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Sample records for psychrophilic enrichment cultures

  1. Phylogenetic analysis of anaerobic psychrophilic enrichment cultures obtained from a greenland glacier ice core

    NASA Technical Reports Server (NTRS)

    Sheridan, Peter P.; Miteva, Vanya I.; Brenchley, Jean E.

    2003-01-01

    The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at -9 degrees C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 x 10(7) cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at -2 degrees C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years.

  2. Phylogenetic analysis of anaerobic psychrophilic enrichment cultures obtained from a greenland glacier ice core

    NASA Technical Reports Server (NTRS)

    Sheridan, Peter P.; Miteva, Vanya I.; Brenchley, Jean E.

    2003-01-01

    The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at -9 degrees C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 x 10(7) cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at -2 degrees C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years.

  3. Phylogenetic analysis of anaerobic psychrophilic enrichment cultures obtained from a greenland glacier ice core.

    PubMed

    Sheridan, Peter P; Miteva, Vanya I; Brenchley, Jean E

    2003-04-01

    The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at -9 degrees C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 x 10(7) cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at -2 degrees C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years.

  4. Phylogenetic Analysis of Anaerobic Psychrophilic Enrichment Cultures Obtained from a Greenland Glacier Ice Core

    PubMed Central

    Sheridan, Peter P.; Miteva, Vanya I.; Brenchley, Jean E.

    2003-01-01

    The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at −9°C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 × 107 cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at −2°C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years. PMID:12676695

  5. Psychrophiles

    NASA Astrophysics Data System (ADS)

    Siddiqui, Khawar S.; Williams, Timothy J.; Wilkins, David; Yau, Sheree; Allen, Michelle A.; Brown, Mark V.; Lauro, Federico M.; Cavicchioli, Ricardo

    2013-05-01

    Psychrophilic (cold-adapted) microorganisms make a major contribution to Earth's biomass and perform critical roles in global biogeochemical cycles. The vast extent and environmental diversity of Earth's cold biosphere has selected for equally diverse microbial assemblages that can include archaea, bacteria, eucarya, and viruses. Underpinning the important ecological roles of psychrophiles are exquisite mechanisms of physiological adaptation. Evolution has also selected for cold-active traits at the level of molecular adaptation, and enzymes from psychrophiles are characterized by specific structural, functional, and stability properties. These characteristics of enzymes from psychrophiles not only manifest in efficient low-temperature activity, but also result in a flexible protein structure that enables biocatalysis in nonaqueous solvents. In this review, we examine the ecology of Antarctic psychrophiles, physiological adaptation of psychrophiles, and properties of cold-adapted proteins, and we provide a view of how these characteristics inform studies of astrobiology.

  6. Origin of Cyanide in Cultures of a Psychrophilic Basidiomycete1

    PubMed Central

    Stevens, Dennis L.; Strobel, Gary A.

    1968-01-01

    An unidentified psychrophilic basidiomycete used valine and isoleucine as precursors to hydrocyanic acid (HCN). As probable intermediates in the pathway from valine and isoleucine two cyanogenic glucosides, linamarin and lotaustralin, were demonstrated in fungus cultures. The fungus contained two β-glucosidases and an oxynitrilase which, acting together, were capable of releasing cyanide from both linamarin and lotaustralin. The two β-glucosidases were purified and compared as to pH optimum, Michaelis constant, energy of activation, thermal stability, and substrate specificity. The products of methyl ethyl ketone cyanohydrin and acetone cyanohydrin dissociation by the oxynitrilase were demonstrated to be HCN together with methyl ethyl ketone and acetone, respectively. The oxynitrilase attacked aliphatic hydroxynitriles, but showed no activity on aromatic hydroxynitriles. Images PMID:5651322

  7. Culture-independent characterization of novel psychrophilic magnetotactic cocci from Antarctic marine sediments.

    PubMed

    Abreu, Fernanda; Carolina, Ana; Araujo, V; Leão, Pedro; Silva, Karen Tavares; Carvalho, Fabíola Marques de; Cunha, Oberdan de Lima; Almeida, Luiz Gonzaga; Geurink, Corey; Farina, Marcos; Rodelli, Daniel; Jovane, Luigi; Pellizari, Vivian H; Vasconcelos, Ana Tereza de; Bazylinski, Dennis A; Lins, Ulysses

    2016-12-01

    Magnetotactic bacteria (MTB) are a heterogeneous group of ubiquitous aquatic microorganisms capable of biomineralizing nano-sized, membrane-bound, magnetic iron-rich mineral particles called magnetosomes. MTB are found in chemically-stratified aquatic sediments and/or water columns with a wide range of salinities, moderate to high temperatures, and pH varying from neutral to strongly alkaline. MTB from very cold environments have not been investigated to any great degree and here we characterize MTB from the low temperature Antarctic maritime region. Sediment samples were collected at nine sampling sites within Admiralty Bay, King George Island (62°23'S 58°27'W) from 2009 to 2013. Samples from five sites contained MTB and those from two of these sites contained large number of magnetotactic cocci that were studied using electron microscopy and molecular techniques. The magnetotactic cocci contained magnetosomes either arranged as two or four chains or as a disorganized cluster. The crystalline habit and composition of all magnetosomes analyzed with high-resolution transmission electron microscopy and energy dispersive X-ray microanalysis were consistent with elongated prismatic crystals of magnetite (Fe3 O4 ). The retrieved 16S rRNA gene sequences from magnetically-enriched magnetotactic cocci clustered into three distinct groups affiliated with the Alphaproteobacteria class of the Proteobacteria. Novel sequences of each phylogenetic cluster were confirmed using fluorescent in situ hybridization. Metagenomic data analysis of magnetically-enriched magnetotactic cocci revealed the presence of mam genes and MTB-specific hypothetical protein coding genes. Sequence homology and phylogenetic analysis indicated that predicted proteins are related to those of cultivated alphaproteobacterial MTB. The consistent and continuous low temperature of the sediment where the magnetotactic cocci are present (always below 1°C) suggests that these MTB from maritime Antarctica are

  8. Cultural Enrichment through Community Action.

    ERIC Educational Resources Information Center

    Wilson, O. J.

    This project was conceived as a technique for helping to eliminate a cultural void in the areas of art, music, and theatre in the service area of Western Kentucky University. To implement this concept, demonstrations were conducted in art, music, theatre, and in library and lecture resources in 16 counties over a four-year period. The attendance…

  9. CUE (CULTURE, UNDERSTANDING, ENRICHMENT)--SCIENCE.

    ERIC Educational Resources Information Center

    BROWN, ROBERT M.; AND OTHERS

    THIS PUBLICATION IS A TEACHING GUIDE TO PROVIDE GUIDANCE FOR INTEGRATING CAREFULLY SELECTED AUDIOVISUAL ITEMS INTO EXISTING NINTH-GRADE CURRICULUMS IN SCIENCE. IT IS ONE OF FIVE GUIDES PREPARED FOR USE IN PROJECT CUE, AN EXPERIMENTAL PROGRAM DESIGNED TO INCREASE CULTURAL UNDERSTANDING AND ENRICHMENT IN THE EDUCATIONAL PROGRAMS OF HIGH SCHOOLS. THE…

  10. CUE (CULTURE, UNDERSTANDING, ENRICHMENT)--INDUSTRIAL ARTS.

    ERIC Educational Resources Information Center

    BROWN, ROBERT M.; AND OTHERS

    THIS PUBLICATION IS A TEACHING GUIDE TO PROVIDE GUIDANCE FOR INTEGRATING CAREFULLY SELECTED AUDIOVISUAL ITEMS INTO EXISTING NINTH-GRADE CURRICULUMS IN INDUSTRIAL ARTS. IT IS ONE OF FIVE GUIDES PREPARED FOR USE IN PROJECT CUE, AN EXPERIMENTAL PROGRAM DESIGNED TO INCREASE CULTURAL UNDERSTANDING AND ENRICHMENT IN THE EDUCATIONAL PROGRAMS OF HIGH…

  11. CUE (CULTURE, UNDERSTANDING, ENRICHMENT)--SOCIAL STUDIES.

    ERIC Educational Resources Information Center

    BROWN, ROBERT M.; AND OTHERS

    THIS PUBLICATION IS A TEACHING GUIDE TO PROVIDE GUIDANCE FOR INTEGRATING CAREFULLY SELECTED AUDIOVISUAL ITEMS INTO EXISTING NINTH-GRADE CURRICULUMS IN SOCIAL STUDIES. IT IS ONE OF FIVE GUIDES PREPARED FOR USE IN PROJECT CUE. AN EXPERIMENTAL PROGRAM DESIGNED TO INCREASE CULTURAL UNDERSTANDING AND ENRICHMENT IN THE EDUCATIONAL PROGRAMS OF HIGH…

  12. CUE (CULTURE, UNDERSTANDING, ENRICHMENT)--SOCIAL STUDIES.

    ERIC Educational Resources Information Center

    BROWN, ROBERT M.; AND OTHERS

    THIS PUBLICATION IS A TEACHING GUIDE TO PROVIDE GUIDANCE FOR INTEGRATING CAREFULLY SELECTED AUDIOVISUAL ITEMS INTO EXISTING NINTH-GRADE CURRICULUMS IN SOCIAL STUDIES. IT IS ONE OF FIVE GUIDES PREPARED FOR USE IN PROJECT CUE. AN EXPERIMENTAL PROGRAM DESIGNED TO INCREASE CULTURAL UNDERSTANDING AND ENRICHMENT IN THE EDUCATIONAL PROGRAMS OF HIGH…

  13. CUE (CULTURE, UNDERSTANDING, ENRICHMENT)--SCIENCE.

    ERIC Educational Resources Information Center

    BROWN, ROBERT M.; AND OTHERS

    THIS PUBLICATION IS A TEACHING GUIDE TO PROVIDE GUIDANCE FOR INTEGRATING CAREFULLY SELECTED AUDIOVISUAL ITEMS INTO EXISTING NINTH-GRADE CURRICULUMS IN SCIENCE. IT IS ONE OF FIVE GUIDES PREPARED FOR USE IN PROJECT CUE, AN EXPERIMENTAL PROGRAM DESIGNED TO INCREASE CULTURAL UNDERSTANDING AND ENRICHMENT IN THE EDUCATIONAL PROGRAMS OF HIGH SCHOOLS. THE…

  14. [Phylogenetic diversity and cold-adaptive hydrolytic enzymes of culturable psychrophilic bacteria associated with sea ice from high latitude ocean, Artic].

    PubMed

    Yu, Yong; Li, Hui-Rong; Chen, Bo; Zeng, Yin-Xin; He, Jian-Feng

    2006-04-01

    The phylogenetic diversity of culturable psychrophilic bacteria associated with sea ice from high latitude sea (77 degrees 30'N - 81 degrees 12'N), Canadian Basin and Greenland sea Arctic, was investigated. A total of 37 psychrophilic strains were isolated using three different methods of ( i ) spread plate method: 100 microL of each dilution ice-melt sample was spreaded onto the surface of Marine 2216 agar (DIFCO laboratories, Detroit, MI) and incubated for 2 to 6 weeks at 4 degrees C; ( ii ) bath culture and spread plate method: 1 mL of sample was added to 9mL of NSW (unamended natural seawater, 0.2 microm prefiltered and autoclaved) and incubated for 1 months at - 1 degrees C, then spread plate method was used to isolate bacterial strains from the pre-cultured samples; ( iii ) cold shock, bath culture and spread plate method: samples were exposed to - 20 degrees C for 24h, then bacterial strains isolated by bath culture and spread plate method under aerobic conditions. Nearly half of psychrophilic strains are isolated by using method iii . 16S rDNA nearly full-length sequence analysis reveal that psychrophilic strains fall in two phylogenetic divisions, gamma-proteobacteria (in the genera Colwellia, Marinobacter, Shewanella, Thalassomonas, Glaciecola, Marinomonas and Pseudoalteromonas) and Cytophaga-Flexibacter-Bacteroides (in the genera Flavobacterium and Psychroflexus). Nine of bacterial isolates (BSi20007, BSi20497, BSi20517, BSi20537, BSi20170, BSi20001, BSi20002, BSi20675 and BSi20101) quite likely represent novel species (16S rDNA sequence similarity below 97%). One of strains (BSi20002) from Canadian Basin shows 100% sequence similarity to the Antarctic Weddell sea ice isolate Marinobacter sp. ANT8277, suggesting bacteria may have a bipolar distribution at the species level. AF283859 sequences were submitted to the BLAST search program of the National Center for Biotechnology Information website (NCBI, http://www. ncbi. nlm.nih. gov). Twenty sequences

  15. 21 CFR 866.2330 - Enriched culture medium.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Enriched culture medium. 866.2330 Section 866.2330...) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2330 Enriched culture medium. (a) Identification. An enriched culture medium is a device that consists primarily of liquid or...

  16. 21 CFR 866.2330 - Enriched culture medium.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Enriched culture medium. 866.2330 Section 866.2330...) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2330 Enriched culture medium. (a) Identification. An enriched culture medium is a device that consists primarily of liquid or...

  17. 21 CFR 866.2330 - Enriched culture medium.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Enriched culture medium. 866.2330 Section 866.2330...) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2330 Enriched culture medium. (a) Identification. An enriched culture medium is a device that consists primarily of liquid...

  18. 21 CFR 866.2330 - Enriched culture medium.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Enriched culture medium. 866.2330 Section 866.2330...) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2330 Enriched culture medium. (a) Identification. An enriched culture medium is a device that consists primarily of liquid...

  19. 21 CFR 866.2330 - Enriched culture medium.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Enriched culture medium. 866.2330 Section 866.2330...) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Microbiology Devices § 866.2330 Enriched culture medium. (a) Identification. An enriched culture medium is a device that consists primarily of liquid...

  20. Psychrophilic and Mesophilic Fungi in Frozen Food Products

    PubMed Central

    Kuehn, Harold H.; Gunderson, Millard F.

    1963-01-01

    The mold flora of certain frozen pastries and chicken pies was investigated. Molds were determined qualitatively or quantitatively, or both, by preparing pour plates of the blended product and incubating the plates at various temperatures. The mesophilic fungal flora developed on plates incubated at 10 and 20 C, whereas psychrophilic fungi were obtained on plates incubated at 0 and 5 C. About 2,000 cultures of fungi, representing about 100 different species, were isolated from various products. Four different brands of blueberry, two brands of cherry pastries, two brands of apple, and one brand of raspberry pastries were examined. In addition, two brands of chicken pies were studied. Blueberry pastries had a much higher total fungal population than the other products, although different brands of blueberry pastries varied considerably. Blueberry pastries had from 347 to 1,586 psychrophilic fungi per g. Cherry pastries had about 70 to 110 psychrophiles per g, and apple pastries had 19 to 92 psychrophiles per g. Chicken pies contained very few psychrophilic fungi, about 15 per g. Aureobasidium pullulans was recovered most frequently. About 90% of the psychrophilic fungi found in blueberry products was A. pullulans. Depending upon the brand of cherry pastry, either Phoma spp. or A. pullulans was the most common fungus present. Apple pastries also displayed brand variation, but were unique in having many mesophilic aspergilli. This genus was generally absent from other products. The Penicillium content of apple pastries was also rather high; 50% of the psychrophilic flora was represented by this genus. The psychrophilic fungal flora of chicken pies was composed primarily of penicillia (50%) and Chrysosporium pannorum (46%). PMID:13927344

  1. Bacteriophages may bias outcome of bacterial enrichment cultures.

    PubMed

    Muniesa, Maite; Blanch, Anicet R; Lucena, Francisco; Jofre, Juan

    2005-08-01

    Enrichment cultures are widely used for the isolation of bacteria in clinical, biotechnological, and environmental studies. However, competition, relative growth rates, or inhibitory effects may alter the outcome of enrichment cultures, causing the phenomenon known as enrichment bias. Bacteriophages are a major component in many microbial systems, and it abounds in natural settings. This abundance means that bacteriophages are likely to be present in many laboratory enrichment cultures. Our hypothesis was that bacteriophages present in the sample might bias the enriched subpopulation, since it can infect and lyse the target bacteria during the enrichment step once the bacteria reach a given density. Here we show that the presence of bacteriophages in Salmonella and Shigella enrichment cultures produced a significant reduction (more than 1 log unit) in the number of these bacteria compared with samples in which bacteriophages had been reduced by filtration through 0.45-microm non-protein-binding membranes. Furthermore, our data indicate that the Salmonella biotypes isolated after the enrichment culture change if bacteriophages are present, thus distorting the results of the analysis.

  2. Bacteriophages May Bias Outcome of Bacterial Enrichment Cultures

    PubMed Central

    Muniesa, Maite; Blanch, Anicet R.; Lucena, Francisco; Jofre, Juan

    2005-01-01

    Enrichment cultures are widely used for the isolation of bacteria in clinical, biotechnological, and environmental studies. However, competition, relative growth rates, or inhibitory effects may alter the outcome of enrichment cultures, causing the phenomenon known as enrichment bias. Bacteriophages are a major component in many microbial systems, and it abounds in natural settings. This abundance means that bacteriophages are likely to be present in many laboratory enrichment cultures. Our hypothesis was that bacteriophages present in the sample might bias the enriched subpopulation, since it can infect and lyse the target bacteria during the enrichment step once the bacteria reach a given density. Here we show that the presence of bacteriophages in Salmonella and Shigella enrichment cultures produced a significant reduction (more than 1 log unit) in the number of these bacteria compared with samples in which bacteriophages had been reduced by filtration through 0.45-μm non-protein-binding membranes. Furthermore, our data indicate that the Salmonella biotypes isolated after the enrichment culture change if bacteriophages are present, thus distorting the results of the analysis. PMID:16085813

  3. Enhanced isolation and culture of highly efficient psychrophilic oil-degrading bacteria from oil-contaminated soils in South Korea.

    PubMed

    Pham, Van H T; Kim, Jaisoo; Jeong, Seung-Woo

    2014-11-01

    It is known that isolation of oil-degrading bacterial strains is difficult at low temperatures, and the biodegradation efficiency of oil-contaminated soil is significantly reduced in cold weather. In this study, 14 strains were isolated from oil-contaminated soil that grew well at 10°C by using a newly developed culture method. 11 of the 14 isolates were successfully cultured in mineral salts medium containing 1,500 ppm of oil components, 500 ppm each kerosene, gasoline, and diesel as carbon sources, at 10°C for 2 weeks. The oil degradation efficiencies of these 11 isolates ranged from 36% to 100%, as measured by total petroleum hydrocarbon (TPH) degradation analyses. Three strains (Pseudomonas simiae G1-10O, P. taiwanensis Y1-4, and P. koreensis Gwa2) displayed complete degradation (100%), and six others (R frederiksbergensis G2-2, P arsenicoxydans Y2-1, R umsongensis Gwa3, P. migulae Gwa5, RhodococcusjialingiaeY 1-l , and R. qingshengii Y2-2) showed relatively high degradation efficiencies (> 70%). This study suggests that these isolates can be effectively utilised in thetreatment of oil-contaminated soil in landfarming, especially during winter.

  4. Mineralization of trichloroethylene by heterotrophic enrichment cultures

    SciTech Connect

    Phelps, T.J.; Ringelberg, D.; Mikell, A.T.; White, D.C. |; Fliermans, C.B.

    1988-12-31

    Microbial consortia capable of aerobically degrading greater than 99% of 50 mg/l exogenous trichloroethylene (TCE) have been enriched from TCE contaminated subsurface sediments. Concentrations of TCE greater than 300 mg/l were not degraded nor was TCE used as a sole energy source. Successful electron donors for growth included tryptone-yeast extract, methanol, methane or propane. The optimum temperature for growth was 22--37 C and the ph optimum was 7.0--8.1. Utilization of TCE occurred only after apparent microbial growth had ceased. The major end products recovered were hydrochloric acid and carbon dioxide. Minor products included dichloroethylene, vinylidine chloride and possibly chloroform.

  5. Biodegradation of mixed pesticides by mixed pesticide enriched cultures.

    PubMed

    Krishna, K Rama; Philip, Ligy

    2009-01-01

    This paper discusses the degradation kinetics of mixed (lindane, methyl parathion and carbofuran) pesticides by mixed pesticide enriched cultures (MEC) under various environmental conditions. The bacterial strains isolated from the mixed microbial consortium were identified as Pseudomonas aeruginosa (MTCC 9236), Bacillus sp. (MTCC 9235) and Chryseobacterium joostei (MTCC 9237). Batch studies were conducted to estimate the biokinetic parameters like the maximum specific growth rate (mu(max)), Yield Coefficient (Y(T)), half saturation concentration (K(s)) and inhibition concentration (Ki) for individual and mixed pesticide enriched cultures. The cultures enriched in a particular pollutant always showed high growth rate and low inhibition in that particular pollutant compared to MEC. After seven weeks of incubation, mixed pesticide enriched cultures were able to degrade 72% lindane, 95% carbofuran and 100% of methyl parathion in facultative co-metabolic conditions. In aerobic systems, degradation efficiencies of lindane methyl parathion and carbofuran were increased by the addition of 2g L(- 1) of dextrose. Though many metabolic compounds of mixed pesticides were observed at different time intervals, none of the metabolites were persistent. Based on the observed metabolites, a degradation pathway was postulated for different pesticides under various environmental conditions.

  6. Protein Chips for Detection of Salmonella spp. from Enrichment Culture

    PubMed Central

    Poltronieri, Palmiro; Cimaglia, Fabio; De Lorenzis, Enrico; Chiesa, Maurizio; Mezzolla, Valeria; Reca, Ida Barbara

    2016-01-01

    Food pathogens are the cause of foodborne epidemics, therefore there is a need to detect the pathogens in food productions rapidly. A pre-enrichment culture followed by selective agar plating are standard detection methods. Molecular methods such as qPCR have provided a first rapid protocol for detection of pathogens within 24 h of enrichment culture. Biosensors also may provide a rapid tool to individuate a source of Salmonella contamination at early times of pre-enrichment culture. Forty mL of Salmonella spp. enrichment culture were processed by immunoseparation using the Pathatrix, as in AFNOR validated qPCR protocols. The Salmonella biosensor combined with immunoseparation showed a limit of detection of 100 bacteria/40 mL, with a 400 fold increase to previous results. qPCR analysis requires processing of bead-bound bacteria with lysis buffer and DNA clean up, with a limit of detection of 2 cfu/50 μL. Finally, a protein chip was developed and tested in screening and identification of 5 common pathogen species, Salmonella spp., E. coli, S. aureus, Campylobacter spp. and Listeria spp. The protein chip, with high specificity in species identification, is proposed to be integrated into a Lab-on-Chip system, for rapid and reproducible screening of Salmonella spp. and other pathogen species contaminating food productions. PMID:27110786

  7. Substrate versatility of polyhydroxyalkanoate producing glycerol grown bacterial enrichment culture.

    PubMed

    Moralejo-Gárate, Helena; Kleerebezem, Robbert; Mosquera-Corral, Anuska; Campos, José Luis; Palmeiro-Sánchez, Tania; van Loosdrecht, Mark C M

    2014-12-01

    Waste-based polyhydroxyalkanoate (PHA) production by bacterial enrichments generally follows a three step strategy in which first the wastewater is converted into a volatile fatty acid rich stream that is subsequently used as substrate in a selector and biopolymer production units. In this work, a bacterial community with high biopolymer production capacity was enriched using glycerol, a non-fermented substrate. The substrate versatility and PHA production capacity of this community was studied using glucose, lactate, acetate and xylitol as substrate. Except for xylitol, very high PHA producing capacities were obtained. The PHA accumulation was comparable or even higher than with glycerol as substrate. This is the first study that established a high PHA content (≈70 wt%) with glucose as substrate in a microbial enrichment culture. The results presented in this study support the development of replacing pure culture based PHA production by bacterial enrichment cultures. A process where mixtures of substrates can be easily handled and the acidification step can potentially be avoided is described.

  8. Optimizing enrichment culture conditions for detecting Helicobacter pylori in foods.

    PubMed

    Jiang, Xiuping; Doyle, Michael P

    2002-12-01

    The survival and growth of Helicobacter pylori under enrichment conditions in fresh, autoclaved and irradiated ground beef were determined. H. pylori grew in autoclaved ground beef at 37 degrees C under microaerobic conditions in brain heart infusion broth with 7% horse serum at pH 7.3 after 3 to 7 days of lag time but did not grow within 7 days in irradiated (10 kGy) ground beef under the same enrichment conditions. Adjustment of the enrichment broth to pH 5.5 enabled the growth (ca. 2 log10 CFU/ml) of H. pylori within 7 days in the presence of irradiated ground beef and the prolific growth (ca. 3 to 4 log10 CFU/ml) of H. pylori within 3 days in the presence of autoclaved beef. H. pylori in fresh ground beef could not be isolated from enrichment media with antibiotics; however. H. pylori ureA could be detected by polymerase chain reaction (PCR) in such enrichment media after 1 to 3 days of incubation at 37 degrees C. The addition of supplements, i.e., 0.3% mucin, 0.05% ferrous sulfate, and 0.05% sodium pyruvate or 0.008 M urea, or the adjustment of the enrichment broth pH to 5.5 or 4.5 enabled the detection of H. pylori ureA in enrichment media incubated for 1, 2, 3, and/or 7 days at 37 degrees C. H. pylori in sterile milk refrigerated at 4 degrees C at an initial level of 10(6) CFU/ml was inactivated to an undetectable level within 6 days; however, H. pylori was not detected either by a PCR assay or by the plating of enrichment cultures of 120 raw bovine milk samples.

  9. [Reductive Dechlorination of Trichloroethylene by Benzoate-Enriched Anaerobic Cultures].

    PubMed

    Li, Jiang-wei; Yang, Xiao-yong; Hu, An-yi; Yu, Chang-ping

    2015-10-01

    Gas chromatography was used to monitor the reductive dechlorination of trichloroethylene (TCE) by anaerobic enrichment cultures with benzoate as the sole carbon source. The 454 pyrosequencing technique was used to investigate the microbial community and the real-time quantitative PCR was used to quantify the gene copies of Dehalococcoides spp. (DHC). The results showed that TCE was dechlorinated to vinyl chloride along with the formation of methane in 94 days. The anaerobic enrichment cultures exhibited a high diversity, which were classified into 16 phyla, 33 classes, 52 orders, 88 families and 129 genera, while 51.2% of them belonged to unclassified group, which inferred that there were a large portion of bacteria with unknown functional in this system. Degradation of TCE was accomplished by reductive dechlorinating and other functional populations, and the DHC which carried tceA gene could be the dominant reductive dechlorinating populations in the system.

  10. Enriching the Student Experience Through a Collaborative Cultural Learning Model.

    PubMed

    McInally, Wendy; Metcalfe, Sharon; Garner, Bonnie

    2015-01-01

    This article provides a knowledge and understanding of an international, collaborative, cultural learning model for students from the United States and Scotland. Internationalizing the student experience has been instrumental for student learning for the past eight years. Both countries have developed programs that have enriched and enhanced the overall student learning experience, mainly through the sharing of evidence-based care in both hospital and community settings. Student learning is at the heart of this international model, and through practice learning, leadership, and reflective practice, student immersion in global health care and practice is immense. Moving forward, we are seeking new opportunities to explore learning partnerships to provide this collaborative cultural learning experience.

  11. Identifying numerically abundant culturable bacteria from complex communities: an example from a lignin enrichment culture.

    PubMed Central

    González, J M; Whitman, W B; Hodson, R E; Moran, M A

    1996-01-01

    Culturable bacteria that were numerically important members of a marine enrichment community were identified and characterized phylogenetically. Selective and nonselective isolation methods were used to obtain 133 culturable bacterial isolates from model marine communities enriched with the high-molecular-weight (lignin-rich) fraction of pulp mill effluent. The culture collection was screened against community DNA from the lignin enrichments by whole-genome hybridization methods, and three marine bacterial isolates were identified as being numerically important in the communities. One isolate was in the alpha-subclass of Proteobacteria, and the other two were in the gamma-subclass of Proteobacteria. Isolate-specific 16S rRNA oligonucleotide probes designed to precisely quantify the isolates in the lignin enrichment communities indicated contributions ranging from 2 to 32% of enrichment DNA, values nearly identical to those originally obtained by the simpler whole-genome hybridization method. Two 16S rRNA sequences closely related to that of one of the isolates, although not identical, were amplified via PCR from the seawater sample originally used to inoculate the enrichment medium. Partial sequences of 14 other isolates revealed significant phylogenetic diversity and unusual sequences among the culturable lignin enrichment bacteria, with the Proteobacteria, Cytophaga-Flavobacterium, and gram-positive groups represented. PMID:8953714

  12. Culture Enriched Molecular Profiling of the Cystic Fibrosis Airway Microbiome

    PubMed Central

    Sibley, Christopher D.; Grinwis, Margot E.; Field, Tyler R.; Eshaghurshan, Christina S.; Faria, Monica M.; Dowd, Scot E.; Parkins, Michael D.; Rabin, Harvey R.; Surette, Michael G.

    2011-01-01

    The microbiome of the respiratory tract, including the nasopharyngeal and oropharyngeal microbiota, is a dynamic community of microorganisms that is highly diverse. The cystic fibrosis (CF) airway microbiome refers to the polymicrobial communities present in the lower airways of CF patients. It is comprised of chronic opportunistic pathogens (such as Pseudomonas aeruginosa) and a variety of organisms derived mostly from the normal microbiota of the upper respiratory tract. The complexity of these communities has been inferred primarily from culture independent molecular profiling. As with most microbial communities it is generally assumed that most of the organisms present are not readily cultured. Our culture collection generated using more extensive cultivation approaches, reveals a more complex microbial community than that obtained by conventional CF culture methods. To directly evaluate the cultivability of the airway microbiome, we examined six samples in depth using culture-enriched molecular profiling which combines culture-based methods with the molecular profiling methods of terminal restriction fragment length polymorphisms and 16S rRNA gene sequencing. We demonstrate that combining culture-dependent and culture-independent approaches enhances the sensitivity of either approach alone. Our techniques were able to cultivate 43 of the 48 families detected by deep sequencing; the five families recovered solely by culture-independent approaches were all present at very low abundance (<0.002% total reads). 46% of the molecular signatures detected by culture from the six patients were only identified in an anaerobic environment, suggesting that a large proportion of the cultured airway community is composed of obligate anaerobes. Most significantly, using 20 growth conditions per specimen, half of which included anaerobic cultivation and extended incubation times we demonstrate that the majority of bacteria present can be cultured. PMID:21829484

  13. Absence of microbial mineralization of lignin in anaerobic enrichment cultures.

    PubMed Central

    Odier, E; Monties, B

    1983-01-01

    The existence of anaerobic biodegradation of lignin was examined in mixed microflora. Egyptian soil samples, in which rapid mineralization of organic matter takes place in the presence of an important anaerobic microflora, were used to obtain the anaerobic enrichment cultures for this study. Specifically, 14CO2 or [14C]lignin wood was used to investigate the release of labeled gaseous or soluble degradation products of lignin in microbial cultures. No conversion of 14C-labeled lignin to 14CO2 or 14CH4 was observed after 6 months of incubation at 30 degrees C in anaerobic conditions with or without NO3-. A small increase in soluble radioactivity was observed in certain cultures, but it could not be related to the release of catabolic products during the anaerobic biodegradation of lignin. PMID:6639020

  14. Anaerobic oxidation of acetylene by estuarine sediments and enrichment cultures

    USGS Publications Warehouse

    Culbertson, Charles W.; Zehnder, Alexander J. B.; Oremland, Ronald S.

    1981-01-01

    Acetylene disappeared from the gas phase of anaerobically incubated estuarine sediment slurries, and loss was accompanied by increased levels of carbon dioxide. Acetylene loss was inhibited by chloramphenicol, air, and autoclaving. Addition of 14C2H2 to slurries resulted in the formation of 14CO2 and the transient appearance of 14C-soluble intermediates, of which acetate was a major component. Acetylene oxidation stimulated sulfate reduction; however, sulfate reduction was not required for the loss of C2H2 to occur. Enrichment cultures were obtained which grew anaerobically at the expense of C2H2.

  15. Anaerobic Oxidation of Acetylene by Estuarine Sediments and Enrichment Cultures

    PubMed Central

    Culbertson, Charles W.; Zehnder, Alexander J. B.; Oremland, Ronald S.

    1981-01-01

    Acetylene disappeared from the gas phase of anaerobically incubated estuarine sediment slurries, and loss was accompanied by increased levels of carbon dioxide. Acetylene loss was inhibited by chloramphenicol, air, and autoclaving. Addition of 14C2H2 to slurries resulted in the formation of 14CO2 and the transient appearance of 14C-soluble intermediates, of which acetate was a major component. Acetylene oxidation stimulated sulfate reduction; however, sulfate reduction was not required for the loss of C2H2 to occur. Enrichment cultures were obtained which grew anaerobically at the expense of C2H2. PMID:16345714

  16. Effect of Enrichment Medium on Real-time Detection of Salmonella enterica from Lettuce and Tomato Enrichment Cultures

    USDA-ARS?s Scientific Manuscript database

    vThree commonly used enrichment broths for detection of Salmonella (Buffered Peptone Water – BPW, Tryptic Soy Broth – TSB, and Universal Preenrichment Broth – UPB) were compared for use in real time SYBR Green PCR detection of Salmonella introduced into enrichment cultures made from store bought let...

  17. Dimethylamine biodegradation by mixed culture enriched from drinking water biofilter.

    PubMed

    Liao, Xiaobin; Chen, Chao; Zhang, Jingxu; Dai, Yu; Zhang, Xiaojian; Xie, Shuguang

    2015-01-01

    Dimethylamine (DMA) is one of the important precursors of drinking water disinfection by-product N-nitrosodimethylamine (NDMA). Reduction of DMA to minimize the formation of carcinogenic NDMA in drinking water is of practical importance. Biodegradation plays a major role in elimination of DMA pollution in the environment, yet information on DMA removal by drinking water biofilter is still lacking. In this study, microcosms with different treatments were constructed to investigate the potential of DMA removal by a mixed culture enriched from a drinking water biofilter and the effects of carbon and nitrogen sources. DMA could be quickly mineralized by the enrichment culture. Amendment of a carbon source, instead of a nitrogen source, had a profound impact on DMA removal. A shift in bacterial community structure was observed with DMA biodegradation, affected by carbon and nitrogen sources. Proteobacteria was the predominant phylum group in DMA-degrading microcosms. Microorganisms from a variety of bacterial genera might be responsible for the rapid DMA mineralization. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Biotransformation of acyclovir by an enriched nitrifying culture.

    PubMed

    Xu, Yifeng; Yuan, Zhiguo; Ni, Bing-Jie

    2017-03-01

    This work evaluates the biodegradation of the antiviral drug acyclovir by an enriched nitrifying culture during ammonia oxidation and without the addition of ammonium. The study on kinetics was accompanied with the structural elucidation of biotransformation products through batch biodegradation experiments at two different initial levels of acyclovir (15 mg L(-1) and 15 μg L(-1)). The pseudo first order kinetic studies of acyclovir in the presence of ammonium indicated the higher degradation rates under higher ammonia oxidation rates than those constant degradation rates in the absence of ammonium. The positive correlation was found between acyclovir degradation rate and ammonia oxidation rate, confirming the cometabolism of acyclovir by the enriched nitrifying culture in the presence of ammonium. Formation of the product carboxy-acyclovir (P239) indicated the main biotransformation pathway was aerobic oxidation of the terminal hydroxyl group, which was independent on the metabolic type (i.e. cometabolism or metabolism). This enzyme-linked reaction might be catalyzed by monooxygenase from ammonia oxidizing bacteria or heterotrophs. The formation of carboxy-acyclovir was demonstrated to be irrelevant to the acyclovir concentrations applied, indicating the revealed biotransformation pathway might be the dominant removal pathway of acyclovir in wastewater treatment.

  19. Enrichment of spinal cord cell cultures with motoneurons

    PubMed Central

    1978-01-01

    Spinal cord cell cultures contain several types of neurons. Two methods are described for enriching such cultures with motoneurons (defined here simply as cholinergic cells that are capable of innervating muscle). In the first method, 7-day embryonic chick spinal cord neurons were separated according to size by 1 g velocity sedimentation. It is assumed that cholinergic motoneurons are among the largest cells present at this stage. The spinal cords were dissociated vigorously so that 95-98% of the cells in the initial suspension were isolated from one another. Cells in leading fractions (large cell fractions: LCFs) contain about seven times as much choline acetyltransferase (CAT) activity per unit cytoplasm as do cells in trailing fractions (small cell fractions: SCFs). Muscle cultures seeded with LCFs develop 10-70 times as much CAT as cultures seeded with SCFs and six times as much CAT as cultures seeded with control (unfractionated) spinal cord cells. More than 20% of the large neurons in LCF-muscle cultures innervate nearby myotubes. In the second method, neurons were gently dissociated from 4-day embryonic spinal cords and maintained in vitro. This approach is based on earlier observations that cholinergic neurons are among the first cells to withdraw form the mitotic cycle in the developing chick embryo (Hamburger, V. 1948. J. Comp. Neurol. 88:221- 283; and Levi-Montalcini, R. 1950. J. Morphol. 86:253-283). 4-Day spinal cord-muscle cultures develop three times as much CAT as do 7-day spinal cord-muscle plates, prepared in the same (gentle) manner. More than 50% of the relatively large 4-day neurons innervate nearby myotubes. Thus, both methods are useful first steps toward the complete isolation of motoneurons. Both methods should facilitate study of the development of cholinergic neurons and of nerve-muscle synapse formation. PMID:566275

  20. Anaerobic naphthalene degradation by a sulfate-reducing enrichment culture.

    PubMed

    Meckenstock, R U; Annweiler, E; Michaelis, W; Richnow, H H; Schink, B

    2000-07-01

    Anaerobic naphthalene degradation by a sulfate-reducing enrichment culture was studied by substrate utilization tests and identification of metabolites by gas chromatography-mass spectrometry. In substrate utilization tests, the culture was able to oxidize naphthalene, 2-methylnaphthalene, 1- and 2-naphthoic acids, phenylacetic acid, benzoic acid, cyclohexanecarboxylic acid, and cyclohex-1-ene-carboxylic acid with sulfate as the electron acceptor. Neither hydroxylated 1- or 2-naphthoic acid derivatives and 1- or 2-naphthol nor the monoaromatic compounds ortho-phthalic acid, 2-carboxy-1-phenylacetic acid, and salicylic acid were utilized by the culture within 100 days. 2-Naphthoic acid accumulated in all naphthalene-grown cultures. Reduced 2-naphthoic acid derivatives could be identified by comparison of mass spectra and coelution with commercial reference compounds such as 1,2,3, 4-tetrahydro-2-naphthoic acid and chemically synthesized decahydro-2-naphthoic acid. 5,6,7,8-Tetrahydro-2-naphthoic acid and octahydro-2-naphthoic acid were tentatively identified by their mass spectra. The metabolites identified suggest a stepwise reduction of the aromatic ring system before ring cleavage. In degradation experiments with [1-(13)C]naphthalene or deuterated D(8)-naphthalene, all metabolites mentioned derived from the introduced labeled naphthalene. When a [(13)C]bicarbonate-buffered growth medium was used in conjunction with unlabeled naphthalene, (13)C incorporation into the carboxylic group of 2-naphthoic acid was shown, indicating that activation of naphthalene by carboxylation was the initial degradation step. No ring fission products were identified.

  1. Identification of toluene degraders in a methanogenic enrichment culture.

    PubMed

    Fowler, S Jane; Gutierrez-Zamora, Maria-Luisa; Manefield, Mike; Gieg, Lisa M

    2014-09-01

    Methanogenic biodegradation involves the cooperative metabolism of syntrophic bacteria that catalyse the initial attack and subsequent degradation of hydrocarbons, and methanogens that convert intermediates such as hydrogen and carbon dioxide, formate, and/or acetate to methane. The identity of syntrophic microbes and the nature of their interactions with other syntrophs and methanogens are not well understood. Furthermore, it is difficult to isolate the organisms responsible for the initial activation and subsequent degradation of hydrocarbon substrates under methanogenic conditions due to the thermodynamic relationships that exist among microbes in methanogenic communities. We used time-resolved RNA stable isotope probing and RT-qPCR to identify the organisms involved in the initial attack on toluene and subsequent degradation reactions in a highly enriched toluene-degrading methanogenic culture. Our results reveal the importance of a Desulfosporosinus sp. in anaerobic toluene activation in the culture. Other organisms that appear to play roles in toluene degradation include Syntrophaceae, Desulfovibrionales and Chloroflexi. The high bacterial diversity observed in this culture and the extensive labelling of different phylogenetic groups over the course of the stable isotope probing experiment highlight the complexity of the relationships that exist in methanogenic ecosystems. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  2. Bordetella avium Antibiotic Resistance, Novel Enrichment Culture, and Antigenic Characterization

    PubMed Central

    Beach, Nathan M.; Thompson, Seth; Mutnick, Rachel; Brown, Lisa; Kettig, Gina; Puffenbarger, Robyn; Miyamoto, David; Temple, Louise

    2012-01-01

    Bordetella avium continues to be an economic issue in the turkey industry as the causative agent of bordetellosis, which often leads to serious secondary infections. This study presents a broad characterization of the antibiotic resistance patterns in this diverse collection of B. avium strains collected over the past thirty years. In addition, the plasmid basis for the antibiotic resistance was characterized. The antibiotic resistance pattern allowed the development of a novel enrichment culture method that was subsequently employed to gather new isolates from diseased turkeys and a healthy sawhet owl. While a healthy turkey flock was shown to seroconvert by four weeks-of-age, attempts to culture B. avium from healthy turkey poults were unsuccessful. Western blot of B. avium strains using pooled serum from diseased and healthy commercial turkey flocks revealed both antigenic similarities and differences between strains. In sum, the work documents the continued exposure of commercial turkey flocks to B. avium and the need for development of an effective, inexpensive vaccine to control spread of the disease. PMID:22721730

  3. Rare bacterium of new genus isolated with prolonged enrichment culture.

    PubMed

    Hashizume, Akiko; Fudou, Ryosuke; Jojima, Yasuko; Nakai, Ryohsuke; Hiraishi, Akira; Tabuchi, Akira; Sen, Kikuo; Shibai, Hiroshiro

    2004-01-01

    Dynamic change in microbial flora was monitored with an oxygen electrode. The 1st phase microorganisms, which first grew well in LB medium, were followed by the 2nd phase microorganisms, which supposedly assimilated microbial cells of the 1st phase and their metabolites. In a similar way, a change in microbial flora was observed from the 1st phase to the 4th phase in 84 hr. Based on this observation, prolonged enrichment culture was done for as long as two months to increase the ratio of existence of rare microorganisms. From these culture liquids, four slow-growing bacteria (provisionally named Shinshu-ah1, -ah2, -ah3, and -ah4), which formed scarcely visible small colonies, were isolated. Sequence analysis of their 16S rDNA showed that Shinshu-ah1 had 97% homology with Bradyrhizobium japonicum and uncultured alpha proteobacterium clone blaii 16, Shinshu-ah2 91% with Rasbo bacterium, Alpha proteobacterium 34619, Bradyrhizobium genosp. P, Afipia felis and an unidentified bacterium, Shinshu-ah3 99% with Methylobacterium mesophilicum, and Shinshu-ah4 95% with Agromyces ramosus DSM 43045. Phylogenetic study indicated that Shinshu-ah2 had a possibility to form a new family, Shinshu-ah1 a new genus, and Shinshu-ah4 a new species.

  4. Selective enrichment media bias the types of Salmonella enterica strains isolated from mixed strain cultures and complex enrichment broths.

    PubMed

    Gorski, Lisa

    2012-01-01

    For foodborne outbreak investigations it can be difficult to isolate the relevant strain from food and/or environmental sources. If the sample is contaminated by more than one strain of the pathogen the relevant strain might be missed. In this study mixed cultures of Salmonella enterica were grown in one set of standard enrichment media to see if culture bias patterns emerged. Nineteen strains representing four serogroups and ten serotypes were compared in four-strain mixtures in Salmonella-only and in cattle fecal culture enrichment backgrounds using Salmonella enrichment media. One or more strain(s) emerged as dominant in each mixture. No serotype was most fit, but strains of serogroups C2 and E were more likely to dominate enrichment culture mixtures than strains of serogroups B or C1. Different versions of Rappaport-Vassiliadis (RV) medium gave different patterns of strain dominance in both Salmonella-only and fecal enrichment culture backgrounds. The fittest strains belonged to serogroups C1, C2, and E, and included strains of S. Infantis, S. Thompson S. Newport, S. 6,8:d:-, and S. Give. Strains of serogroup B, which included serotypes often seen in outbreaks such as S. Typhimurium, S. Saintpaul, and S. Schwarzengrund were less likely to emerge as dominant strains in the mixtures when using standard RV as part of the enrichment. Using a more nutrient-rich version of RV as part of the protocol led to a different pattern of strains emerging, however some were still present in very low numbers in the resulting population. These results indicate that outbreak investigations of food and/or other environmental samples should include multiple enrichment protocols to ensure isolation of target strains of Salmonella.

  5. Biotechnological uses of enzymes from psychrophiles

    PubMed Central

    Cavicchioli, R.; Charlton, T.; Ertan, H.; Omar, S. Mohd; Siddiqui, K. S.; Williams, T. J.

    2011-01-01

    Summary The bulk of the Earth's biosphere is cold (e.g. 90% of the ocean's waters are ≤ 5°C), sustaining a broad diversity of microbial life. The permanently cold environments vary from the deep ocean to alpine reaches and to polar regions. Commensurate with the extent and diversity of the ecosystems that harbour psychrophilic life, the functional capacity of the microorganisms that inhabitat the cold biosphere are equally diverse. As a result, indigenous psychrophilic microorganisms provide an enormous natural resource of enzymes that function effectively in the cold, and these cold‐adapted enzymes have been targeted for their biotechnological potential. In this review we describe the main properties of enzymes from psychrophiles and describe some of their known biotechnological applications and ways to potentially improve their value for biotechnology. The review also covers the use of metagenomics for enzyme screening, the development of psychrophilic gene expression systems and the use of enzymes for cleaning. PMID:21733127

  6. Psychrophilic Lipase from Arctic Bacterium

    PubMed Central

    Ramle, Zakiah; Rahim, Rashidah Abdul

    2016-01-01

    A lipase producer psychrophilic microorganism isolated from Arctic sample was studied. The genomic DNA of the isolate was extracted using modified CTAB method. Identification of the isolate by morphological and 16S rRNA sequence analysis revealed that the isolate is closely related to Arthrobacter gangotriensis (97% similarity). A. gangotriensis was determined as positive lipase producer based on the plate screening using specific and sensitive plate assay of Rhodamine B. The PCR result using Arthrobacter sp.’s full lipase gene sequence as the template primers emphasised a possible lipase gene at 900 bp band size. The gene is further cloned in a suitable vector system for expression of lipase. PMID:27965754

  7. Isolation and Physiological Characterization of Psychrophilic Denitrifying Bacteria from Permanently Cold Arctic Fjord Sediments (Svalbard, Norway)

    NASA Technical Reports Server (NTRS)

    Canion, Andy; Prakash, Om; Green, Stefan J.; Jahnke, Linda; Kuypers, Marcel M. M.; Kostka, Joel E.

    2013-01-01

    A large proportion of reactive nitrogen loss from polar sediments is mediated by denitrification, but microorganisms mediating denitrification in polar environments remain poorly characterized. A combined approach of most-probable-number (MPN) enumeration, cultivation and physiological characterization was used to describe psychrophilic denitrifying bacterial communities in sediments of three Arctic fjords in Svalbard (Norway). A MPN assay showed the presence of 10(sup 3)-10(sup 6) cells of psychrophilic nitrate-respiring bacteria g(sup -1) of sediment. Fifteen strains within the Proteobacteria were isolated using a systematic enrichment approach with organic acids as electron donors and nitrate as an electron acceptor. Isolates belonged to five genera, including Shewanella, Pseudomonas, Psychromonas (Gammaproteobacteria), Arcobacter (Epsilonproteobacteria) and Herminiimonas (Betaproteobacteria). All isolates were denitrifiers, except Shewanella, which exhibited the capacity for dissimilatory nitrate reduction to ammonium (DNRA). Growth from 0 to 40 degC demonstrated that all genera except Shewanella were psychrophiles with optimal growth below 15 degC, and adaptation to low temperature was demonstrated as a shift from primarily C16:0 saturated fatty acids to C16:1 monounsaturated fatty acids at lower temperatures. This study provides the first targeted enrichment and characterization of psychrophilic denitrifying bacteria from polar sediments, and two genera, Arcobacter and Herminiimonas, are isolated for the first time from permanently cold marine sediments.

  8. Isolation and physiological characterization of psychrophilic denitrifying bacteria from permanently cold Arctic fjord sediments (Svalbard, Norway).

    PubMed

    Canion, Andy; Prakash, Om; Green, Stefan J; Jahnke, Linda; Kuypers, Marcel M M; Kostka, Joel E

    2013-05-01

    A large proportion of reactive nitrogen loss from polar sediments is mediated by denitrification, but microorganisms mediating denitrification in polar environments remain poorly characterized. A combined approach of most-probable-number (MPN) enumeration, cultivation and physiological characterization was used to describe psychrophilic denitrifying bacterial communities in sediments of three Arctic fjords in Svalbard (Norway). A MPN assay showed the presence of 10(3) -10(6) cells of psychrophilic nitrate-respiring bacteria g(-1) of sediment. Fifteen strains within the Proteobacteria were isolated using a systematic enrichment approach with organic acids as electron donors and nitrate as an electron acceptor. Isolates belonged to five genera, including Shewanella, Pseudomonas, Psychromonas (Gammaproteobacteria), Arcobacter (Epsilonproteobacteria) and Herminiimonas (Betaproteobacteria). All isolates were denitrifiers, except Shewanella, which exhibited the capacity for dissimilatory nitrate reduction to ammonium (DNRA). Growth from 0 to 40°C demonstrated that all genera except Shewanella were psychrophiles with optimal growth below 15°C, and adaptation to low temperature was demonstrated as a shift from primarily C16:0 saturated fatty acids to C16:1 monounsaturated fatty acids at lower temperatures. This study provides the first targeted enrichment and characterization of psychrophilic denitrifying bacteria from polar sediments, and two genera, Arcobacter and Herminiimonas, are isolated for the first time from permanently cold marine sediments.

  9. Anaerobic biodegradation of pentachlorophenol in mixtures containing cadmium by two physiologically distinct microbial enrichment cultures.

    PubMed

    Kamashwaran, S R; Crawford, D L

    2001-07-01

    Anaerobic biodegradation of pentachlorophenol (PCP), in mixtures containing cadmium (Cd), by sulfidogenic (SRB) and methanogenic (MET) enrichment cultures, was studied. Removal of 91-93% of PCP occurred in both SRB- and MET-enriched cultures, in the absence of Cd, within 82 days. The presence of soluble Cd initially decreased the rate of PCP removal by the enrichment cultures, but PCP removal rates improved as the Cd precipitated. GC-MS, 14C-PCP, and 13C-PCP studies confirmed mineralization of PCP by both enrichment cultures, as well as the incorporation of PCP carbon into specific phospholipid fatty acids (PLFAs) of the cell membranes of PCP-degrading anaerobes. This is the first report on anaerobic biodegradation of PCP by SRB- and MET-enriched cultures in the presence, with simultaneous precipitation, of the toxic heavy metal Cd, and of the incorporation of PCP carbons into specific PLFAs of the anaerobic bacterial cells.

  10. Microbial community and function of enrichment cultures with methane and toluene.

    PubMed

    Su, Yao; Xia, Fang-Fang; Tian, Bao-Hu; Li, Wei; He, Ruo

    2014-04-01

    The interaction effect of co-existence of toluene and CH4 on community and activity of methanotrophs and toluene-degrading bacteria was characterized in three consortia enriched with CH4 and toluene (MT), toluene (T), and CH4 (M), respectively, in this study. The CH4 oxidation activity in the enrichment culture of MT was significantly lower than that of M at the end of the experiment (P = 0.001). The toluene degradation rate could be enhanced by continuous addition of CH4 and toluene in the initial days, but it was inhibited in the later days. Phylogenetic analysis of 16S rRNA genes showed that Proteobacteria and Bacteroidetes were dominant in the three enriched consortia, but the community of methanotrophs and toluene-degrading bacteria was significantly affected by the co-existence of CH4 and toluene. Both Methylosinus (91.8 %) and Methylocystis (8.2 %) were detected in the enrichment culture of MT, while only Methylocystis species were detected in M. The toluene-degrading bacteria including Burkholderia, Flavobacteria, Microbacterium, and Azoarcus were all detected in the enrichment culture of T. However, only Azoarcus was found in the enrichment culture of MT. Significantly higher contents of extracellular polymeric substances polysaccharose and protein in the enrichment culture of MT than that of T and M suggested that a higher environmental stress occurred in the enrichment culture of MT.

  11. Co-Enriching Microflora Associated with Culture Based Methods to Detect Salmonella from Tomato Phyllosphere

    PubMed Central

    Ottesen, Andrea R.; Gonzalez, Antonio; Bell, Rebecca; Arce, Caroline; Rideout, Steven; Allard, Marc; Evans, Peter; Strain, Errol; Musser, Steven; Knight, Rob; Brown, Eric; Pettengill, James B.

    2013-01-01

    The ability to detect a specific organism from a complex environment is vitally important to many fields of public health, including food safety. For example, tomatoes have been implicated numerous times as vehicles of foodborne outbreaks due to strains of Salmonella but few studies have ever recovered Salmonella from a tomato phyllosphere environment. Precision of culturing techniques that target agents associated with outbreaks depend on numerous factors. One important factor to better understand is which species co-enrich during enrichment procedures and how microbial dynamics may impede or enhance detection of target pathogens. We used a shotgun sequence approach to describe taxa associated with samples pre-enrichment and throughout the enrichment steps of the Bacteriological Analytical Manual's (BAM) protocol for detection of Salmonella from environmental tomato samples. Recent work has shown that during efforts to enrich Salmonella (Proteobacteria) from tomato field samples, Firmicute genera are also co-enriched and at least one co-enriching Firmicute genus (Paenibacillus sp.) can inhibit and even kills strains of Salmonella. Here we provide a baseline description of microflora that co-culture during detection efforts and the utility of a bioinformatic approach to detect specific taxa from metagenomic sequence data. We observed that uncultured samples clustered together with distinct taxonomic profiles relative to the three cultured treatments (Universal Pre-enrichment broth (UPB), Tetrathionate (TT), and Rappaport-Vassiliadis (RV)). There was little consistency among samples exposed to the same culturing medias, suggesting significant microbial differences in starting matrices or stochasticity associated with enrichment processes. Interestingly, Paenibacillus sp. (Salmonella inhibitor) was significantly enriched from uncultured to cultured (UPB) samples. Also of interest was the sequence based identification of a number of sequences as Salmonella despite

  12. Continuous enrichment cultures: insights into prokaryotic diversity and metabolic interactions in deep-sea vent chimneys.

    PubMed

    Postec, Anne; Lesongeur, Françoise; Pignet, Patricia; Ollivier, Bernard; Querellou, Joël; Godfroy, Anne

    2007-11-01

    The prokaryotic diversity of culturable thermophilic communities of deep-sea hydrothermal chimneys was analysed using a continuous enrichment culture performed in a gas-lift bioreactor, and compared to classical batch enrichment cultures in vials. Cultures were conducted at 60 degrees C and pH 6.5 using a complex medium containing carbohydrates, peptides and sulphur, and inoculated with a sample of a hydrothermal black chimney collected at the Rainbow field, Mid-Atlantic Ridge, at 2,275 m depth. To assess the relevance of both culture methods, bacterial and archaeal diversity was studied using cloning and sequencing, DGGE, and whole-cell hybridisation of 16S rRNA genes. Sequences of heterotrophic microorganisms belonging to the genera Marinitoga, Thermosipho, Caminicella (Bacteria) and Thermococcus (Archaea) were obtained from both batch and continuous enrichment cultures while sequences of the autotrophic bacterial genera Deferribacter and Thermodesulfatator were only detected in the continuous bioreactor culture. It is presumed that over time constant metabolite exchanges will have occurred in the continuous enrichment culture enabling the development of a more diverse prokaryotic community. In particular, CO(2) and H(2) produced by the heterotrophic population would support the growth of autotrophic populations. Therefore, continuous enrichment culture is a useful technique to grow over time environmentally representative microbial communities and obtain insights into prokaryotic species interactions that play a crucial role in deep hydrothermal environments.

  13. Capturing the diversity of the human gut microbiota through culture-enriched molecular profiling.

    PubMed

    Lau, Jennifer T; Whelan, Fiona J; Herath, Isiri; Lee, Christine H; Collins, Stephen M; Bercik, Premysl; Surette, Michael G

    2016-07-01

    The human gut microbiota has been implicated in most aspects of health and disease; however, most of the bacteria in this community are considered unculturable, so studies have relied on molecular-based methods. These methods generally do not permit the isolation of organisms, which is required to fully explore the functional roles of bacteria for definitive association with host phenotypes. Using a combination of culture and 16S rRNA gene sequencing, referred to as culture-enriched molecular profiling, we show that the majority of the bacteria identified by 16S sequencing of the human gut microbiota can be cultured. Five fresh, anaerobic fecal samples were cultured using 33 media and incubation of plates anaerobically and aerobically resulted in 66 culture conditions for culture-enriched molecular profiling. The cultivable portion of the fecal microbiota was determined by comparing the operational taxonomic units (OTUs) recovered by 16S sequencing of the culture plates to OTUs from culture-independent sequencing of the fecal sample. Targeted isolation of Lachnospiraceae strains using conditions defined by culture-enriched molecular profiling was carried out on two fresh stool samples. We show that culture-enriched molecular profiling, utilizing 66 culture conditions combined with 16S rRNA gene sequencing, allowed for the culturing of an average of 95 % of the OTUs present at greater than 0.1 % abundance in fecal samples. Uncultured OTUs were low abundance in stool. Importantly, comparing culture-enrichment to culture-independent sequencing revealed that the majority of OTUs were detected only by culture, highlighting the advantage of culture for studying the diversity of the gut microbiota. Applying culture-enriched molecular profiling to target Lachnospiraceae strains resulted in the recovery of 79 isolates, 12 of which are on the Human Microbiome Project's "Most Wanted" list. We show that, through culture-enriched molecular profiling, the majority of the

  14. Atrazine and its metabolites degradation in mineral salts medium and soil using an enrichment culture.

    PubMed

    Kumar, Anup; Singh, Neera

    2016-03-01

    An atrazine-degrading enrichment culture was used to study degradation of atrazine metabolites viz. hydroxyatrazine, deethylatrazine, and deisopropylatrazine in mineral salts medium. Results suggested that the enrichment culture was able to degrade only hydroxyatrazine, and it was used as the sole source of carbon and nitrogen. Hydroxyatrazine degradation slowed down when sucrose and/or ammonium hydrogen phosphate were supplemented as the additional sources of carbon and nitrogen, respectively. The enrichment culture could degrade high concentrations of atrazine (up to 110 μg/mL) in mineral salts medium, and neutral pH was optimum for atrazine degradation. Further, except in an acidic soil, enrichment culture was able to degrade atrazine in three soil types having different physico-chemical properties. Raising the pH of acidic soil to neutral or alkaline enabled the enrichment culture to degrade atrazine suggesting that acidic pH inhibited atrazine-degrading ability. The study suggested that the enrichment culture can be successfully utilized to achieve complete degradation of atrazine and its persistent metabolite hydroxyatrazine in the contaminated soil and water.

  15. Co-occurrence of Methanosarcina mazei and Geobacteraceae in an iron (III)-reducing enrichment culture

    PubMed Central

    Zheng, Shiling; Zhang, Hongxia; Li, Ying; Zhang, Hua; Wang, Oumei; Zhang, Jun; Liu, Fanghua

    2015-01-01

    Methanosaeta harundinacea and Methanosarcina barkeri, known as classic acetoclastic methanogens, are capable of directly accepting electrons from Geobacter metallireducens for the reduction of carbon dioxide to methane, having been revealed as direct interspecies electron transfer (DIET) in the laboratory co-cultures. However, whether their co-occurrences are ubiquitous in the iron (III)-reducing environments and the other species of acetoclastic methanogens such as Methanosarcina mazei are capable of DIET are still unknown. Instead of initiating the co-cultures with pure cultures, two-step cultivation was employed to selectively enrich iron (III)-reducing microorganisms in a coastal gold mining river, Jiehe River, with rich iron content in the sediments. First, iron (III) reducers including Geobacteraceae were successfully enriched by 3-months successive culture on amorphous Fe(III) oxides as electron acceptor and acetate as electron donor. High-throughput Illumina sequencing, terminal restriction fragment length polymorphism (T-RFLP) and clone library analysis based on 16S rRNA genes revealed that the enrichment cultures actively contained the bacteria belong to Geobacteraceae and Bacilli, exclusively dominated by the archaea belong to Methanosarcinaceae. Second, the enrichment cultures including methanogens and Geobacteraceae were transferred with ethanol as alternative electron donor. Remarkably, aggregates were successively formed in the enrichments after three transfers. The results revealed by RNA-based analysis demonstrate that the co-occurrence of Methanosarcina mazei and Geobacteraceae in an iron (III)-reducing enrichment culture. Furthermore, the aggregates, as close physical contact, formed in the enrichment culture, indicate that DIET could be a possible option for interspecies electron transfer in the aggregates. PMID:26441876

  16. Indian Culture Master Plan. A Master Plan for Enriching the Background of Public School Personnel in Native American Cultures.

    ERIC Educational Resources Information Center

    Montana State Board of Education, Helena.

    Recognizing that the cultural and historical contributions of Native Americans should be part of the basic education of all citizens in the state, the 1974 Montana legislature directed that an Indian Culture Master Plan be devised to enrich the background of all public school teachers. The plan was to provide means by which all public school…

  17. Characteristics of enriched cultures for bio-huff-`n`-puff tests at Jilin oil field

    SciTech Connect

    Xiu-Yuan Wang; Gang Dai; Yan-Fen Xue; Shu-Hua Xie

    1995-12-31

    Three enriched cultures (48, 15a, and 26a), selected from more than 80 soil and water samples, could grow anaerobically in the presence of crude oil at 30{degrees}C and could ferment molasses to gases and organic acids. Oil recovery by culture 48 in the laboratory model experiment was enhanced by 25.2% over the original reserves and by 53.7% over the residual reserves. Enriched culture 48 was composed of at least 4 species belonging to the genera Eubacterium, Fusobacterium, and Bacteroides. This enriched culture was used as inoculum for MEOR field trials at Jilin oil field with satisfactory results. The importance of the role of these isolates in EOR was confirmed by their presence and behavior in the fluids produced from the microbiologically treated reservoir.

  18. Inupiaq Cultural Enrichment Program. Junior High-High School.

    ERIC Educational Resources Information Center

    Ahngasuk, Margaret; And Others

    Part of a project to teach Inupiaq culture and language, the teacher's guide contains 18 lessons about survival. The lessons focus on survival on ice, water, and land; hunting gear; fall and early winter weather; emergency shelters; landmarks; traditional clothing; land and water transportation; first aid; search and rescue; sewing; traditional…

  19. Enrich Your Kindergarten Program with a Cross-Cultural Connection.

    ERIC Educational Resources Information Center

    Salmon, Michaele; Akaran, Susan Evans

    2001-01-01

    Describes a pen pal connection between a New Jersey kindergarten class and an Alaskan Eskimo first-grade class. Details how they used monthly e-mail and regular mail, and exchanged class projects to heighten respect and understanding about others' cultures. Provides examples of monthly contact during one school year and websites for finding a…

  20. Inupiaq Cultural Enrichment Program. Junior High-High School.

    ERIC Educational Resources Information Center

    Ahngasuk, Margaret; And Others

    Part of a project to teach Inupiaq culture and language, the teacher's guide contains 18 lessons about survival. The lessons focus on survival on ice, water, and land; hunting gear; fall and early winter weather; emergency shelters; landmarks; traditional clothing; land and water transportation; first aid; search and rescue; sewing; traditional…

  1. A study of psychrophilic organisms isolated from the manufacture and assembly areas of spacecraft to be used in the Viking mission

    NASA Technical Reports Server (NTRS)

    Foster, T. L.; Winans, L., Jr.

    1974-01-01

    The ability of psychrophilic microorganisms to grow in some of the environmental conditions suggested for Mars is studied with particular attention given to the effects of moisture and nutrients on growth. Results of growth with the slide culture technique are presented and indicate that this technique can be a rapid and sensitive technique for demonstration of microbial growth under various environmental conditions. Additional soil samples have been obtained from Cape Kennedy, and results of these assays at various low temperatures for psychrophilic populations are presented. The heat resistance of some of the psychrophilic sporeformers have been determined. Psychrophilic organisms were isolated from the teflon ribbons at Cape Kennedy and characterization of these was begun. In addition, heat survivors from the teflon ribbons are being investigated, and partial characterizations of these are presented.

  2. Enrichment of skin-derived neural precursor cells from dermal cell populations by altering culture conditions.

    PubMed

    Bayati, Vahid; Gazor, Rohoullah; Nejatbakhsh, Reza; Negad Dehbashi, Fereshteh

    2016-01-01

    As stem cells play a critical role in tissue repair, their manipulation for being applied in regenerative medicine is of great importance. Skin-derived precursors (SKPs) may be good candidates for use in cell-based therapy as the only neural stem cells which can be isolated from an accessible tissue, skin. Herein, we presented a simple protocol to enrich neural SKPs by monolayer adherent cultivation to prove the efficacy of this method. To enrich neural SKPs from dermal cell populations, we have found that a monolayer adherent cultivation helps to increase the numbers of neural precursor cells. Indeed, we have cultured dermal cells as monolayer under serum-supplemented (control) and serum-supplemented culture, followed by serum free cultivation (test) and compared. Finally, protein markers of SKPs were assessed and compared in both experimental groups and differentiation potential was evaluated in enriched culture. The cells of enriched culture concurrently expressed fibronectin, vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors as compared to control culture. In addition, they possessed a multipotential capacity to differentiate into neurogenic, glial, adipogenic, osteogenic and skeletal myogenic cell lineages. It was concluded that serum-free adherent culture reinforced by growth factors have been shown to be effective on proliferation of skin-derived neural precursor cells (skin-NPCs) and drive their selective and rapid expansion.

  3. Two-stage mineralization of phenanthrene by estuarine enrichment cultures

    SciTech Connect

    Guerin, W.F.; Jones, G.E.

    1988-04-01

    The polycyclic aromatic hydrocarbon phenanthrene was mineralized in two stages by soil, estuarine water, and sediment microbial populations. At high concentrations, phenanthrene was degraded, with the concomitant production of biomass and accumulation of Folin-Ciocalteau-reactive aromatic intermediates. Subsequent consumption of these intermediates resulted in a secondary increase in biomass. Analysis of intermediates by high-performance liquid chromatography, thin-layer chromatography, and UV absorption spectrometry showed 1-hydroxy-2-naphthoic acid (1H2NA) to be the predominant product. A less pronounced two-stage mineralization pattern was also observed by monitoring /sup 14/CO/sub 2/ production from low concentrations (0.5 mg liter/sup -1/) of radiolabeled phenanthrene. Here, mineralization of /sup 14/C-labeled 1H2NA could explain the incremental /sup 14/CO/sub 2/ produced during the later part of the incubations. Accumulation of 1H2NA by isolates obtained from enrichments was dependent on the initial phenanthrene concentration. The production of metabolites during polycyclic aromatic hydrocarbon biodegradation is discussed with regard to its possible adaptive significance and its methodological implications.

  4. Two-Stage Mineralization of Phenanthrene by Estuarine Enrichment Cultures

    PubMed Central

    Guerin, William F.; Jones, Galen E.

    1988-01-01

    The polycyclic aromatic hydrocarbon phenanthrene was mineralized in two stages by soil, estuarine water, and sediment microbial populations. At high concentrations, phenanthrene was degraded, with the concomitant production of biomass and accumulation of Folin-Ciocalteau-reactive aromatic intermediates. Subsequent consumption of these intermediates resulted in a secondary increase in biomass. Analysis of intermediates by high-performance liquid chromatography, thin-layer chromatography, and UV absorption spectrometry showed 1-hydroxy-2-naphthoic acid (1H2NA) to be the predominant product. A less pronounced two-stage mineralization pattern was also observed by monitoring 14CO2 production from low concentrations (0.5 mg liter−1) of radiolabeled phenanthrene. Here, mineralization of 14C-labeled 1H2NA could explain the incremental 14CO2 produced during the later part of the incubations. Accumulation of 1H2NA by isolates obtained from enrichments was dependent on the initial phenanthrene concentration. The production of metabolites during polycyclic aromatic hydrocarbon biodegradation is discussed with regard to its possible adaptive significance and its methodological implications. PMID:16347614

  5. Differential Gene Expression Profiling of Enriched Human Spermatogonia after Short- and Long-Term Culture

    PubMed Central

    Conrad, Sabine; Azizi, Hossein; Hatami, Maryam; Kubista, Mikael; Bonin, Michael; Hennenlotter, Jörg; Renninger, Markus; Skutella, Thomas

    2014-01-01

    This study aimed to provide a molecular signature for enriched adult human stem/progenitor spermatogonia during short-term (<2 weeks) and long-term culture (up to more than 14 months) in comparison to human testicular fibroblasts and human embryonic stem cells. Human spermatogonia were isolated by CD49f magnetic activated cell sorting and collagen−/laminin+ matrix binding from primary testis cultures obtained from ten adult men. For transcriptomic analysis, single spermatogonia-like cells were collected based on their morphology and dimensions using a micromanipulation system from the enriched germ cell cultures. Immunocytochemical, RT-PCR and microarray analyses revealed that the analyzed populations of cells were distinct at the molecular level. The germ- and pluripotency-associated genes and genes of differentiation/spermatogenesis pathway were highly expressed in enriched short-term cultured spermatogonia. After long-term culture, a proportion of cells retained and aggravated the “spermatogonial” gene expression profile with the expression of germ and pluripotency-associated genes, while in the majority of long-term cultured cells this molecular profile, typical for the differentiation pathway, was reduced and more genes related to the extracellular matrix production and attachment were expressed. The approach we provide here to study the molecular status of in vitro cultured spermatogonia may be important to optimize the culture conditions and to evaluate the germ cell plasticity in the future. PMID:24738045

  6. Synergistic degradation of deca-BDE by an enrichment culture and zero-valent iron.

    PubMed

    Chen, Xingjuan; Chen, Guilan; Qiu, Mengde; Sun, Guoping; Guo, Jun; Xu, Meiying

    2014-01-01

    Debromination of decabromodiphenyl ether (deca-BDE) by microbe and by zero-valent iron (ZVI) has been reported previously. However, no study has indicated the presence of microorganisms and their effect on ZVI-mediated reduction of deca-BDE. Synergistic degradation of deca-BDE by an enrichment culture and ZVI was studied. It was found that synergistic effects enhanced the debromination of deca-BDE as well as promoting the reduction of lower brominated products. ZVI stimulated microbial debromination by serving as an electron donor. Correlation analysis also confirmed that ZVI was capable of enhancing microbial population in the debromination of deca-BDE. Conversely, the enrichment culture produced acid which maintained pH stability and stimulated the oxidation of ZVI. The enrichment culture supplied its energy requirements by the oxidation of ZVI and concomitant reduction of deca-BDE, but incapable of growth and reduction of BDE-209 without ZVI and vice versa. Compared to the initial culture, the microbial community of the enrichment culture became dominated by several bacterial genera based on the results of 16S rRNA-gene pyrosequencing.

  7. Alginate immobilized enrichment culture for atrazine degradation in soil and water system.

    PubMed

    Kumar, Anup; Nain, Lata; Singh, Neera

    2017-04-03

    An atrazine degrading enrichment culture, a consortium of bacteria of genus Bacillus along with Pseudomonas and Burkholderia, was immobilized in sodium alginate and was used to study atrazine degradation in mineral salts medium (MSM), soil and wastewater effluent. Sodium alginate immobilized consortium, when stored at room temperature (24 ± 5°C), was effective in degrading atrazine in MSM up to 90 days of storage. The survival of bacteria in alginate beads, based on colony formation unit (CFU) counts, suggested survival up to 90 days and population counts decreased to 1/5(th) on 120 days. Comparison of atrazine degrading ability of the freely suspended enrichment culture and immobilized culture suggested that the immobilized culture took longer time for complete degradation of atrazine as a lag phase of 2 days was observed in the MSM inoculated with alginate immobilized culture. The free cells resulted in complete degradation of atrazine within 6 days, while immobilized cells took 10 days for 100% atrazine degradation. Further, immobilized cultures were able to degrade atrazine in soil and wastewater effluent. Alginate beads were stable and effective in degrading atrazine till 3rd transfer and disintegrated thereafter. The study suggested that immobilized enrichment culture, due to its better storage and application, can be used to degrade atrazine in soil water system.

  8. Newly cultured bacteria with broad diversity isolated from 8 week continuous culture enrichments of cow feces on complex polysaccharides

    USDA-ARS?s Scientific Manuscript database

    One of the fascinating functions of the mammalian intestinal microbiota is the fermentation of plant cell wall components. Eight week continuous culture enrichments of cow feces with cellulose and xylan/pectin were used to isolate bacteria from this community. A total of 459 bacterial isolates were ...

  9. Cooperative catabolic pathways within an atrazine-degrading enrichment culture isolated from soil.

    PubMed

    Smith, Daniel; Alvey, Sam; Crowley, David E

    2005-07-01

    Atrazine degradation previously has been shown to be carried out by individual bacterial species or by relatively simple consortia that have been isolated using enrichment cultures. Here, the degradative pathway for atrazine was examined for a complex 8-membered enrichment culture. The species composition of the culture was determined by PCR-DGGE. The bacterial species included Agrobacterium tumefaciens, Caulobacter crescentus, Pseudomonas putida, Sphingomonas yaniokuyae, Nocardia sp., Rhizobium sp., Flavobacterium oryzihabitans, and Variovorax paradoxus. All of the isolates were screened for the presence of known genes that function for atrazine degradation including atzA,-B,-C,-D,-E,-F and trzD,-N. Dechlorination of atrazine, which was obligatory for complete mineralization, was carried out exclusively by Nocardia sp., which contained the trzN gene. Following dechlorination, the resulting product, hydroxyatrazine was further degraded via two separate pathways. In one pathway Nocardia converted hydroxyatrazine to N-ethylammelide via an unidentified gene product. In the second pathway, hydroxyatrazine generated by Nocardia sp. was hydrolyzed to N-isopropylammelide by Rhizobium sp., which contained the atzB gene. Each member of the enrichment culture contained atzC, which is responsible for ring cleavage, but none of the isolates carried the atzD,-E, or -F genes. Each member further contained either trzD or exhibited urease activity. The enrichment culture was destabilized by loss of Nocardia sp. when grown on ethylamine, ethylammelide, and cyanuric acid, after which the consortium was no longer able to degrade atrazine. The analysis of this enrichment culture highlights the broad level bacterial community interactions that may be involved in atrazine degradation in nature.

  10. Breast Cancer Stem Cell Culture and Enrichment Using Poly(ε-Caprolactone) Scaffolds.

    PubMed

    Palomeras, Sònia; Rabionet, Marc; Ferrer, Inés; Sarrats, Ariadna; Garcia-Romeu, Maria Luisa; Puig, Teresa; Ciurana, Joaquim

    2016-04-23

    The cancer stem cell (CSC) population displays self-renewal capabilities, resistance to conventional therapies, and a tendency to post-treatment recurrence. Increasing knowledge about CSCs' phenotype and functions is needed to investigate new therapeutic strategies against the CSC population. Here, poly(ε-caprolactone) (PCL), a biocompatible polymer free of toxic dye, has been used to fabricate scaffolds, solid structures suitable for 3D cancer cell culture. It has been reported that scaffold cell culture enhances the CSCs population. A RepRap BCN3D+ printer and 3 mm PCL wire were used to fabricate circular scaffolds. PCL design and fabrication parameters were first determined and then optimized considering several measurable variables of the resulting scaffolds. MCF7 breast carcinoma cell line was used to assess scaffolds adequacy for 3D cell culture. To evaluate CSC enrichment, the Mammosphere Forming Index (MFI) was performed in 2D and 3D MCF7 cultures. Results showed that the 60° scaffolds were more suitable for 3D culture than the 45° and 90° ones. Moreover, 3D culture experiments, in adherent and non-adherent conditions, showed a significant increase in MFI compared to 2D cultures (control). Thus, 3D cell culture with PCL scaffolds could be useful to improve cancer cell culture and enrich the CSCs population.

  11. Using Enrichment Clusters to Address the Needs of Culturally and Linguistically Diverse Learners

    ERIC Educational Resources Information Center

    Allen, Jennifer K.; Robbins, Margaret A.; Payne, Yolanda Denise; Brown, Katherine Backes

    2016-01-01

    Using data from teacher interviews, classroom observations, and a professional development workshop, this article explains how one component of the schoolwide enrichment model (SEM) has been implemented at a culturally diverse elementary school serving primarily Latina/o and African American students. Based on a broadened conception of giftedness,…

  12. Broad diversity and newly cultured bacterial isolates from enrichment of pig feces on complex polysaccharides

    USDA-ARS?s Scientific Manuscript database

    One of the fascinating functions of the mammalian intestinal microbiota is the fermentation of plant cell wall components. We used 8 week continuous culture enrichments of pig feces with cellulose and xylan/pectin to isolated bacteria from this community. A total of 575 bacterial isolates were class...

