini, Riccardo; De Filippis, Francesca; Ercolini, Danilo; Gabucci, Claudia; Petruzzelli, Annalisa; Tonucci, Franco; Clementi, Francesca; Aquilanti, Lucia

2018-07-02

The present study aimed to identify the microbiota present in six species of processed edible insects produced in Thailand and marketed worldwide via the internet, namely, giant water bugs (Belostoma lutarium), black ants (Polyrhachis), winged termites (alates, Termitoidae), rhino beetles (Hyboschema contractum), mole crickets (Gryllotalpidae), and silkworm pupae (Bombyx mori). For each species, two samples of boiled, dried and salted insects were purchased. The microbial DNA was extracted from the insect samples and subjected to polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), high-throughput sequencing and qualitative real-time PCR assays. The microbiota of the analyzed samples were widely characterized by the presence of spore-forming bacteria mainly represented by the genera Bacillus and Clostridium. Moreover, the genera Anaerobacillus, Paenibacillus, Geobacillus, Pseudomonas, Stenotrophomonas, Massilia, Delftia, Lactobacillus, Staphylococcus, Streptococcus, Vagococcus, and Vibrio were also detected. Real-time PCR allowed for ascertainment of the absence of Coxiella burnetii, Shiga toxin-producing E. coli (STEC), and Pseudomonas aeruginosa in all samples. The results of this study confirm the importance of combining different molecular techniques to characterize the biodiversity of complex ecosystems such as edible insects. The presence of potential human pathogens suggests the need for a careful application of good manufacturing practices during insect processing. This study provides further data that will be useful in risk analyses of edible insects as a novel food source. Copyright © 2018 Elsevier B.V. All rights reserved.

Characterisation of the bacterial community in expressed prostatic secretions from patients with chronic prostatitis/chronic pelvic pain syndrome and infertile men: a preliminary investigation

PubMed Central

Hou, Dong-Sheng; Long, Wen-Min; Shen, Jian; Zhao, Li-Ping; Pang, Xiao-Yan; Xu, Chen

2012-01-01

The expressed prostatic secretions (EPSs) of men with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), infertile men and normal men were subjected to microbiological study. EPSs were collected from the subjects, which included 26 normal men, 11 infertile patients and 51 CP/CPPS patients. DNA was extracted from each specimen, and the V3 regions of the 16S rRNA genes were amplified using universal bacterial primers. The results showed that the EPS 16S rRNA gene-positive rate in the CP/CPPS and infertile patients was much higher than in the normal men, but without any difference among the three patient groups. The denaturing gradient gel electrophoresis (DGGE) method was used to characterize the EPS bacterial community structure of the prostate fluid from patients with CP/CPPS or infertility issues. Principal component analysis (PCA) and partial least squares (PLS) analyses of PCR-DGGE profiles revealed that the EPS bacterial community structure differed among the three groups. Three bands were identified as the key factors responsible for the discrepancy between CP/CPPS patients and infertile patients (P<0.05). Two bands were identified as priority factors in the discrepancy of category IIIA and category IIIB prostatitis patients (P<0.05). According to this research, the ecological balance of the prostate and low urethra tract, when considered as a microenvironment, might play an important role in the maintenance of a healthy male reproductive tract. PMID:22635162

Diversity of Fungi, Bacteria, and Actinomycetes on Leaves Decomposing in a Stream▿

PubMed Central

Das, Mitali; Royer, Todd V.; Leff, Laura G.

2007-01-01

Although fungi, bacteria, and specific bacterial taxa, such as the actinomycetes, have been studied extensively in various habitats, few studies have examined them simultaneously, especially on decomposing leaves in streams. In this study, sugar maple and white oak leaves were incubated in a stream in northeastern Ohio for 181 days during which samples were collected at regular intervals. Following DNA extraction, PCR-denaturing gradient gel electrophoresis (DGGE) was performed using fungus-, bacterium-, and actinomycete-specific primers. In addition, fungal and bacterial biomass was estimated. Fungal biomass differed on different days but not between leaves of the two species and was always greater than bacterial biomass. There were significant differences in bacterial biomass through time and between leaf types on some days. Generally, on the basis of DGGE, few differences in community structure were found for different leaf types. However, the ribotype richness of fungi was significantly greater than those of the bacteria and actinomycetes, which were similar to each other. Ribotype richness decreased toward the end of the study for each group except bacteria. Lack of differences between the two leaf types suggests that the microorganisms colonizing the leaf biofilm were primarily generalists that could exploit the resources of the leaves of either species equally well. Thus, we conclude that factors, such as the ecological role of the taxa (generalists versus specialists), stage of decay, and time of exposure, appeared to be more important determinants of microbial community structure than leaf quality. PMID:17142366

Depth-resolved abundance and diversity of arsenite-oxidizing bacteria in the groundwater of Beimen, a blackfoot disease endemic area of southwestern Taiwan.

PubMed

Das, Suvendu; Kar, Sandeep; Jean, Jiin-Shuh; Rathod, Jagat; Chakraborty, Sukalyan; Liu, Hsiao-Sheng; Bundschuh, Jochen

2013-12-01

The role of arsenite oxidizers in natural attenuation of arsenic pollution necessitates studies on their abundance and diversity in arsenic-contaminated aquifers. In this study, most probable number-polymerase chain reaction (MPN-PCR) and denaturing gradient gel electrophoresis (DGGE) was applied to monitor depth-wise abundance and diversity of aerobic arsenite oxidizers in arsenic-enriched groundwater of Beimen, southwestern Taiwan. The results revealed that the abundance of arsenite oxidizers ranged from 0.04 to 0.22, and the lowest ratio was observed in the most arsenic-enriched and comparatively more reduced groundwater (depth 200 m) of Beimen 1. The highest ratio was observed in the less arsenic-enriched and less reduced groundwater (depth 60 m) of Beimen 2B. DGGE profiles showed a shift in diversity of arsenite oxidizers, consisting of members of the Betaproteobacteria (61%), Alphaproteobacteria (28%) and Gammaproteobacteria (11%), depending on mainly arsenic concentration and redox level in groundwater. Groundwater with the lowest arsenic and highest dissolved oxygen at Beimen 2B harbored 78% of the arsenite oxidizers communities, while groundwater with the highest arsenic and lowest dissolved oxygen at Beimen 1 and Beimen-Jinhu harbored 17 and 22% of arsenite oxidizers communities, respectively. Pseudomonas sp. was found only in groundwater containing high arsenic at Beimen 1 and Beimen-Jinhu, while arsenite oxidizers belonging to Alpha- and Betaproteobacteria were dominated in groundwater containing low arsenic. Copyright © 2013 Elsevier Ltd. All rights reserved.

Impact of Feed Efficiency and Diet on Adaptive Variations in the Bacterial Community in the Rumen Fluid of Cattle

PubMed Central

Hernandez-Sanabria, Emma; Goonewardene, Laksiri A.; Wang, Zhiquan; Durunna, Obioha N.; Moore, Stephen S.

2012-01-01

Limited knowledge of the structure and activities of the ruminal bacterial community prevents the understanding of the effect of population dynamics on functional bacterial groups and on host productivity. This study aimed to identify particular bacteria associated with host feed efficiency in steers with differing diets and residual feed intake (RFI) using culture-independent methods: PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR analysis. PCR-DGGE profiles were generated from the ruminal fluid of 55 steers fed a low-energy-density diet and then switched to a high-energy-density diet. Bacterial profile comparisons by multivariate statistical analysis showed a trend only for RFI-related clusters on the high-energy diet. When steers (n = 19) belonging to the same RFI group under both diets were used to identify specific bacterial phylotypes related to feed efficiency traits, correlations were detected between dry matter intake, average daily gain, and copy numbers of the 16S rRNA gene of Succinivibrio sp. in low-RFI (efficient) steers, whereas correlations between Robinsoniella sp. and RFI (P < 0.05) were observed for high-RFI (inefficient) animals. Eubacterium sp. differed significantly (P < 0.05) between RFI groups that were only on the high-energy diet. Our work provides a comprehensive framework to understand how particular bacterial phylotypes contribute to differences in feed efficiency and ultimately influence host productivity, which may either depend on or be independent from diet factors. PMID:22156428

Sediment Enzyme Activities and Microbial Community Diversity in an Oligotrophic Drinking Water Reservoir, Eastern China

PubMed Central

Zhang, Haihan; Huang, Tinglin; Liu, Tingting

2013-01-01

Drinking water reservoir plays a vital role in the security of urban water supply, yet little is known about microbial community diversity harbored in the sediment of this oligotrophic freshwater environmental ecosystem. In the present study, integrating community level physiological profiles (CLPPs), nested polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and clone sequence technologies, we examined the sediment urease and protease activities, bacterial community functional diversity, genetic diversity of bacterial and fungal communities in sediments from six sampling sites of Zhou cun drinking water reservoir, eastern China. The results showed that sediment urease activity was markedly distinct along the sites, ranged from 2.48 to 11.81 mg NH3-N/(g·24h). The highest average well color development (AWCD) was found in site C, indicating the highest metabolic activity of heterotrophic bacterial community. Principal component analysis (PCA) revealed tremendous differences in the functional (metabolic) diversity patterns of the sediment bacterial communities from different sites. Meanwhile, DGGE fingerprints also indicated spatial changes of genetic diversity of sediment bacterial and fungal communities. The sequence BLAST analysis of all the sediment samples found that Comamonas sp. was the dominant bacterial species harbored in site A. Alternaria alternate, Allomyces macrogynus and Rhizophydium sp. were most commonly detected fungal species in sediments of the Zhou cun drinking water reservoir. The results from this work provide new insights about the heterogeneity of sediment microbial community metabolic activity and genetic diversity in the oligotrophic drinking water reservoir. PMID:24205265

Effect of Condensed Tannins on Bacterial Diversity and Metabolic Activity in the Rat Gastrointestinal Tract

PubMed Central

Smith, Alexandra H.; Mackie, Roderick I.

2004-01-01

The effect of dietary condensed tannins (proanthocyanidins) on rat fecal bacterial populations was ascertained in order to determine whether the proportion on tannin-resistant bacteria increased and if there was a change in the predominant bacterial populations. After 3 weeks of tannin diets the proportion of tannin-resistant bacteria increased significantly (P < 0.05) from 0.3% ± 5.5% to 25.3% ± 8.3% with a 0.7% tannin diet and to 47.2% ± 5.1% with a 2% tannin diet. The proportion of tannin-resistant bacteria returned to preexposure levels in the absence of dietary tannins. A shift in bacterial populations was confirmed by molecular fingerprinting of fecal bacterial populations by denaturing gradient gel electrophoresis (DGGE). Posttreatment samples were generally still distinguishable from controls after 3.5 weeks. Sequence analysis of DGGE bands and characterization of tannin-resistant isolates indicated that tannins selected for Enterobacteriaceae and Bacteroides species. Dot blot quantification confirmed that these gram-negative bacterial groups predominated in the presence of dietary tannins and that there was a corresponding decrease in the gram-positive Clostridium leptum group and other groups. Metabolic fingerprint patterns revealed that functional activities of culturable fecal bacteria were affected by the presence of tannins. Condensed tannins of Acacia angustissima altered fecal bacterial populations in the rat gastrointestinal tract, resulting in a shift in the predominant bacteria towards tannin-resistant gram-negative Enterobacteriaceae and Bacteroides species. PMID:14766594

Analysis of a microbial community oxidizing inorganic sulfide and mercaptans.

PubMed

Duncan, K E; Sublette, K L; Rider, P A; Stepp, A; Beitle, R R; Conner, J A; Kolhatkar, R

2001-01-01

Successful treatment of refinery spent-sulfidic caustic (which results from the addition of sodium hydroxide solutions to petroleum refinery waste streams) was achieved in a bioreactor containing an enrichment culture immobilized in organic polymer beads with embedded powdered activated carbon (Bio-Sep). The aerobic enrichment culture had previously been selected using a gas mixture of hydrogen sulfide and methyl mercaptan (MeSH) as the sole carbon and energy sources. The starting cultures for the enrichment consisted of several different Thiobacilli spp. (T. thioparus, T. denitrificans, T. thiooxidans, and T. neopolitanus), as well as activated sludge from a refinery aerobic wastewater treatment system and sludge from an industrial anaerobic digester. Microscopic examination (light and SEM) of the beads and of microbial growth on the walls of the bioreactor revealed a great diversity of microorganisms. Further characterization was undertaken starting with culturable aerobic heterotrophic microorganisms (sequencing of PCR-amplified DNA coding for 16S rRNA, Gram staining) and by PCR amplification of DNA coding for 16S rRNA extracted directly from the cell mass, followed by the separation of the PCR products by DGGE (denaturing gradient gel electrophoresis). Eight prominent bands from the DGGE gel were sequenced and found to be closest to sequences of uncultured Cytophagales (3 bands), Gram-positive cocci (Micrococcineae), alpha proteobacteria (3 bands), and an unidentified beta proteobacterium. Culturable microbes included several genera of fungi as well as various Gram-positive and Gram-negative heterotrophic bacteria not seen in techniques using direct DNA extraction.

Arbuscular Mycorrhizal Fungi Community Structure, Abundance and Species Richness Changes in Soil by Different Levels of Heavy Metal and Metalloid Concentration

PubMed Central

Krishnamoorthy, Ramasamy; Kim, Chang-Gi; Subramanian, Parthiban; Kim, Ki-Yoon; Selvakumar, Gopal; Sa, Tong-Min

2015-01-01

Arbuscular Mycorrhizal Fungi (AMF) play major roles in ecosystem functioning such as carbon sequestration, nutrient cycling, and plant growth promotion. It is important to know how this ecologically important soil microbial player is affected by soil abiotic factors particularly heavy metal and metalloid (HMM). The objective of this study was to understand the impact of soil HMM concentration on AMF abundance and community structure in the contaminated sites of South Korea. Soil samples were collected from the vicinity of an abandoned smelter and the samples were subjected to three complementary methods such as spore morphology, terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) for diversity analysis. Spore density was found to be significantly higher in highly contaminated soil compared to less contaminated soil. Spore morphological study revealed that Glomeraceae family was more abundant followed by Acaulosporaceae and Gigasporaceae in the vicinity of the smelter. T-RFLP and DGGE analysis confirmed the dominance of Funneliformis mosseae and Rhizophagus intraradices in all the study sites. Claroideoglomus claroideum, Funneliformis caledonium, Rhizophagus clarus and Funneliformis constrictum were found to be sensitive to high concentration of soil HMM. Richness and diversity of Glomeraceae family increased with significant increase in soil arsenic, cadmium and zinc concentrations. Our results revealed that the soil HMM has a vital impact on AMF community structure, especially with Glomeraceae family abundance, richness and diversity. PMID:26035444

Effect of bioaugmentation and biostimulation on sulfate-reducing column startup captured by functional gene profiling.

PubMed

Pereyra, Luciana P; Hiibel, Sage R; Perrault, Elizabeth M; Reardon, Kenneth F; Pruden, Amy

2012-10-01

Sulfate-reducing permeable reactive zones (SR-PRZs) depend upon a complex microbial community to utilize a lignocellulosic substrate and produce sulfides, which remediate mine drainage by binding heavy metals. To gain insight into the impact of the microbial community composition on the startup time and pseudo-steady-state performance, functional genes corresponding to cellulose-degrading (CD), fermentative, sulfate-reducing, and methanogenic microorganisms were characterized in columns simulating SR-PRZs using quantitative polymerase chain reaction (qPCR) and denaturing gradient gel electrophoresis (DGGE). Duplicate columns were bioaugmented with sulfate-reducing or CD bacteria or biostimulated with ethanol or carboxymethyl cellulose and compared with baseline dairy manure inoculum and uninoculated controls. Sulfate removal began after ~ 15 days for all columns and pseudo-steady state was achieved by Day 30. Despite similar performance, DGGE profiles of 16S rRNA gene and functional genes at pseudo-steady state were distinct among the column treatments, suggesting the potential to control ultimate microbial community composition via bioaugmentation and biostimulation. qPCR revealed enrichment of functional genes in all columns between the initial and pseudo-steady-state time points. This is the first functional gene-based study of CD, fermentative and sulfate-reducing bacteria and methanogenic archaea in a lignocellulose-based environment and provides new qualitative and quantitative insight into startup of a complex microbial system. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

The Freshwater Sponge Ephydatia fluviatilis Harbours Diverse Pseudomonas Species (Gammaproteobacteria, Pseudomonadales) with Broad-Spectrum Antimicrobial Activity

PubMed Central

Keller-Costa, Tina; Jousset, Alexandre; van Overbeek, Leo; van Elsas, Jan Dirk; Costa, Rodrigo

2014-01-01

Bacteria are believed to play an important role in the fitness and biochemistry of sponges (Porifera). Pseudomonas species (Gammaproteobacteria, Pseudomonadales) are capable of colonizing a broad range of eukaryotic hosts, but knowledge of their diversity and function in freshwater invertebrates is rudimentary. We assessed the diversity, structure and antimicrobial activities of Pseudomonas spp. in the freshwater sponge Ephydatia fluviatilis. Polymerase Chain Reaction – Denaturing Gradient Gel Electrophoresis (PCR-DGGE) fingerprints of the global regulator gene gacA revealed distinct structures between sponge-associated and free-living Pseudomonas communities, unveiling previously unsuspected diversity of these assemblages in freshwater. Community structures varied across E. fluviatilis specimens, yet specific gacA phylotypes could be detected by PCR-DGGE in almost all sponge individuals sampled over two consecutive years. By means of whole-genome fingerprinting, 39 distinct genotypes were found within 90 fluorescent Pseudomonas isolates retrieved from E. fluviatilis. High frequency of in vitro antibacterial (49%), antiprotozoan (35%) and anti-oomycetal (32%) activities was found among these isolates, contrasting less-pronounced basidiomycetal (17%) and ascomycetal (8%) antagonism. Culture extracts of highly predation-resistant isolates rapidly caused complete immobility or lysis of cells of the protozoan Colpoda steinii. Isolates tentatively identified as P. jessenii, P. protegens and P. oryzihabitans showed conspicuous inhibitory traits and correspondence with dominant sponge-associated phylotypes registered by cultivation-independent analysis. Our findings suggest that E. fluviatilis hosts both transient and persistent Pseudomonas symbionts displaying antimicrobial activities of potential ecological and biotechnological value. PMID:24533086

Characterization of Microbial Communities in Gas Industry Pipelines

PubMed Central

Zhu, Xiang Y.; Lubeck, John; Kilbane, John J.

2003-01-01

Culture-independent techniques, denaturing gradient gel electrophoresis (DGGE) analysis, and random cloning of 16S rRNA gene sequences amplified from community DNA were used to determine the diversity of microbial communities in gas industry pipelines. Samples obtained from natural gas pipelines were used directly for DNA extraction, inoculated into sulfate-reducing bacterium medium, or used to inoculate a reactor that simulated a natural gas pipeline environment. The variable V2-V3 (average size, 384 bp) and V3-V6 (average size, 648 bp) regions of bacterial and archaeal 16S rRNA genes, respectively, were amplified from genomic DNA isolated from nine natural gas pipeline samples and analyzed. A total of 106 bacterial 16S rDNA sequences were derived from DGGE bands, and these formed three major clusters: beta and gamma subdivisions of Proteobacteria and gram-positive bacteria. The most frequently encountered bacterial species was Comamonas denitrificans, which was not previously reported to be associated with microbial communities found in gas pipelines or with microbially influenced corrosion. The 31 archaeal 16S rDNA sequences obtained in this study were all related to those of methanogens and phylogenetically fall into three clusters: order I, Methanobacteriales; order III, Methanomicrobiales; and order IV, Methanosarcinales. Further microbial ecology studies are needed to better understand the relationship among bacterial and archaeal groups and the involvement of these groups in the process of microbially influenced corrosion in order to develop improved ways of monitoring and controlling microbially influenced corrosion. PMID:12957923

Impact of Nisin-Activated Packaging on Microbiota of Beef Burgers during Storage

PubMed Central

Ferrocino, Ilario; Greppi, Anna; La Storia, Antonietta; Rantsiou, Kalliopi; Ercolini, Danilo

2015-01-01

Beef burgers were stored at 4°C in a vacuum in nisin-activated antimicrobial packaging. Microbial ecology analyses were performed on samples collected between days 0 and 21 of storage to discover the population diversity. Two batches were analyzed using RNA-based denaturing gradient gel electrophoresis (DGGE) and pyrosequencing. The active packaging retarded the growth of the total viable bacteria and lactic acid bacteria. Culture-independent analysis by pyrosequencing of RNA extracted directly from meat showed that Photobacterium phosphoreum, Lactococcus piscium, Lactobacillus sakei, and Leuconostoc carnosum were the major operational taxonomic units (OTUs) shared between control and treated samples. Beta diversity analysis of the 16S rRNA sequence data and RNA-DGGE showed a clear separation between two batches based on the microbiota. Control samples from batch B showed a significant high abundance of some taxa sensitive to nisin, such as Kocuria rhizophila, Staphylococcus xylosus, Leuconostoc carnosum, and Carnobacterium divergens, compared to control samples from batch A. However, only from batch B was it possible to find a significant difference between controls and treated samples during storage due to the active packaging. Predicted metagenomes confirmed differences between the two batches and indicated that the use of nisin-based antimicrobial packaging can determine a reduction in the abundance of specific metabolic pathways related to spoilage. The present study aimed to assess the viable bacterial communities in beef burgers stored in nisin-based antimicrobial packaging, and it highlights the efficacy of this strategy to prolong beef burger shelf life. PMID:26546424

Unraveling the microbiota of teat apices of clinically healthy lactating dairy cows, with special emphasis on coagulase-negative staphylococci.

PubMed

Braem, G; De Vliegher, S; Verbist, B; Piessens, V; Van Coillie, E; De Vuyst, L; Leroy, F

2013-03-01

Swab samples (n=72) obtained from the teat apex of lactating dairy cows without visual signs of inflammation (n=18) were gathered on 2 well-managed Flemish dairy herds (herds 1 and 2) during the same month to assess the bacterial diversity of teat apices before milking. A combination of both culture-dependent [plating and (GTG)(5)-PCR fingerprinting of the colonies] and culture-independent [denaturing gradient gel electrophoresis (PCR-DGGE)] techniques indicated that the teat apices contain a wide diversity of bacterial genera. Despite a low bacterial load, 20 bacterial genera of 3 phyla (Actinobacteria, Firmicutes, and Proteobacteria) were present. The most prevalent bacteria were the coagulase-negative staphylococci (CNS), encompassing a total of 15 species, which were identified to the species level using a combination of (GTG)(5)-PCR fingerprinting, gene sequencing (16S ribosomal RNA and rpoB genes), and a novel PCR-DGGE technique based on the tuf-PCR amplicon. Overall bacterial diversity did not differ significantly between the herds or between noninfected and subclinically infected quarters in herd 1. In herd 1, borderline significant lower CNS species diversity was found on teat apices of noninfected quarters compared with subclinically infected quarters. The most prevalent CNS species were Staphylococcus haemolyticus and Staphylococcus equorum in both herds and Staphylococcus carnosus in herd 2. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Enrichment and Molecular Characterization of a Bacterial Culture That Degrades Methoxy-Methyl Urea Herbicides and Their Aniline Derivatives

PubMed Central

El-Fantroussi, Said

2000-01-01

Soil treated with linuron for more than 10 years showed high biodegradation activity towards methoxy-methyl urea herbicides. Untreated control soil samples taken from the same location did not express any linuron degradation activity, even after 40 days of incubation. Hence, the occurrence in the field of a microbiota having the capacity to degrade a specific herbicide was related to the long-term treatment of the soil. The enrichment culture isolated from treated soil showed specific degradation activity towards methoxy-methyl urea herbicides, such as linuron and metobromuron, while dimethyl urea herbicides, such as diuron, chlorotoluron, and isoproturon, were not transformed. The putative metabolic intermediates of linuron and metobromuron, the aniline derivatives 3,4-dichloroaniline and 4-bromoaniline, were also degraded. The temperature of incubation drastically affected degradation of the aniline derivatives. Whereas linuron was transformed at 28 and 37°C, 3,4-dichloroaniline was transformed only at 28°C. Monitoring the enrichment process by reverse transcription-PCR and denaturing gradient gel electrophoresis (DGGE) showed that a mixture of bacterial species under adequate physiological conditions was required to completely transform linuron. This research indicates that for biodegradation of linuron, several years of adaptation have led to selection of a bacterial consortium capable of completely transforming linuron. Moreover, several of the putative species appear to be difficult to culture since they were detectable by DGGE but were not culturable on agar plates. PMID:11097876

Molecular comparison of the sampling efficiency of four types of airborne bacterial samplers.

PubMed

Li, Kejun

2011-11-15

In the present study, indoor and outdoor air samples were collected using four types of air samplers often used for airborne bacterial sampling. These air samplers included two solid impactors (BioStage and RCS), one liquid impinger (BioSampler), and one filter sampler with two kinds of filters (a gelatin and a cellulose acetate filter). The collected air samples were further processed to analyze the diversity and abundance of culturable bacteria and total bacteria through standard culture techniques, denaturing gradient gel electrophoresis (DGGE) fingerprinting and quantitative polymerase chain reaction (qPCR) analysis. The DGGE analysis indicated that the air samples collected using the BioStage and RCS samplers have higher culturable bacterial diversity, whereas the samples collected using the BioSampler and the cellulose acetate filter sampler have higher total bacterial diversity. To obtain more information on the sampled bacteria, some gel bands were excised and sequenced. In terms of sampling efficiency, results from the qPCR tests indicated that the collected total bacterial concentration was higher in samples collected using the BioSampler and the cellulose acetate filter sampler. In conclusion, the sampling bias and efficiency of four kinds of air sampling systems were compared in the present study and the two solid impactors were concluded to be comparatively efficient for culturable bacterial sampling, whereas the liquid impactor and the cellulose acetate filter sampler were efficient for total bacterial sampling. Copyright © 2011 Elsevier B.V. All rights reserved.

Ecotoxicological assessment of pesticides and their combination on rhizospheric microbial community structure and function of Vigna radiata.

PubMed

Walvekar, Varsha Ashok; Bajaj, Swati; Singh, Dileep K; Sharma, Shilpi

2017-07-01

India is one of the leading countries in production and indiscriminate consumption of pesticides. Owing to their xenobiotic nature, pesticides affect soil microorganisms that serve as mediators in plant growth promotion. Our study aimed to deliver a comprehensive picture, by comparing the effects of synthetic pesticides (chlorpyriphos, cypermethrin, and a combination of both) with a biopesticide (azadirachtin) at their recommended field application level (L), and three times the recommended dosage (H) on structure and function of microbial community in rhizosphere of Vigna radiata. Effect on culturable fraction was assessed by enumeration on selective media, while PCR-denaturing gradient gel electrophoresis (DGGE) was employed to capture total bacterial community diversity. This was followed by a metabolic sketch using community-level physiological profiling (CLPP), to obtain a broader picture of the non-target effects on rhizospheric microbial community. Although plant parameters were not significantly affected by pesticide application, the microbial community structure experienced an undesirable impact as compared to control devoid of pesticide treatment. Examination of DGGE banding patterns through cluster analysis revealed that microbial community structure of pesticide-treated soils had only 70% resemblance to control rhizospheric soil even at 45 days post application. Drastic changes in the metabolic profiles of pesticide-treated soils were also detected in terms of substrate utilization, rhizospheric diversity, and evenness. It is noteworthy that the effects exacerbated by biopesticide were comparable to that of synthetic pesticides, thus emphasizing the significance of ecotoxicological assessments before tagging biopesticides as "safe alternatives."

Microscopic, chemical, and molecular-biological investigation of the decayed medieval stained window glasses of two Catalonian churches

PubMed Central

Piñar, Guadalupe; Garcia-Valles, Maite; Gimeno-Torrente, Domingo; Fernandez-Turiel, Jose Luis; Ettenauer, Jörg; Sterflinger, Katja

2013-01-01

We investigated the decayed historical church window glasses of two Catalonian churches, both under Mediterranean climate. Glass surfaces were studied by scanning electron microscopy (SEM), energy dispersive spectrometry (EDS), and X-ray diffraction (XRD). Their chemical composition was determined by wavelength-dispersive spectrometry (WDS) microprobe analysis. The biodiversity was investigated by molecular methods: DNA extraction from glass, amplification by PCR targeting the16S rRNA and ITS regions, and fingerprint analyses by denaturing gradient gel electrophoresis (DGGE). Clone libraries containing either PCR fragments of the bacterial 16S rDNA or the fungal ITS regions were screened by DGGE. Clone inserts were sequenced and compared with the EMBL database. Similarity values ranged from 89 to 100% to known bacteria and fungi. Biological activity in both sites was evidenced in the form of orange patinas, bio-pitting, and mineral precipitation. Analyses revealed complex bacterial communities consisting of members of the phyla Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. Fungi showed less diversity than bacteria, and species of the genera Cladosporium and Phoma were dominant. The detected Actinobacteria and fungi may be responsible for the observed bio-pitting phenomenon. Moreover, some of the detected bacteria are known for their mineral precipitation capabilities. Sequence results also showed similarities with bacteria commonly found on deteriorated stone monuments, supporting the idea that medieval stained glass biodeterioration in the Mediterranean area shows a pattern comparable to that on stone. PMID:24092957

Shifts of microbial communities of wheat (Triticum aestivum L.) cultivation in a closed artificial ecosystem.

PubMed

Qin, Youcai; Fu, Yuming; Dong, Chen; Jia, Nannan; Liu, Hong

2016-05-01

The microbial communities of plant ecosystems are in relation to plant growing environment, but the alteration in biodiversity of rhizosphere and phyllosphere microbial communities in closed and controlled environments is unknown. The purpose of this study is to analyze the change regularity of microbial communities with wheat plants dependent-cultivated in a closed artificial ecosystem. The microbial community structures in closed-environment treatment plants were investigated by a culture-dependent approach, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), and Illumina Miseq high-throughput sequencing. The results indicated that the number of microbes decreased along with time, and the magnitude of bacteria, fungi, and actinomycetes were 10(7)-10(8), 10(5), and 10(3)-10(4) CFU/g (dry weight), respectively. The analysis of PCR-DGGE and Illumina Miseq revealed that the wheat leaf surface and near-root substrate had different microbial communities at different periods of wheat ecosystem development and showed that the relative highest diversity of microbial communities appeared at late and middle periods of the plant ecosystem, respectively. The results also indicated that the wheat leaf and substrate had different microbial community compositions, and the wheat substrate had higher richness of microbial community than the leaf. Flavobacterium, Pseudomonas, Paenibacillus, Enterobacter, Penicillium, Rhodotorula, Acremonium, and Alternaria were dominant in the wheat leaf samples, and Pedobacter, Flavobacterium, Halomonas, Marinobacter, Salinimicrobium, Lysobacter, Pseudomonas, Halobacillus, Xanthomonas, Acremonium, Monographella, and Penicillium were dominant populations in the wheat near-root substrate samples.

[Analysis of community composition in dental plaque of elder people with root caries].

PubMed

Ma, Shan-fen; Liang, Jing-ping; Jiang, Yun-tao; Zhu, Cai-lian

2011-10-01

To analyze the community in dental plaque of elder people with root caries. Total DNAs were extracted from the root caries dental plaques of nine elders over 60 years of age. Polymerase chaid reaction-based denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze the microbial composition, DGGE bands were excised from the gels for sequencing and identification. The dominant genus in root caries dental plaque of elder people were: Acinetobacte [0.9% (1/114)], Actinobaculum [1.8% (2/114)], Actinomyces [15.8% (18/114)], Aggregatibacter [0.9% (1/114)], Capnocytophaga [14.0% (16/114)], Corynebacterium [0.9% (1/114)], Haemophilus [0.9% (1/114)], Mobiluncus [0.9% (1/114)], Naxibacter [0.9% (1/114)], Neisseriaceae [10.5% (12/114)], Porphyromonas [0.9% (1/114)], Prevotella [12.3% (14/114)], Selenomonas [6.1% (7/114)], Staphylococcus [1.8% (2/114)], Oralis streptococcus [6.1% (7/114)], Mutans streptococcu [7.9% (9/114)], Tannerella [0.9% (1/114)], Treponema [1.8% (2/114)], Veillonella [10.5% (12/114)] and two uncultured unknown genus [1.8% (2/114)]. Uncultred genotypes accounted for 19.30% of the total. Gram-positive bacteria genotype accounted for 31.6% (36/114), and Gram-negative bacteria genotype accounted for 66.7% (76/114). There were many bacteria genotypes in root caries dental plaque in the elderly, which were widely distributed. Gram-negative bacteria accounted for the majority. Genotype-specific pathogenic bacteria were not found.

[Characteristics of soil ammonia-oxidation microbial communities in different subtropical forests, China].

PubMed

Li, Yong-Chun; Liu, Bu-Rong; Guo, Shuai; Wu, Qi-Feng; Qin, Hua; Wu, Jia-Sen; Xu, Qiu-Fang

2014-01-01

To investigate the effects of different forest stands in subtropical China on the communities of soil ammonia-oxidizing microorganisms, we characterized the abundance of ammonia-oxidizing archaea (AOA) and bacteria (AOB), and the community structure of AOA in soils under stands of broad-leaved (BF) , Chinese fir (CF) , Pinus massoniana (PF) and moso bamboo (MB) forests using real-time quantitative PCR and denaturing gradient gel electrophoresis (DGGE). The results showed that the AOA gene copy numbers (1.62 x 10(6)-1.88 x 10(7) per gram of dry soil) were significantly higher than those of AOB genes (2.41 x 10(5)-4.36 x 10(5) per gram of dry soil). Significantly higher soil AOA abundance was detected in the MB than that in the CF (P < 0.05), and the latter was significantly higher than that in the BF and PF soils (P < 0.05). There were no significant differences in the soil AOB abundance among the four forest stands. As indicated by DGGE pattern, soil AOA species varied among the four forest stands. There was a difference in the soil AOA communities between the CF and MB stands. The AOA demonstrated a competitive advantage over the AOB in the soils under these major subtropical forests. Soil pH, concentrations of soil available potassium and organic carbon as well as the forest type were the main factors that influence the variation of AOA community structure and diversity.

Comparison of commercial DNA extraction kits for isolation and purification of bacterial and eukaryotic DNA from PAH-contaminated soils.

PubMed

Mahmoudi, Nagissa; Slater, Greg F; Fulthorpe, Roberta R

2011-08-01

Molecular characterization of the microbial populations of soils and sediments contaminated with polycyclic aromatic hydrocarbons (PAHs) is often a first step in assessing intrinsic biodegradation potential. However, soils are problematic for molecular analysis owing to the presence of organic matter, such as humic acids. Furthermore, the presence of contaminants, such as PAHs, can cause further challenges to DNA extraction, quantification, and amplification. The goal of our study was to compare the effectiveness of four commercial soil DNA extraction kits (UltraClean Soil DNA Isolation kit, PowerSoil DNA Isolation kit, PowerMax Soil DNA Isolation kit, and FastDNA SPIN kit) to extract pure, high-quality bacterial and eukaryotic DNA from PAH-contaminated soils. Six different contaminated soils were used to determine if there were any biases among the kits due to soil properties or level of contamination. Extracted DNA was used as a template for bacterial 16S rDNA and eukaryotic 18S rDNA amplifications, and PCR products were subsequently analyzed using denaturing gel gradient electrophoresis (DGGE). We found that the FastDNA SPIN kit provided significantly higher DNA yields for all soils; however, it also resulted in the highest levels of humic acid contamination. Soil texture and organic carbon content of the soil did not affect the DNA yield of any kit. Moreover, a liquid-liquid extraction of the DNA extracts found no residual PAHs, indicating that all kits were effective at removing contaminants in the extraction process. Although the PowerSoil DNA Isolation kit gave relatively low DNA yields, it provided the highest quality DNA based on successful amplification of both bacterial and eukaryotic DNA for all six soils. DGGE fingerprints among the kits were dramatically different for both bacterial and eukaryotic DNA. The PowerSoil DNA Isolation kit revealed multiple bands for each soil and provided the most consistent DGGE profiles among replicates for both bacterial and eukaryotic DNA.

The dominant microbial community associated with fermentation of Obushera (sorghum and millet beverages) determined by culture-dependent and culture-independent methods.

PubMed

Mukisa, Ivan M; Porcellato, Davide; Byaruhanga, Yusuf B; Muyanja, Charles M B K; Rudi, Knut; Langsrud, Thor; Narvhus, Judith A

2012-11-01

Obushera includes four fermented cereal beverages from Uganda namely: Obutoko, Enturire, Ekitiribita and Obuteire, whose microbial diversity has not hitherto been fully investigated. Knowledge of the microbial diversity and dynamics in these products is crucial for understanding their safety and development of appropriate starter cultures for controlled industrial processing. Culture-dependent and culture-independent techniques including denaturating gradient gel electrophoresis (DGGE) and mixed DNA sequencing of polymerase chain reaction (PCR) amplified ribosomal RNA genes were used to study the bacteria and yeast diversity of Obushera. The pH dropped from 6.0-4.6 to 3.5-4.0 within 1-2 days for Obutoko, Enturire and Obuteire whereas that of Ekitiribita decreased to 4.4 after 4 days. Counts of lactic acid bacteria (LAB) increased from 5.0 to 11.0 log cfug(-1) and yeasts increased from 3.4 to 7.1 log cfug(-1) while coliform counts decreased from 2.0 to <1 log cfug(-1) during four days of fermentation. LAB and yeast isolates were identified by rRNA gene sequence analysis. LAB isolates included: Enterococcus spp., Lactobacillus (Lb.) plantarum, Lb. fermentum, Lb. delbrueckii, Lactococcus lactis, Leuconostoc lactis, Streptococcus (S.) infantarius subsp. infantarius, Pediococcus pentosaceus and Weisella (W.) confusa. DGGE indicated predominance of S. gallolyticus, S. infantarius subsp. infantarius, Lb. fermentum, Lb. delbrueckii, W. confusa, Lb. reuteri, Fructobacillus spp., L. lactis and L. lactis. Yeast isolates included Clavispora lusitaniae, Cyberlindnera fabianii, Issatchenkia orientalis and Saccharomyces cerevisiae. DGGE indicated predominance of S. cerevisiae in Obutoko, Enturire and Obuteire and also detected Pichia spp. and I. orientalis in Obutoko. Obushera produced in the laboratory was initially dominated by Enterobacteriaceae and later by Lactococcus spp. Enterobacteriaceae and Bacillus spp. were also detected in Ekitiribita. Development of starters for Obushera may require combinations of LAB and S. cerevisiae for Obutoko, Enturire and Obuteire and LAB for Ekitiribita. Copyright © 2012 Elsevier B.V. All rights reserved.

Phylogenetically and Spatially Close Marine Sponges Harbour Divergent Bacterial Communities

PubMed Central

Hardoim, Cristiane C. P.; Esteves, Ana I. S.; Pires, Francisco R.; Gonçalves, Jorge M. S.; Cox, Cymon J.; Xavier, Joana R.; Costa, Rodrigo

2012-01-01

Recent studies have unravelled the diversity of sponge-associated bacteria that may play essential roles in sponge health and metabolism. Nevertheless, our understanding of this microbiota remains limited to a few host species found in restricted geographical localities, and the extent to which the sponge host determines the composition of its own microbiome remains a matter of debate. We address bacterial abundance and diversity of two temperate marine sponges belonging to the Irciniidae family - Sarcotragus spinosulus and Ircinia variabilis – in the Northeast Atlantic. Epifluorescence microscopy revealed that S. spinosulus hosted significantly more prokaryotic cells than I. variabilis and that prokaryotic abundance in both species was about 4 orders of magnitude higher than in seawater. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) profiles of S. spinosulus and I. variabilis differed markedly from each other – with higher number of ribotypes observed in S. spinosulus – and from those of seawater. Four PCR-DGGE bands, two specific to S. spinosulus, one specific to I. variabilis, and one present in both sponge species, affiliated with an uncultured sponge-specific phylogenetic cluster in the order Acidimicrobiales (Actinobacteria). Two PCR-DGGE bands present exclusively in S. spinosulus fingerprints affiliated with one sponge-specific phylogenetic cluster in the phylum Chloroflexi and with sponge-derived sequences in the order Chromatiales (Gammaproteobacteria), respectively. One Alphaproteobacteria band specific to S. spinosulus was placed in an uncultured sponge-specific phylogenetic cluster with a close relationship to the genus Rhodovulum. Our results confirm the hypothesized host-specific composition of bacterial communities between phylogenetically and spatially close sponge species in the Irciniidae family, with S. spinosulus displaying higher bacterial community diversity and distinctiveness than I. variabilis. These findings suggest a pivotal host-driven effect on the shape of the marine sponge microbiome, bearing implications to our current understanding of the distribution of microbial genetic resources in the marine realm. PMID:23300853

Variation of Bacterial Community Diversity in Rhizosphere Soil of Sole-Cropped versus Intercropped Wheat Field after Harvest.

PubMed

Yang, Zhenping; Yang, Wenping; Li, Shengcai; Hao, Jiaomin; Su, Zhifeng; Sun, Min; Gao, Zhiqiang; Zhang, Chunlai

2016-01-01

As the major crops in north China, spring crops are usually planted from April through May every spring and harvested in fall. Wheat is also a very common crop traditionally planted in fall or spring and harvested in summer year by year. This continuous cropping system exhibited the disadvantages of reducing the fertility of soil through decreasing microbial diversity. Thus, management of microbial diversity in the rhizosphere plays a vital role in sustainable crop production. In this study, ten common spring crops in north China were chosen sole-cropped and four were chosen intercropped with peanut in wheat fields after harvest. Denaturing gradient gel electrophoresis (DGGE) and DNA sequencing of one 16S rDNA fragment were used to analyze the bacterial diversity and species identification. DGGE profiles showed the bacterial community diversity in rhizosphere soil samples varied among various crops under different cropping systems, more diverse under intercropping system than under sole-cropping. Some intercropping-specific bands in DGGE profiles suggested that several bacterial species were stimulated by intercropping systems specifically. Furthermore, the identification of these dominant and functional bacteria by DNA sequencing indicated that intercropping systems are more beneficial to improve soil fertility. Compared to intercropping systems, we also observed changes in microbial community of rhizosphere soil under sole-crops. The rhizosphere bacterial community structure in spring crops showed a strong crop species-specific pattern. More importantly, Empedobacter brevis, a typical plant pathogen, was only found in the carrot rhizosphere, suggesting carrot should be sown prudently. In conclusion, our study demonstrated that crop species and cropping systems had significant effects on bacterial community diversity in the rhizosphere soils. We strongly suggest sorghum, glutinous millet and buckwheat could be taken into account as intercropping crops with peanut; while hulled oat, mung bean or foxtail millet could be considered for sowing in wheat fields after harvest in North China.

Soil mineral composition matters: response of microbial communities to phenanthrene and plant litter addition in long-term matured artificial soils.

PubMed

Babin, Doreen; Vogel, Cordula; Zühlke, Sebastian; Schloter, Michael; Pronk, Geertje Johanna; Heister, Katja; Spiteller, Michael; Kögel-Knabner, Ingrid; Smalla, Kornelia

2014-01-01

The fate of polycyclic aromatic hydrocarbons (PAHs) in soil is determined by a suite of biotic and abiotic factors, and disentangling their role in the complex soil interaction network remains challenging. Here, we investigate the influence of soil composition on the microbial community structure and its response to the spiked model PAH compound phenanthrene and plant litter. We used long-term matured artificial soils differing in type of clay mineral (illite, montmorillonite) and presence of charcoal or ferrihydrite. The soils received an identical soil microbial fraction and were incubated for more than two years with two sterile manure additions. The matured artificial soils and a natural soil were subjected to the following spiking treatments: (I) phenanthrene, (II) litter, (III) litter + phenanthrene, (IV) unspiked control. Total community DNA was extracted from soil sampled on the day of spiking, 7, 21, and 63 days after spiking. Bacterial 16S rRNA gene and fungal internal transcribed spacer amplicons were quantified by qPCR and subjected to denaturing gradient gel electrophoresis (DGGE). DGGE analysis revealed that the bacterial community composition, which was strongly shaped by clay minerals after more than two years of incubation, changed in response to spiked phenanthrene and added litter. DGGE and qPCR showed that soil composition significantly influenced the microbial response to spiking. While fungal communities responded only in presence of litter to phenanthrene spiking, the response of the bacterial communities to phenanthrene was less pronounced when litter was present. Interestingly, microbial communities in all artificial soils were more strongly affected by spiking than in the natural soil, which might indicate the importance of higher microbial diversity to compensate perturbations. This study showed the influence of soil composition on the microbiota and their response to phenanthrene and litter, which may increase our understanding of complex interactions in soils for bioremediation applications.

Deterioration to extinction of wastewater bacteria by non-thermal atmospheric pressure air plasma as assessed by 16S rDNA-DGGE fingerprinting

PubMed Central

El-Sayed, Wael S.; Ouf, Salama A.; Mohamed, Abdel-Aleam H.

2015-01-01

The use of cold plasma jets for inactivation of a variety of microorganisms has recently been evaluated via culture-based methods. Accordingly, elucidation of the role of cold plasma in decontamination would be inaccurate because most microbial populations within a system remain unexplored owing to the high amount of yet uncultured bacteria. The impact of cold atmospheric plasma on the bacterial community structure of wastewater from two different industries was investigated by metagenomic-based polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) utilizing 16S rRNA genes. Three doses of atmospheric pressure dielectric barrier discharge plasma were applied to wastewater samples on different time scales. DGGE revealed that the bacterial community gradually changed and overall abundance decreased to extinction upon plasma treatment. The bacterial community in food processing wastewater contained 11 key operational taxonomic units that remained almost completely unchanged when exposed to plasma irradiation at 75.5 mA for 30 or 60 s. However, when exposure time was extended to 90 s, only Escherichia coli, Coliforms, Aeromonas sp., Vibrio sp., and Pseudomonas putida survived. Only E. coli, Aeromonas sp., Vibrio sp., and P. putida survived treatment at 81.94 mA for 90 s. Conversely, all bacterial groups were completely eliminated by treatment at 85.34 mA for either 60 or 90 s. Dominant bacterial groups in leather processing wastewater also changed greatly upon exposure to plasma at 75.5 mA for 30 or 60 s, with Enterobacter aerogenes, Klebsiella sp., Pseudomonas stutzeri, and Acidithiobacillus ferrooxidans being sensitive to and eliminated from the community. At 90 s of exposure, all groups were affected except for Pseudomonas sp. and Citrobacter freundii. The same trend was observed for treatment at 81.94 mA. The variability in bacterial community response to different plasma treatment protocols revealed that plasma had a selective impact on bacterial community structure at lower doses and potential bactericidal effects at higher doses. PMID:26500637

Soil Mineral Composition Matters: Response of Microbial Communities to Phenanthrene and Plant Litter Addition in Long-Term Matured Artificial Soils

PubMed Central

Babin, Doreen; Vogel, Cordula; Zühlke, Sebastian; Schloter, Michael; Pronk, Geertje Johanna; Heister, Katja; Spiteller, Michael; Kögel-Knabner, Ingrid; Smalla, Kornelia

2014-01-01

The fate of polycyclic aromatic hydrocarbons (PAHs) in soil is determined by a suite of biotic and abiotic factors, and disentangling their role in the complex soil interaction network remains challenging. Here, we investigate the influence of soil composition on the microbial community structure and its response to the spiked model PAH compound phenanthrene and plant litter. We used long-term matured artificial soils differing in type of clay mineral (illite, montmorillonite) and presence of charcoal or ferrihydrite. The soils received an identical soil microbial fraction and were incubated for more than two years with two sterile manure additions. The matured artificial soils and a natural soil were subjected to the following spiking treatments: (I) phenanthrene, (II) litter, (III) litter + phenanthrene, (IV) unspiked control. Total community DNA was extracted from soil sampled on the day of spiking, 7, 21, and 63 days after spiking. Bacterial 16S rRNA gene and fungal internal transcribed spacer amplicons were quantified by qPCR and subjected to denaturing gradient gel electrophoresis (DGGE). DGGE analysis revealed that the bacterial community composition, which was strongly shaped by clay minerals after more than two years of incubation, changed in response to spiked phenanthrene and added litter. DGGE and qPCR showed that soil composition significantly influenced the microbial response to spiking. While fungal communities responded only in presence of litter to phenanthrene spiking, the response of the bacterial communities to phenanthrene was less pronounced when litter was present. Interestingly, microbial communities in all artificial soils were more strongly affected by spiking than in the natural soil, which might indicate the importance of higher microbial diversity to compensate perturbations. This study showed the influence of soil composition on the microbiota and their response to phenanthrene and litter, which may increase our understanding of complex interactions in soils for bioremediation applications. PMID:25222697

Analysis of bacterial community shifts in the gastrointestinal tract of pigs fed diets supplemented with β-glucan from Laminaria digitata, Laminaria hyperborea and Saccharomyces cerevisiae.

PubMed

Murphy, P; Dal Bello, F; O'Doherty, J; Arendt, E K; Sweeney, T; Coffey, A

2013-07-01

This study was designed to evaluate the effects of algal and yeast β-glucans on the porcine gastrointestinal microbiota, specifically the community of Lactobacillus, Bifidobacterium and coliforms. A total of 48 pigs were fed four diets over a 28-day period to determine the effect that each had on these communities. The control diet consisted of wheat and soya bean meal. The remaining three diets contained wheat and soya bean meal supplemented with β-glucan at 250 g/tonne from Laminaria digitata, Laminaria hyperborea or Saccharomyces cerevisiae. Faecal samples were collected from animals before feeding each diet and after the feeding period. The animals were slaughtered the following day and samples were collected from the stomach, ileum, caecum, proximal colon and distal colon. Alterations in Lactobacillus in the gastrointestinal tract (GIT) were analysed using denaturing gradient gel electrophoresis (DGGE) profiles generated by group-specific 16S rRNA gene PCR amplicons. Plate count analysis was also performed to quantify total coliforms. DGGE profiles indicated that all β-glucan diets provoked the emergence of a richer community of Lactobacillus. The richest community of lactobacilli emerged after feeding L. digitata (LD β-glucan). Plate count analysis revealed that the L. hyperborea (LH β-glucan) diet had a statistically significant effect on the coliform counts in the proximal colon in comparison with the control diet. β-glucan from L. digitata and S. cerevisiae also generally reduced coliforms but to a lesser extent. Nevertheless, the β-glucan diets did not significantly reduce levels of Lactobacillus or Bifidobacterium. DGGE analysis of GIT samples indicated that the three β-glucan diets generally promoted the establishment of a more varied range of Lactobacillus species in the caecum, proximal and distal colon. The LH β-glucan had the most profound reducing effect on coliform counts when compared with the control diet and diets supplemented with L. digitata and S. cerevisiae β-glucans.

The effect of alum addition on microbial communities in poultry litter.

PubMed

Rothrock, M J; Cook, K L; Warren, J G; Sistani, K

2008-08-01

Alum [Al(2)(SO(4))(3).14H(2)O] is a common poultry litter amendment used to decrease water-soluble phosphorus or reduce ammonia volatilization, or both. Although the physiochemical effects of alum addition have been well researched, little attention has been given to the poultry litter microbial communities. The goal of this study was to use molecular biological methods [denaturing gradient gel electrophoresis (DGGE), community cloning, and quantitative real-time PCR] to characterize general, group-specific and pathogenic microbial communities in alum (10% wt/wt) and non-alum-treated litter. According to quantitative real-time PCR analyses, alum addition to the poultry litter resulted in significant reductions in both Campylobacter jejuni and Escherichia coli concentrations by the end of the first month of the experiment (3 log and 2 log, respectively). The concentrations of Salmonella spp. were below detection (<5 x 10(3) cell.g(-1) of litter) for the entire experiment. The DGGE analyses revealed significant reductions in the Clostridium/Eubacterium and low %GC gram-positive groups in the alum-treated litters by the end of the first month, with no bands detectable for either group after 8 wk of incubation. Conversely, minimal effects of alum addition were observed in the Actinomycetes community. The most significant shift in the microbial community (based on DGGE analyses) occurred in the fungal population, with a large increase in diversity and abundance within 1 mo of alum addition (1 dominant band on d 0 to 9 dominant bands at 4 wk). Specifically, the incidence of Aspergillus spp. increased from 0 to 50% of the sequences in fungal clone libraries (n = 80) over the course of the experiment. This suggests that the addition of alum to poultry litter potentially shifts the microbial populations from bacterially dominated to dominated by fungi. The ramifications of this shift in dominance are still unknown, and future work will be aimed at characterizing these fungi and elucidating their role in the acidified litter environment.

Dietary administration of the probiotic SpPdp11: Effects on the intestinal microbiota and immune-related gene expression of farmed Solea senegalensis treated with oxytetracycline.

PubMed

Tapia-Paniagua, S T; Vidal, S; Lobo, C; García de la Banda, I; Esteban, M A; Balebona, M C; Moriñigo, M A

2015-10-01

Few antimicrobials are currently authorised in the aquaculture industry to treat infectious diseases. Among them, oxytetracycline (OTC) is one of the first-choice drugs for nearly all bacterial diseases. The objective of this study was to evaluate the effect of the dietary administration of OTC both alone and jointly with the probiotic Shewanella putrefaciens Pdp11 (SpPdp11) on the intestinal microbiota and hepatic expression of genes related to immunity in Senegalese sole (Solea senegalensis) juveniles. The results demonstrated that the richness and diversity of the intestinal microbiota of fish treated with OTC decreased compared with those of the control group but that these effects were lessened by the simultaneous administration of SpPdp11. In addition, specimens that received OTC and SpPdp11 jointly showed a decreased intensity of the Denaturing Gradient Gel Electrophoresis (DGGE) bands related to Vibrio genus and the presence of DGGE bands related to Lactobacillus and Shewanella genera. The relationship among the intestinal microbiota of fish fed with control and OTC diets and the expression of the NADPH oxidase and CASPASE-6 genes was demonstrated by a Principal Components Analysis (PCA) carried out in this study. In contrast, a close relationship between the transcription of genes, such as NKEF, IGF-β, HSP70 and GP96, and the DGGE bands of fish treated jointly with OTC and SpPdp11 was observed in the PCA study. In summary, the results obtained in this study demonstrate that the administration of OTC results in the up-regulation of genes related to apoptosis but that the joint administration of OTC and S. putrefaciens Pdp11 increases the transcription of genes related to antiapoptotic effects and oxidative stress regulation. Further, a clear relationship between these changes and those detected in the intestinal microbiota is established. Copyright © 2015 Elsevier Ltd. All rights reserved.

Taxonomic structure and stability of the bacterial community in belgian sourdough ecosystems as assessed by culture and population fingerprinting.

PubMed

Scheirlinck, Ilse; Van der Meulen, Roel; Van Schoor, Ann; Vancanneyt, Marc; De Vuyst, Luc; Vandamme, Peter; Huys, Geert

2008-04-01

A total of 39 traditional sourdoughs were sampled at 11 bakeries located throughout Belgium which were visited twice with a 1-year interval. The taxonomic structure and stability of the bacterial communities occurring in these traditional sourdoughs were assessed using both culture-dependent and culture-independent methods. A total of 1,194 potential lactic acid bacterium (LAB) isolates were tentatively grouped and identified by repetitive element sequence-based PCR, followed by sequence-based identification using 16S rRNA and pheS genes from a selection of genotypically unique LAB isolates. In parallel, all samples were analyzed by denaturing gradient gel electrophoresis (DGGE) of V3-16S rRNA gene amplicons. In addition, extensive metabolite target analysis of more than 100 different compounds was performed. Both culturing and DGGE analysis showed that the species Lactobacillus sanfranciscensis, Lactobacillus paralimentarius, Lactobacillus plantarum, and Lactobacillus pontis dominated the LAB population of Belgian type I sourdoughs. In addition, DGGE band sequence analysis demonstrated the presence of Acetobacter sp. and a member of the Erwinia/Enterobacter/Pantoea group in some samples. Overall, the culture-dependent and culture-independent approaches each exhibited intrinsic limitations in assessing bacterial LAB diversity in Belgian sourdoughs. Irrespective of the LAB biodiversity, a large majority of the sugar and amino acid metabolites were detected in all sourdough samples. Principal component-based analysis of biodiversity and metabolic data revealed only little variation among the two samples of the sourdoughs produced at the same bakery. The rare cases of instability observed could generally be linked with variations in technological parameters or differences in detection capacity between culture-dependent and culture-independent approaches. Within a sampling interval of 1 year, this study reinforces previous observations that the bakery environment rather than the type or batch of flour largely determines the development of a stable LAB population in sourdoughs.

Are We Biologically Safe with Snow Precipitation? A Case Study in Beijing

PubMed Central

Shen, Fangxia; Yao, Maosheng

2013-01-01

In this study, the bacterial and fungal abundances, diversities, conductance levels as well as total organic carbon (TOC) were investigated in the snow samples collected from five different snow occurrences in Beijing between January and March, 2010. The collected snow samples were melted and cultured at three different temperatures (4, 26 and 37°C). The culturable bacterial concentrations were manually counted and the resulting colony forming units (CFUs) at 26°C were further studied using V3 region of 16 S rRNA gene-targeted polymerase chain reaction -denaturing gradient gel electrophoresis (PCR-DGGE). The clone library was constructed after the liquid culturing of snow samples at 26°C. And microscopic method was employed to investigate the fungal diversity in the samples. In addition, outdoor air samples were also collected using mixed cellulose ester (MCE) filters and compared with snow samples with respect to described characteristics. The results revealed that snow samples had bacterial concentrations as much as 16000 CFU/ml for those cultured at 26°C, and the conductance levels ranged from 5.6×10−6 to 2.4×10−5 S. PCR-DGGE, sequencing and microscopic analysis revealed remarkable bacterial and fungal diversity differences between the snow samples and the outdoor air samples. In addition, DGGE banding profiles for the snow samples collected were also shown distinctly different from one another. Absent from the outdoor air, certain human, plant, and insect fungal pathogens were found in the snow samples. By calculation, culturable bacteria accounted for an average of 3.38% (±1.96%) of TOC for the snow samples, and 0.01% for that of outdoor air samples. The results here suggest that snow precipitations are important sources of fungal pathogens and ice nucleators, thus could affect local climate, human health and agriculture security. PMID:23762327

Are we biologically safe with snow precipitation? A case study in beijing.

PubMed

Shen, Fangxia; Yao, Maosheng

2013-01-01

In this study, the bacterial and fungal abundances, diversities, conductance levels as well as total organic carbon (TOC) were investigated in the snow samples collected from five different snow occurrences in Beijing between January and March, 2010. The collected snow samples were melted and cultured at three different temperatures (4, 26 and 37°C). The culturable bacterial concentrations were manually counted and the resulting colony forming units (CFUs) at 26°C were further studied using V3 region of 16 S rRNA gene-targeted polymerase chain reaction -denaturing gradient gel electrophoresis (PCR-DGGE). The clone library was constructed after the liquid culturing of snow samples at 26°C. And microscopic method was employed to investigate the fungal diversity in the samples. In addition, outdoor air samples were also collected using mixed cellulose ester (MCE) filters and compared with snow samples with respect to described characteristics. The results revealed that snow samples had bacterial concentrations as much as 16000 CFU/ml for those cultured at 26°C, and the conductance levels ranged from 5.6×10(-6) to 2.4×10(-5) S. PCR-DGGE, sequencing and microscopic analysis revealed remarkable bacterial and fungal diversity differences between the snow samples and the outdoor air samples. In addition, DGGE banding profiles for the snow samples collected were also shown distinctly different from one another. Absent from the outdoor air, certain human, plant, and insect fungal pathogens were found in the snow samples. By calculation, culturable bacteria accounted for an average of 3.38% (±1.96%) of TOC for the snow samples, and 0.01% for that of outdoor air samples. The results here suggest that snow precipitations are important sources of fungal pathogens and ice nucleators, thus could affect local climate, human health and agriculture security.

Taxonomic Structure and Stability of the Bacterial Community in Belgian Sourdough Ecosystems as Assessed by Culture and Population Fingerprinting▿ †

PubMed Central

Scheirlinck, Ilse; Van der Meulen, Roel; Van Schoor, Ann; Vancanneyt, Marc; De Vuyst, Luc; Vandamme, Peter; Huys, Geert

2008-01-01

A total of 39 traditional sourdoughs were sampled at 11 bakeries located throughout Belgium which were visited twice with a 1-year interval. The taxonomic structure and stability of the bacterial communities occurring in these traditional sourdoughs were assessed using both culture-dependent and culture-independent methods. A total of 1,194 potential lactic acid bacterium (LAB) isolates were tentatively grouped and identified by repetitive element sequence-based PCR, followed by sequence-based identification using 16S rRNA and pheS genes from a selection of genotypically unique LAB isolates. In parallel, all samples were analyzed by denaturing gradient gel electrophoresis (DGGE) of V3-16S rRNA gene amplicons. In addition, extensive metabolite target analysis of more than 100 different compounds was performed. Both culturing and DGGE analysis showed that the species Lactobacillus sanfranciscensis, Lactobacillus paralimentarius, Lactobacillus plantarum, and Lactobacillus pontis dominated the LAB population of Belgian type I sourdoughs. In addition, DGGE band sequence analysis demonstrated the presence of Acetobacter sp. and a member of the Erwinia/Enterobacter/Pantoea group in some samples. Overall, the culture-dependent and culture-independent approaches each exhibited intrinsic limitations in assessing bacterial LAB diversity in Belgian sourdoughs. Irrespective of the LAB biodiversity, a large majority of the sugar and amino acid metabolites were detected in all sourdough samples. Principal component-based analysis of biodiversity and metabolic data revealed only little variation among the two samples of the sourdoughs produced at the same bakery. The rare cases of instability observed could generally be linked with variations in technological parameters or differences in detection capacity between culture-dependent and culture-independent approaches. Within a sampling interval of 1 year, this study reinforces previous observations that the bakery environment rather than the type or batch of flour largely determines the development of a stable LAB population in sourdoughs. PMID:18310426

Leaf-associated fungal diversity in acidified streams: insights from combining traditional and molecular approaches.

PubMed

Clivot, Hugues; Cornut, Julien; Chauvet, Eric; Elger, Arnaud; Poupin, Pascal; Guérold, François; Pagnout, Christophe

2014-07-01

We combined microscopic and molecular methods to investigate fungal assemblages on alder leaf litter exposed in the benthic and hyporheic zones of five streams across a gradient of increasing acidification for 4 weeks. The results showed that acidification and elevated Al concentrations strongly depressed sporulating aquatic hyphomycetes diversity in both zones of streams, while fungal diversity assessed by denaturing gradient gel electrophoresis (DGGE) appeared unaffected. Clone library analyses revealed that fungal communities on leaves were dominated by members of Ascomycetes and to a lesser extent by Basidiomycetes and Chytridiomycetes. An important contribution of terrestrial fungi was observed in both zones of the most acidified stream and in the hyporheic zone of the reference circumneutral stream. The highest leaf breakdown rate was observed in the circumneutral stream and occurred in the presence of both the highest diversity of sporulating aquatic hyphomycetes and the highest contribution to clone libraries of sequences affiliated with aquatic hyphomycetes. Both methods underline the major role played by aquatic hyphomycetes in leaf decomposition process. Our findings also bring out new highlights on the identity of leaf-associated fungal communities and their responses to anthropogenic alteration of running water ecosystems. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

A comparison of stable-isotope probing of DNA and phospholipid fatty acids to study prokaryotic functional diversity in sulfate-reducing marine sediment enrichment slurries.