  13. Using Enrichment Clusters to Address the Needs of Culturally and Linguistically Diverse Learners

    ERIC Educational Resources Information Center

    Allen, Jennifer K.; Robbins, Margaret A.; Payne, Yolanda Denise; Brown, Katherine Backes

    2016-01-01

    Using data from teacher interviews, classroom observations, and a professional development workshop, this article explains how one component of the schoolwide enrichment model (SEM) has been implemented at a culturally diverse elementary school serving primarily Latina/o and African American students. Based on a broadened conception of giftedness,…

  14. Biodegradation of Endocrine Disruptors in Solid-Liquid Two-Phase Partitioning Systems by Enrichment Cultures

    PubMed Central

    dos Santos, Silvia Cristina Cunha; Ouellette, Julianne; Juteau, Pierre; Lépine, François; Déziel, Eric

    2013-01-01

    Naturally occurring and synthetic estrogens and other molecules from industrial sources strongly contribute to the endocrine disruption of urban wastewater. Because of the presence of these molecules in low but effective concentrations in wastewaters, these endocrine disruptors (EDs) are only partially removed after most wastewater treatments, reflecting the presence of these molecules in rivers in urban areas. The development of a two-phase partitioning bioreactor (TPPB) might be an effective strategy for the removal of EDs from wastewater plant effluents. Here, we describe the establishment of three ED-degrading microbial enrichment cultures adapted to a solid-liquid two-phase partitioning system using Hytrel as the immiscible water phase and loaded with estrone, estradiol, estriol, ethynylestradiol, nonylphenol, and bisphenol A. All molecules except ethynylestradiol were degraded in the enrichment cultures. The bacterial composition of the three enrichment cultures was determined using 16S rRNA gene sequencing and showed sequences affiliated with bacteria associated with the degradation of these compounds, such as Sphingomonadales. One Rhodococcus isolate capable of degrading estrone, estradiol, and estriol was isolated from one enrichment culture. These results highlight the great potential for the development of TPPB for the degradation of highly diluted EDs in water effluents. PMID:23728808

  15. Tetrachloroethene conversion to ethene by a Dehalococcoides-containing enrichment culture from Bitterfeld.

    PubMed

    Cichocka, Danuta; Nikolausz, Marcell; Haest, Pieter Jan; Nijenhuis, Ivonne

    2010-05-01

    A Dehalococcoides-dominated culture coupling reductive dechlorination of tetrachloroethene (PCE) to ethene to growth was enriched from a European field site for the first time. Microcosms were set up using groundwater from a chlorinated ethene-contaminated anaerobic aquifer in Bitterfeld (Germany). Active, lactate-amended microcosms capable of PCE dechlorination to ethene without the accumulation of intermediates were used for further enrichment. After three transfers on lactate as an electron donor and PCE as an electron acceptor, the enrichment was transferred to parallel cultures with one of the chlorinated ethenes as an electron acceptor and acetate and hydrogen as the carbon and energy source, respectively. After three more transfers, a highly purified culture was derived that was capable of dechlorinating PCE with hydrogen and acetate as the electron donor and carbon source, respectively. PCR, followed by denaturing gradient gel electrophoresis, cloning and sequencing revealed that this culture was dominated by a Dehalococcoides sp. belonging to the Pinellas group. Investigation of substrate specificity in the parallel cultures suggested the presence of a novel Dehalococcoides that can couple all dechlorination steps, from PCE to ethene, to energy conservation. Quantitative real-time PCR confirmed growth with PCE, cis-dichloroethene, 1,1-dichloroethene or vinyl chloride as electron acceptors. The culture was designated BTF08 due to its origin in Bitterfeld.

  16. Enrichment of cancer stem cell-like cells by culture in alginate gel beads.

    PubMed

    Xu, Xiao-xi; Liu, Chang; Liu, Yang; Yang, Li; Li, Nan; Guo, Xin; Sun, Guang-wei; Ma, Xiao-jun

    2014-05-10

    Cancer stem cells (CSCs) are most likely the reason of cancer reoccurrence and metastasis. For further elucidation of the mechanism underlying the characteristics of CSCs, it is necessary to develop efficient culture systems to culture and expand CSCs. In this study, a three-dimensional (3D) culture system based on alginate gel (ALG) beads was reported to enrich CSCs. Two cell lines derived from different histologic origins were encapsulated in ALG beads respectively and the expansion of CSCs was investigated. Compared with two-dimensional (2D) culture, the proportion of cells with CSC-like phenotypes was significantly increased in ALG beads. Expression levels of CSC-related genes were greater in ALG beads than in 2D culture. The increase of CSC proportion after being cultured within ALG beads was further confirmed by enhanced tumorigenicity in vivo. Moreover, increased metastasis ability and higher anti-cancer drug resistance were also observed in 3D-cultured cells. Furthermore, we found that it was hypoxia, through the upregulation of hypoxia-inducible factors (HIFs) that occurred in ALG beads to induce the increasing of CSC proportion. Therefore, ALG bead was an efficient culture system for CSC enrichment, which might provide a useful platform for CSC research and promote the development of new anti-cancer therapies targeting CSCs. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Comparative void-volume analysis of psychrophilic and mesophilic enzymes: Structural bioinformatics of psychrophilic enzymes reveals sources of core flexibility.

    PubMed

    Paredes, Diana I; Watters, Kyle; Pitman, Derek J; Bystroff, Christopher; Dordick, Jonathan S

    2011-10-20

    Psychrophiles, cold-adapted organisms, have adapted to live at low temperatures by using a variety of mechanisms. Their enzymes are active at cold temperatures by being structurally more flexible than mesophilic enzymes. Even though, there are some indications of the possible structural mechanisms by which psychrophilic enzymes are catalytic active at cold temperatures, there is not a generalized structural property common to all psychrophilic enzymes. We examine twenty homologous enzyme pairs from psychrophiles and mesophiles to investigate flexibility as a key characteristic for cold adaptation. B-factors in protein X-ray structures are one way to measure flexibility. Comparing psychrophilic to mesophilic protein B-factors reveals that psychrophilic enzymes are more flexible in 5-turn and strand secondary structures. Enzyme cavities, identified using CASTp at various probe sizes, indicate that psychrophilic enzymes have larger average cavity sizes at probe radii of 1.4-1.5 Å, sufficient for water molecules. Furthermore, amino acid side chains lining these cavities show an increased frequency of acidic groups in psychrophilic enzymes. These findings suggest that embedded water molecules may play a significant role in cavity flexibility, and therefore, overall protein flexibility. Thus, our results point to the important role enzyme flexibility plays in adaptation to cold environments.

  18. Identification of triclosan-degrading bacteria in a triclosan enrichment culture using stable isotope probing.

    PubMed

    Lee, Do Gyun; Cho, Kun-Ching; Chu, Kung-Hui

    2014-02-01

    Triclosan, a widely used antimicrobial agent, is an emerging contaminant in the environment. Despite its antimicrobial character, biodegradation of triclosan has been observed in pure cultures, soils and activated sludge. However, little is known about the microorganisms responsible for the degradation in mixed cultures. In this study, active triclosan degraders in a triclosan-degrading enrichment culture were identified using stable isotope probing (SIP) with universally (13)C-labeled triclosan. Eleven clones contributed from active microorganisms capable of uptake the (13)C in triclosan were identified. None of these clones were similar to known triclosan-degraders/utilizers. These clones distributed among α-, β-, or γ-Proteobacteria: one belonging to Defluvibacter (α-Proteobacteria), seven belonging to Alicycliphilus (β-Proteobacteria), and three belonging to Stenotrophomonas (γ-Proteobacteria). Successive additions of triclosan caused a significant shift in the microbial community structure of the enrichment culture, with dominant ribotypes belonging to the genera Alicycliphilus and Defluvibacter. Application of SIP has successfully identified diverse uncultivable triclosan-degrading microorganisms in an activated sludge enrichment culture. The results of this study not only contributed to our understanding of the microbial ecology of triclosan biodegradation in wastewater, but also suggested that triclosan degraders are more phylogenetically diverse than previously reported.

  19. Prospecting for ice association: characterization of freeze-thaw selected enrichment cultures from latitudinally distant soils.

    PubMed

    Wilson, Sandra L; Grogan, Paul; Walker, Virginia K

    2012-04-01

    Freeze-thaw stress has previously been shown to alter soil community structure and function. We sought to further investigate this stress on enriched microbial consortia with the aim of identifying microbes with ice-associating adaptations that facilitate survival. Enrichments were established to obtain culturable psychrotolerant microbes from soil samples from the latitudinal extremes of the Canadian Shield plateau. The resulting consortia were subjected to consecutive freeze-thaw cycles, and survivors were putatively identified by their 16S rRNA gene sequences. Even though the northerly site was exposed to longer, colder winters and large spring-time temperature fluctuations, the selective regime similarly affected both enriched consortia. Quantitative PCR and metagenomic sequencing were used to determine the frequency of a subset of the resistant microbes in the original enrichments. The metagenomes showed 22 initial genera, only 6 survived and these were not dominant prior to selection. When survivors were assayed for ice recrystallization inhibition and ice nucleation activities, over 60% had at least one of these properties. These phenotypes were not more prevalent in the northern enrichment, indicating that regarding these adaptations, the enrichment strategy yielded seemingly functionally similar consortia from each site.

  20. Cholera Rapid Test with Enrichment Step Has Diagnostic Performance Equivalent to Culture.

    PubMed

    Ontweka, Lameck N; Deng, Lul O; Rauzier, Jean; Debes, Amanda K; Tadesse, Fisseha; Parker, Lucy A; Wamala, Joseph F; Bior, Bior K; Lasuba, Michael; But, Abiem Bona; Grandesso, Francesco; Jamet, Christine; Cohuet, Sandra; Ciglenecki, Iza; Serafini, Micaela; Sack, David A; Quilici, Marie-Laure; Azman, Andrew S; Luquero, Francisco J; Page, Anne-Laure

    2016-01-01

    Cholera rapid diagnostic tests (RDT) could play a central role in outbreak detection and surveillance in low-resource settings, but their modest performance has hindered their broad adoption. The addition of an enrichment step may improve test specificity. We describe the results of a prospective diagnostic evaluation of the Crystal VC RDT (Span Diagnostics, India) with enrichment step and of culture, each compared to polymerase chain reaction (PCR), during a cholera outbreak in South Sudan. RDTs were performed on alkaline peptone water inoculated with stool and incubated for 4-6 hours at ambient temperature. Cholera culture was performed from wet filter paper inoculated with stool. Molecular detection of Vibrio cholerae O1 by PCR was done from dry Whatman 903 filter papers inoculated with stool, and from wet filter paper supernatant. In August and September 2015, 101 consecutive suspected cholera cases were enrolled, of which 36 were confirmed by PCR. The enriched RDT had 86.1% (95% CI: 70.5-95.3) sensitivity and 100% (95% CI: 94.4-100) specificity compared to PCR as the reference standard. The sensitivity of culture versus PCR was 83.3% (95% CI: 67.2-93.6) for culture performed on site and 72.2% (95% CI: 54.8-85.8) at the international reference laboratory, where samples were tested after an average delay of two months after sample collection, and specificity was 98.5% (95% CI: 91.7-100) and 100% (95% CI: 94.5-100), respectively. The RDT with enrichment showed performance comparable to that of culture and could be a sustainable alternative to culture confirmation where laboratory capacity is limited.

  1. Cholera Rapid Test with Enrichment Step Has Diagnostic Performance Equivalent to Culture

    PubMed Central

    Ontweka, Lameck N.; Deng, Lul O.; Rauzier, Jean; Debes, Amanda K.; Tadesse, Fisseha; Parker, Lucy A.; Wamala, Joseph F.; Bior, Bior K.; Lasuba, Michael; But, Abiem Bona; Grandesso, Francesco; Jamet, Christine; Cohuet, Sandra; Ciglenecki, Iza; Serafini, Micaela; Sack, David A.; Quilici, Marie-Laure; Azman, Andrew S.; Luquero, Francisco J.

    2016-01-01

    Cholera rapid diagnostic tests (RDT) could play a central role in outbreak detection and surveillance in low-resource settings, but their modest performance has hindered their broad adoption. The addition of an enrichment step may improve test specificity. We describe the results of a prospective diagnostic evaluation of the Crystal VC RDT (Span Diagnostics, India) with enrichment step and of culture, each compared to polymerase chain reaction (PCR), during a cholera outbreak in South Sudan. RDTs were performed on alkaline peptone water inoculated with stool and incubated for 4–6 hours at ambient temperature. Cholera culture was performed from wet filter paper inoculated with stool. Molecular detection of Vibrio cholerae O1 by PCR was done from dry Whatman 903 filter papers inoculated with stool, and from wet filter paper supernatant. In August and September 2015, 101 consecutive suspected cholera cases were enrolled, of which 36 were confirmed by PCR. The enriched RDT had 86.1% (95% CI: 70.5–95.3) sensitivity and 100% (95% CI: 94.4–100) specificity compared to PCR as the reference standard. The sensitivity of culture versus PCR was 83.3% (95% CI: 67.2–93.6) for culture performed on site and 72.2% (95% CI: 54.8–85.8) at the international reference laboratory, where samples were tested after an average delay of two months after sample collection, and specificity was 98.5% (95% CI: 91.7–100) and 100% (95% CI: 94.5–100), respectively. The RDT with enrichment showed performance comparable to that of culture and could be a sustainable alternative to culture confirmation where laboratory capacity is limited. PMID:27992488

  2. Fermentation of model hemicelluloses by Prevotella strains and Butyrivibrio fibrisolvens in pure culture and in ruminal enrichment cultures.

    PubMed

    Emerson, Elianna L; Weimer, Paul J

    2017-05-01

    Hemicelluloses are major components of plant biomass, but their fermentation in the rumens of cattle and other ruminants is poorly understood. We compared four species of the ruminally dominant genus Prevotella and the well-known hemicellulose utilizer, Butyrivibrio fibrisolvens, with respect to degradation of several isolated hemicelluloses (xylans, glucomannan, and xyloglucan). We also performed Illumina sequencing of the V3/V4 region of 16S rRNA genes to determine the relative proportions of Prevotella and Butyrivibrio in hemicellulose-fed enrichment cultures inoculated from ruminal contents of dairy cattle fed a total mixed ration (TMR) rich in hemicelluloses. Results confirmed the xylan fermentation and butyrate production abilities of B. fibrisolvens. Despite their reputation as generalist fermenters, the Prevotella strains poorly fermented these hemicelluloses but exhibited dramatic differences in fermentation end products. Prevotella was much less abundant in mixed bacterial enrichment cultures fed the same TMR than in the ruminal inoculum, yet Prevotella was again the most abundant genus in enrichment cultures fed xylans. By contrast, glucomannan fermentations were dominated by Streptococcus sp. Genera known for hemicellulose degradation (Butyrivibrio, Ruminococcus, and Fibrobacter) were not significantly enriched on these hemicelluloses. Substantial differences in fermentation end product distribution from the different hemicelluloses were observed, which would likely affect nutrient partitioning in the host animal. Differences in community composition between in vitro hemicellulose enrichments and inoculum samples emerged at every phylogenetic level, suggesting that in vitro conditions provide unique selective pressures on the bacterial community and also that ruminal bacteria exhibit specialization with respect to hemicellulose utilization.

  3. Lipid recovery from a vegetable oil emulsion using microbial enrichment cultures.

    PubMed

    Tamis, Jelmer; Sorokin, Dimitry Y; Jiang, Yang; van Loosdrecht, Mark C M; Kleerebezem, Robbert

    2015-01-01

    Many waste streams have a relatively high vegetable oil content, which is a potential resource that should be recovered. Microbial storage compound production for the recovery of lipids from lipid-water emulsions with open (unsterilized) microbial cultures was investigated in a sequencing batch reactor using a diluted vegetable oil emulsion as model substrate. After feeding, triacylglycerides (TAG) were accumulated intracellular by the microbial enrichment culture and subsequently used for growth in the remainder of the sequencing batch cycle. Roughly 50% of the added TAG could be recovered as intracellular lipids in this culture. The maximum lipid storage capacity of the enrichment culture was 54% on volatile suspended solids (VSS) mass basis in a separate fed-batch accumulation experiment. The microbial community was dominated by a lipolytic fungus, Trichosporon gracile, that was responsible for intracellular lipid accumulation but also a significant fraction of lipolytic and long chain fatty-acid-utilizing bacteria was present. Herewith, we demonstrate an effective strategy for enrichment of a microbial community that can accumulate significant amounts of lipids from wastewaters without the need for sterilization of substrates or equipment. Further optimization of this process will make recovery of lipids from wastewater possible.

  4. Erythrocyte Enrichment in Hematopoietic Progenitor Cell Cultures Based on Magnetic Susceptibility of the Hemoglobin

    PubMed Central

    Jin, Xiaoxia; Abbot, Stewart; Zhang, Xiaokui; Kang, Lin; Voskinarian-Berse, Vanessa; Zhao, Rui; Kameneva, Marina V.; Moore, Lee R.; Chalmers, Jeffrey J.; Zborowski, Maciej

    2012-01-01

    Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes. PMID:22952572

  5. Erythrocyte enrichment in hematopoietic progenitor cell cultures based on magnetic susceptibility of the hemoglobin.

    PubMed

    Jin, Xiaoxia; Abbot, Stewart; Zhang, Xiaokui; Kang, Lin; Voskinarian-Berse, Vanessa; Zhao, Rui; Kameneva, Marina V; Moore, Lee R; Chalmers, Jeffrey J; Zborowski, Maciej

    2012-01-01

    Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes.

  6. High-affinity methane oxidation by a soil enrichment culture containing a type II methanotroph

    SciTech Connect

    Dunfield, P.F. |; Liesack, W.; Henckel, T.; Conrad, R.; Knowles, R.

    1999-03-01

    Methanotrophic bacteria in an organic soil were enriched on gaseous mixing ratios of <275 parts per million of volume (ppmv) of methane (CH{sub 4}). After 4 years of growth and periodic dilution, a mixed culture was obtained which displayed an apparent half-saturation constant [K{sub m(app)}] for CH{sub 4} of 56 to 186 nM (40 to 132 ppmv). This value was the same as that measured in the soil itself and about 1 order of magnitude lower than reported values for pure cultures of methane oxidizers. However, the K{sub m(app)} increased when the culture was transferred to higher mixing ratios of CH{sub 4}. Denaturing gradient gel electrophoresis of the enrichment grown on <275 ppmv of CH{sub 4} revealed a single gene product of pmoA, which codes for a subunit of particulate methane monooxygenase. This suggested that only one methanotroph species was present. This organism was isolated from a sample of the enrichment culture grown on 1% CH{sub 4} and phylogenetically positioned based on its 16S rRNA, pmoA, and mxaF gene sequences as a type II strain of the Methlocystis/Methylosinus group. A coculture of this strain with a Variovorax sp., when grown on <275 ppmv of CH{sub 4}, had a K{sub m(app)} similar to that of the initial enrichment culture. The data suggest that the affinity of methanotrophic bacteria for CH{sub 4} varies with growth conditions and that the oxidation of atmospheric CH{sub 4} observed in this soil is carried out by type II methanotrophic bacteria which are similar to characterized species.

  7. The attractiveness of enriched and impoverished options: culture, self-construal, and regulatory focus.

    PubMed

    Yinlong Zhang; Mittal, Vikas

    2007-04-01

    The authors propose that there is a systematic impact of a person's activated self-construal (interdependent vs. independent) on the evaluation of enriched and impoverished options. This hypothesis is tested using a cross-country comparison of participants from China and the United States (Study 1) and by priming self-construal (Study 2) and regulatory focus (Study 3). Results show that people with an independent self-construal and a promotion focus perceive enriched options as more attractive than impoverished options. Conversely, people with an interdependent self-construal and a prevention focus perceive enriched options as less attractive than impoverished options. Mediation analyses show that the effect of cultural orientation operates through self-construal. In turn, self-construal operates through activated regulatory focus. Analysis of cognitive responses shows that the effect of regulatory focus is mediated by positive/negative thoughts about option attributes.

  8. Microbial characterization of anode-respiring bacteria within biofilms developed from cultures previously enriched in dissimilatory metal-reducing bacteria.

    PubMed

    Pierra, Mélanie; Carmona-Martínez, Alessandro A; Trably, Eric; Godon, Jean-Jacques; Bernet, Nicolas

    2015-11-01

    This work evaluated the use of a culture enriched in DMRB as a strategy to enrich ARB on anodes. DMRB were enriched with Fe(III) as final electron acceptor and then transferred to a potentiostatically-controlled system with an anode as sole final electron acceptor. Three successive iron-enrichment cultures were carried out. The first step of enrichment revealed a successful selection of the high current-producing ARB Geoalkalibacter subterraneus. After few successive enrichment steps, the microbial community analysis in electroactive biofilms showed a significant divergence with an impact on the biofilm electroactivity. Enrichment of ARB in electroactive biofilms through the pre-selection of DMRB should therefore be carefully considered.

  9. Innovative Approaches Using Lichen Enriched Media to Improve Isolation and Culturability of Lichen Associated Bacteria.

    PubMed

    Biosca, Elena G; Flores, Raquel; Santander, Ricardo D; Díez-Gil, José Luis; Barreno, Eva

    2016-01-01

    Lichens, self-supporting mutualistic associations between a fungal partner and one or more photosynthetic partners, also harbor non-photosynthetic bacteria. The diversity and contribution of these bacteria to the functioning of lichen symbiosis have recently begun to be studied, often by culture-independent techniques due to difficulties in their isolation and culture. However, culturing as yet unculturable lichenic bacteria is critical to unravel their potential functional roles in lichen symbiogenesis, to explore and exploit their biotechnological potential and for the description of new taxa. Our objective was to improve the recovery of lichen associated bacteria by developing novel isolation and culture approaches, initially using the lichen Pseudevernia furfuracea. We evaluated the effect of newly developed media enriched with novel lichen extracts, as well as the influence of thalli washing time and different disinfection and processing protocols of thalli. The developed methodology included: i) the use of lichen enriched media to mimic lichen nutrients, supplemented with the fungicide natamycin; ii) an extended washing of thalli to increase the recovery of ectolichenic bacteria, thus allowing the disinfection of thalli to be discarded, hence enhancing endolichenic bacteria recovery; and iii) the use of an antioxidant buffer to prevent or reduce oxidative stress during thalli disruption. The optimized methodology allowed significant increases in the number and diversity of culturable bacteria associated with P. furfuracea, and it was also successfully applied to the lichens Ramalina farinacea and Parmotrema pseudotinctorum. Furthermore, we provide, for the first time, data on the abundance of culturable ecto- and endolichenic bacteria that naturally colonize P. furfuracea, R. farinacea and P. pseudotinctorum, some of which were only able to grow on lichen enriched media. This innovative methodology is also applicable to other microorganisms inhabiting these

  10. Innovative Approaches Using Lichen Enriched Media to Improve Isolation and Culturability of Lichen Associated Bacteria

    PubMed Central

    Biosca, Elena G.; Flores, Raquel; Santander, Ricardo D.; Díez-Gil, José Luis; Barreno, Eva

    2016-01-01

    Lichens, self-supporting mutualistic associations between a fungal partner and one or more photosynthetic partners, also harbor non-photosynthetic bacteria. The diversity and contribution of these bacteria to the functioning of lichen symbiosis have recently begun to be studied, often by culture-independent techniques due to difficulties in their isolation and culture. However, culturing as yet unculturable lichenic bacteria is critical to unravel their potential functional roles in lichen symbiogenesis, to explore and exploit their biotechnological potential and for the description of new taxa. Our objective was to improve the recovery of lichen associated bacteria by developing novel isolation and culture approaches, initially using the lichen Pseudevernia furfuracea. We evaluated the effect of newly developed media enriched with novel lichen extracts, as well as the influence of thalli washing time and different disinfection and processing protocols of thalli. The developed methodology included: i) the use of lichen enriched media to mimic lichen nutrients, supplemented with the fungicide natamycin; ii) an extended washing of thalli to increase the recovery of ectolichenic bacteria, thus allowing the disinfection of thalli to be discarded, hence enhancing endolichenic bacteria recovery; and iii) the use of an antioxidant buffer to prevent or reduce oxidative stress during thalli disruption. The optimized methodology allowed significant increases in the number and diversity of culturable bacteria associated with P. furfuracea, and it was also successfully applied to the lichens Ramalina farinacea and Parmotrema pseudotinctorum. Furthermore, we provide, for the first time, data on the abundance of culturable ecto- and endolichenic bacteria that naturally colonize P. furfuracea, R. farinacea and P. pseudotinctorum, some of which were only able to grow on lichen enriched media. This innovative methodology is also applicable to other microorganisms inhabiting these

  11. Enrichment and characterization of chlorinated organophosphate ester-degrading mixed bacterial cultures.

    PubMed

    Takahashi, Shouji; Kawashima, Koji; Kawasaki, Manami; Kamito, Jun; Endo, Yusuke; Akatsu, Kumiko; Horino, Sadatoshi; Yamada, Ryo-Hei; Kera, Yoshio

    2008-07-01

    Chlorinated organophosphate ester (OPE)-degrading enrichment cultures were obtained using tris(2-chloroethyl) phosphate (TCEP) or tris(1,3-dichloro-2-propyl) phosphate (TDCPP) as the sole phosphorus source. In cultures with 46 environmental samples, significant TCEP and TDCPP degradation was observed in 10 and 3 cultures, respectively, and successive subcultivation markedly increased their degradation rates. 67E and 45D stable enrichment cultures obtained with TCEP and TDCPP, respectively, completely degraded 20 muM of the respective compounds within 6 h and also the other, although the degradation rate of TCEP by 45D was relatively slow. We confirmed chloride ion generation on degradation in both cases and the generation of 2-chloroethanol (2-CE) and 1,3-dichloro-2-propanol (1,3-DCP) as metabolites of TCEP and TDCPP, respectively. 67E and 45D also showed dehalogenation ability toward 2-CE and 1,3-DCP, respectively. Addition of inorganic phosphate did not significantly influence their ability to degrade the chlorinated OPEs but markedly increased their dehalogenation ability, which was maximum at 0.2 mM of inorganic phosphate and decreased at a higher concentration. Denaturing gradient gel electrophoresis analysis showed that dominant bacteria in 67E are related to Acidovorax spp. and Sphingomonas spp. and those in 45D are Acidovorax spp., Aquabacterium spp., and Sphingomonas spp. This analysis indicated the relationship of the Sphingomonas- and Acidovorax-related bacteria with the cleavage of the phosphoester bond and dehalogenation, respectively, in both cultures. This is the first report on bacterial enrichment cultures capable of degrading both TCEP and TDCPP.

  12. Developments in techniques for the isolation, enrichment, main culture conditions and identification of spermatogonial stem cells.

    PubMed

    He, Yanan; Chen, Xiaoli; Zhu, Huabin; Wang, Dong

    2015-12-01

    The in vitro culture system of spermatogonial stem cells (SSCs) provides a basis for studies on spermatogenesis, and also contributes to the development of new methods for the preservation of livestock and animal genetic modification. In vitro culture systems have mainly been established for mouse SSCs, but are lacking for farm animals. We reviewed and analyzed the current progress in SSC techniques such as isolation, purification, cultivation and identification. Based on the published studies, we concluded that two-step enzyme digestion and magnetic-activated cell sorting are fast becoming the main methods for isolation and enrichment of SSCs. With regard to the culture systems, serum and feeders were earlier thought to play an important role in the self-renewal and proliferation of SSCs, but serum- and feeder-free culture systems as a means of overcoming the limitations of SSC differentiation in long-term SSC culture are being explored. However, there is still a need to establish more efficient and ideal culture systems that can also be used for SSC culture in larger mammals. Although the lack of SSC-specific surface markers has seriously affected the efficiency of purification and identification, the transgenic study is helpful for our identification of SSCs. Therefore, future studies on SSC techniques should focus on improving serum- and feeder-free culture techniques, and discovering and identifying specific surface markers of SSCs, which will provide new ideas for the optimization of SSC culture systems for mice and promote related studies in farm animals.

  13. Thermolabile triose phosphate isomerase in a psychrophilic Clostridium.

    NASA Technical Reports Server (NTRS)

    Shing, Y. W.; Akagi, J. M.; Himes, R. H.

    1972-01-01

    It was found that a psychrophilic Clostridium contains a triose phosphate isomerase which is very labile at moderate temperatures. An investigation showed that the optimal growth temperature of the psychrophile was between 15 and 20 deg C. No growth occurred at 25 deg C. The thermostability of the glycolytic enzymes in the cell-free extracts of Clostridium sp. strain 69 was studied. The data obtained show that the triose phosphate isomerase is quite labile at moderate temperatures. The instability of the enzyme is sufficient to explain the low maximum growth temperature of the psychrophile.

  14. Thermolabile triose phosphate isomerase in a psychrophilic Clostridium.

    NASA Technical Reports Server (NTRS)

    Shing, Y. W.; Akagi, J. M.; Himes, R. H.

    1972-01-01

    It was found that a psychrophilic Clostridium contains a triose phosphate isomerase which is very labile at moderate temperatures. An investigation showed that the optimal growth temperature of the psychrophile was between 15 and 20 deg C. No growth occurred at 25 deg C. The thermostability of the glycolytic enzymes in the cell-free extracts of Clostridium sp. strain 69 was studied. The data obtained show that the triose phosphate isomerase is quite labile at moderate temperatures. The instability of the enzyme is sufficient to explain the low maximum growth temperature of the psychrophile.

  15. Impact of non-storing biomass on PHA production: an enrichment culture on acetate and methanol.

    PubMed

    Marang, Leonie; Jiang, Yang; van Loosdrecht, Mark C M; Kleerebezem, Robbert

    2014-11-01

    The use of enrichment cultures for polyhydroxyalkanoate (PHA) production from substrate mixtures such as wastewater inevitably results in the establishment of a non-PHA-storing population besides the PHA-producing bacteria. This reduces the maximum PHA content that can be established, and increases downstream-processing costs. The aim of this study was to investigate the impact of non-storing biomass on the PHA production process. A microbial culture was enriched in a sequencing batch reactor fed with acetate and methanol. Methanol served as model substrate for compounds unsuitable for PHA production. The enrichment was dominated by Plasticicumulans acidivorans, a known PHA producer, and Methylobacillus flagellatus, an obligate methylotroph that cannot store PHA. As expected, the presence of the non-storing population lowered the maximum PHA content of the culture, from more than 80 to 66wt.%. To mimic a nitrogen-rich waste stream, additional accumulation experiments were performed with continuous supply of carbon and ammonium. In these experiments P. acidivorans still accumulated large amounts of PHA, but unrestricted growth of the non-storing, methylotrophic population reduced the maximum overall PHA content to 52wt.%. Besides ammonium limitation, other strategies to restrict the fraction of non-storing biomass should be developed. The mixture of acetate and methanol is a useful model substrate for the development of such strategies. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Effective enrichment of cholangiocarcinoma secretomes using the hollow fiber bioreactor culture system.

    PubMed

    Weeraphan, Churat; Diskul-Na-Ayudthaya, Penchatr; Chiablaem, Khajeelak; Khongmanee, Amnart; Chokchaichamnankit, Daranee; Subhasitanont, Pantipa; Svasti, Jisnuson; Srisomsap, Chantragan

    2012-09-15

    The Northeastern region of Thailand is well known to have high incidence of bile duct cancer known as cholangiocarcinoma. So there is a continued need to improve diagnosis and treatment, and discovery of biomarkers for early detection of bile duct cancer should greatly improve treatment outcome for these patients. The secretome, a collection of proteins secreted from cells, is a useful source for identifying circulating biomarkers in blood secreted from cancer cells. Here a Hollow Fiber Bioreactor culture system was used for enrichment of cholangiocarcinoma secretomes, since this culture system mimics the dense three-dimensional microenvironment of the tumor found in vivo. Two-dimensional fluorescence difference gel electrophoresis using a sensitive Fluor saturation dye staining, followed by LC/MS/MS, was used to compare protein expression in the secretomes of cells cultured in the Hollow Fiber system and cells cultured in the monolayer culture system. For the first time, the 2D-patterns of cholangiocarcinoma secretomes from the two culture systems could be compared. The Hollow Fiber system improved the quality and quantity of cholangiocarcinoma secreted proteins compared to conventional monolayer system, showing less interference by cytoplasmic proteins and yielding more secreted proteins. Overall, 75 spots were analyzed by LC/MS/MS and 106 secreted proteins were identified. Two novel secreted proteins (C19orf10 and cystatin B) were found only in the Hollow Fiber system and were absent from the traditional monolayer culture system. Among the highly expressed proteins, 22 secreted soluble proteins were enriched by 5 fold in Hollow Fiber system compared to monolayer culture system. The Hollow Fiber system is therefore useful for preparing a wide range of proteins from low-abundance cell secretomes. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Comparative analysis of metagenomes from three methanogenic hydrocarbon-degrading enrichment cultures with 41 environmental samples

    PubMed Central

    Tan, Boonfei; Jane Fowler, S; Laban, Nidal Abu; Dong, Xiaoli; Sensen, Christoph W; Foght, Julia; Gieg, Lisa M

    2015-01-01

    Methanogenic hydrocarbon metabolism is a key process in subsurface oil reservoirs and hydrocarbon-contaminated environments and thus warrants greater understanding to improve current technologies for fossil fuel extraction and bioremediation. In this study, three hydrocarbon-degrading methanogenic cultures established from two geographically distinct environments and incubated with different hydrocarbon substrates (added as single hydrocarbons or as mixtures) were subjected to metagenomic and 16S rRNA gene pyrosequencing to test whether these differences affect the genetic potential and composition of the communities. Enrichment of different putative hydrocarbon-degrading bacteria in each culture appeared to be substrate dependent, though all cultures contained both acetate- and H2-utilizing methanogens. Despite differing hydrocarbon substrates and inoculum sources, all three cultures harbored genes for hydrocarbon activation by fumarate addition (bssA, assA, nmsA) and carboxylation (abcA, ancA), along with those for associated downstream pathways (bbs, bcr, bam), though the cultures incubated with hydrocarbon mixtures contained a broader diversity of fumarate addition genes. A comparative metagenomic analysis of the three cultures showed that they were functionally redundant despite their enrichment backgrounds, sharing multiple features associated with syntrophic hydrocarbon conversion to methane. In addition, a comparative analysis of the culture metagenomes with those of 41 environmental samples (containing varying proportions of methanogens) showed that the three cultures were functionally most similar to each other but distinct from other environments, including hydrocarbon-impacted environments (for example, oil sands tailings ponds and oil-affected marine sediments). This study provides a basis for understanding key functions and environmental selection in methanogenic hydrocarbon-associated communities. PMID:25734684

  18. Comparative analysis of metagenomes from three methanogenic hydrocarbon-degrading enrichment cultures with 41 environmental samples.

    PubMed

    Tan, Boonfei; Fowler, S Jane; Abu Laban, Nidal; Dong, Xiaoli; Sensen, Christoph W; Foght, Julia; Gieg, Lisa M

    2015-09-01

    Methanogenic hydrocarbon metabolism is a key process in subsurface oil reservoirs and hydrocarbon-contaminated environments and thus warrants greater understanding to improve current technologies for fossil fuel extraction and bioremediation. In this study, three hydrocarbon-degrading methanogenic cultures established from two geographically distinct environments and incubated with different hydrocarbon substrates (added as single hydrocarbons or as mixtures) were subjected to metagenomic and 16S rRNA gene pyrosequencing to test whether these differences affect the genetic potential and composition of the communities. Enrichment of different putative hydrocarbon-degrading bacteria in each culture appeared to be substrate dependent, though all cultures contained both acetate- and H2-utilizing methanogens. Despite differing hydrocarbon substrates and inoculum sources, all three cultures harbored genes for hydrocarbon activation by fumarate addition (bssA, assA, nmsA) and carboxylation (abcA, ancA), along with those for associated downstream pathways (bbs, bcr, bam), though the cultures incubated with hydrocarbon mixtures contained a broader diversity of fumarate addition genes. A comparative metagenomic analysis of the three cultures showed that they were functionally redundant despite their enrichment backgrounds, sharing multiple features associated with syntrophic hydrocarbon conversion to methane. In addition, a comparative analysis of the culture metagenomes with those of 41 environmental samples (containing varying proportions of methanogens) showed that the three cultures were functionally most similar to each other but distinct from other environments, including hydrocarbon-impacted environments (for example, oil sands tailings ponds and oil-affected marine sediments). This study provides a basis for understanding key functions and environmental selection in methanogenic hydrocarbon-associated communities.

  19. [ATP pool and bioluminescence in psychrophilic bacteria Photobacterium phosphoreum].

    PubMed

    Alekserova, L É; Alenina, K A; Efremenko, E N; Mazhul', M M; Piskunova, N F; Ismailov, A D

    2014-01-01

    Bioluminescence activity and ATP pool were investigated in the culture of psychrophilic bacteria Photobacterium phosphoreum collected-from the exponential and stationary growth phases, as well as immobilized in polyvinyl alcohol (PVA) cryogel. In liquid culture, ATP pool remained at an almost a constant level throughout the luminescence cycle (over 100 h). The ATP pool in the stationary-phase and PVA-immobilizedl cells remained constant throughout their incubation in the medium (over 200 h) and in 3% NaCl solution (over 100 h): Quantitative assessment of integral photon yield and ATP pool indicated that bioluminescence decay in growing or stationary cells was not caused by limitation by the energy substrates of the luciferase reaction. Kinetic and quantitative parameters of emission activity and ATP pool excluded the possibility of formation of the aldehyde substrate for luciferase via reduction of the relevant fatty acids in NADPH and ATP-dependent reductase reaction and its oxidation in the monooxygenase reaction. Our results indicate that the aliphatic aldehyde is not utilized in the process of light emission.

  20. Psychrophiles and astrobiology: microbial life of frozen worlds

    NASA Astrophysics Data System (ADS)

    Pikuta, Elena V.; Hoover, Richard B.

    2003-01-01

    Most bodies of our Solar System are "Frozen Worlds" where the prevailing surface temperature remains at or below freezing. On Earth there are vast permanently frozen regions of permafrost, polar ice sheets, and glaciers and the deep oceans and deep-sea marine sediments have remained at 2 - 4°C for eons. Psychrophilic and psychrotrophic microbiota that inhabit these regimes provide analogs for microbial life that might inhabit ice sheets and permafrost of Mars, comets, or the ice/water interfaces or sediments deep beneath the icy crusts of Europa, Callisto, or Ganymede. Cryopreserved micro-organisms can remain viable (in a deep anabiotic state) for millions of years frozen in permafrost and ice. Psychrophilic and psychrotrophic (cold-loving) microbes can carry out metabolic processes in water films and brine, acidic, or alkaline chanels in permafrost or ice at temperatures far below 0°C. These microbes of the cryosphere help define the thermal and temporal limits of life on Earth and may provide clues to where and how to search for evidence of life elsewhere in the Cosmos. Astrobiologists at the NASA Marshall Space Flight Center have collected microbial extremophiles from the Pleistocene ice wedges and frozen thermokarst ponds from the Fox Permafrost Tunnel of Alaska. Microbes have also been isolated from samples of Magellanic Penguin guano from Patagonia; deep-sea marine muds near hydrothermal vents; snow and permafrost from Siberia, and deep ice cores, ice-bubble and cryoconite rocks of the Central Antarctic Ice Sheet. These samples have yielded microbial extremophiles representing a wide variety of anaerobic bacteria and archaea. These microbes have been isolated, cultured, characterized and analyzed by phylogenetic and genomic methods. Images were obtained by Phase Contrast, Environmental, Field Emission Scanning and Transmission Electron Microscopes to study the ultra-microstructure and elemental distribution in the composition of these micro-organisms. We

  1. Cultivation of mesophilic soil crenarchaeotes in enrichment cultures from plant roots.

    PubMed

    Simon, Holly M; Jahn, Courtney E; Bergerud, Luke T; Sliwinski, Marek K; Weimer, Paul J; Willis, David K; Goodman, Robert M

    2005-08-01

    Because archaea are generally associated with extreme environments, detection of nonthermophilic members belonging to the archaeal division Crenarchaeota over the last decade was unexpected; they are surprisingly ubiquitous and abundant in nonextreme marine and terrestrial habitats. Metabolic characterization of these nonthermophilic crenarchaeotes has been impeded by their intractability toward isolation and growth in culture. From studies employing a combination of cultivation and molecular phylogenetic techniques (PCR-single-strand conformation polymorphism, sequence analysis of 16S rRNA genes, fluorescence in situ hybridization, and real-time PCR), we present evidence here that one of the two dominant phylotypes of Crenarchaeota that colonizes the roots of tomato plants grown in soil from a Wisconsin field is selectively enriched in mixed cultures amended with root extract. Clones recovered from enrichment cultures were found to group phylogenetically with sequences from clade C1b.A1. This work corroborates and extends our recent findings, indicating that the diversity of the crenarchaeal soil assemblage is influenced by the rhizosphere and that mesophilic soil crenarchaeotes are found associated with plant roots, and provides the first evidence for growth of nonthermophilic crenarchaeotes in culture.

  2. Cultivation of Mesophilic Soil Crenarchaeotes in Enrichment Cultures from Plant Roots

    PubMed Central

    Simon, Holly M.; Jahn, Courtney E.; Bergerud, Luke T.; Sliwinski, Marek K.; Weimer, Paul J.; Willis, David K.; Goodman, Robert M.

    2005-01-01

    Because archaea are generally associated with extreme environments, detection of nonthermophilic members belonging to the archaeal division Crenarchaeota over the last decade was unexpected; they are surprisingly ubiquitous and abundant in nonextreme marine and terrestrial habitats. Metabolic characterization of these nonthermophilic crenarchaeotes has been impeded by their intractability toward isolation and growth in culture. From studies employing a combination of cultivation and molecular phylogenetic techniques (PCR-single-strand conformation polymorphism, sequence analysis of 16S rRNA genes, fluorescence in situ hybridization, and real-time PCR), we present evidence here that one of the two dominant phylotypes of Crenarchaeota that colonizes the roots of tomato plants grown in soil from a Wisconsin field is selectively enriched in mixed cultures amended with root extract. Clones recovered from enrichment cultures were found to group phylogenetically with sequences from clade C1b.A1. This work corroborates and extends our recent findings, indicating that the diversity of the crenarchaeal soil assemblage is influenced by the rhizosphere and that mesophilic soil crenarchaeotes are found associated with plant roots, and provides the first evidence for growth of nonthermophilic crenarchaeotes in culture. PMID:16085872

  3. Biodegradation of munitions compounds by a sulfate reducing bacterial enrichment culture

    SciTech Connect

    Boopathy, R.; Manning, J.

    1997-08-01

    The degradation of several munitions compounds was studied. The compounds included 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine, octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine, 2,4,6-trinitrobenzene (TNB), and 2,4-dinitrotoluene. All of the compounds studied were degraded by the sulfate reducing bacterial (SRB) enrichment culture. The SRB culture did not use the munitions compounds as their sole source of carbon. However, all the munitions compounds tested served as the sole source of nitrogen for the SRB culture. Degradation of munitions compounds was achieved by a co-metabolic process. The SRB culture used a variety of carbon sources including pyruvate, ethanol, formate, lactate, and H{sub 2}-CO{sub 2}. The SRB culture was an incomplete oxidizer, unable to carry out the terminal oxidation of organic substrates to CO{sub 2} as the sole product, and it did not use acetate or methanol as a carbon source. In addition to serving as nitrogen sources, the munitions compounds also served as electron acceptors in the absence of sulfate. A soil slurry experiment with 5% and 10% munitions compounds-contaminated soil showed that the contaminant TNT was metabolized by the SRB culture in the presence of pyruvate as electron donor. This culture may be useful in decontaminating munitions compounds-contaminated soil and water under anaerobic conditions.

  4. Bacterial community analysis of cypermethrin enrichment cultures and bioremediation of cypermethrin contaminated soils.

    PubMed

    Akbar, Shamsa; Sultan, Sikander; Kertesz, Michael

    2015-07-01

    Cypermethrin is widely used for insect control; however, its toxicity toward aquatic life requires its complete removal from contaminated areas where the natural degradation ability of microbes can be utilized. Agricultural soil with extensive history of CM application was used to prepare enrichment cultures using cypermethrin as sole carbon source for isolation of cypermethrin degrading bacteria and bacterial community analysis using PCR-DGGE of 16 S rRNA gene. DGGE analysis revealed that dominant members of CM enrichment culture were associated with α-proteobacteria followed by γ-proteobacteria, Firmicutes, and Actinobacteria. Three potential CM-degrading isolates identified as Ochrobactrum anthropi JCm1, Bacillus megaterium JCm2, and Rhodococcus sp. JCm5 degraded 86-100% of CM (100 mg L(-1) ) within 10 days. These isolates were also able to degrade other pyrethroids, carbofuran, and cypermethrin degradation products. Enzyme activity assays revealed that enzymes involved in CM-degradation were inducible and showed activity when strains were grown on cypermethrin. Degradation kinetics of cypermethrin (200 mg kg(-1)) in soils inoculated with isolates JCm1, JCm2, and JCm5 suggested time-dependent disappearance of cypermethrin with rate constants of 0.0516, 0.0425, and 0.0807 d(-1), respectively, following first order rate kinetics. The isolated bacterial strains were among dominant genera selected under CM enriched conditions and represent valuable candidates for in situ bioremediation of contaminated soils and waters.

  5. Tetrachloromethane-Degrading Bacterial Enrichment Cultures and Isolates from a Contaminated Aquifer

    PubMed Central

    Penny, Christian; Gruffaz, Christelle; Nadalig, Thierry; Cauchie, Henry-Michel; Vuilleumier, Stéphane; Bringel, Françoise

    2015-01-01

    Abstract: The prokaryotic community of a groundwater aquifer exposed to high concentrations of tetrachloromethane (CCl4) for more than three decades was followed by terminal restriction fragment length polymorphism (T-RFLP) during pump-and-treat remediation at the contamination source. Bacterial enrichments and isolates were obtained under selective anoxic conditions, and degraded 10 mg·L−1 CCl4, with less than 10% transient formation of chloroform. Dichloromethane and chloromethane were not detected. Several tetrachloromethane-degrading strains were isolated from these enrichments, including bacteria from the Klebsiella and Clostridium genera closely related to previously described CCl4 degrading bacteria, and strain TM1, assigned to the genus Pelosinus, for which this property was not yet described. Pelosinus sp. TM1, an oxygen-tolerant, Gram-positive bacterium with strictly anaerobic metabolism, excreted a thermostable metabolite into the culture medium that allowed extracellular CCl4 transformation. As estimated by T-RFLP, phylotypes of CCl4-degrading enrichment cultures represented less than 7%, and archaeal and Pelosinus strains less than 0.5% of the total prokaryotic groundwater community. PMID:27682092

  6. Dry-heat resistance of selected psychrophiles.

    PubMed

    Winans, L; Pflug, I J; Foster, T L

    1977-08-01

    The dry-heat resistance characteristics of spores of psychrophilic organisms isolated from soil samples from the Viking spacecraft assembly areas at Cape Kennedy Space Flight Center, Cape Canaveral, Fla., were studied. Spore suspensions were produced, and dry-heat D values were determined for the microorganisms that demonstrated growth or survival under a simulated Martian environment. The dry-heat tests were carried out by using the planchet-boat-hot plate system at 110 and 125 degrees C with an ambient relative humidity of 50% at 22 degrees C. The spores evaluated had a relatively low resistance to dry heat. D(110 degrees C) values ranged from 7.5 to 122 min, whereas the D(123 degrees C) values ranged from less than 1.0 to 9.8 min.

  7. High-efficiency hydrogen production by an anaerobic, thermophilic enrichment culture from an Icelandic hot spring.

    PubMed

    Koskinen, Perttu E P; Lay, Chyi-How; Puhakka, Jaakko A; Lin, Ping-Jei; Wu, Shu-Yii; Orlygsson, Jóhann; Lin, Chiu-Yue

    2008-11-01

    Dark fermentative hydrogen production from glucose by a thermophilic culture (33HL), enriched from an Icelandic hot spring sediment sample, was studied in two continuous-flow, completely stirred tank reactors (CSTR1, CSTR2) and in one semi-continuous, anaerobic sequencing batch reactor (ASBR) at 58 degrees C. The 33HL produced H2 yield (HY) of up to 3.2 mol-H2/mol-glucose along with acetate in batch assay. In the CSTR1 with 33HL inoculum, H2 production was unstable. In the ASBR, maintained with 33HL, the H2 production enhanced after the addition of 6 mg/L of FeSO4 x H2O resulting in HY up to 2.51 mol-H2/mol-glucose (H2 production rate (HPR) of 7.85 mmol/h/L). The H2 production increase was associated with an increase in butyrate production. In the CSTR2, with ASBR inoculum and FeSO4 supplementation, stable, high-rate H2 production was obtained with HPR up to 45.8 mmol/h/L (1.1 L/h/L) and HY of 1.54 mol-H2/mol-glucose. The 33HL batch enrichment was dominated by bacterial strains closely affiliated with Thermobrachium celere (99.8-100%). T. celere affiliated strains, however, did not thrive in the three open system bioreactors. Instead, Thermoanaerobacterium aotearoense (98.5-99.6%) affiliated strains, producing H2 along with butyrate and acetate, dominated the reactor cultures. This culture had higher H2 production efficiency (HY and specific HPR) than reported for mesophilic mixed cultures. Further, the thermophilic culture readily formed granules in CSTR and ASBR systems. In summary, the thermophilic culture as characterized by high H2 production efficiency and ready granulation is considered very promising for H2 fermentation from carbohydrates.

  8. Integrated biogas upgrading and hydrogen utilization in an anaerobic reactor containing enriched hydrogenotrophic methanogenic culture.

    PubMed

    Luo, Gang; Angelidaki, Irini

    2012-11-01

    Biogas produced by anaerobic digestion, is mainly used in a gas motor for heat and electricity production. However, after removal of CO(2) , biogas can be upgraded to natural gas quality, giving more utilization possibilities, such as utilization as autogas, or distant utilization by using the existing natural gas grid. The current study presents a new biological method for biogas upgrading in a separate biogas reactor, containing enriched hydrogenotrophic methanogens and fed with biogas and hydrogen. Both mesophilic- and thermophilic anaerobic cultures were enriched to convert CO(2) to CH(4) by addition of H(2) . Enrichment at thermophilic temperature (55°C) resulted in CO(2) and H(2) bioconversion rate of 320 mL CH(4) /(gVSS h), which was more than 60% higher than that under mesophilic temperature (37°C). Different dominant species were found at mesophilic- and thermophilic-enriched cultures, as revealed by PCR-DGGE. Nonetheless, they all belonged to the order Methanobacteriales, which can mediate hydrogenotrophic methanogenesis. Biogas upgrading was then tested in a thermophilic anaerobic reactor under various operation conditions. By continuous addition of hydrogen in the biogas reactor, high degree of biogas upgrading was achieved. The produced biogas had a CH(4) content, around 95% at steady-state, at gas (mixture of biogas and hydrogen) injection rate of 6 L/(L day). The increase of gas injection rate to 12 L/(L day) resulted in the decrease of CH(4) content to around 90%. Further study showed that by decreasing the gas-liquid mass transfer by increasing the stirring speed of the mixture the CH(4) content was increased to around 95%. Finally, the CH(4) content around 90% was achieved in this study with the gas injection rate as high as 24 L/(L day).

  9. Anaerobic Psychrophiles from Alaska, Antarctica, and Patagonia: Implications to Possible Life on Mars and Europa

    NASA Technical Reports Server (NTRS)

    Hoover, Richard B.; Pikuta, Elena V.; Marsic, Damien; Ng, Joseph

    2002-01-01

    Microorganisms preserved within the permafrost, glaciers, and polar ice sheets of planet Earth provide analogs for microbial life forms that may be encountered in ice or permafrost of Mars, Europa, Callisto, Ganymede, asteroids, comets or other frozen worlds in the Cosmos. The psychrophilic and psychrotolerant microbes of the terrestrial cryosphere help establish the thermal and temporal limitations of life on Earth and provide clues to where and how we should search for evidence of life elsewhere in the Universe. For this reason, the cold-loving microorganisms are directly relevant to Astrobiology. Cryopreserved microorganisms can remain viable (in deep anabiosis) in permafrost and ice for millions of years. Permafrost, ice wedges, pingos, glaciers, and polar ice sheets may contain intact ancient DNA, lipids, enzymes, proteins, genes, and even frozen and yet viable ancient microbiota. Some microorganisms carry out metabolic processes in water films and brine, acidic, or alkaline channels in permafrost or ice at temperatures far below 0 C. Complex microbial communities live in snow, ice-bubbles, cryoconite holes on glaciers and ancient microbial ecosystems are cryopreserved within the permafrost, glaciers, and polar caps. In the Astrobiology group of the NASA Marshall Space Flight Center and the University of Alabama at Huntsville, we have employed advanced techniques for the isolation, culture, and phylogenetic analysis of many types of microbial extremophiles. We have also used the Environmental Scanning Electron Microscope to study the morphology, ultra-microstructure and chemical composition of microorganisms in ancient permafrost and ice. We discuss several interesting and novel anaerobic microorganisms that we have isolated and cultured from the Pleistocene ice of the Fox Tunnel of Alaska, guano of the Magellanic Penguin, deep-sea sediments from the vicinity of the Rainbow Hydrothermal Vent and enrichment cultures from ice of the Patriot Hills of Antarctica

  10. Anaerobic Psychrophiles from Alaska, Antarctica, and Patagonia: Implications to Possible Life on Mars and Europa

    NASA Technical Reports Server (NTRS)

    Hoover, Richard B.; Pikuta, Elena V.; Marsic, Damien; Ng, Joseph; Six, N. Frank (Technical Monitor)

    2001-01-01

    Microorganisms preserved within the permafrost, glaciers, and polar ice sheets of planet Earth provide analogs for microbial life forms that may be encountered in ice or permafrost of Mars, Europa, Callisto, Ganymede, asteroids, comets or other frozen worlds in the Cosmos. The psychrophilic and psychrotolerant microbes of the terrestrial cryosphere help establish the thermal and temporal limitations of life on Earth and provide clues to where and how we should search for evidence of life elsewhere in the Universe. For this reason, the cold-loving microorganisms are directly relevant to Astrobiology. Cryo-preserved microorganisms can remain viable (in deep anabiosis) in permafrost and ice for millions of years. Permafrost, ice wedges, pingos, glaciers, and polar ice sheets may contain intact ancient DNA, lipids, enzymes, proteins, genes, and even frozen and yet viable ancient microbiota. Some microorganisms carry out metabolic processes in water films and brine, acidic, or alkaline channels in permafrost or ice at temperatures far below 0 T. Complex microbial communities live in snow, ice-bubbles, cryoconite holes on glaciers and ancient microbial ecosystems are cryopreserved within the permafrost, glaciers, and polar caps. In the Astrobiology group of the NASA Marshall Space Flight Center and the University of Alabama at Huntsville, we have employed advanced techniques for the isolation, culture, and phylogenetic analysis of many types of microbial extremophiles. We have also used the Environmental Scanning Electron Microscope to study the morphology, ultra-microstructure and chemical composition of microorganisms in ancient permafrost and ice. We discuss several interesting and novel anaerobic microorganisms that we have isolated and cultured from the Pleistocene ice of the Fox Tunnel of Alaska, guano of the Magellanic Penguin, deep sea sediments from the vicinity of the Rainbow Hydrothermal Vent and enrichment cultures from ice of the Patriot Hills of Antarctica

  11. Anaerobic Psychrophiles from Alaska, Antarctica, and Patagonia: Implications to Possible Life on Mars and Europa

    NASA Technical Reports Server (NTRS)

    Hoover, Richard B.; Pikuta, Elena V.; Marsic, Damien; Ng, Joseph; Six, N. Frank (Technical Monitor)

    2001-01-01

    Microorganisms preserved within the permafrost, glaciers, and polar ice sheets of planet Earth provide analogs for microbial life forms that may be encountered in ice or permafrost of Mars, Europa, Callisto, Ganymede, asteroids, comets or other frozen worlds in the Cosmos. The psychrophilic and psychrotolerant microbes of the terrestrial cryosphere help establish the thermal and temporal limitations of life on Earth and provide clues to where and how we should search for evidence of life elsewhere in the Universe. For this reason, the cold-loving microorganisms are directly relevant to Astrobiology. Cryo-preserved microorganisms can remain viable (in deep anabiosis) in permafrost and ice for millions of years. Permafrost, ice wedges, pingos, glaciers, and polar ice sheets may contain intact ancient DNA, lipids, enzymes, proteins, genes, and even frozen and yet viable ancient microbiota. Some microorganisms carry out metabolic processes in water films and brine, acidic, or alkaline channels in permafrost or ice at temperatures far below 0 T. Complex microbial communities live in snow, ice-bubbles, cryoconite holes on glaciers and ancient microbial ecosystems are cryopreserved within the permafrost, glaciers, and polar caps. In the Astrobiology group of the NASA Marshall Space Flight Center and the University of Alabama at Huntsville, we have employed advanced techniques for the isolation, culture, and phylogenetic analysis of many types of microbial extremophiles. We have also used the Environmental Scanning Electron Microscope to study the morphology, ultra-microstructure and chemical composition of microorganisms in ancient permafrost and ice. We discuss several interesting and novel anaerobic microorganisms that we have isolated and cultured from the Pleistocene ice of the Fox Tunnel of Alaska, guano of the Magellanic Penguin, deep sea sediments from the vicinity of the Rainbow Hydrothermal Vent and enrichment cultures from ice of the Patriot Hills of Antarctica

  12. Anaerobic Psychrophiles from Alaska, Antarctica, and Patagonia: Implications to Possible Life on Mars and Europa

    NASA Technical Reports Server (NTRS)

    Hoover, Richard B.; Pikuta, Elena V.; Marsic, Damien; Ng, Joseph

    2002-01-01

    Microorganisms preserved within the permafrost, glaciers, and polar ice sheets of planet Earth provide analogs for microbial life forms that may be encountered in ice or permafrost of Mars, Europa, Callisto, Ganymede, asteroids, comets or other frozen worlds in the Cosmos. The psychrophilic and psychrotolerant microbes of the terrestrial cryosphere help establish the thermal and temporal limitations of life on Earth and provide clues to where and how we should search for evidence of life elsewhere in the Universe. For this reason, the cold-loving microorganisms are directly relevant to Astrobiology. Cryopreserved microorganisms can remain viable (in deep anabiosis) in permafrost and ice for millions of years. Permafrost, ice wedges, pingos, glaciers, and polar ice sheets may contain intact ancient DNA, lipids, enzymes, proteins, genes, and even frozen and yet viable ancient microbiota. Some microorganisms carry out metabolic processes in water films and brine, acidic, or alkaline channels in permafrost or ice at temperatures far below 0 C. Complex microbial communities live in snow, ice-bubbles, cryoconite holes on glaciers and ancient microbial ecosystems are cryopreserved within the permafrost, glaciers, and polar caps. In the Astrobiology group of the NASA Marshall Space Flight Center and the University of Alabama at Huntsville, we have employed advanced techniques for the isolation, culture, and phylogenetic analysis of many types of microbial extremophiles. We have also used the Environmental Scanning Electron Microscope to study the morphology, ultra-microstructure and chemical composition of microorganisms in ancient permafrost and ice. We discuss several interesting and novel anaerobic microorganisms that we have isolated and cultured from the Pleistocene ice of the Fox Tunnel of Alaska, guano of the Magellanic Penguin, deep-sea sediments from the vicinity of the Rainbow Hydrothermal Vent and enrichment cultures from ice of the Patriot Hills of Antarctica

  13. Anaerobic psychrophiles from Alaska, Antarctica, and Patagonia: implications to possible life on Mars and Europa

    NASA Astrophysics Data System (ADS)

    Hoover, Richard B.; Pikuta, Elena V.; Marsic, Damien; Ng, Joseph D.

    2002-02-01

    Microorganisms preserved within the permafrost, glaciers, and polar ice sheets of planet Earth provide analogs for microbial life forms that may be encountered in ice or permafrost of Mars, Europa, Callisto, Ganymede, asteroids, comets or other frozen worlds in the Cosmos. The psychrophilic and psychrotolerant microbes of the terrestrial cryosphere help establish the thermal and temporal limitations of life on Earth and provide clues to where and how we should search for evidence of life elsewhere in the Universe. For this reason, the cold-loving microorganisms are directly relevant to Astrobiology. Cryopreserved microorganisms can remain viable (in deep anabiosis) in permafrost and ice for millions of years. Permafrost, ice wedges, pingos, glaciers, and polar ice sheets may contain intact ancient DNA, lipids, enzymes, proteins, genes, and even frozen and yet viable ancient microbiota. Some microorganisms carry out metabolic processes in water films and brine, acidic, or alkaline channels in permafrost or ice at temperatures far below 0 degree(s)C. Complex microbial communities live in snow, ice-bubbles, cryoconite holes on glaciers and ancient microbial ecosystems are cryopreserved within the permafrost, glaciers, and polar caps. In the Astrobiology group of the NASA Marshall Space Flight Center and the University of Alabama at Huntsville, we have employed advanced techniques for the isolation, culture, and phylogenetic analysis of many types of microbial extremophiles. We have also used the Environmental Scanning Electron Microscope to study the morphology, ultra-microstructure and chemical composition of microorganisms in ancient permafrost and ice. We discuss several interesting and novel anaerobic microorganisms that we have isolated and cultured from the Pleistocene ice of the Fox Tunnel of Alaska, guano of the Magellanic Penguin, deep-sea sediments from the vicinity of the Rainbow Hydrothermal Vent and enrichment cultures from ice of the Patriot Hills of

  14. Anaerobic biotransformation of high concentrations of chloroform by an enrichment culture and two bacterial isolates.

    PubMed

    Shan, Huifeng; Kurtz, Harry D; Mykytczuk, Nadia; Trevors, Jack T; Freedman, David L

    2010-10-01

    A fermentative enrichment culture (designated DHM-1) was developed that is capable of cometabolically biotransforming high concentrations of chloroform (CF) to nontoxic end products. Two Pantoea spp. were isolated from DHM-1 that also possess this dechlorination capability. Following acclimation to increasing levels of CF, corn syrup-grown DHM-1 was able to transform over 500 mg/liter CF in the presence of vitamin B(12) (approximately 3% of CF on a molar basis) at a rate as high as 22 mg/liter/day in a mineral salts medium. CO, CO(2), and organic acids were the predominant biodegradation products, suggesting that hydrolytic reactions predominate during CF transformation. DHM-1 was capable of growing on corn syrup in the presence of high concentrations of CF (as may be present near contaminant source zones in groundwater), which makes it a promising culture for bioaugmentation. Strains DHM-1B and DHM-1T transform CF at rates similar to that of the DHM-1 enrichment culture. The ability of these strains to grow in the presence of high concentrations of CF appears to be related to alteration of membrane fluidity or homeoviscous and homeophasic adaptation.

  15. Molecular Detection of Culture-Confirmed Bacterial Bloodstream Infections with Limited Enrichment Time

    PubMed Central

    Moore, Miranda S.; McCann, Chase D.

    2013-01-01

    Conventional blood culturing using automated instrumentation with phenotypic identification requires a significant amount of time to generate results. This study investigated the speed and accuracy of results generated using PCR and pyrosequencing compared to the time required to obtain Gram stain results and final culture identification for cases of culture-confirmed bloodstream infections. Research and physician-ordered blood cultures were drawn concurrently. Aliquots of the incubating research blood culture fluid were removed hourly between 5 and 8 h, at 24 h, and again at 5 days. DNA was extracted from these 6 time point aliquots and analyzed by PCR and pyrosequencing for bacterial rRNA gene targets. These results were then compared to those of the physician-ordered blood culture. PCR and pyrosequencing accurately identified 92% of all culture-confirmed cases after a mean enrichment time of 5.8 ± 2.9 h. When the time needed to complete sample processing was included for PCR and pyrosequencing protocols, the molecular approach yielded results in 11.8 ± 2.9 h compared to means of 27.9 ± 13.6 h to obtain the Gram stain results and 81.6 ± 24.0 h to generate the final culture-based identification. The molecular approach enabled accurate detection of most bacteria present in incubating blood culture bottles on average about 16 h sooner than Gram stain results became available and approximately 3 days sooner than the phenotypic identification was entered in the Laboratory Information System. If implemented, this more rapid molecular approach could minimize the number of doses of unnecessary or ineffective antibiotics administered to patients. PMID:23985915

  16. Metagenomic Analyses of the Autotrophic Fe(II)-Oxidizing, Nitrate-Reducing Enrichment Culture KS

    PubMed Central

    Tominski, Claudia; Kappler, Andreas; Behrens, Sebastian

    2016-01-01

    Nitrate-dependent ferrous iron [Fe(II)] oxidation (NDFO) is a well-recognized chemolithotrophic pathway in anoxic sediments. The neutrophilic chemolithoautotrophic enrichment culture KS originally obtained from a freshwater sediment (K. L. Straub, M. Benz, B. Schink, and F. Widdel, Appl Environ Microbiol 62:1458–1460, 1996) has been used as a model system to study NDFO. However, the primary Fe(II) oxidizer in this culture has not been isolated, despite extensive efforts to do so. Here, we present a metagenomic analysis of this enrichment culture in order to gain insight into electron transfer pathways and the roles of different bacteria in the culture. We obtained a near-complete genome of the primary Fe(II) oxidizer, a species in the family Gallionellaceae, and draft genomes from its flanking community members. A search of the putative extracellular electron transfer pathways in these genomes led to the identification of a homolog of the MtoAB complex [a porin-multiheme cytochrome c system identified in neutrophilic microaerobic Fe(II)-oxidizing Sideroxydans lithotrophicus ES-1] in a Gallionellaceae sp., and findings of other putative genes involving cytochrome c and multicopper oxidases, such as Cyc2 and OmpB. Genome-enabled metabolic reconstruction revealed that this Gallionellaceae sp. lacks nitric oxide and nitrous oxide reductase genes and may partner with flanking populations capable of complete denitrification to avoid toxic metabolite accumulation, which may explain its resistance to growth in pure culture. This and other revealed interspecies interactions and metabolic interdependencies in nitrogen and carbon metabolisms may allow these organisms to cooperate effectively to achieve robust chemolithoautotrophic NDFO. Overall, the results significantly expand our knowledge of NDFO and suggest a range of genetic targets for further exploration. PMID:26896135

  17. Anaerobic Degradation of Pristane in Nitrate-Reducing Microcosms and Enrichment Cultures

    PubMed Central

    Bregnard, T. P.; Haner, A.; Hohener, P.; Zeyer, J.

    1997-01-01

    Microcosm studies were conducted under nitrate-reducing conditions with diesel fuel-contaminated aquifer material from a site treated by in situ bioremediation. In the microcosms, the consumption of nitrate and the production of inorganic carbon were strongly stimulated by the addition of the isoprenoid alkane pristane (2,6,10,14-tetramethylpentadecane). Within 102 days enrichment cultures degraded more than 90% of the pristane supplied as coatings on reticulated sinter glass rings. The study demonstrates that pristane can no longer be regarded as recalcitrant under anaerobic conditions. PMID:16535616

  18. Exploration and isolation of novel thermophiles in frozen enrichment cultures derived from a terrestrial acidic hot spring.

    PubMed

    Sakai, Hiroyuki D; Kurosawa, Norio

    2016-03-01

    An isolation strategy, exploring novel microorganisms in frozen enrichment cultures (ENFE), which uses a combination of enrichment culture and 16S rRNA gene clone analysis, was evaluated for isolating uncultured thermophiles from a terrestrial acidic hot spring. The procedure comprised (a) multiple enrichment cultures under various conditions, (b) cryostorage of all enrichments, (c) microbial community analyses of the enrichments using 16S rRNA gene sequences, and (d) purification of microorganisms from enrichments containing previously uncultured microorganisms. The enrichments were performed under a total of 36 conditions, and 16 of these enrichments yielded positive microbial growth with the detection of three previously uncultured archaea. Two of the three previously uncultured archaea, strains HS-1 and HS-3, were successfully isolated. Strain HS-1 and HS-3 represented a novel lineage of the order Sulfolobales and novel species of the genus Sulfolobus, respectively. Although innovative isolation methods play strategic roles in isolating previously uncultured microorganisms, the ENFE strategy showed potential for characterizing and isolating such microorganisms using conventional media and techniques.

  19. Neonicotinoid Insecticides Alter the Gene Expression Profile of Neuron-Enriched Cultures from Neonatal Rat Cerebellum

    PubMed Central

    Kimura-Kuroda, Junko; Nishito, Yasumasa; Yanagisawa, Hiroko; Kuroda, Yoichiro; Komuta, Yukari; Kawano, Hitoshi; Hayashi, Masaharu

    2016-01-01

    Neonicotinoids are considered safe because of their low affinities to mammalian nicotinic acetylcholine receptors (nAChRs) relative to insect nAChRs. However, because of importance of nAChRs in mammalian brain development, there remains a need to establish the safety of chronic neonicotinoid exposures with regards to children’s health. Here we examined the effects of long-term (14 days) and low dose (1 μM) exposure of neuron-enriched cultures from neonatal rat cerebellum to nicotine and two neonicotinoids: acetamiprid and imidacloprid. Immunocytochemistry revealed no differences in the number or morphology of immature neurons or glial cells in any group versus untreated control cultures. However, a slight disturbance in Purkinje cell dendritic arborization was observed in the exposed cultures. Next we performed transcriptome analysis on total RNAs using microarrays, and identified significant differential expression (p < 0.05, q < 0.05, ≥1.5 fold) between control cultures versus nicotine-, acetamiprid-, or imidacloprid-exposed cultures in 34, 48, and 67 genes, respectively. Common to all exposed groups were nine genes essential for neurodevelopment, suggesting that chronic neonicotinoid exposure alters the transcriptome of the developing mammalian brain in a similar way to nicotine exposure. Our results highlight the need for further careful investigations into the effects of neonicotinoids in the developing mammalian brain. PMID:27782041

  20. Neonicotinoid Insecticides Alter the Gene Expression Profile of Neuron-Enriched Cultures from Neonatal Rat Cerebellum.

    PubMed

    Kimura-Kuroda, Junko; Nishito, Yasumasa; Yanagisawa, Hiroko; Kuroda, Yoichiro; Komuta, Yukari; Kawano, Hitoshi; Hayashi, Masaharu

    2016-10-04

    Neonicotinoids are considered safe because of their low affinities to mammalian nicotinic acetylcholine receptors (nAChRs) relative to insect nAChRs. However, because of importance of nAChRs in mammalian brain development, there remains a need to establish the safety of chronic neonicotinoid exposures with regards to children's health. Here we examined the effects of longterm (14 days) and low dose (1 μM) exposure of neuron-enriched cultures from neonatal rat cerebellum to nicotine and two neonicotinoids: acetamiprid and imidacloprid. Immunocytochemistry revealed no differences in the number or morphology of immature neurons or glial cells in any group versus untreated control cultures. However, a slight disturbance in Purkinje cell dendritic arborization was observed in the exposed cultures. Next we performed transcriptome analysis on total RNAs using microarrays, and identified significant differential expression (p < 0.05, q < 0.05, ≥1.5 fold) between control cultures versus nicotine-, acetamiprid-, or imidacloprid-exposed cultures in 34, 48, and 67 genes, respectively. Common to all exposed groups were nine genes essential for neurodevelopment, suggesting that chronic neonicotinoid exposure alters the transcriptome of the developing mammalian brain in a similar way to nicotine exposure. Our results highlight the need for further careful investigations into the effects of neonicotinoids in the developing mammalian brain.

  1. A rapid, highly sensitive and culture-free detection of pathogens from blood by positive enrichment.

    PubMed

    Vutukuru, Manjula Ramya; Sharma, Divya Khandige; Ragavendar, M S; Schmolke, Susanne; Huang, Yiwei; Gumbrecht, Walter; Mitra, Nivedita

    2016-12-01

    Molecular diagnostics is a promising alternative to culture based methods for the detection of bloodstream infections, notably due to its overall lower turnaround time when starting directly from patient samples. Whole blood is usually the starting diagnostic sample in suspected bloodstream infections. The detection of low concentrations of pathogens in blood using a molecular assay necessitates a fairly high starting volume of blood sample in the range of 5-10mL. This large volume of blood sample has a substantial accompanying human genomic content that interferes with pathogen detection. In this study, we have established a workflow using magnetic beads coated with Apolipoprotein H that makes it possible to concentrate pathogens from a 5.0mL whole blood sample, thereby enriching pathogens from whole blood background and also reducing the sample volume to ~200μL or less. We have also demonstrated that this method of enrichment allows detection of 1CFU/mL of Escherichia coli, Enterococcus gallinarum and Candida tropicalis from 5mL blood using quantitative PCR; a detection limit that is not possible in unenriched samples. The enrichment method demonstrated here took 30min to complete and can be easily integrated with various downstream molecular and microbiological techniques. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Effect of oxygen enrichment on morphology, growth, and heterologous protein production in chemostat cultures of Aspergillus niger B1-D.

    PubMed

    Wongwicharn, A; McNeil, B; Harvey, L M

    1999-11-20

    The response of steady state chemostat cultures of a recombinant Aspergillus niger (B1-D), secreting both a heterologous enzyme (Hen Egg White Lysozyme [HEWL]) and a native enzyme (Glucoamylase), to varying levels of O2 enrichment of the process gas was evaluated. Formation of both the native and the foreign enzyme increased with increasing O2 supply. Conversely, biomass levels and total extracellular protein levels were generally not increased under O2 enriched conditions. Two distinct micromorphologies were apparent in these cultures, one, typically seen under O2 limiting conditions (i. e. at 0 and 10% enrichment levels), tended to be represented by long, sparsely branched hyphal elements, with low percentages of "active" length (i. e. how much of the hypha is cytoplasm filled); whilst, a second micromorphology, typical of O2 enriched cultures at 30 and 50% O2 enrichment, was represented by shorter hyphal elements, with more branching and a higher % "active" length. At these higher O2 levels, formation of a yellow pigment occurred, and signs of culture autolysis were noted. At 50% enrichment, a "stranded" aggregate morphology was apparent, possibly as a response to a hyperoxidant state. Production of both the native enzyme and HEWL correlated well with a simple morphological measure (tip number) or, with % "active" length. It is proposed the morphological changes noted in the cultures were associated with the increased production of both HEWL and glucoamylase.

  3. Genomics of an extreme psychrophile, Psychromonas ingrahamii

    SciTech Connect

    Riley, Monica; Staley, James T.; Danchin, Antoine; Wang, T.; Brettin, Tom; Hauser, Loren John; Land, Miriam L; Thompson, Linda S

    2008-05-01

    Background: The genome sequence of the sea-ice bacterium Psychromonas ingrahamii 37, which grows exponentially at -12C, may reveal features that help to explain how this extreme psychrophile is able to grow at such low temperatures. Determination of the whole genome sequence allows comparison with genes of other psychrophiles and mesophiles. Results: Correspondence analysis of the composition of all P. ingrahamii proteins showed that (1) there are 6 classes of proteins, at least one more than other bacteria, (2) integral inner membrane proteins are not sharply separated from bulk proteins suggesting that, overall, they may have a lower hydrophobic character, and (3) there is strong opposition between asparagine and the oxygen-sensitive amino acids methionine, arginine, cysteine and histidine and (4) one of the previously unseen clusters of proteins has a high proportion of "orphan" hypothetical proteins, raising the possibility these are cold-specific proteins. Based on annotation of proteins by sequence similarity, (1) P. ingrahamii has a large number (61) of regulators of cyclic GDP, suggesting that this bacterium produces an extracellular polysaccharide that may help sequester water or lower the freezing point in the vicinity of the cell. (2) P. ingrahamii has genes for production of the osmolyte, betaine choline, which may balance the osmotic pressure as sea ice freezes. (3) P. ingrahamii has a large number (11) of three-subunit TRAP systems that may play an important role in the transport of nutrients into the cell at low temperatures. (4) Chaperones and stress proteins may play a critical role in transforming nascent polypeptides into 3-dimensional configurations that permit low temperature growth. (5) Metabolic properties of P. ingrahamii were deduced. Finally, a few small sets of proteins of unknown function which may play a role in psychrophily have been singled out as worthy of future study. Conclusion: The results of this genomic analysis provide a

  4. Genomics of an extreme psychrophile, Psychromonas ingrahamii

    PubMed Central

    Riley, Monica; Staley, James T; Danchin, Antoine; Wang, Ting Zhang; Brettin, Thomas S; Hauser, Loren J; Land, Miriam L; Thompson, Linda S

    2008-01-01

    Background The genome sequence of the sea-ice bacterium Psychromonas ingrahamii 37, which grows exponentially at -12C, may reveal features that help to explain how this extreme psychrophile is able to grow at such low temperatures. Determination of the whole genome sequence allows comparison with genes of other psychrophiles and mesophiles. Results Correspondence analysis of the composition of all P. ingrahamii proteins showed that (1) there are 6 classes of proteins, at least one more than other bacteria, (2) integral inner membrane proteins are not sharply separated from bulk proteins suggesting that, overall, they may have a lower hydrophobic character, and (3) there is strong opposition between asparagine and the oxygen-sensitive amino acids methionine, arginine, cysteine and histidine and (4) one of the previously unseen clusters of proteins has a high proportion of "orphan" hypothetical proteins, raising the possibility these are cold-specific proteins. Based on annotation of proteins by sequence similarity, (1) P. ingrahamii has a large number (61) of regulators of cyclic GDP, suggesting that this bacterium produces an extracellular polysaccharide that may help sequester water or lower the freezing point in the vicinity of the cell. (2) P. ingrahamii has genes for production of the osmolyte, betaine choline, which may balance the osmotic pressure as sea ice freezes. (3) P. ingrahamii has a large number (11) of three-subunit TRAP systems that may play an important role in the transport of nutrients into the cell at low temperatures. (4) Chaperones and stress proteins may play a critical role in transforming nascent polypeptides into 3-dimensional configurations that permit low temperature growth. (5) Metabolic properties of P. ingrahamii were deduced. Finally, a few small sets of proteins of unknown function which may play a role in psychrophily have been singled out as worthy of future study. Conclusion The results of this genomic analysis provide a

  5. DEPENDENCE OF A HIGH-RATE, PCE-DECHLORINATING ENRICHMENT CULTURE ON METHANOGENIC ACTIVITY. (R825549C053)

    EPA Science Inventory

    The role served by the presence of methanogenic activity within a tetrachloroethene (PCE)-dechlorinating culture was investigated through a series of supplementation experiments. An acclimated lactate-enrichment culture (LEC 1) capable of rapidly converting PCE to ethene was s...

  6. Effect of liposomes on mineralization in rat osteoblast-enriched cultures.

    PubMed

    Huang, J S; Wu, H F; Hong, J M; Chang, F H; Hong, K

    1999-04-01

    Liposomes, artificial membranous lipid vesicles, have been used as model structures for biological calcification processes. However, there is no definite conclusion that liposomes can be like matrix vesicles for inducing bone calcification and bone-like tissue formation on primary cultured cells. To determine whether liposomes can promote bone cell growth and mineralization by inducing crystal nucleation, liposomes composed of egg phosphatidylcholine, cholesterol, and bovine brain phosphatidylserine were added to 21-day-old Sprague-Dawley fetal rat calvarial cell cultures from day 1. The aims were to observe proliferation and the phenotype of osteoblasts by measuring cell numbers and alkaline phosphatase (ALP) activity and, when added at confluence, to observe calcification. The data were analyzed with two-way ANOVA. During the 16-day culture period, cell numbers were not significantly affected by liposomes (100 mumol/L). However, ALP activity was significantly inhibited by liposomes (p < 0.05) at day 16 and thereafter. Calcified particles were detected by von Kossa's method, and were larger and more abundant (p < 0.05) in the liposomes groups than in the control from days 12-24. This response depended on liposomes dose. These findings suggest that liposomes promote calcification and accelerate the formation of bone-like tissue. Liposomes slightly reduce the expression of the osteoblast phenotype and do not affect cell growth in primary rat osteoblast-enriched cultures.

  7. Impact of arachidonic acid enrichment of live rotifer prey on bacterial communities in rotifer and larval fish cultures.

    PubMed

    Seychelles, Laurent H; Doiron, Kim; Audet, Céline; Tremblay, Réjean; Pernet, Fabrice; Lemarchand, Karine

    2013-03-01

    Rotifers (Brachionus plicatilis), commonly used at first feeding in commercial fish hatcheries, carry a large bacteria load. Because they are relatively poor in essential fatty acids, it is common practice to enrich them with fatty acids, including arachidonic acid (AA). This study aims to determine whether prey enrichment with AA may act as a prebiotic and modify the microbial community composition either in AA-enriched rotifer cultures or in larval-rearing water using winter flounder (Pseudopleuronectes americanus) as a larval fish model. AA enrichment modified the bacterial community composition in both the rotifer culture tanks and the larval-rearing tanks. We observed an increase in the number of cultivable bacteria on TCBS (thiosulfate-citrate-bile salts-sucrose) agar, used as a proxy for the abundance of Vibrio sp. The results suggest that AA may also play an indirect role in larval health.

  8. Biogeochemical insights into microbe-mineral-fluid interactions in hydrothermal chimneys using enrichment culture.

    PubMed

    Callac, Nolwenn; Rouxel, Olivier; Lesongeur, Françoise; Liorzou, Céline; Bollinger, Claire; Pignet, Patricia; Chéron, Sandrine; Fouquet, Yves; Rommevaux-Jestin, Céline; Godfroy, Anne

    2015-05-01

    Active hydrothermal chimneys host diverse microbial communities exhibiting various metabolisms including those involved in various biogeochemical cycles. To investigate microbe-mineral-fluid interactions in hydrothermal chimney and the driver of microbial diversity, a cultural approach using a gas-lift bioreactor was chosen. An enrichment culture was performed using crushed active chimney sample as inoculum and diluted hydrothermal fluid from the same vent as culture medium. Daily sampling provided time-series access to active microbial diversity and medium composition. Active archaeal and bacterial communities consisted mainly of sulfur, sulfate and iron reducers and hydrogen oxidizers with the detection of Thermococcus, Archaeoglobus, Geoglobus, Sulfurimonas and Thermotoga sequences. The simultaneous presence of active Geoglobus sp. and Archaeoglobus sp. argues against competition for available carbon sources and electron donors between sulfate and iron reducers at high temperature. This approach allowed the cultivation of microbial populations that were under-represented in the initial environmental sample. The microbial communities are heterogeneously distributed within the gas-lift bioreactor; it is unlikely that bulk mineralogy or fluid chemistry is the drivers of microbial community structure. Instead, we propose that micro-environmental niche characteristics, created by the interaction between the mineral grains and the fluid chemistry, are the main drivers of microbial diversity in natural systems.

  9. Bacterial oxidation of dibromomethane and methyl bromide in natural waters and enrichment cultures

    USGS Publications Warehouse

    Goodwin, K.D.; Schaefer, J.K.; Oremland, R.S.

    1998-01-01

    Bacterial oxidation of 14CH2Br2 and 14CH3Br was measured in freshwater, estuarine, seawater, and hypersaline-alkaline samples. In general, bacteria from the various sites oxidized similar amounts of 14CH2Br2 and comparatively less 14CH3Br. Bacterial oxidation of 14CH3Br was rapid in freshwater samples compared to bacterial oxidation of 14CH3Br in more saline waters. Freshwater was also the only site in which methyl fluoride-sensitive bacteria (e.g., methanotrophs or nitrifiers) governed brominated methane oxidation. Half-life calculations indicated that bacterial oxidation of CH2Br2 was potentially significant in all of the waters tested. In contrast, only in freshwater was bacterial oxidation of CH3Br as fast as chemical removal. The values calculated for more saline sites suggested that bacterial oxidation of CH3Br was relatively slow compared to chemical and physical loss mechanisms. However, enrichment cultures demonstrated that bacteria in seawater can rapidly oxidize brominated methanes. Two distinct cultures of nonmethanotrophic methylotrophs were recovered; one of these cultures was able to utilize CH2Br2 as a sole carbon source, and the other was able to utilize CH3Br as a sole carbon source.

  10. Persistence of pentolite (PETN and TNT) in soil microcosms and microbial enrichment cultures.

    PubMed

    Arbeli, Ziv; Garcia-Bonilla, Erika; Pardo, Cindy; Hidalgo, Kelly; Velásquez, Trigal; Peña, Luis; C, Eliana Ramos; Avila-Arias, Helena; Molano-Gonzalez, Nicolás; Brandão, Pedro F B; Roldan, Fabio

    2016-05-01

    Pentolite is a mixture (1:1) of 2,4,6-trinitrotoluene (TNT) and pentaerythritol tetranitrate (PETN), and little is known about its fate in the environment. This study was aimed to determine the dissipation of pentolite in soils under laboratory conditions. Microcosm experiments conducted with two soils demonstrated that dissipation rate of PETN was significantly slower than that of TNT. Interestingly, the dissipation of PETN was enhanced by the presence of TNT, while PETN did not enhanced the dissipation of TNT. Pentolite dissipation rate was significantly faster under biostimulation treatment (addition of carbon source) in soil from the artificial wetland, while no such stimulation was observed in soil from detonation field. In addition, the dissipation rate of TNT and PETN in soil from artificial wetland under biostimulation was significantly faster than the equivalent abiotic control, although it seems that non-biological processes might also be important for the dissipation of TNT and PETN. Transformation of PETN was also slower during establishment of enrichment culture using pentolite as the sole nitrogen source. In addition, transformation of these explosives was gradually reduced and practically stopped after the forth cultures transfer (80 days). DGGE analysis of bacterial communities from these cultures indicates that all consortia were dominated by bacteria from the order Burkholderiales and Rhodanobacter. In conclusion, our results suggest that PETN might be more persistent than TNT.

  11. Reductive dechlorination of tetrachloroethene to cis-1, 2-dichloroethene by a thermophilic anaerobic enrichment culture.

    PubMed

    Kengen, S W; Breidenbach, C G; Felske, A; Stams, A J; Schraa, G; de Vos, W M

    1999-06-01

    Thermophilic anaerobic biodegradation of tetrachloroethene (PCE) was investigated with various inocula from geothermal and nongeothermal areas. Only polluted harbor sediment resulted in a stable enrichment culture that converted PCE via trichloroethene to cis-1, 2-dichloroethene at the optimum temperature of 60 to 65 degrees C. After several transfers, methanogens were eliminated from the culture. Dechlorination was supported by lactate, pyruvate, fructose, fumarate, and malate as electron donor but not by H2, formate, or acetate. Fumarate and L-malate led to the highest dechlorination rate. In the absence of PCE, fumarate was fermented to acetate, H2, CO2, and succinate. With PCE, less H2 was formed, suggesting that PCE competed for the reducing equivalents leading to H2. PCE dechlorination, apparently, was not outcompeted by fumarate as electron acceptor. At the optimum dissolved PCE concentration of approximately 60 microM, a high dechlorination rate of 1.1 micromol h-1 mg-1 (dry weight) was found, which indicates that the dechlorination is not a cometabolic activity. Microscopic analysis of the fumarate-grown culture showed the dominance of a long thin rod. Molecular analysis, however, indicated the presence of two dominant species, both belonging to the low-G+C gram positives. The highest similarity was found with the genus Dehalobacter (90%), represented by the halorespiring organism Dehalobacter restrictus, and with the genus Desulfotomaculum (86%).

  12. Reductive Dechlorination of Tetrachloroethene to cis-1,2-Dichloroethene by a Thermophilic Anaerobic Enrichment Culture

    PubMed Central

    Kengen, Servé W. M.; Breidenbach, Caroline G.; Felske, Andreas; Stams, Alfons J. M.; Schraa, Gosse; de Vos, Willem M.

    1999-01-01

    Thermophilic anaerobic biodegradation of tetrachloroethene (PCE) was investigated with various inocula from geothermal and nongeothermal areas. Only polluted harbor sediment resulted in a stable enrichment culture that converted PCE via trichloroethene to cis-1,2-dichloroethene at the optimum temperature of 60 to 65°C. After several transfers, methanogens were eliminated from the culture. Dechlorination was supported by lactate, pyruvate, fructose, fumarate, and malate as electron donor but not by H2, formate, or acetate. Fumarate and l-malate led to the highest dechlorination rate. In the absence of PCE, fumarate was fermented to acetate, H2, CO2, and succinate. With PCE, less H2 was formed, suggesting that PCE competed for the reducing equivalents leading to H2. PCE dechlorination, apparently, was not outcompeted by fumarate as electron acceptor. At the optimum dissolved PCE concentration of ∼60 μM, a high dechlorination rate of 1.1 μmol h−1 mg−1 (dry weight) was found, which indicates that the dechlorination is not a cometabolic activity. Microscopic analysis of the fumarate-grown culture showed the dominance of a long thin rod. Molecular analysis, however, indicated the presence of two dominant species, both belonging to the low-G+C gram positives. The highest similarity was found with the genus Dehalobacter (90%), represented by the halorespiring organism Dehalobacter restrictus, and with the genus Desulfotomaculum (86%). PMID:10347007

  13. Detection of Yersinia spp. in meat products by enrichment culture, immunomagnetic separation and nested PCR.

    PubMed

    Estrada, Cecilia S M Lucero; Velázquez, Lidia Del Carmen; Favier, Gabriela Isabel; Genaro, María Silvia Di; Escudero, María Esther

    2012-05-01

    The prevalence of Yersinia enterocolitica in meat products was assessed by four methods: cold enrichment in trypticase soy broth (A), enrichment in modified Rappaport broth at 25 °C (B), concentration by immunomagnetic separation (C) and yadA nested PCR (D). Furthermore, the pathogenic potentials of the isolates were established by phenotypic and genotypic tests, and their genomic relationships were determined by pulsed-field gel electrophoresis (PFGE). A total of 238 samples were collected at retail level in the city of San Luis, Argentina, during the period 2007-2008. The highest Yersinia prevalence in meat products was observed by method D (92 positive samples), followed by methods A (13 positive samples) and C (5 positive samples); however, no isolation was obtained by method B. Fourteen Y. enterocolitica and 4 Yersinia intermedia strains were recovered by culture. All Y. enterocolitica 2/O:9 strains gave results related to virulence by phenotypic tests and exhibited the genotype virF(+)myfA(+)ail(+)ystA(+). Two biotype 1A strains showed a genotype virF(-)myfA(-)ail(+)ystA(+)ystB(+). The 14 Y. enterocolitica strains isolated during this work plus one reference strain were separated into 11 genomic types by PFGE. This genomic heterogeneity of the isolates shows the diversity of Y. enterocolitica strains in our region. It is the first time that IMS was used to search Y. enterocolitica strains from naturally contaminated meat products.

  14. Selective enrichment and production of highly urease active bacteria by non-sterile (open) chemostat culture.

    PubMed

    Cheng, Liang; Cord-Ruwisch, Ralf

    2013-10-01

    In general, bioprocesses can be subdivided into naturally occurring processes, not requiring sterility (e.g., beer brewing, wine making, lactic acid fermentation, or biogas digestion) and other processes (e.g., the production of enzymes and antibiotics) that typically require a high level of sterility to avoid contaminant microbes overgrowing the production strain. The current paper describes the sustainable, non-sterile production of an industrial enzyme using activated sludge as inoculum. By using selective conditions (high pH, high ammonia concentration, and presence of urea) for the target bacterium, highly active ureolytic bacteria, physiologically resembling Sporosarcina pasteurii were reproducibly enriched and then continuously produced via chemostat operation of the bioreactor. When using a pH of 10 and about 0.2 M urea in a yeast extract-based medium, ureolytic bacteria developed under aerobic chemostat operation at hydraulic retention times of about 10 h with urease levels of about 60 μmol min⁻¹ ml⁻¹ culture. For cost minimization at an industrial scale the costly protein-rich yeast extract medium could be replaced by commercial milk powder or by lysed activated sludge. Glutamate, molasses, or glucose-based media did not result in the enrichment of ureolytic bacteria by the chemostat. The concentration of intracellular urease was sufficiently high such that the produced raw effluent from the reactor could be used directly for biocementation in the field.

  15. Structural investigation and biological activity of the lipooligosaccharide from the psychrophilic bacterium Pseudoalteromonas haloplanktis TAB 23.

    PubMed

    Carillo, Sara; Pieretti, Giuseppina; Parrilli, Ermenegilda; Tutino, Maria L; Gemma, Sabrina; Molteni, Monica; Lanzetta, Rosa; Parrilli, Michelangelo; Corsaro, Maria M

    2011-06-14

    Pseudoalteromonas haloplanktis TAB 23 is a Gram-negative psychrophilic bacterium isolated from the Antarctic coastal sea. To survive in these conditions psychrophilic bacteria have evolved typical membrane lipids and "antifreeze" proteins to protect the inner side of the microorganism. As for Gram-negative bacteria, the outer membrane is mainly constituted by lipopoly- or lipooligosaccharides (LPS or LOS, respectively), which lean towards the external environment. Despite this, very little is known about the peculiarity of LPS from Gram-negative psychrophilic bacteria and what their role is in adaptation to cold temperature. Here we report the complete structure of the LOS from P. haloplanktis TAB 23. The lipid A was characterized by MALDI-TOF MS analysis and was tested in vitro showing a significant inhibitory effect on the LPS-induced pro-inflammatory cytokine production when added in culture with LPS from Escherichia coli. The product obtained after de-O-acylation of the LPS was analyzed by MALDI-TOF MS revealing the presence of several molecular species, differing in phosphorylation degree and oligosaccharide length. The oligosaccharide portion released after strong alkaline hydrolysis was purified by anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) to give three main fractions, characterized by means of 2D NMR spectroscopy, which showed a very short highly phosphorylated saccharidic chain with the following general structure. α-Hepp3R,6R,4R'-(1→5)-α-Kdop4P-(2→6)-β-GlcpN4R-(1→6)-α-GlcpN1P (R=-H(2)PO(3) or -H; R'=α-Galp-(1→4)-β-Galp-(1→ or H-). Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Development of bioconcrete material using an enrichment culture of novel thermophilic anaerobic bacteria.

    PubMed

    Ghosh, P; Mandal, S; Pal, S; Bandyopadhyaya, G; Chattopadhyay, B D

    2006-04-01

    In the biosphere, bacteria can function as geo-chemical agents, promoting the dispersion, fractionation and/or concentration of materials. Microbial mineral precipitation is resulted from metabolic activities of microorganisms. Based on this biomineralogy concept, an attempt has been made to develop bioconcrete material incorporating of an enrichment culture of thermophilic and anaerobic bacteria within cement-sand mortar/concrete. The results showed a significant increase in compressive strength of both cement-sand mortar and concrete due to the development of filler material within the pores of cement sand matrix. Maximum strength was observed at concentration 10(5)cell/ml of water used in mortar/concrete. Addition of Escherichia coil or media composition on mortar showed no such improvement in strength.

  17. Some like it cold: understanding the survival strategies of psychrophiles

    PubMed Central

    De Maayer, Pieter; Anderson, Dominique; Cary, Craig; Cowan, Don A

    2014-01-01

    Much of the Earth’s surface, both marine and terrestrial, is either periodically or permanently cold. Although habitats that are largely or continuously frozen are generally considered to be inhospitable to life, psychrophilic organisms have managed to survive in these environments. This is attributed to their innate adaptive capacity to cope with cold and its associated stresses. Here, we review the various environmental, physiological and molecular adaptations that psychrophilic microorganisms use to thrive under adverse conditions. We also discuss the impact of modern “omic” technologies in developing an improved understanding of these adaptations, highlighting recent work in this growing field. PMID:24671034

  18. Selective enrichment yields robust ethene-producing dechlorinating cultures from microcosms stalled at cis-dichloroethene.

    PubMed

    Delgado, Anca G; Kang, Dae-Wook; Nelson, Katherine G; Fajardo-Williams, Devyn; Miceli, Joseph F; Done, Hansa Y; Popat, Sudeep C; Krajmalnik-Brown, Rosa

    2014-01-01

    Dehalococcoides mccartyi strains are of particular importance for bioremediation due to their unique capability of transforming perchloroethene (PCE) and trichloroethene (TCE) to non-toxic ethene, through the intermediates cis-dichloroethene (cis-DCE) and vinyl chloride (VC). Despite the widespread environmental distribution of Dehalococcoides, biostimulation sometimes fails to promote dechlorination beyond cis-DCE. In our study, microcosms established with garden soil and mangrove sediment also stalled at cis-DCE, albeit Dehalococcoides mccartyi containing the reductive dehalogenase genes tceA, vcrA and bvcA were detected in the soil/sediment inocula. Reductive dechlorination was not promoted beyond cis-DCE, even after multiple biostimulation events with fermentable substrates and a lengthy incubation. However, transfers from microcosms stalled at cis-DCE yielded dechlorination to ethene with subsequent enrichment cultures containing up to 10(9) Dehalococcoides mccartyi cells mL(-1). Proteobacterial classes which dominated the soil/sediment communities became undetectable in the enrichments, and methanogenic activity drastically decreased after the transfers. We hypothesized that biostimulation of Dehalococcoides in the cis-DCE-stalled microcosms was impeded by other microbes present at higher abundances than Dehalococcoides and utilizing terminal electron acceptors from the soil/sediment, hence, outcompeting Dehalococcoides for H2. In support of this hypothesis, we show that garden soil and mangrove sediment microcosms bioaugmented with their respective cultures containing Dehalococcoides in high abundance were able to compete for H2 for reductive dechlorination from one biostimulation event and produced ethene with no obvious stall. Overall, our results provide an alternate explanation to consolidate conflicting observations on the ubiquity of Dehalococcoides mccartyi and occasional stalling of dechlorination at cis-DCE; thus, bringing a new perspective to

  19. Selective Enrichment Yields Robust Ethene-Producing Dechlorinating Cultures from Microcosms Stalled at cis-Dichloroethene

    PubMed Central

    Delgado, Anca G.; Kang, Dae-Wook; Nelson, Katherine G.; Fajardo-Williams, Devyn; Miceli, Joseph F.; Done, Hansa Y.; Popat, Sudeep C.; Krajmalnik-Brown, Rosa

    2014-01-01

    Dehalococcoides mccartyi strains are of particular importance for bioremediation due to their unique capability of transforming perchloroethene (PCE) and trichloroethene (TCE) to non-toxic ethene, through the intermediates cis-dichloroethene (cis-DCE) and vinyl chloride (VC). Despite the widespread environmental distribution of Dehalococcoides, biostimulation sometimes fails to promote dechlorination beyond cis-DCE. In our study, microcosms established with garden soil and mangrove sediment also stalled at cis-DCE, albeit Dehalococcoides mccartyi containing the reductive dehalogenase genes tceA, vcrA and bvcA were detected in the soil/sediment inocula. Reductive dechlorination was not promoted beyond cis-DCE, even after multiple biostimulation events with fermentable substrates and a lengthy incubation. However, transfers from microcosms stalled at cis-DCE yielded dechlorination to ethene with subsequent enrichment cultures containing up to 109 Dehalococcoides mccartyi cells mL−1. Proteobacterial classes which dominated the soil/sediment communities became undetectable in the enrichments, and methanogenic activity drastically decreased after the transfers. We hypothesized that biostimulation of Dehalococcoides in the cis-DCE-stalled microcosms was impeded by other microbes present at higher abundances than Dehalococcoides and utilizing terminal electron acceptors from the soil/sediment, hence, outcompeting Dehalococcoides for H2. In support of this hypothesis, we show that garden soil and mangrove sediment microcosms bioaugmented with their respective cultures containing Dehalococcoides in high abundance were able to compete for H2 for reductive dechlorination from one biostimulation event and produced ethene with no obvious stall. Overall, our results provide an alternate explanation to consolidate conflicting observations on the ubiquity of Dehalococcoides mccartyi and occasional stalling of dechlorination at cis-DCE; thus, bringing a new perspective to better

  20. Effect of dissolved oxygen concentration (microaerobic and aerobic) on selective enrichment culture for bioaugmentation of acidic industrial wastewater.

    PubMed

    Quan, Ying; Han, Hui; Zheng, Shaokui

    2012-09-01

    The successful application of bioaugmentation is largely dependent on the selective enrichment of culture with regards to pH, temperature, salt, or specific toxic organic pollutants. In this study, we investigated the effect of dissolved oxygen (DO) concentrations (aerobic, >2 mg L(-1); microaerobic, <1 mg L(-1)) on yeast enrichment culture for bioaugmentation of acidic industrial wastewater (pH 3.9-4.7). Clone library analyses revealed that the yeast community shifted in response to different DO levels, and that Candida humilis and Candida pseudolambica were individually dominant in the aerobic and microaerobic enrichment cultures. This would significantly influence the isolation results, and further hinder bioaugmentation due to differences in DO environments during the enrichment and application periods. However, differences in the selective enrichment culture cannot be predicted based on differences in pollutant removal performance. Thus, DO concentrations (aerobic/microaerobic) should be considered a secondary selective pressure to achieve successful bioaugmentation. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Inhibitory effect of 5- and 6-ring PAHs on pyrene mineralization by a mixed enrichment culture

    SciTech Connect

    Molina, M.; Agraujo, R.

    1995-12-31

    This research investigates the mineralization of pyrene in mixtures of PAHs to identify potential synergistic or antagonistic interactions that affect the degradation of individual compounds. Mineralization of {sup 14}C pyrene (25 RM) by a mixed enrichment culture was studied in systems containing mixtures of 5- and 6-ring PAHs in minimal salts medium (MSM). In the absence of the High Molecular Weight (HMW)-PAHs, the culture mineralized 62% of the added pyrene. Addition of an equal mixture of benzo(a)pyrene, dibenzanthracene, and benzo(g,h,i)peryiene (25 {micro}M total concentration) reduced pyrene mineralization to 25% after a 9-day lag phase. An increase on the molar concentration of the HMW-PAH mixture to 75 and 125 {micro}M decreased pyrene mineralization to 9.2 and 1%, respectively. Results from treatments containing individual (25 {micro}M each), or pairs of the HMW-compounds demonstrated that none of the three individual compounds caused a significant reduction in the extent of pyrene mineralization. However, the combination of benzo(a)pyrene and benzanthracene significantly inhibited pyrene activity. In addition, the presence of both benzo(a)pyrene and benzo(g,h,i)peryiene reduced mineralization by almost 23%. Determination of bacterial density by epifluorescence microscopy showed that mineralization of pyrene coincides with growth of the bacterial culture; presence of the 5- and 6-ring PAHs delayed growth with a concurrent inhibition of mineralization. When growth resumes, the inhibitory effect is reduced. A decrease of pyrene inhibition was also noted when MSM was replaced with sediment extract, or when sediment (1 {micro}g/ml) was added to the medium. These results suggest a synergistic inhibitory effect by combinations of specific HMW-PAHs rather than inhibition by individual compounds of the mixture on both the growth of the bacterial culture and the extent of pyrene mineralization.

  2. Methylosphaera hansonii gen. nov., sp. nov., a psychrophilic, group I methanotroph from Antarctic marine-salinity, meromictic lakes.

    PubMed

    Bowman, J P; McCammon, S A; Skerratt, J H

    1997-04-01

    Methanotrophic bacteria were enumerated and isolated from the chemocline and surface sediments of marine-salinity Antarctic meromictic lakes located in the Vestfold Hills, Antarctica (68 degrees S 78 degrees E). Most probable number (MPN) analysis indicated that at the chemocline of Ace Lake the methanotroph population made up only a small proportion of the total microbial population and was sharply stratified, with higher populations detected in the surface sediments collected at the edge of Ace Lake and Burton Lake. Methanotrophs were not detected in Pendant Lake. Only a single phenotypic group of methanotrophs was successfully enriched, enumerated and isolated into pure culture from the lake samples. Strains of this group were non-motile, coccoidal in morphology, did not form resting cells, reproduced by constriction, and required seawater for growth. The strains were also psychrophilic, with optimal growth occurring at 10-13 degrees C and maximum growth temperatures of 16-21 degrees C. The ribulose monophosphate pathway but not the serine pathway for incorporation of C1 compounds was detectable in the strains. The guanine plus cytosine (G + C) content of the genomic DNA was 43-46 mol%. Whole-cell fatty acid analysis indicated that 16:1 omega 8c (37-41%), 16:1 omega 6c (17-19%), 16:1 omega 7c (15-19%) and 16:0 (14-15%) were the major fatty acids in the strains. 16s rDNA sequence analysis revealed that the strains form a distinct line of descent in the family Methylococcaceae (group I methanotrophs), with the closest relative being the Louisiana Slope methanotrophic mytilid endosymbiont (91.8-92.3% sequence similarity). On the basis of polyphasic taxonomic characteristics the Antarctic lake isolates represent a novel group I methanotrophic genus with the proposed name Methylosphaera hansonii (type strain ACAM 549).

  3. Desulfoconvexum algidum gen. nov., sp. nov., a psychrophilic sulfate-reducing bacterium isolated from a permanently cold marine sediment.

    PubMed

    Könneke, Martin; Kuever, Jan; Galushko, Alexander; Jørgensen, Bo Barker

    2013-03-01

    A sulfate-reducing bacterium, designated JHA1(T), was isolated from a permanently cold marine sediment sampled in an Artic fjord on the north-west coast of Svalbard. The isolate was originally enriched at 4 °C in a highly diluted liquid culture amended with hydrogen and sulfate. Strain JHA1(T) was a psychrophile, growing fastest between 14 and 16 °C and not growing above 20 °C. Fastest growth was found at neutral pH (pH 7.2-7.4) and at marine concentrations of NaCl (20-30 g l(-1)). Phylogenetic analysis of 16S rRNA gene sequences revealed that strain JHA1(T) was a member of the family Desulfobacteraceae in the Deltaproteobacteria. The isolate shared 99 % 16S rRNA gene sequence similarity with an environmental sequence obtained from permanently cold Antarctic sediment. The closest recognized relatives were Desulfobacula phenolica DSM 3384(T) and Desulfobacula toluolica DSM 7467(T) (both <95 % sequence similarity). In contrast to its closest phylogenetic relatives, strain JHA1(T) grew chemolithoautotrophically with hydrogen as an electron donor. CO dehydrogenase activity indicated the operation of the reductive acetyl-CoA pathway for inorganic carbon assimilation. Beside differences in physiology and morphology, strain JHA1(T) could be distinguished chemotaxonomically from the genus Desulfobacula by the absence of the cellular fatty acid C16 : 0 10-methyl. Phylogenetic differentiation from other genera was further supported by DsrAB and AprBA sequence analysis. Based on the described phylogenetic and phenotypic differences between strain JHA1(T) and its closest relatives, the establishment of a novel genus and a novel species, Desulfoconvexum algidum gen. nov., sp. nov. is proposed. The type strain is JHA1(T) ( = DSM 21856(T)  = JCM 16085(T)).

  4. Co-metabolic degradation of diazo dye- reactive blue 160 by enriched mixed cultures BDN.

    PubMed

    Balapure, Kshama H; Jain, Kunal; Chattaraj, Sananda; Bhatt, Nikhil S; Madamwar, Datta

    2014-08-30

    Mixed cultures BDN (BDN) proficient in decolourizing diazo dye-reactive blue 160 (RB160) consist of eight bacterial strains, was developed through culture enrichment method from soil samples contaminated with anthropogenic activities. The synthrophic interactions of BDN have led to complete decolourization and degradation of RB160 (100mg/L) within 4h along with co-metabolism of yeast extract (0.5%) in minimal medium. BDN microaerophilicaly decolourized even 1500mg/L of RB160 under high saline conditions (20g/L NaCl) at 37°C and pH 7.0. BDN exhibited broad substrate specificity and decolourized 27 structurally different dyes. The reductase enzymes symmetrically cleaved RB160 and oxidative enzymes further metabolised the degraded products and five different intermediates were identified using FTIR, (1)HNMR and GC-MS. The phytotoxicity assay confirmed that intact RB160 was more toxic than dye degraded intermediates. The BDN was able to colonize and decolourized RB160 in soil model system in presence of indigenous miocroflora as well as in sterile soil without any amendment of additional nutrients, which signifies it useful and potential application in bioremediation. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Stable Isotopic Studies of n-Alkane Metabolism by a Sulfate-Reducing Bacterial Enrichment Culture

    PubMed Central

    Davidova, Irene A.; Gieg, Lisa M.; Nanny, Mark; Kropp, Kevin G.; Suflita, Joseph M.

    2005-01-01

    Gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy were used to study the metabolism of deuterated n-alkanes (C6 to C12) and 1-13C-labeled n-hexane by a highly enriched sulfate-reducing bacterial culture. All substrates were activated via fumarate addition to form the corresponding alkylsuccinic acid derivatives as transient metabolites. Formation of d14-hexylsuccinic acid in cell extracts from exogenously added, fully deuterated n-hexane confirmed that this reaction was the initial step in anaerobic alkane metabolism. Analysis of resting cell suspensions amended with 1-13C-labeled n-hexane confirmed that addition of the fumarate occurred at the C-2 carbon of the parent substrate. Subsequent metabolism of hexylsuccinic acid resulted in the formation of 4-methyloctanoic acid, and 3-hydroxy-4-methyloctanoic acid was tentatively identified. We also found that 13C nuclei from 1-13C-labeled n-hexane became incorporated into the succinyl portion of the initial metabolite in a manner that indicated that 13C-labeled fumarate was formed and recycled during alkane metabolism. Collectively, the findings obtained with a sulfate-reducing culture using isotopically labeled alkanes augment and support the previously proposed pathway (H. Wilkes, R. Rabus, T. Fischer, A. Armstroff, A. Behrends, and F. Widdel, Arch. Microbiol. 177:235-243, 2002) for metabolism of deuterated n-hexane by a denitrifying bacterium. PMID:16332800

  6. Stable isotopic studies of n-alkane metabolism by a sulfate-reducing bacterial enrichment culture.

    PubMed

    Davidova, Irene A; Gieg, Lisa M; Nanny, Mark; Kropp, Kevin G; Suflita, Joseph M

    2005-12-01

    Gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy were used to study the metabolism of deuterated n-alkanes (C6 to C12) and 1-13C-labeled n-hexane by a highly enriched sulfate-reducing bacterial culture. All substrates were activated via fumarate addition to form the corresponding alkylsuccinic acid derivatives as transient metabolites. Formation of d14-hexylsuccinic acid in cell extracts from exogenously added, fully deuterated n-hexane confirmed that this reaction was the initial step in anaerobic alkane metabolism. Analysis of resting cell suspensions amended with 1-13C-labeled n-hexane confirmed that addition of the fumarate occurred at the C-2 carbon of the parent substrate. Subsequent metabolism of hexylsuccinic acid resulted in the formation of 4-methyloctanoic acid, and 3-hydroxy-4-methyloctanoic acid was tentatively identified. We also found that 13C nuclei from 1-13C-labeled n-hexane became incorporated into the succinyl portion of the initial metabolite in a manner that indicated that 13C-labeled fumarate was formed and recycled during alkane metabolism. Collectively, the findings obtained with a sulfate-reducing culture using isotopically labeled alkanes augment and support the previously proposed pathway (H. Wilkes, R. Rabus, T. Fischer, A. Armstroff, A. Behrends, and F. Widdel, Arch. Microbiol. 177:235-243, 2002) for metabolism of deuterated n-hexane by a denitrifying bacterium.

  7. Selective enrichment media bias the types of salmonella enterica strains isolated from mixed strain cultures and complex enrichment broths

    USDA-ARS?s Scientific Manuscript database

    For foodborne outbreak investigations it can be difficult to isolate the relevant strain from food and/or environmental sources. If the sample is contaminated by more than one strain of the organism the relevant strain might be missed. In this study mixed cultures of Salmonella enterica were grown...

  8. Chronic impact of tetracycline on nitrification kinetics and the activity of enriched nitrifying microbial culture.

    PubMed

    Katipoglu-Yazan, Tugce; Merlin, Christophe; Pons, Marie-Noëlle; Ubay-Cokgor, Emine; Orhon, Derin

    2015-04-01

    This study evaluated the chronic impact of tetracycline on biomass with enriched nitrifying community sustained in a lab-scale activated sludge system. For this purpose, a fill and draw reactor fed with 100 mg COD/L of peptone mixture and 50 mg N/L of ammonia was sustained at a sludge age of 15 days. At steady-state, the reactor operation was continued with a daily tetracycline dosing of 50 mg/L for more than 40 days, with periodic monitoring of the microbial composition, the nitrifying bacteria abundance, as well as the amoA and 16S rRNA gene activity, using molecular techniques. Changes in the kinetics of nitrification were quantified by modelling concentration profiles of major nitrogen fractions and oxygen uptake rate profiles derived from parallel batch experiments. Activated sludge modeling results indicated inhibitory impact of tetracycline on the growth of nitrifiers with a significant increase of the half saturation coefficients in corresponding rate equations. Tetracycline also inactivated biomass components of the enriched culture at a gradually increasing rate with time of exposure, leading to total collapse of nitrification. Molecular analyses revealed significant changes in the composition of the microbial community throughout the observation period. They also showed that continuous exposure to tetracycline inflicted significant reduction in amoA mRNA and 16S rRNA levels directly affecting nitrification. The chronic impact was much more pronounced on the ammonia oxidizing bacteria (AOB) community. These observations explained the basis of numerical changes identified in the growth kinetics of nitrifiers under stress conditions. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. High molecular weight dissolved organic matter enrichment selects for methylotrophs in dilution to extinction cultures

    PubMed Central

    Sosa, Oscar A; Gifford, Scott M; Repeta, Daniel J; DeLong, Edward F

    2015-01-01

    The role of bacterioplankton in the cycling of marine dissolved organic matter (DOM) is central to the carbon and energy balance in the ocean, yet there are few model organisms available to investigate the genes, metabolic pathways, and biochemical mechanisms involved in the degradation of this globally important carbon pool. To obtain microbial isolates capable of degrading semi-labile DOM for growth, we conducted dilution to extinction cultivation experiments using seawater enriched with high molecular weight (HMW) DOM. In total, 93 isolates were obtained. Amendments using HMW DOM to increase the dissolved organic carbon concentration 4x (280 μM) or 10x (700 μM) the ocean surface water concentrations yielded positive growth in 4–6% of replicate dilutions, whereas <1% scored positive for growth in non-DOM-amended controls. The majority (71%) of isolates displayed a distinct increase in cell yields when grown in increasing concentrations of HMW DOM. Whole-genome sequencing was used to screen the culture collection for purity and to determine the phylogenetic identity of the isolates. Eleven percent of the isolates belonged to the gammaproteobacteria including Alteromonadales (the SAR92 clade) and Vibrio. Surprisingly, 85% of isolates belonged to the methylotrophic OM43 clade of betaproteobacteria, bacteria thought to metabolically specialize in degrading C1 compounds. Growth of these isolates on methanol confirmed their methylotrophic phenotype. Our results indicate that dilution to extinction cultivation enriched with natural sources of organic substrates has a potential to reveal the previously unsuspected relationships between naturally occurring organic nutrients and the microorganisms that consume them. PMID:25978545

  10. High molecular weight dissolved organic matter enrichment selects for methylotrophs in dilution to extinction cultures.

    PubMed

    Sosa, Oscar A; Gifford, Scott M; Repeta, Daniel J; DeLong, Edward F

    2015-12-01

    The role of bacterioplankton in the cycling of marine dissolved organic matter (DOM) is central to the carbon and energy balance in the ocean, yet there are few model organisms available to investigate the genes, metabolic pathways, and biochemical mechanisms involved in the degradation of this globally important carbon pool. To obtain microbial isolates capable of degrading semi-labile DOM for growth, we conducted dilution to extinction cultivation experiments using seawater enriched with high molecular weight (HMW) DOM. In total, 93 isolates were obtained. Amendments using HMW DOM to increase the dissolved organic carbon concentration 4x (280 μM) or 10x (700 μM) the ocean surface water concentrations yielded positive growth in 4-6% of replicate dilutions, whereas <1% scored positive for growth in non-DOM-amended controls. The majority (71%) of isolates displayed a distinct increase in cell yields when grown in increasing concentrations of HMW DOM. Whole-genome sequencing was used to screen the culture collection for purity and to determine the phylogenetic identity of the isolates. Eleven percent of the isolates belonged to the gammaproteobacteria including Alteromonadales (the SAR92 clade) and Vibrio. Surprisingly, 85% of isolates belonged to the methylotrophic OM43 clade of betaproteobacteria, bacteria thought to metabolically specialize in degrading C1 compounds. Growth of these isolates on methanol confirmed their methylotrophic phenotype. Our results indicate that dilution to extinction cultivation enriched with natural sources of organic substrates has a potential to reveal the previously unsuspected relationships between naturally occurring organic nutrients and the microorganisms that consume them.

  11. Psychrophilic Biomass Producers in the Trophic Chain of the Microbial Community of Lake Untersee, Antarctica

    NASA Technical Reports Server (NTRS)

    Pikuta, Elena V.; Hoover, Richard B.

    2010-01-01

    The study of photosynthetic microorganisms from the Lake Untersee samples showed dispersed distribution of phototrophs within 80 m water column. Lake Untersee represents a unique ecosystem that experienced complete isolation: sealed by the Anuchin Glacier for many millennia. Consequently, its biocenosis has evolved over a significant period of time without exchange or external interaction with species from other environments. The major producers of organic matter in Lake Untersee are represented by phototrophic and chemolithotrophic microorganisms. This is the traditional trophic scheme for lacustrine ecosystems on Earth. Among the phototrophs, diatoms were not found, which differentiates this lake from other known ecosystems. The dominant species among phototrophs was Chlamydomonas sp. with typical morphostructure: green chloroplasts, bright red round spot, and two polar flagella near the opening. As expected, the physiology of studied phototrophs was limited by low temperature, which defined them as obligate psychrophilic microorganisms. By the quantity estimation of methanogenesis in this lake, the litho-autotrophic production of organic matter is competitive with phototrophic production. However, pure cultures of methanogens have not yet been obtained. We discuss the primary producers of organic matter and the participation of our novel psychrophilic homoacetogen into the litho-autotrophic link of biomass production in Lake Untersee.

  12. MICROBIAL REDUCTIVE DECHLORINATION OF HEXACHLORO-1,3-BUTADIENE IN A METHANOGENIC ENRICHMENT CULTURE. (R825513C007)

    EPA Science Inventory

    Sequential reductive dechlorination of hexachloro-1,3-butadiene (HCBD) was achieved by a mixed, methanogenic culture enriched from a contaminated estuarine sediment. Both methanol and lactate served as carbon and electron sources. Methanol was stoichiometrically converted to m...

  13. MICROBIAL REDUCTIVE DECHLORINATION OF HEXACHLORO-1,3-BUTADIENE IN A METHANOGENIC ENRICHMENT CULTURE. (R825513C007)

    EPA Science Inventory

    Sequential reductive dechlorination of hexachloro-1,3-butadiene (HCBD) was achieved by a mixed, methanogenic culture enriched from a contaminated estuarine sediment. Both methanol and lactate served as carbon and electron sources. Methanol was stoichiometrically converted to m...

  14. The Learning Effects of an Ecology Enrichment Summer Program on Gifted Students from Mainstream and Diverse Cultural Backgrounds: A Preliminary Study

    ERIC Educational Resources Information Center

    Wang, Wen-Ling; Wu, Jiun-Wei; Lin, Yu-Chin

    2006-01-01

    Enrichment is one of the important educational models for gifted students. However, the research on gifted enrichment programs rarely leads to instructional interventions for culturally diverse students. The purposes of this study were: (a) to propose an ecology enrichment summer program for gifted students from mainstream and diverse cultural…

  15. Sphere Culture of Murine Lung Cancer Cell Lines Are Enriched with Cancer Initiating Cells

    PubMed Central

    Morrison, Brian J.

    2012-01-01

    Cancer initiating cells (CICs) represent a unique cell population essential for the maintenance and growth of tumors. Most in vivo studies of CICs utilize human tumor xenografts in immunodeficient mice. These models provide limited information on the interaction of CICs with the host immune system and are of limited value in assessing therapies targeting CICs, especially immune-based therapies. To assess this, a syngeneic cancer model is needed. We examined the sphere-forming capacity of thirteen murine lung cancer cell lines and identified TC-1 and a metastatic subclone of Lewis lung carcinoma (HM-LLC) as cell lines that readily formed and maintained spheres over multiple passages. TC-1 tumorspheres were not enriched for expression of CD133 or CD44, putative CIC markers, nor did they demonstrate Hoechst 33342 side population staining or Aldefluor activity compared to adherent TC-1 cells. However, in tumorsphere culture, these cells exhibited self-renewal and long-term symmetric division capacity and expressed more Oct-4 compared to adherent cells. HM-LLC sphere-derived cells exhibited increased Oct-4, CD133, and CD44 expression, demonstrated a Hoechst 33342 side population and Aldefluor activity compared to adherent cells or a low metastatic subclone of LLC (LM-LLC). In syngeneic mice, HM-LLC sphere-derived cells required fewer cells to initiate tumorigenesis compared to adherent or LM-LLC cells. Similarly TC-1 sphere-derived cells were more tumorigenic than adherent cells in syngeneic mice. In contrast, in immunocompromised mice, less than 500 sphere or adherent TC-1 cells and less than 1,000 sphere or adherent LLC cells were required to initiate a tumor. We suggest that no single phenotypic marker can identify CICs in murine lung cancer cell lines. Tumorsphere culture may provide an alternative approach to identify and enrich for murine lung CICs. Furthermore, we propose that assessing tumorigenicity of murine lung CICs in syngeneic mice better models the

  16. Kinetics of 1,2-Dichloroethane and 1,2-Dibromoethane Biodegradation in Anaerobic Enrichment Cultures

    PubMed Central

    Yu, Rong; Peethambaram, Hari S.; Falta, Ronald W.; Verce, Matthew F.; Henderson, James K.; Bagwell, Christopher E.; Brigmon, Robin L.

    2013-01-01

    1,2-Dichloroethane (1,2-DCA) and 1,2-dibromoethane (ethylene dibromide [EDB]) contaminate groundwater at many hazardous waste sites. The objectives of this study were to measure yields, maximum specific growth rates (μ̂), and half-saturation coefficients (KS) in enrichment cultures that use 1,2-DCA and EDB as terminal electron acceptors and lactate as the electron donor and to evaluate if the presence of EDB has an effect on the kinetics of 1,2-DCA dehalogenation and vice versa. Biodegradation was evaluated at the high concentrations found at some industrial sites (>10 mg/liter) and at lower concentrations found at former leaded-gasoline sites (1.9 to 3.7 mg/liter). At higher concentrations, the Dehalococcoides yield was 1 order of magnitude higher when bacteria were grown with 1,2-DCA than when they were grown with EDB, while μ̂'s were similar for the two compounds, ranging from 0.19 to 0.52 day−1 with 1,2-DCA to 0.28 to 0.36 day−1 for EDB. KS was larger for 1,2-DCA (15 to 25 mg/liter) than for EDB (1.8 to 3.7 mg/liter). In treatments that received both compounds, EDB was always consumed first and adversely impacted the kinetics of 1,2-DCA utilization. Furthermore, 1,2-DCA dechlorination was interrupted by the addition of EDB at a concentration 100 times lower than that of the remaining 1,2-DCA; use of 1,2-DCA did not resume until the EDB level decreased close to its maximum contaminant level (MCL). In lower-concentration experiments, the preferential consumption of EDB over 1,2-DCA was confirmed; both compounds were eventually dehalogenated to their respective MCLs (5 μg/liter for 1,2-DCA, 0.05 μg/liter for EDB). The enrichment culture grown with 1,2-DCA has the advantage of a more rapid transition to 1,2-DCA after EDB is consumed. PMID:23263950

  17. Sustained growth of explants from Mediterranean sponge Crambe crambe cultured in vitro with enriched RPMI 1640.

    PubMed

    Garcia Camacho, F; Chileh, T; Cerón García, M C; Sanchez Mirón, A; Belarbi, E H; Contreras Gómez, A; Molina Grima, E

    2006-01-01

    Marine sponges are potential sources of many unique metabolites, including cytotoxic and anticancer compounds. Natural sponge populations are insufficient or inaccessible for producing commercial quantities of metabolites of interest. It is commonly accepted that tissue (fragments, explants, and primmorphs) and in vitro cell cultivation show great potential. However, there is little knowledge of the nutritional requirements of marine sponges to carry out efficient and sustained in vitro culture and progress has been slow. In marine invertebrate fila many unsuccessful attempts have been made with in vitro cultures using typical commercial animal cell media based on sources of dissolved organic carbon (DOC) (e.g., DMEM, RPMI, M199, L-15, etc.). One of the reasons for this failure is the use of hardly identifiable growth promoters, based on terrestrial animal sera. An alternative is the use of extracts from marine animals, since they may contain nutrients necessary for growth. In this work we have cultivated in vitro explants of the encrusting marine sponge Crambe crambe. It is one of the most abundant sponges on the Mediterranean coastline and also possesses an array of potentially active metabolites (crambines and crambescidins). Initially a new approach was developed in order to show consumption of DOC by explants. Thus, different initial DOC concentrations (300, 400, 700 and 1200 mg DOC L(-1)) were assayed. Consumption was evident in all four assays and was more marked in the first 6 h. The DOC assimilation data were adjusted to an empirical model widely used for uptake kinetics of organic dissolved compounds in marine invertebrates. Second, a protocol was established to cultivate explants in vitro. Different medium formulations based on RPMI 1640 commercial medium enriched with amino acids and inorganic salts to emulate seawater salinity were assayed. The enrichment of this medium with an Octopus aqueous extract in the proportions of 10% and 20% (v/v) resulted in

  18. The Culture of Cancer Cell Lines as Tumorspheres Does Not Systematically Result in Cancer Stem Cell Enrichment

    PubMed Central

    Calvet, Christophe Y.; André, Franck M.; Mir, Lluis M.

    2014-01-01

    Cancer stem cells (CSC) have raised great excitement during the last decade and are promising targets for an efficient treatment of tumors without relapses and metastases. Among the various methods that enable to enrich cancer cell lines in CSC, tumorspheres culture has been predominantly used. In this report, we attempted to generate tumorspheres from several murine and human cancer cell lines: B16-F10, HT-29, MCF-7 and MDA-MB-231 cells. Tumorspheres were obtained with variable efficiencies from all cell lines except from MDA-MB-231 cells. Then, we studied several CSC characteristics in both tumorspheres and adherent cultures of the B16-F10, HT-29 and MCF-7 cells. Unexpectedly, tumorspheres-forming cells were less clonogenic and, in the case of B16-F10, less proliferative than attached cells. In addition, we did not observe any enrichment in the population expressing CSC surface markers in tumorspheres from B16-F10 (CD133, CD44 and CD24 markers) or MCF-7 (CD44 and CD24 markers) cells. On the contrary, tumorspheres culture of HT-29 cells appeared to enrich in cells expressing colon CSC markers, i.e. CD133 and CD44 proteins. For the B16-F10 cell line, when 1 000 cells were injected in syngenic C57BL/6 mice, tumorspheres-forming cells displayed a significantly lower tumorigenic potential than adherent cells. Finally, tumorspheres culture of B16-F10 cells induced a down-regulation of vimentin which could explain, at least partially, the lower tumorigenicity of tumorspheres-forming cells. All these results, along with the literature, indicate that tumorspheres culture of cancer cell lines can induce an enrichment in CSC but in a cell line-dependent manner. In conclusion, extensive characterization of CSC properties in tumorspheres derived from any cancer cell line or cancer tissue must be performed in order to ensure that the generated tumorspheres are actually enriched in CSC. PMID:24586931

  19. Unique Kinetic Properties of Phenol-Degrading Variovorax Strains Responsible for Efficient Trichloroethylene Degradation in a Chemostat Enrichment Culture

    PubMed Central

    Futamata, Hiroyuki; Nagano, Yayoi; Watanabe, Kazuya; Hiraishi, Akira

    2005-01-01

    A chemostat enrichment of soil bacteria growing on phenol as the sole carbon source has been shown to exhibit quite high trichloroethylene (TCE)-degrading activities (H. Futamata, S. Harayama, and K. Watanabe, Appl. Environ. Microbiol. 67:4671-4677, 2001). To identify the bacterial populations responsible for the high TCE-degrading activity, a multidisciplinary survey of the chemostat enrichment was conducted by employing molecular-ecological and culture-dependent approaches. Three chemostat enrichment cultures were newly developed under different phenol-loading conditions (0.25, 0.75, and 1.25 g liter−1 day−1) in this study, and the TCE-degrading activities of the enrichments were measured. Among them, the enrichment at 0.75 g liter−1 day−1 (enrichment 0.75) expressed the highest activity. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments detected a Variovorax ribotype as the strongest band in enrichment 0.75; however, it was not a major ribotype in the other samples. Bacteria were isolated from enrichment 0.75 by direct plating, and their 16S rRNA genes and genes encoding the largest subunit of phenol hydroxylase (LmPHs) were analyzed. Among the bacteria isolated, several strains were affiliated with the genus Variovorax and were shown to have high-affinity-type LmPHs. The LmPH of the Variovorax strains was also detected as the major genotype in enrichment 0.75. Kinetic analyses of phenol and TCE degradation revealed, however, that these strains exhibited quite low affinity for phenol compared to other phenol-degrading bacteria, while they showed quite high specific TCE-degrading activities and relatively high affinity for TCE. Owing to these unique kinetic traits, the Variovorax strains can obviate competitive inhibition of TCE degradation by the primary substrate of the catabolic enzyme (i.e., phenol), contributing to the high TCE-degrading activity of the chemostat enrichments. On the basis of physiological information

  20. Biodegradation of Various Aromatic Compounds by Enriched Bacterial Cultures: Part A-Monocyclic and Polycyclic Aromatic Hydrocarbons.

    PubMed

    Oberoi, Akashdeep Singh; Philip, Ligy; Bhallamudi, S Murty

    2015-08-01

    Present study focused on the screening of bacterial consortium for biodegradation of monocyclic aromatic hydrocarbon (MAH) and polycyclic aromatic hydrocarbons (PAHs). Target compounds in the present study were naphthalene, acenaphthene, phenanthrene (PAHs), and benzene (MAH). Microbial consortia enriched with the above target compounds were used in screening experiments. Naphthalene-enriched consortium was found to be the most efficient consortium, based on its substrate degradation rate and its ability to degrade other aromatic pollutants with significantly high efficiency. Substrate degradation rate with naphthalene-enriched culture followed the order benzene > naphthalene > acenaphthene > phenanthrene. Chryseobacterium and Rhodobacter were discerned as the predominant species in naphthalene-enriched culture. They are closely associated to the type strain Chryseobacterium arthrosphaerae and Rhodobacter maris, respectively. Single substrate biodegradation studies with naphthalene (PAH) and benzene (MAH) were carried out using naphthalene-enriched microbial consortium (NAPH). Phenol and 2-hydroxybenzaldehyde were identified as the predominant intermediates during benzene and naphthalene degradation, respectively. Biodegradation of toluene, ethyl benzene, xylene, phenol, and indole by NAPH was also investigated. Monod inhibition model was able to simulate biodegradation kinetics for benzene, whereas multiple substrate biodegradation model was able to simulate biodegradation kinetics for naphthalene.

  1. Study of selenocompounds from selenium-enriched culture of edible sprouts.

    PubMed

    Funes-Collado, Virginia; Morell-Garcia, Albert; Rubio, Roser; López-Sánchez, José Fermín

    2013-12-15

    Selenium is recognised as an essential micronutrient for humans and animals. One of the main sources of selenocompounds in the human diet is vegetables. Therefore, this study deals with the Se species present in different edible sprouts grown in Se-enriched media. We grew alfalfa, lentil and soy in a hydroponic system amended with soluble salts, containing the same proportion of Se, in the form of Se(VI) and Se(IV). Total Se in the sprouts was determined by acidic digestion in a microwave system and by ICP/MS. Se speciation was carried out by enzymatic extraction (Protease XIV) and measured by LC-ICP/MS. The study shows that the Se content of plants depends on the content in the growth culture, and that part of the inorganic Se was biotransformed mainly into SeMet. These results contribute to our understanding of the uptake of inorganic Se and its biotransformation by edible plants. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Ultrastructure and Viral Metagenome of Bacteriophages from an Anaerobic Methane Oxidizing Methylomirabilis Bioreactor Enrichment Culture

    PubMed Central

    Gambelli, Lavinia; Cremers, Geert; Mesman, Rob; Guerrero, Simon; Dutilh, Bas E.; Jetten, Mike S. M.; Op den Camp, Huub J. M.; van Niftrik, Laura

    2016-01-01

    With its capacity for anaerobic methane oxidation and denitrification, the bacterium Methylomirabilis oxyfera plays an important role in natural ecosystems. Its unique physiology can be exploited for more sustainable wastewater treatment technologies. However, operational stability of full-scale bioreactors can experience setbacks due to, for example, bacteriophage blooms. By shaping microbial communities through mortality, horizontal gene transfer, and metabolic reprogramming, bacteriophages are important players in most ecosystems. Here, we analyzed an infected Methylomirabilis sp. bioreactor enrichment culture using (advanced) electron microscopy, viral metagenomics and bioinformatics. Electron micrographs revealed four different viral morphotypes, one of which was observed to infect Methylomirabilis cells. The infected cells contained densely packed ~55 nm icosahedral bacteriophage particles with a putative internal membrane. Various stages of virion assembly were observed. Moreover, during the bacteriophage replication, the host cytoplasmic membrane appeared extremely patchy, which suggests that the bacteriophages may use host bacterial lipids to build their own putative internal membrane. The viral metagenome contained 1.87 million base pairs of assembled viral sequences, from which five putative complete viral genomes were assembled and manually annotated. Using bioinformatics analyses, we could not identify which viral genome belonged to the Methylomirabilis- infecting bacteriophage, in part because the obtained viral genome sequences were novel and unique to this reactor system. Taken together these results show that new bacteriophages can be detected in anaerobic cultivation systems and that the effect of bacteriophages on the microbial community in these systems is a topic for further study. PMID:27877158

  3. Anaerobic alkane biodegradation by cultures enriched from oil sands tailings ponds involves multiple species capable of fumarate addition.

    PubMed

    Tan, BoonFei; Semple, Kathleen; Foght, Julia

    2015-05-01

    A methanogenic short-chain alkane-degrading culture (SCADC) was enriched from oil sands tailings and transferred several times with a mixture of C6, C7, C8 and C10 n-alkanes as the predominant organic carbon source, plus 2-methylpentane, 3-methylpentane and methylcyclopentane as minor components. Cultures produced ∼40% of the maximum theoretical methane during 18 months incubation while depleting the n-alkanes, 2-methylpentane and methylcyclopentane. Substrate depletion correlated with detection of metabolites characteristic of fumarate activation of 2-methylpentane and methylcyclopentane, but not n-alkane metabolites. During active methanogenesis with the mixed alkanes, reverse-transcription PCR confirmed the expression of functional genes (assA and bssA) associated with hydrocarbon addition to fumarate. Pyrosequencing of 16S rRNA genes amplified during active alkane degradation revealed enrichment of Clostridia (particularly Peptococcaceae) and methanogenic Archaea (Methanosaetaceae and Methanomicrobiaceae). Methanogenic cultures transferred into medium containing sulphate produced sulphide, depleted n-alkanes and produced the corresponding succinylated alkane metabolites, but were slow to degrade 2-methylpentane and methylcyclopentane; these cultures were enriched in Deltaproteobacteria rather than Clostridia. 3-Methylpentane was not degraded by any cultures. Thus, nominally methanogenic oil sands tailings harbour dynamic and versatile hydrocarbon-degrading fermentative syntrophs and sulphate reducers capable of degrading n-, iso- and cyclo-alkanes by addition to fumarate.

  4. Identity and Substrate Specificity of Reductive Dehalogenases Expressed in Dehalococcoides-Containing Enrichment Cultures Maintained on Different Chlorinated Ethenes

    PubMed Central

    Liang, Xiaoming; Molenda, Olivia; Tang, Shuiquan

    2015-01-01

    Many reductive dehalogenases (RDases) have been identified in organohalide-respiring microorganisms, and yet their substrates, specific activities, and conditions for expression are not well understood. We tested whether RDase expression varied depending on the substrate-exposure history of reductive dechlorinating communities. For this purpose, we used the enrichment culture KB-1 maintained on trichloroethene (TCE), as well as subcultures maintained on the intermediates cis-dichloroethene (cDCE) and vinyl chloride (VC). KB-1 contains a TCE-to-cDCE dechlorinating Geobacter and several Dehalococcoides strains that together harbor many of the known chloroethene reductases. Expressed RDases were identified using blue native polyacrylamide gel electrophoresis, enzyme assays in gel slices, and peptide sequencing. As anticipated but never previously quantified, the RDase from Geobacter was only detected transiently at the beginning of TCE dechlorination. The Dehalococcoides RDase VcrA and smaller amounts of TceA were expressed in the parent KB-1 culture during complete dechlorination of TCE to ethene regardless of time point or amended substrate. The Dehalococcoides RDase BvcA was only detected in enrichments maintained on cDCE as growth substrates, in roughly equal abundance to VcrA. Only VcrA was detected in subcultures enriched on VC. Enzyme assays revealed that 1,1-DCE, a substrate not used for culture enrichment, afforded the highest specific activity. trans-DCE was substantially dechlorinated only by extracts from cDCE enrichments expressing BvcA. RDase gene distribution indicated enrichment of different strains of Dehalococcoides as a function of electron acceptor TCE, cDCE, or VC. Each chloroethene reductase has distinct substrate preferences leading to strain selection in mixed communities. PMID:25934625

  5. Identity and Substrate Specificity of Reductive Dehalogenases Expressed in Dehalococcoides-Containing Enrichment Cultures Maintained on Different Chlorinated Ethenes.

    PubMed

    Liang, Xiaoming; Molenda, Olivia; Tang, Shuiquan; Edwards, Elizabeth A

    2015-07-01

    Many reductive dehalogenases (RDases) have been identified in organohalide-respiring microorganisms, and yet their substrates, specific activities, and conditions for expression are not well understood. We tested whether RDase expression varied depending on the substrate-exposure history of reductive dechlorinating communities. For this purpose, we used the enrichment culture KB-1 maintained on trichloroethene (TCE), as well as subcultures maintained on the intermediates cis-dichloroethene (cDCE) and vinyl chloride (VC). KB-1 contains a TCE-to-cDCE dechlorinating Geobacter and several Dehalococcoides strains that together harbor many of the known chloroethene reductases. Expressed RDases were identified using blue native polyacrylamide gel electrophoresis, enzyme assays in gel slices, and peptide sequencing. As anticipated but never previously quantified, the RDase from Geobacter was only detected transiently at the beginning of TCE dechlorination. The Dehalococcoides RDase VcrA and smaller amounts of TceA were expressed in the parent KB-1 culture during complete dechlorination of TCE to ethene regardless of time point or amended substrate. The Dehalococcoides RDase BvcA was only detected in enrichments maintained on cDCE as growth substrates, in roughly equal abundance to VcrA. Only VcrA was detected in subcultures enriched on VC. Enzyme assays revealed that 1,1-DCE, a substrate not used for culture enrichment, afforded the highest specific activity. trans-DCE was substantially dechlorinated only by extracts from cDCE enrichments expressing BvcA. RDase gene distribution indicated enrichment of different strains of Dehalococcoides as a function of electron acceptor TCE, cDCE, or VC. Each chloroethene reductase has distinct substrate preferences leading to strain selection in mixed communities. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  6. Direct Cloning from Enrichment Cultures, a Reliable Strategy for Isolation of Complete Operons and Genes from Microbial Consortia

    PubMed Central

    Entcheva, Plamena; Liebl, Wolfgang; Johann, Andre; Hartsch, Thomas; Streit, Wolfgang R.

    2001-01-01

    Enrichment cultures of microbial consortia enable the diverse metabolic and catabolic activities of these populations to be studied on a molecular level and to be explored as potential sources for biotechnology processes. We have used a combined approach of enrichment culture and direct cloning to construct cosmid libraries with large (>30-kb) inserts from microbial consortia. Enrichment cultures were inoculated with samples from five environments, and high amounts of avidin were added to the cultures to favor growth of biotin-producing microbes. DNA was extracted from three of these enrichment cultures and used to construct cosmid libraries; each library consisted of between 6,000 and 35,000 clones, with an average insert size of 30 to 40 kb. The inserts contained a diverse population of genomic DNA fragments isolated from the consortia organisms. These three libraries were used to complement the Escherichia coli biotin auxotrophic strain ATCC 33767 Δ(bio-uvrB). Initial screens resulted in the isolation of seven different complementing cosmid clones, carrying biotin biosynthesis operons. Biotin biosynthesis capabilities and growth under defined conditions of four of these clones were studied. Biotin measured in the different culture supernatants ranged from 42 to 3,800 pg/ml/optical density unit. Sequencing the identified biotin synthesis genes revealed high similarities to bio operons from gram-negative bacteria. In addition, random sequencing identified other interesting open reading frames, as well as two operons, the histidine utilization operon (hut), and the cluster of genes involved in biosynthesis of molybdopterin cofactors in bacteria (moaABCDE). PMID:11133432

  7. Phylogenetic analysis of methanogenic enrichment cultures obtained from Lonar Lake in India: isolation of Methanocalculus sp. and Methanoculleus sp.

    PubMed

    Surakasi, Venkata Prasad; Wani, Aijaz Ahmad; Shouche, Yogesh S; Ranade, Dilip R

    2007-11-01

    The diversity of methanogenic archaea in enrichment cultures established from the sediments of Lonar Lake (India), a soda lake having pH approximately 10, was investigated using 16S rDNA molecular phylogenetic approach. Methanogenic enrichment cultures were developed in a medium that simulated conditions of soda lake with three different substrates viz., H(2):CO(2), sodium acetate, and trimethylamine (TMA), at alkaline pH. Archaeal 16S rRNA clone libraries were generated from enrichment cultures and 13 RFLP groups were obtained. Representative sequence analysis of each RFLP group indicated that the majority of the 16S rRNA gene sequences were phylogenetically affiliated with uncultured Archaea. Some of the groups may belong to new archaeal genera or families. Three RFLP groups were related to Methanoculleus sp, while two related to Methanocalculus sp. 16S rRNA gene sequences found in Lonar Lake were different from sequences reported from other soda lakes and more similar to those of oil reservoirs, palm oil waste treatment digesters, and paddy fields. In culture-based studies, three isolates were obtained. Two of these were related to Methanoculleus sp. IIE1 and one to Methanocalculus sp. 01F97C. These results clearly show that the Lonar Lake ecosystem harbors unexplored methanogens.

  8. Biodegradation of high concentrations of halomethanes by a fermentative enrichment culture

    PubMed Central

    2014-01-01

    A fermentative enrichment culture (designated DHM-1) that grows on corn syrup was evaluated for its ability to cometabolically biodegrade high concentrations of chloroform (CF), carbon tetrachloride (CT), and trichlorofluoromethane (CFC-11). When provided with corn syrup and vitamin B12 (0.03 mol B12 per mol CF), DHM-1 grew and biodegraded up to 2,000 mg/L of CF in 180 days, with only minor transient accumulation of dichloromethane and chloromethane. CT (15 mg/L) and CFC-11 (25 mg/L) were also biodegraded without significant accumulation of halomethane daughter products. The rate of CF biodegradation followed a Michaelis-Menten-like pattern with respect to the B12 concentration; one-half the maximum rate (66 mg CF/L/d) occurred at 0.005 mol B12 per mol CF. DHM-1 was able to biodegrade 500 mg/L of CF at an inoculum level as low as 10−8 mg protein/L. The highest rate of CF biodegradation occurred at pH 7.7; activity decreased substantially below pH 6.0. DHM-1 biodegraded mixtures of CT, CFC-11, and CF, although CFC-11 inhibited CF biodegradation. Evidence for compete defluorination of CFC-11 was obtained based on a fluoride mass balance. Overall, the results suggest that DHM-1 may be effective for bioaugmentation in source zones contaminated with thousands of milligrams per liter of CF and tens of milligrams per liter of CT and CFC-11. PMID:25401061

  9. Biodegradation of tributyl phosphate by novel bacteria isolated from enrichment cultures.

    PubMed

    Ahire, Kedar C; Kapadnis, Balu P; Kulkarni, Girish J; Shouche, Yogesh S; Deopurkar, Rajendra L

    2012-02-01

    Tributyl phosphate (TBP) is an organophosphorous compound, used extensively (3000-5000 tonnes/annum) as a solvent for nuclear fuel processing and as a base stock in the formulation of fire-resistant aircraft hydraulic fluids and other applications. Because of its wide applications and relative stability in the natural environment TBP poses the problem of pollution and health hazards. In the present study, fifteen potent bacterial strains capable of using tributyl phosphate (TBP) as sole carbon and phosphorus source were isolated from enrichment cultures. These isolates were identified on the basis of biochemical and morphological characteristics and 16S rRNA gene sequence analysis. Phylogenetic analysis of 16S rRNA gene sequences revealed that two isolates belonged to class Bacilli and thirteen to β and γ-Proteobacteria. All these isolates were found to be members of genera Alcaligenes, Providencia, Delftia, Ralstonia, and Bacillus. These isolates were able to tolerate and degrade up to 5 mM TBP, the highest concentration reported to date. The GC-MS method was developed to monitor TBP degradation. Two strains, Providencia sp. BGW4 and Delftia sp. BGW1 showed respectively, 61.0 ± 2.8% and 57.0 ± 2.0% TBP degradation within 4 days. The degradation rate constants, calculated by first order kinetic model were between 0.0024 and 0.0099 h(-1). These bacterial strains are novel for TBP degradation and could be used as an important bioresource for efficient decontamination of TBP polluted waste streams.

  10. Bacterial community analysis in chlorpyrifos enrichment cultures via DGGE and use of bacterial consortium for CP biodegradation.

    PubMed

    Akbar, Shamsa; Sultan, Sikander; Kertesz, Michael

    2014-10-01

    The organophosphate pesticide chlorpyrifos (CP) has been used extensively since the 1960s for insect control. However, its toxic effects on mammals and persistence in environment necessitate its removal from contaminated sites, biodegradation studies of CP-degrading microbes are therefore of immense importance. Samples from a Pakistani agricultural soil with an extensive history of CP application were used to prepare enrichment cultures using CP as sole carbon source for bacterial community analysis and isolation of CP metabolizing bacteria. Bacterial community analysis (denaturing gradient gel electrophoresis) revealed that the dominant genera enriched under these conditions were Pseudomonas, Acinetobacter and Stenotrophomonas, along with lower numbers of Sphingomonas, Agrobacterium and Burkholderia. Furthermore, it revealed that members of Bacteroidetes, Firmicutes, α- and γ-Proteobacteria and Actinobacteria were present at initial steps of enrichment whereas β-Proteobacteria appeared in later steps and only Proteobacteria were selected by enrichment culturing. However, when CP-degrading strains were isolated from this enrichment culture, the most active organisms were strains of Acinetobacter calcoaceticus, Pseudomonas mendocina and Pseudomonas aeruginosa. These strains degraded 6-7.4 mg L(-1) day(-1) of CP when cultivated in mineral medium, while the consortium of all four strains degraded 9.2 mg L(-1) day(-1) of CP (100 mg L(-1)). Addition of glucose as an additional C source increased the degradation capacity by 8-14 %. After inoculation of contaminated soil with CP (200 mg kg(-1)) disappearance rates were 3.83-4.30 mg kg(-1) day(-1) for individual strains and 4.76 mg kg(-1) day(-1) for the consortium. These results indicate that these organisms are involved in the degradation of CP in soil and represent valuable candidates for in situ bioremediation of contaminated soils and waters.

  11. Diversity of cold-active protease-producing bacteria from arctic terrestrial and marine environments revealed by enrichment culture.

    PubMed

    Kim, Eun Hye; Cho, Kyeung Hee; Lee, Yung Mi; Yim, Joung Han; Lee, Hong Kum; Cho, Jang-Cheon; Hong, Soon Gyu

    2010-08-01

    A new approach for enrichment culture was applied to obtain cold-active protease-producing bacteria for marine and terrestrial samples from Svalbard, Norway. The method was developed for the enrichment of bacteria by long-term incubation at low temperatures in semi-solid agar medium containing meat pieces as the main source of carbon and energy. ZoBell and 0.1x nutrient broth were added for marine and terrestrial microorganisms, respectively, to supply basal elements for growth. One to three types of colonies were observed from each enrichment culture, indicating that specific bacterial species were enriched during the experimental conditions. Among 89 bacterial isolates, protease activity was observed from 48 isolates in the screening media containing skim milk. Good growth was observed at 4 degrees C and 10 degrees C while none of the isolates could grow at 37 degrees C. At low temperatures, enzyme activity was equal to or higher than activity at higher temperatures. Bacterial isolates were included in the genera Pseudoalteromonas (33 isolates), Arthrobacter (24 isolates), Pseudomonas (16 isolates), Psychrobacter (6 isolates), Sphingobacterium (6 isolates), Flavobacterium (2 isolates), Sporosarcina (1 isolate), and Stenotrophomonas (1 isolate). Protease activity was observed from Pseudoalteromonas (33 isolates), Pseudomonas (10 isolates), Arthrobacter (4 isolates), and Flavobacterium (1 isolate).

  12. Assessing the influence of CH4 concentration during culture enrichment on the biodegradation kinetics and population structure.

    PubMed

    López, Juan C; Quijano, Guillermo; Pérez, Rebeca; Muñoz, Raúl

    2014-12-15

    Methanotrophic communities were enriched in three stirred tank reactors continuously supplied with CH4-laden air at 20, 2 and 0.2 gCH4 m(-3) in order to evaluate the influence of CH4 concentration on the biodegradation kinetics, population structure and potential polyhydroxyalkanoate production under sequential nitrogen limitations. The population structure of the enriched cultures, dominated by type I methanotrophs, was influenced by CH4 concentration. No significant correlation between CH4 concentration and the maximum specific degradation rate (qmax) or the half-saturation constant (KS) was recorded, microorganisms enriched at 2 gCH4 m(-3) presenting the highest qmax and those enriched at 20 and 0.2 gCH4 m(-3) exhibiting the lowest KS. Maximum polyhydroxybutyrate (PHB) contents of 1.0% and 12.6% (w/w) were achieved at 20 and 2 g CH4 m(-3), respectively. Polyhydroxyvalerate (PHV) was also detected at PHV:PHB ratios of up to 12:1 and 4:1 in the communities enriched at 20 and 0.2 gCH4 m(-3), respectively. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Evidence for the existence of psychrophilic methanogenic communities in anoxic sediments of deep lakes.

    PubMed

    Nozhevnikova, Alla N; Zepp, Kornelia; Vazquez, Francisco; Zehnder, Alexander J B; Holliger, Christof

    2003-03-01

    In order to obtain evidence for the existence of psychrophilic methanogenic communities in sediments of deep lakes that are low-temperature environments (4 to 5 degrees C), slurries were first incubated at temperatures between 4 and 60 degrees C for several weeks, at which time they were amended, or not, with an additional substrate, such as cellulose, butyrate, propionate, acetate, or hydrogen, and further incubated at 6 degrees C. Initial methane production rates were highest in slurries preincubated at temperatures between 4 and 15 degrees C, with maximal rates in slurries kept at 6 degrees C. Hydrogen-amended cultures were the only exceptions, with the highest methane production rates at 6 degrees C after preincubation at 30 degrees C.

  14. Psychrophilic and Mesophilic Fungi in Fruit-Filled Pastries

    PubMed Central

    Kuehn, Harold H.; Gunderson, Millard F.

    1962-01-01

    Surveys of the mold flora of frozen blueberry and cherry pastries were undertaken. Molds were enumerated by preparing pour plates of the blended product and incubating the plates at 0, 5, 10, and 20 C. In this manner, the total fungal content of the product could be ascertained from the 10 and 20 C plates, and the psychrophilic fungal population was represented by those fungi which grew at 0 and 5 C. The pastry portion, or crust, of the blueberry material was sampled separately from the filling portion. Certain differences in fungal flora were apparent. Aureobasidium pullulans was the dominant fungus in crust at all temperatures of isolation. However, Penicillium thomii proved to be the most common mesophilic fungus in the filling portion, and A. pullulans was the most common psychrophile in the filling. Aspergilli were quite common in the crust, but, in general, were absent from the fruit filling. Cherry pastries had a much smaller total fungal flora than did the blueberry product. However, A. pullulans again was the most prevalent fungus in cherry pastries at all temperatures of isolation. Certain differences in fungal flora were apparent in the two fruit products. Phoma spp. were almost completely absent in blueberries, but represented the second most common fungus in cherry pastries. Blueberry filling had 440 psychrophilic fungi per gram of sample (at 0 C), blueberry crust had 65 per gram, and cherry pastries had 77 per gram. Images FIG. 1 PMID:14460237

  15. Nitrogen source effects on the denitrifying anaerobic methane oxidation culture and anaerobic ammonium oxidation bacteria enrichment process.

    PubMed

    Fu, Liang; Ding, Jing; Lu, Yong-Ze; Ding, Zhao-Wei; Zeng, Raymond J

    2017-05-01

    The co-culture system of denitrifying anaerobic methane oxidation (DAMO) and anaerobic ammonium oxidation (Anammox) has a potential application in wastewater treatment plant. This study explored the effects of permutation and combination of nitrate, nitrite, and ammonium on the culture enrichment from freshwater sediments. The co-existence of NO3(-), NO2(-), and NH4(+) shortened the enrichment time from 75 to 30 days and achieved a total nitrogen removal rate of 106.5 mg/L/day on day 132. Even though ammonium addition led to Anammox bacteria increase and a higher nitrogen removal rate, DAMO bacteria still dominated in different reactors with the highest proportion of 64.7% and the maximum abundance was 3.07 ± 0.25 × 10(8) copies/L (increased by five orders of magnitude) in the nitrite reactor. DAMO bacteria showed greater diversity in the nitrate reactor, and one was similar to M. oxyfera; DAMO bacteria in the nitrite reactor were relatively unified and similar to M. sinica. Interestingly, no DAMO archaea were found in the nitrate reactor. This study will improve the understanding of the impact of nitrogen source on DAMO and Anammox co-culture enrichment.

  16. Enrichment of a mixed microbial culture for polyhydroxyalkanoates production: Effect of pH and N and P concentrations.

    PubMed

    Montiel-Jarillo, Gabriela; Carrera, Julián; Suárez-Ojeda, María Eugenia

    2017-04-01

    Polyhydroxyalkanoates (PHA) are biopolymers that can be an alternative against conventional plastics. The study reported herein evaluated the enrichment of a mixed microbial culture (MMC) operated under feast/famine regime and different pHs in a sequencing batch reactor (SBR) using acetate as sole carbon source to produce polyhydroxyalkanoates (PHAs). The enrichment step was evaluated at controlled pH of 7.5 and also without pH control (averaged value of 9.0). The acetate uptake rate (-qS) of both enrichments at the end of the experimental period exhibited similar behaviour being about 0.18CmolAcCmolX(-1)h(-1) and 0.19CmolAcCmolX(-1)h(-1) for SBR-A and SBR-B, respectively. However, the PHA-storing capacity of the biomass enriched without pH control was better, exhibiting a maximum PHA content of 36% (gPHAg(-1) VSS) with a PHA production rate (qPHA) of 0.16CmolPHACmolX(-1)h(-1). Batch experiments were performed to evaluate PHA-storing capacity of the enriched culture at different pHs and nutrients concentrations. In the pH experiments (without nutrient limitation), it was found that in the absence of controlled pH, the enriched biomass exhibited a PHA content of 44% gPHAg(-1) VSS with -qS and PHA to substrate yield (YPHA/Ac) of 0.57CmolAcCmolX(-1)h(-1) and 0.33CmolPHACmolAc(-1), respectively. Regarding the experiments at variable nutrients concentration (pH ranging 8.8 to 9.2), the results indicate that the PHA content in the enriched biomass is significantly higher being around 51% gPHAg(-1) VSS under nitrogen limitation. This work demonstrated the feasibility of the enrichment of a MMC with PHA storage ability without pH control. Results also suggest that better PHAs contents and substrate uptake rates are obtained without controlling the pH in the accumulation step. Finally, this work also highlights the importance of understanding the role of nutrients concentration during the accumulation step. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Metatranscriptome of an Anaerobic Benzene-Degrading, Nitrate-Reducing Enrichment Culture Reveals Involvement of Carboxylation in Benzene Ring Activation

    PubMed Central

    Luo, Fei; Gitiafroz, Roya; Devine, Cheryl E.; Gong, Yunchen; Hug, Laura A.; Raskin, Lutgarde

    2014-01-01

    The enzymes involved in the initial steps of anaerobic benzene catabolism are not known. To try to elucidate this critical step, a metatranscriptomic analysis was conducted to compare the genes transcribed during the metabolism of benzene and benzoate by an anaerobic benzene-degrading, nitrate-reducing enrichment culture. RNA was extracted from the mixed culture and sequenced without prior mRNA enrichment, allowing simultaneous examination of the active community composition and the differential gene expression between the two treatments. Ribosomal and mRNA sequences attributed to a member of the family Peptococcaceae from the order Clostridiales were essentially only detected in the benzene-amended culture samples, implicating this group in the initial catabolism of benzene. Genes similar to each of two subunits of a proposed benzene-carboxylating enzyme were transcribed when the culture was amended with benzene. Anaerobic benzoate degradation genes from strict anaerobes were transcribed only when the culture was amended with benzene. Genes for other benzoate catabolic enzymes and for nitrate respiration were transcribed in both samples, with those attributed to an Azoarcus species being most abundant. These findings indicate that the mineralization of benzene starts with its activation by a strict anaerobe belonging to the Peptococcaceae, involving a carboxylation step to form benzoate. These data confirm the previously hypothesized syntrophic association between a benzene-degrading Peptococcaceae strain and a benzoate-degrading denitrifying Azoarcus strain for the complete catabolism of benzene with nitrate as the terminal electron acceptor. PMID:24795366

  18. Building Learning Communities for Research Collaboration and Cross-Cultural Enrichment in Science Education

    NASA Astrophysics Data System (ADS)

    Sparrow, E. B.