PubMed

Webster, Gordon; Watt, Lynsey C; Rinna, Joachim; Fry, John C; Evershed, Richard P; Parkes, R John; Weightman, Andrew J

2006-09-01

Marine sediment slurries enriched for anaerobic, sulfate-reducing prokaryotic communities utilizing glucose and acetate were used to provide the first comparison between stable-isotope probing (SIP) of phospholipid fatty acids (PLFA) and DNA (16S rRNA and dsrA genes) biomarkers. Different 13C-labelled substrates (glucose, acetate and pyruvate) at low concentrations (100 microM) were used over a 7-day incubation to follow and identify carbon flow into different members of the community. Limited changes in total PLFA and bacterial 16S rRNA gene DGGE profiles over 7 days suggested the presence of a stable bacterial community. A broad range of PLFA were rapidly labelled (within 12 h) in the 13C-glucose slurry but this changed with time, suggesting the presence of an active glucose-utilizing population and later development of another population able to utilize glucose metabolites. The identity of the major glucose-utilizers was unclear as 13C-enriched PLFA were common (16:0, 16:1, 18:1omega7, highest incorporation) and there was little difference between 12C- and 13C-DNA 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) profiles. Seemingly glucose, a readily utilizable substrate, resulted in widespread incorporation consistent with the higher extent of 13C-incorporation (approximately 10 times) into PLFA compared with 13C-acetate or 13C-pyruvate. 13C-PLFA in the 13C-acetate and 13C-pyruvate slurries were similar to each other and to those that developed in the 13C-glucose slurry after 4 days. These were more diagnostic, with branched odd-chain fatty acids (i15:0, a15:0 and 15:1omega6) possibly indicating the presence of Desulfococcus or Desulfosarcina sulfate-reducing bacteria (SRB) and sequences related to these SRB were in the 13C-acetate-DNA dsrA gene library. The 13C-acetate-DNA 16S rRNA gene library also contained sequences closely related to SRB, but these were the acetate-utilizing Desulfobacter sp., as well as a broad range of uncultured Bacteria. In contrast, analysis of DGGE bands from 13C-DNA demonstrated that the candidate division JS1 and Firmicutes were actively assimilating 13C-acetate. Denaturing gradient gel electrophoresis also confirmed the presence of JS1 in the 13C-DNA from the 13C-glucose slurry. These results demonstrate that JS1, originally found in deep subsurface sediments, is more widely distributed in marine sediments and provides the first indication of its metabolism; incorporation of acetate and glucose (or glucose metabolites) under anaerobic, sulfate-reducing conditions. Here we demonstrate that PLFA- and DNA-SIP can be used together in a sedimentary system, with low concentrations of 13C-substrate and overlapping incubation times (up to 7 days) to provide complementary, although not identical, information on carbon flow and the identity of active members of an anaerobic prokaryotic community.

Effects of organic amendments and mulches on soil microbial communities in quarry restoration under semiarid climate

NASA Astrophysics Data System (ADS)

Luna Ramos, Lourdes; Pastorelli, Roberta; Miralles Mellado, Isabel; Fabiani, Arturo; Bastida López, Felipe; Hernández Fernández, María Teresa; García Izquierdo, Carlos; Solé Benet, Albert

2015-04-01

Mining activities generate loss of the quality of the environment and landscape specially in arid and semiarid Mediterranean regions. A precondition for ecosystem reclamation in such highly disturbed mining areas is the development of functional soils with appropriate levels of organic matter. In an experimental soil restoration in limestone quarries from Sierra de Gádor (Almería), SE Spain, 9 plots 15 x 5 m were prepared to test organic amendments (compost from solid urban residues-DOW-, sludge from urban water treatment-SS-, control-NA-) and different mulches (fine gravel-GM-, wood chips-WM-, control-NM-) with the aim to improve soil/substrate properties and to reduce evaporation and erosion. In each experimental plot, 75 native plants (Macrochloa tenacissima, Anthyllis terniflora and Anthyllis cytisoides) were planted. After 5 years from the start of the experiment, we evaluated how microbial community composition responded to the organic amendments and mulches. Microbial community composition of both bacteria and fungi was determined by phospholipid fatty acid (PLFA) and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprinting. The results of the two-way ANOVA showed that PLFAs were significantly affected by organic amendments but not by the mulches or interaction of both factors. Experimental plots with DOW showed significantly higher level of fungal PLFAs than those with SS and NA, even higher than the reference undisturbed soil. However, any plot with organic amendments did not reach the content of bacterial PLFAs of the reference soils. The bacterial diversity (evaluated by diversity indices calculated from DGGE profiles) was greater in soil samples taken under NA and GM. Comparing these indices in fungal DGGE, we found greater values for soil samples taken under DOW and without mulches. Results from UPGMA analysis showed significant differences in the structure of soil bacterial communities from the different treatments respect to that of reference soil. Fungal communities could be divided into main groups according to the organic amendment. Within each group, GM amendment generated fungal community structures with lower similarities with respect to the other mulch treatments. In contrast to PLFA results, DGGE fingerprints revealed significant influence of the combination of organic amendments and mulches on diversity and composition of soil microbial communities.

Comparison of vaginal microbial community structure in healthy and endometritis dairy cows by PCR-DGGE and real-time PCR.

PubMed

Wang, Jun; Sun, Chengtao; Liu, Chang; Yang, Yujiang; Lu, Wenfa

2016-04-01

The normal vaginal microflora provides protection against infections of the reproductive tract. Previous studies have focused on the isolation and screening of probiotic strains from the vagina of cows; however, the vaginal microflora of postpartum cows is poorly characterized. The present study was conducted to evaluate and characterize the vaginal microflora of healthy postpartum cows in relation to postpartum cows with endometritis by using PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) and Real-time PCR. The study population comprised 5 healthy cows and 5 cows with endometritis. The results indicated that the vaginal bacterial microflora of healthy postpartum cows was dominated by Lactobacillus sakei subsp. and Weissella koreensis, while there were no dominant bacterial species in the vaginal microflora of postpartum cows with endometritis. Common microorganisms such as Bacteroides spp., Fusobacterium spp., Enterococcus spp., Prevotella spp., Clostridium perfringens strains, and Escherichia coli were detected in both groups of cows by Real-time PCR. The bacterial diversity in the vagina of cows with endometritis was significantly higher than that in healthy cows. The results indicated that the vaginal microflora of cows with endometritis was more diverse and lacked dominant bacterial species as compared to that of the healthy cows, suggesting that disruption of the normal vaginal microflora may contribute to the onset of endometritis. This microbial community analysis provided information that might be used to develop probiotics to treat endometritis in cows; however, further investigation is needed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structural shifts of fecal microbial communities in rats with acute rejection after liver transplantation.

PubMed

Xie, Yirui; Luo, Zhuanbo; Li, Zhengfeng; Deng, Min; Liu, Hao; Zhu, Biao; Ruan, Bing; Li, Lanjuan

2012-08-01

Bacterial translocation and the development of sepsis after orthotopic liver transplantation (OLT) may be promoted by immunological damage to the intestinal mucosa or by quantitative and qualitative changes in intestinal microbiota. This study monitored structural shifts of gut microbiota in rats with OLT using PCR-denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR (RT-qPCR). RT-qPCR targets six major microorganisms (Domain Bacteria, Bacteroides, Bifidobacteria, Enterobacteriaceae, Lactobacillus and Clostridium leptum subgroup). Isograft, Allograft and Sham model were studied. Bacterial translocation to host organs and plasma endotoxin were determined. Alteration in gut microbiota was associated with the elevation of plasma endotoxin and a higher rate of bacterial translocation (BT) to liver in rats with acute rejection. Dynamic analysis of DGGE fingerprints showed that the gut microbiota structure of animals in the three groups was similar before the operation. But significant alterations in the composition of fecal microbiota in Allograft group were observed at 1 and 2 weeks after the OLT. The acute rejection was accompanied by the shifts of gut microbiota towards members of Bacteroides and Ruminococcus. Results from RT-qPCR indicated that Bacteroides significantly increased at 2 weeks after the OLT, whereas numbers of Bifidobacterium spp. decreased at 1 week and recovered at 2 weeks after the OLT. In summary, our data showed that rats with acute rejection after OLT exhibited significant structure shifts in the gut microbiota which dominant by overgrowth of Bacteroides and Ruminococcus, and these were associated with elevation of plasma endotoxin and higher rate of BT.

Molecular analysis of the biomass of a fluidized bed reactor treating synthetic vinasse at anaerobic and micro-aerobic conditions.

PubMed

Rodríguez, Elisa; Lopes, Alexandre; Fdz-Polanco, María; Stams, Alfons J M; García-Encina, Pedro A

2012-03-01

The microbial communities (Bacteria and Archaea) established in an anaerobic fluidized bed reactor used to treat synthetic vinasse (betaine, glucose, acetate, propionate, and butyrate) were characterized by denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis. This study was focused on the competitive and syntrophic interactions between the different microbial groups at varying influent substrate to sulfate ratios of 8, 4, and 2 and anaerobic or micro-aerobic conditions. Acetogens detected along the anaerobic phases at substrate to sulfate ratios of 8 and 4 seemed to be mainly involved in the fermentation of glucose and betaine, but they were substituted by other sugar or betaine degraders after oxygen application. Typical fatty acid degraders that grow in syntrophy with methanogens were not detected during the entire reactor run. Likely, sugar and betaine degraders outnumbered them in the DGGE analysis. The detected sulfate-reducing bacteria (SRB) belonged to the hydrogen-utilizing Desulfovibrio. The introduction of oxygen led to the formation of elemental sulfur (S(0)) and probably other sulfur compounds by sulfide-oxidizing bacteria (γ-Proteobacteria). It is likely that the sulfur intermediates produced from sulfide oxidation were used by SRB and other microorganisms as electron acceptors, as was supported by the detection of the sulfur respiring Wolinella succinogenes. Within the Archaea population, members of Methanomethylovorans and Methanosaeta were detected throughout the entire reactor operation. Hydrogenotrophic methanogens mainly belonging to the genus Methanobacterium were detected at the highest substrate to sulfate ratio but rapidly disappeared by increasing the sulfate concentration.

Polyphasic Study of the Spatial Distribution of Microorganisms in Mexican Pozol, a Fermented Maize Dough, Demonstrates the Need for Cultivation-Independent Methods To Investigate Traditional Fermentations

PubMed Central

Ampe, Frédéric; ben Omar, Nabil; Moizan, Claire; Wacher, Carmen; Guyot, Jean-Pierre

1999-01-01

The distribution of microorganisms in pozol balls, a fermented maize dough, was investigated by a polyphasic approach in which we used both culture-dependent and culture-independent methods, including microbial enumeration, fermentation product analysis, quantification of microbial taxa with 16S rRNA-targeted oligonucleotide probes, determination of microbial fingerprints by denaturing gradient gel electrophoresis (DGGE), and 16S ribosomal DNA gene sequencing. Our results demonstrate that DGGE fingerprinting and rRNA quantification should allow workers to precisely and rapidly characterize the microbial assemblage in a spontaneous lactic acid fermented food. Lactic acid bacteria (LAB) accounted for 90 to 97% of the total active microflora; no streptococci were isolated, although members of the genus Streptococcus accounted for 25 to 50% of the microflora. Lactobacillus plantarum and Lactobacillus fermentum, together with members of the genera Leuconostoc and Weissella, were the other dominant organisms. The overall activity was more important at the periphery of a ball, where eucaryotes, enterobacteria, and bacterial exopolysacharide producers developed. Our results also showed that the metabolism of heterofermentative LAB was influenced in situ by the distribution of the LAB in the pozol ball, whereas homolactic fermentation was controlled primarily by sugar limitation. We propose that starch is first degraded by amylases from LAB and that the resulting sugars, together with the lactate produced, allow a secondary flora to develop in the presence of oxygen. Our results strongly suggest that cultivation-independent methods should be used to study traditional fermented foods. PMID:10584005

Diverse growth hormone receptor gene mutations in Laron syndrome.

PubMed Central

Berg, M A; Argente, J; Chernausek, S; Gracia, R; Guevara-Aguirre, J; Hopp, M; Pérez-Jurado, L; Rosenbloom, A; Toledo, S P; Francke, U

1993-01-01

To better understand the molecular genetic basis and genetic epidemiology of Laron syndrome (growth-hormone insensitivity syndrome), we analyzed the growth-hormone receptor (GHR) genes of seven unrelated affected individuals from the United States, South America, Europe, and Africa. We amplified all nine GHR gene exons and splice junctions from these individuals by PCR and screened the products for mutations by using denaturing gradient gel electrophoresis (DGGE). We identified a single GHR gene fragment with abnormal DGGE results for each affected individual, sequenced this fragment, and, in each case, identified a mutation likely to cause Laron syndrome, including two nonsense mutations (R43X and R217X), two splice-junction mutations, (189-1 G to T and 71 + 1 G to A), and two frameshift mutations (46 del TT and 230 del TA or AT). Only one of these mutations, R43X, has been previously reported. Using haplotype analysis, we determined that this mutation, which involves a CpG dinucleotide hot spot, likely arose as a separate event in this case, relative to the two prior reports of R43X. Aside from R43X, the mutations we identified are unique to patients from particular geographic regions. Ten GHR gene mutations have now been described in this disorder. We conclude that Laron syndrome is caused by diverse GHR gene mutations, including deletions, RNA processing defects, translational stop codons, and missense codons. All the identified mutations involve the extracellular domain of the receptor, and most are unique to particular families or geographic areas. Images Figure 1 Figure 2 PMID:8488849

Effect of biostimulants on 2,4,6-trinitrotoluene (TNT) degradation and bacterial community composition in contaminated aquifer sediment enrichments.

PubMed

Fahrenfeld, Nicole; Zoeckler, Jeffrey; Widdowson, Mark A; Pruden, Amy

2013-04-01

2,4,6-Trinitrotoluene (TNT) is a toxic and persistent explosive compound occurring as a contaminant at numerous sites worldwide. Knowledge of the microbial dynamics driving TNT biodegradation is limited, particularly in native aquifer sediments where it poses a threat to water resources. The purpose of this study was to quantify the effect of organic amendments on anaerobic TNT biodegradation rate and pathway in an enrichment culture obtained from historically contaminated aquifer sediment and to compare the bacterial community dynamics. TNT readily biodegraded in all microcosms, with the highest biodegradation rate obtained under the lactate amended condition followed by ethanol amended and naturally occurring organic matter (extracted from site sediment) amended conditions. Although a reductive pathway of TNT degradation was observed across all conditions, denaturing gradient gel electrophoresis (DGGE) analysis revealed distinct bacterial community compositions. In all microcosms, Gram-negative γ- or β-Proteobacteria and Gram-positive Negativicutes or Clostridia were observed. A Pseudomonas sp. in particular was observed to be stimulated under all conditions. According to non-metric multidimensional scaling analysis of DGGE profiles, the microcosm communities were most similar to heavily TNT-contaminated field site sediment, relative to moderately and uncontaminated sediments, suggesting that TNT contamination itself is a major driver of microbial community structure. Overall these results provide a new line of evidence of the key bacteria driving TNT degradation in aquifer sediments and their dynamics in response to organic carbon amendment, supporting this approach as a promising technology for stimulating in situ TNT bioremediation in the subsurface.

Microbial succession in a compost-packed biofilter treating benzene-contaminated air.

PubMed

Borin, Sara; Marzorati, Massimo; Brusetti, Lorenzo; Zilli, Mario; Cherif, Hanene; Hassen, Abdennaceur; Converti, Attilio; Sorlini, Claudia; Daffonchio, Daniele

2006-03-01

Air artificially contaminated with increasing concentrations of benzene was treated in a laboratory scale compost-packed biofilter for 240 days with a removal efficiency of 81-100%. The bacterial community in the packing material (PM) at different heights of the biofilter was analysed every 60 days. Bacterial plate counts and ribosomal intergenic spacer analysis (RISA) of the isolated strains showed that the number of cultivable aerobic heterotrophic bacteria and the species diversity increased with benzene availability. Identification of the isolated species and the main bands in denaturing gradient gel electrophoresis (DGGE) profiles from total compost DNA during the treatment revealed that, at a relatively low volumetric benzene load (1.2< or =VBL< or =6.4 g m(-3) (PM) h(-1)), besides low G+C Gram positive bacteria, originally present in the packing compost, bacteroidetes and beta- and gamma-proteobacteria became detectable in the colonising population. At the VBL value (24.8 g m(-3) (PM) h(-1)) ensuring the maximum elimination capacity of the biofilter (20.1 g m(-3) (PM) h(-1)), strains affiliated to the genus Rhodococcus dominated the microflora, followed by beta-proteobacteria comprising the genera Bordetella and Neisseria. Under these conditions, more than 35% of the isolated strains were able to grow on benzene as the sole carbon source. Comparison of DGGE and automated RISA profiles of the total community and isolated strains showed that a complex bacterial succession occurred in the reactor in response to the increasing concentrations of the pollutant and that cultivable bacteria played a major role in benzene degradation under the adopted conditions.

Altamira cave Paleolithic paintings harbor partly unknown bacterial communities.

PubMed

Schabereiter-Gurtner, Claudia; Saiz-Jimenez, Cesareo; Piñar, Guadalupe; Lubitz, Werner; Rölleke, Sabine

2002-05-21

Since it has been reported that microorganisms can affect painting pigments, Paleolithic painting microbiology deserves attention. The present study is the first report on the bacterial colonization of the valuable Paleolithic paintings in the famous Altamira cave (Spain). One sample taken from a painting area in the Polychromes Hall was analyzed culture-independently. This was the first time microbiologists were allowed to take sample material directly from Altamira paintings. Identification methods included PCR amplification of 16S rRNA genes (16S rDNA) and community fingerprinting by denaturing gradient gel electrophoresis (DGGE). The applied approach gave insight into a great bacterial taxonomic diversity, and allowed the detection of unexpected and unknown bacteria with potential effects on the conservation of the painting. Regarding the number of 29 visible DGGE bands in the community fingerprint, the numbers of analyzed clones described about 72% of the phylogenetic diversity present in the sample. Thirty-eight percent of the sequences analyzed were phylogenetically most closely related to cultivated bacteria, while the majority (62%) were most closely related to environmental 16S rDNA clones. Bacteria identified in Altamira were related with sequence similarities between 84.8 and 99.4% to members of the cosmopolitan Proteobacteria (52.3%), to members of the Acidobacterium division (23.8%), Cytophaga/Flexibacter/Bacteroides phylum (9.5%), green non-sulfur bacteria (4.8%), Planctomycetales (4.8%) and Actinobacteria (4.8%). The high number of clones most closely related to environmental 16S rDNA clones showed the broad spectrum of unknown and yet to be cultivated bacteria in Altamira cave.

Anaerobic Oxidation of Methane at a Marine Methane Seep in a Forearc Sediment Basin off Sumatra, Indian Ocean

PubMed Central

Siegert, Michael; Krüger, Martin; Teichert, Barbara; Wiedicke, Michael; Schippers, Axel

2011-01-01

A cold methane seep was discovered in a forearc sediment basin off the island Sumatra, exhibiting a methane-seep adapted microbial community. A defined seep center of activity, like in mud volcanoes, was not discovered. The seep area was rather characterized by a patchy distribution of active spots. The relevance of anaerobic oxidation of methane (AOM) was reflected by 13C-depleted isotopic signatures of dissolved inorganic carbon. The anaerobic conversion of methane to CO2 was confirmed in a 13C-labeling experiment. Methane fueled a vital microbial community with cell numbers of up to 4 × 109 cells cm−3 sediment. The microbial community was analyzed by total cell counting, catalyzed reporter deposition–fluorescence in situ hybridization (CARD–FISH), quantitative real-time PCR (qPCR), and denaturing gradient gel electrophoresis (DGGE). CARD–FISH cell counts and qPCR measurements showed the presence of Bacteria and Archaea, but only small numbers of Eukarya. The archaeal community comprised largely members of ANME-1 and ANME-2. Furthermore, members of the Crenarchaeota were frequently detected in the DGGE analysis. Three major bacterial phylogenetic groups (δ-Proteobacteria, candidate division OP9, and Anaerolineaceae) were abundant across the study area. Several of these sequences were closely related to the genus Desulfococcus of the family Desulfobacteraceae, which is in good agreement with previously described AOM sites. In conclusion, the majority of the microbial community at the seep consisted of AOM-related microorganisms, while the relevance of higher hydrocarbons as microbial substrates was negligible. PMID:22207865

Bacterial Population in Intestines of the Black Tiger Shrimp (Penaeus monodon) under Different Growth Stages

PubMed Central

Rungrassamee, Wanilada; Klanchui, Amornpan; Chaiyapechara, Sage; Maibunkaew, Sawarot; Tangphatsornruang, Sithichoke; Jiravanichpaisal, Pikul; Karoonuthaisiri, Nitsara

2013-01-01

Intestinal bacterial communities in aquaculture have been drawn to attention due to potential benefit to their hosts. To identify core intestinal bacteria in the black tiger shrimp (Penaeus monodon), bacterial populations of disease-free shrimp were characterized from intestines of four developmental stages (15-day-old post larvae (PL15), 1- (J1), 2- (J2), and 3-month-old (J3) juveniles) using pyrosequencing, real-time PCR and denaturing gradient gel electrophoresis (DGGE) approaches. A total of 25,121 pyrosequencing reads (reading length = 442±24 bases) were obtained, which were categorized by barcode for PL15 (7,045 sequences), J1 (3,055 sequences), J2 (13,130 sequences) and J3 (1,890 sequences). Bacteria in the phyla Bacteroides, Firmicutes and Proteobacteria were found in intestines at all four growth stages. There were 88, 14, 27, and 20 bacterial genera associated with the intestinal tract of PL15, J1, J2 and J3, respectively. Pyrosequencing analysis revealed that Proteobacteria (class Gammaproteobacteria) was a dominant bacteria group with a relative abundance of 89% for PL15 and 99% for J1, J2 and J3. Real-time PCR assay also confirmed that Gammaproteobacteria had the highest relative abundance in intestines from all growth stages. Intestinal bacterial communities from the three juvenile stages were more similar to each other than that of the PL shrimp based on PCA analyses of pyrosequencing results and their DGGE profiles. This study provides descriptive bacterial communities associated to the black tiger shrimp intestines during these growth development stages in rearing facilities. PMID:23577162

Bacterial population in intestines of the black tiger shrimp (Penaeus monodon) under different growth stages.

PubMed

Rungrassamee, Wanilada; Klanchui, Amornpan; Chaiyapechara, Sage; Maibunkaew, Sawarot; Tangphatsornruang, Sithichoke; Jiravanichpaisal, Pikul; Karoonuthaisiri, Nitsara

2013-01-01

Intestinal bacterial communities in aquaculture have been drawn to attention due to potential benefit to their hosts. To identify core intestinal bacteria in the black tiger shrimp (Penaeus monodon), bacterial populations of disease-free shrimp were characterized from intestines of four developmental stages (15-day-old post larvae (PL15), 1- (J1), 2- (J2), and 3-month-old (J3) juveniles) using pyrosequencing, real-time PCR and denaturing gradient gel electrophoresis (DGGE) approaches. A total of 25,121 pyrosequencing reads (reading length = 442±24 bases) were obtained, which were categorized by barcode for PL15 (7,045 sequences), J1 (3,055 sequences), J2 (13,130 sequences) and J3 (1,890 sequences). Bacteria in the phyla Bacteroides, Firmicutes and Proteobacteria were found in intestines at all four growth stages. There were 88, 14, 27, and 20 bacterial genera associated with the intestinal tract of PL15, J1, J2 and J3, respectively. Pyrosequencing analysis revealed that Proteobacteria (class Gammaproteobacteria) was a dominant bacteria group with a relative abundance of 89% for PL15 and 99% for J1, J2 and J3. Real-time PCR assay also confirmed that Gammaproteobacteria had the highest relative abundance in intestines from all growth stages. Intestinal bacterial communities from the three juvenile stages were more similar to each other than that of the PL shrimp based on PCA analyses of pyrosequencing results and their DGGE profiles. This study provides descriptive bacterial communities associated to the black tiger shrimp intestines during these growth development stages in rearing facilities.

The effect of a multispecies probiotic on the composition of the faecal microbiota and bowel habits in chronic obstructive pulmonary disease patients treated with antibiotics.

PubMed

Koning, Catherina J M; Jonkers, Daisy; Smidt, Hauke; Rombouts, Frans; Pennings, Herman-Jan; Wouters, Emiel; Stobberingh, Ellen; Stockbrügger, Reinhold

2010-05-01

Short-term antibiotic treatment profoundly affects the intestinal microbiota, which may lead to sustained changes in microbiota composition. Probiotics may restore such a disturbance. The objective of the present study was to investigate the effect of a multispecies probiotic on the faecal microbiota during and after antibiotic intake in patients with a history of frequent antibiotic use. In this randomised, placebo-controlled, double-blind study, thirty chronic obstructive pulmonary disease (COPD) patients treated with antibiotics for a respiratory tract infection received 5 g of a multispecies probiotic or placebo twice daily for 2 weeks. Faecal samples were collected at 0, 7, 14 and 63 d. Changes in the composition of the dominant faecal microbiota were determined by PCR-denaturing gradient gel electrophoresis (DGGE). Changes in bacterial subgroups were determined by quantitative PCR and culture. Bowel movements were scored daily according to the Bristol stool form scale. During and after antibiotic treatment, DGGE-based similarity indices (SI) were high ( >/= 84 %) and band richness was relatively low, both remaining stable over time. No difference in SI was observed between patients with and without diarrhoea-like bowel movements. The multispecies probiotic had a modest effect on the bacterial subgroups. Nevertheless, it affected neither the composition of the dominant faecal microbiota nor the occurrence of diarrhoea-like bowel movements. The dominant faecal microbiota was not affected by antibiotics in this COPD population, suggesting an existing imbalance of the microbiota, which may also have contributed to the lack of effect by probiotic intake.

Molecular characterization of intergeneric hybrid between Aspergillus oryzae and Trichoderma harzianum by protoplast fusion.

PubMed

Patil, N S; Patil, S M; Govindwar, S P; Jadhav, J P

2015-02-01

Protoplast fusion between Aspergillus oryzae and Trichoderma harzianum and application of fusant in degradation of shellfish waste. The filamentous chitinolytic fungal strains A. oryzae NCIM 1272 and T. harzianum NCIM 1185 were selected as parents for protoplast fusion. Viable protoplasts were released from fungal mycelium using enzyme cocktail containing 5 mg ml(-1) lysing enzymes from T. harzianum, 0.06 mg ml(-1) β-glucuronidase from Helix pomatia and 1 mg ml(-1) purified Penicillium ochrochloron chitinase in 0.8 mol l(-1) sorbitol as an osmotic stabilizer. Intergeneric protoplast fusion was carried out using 60% polyethylene glycol as a fusogen. At optimum conditions, the regeneration frequency of the fused protoplasts on colloidal chitin medium and fusion frequency were calculated. Fusant showed higher rate of growth pattern, chitinase activity and protein content than parents. Fusant formation was confirmed by morphological markers, viz. colony morphology and spore size and denaturation gradient gel electrophoresis (DGGE). This study revealed protoplast fusion between A. oryzae and T. harzianum significantly enhanced chitinase activity which ultimately provides potential strain for degradation of shellfish waste. Consistency in the molecular characterization results using DGGE is the major outcome of this study which can be emerged as a fundamental step in fusant identification. Now it is need to provide attention over effective chitin degradation to manage shrimp processing issues. In this aspect, ability of fusant to degrade shellfish waste efficiently in short incubation time revealed discovery of potential strain in the reclamation of seafood processing crustacean bio-waste. © 2014 The Society for Applied Microbiology.

Differences between bacterial communities in the gut of a soil-feeding termite (Cubitermes niokoloensis) and its mounds.

PubMed

Fall, Saliou; Hamelin, Jérôme; Ndiaye, Farma; Assigbetse, Komi; Aragno, Michel; Chotte, Jean Luc; Brauman, Alain

2007-08-01

In tropical ecosystems, termite mound soils constitute an important soil compartment covering around 10% of African soils. Previous studies have shown (S. Fall, S. Nazaret, J. L. Chotte, and A. Brauman, Microb. Ecol. 28:191-199, 2004) that the bacterial genetic structure of the mounds of soil-feeding termites (Cubitermes niokoloensis) is different from that of their surrounding soil. The aim of this study was to characterize the specificity of bacterial communities within mounds with respect to the digestive and soil origins of the mound. We have compared the bacterial community structures of a termite mound, termite gut sections, and surrounding soil using PCR-denaturing gradient gel electrophoresis (DGGE) analysis and cloning and sequencing of PCR-amplified 16S rRNA gene fragments. DGGE analysis revealed a drastic difference between the genetic structures of the bacterial communities of the termite gut and the mound. Analysis of 266 clones, including 54 from excised bands, revealed a high level of diversity in each biota investigated. The soil-feeding termite mound was dominated by the Actinobacteria phylum, whereas the Firmicutes and Proteobacteria phyla dominate the gut sections of termites and the surrounding soil, respectively. Phylogenetic analyses revealed a distinct clustering of Actinobacteria phylotypes between the mound and the surrounding soil. The Actinobacteria clones of the termite mound were diverse, distributed among 10 distinct families, and like those in the termite gut environment lightly dominated by the Nocardioidaceae family. Our findings confirmed that the soil-feeding termite mound (C. niokoloensis) represents a specific bacterial habitat in the tropics.

Comparison of Microbial Community Compositions of Injection and Production Well Samples in a Long-Term Water-Flooded Petroleum Reservoir

PubMed Central

Ren, Hong-Yan; Zhang, Xiao-Jun; Song, Zhi-yong; Rupert, Wieger; Gao, Guang-Jun; Guo, Sheng-xue; Zhao, Li-Ping

2011-01-01

Water flooding plays an important role in recovering oil from depleted petroleum reservoirs. Exactly how the microbial communities of production wells are affected by microorganisms introduced with injected water has previously not been adequately studied. Using denaturing gradient gel electrophoresis (DGGE) approach and 16S rRNA gene clone library analysis, the comparison of microbial communities is carried out between one injection water and two production waters collected from a working block of the water-flooded Gudao petroleum reservoir located in the Yellow River Delta. DGGE fingerprints showed that the similarities of the bacterial communities between the injection water and production waters were lower than between the two production waters. It was also observed that the archaeal composition among these three samples showed no significant difference. Analysis of the 16S rRNA gene clone libraries showed that the dominant groups within the injection water were Betaproteobacteria, Gammaproteobacteria and Methanomicrobia, while the dominant groups in the production waters were Gammaproteobacteria and Methanobacteria. Only 2 out of 54 bacterial operational taxonomic units (OTUs) and 5 out of 17 archaeal OTUs in the injection water were detected in the production waters, indicating that most of the microorganisms introduced by the injection water may not survive to be detected in the production waters. Additionally, there were 55.6% and 82.6% unique OTUs in the two production waters respectively, suggesting that each production well has its specific microbial composition, despite both wells being flooded with the same injection water. PMID:21858049

Characterization of sulfur oxidizing bacteria related to biogenic sulfuric acid corrosion in sludge digesters.

PubMed

Huber, Bettina; Herzog, Bastian; Drewes, Jörg E; Koch, Konrad; Müller, Elisabeth

2016-07-18

Biogenic sulfuric acid (BSA) corrosion damages sewerage and wastewater treatment facilities but is not well investigated in sludge digesters. Sulfur/sulfide oxidizing bacteria (SOB) oxidize sulfur compounds to sulfuric acid, inducing BSA corrosion. To obtain more information on BSA corrosion in sludge digesters, microbial communities from six different, BSA-damaged, digesters were analyzed using culture dependent methods and subsequent polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). BSA production was determined in laboratory scale systems with mixed and pure cultures, and in-situ with concrete specimens from the digester headspace and sludge zones. The SOB Acidithiobacillus thiooxidans, Thiomonas intermedia, and Thiomonas perometabolis were cultivated and compared to PCR-DGGE results, revealing the presence of additional acidophilic and neutrophilic SOB. Sulfate concentrations of 10-87 mmol/L after 6-21 days of incubation (final pH 1.0-2.0) in mixed cultures, and up to 433 mmol/L after 42 days (final pH <1.0) in pure A. thiooxidans cultures showed huge sulfuric acid production potentials. Additionally, elevated sulfate concentrations in the corroded concrete of the digester headspace in contrast to the concrete of the sludge zone indicated biological sulfur/sulfide oxidation. The presence of SOB and confirmation of their sulfuric acid production under laboratory conditions reveal that these organisms might contribute to BSA corrosion within sludge digesters. Elevated sulfate concentrations on the corroded concrete wall in the digester headspace (compared to the sludge zone) further indicate biological sulfur/sulfide oxidation in-situ. For the first time, SOB presence and activity is directly relatable to BSA corrosion in sludge digesters.