    2003-12-01

    The GLOBE program has provided opportunities for environmental science research and education collaborations among scientists, teachers and K-12 students, and for cross-cultural enrichment nationally and abroad. In Alaska, GLOBE has also provided funding leverage in some cases, and a base for several other science education programs that share a common goal of increasing student interest, understanding, process skills and achievement in science, through involvement in ongoing research investigations. These programs that use GLOBE methodologies (standardized scientific measurements and learning activities developed by scientists and educators) are: Global Change Education Using Western Science and Native Knowledge also known as "Observing Locally, Connecting Globally" (OLCG); Alaska Earth System Science Education Alliance: Improving Understanding of Climate Variability and Its Relevance to Rural Alaska; Schoolyard Long Term Ecological Research; Alaska Rural Research Partnership; Alaska Partnership for Teacher Enhancement; Alaska Lake Ice and Snow Observatory Network; Alaska Boreal Forest Council Education Outreach; Calypso Farm and Ecology Center; Environmental Education Outreach; and also GLOBE Arctic POPs (persistent organic pollutants) a program that involves countries in the circumpolar North. The University of Alaska GLOBE Partnership has collaborated with the BLM Campbell Creek Science Center Globe Partnership in facilitating GLOBE Training Workshops and providing teacher support. GLOBE's extensive website including data entry, archive, analysis and visualization capabilities; GLOBE Teacher Guide, videos and other materials provided; excellent GLOBE science research and education staff, training support office, GLOBE help desk, alignment of GLOBE curriculum with national science education standards and GLOBE certification of teachers trained on even just one GLOBE investigation, have made it easier to implement GLOBE in the classroom. Using GLOBE, whole

  19. Molecular and carbon isotopic characterization of an anaerobic stable enrichment culture containing Dehalobacterium sp. during dichloromethane fermentation.

    PubMed

    Trueba-Santiso, Alba; Parladé, Eloi; Rosell, Mònica; Lliros, Marc; Mortan, Siti Hatijah; Martínez-Alonso, Maira; Gaju, Nuria; Martín-González, Lucía; Vicent, Teresa; Marco-Urrea, Ernest

    2017-03-01

    Biodegradation of dichloromethane (DCM) under reducing conditions is of major concern due to its widespread detection in contaminated groundwaters. Here, we report an anaerobic enrichment culture derived from a membrane bioreactor operating in an industrial wastewater treatment plant, capable of fermenting DCM and the brominated analogue dibromomethane (DBM). Comparative analysis of bacterial 16S rDNA-DGGE profiles from fresh liquid medium inoculated with single colonies picked from serial dilution-to-extinction agar vials showed that cultures degrading DCM contained a predominant band belonging to Dehalobacterium, however this band was absent in cultures unable to degrade DCM. Analysis of the microbial composition of the enrichment by bacterial 16S rRNA gene amplicon paired-end sequencing confirmed the presence of Dehalobacterium together with three additional phylotypes belonging to Acetobacterium, Desulfovibrio, and Wolinella, representing all four operational taxonomic units >99.9% of the retrieved sequences. The carbon isotopic fractionation (ε) determined for DCM degradation in this culture was -27±2‰. This value differs from the ε previously reported for the DCM-fermentative bacteria Dehalobacter (-15.5±1.5‰) but they are both significantly different from those reported for facultative methylotrophic organisms (ranging from -45 to -61‰). This significant difference in the ε allows differentiating between hydrolytic transformation of DCM via glutathione-dependent dehalogenases and fermentation pathway. The carbon isotopic fractionation of dichloromethane by an enriched Dehalobacterium-containing culture has significant potential to monitor biodegradation of DCM in groundwaters. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Degradative capacities and 16S rRNA-targeted whole-cell hybridization of sulfate-reducing bacteria in an anaerobic enrichment culture utilizing alkylbenzenes from crude oil

    SciTech Connect

    Rabus, R.; Widdel, F.; Fukui, Manabu

    1996-10-01

    Production of sulfide in oil field waters, a process which is referred to as souring, has been of concern. Hydrogen sulfide may lead to poisoning, contamination of oil and gas, corrosion of pipelines, conversion of iron mineral to ferrous sulfide. This study used a previously established sulfate-reducing enrichment culture on crude oil as a model system of bacterial habitats in which crude oil is the only potential source of organic substrates, and the enrichment culture was studied in detail including substrate preferences and major nutritional types of sulfate-reducing bacteria in the enrichment culture. 74 refs., 2 figs., 2 tabs.

  1. Detection of Campylobacter jejuni and Campylobacter coli in foods by enrichment culture and polymerase chain reaction enzyme-linked immunosorbent assay.

    PubMed

    Bolton, F J; Sails, A D; Fox, A J; Wareing, D R A; Greenway, D L A

    2002-05-01

    A polymerase chain reaction (PCR) assay based on a solution hybridization format with colorimetric end-point detection (PCR ELISA) was investigated for the specific detection of Campylobacter jejuni and Campylobacter coli in food samples following enrichment culture. One hundred fifteen samples of raw meat and offal (poultry, porcine, ovine, and bovine), raw shellfish, and artificially contaminated milk were enriched in blood-free Campylobacter Enrichment Broth for 48 h. Enrichment cultures were subcultured to Campylobacter blood-free selective agar plates, and presumptive isolates were identified by phenotypic methods. DNA was extracted from 1-ml aliquots of the enrichment cultures using a rapid extraction method, and the DNA was used as the template in a PCR ELISA. A comparison of the PCR ELISA with the enrichment culture and subculture to selective agar method showed that the results of 112 of the 115 samples tested were in agreement by both methods. Seventy-one of the various food samples were positive in the PCR ELISA, and 70 samples were positive by culture. The PCR ELISA had a sensitivity of 99% and a specificity of 96%, with a positive predictive value of 97% and a negative predictive value of 98%. The PCR ELISA is a rapid, sensitive, and specific method for the detection of C. jejuni and C. coli in foods following enrichment culture and significantly reduces the time required for their detection.

  2. Effect of free ammonia and free nitrous acid concentration on the anabolic and catabolic processes of an enriched Nitrosomonas culture.

    PubMed

    Vadivelu, Vel M; Keller, Jurg; Yuan, Zhiguo

    2006-12-05

    The effects of free ammonia (FA; NH(3)) and free nitrous acid (FNA; HNO(2)) concentrations on the metabolisms of an enriched ammonia oxidizing bacteria (AOB) culture were investigated using a method allowing the decoupling of growth and energy generation processes. A lab-scale sequencing batch reactor (SBR) was operated for the enrichment of an AOB culture. Fluorescent in-situ hybridization (FISH) analysis showed that 82% of the bacterial population in the SBR bound to the NEU probe specifically designed for Nitrosomonas europaea. Batch tests were carried out to measure the oxygen and ammonium consumption rates by the culture at various FA and FNA levels, in the presence or absence of inorganic carbon (CO(2), HCO(3) (-), and CO(3) (2-)). It was revealed that FA of up to 16.0 mgNH(3)-N . L(-1), which was the highest concentration used in this study, did not have any inhibitory effect on either the catabolic or anabolic processes of the Nitrosomonas culture. In contrast, FNA inhibited both the growth and energy production capabilities of the Nitrosomonas culture. The inhibition on growth initiated at approximately 0.10 mgHNO(2)-N . L(-1), and the data suggested that the biosynthesis was completely stopped at an FNA concentration of 0.40 mgHNO(2)-N . L(-1). The inhibition on energy generation initiated at a slightly lower level but the Nitrosomonas culture was still oxidizing ammonia at half of the maximum rate at an FNA concentration of 0.50-0.63 mgHNO(2)-N . L(-1). The affinity constant of the Nitrosomonas culture with respect to ammonia was determined to be 0.36 mgNH(3)-N . L(-1), independent of the presence or absence of inorganic carbon.

  3. Study of interferon-β antiviral activity against Herpes simplex virus type 1 in neuron-enriched trigeminal ganglia cultures.

    PubMed

    Low-Calle, Ana Maria; Prada-Arismendy, Jeanette; Castellanos, Jaime E

    2014-02-13

    Herpes simplex virus type 1 (HSV-1) causes a lytic infection in epithelial cells before being captured and moved via retrograde axonal transport to the nuclei of the sensory neurons of the trigeminal ganglion or dorsal root, where it establishes a latent infection. HSV-1 infection induces an antiviral response through the production of Beta Interferon (IFN-β) in infected trigeminal ganglia. The aim of this work was to characterize the response induced by IFN-β in neuron-enriched trigeminal ganglia primary cultures infected with HSV-1. An antiviral effect of IFN-β in these cultures was observed, including reduced viral production and increased cell survival. In contrast, viral infection significantly decreased both double stranded RNA dependent protein kinase (Pkr) transcription and Jak-1 and Stat-1 phosphorylation, suggesting a possible HSV-1 immune evasion mechanism in trigeminal cells. Additionally, HSV-1 infection upregulated Suppressor of Cytokine Signaling-3 (Socs3) mRNA; upregulation of socs3 was inhibited in IFN-β treated cultures. HSV-1 infection increased the number of Socs3 positive cells and modified the intracellular distribution of Socs3 protein, in infected cells. This neuron-enriched trigeminal ganglia culture model could be used to elucidate the HSV-1 viral cycle in sensory neurons and to study cellular antiviral responses and possible viral evasion mechanisms that underlie the choice between viral replication and latency.

  4. A Culture-Independent Approach to Enrich Endophytic Bacterial Cells from Sugarcane Stems for Community Characterization.

    PubMed

    Dos-Santos, Carlos M; de Souza, Daniel G; Balsanelli, Eduardo; Cruz, Leonardo Magalhães; de Souza, Emanuel M; Baldani, José I; Schwab, Stefan

    2017-08-01

    Bacterial endophytes constitute a very diverse community and they confer important benefits which help to improve agricultural yield. Some of these benefits remain underexplored or little understood, mainly due to the bottlenecks associated with the plant feature, a low number of endophytic bacterial cells in relation to the plant, and difficulties in accessing these bacteria using cultivation-independent methods. Enriching endophytic bacterial cells from plant tissues, based on a non-biased, cultivation-independent physical enrichment method, may help to circumvent those problems, especially in the case of sugarcane stems, which have a high degree of interfering factors, such as polysaccharides, phenolic compounds, nucleases, and fibers. In the present study, an enrichment approach for endophytic bacterial cells from sugarcane lower stems is described. The results demonstrate that the enriched bacterial cells are suitable for endophytic community characterization. A community analysis revealed the presence of previously well-described but also novel endophytic bacteria in sugarcane tissues which may exert functions such as plant growth promotion and biological control, with a predominance of the Proteobacterial phylum, but also Actinobacteria, Bacteroidetes, and Firmicutes, among others. In addition, by comparing the present and literature data, it was possible to list the most frequently detected bacterial endophyte genera in sugarcane tissues. The presented enrichment approach paves the way for improved future research toward the assessment of endophytic bacterial community in sugarcane and other biofuel crops.

  5. Improved enrichment culture technique for methane-oxidizing bacteria from marine ecosystems: the effect of adhesion material and gas composition.

    PubMed

    Vekeman, Bram; Dumolin, Charles; De Vos, Paul; Heylen, Kim

    2017-02-01

    Cultivation of microbial representatives of specific functional guilds from environmental samples depends largely on the suitability of the applied growth conditions. Especially the cultivation of marine methanotrophs has received little attention, resulting in only a limited number of ex situ cultures available. In this study we investigated the effect of adhesion material and headspace composition on the methane oxidation activity in methanotrophic enrichments obtained from marine sediment. Addition of sterilized natural sediment or alternatively the addition of acid-washed silicon dioxide significantly increased methane oxidation. This positive effect was attributed to bacterial adhesion on the particles via extracellular compounds, with a minimum amount of particles required for effect. As a result, the particles were immobilized, thus creating a stratified environment in which a limited diffusive gas gradients could build up and various microniches were formed. Such diffusive gas gradient might necessitate high headspace concentrations of CH4 and CO2 for sufficient concentrations to reach the methane-oxidizing bacteria in the enrichment culture technique. Therefore, high concentrations of methane and carbon dioxide, in addition to the addition of adhesion material, were tested and indeed further stimulated methane oxidation. Use of adhesion material in combination with high concentrations of methane and carbon dioxide might thus facilitate the cultivation and subsequent enrichment of environmentally important members of this functional guild. The exact mechanism of the observed positive effects on methane oxidation and the differential effect on methanotrophic diversity still needs to be explored.

  6. Development and evaluation of a rapid, simple, and sensitive immunochromatographic assay to detect thermostable direct hemolysin produced by Vibrio parahaemolyticus in enrichment cultures of stool specimens.

    PubMed

    Kawatsu, Kentaro; Ishibashi, Masanori; Tsukamoto, Teizo

    2006-05-01

    Thermostable direct hemolysin (TDH) is considered to be a major virulence factor in Vibrio parahaemolyticus, and most cases of V. parahaemolyticus diarrhea in humans are caused by tdh gene-positive strains. In the present study, we developed an immunochromatographic assay to detect TDH (TDH-ICA) and evaluated the utility of TDH-ICA for the diagnosis of V. parahaemolyticus diarrhea. TDH-ICA allowed the detection of 0.2 ng/ml of TDH within 10 min. Fecal homogenates were spiked with various numbers of tdh-positive V. parahaemolyticus organisms, and their enrichment cultures were tested with TDH-ICA. The results of detection of TDH in the enrichment cultures by TDH-ICA were in accord with the results of recovery of the spiked V. parahaemolyticus organisms from the enrichment cultures by plating onto thiosulfate-citrate-bile salts-sucrose agar. When enrichment cultures of 217 stool specimens from patients with diarrhea were tested with TDH-ICA, the TDH-ICA results showed 100% sensitivity and specificity compared to the results of isolation of V. parahaemolyticus from the stool specimens by a conventional bacterial culture test. Since TDH-ICA was able to detect TDH in a fecal enrichment culture within 10 min, TDH-ICA testing of a fecal enrichment culture could be completed rapidly and easily within approximately 16 h, including incubation time for the fecal enrichment culture. These results indicate that TDH-ICA is a rapid, simple, and sensitive TDH detection method and that TDH-ICA testing of a fecal enrichment culture is useful as an adjunct to facilitate the early diagnosis of V. parahaemolyticus diarrhea.

  7. 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.

  8. Psychrophilic dry anaerobic digestion of dairy cow feces: Long-term operation

    SciTech Connect

    Massé, Daniel I. Cata Saady, Noori M.

    2015-02-15

    Highlights: • Psychrophilic dry anaerobic digestion (PDAD) of cow feces (CF) is feasible. • PDAD of CF is as efficient as mesophilic and thermophilic AD at TCL 21 days. • CF (13–16% TS at OLR 5.0 g TCOD{sub fed} kg{sup −1} inoculum d{sup −1}) yielded 222 ± 27 {sub N}L CH{sub 4} kg{sup −1} VS fed. - Abstract: This paper reports experimental results which demonstrate psychrophilic dry anaerobic digestion of cow feces during long-term operation in sequence batch reactor. Cow feces (13–16% total solids) has been anaerobically digested in 12 successive cycles (252 days) at 21 days treatment cycle length (TCL) and temperature of 20 °C using psychrotrophic anaerobic mixed culture. An average specific methane yield (SMY) of 184.9 ± 24.0, 189.9 ± 27.3, and 222 ± 27.7 {sub N}L CH{sub 4} kg{sup −1} of VS fed has been achieved at an organic loading rate of 3.0, 4.0, and 5.0 g TCOD kg{sup −1} inoculum d{sup −1} and TCL of 21 days, respectively. The corresponding substrate to inoculum ratio (SIR) was 0.39 ± 0.06, 0.48 ± .02, 0.53 ± 0.05, respectively. Average methane production rate of 10 ± 1.4 {sub N}L CH{sub 4} kg{sup −1} VS fed d{sup −1} has been obtained. The low concentration of volatile fatty acids indicated that hydrolysis was the reaction limiting step.

  9. Novel Psychrophilic and Thermolabile l-Threonine Dehydrogenase from Psychrophilic Cytophaga sp. Strain KUC-1

    PubMed Central

    Kazuoka, Takayuki; Takigawa, Shouhei; Arakawa, Noriaki; Hizukuri, Yoshiyuki; Muraoka, Ikuo; Oikawa, Tadao; Soda, Kenji

    2003-01-01

    A psychrophilic bacterium, Cytophaga sp. strain KUC-1, that abundantly produces a NAD+-dependent l-threonine dehydrogenase was isolated from Antarctic seawater, and the enzyme was purified. The molecular weight of the enzyme was estimated to be 139,000, and that of the subunit was determined to be 35,000. The enzyme is a homotetramer. Atomic absorption analysis showed that the enzyme contains no metals. In these respects, the Cytophaga enzyme is distinct from other l-threonine dehydrogenases that have thus far been studied. l-Threonine and dl-threo-3-hydroxynorvaline were the substrates, and NAD+ and some of its analogs served as coenzymes. The enzyme showed maximum activity at pH 9.5 and at 45°C. The kinetic parameters of the enzyme are highly influenced by temperatures. The Km for l-threonine was lowest at 20°C. Dead-end inhibition studies with pyruvate and adenosine-5′-diphosphoribose showed that the enzyme reaction proceeds via the ordered Bi Bi mechanism in which NAD+ binds to an enzyme prior to l-threonine and 2-amino-3-oxobutyrate is released from the enzyme prior to NADH. The enzyme gene was cloned into Escherichia coli, and its nucleotides were sequenced. The enzyme gene contains an open reading frame of 939 bp encoding a protein of 312 amino acid residues. The amino acid sequence of the enzyme showed a significant similarity to that of UDP-glucose 4-epimerase from Staphylococcus aureus and belongs to the short-chain dehydrogenase-reductase superfamily. In contrast, l-threonine dehydrogenase from E. coli belongs to the medium-chain alcohol dehydrogenase family, and its amino acid sequence is not at all similar to that of the Cytophaga enzyme. l-Threonine dehydrogenase is significantly similar to an epimerase, which was shown for the first time. The amino acid residues playing an important role in the catalysis of the E. coli and human UDP-glucose 4-epimerases are highly conserved in the Cytophaga enzyme, except for the residues participating in the

  10. Embedded Voices: Building a Non-Learning Culture within a Learning Enrichment Programme

    ERIC Educational Resources Information Center

    Hymer, Barry; Watkins, Chris; Dawson, Elizabeth; Buxton, Ruth

    2015-01-01

    The researchers examined transcripts of comments made and dialogues engaged in by children, teachers and student teaching assistants during a 10-week enrichment programme for gifted and talented children aged 7-9 years. Attempts were made to match these utterances with the programme's aims and aspirations as expressed in a promotional document.…

  11. Anaerobic Biotransformation of High Concentrations of Chloroform by an Enrichment Culture and Two Bacterial Isolates ▿ †

    PubMed Central

    Shan, Huifeng; Kurtz, Harry D.; Mykytczuk, Nadia; Trevors, Jack T.; Freedman, David L.

    2010-01-01

    A fermentative enrichment culture (designated DHM-1) was developed that is capable of cometabolically biotransforming high concentrations of chloroform (CF) to nontoxic end products. Two Pantoea spp. were isolated from DHM-1 that also possess this dechlorination capability. Following acclimation to increasing levels of CF, corn syrup-grown DHM-1 was able to transform over 500 mg/liter CF in the presence of vitamin B12 (approximately 3% of CF on a molar basis) at a rate as high as 22 mg/liter/day in a mineral salts medium. CO, CO2, and organic acids were the predominant biodegradation products, suggesting that hydrolytic reactions predominate during CF transformation. DHM-1 was capable of growing on corn syrup in the presence of high concentrations of CF (as may be present near contaminant source zones in groundwater), which makes it a promising culture for bioaugmentation. Strains DHM-1B and DHM-1T transform CF at rates similar to that of the DHM-1 enrichment culture. The ability of these strains to grow in the presence of high concentrations of CF appears to be related to alteration of membrane fluidity or homeoviscous and homeophasic adaptation. PMID:20693443

  12. Microbial Diversity in Sulfate-Reducing Marine Sediment Enrichment Cultures Associated with Anaerobic Biotransformation of Coastal Stockpiled Phosphogypsum (Sfax, Tunisia).

    PubMed

    Zouch, Hana; Karray, Fatma; Armougom, Fabrice; Chifflet, Sandrine; Hirschler-Réa, Agnès; Kharrat, Hanen; Kamoun, Lotfi; Ben Hania, Wajdi; Ollivier, Bernard; Sayadi, Sami; Quéméneur, Marianne

    2017-01-01

    Anaerobic biotechnology using sulfate-reducing bacteria (SRB) is a promising alternative for reducing long-term stockpiling of phosphogypsum (PG), an acidic (pH ~3) by-product of the phosphate fertilizer industries containing high amounts of sulfate. The main objective of this study was to evaluate, for the first time, the diversity and ability of anaerobic marine microorganisms to convert sulfate from PG into sulfide, in order to look for marine SRB of biotechnological interest. A series of sulfate-reducing enrichment cultures were performed using different electron donors (i.e., acetate, formate, or lactate) and sulfate sources (i.e., sodium sulfate or PG) as electron acceptors. Significant sulfide production was observed from enrichment cultures inoculated with marine sediments, collected near the effluent discharge point of a Tunisian fertilizer industry (Sfax, Tunisia). Sulfate sources impacted sulfide production rates from marine sediments as well as the diversity of SRB species belonging to Deltaproteobacteria. When PG was used as sulfate source, Desulfovibrio species dominated microbial communities of marine sediments, while Desulfobacter species were mainly detected using sodium sulfate. Sulfide production was also affected depending on the electron donor used, with the highest production obtained using formate. In contrast, low sulfide production (acetate-containing cultures) was associated with an increase in the population of Firmicutes. These results suggested that marine Desulfovibrio species, to be further isolated, are potential candidates for bioremediation of PG by immobilizing metals and metalloids thanks to sulfide production by these SRB.

  13. Feasibility study of an alkaline-based chemical treatment for the purification of polyhydroxybutyrate produced by a mixed enriched culture.

    PubMed

    Jiang, Yang; Mikova, Gizela; Kleerebezem, Robbert; van der Wielen, Luuk Am; Cuellar, Maria C

    2015-12-01

    This study focused on investigating the feasibility of purifying polyhydroxybutyrate (PHB) from mixed culture biomass by alkaline-based chemical treatment. The PHB-containing biomass was enriched on acetate under non-sterile conditions. Alkaline treatment (0.2 M NaOH) together with surfactant SDS (0.2 w/v% SDS) could reach 99% purity, with more than 90% recovery. The lost PHB could be mostly attributed to PHB hydrolysis during the alkaline treatment. PHB hydrolysis could be moderated by increasing the crystallinity of the PHB granules, for example, by biomass pretreatment (e.g. freezing or lyophilization) or by effective cell lysis (e.g. adjusting alkali concentration). The suitability of the purified PHB by alkaline treatment for polymer applications was evaluated by molecular weight and thermal stability. A solvent based purification method was also performed for comparison purposes. As result, PHB produced by mixed enriched cultures was found suitable for thermoplastic applications when purified by the solvent method. While the alkaline method resulted in purity, recovery yield and molecular weight comparable to values reported in literature for PHB produced by pure cultures, it was found unsuitable for thermoplastic applications. Given the potential low cost and favorable environmental impact of this method, it is expected that PHB purified by alkaline method may be suitable for other non-thermal polymer applications, and as a platform chemical.

  14. An agarose gel-based neurosphere culture system leads to enrichment of neuronal lineage cells in vitro.

    PubMed

    Park, Kyuhee; Nam, Yeonju; Choi, Yongmun

    2015-05-01

    Stem cell-based therapy holds great potential especially for neurological disorders. However, clinical applications await further understanding of many aspects of stem cell differentiation and development of technology enabling manipulation of stem cells into desired cell types in the central nervous system. Here, we developed a new method that leads to enrichment of neuronal lineage cells in neural stem cell cultures. The protocol involves cultivation of primary cells derived from the forebrains of rat E18 embryos above a layer of nonadhesive hard agarose gel in the form of neurospheres. In contrast to the neurospheres that were cultured above an anti-adhesive hydrogel layer, the primary cells that were cultured above a layer of agarose gel preferentially differentiated into β-III tubulin-positive neurons when allowed to undergo differentiation in vitro.In an effort to investigate the mechanism behind this observation, we found that the gene expression of a vertebrate neuronal determination gene (neurogenin1) was enhanced in the neurospheres that proliferated above a layer of agarose gel as compared with the control, and the gene expression level of neurogenin1 was quite well correlated with the rigidity of agarose gel. These results indicate that agarose gel can contribute, at least in part, to enrich neuronal progenitors and immature postmitotic neurons during neurosphere formation and may provide additional information to establish efficient protocols for the neural stem cell-based study.

  15. 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

  16. Effects of carbon sources on the enrichment of halophilic polyhydroxyalkanoate-storing mixed microbial culture in an aerobic dynamic feeding process

    NASA Astrophysics Data System (ADS)

    Cui, You-Wei; Zhang, Hong-Yu; Lu, Peng-Fei; Peng, Yong-Zhen

    2016-08-01

    Microbial polyhydroxyalkanoate (PHA) production serves as a substitute for petroleum-based plastics. Enriching mixed microbial cultures (MMCs) with the capacity to store PHA is a key precursor for low-cost PHA production. This study investigated the impact of carbon types on enrichment outcomes. Three MMCs were separately fed by acetate sodium, glucose, and starch as an enriching carbon source, and were exposed to long-term aerobic dynamic feeding (ADF) periods. The PHA production capacity, kinetics and stoichiometry of the enrichments, the PHA composition, and the microbial diversity and community composition were explored to determine carbon and enrichment correlations. After 350-cycle enriching periods under feast-famine (F-F) regimes, the MMCs enriched by acetate sodium and glucose contained a maximum PHA content of 64.7% and 60.5% cell dry weight (CDW). The starch-enriched MMC only had 27.3% CDW of PHA. High-throughput sequencing revealed that non-PHA bacteria survived alongside PHA storing bacteria, even under severe F-F selective pressure. Genus of Pseudomonas and Stappia were the possible PHA accumulating bacteria in acetate-enriched MMC. Genus of Oceanicella, Piscicoccus and Vibrio were found as PHA accumulating bacteria in glucose-enriched MMC. Vibrio genus was the only PHA accumulating bacteria in starch-enriched MMC. The community diversity and composition were regulated by the substrate types.

  17. Effects of carbon sources on the enrichment of halophilic polyhydroxyalkanoate-storing mixed microbial culture in an aerobic dynamic feeding process

    PubMed Central

    Cui, You-Wei; Zhang, Hong-Yu; Lu, Peng-Fei; Peng, Yong-Zhen

    2016-01-01

    Microbial polyhydroxyalkanoate (PHA) production serves as a substitute for petroleum-based plastics. Enriching mixed microbial cultures (MMCs) with the capacity to store PHA is a key precursor for low-cost PHA production. This study investigated the impact of carbon types on enrichment outcomes. Three MMCs were separately fed by acetate sodium, glucose, and starch as an enriching carbon source, and were exposed to long-term aerobic dynamic feeding (ADF) periods. The PHA production capacity, kinetics and stoichiometry of the enrichments, the PHA composition, and the microbial diversity and community composition were explored to determine carbon and enrichment correlations. After 350-cycle enriching periods under feast-famine (F-F) regimes, the MMCs enriched by acetate sodium and glucose contained a maximum PHA content of 64.7% and 60.5% cell dry weight (CDW). The starch-enriched MMC only had 27.3% CDW of PHA. High-throughput sequencing revealed that non-PHA bacteria survived alongside PHA storing bacteria, even under severe F-F selective pressure. Genus of Pseudomonas and Stappia were the possible PHA accumulating bacteria in acetate-enriched MMC. Genus of Oceanicella, Piscicoccus and Vibrio were found as PHA accumulating bacteria in glucose-enriched MMC. Vibrio genus was the only PHA accumulating bacteria in starch-enriched MMC. The community diversity and composition were regulated by the substrate types. PMID:27485896

  18. Conversion of methane-derived carbon and microbial community in enrichment cultures in response to O2 availability.

    PubMed

    Wei, Xiao-Meng; He, Ruo; Chen, Min; Su, Yao; Ma, Ruo-Chan

    2016-04-01

    Methanotrophs not only play an important role in mitigating CH4 emissions from the environment, but also provide a large quantity of CH4-derived carbon to their habitats. In this study, the distribution of CH4-derived carbon and microbial community was investigated in a consortium enriched at three O2 tensions, i.e., the initial O2 concentrations of 2.5 % (LO-2), 5 % (LO-1), and 21 % (v/v) (HO). The results showed that compared with the O2-limiting environments (2.5 and 5 %), more CH4-derived carbon was converted into CO2 and biomass under the O2 sufficient condition (21 %). Besides biomass and CO2, a high conversion efficiency of CH4-derived carbon to dissolved organic carbon was detected in the cultures, especially in LO-2. Quantitative PCR and Miseq sequencing both showed that the abundance of methanotroph increased with the increasing O2 concentrations. Type II methanotroph Methylocystis dominated in the enrichment cultures, accounting for 54.8, 48.1, and 36.9 % of the total bacterial 16S rRNA gene sequencing reads in HO, LO-1, and LO-2, respectively. Methylotrophs, mainly including Methylophilus, Methylovorus, Hyphomicrobium, and Methylobacillus, were also abundant in the cultures. Compared with the O2 sufficient condition (21 %), higher microbial biodiversity (i.e., higher Simpson and lower Shannon indexes) was detected in LO-2 enriched at the initial O2 concentration of 2.5 %. These findings indicated that compared with the O2 sufficient condition, more CH4-derived carbon was exuded into the environments and promoted the growth of non-methanotrophic microbes in O2-limiting environments.

  19. Diversity of bacteria and Archaea in sulphate-reducing enrichment cultures inoculated from serial dilution of Zostera noltii rhizosphere samples.

    PubMed

    Cifuentes, Ana; Antón, Josefa; De Wit, Rutger; Rodríguez-Valera, Francisco

    2003-09-01

    We have analysed the diversity of culturable sulphate-reducing bacteria (SRB) in Zostera noltii colonized sediments from Bassin d'Arcachon (France). Four organic substrates have been tested as well as the combination of H2 and CO2 to select for lithotrophic SRB. All energy sources were supplied in parallel cultures that were amended with yeast extract plus NH4+ and prepared without a source of combined nitrogen, the latter to select for diazotrophic SRB. The 10 different enrichment media were inoculated from serial dilution of rhizosphere samples. The highest dilution cultures yielding positive growth (i.e. 10-7) were studied by molecular techniques (16S rDNA clone libraries, RISA and ARDRA). Lactate as a single organic substrate in combination with a source of combined nitrogen resulted in selection of members of the Desulfovibrionaceae. Surprisingly, when lactate was added without a source of combined nitrogen, Desulfobacteriaceae were selected. A strong influence of the presence or absence of combined nitrogen was also observed for the substrates sucrose and fructose. Whereas the liquid culture growing on sucrose and NH4+ systematically yielded 16S rDNA clones related to an environmental unidentified green sulphur bacterium (OPS185), on plates we were able to isolate a SRB related to Desulfovibrio dechloracetivorans, which likely represents a non-described species. Under diazotrophic conditions, sucrose selected for SRB clones related to the cluster formed by Desulfovibrio zosterae, Desulfovibrio salexigens and Desulfovibrio bastinii. The corresponding isolate obtained on plates showed only low sequence similarity with this closest neighbour (93.8%), and we suggest that it also represents a non-described species. Surprisingly, a 16S rDNA sequence corresponding to an archaeon, i.e. a non-extremophile Crenoarchaeota, was retrieved from several of the SRB enrichment cultures even after subsequent transfers.

  20. Anaerobic Psychrophiles from Lake Zub and Lake Untersee, Antarctica

    NASA Technical Reports Server (NTRS)

    Townsend, Alisa; Pikuta, Elena V.; Guisler, Melissa; Stahl, Sarah; Hoover, Richard B.

    2009-01-01

    The study of samples from Antarctica 2008 and 2009 expeditions organized and successfully conducted by Richard Hoover led to the isolation of diverse anaerobic strains with psychrotolerant and psychrophilic physiology. Due to the fact that Lake Untersee has never been subject to microbiological study, this work with the samples has significant and pioneering impact to the knowledge about the biology of this unique ecosystem. Also, the astrobiological significance for the study of these ecosystems is based on new findings of ice covered water systems on other bodies of our solar system. Anaerobic psychrotolerant strain LZ-22 was isolated from a frozen sample of green moss with soils around the rhizosphere collected near Lake Zub in Antarctica. Morphology of strain LZ-22 was observed to be motile, rod shaped and spore-forming cells with sizes 1 x 5-10 micron. This new isolate is a mesophile with the maximum temperature of growth at 40C. Strain LZ-22 is able to live on media without NaCl and in media with up to 7% (w/v) NaCl. It is catalase negative and grows only on sugars with the best growth rate being on lactose. The strain is a neutrophile and grows between pH 5 and 9.0 with the optimum at 7.8. Another two strains UL7-96mG and LU-96m7P were isolated from deep water samples of Lake Untersee. Proteolytic strain LU-96m7P had a truly psychrophilic nature and refused to grow at room temperature. Sugarlytic strain UL7-96mG was found to be psychrotolerant, but its rate of growth at 3C was very high compared with other mesophiles. Two homoacetogenic psychrophilic strains A7AC-96m and AC-DS7 were isolated and purified from samples of Lake Untersee; both of them are able to grow chemolithotrophically on H2+CO2. In the presence of lactate, these strains are able to grow only at 0-18C, and growth at 22C was observed only with yeast extract stimulation. In this paper, physiological and morphological characteristics of novel psychrophilic and psychrotolerant isolates from

  1. Anaerobic psychrophiles from Lake Zub and Lake Untersee, Antarctica

    NASA Astrophysics Data System (ADS)

    Townsend, Alisa; Pikuta, Elena V.; Guisler, Melissa; Stahl, Sarah; Hoover, Richard B.

    2009-08-01

    The study of samples from Antarctica 2008 and 2009 expeditions organized and successfully conducted by Richard Hoover led to the isolation of diverse anaerobic strains with psychrotolerant and psychrophilic physiology. Due to the fact that Lake Untersee has never been subject to microbiological study, this work with the samples has significant and pioneering impact to the knowledge about the biology of this unique ecosystem. Also, the astrobiological significance for the study of these ecosystems is based on new findings of ice covered water systems on other bodies of our solar system. Anaerobic psychrotolerant strain LZ-22 was isolated from a frozen sample of green moss with soils around the rhizosphere collected near Lake Zub in Antarctica. Morphology of strain LZ-22 was observed to be motile, rod shaped and spore-forming cells with sizes 1 x 5-10 μm. This new isolate is a mesophile with the maximum temperature of growth at 40°C. Strain LZ-22 is able to live on media without NaCl and in media with up to 7 % (w/v) NaCl. It is catalase negative and grows only on sugars with the best growth rate being on lactose. The strain is a neutrophile and grows between pH 5 and 9.0 with the optimum at 7.8. Another two strains UL7-96mG and LU-96m7P were isolated from deep water samples of Lake Untersee. Proteolytic strain LU-96m7P had a truly psychrophilic nature and refused to grow at room temperature. Sugarlytic strain UL7-96mG was found to be psychrotolerant, but its rate of growth at 3°C was very high compared with other mesophiles. Two homoacetogenic psychrophilic strains A7AC-96m and AC-DS7 were isolated and purified from samples of Lake Untersee; both of them are able to grow chemolithotrophically on H2+CO2. In the presence of lactate, these strains are able to grow only at 0-18 °C, and growth at 22 °C was observed only with yeast extract stimulation. In this paper, physiological and morphological characteristics of novel psychrophilic and psychrotolerant isolates

  2. Enrichment and identification of Δ(9)-Tetrahydrocannabinolic acid synthase from Pichia pastoris culture supernatants.

    PubMed

    Lange, Kerstin; Poetsch, Ansgar; Schmid, Andreas; Julsing, Mattijs K

    2015-09-01

    This data article refers to the report Δ(9)-Tetrahydrocannabinolic acid synthase (THCAS) production in Pichia pastoris enables chemical synthesis of cannabinoids (Lange et. al. 2015) [2]. THCAS was produced on a 2 L lab scale using recombinant P. pastoris KM71 KE1. Enrichment of THCAS as a technically pure enzyme was realized using dialysis and cationic exchange chromatography. nLC-ESI-MS/MS analysis identified THCAS in different fractions obtained by cationic exchange chromatography.

  3. Investigation and characterization of the duct cell-enriching process during serum-free suspension and monolayer culture using the human exocrine pancreas fraction.

    PubMed

    Klein, Tino; Heremans, Yves; Heimberg, Harry; Pipeleers, Daniel; Madsen, Ole D; Serup, Palle; Heller, R Scott

    2009-01-01

    We aimed to characterize a serum-free culture system resulting in highly enriched duct cells from human exocrine pancreas. In addition, we tested the effect of vascular endothelial growth factor (VEGF) on endothelial cell proliferation and endocrine differentiation of the duct cells. The exocrine pellet fraction was cultivated in suspension followed by monolayer culture. Time course analysis of multiple acinar and duct cell markers was performed using reverse transcription-polymerase chain reaction and immunocytochemistry. The effects of VEGF and placental growth factor on the quantities of endothelial, duct, and endocrine cells and fibroblasts were investigated using computerized imaging analysis. Suspension culture of the exocrine material efficiently enriched the cultures for duct cells. Frequent acinar cell death as well as cell selective adherence of acinar cells to the culture dish was the underlying cause of the enrichment. Confocal microscopy demonstrated the virtual absence of cells coexpressing duct cell- and acinar cell-specific markers. The endothelial immunoreactivity of the suspension culture system could be increased 2-fold by VEGF treatment, yet no effect was observed on endocrine cell numbers. We have characterized a serum-free in vitro culture system to enrich human duct cells and further show that the contribution of acinoductal transdifferentiation to the enrichment of duct cells is negligible.

  4. Microbial succession in response to pollutants in batch-enrichment culture

    PubMed Central

    Jiao, Shuo; Chen, Weimin; Wang, Entao; Wang, Junman; Liu, Zhenshan; Li, Yining; Wei, Gehong

    2016-01-01

    As a global problem, environmental pollution is an important factor to shape the microbial communities. The elucidation of the succession of microbial communities in response to pollutants is essential for developing bioremediation procedures. In the present study, ten batches of soil-enrichment subcultures were subjected to four treatments: phenanthrene, n-octadecane, phenanthrene + n-octadecane, or phenanthrene + n-octadecane + CdCl2. Forty pollutant-degrading consortia, corresponding to each batch of the four treatments were obtained. High-throughput sequencing of the 16S rRNA gene revealed that the diversity, richness and evenness of the consortia decreased throughout the subculturing procedure. The well-known hydrocarbon degraders Acinetobacter, Gordonia, Sphingobium, Sphingopyxis, and Castellaniella and several other genera, including Niabella and Naxibacter, were detected in the enriched consortia. The predominant microbes varied and the microbial community in the consortia gradually changed during the successive subculturing depending on the treatment, indicating that the pollutants influenced the microbial successions. Comparison of the networks in the treatments indicated that organic pollutants and CdCl2 affected the co-occurrence patterns in enriched consortia. In conclusion, single environmental factors, such as the addition of nutrients or selection pressure, can shape microbial communities and partially explain the extensive differences in microbial community structures among diverse environments. PMID:26905741

  5. Microbial succession in response to pollutants in batch-enrichment culture.

    PubMed

    Jiao, Shuo; Chen, Weimin; Wang, Entao; Wang, Junman; Liu, Zhenshan; Li, Yining; Wei, Gehong

    2016-02-24

    As a global problem, environmental pollution is an important factor to shape the microbial communities. The elucidation of the succession of microbial communities in response to pollutants is essential for developing bioremediation procedures. In the present study, ten batches of soil-enrichment subcultures were subjected to four treatments: phenanthrene, n-octadecane, phenanthrene + n-octadecane, or phenanthrene + n-octadecane + CdCl2. Forty pollutant-degrading consortia, corresponding to each batch of the four treatments were obtained. High-throughput sequencing of the 16S rRNA gene revealed that the diversity, richness and evenness of the consortia decreased throughout the subculturing procedure. The well-known hydrocarbon degraders Acinetobacter, Gordonia, Sphingobium, Sphingopyxis, and Castellaniella and several other genera, including Niabella and Naxibacter, were detected in the enriched consortia. The predominant microbes varied and the microbial community in the consortia gradually changed during the successive subculturing depending on the treatment, indicating that the pollutants influenced the microbial successions. Comparison of the networks in the treatments indicated that organic pollutants and CdCl2 affected the co-occurrence patterns in enriched consortia. In conclusion, single environmental factors, such as the addition of nutrients or selection pressure, can shape microbial communities and partially explain the extensive differences in microbial community structures among diverse environments.

  6. Extracellular matrix-enriched polymeric scaffolds as a substrate for hepatocyte cultures: in vitro and in vivo studies.

    PubMed

    Zavan, B; Brun, P; Vindigni, V; Amadori, A; Habeler, W; Pontisso, P; Montemurro, D; Abatangelo, G; Cortivo, R

    2005-12-01

    Tissue engineering is a promising approach to developing hepatic tissue suitable for the functional replacement of a failing liver. The aim of the present study was to investigate whether an extracellular cell matrix obtained from fibroblasts-cultured within scaffolds of hyaluronic acid (HYAFF) could influence the proliferation rate and survival of rat hepatocytes both during long-term culture and after in vivo transplantation. Cultures were evaluated by histological and morphological analysis, a proliferation assay and metabolic activity (albumin secretion). Hepatocytes cultured in extracellular matrix-enriched scaffolds exhibited a round cellular morphology and re-established cell-cell contacts, growing into aggregates of several cells along and/or among fibers in the fabric. Hepatocytes were able to secrete albumin up to 14 days in culture. In vivo results demonstrated the biocompatibility of HYAFF-11 implanted in nude mice, in which hepatocytes maintained small well-organised aggregates until the 35th day. In conclusion, the presence of a fibroblast-secreted extracellular matrix improved the biological properties of the hyaluronan scaffold, favoring the survival and morphological integrity of hepatocytes in vitro and in vivo.

  7. Passport to Cultural Enrichment: The Peace Corps World Wise Schools Experience

    ERIC Educational Resources Information Center

    Carano, Kenneth T.

    2009-01-01

    In recent studies, youths in the United States have demonstrated a remarkable lack of cultural literacy. As the world is becoming increasingly interconnected, it is imperative that students enhance their understanding of other cultures. A classroom correspondence match with a Peace Corps volunteer through the Coverdell Peace Corps World Wise…

  8. Passport to Cultural Enrichment: The Peace Corps World Wise Schools Experience

    ERIC Educational Resources Information Center

    Carano, Kenneth T.

    2009-01-01

    In recent studies, youths in the United States have demonstrated a remarkable lack of cultural literacy. As the world is becoming increasingly interconnected, it is imperative that students enhance their understanding of other cultures. A classroom correspondence match with a Peace Corps volunteer through the Coverdell Peace Corps World Wise…

  9. Characterization of an isoproturon mineralizing bacterial culture enriched from a French agricultural soil.

    PubMed

    Hussain, Sabir; Sørensen, Sebastian R; Devers-Lamrani, Marion; El-Sebai, Talaat; Martin-Laurent, Fabrice

    2009-11-01

    The phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was found to be rapidly mineralized by a bacterial culture isolated from an agricultural soil regularly exposed to IPU. Molecular analysis of the bacterial culture by DNA fingerprinting, cloning and sequencing of the 16S rRNA genes revealed that it consisted of six different members among whom the dominant was related to Sphingomonas sp. Six bacterial strains belonging to genera Ancylobacter, Pseudomonas, Stenotrophomonas, Methylobacterium, Variovorax and Agrobacterium were isolated from the IPU-degrading culture. None of these were able to degrade IPU in pure culture and only the intact culture sustained the ability to mineralize IPU. The composition of the culture appeared stable suggesting that yet unknown interactions are involved in the IPU mineralization. IPU degradation involved the transitory accumulation of three known IPU metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, and 4-isopropylaniline and their further degradation. Thus, it indicates a metabolic pathway initiated by two successive N-demethylations, followed by cleavage of the urea side chain. This culture did not degrade other structurally related phenylurea herbicides. The degrading activity of the bacterial culture was deeply influenced by the pH, being completely inhibited at pH 5.5 and optimal at pH 7.5.

  10. The confounding effect of nitrite on N2O production by an enriched ammonia-oxidizing culture.

    PubMed

    Law, Yingyu; Lant, Paul; Yuan, Zhiguo

    2013-07-02

    The effect of nitrite (NO2(-)) on the nitrous oxide (N2O) production rate of an enriched ammonia-oxidizing bacteria (AOB) culture was characterized over a concentration range of 0-1000 mg N/L. The AOB culture was enriched in a nitritation system fed with synthetic anaerobic digester liquor. The N2O production rate was highest at NO2(-) concentrations of less than 50 mg N/L. At dissolved oxygen (DO) concentration of 0.55 mg O2/L, further increases in NO2(-) concentration from 50 to 500 mg N/L resulted in a gradual decrease in N2O production rate, which maintained at its lowest level of 0.20 mg N2O-N/h/g VSS in the NO2(-) concentration range of 500-1000 mg N/L. The observed NO2(-)-induced decrease in N2O production was even more apparent at increased DO concentration. At DO concentrations of 1.30 and 2.30 mg O2/L, the lowest N2O production rate (0.25 mg N2O-N/h/g VSS) was attained at a lower NO2(-) concentration of 200-250 mg N/L. These observations suggest that N2O production by the culture is diminished by both high NO2(-) and high DO concentrations. Collectively, the findings show that exceedingly high NO2(-) concentrations in nitritation systems could lead to decreased N2O production. Further studies are required to determine the extent to which the same response to NO2(-) is observed across different AOB cultures.

  11. mRNA differential display in a microbial enrichment culture: simultaneous identification of three cyclohexanone monooxygenases from three species.

    PubMed

    Brzostowicz, Patricia C; Walters, Dana M; Thomas, Stuart M; Nagarajan, Vasantha; Rouvière, Pierre E

    2003-01-01

    mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. Furthermore, in the case of Arthrobacter strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouvière, Gene 273:305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in Escherichia coli.

  12. mRNA Differential Display in a Microbial Enrichment Culture: Simultaneous Identification of Three Cyclohexanone Monooxygenases from Three Species

    PubMed Central

    Brzostowicz, Patricia C.; Walters, Dana M.; Thomas, Stuart M.; Nagarajan, Vasantha; Rouvière, Pierre E.

    2003-01-01

    mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. Furthermore, in the case of Arthrobacter strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouvière, Gene 273:305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in Escherichia coli. PMID:12514013

  13. N2O production rate of an enriched ammonia-oxidising bacteria culture exponentially correlates to its ammonia oxidation rate.

    PubMed

    Law, Yingyu; Ni, Bing-Jie; Lant, Paul; Yuan, Zhiguo

    2012-06-15

    The relationship between the ammonia oxidation rate (AOR) and nitrous oxide production rate (N(2)OR) of an enriched ammonia-oxidising bacteria (AOB) culture was investigated. The AOB culture was enriched in a nitritation system fed with synthetic anaerobic digester liquor. The AOR was controlled by adjusting the dissolved oxygen (DO) and pH levels and also by varying the initial ammonium (NH(4)(+)) concentration in batch experiments. Tests were also performed directly on the parent reactor where a stepwise decrease/increase in DO was implemented to alter AOR. The experimental data indicated a clear exponential relationship between the biomass specific N(2)OR and AOR. Four metabolic models were used to analyse the experimental data. The metabolic model formulated based on aerobic N(2)O production from the decomposition of nitrosyl radical (NOH) predicted the exponential correlation observed experimentally. The experimental data could not be reproduced by models developed on the basis of N(2)O production through nitrite (NO(2)(-)) and nitric oxide (NO) reduction by AOB.

  14. Psychrophilic Enzymes: From Folding to Function and Biotechnology

    PubMed Central

    Feller, Georges

    2013-01-01

    Psychrophiles thriving permanently at near-zero temperatures synthesize cold-active enzymes to sustain their cell cycle. Genome sequences, proteomic, and transcriptomic studies suggest various adaptive features to maintain adequate translation and proper protein folding under cold conditions. Most psychrophilic enzymes optimize a high activity at low temperature at the expense of substrate affinity, therefore reducing the free energy barrier of the transition state. Furthermore, a weak temperature dependence of activity ensures moderate reduction of the catalytic activity in the cold. In these naturally evolved enzymes, the optimization to low temperature activity is reached via destabilization of the structures bearing the active site or by destabilization of the whole molecule. This involves a reduction in the number and strength of all types of weak interactions or the disappearance of stability factors, resulting in improved dynamics of active site residues in the cold. These enzymes are already used in many biotechnological applications requiring high activity at mild temperatures or fast heat-inactivation rate. Several open questions in the field are also highlighted. PMID:24278781

  15. Optimization to Low Temperature Activity in Psychrophilic Enzymes

    PubMed Central

    Struvay, Caroline; Feller, Georges

    2012-01-01

    Psychrophiles, i.e., organisms thriving permanently at near-zero temperatures, synthesize cold-active enzymes to sustain their cell cycle. These enzymes are already used in many biotechnological applications requiring high activity at mild temperatures or fast heat-inactivation rate. Most psychrophilic enzymes optimize a high activity at low temperature at the expense of substrate affinity, therefore reducing the free energy barrier of the transition state. Furthermore, a weak temperature dependence of activity ensures moderate reduction of the catalytic activity in the cold. In these naturally evolved enzymes, the optimization to low temperature activity is reached via destabilization of the structures bearing the active site or by destabilization of the whole molecule. This involves a reduction in the number and strength of all types of weak interactions or the disappearance of stability factors, resulting in improved dynamics of active site residues in the cold. Considering the subtle structural adjustments required for low temperature activity, directed evolution appears to be the most suitable methodology to engineer cold activity in biological catalysts. PMID:23109875

  16. Psychrophilic enzymes: from folding to function and biotechnology.

    PubMed

    Feller, Georges

    2013-01-01

    Psychrophiles thriving permanently at near-zero temperatures synthesize cold-active enzymes to sustain their cell cycle. Genome sequences, proteomic, and transcriptomic studies suggest various adaptive features to maintain adequate translation and proper protein folding under cold conditions. Most psychrophilic enzymes optimize a high activity at low temperature at the expense of substrate affinity, therefore reducing the free energy barrier of the transition state. Furthermore, a weak temperature dependence of activity ensures moderate reduction of the catalytic activity in the cold. In these naturally evolved enzymes, the optimization to low temperature activity is reached via destabilization of the structures bearing the active site or by destabilization of the whole molecule. This involves a reduction in the number and strength of all types of weak interactions or the disappearance of stability factors, resulting in improved dynamics of active site residues in the cold. These enzymes are already used in many biotechnological applications requiring high activity at mild temperatures or fast heat-inactivation rate. Several open questions in the field are also highlighted.

  17. Evaluation of liquid and solid culture media for the recovery and enrichment of Burkholderia cenocepacia from distilled water.

    PubMed

    Ahn, Youngbeom; Kim, Jeong Myeong; Ahn, Hyeri; Lee, Yong-Jin; LiPuma, John J; Hussong, David; Cerniglia, Carl E

    2014-07-01

    Burkholderia cepacia complex (BCC) presence has been the cause of recalls of both sterile and non-sterile pharmaceutical products since these opportunistic pathogens have been implicated to cause infections to susceptible individuals. BCC are ubiquitous in nature, but in pharmaceutical settings the most common source is contaminated water systems. Some strains of BCC, previously described as Pseudomonas cepacia, were not readily detected by standard culture methods. We have explored different strategies to recover and enrich Burkholderia cenocepacia previously cultured in distilled water for 40 days. Enrichment media of varied nutrient concentrations and composition were used, including modified Tryptic Soy Agar or Broth (TSA or TSB), Reasoner's 2nd Agar or Broth (R2A or R2AB), Brain-Heart Infusion Broth (BHIB), Mueller-Hinton Broth (MHB), and Ashdown's (ASH) medium. Of the various broth media tested, cell growth was significantly greater in TSB and R2AB than in BHIB, MHB, or ASH broth. TSB and R2AB were also compared for their recovery efficiency. Generally, there was no significant difference between the numbers of B. cenocepacia grown on 15 differently modified TSA and five modified R2A solid media. Overall, however, diluted TSA and TSB media, and R2A and R2AB showed better recovery efficiency than TSA and TSB for inocula containing small numbers of cells. All strains persisted in distilled water for 40 days. Broth media were more effective than solid media for recovery of B. cenocepacia from distilled water. These results may assist in improving detection assays with recovery and enrichment strategies to maximize recovery of these fastidious organisms.

  18. Microbial Community Response of an Organohalide Respiring Enrichment Culture to Permanganate Oxidation

    PubMed Central

    Sutton, Nora B.; Atashgahi, Siavash; Saccenti, Edoardo; Grotenhuis, Tim; Smidt, Hauke; Rijnaarts, Huub H. M.

    2015-01-01

    While in situ chemical oxidation is often used to remediate tetrachloroethene (PCE) contaminated locations, very little is known about its influence on microbial composition and organohalide respiration (OHR) activity. Here, we investigate the impact of oxidation with permanganate on OHR rates, the abundance of organohalide respiring bacteria (OHRB) and reductive dehalogenase (rdh) genes using quantitative PCR, and microbial community composition through sequencing of 16S rRNA genes. A PCE degrading enrichment was repeatedly treated with low (25 μmol), medium (50 μmol), or high (100 μmol) permanganate doses, or no oxidant treatment (biotic control). Low and medium treatments led to higher OHR rates and enrichment of several OHRB and rdh genes, as compared to the biotic control. Improved degradation rates can be attributed to enrichment of (1) OHRB able to also utilize Mn oxides as a terminal electron acceptor and (2) non-dechlorinating community members of the Clostridiales and Deltaproteobacteria possibly supporting OHRB by providing essential co-factors. In contrast, high permanganate treatment disrupted dechlorination beyond cis-dichloroethene and caused at least a 2–4 orders of magnitude reduction in the abundance of all measured OHRB and rdh genes, as compared to the biotic control. High permanganate treatments resulted in a notably divergent microbial community, with increased abundances of organisms affiliated with Campylobacterales and Oceanospirillales capable of dissimilatory Mn reduction, and decreased abundance of presumed supporters of OHRB. Although OTUs classified within the OHR-supportive order Clostridiales and OHRB increased in abundance over the course of 213 days following the final 100 μmol permanganate treatment, only limited regeneration of PCE dechlorination was observed in one of three microcosms, suggesting strong chemical oxidation treatments can irreversibly disrupt OHR. Overall, this detailed investigation into dose

  19. Microbial Community Response of an Organohalide Respiring Enrichment Culture to Permanganate Oxidation.

    PubMed

    Sutton, Nora B; Atashgahi, Siavash; Saccenti, Edoardo; Grotenhuis, Tim; Smidt, Hauke; Rijnaarts, Huub H M

    2015-01-01

    While in situ chemical oxidation is often used to remediate tetrachloroethene (PCE) contaminated locations, very little is known about its influence on microbial composition and organohalide respiration (OHR) activity. Here, we investigate the impact of oxidation with permanganate on OHR rates, the abundance of organohalide respiring bacteria (OHRB) and reductive dehalogenase (rdh) genes using quantitative PCR, and microbial community composition through sequencing of 16S rRNA genes. A PCE degrading enrichment was repeatedly treated with low (25 μmol), medium (50 μmol), or high (100 μmol) permanganate doses, or no oxidant treatment (biotic control). Low and medium treatments led to higher OHR rates and enrichment of several OHRB and rdh genes, as compared to the biotic control. Improved degradation rates can be attributed to enrichment of (1) OHRB able to also utilize Mn oxides as a terminal electron acceptor and (2) non-dechlorinating community members of the Clostridiales and Deltaproteobacteria possibly supporting OHRB by providing essential co-factors. In contrast, high permanganate treatment disrupted dechlorination beyond cis-dichloroethene and caused at least a 2-4 orders of magnitude reduction in the abundance of all measured OHRB and rdh genes, as compared to the biotic control. High permanganate treatments resulted in a notably divergent microbial community, with increased abundances of organisms affiliated with Campylobacterales and Oceanospirillales capable of dissimilatory Mn reduction, and decreased abundance of presumed supporters of OHRB. Although OTUs classified within the OHR-supportive order Clostridiales and OHRB increased in abundance over the course of 213 days following the final 100 μmol permanganate treatment, only limited regeneration of PCE dechlorination was observed in one of three microcosms, suggesting strong chemical oxidation treatments can irreversibly disrupt OHR. Overall, this detailed investigation into dose

  20. A new protocol to detect multiple foodborne pathogens with PCR dipstick DNA chromatography after a six-hour enrichment culture in a broad-range food pathogen enrichment broth.

    PubMed

    Hayashi, Masahiro; Natori, Tatsuya; Kubota-Hayashi, Sayoko; Miyata, Machiko; Ohkusu, Kiyofumi; Kawamoto, Keiko; Kurazono, Hisao; Makino, Souichi; Ezaki, Takayuki

    2013-01-01

    A quick foodborne pathogen screening method after six-hour enrichment culture with a broad-range food pathogen enrichment broth is described. Pathogenic factors of Salmonella enterica, Shigella spp., enteroinvasive Escherichia coli, and enterohemorrhagic E. coli are amplified with a cocktail primer and rapid polymerase chain reaction (PCR), which finishes amplification in 30 min. The PCR amplicon was differentiated with a dipstick DNA chromatography assay in 5-10 min. Starting from a four- to six-hour enrichment culture, this assay was finished within 45 min. Detection sensitivity of this protocol was less than 2.5 CFU/25 g for S. enterica and 3.3 CFU/25 g for enterohemorrhagic E. coli in spiked ground meat experiments.

  1. Culturing bias in marine heterotrophic flagellates analyzed through seawater enrichment incubations.

    PubMed

    del Campo, Javier; Balagué, Vanessa; Forn, Irene; Lekunberri, Itziar; Massana, Ramon

    2013-10-01

    The diversity of heterotrophic flagellates is generally based on cultivated strains, on which ultrastructural, physiological, and molecular studies have been performed. However, the relevance of these cultured strains as models of the dominant heterotrophic flagellates in the marine planktonic environment is unclear. In fact, molecular surveys typically recover novel eukaryotic lineages that have refused cultivation so far. This study was designed to directly address the culturing bias in planktonic marine heterotrophic flagellates. Several microcosms were established adding increasing amounts and sources of organic matter to a confined natural microbial community pre-filtered by 3 μm. Growth dynamics were followed by epifluorescence microscopy and showed the expected higher yield of bacteria and heterotrophic flagellates at increased organic matter additions. Moreover, protist diversity analyzed by molecular tools showed a clear substitution in the community, which differed more and more from the initial sample as the organic matter increased. Within this gradient, there was also an increase of sequences related to cultured organisms as well as a decrease in diversity. Culturing bias is partly explained by the use of organic matter in the isolation process, which drives a shift in the community to conditions closer to laboratory cultures. An intensive culturing effort using alternative isolation methods is necessary to allow the access to the missing heterotrophic flagellates that constitute the abundant and active taxa in marine systems.

  2. Applicability of a Lactobacillus amylovorus strain as co-culture for natural folate bio-enrichment of fermented milk.

    PubMed

    Laiño, Jonathan Emiliano; Juarez del Valle, Marianela; Savoy de Giori, Graciela; LeBlanc, Jean Guy Joseph

    2014-11-17

    The ability of 55 strains from different Lactobacillus species to produce folate was investigated. In order to evaluate folic acid productivity, lactobacilli were cultivated in the folate-free culture medium (FACM). Most of the tested strains needed folate for growth. The production and the extent of vitamin accumulation were distinctive features of individual strains. Lactobacillus amylovorus CRL887 was selected for further studies because of its ability to produce significantly higher concentrations of vitamin (81.2 ± 5.4 μg/L). The safety of this newly identified folate producing strain was evaluated through healthy experimental mice. No bacterial translocation was detected in liver and spleen after consumption of CRL887 during 7 days and no undesirable side effects were observed in the animals that received this strain. This strain in co-culture with previously selected folate producing starter cultures (Lactobacillus bulgaricus CRL871, and Streptococcus thermophilus CRL803 and CRL415) yielded a yogurt containing high folate concentrations (263.1 ± 2.4 μg/L); a single portion of which would provide 15% of the recommended dietary allowance. This is the first report where a Lactobacillus amylovorus strain was successfully used as co-culture for natural folate bio-enrichment of fermented milk.

  3. Preparation of Membrane Vesicles Enriched in ATP-Dependent Proton Transport from Suspension Cultures of Tomato Cells

    PubMed Central

    Dupont, Frances M.; Zabala, Maria De Gracia

    1985-01-01

    Membranes enriched in ATP-dependent proton transport were prepared from suspension cultures of tomato cells (Lycopersicon esculentum Mill cv VF36). Suspension cultures were a source of large quantities of membranes from rapidly growing, undifferentiated cells. Proton transport activity was assayed as quench of acridine orange fluorescence. The activity of the proton translocating ATPase and of several other membrane enzymes was measured as a function of the cell culture cycle. The relative distribution of the enzymes between the 3,000, 10,000, and 100,000g pellets remained the same throughout the cell culture cycle, but yield of total activity and activity per gram fresh weight with time had a unique profile for each enzyme tested. Maximal yield of the proton translocating ATPase activity was obtained from cells in the middle logarithmic phase of growth, and from 50 to 90% of the activity was found in the 10,000g pellet. The proton translocating ATPase activity was separable from NADPH cytochrome c reductase and cytochrome c oxidase on a sucrose gradient. Proton transport activity had a broad pH optimum (7.0-8.0), was stimulated by KCl with a Km of 5 to 10 millimolar, stimulation being due to the anion, Cl−, and not the cation, K+, and was not inhibited by vanadate, but was inhibited by NO3−. The activity is tentatively identified as the tonoplast ATPase. PMID:16664030

  4. Culture enrichment assists the diagnosis of cattle botulism by a monoclonal antibody based sandwich ELISA.

    PubMed

    Brooks, C E; Clarke, H J; Finlay, D A; McConnell, W; Graham, D A; Ball, H J

    2010-07-29

    Monoclonal antibodies (MAbs) obtained from a mouse immunised with Clostridium botulinum type D toxoid were developed into a sandwich ELISA (sELISA) format that was able to detect type D toxin and types C and D toxin complexes. The sELISA was examined for its potential to replace the mouse bioassay as an alternative in vitro assay for the diagnosis of cattle botulism. Its application directly to intestinal samples collected from suspect cattle botulism cases and prepared for testing for the standard mouse bioassay showed poor correlation and sensitivity with the mouse bioassay results. However, anaerobic pre-enrichment of the samples after heat treatment at 80 degrees C for 10 min to activate any residual C. botulinum spores greatly improved the sELISA detection rate of the toxin by increasing the sample toxin levels. All of the mouse bioassay positive cattle cases tested were detected by the sELISA from the heated and pre-enriched samples tested after 24h incubation. Toxin was detected by sELISA and subsequently confirmed by mouse bioassay in samples from an additional 3 cases that had been originally mouse bioassay negative. The results indicate that the application of this procedure for screening intestinal samples for C. botulinum strains that produce types C and D toxins from suspect cattle botulism cases would improve the diagnostic rate as well as significantly reduce the number of mice involved in diagnosis.

  5. Autologous fibrin scaffolds cultured dermal fibroblasts and enriched with encapsulated bFGF for tissue engineering.

    PubMed

    de la Puente, Pilar; Ludeña, Dolores; Fernández, Ana; Aranda, Jose L; Varela, Gonzalo; Iglesias, Javier

    2011-12-15

    Autologous fibrin scaffolds (AFSs) enriched with cells and specific growth factors represent a promising biocompatible scaffold for tissue engineering. Here, we analyzed the in vitro behavior of dermal fibroblasts (DFs) (cellular attachment, distribution, viability and proliferation, histological and immunohistochemical changes), comparing AFS with and without alginate microcapsules loaded with basic fibroblast growth factor (bFGF), to validate our scaffold in a future animal model in vivo. In all cases, DFs showed good adhesion and normal distribution, while in scaffolds with bFGF at 14 days, the cell counts detected in proliferation and viability assays were greatly improved, as was the proliferative state, and there was a decrease in muscle specific actin expression and collagen synthesis in comparison with the scaffolds without bFGF. In addition, the use of plasma without fibrinogen concentration methods, together with the maximum controlled release of bFGF at 14 days, favored cell proliferation. To conclude, we have been able to create an AFS enriched with fully functional DFs and release-controlled bFGF that could be used in multiple applications for tissue engineering.

  6. Atrazine biodegradation efficiency, metabolite detection, and trzD gene expression by enrichment bacterial cultures from agricultural soil

    PubMed Central

    Solomon, Robinson David Jebakumar; Kumar, Amit; Satheeja Santhi, Velayudhan

    2013-01-01

    Atrazine is a selective herbicide used in agricultural fields to control the emergence of broadleaf and grassy weeds. The persistence of this herbicide is influenced by the metabolic action of habituated native microorganisms. This study provides information on the occurrence of atrazine mineralizing bacterial strains with faster metabolizing ability. The enrichment cultures were tested for the biodegradation of atrazine by high-performance liquid chromatography (HPLC) and mass spectrometry. Nine cultures JS01.Deg01 to JS09.Deg01 were identified as the degrader of atrazine in the enrichment culture. The three isolates JS04.Deg01, JS07.Deg01, and JS08.Deg01 were identified as efficient atrazine metabolizers. Isolates JS04.Deg01 and JS07.Deg01 produced hydroxyatrazine (HA) N-isopropylammelide and cyanuric acid by dealkylation reaction. The isolate JS08.Deg01 generated deethylatrazine (DEA), deisopropylatrazine (DIA), and cyanuric acid by N-dealkylation in the upper degradation pathway and later it incorporated cyanuric acid in their biomass by the lower degradation pathway. The optimum pH for degrading atrazine by JS08.Deg01 was 7.0 and 16S rDNA phylogenetic typing identified it as Enterobacter cloacae strain JS08.Deg01. The highest atrazine mineralization was observed in case of isolate JS08.Deg01, where an ample amount of trzD mRNA was quantified at 72 h of incubation with atrazine. Atrazine bioremediating isolate E. cloacae strain JS08.Deg01 could be the better environmental remediator of agricultural soils and the crop fields contaminated with atrazine could be the source of the efficient biodegrading microbial strains for the environmental cleanup process. PMID:24302716

  7. Atrazine biodegradation efficiency, metabolite detection, and trzD gene expression by enrichment bacterial cultures from agricultural soil.

    PubMed

    Solomon, Robinson David Jebakumar; Kumar, Amit; Satheeja Santhi, Velayudhan

    2013-12-01

    Atrazine is a selective herbicide used in agricultural fields to control the emergence of broadleaf and grassy weeds. The persistence of this herbicide is influenced by the metabolic action of habituated native microorganisms. This study provides information on the occurrence of atrazine mineralizing bacterial strains with faster metabolizing ability. The enrichment cultures were tested for the biodegradation of atrazine by high-performance liquid chromatography (HPLC) and mass spectrometry. Nine cultures JS01.Deg01 to JS09.Deg01 were identified as the degrader of atrazine in the enrichment culture. The three isolates JS04.Deg01, JS07.Deg01, and JS08.Deg01 were identified as efficient atrazine metabolizers. Isolates JS04.Deg01 and JS07.Deg01 produced hydroxyatrazine (HA) N-isopropylammelide and cyanuric acid by dealkylation reaction. The isolate JS08.Deg01 generated deethylatrazine (DEA), deisopropylatrazine (DIA), and cyanuric acid by N-dealkylation in the upper degradation pathway and later it incorporated cyanuric acid in their biomass by the lower degradation pathway. The optimum pH for degrading atrazine by JS08.Deg01 was 7.0 and 16S rDNA phylogenetic typing identified it as Enterobacter cloacae strain JS08.Deg01. The highest atrazine mineralization was observed in case of isolate JS08.Deg01, where an ample amount of trzD mRNA was quantified at 72 h of incubation with atrazine. Atrazine bioremediating isolate E. cloacae strain JS08.Deg01 could be the better environmental remediator of agricultural soils and the crop fields contaminated with atrazine could be the source of the efficient biodegrading microbial strains for the environmental cleanup process.

  8. Lactate induces tumour necrosis factor-alpha, interleukin-6 and interleukin-1beta release in microglial- and astroglial-enriched primary cultures.

    PubMed

    Andersson, Anna K; Rönnbäck, Lars; Hansson, Elisabeth

    2005-06-01

    Hyperammonaemia has deleterious effects on the CNS in patients with liver dysfunction. Cellular mechanisms underlying the effects of hyperammonaemia are largely unknown, although astrocytes have been the main target of interest. This study investigated how treatment with NH4Cl and lactate, which increase in the brain as a consequence of hyperammonaemia, affects cells in primary rat cultures enriched in either astrocytes or microglia. Morphological changes were studied over time using light microscopy. Release of the proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and IL-1beta was measured using ELISA. NH4Cl was found to induce vacuole formation in both culture systems. Lactate treatment altered astrocytic appearance, resulting in increased space between individual cells. Microglia adopted a round morphology with either NH4Cl or lactate treatment. Lactate, but not NH4Cl, induced release of TNF-alpha and IL-6 in both astroglial- and microglial-enriched cultures, while IL-1beta was released only in microglial cultures. Cytokine release was higher in the microglial- than in the astroglial-enriched cultures. Additionally, the astroglial-enriched cultures containing approximately 10% microglial cells released more cytokines than cultures containing about 5% microglial cells. Taken together, our data suggest that most TNF-alpha, IL-6 and IL-1beta release comes from microglia. Thus, microglia could play an important role in the pathological process of hyperammonaemia.

  9. One health from a social-ecological systems perspective: enriching social and cultural dimensions.

    PubMed

    Ross, Helen

    2013-01-01

    This chapter offers insights from the environmental management paradigm of 'social-ecological systems' and related bodies of theory on people-environment relationships to assist the evolution of the One Health interdisciplinary endeavour of health promotion across human-animal ecosystem relationships. It also seeks to expand thinking about the social and cultural dimensions that are likely to prove important in the development of thinking and practice in the One Health field. It advocates consideration of cultural and economic relationships affecting people's interactions with domesticated and wild animal species and ecosystems, and exploration of the cognitive and behavioural aspects of these interactions.

  10. Culturally Diverse Literature: Enriching Variety in an Era of Common Core State Standards

    ERIC Educational Resources Information Center

    Boyd, Fenice B.; Causey, Lauren L.; Galda, Lee

    2015-01-01

    The authors argue for the overwhelming importance of finding and including culturally diverse literature into the curricula teachers are authorized to teach. They discuss the implications of use and offer ideas on how to identify quality literature to include in classroom and school libraries.

  11. Culturally Diverse Literature: Enriching Variety in an Era of Common Core State Standards

    ERIC Educational Resources Information Center

    Boyd, Fenice B.; Causey, Lauren L.; Galda, Lee

    2015-01-01

    The authors argue for the overwhelming importance of finding and including culturally diverse literature into the curricula teachers are authorized to teach. They discuss the implications of use and offer ideas on how to identify quality literature to include in classroom and school libraries.

  12. Afro-Hispanic Literature: Cultural and Literary Enrichment for the Foreign Language Classroom.

    ERIC Educational Resources Information Center

    Cyrus, Stanley A.; Legge, June M.

    Millions of people of African descent in Spanish-speaking countries are commonly omitted from the cultural, literary, and linguistic content of Spanish classes. Afro-Spanish literature can be integrated into the Spanish curriculum from the first year. This literature is not easily defined, but does reflect and aid in understanding the black…

  13. Halophilic-Psychrophilic Bacteria from Tirich Mir Glacier, Pakistan, as Potential Candidate for Astrobiological Studies

    NASA Astrophysics Data System (ADS)

    Rafiq, M. R.; Anesio, A. M. A.; Hayat, M. H.; Zada, S. Z.; Sajjad, W. S.; Shah, A. A. S.; Hasan, F. H.

    2016-09-01

    Hindu Kush, Karakoram, and Himalaya region is referred to as 'third pole' and could be suitable as a terrestrial analog of Mars and increased possibility of finding polyextremophiles. Study is focused on halophilic psychrophiles.

  14. Psychrophilic yeasts from worldwide glacial habitats: diversity, adaptation strategies and biotechnological potential.

    PubMed

    Buzzini, Pietro; Branda, Eva; Goretti, Marta; Turchetti, Benedetta

    2012-11-01

    Glacial habitats (cryosphere) include some of the largest unexplored and extreme biospheres on Earth. These habitats harbor a wide diversity of psychrophilic prokaryotic and eukaryotic microorganisms. These highly specialized microorganisms have developed adaptation strategies to overcome the direct and indirect life-endangering influence of low temperatures. For many years Antarctica has been the geographic area preferred by microbiologists for studying the diversity of psychrophilic microorganisms (including yeasts). However, there have been an increasing number of studies on psychrophilic yeasts sharing the non-Antarctic cryosphere. The present paper provides an overview of the distribution and adaptation strategies of psychrophilic yeasts worldwide. Attention is also focused on their biotechnological potential, especially on their exploitation as a source of cold-active enzymes and for bioremediation purposes.

  15. An extracellular ice-binding glycoprotein from an Arctic psychrophilic yeast.

    PubMed

    Lee, Jong Kyu; Park, Kyoung Sun; Park, Seungil; Park, Hyun; Song, Young Hwan; Kang, Sung-Ho; Kim, Hak Jun

    2010-04-01

    A psychrophilic yeast was isolated from an Arctic pond and its culture supernatant showed ice-binding activity. This isolate, identified as Leucosporidium sp. based on an analysis of the D1/D2 and ITS regions of its ribosomal DNA, produced a secretory ice-binding protein (IBP). Yeast IBP was purified from the culture medium to near homogeneity by the ice affinity method and appeared to be glycosylated with a molecular mass of approximately 26 kDa. In addition, the yeast IBP was shown to have thermal hysteresis (TH) and recrystallization inhibition (RI) activities. The full-length cDNA for yeast IBP was determined and was found to encode a 261 amino acid protein with molecular weight of 26.8 kDa that includes an N-terminal signal peptide and one potential N-glycosylation site. The deduced protein showed high sequence identity with other IBPs and hypothetical IBPs from fungi, diatoms, and bacteria, clustering with a class of ice-active proteins.

  16. Growth enhancement of fowls by dietary administration of recombinant yeast cultures containing enriched growth hormone.

    PubMed

    Chen, C M; Cheng, W T; Chang, Y C; Chang, T J; Chen, H L

    2000-09-15

    In present study the methylotrophic yeast, Pichia pastoris, was used to express a recombinant growth hormone (rGH) gene of swine. A synthetic secretion cassette was constructed using the promoter of the alcohol oxidase1 gene (AOX1), and a alpha-factor signal peptide. After electroporatic transformation and zeocin selection, several clones exhibited high levels of rGH protein expression constituting more than 20% of total yeast protein. Over 95% of rGH was shown to be export into the culture supernatant. Yeast transformant containing the highest recombinant growth hormone level (rGH yeast) and native GS115 Pichia pastoris (non-rGH yeast, as a control) were separately cultured, harvested and adsorbed by wheat bran. Yeast cultures of four dosages (0.05, 0.1, 0.2, and 0.4%) were mixed respectively with chick basal diet and fed to simulated country chickens for 9 weeks. The results showed that, when compared to control chicks, the percentage of body weight gain was improved significantly (P<0.05) in chicks fed with diets containing 0.1 or 0.2% rGH-rich yeast culture at brooding stage, and in chicks fed with 0.4% rGH-rich yeast culture at growing stage. The average weight gain in rGH yeast treated groups for the full-term (0 to 63d) and short term (43 to 63d) of growth were 10.6 and 9.4%, respectively, better than the non-rGH yeast control group. These experimental data suggest that the use of rGH-containing yeast as a supplement in fed provided an alternative approach for growth improvement in simulated country chickens.

  17. Dechlorination of three tetrachlorobenzene isomers by contaminated harbor sludge-derived enrichment cultures follows thermodynamically favorable reactions.

    PubMed

    Lu, Yue; Ramiro-Garcia, Javier; Vandermeeren, Pieter; Herrmann, Steffi; Cichocka, Danuta; Springael, Dirk; Atashgahi, Siavash; Smidt, Hauke

    2017-03-01

    Dechlorination patterns of three tetrachlorobenzene isomers, 1,2,3,4-, 1,2,3,5-, and 1,2,4,5-TeCB, were studied in anoxic microcosms derived from contaminated harbor sludge. The removal of doubly, singly, and un-flanked chlorine atoms was noted in 1,2,3,4- and 1,2,3,5-TeCB fed microcosms, whereas only singly flanked chlorine was removed in 1,2,4,5-TeCB microcosms. The thermodynamically more favorable reactions were selectively followed by the enriched cultures with di- and/or mono-chlorobenzene as the main end products of the reductive dechlorination of all three isomers. Based on quantitative PCR analysis targeting 16S rRNA genes of known organohalide-respiring bacteria, the growth of Dehalococcoides was found to be associated with the reductive dechlorination of all three isomers, while growth of Dehalobacter, another known TeCB dechlorinator, was only observed in one 1,2,3,5-TeCB enriched microcosm among biological triplicates. Numbers of Desulfitobacterium and Geobacter as facultative dechlorinators were rather stable suggesting that they were not (directly) involved in the observed TeCB dechlorination. Bacterial community profiling suggested bacteria belonging to the phylum Bacteroidetes and the order Clostridiales as well as sulfate-reducing members of the class Deltaproteobacteria as putative stimulating guilds that provide electron donor and/or organic cofactors to fastidious dechlorinators. Our results provide a better understanding of thermodynamically preferred TeCB dechlorinating pathways in harbor environments and microbial guilds enriched and active in anoxic TeCB dechlorinating microcosms.

  18. Stable acetate production in extreme-thermophilic (70°C) mixed culture fermentation by selective enrichment of hydrogenotrophic methanogens.

    PubMed

    Zhang, Fang; Zhang, Yan; Ding, Jing; Dai, Kun; van Loosdrecht, Mark C M; Zeng, Raymond J

    2014-06-12

    The control of metabolite production is difficult in mixed culture fermentation. This is particularly related to hydrogen inhibition. In this work, hydrogenotrophic methanogens were selectively enriched to reduce the hydrogen partial pressure and to realize efficient acetate production in extreme-thermophilic (70°C) mixed culture fermentation. The continuous stirred tank reactor (CSTR) was stable operated during 100 days, in which acetate accounted for more than 90% of metabolites in liquid solutions. The yields of acetate, methane and biomass in CSTR were 1.5 ± 0.06, 1.0 ± 0.13 and 0.4 ± 0.05 mol/mol glucose, respectively, close to the theoretical expected values. The CSTR effluent was stable and no further conversion occurred when incubated for 14 days in a batch reactor. In fed-batch experiments, acetate could be produced up to 34.4 g/L, significantly higher than observed in common hydrogen producing fermentations. Acetate also accounted for more than 90% of soluble products formed in these fed-batch fermentations. The microbial community analysis revealed hydrogenotrophic methanogens (mainly Methanothermobacter thermautotrophicus and Methanobacterium thermoaggregans) as 98% of Archaea, confirming that high temperature will select hydrogenotrophic methanogens over aceticlastic methanogens effectively. This work demonstrated a potential application to effectively produce acetate as a value chemical and methane as an energy gas together via mixed culture fermentation.

  19. Stable acetate production in extreme-thermophilic (70°C) mixed culture fermentation by selective enrichment of hydrogenotrophic methanogens

    NASA Astrophysics Data System (ADS)

    Zhang, Fang; Zhang, Yan; Ding, Jing; Dai, Kun; van Loosdrecht, Mark C. M.; Zeng, Raymond J.

    2014-06-01

    The control of metabolite production is difficult in mixed culture fermentation. This is particularly related to hydrogen inhibition. In this work, hydrogenotrophic methanogens were selectively enriched to reduce the hydrogen partial pressure and to realize efficient acetate production in extreme-thermophilic (70°C) mixed culture fermentation. The continuous stirred tank reactor (CSTR) was stable operated during 100 days, in which acetate accounted for more than 90% of metabolites in liquid solutions. The yields of acetate, methane and biomass in CSTR were 1.5 +/- 0.06, 1.0 +/- 0.13 and 0.4 +/- 0.05 mol/mol glucose, respectively, close to the theoretical expected values. The CSTR effluent was stable and no further conversion occurred when incubated for 14 days in a batch reactor. In fed-batch experiments, acetate could be produced up to 34.4 g/L, significantly higher than observed in common hydrogen producing fermentations. Acetate also accounted for more than 90% of soluble products formed in these fed-batch fermentations. The microbial community analysis revealed hydrogenotrophic methanogens (mainly Methanothermobacter thermautotrophicus and Methanobacterium thermoaggregans) as 98% of Archaea, confirming that high temperature will select hydrogenotrophic methanogens over aceticlastic methanogens effectively. This work demonstrated a potential application to effectively produce acetate as a value chemical and methane as an energy gas together via mixed culture fermentation.

  20. pH-Mediated Microbial and Metabolic Interactions in Fecal Enrichment Cultures

    PubMed Central

    Ilhan, Zehra Esra; Marcus, Andrew K.; Kang, Dae-Wook; Rittmann, Bruce E.

    2017-01-01

    ABSTRACT pH and fermentable substrates impose selective pressures on gut microbial communities and their metabolisms. We evaluated the relative contributions of pH, alkalinity, and substrate on microbial community structure, metabolism, and functional interactions using triplicate batch cultures started from fecal slurry and incubated with an initial pH of 6.0, 6.5, or 6.9 and 10 mM glucose, fructose, or cellobiose as the carbon substrate. We analyzed 16S rRNA gene sequences and fermentation products. Microbial diversity was driven by both pH and substrate type. Due to insufficient alkalinity, a drop in pH from 6.0 to ~4.5 clustered pH 6.0 cultures together and distant from pH 6.5 and 6.9 cultures, which experienced only small pH drops. Cellobiose yielded more acidity than alkalinity due to the amount of fermentable carbon, which moved cellobiose pH 6.5 cultures away from other pH 6.5 cultures. The impact of pH on microbial community structure was reflected by fermentative metabolism. Lactate accumulation occurred in pH 6.0 cultures, whereas propionate and acetate accumulations were observed in pH 6.5 and 6.9 cultures and independently from the type of substrate provided. Finally, pH had an impact on the interactions between lactate-producing and -consuming communities. Lactate-producing Streptococcus dominated pH 6.0 cultures, and acetate- and propionate-producing Veillonella, Bacteroides, and Escherichia dominated the cultures started at pH 6.5 and 6.9. Acid inhibition on lactate-consuming species led to lactate accumulation. Our results provide insights into pH-derived changes in fermenting microbiota and metabolisms in the human gut. IMPORTANCE The human gut is a dynamic environment in which microorganisms consistently interact with the host via their metabolic products. Some of the most important microbial metabolic products are fermentation products such as short-chain fatty acids. Production of these fermentation products and the prevalence of fermenting

  1. Isolation and Identification of Psychrophilic Species of Bacillus from Milk

    PubMed Central

    Shehata, T. E.; Collins, E. B.

    1971-01-01

    Forty isolates from 97 raw milk samples (heated to 80 C for 10 min and stored at 4 to 7 C for 3 to 4 weeks) were sporeforming, aerobic, gram-positive or gram-variable, rod-shaped bacteria. Fifteen isolates that were identified had characteristics similar to species of Bacillus, except that they had lower growth temperature ranges, were gram-variable, and were somewhat different in sugar fermentations. Four isolates grew well within 2 weeks at 0 C, but they grew faster at 20 to 25 C. These psychrophilic sporeforming bacteria, the importance of which is discussed, are considered to be variant strains of mesophilic bacilli adapted to low temperatures. PMID:5108104

  2. Enrichment of highly settleable microalgal consortia in mixed cultures for effluent polishing and low-cost biomass production.

    PubMed

    Hu, Yuansheng; Hao, Xiaodi; van Loosdrecht, Mark; Chen, Huiqin

    2017-08-15

    Microalgae cultivation is a promising technology for integrated effluent polishing and biofuel production, but poor separability of microalgal cells hinders its industrial application. This study intended to selectively enrich settleable microalgal consortia in mixed culture by applying "wash-out" pressure, which was realized by controlling settling time (ST) and volume exchange ratio (VER) in photo-SBRs. The results demonstrated that highly settleable microalgal consortia (settling efficiency>97%; SVI = 17-50 mL/g) could be enriched from indigenous algal cultures developed in WWTP's effluent. High VER was the key factor for the fast development of settleable microalgae. VER was also a controlling factor of the algal community structure. High VERs (0.5 and 0.7) resulted in the dominance of diatom, while low VER (0.2) facilitated the dominance of cyanobacteria. The settleable microalgal consortia were very efficient in phosphorus removal (effluent PO4(3-)-P<0.1 mg/L; removal efficiency>99%), which was largely attributed to intensive chemical precipitation of phosphate induced by high pH (8.5-10). However, the high pH decreased the bioavailable inorganic carbon, resulting in incomplete nitrate removal (effluent NO3(-)-N = 2.2-4 mg/L; removal efficiency = 61-79%) under high VERs and low lipid content (up to 10%) in the settleable microalgae. This problem could be resolved by sparging CO2 or controlling pH. Overall, this study demonstrated a simple and effective method to overcome the separation challenge in scale-up of microalgae biotechnology for advanced wastewater purification and biofuel production. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Real-time PCR detection of Campylobacter spp.: A comparison to classic culturing and enrichment.

    PubMed

    de Boer, P; Rahaoui, H; Leer, R J; Montijn, R C; van der Vossen, J M B M

    2015-10-01

    The major disadvantage of the current gold standard for detection of the food pathogen Campylobacter, i.e. culturing, is the lengthy procedure. In this study we assessed the use of real-time PCR for detection of Campylobacter. To this end, 926 poultry samples, taken from transport containers and broiler caeca in The Netherlands in 2007, were subjected to three different real-time PCR detection methods: one targeting the Campylobacter jejuni hipO gene, one targeting the Campylobacter coli glyA gene, and one generically targeting Campylobacter spp. 16S rDNA sequence. The PCR results from the three different PCR protocols were compared to the work of Nauta et al. (2009) who analyzed the same set of samples collected from 62 broiler flocks by means of enrichment culturing. The results indicate that the generic 16S campylobacter PCR detection is equally reliable but much faster (4 h instead of ≥2 days) than detection by means of culturing. Moreover, PCR detection targeting the hipO and the glyA gene provide the possibility of C. jejuni and C. coli species discrimination. The generic Campylobacter spp. PCR analysis also confirmed the high incidence of Campylobacter spp. in poultry samples (∼90%) and the species specific PCR showed the simultaneous presence of C. jejuni and C. coli in ∼24% of the samples. Furthermore, the results from the three PCR analyses suggested the occurrence of alternative Campylobacter species in almost 10% of the samples. The campylobacter PCR detection methods reported here can replace traditional culturing because of being quicker and more reliable. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. High Yield Derivation of Enriched Glutamatergic Neurons from Suspension-Cultured Mouse ESCs for Neurotoxicology Research

    DTIC Science & Technology

    2012-10-24

    and without LIF) in a 10-cm ultra-low attachment culture dish ( Corning , Lowell, MA). Differentiating aggregates were maintained on a rotary shaker at 45...Wold B: Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 2008, 5(7):621–628. 16. da Huang W, Sherman BT , Tan Q, Collins JR...algorithm to functionally analyze large gene lists. Genome Biol 2007, 8(9):R183. 17. Huang da W, Sherman BT , Zheng X, Yang J, Imamichi T, Stephens R

  5. Enrichment, Isolation, and Cultural Characteristics of Marine Strains of Clostridium botulinum Type C

    PubMed Central

    Segner, W. P.; Schmidt, C. F.; Boltz, J. K.

    1971-01-01

    Terrestrial strains of Clostridium botulinum type C, designated 468 and 571, were used to screen various media for growth and sporulation at 30 C. Of the various formulations tested, only egg meat medium fortified with 1% additions of yeast extract, ammonium sulfate, and glucose (FEM medium) gave good growth and satisfactory sporulation. FEM medium was used to recover four marine type C isolates from inshore sediments collected along the Atlantic, the Gulf of Mexico, and the Pacific coasts of the United States. The isolation techniques involved repeated transfer of cultures showing type C toxin in FEM medium and purification by a deep tube method. The medium used for purification was beef infusion-agar supplemented with 0.14% sodium bicarbonate and 0.1% l-cysteine hydrochloride. l-Cysteine was adopted in preference to sodium thioglycolate, because some lots of the latter were definitely inhibitory for growth. The addition of bicarbonate markedly increased viable spore counts of both the marine and terrestrial strains. Various cultural and biochemical characteristics of the marine and the terrestrial strains were compared. With the exception of some variations in their fermentation patterns, both groups showed similar characteristics. Of 23 fermentable compounds tested, the terrestrial strains attacked only glucose and mannose. The marine strains fermented glucose, mannose, galactose, and ribose actively; dextrin, inositol, maltose, and melibiose were weakly fermented. PMID:4944800

  6. Antitumoural activity of viniferin-enriched extracts from Vitis vinifera L. cell cultures.

    PubMed

    Giovannelli, L; Innocenti, M; Santamaria, A R; Bigagli, E; Pasqua, G; Mulinacci, N

    2014-01-01

    The aim of this work was to evaluate the effect of stilbenes from different cultivars of Vitis vinifera on tumour proliferation. Extracts were obtained from elicited V. vinifera cell cultures and characterised by HPLC/DAD/MS. Cell growth was evaluated in four human cancer cell lines and in normal human fibroblasts. The cells were exposed to the extracts or to trans-resveratrol, used as reference molecule, for 48 h, at 1-10 μM concentrations of total stilbenoids. All the extracts exhibited antiproliferative activity, mediated by modulation of the cell cycle and induction of cytotoxicity in cancer but not in normal cell lines, and positively correlated with the content in dimeric stilbenoids. The Alphonse Lavallée extract was the most active, and the obtained stilbenoid fraction resulted 8-10 times more active than trans-resveratrol. Extracts from V. vinifera cell cultures could represent new sources of active stilbenoid compounds to be further assayed in in vivo studies for their antitumoural properties.

  7. Pilot-scale chitin extraction from shrimp shell waste by deproteination and decalcification with bacterial enrichment cultures.

    PubMed

    Bajaj, Mini; Freiberg, Andrea; Winter, Josef; Xu, Youmei; Gallert, Claudia

    2015-11-01

    Extraction of chitin from mechanically pre-purified shrimp shells can be achieved by successive NaOH/HCl treatment, protease/HCl treatment or by environmentally friendly fermentation with proteolytic/lactic acid bacteria (LAB). For the last mentioned alternative, scale-up of shrimp shell chitin purification was investigated in 0.25 L (F1), 10 L (F2), and 300 L (F3) fermenters using an anaerobic, chitinase-deficient, proteolytic enrichment culture from ground meat for deproteination and a mixed culture of LAB from bio-yoghurt for decalcification. Protein removal in F1, F2, and F3 proceeded in parallel within 40 h at an efficiency of 89-91 %. Between 85 and 90 % of the calcit was removed from the shells by LAB in another 40 h in F1, F2, and F3. After deproteination of shrimp shells in F3, spent fermentation liquor was re-used for a next batch of 30-kg shrimp shells in F4 (300 L) which eliminated 85.5 % protein. The purity of the resulting chitin was comparable in F1, F2, F3, and F4. Viscosities of chitosan, obtained after chitin deacetylation and of chitin, prepared biologically or chemically in the laboratory, were much higher than those of commercially available chitin and chitosan.

  8. Enriching the Content Provided by Cultural Catalogues with Data from Institutional Repositories

    NASA Astrophysics Data System (ADS)

    Rodríguez Miranda, Á.; Valle Melón, J. M.; Porcal-Gonzalo, M. C.

    2013-07-01

    Institutional repositories play a key role in universities and research centers for the preservation and dissemination of the knowledge generated or collected by these agents. Part of the information contained is related with Cultural Heritage and, therefore, it could be taken into account by thematic catalogues such as Europeana. In this paper, the opportunities and limitations of this connection are considered and presented by means of an ongoing case at the University of the Basque Country's institutional repository. In particular, we will deal with the information gathered about San Prudencio's Monastery (Clavijo, Spain), which includes a wide range of data from original datasets (photographs, pointclouds, coordinates, sketches and so on) up to finals results (reports, plans, 3D models, papers and so on).

  9. Impact of estuarine gradients on reductive dechlorination of 1,2,3,4-tetrachlorodibenzo-p-dioxin in river sediment enrichment cultures.

    PubMed

    Dam, Hang T; Häggblom, Max M

    2017-02-01

    Polychlorinated dibenzo-p-dioxins (PCDDs) are among the most persistent organic pollutants. Although the total input of PCDDs into the environment has decreased substantially over the past four decades, their input via non-point sources is still increasing, especially in estuarine metropolitan areas. Here we report on the microbially mediated reductive dechlorination of PCDDs in anaerobic enrichment cultures established from sediments collected from five locations along the Hackensack River, NJ and investigate the impacts of sediment physicochemical characteristics on dechlorination activity. Dechlorination of 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TeCDD) and abundance of Dehalococcoides spp. negatively correlated with salinity and sulfate concentration in sediments used to establish the cultures. 1,2,3,4-TeCDD was dechlorinated to a lesser extent in cultures established from sediments from the tidally influenced estuarine mouth of the river. In cultures established from low salinity sediments, 1,2,3,4-TeCDD was reductively dechlorinated with the accumulation of 2-monochlorodibenzo-p-dioxin as the major product. Sulfate concentrations above 2 mM inhibited 1,2,3,4-TecDD dechlorination activity. Consecutive lateral- and peri- dechlorination took place in enrichment cultures with a minimal accumulation of 2,3-dichlorodibenzo-p-dioxin in active cultures. A Dehalococcoides spp. community was enriched and accounted for up to 64% of Chloroflexi detected in these sediment cultures.

  10. Survival of a Psychrophilic Marine Vibrio Under Long-Term Nutrient Starvation 1

    PubMed Central

    Novitsky, James A.; Morita, Richard Y.

    1977-01-01

    Ant-300, a psychrophilic marine vibrio isolated from the surface water of the Antarctic convergence, was starved for periods of more than 1 year. During the first week of starvation, cell numbers increased from 100 to 800% of the initial number of cells. Fifty percent of the starved cells remained viable for 6 to 7 weeks while a portion of the population remained viable for more than 1 year. During the first 2 days of starvation, the endogenous respiration of the cells decreased over 80%. After 7 days, respiration had been reduced to 0.0071% total carbon respired per hour and remained constant thereafter. After 6 weeks of starvation, 46% of the cellular deoxyribonucleic acid had been degraded. Observation of the cellular deoxyribonucleic acid with Feulgen staining before starvation showed the average number of nuclear bodies per cell varied from 1.44 to 4.02 depending on the age of the culture. A linear relationship was found between the number of nuclear bodies per cell and the increase in cell numbers upon starvation. Our data suggest that Ant-300 is capable of surviving long periods of time with little or no nutrients and is therefore well adapted for the sparse nutrient conditions of the colder portions of the open ocean. PMID:16345219

  11. Glutathionylation of the iron superoxide dismutase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis.

    PubMed

    Castellano, Immacolata; Ruocco, Maria Rosaria; Cecere, Francesca; Di Maro, Antimo; Chambery, Angela; Michniewicz, Andzelika; Parlato, Giuseppe; Masullo, Mariorosario; De Vendittis, Emmanuele

    2008-05-01

    Our previous work showed that the adduct between beta-mercaptoethanol and the single cysteine residue (Cys57) in superoxide dismutase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis (PhSOD) reduces the enzyme inactivation by peroxynitrite. In this work, immunoblotting experiments prove that peroxynitrite inactivation of PhSOD involves formation of nitrotyrosine residue(s). In order to study the role of Cys57 as a redox-sensor residue modifiable by cellular thiols, a recombinant PhSOD and two Cys57 mutants were produced and characterized. Recombinant and mutant enzymes share similar activity and peroxynitrite inactivation, but different reactivity towards three glutathione forms. Indeed, oxidized glutathione and S-nitrosoglutathione, but reduced glutathione, lead to S-glutathionylation of recombinant PhSOD. This new covalent modification for a Fe-SOD does not occur in both Cys57 mutants, thus indicating that its target is Cys57. Moreover, mass spectrometry analysis confirmed that S-glutathionylation of Cys57 takes place also with endogenous PhSOD. Formation of this mixed disulfide in PhSOD protects the enzyme from tyrosine nitration and peroxynitrite inactivation. PhSOD undergoes S-glutathionylation during its overproduction in E. coli cells and in a growing culture of P. haloplanktis. In both cases the extent of glutathionylated PhSOD is enhanced upon cell exposure to oxidative agents. We suggest that S-glutathionylation of PhSOD could represent a further cold-adaptation strategy to improve the antioxidant cellular defence mechanism.

  12. Psychrophilic dry anaerobic digestion of dairy cow feces: long-term operation.

    PubMed

    Massé, Daniel I; Cata Saady, Noori M

    2015-02-01

    This paper reports experimental results which demonstrate psychrophilic dry anaerobic digestion of cow feces during long-term operation in sequence batch reactor. Cow feces (13-16% total solids) has been anaerobically digested in 12 successive cycles (252 days) at 21 days treatment cycle length (TCL) and temperature of 20 °C using psychrotrophic anaerobic mixed culture. An average specific methane yield (SMY) of 184.9 ± 24.0, 189.9 ± 27.3, and 222 ± 27.7 (N)L CH4 kg(-1) of VS fed has been achieved at an organic loading rate of 3.0, 4.0, and 5.0 g TCOD kg(-1) inoculum d(-1) and TCL of 21 days, respectively. The corresponding substrate to inoculum ratio (SIR) was 0.39 ± 0.06, 0.48 ± .02, 0.53 ± 0.05, respectively. Average methane production rate of 10 ± 1.4(N)L CH4 kg(-1) VS fed d(-1) has been obtained. The low concentration of volatile fatty acids indicated that hydrolysis was the reaction limiting step. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

  13. Enrichment of hepatocytes in a HepaRG culture using spatially selective photodynamic treatment

    NASA Astrophysics Data System (ADS)

    Bednarkiewicz, Artur; Rodrigues, Robim M.; Whelan, Maurice P.

    2010-03-01

    The human hepatoma HepaRG cell line is an in vitro cell model that is becoming an important tool in drug metabolism, hepatotoxicity, genotoxicity, and enzyme induction studies. The cells are highly proliferative during their undifferentiated state but once committed, they differentiate into two distinctly different cell types, namely, hepatocyte-like and biliary epithelial-like cells. The presence of the latter in the cell culture is considered to be a drawback of the cell model. Since the proliferating undifferentiated HepaRG cells have a bipotent character, the only way to improve the content ratio of hepatic versus biliary cells of differentiated HepaRG cells is to eradicate biliary cells in situ, in a way that free surface space does not become available and thus no transdifferentiation can occur. Spatially selective photodynamic therapy has proven to be effective for that purpose. First, all the cells were administered aminolevulinic acid (δ-ALA) to stimulate the synthesis of protoporphyrin IX (PpIX), a naturally occurring photosensitizer. Then, the biliary cells were automatically identified and outlined by bright-field image processing. Last, UV light patterns were projected onto the epithelial cells alone by a spatial light modulation device connected to an optical microscope; therefore, only these cells were destroyed by photodynamic therapy.

  14. Methylation is the initial reaction in anaerobic naphthalene degradation by a sulfate-reducing enrichment culture.

    PubMed

    Safinowski, Michael; Meckenstock, Rainer U

    2006-02-01

    The sulfate-reducing culture N47 can utilize naphthalene or 2-methylnaphthalene as the sole carbon source and electron donor. Here we show that the initial reaction in the naphthalene degradation pathway is a methylation to 2-methylnaphthalene which then undergoes the subsequent oxidation to the central metabolite 2-naphthoic acid, ring reduction and cleavage. Specific metabolites occurring exclusively during anaerobic degradation of 2-methylnaphthalene were detected during growth on naphthalene, i.e. naphthyl-2-methyl-succinate and naphthyl-2-methylene-succinate. Additionally, all three enzymes involved in anaerobic degradation of 2-methylnaphthalene to 2-naphthoic acid that could be measured in vitro so far, i.e. naphthyl-2-methyl-succinate synthase, succinyl-CoA:naphthyl-2-methyl-succinate CoA-transferase and naphthyl-2-methyl-succinyl-CoA dehydrogenase were also detected in naphthalene-grown cells with similar activities. Induction experiments were performed to study the growth behaviour of the cell when transferred from naphthalene to 2-methylnaphthalene or vice versa. When the cells were transferred from naphthalene to 2-methylnaphthalene they grew immediately, indicating that no new enzymes had to be induced. On the contrary, the transfer of cells from 2-methylnaphthalene to naphthalene caused a lag-phase of almost 100 days demonstrating that an additional catabolic enzyme has to be activated in this case. We propose the methylation as a novel general mechanism of activation reactions in anaerobic degradation of unsubstituted aromatic hydrocarbons.

  15. The effect of pre-enrichment protocol on the sensitivity and specificity of PCR for detection of naturally contaminated Salmonella in raw poultry compared to conventional culture.

    PubMed

    Myint, M S; Johnson, Y J; Tablante, N L; Heckert, R A

    2006-09-01

    Salmonella spp. are the leading cause of foodborne illness worldwide. Conventional culture techniques for the detection of Salmonella spp. are labor intensive and time consuming. Several rapid detection methods have been developed over the past few years. However, standard methods for sample handling and preparation have not been established and limited data are available on the sensitivity and specificity of these methods for detection of Salmonella in naturally contaminated retail meat. Using culture as the gold standard for Salmonella detection in naturally contaminated raw poultry products, the sensitivity and specificity of a polymerase chain reaction (PCR) detection method was determined under varying enrichment protocols. Chicken meat samples (ground, boneless/skinless breast meat, and bone-in breast meat with skin) from retail grocery stores were pre-enriched in buffered peptone water (BPW) and Salmonella specific primers ST 11 and ST 15 were used to amplify a 429 bp region of random fragment target specific to all Salmonella spp. There was a significant decrease (P-value<0.001) in the sensitivity of the PCR test when BPW pre-enrichment alone (85%) was used compared to the sensitivity achieved after both BPW enrichment and selective enrichment with RV and TT-H (100%). PCR failed to detect any positive samples when no pre-enrichment was conducted. A minimum of 12h pre-enrichment was required for detection of Salmonella by PCR at a limit of 100 colony forming unit (cfu)/1 ml of sample. No detectable amplification product was seen in those naturally contaminated meat samples testing negative by culture methods.

  16. Degradation properties of various macromolecules of cultivable psychrophilic bacteria from the deep-sea water of the South Pacific Gyre.

    PubMed

    Zhang, Li; Wang, Yan; Liang, Jing; Song, Qinghao; Zhang, Xiao-Hua

    2016-09-01

    The deep-sea water of the South Pacific Gyre (SPG, 20°S-45°S) is a cold and ultra-oligotrophic environment that is the source of cold-adapted enzymes. However, the characteristic features of psychrophilic enzymes derived from culturable microbes in the SPG remained largely unknown. In this study, the degradation properties of 174 cultures from the deep water of the SPG were used to determine the diversity of cold-adapted enzymes. Thus, the abilities to degrade polysaccharides, proteins, lipids, and DNA at 4, 16, and 28 °C were investigated. Most of the isolates showed one or more extracellular enzyme activities, including amylase, chitinase, cellulase, lipase, lecithinase, caseinase, gelatinase, and DNase at 4, 16, and 28 °C. Moreover, nearly 85.6 % of the isolates produced cold-adapted enzymes at 4 °C. The psychrophilic enzyme-producing isolates distributed primarily in Alteromonas and Pseudoalteromonas genera of the Gammaproteobacteria. Pseudoalteromonas degraded 9 types of macromolecules but not cellulose, Alteromonas secreted 8 enzymes except for cellulase and chitinase. Interestingly, the enzymatic activities of Gammaproteobacteria isolates at 4 °C were higher than those observed at 16 or 28 °C. In addition, we cloned and expressed a gene encoding an α-amylase (Amy2235) from Luteimonas abyssi XH031(T), and examined the properties of the recombinant protein. These cold-active enzymes may have huge potential for academic research and industrial applications. In addition, the capacity of the isolates to degrade various types of organic matter may indicate their unique ecological roles in the elemental biogeochemical cycling of the deep biosphere.

  17. Enrichment culture and identification of endophytic methanotrophs isolated from peatland plants.

    PubMed

    Stępniewska, Zofia; Goraj, Weronika; Kuźniar, Agnieszka; Łopacka, Natalia; Małysza, Magdalena

    2017-03-09

    Aerobic methane-oxidizing bacteria (MOB) are an environmentally significant group of microorganisms due to their role in the global carbon cycle. Research conducted over the past few decades has increased the interest in discovering novel genera of methane-degrading bacteria, which efficiently utilize methane and decrease the global warming effect. Moreover, methanotrophs have more promising applications in environmental bioengineering, biotechnology, and pharmacy. The investigations were undertaken to recognize the variety of endophytic methanotrophic bacteria associated with Carex nigra, Vaccinium oxycoccus, and Eriophorum vaginatum originating from Moszne peatland (East Poland). Methanotrophic bacteria were isolated from plants by adding sterile fragments of different parts of plants (roots and stems) to agar mineral medium (nitrate mineral salts (NMS)) and incubated at different methane values (1-20% CH4). Single colonies were streaked on new NMS agar media and, after incubation, transferred to liquid NMS medium. Bacterial growth dynamics in the culture solution was studied by optical density-OD600 and methane consumption. Changes in the methane concentration during incubation were controlled by the gas chromatography technique. Characterization of methanotrophs was made by fluorescence in situ hybridization (FISH) with Mg705 and Mg84 for type I methanotrophs and Ma450 for type II methanotrophs. Identification of endophytes was performed after 16S ribosomal RNA (rRNA) and mmoX gene amplification. Our study confirmed the presence of both types of methanotrophic bacteria (types I and II) with the predominance of type I methanotrophs. Among cultivable methanotrophs, there were different strains of the genus Methylomonas and Methylosinus. Furthermore, we determined the potential of the examined bacteria for methane oxidation, which ranged from 0.463 ± 0.067 to 5.928 ± 0.169 μmol/L CH4/mL/day.

  18. Predator-specific enrichment of actinobacteria from a cosmopolitan freshwater clade in mixed continuous culture.

    PubMed

    Pernthaler, J; Posch, T; Simek, K; Vrba, J; Pernthaler, A; Glöckner, F O; Nübel, U; Psenner, R; Amann, R

    2001-05-01

    We investigated whether individual populations of freshwater bacteria in mixed experimental communities may exhibit specific responses to the presence of different bacterivorous protists. In two successive experiments, a two-stage continuous cultivation system was inoculated with nonaxenic batch cultures of the cryptophyte Cryptomonas sp. Algal exudates provided the sole source of organic carbon for growth of the accompanying microflora. The dynamics of several 16S rRNA-defined bacterial populations were followed in the experimental communities. Although the composition and stability of the two microbial communities differed, numerous members of the first assemblage could again be detected during the second experiment. The introduction of a size-selectively feeding mixotrophic nanoflagellate (Ochromonas sp.) always resulted in an immediate bloom of a single phylotype population of members of the class Actinobacteria (Ac1). These bacteria were phylogenetically affiliated with an uncultured lineage of gram-positive bacteria that have been found in freshwater habitats only. The Ac1 cells were close to the average size of freshwater bacterioplankton and significantly smaller than any of the other experimental community members. In contrast, no increase of the Ac1 population was observed in vessels exposed to the bacterivorous ciliate Cyclidium glaucoma. However, when the Ochromonas sp. was added after the establishment of C. glaucoma, the proportion of population Ac1 within the microbial community rapidly increased. Populations of a beta proteobacterial phylotype related to an Aquabacterium sp. decreased relative to the total bacterial communities following the addition of either predator, albeit to different extents. The community structure of pelagic microbial assemblages can therefore be influenced by the taxonomic composition of the predator community.

  19. Predator-Specific Enrichment of Actinobacteria from a Cosmopolitan Freshwater Clade in Mixed Continuous Culture

    PubMed Central

    Pernthaler, Jakob; Posch, Thomas; S̆imek, Karel; Vrba, Jaroslav; Pernthaler, Annelie; Glöckner, Frank Oliver; Nübel, Ulrich; Psenner, Roland; Amann, Rudolf

    2001-01-01

    We investigated whether individual populations of freshwater bacteria in mixed experimental communities may exhibit specific responses to the presence of different bacterivorous protists. In two successive experiments, a two-stage continuous cultivation system was inoculated with nonaxenic batch cultures of the cryptophyte Cryptomonas sp. Algal exudates provided the sole source of organic carbon for growth of the accompanying microflora. The dynamics of several 16S rRNA-defined bacterial populations were followed in the experimental communities. Although the composition and stability of the two microbial communities differed, numerous members of the first assemblage could again be detected during the second experiment. The introduction of a size-selectively feeding mixotrophic nanoflagellate (Ochromonas sp.) always resulted in an immediate bloom of a single phylotype population of members of the class Actinobacteria (Ac1). These bacteria were phylogenetically affiliated with an uncultured lineage of gram-positive bacteria that have been found in freshwater habitats only. The Ac1 cells were close to the average size of freshwater bacterioplankton and significantly smaller than any of the other experimental community members. In contrast, no increase of the Ac1 population was observed in vessels exposed to the bacterivorous ciliate Cyclidium glaucoma. However, when the Ochromonas sp. was added after the establishment of C. glaucoma, the proportion of population Ac1 within the microbial community rapidly increased. Populations of a beta proteobacterial phylotype related to an Aquabacterium sp. decreased relative to the total bacterial communities following the addition of either predator, albeit to different extents. The community structure of pelagic microbial assemblages can therefore be influenced by the taxonomic composition of the predator community. PMID:11319094

  20. Using Culture beyond Its Borders: The Use of Content-Enriched Instruction and the Effects of Input Enhancement on Learning in High School French Classes

    ERIC Educational Resources Information Center

    Grim, Frédérique

    2014-01-01

    The American Council on the Teaching of Foreign Languages Standards emphasizes the integration of Communication, Cultures, Connections, Comparisons, and Communities within teaching. "Content-enriched instruction" aims at teaching linguistic forms within content and eases the implementation of the five Cs. The focus is at beginning levels…

  1. Characterization of cultures enriched from acidic polycyclic aromatic hydrocarbon-contaminated soil for growth on pyrene at low pH.

    PubMed

    Uyttebroek, Maarten; Vermeir, Steven; Wattiau, Pierre; Ryngaert, Annemie; Springael, Dirk

    2007-05-01

    Two polycyclic aromatic hydrocarbon (PAH)-contaminated soils of pH 2 were successfully used as inoculum to enrich cultures growing on phenanthrene and pyrene at different pHs, including pH 3. Selected pyrene-utilizing cultures obtained at pH 3, pH 5, and pH 7 were further characterized. All showed rapid [14C]pyrene mineralization at pH 3 and pH 5 and grew on pyrene at pH values ranging from 2 to 6. Eubacterial and mycobacterial 16S rRNA gene denaturing gradient gel electrophoresis fingerprinting and sequencing indicated that the cultures were dominated by a single bacterium closely related to Mycobacterium montefiorense, belonging to the slow-growing Mycobacterium sp. In contrast, a culture enriched on pyrene at pH 7 from a slightly alkaline soil sampled at the same site was dominated by Pseudomonas putida and a fast-growing Mycobacterium sp. The M. montefiorense-related species dominating the pyrene-utilizing cultures enriched from the acidic soils was also the dominant Mycobacterium species in the acidic soils. Our data indicate that a slow-growing Mycobacterium species is involved in PAH degradation in that culture and show that bacteria able to degrade high-molecular-weight PAHs at low pH are present in acidic PAH-contaminated soil.

  2. Rapid Identification of Shiga Toxin-Producing Escherichia coli O Serogroups from Fresh Produce and Raw Milk Enrichment Cultures by Luminex Bead-Based Suspension Array.

    PubMed

    Kase, Julie A; Maounounen-Laasri, Anna; Lin, Andrew

    2016-09-01

    The U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) Chapter 4a describes a Luminex microbead-based suspension array used to screen colonies for 11 clinically relevant Shiga toxin-producing Escherichia coli (STEC) serogroups: O26, O45, O91, O103, O104, O111, O113, O121, O128, O145, and O157. We evaluated the usefulness of this method to identify STEC-positive enrichment samples before agar plating. Twelve E. coli strains were added to three types of fresh produce (bagged baby spinach, alfalfa sprouts, and cilantro) at levels near the detection limit of the test. A subset of these strains (six O serogroups) was similarly evaluated in raw milk. For comparison, portions of each of the 168 enrichment cultures were analyzed for serogroup by a real-time PCR assay and a Bio-Plex 200 assay with the bead-based suspensions. No false-positive results were obtained. Of the 112 samples with a reported cycle threshold (CT) value, 101 undiluted, diluted, or extracted enrichment cultures also produced ratios above 5.0 in the Bio-Plex assay. When PCR CT values approached or were greater than 35, Bio-Plex detection became less reliable. Using undiluted or extracted enrichment cultures resulted in a significantly larger number of positive results. With the same enrichment material prepared for real-time PCR analysis as described in the BAM Chapter 4a, the STEC microbead-based suspension array can accurately screen food enrichment cultures.

  3. Characterization of bacterial diversity in an atrazine degrading enrichment culture and degradation of atrazine, cyanuric acid and biuret in industrial wastewater.

    PubMed

    Dutta, Anirban; Vasudevan, Venugopal; Nain, Lata; Singh, Neera

    2016-01-01

    An enrichment culture was used to study atrazine degradation in mineral salt medium (MSM) (T1), MSM+soil extract (1:1, v/v) (T2) and soil extract (T3). Results suggested that enrichment culture required soil extract to degrade atrazine, as after second sequential transfer only partial atrazine degradation was observed in T1 treatment while atrazine was completely degraded in T2 and T3 treatments even after fourth transfer. Culture independent polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique confirmed selective enrichment of genus Bacillus along with Pseudomonas and Burkholderia. Degradation of atrazine/metabolites in the industrial wastewater was studied at different initial concentrations of the contaminants [wastewater-water (v/v) ratio: T1, 1:9; T2, 2:8; T3, 3:7; T4, 5:5 and T5, undiluted effluent]. The initial concentrations of atrazine, cyanuric acid and biuret ranged between 5.32 and 53.92 µg mL(-1), 265.6 and 1805.2 µg mL(-1) and 1.85 and 16.12 µg mL(-1), respectively. The enrichment culture was able to completely degrade atrazine, cyanuric acid and biuret up to T4 treatment, while no appreciable degradation of contaminants was observed in the undiluted effluent (T5). Inability of enrichment culture to degrade atrazine/metabolites might be due to high concentrations of cyanuric acid. Therefore, a separate study on cyanuric acid degradation suggested: (i) no appreciable cyanuric acid degradation with accumulation of an unidentified metabolite in the medium where cyanuric acid was supplemented as the sole source of carbon and nitrogen; (ii) partial cyanuric acid degradation with accumulation of unidentified metabolite in the medium containing additional nitrogen source; and (iii) complete cyanuric acid degradation in the medium supplemented with an additional carbon source. This unidentified metabolite observed during cyanuric acid degradation and also detected in the enrichment culture inoculated wastewater samples

  4. Establishment of microbial eukaryotic enrichment cultures from a chemically stratified antarctic lake and assessment of carbon fixation potential.

    PubMed

    Dolhi, Jenna M; Ketchum, Nicholas; Morgan-Kiss, Rachael M

    2012-04-20

    Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a chemically stratified water column and unlike other inland bodies of water, largely prevents external input of carbon and nutrients from streams. Biota are exposed to numerous environmental stresses, including year-round severe nutrient deficiency, low temperatures, extreme shade, hypersalinity, and 24-hour darkness during the winter (1). These extreme environmental conditions limit the biota in Lake Bonney almost exclusively to microorganisms (2). Single-celled microbial eukaryotes (called "protists") are important players in global biogeochemical cycling (3) and play important ecological roles in the cycling of carbon in the dry valley lakes, occupying both primary and tertiary roles in the aquatic food web. In the dry valley aquatic food web, protists that fix inorganic carbon (autotrophy) are the major producers of organic carbon for organotrophic organisms (4, 2). Phagotrophic or heterotrophic protists capable of ingesting bacteria and smaller protists act as the top predators in the food web (5). Last, an unknown proportion of the protist population is capable of combined mixotrophic metabolism (6, 7). Mixotrophy in protists involves the ability to combine photosynthetic capability with phagotrophic ingestion of prey microorganisms. This form of mixotrophy differs from mixotrophic metabolism in bacterial species, which generally involves uptake dissolved carbon molecules. There are currently very few protist isolates from permanently ice-capped polar lakes, and studies of protist diversity and ecology in this extreme environment have been limited (8, 4, 9, 10, 5). A better understanding of protist metabolic versatility in the simple dry valley lake food web will aid in the development of models for the role of protists in the global carbon cycle. We employed an enrichment culture approach to isolate potentially

  5. Photosynthetic and Respiratory Rates of Two Psychrophilic Diatoms 1

    PubMed Central

    Van Baalen, Chase

    1985-01-01

    The photosynthetic rates in two psychrophilic diatoms, Chaetoceros sp. strain K3-10 and Nitzschia sp. K3-3 for cells grown at 0°C were 8 to 10 microliters O2 evolved per milligram dry weight per hour, and 10-fold higher, about 80 for cells grown at 10°C. The respiration rates followed the same pattern, with a value of around 1 microliter dark uptake per milligram dry weight per hour for both organisms grown at 0°C, and 6 to 10 for cells grown at 10°C. When cells grown at 0°C were immediately shifted to 10°C or cells grown at 10°C were shifted to 0°C, the respiratory rates quickly adapted to values characteristic of cells grown at the shift temperature. On the other hand, the light-saturated rate of O2 evolution showed much less immediate adaptation, especially on the up shift, 0° to 10°C. The chlorophyll a content of 0°C grown cells was about 0.5% of dry weight, in 10°C grown cells 1.3% (strain K3-10) and 2.2% (strain K3-3). In addition to a diminished chlorophyll a content in 0°C grown cells, there seemed proportionally (by absorbance and calculation) less c to a than in 10°C grown cells. The relative fluorescence excitation spectra of 680-nm emission also showed a lower contribution by both chlorophyll c and fucoxanthin in 0°C grown cells of Chaetoceros sp. strain K3-10 as compared to 10°C grown cells. The data at hand suggest that in psychrophilic diatoms continuously growing at 0°C there may be problems associated with synthesis of an effective accessory pigment system, and as a working hypothesis it is suggested this is related to restriction of synthesis of one or several accessory pigment proteins. PMID:16664328

  6. Psychrophilic and Psychrotolerant Microbial Extremophiles in Polar Environments

    NASA Technical Reports Server (NTRS)

    Hoover, Richard B.; Pikuta, Elena V.

    2010-01-01

    The microbial extremophiles that inhabit the polar regions of our planet are of tremendous significance. The psychrophilic and psychrotolerant microorganisms, which inhabit all of the cold environments on Earth have important applications to Bioremediation, Medicine, Pharmaceuticals, and many other areas of Biotechnology. Until recently, most of the research on polar microorganisms was confined to studies of polar diatoms, yeast, fungi and cyanobacteria. However, within the past three decades, extensive studies have been conducted to understand the bacteria and archaea that inhabit the Arctic and Antarctic sea-ice, glaciers, ice sheets, permafrost and the cryptoendolithic, cryoconite and ice-bubble environments. These investigations have resulted in the discovery of many new genera and species of anaerobic and aerobic microbial extremophiles. Exotic enzymes, cold-shock proteins and pigments produced by some of the extremophiles from polar environments have the potential to be of great benefit to Mankind. Knowledge about microbial life in the polar regions is crucial to understanding the limitations and biodiversity of life on Earth and may provide valuable clues to the Origin of Life on Earth. The discovery of viable microorganisms in ancient ice from the Fox Tunnel, Alaska and the deep Vostok Ice has shown that microorganisms can remain alive while cryopreserved in ancient ice. The psychrophilic lithoautotrophic homoacetogen isolated from the deep anoxic trough of Lake Untersee is an ideal candidate for life that might inhabit comets or the polar caps of Mars. The spontaneous release of gas from within the Anuchin Glacier above Lake Untersee may provide clues to the ice geysers that erupt from the tiger stripe regions of Saturn s moon Enceladus. The methane productivity in the lower regimes of Lake Untersee may also provide insights into possible mechanisms for the recently discovered methane releases on Mars. Since most of the other water bearing bodies of our

  7. The metabolism of neonicotinoid insecticide thiamethoxam by soil enrichment cultures, and the bacterial diversity and plant growth-promoting properties of the cultured isolates.

    PubMed

    Zhou, Guang-Can; Wang, Ying; Ma, Yuan; Zhai, Shan; Zhou, Ling-Yan; Dai, Yi-Jun; Yuan, Sheng

    2014-01-01

    A soil enrichment culture (SEC) rapidly degraded 96% of 200 mg L(-1) neonicotinoid insecticide thiamethoxam (TMX) in MSM broth within 30 d; therefore, its metabolic pathway of TMX, bacterial diversity and plant growth-promoting rhizobacteria (PGPR) activities of the cultured isolates were studied. The SEC transformed TMX via the nitro reduction pathway to form nitrso, urea metabolites and via cleavage of the oxadiazine cycle to form a new metabolite, hydroxyl CLO-tri. In addition, 16S rRNA gene-denaturing gradient gel electrophoresis analysis revealed that uncultured rhizobacteria are predominant in the SEC broth and that 77.8% of the identified bacteria belonged to uncultured bacteria. A total of 31 cultured bacterial strains including six genera (Achromobacter, Agromyces, Ensifer, Mesorhizobium, Microbacterium and Pseudoxanthomonas) were isolated from the SEC broth. The 12 strains of Ensifer adhaerens have the ability to degrade TMX. All six selected bacteria showed PGPR activities. E. adhaerens TMX-23 and Agromyces mediolanus TMX-25 produced indole-3-acetic acid, whereas E. adhaerens TMX-23 and Mesorhizobium alhagi TMX-36 are N2-fixing bacteria. The six-isolated microbes were tolerant to 200 mg L(-1) TMX, and the growth of E. adhaerens was significantly enhanced by TMX, whereas that of Achromobacter sp. TMX-5 and Microbacterium sp.TMX-6 were enhanced slightly. The present study will help to explain the fate of TMX in the environment and its microbial degradation mechanism, as well as to facilitate future investigations of the mechanism through which TMX enhances plant vigor.

  8. Comparative and multilaboratory studies of two immunodiffusion method enrichment protocols and the AOAC/Bacteriological Analytical Manual culture method for detection of Salmonella in all foods.

    PubMed

    Feldsine, P T; Falbo-Nelson, M T; Hustead, D L; Flowers, R S; Flowers, M J

    1995-01-01

    The single enrichment immunodiffusion (1-step), the preenrichment and selective enrichment immunodiffusion (2-step), and the AOAC/Bacteriological Analytical Manual culture methods for Salmonella were evaluated for equivalence in 2 separate studies, a comparative evaluation and a multilaboratory dilution study. In the comparative study, all 3 methods were performed on 10 food types. For 550 samples, analyses resulted in 99.3 and 99.6% agreement between the culture method and the 1-step and 2-step methods, respectively. False negative rates were 0.9 and 0.3% for 1-step and culture, and 0.0% and 0.6% for the 2-step and culture, respectively. Subsequently, 6 food types were included in a multilaboratory dilution-to-extinction study. A sequential dilution series of Salmonella in foods was analyzed by the 3 methods to determine their lower limits of detection for Salmonella. A total of 1185 samples analyzed resulted in 98.9% agreement between 1-step and culture, and 99.7% agreement between 2-step and culture. False negative rates were 1.8 and 0.1% for 1-step and culture, and 0.4 and 0.1% for 2-step and culture, respectively. During these evaluations, 1735 samples and controls representing 10 different naturally contaminated and inoculated foods were tested. The data indicate statistical equivalence of all 3 methods when analyzing all food types.

  9. Effect of nitrogen source on methanol oxidation and genetic diversity of methylotrophic mixed cultures enriched from pulp and paper mill biofilms.

    PubMed

    Babbitt, Callie W; Lindner, Angela S

    2011-04-01

    Methanol-oxidizing bacteria may play an important role in the development and use of biological treatment systems for the removal of methanol from industrial effluents. Optimization of methanol degradation potential in such systems is contingent on availability of nutrients, such as nitrogen, in the most favorable form and concentration. To that end, this study examined the variation in growth, methanol degradation, and bacterial diversity of two mixed methylotrophic cultures that were provided nitrogen either as ammonium or nitrate and in three different concentrations. Methanol-degrading cultures were enriched from biofilms sampled at a pulp and paper mill and grown in liquid batch culture with methanol as the only carbon source and either ammonium or nitrate as the only added nitrogen source. Results indicate that growth and methanol removal of the mixed cultures increase directly with increased nitrogen, added in either form. However, methanol removal and bacterial diversity, as observed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) methods, were higher when using nitrate as the nitrogen source for enrichment and growth, rather than ammonium. Based on results described here, nitrate may potentially be a better nitrogen source when enriching or working with mixed methylotrophic cultures, and possibly more effective when used as a nutrient addition to biofilters.

  10. Biodegradation of Various Aromatic Compounds by Enriched Bacterial Cultures: Part B--Nitrogen-, Sulfur-, and Oxygen-Containing Heterocyclic Aromatic Compounds.

    PubMed

    Oberoi, Akashdeep Singh; Philip, Ligy; Bhallamudi, S Murty

    2015-07-01

    Present study focused on the biodegradation of various heterocyclic nitrogen, sulfur, and oxygen (NSO) compounds using naphthalene-enriched culture. Target compounds in the study were pyridine, quinoline, benzothiophene, and benzofuran. Screening studies were carried out using different microbial consortia enriched with specific polycyclic aromatic hydrocarbon (PAH) and NSO compounds. Among different microbial consortia, naphthalene-enriched culture was the most efficient consortium based on high substrate degradation rate. Substrate degradation rate with naphthalene-enriched culture followed the order pyridine > quinoline > benzofuran > benzothiophene. Benzothiophene and benzofuran were found to be highly recalcitrant pollutants. Benzothiophene could not be biodegraded when concentration was above 50 mg/l. It was observed that 2-(1H)-quinolinone, benzothiophene-2-one, and benzofuran-2,3-dione were formed as metabolic intermediates during biodegradation of quinoline, benzothiophene, and benzofuran, respectively. Quinoline-N and pyridine-N were transformed into free ammonium ions during the biodegradation process. Biodegradation pathways for various NSO compounds are proposed. Monod inhibition model was able to simulate single substrate biodegradation kinetics satisfactorily. Benzothiophene and benzofuran biodegradation kinetics, in presence of acetone, was simulated using a generalized multi-substrate model.

  11. High rate psychrophilic anaerobic digestion of undiluted dairy cow feces.

    PubMed

    Massé, Daniel I; Saady, Noori M Cata

    2015-01-01

    Novel high rate psychrophilic (20°C) anaerobic digestion (PAD) of undiluted cow feces (11.5-13.5% total solids) was demonstrated using sequence batch reactor in long-term operation with successive cycles of 21days treatment cycle length (TCL). At organic loading rates (OLR) 9.0, 10.0, 11.0 and 12.0g TCOD kg(-1) inoculum d(-1) average specific methane yield (SMY) was 154.0±11.7, 152.1±12.2, 126.0±2.8 and 116.0±6.1NL CH4 per kg of VS fed, respectively. Volatile solids removal averaged around 31.7±3.3%, 32.2±1.0%, 27.9±2.2% and 23.4±0.5%, respectively. Substrate-to-inoculum ratio (SIR; wet-mass basis) ranged between 1.17±0.06 and 1.43±0.05. Concentration of volatile fatty acids in the bioreactors during the TCL indicated that hydrolysis was the rate limiting reaction. High rate PAD of undiluted cow feces is possible at OLR (g TCOD kg(-1) inoculum d(-1)) 9.0 and 10.0 with a TCL of 21days; however, OLR of 11.0 and 12.0 are also possible but require longer TCL to maintain the SMY.

  12. Characterization of psychrophilic alanine racemase from Bacillus psychrosaccharolyticus.

    PubMed

    Okubo, Y; Yokoigawa, K; Esaki, N; Soda, K; Kawai, H

    1999-03-16

    A psychrophilic alanine racemase gene from Bacillus psychrosaccharolyticus was cloned and expressed in Escherichia coli SOLR with a plasmid pYOK3. The gene starting with the unusual initiation codon GTG showed higher preference for codons ending in A or T. The enzyme purified to homogeneity showed the high catalytic activity even at 0 degrees C and was extremely labile over 35 degrees C. The enzyme was found to have a markedly large Km value (5.0 microM) for the pyridoxal 5'-phosphate (PLP) cofactor in comparison with other reported alanine racemases, and was stabilized up to 50 degrees C in the presence of excess amounts of PLP. The low affinity of the enzyme for PLP may be related to the thermolability, and may be related to the high catalytic activity, initiated by the transaldimination reaction, at low temperature. The enzyme has a distinguishing hydrophilic region around the residue no. 150 in the deduced amino acid sequence (383 residues), whereas the corresponding regions of other Bacillus alanine racemases are hydrophobic. The position of the region in the three dimensional structure of C atoms of the enzyme was predicted to be in a surface loop surrounding the active site. The region may interact with solvent and reduce the compactness of the active site.

  13. Psychrophilic yeasts in glacial environments of Alpine glaciers.

    PubMed

    Turchetti, Benedetta; Buzzini, Pietro; Goretti, Marta; Branda, Eva; Diolaiuti, Guglielmina; D'Agata, Carlo; Smiraglia, Claudio; Vaughan-Martini, Ann

    2008-01-01

    The presence of psychrophilic yeasts in supra- and subglacial sediments, ice and meltwater collected from two glaciers of the Italian Alps (Forni and Sforzellina-Ortles-Cevedale group) was investigated. After incubation at 4 degrees C, subglacial sediments contained from 1.3 x 10(3) to 9.6 x 10(3) CFU of yeasts g(-1). The number of yeast cells in supraglacial sediments was c. 10-100-fold lower. A significant proportion of isolated yeasts exhibited one or more extracellular enzymatic activities (starch-degrading, lipolytic, esterolytic, proteolytic and pectinolytic activity) at 4 degrees C. Selected isolates were able to grow at 2 degrees C under laboratory-simulated in situ conditions. In all, 106 isolated yeasts were identified by MSP-PCR fingerprinting and 26S rRNA gene sequencing of the D1/D2 region as belonging to 10 species: Aureobasidium pullulans, Cryptococcus gilvescens (over 50% of the total), Cryptococcus terricolus, Mrakia gelida, Naganishia globosa, Rhodotorula glacialis, Rhodotorula psychrophenolica, Rhodotorula bacarum, Rhodotorula creatinivora and Rhodotorula laryngis. Four strains, all belonging to a new yeast species, yet to be described, were also isolated.

  14. A habitat for psychrophiles in deep Antarctic ice

    PubMed Central

    Price, P. Buford

    2000-01-01

    Microbes, some of which may be viable, have been found in ice cores drilled at Vostok Station at depths down to ≈3,600 m, close to the surface of the huge subglacial Lake Vostok. Two types of ice have been found. The upper 3,500 m comprises glacial ice containing traces of nutrients of aeolian origin including sulfuric acid, nitric acid, methanosulfonic acid (MSA), formic acid, sea salts, and mineral grains. Ice below ≈3,500 m comprises refrozen water from Lake Vostok, accreted to the bottom of the glacial ice. Nutrients in the accretion ice include salts and dissolved organic carbon. There is great interest in searching for living microbes and especially for new species in deepest Antarctic ice. I propose a habitat consisting of interconnected liquid veins along three-grain boundaries in ice in which psychrophilic bacteria can move and obtain energy and carbon from ions in solution. In the accretion ice, with an age of a few 104 years and a temperature a few degrees below freezing, the carbon and energy sources in the veins can maintain significant numbers of cells per cubic centimeter that are metabolizing but not multiplying. In the 4 × 105-year-old colder glacial ice, at least 1 cell per cm3 in acid veins can be maintained. With fluorescence microscopy tuned to detect NADH in live organisms, motile bacteria could be detected by direct scanning of the veins in ice samples. PMID:10655516

  15. Bayesian techniques for comparison of the test performance of PCR and culture for the identification of Campylobacter in enriched comminuted chicken samples.

    PubMed

    Ebel, E D; Williams, M S; Golden, N J; Tankson, J

    2016-05-01

    Using Bayesian methods that do not require the definition of a gold standard, the diagnostic sensitivity and specificity of a real-time polymerase chain reaction (PCR) assay are compared to those of an enriched culture assay for detection of Campylobacter in enriched comminuted chicken samples. Food Safety and Inspection Service comminuted chicken samples were collected from production facilities across the United States. Enriched samples were examined using a commercial real-time PCR kit and plated for culture. Allowing for conditional dependence between these approaches and defining relatively uninformed prior distributions, the 'no gold standard' Bayesian methods generated estimates of the means (95% credible interval) of the posterior distributions for sensitivity and specificity of the PCR as 93% (79, 100%) and 95% (87, 100%) respectively. The estimated sensitivity implies a mean false negative frequency of 7%. The estimated means of the posterior distributions for sensitivity and specificity of the culture assay were 91% (76, 100%) and 96% (88, 100%) respectively. In this case, the mean false negative frequency is 9%. Graphical comparisons of the posterior distributions with their corresponding prior distributions suggested only subtle differences in the sensitivities of both tests, but the posterior distributions for specificities are substantially more certain than the prior distributions. The study suggests that the commercial real-time PCR assay is a more sensitive screening test that would provide timelier negative test results. The modest 1% reduction in specificity of this PCR assay, as compared to an enriched culture assay, is less of a concern for regulatory testing programs if a culture-based confirmatory assay is applied to all presumptive positive samples. The sensitivity and specificity of a PCR assay and a culture assay for Campylobacter in comminuted poultry produced in the United States were estimated. The PCR assay was shown to be an

  16. Rapid and Specific Enrichment of Culturable Gram Negative Bacteria Using Non-Lethal Copper-Free Click Chemistry Coupled with Magnetic Beads Separation

    PubMed Central

    Fugier, Emilie; Dumont, Audrey; Malleron, Annie; Poquet, Enora; Mas Pons, Jordi; Baron, Aurélie; Vauzeilles, Boris; Dukan, Sam

    2015-01-01

    Currently, identification of pathogenic bacteria present at very low concentration requires a preliminary culture-based enrichment step. Many research efforts focus on the possibility to shorten this pre-enrichment step which is needed to reach the minimal number of cells that allows efficient identification. Rapid microbiological controls are a real public health issue and are required in food processing, water quality assessment or clinical pathology. Thus, the development of new methods for faster detection and isolation of pathogenic culturable bacteria is necessary. Here we describe a specific enrichment technique for culturable Gram negative bacteria, based on non-lethal click chemistry and the use of magnetic beads that allows fast detection and isolation. The assimilation and incorporation of an analog of Kdo, an essential component of lipopolysaccharides, possessing a bio-orthogonal azido function (Kdo-N3), allow functionalization of almost all Gram negative bacteria at the membrane level. Detection can be realized through strain-promoted azide-cyclooctyne cycloaddition, an example of click chemistry, which interestingly does not affect bacterial growth. Using E. coli as an example of Gram negative bacterium, we demonstrate the excellent specificity of the technique to detect culturable E. coli among bacterial mixtures also containing either dead E. coli, or live B. subtilis (as a model of microorganism not containing Kdo). Finally, in order to specifically isolate and concentrate culturable E. coli cells, we performed separation using magnetic beads in combination with click chemistry. This work highlights the efficiency of our technique to rapidly enrich and concentrate culturable Gram negative bacteria among other microorganisms that do not possess Kdo within their cell envelope. PMID:26061695

  17. Deep-sea oil plume enriches psychrophilic oil-degrading bacteria

    SciTech Connect

    Hazen, T.C.; Dubinsky, E.A.; DeSantis, T.Z.; Andersen, G.L.; Piceno, Y.M.; Singh, N.; Jansson, J.K.; Probst, A.; Borglin, S.E.; Fortney, J.L.; Stringfellow, W.T.; Bill, M.; Conrad, M.S.; Tom, L.M.; Chavarria, K.L.; Alusi, T.R.; Lamendella, R.; Joyner, D.C.; Spier, C.; Auer, M.; Zemla, M.L.; Chakraborty, R.; Sonnenthal, E.L.; D'haeseleer, P.; Holman, H.-Y. N.; Osman, S.; Lu, Z.; Van Nostrand, J.D.; Deng, Y.; Zhou, J.; Mason, O.U.

    2010-09-01

    The biological effects and expected fate of the vast amount of oil in the Gulf of Mexico from the Deepwater Horizon blowout are unknown owing to the depth and magnitude of this event. Here, we report that the dispersed hydrocarbon plume stimulated deep-sea indigenous {gamma}-Proteobacteria that are closely related to known petroleum degraders. Hydrocarbon-degrading genes coincided with the concentration of various oil contaminants. Changes in hydrocarbon composition with distance from the source and incubation experiments with environmental isolates demonstrated faster-than-expected hydrocarbon biodegradation rates at 5 C. Based on these results, the potential exists for intrinsic bioremediation of the oil plume in the deep-water column without substantial oxygen drawdown.

  18. Detection of Mycoplasma ovipneumoniae and M. arginini in bighorn sheep using enrichment culture coupled with genus- and species-specific polymerase chain reaction.

    PubMed

    Weiser, Glen C; Drew, Mark L; Cassirer, E Frances; Ward, Alton C S

    2012-04-01

    Mycoplasma species are of interest as possible primary pathogens in the pneumonia complex of bighorn sheep (Ovis canadensis). Previous investigations have not commonly detected low frequencies of Mycoplasma spp. from free-ranging bighorn sheep, possibly due to the fastidious and slow growth of these organisms. We developed a culture protocol that employed an average initial 3-day enrichment culture in liquid Hayflick broth in a CO(2)-enhanced atmosphere. The broth was plated to solid Hayflick medium and the cultures observed for growth for up to 30 days. Polymerase chain reaction (PCR) was performed on DNA isolated from the enrichment broth and on isolates obtained from culture using Mycoplasma genus-specific PCR assays and species-specific PCR assays for M. arginini and M. ovipneumoniae. Some cultures that grew on Hayflick plates were picked as single colonies but were mixed because two organisms may grow together and appear as a single colony. Culture and PCR tests produced similar results for M. arginini, but for M. ovipneumoniae, culture alone was less accurate than PCR. Use of genus-specific primers also may allow detection of other species in samples negative for M. arginini and M. ovipneumoniae. Two methods of transport from field to laboratory (Port-a-Cul™ tubes, cryoprotectant in liquid N(2) and Fisher Transport System) gave similar results under our study conditions.

  19. Cold adaptation of a psychrophilic chaperonin from Psychrobacter sp. and its application for heterologous protein expression.

    PubMed

    Kim, Han-Woo; Wi, Ah Ram; Jeon, Byoung Wook; Lee, Jun Hyuck; Shin, Seung Chul; Park, Hyun; Jeon, Sung-Jong

    2015-09-01

    A chaperonin, PsyGroELS, from the Antarctic psychrophilic bacterium Psychrobacter sp. PAMC21119, was examined for its role in cold adaptation when expressed in a mesophilic Escherichia coli strain. Growth of E. coli harboring PsyGroELS at 10 °C was increased compared to the control strain. A co-expression system using PsyGroELS was developed to increase productivity of the psychrophilic enzyme PsyEst9. PsyEst9 was cloned and expressed using three E. coli variants that co-expressed GroELS from PAMC21119, E. coli, or Oleispira antarctica RB8(T). Co-expression with PsyGroELS was more effective for the production of PsyEst9 compared tothe other chaperonins. PsyGroELS confers cold tolerance to E. coli, and shows potential as an effective co-expression system for the stable production of psychrophilic proteins.

  20. Structural and functional analysis of a novel psychrophilic β-mannanase from Glaciozyma antarctica PI12

    NASA Astrophysics Data System (ADS)

    Parvizpour, Sepideh; Razmara, Jafar; Ramli, Aizi Nor Mazila; Md Illias, Rosli; Shamsir, Mohd Shahir

    2014-06-01

    The structure of a novel psychrophilic β-mannanase enzyme from Glaciozyma antarctica PI12 yeast has been modelled and analysed in detail. To our knowledge, this is the first attempt to model a psychrophilic β-mannanase from yeast. To this end, a 3D structure of the enzyme was first predicted using a threading method because of the low sequence identity (<30 %) using MODELLER9v12 and simulated using GROMACS at varying low temperatures for structure refinement. Comparisons with mesophilic and thermophilic mannanases revealed that the psychrophilic mannanase contains longer loops and shorter helices, increases in the number of aromatic and hydrophobic residues, reductions in the number of hydrogen bonds and salt bridges and numerous amino acid substitutions on the surface that increased the flexibility and its efficiency for catalytic reactions at low temperatures.

  1. The structure of ferricytochrome c552 from the psychrophilic marine bacterium Colwellia psychrerythraea 34H

    PubMed Central

    Harvilla, Paul B.; Wolcott, Holly N.

    2014-01-01

    Approximately 40% of all proteins are metalloproteins, and approximately 80% of Earth’s ecosystems are at temperatures ≤ 5 °C, including 90% of the global ocean. Thus, an essential aspect of marine metallobiochemistry is an understanding of the structure, dynamics, and mechanisms of cold adaptation of metalloproteins from marine microorganisms. Here, the molecular structure of the electron-transfer protein cytochrome c552 from the psychrophilic marine bacterium Colwellia psychrerythraea 34H has been determined by X-ray crystallography (PDB: 4O1W). The structure is highly superimposable with that of the homologous cytochrome from the mesophile Marinobacter hydrocarbonoclasticus. Based on structural analysis and comparison of psychrophilic, psychrotolerant, and mesophilic sequences, a methionine-based ligand-substitution mechanism for psychrophilic protein stabilization is proposed. PMID:24727932

  2. Comparison of polymerase chain reaction methods and plating for analysis of enriched cultures of Listeria monocytogenes when using the ISO11290-1 method.

    PubMed

    Dalmasso, Marion; Bolocan, Andrei Sorin; Hernandez, Marta; Kapetanakou, Anastasia E; Kuchta, Tomáš; Manios, Stavros G; Melero, Beatriz; Minarovičová, Jana; Muhterem, Meryem; Nicolau, Anca Ioana; Rovira, Jordi; Skandamis, Panagiotis N; Stessl, Beatrix; Wagner, Martin; Jordan, Kieran; Rodríguez-Lázaro, David

    2014-03-01

    Analysis for Listeria monocytogenes by ISO11290-1 is time-consuming, entailing two enrichment steps and subsequent plating on agar plates, taking five days without isolate confirmation. The aim of this study was to determine if a polymerase chain reaction (PCR) assay could be used for analysis of the first and second enrichment broths, saving four or two days, respectively. In a comprehensive approach involving six European laboratories, PCR and traditional plating of both enrichment broths from the ISO11290-1 method were compared for the detection of L. monocytogenes in 872 food, raw material and processing environment samples from 13 different dairy and meat food chains. After the first and second enrichments, total DNA was extracted from the enriched cultures and analysed for the presence of L. monocytogenes DNA by PCR. DNA extraction by chaotropic solid-phase extraction (spin column-based silica) combined with real-time PCR (RTi-PCR) was required as it was shown that crude DNA extraction applying sonication lysis and boiling followed by traditional gel-based PCR resulted in fewer positive results than plating. The RTi-PCR results were compared to plating, as defined by the ISO11290-1 method. For first and second enrichments, 90% of the samples gave the same results by RTi-PCR and plating, whatever the RTi-PCR method used. For the samples that gave different results, plating was significantly more accurate for detection of positive samples than RTi-PCR from the first enrichment, but RTi-PCR detected a greater number of positive samples than plating from the second enrichment, regardless of the RTi-PCR method used. RTi-PCR was more accurate for non-food contact surface and food contact surface samples than for food and raw material samples especially from the first enrichment, probably because of sample matrix interference. Even though RTi-PCR analysis of the first enrichment showed less positive results than plating, in outbreak scenarios where a rapid result is

  3. Psychrophilic versus psychrotolerant bacteria--occurrence and significance in polar and temperate marine habitats.

    PubMed

    Helmke, E; Weyland, H

    2004-07-01

    The numerical dominance and ecological role of psychrophilic bacteria in bottom sediments, sea ice, surface water and melt pools of the polar oceans were investigated using isolates, colony forming units (CFU) and metabolic activities. All sediment samples of the Southern Ocean studied showed a clear numerical dominance of cold-loving bacteria. In Arctic sediments underlying the influence of cold polar water bodies psychrophiles prevailed also but they were less dominant in sediments influenced by the warm Atlantic Water. A predominance of psychrophiles was further found in consolidated Antarctic sea ice as well as in multiyear Arctic sea ice and in melt pools on top of Arctic ice floes. A less uniform adaptation response was, however, met in polar surface waters. In the very northern part of the Fram Strait (Arctic Ocean) we found bacterial counts and activities at 1 degree C exceeding those at 22 degrees C. In surface water of the Weddell Sea (Southern Ocean) psychrophiles also dominated numerically in early autumn but the dominance declined obviously with the onset of winter-water and a decrease of chlorphyll a. Otherwise in surface water of the Southern Ocean CFUs were higher at 22 degrees C than at 1 degree C while activities were vice versa indicating at least a functional dominance of psychrophiles. Even in the temperate sediments of the German Bight true psychrophiles were present and a clear shift towards cold adapted communities in winter observed. Among the polar bacteria a more pronounced cold adaptation of Antarctic in comparison with Arctic isolates was obtained. The results and literature data indicate that stenothermic cold adapted bacteria play a significant role in the global marine environment. On the basis of the temperature response of our isolates from different habitats it is suggested to expand the definition of Morita in order to meet the cold adaptation strategies of the bacteria in the various cold habitats.

  4. Diversity of microbial eukaryotes in sediment at a deep-sea methane cold seep: surveys of ribosomal DNA libraries from raw sediment samples and two enrichment cultures.

    PubMed

    Takishita, Kiyotaka; Yubuki, Naoji; Kakizoe, Natsuki; Inagaki, Yuji; Maruyama, Tadashi

    2007-07-01

    Recent culture-independent surveys of eukaryotic small-subunit ribosomal DNA (SSU rDNA) from many environments have unveiled unexpectedly high diversity of microbial eukaryotes (microeukaryotes) at various taxonomic levels. However, such surveys were most probably biased by various technical difficulties, resulting in underestimation of microeukaryotic diversity. In the present study on oxygen-depleted sediment from a deep-sea methane cold seep of Sagami Bay, Japan, we surveyed the diversity of eukaryotic rDNA in raw sediment samples and in two enrichment cultures. More than half of all clones recovered from the raw sediment samples were of the basidiomycetous fungus Cryptococcus curvatus. Among other clones, phylotypes of eukaryotic parasites, such as Apicomplexa, Ichthyosporea, and Phytomyxea, were identified. On the other hand, we observed a marked difference in phylotype composition in the enrichment samples. Several phylotypes belonging to heterotrophic stramenopiles were frequently found in one enrichment culture, while a phylotype of Excavata previously detected at a deep-sea hydrothermal vent dominated the other. We successfully established a clonal culture of this excavate flagellate. Since these phylotypes were not identified in the raw sediment samples, the approach incorporating a cultivation step successfully found at least a fraction of the "hidden" microeukaryotic diversity in the environment examined.

  5. Enhanced enrichment of prostate cancer stem-like cells with miniaturized 3D culture in liquid core-hydrogel shell microcapsules

    PubMed Central

    Yu, Jianhua; Lu, Xiongbin; Zynger, Debra L.; He, Xiaoming

    2015-01-01

    Cancer stem-like cells (CSCs) are rare subpopulations of cancer cells that are reported to be responsible for cancer resistance and metastasis associated with conventional cancer therapies. Therefore, effective enrichment/culture of CSCs is of importance to both the understanding and treatment of cancer. However, it usually takes approximately 10 days for the widely used conventional approach to enrich CSCs through the formation of CSC-containing aggregates. Here we report the time can be shortened to 2 days while obtaining prostate CSC-containing aggregates with better quality based on the expression of surface receptor markers, dye exclusion, gene and protein expression, and in vivo tumorigenicity. This is achieved by encapsulating and culturing human prostate cancer cells in the miniaturized 3D liquid core of microcapsules with an alginate hydrogel shell. The miniaturized 3D culture in core–shell microcapsules is an effective strategy for enriching/culturing CSCs in vitro to facilitate cancer research and therapy development. PMID:24952981

  6. Psychrophilic and psychrotolerant fungi on bats and the presence of Geomyces spp. on bat wings prior to the arrival of white nose syndrome.

    PubMed

    Johnson, Lynnaun J A N; Miller, Andrew N; McCleery, Robert A; McClanahan, Rod; Kath, Joseph A; Lueschow, Shiloh; Porras-Alfaro, Andrea

    2013-09-01

    Since 2006, Geomyces destructans, the causative agent of white nose syndrome (WNS), has killed over 5.7 million bats in North America. The current hypothesis suggests that this novel fungus is an invasive species from Europe, but little is known about the diversity within the genus Geomyces and its distribution on bats in the United States. We documented the psychrophilic and psychrotolerant fungal flora of hibernating bats prior to the arrival of WNS using culture-based techniques. A total of 149 cultures, which were obtained from 30 bats in five bat hibernacula located in four caves and one mine, were sequenced for the entire internal transcribed spacer (ITS) nuclear ribosomal DNA (nrDNA) region. Approximately 53 operational taxonomic units (OTUs) at 97% similarity were recovered from bat wings, with the community dominated by fungi within the genera Cladosporium, Fusarium, Geomyces, Mortierella, Penicillium, and Trichosporon. Eleven Geomyces isolates were obtained and placed in at least seven distinct Geomyces clades based on maximum-likelihood phylogenetic analyses. Temperature experiments revealed that all Geomyces strains isolated are psychrotolerant, unlike G. destructans, which is a true psychrophile. Our results confirm that a large diversity of fungi, including several Geomyces isolates, occurs on bats prior to the arrival of WNS. Most of these isolates were obtained from damaged wings. Additional studies need to be conducted to determine potential ecological roles of these abundant Geomyces strains isolated from bats.

  7. Coliforms, Enterococci, Thermodurics, Thermophiles, and Psychrophiles in Untreated Farm Pond Waters

    PubMed Central

    Malaney, G. W.; Weiser, H. H.; Turner, R. O.; Van Horn, Marilyn

    1962-01-01

    Untreated waters from ten farm ponds located in central, north central, southeastern, and southwestern Ohio were examined for numbers of coliforms, enterococci, thermodurics, thermophiles, and psychrophiles. The median population densities per 100 ml water for all ponds were: coliforms, 23; enterococci, 3.6; thermodurics, 6,000; thermophiles, 450; psychrophiles, 1,000. The results indicate that these farm pond waters were only lightly polluted and suggest that farm ponds, properly maintained, are a source of raw water of high bacteriological quality, requiring a minimum of treatment to be made suitable for domestic and livestock purposes. PMID:14468809

  8. Overexpression, purification, and enthalpy of unfolding of ferricytochrome c552 from a psychrophilic microorganism.

    PubMed

    Oswald, Victoria F; Chen, WeiTing; Harvilla, Paul B; Magyar, John S

    2014-02-01

    The psychrophilic, hydrocarbonoclastic microorganism Colwellia psychrerythraea is important in global nutrient cycling and bioremediation. In order to investigate how this organism can live so efficiently at low temperatures (~4°C), thermal denaturation studies of a small electron transfer protein from Colwellia were performed. Colwellia cytochrome c552 was overexpressed in Escherichia coli, isolated, purified, and characterized by UV-visible absorption spectroscopy. The melting temperature (Tm) and the van't Hoff enthalpy (ΔHvH) were determined. These values suggest an unexpectedly high stability for this psychrophilic cytochrome. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Changes in glucose fermentation pathways by an enriched bacterial culture in response to regulated dissolved H2 concentrations.

    PubMed

    Zheng, Hang; Zeng, Raymond J; Duke, Mikel C; O'Sullivan, Cathryn A; Clarke, William P

    2015-06-01

    It is well established that metabolic pathways in the fermentation of organic waste are primarily controlled by dissolved H2 concentrations, but there is no reported study that compares observed and predicted shifts in fermentation pathways induced by manipulating the dissolved H2 concentration. A perfusion system is presented that was developed to control dissolved H2 concentrations in the continuous fermentation of glucose by a culture highly enriched towards Thermoanaerobacterium thermosaccharolyticum (86 ± 9% relative abundance) from an originally diverse consortia in the leachate of a laboratory digester fed with municipal solid waste. Media from a 2.5 L CSTR was drawn through sintered steel membrane filters to retain biomass, allowing vigorous sparging in a separate chamber without cellular disruption. Through a combination of sparging and variations in glucose feeding rate from 0.8 to 0.2 g/L/d, a range of steady state fermentations were performed with dissolved H2 concentrations as low as an equivalent equilibrated H2 partial pressure of 3 kPa. Trends in product formation rates were simulated using a H2 regulation partitioning model. The model correctly predicted the direction of products redistribution in response to H2 concentration changes and the acetate and butyrate formation rates when H2 concentrations were less than 6 kPa. However, the model over-estimated acetate, ethanol and butanol productions at the expense of butyrate production at higher H2 concentrations. The H2 yield at the lowest dissolved H2 concentration was 2.67 ± 0.08 mol H2 /mol glucose, over 300% higher than the yield achieved in a CSTR operated without sparging. © 2014 Wiley Periodicals, Inc.

  10. Isolation and culture of highly enriched populations of Leydig cells from guinea-pig (Cavia porcellus) testes.

    PubMed

    Kukucka, M A; Misra, H P

    1994-01-01

    Leydig cells were isolated from adult male guinea-pig testes using a multi-step procedure involving enzymatic dissociation and Percoll-gradient centrifugation. The following description is the first account of a successful isolation of adolescent guinea-pig Leydig cells. The enriched Leydig-cell preparation routinely isolated from six intact testicles yielded approximately 5.0 x 10(6) +/- 0.7 x 10(6) (+/- SEM) Leydig cells with a viability of 98.0 +/- 0.4% as determined using the trypan-blue exclusion method. The purity of the isolated cell population as assessed by 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) staining averaged 82.5 +/- 0.8%. Under light microscopy, guinea-pig Leydig cells were polyhedral in shape with a large prominent nucleus and a distinct nucleolus. The acidophilic cytoplasm contained numerous lipid-filled vesicles. Ultrastructurally, guinea-pig Leydig cells displayed an eccentrically located ovoid nucleus with dark-staining peripheral heterochromatin. Large quantities of mitochondria, smooth endoplasmic reticulum and particulate-laden lipid droplets were also evident. The steroidogenic potential of the isolated Leydig cells was verified using a maximally stimulating dose of ovine LH (100 ng ml-1) and human CG (200 mIU ml-1). Leydig cells incubated in a shaking (120 cycles min-1) water bath for 3 h at 37 degrees C in capped polypropylene microcentrifuge tubes produced 233 +/- 21 ng and 223 +/- 18 ng testosterone per 1 x 10(6) cells when maximally stimulated with oLH or hCG, respectively. The inclusion of low (1-5 microM) levels of sodium ascorbate during culture enhanced significantly Leydig-cell viability vs. control values.