Effect of Phenotypic Residual Feed Intake and Dietary Forage Content on the Rumen Microbial Community of Beef Cattle

PubMed Central

Carberry, Ciara A.; Kenny, David A.; Han, Sukkyan; McCabe, Matthew S.

2012-01-01

Feed-efficient animals have lower production costs and reduced environmental impact. Given that rumen microbial fermentation plays a pivotal role in host nutrition, the premise that rumen microbiota may contribute to host feed efficiency is gaining momentum. Since diet is a major factor in determining rumen community structure and fermentation patterns, we investigated the effect of divergence in phenotypic residual feed intake (RFI) on ruminal community structure of beef cattle across two contrasting diets. PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR) were performed to profile the rumen bacterial population and to quantify the ruminal populations of Entodinium spp., protozoa, Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminococcus albus, Prevotella brevis, the genus Prevotella, and fungi in 14 low (efficient)- and 14 high (inefficient)-RFI animals offered a low-energy, high-forage diet, followed by a high-energy, low-forage diet. Canonical correspondence and Spearman correlation analyses were used to investigate associations between physiological variables and rumen microbial structure and specific microbial populations, respectively. The effect of RFI on bacterial profiles was influenced by diet, with the association between RFI group and PCR-DGGE profiles stronger for the higher forage diet. qPCR showed that Prevotella abundance was higher (P < 0.0001) in inefficient animals. A higher (P < 0.0001) abundance of Entodinium and Prevotella spp. and a lower (P < 0.0001) abundance of Fibrobacter succinogenes were observed when animals were offered the low-forage diet. Thus, differences in the ruminal microflora may contribute to host feed efficiency, although this effect may also be modulated by the diet offered. PMID:22562991

Effect of phenotypic residual feed intake and dietary forage content on the rumen microbial community of beef cattle.

PubMed

Carberry, Ciara A; Kenny, David A; Han, Sukkyan; McCabe, Matthew S; Waters, Sinead M

2012-07-01

Feed-efficient animals have lower production costs and reduced environmental impact. Given that rumen microbial fermentation plays a pivotal role in host nutrition, the premise that rumen microbiota may contribute to host feed efficiency is gaining momentum. Since diet is a major factor in determining rumen community structure and fermentation patterns, we investigated the effect of divergence in phenotypic residual feed intake (RFI) on ruminal community structure of beef cattle across two contrasting diets. PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR) were performed to profile the rumen bacterial population and to quantify the ruminal populations of Entodinium spp., protozoa, Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminococcus albus, Prevotella brevis, the genus Prevotella, and fungi in 14 low (efficient)- and 14 high (inefficient)-RFI animals offered a low-energy, high-forage diet, followed by a high-energy, low-forage diet. Canonical correspondence and Spearman correlation analyses were used to investigate associations between physiological variables and rumen microbial structure and specific microbial populations, respectively. The effect of RFI on bacterial profiles was influenced by diet, with the association between RFI group and PCR-DGGE profiles stronger for the higher forage diet. qPCR showed that Prevotella abundance was higher (P < 0.0001) in inefficient animals. A higher (P < 0.0001) abundance of Entodinium and Prevotella spp. and a lower (P < 0.0001) abundance of Fibrobacter succinogenes were observed when animals were offered the low-forage diet. Thus, differences in the ruminal microflora may contribute to host feed efficiency, although this effect may also be modulated by the diet offered.

The glucokinase mutation p.T206P is common among MODY patients of Jewish Ashkenazi descent.

PubMed

Gozlan, Yael; Tenenbaum, Ariel; Shalitin, Shlomit; Lebenthal, Yael; Oron, Tal; Cohen, Ohad; Phillip, Moshe; Gat-Yablonski, Galia

2012-09-01

Maturity-onset diabetes of the young (MODY) is characterized by an autosomal dominant mode of inheritance; a primary defect in insulin secretion with non-ketotic hyperglycemia, age of onset under 25 yr; and lack of autoantibodies. Heterozygous mutations in glucokinase (GCK) are associated with mild fasting hyperglycemia and gestational diabetes mellitus while homozygous or compound heterozygous GCK mutations result in permanent neonatal diabetes mellitus. Given that both the Israeli-Arabic and the various Israeli-Jewish communities tend to maintain ethnic seclusion, we speculated that it would be possible to identify a relatively narrow spectrum of mutations in the Israeli population. To characterize the genetic basis of GCK-MODY in the different ethnic groups of the Israeli population. Patients with clinically identified GCK-MODY and their first degree family members. Molecular analysis of GCK was performed on genomic DNA using polymerase chain reaction, denaturing gradient gel electrophoresis (DGGE), and sequencing. Bioinformatic model was preformed using the NEST program. Mutations in GCK were identified in 25 families and were all family-specific, except c.616A>C. p.T206P. This mutation was identified in six unrelated families, all patients from a Jewish-Ashkenazi descent, thus indicating an ethno-genetic correlation. A simple, fast, and relatively cheap DGGE/restriction-digestion assay was developed. The high incidence of the mutant allele in GCK-MODY patients of Jewish-Ashkenazi descent suggests a founder effect. We propose that clinically identified GCK-MODY patients of Jewish-Ashkenazi origin be first tested for this mutation. © 2011 John Wiley & Sons A/S.

Glyphosate application increased catabolic activity of gram-negative bacteria but impaired soil fungal community.

PubMed

Liu, Yehao; Li, Yongchun; Hua, Xiaomei; Müller, Karin; Wang, Hailong; Yang, Tongyi; Wang, Qiong; Peng, Xin; Wang, Mengcheng; Pang, Yanjun; Qi, Jinliang; Yang, Yonghua

2018-05-01

Glyphosate is a non-selective organophosphate herbicide that is widely used in agriculture, but its effects on soil microbial communities are highly variable and often contradictory, especially for high dose applications. We applied glyphosate at two rates: the recommended rate of 50 mg active ingredient kg -1 soil and 10-fold this rate to simulate multiple glyphosate applications during a growing season. After 6 months, we investigated the effects on the composition of soil microbial community, the catabolic activity and the genetic diversity of the bacterial community using phospholipid fatty acids (PLFAs), community level catabolic profiles (CLCPs), and 16S rRNA denaturing gradient gel electrophoresis (DGGE). Microbial biomass carbon (C mic ) was reduced by 45%, and the numbers of the cultivable bacteria and fungi were decreased by 84 and 63%, respectively, under the higher glyphosate application rate. According to the PLFA analysis, the fungal biomass was reduced by 29% under both application rates. However, the CLCPs showed that the catabolic activity of the gram-negative (G-) bacterial community was significantly increased under the high glyphosate application rate. Furthermore, the DGGE analysis indicated that the bacterial community in the soil that had received the high glyphosate application rate was dominated by G- bacteria. Real-time PCR results suggested that copies of the glyphosate tolerance gene (EPSPS) increased significantly in the treatment with the high glyphosate application rate. Our results indicated that fungi were impaired through glyphosate while G- bacteria played an important role in the tolerance of microbiota to glyphosate applications.

Bacaba beverage produced by Umutina Brazilian Amerindians: Microbiological and chemical characterization

PubMed Central

Puerari, Cláudia; Magalhães-Guedes, Karina Teixeira; Schwan, Rosane Freitas

2015-01-01

Bacaba chicha is a beverage prepared by the indigenous Umutina people from the bacaba fruit (Oenocarpus bacaba), a purple berry that is rich in fat and carbohydrates, as well as a source of phenolic compounds. In this study, samples of bacaba chicha beverage were collected, and the microbial community was assessed using culture-dependent and -independent techniques. The nutritional composition and metabolite profiles were analyzed, and species belonging to lactic acid bacteria (LAB) and yeasts were detected. The LAB group detected by culture-dependent analysis included Enterococcus hormaechei and Leuconostoc lactis. Polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) detected additional Propionibacterium avidum, Acetobacter spp., and uncultured bacteria. Pichia caribbica and Pichia guilliermondii were detected in a culture-dependent method, and Pichia caribbica was confirmed by PCR-DGGE analysis. The pH value of the beverage was 6.2. The nutritional composition was as follows: 16.47 ± 0.73 g 100 mL-1 dry matter, 2.2 ± 0.0 g 100 mL-1 fat, 3.36 ± 0.44 g 100 mL-1 protein, and 10.87 ± 0.26 g 100 mL-1 carbohydrate. The metabolites detected were 2.69 g L-1 succinic acid, 0.9 g L-1 acetic acid, 0.49 g L-1 citric acid, 0.52 g L-1 ethanol, and 0.4 g L-1 glycerol. This is the first study to identify microbial diversity in bacaba chicha spontaneous fermentation. This study is also the starting step in the immaterial record of this Brazilian indigenous beverage prepared from bacaba fruit. PMID:26691483

Bacaba beverage produced by Umutina Brazilian Amerindians: Microbiological and chemical characterization.

PubMed

Puerari, Cláudia; Magalhães-Guedes, Karina Teixeira; Schwan, Rosane Freitas

2015-01-01

Bacaba chicha is a beverage prepared by the indigenous Umutina people from the bacaba fruit (Oenocarpus bacaba), a purple berry that is rich in fat and carbohydrates, as well as a source of phenolic compounds. In this study, samples of bacaba chicha beverage were collected, and the microbial community was assessed using culture-dependent and -independent techniques. The nutritional composition and metabolite profiles were analyzed, and species belonging to lactic acid bacteria (LAB) and yeasts were detected. The LAB group detected by culture-dependent analysis included Enterococcus hormaechei and Leuconostoc lactis. Polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) detected additional Propionibacterium avidum, Acetobacter spp., and uncultured bacteria. Pichia caribbica and Pichia guilliermondii were detected in a culture-dependent method, and Pichia caribbica was confirmed by PCR-DGGE analysis. The pH value of the beverage was 6.2. The nutritional composition was as follows: 16.47 ± 0.73 g 100 mL-1 dry matter, 2.2 ± 0.0 g 100 mL-1 fat, 3.36 ± 0.44 g 100 mL-1 protein, and 10.87 ± 0.26 g 100 mL-1 carbohydrate. The metabolites detected were 2.69 g L-1 succinic acid, 0.9 g L-1 acetic acid, 0.49 g L-1 citric acid, 0.52 g L-1 ethanol, and 0.4 g L-1 glycerol. This is the first study to identify microbial diversity in bacaba chicha spontaneous fermentation. This study is also the starting step in the immaterial record of this Brazilian indigenous beverage prepared from bacaba fruit.

The long-term effects of UV exclusion on the microbial composition and photosynthetic competence of bacteria in hot-spring microbial mats.

PubMed

Norris, Tracy B; McDermott, Timothy R; Castenholz, Richard W

2002-03-01

The primary objective of this study was to determine whether the long-term exclusion of ultraviolet (UV) radiation (UVR) from hot-spring microbial mats resulted in an alteration of microbial composition, such as a shift to more UV-sensitive species. Over a 1-3-month period, microbial mats in two alkaline geothermal streams in Yellowstone National Park were covered with filters that excluded or transmitted UVR. Over some, 25% transmission neutral density screens were also used. In the 40-47 degrees C range, there were no apparent changes in community composition during the summer with or without high or low UVR, as assessed by denaturing gradient gel electrophoresis (DGGE) profiles after polymerase chain reaction amplification of 16S-rRNA genes with general Bacteria and Cyanobacteria primers. Major bands were purified from the DGGE gels and sequenced. Only one of the cyanobacterial sequences matched known strains in the database; the others appear to be unique. Although the bacterial composition of these communities was apparently stable, surface layers of cyanobacteria protected from UVR were not as competent photosynthetically as those that had been maintained under UVR. This decrease in competence was expressed as a loss of the ability to perform at a maximum rate under full UVR plus visible irradiance. However, even +UV-maintained cyanobacteria performed better when UVR was excluded during the photosynthesis tests. It is probable that the large differences in photosynthetic competence observed reflect changes at the level of gene expression in the dominant species rather than changes in species composition.

Effects of Crude Oil, Dispersant, and Oil-Dispersant Mixtures on Human Fecal Microbiota in an In Vitro Culture System

PubMed Central

Kim, Jong Nam; Kim, Bong-Soo; Kim, Seong-Jae; Cerniglia, Carl E.

2012-01-01

ABSTRACT The Deepwater Horizon oil spill of 2010 raised concerns that dispersant and dispersed oil, as well as crude oil itself, could contaminate shellfish and seafood habitats with hazardous residues that had potential implications for human health and the ecosystem. However, little is known about the effects of crude oil and dispersant on the human fecal microbiota. The aim of this research was to evaluate the potential effects of Deepwater Horizon crude oil, Corexit 9500 dispersant, and their combination on human fecal microbial communities, using an in vitro culture test system. Fecal specimens from healthy adult volunteers were made into suspensions, which were then treated with oil, dispersant, or oil-dispersant mixtures under anaerobic conditions in an in vitro culture test system. Perturbations of the microbial community, compared to untreated control cultures, were assessed using denaturing gradient gel electrophoresis (DGGE), real-time PCR, and pyrosequencing methods. DGGE and pyrosequencing analysis showed that oil-dispersant mixtures reduced the diversity of fecal microbiota from all individuals. Real-time PCR results indicated that the copy numbers of 16S rRNA genes in cultures treated with dispersed oil or oil alone were significantly lower than those in control incubations. The abundance of the Bacteroidetes decreased in crude oil-treated and dispersed-oil-treated cultures, while the Proteobacteria increased in cultures treated with dispersed oil. In conclusion, the human fecal microbiota was affected differently by oil and dispersed oil, and the influence of dispersed oil was significantly greater than that of either oil or dispersant alone compared to control cultures. PMID:23093387

Fish-mediated changes in bacterioplankton community composition: an in situ mesocosm experiment

NASA Astrophysics Data System (ADS)

Luo, Congqiang; Yi, Chunlong; Ni, Leyi; Guo, Longgen

2017-06-01

We characterized variations in bacterioplankton community composition (BCC) in mesocosms subject to three different treatments. Two groups contained fish (group one: Cyprinus carpio; group two: Hypophthalmichthys molitrix); and group three, the untreated mesocosm, was the control. Samples were taken seven times over a 49-day period, and BCC was analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR (qPCR). Results revealed that introduction of C. carpio and H. molitrix had a remarkable impact on the composition of bacterioplankton communities, and the BCC was significantly different between each treatment. Sequencing of DGGE bands revealed that the bacterioplankton community in the different treatment groups was consistent at a taxonomic level, but differed in its abundance. H. molitrix promoted the richness of Alphaproteobacteria and Actinobacteria, while more bands affiliated to Cyanobacteria were detected inC. carpio mesocosms. The redundancy analysis (RDA) result demonstrated that the BCC was closely related to the bottom-up (total phosphorus, chlorophyll a, phytoplankton biomass) and top-down forces (biomass of copepods and cladocera) in C. carpio and control mesocosms, respectively. We found no evidence for top-down regulation of BCC by zooplankton in H. molitrix mesocosms, while grazing by protozoa (heterotrophic nanoflagellates, ciliates) became the major way to regulate BCC. Total bacterioplankton abundances were significantly higher in C. carpio mesocosms because of high nutrient concentration and suspended solids. Our study provided insights into the relationship between fish and bacterioplankton at species level, leading to a deep understanding of the function of the microbial loop and the aquatic ecosystem.

Comparison of oral microbiota in tumor and non-tumor tissues of patients with oral squamous cell carcinoma

PubMed Central

2012-01-01

Background Bacterial infections have been linked to malignancies due to their ability to induce chronic inflammation. We investigated the association of oral bacteria in oral squamous cell carcinoma (OSCC/tumor) tissues and compared with adjacent non-tumor mucosa sampled 5 cm distant from the same patient (n = 10). By using culture-independent 16S rRNA approaches, denaturing gradient gel electrophoresis (DGGE) and cloning and sequencing, we assessed the total bacterial diversity in these clinical samples. Results DGGE fingerprints showed variations in the band intensity profiles within non-tumor and tumor tissues of the same patient and among the two groups. The clonal analysis indicated that from a total of 1200 sequences characterized, 80 bacterial species/phylotypes were detected representing six phyla, Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, Actinobacteria and uncultivated TM7 in non-tumor and tumor libraries. In combined library, 12 classes, 16 order, 26 families and 40 genera were observed. Bacterial species, Streptococcus sp. oral taxon 058, Peptostreptococcus stomatis, Streptococcus salivarius, Streptococcus gordonii, Gemella haemolysans, Gemella morbillorum, Johnsonella ignava and Streptococcus parasanguinis I were highly associated with tumor site where as Granulicatella adiacens was prevalent at non-tumor site. Streptococcus intermedius was present in 70% of both non-tumor and tumor sites. Conclusions The underlying changes in the bacterial diversity in the oral mucosal tissues from non-tumor and tumor sites of OSCC subjects indicated a shift in bacterial colonization. These most prevalent or unique bacterial species/phylotypes present in tumor tissues may be associated with OSCC and needs to be further investigated with a larger sample size. PMID:22817758

Aerobic biological treatment of low-strength synthetic wastewater in membrane-coupled bioreactors: the structure and function of bacterial enrichment cultures as the net growth rate approaches zero.

PubMed

Chen, Ruoyu; LaPara, Timothy M

2006-01-01

The goal of the current research was to determine if the stringent nutrient limitation imposed by membrane-coupled bioreactors (MBRs) could be used to force mixed bacterial communities to exhibit a zero net growth rate over an extended time period. Mechanistically, this zero net growth rate could be achieved when the amount of energy available for growth is balanced by the maintenance requirements of the bacterial community. Bench-scale MBRs were fed synthetic feed medium containing gelatin as the major organic substrate. Biomass concentrations initially increased rapidly, but subsequently declined until an asymptote was reached. Leucine aminopeptidase activities concomitantly increased by at least 10-fold, suggesting that bacterial catabolic activity remained high even while growth rates became negligible. In contrast, alpha-glucosidase and heptanoate esterase activities decreased, indicating that the bacterial community specifically adapted to the carbon source in the feed medium. Bacterial community analysis by denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments (PCR-DGGE) suggested that the bacterial community structure completely changed from the beginning to the end of each MBR. Excision and nucleotide sequence analysis of prominent PCR-DGGE bands suggested that many of the dominant populations were similar to novel bacterial strains that were previously uncultivated or recently cultivated during studies specifically targeting these novel populations. This research demonstrates that MBRs have substantial practical applications for biological wastewater treatment; in addition, MBRs are a useful tool to study the ecology of slow-growing bacteria.

Anaerobic Oxidation of Methane at a Marine Methane Seep in a Forearc Sediment Basin off Sumatra, Indian Ocean.

PubMed

Siegert, Michael; Krüger, Martin; Teichert, Barbara; Wiedicke, Michael; Schippers, Axel

2011-01-01

A cold methane seep was discovered in a forearc sediment basin off the island Sumatra, exhibiting a methane-seep adapted microbial community. A defined seep center of activity, like in mud volcanoes, was not discovered. The seep area was rather characterized by a patchy distribution of active spots. The relevance of anaerobic oxidation of methane (AOM) was reflected by (13)C-depleted isotopic signatures of dissolved inorganic carbon. The anaerobic conversion of methane to CO(2) was confirmed in a (13)C-labeling experiment. Methane fueled a vital microbial community with cell numbers of up to 4 × 10(9) cells cm(-3) sediment. The microbial community was analyzed by total cell counting, catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH), quantitative real-time PCR (qPCR), and denaturing gradient gel electrophoresis (DGGE). CARD-FISH cell counts and qPCR measurements showed the presence of Bacteria and Archaea, but only small numbers of Eukarya. The archaeal community comprised largely members of ANME-1 and ANME-2. Furthermore, members of the Crenarchaeota were frequently detected in the DGGE analysis. Three major bacterial phylogenetic groups (δ-Proteobacteria, candidate division OP9, and Anaerolineaceae) were abundant across the study area. Several of these sequences were closely related to the genus Desulfococcus of the family Desulfobacteraceae, which is in good agreement with previously described AOM sites. In conclusion, the majority of the microbial community at the seep consisted of AOM-related microorganisms, while the relevance of higher hydrocarbons as microbial substrates was negligible.

A viable microbial community in a subglacial volcanic crater lake, Iceland.

PubMed

Gaidos, Eric; Lanoil, Brian; Thorsteinsson, Thorsteinn; Graham, Andrew; Skidmore, Mark; Han, Suk-Kyun; Rust, Terri; Popp, Brian

2004-01-01

We describe a viable microbial community in a subglacial lake within the Grímsvötn volcanic caldera, Iceland. We used a hot water drill to penetrate the 300-m ice shelf and retrieved lake water and volcanic tephra sediments. We also acquired samples of borehole water before and after penetration to the lake, overlying glacial ice and snow, and water from a nearby subaerial geothermal lake for comparative analyses. Lake water is at the freezing point and fresh (total dissolved solids = 260 mg L(-1)). Detectable numbers of cells were found in samples of the lake water column and tephra sediments: 2 x 10(4) ml(-1) and 4 x 10(7) g(-1), respectively. Plate counts document abundant cold-adapted cultivable organisms in the lake water, but not in the borehole (before penetration) or glacial ice. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments amplified from genomic DNA extracted from Grímsvötn samples indicates that the lake community is distinct from the assemblages of organisms in borehole water (before penetration) and the overlying ice and snow. Sequencing of selected DGGE bands revealed that many sequences are highly similar to known psychrophilic organisms or cloned DNA from other cold environments. Significant uptake of 14C-labeled bicarbonate occurred in dark, low-temperature incubations of lake water samples, indicating the presence of autotrophs. Acetylene reduction assays under similar incubation conditions showed no significant nitrogen fixation potential by lake water samples. This may be a consequence of the inhibition of diazotrophy by nitrogen in the lake.

Comparison of PCR primer-based strategies for characterization of ammonia oxidizer communities in environmental samples.

PubMed

Mahmood, Shahid; Freitag, Thomas E; Prosser, James I

2006-06-01

PCR-based techniques are commonly used to characterize microbial communities, but are subject to bias that is difficult to assess. This study aimed to evaluate bias of several PCR primer-based strategies used to study diversity of autotrophic ammonia oxidizers. 16S rRNA genes from soil- or sediment-DNA were amplified using primers considered either selective or specific for betaproteobacterial ammonia oxidizers. Five approaches were assessed: (a) amplification with primers betaAMO143f-betaAMO1315r; (b) amplification with primers CTO189f-CTO654r; (c) nested amplification with betaAMO143f-betaAMO1315r followed by CTO189f-CTO654r primers; (d) nested amplification with betaAMO143f-betaAMO1315r and CTO189f-Pf1053r primers; (e) nested amplification with 27f-1492r and CTO189f-CTO654r primers. Amplification products were characterized by denaturing gradient gel electrophoresis (DGGE) analysis after further amplification with 357f-GC-518r primers. DGGE profiles of soil communities were heterogeneous and depended on the approach followed. Ammonia oxidizer diversity was higher using approaches (b), (c) and (e) than using (a) and (d), where sequences of the most prominent bands showed similarities to nonammonia oxidizers. Profiles from marine sediments were more consistent, regardless of the approach adopted, and sequence analysis of excised bands indicated that these consisted of ammonia oxidizers only. The study demonstrates the importance of choice of primer, of screening for sequences of nontarget organisms and use of several approaches when characterizing microbial communities in natural environments.

Bacterial community structure and abundances of antibiotic resistance genes in heavy metals contaminated agricultural soil.

PubMed

Zhang, Fengli; Zhao, Xiaoxue; Li, Qingbo; Liu, Jia; Ding, Jizhe; Wu, Huiying; Zhao, Zongsheng; Ba, Yue; Cheng, Xuemin; Cui, Liuxin; Li, Hongping; Zhu, Jingyuan

2018-04-01

Soil contamination with heavy metals is a worldwide problem especially in China. The interrelation of soil bacterial community structure, antibiotic resistance genes, and heavy metal contamination in soil is still unclear. Here, seven agricultural areas (G1-G7) with heavy metal contamination were sampled with different distances (741 to 2556 m) to the factory. Denaturing gradient gel electrophoresis (DGGE) and Shannon index were used to analyze bacterial community diversity. Real-time fluorescence quantitative PCR was used to detect the relative abundance of ARGs sul1, sul2, tetA, tetM, tetW, one mobile genetic elements (MGE) inti1. Results showed that all samples were polluted by Cadmium (Cd), and some of them were polluted by lead (Pb), mercury (Hg), arsenic (As), copper (Cu), and zinc (Zn). DGGE showed that the most abundant bacterial species were found in G7 with the lightest heavy metal contamination. The results of the principal component analysis and clustering analysis both showed that G7 could not be classified with other samples. The relative abundance of sul1 was correlated with Cu, Zn concentration. Gene sul2 are positively related with total phosphorus, and tetM was associated with organic matter. Total gene abundances and relative abundance of inti1 both correlated with organic matter. Redundancy analysis showed that Zn and sul2 were significantly related with bacterial community structure. Together, our results indicate a complex linkage between soil heavy metal concentration, bacterial community composition, and some global disseminated ARG abundance.

Fish-mediated changes in bacterioplankton community composition: an in situ mesocosm experiment

NASA Astrophysics Data System (ADS)

Luo, Congqiang; Yi, Chunlong; Ni, Leyi; Guo, Longgen

2018-03-01

We characterized variations in bacterioplankton community composition (BCC) in mesocosms subject to three different treatments. Two groups contained fish (group one: Cyprinus carpio; group two: Hypophthalmichthys molitrix); and group three, the untreated mesocosm, was the control. Samples were taken seven times over a 49-d period, and BCC was analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR (qPCR). Results revealed that introduction of C. carpio and H. molitrix had a remarkable impact on the composition of bacterioplankton communities, and the BCC was significantly different between each treatment. Sequencing of DGGE bands revealed that the bacterioplankton community in the different treatment groups was consistent at a taxonomic level, but differed in its abundance. H. molitrix promoted the richness of Alphaproteobacteria and Actinobacteria, while more bands affiliated to Cyanobacteria were detected in C. carpio mesocosms. The redundancy analysis (RDA) result demonstrated that the BCC was closely related to the bottom-up (total phosphorus, chlorophyll a, phytoplankton biomass) and top-down forces (biomass of copepods and cladocera) in C. carpio and control mesocosms, respectively. We found no evidence for top-down regulation of BCC by zooplankton in H. molitrix mesocosms, while grazing by protozoa (heterotrophic nanoflagellates, ciliates) became the major way to regulate BCC. Total bacterioplankton abundances were significantly higher in C. carpio mesocosms because of high nutrient concentration and suspended solids. Our study provided insights into the relationship between fish and bacterioplankton at species level, leading to a deep understanding of the function of the microbial loop and the aquatic ecosystem.

Impact of Nisin-Activated Packaging on Microbiota of Beef Burgers during Storage.

PubMed

Ferrocino, Ilario; Greppi, Anna; La Storia, Antonietta; Rantsiou, Kalliopi; Ercolini, Danilo; Cocolin, Luca

2016-01-15

Beef burgers were stored at 4°C in a vacuum in nisin-activated antimicrobial packaging. Microbial ecology analyses were performed on samples collected between days 0 and 21 of storage to discover the population diversity. Two batches were analyzed using RNA-based denaturing gradient gel electrophoresis (DGGE) and pyrosequencing. The active packaging retarded the growth of the total viable bacteria and lactic acid bacteria. Culture-independent analysis by pyrosequencing of RNA extracted directly from meat showed that Photobacterium phosphoreum, Lactococcus piscium, Lactobacillus sakei, and Leuconostoc carnosum were the major operational taxonomic units (OTUs) shared between control and treated samples. Beta diversity analysis of the 16S rRNA sequence data and RNA-DGGE showed a clear separation between two batches based on the microbiota. Control samples from batch B showed a significant high abundance of some taxa sensitive to nisin, such as Kocuria rhizophila, Staphylococcus xylosus, Leuconostoc carnosum, and Carnobacterium divergens, compared to control samples from batch A. However, only from batch B was it possible to find a significant difference between controls and treated samples during storage due to the active packaging. Predicted metagenomes confirmed differences between the two batches and indicated that the use of nisin-based antimicrobial packaging can determine a reduction in the abundance of specific metabolic pathways related to spoilage. The present study aimed to assess the viable bacterial communities in beef burgers stored in nisin-based antimicrobial packaging, and it highlights the efficacy of this strategy to prolong beef burger shelf life. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The synergy of permeable pavements and geothermal heat pumps for stormwater treatment and reuse.

PubMed

Tota-Maharaj, K; Scholz, M; Ahmed, T; French, C; Pagaling, E

2010-12-14

The use of permeable pavement systems with integrated geothermal heat pumps for the treatment and recycling of urban runoff is novel and timely. This study assesses the efficiency of the combined technology for controlled indoor and uncontrolled outdoor experimental rigs. Water quality parameters such as biochemical oxygen demand, nutrients, total viable heterotrophic bacteria and total coliforms were tested before and after treatment in both rigs. The water borne bacterial community genomic deoxyribonucleic acid (DNA) was analyzed by polymerase chain reaction (PCR) amplification followed by denaturing gradient gel electrophoresis (DGGE) and was further confirmed by DNA sequencing techniques. Despite the relatively high temperatures in the indirectly heated sub-base of the pavement, potentially pathogenic organisms such as Salmonella spp., Escherichia coli, faecal Streptococci and Legionella were not detected. Moreover, mean removal rates of 99% for biochemical oxygen demand, 97% for ammonia-nitrogen and 95% for orthophosphate-phosphates were recorded. This research also supports decision-makers in assessing public health risks based on qualitative molecular microbiological data associated with the recycling of treated urban runoff.

Molecular identification of Mango, Mangifera indica L.var. totupura

PubMed Central

Jagarlamudi, Sankar; G, Rosaiah; Kurapati, Ravi Kumar; Pinnamaneni, Rajasekhar

2011-01-01

Mango (>Mangifera indica) belonging to Anacardiaceae family is a fruit that grows in tropical regions. It is considered as the King of fruits. The present work was taken up to identify a tool in identifying the mango species at the molecular level. The chloroplast trnL-F region was amplified from extracted total genomic DNA using the polymerase chain reaction (PCR) and sequenced. Sequence of the dominant DGGE band revealed that Mangifera indica in tested leaves was Mangifera indica (100% similarity to the ITS sequences of Mangifera indica). This sequence was deposited in NCBI with the accession no. GQ927757. Abbreviations AFLP - Amplified fragment length polymorphism , cpDNA - Chloroplast DNA, DDGE - Denaturing gradient gel electrophoresis, DNA - Deoxyribo nucleic acid, EDTA - Ethylenediamine tetraacetic acid, HCl - Hydrochloric acid, ISSR - Inter simple sequence repeats, ITS - Internal transcribed spacer, MATAB - Methyl Ammonium Bromide, Na2SO3 - Sodium sulphite, NaCl - Sodium chloride, NCBI - National Centre for Biotechnology Information, PCR - Polymerase chain reaction, PEG - Polyethylene glycol, RAPD - Randomly amplified polymorphic DNA, trnL-F - Transfer RNA genes start codon- termination codon. PMID:21423885

Effect of organic matter strength on anammox for modified greenhouse turtle breeding wastewater treatment.

PubMed

Chen, Chongjun; Huang, Xiaoxiao; Lei, Chenxiao; Zhang, Tian C; Wu, Weixiang

2013-11-01

Anaerobic ammonium-N removal from modified greenhouse turtle breeding wastewater with different chemical oxygen demand (COD) strengths (194.0-577.8 mg L(-1)) at relatively fixed C/N ratios (≈ 2) was investigated using a lab-scale up-flow anaerobic sludge blanket (UASB) anammox reactor. During the entire experiment, the total nitrogen (TN) removal efficiency was about 85% or higher, while the average COD removal efficiency was around 56.5 ± 7.9%. Based on the nitrogen and carbon balance, the nitrogen removal contribution was 79.6 ± 4.2% for anammox, 12.7 ± 3.0% for denitrification+denitritation and 7.7 ± 4.9% for other mechanisms. Denaturing gradient gel electrophoresis (DGGE) analyses revealed that Planctomycete, Proteobacteria and Chloroflexi bacteria were coexisted in the reactor. Anammox was always dominant when the reactor was fed with different COD concentrations, which indicated the stability of the anammox process with the coexistence of the denitrification process in treating greenhouse turtle breeding wastewater. Copyright © 2013 Elsevier Ltd. All rights reserved.

Bioleaching of a low-grade nickel-copper sulfide by mixture of four thermophiles.

PubMed

Li, Shuzhen; Zhong, Hui; Hu, Yuehua; Zhao, Jiancun; He, Zhiguo; Gu, Guohua

2014-02-01

This study investigated thermophilic bioleaching of a low grade nickel-copper sulfide using mixture of four acidophilic thermophiles. Effects of 0.2g/L l-cysteine on the bioleaching process were further evaluated. It aimed at offering new alternatives for enhancing metal recoveries from nickel-copper sulfide. Results showed a recovery of 80.4% nickel and 68.2% copper in 16-day bioleaching without l-cysteine; while 83.7% nickel and 81.4% copper were recovered in the presence of l-cysteine. Moreover, nickel recovery was always higher than copper recovery. l-Cysteine was found contributing to lower pH value, faster microbial growth, higher Oxidation-Reduction Potential (ORP), higher zeta potential and absorbing on the sulfide surfaces through amino, carboxyl and sulfhydryl groups. X-ray Diffraction (XRD) patterns of leached residues showed generation of S, jarosite and ammoniojarosite. Denaturing Gradient Gel Electrophoresis (DGGE) results revealed that l-cysteine could have variant impacts on different microorganisms and changed the microbial community composition dramatically during nickel-copper sulfide bioleaching. Copyright © 2013 Elsevier Ltd. All rights reserved.

Mutations in the PDE6B gene in autosomal recessive retinitis pigmentosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Danciger, M.; Blaney, J.; Gao, Y.Q.

1995-11-01

We have studied 24 small families with presumed autosomal recessive inheritance of retinitis pigmentosa by a combination of haplotype analysis and exon screening. Initial analysis of the families was made with a dinucleotide repeat polymorphism adjacent to the gene for rod cGMP-phosphodiesterase (PDE6B). This was followed by denaturing gradient gel electrophoresis (DGGE) and single-strand conformation polymorphism electrophoresis (SSCPE) of the 22 exons and a portion of the 5{prime} untranslated region of the PDE6B gene in the probands of each family in which the PDE6B locus could not be ruled out from segregating with disease. Two probands were found with compoundmore » heterozygous mutations: Gly576Asp and His620(1-bp del) mutations were present in one proband, and a Lys706X null mutation and an AG to AT splice acceptor site mutation in intron 2 were present in the other. Only the affecteds of each of the two families carried both corresponding mutations. 29 refs., 3 figs., 1 tab.« less

Molecular investigation of bacterial communities during the manufacturing and ripening of semi-hard Iranian Liqvan cheese.

PubMed

Ramezani, M; Hosseini, S M; Ferrocino, I; Amoozegar, M A; Cocolin, L

2017-09-01

Liqvan (or Lighvan) is a traditional Iranian cheese from the East Azerbaijan province of Iran, which is made of raw ewe's milk without the addition of a starter. The grazing pastures, environmental conditions and the ancient regional production methods allocate a distinctive microbial ecology to this type of cheese, and these factors are consequently associated with the quality of the product. In this study, the microbiota of the milk, curd and cheese has been investigated using culture independent approaches. Denaturing gradient gel electrophoresis (DGGE) of the bacteria, 16S rRNA based high-throughput sequencing and enumeration of the live bacterial community by means of quantitative PCR (qPCR) have been used for this purpose. The results showed that the main bacterial population in the milk belonged to both microbial contaminants and lactic acid bacteria (LAB). However, both of these populations were totally replaced by LAB during ripening. The present survey contributes by describing the microbiota of this ancient cheese in more detail during fermentation and ripening. Copyright © 2017 Elsevier Ltd. All rights reserved.

Diversity of bacteria and archaea from two shallow marine hydrothermal vents from Vulcano Island.

PubMed

Antranikian, Garabed; Suleiman, Marcel; Schäfers, Christian; Adams, Michael W W; Bartolucci, Simonetta; Blamey, Jenny M; Birkeland, Nils-Kåre; Bonch-Osmolovskaya, Elizaveta; da Costa, Milton S; Cowan, Don; Danson, Michael; Forterre, Patrick; Kelly, Robert; Ishino, Yoshizumi; Littlechild, Jennifer; Moracci, Marco; Noll, Kenneth; Oshima, Tairo; Robb, Frank; Rossi, Mosè; Santos, Helena; Schönheit, Peter; Sterner, Reinhard; Thauer, Rudolf; Thomm, Michael; Wiegel, Jürgen; Stetter, Karl Otto

2017-07-01

To obtain new insights into community compositions of hyperthermophilic microorganisms, defined as having optimal growth temperatures of 80 °C and above, sediment and water samples were taken from two shallow marine hydrothermal vents (I and II) with temperatures of 100 °C at Vulcano Island, Italy. A combinatorial approach of denaturant gradient gel electrophoresis (DGGE) and metagenomic sequencing was used for microbial community analyses of the samples. In addition, enrichment cultures, growing anaerobically on selected polysaccharides such as starch and cellulose, were also analyzed by the combinatorial approach. Our results showed a high abundance of hyperthermophilic archaea, especially in sample II, and a comparable diverse archaeal community composition in both samples. In particular, the strains of the hyperthermophilic anaerobic genera Staphylothermus and Thermococcus, and strains of the aerobic hyperthermophilic genus Aeropyrum, were abundant. Regarding the bacterial community, ε-Proteobacteria, especially the genera Sulfurimonas and Sulfurovum, were highly abundant. The microbial diversity of the enrichment cultures changed significantly by showing a high dominance of archaea, particularly the genera Thermococcus and Palaeococcus, depending on the carbon source and the selected temperature.

Enhanced biofiltration using cell attachment promotors.

PubMed

Goncalves, Juan J; Govind, Rakesh

2009-02-15

H2S polluted airstreams were treated in two biotrickling filter columns packed with polyurethane (PU) foam cubes, one with cubes coated with a solution of 25 mg/L of polyethyleneimine (PEI, coated reactor) and the other containing just plain PU cubes (uncoated reactor) at empty bed residence times (EBRT) ranging from 6 to 60 s. and inlet H2S concentrations ranging from 30 to 235 ppm, (overall loads of up to 44 gH2S/m3bed/h), with overall removal efficiencies (RE) in the range of 90-100% over 125 days. The acclimatization characteristics of the coated reactor outperformed those of the uncoated one, and both the observed elimination capacity (EC) of 77 gH2S/m3bed/h and retention of volatile solids (VS) of 42 mgVS/cube were maxima in the coated reactor. Insights into the controlling removal mechanisms were also provided by means of dimensionless analysis of the experimental data. Denaturing gradient gel electrophoresis (DGGE) showed that the dominant surviving species in both units belonged to the genus Acidithiobacillus.

Illumina sequencing-based analysis of a microbial community enriched under anaerobic methane oxidation condition coupled to denitrification revealed coexistence of aerobic and anaerobic methanotrophs.

PubMed

Siniscalchi, Luciene Alves Batista; Leite, Laura Rabelo; Oliveira, Guilherme; Chernicharo, Carlos Augusto Lemos; de Araújo, Juliana Calabria

2017-07-01

Methane is produced in anaerobic environments, such as reactors used to treat wastewaters, and can be consumed by methanotrophs. The composition and structure of a microbial community enriched from anaerobic sewage sludge under methane-oxidation condition coupled to denitrification were investigated. Denaturing gradient gel electrophoresis (DGGE) analysis retrieved sequences of Methylocaldum and Chloroflexi. Deep sequencing analysis revealed a complex community that changed over time and was affected by methane concentration. Methylocaldum (8.2%), Methylosinus (2.3%), Methylomonas (0.02%), Methylacidiphilales (0.45%), Nitrospirales (0.18%), and Methanosarcinales (0.3%) were detected. Despite denitrifying conditions provided, Nitrospirales and Methanosarcinales, known to perform anaerobic methane oxidation coupled to denitrification (DAMO) process, were in very low abundance. Results demonstrated that aerobic and anaerobic methanotrophs coexisted in the reactor together with heterotrophic microorganisms, suggesting that a diverse microbial community was important to sustain methanotrophic activity. The methanogenic sludge was a good inoculum to enrich methanotrophs, and cultivation conditions play a selective role in determining community composition.

In vitro fermentation behaviors of fucosylated chondroitin sulfate from Pearsonothuria graeffei by human gut microflora.

PubMed

Wei, Chao-Yang; Liao, Ning-Bo; Zhang, Yu; Ye, Xing-Qian; Li, Shan; Hu, Ya-Qin; Liu, Dong-Hong; Linhardt, Robert J; Wang, Xin; Chen, Shi-Guo

2017-09-01

A fucosylated chondroitin sulfate (FCS-pg) with highly repeated structure from Pearsonothuria graeffei was subjected to a in vitro fermentation model to investigate its fermentability and effects on human gut microflora. High performance liquid chromatography (HPLC) measurement found FCS-pg can be fermented to short chain fatty acids (SCFAs) by gut microflora from partial human fecal samples. 16S rRNA gene-based polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-DGGE) profiling and real-time quantitative PCR analysis showed that FCS-pg mainly increased the proportions of Clostridium cluster XI, Bacteriodes prevotella group, Bifidobacterium genus, Clostridium cluster I and Clostridium cluster XIVab, whereas the numbers of the Enterobacteriaceae and Lactobacillus decreased. These results indicated that FCS-pg was mainly fermented by Bacteroides, Bifidobacterium and Clostridium. It increased the content of probiotics bacteria in achieving health-enhancing effect, was slightly different than most sulfated polysaccharides from marine animals. The current study provides useful new information on the mechanism of absorption and functional activity on FCS-pg within the gastrointestinal tract of the human body. Copyright © 2017. Published by Elsevier B.V.

Ignimbrite textural properties as determinants of endolithic colonization patterns from hyper-arid Atacama Desert.

PubMed

Cámara, Beatriz; Suzuki, Shino; Nealson, Kenneth H; Wierzchos, Jacek; Ascaso, Carmen; Artieda, Octavio; de los Ríos, Asunción

2014-12-01

This study explores the photosynthetic microbial colonization of rhyolitic ignimbrites in Lomas de Tilocalar, a hyper-arid region of the Atacama Desert, Chile. Colonization appeared in the form of a green layer a few millimeters beneath the ignimbrite surface. Some ignimbrite rocks revealed two distinct micromorphological areas of identical mineralogical and chemical composition but different textural properties. According to texture, colonization patterns varied in terms of the extension and depth of colonization. The diversity of photosynthetic microorganisms was assessed by denaturing gradient gel electrophoresis (DGGE) of the 23S rRNA gene and by generating clone libraries of the 16S rRNA gene. We observed a low diversity of photosynthetic microorganisms colonizing the ignimbrite microhabitat. Most rRNA gene sequences recovered greatly resembled those of Chroococcidiopsis hypolith clones from arid deserts. These results point to highly restrictive conditions of the hyper-arid Atacama Desert conditioning the diversity of cyanobacteria, and suggest that microbial colonization and composition patterns might be determined by the microscale physico-chemical properties of the ignimbrite rocks. Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.

Microbial ecology of anaerobic digesters: the key players of anaerobiosis.

PubMed

Ali Shah, Fayyaz; Mahmood, Qaisar; Maroof Shah, Mohammad; Pervez, Arshid; Ahmad Asad, Saeed

2014-01-01

Anaerobic digestion is the method of wastes treatment aimed at a reduction of their hazardous effects on the biosphere. The mutualistic behavior of various anaerobic microorganisms results in the decomposition of complex organic substances into simple, chemically stabilized compounds, mainly methane and CO2. The conversions of complex organic compounds to CH4 and CO2 are possible due to the cooperation of four different groups of microorganisms, that is, fermentative, syntrophic, acetogenic, and methanogenic bacteria. Microbes adopt various pathways to evade from the unfavorable conditions in the anaerobic digester like competition between sulfate reducing bacteria (SRB) and methane forming bacteria for the same substrate. Methanosarcina are able to use both acetoclastic and hydrogenotrophic pathways for methane production. This review highlights the cellulosic microorganisms, structure of cellulose, inoculum to substrate ratio, and source of inoculum and its effect on methanogenesis. The molecular techniques such as DGGE (denaturing gradient gel electrophoresis) utilized for dynamic changes in microbial communities and FISH (fluorescent in situ hybridization) that deal with taxonomy and interaction and distribution of tropic groups used are also discussed.

Comparison of microbially enhanced compost extracts produced from composted cattle rumen content material and from commercially available inocula.

PubMed

Shrestha, Karuna; Adetutu, Eric M; Shrestha, Pramod; Walsh, Kerry B; Harrower, Keith M; Ball, Andrew S; Midmore, David J

2011-09-01

A comparative study was performed on compost extracts prepared from cattle rumen content composted for three and nine months, nine month old compost inoculated with a Nutri-Life 4/20™ inoculum, and two commercial preparations (LivingSoil™ and Nutri-Life 4/20™), all incubated for 48h. Nutri-Life 4/20™ had the highest concentrations of NO(3)(-)-N and K(+)-K, while rumen compost extract had higher humic and fulvic acids concentration. The bacterial and fungal community level functional diversity of three month old compost extract and of LivingSoil™, assessed with Biolog™, were higher than that of nine month old rumen compost extract, with or without Nutri-Life 4/20™ inoculum, or Nutri-Life 4/20™. No difference in fungal diversity was observed between treatments, as indicated by Denaturing Gradient Gel Electrophoresis (DGGE) analysis, however, bacterial diversity was higher in all compost extracts and LivingSoil™ compared to the Nutri-Life 4/20™. Criteria for judging the quality of a microbially enhanced extract are discussed. Copyright © 2011 Elsevier Ltd. All rights reserved.

Arbuscular mycorrhizal fungal communities in the rhizosphere of a continuous cropping soybean system at the seedling stage.

PubMed

Cui, Jiaqi; Bai, Li; Liu, Xiaorui; Jie, Weiguang; Cai, Baiyan

Arbuscular mycorrhizae (AM) fungi play a crucial role in the growth of soybean; however, the planting system employed is thought to have an effect on AM fungal communities in the rhizosphere. This study was performed to explore the influence of continuous soybean cropping on the diversity of Arbuscular mycorrhizal (AM) fungi, and to identify the dominant AM fungus during the seedling stage. Three soybean cultivars were planted under two and three years continuous cropping, respectively. The diversity of AM fungi in the rhizosphere soil at the seedling stage was subsequently analyzed using polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE). The results showed that an increase in cropping years improved the colonization rate of AM in all three soybean cultivars. Moreover, the dominant species were found to be Funneliformis mosseae and Glomus species. The results of cluster analysis further confirmed that the number of years of continuous cropping significantly affected the composition of rhizospheric AM fungal communities in different soybean cultivars. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Microbial Ecology of Anaerobic Digesters: The Key Players of Anaerobiosis

PubMed Central

Ali Shah, Fayyaz; Mahmood, Qaisar; Maroof Shah, Mohammad; Pervez, Arshid; Ahmad Asad, Saeed

2014-01-01

Anaerobic digestion is the method of wastes treatment aimed at a reduction of their hazardous effects on the biosphere. The mutualistic behavior of various anaerobic microorganisms results in the decomposition of complex organic substances into simple, chemically stabilized compounds, mainly methane and CO2. The conversions of complex organic compounds to CH4 and CO2 are possible due to the cooperation of four different groups of microorganisms, that is, fermentative, syntrophic, acetogenic, and methanogenic bacteria. Microbes adopt various pathways to evade from the unfavorable conditions in the anaerobic digester like competition between sulfate reducing bacteria (SRB) and methane forming bacteria for the same substrate. Methanosarcina are able to use both acetoclastic and hydrogenotrophic pathways for methane production. This review highlights the cellulosic microorganisms, structure of cellulose, inoculum to substrate ratio, and source of inoculum and its effect on methanogenesis. The molecular techniques such as DGGE (denaturing gradient gel electrophoresis) utilized for dynamic changes in microbial communities and FISH (fluorescent in situ hybridization) that deal with taxonomy and interaction and distribution of tropic groups used are also discussed. PMID:24701142

Inhibitory effects of soybean oligosaccharides and water-soluble soybean fibre on formation of putrefactive compounds from soy protein by gut microbiota.

PubMed

Nakata, Toru; Kyoui, Daisuke; Takahashi, Hajime; Kimura, Bon; Kuda, Takashi

2017-04-01

Soybeans are part of the traditional food consumed in Asia countries. In this study, we investigated inhibitory effects of soybean oligosaccharides and water-soluble soybean fibre (Soyafibe) on putrefactive compounds from soy protein by gut microbiota in rats. Caecal microbial fermentation products and microbiota in rats fed 20% soy protein (SP-1) and whole soybean flour (SFL: protein content was 20%) diets were determined. The caecal environment in rats fed 20% soy protein without dietary fibre (SP-2) or with 2% Soyafibe (SFB) was also determined. Compared to SP-1 and SP-2 group, low indole content with high lactic acid was shown in SFL and SFB group, respectively. Using the 16S rRNA genes polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing. Prevotella, Gram-negative anaerobic rods, were detected as dominant in both SFL and SFB groups. Our findings indicated that fermentable polysaccharides in soybeans have inhibitory effects on the formation of putrefactive compounds generated from soy protein by the microbiota. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of the anode feeding composition on the performance of a continuous-flow methane-producing microbial electrolysis cell.

PubMed

Zeppilli, Marco; Villano, Marianna; Aulenta, Federico; Lampis, Silvia; Vallini, Giovanni; Majone, Mauro

2015-05-01

A methane-producing microbial electrolysis cell (MEC) was continuously fed at the anode with a synthetic solution of soluble organic compounds simulating the composition of the soluble fraction of a municipal wastewater. The MEC performance was assessed at different anode potentials in terms of chemical oxygen demand (COD) removal efficiency, methane production, and energy efficiency. As a main result, about 72-80% of the removed substrate was converted into current at the anode, and about 84-86% of the current was converted into methane at the cathode. Moreover, even though both COD removed and methane production slightly decreased as the applied anode potential decreased, the energy efficiency (i.e., the energy recovered as methane with respect to the energy input into the system) increased from 54 to 63%. Denaturing gradient gel electrophoresis (DGGE) analyses revealed a high diversity in the anodic bacterial community with the presence of both fermentative (Proteiniphilum acetatigenes and Petrimonas sulphurifila) and aerobic (Rhodococcus qingshengii) microorganisms, whereas only two microorganisms (Methanobrevibacter arboriphilus and Methanosarcina mazei), both assignable to methanogens, were observed in the cathodic community.

Disconnect of microbial structure and function: enzyme activities and bacterial communities in nascent stream corridors.

PubMed

Frossard, Aline; Gerull, Linda; Mutz, Michael; Gessner, Mark O

2012-03-01

A fundamental issue in microbial and general ecology is the question to what extent environmental conditions dictate the structure of communities and the linkages with functional properties of ecosystems (that is, ecosystem function). We approached this question by taking advantage of environmental gradients established in soil and sediments of small stream corridors in a recently created, early successional catchment. Specifically, we determined spatial and temporal patterns of bacterial community structure and their linkages with potential microbial enzyme activities along the hydrological flow paths of the catchment. Soil and sediments were sampled in a total of 15 sites on four occasions spread throughout a year. Denaturing gradient gel electrophoresis (DGGE) was used to characterize bacterial communities, and substrate analogs linked to fluorescent molecules served to track 10 different enzymes as specific measures of ecosystem function. Potential enzyme activities varied little among sites, despite contrasting environmental conditions, especially in terms of water availability. Temporal changes, in contrast, were pronounced and remarkably variable among the enzymes tested. This suggests much greater importance of temporal dynamics than spatial heterogeneity in affecting specific ecosystem functions. Most strikingly, bacterial community structure revealed neither temporal nor spatial patterns. The resulting disconnect between bacterial community structure and potential enzyme activities indicates high functional redundancy within microbial communities even in the physically and biologically simplified stream corridors of early successional landscapes.

Changes in coral-associated microbial communities during a bleaching event.

PubMed

Bourne, David; Iida, Yuki; Uthicke, Sven; Smith-Keune, Carolyn

2008-04-01

Environmental stressors such as increased sea surface temperatures are well-known for contributing to coral bleaching; however, the effect of increased temperatures and subsequent bleaching on coral-associated microbial communities is poorly understood. Colonies of the hard coral Acropora millepora were tagged on a reef flat off Magnetic Island (Great Barrier Reef) and surveyed over 2.5 years, which included a severe bleaching event in January/February 2002. Daily average water temperatures exceeded the previous 10-year average by more than 1 degrees C for extended periods with field-based visual surveys recording all tagged colonies displaying signs of bleaching. During the bleaching period, direct counts of coral zooxanthellae densities decreased by approximately 64%, before recovery to pre-bleaching levels after the thermal stress event. A subset of three tagged coral colonies were sampled through the bleaching event and changes in the microbial community elucidated. Denaturing gradient gel electrophoresis (DGGE) analysis demonstrated conserved bacterial banding profiles between the three coral colonies, confirming previous studies highlighting specific microbial associations. As coral colonies bleached, the microbial community shifted and redundancy analysis (RDA) of DGGE banding patterns revealed a correlation of increasing temperature with the appearance of Vibrio-affiliated sequences. Interestingly, this shift to a Vibrio-dominated community commenced prior to visual signs of bleaching. Clone libraries hybridized with Vibrio-specific oligonucleotide probes confirmed an increase in the fraction of Vibrio-affiliated clones during the bleaching period. Post bleaching, the coral microbial associations again shifted, returning to a profile similar to the fingerprints prior to bleaching. This provided further evidence for corals selecting and shaping their microbial partners. For non-bleached samples, a close association with Spongiobacter-related sequences were revealed by both clone libraries and DGGE profiling. Despite Vibrio species being previously implicated in bleaching of specific coral species, it is unsure if the relative increase in retrieved Vibrio sequences is due to bacterial infection or an opportunistic response to compromised health and changing environmental parameters of the coral host. This study provides the first molecular-based study demonstrating changes in coral-associated bacterial assemblages during a bleaching event on a natural reef system.

[Metabolic pattern of pig hindgut bacteria on aromatic amino acids by an in vitro fermentation method].

PubMed

Ma, Meilei; He, Xiangyu; Zhu, Weiyun

2016-11-04

This experiment was conducted to study different metabolic patterns of pig hindgut bacteria on aromatic amino acids by an in vitro fermentation method. Ileum, cecum and colon chyme in Duroc, Landrace and Yorkshire goods hybridization pigs were taken as inoculum. The single aromatic amino acid concentration was kept 10 mmol/L in fermentation flask. Then the fermentation flask was incubated at 37℃ for 24 h. Gas production was measured at 4, 8, 12, 16 and 24 h, and samples of fermentation collected at 0 h and 24 h were used to measure ammonia nitrogen NH3-N and microbial crude protein (MCP). Denaturing gradient gel electrophoresis (DGGE) and real-time PCR were used to monitor and quantify the development of bacteria community in zymotic fluid.[ The concentrations of NH3-N and MCP were significantly affected by aromatic amino acids and intestinal segments (P<0.01). Intestinal segments also affected gas production (GP) significantly (P0.01). NH3-N, MCP and GP were affected by interaction of aromatic amino acids and intestinal segments. DGGE analysis showed bacteria of aromatic amino acids shared amount of bands together, especially similarity analysis of DGGE profile of Phe and Tyr in ileum, Tyr and Trp in colon were 87.9% and 80.5% separately. Shannon diversity indices analysis revealed that aromatic amino acids in cecum and colon varied significantly (P<0.05). Real-time PCR results showed that the quantity of total bacteria were affected by aromatic amino acids and intestinal segments significantly (P<0.05). The potential as proportion of different aromatic amino acids are different. Compared with Trp and Phe, the diversity of bacteria utilizing Tyr in cecum or colon is low; compared with Tyr and Trp, a large number of Phe participated in synthesizing bacteria.The fermentation pattern of specific aromatic amino acids in different intestinal segment was unique. Compared with ileum and cecum, much more aromatic amino acids participated in the synthesis of bacteria in colon.

Effect of biostimulation and bioaugmentation on degradation of polyurethane buried in soil.

PubMed

Cosgrove, L; McGeechan, P L; Handley, P S; Robson, G D

2010-02-01

This work investigated biostimulation and bioaugmentation as strategies for removing polyurethane (PU) waste in soil. Soil microcosms were biostimulated with the PU dispersion agent "Impranil" and/or yeast extract or were bioaugmented with PU-degrading fungi, and the degradation of subsequently buried PU was determined. Fungal communities in the soil and colonizing buried PU were enumerated on solid media and were analyzed using denaturing gradient gel electrophoresis (DGGE). Biostimulation with yeast extract alone or in conjunction with Impranil increased PU degradation 62% compared to the degradation in untreated control soil and was associated with a 45% increase in putative PU degraders colonizing PU. Specific fungi were enriched in soil following biostimulation; however, few of these fungi colonized the surface of buried PU. Fungi used for soil bioaugmentation were cultivated on the surface of sterile wheat to form a mycelium-rich inoculum. Wheat, when added alone to soil, increased PU degradation by 28%, suggesting that wheat biomass had a biostimulating effect. Addition of wheat colonized with Nectria haematococca, Penicillium viridicatum, Penicillium ochrochloron, or an unidentified Mucormycotina sp. increased PU degradation a further 30 to 70%, suggesting that biostimulation and bioaugmentation were operating in concert to enhance PU degradation. Interestingly, few of the inoculated fungi could be detected by DGGE in the soil or on the surface of the PU 4 weeks after inoculation. Bioaugmentation did, however, increase the numbers of indigenous PU-degrading fungi and caused an inoculum-dependent change in the composition of the native fungal populations, which may explain the increased degradation observed. These results demonstrate that both biostimulation and bioaugmentation may be viable tools for the remediation of environments contaminated with polyurethane waste.

Endophytic bacterial diversity in the phyllosphere of Amazon Paullinia cupana associated with asymptomatic and symptomatic anthracnose.

PubMed

Bogas, Andréa Cristina; Ferreira, Almir José; Araújo, Welington Luiz; Astolfi-Filho, Spartaco; Kitajima, Elliot Watanabe; Lacava, Paulo Teixeira; Azevedo, João Lúcio

2015-01-01

Endophytes colonize an ecological niche similar to that of phytopathogens, which make them candidate for disease suppression. Anthracnose is a disease caused by Colletotrichum spp., a phytopathogen that can infect guarana (Paullinia cupana), an important commercial crop in the Brazilian Amazon. We investigated the diversity of endophytic bacteria inhabiting the phyllosphere of asymptomatic and symptomatic anthracnose guarana plants. The PCR-denaturation gradient gel electrophoresis (PCR-DGGE) fingerprints revealed differences in the structure of the evaluated communities. Detailed analysis of endophytic bacteria composition using culture-dependent and 16S rRNA clone libraries revealed the presence of Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Acidobacteria phyla. Firmicutes comprised the majority of isolates in asymptomatic plants (2.40E(-4)). However, cloning and sequencing of 16S rRNA revealed differences at the genus level for Neisseria (1.4E(-4)), Haemophilus (2.1E(-3)) and Arsenophonus (3.6E(-5)) in asymptomatic plants, Aquicella (3.5E(-3)) in symptomatic anthracnose plants, and Pseudomonas (1.1E(-3)), which was mainly identified in asymptomatic plants. In cross-comparisons of the endophytic bacterial communities as a whole, symptomatic anthracnose plants contained higher diversity, as reflected in the Shannon-Weaver and Simpson indices estimation (P < 0.05). Similarly, comparisons using LIBSHUFF and heatmap analysis for the relative abundance of operational taxonomic units (OTUs) showed differences between endophytic bacterial communities. These data are in agreement with the NMSD and ANOSIM analysis of DGGE profiles. Our results suggest that anthracnose can restructure endophytic bacterial communities by selecting certain strains in the phyllosphere of P. cupana. The understanding of these interactions is important for the development of strategies of biocontrol for Colletotrichum.

Characterization of Intestinal Bacteria in Wild and Domesticated Adult Black Tiger Shrimp (Penaeus monodon)

PubMed Central

Rungrassamee, Wanilada; Klanchui, Amornpan; Maibunkaew, Sawarot; Chaiyapechara, Sage; Jiravanichpaisal, Pikul; Karoonuthaisiri, Nitsara

2014-01-01

The black tiger shrimp (Penaeus monodon) is a marine crustacean of economic importance in the world market. To ensure sustainability of the shrimp industry, production capacity and disease outbreak prevention must be improved. Understanding healthy microbial balance inside the shrimp intestine can provide an initial step toward better farming practice and probiotic applications. In this study, we employed a barcode pyrosequencing analysis of V3-4 regions of 16S rRNA genes to examine intestinal bacteria communities in wild-caught and domesticated P. monodon broodstock. Shrimp faeces were removed from intestines prior to further analysis in attempt to identify mucosal bacterial population. Five phyla, Actinobacteria, Fusobacteria, Proteobacteria, Firmicutes and Bacteroidetes, were found in all shrimp from both wild and domesticated environments. The operational taxonomic unit (OTU) was assigned at 97% sequence identity, and our pyrosequencing results identified 18 OTUs commonly found in both groups. Sequences of the shared OTUs were similar to bacteria in three phyla, namely i) Proteobacteria (Vibrio, Photobacterium, Novosphingobium, Pseudomonas, Sphingomonas and Undibacterium), ii) Firmicutes (Fusibacter), and iii) Bacteroidetes (Cloacibacterium). The shared bacterial members in P. monodon from two different habitats provide evidence that the internal environments within the host shrimp also exerts selective pressure on bacterial members. Intestinal bacterial profiles were compared using denaturing gradient gel electrophoresis (DGGE). The sequences from DGGE bands were similar to those of Vibrio and Photobacterium in all shrimp, consistent with pyrosequencing results. This work provides the first comprehensive report on bacterial populations in the intestine of adult black tiger shrimp and reveals some similar bacterial members between the intestine of wild-caught and domesticated shrimp. PMID:24618668

Community analysis of a full-scale anaerobic bioreactor treating paper mill wastewater.

PubMed

Roest, Kees; Heilig, Hans G H J; Smidt, Hauke; de Vos, Willem M; Stams, Alfons J M; Akkermans, Antoon D L

2005-03-01

To get insight into the microbial community of an Upflow Anaerobic Sludge Blanket reactor treating paper mill wastewater, conventional microbiological methods were combined with 16S rRNA gene analyses. Particular attention was paid to microorganisms able to degrade propionate or butyrate in the presence or absence of sulphate. Serial enrichment dilutions allowed estimating the number of microorganisms per ml sludge that could use butyrate with or without sulphate (10(5)), propionate without sulphate (10(6)), or propionate and sulphate (10(8)). Quantitative RNA dot-blot hybridisation indicated that Archaea were two-times more abundant in the microbial community of anaerobic sludge than Bacteria. The microbial community composition was further characterised by 16S rRNA-gene-targeted Denaturing Gradient Gel Electrophoresis (DGGE) fingerprinting, and via cloning and sequencing of dominant amplicons from the bacterial and archaeal patterns. Most of the nearly full length (approximately 1.45 kb) bacterial 16S rRNA gene sequences showed less than 97% similarity to sequences present in public databases, in contrast to the archaeal clones (approximately. 1.3 kb) that were highly similar to known sequences. While Methanosaeta was found as the most abundant genus, also Crenarchaeote-relatives were identified. The microbial community was relatively stable over a period of 3 years (samples taken in July 1999, May 2001, March 2002 and June 2002) as indicated by the high similarity index calculated from DGGE profiles (81.9+/-2.7% for Bacteria and 75.1+/-3.1% for Archaea). 16S rRNA gene sequence analysis indicated the presence of unknown and yet uncultured microorganisms, but also showed that known sulphate-reducing bacteria and syntrophic fatty acid-oxidising microorganisms dominated the enrichments.