  11. Preparation of Glycerol-Enriched Yeast Culture and Its Effect on Blood Metabolites and Ruminal Fermentation in Goats

    PubMed Central

    Ye, Gengping; Zhu, Yongxing; Liu, Jin; Chen, Xingxiang; Huang, Kehe

    2014-01-01

    The aim of this study was to isolate a glycerol-producing yeast strain from nature to prepare glycerol-enriched yeast culture (GY), and preliminarily evaluate the effects of GY on blood metabolites and ruminal fermentation in goats. During the trial, six isolates were isolated from unprocessed honey, and only two isolates with higher glycerol yield were identified by analysis of 26S ribosomal DNA sequences. One of the two isolates was identified as Saccharomyces cerevisiae, a direct-fed microbe permitted by the FDA. This isolate was used to prepare GY. The fermentation parameters were optimized through single-factor and orthogonal design methods to maximize the glycerol yield and biomass. The final GY contained 38.7±0.6 g/L glycerol and 12.6±0.5 g/L biomass. In vivo, eight castrated male goats with ruminal fistula were used in a replicated 4×4 Latin square experiment with four consecutive periods of 15 d. Treatments were as follows: control, LGY, MGY, and HGY with 0, 100, 200, and 300 mL GY per goat per day, respectively. The GY was added in two equal portions at 08∶00 and 17∶00 through ruminal fistula. Samples of blood and ruminal fluid were collected on the last one and two days of each period, respectively. Results showed that the plasma concentrations of triglyceride and total cholesterol were not affected by the supplemented GY. Compared with the control, goats supplemented with MGY and HGY had significantly higher (P<0.05) concentrations of plasma glucose and total protein, ruminal volatile fatty acid and molar proportion of propionate, and significantly lower (P<0.05) ruminal pH and ammonia nitrogen. These parameters changed linearly with increasing GY supplementation level (P<0.05). In conclusion, GY has great potential to be developed as a feed additive with dual effects of glycerol and yeast for ruminants. PMID:24709881

  12. Growth of in vitro Oncidesa plantlets cultured under cold cathode fluorescent lamps with super-elevated CO2 enrichment

    PubMed Central

    Norikane, Atsushi; Teixeira da Silva, Jaime A.; Tanaka, Michio

    2013-01-01

    As interest in how to increase biomass production through biotechnological means gains traction, focus is turning towards the use of photoautotrophic micropropagation under elevated levels of carbon dioxide (CO2) to maximize plant growth and productivity. The effect of super-elevated CO2 with cold cathode fluorescent lamps (CCFLs) on the photoautotrophic growth of Oncidesa in vitro has been studied using a gas-permeable film culture vessel, the ‘Vitron’. The growth of Oncidesa (formerly Oncidesa Gower Ramsey ‘U-1’) plantlets on Vacin and Went (VW) medium was stimulated by 10 000 μmol mol−1 CO2. In particular, increasing the photosynthetic photon flux density (PPFD) from 45 to 60 μmol m−2 s−1 under 10 000 μmol mol−1 CO2 in the growth chamber remarkably increased the number of leaves and roots, and shoot and root fresh and dry weights compared with plantlets under the same level of CO2 under low PPFD (45 μmol m−2 s−1). However, there was a remarkable decrease in photosynthetic capacity, and chlorosis and browning of leaves. In stark contrast, plantlets grown on Kyoto medium at 10 000 μmol mol−1 CO2 under high PPFD had a higher photosynthetic rate than plantlets grown on VW medium, and no chlorosis or browning was observed. Furthermore, shoot growth was remarkably enhanced. Therefore, super-elevated CO2 (10 000 μmol mol−1) enrichment and growth under CCFLs can positively affect the efficiency and quality of commercial production of clonal Oncidesa plantlets.

  13. Preparation of glycerol-enriched yeast culture and its effect on blood metabolites and ruminal fermentation in goats.

    PubMed

    Ye, Gengping; Zhu, Yongxing; Liu, Jin; Chen, Xingxiang; Huang, Kehe

    2014-01-01

    The aim of this study was to isolate a glycerol-producing yeast strain from nature to prepare glycerol-enriched yeast culture (GY), and preliminarily evaluate the effects of GY on blood metabolites and ruminal fermentation in goats. During the trial, six isolates were isolated from unprocessed honey, and only two isolates with higher glycerol yield were identified by analysis of 26S ribosomal DNA sequences. One of the two isolates was identified as Saccharomyces cerevisiae, a direct-fed microbe permitted by the FDA. This isolate was used to prepare GY. The fermentation parameters were optimized through single-factor and orthogonal design methods to maximize the glycerol yield and biomass. The final GY contained 38.7±0.6 g/L glycerol and 12.6±0.5 g/L biomass. In vivo, eight castrated male goats with ruminal fistula were used in a replicated 4×4 Latin square experiment with four consecutive periods of 15 d. Treatments were as follows: control, LGY, MGY, and HGY with 0, 100, 200, and 300 mL GY per goat per day, respectively. The GY was added in two equal portions at 08∶00 and 17∶00 through ruminal fistula. Samples of blood and ruminal fluid were collected on the last one and two days of each period, respectively. Results showed that the plasma concentrations of triglyceride and total cholesterol were not affected by the supplemented GY. Compared with the control, goats supplemented with MGY and HGY had significantly higher (P<0.05) concentrations of plasma glucose and total protein, ruminal volatile fatty acid and molar proportion of propionate, and significantly lower (P<0.05) ruminal pH and ammonia nitrogen. These parameters changed linearly with increasing GY supplementation level (P<0.05). In conclusion, GY has great potential to be developed as a feed additive with dual effects of glycerol and yeast for ruminants.

  14. Lipidomic analysis of psychrophilic yeasts cultivated at different temperatures.

    PubMed

    Řezanka, Tomáš; Kolouchová, Irena; Sigler, Karel

    2016-11-01

    Analysis of polar lipids from eight psychrophilic yeasts (Cryptococcus victoriae, Cystofilobasidium capitatum, Holtermaniella wattica, Mrakiella aquatica, M. cryoconiti, Rhodotorula lignophila, Kondoa malvinella and Trichosporon aggtelekiense) grown at 4-28°C by hydrophilic interaction liquid chromatography/high resolution electrospray ionization tandem mass spectrometry determined 17 classes of lipids and identified dozens of molecular species of phospholipids including their regioisomers. Most of the yeasts were able to grow over the whole temperature range, reaching the highest biomass at 4 or 10°C. On temperature drop to 4°C, all eight strains showed a significant decrease of MUFA and a simultaneous increase of PUFA such as α-linolenic acid, the content of which in the biomass reached up to 20%. We also found alterations in the proportions of individual phospholipids (PI, PE and PC), the PC/PE-ratio decreasing with decreasing temperature. With increasing temperature the content of PoO-PC rose while that of LL-PC decreased, the drop in the content of LL-PC being nearly 100-fold while the content of PoO-PC increased more than twice. A change in temperature brought about changes in molecular species of PC (molecular species PO-PC versus OP-PC) as well as PE, i.e. PO-PE and OP-PE. The phase transition temperature of PO-PC differs from OP-PC by 7°C and the difference between PO-PE and OP-PE is some 10°C; we thus assume that the cell compensates for the adverse temperature effect by changing the fatty acids in the sn-1 and sn-2 positions. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Characterization of Fe (III)-reducing enrichment culture and isolation of Fe (III)-reducing bacterium Enterobacter sp. L6 from marine sediment.

    PubMed

    Liu, Hongyan; Wang, Hongyu

    2016-07-01

    To enrich the Fe (III)-reducing bacteria, sludge from marine sediment was inoculated into the medium using Fe (OH)3 as the sole electron acceptor. Efficiency of Fe (III) reduction and composition of Fe (III)-reducing enrichment culture were analyzed. The results indicated that the Fe (III)-reducing enrichment culture with the dominant bacteria relating to Clostridium and Enterobacter sp. had high Fe (III) reduction of (2.73 ± 0.13) mmol/L-Fe (II). A new Fe (III)-reducing bacterium was isolated from the Fe (III)-reducing enrichment culture and identified as Enterobacter sp. L6 by 16S rRNA gene sequence analysis. The Fe (III)-reducing ability of strain L6 under different culture conditions was investigated. The results indicated that strain L6 had high Fe (III)-reducing activity using glucose and pyruvate as carbon sources. Strain L6 could reduce Fe (III) at the range of NaCl concentrations tested and had the highest Fe (III) reduction of (4.63 ± 0.27) mmol/L Fe (II) at the NaCl concentration of 4 g/L. This strain L6 could reduce Fe (III) with unique properties in adaptability to salt variation, which indicated that it can be used as a model organism to study Fe (III)-reducing activity isolated from marine environment. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  16. Improved detection of Burkholderia pseudomallei from non-blood clinical specimens using enrichment culture and PCR: narrowing diagnostic gap in resource-constrained settings.

    PubMed

    Tellapragada, Chaitanya; Shaw, Tushar; D'Souza, Annet; Eshwara, Vandana Kalwaje; Mukhopadhyay, Chiranjay

    2017-07-01

    To evaluate the diagnostic utility of enrichment culture and PCR for improved case detection rates of non-bacteraemic form of melioidosis in limited resource settings. Clinical specimens (n = 525) obtained from patients presenting at a tertiary care hospital of South India with clinical symptoms suggestive of community-acquired pneumonia, lower respiratory tract infections, superficial or internal abscesses, chronic skin ulcers and bone or joint infections were tested for the presence of Burkholderia pseudomallei using conventional culture (CC), enrichment culture (EC) and PCR. Sensitivity, specificity, positive and negative predictive values of CC and PCR were initially deduced using EC as the gold standard method. Further, diagnostic accuracies of all the three methods were analysed using Bayesian latent class modelling (BLCM). Detection rates of B. pseudomallei using CC, EC and PCR were 3.8%, 5.3% and 6%, respectively. Diagnostic sensitivities and specificities of CC and PCR were 71.4, 98.4% and 100 and 99.4%, respectively in comparison with EC as the gold standard test. With Bayesian latent class modelling, EC and PCR demonstrated sensitivities of 98.7 and 99.3%, respectively, while CC showed a sensitivity of 70.3% for detection of B. pseudomallei. An increase of 1.6% (95% CI: 1.08-4.32%) in the case detection rate of melioidosis was observed in the study population when EC and/or PCR were used in adjunct to the conventional culture technique. Our study findings underscore the diagnostic superiority of enrichment culture and/or PCR over conventional microbiological culture for improved case detection of melioidosis from non-blood clinical specimens. © 2017 John Wiley & Sons Ltd.

  17. Efficient aspartic acid production by a psychrophile-based simple biocatalyst.

    PubMed

    Tajima, Takahisa; Hamada, Mai; Nakashimada, Yutaka; Kato, Junichi

    2015-10-01

    We previously constructed a Psychrophile-based Simple bioCatalyst (PSCat) reaction system, in which psychrophilic metabolic enzymes are inactivated by heat treatment, and used it here to study the conversion of aspartic acid from fumaric acid mediated by the activity of aspartate ammonia-lyase (aspartase). In Escherichia coli, the biosynthesis of aspartic acid competes with that of L-malic acid produced from fumaric acid by fumarase. In this study, E. coli aspartase was expressed in psychrophilic Shewanella livingstonensis Ac10 heat treated at 50 °C for 15 min. The resultant PSCat could convert fumaric acid to aspartic acid without the formation of L-malic acid because of heat inactivation of psychrophilic fumarase activity. Furthermore, alginate-immobilized PSCat produced high yields of aspartic acid and could be re-used nine times. The results of our study suggest that PSCat can be applied in biotechnological production as a new approach to increase the yield of target compounds.

  18. Metabolic Influence of Psychrophilic Diatoms on Travertines at the Huanglong Natural Scenic District of China

    PubMed Central

    Sun, Shiyong; Dong, Faqin; Ehrlich, Hermann; Zhao, Xueqing; Liu, Mingxue; Dai, Qunwei; Li, Qiongfang; An, Dejun; Dong, Hailiang

    2014-01-01

    Diatoms are a highly diversified group of algae that are widely distributed in aquatic ecosystems, and various species have different nutrient and temperature requirements for optimal growth. Here, we describe unusual psychrophilic diatoms of Cymbella in a travertine deposition environment in southwestern China in winter season. Travertine surfaces are colonized by these psychrophilic diatoms, which form biofilms of extracellular polysaccharide substances (EPS) with active metabolic activities in extremely cold conditions. The travertine in Huanglong, is a typical single crystalline calcite with anisotropic lattice distortions of unit cell parameters along axes of a and c, and this structure is suggestive of some level of metabolic mediation on mineralization. Near-edge X-ray absorption fine structure spectroscopy (NEXAFS) results further confirmed the occurrence of biogenic distortion of the crystal lattice of travertine calcite. Overall, our results imply that the metabolic influence of psychrophilic diatoms may be particularly important for promoting formation and dissolution of travertine in extremely cold environments of Huanglong. The EPS of psychrophilic diatoms will protect travertine from HCO3− etching and provide template for forming travertine when water re-flowing, in warm season. PMID:25522049

  19. Metabolic influence of psychrophilic diatoms on travertines at the Huanglong Natural Scenic District of China.

    PubMed

    Sun, Shiyong; Dong, Faqin; Ehrlich, Hermann; Zhao, Xueqing; Liu, Mingxue; Dai, Qunwei; Li, Qiongfang; An, Dejun; Dong, Hailiang

    2014-12-01

    Diatoms are a highly diversified group of algae that are widely distributed in aquatic ecosystems, and various species have different nutrient and temperature requirements for optimal growth. Here, we describe unusual psychrophilic diatoms of Cymbella in a travertine deposition environment in southwestern China in winter season. Travertine surfaces are colonized by these psychrophilic diatoms, which form biofilms of extracellular polysaccharide substances (EPS) with active metabolic activities in extremely cold conditions. The travertine in Huanglong, is a typical single crystalline calcite with anisotropic lattice distortions of unit cell parameters along axes of a and c, and this structure is suggestive of some level of metabolic mediation on mineralization. Near-edge X-ray absorption fine structure spectroscopy (NEXAFS) results further confirmed the occurrence of biogenic distortion of the crystal lattice of travertine calcite. Overall, our results imply that the metabolic influence of psychrophilic diatoms may be particularly important for promoting formation and dissolution of travertine in extremely cold environments of Huanglong. The EPS of psychrophilic diatoms will protect travertine from HCO3- etching and provide template for forming travertine when water re-flowing, in warm season.

  20. Complete Genome Sequence of Psychrobacter alimentarius PAMC 27889, a Psychrophile Isolated from an Antarctic Rock Sample

    PubMed Central

    Lee, Jaejin; Kwon, Miye; Yang, Jae Young; Woo, Jusun; Lee, Hong Kum; Hong, Soon Gyu

    2016-01-01

    Psychrobacter alimentarius PAMC 27889, a Gram-negative, psychrophilic bacterium, was isolated from an Antarctic rock sample. Here, we report the complete genome of P. alimentarius PAMC 27889, which has the nonmevalonate methylerythritol phosphate pathway of terpenoid biosynthesis and a complete gene cluster for benzoate degradation. PMID:27445386

  1. Thermal adaptation in yeast: obligate psychrophiles are obligate aerobes, and obligate thermophiles are facultative anaerobes.

    PubMed Central

    Watson, K; Arthur, H; Morton, H

    1978-01-01

    The obligate psychrophilic yeasts Torulopsis psychrophila, T. austromarina, Leucosporidium frigidum, L. gelidum, and L. nivalis were obligate aerobes and were unable to grow anaerobically. In contrast, the obligate thermophilic yeasts T. bovina, T. pintolopesii, Candida slooffii, and Saccharomyces telluris were facultative anaerobes. PMID:568620

  2. Genome Sequence of the Antarctic Psychrophile Bacterium Planococcus antarcticus DSM 14505

    PubMed Central

    Margolles, Abelardo; Gueimonde, Miguel

    2012-01-01

    Planococcus antarcticus DSM 14505 is a psychrophile bacterium that was isolated from cyanobacterial mat samples, originally collected from ponds in McMurdo, Antarctica. This orange-pigmented bacterium grows at 4°C and may possess interesting enzymatic activities at low temperatures. Here we report the first genomic sequence of P. antarcticus DSM 14505. PMID:22843594

  3. High Frequency of Thermodesulfovibrio spp. and Anaerolineaceae in Association with Methanoculleus spp. in a Long-Term Incubation of n-Alkanes-Degrading Methanogenic Enrichment Culture

    PubMed Central

    Liang, Bo; Wang, Li-Ying; Zhou, Zhichao; Mbadinga, Serge M.; Zhou, Lei; Liu, Jin-Feng; Yang, Shi-Zhong; Gu, Ji-Dong; Mu, Bo-Zhong

    2016-01-01

    In the present study, the microbial community and functional gene composition of a long-term active alkane-degrading methanogenic culture was established after two successive enrichment culture transfers and incubated for a total period of 1750 days. Molecular analysis was conducted after the second transfer (incubated for 750 days) for both the active alkanes-degrading methanogenic enrichment cultures (T2-AE) and the background control (T2-BC). A net increase of methane as the end product was detected in the headspace of the enrichment cultures amended with long-chain n-alkanes and intermediate metabolites, including octadecanoate, hexadecanoate, isocaprylate, butyrate, isobutyrate, propionate, acetate, and formate were measured in the liquid cultures. The composition of microbial community shifted through the successive transfers over time of incubation. Sequences of bacterial and archaeal 16S rRNA gene (16S rDNA) and mcrA functional gene indicated that bacterial sequences affiliated to Thermodesulfovibrio spp. and Anaerolineaceae and archaeal sequences falling within the genus Methanoculleus were the most frequently encountered and thus represented the dominant members performing the anaerobic degradation of long-chain n-alkanes and methanogenesis. In addition, the presence of assA functional genes encoding the alkylsuccinate synthase α subunit indicated that fumarate addition mechanism could be considered as a possible initial activation step of n-alkanes in the present study. The succession pattern of microbial communities indicates that Thermodesulfovibrio spp. could be a generalist participating in the metabolism of intermediates, while Anaerolineaceae plays a key role in the initial activation of long-chain n-alkane biodegradation. PMID:27695441

  4. Effect of temperature on sulphate reduction, growth rate and growth yield in five psychrophilic sulphate-reducing bacteria from Arctic sediments.

    PubMed

    Knoblauch, C; Jørgensen, B B

    1999-10-01

    Five psychrophilic sulphate-reducing bacteria (strains ASv26, LSv21, PSv29, LSv54 and LSv514) isolated from Arctic sediments were examined for their adaptation to permanently low temperatures. All strains grew at -1.8 degrees C, the freezing point of sea water, but their optimum temperature for growth (T(opt)) were 7 degrees C (PSv29), 10 degrees C (ASv26, LSv54) and 18 degrees C (LSv21, LSv514). Although T(opt) was considerably above the in situ temperatures of their habitats (-1.7 degrees C and 2.6 degrees C), relative growth rates were still high at 0 degrees C, accounting for 25-41% of those at T(opt). Short-term incubations of exponentially growing cultures showed that the highest sulphate reduction rates occurred 2-9 degrees C above T(opt). In contrast to growth and sulphate reduction rates, growth yields of strains ASv26, LSv54 and PSv29 were almost constant between -1.8 degrees C and T(opt). For strains LSv21 and LSv514, however, growth yields were highest at the lowest temperatures, around 0 degrees C. The results indicate that psychrophilic sulphate-reducing bacteria are specially adapted to permanently low temperatures by high relative growth rates and high growth yields at in situ conditions.

  5. Composting-Like Conditions Are More Efficient for Enrichment and Diversity of Organisms Containing Cellulase-Encoding Genes than Submerged Cultures

    PubMed Central

    Fayolle-Guichard, Françoise; Lombard, Vincent; Hébert, Agnès; Coutinho, Pedro M.; Groppi, Alexis; Barre, Aurélien; Henrissat, Bernard

    2016-01-01

    Cost-effective biofuel production from lignocellulosic biomass depends on efficient degradation of the plant cell wall. One of the major obstacles for the development of a cost-efficient process is the lack of resistance of currently used fungal enzymes to harsh conditions such as high temperature. Adapted, thermophilic microbial communities provide a huge reservoir of potentially interesting lignocellulose-degrading enzymes for improvement of the cellulose hydrolysis step. In order to identify such enzymes, a leaf and wood chip compost was enriched on a mixture of thermo-chemically pretreated wheat straw, poplar and Miscanthus under thermophile conditions, but in two different set-ups. Unexpectedly, metagenome sequencing revealed that incubation of the lignocellulosic substrate with compost as inoculum in a suspension culture resulted in an impoverishment of putative cellulase- and hemicellulase-encoding genes. However, mimicking composting conditions without liquid phase yielded a high number and diversity of glycoside hydrolase genes and an enrichment of genes encoding cellulose binding domains. These identified genes were most closely related to species from Actinobacteria, which seem to constitute important players of lignocellulose degradation under the applied conditions. The study highlights that subtle changes in an enrichment set-up can have an important impact on composition and functions of the microcosm. Composting-like conditions were found to be the most successful method for enrichment in species with high biomass degrading capacity. PMID:27936240

  6. Composting-Like Conditions Are More Efficient for Enrichment and Diversity of Organisms Containing Cellulase-Encoding Genes than Submerged Cultures.

    PubMed

    Heiss-Blanquet, Senta; Fayolle-Guichard, Françoise; Lombard, Vincent; Hébert, Agnès; Coutinho, Pedro M; Groppi, Alexis; Barre, Aurélien; Henrissat, Bernard

    2016-01-01

    Cost-effective biofuel production from lignocellulosic biomass depends on efficient degradation of the plant cell wall. One of the major obstacles for the development of a cost-efficient process is the lack of resistance of currently used fungal enzymes to harsh conditions such as high temperature. Adapted, thermophilic microbial communities provide a huge reservoir of potentially interesting lignocellulose-degrading enzymes for improvement of the cellulose hydrolysis step. In order to identify such enzymes, a leaf and wood chip compost was enriched on a mixture of thermo-chemically pretreated wheat straw, poplar and Miscanthus under thermophile conditions, but in two different set-ups. Unexpectedly, metagenome sequencing revealed that incubation of the lignocellulosic substrate with compost as inoculum in a suspension culture resulted in an impoverishment of putative cellulase- and hemicellulase-encoding genes. However, mimicking composting conditions without liquid phase yielded a high number and diversity of glycoside hydrolase genes and an enrichment of genes encoding cellulose binding domains. These identified genes were most closely related to species from Actinobacteria, which seem to constitute important players of lignocellulose degradation under the applied conditions. The study highlights that subtle changes in an enrichment set-up can have an important impact on composition and functions of the microcosm. Composting-like conditions were found to be the most successful method for enrichment in species with high biomass degrading capacity.

  7. Multiplexed Single Intact Cell Droplet Digital PCR (MuSIC ddPCR) Method for Specific Detection of Enterohemorrhagic E. coli (EHEC) in Food Enrichment Cultures.

    PubMed

    McMahon, Tanis C; Blais, Burton W; Wong, Alex; Carrillo, Catherine D

    2017-01-01

    Foodborne illness attributed to enterohemorrhagic E. coli (EHEC), a highly pathogenic subset of Shiga toxin-producing E. coli (STEC), is increasingly recognized as a significant public health issue. Current microbiological methods for identification of EHEC in foods often use PCR-based approaches to screen enrichment broth cultures for characteristic gene markers [i.e., Shiga toxin (stx) and intimin (eae)]. However, false positives arise when complex food matrices, such as beef, contain mixtures of eae-negative STEC and eae-positive E. coli, but no EHEC with both markers in a single cell. To reduce false-positive detection of EHEC in food enrichment samples, a Multiplexed, Single Intact Cell droplet digital PCR (MuSIC ddPCR) assay capable of detecting the co-occurrence of the stx and eae genes in a single bacterial cell was developed. This method requires: (1) dispersal of intact bacteria into droplets; (2) release of genomic DNA (gDNA) by heat lysis; and (3) amplification and detection of genetic targets (stx and eae) using standard TaqMan chemistries with ddPCR. Performance of the method was tested with panels of EHEC and non-target E. coli. By determining the linkage (i.e., the proportion of droplets in which stx and eae targets were both amplified), samples containing EHEC (typically greater than 20% linkage) could be distinguished from samples containing mixtures of eae-negative STEC and eae-positive E. coli (0-2% linkage). The use of intact cells was necessary as this linkage was not observed with gDNA extracts. EHEC could be accurately identified in enrichment broth cultures containing excess amounts of background E. coli and in enrichment cultures derived from ground beef/pork and leafy-green produce samples. To our knowledge, this is the first report of dual-target detection in single bacterial cells using ddPCR. The application of MuSIC ddPCR to enrichment-culture screening would reduce false-positives, thereby improving the cost, speed, and accuracy of

  8. Multiplexed Single Intact Cell Droplet Digital PCR (MuSIC ddPCR) Method for Specific Detection of Enterohemorrhagic E. coli (EHEC) in Food Enrichment Cultures

    PubMed Central

    McMahon, Tanis C.; Blais, Burton W.; Wong, Alex; Carrillo, Catherine D.

    2017-01-01

    Foodborne illness attributed to enterohemorrhagic E. coli (EHEC), a highly pathogenic subset of Shiga toxin-producing E. coli (STEC), is increasingly recognized as a significant public health issue. Current microbiological methods for identification of EHEC in foods often use PCR-based approaches to screen enrichment broth cultures for characteristic gene markers [i.e., Shiga toxin (stx) and intimin (eae)]. However, false positives arise when complex food matrices, such as beef, contain mixtures of eae-negative STEC and eae-positive E. coli, but no EHEC with both markers in a single cell. To reduce false-positive detection of EHEC in food enrichment samples, a Multiplexed, Single Intact Cell droplet digital PCR (MuSIC ddPCR) assay capable of detecting the co-occurrence of the stx and eae genes in a single bacterial cell was developed. This method requires: (1) dispersal of intact bacteria into droplets; (2) release of genomic DNA (gDNA) by heat lysis; and (3) amplification and detection of genetic targets (stx and eae) using standard TaqMan chemistries with ddPCR. Performance of the method was tested with panels of EHEC and non-target E. coli. By determining the linkage (i.e., the proportion of droplets in which stx and eae targets were both amplified), samples containing EHEC (typically greater than 20% linkage) could be distinguished from samples containing mixtures of eae-negative STEC and eae-positive E. coli (0–2% linkage). The use of intact cells was necessary as this linkage was not observed with gDNA extracts. EHEC could be accurately identified in enrichment broth cultures containing excess amounts of background E. coli and in enrichment cultures derived from ground beef/pork and leafy-green produce samples. To our knowledge, this is the first report of dual-target detection in single bacterial cells using ddPCR. The application of MuSIC ddPCR to enrichment-culture screening would reduce false-positives, thereby improving the cost, speed, and accuracy of

  9. Enrichment of prostate cancer stem-like cells from human prostate cancer cell lines by culture in serum-free medium and chemoradiotherapy.

    PubMed

    Wang, Lei; Huang, Xing; Zheng, Xinmin; Wang, Xinghuan; Li, Shiwen; Zhang, Lin; Yang, Zhonghua; Xia, Zhiping

    2013-01-01

    The discovery of rare subpopulations of cancer stem cells (CSCs) has created a new focus in cancer research. As CSCs demonstrate resistance to chemoradiation therapy relative to other cancer cells, this allows the enrichment of CSC populations by killing apoptosis-susceptible cancer cells. In this study, three commonly used human prostate cancer (PCa) cell lines (DU145, PC-3 and LNCaP) were examined for their expression of the putative stem cell markers CD133 and CD44 via flow cytometric analysis. Under normal culture conditions, CD133(+)/CD44(+) cells were only present in the DU145 cell line, and comprised only a minor percentage (0.1% ± 0.01%) of the total population. However, the proportion of these CD133(+)/CD44(+) prostate CSCs could be increased in these cell lines via culture in serum-free medium (SFM), or through chemotherapy or radiotherapy. Indeed, after culture in SFM, the proportion of CD133(+)/CD44(+) cells in DU145 and PC-3 had increased to 10.3% and 3.0%, respectively. Moreover, the proportion had increased to 9.8% enriched by chemotherapy and 3.5% by radiotherapy in DU145. Colony-formation tests, cell invasion assays, and tumor xenografts in BALB/c nude mice were used to evaluate the stem cell properties of CD133(+)/CD44(+) PCa cells that were isolated via fluorescence-activated cell sorting (FACS). CD133(+)/CD44(+) cells had an enhanced colony-formation capability and invasive ability in vitro, and displayed greater tumorigenic properties in vivo. These results demonstrate the presence of CD133(+)/CD44(+) prostate CSCs in established PCa cell lines and that populations of these cells can be enriched by culture in SFM or chemoradiotherapy. Finding novel therapies to override chemoradiation resistance in the prostate CSCs is the key to improve long-term results in PCa management.

  10. Evidence for cooperative mineralization of diuron by Arthrobacter sp. BS2 and Achromobacter sp. SP1 isolated from a mixed culture enriched from diuron exposed environments.

    PubMed

    Devers-Lamrani, Marion; Pesce, Stéphane; Rouard, Nadine; Martin-Laurent, Fabrice

    2014-12-01

    Diuron was found to be mineralized in buffer strip soil (BS) and in the sediments (SED) of the Morcille river in the Beaujolais vineyard repeatedly treated with this herbicide. Enrichment cultures from BS and SED samples led to the isolation of three bacterial strains transforming diuron to 3,4-dichloroaniline (3,4-DCA) its aniline derivative. 16S rRNA sequencing revealed that they belonged to the genus Arthrobacter (99% of similarity to Arthrobacter globiformis strain K01-01) and were designated as Arthrobacter sp. BS1, BS2 and SED1. Diuron-degrading potential characterized by sequencing of the puhA gene, characterizing the diuron-degradaing potential, revealed 99% similarity to A. globiformis strain D47 puhA gene isolated a decade ago in the UK. These isolates were also able to use chlorotoluron for their growth. Although able to degrade linuron and monolinuron to related aniline derivatives they were not growing on them. Enrichment cultures led to the isolation of a strain from the sediments entirely degrading 3,4-DCA. 16S rRNA sequence analysis showed that it was affiliated to the genus Achromobacter (99% of similarity to Achromobacter sp. CH1) and was designated as Achromobacter sp. SP1. The dcaQ gene encoding enzyme responsible for the transformation of 3,4-DCA to chlorocatechol was found in SP1 with 99% similarity to that of Comamonas testosteroni WDL7. This isolate also used for its growth a range of anilines (3-chloro-4-methyl-aniline, 4-isopropylaniline, 4-chloroaniline, 3-chloroaniline, 4-bromoaniline). The mixed culture composed of BS2 and SP1 strains entirely mineralizes (14)C-diuron to (14)CO2. Diuron-mineralization observed in the enrichment culture could result from the metabolic cooperation between these two populations. Copyright © 2014. Published by Elsevier Ltd.

  11. Comparison of culture media for enrichment and isolation of Salmonella spp. from frozen Channel catfish and Vietnamese basa fillets.

    PubMed

    Pal, Amit; Marshall, Douglas L

    2009-05-01

    Frozen fillets of Channel catfish and Vietnamese basa fish were used to compare Salmonella spp. recovery effectiveness of selective enrichment in Rappaport-Vassiliadis (RV) broth and tetrathionate broth (TT) and selective isolation on Hekteon enteric (HE) agar, xylose lysine deoxycholate (XLD) agar, and bismuth sulfite (BS) agar. Isolate confirmation was through fatty acid methyl ester analysis. Of 60 samples analyzed, 25 were found contaminated with Salmonella (42% incidence). Salmonella spp. recovery after enrichment in RV medium was 35% on HE agar, 30% on XLD agar, and 42% on BS agar. Similarly, after enrichment in TT broth, HE and XLD agars recovered 22% each and BS agar recovered 37%. No performance difference (p>0.05) was observed in the recovery of Salmonella using the combinations of BS, HE, and XLD agars with RV broth and BS agar with TT broth. The combination of selective enrichment in RV and selective isolation on BS gave numerically greatest isolation of Salmonella from Channel catfish and Vietnamese basa fish compared to other isolation combinations.

  12. Bioconversion of H2/CO 2 by acetogen enriched cultures for acetate and ethanol production: the impact of pH.

    PubMed

    Xu, Shuyun; Fu, Bo; Zhang, Lijuan; Liu, He

    2015-06-01

    Syngas fermentation into ethanol and other bioproducts by mixed cultures is considered a promising biotechnology. Effects of pH on product generation and microbial community during H2/CO2 utilization by acetogen enrichment cultures were investigated in this work. The maximum acetate concentration reached 95.41 mmol L(-1) at pH 7, which was 71.7, 21.8 and 50.9% higher than at pH 5, 9 and 11, respectively. The maximum ethanol concentration at pH 7 was 45.7, 50, 72% higher than that at pH 5, 9 and 11, respectively. The CO dehydrogenase (CODH) gene copy number was highest at pH 7, indicating that metabolically active acetogens reached their highest level at pH 7. The CODH gene copy number at pH 9 was lower than at pH 7, but higher than at pH 5 and 11. Correspondingly, the enrichment cultures at pH 7 had the highest species richness and diversity, while those at pH 9 had the second highest diversity, and those at pH 5 and 11 had the lowest diversity. The shift in microbial community structure and the different active acetogen contents resulting from different pHs were responsible for the differences in acetate and ethanol production.

  13. The influence of IgM-enriched immunoglobulin therapy on neonatal mortality and hematological variables in newborn infants with blood culture-proven sepsis.

    PubMed

    Abbasoğlu, Aslıhan; Ecevit, Ayşe; Tuğcu, Ali Ulaş; Yapakçı, Ece; Tekindal, Mustafa Agah; Tarcan, Aylin; Ecevit, Zafer

    2014-01-01

    The aim of this study was to determine the effects of adjuvant immunoglobulin M (IgM)-enriched intravenous immunoglobulin (IVIG) therapy on mortality rate, hematological variables and length of hospital stay in newborn infants with blood culture-proven sepsis. Demographic and clinical features and outcome measures of 63 newborn infants with blood culture-proven sepsis were documented retrospectively from the medical records. The patients were divided into two groups according to their treatment history. The patients in Group 1 received antibiotic therapy only and the patients in Group 2 received both antibiotic and adjuvant IgMenriched IVIG. The study revealed that mortality rates were 28.1% and 12.9% in Group 1 and Group 2, respectively. The mortality rate was lower in Group 2, but the difference between the two groups was not statistically significant (p=0.21). Coagulase-negative Staphylococcus was the most common type of bacteria isolated from the blood culture in both groups. When changing laboratory results were compared between the two groups, hemoglobin, leukocyte count and C-reactive protein levels were different during the first three days of antibiotic treatment. Our study revealed that if diagnosed at an early stage and treated aggressively with appropriate and effective antibiotics, adjuvant IgM-enriched IVIG treatment has no additional benefits in neonatal sepsis.

  14. Biosynthesis of highly enriched 13C-lycopene for human metabolic studies using repeated batch tomato cell culturing with 13C-glucose.

    PubMed

    Moran, Nancy Engelmann; Rogers, Randy B; Lu, Chi-Hua; Conlon, Lauren E; Lila, Mary Ann; Clinton, Steven K; Erdman, John W

    2013-08-15

    While putative disease-preventing lycopene metabolites are found in both tomato (Solanum lycopersicum) products and in their consumers, mammalian lycopene metabolism is poorly understood. Advances in tomato cell culturing techniques offer an economical tool for generation of highly-enriched (13)C-lycopene for human bioavailability and metabolism studies. To enhance the (13)C-enrichment and yields of labelled lycopene from the hp-1 tomato cell line, cultures were first grown in (13)C-glucose media for three serial batches and produced increasing proportions of uniformly labelled lycopene (14.3±1.2%, 39.6±0.5%, and 48.9±1.5%) with consistent yields (from 5.8 to 9 mg/L). An optimised 9-day-long (13)C-loading and 18-day-long labelling strategy developed based on glucose utilisation and lycopene yields, yielded (13)C-lycopene with 93% (13)C isotopic purity, and 55% of isotopomers were uniformly labelled. Furthermore, an optimised acetone and hexane extraction led to a fourfold increase in lycopene recovery from cultures compared to a standard extraction.

  15. Epidemiology of Salmonella sp. in California cull dairy cattle: prevalence of fecal shedding and diagnostic accuracy of pooled enriched broth culture of fecal samples

    PubMed Central

    Abu Aboud, Omran A.; Adaska, John M.; Williams, Deniece R.; Rossitto, Paul V.; Champagne, John D.; Lehenbauer, Terry W.; Atwill, Robert; Li, Xunde

    2016-01-01

    Background The primary objective of this cross-sectional study was to estimate the crude, seasonal and cull-reason stratified prevalence of Salmonella fecal shedding in cull dairy cattle on seven California dairies. A secondary objective was to estimate and compare the relative sensitivity (Se) and specificity (Sp) for pools of 5 and 10 enriched broth cultures of fecal samples for Salmonella sp. detection. Methods Seven dairy farms located in the San Joaquin Valley of California were identified and enrolled in the study as a convenience sample. Cull cows were identified for fecal sampling once during each season between 2014 and 2015, specifically during spring, summer, fall, and winter, and 10 cows were randomly selected for fecal sampling at the day of their sale. In addition, study personnel completed a survey based on responses of the herd manager to questions related to the previous four month’s herd management. Fecal samples were frozen until testing for Salmonella. After overnight enrichment in liquid broth, pools of enrichment broth (EBP) were created for 5 and 10 samples. All individual and pooled broths were cultured on selective media with putative Salmonella colonies confirmed by biochemical testing before being serogrouped and serotyped. Results A total of 249 cull cows were enrolled into the study and their fecal samples tested for Salmonella. The survey-weighted period prevalence of fecal shedding of all Salmonella sp. in the cull cow samples across all study herds and the entire study period was 3.42% (N = 249; SE 1.07). The within herd prevalence of Salmonella shed in feces did not differ over the four study seasons (P = 0.074). The Se of culture of EBP of five samples was 62.5% (SE = 17.12), which was not statistically different from the Se of culture of EBP of 10 (37.5%, SE = 17.12, P = 0.48). The Sp of culture of EBP of five samples was 95.24% (SE = 3.29) and for pools of 10 samples was 100.00% (SE = 0). There was no statistical difference

  16. Degradative capacities and 16S rRNA-targeted whole-cell hybridization of sulfate-reducing bacteria in an anaerobic enrichment culture utilizing alkylbenzenes from crude oil.

    PubMed Central

    Rabus, R; Fukui, M; Wilkes, H; Widdle, F

    1996-01-01

    A mesophilic sulfate-reducing enrichment culture growing anaerobically on crude oil was used as a model system to study which nutritional types of sulfate-reducing bacteria may develop on original petroleum constituents in oil wells, tanks, and pipelines. Chemical analysis of oil hydrocarbons during growth revealed depletion of toluene and o-xylene within 1 month and of m-xylene, o-ethyltoluene, m-ethyltoluene, m-propyltoluene, and m-isopropyltoluene within approximately 2 months. In anaerobic counting series, the highest numbers of CFU (6 x 10(6) to 8 x 10(6) CFU ml-1) were obtained with toluene and benzoate. Almost the same numbers were obtained with lactate, a substrate often used for detection of the vibrio-shaped, incompletely oxidizing Desulfovibrio sp. In the present study, however, lactate yielded mostly colonies of oval to rod-shaped, completely oxidizing, sulfate-reducing bacteria which were able to grow slowly on toluene or crude oil. Desulfovibrio species were detected only at low numbers (3 x 10(5) CFU ml-1). In agreement with this finding, a fluorescently labeled, 16S rRNA-targeted oligonucleotide probe described in the literature as specific for members of the Desulfovibrionaceae (suggested family) hybridized only with a small portion (< 5%) of the cells in the enrichment culture. These results are consistent with the observation that known Desulfovibrio species do not utilize aromatic hydrocarbons, the predominant substrates in the enrichment culture. All known sulfate-reducing bacteria which utilize aromatic compounds belong to a separate branch, the Desulfobacteriaceae (suggested family). Most members of this family are complete oxidizers. For specific hybridization with members of this branch, the probe had to be modified by a nucleotide exchange. Indeed, this modified probe hybridized with more than 95% of the cells in the enrichment culture. The results show that completely oxidizing, alkylbenzene-utilizing sulfate-reducing bacteria rather than

  17. The effects of the ratio of propionate to acetate on the transformation and composition of polyhydroxyalkanoates with enriched cultures of glycogen-accumulating organisms.

    PubMed

    Jiang, Yamin; Chen, Yinguang

    2009-03-01

    Studies on the synthesis of polyhydroxyalkanoates (PHA) mainly focus on the enriched cultures of phosphorus-accumulating (PAO) or glycogen-accumulating organisms (GAO) with acetate or propionate as the sole carbon source. However, PHA production with a mixture of propionate and acetate by enriched cultures of GAO has not been reported. It is well known that the physical and mechanical properties of PHA can be improved by incorporating 3-hydroxyvalerate (3HV) into the polymer. To synthesize PHA with a high 3HV percentage, five laboratory-scale sequencing batch reactors were operated with the enriched cultures of GAO using different ratios of propionate and acetate (P/A) as the carbon sources in this study. The effects of P/A ratio on the anaerobic and aerobic transformations, composition and synthesis rate of PHA were studied. The results showed that with the increase in the P/A ratio, the anaerobic yields of total PHA declined from 4.226 to 2.469 mmol-C per gram of volatile suspended solids (VSS), and the 3-hydroxybutyrate (3HB) fraction in PHA decreased from approximately 70 to 10%, along with an increase in 3HV and 3-hydroxy-2-methylvalerate (3H2MV) percentages. When there were equal quantities (mmol-C L(-1)) ofpropionate and acetate, 3HB was produced more quickly than 3H2MV. By increasing P/A ratio from 1/10 to 10/1, the initial synthesis rate of 3H2MV rose from 0.002 to 0.029, while 3HB and the total PHA declined from 0.083 to 0.007 and 0.110 to 0.071 mmol-C g-VSS(-1) min(-1), respectively. Also, it was observed that more 3HV was incorporated into PHA with increasing P/A ratio. This study suggested that when the enriched cultures of GAO were applied to the synthesis of PHA, the composition, yield and synthesis rate of PHA could be manipulated by varying the feed composition.

  18. Alternative anaerobic enrichments to the bacteriological analytical manual culture method for isolation of Shigella sonnei from selected types of fresh produce.

    PubMed

    Jacobson, Andrew P; Thunberg, Richard L; Johnson, Mildred L; Hammack, Thomas S; Andrews, Wallace H

    2004-01-01

    Alternative methods of reducing oxygen during anaerobic enrichment in the Bacteriological Analytical Manual (BAM) Shigella culture method were evaluated and compared to the current and less practical GasPak method. The alternative anaerobic methods included the use of reducing agents in Shigella broth and reducing culture container headspace volume to minimize atmospheric effects on oxygen concentration in Shigella broth during enrichment. The reducing agents evaluated were sodium thioglycollate, L-cystine, L-cysteine, titanium(III) citrate, and dithiothreitol, each at concentrations of 0.1, 0.05, and 0.01%. The use of Oxyrase for Broth with the enrichment medium (Shigella broth) was evaluated at concentrations of 10, 20 and 30 microL/mL. Recoveries of chill- and freeze-stressed S. sonnei strains 357 and 20143 were determined with each anaerobic method, including the GasPak method, using inoculation levels ranging from 10(0)to 10(3) cells. For each anaerobic method, strain, inoculation level, and stress type, 5 replicate enrichments were evaluated by streaking to MacConkey agar for isolation. The numbers of cultures with each method from which S. sonnei was isolated were used to compare the alternative anaerobic methods to the GasPak method. The alternative anaerobic method with which chill- and freeze-stressed S. sonnei strains 357 and 20143 were isolated most consistently was the use of Oxyrase for Broth in Shigella broth at a concentration of 20 microL/mL. This method was compared to the GasPak anaerobic method in evaluations on the recovery of S. sonnei strains 357 and 20143 from artificially contaminated test portions of parsley, cilantro, green onions, strawberries, carrots, and celery. A third anaerobic method included the use of 0.5 cm mineral oil overlay on cultures containing Oxyrase for Broth at concentrations of 20 microL/mL. Recovery rates of strain 357 were significantly greater (p < 0.05) with the GasPak method than with Oxyrase for Broth, with and

  19. Sensitivity of Direct Culture, Enrichment and PCR for Detection of Campylobacter jejuni and C. coli in Broiler Flocks at Slaughter.

    PubMed

    Rodgers, J D; Simpkin, E; Lee, R; Clifton-Hadley, F A; Vidal, A B

    2017-06-01

    Broiler chicken flocks are a significant source of Campylobacter jejuni and Campylobacter coli that result in the major public health problem of campylobacteriosis. Accurate estimates of the prevalence of both C. coli and C. jejuni in flocks would enhance epidemiological understanding, risk assessment and control options. This study combined results from a panel of 10 detection tests (direct culture, enrichment and PCR) on caecal samples from flocks at slaughter. A parallel interpretation approach was used to determine the presence of Campylobacter spp. and for C. jejuni and C. coli individually. The sample was considered positive if at least one method detected the target and this interpretation was taken to represent a 'proxy gold standard' for detection in the absence of a gold standard reference test. The sensitivity of each individual method to detect Campylobacter spp., C. jejuni and C. coli was then estimated relative to the proxy gold standard. Enrichment in adapted Exeter broth (deficient in polymyxin B) with a resuscitation step was 100% sensitive, whilst direct culture on modified charcoal cefoperazone deoxycholate agar (mCCDA) was highly sensitive (97.9%). Enrichment methods using Preston broth and Bolton broth were significantly less sensitive. Enrichment in Exeter broth promoted the recovery of C. jejuni, whilst enrichment in Bolton broth favoured C. coli. A RT-PCR detection test could identify 80% of flocks that were co-colonised with both species. This study found that 76.3% (n = 127) of flocks were colonised with Campylobacter spp. The majority (95.9%) of Campylobacter-positive flocks were colonised with C. jejuni; however, approximately one-third of positive flocks were simultaneously colonised with both C. jejuni and C. coli. The findings highlight the impact of different detection methodologies on the accuracy of the estimated incidence of both C. jejuni and C. coli entering the abattoir within broiler flocks and the associated

  20. DENATURATION AND RENATURATION OF MALIC DEHYDROGENASE IN A CELL-FREE EXTRACT FROM A MARINE PSYCHROPHILE.

    PubMed

    BURTON, S D; MORITA, R Y

    1963-11-01

    Burton, Sheril D. (Oregon State University, Corvallis), and Richard Y. Morita. Denaturation and renaturation of malic dehydrogenase in a cell-free extract from a marine psychrophile. J. Bacteriol. 86:1019-1024. 1963.-Malic dehydrogenase from a marine psychrophilic vibrio (PS 207) was found to be heat-sensitive at 30 C, the maximal growth temperature for the organism. Initial denaturation was reversible, with maximal renaturation occurring when the denatured enzyme was slowly cooled in the presence of mercaptoethanol, reduced nicotinamide adenine dinucleotide, and malate. No renaturation occurred when these compounds were added after slow cooling, or when the renaturation mixture was rapidly cooled. Mercaptoethylamine, cysteine, glutathione, or mercaptoacetic acid could not replace mercaptoethanol. The kinetics of denaturation and renaturation suggest the presence of several malic isozymes each with different heat labilities, or that these processes are occurring in several distinct steps.

  1. Cloning and expression of phosphoglycerate mutase from the psychrophilic yeast, Glaciozyma antarctica PI12

    NASA Astrophysics Data System (ADS)

    Jaafar, Nardiah Rizwana; Bakar, Farah Diba Abu; Murad, Abdul Munir Abdul; Mahadi, Nor Muhammad

    2015-09-01

    The conversion of 3-phosphoglycerate to 2-phosphoglycerate during glycolysis and gluconeogenesis is catalyzed by phosphoglycerate mutase (PGM). Better understanding of metabolic reactions performed by this enzyme has been studied extensively in prokaryotes and eukaryotes. Here, we report a phosphoglycerate mutase from the psychrophilic yeast, Glaciozyma antarctica. cDNA encoding for PGM from G. antarctica PI12, a psychrophilic yeast isolated from sea ice at Casey Station, Antarctica was amplified. The gene was then cloned into a cloning vector and sequenced, which verified its identity as the gene putatively encoding for PGM. The recombinant protein was expressed in Escherichia coli BL21 (DE3) as inclusion bodies and this was confirmed by SDS-PAGE and Western blot.

  2. Neuron-enriched cultures of adult rat dorsal root ganglia: establishment, characterization, survival, and neuropeptide expression in response to trophic factors.

    PubMed

    Grothe, C; Unsicker, K

    1987-01-01

    It is unknown whether adult dorsal root ganglion (DRG) neurons require trophic factors for their survival and maintenance of neuropeptide phenotypes. We have established and characterized neuron-enriched cultures of adult rat DRGs and investigated their responses to nerve growth factor (NGF), ciliary neuronotrophic factor (CNTF), pig brain extract (PBE, crude fraction of brain-derived neuronotrophic factor, BDNF), and laminin (LN). DRGs were dissected from levels C1 through L6 and dissociated and freed from myelin fragments and most satellite (S-100-immunoreactive) cells by centrifugation on Percoll and preplating. The enriched neurons, characterized by their morphology and immunoreactivity for neuron-specific enolase, constituted a population representative of the in vivo situation with regard to expression of substance P (SP), somatostatin (SOM), and cholecystokinin-8 (CCK) immunoreactivities. In the absence of trophic factors and using polyornithine (PORN) as a substratum, 60-70% of the neurons present initially (0.5 days) had died after 7 days. LN as a substratum did not prevent a 30% loss of neurons up to day 4.5, but it subsequently maintained DRG neurons at a plateau. This behavior might reflect a cotrophic effect of LN and factors provided by non-neuronal cells, whose proliferation between 4.5 and 7 days could not be prevented by addition of mitotic inhibitors of gamma-irradiation. CNTF, but not NGF, slightly enhanced survival at 7 days on either PORN or LN. No neuronal losses were found in non-enriched cultures or when enriched neurons were supplemented with PBE, indicating that non-neuronal cells and PBE provide factor(s) essential for adult DRG neuron survival. Proportions of SP-, SOM-, and CCK-immunoreactive cells were unaltered under any experimental condition, with the exception of a numerical decline in SP cells in 7-day cultures with LN, but not PORN, as the substratum. Our data, considered in the context of recent in vivo and vitro studies, suggest

  3. Optimization of cold-adapted lysozyme production from the psychrophilic yeast Debaryomyces hansenii using statistical experimental methods.

    PubMed

    Wang, Quanfu; Hou, Yanhua; Yan, Peisheng

    2012-06-01

    Statistical experimental designs were employed to optimize culture conditions for cold-adapted lysozyme production of a psychrophilic yeast Debaryomyces hansenii. In the first step of optimization using Plackett-Burman design (PBD), peptone, glucose, temperature, and NaCl were identified as significant variables that affected lysozyme production, the formula was further optimized using a four factor central composite design (CCD) to understand their interaction and to determine their optimal levels. A quadratic model was developed and validated. Compared to the initial level (18.8 U/mL), the maximum lysozyme production (65.8 U/mL) observed was approximately increased by 3.5-fold under the optimized conditions. Cold-adapted lysozymes production was first optimized using statistical experimental methods. A 3.5-fold enhancement of microbial lysozyme was gained after optimization. Such an improved production will facilitate the application of microbial lysozyme. Thus, D. hansenii lysozyme may be a good and new resource for the industrial production of cold-adapted lysozymes. © 2012 Institute of Food Technologists®

  4. Dioszegia antarctica sp. nov. and Dioszegia cryoxerica sp. nov., psychrophilic basidiomycetous yeasts from polar desert soils in Antarctica

    USGS Publications Warehouse

    Rodriguez, Russell J.; Connell, L.; Redman, R.; Barrett, A.; Iszard, M.; Fonseca, A.

    2010-01-01

    During a survey of the culturable soil fungal population in samples collected in Taylor Valley, South Victoria Land, Antarctica, 13 basidiomycetous yeast strains with orange-coloured colonies were isolated. Phylogenetic analyses of internal transcribed spacer (ITS) and partial LSU rRNA gene sequences showed that the strains belong to the Dioszegia clade of the Tremellales (Tremellomycetes, Agaricomycotina), but did not correspond to any of the hitherto recognized species. Two novel species, Dioszegia antarctica sp. nov. (type strain ANT-03-116T =CBS 10920T =PYCC 5970T) and Dioszegia cryoxerica sp. nov. (type strain ANT-03-071T =CBS 10919T =PYCC 5967T), are described to accommodate ten and three of these strains, respectively. Analysis of ITS sequences demonstrated intrastrain sequence heterogeneity in D. cryoxerica. The latter species is also notable for producing true hyphae with clamp connections and haustoria. However, no sexual structures were observed. The two novel species can be considered obligate psychrophiles, since they failed to grow above 20 °C and grew best between 10 and 15 °C.

  5. Start-up of a sequential dry anaerobic digestion of paunch under psychrophilic and mesophilic temperatures.

    PubMed

    Nkemka, Valentine Nkongndem; Hao, Xiying

    2016-06-21

    The present laboratory study evaluated the sequential leach bed dry anaerobic digestion (DAD) of paunch under psychrophilic (22°C) and mesophilic (40°C) temperatures. Three leach bed reactors were operated under the mesophilic temperature in sequence at a solid retention time (SRT) of 40d with a new batch started 27d into the run of the previous one. A total of six batches were operated for 135d. The results showed that the mesophilic DAD of paunch was efficient, reaching methane yields of 126.9-212.1mLg(-1) volatile solid (VS) and a VS reduction of 32.9-55.5%. The average daily methane production rate increased from 334.3mLd(-1) to 571.4mLd(-1) and 825.7mLd(-1) when one, two and three leach bed reactors were in operation, respectively. The psychrophilic DAD of paunch was operated under a SRT of 100d and a total of three batches were performed in sequence for 300d with each batch starting after completion of the previous one. Improvements in the methane yield from 93.9 to 107.3 and 148.3mLg(-1) VS and VS reductions of 24.8, 30.2 and 38.6% were obtained in the consecutive runs, indicating the adaptation of anaerobic microbes from mesophilic to psychrophilic temperatures. In addition, it took three runs for anaerobic microbes to reduce the volatile fatty acid accumulation observed in the first and second trials. This study demonstrates the potential of renewable energy recovery from paunch under psychrophilic and mesophilic temperatures. Copyright © 2016. Published by Elsevier Ltd.

  6. Study of psychrophilic and psychrotolerant micro-organisms isolated in cold rooms used for pharmaceutical processing.

    PubMed

    Sandle, T; Skinner, K

    2013-04-01

    To examine for psychrophilic or psychrotolerant micro-organisms in pharmaceutical cold rooms (in relation to numbers, incidents and species) and to determine, where such micro-organisms are present, whether standard microbiological environmental monitoring regimes require modification. This is presented as a case study. Comparative environmental monitoring within different pharmaceutical facility cold rooms (using standard mesophilic and low temperature incubation). Data were collected over two periods, 5 years apart. The results indicated that psychrophilic micro-organisms were not present and that those micro-organisms deemed psychrotolerant, primarily pseudomonads, could be grown on standard media under mesophilic conditions. Psychrophilic micro-organisms were not detected and those considered to be psychrotolerant were only found in low numbers. Pyschrotolerant organisms were recovered under both low temperature incubation conditions and under standard conditions (between 20 and 35°C). Further evaluation may be required, using alternative agar, and microbiologists should regularly review the species recovered to note differences between different environments. The study came about from requests made by US and UK regulators concerning the risk of any extremophiles present in pharmaceutical manufacturing facilities upon product safety. Regulators expressed concerns about whether standard, and accepted, environmental monitoring regimes were capable of detecting such micro-organisms. The data provide a benchmark to support pharmaceutical manufacturers in relation to their existing monitoring programmes or as a case study with which to undertake a similar study. © 2012 The Society for Applied Microbiology.

  7. Endotoxin structures in the psychrophiles Psychromonas marina and Psychrobacter cryohalolentis contain distinctive acyl features.

    PubMed

    Sweet, Charles R; Alpuche, Giancarlo M; Landis, Corinne A; Sandman, Benjamin C

    2014-07-09

    Lipid A is the essential component of endotoxin (Gram-negative lipopolysaccharide), a potent immunostimulatory compound. As the outer surface of the outer membrane, the details of lipid A structure are crucial not only to bacterial pathogenesis but also to membrane integrity. This work characterizes the structure of lipid A in two psychrophiles, Psychromonas marina and Psychrobacter cryohalolentis, and also two mesophiles to which they are related using MALDI-TOF MS and fatty acid methyl ester (FAME) GC-MS. P. marina lipid A is strikingly similar to that of Escherichia coli in organization and total acyl size, but incorporates an unusual doubly unsaturated tetradecadienoyl acyl residue. P. cryohalolentis also shows structural organization similar to a closely related mesophile, Acinetobacter baumannii, however it has generally shorter acyl constituents and shows many acyl variants differing by single methylene (-CH2-) units, a characteristic it shares with the one previously reported psychrotolerant lipid A structure. This work is the first detailed structural characterization of lipid A from an obligate psychrophile and the second from a psychrotolerant species. It reveals distinctive structural features of psychrophilic lipid A in comparison to that of related mesophiles which suggest constitutive adaptations to maintain outer membrane fluidity in cold environments.

  8. Comparison of Enrichment Procedures for Fluorescent Antibody and Cultural Detection of Salmonellae in Raw Meat and Poultry

    PubMed Central

    Thomason, Berenice M.; Dodd, David J.

    1976-01-01

    No advantage was shown in preenriching raw meat samples for detecting salmonellae by fluorescent antibodies or culture. Trypticase soy-tryptose (Edwards and Ewing, 1972) was equal to or better than selenite-cystine as a postenrichment broth. PMID:776088

  9. Relative binding and biochemical effects of heterodimeric and homodimeric isoforms of platelet-derived growth factor in osteoblast-enriched cultures from fetal rat bone

    SciTech Connect

    Centrella, M.; McCarthy, T.L.; Kusmik, W.F.; Canalis, E. )

    1991-06-01

    Platelet-derived growth factor (PDGF) exists as a homodimer or a heterodimer comprising either PDGF-A or PDGF-B subunits, and each isoform occurs in various tissues, including bone. Although the stimulatory effects of PDGF-BB have been studied in cultures of bone cells and intact bone fragments, the influence of other isoforms that may arise locally or systematically in vivo, has not been reported. Therefore recombinant human PDGF-BB, PDGF-AB, and PDGF-AA were evaluated in osteoblast-enriched cultures from fetal rat bone. Within 24 hours these factors produced a graded response in bone cell DNA and protein synthesis, with half-maximal effects at approximately 0.6, 2.1, and 4.8 nM PDGF-BB, PDGF-AB, and PDGF-AA, respectively. Increases in collagen and noncollagen protein synthesis were abrogated when DNA synthesis was blocked with hydroxyurea. Furthermore, each factor reduced alkaline phosphatase activity, PDGF-BB being the most inhibitory. Binding studies with 125I-PDGF-BB or 125I-PDGF-AA and each unlabeled PDGF isoform produced discrete ligand binding and displacement patterns: 125I-PDGF-BB binding was preferentially displaced by PDGF-BB (Ki approximately 0.7 nM), less by PDGF-AB (Ki approximately 2.3 nM) and poorly by PDGF-AA. In contrast, 125I-PDGF-AA binding was measurably reduced by PDGF-AA (Ki approximately 4.0 nM), but was more effectively displaced by PDGF-BB or PDGF-AB (each with Ki approximately 0.7 nM). These studies indicate that each PDGF isoform produces biochemical effects proportional to binding site occupancy and suggest that receptors that favor PDGF-B subunit binding preferentially mediate these results in osteoblast-enriched bone cell cultures.

  10. Comparative analysis of 16S rRNA and amoA genes from archaea selected with organic and inorganic amendments in enrichment culture.

    PubMed

    Xu, Mouzhong; Schnorr, Jon; Keibler, Brandon; Simon, Holly M

    2012-04-01

    We took advantage of a plant-root enrichment culture system to characterize mesophilic soil archaea selected through the use of organic and inorganic amendments. Comparative analysis of 16S rRNA and amoA genes indicated that specific archaeal clades were selected under different conditions. Three amoA sequence clades were identified, while for a fourth group, identified by 16S rRNA gene analysis alone and referred to as the "root" clade, we detected no corresponding amoA gene. The amoA-containing archaea were present in media with either organic or inorganic amendments, whereas archaea representing the root clade were present only when organic amendment was used. Analysis of amoA gene abundance and expression, together with nitrification-coupled growth assays, indicated potential growth by autotrophic ammonia oxidation for members of two group 1.1b clades. Increased abundance of one of these clades, however, also occurred upon the addition of organic amendment. Finally, although amoA-containing group 1.1a archaea were present in enrichments, we detected neither expression of amoA genes nor evidence for nitrification-coupled growth of these organisms. These data support a model of a diverse metabolic community in mesophilic soil archaea that is just beginning to be characterized.

  11. Comparative Analysis of 16S rRNA and amoA Genes from Archaea Selected with Organic and Inorganic Amendments in Enrichment Culture

    PubMed Central

    Xu, Mouzhong; Schnorr, Jon; Keibler, Brandon

    2012-01-01

    We took advantage of a plant-root enrichment culture system to characterize mesophilic soil archaea selected through the use of organic and inorganic amendments. Comparative analysis of 16S rRNA and amoA genes indicated that specific archaeal clades were selected under different conditions. Three amoA sequence clades were identified, while for a fourth group, identified by 16S rRNA gene analysis alone and referred to as the “root” clade, we detected no corresponding amoA gene. The amoA-containing archaea were present in media with either organic or inorganic amendments, whereas archaea representing the root clade were present only when organic amendment was used. Analysis of amoA gene abundance and expression, together with nitrification-coupled growth assays, indicated potential growth by autotrophic ammonia oxidation for members of two group 1.1b clades. Increased abundance of one of these clades, however, also occurred upon the addition of organic amendment. Finally, although amoA-containing group 1.1a archaea were present in enrichments, we detected neither expression of amoA genes nor evidence for nitrification-coupled growth of these organisms. These data support a model of a diverse metabolic community in mesophilic soil archaea that is just beginning to be characterized. PMID:22267662

  12. Insights into Nitrate-Reducing Fe(II) Oxidation Mechanisms through Analysis of Cell-Mineral Associations, Cell Encrustation, and Mineralogy in the Chemolithoautotrophic Enrichment Culture KS.

    PubMed

    Nordhoff, M; Tominski, C; Halama, M; Byrne, J M; Obst, M; Kleindienst, S; Behrens, S; Kappler, A

    2017-07-01

    Most described nitrate-reducing Fe(II)-oxidizing bacteria (NRFeOB) are mixotrophic and depend on organic cosubstrates for growth. Encrustation of cells in Fe(III) minerals has been observed for mixotrophic NRFeOB but not for autotrophic phototrophic and microaerophilic Fe(II) oxidizers. So far, little is known about cell-mineral associations in the few existing autotrophic NRFeOB. Here, we investigate whether the designated autotrophic Fe(II)-oxidizing strain (closely related to Gallionella and Sideroxydans) or the heterotrophic nitrate reducers that are present in the autotrophic nitrate-reducing Fe(II)-oxidizing enrichment culture KS form mineral crusts during Fe(II) oxidation under autotrophic and mixotrophic conditions. In the mixed culture, we found no significant encrustation of any of the cells both during autotrophic oxidation of 8 to 10 mM Fe(II) coupled to nitrate reduction and during cultivation under mixotrophic conditions with 8 to 10 mM Fe(II), 5 mM acetate, and 4 mM nitrate, where higher numbers of heterotrophic nitrate reducers were present. Two pure cultures of heterotrophic nitrate reducers (Nocardioides and Rhodanobacter) isolated from culture KS were analyzed under mixotrophic growth conditions. We found green rust formation, no cell encrustation, and only a few mineral particles on some cell surfaces with 5 mM Fe(II) and some encrustation with 10 mM Fe(II). Our findings suggest that enzymatic, autotrophic Fe(II) oxidation coupled to nitrate reduction forms poorly crystalline Fe(III) oxyhydroxides and proceeds without cellular encrustation while indirect Fe(II) oxidation via heterotrophic nitrate-reduction-derived nitrite can lead to green rust as an intermediate mineral and significant cell encrustation. The extent of encrustation caused by indirect Fe(II) oxidation by reactive nitrogen species depends on Fe(II) concentrations and is probably negligible under environmental conditions in most habitats.IMPORTANCE Most described nitrate

  13. Protein enrichment of grain sorghum by submerged culture of the amylolytic yeastsSchwanniomyces occidentalis andLipomyces kononenkoae.

    PubMed

    Horn, C H; du Preez, J C; Kilian, S G

    1992-07-01

    Cultivation of aSchwanniomyces occidentalis derepressed mutant in a 10% (w/v) gelatinized grain sorghum slurry increased the crude protein content of the biomass from an initial value of 12% to 41% (dry) within 20 h, with no detectable residual starch. Co-cultivation ofCandida utilis with theS. occidentalis mutant improved the final crude protein content to 47% within 18 h, whereas a co-culture ofC. utilis with aLipomyces kononenkoae mutant resulted in a cultivation time of 50 h with a significantly lower protein content and a low final α-amylase activity. In a 15% (w/v) grain sorghum slurry aC. utilis/S. occidentalis co-culture increased the protein content to about 44% within 30 h. Yeast cultivation increased the lysine and threonine content of the final biomass considerably.

  14. Methanotrophic diversity in high arctic wetlands on the islands of Svalbard (Norway)--denaturing gradient gel electrophoresis analysis of soil DNA and enrichment cultures.

    PubMed

    Wartiainen, Ingvild; Hestnes, Anne Grethe; Svenning, Mette M

    2003-10-01

    The methanotrophic community in arctic soil from the islands of Svalbard, Norway (78 degrees N) was analysed by combining group-specific PCR with PCR of the highly variable V3 region of the 16S rRNA gene and then by denaturing gradient gel electrophoresis (DGGE). Selected bands were sequenced for identification. The analyses were performed with DNA extracted directly from soil and from enrichment cultures at 10 and 20 degrees C. The two genera Methylobacter and Methylosinus were found in all localities studied. The DGGE band patterns were simple, and DNA fragments with single base differences were separated. The arctic tundra is a potential source of extensive methane emission due to climatic warming because of its large reservoirs of stored organic carbon. Higher temperatures due to climatic warming can cause increased methane production, and the abundance and activity of methane-oxidizing bacteria in the arctic soil may be important regulators for methane emission to the atmosphere.

  15. Bacterial diversity of autotrophic enriched cultures from remote, glacial Antarctic, Alpine and Andean aerosol, snow and soil samples

    NASA Astrophysics Data System (ADS)

    González-Toril, E.; Amils, R.; Delmas, R. J.; Petit, J.-R.; Komárek, J.; Elster, J.

    2009-01-01

    Four different communities and one culture of autotrophic microbial assemblages were obtained by incubation of samples collected from high elevation snow in the Alps (Mt. Blanc area) and the Andes (Nevado Illimani summit, Bolivia), from Antarctic aerosol (French station Dumont d'Urville) and a maritime Antarctic soil (King George Island, South Shetlands, Uruguay Station Artigas), in a minimal mineral (oligotrophic) media. Molecular analysis of more than 200 16S rRNA gene sequences showed that all cultured cells belong to the Bacteria domain. Phylogenetic comparison with the currently available rDNA database allowed sequences belonging to Proteobacteria Alpha-, Beta- and Gamma-proteobacteria), Actinobacteria and Bacteroidetes phyla to be identified. The Andes snow culture was the richest in bacterial diversity (eight microorganisms identified) and the marine Antarctic soil the poorest (only one). Snow samples from Col du Midi (Alps) and the Andes shared the highest number of identified microorganisms (Agrobacterium, Limnobacter, Aquiflexus and two uncultured Alphaproteobacteria clones). These two sampling sites also shared four sequences with the Antarctic aerosol sample (Limnobacter, Pseudonocardia and an uncultured Alphaproteobacteriaclone). The only microorganism identified in the Antarctica soil (Brevundimonas sp.) was also detected in the Antarctic aerosol. Most of the identified microorganisms had been detected previously in cold environments, marine sediments soils and rocks. Air current dispersal is the best model to explain the presence of very specific microorganisms, like those identified in this work, in environments very distant and very different from each other.

  16. Morphological and molecular characterizations of psychrophilic fungus Geomyces destructans from New York bats with White Nose Syndrome (WNS).

    PubMed

    Chaturvedi, Vishnu; Springer, Deborah J; Behr, Melissa J; Ramani, Rama; Li, Xiaojiang; Peck, Marcia K; Ren, Ping; Bopp, Dianna J; Wood, Britta; Samsonoff, William A; Butchkoski, Calvin M; Hicks, Alan C; Stone, Ward B; Rudd, Robert J; Chaturvedi, Sudha

    2010-05-24

    Massive die-offs of little brown bats (Myotis lucifugus) have been occurring since 2006 in hibernation sites around Albany, New York, and this problem has spread to other States in the Northeastern United States. White cottony fungal growth is seen on the snouts of affected animals, a prominent sign of White Nose Syndrome (WNS). A previous report described the involvement of the fungus Geomyces destructans in WNS, but an identical fungus was recently isolated in France from a bat that was evidently healthy. The fungus has been recovered sparsely despite plentiful availability of afflicted animals. We have investigated 100 bat and environmental samples from eight affected sites in 2008. Our findings provide strong evidence for an etiologic role of G. destructans in bat WNS. (i) Direct smears from bat snouts, Periodic Acid Schiff-stained tissue sections from infected tissues, and scanning electron micrographs of bat tissues all showed fungal structures similar to those of G. destructans. (ii) G. destructans DNA was directly amplified from infected bat tissues, (iii) Isolations of G. destructans in cultures from infected bat tissues showed 100% DNA match with the fungus present in positive tissue samples. (iv) RAPD patterns for all G. destructans cultures isolated from two sites were indistinguishable. (v) The fungal isolates showed psychrophilic growth. (vi) We identified in vitro proteolytic activities suggestive of known fungal pathogenic traits in G. destructans. Further studies are needed to understand whether G. destructans WNS is a symptom or a trigger for bat mass mortality. The availability of well-characterized G. destructans strains should promote an understanding of bat-fungus relationships, and should aid in the screening of biological and chemical control agents.

  17. Identification of bacteria in enrichment cultures of sulfate reducers in the Cariaco Basin water column employing Denaturing Gradient Gel Electrophoresis of 16S ribosomal RNA gene fragments

    PubMed Central

    2013-01-01

    Background The Cariaco Basin is characterized by pronounced and predictable vertical layering of microbial communities dominated by reduced sulfur species at and below the redox transition zone. Marine water samples were collected in May, 2005 and 2006, at the sampling stations A (10°30′ N, 64°40′ W), B (10°40′ N, 64°45′ W) and D (10°43’N, 64°32’W) from different depths, including surface, redox interface, and anoxic zones. In order to enrich for sulfate reducing bacteria (SRB), water samples were inoculated into anaerobic media amended with lactate or acetate as carbon source. To analyze the composition of enrichment cultures, we performed DNA extraction, PCR-DGGE, and sequencing of selected bands. Results DGGE results indicate that many bacterial genera were present that are associated with the sulfur cycle, including Desulfovibrio spp., as well as heterotrophs belonging to Vibrio, Enterobacter, Shewanella, Fusobacterium, Marinifilum, Mariniliabilia, and Spirochaeta. These bacterial populations are related to sulfur coupling and carbon cycles in an environment of variable redox conditions and oxygen availability. Conclusions In our studies, we found an association of SRB-like Desulfovibrio with Vibrio species and other genera that have a previously defined relevant role in sulfur transformation and coupling of carbon and sulfur cycles in an environment where there are variable redox conditions and oxygen availability. This study provides new information about microbial species that were culturable on media for SRB at anaerobic conditions at several locations and water depths in the Cariaco Basin. PMID:23981583

  18. Identification of in vivo HSP90-interacting proteins reveals modularity of HSP90 complexes is dependent on the environment in psychrophilic bacteria

    PubMed Central

    García-Descalzo, Laura; Alcazar, Alberto; Baquero, Fernando

    2010-01-01

    Heat shock protein 90 (HSP90) is a conserved molecular chaperone that functions as part of complexes in which different client proteins target it to diverse sets of substrates. In this paper, HSP90 complexes were investigated in γ-proteobacteria from mild (Shewanella oneidensis) and cold environments (Shewanella frigidimarina and Psychrobacter frigidicola), to determine changes in HSP90 interactions with client proteins in response to the adaptation to cold environments. HSP90 participation in cold adaptation was determined using the specific inhibitor 17-allylamino-geldanamycin. Then, HSP90 was immunoprecipitated from bacterial cultures, and the proteins in HSP90 complexes were analyzed by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. According to HSP90-associated protein analysis, only 15 common proteins were found in both species from the same genus, S. oneidensis and S. frigidimarina, whereas a significant higher number of common proteins were found in both psychrophilic species S. frigidimarina and P. frigidicola 21 (p < 0.001). Only two HSP90-interacting proteins, the chaperone proteins DnaK and GroEL, were common to the three species. Interestingly, some proteins related to energy metabolism (isocitrate lyase, succinyl-CoA synthetase, alcohol dehydrogenase, NAD(+) synthase, and malate dehydrogenase) and some translation factors only interacted with HSP90 in psychrophilic bacteria. We can conclude that HSP90 and HSP90-associated proteins might take part in the mechanism of adaptation to cold environments, and interestingly, organisms living in similar environments conserve similar potential HSP90 interactors in opposition to phylogenetically closely related organisms of the same genus but from different environments. PMID:20890740

  19. Stable Carbon Isotope Fractionation During 1,2-Dichloropropane-to-Propene Transformation by an Enrichment Culture Containing Dehalogenimonas Strains and a dcpA Gene.

    PubMed

    Martín-González, L; Mortan, S Hatijah; Rosell, M; Parladé, E; Martínez-Alonso, M; Gaju, N; Caminal, G; Adrian, L; Marco-Urrea, E

    2015-07-21

    A stable enrichment culture derived from Besòs river estuary sediments stoichiometrically dechlorinated 1,2-dichloropropane (1,2-DCP) to propene. Sequential transfers in defined anaerobic medium with the inhibitor bromoethanesulfonate produced a sediment-free culture dechlorinating 1,2-DCP in the absence of methanogenesis. Application of previously published genus-specific primers targeting 16S rRNA gene sequences revealed the presence of a Dehalogenimonas strain, and no amplification was obtained with Dehalococcoides-specific primers. The partial sequence of the 16S rRNA amplicon was 100% identical with Dehalogenimonas alkenigignens strain IP3-3. Also, dcpA, a gene described to encode a corrinoid-containing 1,2-DCP reductive dehalogenase was detected. Resistance of the dehalogenating activity to vancomycin, exclusive conversion of vicinally chlorinated alkanes, and tolerance to short-term oxygen exposure is consistent with the hypothesis that a Dehalogenimonas strain is responsible for 1,2-DCP conversion in the culture. Quantitative PCR showed a positive correlation between the number of Dehalogenimonas 16S rRNA genes copies in the culture and consumption of 1,2-DCP. Compound specific isotope analysis revealed that the Dehalogenimonas-catalyzed carbon isotopic fractionation (εC(bulk)) of the 1,2-DCP-to-propene reaction was -15.0 ± 0.7‰ under both methanogenic and nonmethanogenic conditions. This study demonstrates that carbon isotope fractionation is a valuable approach for monitoring in situ 1,2-DCP reductive dechlorination by Dehalogenimonas strains.

  20. Structural and chemical modification of Fe-rich smectite associated with microbial Fe-respiration by psychrophilic bacteria in King George Island, West Antarctica

    NASA Astrophysics Data System (ADS)

    Jung, J.; Kim, J. Y.; Lim, H. S.; Lee, Y. K.; Kim, O. S.; Park, K.; Lee, J.; Yoon, H.; Kim, J. W.

    2015-12-01

    Biotic/abiotic redox reaction is a ubiquitous process in a mineral alteration and an elemental cycling in the sediments/ocean. The role of psychrophiles in clay mineral alteration was tested in the soil for the seven sites from the coast to the inland at Barton Peninsula. Batch experiments of microbe-mineral interaction under the various temperatures (4 ℃, 15 ℃) that mimics the Antarctic condition were performed to understand the mechanism of biogeochemical alteration of clay minerals. After 12 months incubation of the bulk surface soil samples in the M1 minimal medium, the extent of Fe reduction was reached up to 49 and 42 % at 4 ℃ and 15 ℃. The increase in CEC corresponds to the extent of Fe reduction. Moreover, precipitations of secondary phase mineral such as vivianite were observed only in 12 months enrichment samples at 4 ℃ and 15 ℃. Sulfate reducing bacteria and Fe-reducing bacteria capable of reducing Fe were identified by 16S rRNA pyrosequencing. The Fe reduction coupled to oxidation of organic matter might be enhanced by cooperation of a consortium of Sulfate reducing bacteria and Fe-reducing bacteria. Moreover, Nitrate reducing bacteria which have an ability to oxidize ferrous iron anaerobically with nitrate reduction were identified at 15 ℃. The lower values observed in the extent of Fe reduction at 15 ℃ may be associated with Fe-oxidation induced by nitrate reduction.In order to verify the mechanism of microbial Fe reduction in clay minerals at low temperatures (4 and 15 ℃), Fe-rich Nontronite (NAu-1) and Psychrophilic bacteria were incubated for 4 months in anaerobic condition. Total structural Fe in NAu-1 is 16.4 % and 99.6 % of the total Fe is ferric. The extent of Fe reduction in nontronite was reached up to 11.5 % and 11 % at 4 ℃ and 15 ℃, respectively. The structural modification of biologically Fe-reduced nontronite was observed in the (001) peak shift to the lower 2 theta indicating the layer collapse associated with K

  1. Short communication: Viability of culture organisms in honey-enriched acidophilus-bifidus-thermophilus (ABT)-type fermented camel milk.