Effects of cadmium and resource quality on freshwater detritus processing chains: a microcosm approach with two insect species.

PubMed

Campos, Diana; Alves, Artur; Lemos, Marco F L; Correia, António; Soares, Amadeu M V M; Pestana, João L T

2014-07-01

Detritus processing is vital for freshwater ecosystems that depend on the leaf litter from riparian vegetation and is mediated by microorganisms and aquatic invertebrates. Shredder invertebrates transform coarse particulate organic matter into fine particulate organic matter used as food by collector species. Direct and indirect effects of contaminants can impair detritus processing and thus affect the functioning of these ecosystems. Here, we assessed the combined effects of a toxic metal (cadmium) and resource quality (leaf species) on detritus processing and shredder-collector interactions. We considered two types of leaves, alder and eucalyptus that were microbially conditioned under different Cd concentrations in the laboratory. The microbial communities present on leaves were analyzed by Denaturing Gradient Gel Electrophoresis (DGGE), and we also measured microbial respiration rates. Sericostoma vittatum (a caddisfly shredder) and Chironomus riparius (a midge collector) were also exposed to Cd and allowed to consume the corresponding alder or eucalyptus leaves. We evaluated C. riparius growth and leaf mass loss in multispecies microcosms. Cadmium exposure affected leaf conditioning and fungal diversity on both leaf species, as assessed by DGGE. Cadmium exposure also affected the mass loss of alder leaves by reductions in detritivore feeding, and impaired C. riparius growth. Chironomus riparius consumed alder leaf discs in the absence of shredders, but S. vittatum appear to promote C. riparius growth in treatments containing eucalyptus. These results show that indirect effects of contaminants along detritus-processing chains can occur through effects on shredder-collector interactions such as facilitation but they also depend on the nutritional quality of detritus and on sensitivity and feeding plasticity of detritivore species.

Rhizosphere Microbial Community Structure in Relation to Root Location and Plant Iron Nutritional Status

PubMed Central

Yang, Ching-Hong; Crowley, David E.

2000-01-01

Root exudate composition and quantity vary in relation to plant nutritional status, but the impact of the differences on rhizosphere microbial communities is not known. To examine this question, we performed an experiment with barley (Hordeum vulgare) plants under iron-limiting and iron-sufficient growth conditions. Plants were grown in an iron-limiting soil in root box microcosms. One-half of the plants were treated with foliar iron every day to inhibit phytosiderophore production and to alter root exudate composition. After 30 days, the bacterial communities associated with different root zones, including the primary root tips, nonelongating secondary root tips, sites of lateral root emergence, and older roots distal from the tip, were characterized by using 16S ribosomal DNA (rDNA) fingerprints generated by PCR-denaturing gradient gel electrophoresis (DGGE). Our results showed that the microbial communities associated with the different root locations produced many common 16S rDNA bands but that the communities could be distinguished by using correspondence analysis. Approximately 40% of the variation between communities could be attributed to plant iron nutritional status. A sequence analysis of clones generated from a single 16S rDNA band obtained at all of the root locations revealed that there were taxonomically different species in the same band, suggesting that the resolving power of DGGE for characterization of community structure at the species level is limited. Our results suggest that the bacterial communities in the rhizosphere are substantially different in different root zones and that a rhizosphere community may be altered by changes in root exudate composition caused by changes in plant iron nutritional status. PMID:10618246

[Predominant strains of polycyclic aromatic hydrocarbon-degrading consortia from deep sea of the Middle Atlantic Ridge].

PubMed

Cui, Zhisong; Shao, Zongze

2009-07-01

In order to identify the predominant strains of polycyclic aromatic hydrocarbon (PAH)-degrading consortia harboring in sea water and surface sediment collected from deep sea of the Middle Atlantic Ridge. We employed enrichment method and spread-plate method to isolate cultivable bacteria and PAHs degraders from deep sea samples. Phylogenetic analysis was conducted by 16S rRNA gene sequencing of the bacteria. Then we analyzed the dominant bacteria in the PAHs-degrading consortia by denaturing gradient gel electrophoresis (DGGE) combined with DNA sequencing. Altogether 16 cultivable bacteria were obtained, including one PAHs degrader Novosphingobium sp. 4D. Phylogenetic analysis showed that strains closely related to Alcanivorax dieselolei NO1A (5/16) and Tistrella mobilis TISTR 1108T (5/16) constituted two biggest groups among the cultivable bacteria. DGGE analysis showed that strain 4L (also 4M and 4N, Alcanivorax dieselolei NO1A, 99.21%), 4D (Novosphingobium pentaromativorans US6-1(T), 97.07%) and 4B (also 4E, 4H and 4K, Tistrella mobilis TISTR 1108T, > 99%) dominated the consortium MC2D. While in consortium MC3CO, the predominant strains were strain 5C (also 5H, Alcanivorax dieselolei NO1A, > 99%), uncultivable strain represented by band 5-8 (Novosphingobium aromaticivorans DSM 12444T, 99.41%), 5J (Tistrella mobilis TISTR 1108T, 99.52%) and 5F (also 5G, Thalassospira lucentensis DSM 14000T, < 97%). We found that strains of genus Alcanivorax, Novosphingobium, Tistrella and Thalassospira were predominant bacteria of PAHs-degrading consortia in sea water and surface sediment of Middle Atlantic Ridge deep sea, with Novosphingobium spp. as their main PAHs degraders.

Response of microbial activities and diversity to PAHs contamination at coal tar contaminated land

NASA Astrophysics Data System (ADS)

Zhao, Xiaohui; Sun, Yujiao; Ding, Aizhong; Zhang, Dan; Zhang, Dayi

2015-04-01

Coal tar is one of the most hazardous and concerned organic pollutants and the main hazards are polycyclic aromatic hydrocarbons (PAHs). The indigenous microorganisms in soils are capable to degrade PAHs, with essential roles in biochemical process for PAHs natural attenuation. This study investigated 48 soil samples (from 8 depths of 6 boreholes) in Beijing coking and chemistry plant (China) and revealed the correlation between PAHs contamination, soil enzyme activities and microbial community structure, by 16S rRNA denaturing gradient gel electrophoresis (DGGE). At the site, the key contaminants were identified as naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene and anthracene, and the total PAHs concentration ranged from 0.1 to 923.9 mg/kg dry soil. The total PAHs contamination level was positively correlated (p<0.05) with the bacteria count (0.9×107-14.2×107 CFU/mL), catalase activities (0.554-6.230 mL 0.02 M KMnO4/g•h) and dehydrogenase activities (1.9-30.4 TF μg/g•h soil), showing the significant response of microbial population and degrading functions to the organic contamination in soils. The PAHs contamination stimulated the PAHs degrading microbes and promoted their biochemical roles in situ. The positive relationship between bacteria count and dehydrogenase activities (p<0.05) suggested the dominancy of PAHs degrading bacteria in the microbial community. More interestingly, the microbial community deterioration was uncovered via the decline of microbial biodiversity (richness from 16S rRNA DGGE) against total PAHs concentration (p<0.05). Our research described the spatial profiles of PAHs contamination and soil microbial functions at the PAHs heavily contaminated sites, offering deeper understanding on the roles of indigenous microbial community in natural attenuation process.

Histamine development and bacterial diversity in microbially-challenged tonggol (Thunnus tonggol) under temperature abuse during canning manufacture.

PubMed

Hongpattarakere, Tipparat; Buntin, Nirunya; Nuylert, Aem

2016-01-01

Histamine formation and bacteriological changes caused by temperature abuse commonly occurring in the manufacturing process of standard canned tuna was assessed in microbiologically challenged tonggol (Thunnus tonggol). The in situ challenge was performed by water-soaking at 26-28 °C for 7 h to ensure the multiplication and active phase of fish microflora. Right after pre-cooking to back-bone temperature (BBT) of 50-52 °C, histamine dropped to 5.17 ± 2.71 ppm, and slowly reached 6.84 ± 1.69 ppm at 16 h abuse. On the contrary, histamine was reduced to 2.87 ± 1.23 ppm and eventually reached 5.01 ± 1.32 ppm at 24 h abuse in the pre-cooked fish previously frozen. The numbers of total aerobic bacteria, Enterobactericeae, psychrotroph, histamine forming bacteria (HFB) and diversity of fish microflora were revealed by cultural and nested PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) techniques. Interestingly, frozen storage effectively halted histamine formation in raw fish throughout 16 h abuse despite the presence of HFB. These included the prolific strains of Morganella morganii, Proteus penneri, Proteus mirabilin, Citrobacter spp. The nested PCR-DGGE profile confirmed the presence of M. morganii and Citrobacter spp. in raw fish. These prolific strains were hardly observed in the precooked fish previously frozen. Frozen storage did not only promote even histamine distribution throughout fish muscle but also enhanced histamine loss during thawing and pre-cooking. Therefore, pre-cooking and frozen storage were proven to be the effective combined hurdles not only to reduce but also prolong histamine formation of the challenged toggol throughout 24 h of temperature abuse during canning process.

Biodiversity within hot spring microbial mat communities: molecular monitoring of enrichment cultures

NASA Technical Reports Server (NTRS)

Ward, D. M.; Santegoeds, C. M.; Nold, S. C.; Ramsing, N. B.; Ferris, M. J.; Bateson, M. M.

1997-01-01

We have begun to examine the basis for incongruence between hot spring microbial mat populations detected by cultivation or by 16S rRNA methods. We used denaturing gradient gel electrophoresis (DGGE) to monitor enrichments and isolates plated therefrom. At near extincting inoculum dilutions we observed Chloroflexus-like and cyanobacterial populations whose 16S rRNA sequences have been detected in the 'New Pit' Spring Chloroflexus mat and the Octopus Spring cyanobacterial mat. Cyanobacterial populations enriched from 44 to 54 degrees C and 56 to 63 degrees C samples at near habitat temperatures were similar to those previously detected in mat samples of comparable temperatures. However, a lower temperature enrichment from the higher temperature sample selected for the populations found in the lower temperature sample. Three Thermus populations detected by both DGGE and isolation exemplify even more how enrichment may bias our view of community structure. The most abundant population was adapted to the habitat temperature (50 degrees C), while populations adapted to 65 degrees C and 70 degrees C were 10(2)- and 10(4)-fold less abundant, respectively. However, enrichment at 70 degrees C favored the least abundant strain. Inoculum dilution and incubation at the habitat temperature favored the more numerically relevant populations. We enriched many other aerobic chemoorganotrophic populations at various inoculum dilutions and substrate concentrations, most of whose 16S rRNA sequences have not been detected in mats. A common feature of numerically relevant cyanobacterial, Chloroflexus-like and aerobic chemorganotrophic populations, is that they grow poorly and resist cultivation on solidified medium, suggesting plating bias, and that the medium composition and incubation conditions may not reflect the natural microenvironments these populations inhabit.

Metabolic markers and microecological characteristics of tongue coating in patients with chronic gastritis

PubMed Central

2013-01-01

Background In Traditional Chinese Medicine (TCM), tongue diagnosis has been an important diagnostic method for the last 3000 years. Tongue diagnosis is a non-invasive, simple and valuable diagnostic tool. TCM treats the tongue coating on a very sensitive scale that reflects physiological and pathological changes in the organs, especially the spleen and stomach. Tongue coating can diagnose disease severity and determine the TCM syndrome (“Zheng” in Chinese). The biological bases of different tongue coating appearances are still poorly understood and lack systematic investigation at the molecular level. Methods Tongue coating samples were collected from 70 chronic gastritis patients and 20 normal controls. 16S rRNA denatured gradient gel electrophoresis (16S rRNA–DGGE) and liquid chromatography and mass spectrometry (LC–MS) were designed to profile tongue coatings. The statistical techniques used were principal component analysis and partial least squares–discriminate analysis. Results Ten potential metabolites or markers were found in chronic gastritis patients, including UDP-D-galactose, 3-ketolactose, and vitamin D2, based on LC–MS. Eight significantly different strips were observed in samples from chronic gastritis patients based on 16S rRNA–DGGE. Two strips, Strips 8 and 10, were selected for gene sequencing. Strip 10 sequencing showed a 100% similarity to Rothia mucilaginosa. Strip 8 sequencing showed a 96.2% similarity to Moraxella catarrhalis. Conclusions Changes in glucose metabolism could possibly form the basis of tongue coating conformation in chronic gastritis patients. The study revealed important connections between metabolic components, microecological components and tongue coating in chronic gastritis patients. Compared with other diagnostic regimens, such as blood tests or tissue biopsies, tongue coating is more amenable to, and more convenient for, both patients and doctors. PMID:24041039

Microbial sulfate reduction and metal attenuation in pH 4 acid mine water

USGS Publications Warehouse

Church, C.D.; Wilkin, R.T.; Alpers, Charles N.; Rye, R.O.; Blaine, R.B.

2007-01-01

Sediments recovered from the flooded mine workings of the Penn Mine, a Cu-Zn mine abandoned since the early 1960s, were cultured for anaerobic bacteria over a range of pH (4.0 to 7.5). The molecular biology of sediments and cultures was studied to determine whether sulfate-reducing bacteria (SRB) were active in moderately acidic conditions present in the underground mine workings. Here we document multiple, independent analyses and show evidence that sulfate reduction and associated metal attenuation are occurring in the pH-4 mine environment. Water-chemistry analyses of the mine water reveal: (1) preferential complexation and precipitation by H2S of Cu and Cd, relative to Zn; (2) stable isotope ratios of 34S/32S and 18O/16O in dissolved SO4 that are 2-3 ??? heavier in the mine water, relative to those in surface waters; (3) reduction/oxidation conditions and dissolved gas concentrations consistent with conditions to support anaerobic processes such as sulfate reduction. Scanning electron microscope (SEM) analyses of sediment show 1.5-micrometer, spherical ZnS precipitates. Phospholipid fatty acid (PLFA) and denaturing gradient gel electrophoresis (DGGE) analyses of Penn Mine sediment show a high biomass level with a moderately diverse community structure composed primarily of iron- and sulfate-reducing bacteria. Cultures of sediment from the mine produced dissolved sulfide at pH values near 7 and near 4, forming precipitates of either iron sulfide or elemental sulfur. DGGE coupled with sequence and phylogenetic analysis of 16S rDNA gene segments showed populations of Desulfosporosinus and Desulfitobacterium in Penn Mine sediment and laboratory cultures. ?? 2007 Church et al; licensee BioMed Central Ltd.

Bacterial Community Structure and Physiological State within an Industrial Phenol Bioremediation System

PubMed Central

Whiteley, Andrew S.; Bailey, Mark J.

2000-01-01

The structure of bacterial populations in specific compartments of an operational industrial phenol remediation system was assessed to examine bacterial community diversity, distribution, and physiological state with respect to the remediation of phenolic polluted wastewater. Rapid community fingerprinting by PCR-based denaturing gradient gel electrophoresis (DGGE) of 16S rDNA indicated highly structured bacterial communities residing in all nine compartments of the treatment plant and not exclusively within the Vitox biological reactor. Whole-cell targeting by fluorescent in situ hybridization with specific oligonucleotides (directed to the α, β and γ subclasses of the class Proteobacteria [α-, β-, and γ-Proteobacteria, respectively], the Cytophaga-Flavobacterium group, and the Pseudomonas group) tended to mirror gross changes in bacterial community composition when compared with DGGE community fingerprinting. At the whole-cell level, the treatment compartments were numerically dominated by cells assigned to the Cytophaga-Flavobacterium group and to the γ-Proteobacteria. The α subclass Proteobacteria were of low relative abundance throughout the treatment system whilst the β subclass of the Proteobacteria exhibited local dominance in several of the processing compartments. Quantitative image analyses of cellular fluorescence was used as an indicator of physiological state within the populations probed with rDNA. For cells hybridized with EUB338, the mean fluorescence per cell decreased with increasing phenolic concentration, indicating the strong influence of the primary pollutant upon cellular rRNA content. The γ subclass of the Proteobacteria had a ribosome content which correlated positively with total phenolics and thiocyanate. While members of the Cytophaga-Flavobacterium group were numerically dominant in the processing system, their abundance and ribosome content data for individual populations did not correlate with any of the measured chemical parameters. The potential importance of the γ-Proteobacteria and the Cytophaga-Flavobacteria during this bioremediation process was highlighted. PMID:10831417

Different fecal microbiotas and volatile organic compounds in treated and untreated children with celiac disease.

PubMed

Di Cagno, Raffaella; Rizzello, Carlo G; Gagliardi, Francesca; Ricciuti, Patrizia; Ndagijimana, Maurice; Francavilla, Ruggiero; Guerzoni, M Elisabetta; Crecchio, Carmine; Gobbetti, Marco; De Angelis, Maria

2009-06-01

This study aimed at investigating the fecal microbiotas of children with celiac disease (CD) before (U-CD) and after (T-CD) they were fed a gluten-free diet and of healthy children (HC). Brothers or sisters of T-CD were enrolled as HC. Each group consisted of seven children. PCR-denaturing gradient gel electrophoresis (DGGE) analysis with V3 universal primers revealed a unique profile for each fecal sample. PCR-DGGE analysis with group- or genus-specific 16S rRNA gene primers showed that the Lactobacillus community of U-CD changed significantly, while the diversity of the Lactobacillus community of T-CD was quite comparable to that of HC. Compared to HC, the ratio of cultivable lactic acid bacteria and Bifidobacterium to Bacteroides and enterobacteria was lower in T-CD and even lower in U-CD. The percentages of strains identified as lactobacilli differed as follows: HC (ca. 38%) > T-CD (ca. 17%) > U-CD (ca. 10%). Lactobacillus brevis, Lactobacillus rossiae, and Lactobacillus pentosus were identified only in fecal samples from T-CD and HC. Lactobacillus fermentum, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus gasseri were identified only in several fecal samples from HC. Compared to HC, the composition of Bifidobacterium species of T-CD varied, and it varied even more for U-CD. Forty-seven volatile organic compounds (VOCs) belonging to different chemical classes were identified using gas-chromatography mass spectrometry-solid-phase microextraction analysis. The median concentrations varied markedly for HC, T-CD, and U-CD. Overall, the r(2) values for VOC data for brothers and sisters were equal to or lower than those for unrelated HC and T-CD. This study shows the effect of CD pathology on the fecal microbiotas of children.

Characterization of a Highly Enriched Microbial Consortium Reductively Dechlorinating 2,3-Dichlorophenol and 2,4,6-Trichlorophenol and the Corresponding cprA Genes from River Sediment.

PubMed

El-Sayed, Wael S

2016-08-26

Anaerobic reductive dechlorination of 2,3-dichlorophenol (2,3DCP) and 2,4,6-trichlorophenol (2,4,6TCP) was investigated in microcosms from River Nile sediment. A stable sediment-free anaerobic microbial consortium reductively dechlorinating 2,3DCP and 2,4,6TCP was established. Defined sediment-free cultures showing stable dechlorination were restricted to ortho chlorine when enriched with hydrogen as the electron donor, acetate as the carbon source, and either 2,3-DCP or 2,4,6-TCP as electron acceptors. When acetate, formate, or pyruvate were used as electron donors, dechlorination activity was lost. Only lactate can replace dihydrogen as an electron donor. However, the dechlorination potential was decreased after successive transfers. To reveal chlororespiring species, the microbial community structure of chlorophenol-reductive dechlorinating enrichment cultures was analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Eight dominant bacteria were detected in the dechlorinating microcosms including members of the genera Citrobacter, Geobacter, Pseudomonas, Desulfitobacterium, Desulfovibrio and Clostridium. Highly enriched dechlorinating cultures were dominated by four bacterial species belonging to the genera Pseudomonas, Desulfitobacterium, and Clostridium. Desulfitobacterium represented the major fraction in DGGE profiles indicating its importance in dechlorination activity, which was further confirmed by its absence resulting in complete loss of dechlorination. Reductive dechlorination was confirmed by the stoichiometric dechlorination of 2,3DCP and 2,4,6TCP to metabolites with less chloride groups and by the detection of chlorophenol RD cprA gene fragments in dechlorinating cultures. PCR amplified cprA gene fragments were cloned and sequenced and found to cluster with the cprA/pceA type genes of Dehalobacter restrictus.

Relationships between and formation dynamics of the microbiota of consumers, producers, and the environment in an abalone aquatic system

PubMed Central

Zhao, Wang; Liu, Guang-Feng; Wang, Jiang-Yong

2017-01-01

An ecosystem is a community comprising living and nonliving components of the environment. Microbes are ubiquitous elements in each of these components. The dynamics of microbiota formation in an ecosystem is important to elucidate, because how the different components of a system exchange microbes, and how the microbes control ecological processes remain unresolved. In this study, an abalone, Haliotis diversicolor, seed-nursing pond was used as a model system. We first examined changes in bacterial communities during the seedling cultivation of this herbivorous juvenile aquatic invertebrate animal. Denaturing gradient gel electrophoresis (DGGE) and pyrosequencing were used to analyze bacterial community dynamics and spatio-temporal interactions of different system components: consumers (abalone), producers (algae or a substrate), and the environment (water). DGGE fingerprints revealed that the developmental stages of abalone influences bacterial communities of both the abalone and substrate. Although the communities in water fluctuated daily, they could be divided into two clusters that coincided with abalone stages, reflecting the transition from larva to juvenile at around day 21. Pyrosequencing showed that the microbiota in the abalone and substrate had more operational taxonomic units in common than that of either with water. The Bray-Curtis similarity index was used to quantify the formation dynamics of microbiota among the various components of the system. The bacterial communities in producers and consumers showed similar changes. These communities were unstable at the beginning and then slowly stabilized over time. The environmental bacterial community was more stable than the bacterial communities in consumers and producers, and may have been the basis for stability in the system. Our research provides insights into the dynamics of microbiota formation in various biotic elements of a system that will contribute to predictive systems modeling. PMID:28787009

Bio-protective potential of lactic acid bacteria: Effect of Lactobacillus sakei and Lactobacillus curvatus on changes of the microbial community in vacuum-packaged chilled beef.

PubMed

Zhang, Yimin; Zhu, Lixian; Dong, Pengcheng; Liang, Rongrong; Mao, Yanwei; Qiu, Shubing; Luo, Xin

2018-04-01

This study was to determine the bacterial diversity and monitor the community dynamic changes during storage of vacuum-packaged sliced raw beef as affected by Lactobacillus sakei and Lactobacillus curvatus . L. sakei and L. curvatus were separately incubated in vacuumed-packaged raw beef as bio-protective cultures to inhibit the naturally contaminating microbial load. Dynamic changes of the microbial diversity of inoculated or non-inoculated (control) samples were monitored at 4°C for 0 to 38 days, using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The DGGE profiles of DNA directly extracted from non-inoculated control samples highlighted the order of appearance of spoilage bacteria during storage, showing that Enterbacteriaceae and Pseudomonas fragi emerged early, then Brochothrix thermosphacta shared the dominant position, and finally, Pseudomonas putida showed up became predominant. Compared with control, the inoculation of either L. sakei or L. curvatus significantly lowered the complexity of microbial diversity and inhibited the growth of spoilage bacteria (p<0.05). Interestingly, we also found that the dominant position of L. curvatus was replaced by indigenous L. sakei after 13 d for L. curvatus -inoculated samples. Plate counts on selective agars further showed that inoculation with L. sakei or L. curvatus obviously reduced the viable counts of Enterbacteraceae , Pseudomonas spp. and B. thermosphacta during later storage (p< 0.05), with L. sakei exerting greater inhibitory effect. Inoculation with both bio-protective cultures also significantly decreased the total volatile basic nitrogen values of stored samples (p<0.05). Taken together, the results proved the benefits of inoculation with lactic acid bacteria especially L. sakei as a potential way to inhibit growth of spoilage-related bacteria and improve the shelf life of vacuum-packaged raw beef.

Synergetic effect of pH and biochemical components on bacterial diversity during mesophilic anaerobic fermentation of biomass-origin waste.

PubMed

Lü, F; Shao, L M; Bru, V; Godon, J J; He, P J

2009-02-01

To investigate the synergetic effect of pH and biochemical components on bacterial community structure during mesophilic anaerobic degradation of solid wastes with different origins, and under acidic or neutral conditions. The bacterial community in 16 samples of solid wastes with different biochemical compositions and origins was evaluated during mesophilic anaerobic degradation at acidic and neutral pH. Denaturing gradient gel electrophoresis (DGGE) and single-strand conformation polymorphism (SSCP) were used to compare the communities. Multivariate analysis of the DGGE and SSCP results revealed that most of the dominant microbes were dependent on the content of easily degradable carbohydrates in the samples. Furthermore, the dominant microbes were divided into two types, those that preferred an acid environment and those that preferred a neutral environment. A shift in pH was found to change their preference for medium substrates. Although most of the substrates with similar origin and biochemical composition had similar microbial diversity during fermentation, some microbes were found only in substrates with specific origins. For example, two microbes were only found in substrate that contained lignocellulose and animal protein without starch. These microbes were related to micro-organisms that are found in swine manure, as well as in other intestinal or oral niches. In addition, the distribution of fermentation products was less sensitive to the changes in pH and biochemical components than the microbial community. Bacterial diversity during anaerobic degradation of organic wastes was affected by both pH and biochemical components; however, pH exerted a greater effect. The results of this study reveal that control of pH may be an effective method to produce a stable bacterial community and relatively similar product distribution during anaerobic digestion of waste, regardless of variation in the waste feedstocks.

Response of microbial community and catabolic genes to simulated petroleum hydrocarbon spills in soils/sediments from different geographic locations.

PubMed

Liu, Q; Tang, J; Liu, X; Song, B; Zhen, M; Ashbolt, N J

2017-10-01

Study the response of microbial communities and selected petroleum hydrocarbon (PH)-degrading genes on simulated PH spills in soils/sediments from different geographic locations. A microcosm experiment was conducted by spiking mixtures of petroleum hydrocarbons (PHs) to soils/sediments collected from four different regions of China, including the Dagang Oilfield (DG), Sand of Bohai Sea (SS), Northeast China (NE) and Xiamen (XM). Changes in bacterial community and the abundance of PH-degrading genes (alkB, nah and phe) were analysed by denaturing gradient electrophoresis (DGGE) and qPCR, respectively. Degradation of alkanes and PAHs in SS and NE materials were greater (P < 0·05) than those in DG and XM. Clay content was negatively correlated with the degradation of total alkanes by 112 days and PAHs by 56 days, while total organic carbon content was negatively correlated with initial degradation of total alkanes as well as PAHs. Abundances of alkB, nah and phe genes increased 10- to 100-fold and varied by soil type over the incubation period. DGGE fingerprints identified the dominance of α-, β- and γ-Proteobacteria (Gram -ve) and Actinobacteria (Gram +ve) bacteria associated with degradation of PHs in the materials studied. The geographic divergence resulting from the heterogeneity of physicochemical properties of soils/sediments appeared to influence the abundance of metabolic genes and community structure of microbes capable of degrading PHs. When developing practical in-situ bioremediation approaches for PHs contamination of soils/sediment, appropriate microbial community structures and the abundance of PH-degrading genes appear to be influenced by geographic location. © 2017 The Society for Applied Microbiology.

Molecular detection and ecological significance of the cyanobacterial genera Geitlerinema and Leptolyngbya in black band disease of corals.

PubMed

Myers, Jamie L; Sekar, Raju; Richardson, Laurie L

2007-08-01

Black band disease (BBD) is a pathogenic, sulfide-rich microbial mat dominated by filamentous cyanobacteria that infect corals worldwide. We isolated cyanobacteria from BBD into culture, confirmed their presence in the BBD community by using denaturing gradient gel electrophoresis (DGGE), and demonstrated their ecological significance in terms of physiological sulfide tolerance and photosynthesis-versus-irradiance values. Twenty-nine BBD samples were collected from nine host coral species, four of which have not previously been investigated, from reefs of the Florida Keys, the Bahamas, St. Croix, and the Philippines. From these samples, seven cyanobacteria were isolated into culture. Cloning and sequencing of the 16S rRNA gene using universal primers indicated that four isolates were related to the genus Geitlerinema and three to the genus Leptolyngbya. DGGE results, obtained using Cyanobacteria-specific 16S rRNA primers, revealed that the most common BBD cyanobacterial sequence, detected in 26 BBD field samples, was related to that of an Oscillatoria sp. The next most common sequence, 99% similar to that of the Geitlerinema BBD isolate, was present in three samples. One Leptolyngbya- and one Phormidium-related sequence were also found. Laboratory experiments using isolates of BBD Geitlerinema and Leptolyngbya revealed that they could carry out sulfide-resistant oxygenic photosynthesis, a relatively rare characteristic among cyanobacteria, and that they are adapted to the sulfide-rich, low-light BBD environment. The presence of the cyanotoxin microcystin in these cultures and in BBD suggests a role in BBD pathogenicity. Our results confirm the presence of Geitlerinema in the BBD microbial community and its ecological significance, which have been challenged, and provide evidence of a second ecologically significant BBD cyanobacterium, Leptolyngbya.

Molecular Detection and Ecological Significance of the Cyanobacterial Genera Geitlerinema and Leptolyngbya in Black Band Disease of Corals▿ †

PubMed Central

Myers, Jamie L.; Sekar, Raju; Richardson, Laurie L.

2007-01-01

Black band disease (BBD) is a pathogenic, sulfide-rich microbial mat dominated by filamentous cyanobacteria that infect corals worldwide. We isolated cyanobacteria from BBD into culture, confirmed their presence in the BBD community by using denaturing gradient gel electrophoresis (DGGE), and demonstrated their ecological significance in terms of physiological sulfide tolerance and photosynthesis-versus-irradiance values. Twenty-nine BBD samples were collected from nine host coral species, four of which have not previously been investigated, from reefs of the Florida Keys, the Bahamas, St. Croix, and the Philippines. From these samples, seven cyanobacteria were isolated into culture. Cloning and sequencing of the 16S rRNA gene using universal primers indicated that four isolates were related to the genus Geitlerinema and three to the genus Leptolyngbya. DGGE results, obtained using Cyanobacteria-specific 16S rRNA primers, revealed that the most common BBD cyanobacterial sequence, detected in 26 BBD field samples, was related to that of an Oscillatoria sp. The next most common sequence, 99% similar to that of the Geitlerinema BBD isolate, was present in three samples. One Leptolyngbya- and one Phormidium-related sequence were also found. Laboratory experiments using isolates of BBD Geitlerinema and Leptolyngbya revealed that they could carry out sulfide-resistant oxygenic photosynthesis, a relatively rare characteristic among cyanobacteria, and that they are adapted to the sulfide-rich, low-light BBD environment. The presence of the cyanotoxin microcystin in these cultures and in BBD suggests a role in BBD pathogenicity. Our results confirm the presence of Geitlerinema in the BBD microbial community and its ecological significance, which have been challenged, and provide evidence of a second ecologically significant BBD cyanobacterium, Leptolyngbya. PMID:17601818

Characterization of microbial arsenate reduction in the anoxic bottom waters of Mono Lake, California

USGS Publications Warehouse

Hoeft, S.E.; Lucas, F.; Hollibaugh, J.T.; Oremland, R.S.