    PubMed

    Varga, L; Süle, J; Nagy, P

    2014-11-01

    The aim of this research was to monitor the survival during refrigerated storage of Lactobacillus acidophilus LA-5 (A), Bifidobacterium animalis ssp. lactis BB-12 (B), and Streptococcus thermophilus CHCC 742/2130 (T) in cultured dairy foods made from camel and, for comparison, cow milks supplemented with black locust (Robinia pseudoacacia L.) honey and fermented by an acidophilus-bifidus-thermophilus (ABT)-type culture. Two liters of dromedary camel milk and 2 L of cow milk were heated to 90 °C and held for 10 min, then cooled to 40 °C. One half of both types of milk was fortified with black locust honey at the rate of 5.0% (wt/vol), whereas the other half was devoid of honey and served as a control. The camel and cow milks with and without honey were subsequently inoculated with ABT-5 culture and were fermented at 37 °C until a pH value of 4.6 was reached. Thereafter, the probiotic fermented milks were cooled to 15 °C in ice water and were each separated into 18 fractions that were transferred in sterile, tightly capped centrifuge tubes. After 24 h of cooling at 8 °C (d 0), the samples were stored at refrigeration temperature (4 °C). Three tubes of all 4 products (i.e., fermented camel and cow milks with and without honey) were taken at each sampling time (i.e., following 0, 7, 14, 21, 28, and 35 d of storage), and the counts of characteristic microorganisms and those of certain spoilage microbes (yeasts, molds, coliforms, Escherichia coli) were enumerated. The entire experimental program was repeated twice. The results showed that addition of black locust honey at 5% to heat-treated camel and cow milks did not influence the growth and survival of starter streptococci during production and subsequent refrigerated storage of fermented ABT milks. In contrast, honey improved retention of viability of B. animalis ssp. lactis BB-12 in the camel milk-based product during storage at 4 °C up to 5 wk. No spoilage organisms were detected in any of the samples tested

  2. Serial enrichment of spermatogonial stem and progenitor cells (SSCs) in culture for derivation of long-term adult mouse SSC lines.

    PubMed

    Martin, Laura A; Seandel, Marco

    2013-02-25

    Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells(1-3). Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines(4). However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state. Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors(5,6). Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies(7). Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types(8). Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC(2

  3. Psychrophilic pseudomonas in antarctic freshwater lake at stornes peninsula, larsemann hills over east Antarctica.

    PubMed

    Chauhan, Abhishek; Bharti, Pawan K; Goyal, Pankaj; Varma, Ajit; Jindal, Tanu

    2015-01-01

    The Larsemann Hills is an ice-free area of approximately 50 km(2), located halfway between the Vestfold Hills and the Amery Ice Shelf on the south-eastern coast of Prydz Bay, Princess Elizabeth Land, East Antarctica (69º30'S, 76º19'58″E). The ice-free area consists of two major peninsulas (Stornes and Broknes), four minor peninsulas, and approximately 130 islands. The Larsemann Hills area contains more than 150 lakes at different Islands and Peninsulas. Nine lake water samples were collected in a gamma sterilized bottles and were kept in an ice pack to prevent any changes in the microbial flora of the samples during the transportation. The water samples were transported to the lab in vertical position maintaining the temperature 1-4 °C with ice pack enveloped conditions. Samples were studied for Psychrophilic bacterial count, Pseudomonas spp., Staphylococcus aureus, Salmonella and Total MPN Coliform per 100 ml. Psychrophillic counts were found in the range of 12 cfu to 1.6 × 10(2) cfu in all the samples. MPN Coliform per 100 ml was found to be absent in all the samples. No growth and characteristics colonies observed when tested for Salmonella and S.aureus. Pseudomonas sp. was found in ST-2 lake water sample as characteristics colonies (Optimum Growth) were observed on selective media at 22 and 25 °C. Further several biochemical tests were also performed to confirm the presence of this Potential Psychrophilic Pseudomonas sp. for its further application in Science and Technology.

  4. [Distribution of psychrophilic microorganisms in terrestrial biotopes of the Antarctic Region].

    PubMed

    Romanovskaia, V A; Tashirev, A B; Shilin, S O; Gladka, G V

    2012-01-01

    It is shown that the total number of chemoorganotrophic aerobic microorganisms in the Antarctic Region revealed at 1 degree C and 5 degrees C made from 10(4) up to 10(6) cells/g of plant-soil sample of biotopes: grass Deschampcia antarctica, grass Colobanthus, green mosses, crustose black lichens and a biofilm of accretion on vertical rocks. From 10(6) up to 10(8) cells/g of samples were revealed in the same Antarctic samples at 30 degrees C. At 42 degrees C thermotolerant bacteria were either absent, or their quantity was less than 10(4) cells/g of samples. Thus the fraction (part) of the Antarctic microorganisms, which grow at different temperatures, varied: at 1-5 degrees C their part made from 5 to 15%, and at 30 degrees C--from 10 to 45%. At 15-20 degrees C the growth of both psychrophilic/psychrotolerant, and mesophilic microorganisms was observed. When comparing the results of plating of samples from different climatic zones (the Antarctic Region and Ukraine), it is shown that in the Antarctic biotopes in comparison with biotopes of the zone with temperate climate: (1) the total number of microorganisms is lower, (2) quantity of psychrophilic/psychrotolerant bacteria is higher, (3) quantity of mesophilic microorganisms is less, (4) as a result the part psychrophilic/psychrotolerant microorganisms in the total number of microorganisms is much higher. It is evident, that low temperatures, and also daily cycles of freezing and thawing are factors which limit microbial colonization ofAntarctic biotopes.

  5. Vibrio psychroerythrus sp. n.: Classification of the Psychrophilic Marine Bacterium, NRC 1004

    PubMed Central

    D'aoust, J. Y.; Kushner, D. J.

    1972-01-01

    A red-pigmented organism, formerly known as marine psychrophile NRC 1004, has been classified as Vibrio psychroerythrus sp. n. Classification was mainly based on morphology, the ability of the organism to oxidize and ferment glucose, its sensitivity to vibriostat 0/129, and its deoxyribonucleic acid base composition of 40.0 moles% guanine plus cytosine, determined by thermal denaturation. The organism gave positive reactions for catalase, oxidase, and starch hydrolysis and produced acid from maltose and dextrin but not from arabinose. It was indole- and citrate-negative and reduced nitrate to nitrite without producing gas. PMID:5053463

  6. UV Radiation and Visible Light Induce hsp70 Gene Expression in the Antarctic Psychrophilic Ciliate Euplotes focardii.

    PubMed

    Fulgentini, Lorenzo; Passini, Valerio; Colombetti, Giuliano; Miceli, Cristina; La Terza, Antonietta; Marangoni, Roberto

    2015-08-01

    The psychrophilic ciliate Euplotes focardii inhabits the shallow marine coastal sediments of Antarctica, where, over millions of years of evolution, it has reached a strict molecular adaptation to such a constant-temperature environment (about -2 °C). This long evolution at sub-zero temperatures has made E. focardii unable to respond to heat stress with the activation of its heat shock protein (hsp) 70 genes. These genes can, however, be expressed in response to other stresses, like the oxidative one, thus indicating that the molecular adaptation has exclusively altered the heat stress signaling pathways, while it has preserved hsp70 gene activation in response to other environmental stressors. Since radiative stress has proved to be affine to oxidative stress in several organisms, we investigated the capability of UV radiation to induce hsp70 transcription. E. focardii cell cultures were exposed to several different irradiation regimes, ranging from visible only to a mixture of visible, UV-A and UV-B. The irradiation values of each spectral band have been set to be comparable with those recorded in a typical Antarctic spring. Using Northern blot analysis, we measured the expression level of hsp70 immediately after irradiation (0-h-labeled samples), 1 h, and 2 h from the end of the irradiation. Surprisingly, our results showed that besides UV radiation, the visible light was also able to induce hsp70 expression in E. focardii. Moreover, spectrophotometric measurements have revealed no detectable endogenous pigments in E. focardii, making it difficult to propose a possible explanation for the visible light induction of its hsp70 genes. Further research is needed to conclusively clarify this point.

  7. Suppression of VLDL secretion by cultured hepatocytes incubated with chylomicron remnants enriched in n-3 polyunsaturated fatty acids is regulated by hepatic nuclear factor-4alpha.

    PubMed

    López-Soldado, Iliana; Avella, Michael; Botham, Kathleen M

    2009-12-01

    Dietary n-3 polyunsaturated fatty acids (PUFA) suppress the secretion of very low density lipoprotein (VLDL) directly when delivered to the liver in chylomicron remnants (CMR). The role of sterol regulatory element-binding proteins (SREBPs) and hepatic nuclear factor-4alpha (HNF-4alpha) in the regulation of this effect was investigated. Chylomicron remnant-like particles (CRLPs) containing triacylglycerol (TG) from palm (rich in saturated fatty acids (SFA)) or fish (rich in n-3 PUFA) oil were incubated with cultured rat hepatocytes (24h) and the expression of protein and mRNA for SREBP-1, SREBP-2 and HNF-4alpha, and levels of mRNA for their target genes were determined. SREBP-1 and -2 protein expression in the membrane and nuclear fractions was unaffected by either type of CRLPs. mRNA abundance for SREBP-1c and -2 was also unchanged by CRLP-treatment, as were levels of mRNA for target genes of SREBP-1, including steroyl CoA desaturase, acetyl CoA carboxylase, fatty acid synthase and ATP citrate lyase, and SREBP-2 (3-hydroxy-3-methylglutaryl CoA reductase). In contrast, HNF-4alpha protein and mRNA levels were significantly decreased by CRLPs enriched in n-3 PUFA, but not SFA, and the expression of mRNA for HNF-4alpha target genes, including HNF-1alpha, apolipoprotein B and the microsomal TG transfer protein, was also lowered by n-3 PUFA-, but not SFA-enriched CRLPs. These findings suggest that the direct suppression of VLDL secretion by dietary n-3 PUFA delivered to the liver in CMR is mediated via decreased expression of HNF-4alpha.

  8. The social cost of coastal erosion. Using cultural theory to enrich the interpretation of stated preference data.

    NASA Astrophysics Data System (ADS)

    Kontogianni, A.; Tourkolias, C.; Vousdoukas, M.; Skourtos, M.

    2012-04-01

    Natural coastal processes are to a great extent modified by proximity to man-made structures. Engineered interventions, port facilities, housing and industrial infrastructure, all can increase the coastline fluctuations significantly relative to those along a long unobstructed coastline. As a consequence, coastlines are increasingly exposed to coastal erosion, a phenomenon defined as the encroachment of land by the sea after averaging over a period, which is sufficiently long to eliminate the impacts of weather, storm events and local sediment dynamics. In order to provide cost effective management of coastal erosion it is crucial to estimate both the benefits and costs associated with various management alternatives. The initiatives on Integrated Coastal Zone Manegment in Europe, but also the upcoming Marine Strategy Framwork Directive would benefit greatly from a proliferation of socioeconomic information to assist decision makers who must weigh the impacts of various types of coastal improvement and the cost of beach protection/restoration. In that spirit, the objective of the present research is to report the results of a survey undertaken in two resort beaches on the island of Lesvos (Greece), designed to estimate public preferences for avoiding coastal erosion. A mixed methodological approach is employed by combining an open-ended contingent valuation survey with cultural theory of risk perception. The empirical models to analyze individual choices of erosion control programs and the associated welfare measures are presented, followed by the discussion of model specification and estimation issues, and the results of the data analysis. Some concluding remarks are then presented. By choosing this approach we aim at improving our understanding of preference structure for avoiding public risk, accepted level of risk and perceptions thereof. The framework can also be used for assessing the social cost of extreme weather events such as storm surges in the coastal

  9. Biodegradation of 1,2,3- and 1,2,4-trichlorobenzene in soil and in liquid enrichment culture.

    PubMed

    Marinucci, A C; Bartha, R

    1979-11-01

    The biodegradation of radiochemically pure (99%) 1,2,3- and 1,2,4-trichlorobenzene (TCB) in soil was investigated. Experimental difficulties posed by the high volatility and slow biodegradation rate of the TCBs were partially overcome by using a specially designed incubation and trapping apparatus. Evolution of (14)CO(2) from active versus poisoned soil dosed with 50 mug of the individual TCBs per g gave conclusive proof that both isomers are biodegradable. At 20 degrees C, 1,2,4-TCB was mineralized at an approximate rate of 1 nmol/day per 20 g of soil sample, and 1,2,3-TCB was mineralized at one-half to one-third that rate. Mineral fertilizers or cosubstrates failed to increase TCB mineralization rates in soil. Anaerobic conditions had a negative effect on mineralization, and increased temperatures had a positive effect. With increasing 1,2,4-TCB concentrations, (14)CO(2) evolution exhibited saturation kinetics with an apparent K(m) of 55.5 nmol per g of soil. Recovery of total radioactivity was good from soil containing high organic matter concentrations. From low-organic-matter soil, some of the radioactivity was recovered only on combustion, and overall recovery was lower. In soil-inoculated liquid culture, the cosubstrates glucose and benzene caused a slight stimulation of 1,2,4-TCB mineralization. Cochromatography of known standards with the extracts of soil pretreated with [(14)C]TCBs indicated that 3,4,5-trichlorophenol, 2,6-dichlorophenol and, to a lesser degree, 2,3-dichlorophenol were present in soils incubated with 1,2,3-TCB. 2,4-, 2,5-, and 3,4-dichlorophenol were present in soils incubated with 1,2,4-TCB.

  10. Biodegradation of 1,2,3- and 1,2,4-Trichlorobenzene in Soil and in Liquid Enrichment Culture

    PubMed Central

    Marinucci, A. C.; Bartha, R.

    1979-01-01

    The biodegradation of radiochemically pure (99%) 1,2,3- and 1,2,4-trichlorobenzene (TCB) in soil was investigated. Experimental difficulties posed by the high volatility and slow biodegradation rate of the TCBs were partially overcome by using a specially designed incubation and trapping apparatus. Evolution of 14CO2 from active versus poisoned soil dosed with 50 μg of the individual TCBs per g gave conclusive proof that both isomers are biodegradable. At 20°C, 1,2,4-TCB was mineralized at an approximate rate of 1 nmol/day per 20 g of soil sample, and 1,2,3-TCB was mineralized at one-half to one-third that rate. Mineral fertilizers or cosubstrates failed to increase TCB mineralization rates in soil. Anaerobic conditions had a negative effect on mineralization, and increased temperatures had a positive effect. With increasing 1,2,4-TCB concentrations, 14CO2 evolution exhibited saturation kinetics with an apparent Km of 55.5 nmol per g of soil. Recovery of total radioactivity was good from soil containing high organic matter concentrations. From low-organic-matter soil, some of the radioactivity was recovered only on combustion, and overall recovery was lower. In soil-inoculated liquid culture, the cosubstrates glucose and benzene caused a slight stimulation of 1,2,4-TCB mineralization. Cochromatography of known standards with the extracts of soil pretreated with [14C]TCBs indicated that 3,4,5-trichlorophenol, 2,6-dichlorophenol and, to a lesser degree, 2,3-dichlorophenol were present in soils incubated with 1,2,3-TCB. 2,4-, 2,5-, and 3,4-dichlorophenol were present in soils incubated with 1,2,4-TCB. PMID:120698

  11. Structural thermal adaptation of β-tubulins from the Antarctic psychrophilic protozoan Euplotes focardii.

    PubMed

    Chiappori, Federica; Pucciarelli, Sandra; Merelli, Ivan; Ballarini, Patrizia; Miceli, Cristina; Milanesi, Luciano

    2012-04-01

    Tubulin dimers of psychrophilic eukaryotes can polymerize into microtubules at 4°C, a temperature at which microtubules from mesophiles disassemble. This unique capability requires changes in the primary structure and/or in post-translational modifications of the tubulin subunits. To contribute to the understanding of mechanisms responsible for microtubule cold stability, here we present a computational structural analysis based on molecular dynamics (MD) and experimental data of three β-tubulin isotypes, named EFBT2, EFBT3, and EFBT4, from the Antarctic protozoon Euplotes focardii that optimal temperature for growth and reproduction is 4°C. In comparison to the β-tubulin from E. crassus, a mesophilic Euplotes species, EFBT2, EFBT3, and EFBT4 possess unique amino acid substitutions that confer different flexible properties of the polypeptide, as well as an increased hydrophobicity of the regions involved in microtubule interdimeric contacts that may overcome the microtubule destabilizing effect of cold temperatures. The structural analysis based on MD indicated that all isotypes display different flexibility properties in the regions involved in the formation of longitudinal and lateral contacts during microtubule polymerization. We also investigated the role of E. focardii β-tubulin isotypes during the process of cilia formation. The unique characteristics of the primary and tertiary structures of psychrophilic β-tubulin isotypes seem responsible for the formation of microtubules with distinct dynamic and functional properties. Copyright © 2011 Wiley Periodicals, Inc.

  12. Enzyme activity determination on macromolecular substrates by isothermal titration calorimetry: application to mesophilic and psychrophilic chitinases.

    PubMed

    Lonhienne, T; Baise, E; Feller, G; Bouriotis, V; Gerday, C

    2001-02-09

    Isothermal titration calorimetry has been applied to the determination of the kinetic parameters of chitinases (EC 3.2.1.14) by monitoring the heat released during the hydrolysis of chitin glycosidic bonds. Experiments were carried out using two different macromolecular substrates: a soluble polymer of N-acetylglucosamine and the insoluble chitin from crab shells. Different experimental temperatures were used in order to compare the thermodependence of the activity of two chitinases from the psychrophile Arthrobacter sp. TAD20 and of chitinase A from the mesophile Serratia marcescens. The method allowed to determine unequivocally the catalytic rate constant k(cat), the activation energy (E(a)) and the thermodynamic activation parameters (DeltaG(#), DeltaH(#), DeltaS(#)) of the chitinolytic reaction on the soluble substrate. The catalytic activity has also been determined on insoluble chitin, which displays an effect of substrate saturation by chitinases. On both substrates, the thermodependence of the activity of the psychrophilic chitinases was lower than that observed with the mesophilic counterpart.

  13. Purification and characterization of cold-adapted beta-agarase from an Antarctic psychrophilic strain

    PubMed Central

    Li, Jiang; Hu, Qiushi; Li, Yuquan; Xu, Yuan

    2015-01-01

    An extracellular β-agarase was purified from Pseudoalteromonas sp. NJ21, a Psychrophilic agar-degrading bacterium isolated from Antarctic Prydz Bay sediments. The purified agarase (Aga21) revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 80 kDa. The optimum pH and temperature of the agarase were 8.0 and 30 °C, respectively. However, it maintained as much as 85% of the maximum activities at 10 °C. Significant activation of the agarase was observed in the presence of Mg2+, Mn2+, K+; Ca2+, Na+, Ba2+, Zn2+, Cu2+, Co2+, Fe2+, Sr2+ and EDTA inhibited the enzyme activity. The enzymatic hydrolyzed product of agar was characterized as neoagarobiose. Furthermore, this work is the first evidence of cold-adapted agarase in Antarctic psychrophilic bacteria and these results indicate the potential for the Antarctic agarase as a catalyst in medicine, food and cosmetic industries. PMID:26413048

  14. Temperature-Sensitive Salmonella enterica Serovar Enteritidis PT13a Expressing Essential Proteins of Psychrophilic Bacteria

    PubMed Central

    Duplantis, Barry N.; Puckett, Stephanie M.; Rosey, Everett L.; Ameiss, Keith A.; Hartman, Angela D.; Pearce, Stephanie C.

    2015-01-01

    Synthetic genes based on deduced amino acid sequences of the NAD-dependent DNA ligase (ligA) and CTP synthetase (pyrG) of psychrophilic bacteria were substituted for their native homologues in the genome of Salmonella enterica serovar Enteritidis phage type 13a (PT13a). The resulting strains were rendered temperature sensitive (TS) and did not revert to temperature resistance at a detectable level. At permissive temperatures, TS strains grew like the parental strain in broth medium and in macrophage-like cells, but their growth was slowed or stopped when they were shifted to a restrictive temperature. When injected into BALB/c mice at the base of the tail, representing a cool site of the body, the strains with restrictive temperatures of 37, 38.5, and 39°C persisted for less than 1 day, 4 to 7 days, and 20 to 28 days, respectively. The wild-type strain persisted at the site of inoculation for at least 28 days. The wild-type strain, but not the TS strains, was also found in spleen-plus-liver homogenates within 1 day of inoculation of the tail and was detectable in these organs for at least 28 days. Intramuscular vaccination of White Leghorn chickens with the PT13a strain carrying the psychrophilic pyrG gene provided some protection against colonization of the reproductive tract and induced an anti-S. enterica antibody response. PMID:26187965

  15. Antarctomyces pellizariae sp. nov., a new, endemic, blue, snow resident psychrophilic ascomycete fungus from Antarctica.

    PubMed

    de Menezes, Graciéle C A; Godinho, Valéria M; Porto, Bárbara A; Gonçalves, Vívian N; Rosa, Luiz H

    2017-03-01

    In the present study, we have identified and characterised a new snow resident ascomycete blue stain fungus from Antarctica named Antarctomyces pellizariae sp. nov. Menezes, Godinho, Porto, Gonçalves and Rosa, using polyphasic taxonomy techniques. This fungal species was recovered from the seasonal snow of the Antarctic Peninsula. Antarctomyces pellizariae displayed different macro- and micromorphology when compared with A. psychrotrophicus Stchigel and Guarro, the only other Antarctomyces species reported until date. Antarctomyces pellizariae showed psychrophilic behavior and very low growth rate at 22-25 °C, quite different from A. psychrotrophicus that has a higher growth rate at mesophilic temperatures. In addition, micromorphological characteristics and the analysis of the nuclear rDNA internal transcribed spacer, β-tubulin, and RNA polymerase II regions revealed that A. pellizariae is a new species that is related to A. psychrotrophicus and Thelebolus species. Since the Antarctic Peninsula is reported to be one of the main regions of the earth experiencing the effects of global change in climate, species, such as A. pellizariae, might provide information about these effects on the endemic Antarctic biota. In addition, A. pellizariae displayed psychrophilic behavior and might be a source of interesting anti-freeze compounds that might prove useful in biotechnological processes.

  16. INFLUENCE OF MODERATE TEMPERATURE ON GROWTH AND MALIC DEHYDROGENASE ACTIVITY OF A MARINE PSYCHROPHILE.

    PubMed

    MORITA, R Y; BURTON, S D

    1963-11-01

    Morita, Richard Y. (Oregon State University, Corvallis), and Sheril D. Burton. Influence of moderate temperature on growth and malic dehydrogenase activity of a marine psychrophile. J. Bacteriol. 86:1025-1029. 1963.-The maximal and optimal growth temperatures for a marine psychrophilic vibrio (PS 207) were determined to be 30 and 24.5 C, respectively. Malic dehydrogenase was found to be functioning in whole cells at about 1/20 of its observed maximum. Incubation of the cells, prior to or during the assay, at temperatures above the maximal growth temperature permitted the malic dehydrogenase to operate nearer its maximum, but this also inactivated the intracellular enzyme. The heating of whole cells gave an apparent effect of increasing malic dehydrogenase activity. Lysis of the cells permitted the enzyme to function at its full potential but rendered the enzyme more sensitive to heat denaturation. Lysis of the cells also caused the enzyme to lose approximately one-half of its malic dehydrogenase activity with each 10 C drop in temperature, whereas whole cells only lose approximately 1/5 of their enzyme activity at low temperatures with each 10 C drop.

  17. Production of gamma-decalactone by a psychrophilic and a mesophilic strain of the yeast Rhodotorula aurantiaca.

    PubMed

    Alchihab, Mohamed; Destain, Jacqueline; Aguedo, Mario; Majad, Lamia; Ghalfi, Hakim; Wathelet, Jean-Paul; Thonart, Philippe

    2009-07-01

    Among 18 psychrophilic strains isolated near the Antarctic Station, the psychrophilic strain Rhodotorula aurantiaca A19 was selected for its ability of growth and gamma-decalactone production at low temperatures. The effects of temperature, initial pH, and castor oil concentration on the growth and gamma-decalactone production by a psychrophilic and a mesophilic strain of R. aurantiaca were investigated. The highest gamma-decalactone production in flasks (5.8 g/l) was obtained with the strain A19 at 14 degrees C and initial pH 7.0 in medium containing 20 g/l castor oil. On the other hand, these factors did not affect the production of gamma-decalactone by the mesophilic strain. In fermentor, a gamma-decalactone concentration of 6.6 g/l was reached with the strain A19, whereas a maximum of 0.1 g/l was obtained with the mesophilic strain. Our results suggest that the ability to synthesize gamma-decalactone is a particularity of the strain A19, since the mesophilic strain (no. 30645) produced small amounts, and the other (no. 31354) did not exhibit this property. It is, to our knowledge, the first report of gamma-decalactone production by R. aurantiaca and furthermore by a psychrophilic yeast strain. Moreover, the amount of gamma-decalactone obtained in fermentor with the strain 19 was on the order of concentrations usually described in patents.

  18. Draft genome of Cryobacterium sp. MLB-32, an obligate psychrophile from glacier cryoconite holes of high Arctic.

    PubMed

    Singh, Purnima; Kapse, Neelam; Arora, Preeti; Singh, Shiv Mohan; Dhakephalkar, Prashant K

    2015-06-01

    Obligate psychrophilic, Cryobacterium sp. MLB-32, was isolated from cryoconite holes of high Arctic glaciers. Here, we report the first draft genome sequence of the putative novel species of the genus Cryobacterium, providing opportunities for biotechnological and agricultural exploitation of its genome features.

  19. Combined extrinsic and intrinsic manipulations exert complementary neuronal enrichment in embryonic rat neural precursor cultures: an in vitro and in vivo analysis.

    PubMed

    Furmanski, Orion; Gajavelli, Shyam; Lee, Jeung Woon; Collado, Maria E; Jergova, Stanislava; Sagen, Jacqueline

    2009-07-01

    Numerous central nervous system (CNS) disorders share a common pathology in dysregulation of gamma-aminobutyric acid (GABA) inhibitory signaling. Transplantation of GABA-releasing cells at the site of disinhibition holds promise for alleviating disease symptoms with fewer side effects than traditional drug therapies. We manipulated fibroblast growth factor (FGF)-2 deprivation and mammalian achaete-scute homolog (MASH)1 transcription factor levels in an attempt to amplify the default GABAergic neuronal fate in cultured rat embryonic neural precursor cells (NPCs) for use in transplantation studies. Naïve and MASH1 lentivirus-transduced NPCs were maintained in FGF-2 or deprived of FGF-2 for varying lengths of time. Immunostaining and quantitative analysis showed that GABA- and beta-III-tubulin-immunoreactive cells generally decreased through successive passages, suggesting a loss of neurogenic potential in rat neurospheres expanded in vitro. However, FGF-2 deprivation resulted in a small, but significantly increased population of GABAergic cells derived from passaged neurospheres. In contrast to naïve and GFP lentivirus-transduced clones, MASH1 transduction resulted in increased bromodeoxyuridine (BrdU) incorporation and clonal colony size. Western blotting showed that MASH1 overexpression and FGF-2 deprivation additively increased beta-III-tubulin and decreased cyclic nucleotide phosphodiesterase (CNPase) expression, whereas FGF-2 deprivation alone attenuated glial fibrillary acidic protein (GFAP) expression. These results suggest that low FGF-2 signaling and MASH1 activity can operate in concert to enrich NPC cultures for a GABA neuronal phenotype. When transplanted into the adult rat spinal cord, this combination also yielded GABAergic neurons. These findings indicate that, even for successful utilization of the default GABAergic neuronal precursor fate, a combination of both extrinsic and intrinsic manipulations will likely be necessary to realize the full

  20. DNRA and Denitrification Coexist over a Broad Range of Acetate/N-NO3− Ratios, in a Chemostat Enrichment Culture

    PubMed Central

    van den Berg, Eveline M.; Boleij, Marissa; Kuenen, J. Gijs; Kleerebezem, Robbert; van Loosdrecht, Mark C. M.

    2016-01-01

    Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) compete for nitrate in natural and engineered environments. A known important factor in this microbial competition is the ratio of available electron donor and elector acceptor, here expressed as Ac/N ratio (acetate/nitrate-nitrogen). We studied the impact of the Ac/N ratio on the nitrate reduction pathways in chemostat enrichment cultures, grown on acetate mineral medium. Stepwise, conditions were changed from nitrate limitation to nitrate excess in the system by applying a variable Ac/N ratio in the feed. We observed a clear correlation between Ac/N ratio and DNRA activity and the DNRA population in our reactor. The DNRA bacteria dominated under nitrate limiting conditions in the reactor and were outcompeted by denitrifiers under limitation of acetate. Interestingly, in a broad range of Ac/N ratios a dual limitation of acetate and nitrate occurred with co-occurrence of DNRA bacteria and denitrifiers. To explain these observations, the system was described using a kinetic model. The model illustrates that the Ac/N effect and concomitant broad dual limitation range related to the difference in stoichiometry between both processes, as well as the differences in electron donor and acceptor affinities. Population analysis showed that the presumed DRNA-performing bacteria were the same under nitrate limitation and under dual limiting conditions, whereas the presumed denitrifying population changed under single and dual limitation conditions. PMID:27933040

  1. Isolation and characterization of a mesophilic heavy-metals-tolerant sulfate-reducing bacterium Desulfomicrobium sp. from an enrichment culture using phosphogypsum as a sulfate source.

    PubMed

    Azabou, Samia; Mechichi, Tahar; Patel, Bharat K C; Sayadi, Sami

    2007-02-09

    A sulfate-reducing bacterium, was isolated from a 6 month trained enrichment culture in an anaerobic media containing phosphogypsum as a sulfate source, and, designated strain SA2. Cells of strain SA2 were rod-shaped, did not form spores and stained Gram-negative. Phylogenetic analysis of the 16S rRNA gene sequence of the isolate revealed that it was related to members of the genus Desulfomicrobium (average sequence similarity of 98%) with Desulfomicrobium baculatum being the most closely related (sequence similarity of 99%). Strain SA2 used thiosulfate, sulfate, sulfite and elemental sulfur as electron acceptors and produced sulfide. Strain SA2 reduced sulfate contained in 1-20g/L phosphogypsum to sulfide with reduction of sulfate contained in 2g/L phosphogypsum being the optimum concentration. Strain SA2 grew with metalloid, halogenated and non-metal ions present in phosphogypsum and with added high concentrations of heavy metals (125ppm Zn and 100ppm Ni, W, Li and Al). The relative order for the inhibitory metal concentrations, based on the IC(50) values, was Cu, Te>Cd>Fe, Co, Mn>F, Se>Ni, Al, Li>Zn.

  2. DNA stable-isotope probing of oil sands tailings pond enrichment cultures reveals different key players for toluene degradation under methanogenic and sulfidogenic conditions.

    PubMed

    Laban, Nidal Abu; Dao, Anh; Foght, Julia

    2015-05-01

    Oil sands tailings ponds are anaerobic repositories of fluid wastes produced by extraction of bitumen from oil sands ores. Diverse indigenous microbiota biodegrade hydrocarbons (including toluene) in situ, producing methane, carbon dioxide and/or hydrogen sulfide, depending on electron acceptor availability. Stable-isotope probing of cultures enriched from tailings associated specific taxa and functional genes to (13)C6- and (12)C7-toluene degradation under methanogenic and sulfate-reducing conditions. Total DNA was subjected to isopycnic ultracentrifugation followed by gradient fraction analysis using terminal restriction fragment length polymorphism (T-RFLP) and construction of 16S rRNA, benzylsuccinate synthase (bssA) and dissimilatory sulfite reductase (dsrB) gene clone libraries. T-RFLP analysis plus sequencing and in silico digestion of cloned taxonomic and functional genes revealed that Clostridiales, particularly Desulfosporosinus (136 bp T-RF) contained bssA genes and were key toluene degraders during methanogenesis dominated by Methanosaeta. Deltaproteobacterial Desulfobulbaceae (157 bp T-RF) became dominant under sulfidogenic conditions, likely because the Desulfosporosinus T-RF 136 apparently lacks dsrB and therefore, unlike its close relatives, is presumed incapable of dissimilatory sulfate reduction. We infer incomplete oxidation of toluene by Desulfosporosinus in syntrophic association with Methanosaeta under methanogenic conditions, and complete toluene oxidation by Desulfobulbaceae during sulfate reduction.

  3. Enrichment of the Cancer Stem Phenotype in Sphere Cultures of Prostate Cancer Cell Lines Occurs through Activation of Developmental Pathways Mediated by the Transcriptional Regulator ΔNp63α

    PubMed Central

    Portillo-Lara, Roberto; Alvarez, Mario Moisés

    2015-01-01

    Background Cancer stem cells (CSC) drive prostate cancer tumor survival and metastasis. Nevertheless, the development of specific therapies against CSCs is hindered by the scarcity of these cells in prostate tissues. Suspension culture systems have been reported to enrich CSCs in primary cultures and cell lines. However, the molecular mechanisms underlying this phenomenon have not been fully explored. Methodology/Principal Findings We describe a prostasphere assay for the enrichment of CD133+ CSCs in four commercial PCa cell lines: 22Rv1, DU145, LNCaP, and PC3. Overexpression of CD133, as determined by flow cytometric analysis, correlated with an increased clonogenic, chemoresistant, and invasive potential in vitro. This phenotype is concordant to that of CSCs in vivo. Gene expression profiling was then carried out using the Cancer Reference panel and the nCounter system from NanoString Technologies. This analysis revealed several upregulated transcripts that can be further explored as potential diagnostic markers or therapeutic targets. Furthermore, functional annotation analysis suggests that ΔNp63α modulates the activation of developmental pathways responsible for the increased stem identity of cells growing in suspension cultures. Conclusions/Significance We conclude that profiling the genetic mechanisms involved in CSC enrichment will help us to better understand the molecular pathways that underlie CSC pathophysiology. This platform can be readily adapted to enrich and assay actual patient samples, in order to design patient-specific therapies that are aimed particularly against CSCs. PMID:26110651

  4. Enrichment of the Cancer Stem Phenotype in Sphere Cultures of Prostate Cancer Cell Lines Occurs through Activation of Developmental Pathways Mediated by the Transcriptional Regulator ΔNp63α.

    PubMed

    Portillo-Lara, Roberto; Alvarez, Mario Moisés

    2015-01-01

    Cancer stem cells (CSC) drive prostate cancer tumor survival and metastasis. Nevertheless, the development of specific therapies against CSCs is hindered by the scarcity of these cells in prostate tissues. Suspension culture systems have been reported to enrich CSCs in primary cultures and cell lines. However, the molecular mechanisms underlying this phenomenon have not been fully explored. We describe a prostasphere assay for the enrichment of CD133+ CSCs in four commercial PCa cell lines: 22Rv1, DU145, LNCaP, and PC3. Overexpression of CD133, as determined by flow cytometric analysis, correlated with an increased clonogenic, chemoresistant, and invasive potential in vitro. This phenotype is concordant to that of CSCs in vivo. Gene expression profiling was then carried out using the Cancer Reference panel and the nCounter system from NanoString Technologies. This analysis revealed several upregulated transcripts that can be further explored as potential diagnostic markers or therapeutic targets. Furthermore, functional annotation analysis suggests that ΔNp63α modulates the activation of developmental pathways responsible for the increased stem identity of cells growing in suspension cultures. We conclude that profiling the genetic mechanisms involved in CSC enrichment will help us to better understand the molecular pathways that underlie CSC pathophysiology. This platform can be readily adapted to enrich and assay actual patient samples, in order to design patient-specific therapies that are aimed particularly against CSCs.

  5. Feeding glycerol-enriched yeast culture improves performance, energy status, and heat shock protein gene expression of lactating Holstein cows under heat stress.

    PubMed

    Liu, J; Ye, G; Zhou, Y; Liu, Y; Zhao, L; Liu, Y; Chen, X; Huang, D; Liao, S F; Huang, K

    2014-06-01

    This study was conducted to evaluate the effects of supplemental common yeast culture (CY) and glycerol-enriched yeast culture (GY) on performance, plasma metabolites, antioxidant status, and heat shock protein 70 (HSP70) mRNA expression in lactating Holstein cows under heat stress. During summer months, 30 healthy multiparous lactating cows (parity 3.25 ± 0.48; 60 ± 13 d in milk [DIM]; 648 ± 57 kg BW; an average milk yield of 33.8 ± 1.6 kg/d) were blocked by parity, previous milk yield, and DIM and randomly allocated to 3 dietary treatments: no supplemental yeast culture (Control), 1 L/d of CY (33.1 g yeast) per cow, and 2 L/d of GY (153.2 g glycerol and 31.6 g yeast) per cow. During the 60-d experiment, values of air temperature and relative humidity inside the barn were recorded hourly every 3 d to calculate temperature-humidity index (THI). Weekly rectal temperatures (RT) and respiration rates and daily DMI and milk yield were recorded for all cows. Milk and blood samples were taken twice monthly, and BW and BCS were obtained on d 0 and 60. In this experiment, THI values indicated cows experienced a moderate heat stress. Cows supplemented with CY and GY had greater yields of milk, energy-corrected milk and milk fat, and milk fat percent but lower HSP70 mRNA expression in peripheral blood lymphocytes than Control cows (P < 0.05). Supplementing CY and GY tended (P < 0.15) to decrease RT at 1400 h, increase milk protein yield and erythrocyte glutathione, and reduce plasma urea nitrogen compared with Control. Lower plasma NEFA concentration and HSP70 mRNA expression in peripheral blood lymphocytes (P < 0.05) and tendencies towards greater plasma glucose concentration (P = 0.11) but less BW loss (P = 0.14) were observed in GY relative to CY cows. In conclusion, either CY or GY supplementation partially mitigated the negative effects of heat stress on performance and HSP70 mRNA expression of lactating cows, and GY supplementation provided additional improvements

  6. Microbial diversity in methanogenic hydrocarbon-degrading enrichment cultures isolated from a water-flooded oil reservoir (Dagang oil field, China)

    NASA Astrophysics Data System (ADS)

    Jiménez, Núria; Cai, Minmin; Straaten, Nontje; Yao, Jun; Richnow, Hans H.; Krüger, Martin

    2015-04-01

    Microbial transformation of oil to methane is one of the main degradation processes taking place in oil reservoirs, and it has important consequences as it negatively affects the quality and economic value of the oil. Nevertheless, methane could constitute a recovery method of carbon from exhausted reservoirs. Previous studies combining geochemical and isotopic analysis with molecular methods showed evidence for in situ methanogenic oil degradation in the Dagang oil field, China (Jiménez et al., 2012). However, the main key microbial players and the underlying mechanisms are still relatively unknown. In order to better characterize these processes and identify the main microorganisms involved, laboratory biodegradation experiments under methanogenic conditions were performed. Microcosms were inoculated with production and injection waters from the reservoir, and oil or 13C-labelled single hydrocarbons (e.g. n-hexadecane or 2-methylnaphthalene) were added as sole substrates. Indigenous microbiota were able to extensively degrade oil within months, depleting most of the n-alkanes in 200 days, and producing methane at a rate of 76 ± 6 µmol day-1 g-1 oil added. They could also produce heavy methane from 13C-labeled 2-methylnaphthalene, suggesting that further methanogenesis may occur from the aromatic and polyaromatic fractions of Dagang reservoir fluids. Microbial communities from oil and 2-methyl-naphthalene enrichment cultures were slightly different. Although, in both cases Deltaproteobacteria, mainly belonging to Syntrophobacterales (e.g. Syntrophobacter, Smithella or Syntrophus) and Clostridia, mostly Clostridiales, were among the most represented taxa, Gammaproteobacteria could be only identified in oil-degrading cultures. The proportion of Chloroflexi, exclusively belonging to Anaerolineales (e.g. Leptolinea, Bellilinea) was considerably higher in 2-methyl-naphthalene degrading cultures. Archaeal communities consisted almost exclusively of representatives of

  7. Project ENRICH.

    ERIC Educational Resources Information Center

    Gwaley, Elizabeth; And Others

    Project ENRICH was conceived in Beaver County, Pennsylvania, to: (1) identify preschool children with learning disabilities, and (2) to develop a program geared to the remediation of the learning disabilities within a school year, while allowing the child to be enrolled in a regular class situation for the following school year. Through…

  8. Job Enrichment

    ERIC Educational Resources Information Center

    Sanders, Rick

    1970-01-01

    Job enrichment means giving people more decision-making power, more responsibility, more grasp of the totality of the job, and a sense of their own importance in the company. This article presents evidence of the successful working of this approach (Donnelly Mirrors), and the lack of success with an opposing approach (General Motors). (NL)

  9. A study of psychrophilic organisms isolated from the manufacture and assembly areas of spacecraft to be used in the Viking mission

    NASA Technical Reports Server (NTRS)

    Foster, T. L.; Winans, L., Jr.

    1973-01-01

    The sampling of soils from the manufacture and assembly areas of the Viking spacecraft is reported and the methodology employed in the analysis of these samples for psychrophilic microorganisms, and temperature studies on these organisms is outlined. Results showing the major types of organisms and the percentage of obligate psychrophiles in each sample are given and discussed. Emphasis in all areas is toward application of these results to the objectives of the planetary quarantine program.

  10. Identification of miniature plasmids in psychrophilic Arctic bacteria of the genus Variovorax.

    PubMed

    Ciok, Anna; Dziewit, Lukasz; Grzesiak, Jakub; Budzik, Karol; Gorniak, Dorota; Zdanowski, Marek K; Bartosik, Dariusz

    2016-04-01

    The Svalbard archipelago (Spitsbergen Island) is the northernmost landmass in the European Arctic and has a variety of small- and medium-sized glaciers. The plasmidome of eleven psychrophilic strains of Variovorax spp. isolated from the ice surface of Hans and Werenskiold Glaciers of Spitsbergen Island, was defined. This analysis revealed the presence of six plasmids whose nucleotide sequences have been determined. Four of them, exhibiting high reciprocal sequence similarity, possess unique structures, since their genomes lack any recognized genes. These miniature replicons, not exceeding 1 kb in size, include pHW69V1 (746 bp), which is the smallest autonomous replicon so far identified in free-living bacteria. The miniature plasmids share no similarity with known sequences present in the databases. In silico and experimental analyses identified conserved DNA regions essential for the initiation of replication of these replicons.

  11. Kinetics of psychrophilic anaerobic sequencing batch reactor treating flushed dairy manure.

    PubMed

    Ma, Jingwei; Yu, Liang; Frear, Craig; Zhao, Quanbao; Li, Xiujin; Chen, Shulin

    2013-03-01

    In this study, a new strategy, improving biomass retention with fiber material present within the dairy manure as biofilm carriers, was evaluated for treating flushed dairy manure in a psychrophilic anaerobic sequencing batch reactor (ASBR). A kinetic study was carried out for process control and design by comparing four microbial growth kinetic models, i.e. first order, Grau, Monod and Chen and Hashimoto models. A volumetric methane production rate of 0.24L/L/d of and a specific methane productivity of 0.19L/gVSloaded were achieved at 6days HRT. It was proved that an ASBR using manure fiber as support media not only improved methane production but also reduced the necessary HRT and temperature to achieve a similar treating efficiency compared with current technologies. The kinetic model can be used for design and optimization of the process. Copyright © 2012. Published by Elsevier Ltd.

  12. Temperature adaptations in psychrophilic, mesophilic and thermophilic chloride-dependent alpha-amylases.

    PubMed

    Cipolla, Alexandre; Delbrassine, François; Da Lage, Jean-Luc; Feller, Georges

    2012-09-01

    The functional and structural adaptations to temperature have been addressed in homologous chloride-dependent α-amylases from a psychrophilic Antarctic bacterium, the ectothermic fruit fly, the homeothermic pig and from a thermophilic actinomycete. This series covers nearly all temperatures encountered by living organisms. We report a striking continuum in the functional properties of these enzymes coupled to their structural stability and related to the thermal regime of the source organism. In particular, thermal stability recorded by intrinsic fluorescence, circular dichroism and differential scanning calorimetry appears to be a compromise between the requirement for a stable native state and the proper structural dynamics to sustain the function at the environmental/physiological temperatures. The thermodependence of activity, the kinetic parameters, the activations parameters and fluorescence quenching support these activity-stability relationships in the investigated α-amylases.

  13. Isolation and characterization of marine psychrophilic phage-host systems from Arctic sea ice.

    PubMed

    Borriss, Michael; Helmke, Elisabeth; Hanschke, Renate; Schweder, Thomas

    2003-10-01

    Phage-host systems from extreme cold environments have rarely been surveyed. This study is concerned with the isolation and characterization of three different phage-host systems from Arctic sea ice and melt pond samples collected north-west of Svalbard (Arctic). On the basis of 16S rDNA sequences, the three bacterial phage hosts exhibited the greatest similarity to the species Shewanella frigidimarina (96.0%), Flavobacterium hibernum (94.0%), and Colwellia psychrerythraea (98.4%), respectively. The host bacteria are psychrophilic with good growth at 0 degrees C, resulting in a rapid formation of visible colonies at this temperature. The phages showed an even more pronounced adaptation to cold temperatures than the bacteria, with growth maxima below 14 degrees C and good plaque formation at 0 degrees C. Transmission electron microscopy (TEM) examinations revealed that the bacteriophages belonged to the tailed, double-stranded DNA phage families Siphoviridae and Myoviridae. All three phages were host-specific.

  14. Paenibacillus tibetensis sp. nov., a psychrophilic bacterium isolated from alpine swamp meadow soil.

    PubMed

    Han, Li-Li; He, Ji-Zheng; Zheng, Yuan-Ming; Zeng, Jun; Zhang, Li-Mei

    2015-05-01

    A novel psychrophilic strain, SSB001(T), was isolated from an alpine swamp meadow soil in Tibet, China, and identified as a representative of a novel phylogenetic subclade in the genus Paenibacillus , with Paenibacillus antarcticus (96.2%), Paenibacillus macquariensis (96.53%) and Paenibacillus glacialis (96.2%) as the most closely related species on the basis of 16S rRNA gene sequence analyses. The strain was distinguished from defined species of the genus Paenibacillus by further study of rpoB gene sequences, phenotypic characterization, cellular fatty acid composition, quinones, polar lipids and meso-diaminopimelic acid in the peptidoglycan. Based upon these results, we propose the strain as a representative of a novel species named Paenibacillus tibetensis sp. nov., with SSB001(T) ( =ACCC 19728(T) =DSM 29321(T)) as the type strain. The DNA G+C content (mol%) of strain SSB001(T) was 40.18 mol% (HPLC).

  15. Dry-heat resistance of selected psychrophiles. [Viking lander in spacecraft sterilization

    NASA Technical Reports Server (NTRS)

    Winans, L.; Pflug, I. J.; Foster, T. L.

    1977-01-01

    The dry-heat resistance characteristics of spores of psychrophilic organisms isolated from soil samples from the Viking spacecraft assembly areas at Cape Kennedy Space Flight Center, Cape Canaveral, Fla., were studied. Spore suspensions were produced, and dry-heat D values were determined for the microorganisms that demonstrated growth or survival under a simulated Martian environment. The dry-heat tests were carried out by using the planchet-boat-hot plate system at 110 and 125 C with an ambient relative humidity of 50% at 22 C. The spores evaluated had a relatively low resistance to dry heat. D (110 C) values ranged from 7.5 to 122 min, whereas the D (125 C) values ranged from less than 1.0 to 9.8 min.

  16. In silico analysis of the thermodynamic stability changes of psychrophilic and mesophilic alpha-amylases upon exhaustive single-site mutations.

    PubMed

    Gilis, Dimitri

    2006-01-01

    Identifying sequence modifications that distinguish psychrophilic from mesophilic proteins is important for designing enzymes with different thermodynamic stabilities and to understand the underlying mechanisms. The PoPMuSiC algorithm is used to introduce, in silico, all the single-site mutations in four mesophilic and one psychrophilic chloride-dependent alpha-amylases and to evaluate the changes in thermodynamic stability. The analysis of the distribution of the sequence positions that could be stabilized upon mutation shows a clear difference between the three domains of psychrophilic and mesophilic alpha-amylases. Most of the mutations stabilizing the psychrophilic enzyme are found in domains B and C, contrary to the mesophilic proteins where they are preferentially situated in the catalytic domain A. Moreover, the calculations show that the environment of some residues responsible for the activity of the psychrophilic protein has evolved to reinforce favorable interactions with these residues. In the second part, these results are exploited to propose rationally designed mutations that are predicted to confer to the psychrophilic enzyme mesophilic-like thermodynamic properties. Interestingly, most of the mutations found in domain C strengthen the interactions with domain A, in agreement with suggestions made on the basis of structural analyses. Although this study focuses on single-site mutations, the thermodynamic effects of the recommended mutations should be additive if the mutated residues are not close in space.

  17. Role of fatty acids in cold adaptation of Antarctic psychrophilic Flavobacterium spp.

    PubMed

    Králová, Stanislava

    2017-09-01

    Cold-loving microorganisms developed numerous adaptation mechanisms allowing them to survive in extremely cold habitats, such as adaptation of the cell membrane. The focus of this study was on the membrane fatty acids of Antarctic Flavobacterium spp., and their adaptation response to cold-stress. Fatty acids and cold-response of Antarctic flavobacteria was also compared to mesophilic and thermophilic members of the genus Flavobacterium. The results showed that the psychrophiles produced more types of major fatty acids than meso- and thermophilic members of this genus, namely C15:1 iso G, C15:0 iso, C15:0 anteiso, C15:1ω6c, C15:0 iso 3OH, C17:1ω6c, C16:0 iso 3OH and C17:0 iso 3OH, summed features 3 (C16:1ω7cand/or C16:1ω6c) and 9 (C16:0 10-methyl and/or C17:1 iso ω9c). It was shown that the cell membrane of psychrophiles was composed mainly of branched and unsaturated fatty acids. The results also implied that Antarctic flavobacteria mainly used two mechanisms of membrane fluidity alteration in their cold-adaptive response. The first mechanism was based on unsaturation of fatty acids, and the second mechanism on de novo synthesis of branched fatty acids. The alteration of the cell membrane was shown to be similar for all thermotypes of members of the genus Flavobacterium. Copyright © 2017 Elsevier GmbH. All rights reserved.

  18. Plasmids of psychrophilic and psychrotolerant bacteria and their role in adaptation to cold environments

    PubMed Central

    Dziewit, Lukasz; Bartosik, Dariusz

    2014-01-01

    Extremely cold environments are a challenge for all organisms. They are mostly inhabited by psychrophilic and psychrotolerant bacteria, which employ various strategies to cope with the cold. Such harsh environments are often highly vulnerable to the influence of external factors and may undergo frequent dynamic changes. The rapid adjustment of bacteria to changing environmental conditions is crucial for their survival. Such “short-term” evolution is often enabled by plasmids—extrachromosomal replicons that represent major players in horizontal gene transfer. The genomic sequences of thousands of microorganisms, including those of many cold-active bacteria have been obtained over the last decade, but the collected data have yet to be thoroughly analyzed. This report describes the results of a meta-analysis of the NCBI sequence databases to identify and characterize plasmids of psychrophilic and psychrotolerant bacteria. We have performed in-depth analyses of 66 plasmids, almost half of which are cryptic replicons not exceeding 10 kb in size. Our analyses of the larger plasmids revealed the presence of numerous genes, which may increase the phenotypic flexibility of their host strains. These genes encode enzymes possibly involved in (i) protection against cold and ultraviolet radiation, (ii) scavenging of reactive oxygen species, (iii) metabolism of amino acids, carbohydrates, nucleotides and lipids, (iv) energy production and conversion, (v) utilization of toxic organic compounds (e.g., naphthalene), and (vi) resistance to heavy metals, metalloids and antibiotics. Some of the plasmids also contain type II restriction-modification systems, which are involved in both plasmid stabilization and protection against foreign DNA. Moreover, approx. 50% of the analyzed plasmids carry genetic modules responsible for conjugal transfer or mobilization for transfer, which may facilitate the spread of these replicons among various bacteria, including across species

  19. Toward understanding life under subzero conditions: the significance of exploring psychrophilic "cold-shock" proteins.

    PubMed

    Kuhn, Emanuele

    2012-11-01

    Understanding the behavior of proteins under freezing conditions is vital for detecting and locating extraterrestrial life in cold environments, such as those found on Mars and the icy moons of Jupiter and Saturn. This review highlights the importance of studying psychrophilic "cold-shock" proteins, a topic that has yet to be explored. A strategy for analyzing the psychrophilic RNA helicase protein CsdA (Psyc_1082) from Psychrobacter arcticus 273-4 as a key protein for life under freezing temperatures is proposed. The experimental model presented here was developed based on previous data from investigations of Escherichia coli, P. arcticus 273-4, and RNA helicases. P. arcticus 273-4 is considered a model for life in freezing environments. It is capable of growing in temperatures as cold as -10°C by using physiological strategies to survive not only in freezing temperatures but also under low-water-activity and limited-nutrient-availability conditions. The analyses of its genome, transcriptome, and proteome revealed specific adaptations that allow it to inhabit freezing environments by adopting a slow metabolic strategy rather than a cellular dormancy state. During growth at subzero temperatures, P. arcticus 273-4 genes related to energy metabolism and carbon substrate incorporation are downregulated, and genes for maintenance of membranes, cell walls, and nucleic acid motion are upregulated. At -6°C, P. arcticus 273-4 does not upregulate the expression of either RNA or protein chaperones; however, it upregulates the expression of its cold-shock induced DEAD-box RNA helicase protein A (CsdA - Psyc_1082). CsdA - Psyc_1082 was investigated as a key helper protein for sustaining life in subzero conditions. Proving CsdA - Psyc_1082 to be functional as a key protein for life under freezing temperatures may extend the known minimum growth temperature of a mesophilic cell and provide key information about the mechanisms that underlie cold-induced biological systems in

  20. Psychrobacter submarinus sp. nov. and Psychrobacter marincola sp. nov., psychrophilic halophiles from marine environments.

    PubMed

    Romanenko, Lyudmila A; Schumann, Peter; Rohde, Manfred; Lysenko, Anatoly M; Mikhailov, Valery V; Stackebrandt, Erko

    2002-07-01

    Two novel psychrophilic, halophilic, Psychrobacter-like bacteria, strains KMM 225T and KMM 277T, were isolated from sea water and the internal tissues of an ascidian Polysyncraton sp. specimen, respectively, and characterized using a polyphasic approach, which included phenotypic, genotypic, chemotaxonomic and phylogenetic analyses. The novel marine isolates were Gram-negative, aerobic, coccoid, oxidase- and catalase-positive, non-pigmented, non-motile, psychrophilic and halophilic and they utilized a restricted spectrum of carbon sources. Strains KMM 225T and KMM 277T required sea water or sodium ions for growth and were tolerant of up to 12-15% (w/v) NaCl. Growth of strains KMM 225T and KMM 277T was observed at 4-35 and 7-35 degrees C, respectively. The DNA G+C contents of KMM 225T and KMM 277T were respectively 46-8 and 50.7 mol %. Comparison of almost complete 16S rDNA sequences of strains KMM 225T and KMM 277T revealed that both strains were phylogenetically most closely related to each other (99.9% sequence similarity) and slightly less related to Psychrobacter glacincola, with 97.2 and 97.8% similarity, respectively. DNA-DNA reassociation between KMM 225T and KMM 277T revealed 15% similarity, whereas similarity to other Psychrobacter species was 14-25%. Strains KMM 225T and KMM 277T differed from one another in their growth temperature, organic substrate utilization, antibiotic sensitivity and DNA G+C content. Both strains examined could be distinguished from all previously described Psychrobacter species by their physiological, genotypic and phylogenetic characteristics. On the basis of the physiological and molecular properties of the novel isolates, the names Psychrobacter submarinus sp. nov. (type strain KMM 225T = DSM 14161T) and Psychrobacter marincola sp. nov. (type strain KMM 277T = DSM 14160T) are proposed.

  1. Metaproteogenomic analysis of a sulfate-reducing enrichment culture reveals genomic organization of key enzymes in the m-xylene degradation pathway and metabolic activity of proteobacteria.

    PubMed

    Bozinovski, Dragana; Taubert, Martin; Kleinsteuber, Sabine; Richnow, Hans-Hermann; von Bergen, Martin; Vogt, Carsten; Seifert, Jana

    2014-10-01

    This study aimed to ascertain the functional and phylogenetic relationships within an m-xylene degrading sulfate-reducing enrichment culture, which had been maintained for several years in the laboratory with m-xylene as the sole source of carbon and energy. Previous studies indicated that a phylotype affiliated to the Desulfobacteraceae was the main m-xylene assimilating organism. In the present study, genes and gene products were identified by a metaproteogenomic approach using LC-MS/MS analysis of the microbial community, and 2426 peptides were identified from 576 proteins. In the metagenome of the community, gene clusters encoding enzymes involved in fumarate addition to a methyl moiety of m-xylene (nms, bss), as well as gene clusters coding for enzymes involved in modified beta-oxidation to (3-methyl)benzoyl-CoA (bns), were identified in two separate contigs. Additionally, gene clusters containing homologues to bam genes encoding benzoyl-CoA reductase (Bcr) class II, catalyzing the dearomatization of (3-methyl)benzoyl-CoA, were identified. Time-resolved protein stable isotope probing (protein-SIP) experiments using (13)C-labeled m-xylene showed that the respective gene products were highly (13)C-labeled. The present data suggested the identification of gene products that were similar to those involved in methylnaphthalene degradation even though the consortium was not capable of growing in the presence of naphthalene, methylnaphthalene or toluene as substrates. Thus, a novel branch of enzymes was found that was probably specific for anaerobic m-xylene degradation. Copyright © 2014 Elsevier GmbH. All rights reserved.

  2. [NO3-/NO2- inhibits sulfate-reducing activity of the enrichment culture of sulfate-reducing prokaryotes from an off-shore oil reservoir at Bohai Bay, China].

    PubMed

    Liu, Hong-Yu; Shi, Rong-Jiu; Zhang, Ying; Shi, Zhen-Guo; Zhang, Ying-Yue; Yu, Liang; Zhang, Xiao-Bo; Tan, Tao

    2014-08-01

    Long-term injection of sulfate-rich water into oil reservoirs stimulates the proliferation of sulfate-reducing prokaryotes (SRP) therein and results in production of a great amount of H2S, leading to souring in oil reservoirs and related environmental problems. In this study, we first, using modified API RP 38 medium, enriched SRP present in production water from a producing well at Bohai Bay, China, and then examined the inhibitory effects of nitrate or nitrite on sulfate reduction activity of the SRP. Results showed that the enriched SRP culture exhibited a high sulfate reduction activity as indicated by a sulfate-reducing rate of 10.4 mmol SO4(2-) x d(-1) x g(-1) dry cell. In presence of 0.4, 0.8, 1.8, and 4.2 mmol x L(-1) nitrate, sulfate reduction was inhibited for 5, 9, 20, and over 35 days, respectively. With the addition of 0.6, 0.9, 1.4, 2.6 and 4.6 mmol x L(-1) of nitrite, the inhibitory period lasted 3, 12, 22, and over 39 days, respectively. The SRP enrichment culture could dissimilatorily reduce nitrate to ammonium. When sulfate, nitrate and nitrite coexisted, nitrate or nitrite was preferentially used over sulfate as electron acceptor by the enriched SRP. This competitive use of electron acceptor and the strong inhibitory effect of nitrite possibly accounted for the suppression of nitrate and nitrite on the sulfate-reducing activity of the enriched SRP cultures from offshore oil reservoir at Bohai Bay.

  3. Feeding glycerol-enriched yeast culture improves lactation performance, energy status, and hepatic gluconeogenic enzyme expression of dairy cows during the transition period.

    PubMed

    Ye, G; Liu, J; Liu, Y; Chen, X; Liao, S F; Huang, D; Huang, K

    2016-06-01

    This study aimed to evaluate the effects of feeding glycerol-enriched yeast culture (GY) on feed intake, lactation performance, blood metabolites, and expression of some key hepatic gluconeogenic enzymes in dairy cows during the transition period. Forty-four multiparous transition Holstein cows were blocked by parity, previous 305-d mature equivalent milk yield, and expected calving date and randomly allocated to 4 dietary treatments: Control (no additive), 2 L/d of GY (75.8 g/L glycerol and 15.3 g/L yeast), 150 g/d of glycerol (G; 0.998 g/g glycerol), and 1 L/d of yeast culture (Y; 31.1 g/L yeast). All additives were top-dressed and hand mixed into the upper one-third of the total mixed ration in the morning from -14 to +28 d relative to calving. Results indicated that the DMI, NE intake, change of BCS, and milk yields were not affected by the treatments ( > 0.05). Supplementation of GY or Y increased milk fat percentages, milk protein percentages, and milk protein yields relative to the Control or G group ( < 0.05). Cows fed GY or G had higher glucose levels and lower β-hydroxybutyric acid (BHBA) and NEFA levels in plasma than cows fed the Control ( < 0.05) and had lower NEFA levels than cows fed Y ( < 0.05). On 14 d postpartum, cows fed GY or G had higher enzyme activities, mRNA, and protein expression of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C; < 0.05); higher enzyme activities ( < 0.05) and a tendency toward higher mRNA expression ( < 0.10) of glycerol kinase (GK); and a tendency toward higher enzyme activities of pyruvate carboxylase (PC) in the liver ( < 0.10) when compared with cows fed Control or Y. The enzyme activities, mRNA, and protein expression of PEPCK-C, PC, and GK did not differ between cows fed GY and G ( > 0.10). In conclusion, dietary GY or Y supplementation increased the milk fat and protein content of the cows in early lactation and GY or G supplementation improved the energy status as indicated by greater plasma glucose and

  4. Mungo bean sprout microbiome and changes associated with culture based enrichment protocols used in detection of Gram-negative foodborne pathogens.

    PubMed

    Margot, Heike; Stephan, Roger; Tasara, Taurai

    2016-09-06

    Fresh sprouted seeds have been associated with a number of large outbreaks caused by Salmonella and Shiga toxin-producing E. coli. However, the high number of commensal bacteria found on sprouted seeds hampers the detection of these pathogens. Knowledge about the composition of the sprout microbiome is limited. In this study, the microbiome of mungo bean sprouts and the impact of buffered peptone water (BPW) and Enterobacteriaceae enrichment broth (EE-broth)-based enrichment protocols on this microbiome were investigated. Assessments based on aerobic mesophilic colony counts showed similar increases in mungo bean sprout background flora levels independent of the enrichment protocol used. 16S rRNA sequencing revealed a mungo bean sprout microbiome dominated by Proteobacteria and Bacteroidetes. EE-broth enrichment of such samples preserved and increased Proteobacteria dominance while reducing Bacteroidetes and Firmicutes relative abundances. BPW enrichment, however, increased Firmicutes relative abundance while decreasing Proteobacteria and Bacteroidetes levels. Both enrichments also lead to various genus level changes within the Protobacteria and Firmicutes phyla. New insights into the microbiome associated with mungo bean sprout and how it is influenced through BPW and EE-broth-based enrichment strategies used for detecting Gram-negative pathogens were generated. BPW enrichment leads to Firmicutes and Proteobacteria dominance, whereas EE-broth enrichment preserves Proteobacteria dominance in the mungo bean sprout samples. By increasing the relative abundance of Firmicutes, BPW also increases the abundance of Gram-positive organisms including some that might inhibit recovery of Gram-negative pathogens. The use of EE-broth, although preserving and increasing the dominance of Proteobacteria, can also hamper the detection of lowly abundant Gram-negative target pathogens due to outgrowth of such organisms by the highly abundant non-target Proteobacteria genera

  5. The effects of co-contaminants and native wetland sediments on the activity and dominant transformation mechanisms of a 1,1,2,2-tetrachloroethane (TeCA)-degrading enrichment culture

    USGS Publications Warehouse

    Lorah, Michelle M.; Schiffmacher, Emily N.; Becker, Jennifer G.; Voytek, Mary A.

    2016-01-01

    Bioremediation strategies, including bioaugmentation with chlorinated ethene-degrading enrichment cultures, have been successfully applied in the cleanup of subsurface environments contaminated with tetrachloroethene (PCE) and/or trichloroethene (TCE). However, these compounds are frequently found in the environment as components of mixtures that may also contain chlorinated ethanes and methanes. Under these conditions, the implementation of bioremediation may be complicated by inhibition effects, particularly when multiple dehalorespirers are present. We investigated the ability of the 1,1,2,2-tetrachloroethane (TeCA)-dechlorinating culture WBC-2 to biotransform TeCA alone, or a mixture of TeCA plus PCE and carbon tetrachloride (CT), in microcosms. The microcosms contained electron donors provided to biostimulate the added culture and sediment collected from a wetland where numerous “hotspots” of contamination with chlorinated solvent mixtures exist. The dominant TeCA biodegradation mechanism mediated by the WBC-2 culture in the microcosms was different in the presence of these wetland sediments than in the sediment-free enrichment culture or in previous WBC-2 bioaugmented microcosms and column tests conducted with wetland sediment collected at nearby sites. The co-contaminants and their daughter products also inhibited TeCA biodegradation by WBC-2. These results highlight the need to conduct biodegradability assays at new sites, particularly when multiple contaminants and dehalorespiring populations are present.

  6. Temperature Sensitivity Conferred by ligA Alleles from Psychrophilic Bacteria upon Substitution in Mesophilic Bacteria and a Yeast Species

    PubMed Central

    Pankowski, Jarosław A.; Puckett, Stephanie M.

    2016-01-01

    We have assembled a collection of 13 psychrophilic ligA alleles that can serve as genetic elements for engineering mesophiles to a temperature-sensitive (TS) phenotype. When these ligA alleles were substituted into Francisella novicida, they conferred a TS phenotype with restrictive temperatures between 33 and 39°C. When the F. novicida ligA hybrid strains were plated above their restrictive temperatures, eight of them generated temperature-resistant variants. For two alleles, the mutations that led to temperature resistance clustered near the 5′ end of the gene, and the mutations increased the predicted strength of the ribosome binding site at least 3-fold. Four F. novicida ligA hybrid strains generated no temperature-resistant variants at a detectable level. These results suggest that multiple mutations are needed to create temperature-resistant variants of these ligA gene products. One ligA allele was isolated from a Colwellia species that has a maximal growth temperature of 12°C, and this allele supported growth of F. novicida only as a hybrid between the psychrophilic and the F. novicida ligA genes. However, the full psychrophilic gene alone supported the growth of Salmonella enterica, imparting a restrictive temperature of 27°C. We also tested two ligA alleles from two Pseudoalteromonas strains for their ability to support the viability of a Saccharomyces cerevisiae strain that lacked its essential gene, CDC9, encoding an ATP-dependent DNA ligase. In both cases, the psychrophilic bacterial alleles supported yeast viability and their expression generated TS phenotypes. This collection of ligA alleles should be useful in engineering bacteria, and possibly eukaryotic microbes, to predictable TS phenotypes. PMID:26773080

  7. Genomic, Transcriptomic, and Proteomic Analysis Provide Insights Into the Cold Adaptation Mechanism of the Obligate Psychrophilic Fungus Mrakia psychrophila

    PubMed Central

    Su, Yao; Jiang, Xianzhi; Wu, Wenping; Wang, Manman; Hamid, M. Imran; Xiang, Meichun; Liu, Xingzhong

    2016-01-01

    Mrakia psychrophila is an obligate psychrophilic fungus. The cold adaptation mechanism of psychrophilic fungi remains unknown. Comparative genomics analysis indicated that M. psychrophila had a specific codon usage preference, especially for codons of Gly and Arg and its major facilitator superfamily (MFS) transporter gene family was expanded. Transcriptomic analysis revealed that genes involved in ribosome and energy metabolism were upregulated at 4°, while genes involved in unfolded protein binding, protein processing in the endoplasmic reticulum, proteasome, spliceosome, and mRNA surveillance were upregulated at 20°. In addition, genes related to unfolded protein binding were alternatively spliced. Consistent with other psychrophiles, desaturase and glycerol 3-phosphate dehydrogenase, which are involved in biosynthesis of unsaturated fatty acid and glycerol respectively, were upregulated at 4°. Cold adaptation of M. psychrophila is mediated by synthesizing unsaturated fatty acids to maintain membrane fluidity and accumulating glycerol as a cryoprotectant. The proteomic analysis indicated that the correlations between the dynamic patterns between transcript level changes and protein level changes for some pathways were positive at 4°, but negative at 20°. The death of M. psychrophila above 20° might be caused by an unfolded protein response. PMID:27633791

  8. Detection of psychrophilic and psychrotolerant Clostridium spp. in chilled fresh vacuum-packed meat using different PCR methods.

    PubMed

    Bonke, R; Drees, N; Gareis, M

    2016-01-01

    Since 1989, blown pack spoilage has been recognized as a special form of spoilage in vacuum-packed raw and cooked beef. However, only limited information concerning the occurrences of bacteria causing blown pack spoilage on chilled fresh meat is available. In this study, a total of 63 beef and 33 lamb commercially available samples from different countries and without any signs of spoilage were examined for contamination with psychrophilic and psychrotolerant Clostridium spp. using different PCR systems. In total, 34.4% of the chilled fresh vacuum-packed meats were PCR positive. A higher number of lamb samples were identified as PCR positive compared with beef. A geographical relationship between positive results and the origin of the samples could not be determined. PCR system described by Brightwell and Clemens (Development and validation of a real-time PCR assay specific for Clostridium estertheticum and C. estertheticum-like psychrotolerant bacteria. Meat Sci 2012;92:697-703) gave the highest number of positive detections compared with the Broda, Boerema and Bell PCR system (PCR detection of psychrophilic Clostridium spp. causing 'blown pack' spoilage of vacuum-packed chilled meats. J Appl Microbiol 2003;94:515-22). Eight clostridia isolates from two German beef and four Welsh lamb samples were isolated overall. Three of these clostridia isolates were identified as Clostridium estertheticum whereas five clostridia isolates remain unidentified. The study shows that psychrophilic and psychrotolerant Clostridium spp. are more prevalent in retail samples than previously suspected.

  9. Rapid identification of Salmonella serovars in feces by specific detection of virulence genes, invA and spvC, by an enrichment broth culture-multiplex PCR combination assay.

    PubMed Central

    Chiu, C H; Ou, J T

    1996-01-01

    In order to make a rapid and definite diagnosis of Salmonella enteritis in children, an enrichment broth culture-multiplex PCR combination assay was devised to identify Salmonella serovars directly from fecal samples. Two pairs of oligonucleotide primers were prepared according to the sequences of the chromosomal invA and plasmid spvC genes. PCR with these two primers would produce either one amplicon (from the invA gene) or two amplicons (from the invA and spvC genes), depending on whether or not the Salmonella bacteria contained a virulence plasmid. The fecal sample was diluted 10- to 20-fold into gram-negative enrichment broth and incubated to eliminate inhibitory compounds and also to allow selective enrichment of the bacteria. One or two amplicons were obtained, the expected result if Salmonella bacteria were present. The detection limit of this PCR was about 200 bacteria per reaction mixture. The primers were specific, as no amplification products were obtained with 18 species and 22 isolates of non-Salmonella bacteria tested which could be present in the feces or cause contamination. In contrast, when 23 commonly seen Salmonella serovars (38 isolates) were tested, all were shown to carry the invA gene and seven concomitantly harbored the spvC gene of the virulence plasmid. This assay was applied to the diagnosis of Salmonella enteritis in 57 children who were suffering from mucoid and/or bloody diarrhea. Of the 57 children, 38 were PCR positive and 22 were culture positive. There were two culture-positive samples that were not detected by PCR. Thus, this PCR assay showed an efficiency of 95% (38 of 40), which is much higher than the 60% (24 of 40) by culture alone. Not only is this method more sensitive, rapid, and efficient but it will cause only an incremental increase in the cost of stool processing, since enrichment cultivation of fecal samples from diarrheal patients using gram-negative enrichment broth is a routine practice for identification in many

  10. Accelerated methanogenesis from effluents of hydrogen-producing stage in anaerobic digestion by mixed cultures enriched with acetate and nano-sized magnetite particles.

    PubMed

    Yang, Zhiman; Xu, Xiaohui; Guo, Rongbo; Fan, Xiaolei; Zhao, Xiaoxian

    2015-08-01

    Potential for paddy soil enrichments obtained in the presence of nano-sized magnetite particles (named as PSEM) to promote methane production from effluents of hydrogen-producing stage in two-stage anaerobic digestion was investigated. The results showed that the addition of magnetite significantly accelerated methane production from acetate in a dose-independent manner. The results from high-throughput sequencing analysis revealed that Rhodocyclaceae-related species were selectively enriched, which were likely the key players for conversion of acetate to methane in PSEM. Compared to the paddy soil enrichments obtained in the absence of magnetite (named as PSEC), the maximum methane production rate in PSEM was significantly higher (1.5-5.5times higher for the artificial medium and 0.2-1.7times higher for the effluents). The accelerated methane production from the effluents indicated remarkably application potential of PSEM for improving performance of anaerobic digestion.

  11. Structural prediction and comparative docking studies of psychrophilic β- Galactosidase with lactose, ONPG and PNPG against its counter parts of mesophilic and thermophilic enzymes.

    PubMed

    Kumar, Ponnada Suresh; Pulicherla, Kk; Ghosh, Mrinmoy; Kumar, Anmol; Rao, Krs Sambasiva

    2011-01-01

    Enzymes from psychrophiles catalyze the reactions at low temperatures with higher specific activity. Among all the psychrophilic enzymes produced, cold active β-galactosidase from marine psychrophiles revalorizes a new arena in numerous areas at industrial level. The hydrolysis of lactose in to glucose and galactose by cold active β-galactosidase offers a new promising approach in removal of lactose from milk to overcome the problem of lactose intolerance. Herein we propose, a 3D structure of cold active β-galactosidase enzyme sourced from Pseudoalteromonas haloplanktis by using Modeler 9v8 and best model was developed having 88% of favourable region in ramachandran plot. Modelling was followed by docking studies with the help of Auto dock 4.0 against the three substrates lactose, ONPG and PNPG. In addition, comparative docking studies were also performed for the 3D model of psychrophilic β-galactosidase with mesophilic and thermophilic enzymes. Docking studies revealed that binding affinity of enzyme towards the three different substrates is more for psychrophilic enzyme when compared with mesophilic and thermophilic enzymes. It indicates that the enzyme has high specific activity at low temperature when compared with mesophilic and thermophilic enzymes.

  12. THE ANAEROBIC BIODEGRADATION OF O-,M- AND P-CRESOL BY SULFATE-REDUCING BACTERIAL ENRICHMENT CULTURES OBTAINED FROM A SHALLOW ANOXIC AQUIFER

    EPA Science Inventory

    Sulfate-reducing bacterial enrichments were obtained from a shallow anoxic aquifer for their ability to metabolize either o-, m-, orp-cresol. GC/MS and simultaneous adaptation experiments suggested that the anaerobic decomposition of p-cresol proceeds ...

  13. THE ANAEROBIC BIODEGRADATION OF O-,M- AND P-CRESOL BY SULFATE-REDUCING BACTERIAL ENRICHMENT CULTURES OBTAINED FROM A SHALLOW ANOXIC AQUIFER

    EPA Science Inventory

    Sulfate-reducing bacterial enrichments were obtained from a shallow anoxic aquifer for their ability to metabolize either o-, m-, orp-cresol. GC/MS and simultaneous adaptation experiments suggested that the anaerobic decomposition of p-cresol proceeds ...

  14. Culturable prokaryotic diversity of deep, gas hydrate sediments: first use of a continuous high-pressure, anaerobic, enrichment and isolation system for subseafloor sediments (DeepIsoBUG)

    PubMed Central

    Parkes, R John; Sellek, Gerard; Webster, Gordon; Martin, Derek; Anders, Erik; Weightman, Andrew J; Sass, Henrik

    2009-01-01

    Deep subseafloor sediments may contain depressurization-sensitive, anaerobic, piezophilic prokaryotes. To test this we developed the DeepIsoBUG system, which when coupled with the HYACINTH pressure-retaining drilling and core storage system and the PRESS core cutting and processing system, enables deep sediments to be handled without depressurization (up to 25 MPa) and anaerobic prokaryotic enrichments and isolation to be conducted up to 100 MPa. Here, we describe the system and its first use with subsurface gas hydrate sediments from the Indian Continental Shelf, Cascadia Margin and Gulf of Mexico. Generally, highest cell concentrations in enrichments occurred close to in situ pressures (14 MPa) in a variety of media, although growth continued up to at least 80 MPa. Predominant sequences in enrichments were Carnobacterium, Clostridium, Marinilactibacillus and Pseudomonas, plus Acetobacterium and Bacteroidetes in Indian samples, largely independent of media and pressures. Related 16S rRNA gene sequences for all of these Bacteria have been detected in deep, subsurface environments, although isolated strains were piezotolerant, being able to grow at atmospheric pressure. Only the Clostridium and Acetobacterium were obligate anaerobes. No Archaea were enriched. It may be that these sediment samples were not deep enough (total depth 1126–1527 m) to obtain obligate piezophiles. PMID:19694787

  15. NADP(+)-dependent isocitrate dehydrogenase from a psychrophilic bacterium, Psychromonas marina.

    PubMed

    Hirota, Ryo; Tsubouchi, Kango; Takada, Yasuhiro

    2017-07-01

    The gene encoding NADP(+)-dependent isocitrate dehydrogenase (IDH; EC 1.1.1.42) of a psychrophilic bacterium, Psychromonas marina, was cloned and sequenced. The open reading frame of the gene encoding IDH of P. marina (PmIDH) was 2229 bp in length and corresponded to a polypeptide composed of 742 amino acids. The molecular mass of IDH was calculated as 80,426 Da. The deduced amino acid sequence of PmIDH exhibited high degrees of homology with the monomeric IDH from other bacteria such as Colwellia maris (62% identity) and Azotobacter vinelandii (AvIDH) (64%). His-tagged PmIDH overexpressed in Escherichia coli cells was purified and characterized. The optimum temperature of PmIDH activity was about 35 °C; however, the enzyme lost 74% of the activity after incubation for 10 min at 30 °C, indicating that this enzyme is thermolabile. Chimeric enzymes produced through domain swapping between PmIDH and mesophilic AvIDH were constructed and their optimum temperatures and thermostability were determined. The results suggest that regions 2 and 3, especially region 3, of the two IDHs are involved in their catalytic activities and optimum temperature and thermostability for activity.

  16. Massilia eurypsychrophila sp. nov. a facultatively psychrophilic bacteria isolated from ice core.

    PubMed

    Shen, Liang; Liu, Yongqin; Gu, Zhengquan; Xu, Baiqing; Wang, Ninglian; Jiao, Nianzhi; Liu, Hongcan; Zhou, Yuguang

    2015-07-01

    Strain B528-3(T), a Gram-stain-negative, rod-shaped, aerobic, facultatively psychrophilic bacterium with polar flagella, was isolated from an ice core drilled from Muztagh Glacier, Xinjiang, China. The novel isolate was classified into the genus Massilia. The 16S rRNA gene sequence of the novel isolate shares a pairwise similarity of less than 97% with those of all the type strains of the genus Massilia. The major fatty acids of strain B528-3(T) were summed feature 3 (C16:1ω7c and/or iso-C15:0 2-OH) (57.31%), C16:0 (11.46%) and C18:1ω7c (14.72%). The predominant isoprenoid quinone was Q-8. The DNA G + C content was 62.2 mol% (Tm). The major polar lipids of this bacterium were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. From the genotypic and phenotypic data, it is evident that strain B528-3(T) represents a novel species of the genus Massilia, for which the name Massilia eurypsychrophila sp. nov. is proposed. The type strain is B528-3(T) ( = JCM 30074(T) = CGMCC 1.12828(T)).