2002-01-01

Dissimilatory reduction of arsenate (DAsR) occurs in the arsenic-rich, anoxic water column of Mono Lake, California, yet the microorganisms responsible for this observed in situ activity have not been identified. To gain insight as to which microorganisms mediate this phenomenon, as well as to some of the biogeochemical constraints on this activity, we conducted incubations of arsenate-enriched bottom water coupled with inhibition/amendment studies and Denaturing Gradient Gel Electrophoresis (DGGE) characterization techniques. DAsR was totally inhibited by filter-sterilization and by nitrate, partially inhibited (~50%) by selenate, but only slightly (~25%) inhibited by oxyanions that block sulfate-reduction (molybdate and tungstate). The apparent inhibition by nitrate, however, was not due to action as a preferred electron acceptor to arsenate. Rather, nitrate addition caused a rapid, microbial re-oxidation of arsenite to arsenate, which gave the overall appearance of no arsenate loss. A similar microbial oxidation of As(III) was also found with Fe(III), a fact that has implications for the recycling of As(V) in Mono Lake's anoxic bottom waters. DAsR could be slightly (10%) stimulated by substrate amendments of lactate, succinate, malate, or glucose, but not by acetate, suggesting that the DAsR microflora is not electron donor limited. DGGE analysis of amplified 16S rDNA gene fragments from incubated arsenate-enriched bottom waters revealed the presence of two bands that were not present in controls without added arsenate. The resolved sequences of these excised bands indicated the presence of members of the epsilon (Sulfurospirillum) and delta (Desulfovibrio) subgroups of the Proteobacteria, both of which have representative species that are capable of anaerobic growth using arsenate as their electron acceptor.

Relationships between and formation dynamics of the microbiota of consumers, producers, and the environment in an abalone aquatic system.

PubMed

Jiang, Jing-Zhe; Zhao, Wang; Liu, Guang-Feng; Wang, Jiang-Yong

2017-01-01

An ecosystem is a community comprising living and nonliving components of the environment. Microbes are ubiquitous elements in each of these components. The dynamics of microbiota formation in an ecosystem is important to elucidate, because how the different components of a system exchange microbes, and how the microbes control ecological processes remain unresolved. In this study, an abalone, Haliotis diversicolor, seed-nursing pond was used as a model system. We first examined changes in bacterial communities during the seedling cultivation of this herbivorous juvenile aquatic invertebrate animal. Denaturing gradient gel electrophoresis (DGGE) and pyrosequencing were used to analyze bacterial community dynamics and spatio-temporal interactions of different system components: consumers (abalone), producers (algae or a substrate), and the environment (water). DGGE fingerprints revealed that the developmental stages of abalone influences bacterial communities of both the abalone and substrate. Although the communities in water fluctuated daily, they could be divided into two clusters that coincided with abalone stages, reflecting the transition from larva to juvenile at around day 21. Pyrosequencing showed that the microbiota in the abalone and substrate had more operational taxonomic units in common than that of either with water. The Bray-Curtis similarity index was used to quantify the formation dynamics of microbiota among the various components of the system. The bacterial communities in producers and consumers showed similar changes. These communities were unstable at the beginning and then slowly stabilized over time. The environmental bacterial community was more stable than the bacterial communities in consumers and producers, and may have been the basis for stability in the system. Our research provides insights into the dynamics of microbiota formation in various biotic elements of a system that will contribute to predictive systems modeling.

Investigation of physico-chemical properties and microbial community during poultry manure co-composting process.

PubMed

Farah Nadia, Omar; Xiang, Loo Yu; Lie, Lee Yei; Chairil Anuar, Dzulkornain; Mohd Afandi, Mohammed P; Azhari Baharuddin, Samsu

2015-02-01

Co-composting of poultry manure and rubber wood sawdust was performed with the ratio of 2:1 (V/V) for a period of 60 days. An investigation was carried out to study the extracellular enzymatic activities and structural degradation utilizing Fourier transform infrared spectroscopy (FT-IR), thermogravimetry and differential thermal analysis (TG/DTA) and scanning electron microscopy (SEM). The microbial succession was also determined by using denaturing gel gradient electrophoresis (DGGE). The compost was able to reach its highest temperature of 71°C at day 3 and stabilized between 30 and 40°C for 8 weeks. CMCase, FPase and β-glucosidase acted synergistically in order to degrade the cellulosic substrate. The xylanase activities increased gradually during the composting and reached the peak value of 11.637 U/g on day 35, followed by a sharp decline. Both LiP and MnP activities reached their peak values on day 35 with 0.431 and 0.132 U/g respectively. The FT-IR spectra revealed an increase in aromaticity and a decrease in aliphatic compounds such as carbohydrates as decomposition proceeded. TGA/DTG data exhibited significant changes in weight loss in compost samples, indicating degradation of organic matter. SEM micrographs showed higher amounts of parenchyma exposed on the surface of rubber wood sawdust at day 60, showing significant degradation. DGGE and 16S rDNA analyses showed that Burkholderia sp., Pandoraea sp., and Pseudomonas sp. were present throughout the composting process. Ornithinibacillus sp. and Castellaniella ginsengisoli were only found in the initial stage of the composting, while different strains of Burkholderia sp. also occurred in the later stage of composting. Copyright © 2014. Published by Elsevier B.V.

Importance of inoculum properties on the structure and growth of bacterial communities during Recolonisation of humus soil with different pH.

PubMed

Pettersson, Marie; Bååth, Erland

2013-08-01

The relationship between community structure and growth and pH tolerance of a soil bacterial community was studied after liming in a reciprocal inoculum study. An unlimed (UL) humus soil with a pH of 4.0 was fumigated with chloroform for 4 h, after which < 1 % of the initial bacterial activity remained. Half of the fumigated soil was experimentally limed (EL) to a pH of 7.6. Both the UL and the EL soil were then reciprocally inoculated with UL soil or field limed (FL) soil with a pH of 6.2. The FL soil was from a 15-year-old experiment. The structural changes were measured on both bacteria in soil and on bacteria able to grow on agar plates using phospholipids fatty acid (PLFA) and denaturing gradient gel electrophoresis (DGGE) analysis. The developing community pH tolerance and bacterial growth were also monitored over time using thymidine incorporation. The inoculum source had a significant impact on both growth and pH tolerance of the bacterial community in the EL soil. These differences between the EL soil inoculated with UL soil and FL soil were correlated to structural changes, as evidenced by both PLFA and DGGE analyses on the soil. Similar correlations were seen to the fraction of the community growing on agar plates. There were, however, no differences between the soil bacterial communities in the unlimed soils with different inocula. This study showed the connection between the development of function (growth), community properties (pH tolerance) and the structure of the bacterial community. It also highlighted the importance of both the initial properties of the community and the selection pressure after environmental changes in shaping the resulting microbial community.

Diversity of microbial communities correlated to physiochemical parameters in a digestion basin of a zero-discharge mariculture system.

PubMed

Cytryn, Eddie; Gelfand, Ilya; Barak, Yoram; van Rijn, Jaap; Minz, Dror

2003-01-01

Bacterial community structure and physiochemical parameters were examined in a sedimentation basin of a zero-discharge mariculture system. The system consisted of an intensively stocked fish basin from which water was recirculated through two separate treatment loops. Surface water from the basin was pumped over a trickling filter in one loop while bottom-water was recirculated through a sedimentation basin followed by a fluidized bed reactor in the other. Ammonia oxidation to nitrate in the trickling filter and organic matter digestion together with nitrate reduction in the sedimentation basin and fluidized bed reactor, allowed zero-discharge operation of the system. Relatively high concentrations of oxygen, nitrate, sulphate and organic matter detected simultaneously in the digestion basin suggested the potential for a wide range of microbially-mediated transformation processes. In this study, physiochemical parameters were correlated to bacterial diversity and distribution in horizontal and vertical profiles within this basin in an effort to obtain a basic understanding of the chemical and microbial processes in this system. Chemical activity and microbial diversity, the latter measured by denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction (PCR) amplified 16S rDNA fragments, were higher in the sludge layer than in the overlying aqueous layer of the basin. Chemical parameters in sludge samples close to the basin inlet suggested enhanced microbial activity relative to other sampling areas with evidence of both nitrate and sulphate reduction. Four of the nine DGGE bands identified in this zone were affiliated with the Bacteroidetes phylum. Detected sequences closely related to sequences of organisms involved in the sulphur cycle included Desulfovibrio, Dethiosulfovibrio and apparent sulphur oxidizers from the gamma-proteobacteria. In addition, a number of sequences from the beta and alpha-proteobacteria were identified.

Gut Microbiota in Children Hospitalized with Oedematous and Non-Oedematous Severe Acute Malnutrition in Uganda.

PubMed

Kristensen, Kia Hee Schultz; Wiese, Maria; Rytter, Maren Johanne Heilskov; Özçam, Mustafa; Hansen, Lars Hestbjerg; Namusoke, Hanifa; Friis, Henrik; Nielsen, Dennis Sandris

2016-01-01

Severe acute malnutrition (SAM) among children remains a major health problem in many developing countries. SAM manifests in both an oedematous and a non-oedematous form, with oedematous malnutrition in its most severe form also known as kwashiorkor. The pathogenesis of both types of malnutrition in children remains largely unknown, but gut microbiota (GM) dysbiosis has recently been linked to oedematous malnutrition. In the present study we aimed to assess whether GM composition differed between Ugandan children suffering from either oedematous or non-oedematous malnutrition. As part of an observational study among children hospitalized with SAM aged 6-24 months in Uganda, fecal samples were collected at admission. Total genomic DNA was extracted from fecal samples, and PCR amplification was performed followed by Denaturing Gradient Gel Electrophoresis (DGGE) and tag-encoded 16S rRNA gene-targeted high throughput amplicon sequencing. Alpha and beta diversity measures were determined along with ANOVA mean relative abundance and G-test of independence followed by comparisons between groups. Of the 87 SAM children included, 62% suffered from oedematous malnutrition, 66% were boys and the mean age was 16.1 months. GM composition was found to differ between the two groups of children as determined by DGGE (p = 0.0317) and by high-throughput sequencing, with non-oedematous children having lower GM alpha diversity (p = 0.036). However, beta diversity analysis did not reveal larger differences between the GM of children with oedematous and non-oedematous SAM (ANOSIM analysis, weighted UniFrac, R = -0.0085, p = 0.584; unweighted UniFrac, R = 0.0719, p = 0.011). Our results indicate that non-oedematous SAM children have lower GM diversity compared to oedematous SAM children, however no clear compositional differences were identified.

Habitat heterogeneity and associated microbial community structure in a small-scale floodplain hyporheic flow path.

PubMed

Lowell, Jennifer L; Gordon, Nathan; Engstrom, Dale; Stanford, Jack A; Holben, William E; Gannon, James E

2009-10-01

The Nyack floodplain is located on the Middle Fork of the Flathead River, an unregulated, pristine, fifth-order stream in Montana, USA, bordering Glacier National Park. The hyporheic zone is a nutritionally heterogeneous floodplain component harboring a diverse array of microbial assemblages essential in fluvial biogeochemical cycling, riverine ecosystem productivity, and trophic interactions. Despite these functions, microbial community structure in pristine hyporheic systems is not well characterized. The current study was designed to assess whether physical habitat heterogeneity within the hyporheic zone of the Nyack floodplain was sufficient to drive bacterial beta diversity between three different hyporheic flow path locations. Habitat heterogeneity was assessed by measuring soluble reactive phosphorous, nitrate, dissolved organic carbon, dissolved oxygen, and soluble total nitrogen levels seasonally at surface water infiltration, advection, and exfiltration zones. Significant spatial differences were detected in dissolved oxygen and nitrate levels, and seasonal differences were detected in dissolved oxygen, nitrate, and dissolved organic carbon levels. Denaturing gradient gel electrophoresis (DGGE) and cell counts indicated that bacterial diversity increased with abundance, and DGGE fingerprints covaried with nitrate levels where water infiltrated the hyporheic zone. The ribosomal gene phylogeny revealed that hyporheic habitat heterogeneity was sufficient to drive beta diversity between bacterial assemblages. Phylogenetic (P) tests detected sequence disparity between the flow path locations. Small distinct lineages of Firmicutes, Actinomycetes, Planctomycetes, and Acidobacteria defined the infiltration zone and alpha- and beta-proteobacterial lineages delineated the exfiltration and advection zone communities. These data suggest that spatial habitat heterogeneity drives hyporheic microbial community development and that attempts to understand functional differences between bacteria inhabiting nutritionally heterogeneous hyporheic environments might begin by focusing on the biology of these taxa.

Relative Abundance in Bacterial and Fungal Gut Microbes in Obese Children: A Case Control Study.

PubMed

Borgo, Francesca; Verduci, Elvira; Riva, Alessandra; Lassandro, Carlotta; Riva, Enrica; Morace, Giulia; Borghi, Elisa

2017-02-01

Differences in relative proportions of gut microbial communities in adults have been correlated with intestinal diseases and obesity. In this study we evaluated the gut microbiota biodiversity, both bacterial and fungal, in obese and normal-weight school-aged children. We studied 28 obese (mean age 10.03 ± 0.68) and 33 age- and sex-matched normal-weight children. BMI z-scores were calculated, and the obesity condition was defined according to the WHO criteria. Fecal samples were analyzed by 16S rRNA amplification followed by denaturing gradient gel electrophoresis (DGGE) analysis and sequencing. Real-time polymerase chain reaction (PCR) was performed to quantify the most representative microbial species and genera. DGGE profiles showed high bacterial biodiversity without significant correlations with BMI z-score groups. Compared to bacterial profiles, we observed lower richness in yeast species. Sequence of the most representative bands gave back Eubacterium rectale, Saccharomyces cerevisiae, Candida albicans, and C. glabrata as present in all samples. Debaryomyces hansenii was present only in two obese children. Obese children revealed a significantly lower abundance in Akkermansia muciniphyla, Faecalibacterium prausnitzii, Bacteroides/Prevotella group, Candida spp., and Saccharomyces spp. (P = 0.031, P = 0.044, P = 0.003, P = 0.047, and P = 0.034, respectively). Taking into account the complexity of obesity, our data suggest that differences in relative abundance of some core microbial species, preexisting or diet driven, could actively be part of its etiology. This study improved our knowledge about the fungal population in the pediatric school-age population and highlighted the need to consider the influence of cross-kingdom relationships.

Intestinal microbiota and innate immunity-related gene alteration in cirrhotic rats with liver transplantation.

PubMed

Xie, Y R; Liu, S L; Liu, X; Luo, Z B; Zhu, B; Li, Z F; Li, L J; He, Y; Jiang, L; Li, H; Ruan, B

2011-12-01

The present study investigated the alteration of intestinal microbiota, innate immunity-related genes, and bacterial translocation in rats with cirrhosis and liver transplantation. Specific pathogen-free Sprague-Dawley rats were randomized into 4 groups: (1) normal controls (N); (2) liver cirrhosis (LC); (3) normal control groups with liver transplantation (LTN); and (4) liver cirrhosis with liver transplantation (LTC). We examined plasma endotoxin, bacterial tacslocation, denaturing gradient gel electrophoresis (DGGE) profile of intestinal mucosa-associated bacteria, abundance of key bacterial populations, and expression of innate immunity-related gene. The LTC and LC group, showed higher endotoxin levels (1.08±0.73 EU/mL and 0.74±0.70 EU/mL, respectively) than the N group (0.27±0.13 EU/mL; P<.05). the incidence of bacterial translocation (BT) to liver and mesenteric lymph nodes (MLN), and the number of total bacteria were increased significantly in the LTC and LC groups compared with the N group (P<.05). The counts of Lactobacilli and Bacteroides were lower, whereas Enterobacteria were higher in the LC than the N group (P<.05). Mucins (MUC2, MUC3) and Toll-like receptors (TLR2, TLR4) messenger RNA (mRNA) expression were significantly higher in the LC and LTC groups than the N group (P<.05). The marked difference between the groups in the overall structure of the bacterial community was also generated by DGGE profiles. Liver cirrhosis disturbs intestinal microbiota and innate immunity-related genes, which contributes to endotoxemia and bacterial translocation. These had not completely recovered in cirrhotic rats until 1 month after orthotopic liver transplantation. Copyright © 2011 Elsevier Inc. All rights reserved.

Changes in Bacterial Diversity Associated with Epithelial Tissue in the Beef Cow Rumen during the Transition to a High-Grain Diet ▿

PubMed Central

Chen, Yanhong; Penner, Gregory B.; Li, Meiju; Oba, Masahito; Guan, Le Luo

2011-01-01

Our understanding of the ruminal epithelial tissue-associated bacterial (defined as epimural bacteria in this study) community is limited. In this study, we aimed to determine whether diet influences the diversity of the epimural bacterial community in the bovine rumen. Twenty-four beef heifers were randomly assigned to either a rapid grain adaptation (RGA) treatment (n = 18) in which the heifers were allowed to adapt from a diet containing 97% hay to a diet containing 8% hay over 29 days or to the control group (n = 6), which was fed 97% hay. Rumen papillae were collected when the heifers were fed 97%, 25%, and 8% hay diets. PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR analysis were used to characterize rumen epimural bacterial diversity and to estimate the total epimural bacterial population (copy numbers of the 16S rRNA gene). The epimural bacterial diversity from RGA heifers changed (P = 0.01) in response to the rapid dietary transition, whereas it was not affected in control heifers. A total of 88 PCR-DGGE bands were detected, and 44 were identified from phyla including Firmicutes, Bacteroidetes, and Proteobacteria. The bacteria Treponema sp., Ruminobacter sp., and Lachnospiraceae sp. were detected only when heifers were fed 25% and 8% hay diets, suggesting the presence of these bacteria is the result of adaptation to the high-grain diets. In addition, the total estimated population of rumen epimural bacteria was positively correlated with molar proportions of acetate, isobutyrate, and isovalerate, suggesting that they may play a role in volatile fatty acid metabolism in the rumen. PMID:21705529

Microbial sulfate reduction and metal attenuation in pH 4 acid mine water

PubMed Central

Church, Clinton D; Wilkin, Richard T; Alpers, Charles N; Rye, Robert O; McCleskey, R Blaine

2007-01-01

Sediments recovered from the flooded mine workings of the Penn Mine, a Cu-Zn mine abandoned since the early 1960s, were cultured for anaerobic bacteria over a range of pH (4.0 to 7.5). The molecular biology of sediments and cultures was studied to determine whether sulfate-reducing bacteria (SRB) were active in moderately acidic conditions present in the underground mine workings. Here we document multiple, independent analyses and show evidence that sulfate reduction and associated metal attenuation are occurring in the pH-4 mine environment. Water-chemistry analyses of the mine water reveal: (1) preferential complexation and precipitation by H2S of Cu and Cd, relative to Zn; (2) stable isotope ratios of 34S/32S and 18O/16O in dissolved SO4 that are 2–3 ‰ heavier in the mine water, relative to those in surface waters; (3) reduction/oxidation conditions and dissolved gas concentrations consistent with conditions to support anaerobic processes such as sulfate reduction. Scanning electron microscope (SEM) analyses of sediment show 1.5-micrometer, spherical ZnS precipitates. Phospholipid fatty acid (PLFA) and denaturing gradient gel electrophoresis (DGGE) analyses of Penn Mine sediment show a high biomass level with a moderately diverse community structure composed primarily of iron- and sulfate-reducing bacteria. Cultures of sediment from the mine produced dissolved sulfide at pH values near 7 and near 4, forming precipitates of either iron sulfide or elemental sulfur. DGGE coupled with sequence and phylogenetic analysis of 16S rDNA gene segments showed populations of Desulfosporosinus and Desulfitobacterium in Penn Mine sediment and laboratory cultures. PMID:17956615

Changes in the rumen epimural bacterial diversity of beef cattle as affected by diet and induced ruminal acidosis.

PubMed

Petri, R M; Schwaiger, T; Penner, G B; Beauchemin, K A; Forster, R J; McKinnon, J J; McAllister, T A

2013-06-01

Little is known about the nature of the rumen epithelial adherent (epimural) microbiome in cattle fed different diets. Using denaturing gradient gel electrophoresis (DGGE), quantitative real-time PCR (qPCR), and pyrosequencing of the V3 hypervariable coding region of 16S rRNA, epimural bacterial communities of 8 cattle were profiled during the transition from a forage to a high-concentrate diet, during acidosis, and after recovery. A total of 153,621 high-quality gene sequences were obtained, with populations exhibiting less taxonomic variability among individuals than across diets. The bacterial community composition exhibited clustering (P < 0.03) by diet, with only 14 genera, representing >1% of the rumen epimural population, differing (P ≤ 0.05) among diets. During acidosis, levels of Atopobium, Desulfocurvus, Fervidicola, Lactobacillus, and Olsenella increased, while during the recovery, Desulfocurvus, Lactobacillus, and Olsenella reverted to levels similar to those with the high-grain diet and Sharpea and Succinivibrio reverted to levels similar to those with the forage diet. The relative abundances of bacterial populations changed during diet transition for all qPCR targets except Streptococcus spp. Less than 5% of total operational taxonomic units (OTUs) identified exhibited significant variability across diets. Based on DGGE, the community structures of epithelial populations differed (P ≤ 0.10); segregation was most prominent for the mixed forage diet versus the grain, acidotic challenge, and recovery diets. Atopobium, cc142, Lactobacillus, Olsenella, RC39, Sharpea, Solobacterium, Succiniclasticum, and Syntrophococcus were particularly prevalent during acidosis. Determining the metabolic roles of these key genera in the rumens of cattle fed high-grain diets could define a clinical microbial profile associated with ruminal acidosis.

Changes in the Rumen Epimural Bacterial Diversity of Beef Cattle as Affected by Diet and Induced Ruminal Acidosis

PubMed Central

Petri, R. M.; Schwaiger, T.; Penner, G. B.; Beauchemin, K. A.; Forster, R. J.; McKinnon, J. J.

2013-01-01

Little is known about the nature of the rumen epithelial adherent (epimural) microbiome in cattle fed different diets. Using denaturing gradient gel electrophoresis (DGGE), quantitative real-time PCR (qPCR), and pyrosequencing of the V3 hypervariable coding region of 16S rRNA, epimural bacterial communities of 8 cattle were profiled during the transition from a forage to a high-concentrate diet, during acidosis, and after recovery. A total of 153,621 high-quality gene sequences were obtained, with populations exhibiting less taxonomic variability among individuals than across diets. The bacterial community composition exhibited clustering (P < 0.03) by diet, with only 14 genera, representing >1% of the rumen epimural population, differing (P ≤ 0.05) among diets. During acidosis, levels of Atopobium, Desulfocurvus, Fervidicola, Lactobacillus, and Olsenella increased, while during the recovery, Desulfocurvus, Lactobacillus, and Olsenella reverted to levels similar to those with the high-grain diet and Sharpea and Succinivibrio reverted to levels similar to those with the forage diet. The relative abundances of bacterial populations changed during diet transition for all qPCR targets except Streptococcus spp. Less than 5% of total operational taxonomic units (OTUs) identified exhibited significant variability across diets. Based on DGGE, the community structures of epithelial populations differed (P ≤ 0.10); segregation was most prominent for the mixed forage diet versus the grain, acidotic challenge, and recovery diets. Atopobium, cc142, Lactobacillus, Olsenella, RC39, Sharpea, Solobacterium, Succiniclasticum, and Syntrophococcus were particularly prevalent during acidosis. Determining the metabolic roles of these key genera in the rumens of cattle fed high-grain diets could define a clinical microbial profile associated with ruminal acidosis. PMID:23584771

Gut Microbiota in Children Hospitalized with Oedematous and Non-Oedematous Severe Acute Malnutrition in Uganda

PubMed Central

Kristensen, Kia Hee Schultz; Wiese, Maria; Rytter, Maren Johanne Heilskov; Özçam, Mustafa; Hansen, Lars Hestbjerg; Namusoke, Hanifa; Friis, Henrik; Nielsen, Dennis Sandris

2016-01-01

Background Severe acute malnutrition (SAM) among children remains a major health problem in many developing countries. SAM manifests in both an oedematous and a non-oedematous form, with oedematous malnutrition in its most severe form also known as kwashiorkor. The pathogenesis of both types of malnutrition in children remains largely unknown, but gut microbiota (GM) dysbiosis has recently been linked to oedematous malnutrition. In the present study we aimed to assess whether GM composition differed between Ugandan children suffering from either oedematous or non-oedematous malnutrition. Methodology/Principal Findings As part of an observational study among children hospitalized with SAM aged 6–24 months in Uganda, fecal samples were collected at admission. Total genomic DNA was extracted from fecal samples, and PCR amplification was performed followed by Denaturing Gradient Gel Electrophoresis (DGGE) and tag-encoded 16S rRNA gene-targeted high throughput amplicon sequencing. Alpha and beta diversity measures were determined along with ANOVA mean relative abundance and G-test of independence followed by comparisons between groups. Of the 87 SAM children included, 62% suffered from oedematous malnutrition, 66% were boys and the mean age was 16.1 months. GM composition was found to differ between the two groups of children as determined by DGGE (p = 0.0317) and by high-throughput sequencing, with non-oedematous children having lower GM alpha diversity (p = 0.036). However, beta diversity analysis did not reveal larger differences between the GM of children with oedematous and non-oedematous SAM (ANOSIM analysis, weighted UniFrac, R = -0.0085, p = 0.584; unweighted UniFrac, R = 0.0719, p = 0.011). Conclusions/Significance Our results indicate that non-oedematous SAM children have lower GM diversity compared to oedematous SAM children, however no clear compositional differences were identified. PMID:26771456

Microorganisms with Novel Dissimilatory (Bi)Sulfite Reductase Genes Are Widespread and Part of the Core Microbiota in Low-Sulfate Peatlands ▿ †

PubMed Central

Steger, Doris; Wentrup, Cecilia; Braunegger, Christina; Deevong, Pinsurang; Hofer, Manuel; Richter, Andreas; Baranyi, Christian; Pester, Michael; Wagner, Michael; Loy, Alexander

2011-01-01

Peatlands of the Lehstenbach catchment (Germany) house as-yet-unidentified microorganisms with phylogenetically novel variants of the dissimilatory (bi)sulfite reductase genes dsrAB. These genes are characteristic of microorganisms that reduce sulfate, sulfite, or some organosulfonates for energy conservation but can also be present in anaerobic syntrophs. However, nothing is currently known regarding the abundance, community dynamics, and biogeography of these dsrAB-carrying microorganisms in peatlands. To tackle these issues, soils from a Lehstenbach catchment site (Schlöppnerbrunnen II fen) from different depths were sampled at three time points over a 6-year period to analyze the diversity and distribution of dsrAB-containing microorganisms by a newly developed functional gene microarray and quantitative PCR assays. Members of novel, uncultivated dsrAB lineages (approximately representing species-level groups) (i) dominated a temporally stable but spatially structured dsrAB community and (ii) represented “core” members (up to 1% to 1.7% relative abundance) of the autochthonous microbial community in this fen. In addition, denaturing gradient gel electrophoresis (DGGE)- and clone library-based comparisons of the dsrAB diversity in soils from a wet meadow, three bogs, and five fens of various geographic locations (distance of ∼1 to 400 km) identified that one Syntrophobacter-related and nine novel dsrAB lineages are widespread in low-sulfate peatlands. Signatures of biogeography in dsrB-based DGGE data were not correlated with geographic distance but could be explained largely by soil pH and wetland type, implying that the distribution of dsrAB-carrying microorganisms in wetlands on the scale of a few hundred kilometers is not limited by dispersal but determined by local environmental conditions. PMID:21169452

Continuous volatile fatty acid production from lignocellulosic biomass by a novel rumen-mimetic bioprocess.

PubMed

Agematu, Hitosi; Takahashi, Takehiko; Hamano, Yoshio

2017-11-01

Lignocellulosic biomass is an attractive source of biofuels and biochemicals, being abundant in various plant sources. However, processing this type of biomass requires hydrolysis of cellulose. The proposed rumen-mimetic bioprocess consists of dry-pulverization of lignocellulosic biomass and pH-controlled continuous cultivation of ruminal bacteria using ammonium as a nitrogen source. In this study, ruminal bacteria were continuously cultivated for over 60 days and used to digest microcrystalline cellulose, rice straw, and Japanese cedar to produce volatile fatty acids (VFAs). The ruminal bacteria grew well in the chemically defined medium. The amounts of VFAs produced from 20 g of cellulose, rice straw, and Japanese cedar were 183 ± 29.7, 69.6 ± 12.2, and 21.8 ± 12.9 mmol, respectively. Each digestion completed within 24 h. The carbon yield was 60.6% when 180 mmol of VFAs was produced from 20 g of cellulose. During the cultivation, the bacteria were observed to form flocs that enfolded the feed particles. These flocs likely contain all of the bacterial species necessary to convert lignocellulosic biomass to VFAs and microbial protein symbiotically. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rDNA fragments revealed that the bacterial community was relatively stable after 1 week in cultivation, though it was different from the original community structure. Furthermore, sequence analysis of the DGGE bands indicates that the microbial community includes a cellulolytic bacterium, a bacterium acting synergistically with cellulolytic bacteria, and a propionate-producing bacterium, as well as other anaerobic bacteria. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Nitrogen Cycling and Community Structure of Proteobacterial β-Subgroup Ammonia-Oxidizing Bacteria within Polluted Marine Fish Farm Sediments

PubMed Central

McCaig, Allison E.; Phillips, Carol J.; Stephen, John R.; Kowalchuk, George A.; Harvey, S. Martyn; Herbert, Rodney A.; Embley, T. Martin; Prosser, James I.

1999-01-01

A multidisciplinary approach was used to study the effects of pollution from a marine fish farm on nitrification rates and on the community structure of ammonia-oxidizing bacteria in the underlying sediment. Organic content, ammonium concentrations, nitrification rates, and ammonia oxidizer most-probable-number counts were determined in samples of sediment collected from beneath a fish cage and on a transect at 20 and 40 m from the cage. The data suggest that nitrogen cycling was significantly disrupted directly beneath the fish cage, with inhibition of nitrification and denitrification. Although visual examination indicated some slight changes in sediment appearance at 20 m, all other measurements were similar to those obtained at 40 m, where the sediment was considered pristine. The community structures of proteobacterial β-subgroup ammonia-oxidizing bacteria at the sampling sites were compared by PCR amplification of 16S ribosomal DNA (rDNA), using primers which target this group. PCR products were analyzed by denaturing gradient gel electrophoresis (DGGE) and with oligonucleotide hybridization probes specific for different ammonia oxidizers. A DGGE doublet observed in PCR products from the highly polluted fish cage sediment sample was present at a lower intensity in the 20-m sample but was absent from the pristine 40-m sample station. Band migration, hybridization, and sequencing demonstrated that the doublet corresponded to a marine Nitrosomonas group which was originally observed in 16S rDNA clone libraries prepared from the same sediment samples but with different PCR primers. Our data suggest that this novel Nitrosomonas subgroup was selected for within polluted fish farm sediments and that the relative abundance of this group was influenced by the extent of pollution. PMID:9872782

Physicochemical and microbiological characterization of chicha, a rice-based fermented beverage produced by Umutina Brazilian Amerindians.

PubMed

Puerari, Cláudia; Magalhães-Guedes, Karina Teixeira; Schwan, Rosane Freitas

2015-04-01

Chicha is a traditional, fermented rice beverage produced by the indigenous Umutina people in Brazil. Culture-dependent and independent approaches were used to investigate the microbial community dynamic. The bacterial population ranged from 0.1 to 6.83 log mL(-1). Lactic acid bacteria (LAB) and Bacillus dominated throughout the fermentation process. Representative colonies were grouped by Repetitive Extragenic Palindromic and Polymerase Chain Reaction (Rep-PCR) and by biochemical features. Genera of Lactobacillus, Bacillus, Leuconostoc, Enterococcus, Streptomyces, Enterobacter, Acinetobacter, Escherichia, Cronobacter, and Klebsiella were identified by partial 16S rRNA gene sequence. As shown by Polimerase and Denaturing Gradient Gel Electrophoresis (PCR-DGGE) analysis, uncultivable Bifidobacterium and Propioniobacterium were found throughout fermentation. Uncultured fungi composed the fungal PCR-DGGE profile. The pH values decreased from 5.2 (time 0) to 3.9 at 36 h of fermentation. Ethanol was not found. The lactic acid concentration increased rapidly throughout fermentation until it reached a high final value (1.4 g L(-1)) and the average glycerol content in the beverage was 0.425 g L(-1). Chicha fermentation might be described by the following phenomena: (i) increasing bacterial population, with lactic acid bacteria (LAB) as the largest group detected; (ii) increasing concentrations of lactic and citric acids; and (iii) the final product is characterized by a high content of acids and the absence of ethanol, therefore characterizing rice chicha an acidic and nonalcoholic beverage. First, this study characterizes the microbial population involved in the nonalcoholic fermentation of chicha, which is produced from rice by Amerindians in Brazil. This study is important for promoting the appreciation of and safeguarding this Brazilian indigenous beverage as an immaterial cultural heritage. Copyright © 2014 Elsevier Ltd. All rights reserved.

Microbiological and molecular characterization of commercially available probiotics containing Bacillus clausii from India and Pakistan.

PubMed

Patrone, Vania; Molinari, Paola; Morelli, Lorenzo

2016-11-21

Probiotics are actively used for treatment of diarrhoea, respiratory infections, and prevention of infectious gastrointestinal diseases. The efficacy of probiotics is due to strain-specific features and the number of viable cells; however, several reports of deviations from the label in the actual content of strains in probiotic products are a matter of concern. Most of the available data on quality focuses on probiotic products containing lactobacilli and/or bifidobacteria, while very few data are available on spore-forming probiotics. The present study evaluates the label claims for spore count and species identification in five commercial probiotic products marketed in India and Pakistan that claim to contain Bacillus clausii: Tufpro, Ecogro, Enterogermina, Entromax, and Ospor. Bacterial enumeration from three batches was done by microbiological plating methods by two independent operators. Species identification was done using PCR amplification and sequence analysis of the 16S rRNA gene, and determination of the total amount of species present in the products was done using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis followed by DNA sequencing of the excised bands. Plate count methods demonstrated poor correlations between quantitative label indications and bacteria recovered from plates for Tufpro, Ecogro, and Ospor. The 16S rRNA analysis performed on bacteria isolated from plate counts showed that only Enterogermina and Ospor contained homogenous B. clausii. PCR-DGGE analysis revealed that only Enterogermina had a homogenous B. clausii population while other products had mixed bacterial populations. In conclusion, the current analysis clearly demonstrates that of the five analysed commercial probiotics, only Enterogermina followed the label claims. Copyright © 2016 Elsevier B.V. All rights reserved.

Eukarya associated with the stony coral Oculina patagonica from the Mediterranean Sea.

PubMed

Rubio-Portillo, Esther; Souza-Egipsy, Virginia; Ascaso, Carmen; de Los Rios Murillo, Asunción; Ramos-Esplá, Alfonso A; Antón, Josefa

2014-10-01

Oculina patagonica is a putative alien scleractinian coral from the Southwest Atlantic that inhabits across the Mediterranean Sea. Here, we have addressed the diversity of Eukarya associated with this coral and its changes related to the environmental conditions and coral status. A total of 46 colonies of O. patagonica were taken from Alicante coast (Spain) and Pietra Ligure coast (Italy) and analyzed using denaturing gradient gel electrophoresis (DGGE) of the small-subunit 18S rRNA and 16S plastid rRNA genes, internal transcribed spacer region 2 (ITS 2) analyses, and electron microscopy. Our results show that Eukarya and plastid community associated to O. patagonica change with environmental conditions and coral status. Cryptic species, which can be difficult to identify by optical methods, were distinguished by 18S rRNA gene DGGE: the barnacle Megatrema anglicum, which was detected at two locations, and two boring sponges related to Cliona sp. and Siphonodictyon coralliphagum detected in samples from Tabarca and Alicante Harbour, respectively. Eukaryotic phototrophic community from the skeletal matrix of healthy corals was dominated by Ochrosphaera sp. while bleached corals from the Harbour and Tabarca were associated to different uncultured phototrophic organism. Differences in ultrastructural morphologies of the zooxanthellae between healthy and bleached corals were observed. Nevertheless, no differences were found in Symbiodinium community among time, environments, coral status and location, showing that O. patagonica hosted only one genotype of Symbiodinium belonging to clade B2. The fact that this clade has not been previously detected in other Mediterranean corals and is more frequent in the tropical Western Atlantic, is a new evidence that O. patagonica is an alien species in the Mediterranean Sea. Copyright © 2014 Elsevier B.V. All rights reserved.

Probiotics Can Induce Follicle Maturational Competence: The Danio rerio Case1

PubMed Central

Gioacchini, Giorgia; Giorgini, Elisabetta; Merrifield, Daniel L.; Hardiman, Gary; Borini, Andrea; Vaccari, Lisa; Carnevali, Oliana

2011-01-01

ABSTRACT In the present study, the effects of the probiotic Lactobacillus rhamnosus IMC 501 on the acquisition of oocyte maturational competence was examined in zebrafish (Danio rerio). L. rhamnosus administration induced the responsiveness of incompetent follicles (stage IIIa) to 17,20-dihydroxy-4-pregnen-3-one and their in vitro maturation. Acquisition of competence by the stage IIIa follicles was further validated by changes of lhr, mprb, inhbaa (activin betaA1), tgfb1, and gdf9 gene expression, which have recently emerged as key regulators of oocyte acquisition of maturational competence, and pou5f1 gene expression, which in other models has been shown to govern the establishment of developmental competence of oocytes. In addition, a DNA microarray experiment was conducted using the same follicles, and with relative gene ontology (GO) data analysis, the molecular effects of probiotic administration emerged. Molecular analysis using PCR-DGGE (denaturing gradient gel electrophoresis) approach, providing information about only the most abundant bacterial members of the microbial community, revealed that the probiotic was able to populate the gastrointestinal tract and modulate the microbial communities, causing a clear shift in them and specifically enhancing the presence of the lactic acid bacteria Streptococcus thermophilus. At the same time, PCR-DGGE analysis revealed that the probiotic was not directly associated with the ovaries. Finally, the effects of probiotic treatment on zebrafish follicle development were also analyzed by FPA (focal plane array) Fourier transform-infrared (FT-IR) imaging, a technique that provides the overall biochemical composition of samples. Changes were found above all in stage IIIa follicles from probiotic-exposed females; the modifications, observed in protein secondary structures as well as in hydration and in bands related to phosphate moieties, allowed us to hypothesize that probiotics act at this follicle stage, affecting the maturation phase. PMID:22088919

Dissimilatory arsenate and sulfate reduction in sediments of two hypersaline, arsenic-rich soda lakes: Mono and Searles Lakes, California

USGS Publications Warehouse

Kulp, T.R.; Hoeft, S.E.; Miller, L.G.; Saltikov, C.; Murphy, J.N.; Han, S.; Lanoil, B.; Oremland, R.S.

2006-01-01

A radioisotope method was devised to study bacterial respiratory reduction of arsenate in sediments. The following two arsenic-rich soda lakes in California were chosen for comparison on the basis of their different salinities: Mono Lake (???90 g/liter) and Searles Lake (???340 g/liter). Profiles of arsenate reduction and sulfate reduction were constructed for both lakes. Reduction of [73As] arsenate occurred at all depth intervals in the cores from Mono Lake (rate constant [k] = 0.103 to 0.04 h-1) and Searles Lake (k = 0.012 to 0.002 h-1), and the highest activities occurred in the top sections of each core. In contrast, [35S] sulfate reduction was measurable in Mono Lake (k = 7.6 ?? 104 to 3.2 ?? 10-6 h-1) but not in Searles Lake. Sediment DNA was extracted, PCR amplified, and separated by denaturing gradient gel electrophoresis (DGGE) to obtain phylogenetic markers (i.e., 16S rRNA genes) and a partial functional gene for dissimilatory arsenate reduction (arrA). The amplified arrA gene product showed a similar trend in both lakes; the signal was strongest in surface sediments and decreased to undetectable levels deeper in the sediments. More arrA gene signal was observed in Mono Lake and was detectable at a greater depth, despite the higher arsenate reduction activity observed in Searles Lake. A partial sequence (about 900 bp) was obtained for a clone (SLAS-3) that matched the dominant DGGE band found in deeper parts of the Searles Lake sample (below 3 cm), and this clone was found to be closely related to SLAS-1, a novel extremophilic arsenate respirer previously cultivated from Searles Lake. Copyright ?? 2006, American Society for Microbiology. All Rights Reserved.

Activity and Phylogenetic Diversity of Bacterial Cells with High and Low Nucleic Acid Content and Electron Transport System Activity in an Upwelling Ecosystem

PubMed Central

Longnecker, K.; Sherr, B. F.; Sherr, E. B.

2005-01-01

We evaluated whether bacteria with higher cell-specific nucleic acid content (HNA) or an active electron transport system, i.e., positive for reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), were responsible for the bulk of bacterioplankton metabolic activity. We also examined whether the phylogenetic diversity of HNA and CTC-positive cells differed from the diversity of Bacteria with low nucleic acid content (LNA). Bacterial assemblages were sampled both in eutrophic shelf waters and in mesotrophic offshore waters in the Oregon coastal upwelling region. Cytometrically sorted HNA, LNA, and CTC-positive cells were assayed for their cell-specific [3H]leucine incorporation rates. Phylogenetic diversity in sorted non-radioactively labeled samples was assayed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Cell-specific rates of leucine incorporation of HNA and CTC-positive cells were on average only slightly greater than the cell-specific rates of LNA cells. HNA cells accounted for most bacterioplankton substrate incorporation due to high abundances, while the low abundances of CTC-positive cells resulted in only a small contribution by these cells to total bacterial activity. The proportion of the total bacterial leucine incorporation attributable to LNA cells was higher in offshore regions than in shelf waters. Sequence data obtained from DGGE bands showed broadly similar phylogenetic diversity across HNA, LNA, and CTC-positive cells, with between-sample and between-region variability in the distribution of phylotypes. Our results suggest that LNA bacteria are not substantially different from HNA bacteria in either cell-specific rates of substrate incorporation or phylogenetic composition and that they can be significant contributors to bacterial metabolism in the sea. PMID:16332746

Responses of Baltic Sea Ice and Open-Water Natural Bacterial Communities to Salinity Change

PubMed Central

Kaartokallio, Hermanni; Laamanen, Maria; Sivonen, Kaarina

2005-01-01

To investigate the responses of Baltic Sea wintertime bacterial communities to changing salinity (5 to 26 practical salinity units), an experimental study was conducted. Bacterial communities of Baltic seawater and sea ice from a coastal site in southwest Finland were used in two batch culture experiments run for 17 or 18 days at 0°C. Bacterial abundance, cell volume, and leucine and thymidine incorporation were measured during the experiments. The bacterial community structure was assessed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified partial 16S rRNA genes with sequencing of DGGE bands from initial communities and communities of day 10 or 13 of the experiment. The sea ice-derived bacterial community was metabolically more active than the open-water community at the start of the experiment. Ice-derived bacterial communities were able to adapt to salinity change with smaller effects on physiology and community structure, whereas in the open-water bacterial communities, the bacterial cell volume evolution, bacterial abundance, and community structure responses indicated the presence of salinity stress. The closest relatives for all eight partial 16S rRNA gene sequences obtained were either organisms found in polar sea ice and other cold habitats or those found in summertime Baltic seawater. All sequences except one were associated with the α- and γ-proteobacteria or the Cytophaga-Flavobacterium-Bacteroides group. The overall physiological and community structure responses were parallel in ice-derived and open-water bacterial assemblages, which points to a linkage between community structure and physiology. These results support previous assumptions of the role of salinity fluctuation as a major selective factor shaping the sea ice bacterial community structure. PMID:16085826

Activity and phylogenetic diversity of bacterial cells with high and low nucleic acid content and electron transport system activity in an upwelling ecosystem.

PubMed

Longnecker, K; Sherr, B F; Sherr, E B

2005-12-01

We evaluated whether bacteria with higher cell-specific nucleic acid content (HNA) or an active electron transport system, i.e., positive for reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), were responsible for the bulk of bacterioplankton metabolic activity. We also examined whether the phylogenetic diversity of HNA and CTC-positive cells differed from the diversity of Bacteria with low nucleic acid content (LNA). Bacterial assemblages were sampled both in eutrophic shelf waters and in mesotrophic offshore waters in the Oregon coastal upwelling region. Cytometrically sorted HNA, LNA, and CTC-positive cells were assayed for their cell-specific [3H]leucine incorporation rates. Phylogenetic diversity in sorted non-radioactively labeled samples was assayed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Cell-specific rates of leucine incorporation of HNA and CTC-positive cells were on average only slightly greater than the cell-specific rates of LNA cells. HNA cells accounted for most bacterioplankton substrate incorporation due to high abundances, while the low abundances of CTC-positive cells resulted in only a small contribution by these cells to total bacterial activity. The proportion of the total bacterial leucine incorporation attributable to LNA cells was higher in offshore regions than in shelf waters. Sequence data obtained from DGGE bands showed broadly similar phylogenetic diversity across HNA, LNA, and CTC-positive cells, with between-sample and between-region variability in the distribution of phylotypes. Our results suggest that LNA bacteria are not substantially different from HNA bacteria in either cell-specific rates of substrate incorporation or phylogenetic composition and that they can be significant contributors to bacterial metabolism in the sea.

[Effects of continuous application of bio-organic fertilizer on banana production and cultural microflora of bulk soil in orchard with serious disease incidence].

PubMed

Zhong, Shu-tang; Shen, Zong-zhuan; Sun, Yi-fei; Lyu, Na-na; Ruan, Yun-ze; Li, Rong; Shen, Qi-rong

2015-02-01

A field experiment was conducted for two years to investigate the effects of different fertilization applications on the suppression of banana fusarium wilt disease, crop yield, fruit quality and culturable microflora in a banana orchard which has been monocultured with banana for 12 years and suffered serious banana fusarium wilt disease. The fertilizers included chemical fertilizer (CF), cow manure compost (CM), pig manure compost (PM) and bio-organic fertilizer (BIO). The banana soil microflora was invested using plate-counting method and culture-dependent polymerase chain reaction denaturing gradient gel electrophoresis method (CD PCR-DGGE). Results showed that, compared with the other treatments, 2-year consecutive application of BIO significantly reduced the banana fusarium wilt disease incidence, and improved the banana mass per tree, crop yield, total soluble sugar content and the ratio of total soluble sugar to titratable acidity of fruits (sugar/acid ratio). Moreover, the analysis of culturable microflora showed that BIO application significantly increased the soil microbial biomass, soil culturable bacteria, bacillus and actinomycetes, and the ratio of bacteria to fungi (B/F) , while decreased the Fusarium oxysporum. Based on the CD PCR-DGGE results, the BIO application significantly altered the soil culturable bacterial structure and showed highest richness and diversity after 2 years of BIO application. The phylogenetic analysis of the selected bands showed that BIO application enriched the soil with the species of Paenibacillus sp., Burkholderia sp., uncultured Verrucomicrobia sp. and Bacillus aryabhattai, and depressed the species of Ralstonia sp., Chryseobacterium gleum, Fluviicola taffensis, Enterobacter sp. and Bacillus megaterium. These results confirmed that the continuous application of BIO effectively controlled the fusarium wilt disease, improved the crop yield and fruit quality, and modulated the soil culturable microflora under field condition.

Characterization of bacteria isolated from palaeoproterozoic metasediments for sequestration of carbon dioxide and formation of calcium carbonate.

PubMed

Srivastava, Shaili; Bharti, Randhir K; Thakur, Indu Shekhar

2015-01-01

Bacterial community of palaeoproterozoic metasediments was enriched in the chemostat in the presence of different concentrations of NaHCO3. Six bacterial isolates were isolated from the chemostat on nutrient agar plates on the basis of distinct morphology. Denaturing gradient gel electrophoresis (DGGE) proved the presence of six operational taxonomic units (OTUs) at 50 and 100 mM NaHCO3. The OTU was reduced to three and one at enrichment concentration of 150 and 200 mM NaHCO3 respectively. These six isolates were tested for sequestration of carbon dioxide by (14)C metabolic labeling of NaH(14)CO3. Among the six isolates, one of the bacterium showed better potency to fix radiolabeled NaH(14)CO3. The isolate (ISTD04) was identified as Serratia sp. by 16S ribosomal RNA (16S rRNA) sequence analysis and was found to be same as the DGGE OTU sequence at 200-mM NaHCO3 concentration. The bacterium was tested for product formation in form of calcite crystals in presence of 5 % CO2. Scanning electron microscopy (SEM) of product formed by the bacterium revealed defined faceted rhombohedral structure which resembled calcite and vaterite phases of the crystal. Formation of calcium carbonate crystals was further confirmed by Fourier transform infrared (FTIR) spectroscopy as carbonate group showing strong vibration at 1,456 cm(-1). Major calcite phase diffraction peaks were determined by X-ray diffraction (XRD) analysis, and energy-dispersive X-ray (EDX) analysis showed the presence of CaO (72 %) and carbon (18 %). Bacterium use bicarbonate as carbon source for their growth as well as by-product formation in form of calcite shows carbon circulation and storage.

Soil bacterial and fungal community successions under the stress of chlorpyrifos application and molecular characterization of chlorpyrifos-degrading isolates using ERIC-PCR*

PubMed Central

Chen, Lie-zhong; Li, Yan-li; Yu, Yun-long

2014-01-01

Chlorpyrifos is a widely used insecticide in recent years, and it will produce adverse effects on soil when applied on crops or mixed with soil. In this study, nested polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) were combined to explore the bacterial and fungal community successions in soil treated with 5 and 20 mg/kg of chlorpyrifos. Furthermore, isolates capable of efficiently decomposing chlorpyrifos were molecular-typed using enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). Under the experimental conditions, degradation of chlorpyrifos in soil was interpreted with the first-order kinetics, and the half-lives of chlorpyrifos at 5 and 20 mg/kg doses were calculated to be 8.25 and 8.29 d, respectively. DGGE fingerprint and principal component analysis (PCA) indicated that the composition of the fungal community was obviously changed with the chlorpyrifos treatment, and that samples of chlorpyrifos treatment were significantly separated from those of the control from the beginning to the end. While for the bacterial community, chlorpyrifos-treated soil samples were apparently different in the first 30 d and recovered to a similar level of the control up until 60 d, and the distance in the PCA between the chlorpyrifos-treated samples and the control was getting shorter through time and was finally clustered into one group. Together, our results demonstrated that the application of chlorpyrifos could affect the fungal community structure in a quick and lasting way, while only affecting the bacterial community in a temporary way. Finally, nine typical ERIC types of chlorpyrifos-degrading isolates were screened. PMID:24711353

Microbial community structure and activity in trace element-contaminated soils phytomanaged by Gentle Remediation Options (GRO).

PubMed

Touceda-González, M; Prieto-Fernández, Á; Renella, G; Giagnoni, L; Sessitsch, A; Brader, G; Kumpiene, J; Dimitriou, I; Eriksson, J; Friesl-Hanl, W; Galazka, R; Janssen, J; Mench, M; Müller, I; Neu, S; Puschenreiter, M; Siebielec, G; Vangronsveld, J; Kidd, P S

2017-12-01

Gentle remediation options (GRO) are based on the combined use of plants, associated microorganisms and soil amendments, which can potentially restore soil functions and quality. We studied the effects of three GRO (aided-phytostabilisation, in situ stabilisation and phytoexclusion, and aided-phytoextraction) on the soil microbial biomass and respiration, the activities of hydrolase enzymes involved in the biogeochemical cycles of C, N, P, and S, and bacterial community structure of trace element contaminated soils (TECS) from six field trials across Europe. Community structure was studied using denaturing gradient gel electrophoresis (DGGE) fingerprinting of Bacteria, α- and β-Proteobacteria, Actinobacteria and Streptomycetaceae, and sequencing of DGGE bands characteristic of specific treatments. The number of copies of genes involved in ammonia oxidation and denitrification were determined by qPCR. Phytomanagement increased soil microbial biomass at three sites and respiration at the Biogeco site (France). Enzyme activities were consistently higher in treated soils compared to untreated soils at the Biogeco site. At this site, microbial biomass increased from 696 to 2352 mg ATP kg -1 soil, respiration increased from 7.4 to 40.1 mg C-CO 2 kg -1 soil d -1 , and enzyme activities were 2-11-fold higher in treated soils compared to untreated soil. Phytomanagement induced shifts in the bacterial community structure at both, the total community and functional group levels, and generally increased the number of copies of genes involved in the N cycle (nirK, nirS, nosZ, and amoA). The influence of the main soil physico-chemical properties and trace element availability were assessed and eventual site-specific effects elucidated. Overall, our results demonstrate that phytomanagement of TECS influences soil biological activity in the long term. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development of a systematic feedback isolation approach for targeted strains from mixed culture systems.

PubMed

Poudel, Pramod; Tashiro, Yukihiro; Miyamoto, Hirokuni; Miyamoto, Hisashi; Okugawa, Yuki; Sakai, Kenji

2017-01-01

Elucidation of functions of bacteria in a mixed culture system (MCS) such as composting, activated sludge system is difficult, since the system is complicating with many unisolated bacteria. Here, we developed a systematic feedback isolation strategy for the isolation and rapid screening of multiple targeted strains from MCS. Six major strains (Corynebacterium sphenisci, Bacillus thermocloacae, Bacillus thermoamylovorans, Bacillus smithii, Bacillus humi, and Bacillus coagulans), which are detected by denaturing gradient gel electrophoresis (DGGE) analysis in our previous study on MCS for l-lactic acid production, were targeted for isolation. Based on information of suitable cultivation conditions (e.g., media, pH, temperature) from the literature, feedback isolation was performed to form 136 colonies. The following direct colony matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was optimised as the second screening to narrow down 20 candidate colonies from similar spectra patterns with six closest type strains. This step could distinguish bacteria at the species level with distance similarity scores ≥0.55 corresponding to 16S rRNA gene sequence similarity ≥98.2%, suggesting that this is an effective technique to minimize isolates close to targeted type strains. Analysis of 16S rRNA gene sequences indicated that two targeted strains and one strain related to the target had successfully been isolated, showing high similarities (99.5-100%) with the sequences from the DGGE bands, and that the other candidates were affiliated with three strains that were closely related to the target species. This study proposes a new method for systematic feedback isolation that may be useful for isolating targeted strains from MCS for further investigation. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Effect of Elevated Salt Concentrations on the Aerobic Granular Sludge Process: Linking Microbial Activity with Microbial Community Structure▿

PubMed Central

Bassin, J. P.; Pronk, M.; Muyzer, G.; Kleerebezem, R.; Dezotti, M.; van Loosdrecht, M. C. M.

2011-01-01

The long- and short-term effects of salt on biological nitrogen and phosphorus removal processes were studied in an aerobic granular sludge reactor. The microbial community structure was investigated by PCR-denaturing gradient gel electrophoresis (DGGE) on 16S rRNA and amoA genes. PCR products obtained from genomic DNA and from rRNA after reverse transcription were compared to determine the presence of bacteria as well as the metabolically active fraction of bacteria. Fluorescence in situ hybridization (FISH) was used to validate the PCR-based results and to quantify the dominant bacterial populations. The results demonstrated that ammonium removal efficiency was not affected by salt concentrations up to 33 g/liter NaCl. Conversely, a high accumulation of nitrite was observed above 22 g/liter NaCl, which coincided with the disappearance of Nitrospira sp. Phosphorus removal was severely affected by gradual salt increase. No P release or uptake was observed at steady-state operation at 33 g/liter NaCl, exactly when the polyphosphate-accumulating organisms (PAOs), “Candidatus Accumulibacter phosphatis” bacteria, were no longer detected by PCR-DGGE or FISH. Batch experiments confirmed that P removal still could occur at 30 g/liter NaCl, but the long exposure of the biomass to this salinity level was detrimental for PAOs, which were outcompeted by glycogen-accumulating organisms (GAOs) in the bioreactor. GAOs became the dominant microorganisms at increasing salt concentrations, especially at 33 g/liter NaCl. In the comparative analysis of the diversity (DNA-derived pattern) and the activity (cDNA-derived pattern) of the microbial population, the highly metabolically active microorganisms were observed to be those related to ammonia (Nitrosomonas sp.) and phosphate removal (“Candidatus Accumulibacter”). PMID:21926194

Molecular Analysis of Ammonia-Oxidizing Bacteria of the β Subdivision of the Class Proteobacteria in Compost and Composted Materials

PubMed Central

Kowalchuk, George A.; Naoumenko, Zinaida S.; Derikx, Piet J. L.; Felske, Andreas; Stephen, John R.; Arkhipchenko, Irina A.

1999-01-01

Although the practice of composting animal wastes for use as biofertilizers has increased in recent years, little is known about the microorganisms responsible for the nitrogen transformations which occur in compost and during the composting process. Ammonia is the principle available nitrogenous compound in composting material, and the conversion of this compound to nitrite in the environment by chemolithotrophic ammonia-oxidizing bacteria is an essential step in nitrogen cycling. Therefore, the distribution of ammonia-oxidizing members of the β subdivision of the class Proteobacteria in a variety of composting materials was assessed by amplifying 16S ribosomal DNA (rDNA) and 16S rRNA by PCR and reverse transcriptase PCR (RT-PCR), respectively. The PCR and RT-PCR products were separated by denaturing gradient gel electrophoresis (DGGE) and were identified by hybridization with a hierarchical set of oligonucleotide probes designed to detect ammonia oxidizer-like sequence clusters in the genera Nitrosospira and Nitrosomonas. Ammonia oxidizer-like 16S rDNA was detected in almost all of the materials tested, including industrial and experimental composts, manure, and commercial biofertilizers. A comparison of the DGGE and hybridization results after specific PCR and RT-PCR suggested that not all of the different ammonia oxidizer groups detected in compost are equally active. amoA, the gene encoding the active-site-containing subunit of ammonia monooxygenase, was also targeted by PCR, and template concentrations were estimated by competitive PCR. Detection of ammonia-oxidizing bacteria in the composts tested suggested that such materials may not be biologically inert with respect to nitrification and that the fate of nitrogen during composting and compost storage may be affected by the presence of these organisms. PMID:9925559

Characterization of the Prokaryotic Diversity in Cold Saline Perennial Springs of the Canadian High Arctic▿

PubMed Central

Perreault, Nancy N.; Andersen, Dale T.; Pollard, Wayne H.; Greer, Charles W.; Whyte, Lyle G.

2007-01-01

The springs at Gypsum Hill and Colour Peak on Axel Heiberg Island in the Canadian Arctic originate from deep salt aquifers and are among the few known examples of cold springs in thick permafrost on Earth. The springs discharge cold anoxic brines (7.5 to 15.8% salts), with a mean oxidoreduction potential of −325 mV, and contain high concentrations of sulfate and sulfide. We surveyed the microbial diversity in the sediments of seven springs by denaturing gradient gel electrophoresis (DGGE) and analyzing clone libraries of 16S rRNA genes amplified with Bacteria and Archaea-specific primers. Dendrogram analysis of the DGGE banding patterns divided the springs into two clusters based on their geographic origin. Bacterial 16S rRNA clone sequences from the Gypsum Hill library (spring GH-4) were classified into seven phyla (Actinobacteria, Bacteroidetes, Firmicutes, Gemmatimonadetes, Proteobacteria, Spirochaetes, and Verrucomicrobia); Deltaproteobacteria and Gammaproteobacteria sequences represented half of the clone library. Sequences related to Proteobacteria (82%), Firmicutes (9%), and Bacteroidetes (6%) constituted 97% of the bacterial clone library from Colour Peak (spring CP-1). Most GH-4 archaeal clone sequences (79%) were related to the Crenarchaeota while half of the CP-1 sequences were related to orders Halobacteriales and Methanosarcinales of the Euryarchaeota. Sequences related to the sulfur-oxidizing bacterium Thiomicrospira psychrophila dominated both the GH-4 (19%) and CP-1 (45%) bacterial libraries, and 56 to 76% of the bacterial sequences were from potential sulfur-metabolizing bacteria. These results suggest that the utilization and cycling of sulfur compounds may play a major role in the energy production and maintenance of microbial communities in these unique, cold environments. PMID:17220254

Epilithic Cyanobacterial Communities of a Marine Tropical Beach Rock (Heron Island, Great Barrier Reef): Diversity and Diazotrophy▿

PubMed Central

Díez, Beatriz; Bauer, Karolina; Bergman, Birgitta

2007-01-01

The diversity and nitrogenase activity of epilithic marine microbes in a Holocene beach rock (Heron Island, Great Barrier Reef, Australia) with a proposed biological calcification “microbialite” origin were examined. Partial 16S rRNA gene sequences from the dominant mat (a coherent and layered pink-pigmented community spread over the beach rock) and biofilms (nonstratified, differently pigmented microbial communities of small shallow depressions) were retrieved using denaturing gradient gel electrophoresis (DGGE), and a clone library was retrieved from the dominant mat. The 16S rRNA gene sequences and morphological analyses revealed heterogeneity in the cyanobacterial distribution patterns. The nonheterocystous filamentous genus Blennothrix sp., phylogenetically related to Lyngbya, dominated the mat together with unidentified nonheterocystous filaments of members of the Pseudanabaenaceae and the unicellular genus Chroococcidiopsis. The dominance and three-dimensional intertwined distribution of these organisms were confirmed by nonintrusive scanning microscopy. In contrast, the less pronounced biofilms were dominated by the heterocystous cyanobacterial genus Calothrix, two unicellular Entophysalis morphotypes, Lyngbya spp., and members of the Pseudanabaenaceae family. Cytophaga-Flavobacterium-Bacteroides and Alphaproteobacteria phylotypes were also retrieved from the beach rock. The microbial diversity of the dominant mat was accompanied by high nocturnal nitrogenase activities (as determined by in situ acetylene reduction assays). A new DGGE nifH gene optimization approach for cyanobacterial nitrogen fixers showed that the sequences retrieved from the dominant mat were related to nonheterocystous uncultured cyanobacterial phylotypes, only distantly related to sequences of nitrogen-fixing cultured cyanobacteria. These data stress the occurrence and importance of nonheterocystous epilithic cyanobacteria, and it is hypothesized that such epilithic cyanobacteria are the principal nitrogen fixers of the Heron Island beach rock. PMID:17416688

Drinking habits are associated with changes in the dental plaque microbial community.

PubMed

Signoretto, Caterina; Bianchi, Franco; Burlacchini, Gloria; Sivieri, Francesca; Spratt, Dave; Canepari, Pietro

2010-02-01

Caries and gingivitis are the most prevalent oral infectious diseases of humans and are due to the accumulation of dental plaque (a microbial biofilm) on the tooth surface and at the gingival margin, respectively. Several in vitro and in vivo studies have shown that many natural components of foods and beverages inhibit the adhesion of and/or exert activity against oral bacteria. These biological activities have mainly been attributed to the polyphenol fraction. In order to explore the possibility that diet can alter the dental plaque community, in this study we evaluated the composition of the microbiota of supra- and subgingival plaque samples collected from 75 adult subjects with different drinking habits (drinkers of coffee, red wine, or water for at least 2 years) by analyzing the microbial population through the separation of PCR-amplified fragments using the denaturing gradient gel electrophoresis (DGGE) technique. The mean numbers of bands of the DGGE profiles from all three categories were evaluated. There were no significant differences between the two kinds of plaque collected from the control group (water drinkers), and this group showed the highest number of bands (supragingival plaque, 18.98 +/- 3.16 bands; subgingival plaque, 18.7 +/- 3.23 bands). The coffee and wine drinker groups generated the lowest numbers of bands for both supragingival plaque (coffee drinkers, 8.25 +/- 3.53 bands; wine drinkers, 7.93 +/- 2.55 bands) and subgingival plaque (coffee drinkers, 8.3 +/- 3.03 bands; wine drinkers, 7.65 +/- 1.68 bands). The differences between coffee drinkers or wine drinkers and the control group (water drinkers) were statistically significant. A total of 34 microorganisms were identified, and the frequency of their distribution in the three subject categories was analyzed. A greater percentage of subjects were positive for facultative aerobes when supragingival plaque was analyzed, while anaerobes were more frequent in subgingival plaque samples. It is noteworthy that the frequency of identification of anaerobes was significantly reduced when the frequencies for coffee and wine drinkers were compared with the frequencies for subjects in the control group. The DGGE profiles of the organisms in both plaque samples from all groups were generated and were used to construct dendrograms. A number of distinct clusters of organisms from water, coffee, and wine drinkers were formed. The clustering of some of the DGGE results into cohort-specific clusters implies similarities in the microbiotas within these groups and relevant differences in the microbiotas between cohorts. This supports the notion that the drinking habits of the subjects may influence the microbiota at both the supragingival and the subgingival levels.