  17. Rhodoferax antarcticus sp. nov., a moderately psychrophilic purple nonsulfur bacterium isolated from an Antarctic microbial mat

    NASA Technical Reports Server (NTRS)

    Madigan, M. T.; Jung, D. O.; Woese, C. R.; Achenbach, L. A.

    2000-01-01

    A new species of purple nonsulfur bacteria isolated from an Antarctic microbial mat is described. The organism, designated strain ANT.BR, was mildly psychrophilic, growing optimally at 15-18 degrees C with a growth temperature range of 0-25 degrees C. Cells of strain ANT.BR were highly motile curved rods and spirals, contained bacteriochlorophyll a, and showed a multicomponent in vivo absorption spectrum. A specific phylogenetic relationship was observed between strain ANT.BR and the purple bacterium Rhodoferax fermentans FR2T, and the two organisms shared several physiological and other phenotypic properties, with the notable exception of growth temperature optimum. Tests of genomic DNA hybridization, however, showed Rfx. fermentans FR2T and strain ANT.BR to be genetically distinct bacteria. Because of its unique set of properties, especially its requirement for low growth temperatures, we propose to recognize strain ANT.BR as a new species of the genus Rhodoferax, Rhodoferax antarcticus, named for its known habitat, the Antarctic.

  18. Influence of hydraulic retention time on the psychrophilic hydrolysis/acidogenesis of proteins.

    PubMed

    Poirrier, Paola; Schiappacasse, María Cristina; Carballa, Marta; Lema, Juan M

    2016-11-01

    The influence of the hydraulic retention time (HRT) on the anaerobic hydrolysis of complex substrates has been studied under psychrophilic conditions. For this purpose, a continuous stirred tank reactor was operated at 15 °C and neutral pH and gelatin was considered as a model protein. Three HRTs have been tested: 12, 21 and 36 h. Gelatin hydrolysis was greatly dependent on HRT, increasing from 40% at 12 h-HRT to a maximum of 65% at 36 h-HRT. Molecular size distribution analyses of the effluent showed that hydrolysation of compounds larger than 10 kDa was poor at 12 h-HRT, whereas the fraction of 1-10 kDa was completely transformed into compounds smaller than 1 kDa. Higher HRT (36 h) improved the degradation of the recalcitrant fraction (>10 kDa), obtaining an effluent with around 95% of soluble molecules (<1 kDa). In that way, the use of membrane bioreactors for the treatment of this type of macromolecules could improve the degradation efficiencies by enabling to increase the residence time of the non-hydrolyzed molecules, with what would be possible to achieve higher organic loading rate operation.

  19. Expression, purification, and characterization of cold-adapted inorganic pyrophosphatase from psychrophilic Shewanella sp. AS-11.

    PubMed

    Ginting, Elvy Like; Iwasaki, Syouhei; Maeganeku, Chihiro; Motoshima, Hiroyuki; Watanabe, Keiichi

    2014-01-01

    In the presence of divalent cations, inorganic pyrophosphatase is activated to hydrolyze inorganic pyrophosphate to inorganic phosphate. Here, we clone, express, purify, and characterize inorganic pyrophosphatase from the psychrophilic Shewanella sp. AS-11 (Sh-PPase). The recombinant Sh-PPase was expressed in Escherichia coli BL21 (DE3) at 20°C using pET16b as an expression vector and purified from the cell extracts by a combination of ammonium sulfate fractionation and anion-exchange chromatography. Sh-PPase was found to be a family II PPase with a subunit molecular mass of 34 kD that preferentially utilizes Mn²⁺ over Mg²⁺ ions for activity. The functional characteristics of Sh-PPase, such as activity, temperature dependency, and thermal inactivation, were greatly influenced by manganese ions. Manganese ion activation increased the enzyme's activity at low temperatures; therefore, it was required to gain the cold-adapted characteristics of Sh-PPase.

  20. Marinobacter psychrophilus sp. nov., a psychrophilic bacterium isolated from the Arctic.

    PubMed

    Zhang, De-Chao; Li, Hui-Rong; Xin, Yu-Hua; Chi, Zhen-Ming; Zhou, Pei-Jin; Yu, Yong

    2008-06-01

    Strain 20041(T) was isolated from sea-ice of the Canadian Basin (7 degrees 23' 14'' N 14 degrees 06' 55'' W). Phylogenetic analysis based on 16S rRNA gene homology showed that strain 20041(T) was related to members of the genus Marinobacter and had highest 16S rRNA gene sequence similarity with the type strain of Marinobacter maritimus. Cells were Gram-negative, rod-shaped, psychrophilic and motile. The temperature range for growth was 0-22 degrees C, with optimum growth occurring at 16-18 degrees C and at approximately pH 6.0-9.0. Strain 20041(T) had ubiquinone-9 as the major respiratory quinone and iso-C(15 : 0) 2OH and/or C(16 : 1)omega7c, C(16 : 0), C(18 : 1)omega9c and C(12 : 0) 3OH as major fatty acids. The genomic DNA G+C content was 55.4 mol%. On the basis of the phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness data, strain 20041(T) is considered to represent a novel species, for which the name Marinobacter psychrophilus sp. nov. is proposed. The type strain is 20041(T) (=CGMCC 1.6499(T)=JCM 14643(T)).

  1. Moritella dasanensis sp. nov., a psychrophilic bacterium isolated from the Arctic ocean.

    PubMed

    Kim, Hak Jun; Park, Soyoung; Lee, Jung Min; Park, Seungil; Jung, Woongsic; Kang, Jae-Shin; Joo, Hyung Min; Seo, Ki-Won; Kang, Sung-Ho

    2008-04-01

    An aerobic, motile, Gram-negative, ice-active substance-producing, rod-shaped psychrophile, designated strain ArB 0140T, was isolated from seawater collected from near a glacier in Kongsfjorden, Svalbard Archipelago, Norway. Phylogenetic analysis using 16S rRNA gene sequences indicated that strain ArB 0140T showed a distinct phyletic line within the genus Moritella. Characteristic chemotaxonomic data [predominant isoprenoid quinone, Q8; major fatty acids, C14 : 0, C14 : 1, C16 : 0, C16 : 1 and C22 : 6 (docosahexaenoic acid; DHA)] also corroborated the affiliation of strain ArB 0140T to the genus Moritella. The maximal growth rate of the novel strain was observed at 9 degrees C, with a maximum temperature for growth of 18 degrees C. The genomic DNA G+C content was 46.9 mol%. Based on the data obtained from this polyphasic study, including DNA-DNA relatedness, physiological and biochemical tests and ice-controlling activity, strain ArB 0140T was found to be genetically and phenotypically different from other recognized species of the genus Moritella. Therefore strain ArB 0140T represents a novel species, for which the name Moritella dasanensis sp. nov. is proposed. The type strain is ArB 0140T (=KCTC 10814T=KCCM 42845T=JCM 14759T).

  2. Rhodoferax antarcticus sp. nov., a moderately psychrophilic purple nonsulfur bacterium isolated from an Antarctic microbial mat.

    PubMed

    Madigan, M T; Jung, D O; Woese, C R; Achenbach, L A

    2000-04-01

    A new species of purple nonsulfur bacteria isolated from an Antarctic microbial mat is described. The organism, designated strain ANT.BR, was mildly psychrophilic, growing optimally at 15-18 degrees C with a growth temperature range of 0-25 degrees C. Cells of strain ANT.BR were highly motile curved rods and spirals, contained bacteriochlorophyll a, and showed a multicomponent in vivo absorption spectrum. A specific phylogenetic relationship was observed between strain ANT.BR and the purple bacterium Rhodoferax fermentans FR2T, and the two organisms shared several physiological and other phenotypic properties, with the notable exception of growth temperature optimum. Tests of genomic DNA hybridization, however, showed Rfx. fermentans FR2T and strain ANT.BR to be genetically distinct bacteria. Because of its unique set of properties, especially its requirement for low growth temperatures, we propose to recognize strain ANT.BR as a new species of the genus Rhodoferax, Rhodoferax antarcticus, named for its known habitat, the Antarctic.

  3. Sporosarcina antarctica sp. nov., a psychrophilic bacterium isolated from the Antarctic.

    PubMed

    Yu, Yong; Xin, Yu-Hua; Liu, Hong-Can; Chen, Bo; Sheng, Jun; Chi, Zhen-Ming; Zhou, Pei-Jin; Zhang, De-Chao

    2008-09-01

    A Gram-positive, psychrophilic, rod-shaped bacterium, designated strain N-05(T), was isolated from soil samples collected off King George Island, west Antarctica (6 degrees 13' 31'' S 5 degrees 57' 08'' W). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain N-05(T) was related to members of the genus Sporosarcina and had highest 16S rRNA gene sequence similarity with the type strain of Sporosarcina macmurdoensis (98.0%). The temperature range for growth of strain N-05(T) was 0-23 degrees C, with optimum growth occurring at 17-18 degrees C and approximately pH 6.0-8.0. Strain N-05(T) had MK-7 as the major menaquinone and anteiso-C(15:0) and C(16:1)omega7c alcohol as major fatty acids. The genomic DNA G+C content was 39.2 mol%. On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness data, strain N-05(T) is considered to represent a novel species of the genus Sporosarcina, for which the name Sporosarcina antarctica is proposed. The type strain is N-05(T) (=CGMCC 1.6503(T)=JCM 14646(T)).

  4. Cryobacterium levicorallinum sp. nov., a psychrophilic bacterium isolated from glacier ice.

    PubMed

    Liu, Qing; Liu, Hongcan; Zhang, Jianli; Zhou, Yuguang; Xin, Yuhua

    2013-08-01

    In this study, two psychrophilic bacterial strains were isolated from the China No. 1 glacier in Xinjiang, north-west China. Cells were Gram-positive rods. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strains belonged to the genus Cryobacterium. Phylogenetic analysis showed that they clustered together and are most closely related to Cryobacterium luteum CGMCC 1.11210(T), Cryobacterium flavum CGMCC 1.11215(T), Cryobacterium psychrophilum CGMCC 1.4292(T), Cryobacterium psychrotolerans CGMCC 1.5382(T) and Cryobacterium roopkundense CGMCC 1.10672(T). The major cellular fatty acids of the novel strains were anteiso-C15 : 0, anteiso-C15 : 1 A, iso-C16 : 0 and iso-C15 : 0. Both strains contained diphosphatidylglycerol, phosphatidylglycerol and one unidentified glycolipid in the cell membrane. The results of DNA-DNA hybridization and physiological tests allowed the genotypic and phenotypic differentiation of strains Hh34(T) and Hh28 from related species. However, their high DNA-DNA relatedness showed that they belong to the same novel species. Strain Hh34(T) (= NBRC 107883(T) = CGMCC 1.11211(T)) was selected as the type strain to represent this novel species, for which the name Cryobacterium levicorallinum sp. nov. is proposed.

  5. Rhodoferax antarcticus sp. nov., a moderately psychrophilic purple nonsulfur bacterium isolated from an Antarctic microbial mat

    NASA Technical Reports Server (NTRS)

    Madigan, M. T.; Jung, D. O.; Woese, C. R.; Achenbach, L. A.

    2000-01-01

    A new species of purple nonsulfur bacteria isolated from an Antarctic microbial mat is described. The organism, designated strain ANT.BR, was mildly psychrophilic, growing optimally at 15-18 degrees C with a growth temperature range of 0-25 degrees C. Cells of strain ANT.BR were highly motile curved rods and spirals, contained bacteriochlorophyll a, and showed a multicomponent in vivo absorption spectrum. A specific phylogenetic relationship was observed between strain ANT.BR and the purple bacterium Rhodoferax fermentans FR2T, and the two organisms shared several physiological and other phenotypic properties, with the notable exception of growth temperature optimum. Tests of genomic DNA hybridization, however, showed Rfx. fermentans FR2T and strain ANT.BR to be genetically distinct bacteria. Because of its unique set of properties, especially its requirement for low growth temperatures, we propose to recognize strain ANT.BR as a new species of the genus Rhodoferax, Rhodoferax antarcticus, named for its known habitat, the Antarctic.

  6. Improvement of sensitivity for Mycobacterium avium subsp. paratuberculosis (MAP) detection in bovine fecal samples by specific duplex F57/IC real-time and conventional IS900 PCRs after solid culture enrichment.

    PubMed

    Fawzy, Ahmad; Eisenberg, Tobias; El-Sayed, Amr; Zschöck, Michael

    2015-04-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease in ruminants and a probable pathogen of Crohn's disease in humans. Accurate, cost-effective, and time-relevant diagnostics are the basis for efficient control programs. This study was conducted as an attempt to re-evaluate MAP detection improvement by coupling solid media enrichment to a more specific IS900 conventional PCR and a very specific F57/IC real-time PCR. In a spiking experiment, we investigated the improvement of molecular-based MAP detection in feces after a culture-based enrichment step into Herrold's egg yolk media with mycobactin J (HEYM-MJ) for different time intervals, when compared to traditional culture. Detection limit of culture was 0.33 × 10(4) bacteria × g(-1) (33 CFU g(-1)), while that of IS900 PCR when coupled with an enrichment step for 2, 4, and 6 weeks was 0.33 × 10(5) (0.33 × 10(3) CFU g(-1)), 0.33 × 10(4) (33 CFU g(-1)), and 33 (>3.3 CFU g(-1)) bacteria × g(-1), respectively. Whereas the detection limits of F57/IC real-time PCR after the enrichment step for the same time intervals were 0.33 × 10(5) (0.33 × 10(3) CFU g(-1)), 0.33 × 10(3) (3.3 CFU g(-1)), and 33 (>3.3 CFU g(-1)) bacteria × g(-1), respectively. Altogether, enrichment of bovine fecal samples into solid media increased the sensitivity of specific molecular detection of MAP using IS900 conventional PCR and duplex F57/IC real-time PCR and offers an expedited and accurate alternative for MAP detection in bovine feces. Validation of these results is further recommended using field bovine fecal samples.

  7. Multiplex Real-Time PCR Assays for Screening of Shiga Toxin 1 and 2 Genes, Including All Known Subtypes, and Escherichia coli O26-, O111-, and O157-Specific Genes in Beef and Sprout Enrichment Cultures.

    PubMed

    Harada, Tetsuya; Iguchi, Atsushi; Iyoda, Sunao; Seto, Kazuko; Taguchi, Masumi; Kumeda, Yuko

    2015-10-01

    Shiga toxin family members have recently been classified using a new nomenclature into three Stx1 subtypes (Stx1a, Stx1c, and Stx1d) and seven Stx2 subtypes (Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g). To develop screening methods for Stx genes, including all of these subtype genes, and Escherichia coli O26-, O111-, and O157-specific genes in laboratory investigations of Shiga toxin-producing E. coli (STEC) foodborne cases, we developed multiplex real-time PCR assays and evaluated their specificity and quantitative accuracy using STEC and non-STEC isolates, recombinant plasmids, and food enrichment cultures and by performing STEC spiking experiments with beef and sprout enrichment cultures. In addition, we evaluated the relationship between the recovery rates of the target strains by direct plating and immunomagnetic separation and the cycle threshold (CT) values of the real-time PCR assays for the Stx subtypes and STEC O26, O111, and O157 serogroups. All three stx1- and seven stx2-subtype genes were detected by real-time PCR with high sensitivity and specificity, and the quantitative accuracy of this assay was confirmed using control plasmids and STEC spiking experiments. The results of the STEC spiking experiments suggest that it is not routinely possible to isolate STEC from enrichment cultures with real-time PCR CT values greater than 30 by direct plating on MacConkey agar, although highly selective media and immunomagnetic beads were able to isolate the inoculated strains from the enrichment cultures. These data suggest that CT values obtained from the highly quantitative real-time PCR assays developed in this study provide useful information to develop effective isolation strategies for STEC from food samples. The real-time PCR assays developed here are expected to aid in investigations of infections or outbreaks caused by STEC harboring any of the stx-subtype genes in the new Stx nomenclature, as well as STEC O26, O111, and O157.

  8. Anaerobic cultures from preserved tissues of baby mammoth

    NASA Astrophysics Data System (ADS)

    Pikuta, Elena V.; Fisher, Daniel; Hoover, Richard B.

    2011-10-01

    Microbiological analysis of several cold-preserved tissue samples from the Siberian baby mammoth known as Lyuba revealed a number of culturable bacterial strains that were grown on anaerobic media at 3 oC. Lactic acid produced by LAB (lactic acid bacteria) group, usually by members of the genera Carnobacterium and Lactosphera, appears to be a wonderful preservative that keeps other bacteria from colonizing a system. Permafrost and lactic acid preserved the body of this one month-old baby mammoth and kept it in exceptionally good condition, resulting in this mammoth being the most complete sample of the species ever recovered. The diversity of novel psychrophilic anaerobic isolates was expressed on morphological, physiological and phylogenetic levels. Here, we discuss the specifics of the isolation of new psychrophilic strains, differentiation from trivial contamination, and preliminary results for characterization of the cultures.

  9. Culture.

    PubMed

    Smith, Timothy B; Rodríguez, Melanie Domenech; Bernal, Guillermo

    2011-02-01

    This article summarizes the definitions, means, and research of adapting psychotherapy to clients' cultural backgrounds. We begin by reviewing the prevailing definitions of cultural adaptation and providing a clinical example. We present an original meta-analysis of 65 experimental and quasi-experimental studies involving 8,620 participants. The omnibus effect size of d = .46 indicates that treatments specifically adapted for clients of color were moderately more effective with that clientele than traditional treatments. The most effective treatments tended to be those with greater numbers of cultural adaptations. Mental health services targeted to a specific cultural group were several times more effective than those provided to clients from a variety of cultural backgrounds. We recommend a series of research-supported therapeutic practices that account for clients' culture, with culture-specific treatments being more effective than generally culture-sensitive treatments. © 2010 Wiley Periodicals, Inc.

  10. Quantitative ecology and dry-heat resistance of psychrophiles. M.S. Thesis; [in soil samples from Viking spacecraft manufacturing areas

    NASA Technical Reports Server (NTRS)

    Winans, L., Jr.

    1974-01-01

    Microorganisms capable of growth at 7 C were enumerated and isolated from soil samples from the manufacture area (Denver, Colorado) and assembly area (Cape Kennedy, Florida) of the Viking spacecraft. Temperature requirements were determined for these isolates, and those growing at 3 C, but not at 32 C were designated as obligate psychrophiles in this investigation. These were identified to major generic groups, and the population density of obligate psychrophiles from the various groups was determined. Dry heat D-values were found for those spores that demonstrated growth or survival under a simulated Martian environment.

  11. The Genome Sequence of the psychrophilic archaeon, Methanococcoides burtonii: the Role of Genome Evolution in Cold-adaptation

    SciTech Connect

    Allen, Michelle A.; Lauro, Federico M.; Williams, Timothy J.; Burg, Dominic; Siddiqui, Khawar S.; De Francisci, David; Chong, Kevin W.Y.; Pilak, Oliver; Chew, Hwee H.; De Maere, Matthew Z.; Ting, Lily; Katrib, Marilyn; Ng, Charmaine; Sowers, Kevin R.; Galperin, Michael Y.; Anderson, Iain J.; Ivanova, Natalia; Dalin, Eileen; Martinez, Michelle; Lapidus, Alla; Hauser, Loren; Land, Miriam; Thomas, Torsten; Cavicchioli, Ricardo

    2009-04-01

    Psychrophilic archaea are abundant and perform critical roles throughout the Earth's expansive cold biosphere. Here we report the first complete genome sequence for a psychrophilic methanogenic archaeon, Methanococcoides burtonii. The genome sequence was manually annotated including the use of a five tiered Evidence Rating system that ranked annotations from Evidence Rating (ER) 1 (gene product experimentally characterized from the parent organism) to ER5 (hypothetical gene product) to provide a rapid means of assessing the certainty of gene function predictions. The genome is characterized by a higher level of aberrant sequence composition (51%) than any other archaeon. In comparison to hyper/thermophilic archaea which are subject to selection of synonymous codon usage, M. burtonii has evolved cold adaptation through a genomic capacity to accommodate highly skewed amino acid content, while retaining codon usage in common with its mesophilic Methanosarcina cousins. Polysaccharide biosynthesis genes comprise at least 3.3% of protein coding genes in the genome, and Cell wall/membrane/envelope biogenesis COG genes are over-represented. Likewise, signal transduction (COG category T) genes are over-represented and M. burtonii has a high 'IQ' (a measure of adaptive potential) compared to many methanogens. Numerous genes in these two over-represented COG categories appear to have been acquired from {var_epsilon}- and {delta}-proteobacteria, as do specific genes involved in central metabolism such as a novel B form of aconitase. Transposases also distinguish M. burtonii from other archaea, and their genomic characteristics indicate they play an important role in evolving the M. burtonii genome. Our study reveals a capacity for this model psychrophile to evolve through genome plasticity (including nucleotide skew, horizontal gene transfer and transposase activity) that enables adaptation to the cold, and to the biological and physical changes that have occurred over the

  12. Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic β-Glucosidase BglU in Micrococcus antarcticus

    PubMed Central

    Miao, Li-Li; Hou, Yan-Jie; Fan, Hong-Xia; Qu, Jie; Qi, Chao; Liu, Ying

    2016-01-01

    Psychrophilic enzymes play crucial roles in cold adaptation of microbes and provide useful models for studies of protein evolution, folding, and dynamic properties. We examined the crystal structure (2.2-Å resolution) of the psychrophilic β-glucosidase BglU, a member of the glycosyl hydrolase 1 (GH1) enzyme family found in the cold-adapted bacterium Micrococcus antarcticus. Structural comparison and sequence alignment between BglU and its mesophilic and thermophilic counterpart enzymes (BglB and GlyTn, respectively) revealed two notable features distinct to BglU: (i) a unique long-loop L3 (35 versus 7 amino acids in others) involved in substrate binding and (ii) a unique amino acid, His299 (Tyr in others), involved in the stabilization of an ordered water molecule chain. Shortening of loop L3 to 25 amino acids reduced low-temperature catalytic activity, substrate-binding ability, the optimal temperature, and the melting temperature (Tm). Mutation of His299 to Tyr increased the optimal temperature, the Tm, and the catalytic activity. Conversely, mutation of Tyr301 to His in BglB caused a reduction in catalytic activity, thermostability, and the optimal temperature (45 to 35°C). Loop L3 shortening and H299Y substitution jointly restored enzyme activity to the level of BglU, but at moderate temperatures. Our findings indicate that loop L3 controls the level of catalytic activity at low temperatures, residue His299 is responsible for thermolability (particularly heat lability of the active center), and long-loop L3 and His299 are jointly responsible for the psychrophilic properties. The described structural basis for the cold adaptedness of BglU will be helpful for structure-based engineering of new cold-adapted enzymes and for the production of mutants useful in a variety of industrial processes at different temperatures. PMID:26801571

  13. The genome sequence of the psychrophilic archaeon, Methanococcoides burtonii: the role of genome evolution in cold adaptation

    SciTech Connect

    Allen, Michele A; Lauro, Federico M; Williams, Timothy J; Burg, Dominic; Siddiqui, Khawar S; DeFrancisci, Davide; Chong, Kevin WY; Pilak, Oliver; Chew, Hwee H; DeMaere, Matthew Z; Ting, Lily; Katrib, Marilyn; Ng, Charmaine; Sowers, Kevin R; Galperin, Michael Y.; Anderson, Iain; Ivanova, N; Dalin, Eileen; Martinez, Michele; Lapidus, Alla L.; Hauser, Loren John; Land, Miriam L; Thomas, Torsten; Cavicchioli, Ricardo

    2009-01-01

    Psychrophilic archaea are abundant and perform critical roles throughout the Earth's expansive cold biosphere. Here we report the first complete genome sequence for a psychrophilic methanogenic archaeon, Methanococcoides burtonii. The genome sequence was manually annotated including the use of a five-tiered evidence rating (ER) system that ranked annotations from ER1 (gene product experimentally characterized from the parent organism) to ER5 (hypothetical gene product) to provide a rapid means of assessing the certainty of gene function predictions. The genome is characterized by a higher level of aberrant sequence composition (51%) than any other archaeon. In comparison to hyper/thermophilic archaea, which are subject to selection of synonymous codon usage, M. burtonii has evolved cold adaptation through a genomic capacity to accommodate highly skewed amino-acid content, while retaining codon usage in common with its mesophilic Methanosarcina cousins. Polysaccharide biosynthesis genes comprise at least 3.3% of protein coding genes in the genome, and Cell wall, membrane, envelope biogenesis COG genes are overrepresented. Likewise, signal transduction (COG category T) genes are overrepresented and M. burtonii has a high 'IQ' (a measure of adaptive potential) compared to many methanogens. Numerous genes in these two overrepresented COG categories appear to have been acquired from - and -Proteobacteria, as do specific genes involved in central metabolism such as a novel B form of aconitase. Transposases also distinguish M. burtonii from other archaea, and their genomic characteristics indicate they have an important role in evolving the M. burtonii genome. Our study reveals a capacity for this model psychrophile to evolve through genome plasticity (including nucleotide skew, horizontal gene transfer and transposase activity) that enables adaptation to the cold, and to the biological and physical changes that have occurred over the last several thousand years as it

  14. Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic β-Glucosidase BglU in Micrococcus antarcticus.

    PubMed

    Miao, Li-Li; Hou, Yan-Jie; Fan, Hong-Xia; Qu, Jie; Qi, Chao; Liu, Ying; Li, De-Feng; Liu, Zhi-Pei

    2016-01-22

    Psychrophilic enzymes play crucial roles in cold adaptation of microbes and provide useful models for studies of protein evolution, folding, and dynamic properties. We examined the crystal structure (2.2-Å resolution) of the psychrophilic β-glucosidase BglU, a member of the glycosyl hydrolase 1 (GH1) enzyme family found in the cold-adapted bacterium Micrococcus antarcticus. Structural comparison and sequence alignment between BglU and its mesophilic and thermophilic counterpart enzymes (BglB and GlyTn, respectively) revealed two notable features distinct to BglU: (i) a unique long-loop L3 (35 versus 7 amino acids in others) involved in substrate binding and (ii) a unique amino acid, His299 (Tyr in others), involved in the stabilization of an ordered water molecule chain. Shortening of loop L3 to 25 amino acids reduced low-temperature catalytic activity, substrate-binding ability, the optimal temperature, and the melting temperature (Tm). Mutation of His299 to Tyr increased the optimal temperature, the Tm, and the catalytic activity. Conversely, mutation of Tyr301 to His in BglB caused a reduction in catalytic activity, thermostability, and the optimal temperature (45 to 35°C). Loop L3 shortening and H299Y substitution jointly restored enzyme activity to the level of BglU, but at moderate temperatures. Our findings indicate that loop L3 controls the level of catalytic activity at low temperatures, residue His299 is responsible for thermolability (particularly heat lability of the active center), and long-loop L3 and His299 are jointly responsible for the psychrophilic properties. The described structural basis for the cold adaptedness of BglU will be helpful for structure-based engineering of new cold-adapted enzymes and for the production of mutants useful in a variety of industrial processes at different temperatures. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  15. Cytochrome f from the Antarctic psychrophile, Chlamydomonas raudensis UWO 241: structure, sequence, and complementation in the mesophile, Chlamydomonas reinhardtii.

    PubMed

    Gudynaite-Savitch, Loreta; Gretes, Michael; Morgan-Kiss, Rachael M; Savitch, Leonid V; Simmonds, John; Kohalmi, Susanne E; Hüner, Norman P A

    2006-04-01

    Although cytochrome f from the Antarctic psychrophile, Chlamydomonas raudensis UWO 241, exhibits a lower apparent molecular mass (34 kD) than that of the mesophile C. reinhardtii (41 kD) based on SDS-PAGE, both proteins are comparable in calculated molecular mass and show 79% identity in amino acid sequence. The difference in apparent molecular mass was maintained after expression of petA from both Chlamydomonas species in either E. coli or a C. reinhardtii DeltapetA mutant and after substitution of a unique third cysteine-292 to phenylalanine in the psychrophilic cytochrome f. Moreover, the heme of the psychrophilic form of cytochrome f was less stable upon heating than that of the mesophile. In contrast to C. raudensis, a C. reinhardtii DeltapetA mutant transformed with petA from C. raudensis exhibited the ability to undergo state transitions and a capacity for intersystem electron transport comparable to that of C. reinhardtii wild type. However, the C. reinhardtii petA transformants accumulated lower levels of cytochrome b ( 6 ) /f complexes and exhibited lower light saturated rates of O(2) evolution than C. reinhardtii wild type. We show that the presence of an altered form of cytochrome f in C. raudensis does not account for its inability to undergo state transitions or its impaired capacity for intersystem electron transport as previously suggested. A combined survey of the apparent molecular mass, thermal stability and amino acid sequences of cytochrome f from a broad range of mesophilic species shows unequivocally that the observed differences in cytochrome f structure are not related to psychrophilly. Thus, caution must be exercised in relating differences in amino acid sequence and thermal stability to adaptation to cold environments.

  16. Extensive gene acquisition in the extremely psychrophilic bacterial species Psychroflexus torquis and the link to sea-ice ecosystem specialism.

    PubMed

    Feng, Shi; Powell, Shane M; Wilson, Richard; Bowman, John P

    2014-01-01

    Sea ice is a highly dynamic and productive environment that includes a diverse array of psychrophilic prokaryotic and eukaryotic taxa distinct from the underlying water column. Because sea ice has only been extensive on Earth since the mid-Eocene, it has been hypothesized that bacteria highly adapted to inhabit sea ice have traits that have been acquired through horizontal gene transfer (HGT). Here we compared the genomes of the psychrophilic bacterium Psychroflexus torquis ATCC 700755(T), associated with both Antarctic and Arctic sea ice, and its closely related nonpsychrophilic sister species, P. gondwanensis ACAM 44(T). Results show that HGT has occurred much more extensively in P. torquis in comparison to P. gondwanensis. Genetic features that can be linked to the psychrophilic and sea ice-specific lifestyle of P. torquis include genes for exopolysaccharide (EPS) and polyunsaturated fatty acid (PUFA) biosynthesis, numerous specific modes of nutrient acquisition, and proteins putatively associated with ice-binding, light-sensing (bacteriophytochromes), and programmed cell death (metacaspases). Proteomic analysis showed that several genes associated with these traits are highly translated, especially those involved with EPS and PUFA production. Because most of the genes relating to the ability of P. torquis to dwell in sea-ice ecosystems occur on genomic islands that are absent in closely related P. gondwanensis, its adaptation to the sea-ice environment appears driven mainly by HGT. The genomic islands are rich in pseudogenes, insertional elements, and addiction modules, suggesting that gene acquisition is being followed by a process of genome reduction potentially indicative of evolving ecosystem specialism.

  17. Complete genome sequence of the Sporosarcina psychrophila DSM 6497, a psychrophilic Bacillus strain that mediates the calcium carbonate precipitation.

    PubMed

    Yan, Wenkai; Xiao, Xiang; Zhang, Yu

    2016-05-20

    Sporosarcina psychrophila DSM 6497 is a gram positive, spore-formation psychrophilic bacterial strain, widely distributed in terrestrial and aquatic environments. Here we report its complete sequence including one circular chromosome of 4674191bp with a GC content of 40.3%. Genes encoding urease are predicted in the genome, which provide insight information on the microbiologically mediated urea hydrolysis process. This urea hydrolysis can further lead to an increase of carbonate anion and alkalinity in the environment, which promotes the microbiologically induced carbonate precipitation with various applications, such as the bioremediation of calcium rich wastewater and bio-reservation of architectural patrimony. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Phosphate-independent utilization of phosphonoacetic acid as sole phosphorus source by a psychrophilic strain of Geomyces pannorum P15.

    PubMed

    Klimek-Ochab, Magdalena

    2014-09-01

    A psychrophilic fungal strain of Geomyces pannorum P15 was screened for its ability to utilize a range of synthetic and natural organophosphonate compounds as the sole source of phosphorus, nitrogen, or carbon. Only phosphonoacetic acid served as a phosphorus source for microbial growth in phosphate-independent manner. Substrate metabolism did not lead to extracellular release of inorganic phosphate. No phosphonate metabolizing enzyme activity was detectable in cell-free extracts prepared from Geomyces biomass pregrown on 2 mmol/L phosphonoacetic acid.

  19. Local entropy difference upon a substrate binding of a psychrophilic α-amylase and a mesophilic homologue

    NASA Astrophysics Data System (ADS)

    Kosugi, Takahiro; Hayashi, Shigehiko

    2011-01-01

    Psychrophilic α-amylase from the antarctic bacterium pseudoalteromonashaloplanktis (AHA) and its mesophilic homologue, porcine pancreatic α-amylase (PPA) are theoretically investigated with molecular dynamics (MD) simulations. We carried out 240-ns MD simulations for four systems, AHA and PPA with/without the bound substrate, and examined protein conformational entropy changes upon the substrate binding. We developed an analysis that decomposes the entropy changes into contributions of individual amino acids, and successfully identified protein regions responsible for the entropy changes. The results provide a molecular insight into the structural flexibilities of those enzymes related to the temperature dependences of the enzymatic activity.

  20. Sphingobacterium psychroaquaticum sp. nov., a psychrophilic bacterium isolated from Lake Michigan water.

    PubMed

    Albert, Richard A; Waas, Nancy E; Pavlons, Shawn C; Pearson, Jamie L; Ketelboeter, Laura; Rosselló-Móra, Ramon; Busse, Hans-Jürgen

    2013-03-01

    A psychrophilic, Gram-negative bacterium, designated MOL-1(T), was isolated from water of Lake Michigan. 16S rRNA gene sequence analysis revealed that the sequence of strain MOL-1(T) has sequence similarity of 95.6, 94.8, 94.3, 94.3, 94.2 and 93.9 %, respectively, to the 16S rRNA gene sequences of Sphingobacterium shayense HS39(T), S. lactis WCC 4512(T), S. composti T5-12(T), S. daejeonense TR6-04(T), S. bambusae IBFC2009(T) and S. alimentarium WCC 4521(T). The major cellular fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c). Menaquinone MK-7 is the predominant respiratory quinone, while sym-homospermidine is the predominant polyamine. The polar lipid profile is composed of the predominant lipids phosphatidylethanolamine and unidentified polar lipid L2, with moderate amounts of unidentified polar lipids L1, L5 and L6 and unidentified aminophospholipids APL1 and APL2 and minor to trace amounts of unidentified polar lipids L3, L4, L7, L8, L9 and L10, unidentified phospholipid PL4 and unidentified aminophospholipid APL3. After molecular and phenotypic studies, including chemotaxonomic analyses, it was concluded that strain MOL-1(T) represents a novel Sphingobacterium species, for which the name Sphingobacterium psychroaquaticum sp. nov. is proposed. The type strain is MOL-1(T) ( = NRRL B-59232(T)  = DSM 22418(T)).

  1. Low temperature reduction of hexavalent chromium by a microbial enrichment consortium and a novel strain of Arthrobacter aurescens

    PubMed Central

    Horton, Rene' N; Apel, William A; Thompson, Vicki S; Sheridan, Peter P

    2006-01-01

    Background Chromium is a transition metal most commonly found in the environment in its trivalent [Cr(III)] and hexavalent [Cr(VI)] forms. The EPA maximum total chromium contaminant level for drinking water is 0.1 mg/l (0.1 ppm). Many water sources, especially underground sources, are at low temperatures (less than or equal to 15 Centigrade) year round. It is important to evaluate the possibility of microbial remediation of Cr(VI) contamination using microorganisms adapted to these low temperatures (psychrophiles). Results Core samples obtained from a Cr(VI) contaminated aquifer at the Hanford facility in Washington were enriched in Vogel Bonner medium at 10 Centigrade with 0, 25, 50, 100, 200, 400 and 1000 mg/l Cr(VI). The extent of Cr(VI) reduction was evaluated using the diphenyl carbazide assay. Resistance to Cr(VI) up to and including 1000 mg/l Cr(VI) was observed in the consortium experiments. Reduction was slow or not observed at and above 100 mg/l Cr(VI) using the enrichment consortium. Average time to complete reduction of Cr(VI) in the 30 and 60 mg/l Cr(VI) cultures of the consortium was 8 and 17 days, respectively at 10 Centigrade. Lyophilized consortium cells did not demonstrate adsorption of Cr(VI) over a 24 hour period. Successful isolation of a Cr(VI) reducing organism (designated P4) from the consortium was confirmed by 16S rDNA amplification and sequencing. Average time to complete reduction of Cr(VI) at 10 Centigrade in the 25 and 50 mg/l Cr(VI) cultures of the isolate P4 was 3 and 5 days, respectively. The 16S rDNA sequence from isolate P4 identified this organism as a strain of Arthrobacter aurescens, a species that has not previously been shown to be capable of low temperature Cr(VI) reduction. Conclusion A. aurescens, indigenous to the subsurface, has the potential to be a predominant metal reducer in enhanced, in situ subsurface bioremediation efforts involving Cr(VI) and possibly other heavy metals and radionuclides. PMID:16436214

  2. Biodegradation of isopropanol in a three phase fixed bed bioreactor: start up and acclimation using a previously-enriched microbial culture.

    PubMed

    Bustard, M T; Meeyoo, V; Wright, P C

    2001-10-01

    The aerobic biodegradation of high liquid phase concentrations of 2-propanol (IPA) by a previously enriched solvent-tolerant bacterial consortium within a 1.9 l fed-batch three phase fixed bed bioreactor was investigated. Solvent concentrations of up to 7.9 g l(-1) were investigated. Previously enriched solvent-tolerant bacterial cells were immobilised onto porous glass cylinders as a means obioprocess intensification. Bioreactor start-up and acclimation was studied anacetone concentration tracked as an indicator of IPA utilization, as the sole carbon source within a minimal salts medium (MSM). The initial batch treatment of IPA exhibited a biodegradation rate of 0.11 g l(-1) h(-1) prior to biofilm formation Biofilm growth during the second batch treatment was consistent with an increase in metabolic activity and an IPA biodegradation rate of 0.34 g l(-1), followed by a reduction of biodegradation rate to a constant value of 0.078 g l(-1) h(-1) after 650 h. A maximum acetone generation rate of 1.3 g l(-1) h(-1) was obtained during the fourth IPA addition although the maximum acetone biodegradation rate of 0.38 g l(-1) h(-1) was observed during the initial IPA addition. It is proposed that the metabolic lag resulting from switching from alcohol dehydrogenase to acetone carboxylase is a major rate-limiting step in the deep oxidation of IPA to acetone. The results demonstrate the potential of a previously enriched solvent-tolerant bacterial consortium in fixed bed bioreactor systems, for the aerobic treatment of concentrated solvent-containing wastestreams.

  3. Psychrophilic proteases dramatically reduce single-cell RNA-seq artifacts: a molecular atlas of kidney development.

    PubMed

    Adam, Mike; Potter, Andrew S; Potter, S Steven

    2017-10-01

    Single-cell RNA-seq is a powerful technique. Nevertheless, there are important limitations, including the technical challenges of breaking down an organ or tissue into a single-cell suspension. Invariably, this has required enzymatic incubation at 37°C, which can be expected to result in artifactual changes in gene expression patterns. Here, we describe a dissociation method that uses a protease with high activity in the cold, purified from a psychrophilic microorganism. The entire procedure is carried out at 6°C or colder, at which temperature mammalian transcriptional machinery is largely inactive, thereby effectively 'freezing in' the in vivo gene expression patterns. To test this method, we carried out RNA-seq on 20,424 single cells from postnatal day 1 mouse kidneys, comparing the results of the psychrophilic protease method with procedures using 37°C incubation. We show that the cold protease method provides a great reduction in gene expression artifacts. In addition, the results produce a single-cell resolution gene expression atlas of the newborn mouse kidney, an interesting time in development when mature nephrons are present yet nephrogenesis remains extremely active. © 2017. Published by The Company of Biologists Ltd.

  4. Cloning and in-silico analysis of beta-1,3-xylanase from psychrophilic yeast, Glaciozyma antarctica PI12

    NASA Astrophysics Data System (ADS)

    Nor, Nooraisyah Mohamad; Bakar, Farah Diba Abu; Mahadi, Nor Muhammad; Murad, Abdul Munir Abdul

    2015-09-01

    A beta-1,3-xylanase (EC 3.2.1.32) gene from psychrophilic yeast, Glaciozyma antarctica has been identified via genome data mining. The enzyme was grouped into GH26 family based on Carbohydrate Active Enzyme (CaZY) database. The molecular weight of this protein was predicted to be 42 kDa and is expected to be soluble for expression. The presence of signal peptide suggested that this enzyme may be released extracellularly into the marine environment of the host's habitat. This supports the theory that such enzymatic activity is required for degradation of nutrients of polysaccharide origins into simpler carbohydrates outside the environment before it could be taken up inside the cell. The sequence for this protein showed very little conservation (< 30%) with other beta-1,3-xylanases from available databases. Based on the phylogenetic analysis, this protein also showed distant relationship to other xylanases from eukaryotic origin. The protein may have undergone major substitution in its gene sequence order to adapt to the cold climate. This is the first report of beta-1,3-xylanase gene isolated from a psychrophilic yeast.

  5. Culture.

    ERIC Educational Resources Information Center

    1997

    Twelve conference papers on cultural aspects of second language instruction include: "Towards True Multiculturalism: Ideas for Teachers" (Brian McVeigh); Comparing Cultures Through Critical Thinking: Development and Interpretations of Meaningful Observations" (Laurel D. Kamada); "Authority and Individualism in Japan and the…

  6. A study of psychrophilic organisms isolated from the manufacture and assembly areas of spacecraft to be used in the Viking mission, 1 January - 30 June 1973

    NASA Technical Reports Server (NTRS)

    Foster, T. L.; Winans, L., Jr.

    1973-01-01

    Soil samples from the areas associated with the Viking spacecraft were analyzed for major generic groups of microorganisms and the percentage of obligate psychrophiles. Results are presented which show the distribution of organisms isolated at low temperatures and the methods employed for subjecting samples to simulated Martian conditions. Emphasis is placed on application of these results to the objectives of the quarantine program.

  7. Enrichment of Secondary Wastewater Sludge for Production of Hydrogen from Crude Glycerol and Comparative Evaluation of Mono-, Co- and Mixed-Culture Systems.

    PubMed

    Pachapur, Vinayak Laxman; Kutty, Prianka; Brar, Satinder Kaur; Ramirez, Antonio Avalos

    2016-01-13

    Anaerobic digestion using mixed-culture with broader choice of pretreatments for hydrogen (H₂) production was investigated. Pretreatment of wastewater sludge by five methods, such as heat, acid, base, microwave and chloroform was conducted using crude glycerol (CG) as substrate. Results for heat treatment (100 °C for 15 min) showed the highest H₂ production across the pretreatment methods with 15.18 ± 0.26 mmol/L of medium at 30 °C in absence of complex media and nutrient solution. The heat-pretreated inoculum eliminated H₂ consuming bacteria and produced twice as much as H₂ as compared to other pretreatment methods. The fermentation conditions, such as CG concentration (1.23 to 24 g/L), percentage of inoculum size (InS) (1.23% to 24% v/v) along with initial pH (2.98 to 8.02) was tested using central composite design (CCD) with H₂ production as response parameter. The maximum H₂ production of 29.43 ± 0.71 mmol/L obtained at optimum conditions of 20 g/L CG, 20% InS and pH 7. Symbiotic correlation of pH over CG and InS had a significant (p-value: 0.0011) contribution to H₂ production. The mixed-culture possessed better natural acclimatization activity for degrading CG, at substrate inhibition concentration and provided efficient inoculum conditions in comparison to mono- and co-culture systems. The heat pretreatment step used across mixed-culture system is simple, cheap and industrially applicable in comparison to mono-/co-culture systems for H₂ production.

  8. Enrichment of Secondary Wastewater Sludge for Production of Hydrogen from Crude Glycerol and Comparative Evaluation of Mono-, Co- and Mixed-Culture Systems

    PubMed Central

    Pachapur, Vinayak Laxman; Kutty, Prianka; Brar, Satinder Kaur; Ramirez, Antonio Avalos

    2016-01-01

    Anaerobic digestion using mixed-culture with broader choice of pretreatments for hydrogen (H2) production was investigated. Pretreatment of wastewater sludge by five methods, such as heat, acid, base, microwave and chloroform was conducted using crude glycerol (CG) as substrate. Results for heat treatment (100 °C for 15 min) showed the highest H2 production across the pretreatment methods with 15.18 ± 0.26 mmol/L of medium at 30 °C in absence of complex media and nutrient solution. The heat-pretreated inoculum eliminated H2 consuming bacteria and produced twice as much as H2 as compared to other pretreatment methods. The fermentation conditions, such as CG concentration (1.23 to 24 g/L), percentage of inoculum size (InS) (1.23% to 24% v/v) along with initial pH (2.98 to 8.02) was tested using central composite design (CCD) with H2 production as response parameter. The maximum H2 production of 29.43 ± 0.71 mmol/L obtained at optimum conditions of 20 g/L CG, 20% InS and pH 7. Symbiotic correlation of pH over CG and InS had a significant (p-value: 0.0011) contribution to H2 production. The mixed-culture possessed better natural acclimatization activity for degrading CG, at substrate inhibition concentration and provided efficient inoculum conditions in comparison to mono- and co-culture systems. The heat pretreatment step used across mixed-culture system is simple, cheap and industrially applicable in comparison to mono-/co-culture systems for H2 production. PMID:26771607

  9. Effect of a Semi-Purified Oligosaccharide-Enriched Fraction from Caprine Milk on Barrier Integrity and Mucin Production of Co-Culture Models of the Small and Large Intestinal Epithelium

    PubMed Central

    Barnett, Alicia M.; Roy, Nicole C.; McNabb, Warren C.; Cookson, Adrian L.

    2016-01-01

    Caprine milk contains the highest amount of oligosaccharides among domestic animals, which are structurally similar to human milk oligosaccharides (HMOs). This suggests caprine milk oligosaccharides may offer similar protective and developmental effects to that of HMOs. However, to date, studies using oligosaccharides from caprine milk have been limited. Thus, this study aimed to examine the impact of a caprine milk oligosaccharide-enriched fraction (CMOF) on barrier function of epithelial cell co-cultures of absorptive enterocytes (Caco-2 cells) and mucus-secreting goblet cells (HT29-MTX cells), that more closely simulate the cell proportions found in the small (90:10) and large intestine (75:25). Treatment of epithelial co-cultures with 0.4, 1.0, 2.0 and 4.0 mg/mL of CMOF was shown to have no effect on metabolic activity but did enhance cell epithelial barrier integrity as measured by trans-epithelial electrical resistance (TEER), in a dose-dependent manner. The CMOF at the maximum concentration tested (4.0 mg/mL) enhanced TEER, mucin gene expression and mucin protein abundance of epithelial co-cultures, all of which are essential components of intestinal barrier function. PMID:27164134

  10. Predominance of Viable Spore-Forming Piezophilic Bacteria in High-Pressure Enrichment Cultures from ~1.5 to 2.4 km-Deep Coal-Bearing Sediments below the Ocean Floor.

    PubMed

    Fang, Jiasong; Kato, Chiaki; Runko, Gabriella M; Nogi, Yuichi; Hori, Tomoyuki; Li, Jiangtao; Morono, Yuki; Inagaki, Fumio

    2017-01-01

    Phylogenetically diverse microorganisms have been observed in marine subsurface sediments down to ~2.5 km below the seafloor (kmbsf). However, very little is known about the pressure-adapted and/or pressure-loving microorganisms, the so called piezophiles, in the deep subseafloor biosphere, despite that pressure directly affects microbial physiology, metabolism, and biogeochemical processes of carbon and other elements in situ. In this study, we studied taxonomic compositions of microbial communities in high-pressure incubated sediment, obtained during the Integrated Ocean Drilling Program (IODP) Expedition 337 off the Shimokita Peninsula, Japan. Analysis of 16S rRNA gene-tagged sequences showed that members of spore-forming bacteria within Firmicutes and Actinobacteria were predominantly detected in all enrichment cultures from ~1.5 to 2.4 km-deep sediment samples, followed by members of Proteobacteria, Acidobacteria, and Bacteroidetes according to the sequence frequency. To further study the physiology of the deep subseafloor sedimentary piezophilic bacteria, we isolated and characterized two bacterial strains, 19R1-5 and 29R7-12, from 1.9 and 2.4 km-deep sediment samples, respectively. The isolates were both low G+C content, gram-positive, endospore-forming and facultative anaerobic piezophilic bacteria, closely related to Virgibacillus pantothenticus and Bacillus subtilis within the phylum Firmicutes, respectively. The optimal pressure and temperature conditions for growth were 20 MPa and 42°C for strain 19R1-5, and 10 MPa and 43°C for strain 29R7-12. Bacterial (endo)spores were observed in both the enrichment and pure cultures examined, suggesting that these piezophilic members were derived from microbial communities buried in the ~20 million-year-old coal-bearing sediments after the long-term survival as spores and that the deep biosphere may host more abundant gram-positive spore-forming bacteria and their spores than hitherto recognized.

  11. Predominance of Viable Spore-Forming Piezophilic Bacteria in High-Pressure Enrichment Cultures from ~1.5 to 2.4 km-Deep Coal-Bearing Sediments below the Ocean Floor

    PubMed Central

    Fang, Jiasong; Kato, Chiaki; Runko, Gabriella M.; Nogi, Yuichi; Hori, Tomoyuki; Li, Jiangtao; Morono, Yuki; Inagaki, Fumio

    2017-01-01

    Phylogenetically diverse microorganisms have been observed in marine subsurface sediments down to ~2.5 km below the seafloor (kmbsf). However, very little is known about the pressure-adapted and/or pressure-loving microorganisms, the so called piezophiles, in the deep subseafloor biosphere, despite that pressure directly affects microbial physiology, metabolism, and biogeochemical processes of carbon and other elements in situ. In this study, we studied taxonomic compositions of microbial communities in high-pressure incubated sediment, obtained during the Integrated Ocean Drilling Program (IODP) Expedition 337 off the Shimokita Peninsula, Japan. Analysis of 16S rRNA gene-tagged sequences showed that members of spore-forming bacteria within Firmicutes and Actinobacteria were predominantly detected in all enrichment cultures from ~1.5 to 2.4 km-deep sediment samples, followed by members of Proteobacteria, Acidobacteria, and Bacteroidetes according to the sequence frequency. To further study the physiology of the deep subseafloor sedimentary piezophilic bacteria, we isolated and characterized two bacterial strains, 19R1-5 and 29R7-12, from 1.9 and 2.4 km-deep sediment samples, respectively. The isolates were both low G+C content, gram-positive, endospore-forming and facultative anaerobic piezophilic bacteria, closely related to Virgibacillus pantothenticus and Bacillus subtilis within the phylum Firmicutes, respectively. The optimal pressure and temperature conditions for growth were 20 MPa and 42°C for strain 19R1-5, and 10 MPa and 43°C for strain 29R7-12. Bacterial (endo)spores were observed in both the enrichment and pure cultures examined, suggesting that these piezophilic members were derived from microbial communities buried in the ~20 million-year-old coal-bearing sediments after the long-term survival as spores and that the deep biosphere may host more abundant gram-positive spore-forming bacteria and their spores than hitherto recognized. PMID:28220112

  12. Immunoaffinity enrichment and liquid chromatography-selected reaction monitoring mass spectrometry for quantitation of carbonic anhydrase 12 in cultured renal carcinoma cells

    PubMed Central

    Rafalko, Agnes; Iliopoulos, Othon; Fusaro, Vincent A.; Hancock, William; Hincapie, Marina

    2010-01-01

    Liquid chromatography-selected reaction monitoring (LC-SRM) is a highly specific and sensitive mass spectrometry (MS) technique that is widely being applied to selectively qualify and validate candidate markers within complex biological samples. However, in order for LC-SRM methods to take on these attributes, target-specific optimization of sample processing is required, in order to reduce analyte complexity, prior to LC-SRM. In this study, we have developed a targeted platform consisting of protein immunoaffinity enrichment on magnetic beads and LC-SRM for measuring carbonic anhydrase 12 (CA12) protein in a renal cell carcinoma (RCC) cell line (PRC3), a candidate biomarker for RCC whose expression at the protein level has not been previously reported. Sample processing and LC-SRM assay were optimized for signature peptides selected as surrogate markers of CA12 protein. Using LC-SRM coupled with stable isotope dilution, we achieved limits of quantitation in the low fmol range sufficient for measuring clinically relevant biomarkers with good intra- and inter-assay accuracy and precision (≤17%). Our results show that using a quantitative immunoaffinity capture approach provides specific, accurate, and robust assays amenable to high-throughput verification of potential biomarkers. PMID:20936840

  13. Differential cold-adaptation among protein components of the thioredoxin system in the psychrophilic eubacterium Pseudoalteromonas haloplanktis TAC 125.

    PubMed

    Cotugno, Roberta; Rosaria Ruocco, Maria; Marco, Salvatore; Falasca, Patrizia; Evangelista, Giovanna; Raimo, Gennaro; Chambery, Angela; Di Maro, Antimo; Masullo, Mariorosario; De Vendittis, Emmanuele

    2009-05-01

    Thioredoxin and thioredoxin reductase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis were obtained as recombinant His-tagged proteins (rPhTrx and rPhTrxR, respectively). rPhTrxR is organised as a homodimeric flavoenzyme, whereas rPhTrx is a small monomeric protein, both containing a functional disulfide bridge. However, three additional cysteines are present as free thiols in purified rPhTrxR. When individually tested in specific assays, rPhTrxR and rPhTrx display a full activity at low temperatures, an indispensable requirement for cold-adapted proteins. In particular, rPhTrxR catalyses the NADPH dependent reduction of DTNB and rPhTrx provokes the insulin precipitation in the presence of DTT. The analysis of the effect of temperature on these reactions indicates that rPhTrxR is more cold-adapted than rPhTrx, having a higher psychrophilicity. The combined activity of rPhTrxR and rPhTrx, tested in a reconstituted assay containing NADPH as electrons donor and human insulin as the thioredoxin substrate, demonstrates a direct functional interaction between the purified recombinant components of the thioredoxin system of P. haloplanktis. Furthermore, the NADPH-dependent reduction of rPhTrx catalysed by rPhTrxR is fully reversible and allows the determination of its redox potential, whose value is in the range of other bacterial and archaeal thioredoxins. The analysis of the thermostability of rPhTrxR points to its discrete heat resistance. However, rPhTrx is much more heat resistant, with a half inactivation time of about 4 h at 95 degrees C. This exceptional heat resistance for a psychrophilic protein is significantly decreased by the reduction of the disulfide bridge of rPhTrx. Functionality, thermodependence and thermostability of the P. haloplanktis thioredoxin system point to the relevance of this key mechanism for the preservation of the reduced state of cytoplasmic proteins even in a cold-adapted source.

  14. Beyond Job Enrichment to Employment Enrichment

    ERIC Educational Resources Information Center

    Werther, William B., Jr.

    1975-01-01

    Employment enrichment views the total work environment confronting employees as a system consisting of two overlapping areas: worker-job and worker-organization subsystems. Job enrichment has improved the worker-job subsystem. The focus of this article is on methods of improving the worker-organization relationship. (Author/JB)

  15. Cultural

    Treesearch

    Wilbur F. LaPage

    1971-01-01

    A critical look at outdoor recreation research and some underlying premises. The author focuses on the concept of culture as communication and how it influences our perception of problems and our search for solutions. Both outdoor recreation and science are viewed as subcultures that have their own bodies of mythology, making recreation problems more difficult to...

  16. Survival of the North American strain of viral hemorrhagic septicemia virus (VHSV) in filtered seawater and seawater containing ovarian fluid, crude oil and serum-enriched culture medium

    USGS Publications Warehouse

    Kocan, R.M.; Hershberger, P.K.; Elder, N.E.

    2001-01-01

     The North American strain of viral hemorrhagic septicemia virus (NA-VHSV) could be recovered for up to 40 h in natural filtered seawater (27 ppt) with a 50% loss of infectivity after approximately 10 h at 15°C. Addition of 10 ppb North Slope crude oil to the seawater had no effect on virus survival. However, when various concentrations of teleost ovarian fluid were added to seawater, virus could be recovered after 72 h at 0.01% ovarian fluid and after 96 h at 1.0%. When cell culture medium supplemented with 10% fetal bovine serum was added to the seawater, 100% of the virus could be recovered for the first 15 d and 60% of the virus remained after 36 d. These findings quantify NA-VHSV infectivity in natural seawater and demonstrate that ovarian fluid, which occurs naturally during spawning events, significantly prolongs the survival and infectivity of the virus. The extended stabilization of virus in culture medium supplemented with serum allows for low titer field samples to be collected and transported in an unfrozen state without significant loss of virus titer.

  17. Dry anaerobic digestion of high solids content dairy manure at high organic loading rates in psychrophilic sequence batch reactor.

    PubMed

    Massé, Daniel I; Saady, Noori M Cata

    2015-05-01

    Cow manure with bedding is renewable organic biomass available around the year on dairy farms. Developing efficient and cost-effective psychrophilic dry anaerobic digestion (PDAD) processes could contribute to solving farm-related environmental, energy, and manure management problems in cold-climate regions. This study was to increase the organic loading rate (OLR), fed to a novel psychrophilic (20 °C) dry anaerobic digestion of 27% total solid dairy manure (cow feces and wheat straw) in sequence batch reactor (PDAD-SBR), by 133 to 160%. The PDAD-SBR process operated at treatment cycle length of 21 days and OLR of 7.0 and 8.0 g total chemical oxygen demand (TCOD) kg(-1) inoculum day(-1) (5.2 ± 0.1 and 5.8 ± 0.0 g volatile solids (VS) kg(-1) inoculum day(-1)) for four successive cycles (84 days) produced average specific methane yields (SMYs) of 147.1 ± 17.2 and 143.2 ± 11.7 normalized liters (NL) CH4 kg(-1) VS fed, respectively. PDAD of cow feces and wheat straw is possible with VS-based inoculum-to-substrate ratio of 1.45 at OLR of 8.0 g TCOD kg(-1) inoculum day(-1). Hydrolysis was the limiting step reaction. The VS removal averaged around 57.4 ± 0.5 and 60.5 ± 5.7% at OLR 7.0 and 8.0 g TCOD kg(-1) inoculum day(-1), respectively.

  18. Effect of CO sub 2 enrichment and high photosynthetic photon flux densities (PPFD) on rubisco and PEP-case activities of in vitro cultured strawberry plants

    SciTech Connect

    Desjardins, Y.; Beeson, R.; Gosselin, A. )

    1989-04-01

    Standard growing conditions in vitro (low light and CO{sub 2}) are not conducive to autotrophy. An experiment was conducted to improve photosynthesis in vitro in the hope of increasing survival in acclimatization. A factorial experiment was elaborated where CO{sub 2} and PPFD were supplied to in vitro cultured strawberry plants in the rooting stage. Activities of carboxylating enzymes were determined after 4 weeks of culture. The activities of non-activated and activated rubisco and PEP-Case were measured after extraction of the enzymes and a reaction with NaH{sup 14}CO{sub 3} followed by scintillation counting spectroscopy. High CO{sub 2} concentration significantly increased net assimilation rates (NAR) by 165% over the control for both 1650 and 3000 ppm CO{sub 2}. High PPFD only increased NAR by 12 and 35% for 150 and 250 {mu}mol{center dot}m{sup {minus}2}{center dot}s{sup {minus}1} respectively over the control. Plants grown at 3000 ppm CO{sub 2} had the highest level of chlorophyll/g FW with 97% more than the control. The activity of PEP-Case was the highest at high light levels and high CO{sub 2} with rates of 1.65 for 1650 ppm versus 1.22 mmol CO{sub 2} mg{sup {minus}1} chl. h{sup {minus}1} at 250 {mu}mol{center dot}m{sup {minus}2}{center dot}s{sup {minus}1}. There was no difference in PEP activity at low light levels. The rubisco activity was lower at 1650 and 3000 ppm CO{sub 2}. Increases in NAR correlate more closely to the PEP-Case than to Rubisco activity. Physiological significance of high activity of PEP-Case over rubisco will be discussed.

  19. Derived enriched uranium market

    SciTech Connect

    Rutkowski, E.

    1996-12-01

    The potential impact on the uranium market of highly enriched uranium from nuclear weapons dismantling in the Russian Federation and the USA is analyzed. Uranium supply, conversion, and enrichment factors are outlined for each country; inventories are also listed. The enrichment component and conversion components are expected to cause little disruption to uranium markets. The uranium component of Russian derived enriched uranium hexafluoride is unresolved; US legislation places constraints on its introduction into the US market.

  20. Development of a rapid lateral flow immunoassay test for detection of exosomes previously enriched from cell culture medium and body fluids.

    PubMed

    Oliveira-Rodríguez, Myriam; López-Cobo, Sheila; Reyburn, Hugh T; Costa-García, Agustín; López-Martín, Soraya; Yáñez-Mó, María; Cernuda-Morollón, Eva; Paschen, Annette; Valés-Gómez, Mar; Blanco-López, Maria Carmen

    2016-01-01

    Exosomes are cell-secreted nanovesicles (40-200 nm) that represent a rich source of novel biomarkers in the diagnosis and prognosis of certain diseases. Despite the increasingly recognized relevance of these vesicles as biomarkers, their detection has been limited due in part to current technical challenges in the rapid isolation and analysis of exosomes. The complexity of the development of analytical platforms relies on the heterogeneous composition of the exosome membrane. One of the most attractive tests is the inmunochromatographic strips, which allow rapid detection by unskilled operators. We have successfully developed a novel lateral flow immunoassay (LFIA) for the detection of exosomes based on the use of tetraspanins as targets. We have applied this platform for the detection of exosomes purified from different sources: cell culture supernatants, human plasma and urine. As proof of concept, we explored the analytical potential of this LFIA platform to accurately quantify exosomes purified from a human metastatic melanoma cell line. The one-step assay can be completed in 15 min, with a limit of detection of 8.54×10(5) exosomes/µL when a blend of anti-CD9 and anti-CD81 were selected as capture antibodies and anti-CD63 labelled with gold nanoparticles as detection antibody. Based on our results, this platform could be well suited to be used as a rapid exosome quantification tool, with promising diagnostic applications, bearing in mind that the detection of exosomes from different sources may require adaptation of the analytical settings to their specific composition.

  1. Development of a rapid lateral flow immunoassay test for detection of exosomes previously enriched from cell culture medium and body fluids

    PubMed Central

    Oliveira-Rodríguez, Myriam; López-Cobo, Sheila; Reyburn, Hugh T.; Costa-García, Agustín; López-Martín, Soraya; Yáñez-Mó, María; Cernuda-Morollón, Eva; Paschen, Annette; Valés-Gómez, Mar; Blanco-López, Maria Carmen

    2016-01-01

    Exosomes are cell-secreted nanovesicles (40–200 nm) that represent a rich source of novel biomarkers in the diagnosis and prognosis of certain diseases. Despite the increasingly recognized relevance of these vesicles as biomarkers, their detection has been limited due in part to current technical challenges in the rapid isolation and analysis of exosomes. The complexity of the development of analytical platforms relies on the heterogeneous composition of the exosome membrane. One of the most attractive tests is the inmunochromatographic strips, which allow rapid detection by unskilled operators. We have successfully developed a novel lateral flow immunoassay (LFIA) for the detection of exosomes based on the use of tetraspanins as targets. We have applied this platform for the detection of exosomes purified from different sources: cell culture supernatants, human plasma and urine. As proof of concept, we explored the analytical potential of this LFIA platform to accurately quantify exosomes purified from a human metastatic melanoma cell line. The one-step assay can be completed in 15 min, with a limit of detection of 8.54×105 exosomes/µL when a blend of anti-CD9 and anti-CD81 were selected as capture antibodies and anti-CD63 labelled with gold nanoparticles as detection antibody. Based on our results, this platform could be well suited to be used as a rapid exosome quantification tool, with promising diagnostic applications, bearing in mind that the detection of exosomes from different sources may require adaptation of the analytical settings to their specific composition. PMID:27527605

  2. Crystallization and preliminary X-ray diffraction studies of tetrameric malate dehydrogenase from the novel Antarctic psychrophile Flavobacterium frigidimaris KUC-1

    SciTech Connect

    Fujii, Tomomi; Oikawa, Tadao; Muraoka, Ikuo; Soda, Kenji; Hata, Yasuo

    2007-11-01

    A psychrophilic malate dehydrogenase from the novel Antarctic bacterium F. frigidimaris KUC-1 was crystallized using the hanging-drop vapour-diffusion method. The crystals contained one tetrameric molecule per asymmetric unit. The best crystal diffracted to 1.8 Å resolution. Flavobacterium frigidimaris KUC-1 is a novel psychrotolerant bacterium isolated from Antarctic seawater. Malate dehydrogenase (MDH) is an essential metabolic enzyme in the citric acid cycle and has been cloned, overexpressed and purified from F. frigidimaris KUC-1. In contrast to the already known dimeric form of MDH from the psychrophile Aquaspirillium arcticum, F. frigidimaris MDH exists as a tetramer. It was crystallized at 288 K by the hanging-drop vapour-diffusion method using ammonium sulfate as the precipitating agent. The crystal diffracted to a maximum resolution of 1.80 Å. It contains one tetrameric molecule in the asymmetric unit.

  3. A study of psychrophilic organisms isolated from the manufacture and assembly areas of spacecraft to be used in the Viking mission

    NASA Technical Reports Server (NTRS)

    Foster, T. L.

    1974-01-01

    The effect of storage of dry heat treated Teflon ribbons under nitrogen gas followed by high vacuum on the recovery of hardy organisms from the ribbons was studied. A similar experiment was performed on spore crops of hardy organisms recovered previously from Cape Canaveral. Hardy organisms have been inoculated onto slides and subjected to an artificial Martian environment in an attempt to demonstrate their growth in this environment. Additional experiments using the artificial Martian environment include response of soil samples from the VAB with both constant temperature and freeze-thaw cycles. These experiments were performed with dried soil and soil containing added water. Other investigations included the effect of heatshock on soil samples, psychrophilic counts of new soil samples from the manufacture area of the Viking spacecraft, effect of pour plate versus spread plate on psychrophilic counts, and preparation of spore crops of hardy organisms from Cape Canaveral.

  4. Identity and physiology of a new psychrophilic eukaryotic green alga, Chlorella sp., strain BI, isolated from a transitory pond near Bratina Island, Antarctica

    USGS Publications Warehouse

    Morgan-Kiss, R. M.; Ivanov, A.G.; Modla, S.; Czymmek, K.; Huner, N.P.A.; Priscu, J.C.; Lisle, J.T.; Hanson, T.E.

    2008-01-01

    Permanently low temperature environments are one of the most abundant microbial habitats on earth. As in most ecosystems, photosynthetic organisms drive primary production in low temperature food webs. Many of these phototrophic microorganisms are psychrophilic; however, functioning of the photosynthetic processes of these enigmatic psychrophiles (the 'photopsychrophiles') in cold environments is not well understood. Here we describe a new chlorophyte isolated from a low temperature pond, on the Ross Ice Shelf near Bratina Island, Antarctica. Phylogenetic and morphological analyses place this strain in the Chlorella clade, and we have named this new chlorophyte Chlorella BI. Chlorella BI is a psychrophilic species, exhibiting optimum temperature for growth at around 10??C. However, psychrophily in the Antarctic Chlorella was not linked to high levels of membrane-associated poly-unsaturated fatty acids. Unlike the model Antarctic lake alga, Chlamydomonas raudensis UWO241, Chlorella BI has retained the ability for dynamic short term adjustment of light energy distribution between photosystem II (PS II) and photosystem I (PS I). In addition, Chlorella BI can grow under a variety of trophic modes, including heterotrophic growth in the dark. Thus, this newly isolated photopsychrophile has retained a higher versatility in response to environmental change than other well studied cold-adapted chlorophytes. ?? 2008 Springer.

  5. Identity and physiology of a new psychrophilic eukaryotic green alga, Chlorella sp., strain BI, isolated from a transitory pond near Bratina Island, Antarctica.

    PubMed

    Morgan-Kiss, Rachael M; Ivanov, Alexander G; Modla, Shannon; Czymmek, Kirk; Hüner, Norman P A; Priscu, John C; Lisle, John T; Hanson, Thomas E

    2008-09-01

    Permanently low temperature environments are one of the most abundant microbial habitats on earth. As in most ecosystems, photosynthetic organisms drive primary production in low temperature food webs. Many of these phototrophic microorganisms are psychrophilic; however, functioning of the photosynthetic processes of these enigmatic psychrophiles (the "photopsychrophiles") in cold environments is not well understood. Here we describe a new chlorophyte isolated from a low temperature pond, on the Ross Ice Shelf near Bratina Island, Antarctica. Phylogenetic and morphological analyses place this strain in the Chlorella clade, and we have named this new chlorophyte Chlorella BI. Chlorella BI is a psychrophilic species, exhibiting optimum temperature for growth at around 10 degrees C. However, psychrophily in the Antarctic Chlorella was not linked to high levels of membrane-associated poly-unsaturated fatty acids. Unlike the model Antarctic lake alga, Chlamydomonas raudensis UWO241, Chlorella BI has retained the ability for dynamic short term adjustment of light energy distribution between photosystem II (PS II) and photosystem I (PS I). In addition, Chlorella BI can grow under a variety of trophic modes, including heterotrophic growth in the dark. Thus, this newly isolated photopsychrophile has retained a higher versatility in response to environmental change than other well studied cold-adapted chlorophytes.

  6. Gravimetric enrichment of high lipid and starch accumulating microalgae.

    PubMed

    Hassanpour, Morteza; Abbasabadi, Mahsa; Ebrahimi, Sirous; Hosseini, Maryam; Sheikhbaglou, Ahmad

    2015-11-01

    This study presents gravimetric enrichment of mixed culture to screen starch and lipid producing species separately in a sequencing batch reactor. In the enriched starch-producing mixed culture photobioreactor, the starch content at the end of steady state batch became 3.42 times the beginning of depletion. Whereas in the enriched lipid-producing photobioreactor, the lipid content at the end of steady state batch became 3 times the beginning of famine phase. The obtained results revealed that the gravimetric enrichment is a suitable screening method for specific production of storage compounds in none-sterile large-scaled condition.

  7. Enrichment through Creative Arts.

    ERIC Educational Resources Information Center

    Krause, Claire S.

    The CREST (Creative Resources Enriching Student Talents) Project, an enrichment approach for elementary gifted, talented, and creative students, is described. The project is explained to incorporate an interdisciplinary approach to instruction in art and science using resources within the community. Chapter 1 outlines the project philosophy,…

  8. Spectroscopic analyses of manganese ions effects on the conformational changes of inorganic pyrophosphatase from psychrophilic Shewanella sp. AS-11.

    PubMed

    Ginting, Elvy Like; Maeganeku, Chihiro; Motoshima, Hiroyuki; Watanabe, Keiichi

    2014-02-01

    Mn²⁺ ions influence the activity, temperature dependence, and thermostability of the psychrophilic Shewanella-PPase (Sh-PPase), and are required to function in cold environments. The functional characteristics of Sh-PPase on activation with Mn²⁺ ions are possibly related to conformational changes in the molecule. In this study, conformational changes of Sh-PPase on activation with Mn²⁺ ions were analyzed in solution by fluorescence spectroscopy analysis of intrinsic tryptophan residues, 1-anilino-8-naphthalene sulfonate fluorescence, and circular dichroism spectroscopy. For Sh-PPase, Mn²⁺ ions did not affect the flexibility of the tryptophan residues and secondary structure of the enzyme. However, the microenvironment of the tryptophan residues and surface area of Sh-PPase were more hydrophilic on activation with Mn²⁺ ions. These results indicate that activation with Mn²⁺ ions causes conformational changes around the aromatic amino acid residues and affects the hydrophobicity of the enzyme surface, which results in conformational changes. Substrate-induced conformational changes reflect that metal-free Sh-PPase in solution indicated an open structure and will be a close structure when binding substrate. In combination of our spectroscopic analyses on Sh-PPase, it can be concluded that activation with Mn²⁺ ions changes some conformation of Sh-PPase molecule in solution.

  9. Structure prediction of Fe(II) 2-oxoglutarate dioxygenase from a psychrophilic yeast Glaciozyma antarctica PI12

    NASA Astrophysics Data System (ADS)

    Yusof, Nik Yusnoraini; Bakar, Farah Diba Abu; Mahadi, Nor Muhammad; Raih, Mohd Firdaus; Murad, Abdul Munir Abdul

    2015-09-01

    A cDNA encoding Fe(II) 2-oxoglutarate (2OG) dependent dioxygenases was isolated from psychrophilic yeast, Glaciozyma antarctica PI12. We have successfully amplified 1,029 bp cDNA sequence that encodes 342 amino acid with predicted molecular weight 38 kDa. The prediction protein was analysed using various bioinformatics tools to explore the properties of the protein. Based on a BLAST search analysis, the Fe2OX amino acid sequence showed 61% identity to the sequence of oxoglutarate/iron-dependent oxygenase from Rhodosporidium toruloides NP11. SignalP prediction showed that the Fe2OX protein contains no putative signal peptide, which suggests that this enzyme most probably localised intracellularly.The structure of Fe2OX was predicted by homology modelling using MODELLER9v11. The model with the lowest objective function was selected from hundred models generated using MODELLER9v11. Analysis of the structure revealed the longer loop at Fe2OX from G.antarctica that might be responsible for the flexibility of the structure, which contributes to its adaptation to low temperatures. Fe2OX hold a highly conserved Fe(II) binding HXD/E…H triad motif. The binding site for 2-oxoglutarate was found conserved for Arg280 among reported studies, however the Phe268 was found to be different in Fe2OX.

  10. Desulfuromonas svalbardensis sp. nov. and Desulfuromusa ferrireducens sp. nov., psychrophilic, Fe(III)-reducing bacteria isolated from Arctic sediments, Svalbard.

    PubMed

    Vandieken, Verona; Mussmann, Marc; Niemann, Helge; Jørgensen, Bo Barker

    2006-05-01

    Two psychrophilic, Gram-negative, rod-shaped, motile bacteria (strains 112T and 102T) that conserved energy from dissimilatory Fe(III) reduction concomitant with acetate oxidation were isolated from permanently cold Arctic marine sediments. Both strains grew at temperatures down to -2 degrees C, with respective temperature optima of 14 degrees C and 14-17 degrees C for strains 112T and 102T. The isolated strains reduced Fe(III) using common fermentation products such as acetate, lactate, propionate, formate or hydrogen as electron donors, and they also grew with fumarate as the sole substrate. As alternatives to Fe(III), they reduced fumarate, S0 and Mn(IV). Based on 16S rRNA gene sequence similarity, strain 112T was most closely related to Desulfuromonas acetoxidans (97.0 %) and Desulfuromonas thiophila NZ27T (95.5 %), and strain 102T to Malonomonas rubra Gra Mal 1T (96.3 %) and Desulfuromusa succinoxidans GylacT (95.9 %) within the Deltaproteobacteria. Strains 112T and 102T therefore represent novel species, for which the names Desulfuromonas svalbardensis sp. nov. (type strain 112T=DSM 16958T=JCM 12927T) and Desulfuromusa ferrireducens sp. nov. (type strain 102T=DSM 16956T=JCM 12926T) are proposed.

  11. In silico analysis of β-1,3-glucanase from a psychrophilic yeast, Glaciozyma antarctica PI12

    NASA Astrophysics Data System (ADS)

    Mohammadi, Salimeh; Bakar, Farah Diba Abu; Rabu, Amir; Murad, Abdul Munir Abdul

    2014-09-01

    1,3-beta-glucanase is an industrially important enzyme having wide range of applications especially in food industry. It is crucial to gain an understanding about the structure and functional aspects of various beta-1,3-glucanase produced from diverse sources. In this, study a cDNA encoding β-1,3-glucanase (GaExg55) was isolated from a psychrophilic yeast, Glaciozyma antarctica PI12. The cDNA sequence has been submitted to Genbank with an accession number (KJ436377). Subsequently, the perdition protein was analyzed using various bioinformatics tools to explore the properties of the protein. GaEXG55 is consisting of 1,440-bp nucleotides encoding 480 amino acid residues. Alignment of the deduced amino acid for GaExg55 with other exo-β-1,3-glucanase available at the NCBI database indicate that deduced amino acids shared a consensus motif NEP, which is signature pattern of GH5 hydrolases. Predicted molecular weight of GaExg55 is 53.66 kDa. GaExg55 sequences possesses signal peptide sequence and it is highly conserved with other fungal exo-beta-1,3 glucanase.

  12. Cytoplasmic and Periplasmic Proteomic Signatures of Exponentially Growing Cells of the Psychrophilic Bacterium Pseudoalteromonas haloplanktis TAC125 ▿ †

    PubMed Central

    Wilmes, Boris; Kock, Holger; Glagla, Susanne; Albrecht, Dirk; Voigt, Birgit; Markert, Stephanie; Gardebrecht, Antje; Bode, Rüdiger; Danchin, Antoine; Feller, Georges; Hecker, Michael; Schweder, Thomas

    2011-01-01

    The psychrophilic model bacterium Pseudoalteromonas haloplanktis is characterized by remarkably fast growth rates under low-temperature conditions in a range from 5°C to 20°C. In this study the proteome of cellular compartments, the cytoplasm and periplasm, of P. haloplanktis strain TAC125 was analyzed under exponential growth conditions at a permissive temperature of 16°C. By means of two-dimensional protein gel electrophoresis and mass spectrometry, a first inventory of the most abundant cytoplasmic and periplasmic proteins expressed in a peptone-supplemented minimal medium was established. By this approach major enzymes of the amino acid catabolism of this marine bacterium could be functionally deduced. The cytoplasmic proteome showed a predominance of amino acid degradation pathways and tricarboxylic acid (TCA) cycle enzymes but also the protein synthesis machinery. Furthermore, high levels of cold acclimation and oxidative stress proteins could be detected at this moderate growth temperature. The periplasmic proteome was characterized by a significant abundance of transporters, especially of highly expressed putative TonB-dependent receptors. This high capacity for protein synthesis, efficient amino acid utilization, and substrate transport may contribute to the fast growth rates of the copiotrophic bacterium P. haloplanktis in its natural environments. PMID:21183643

  13. Characterization of a salt-tolerant family 42 beta-galactosidase from a psychrophilic antarctic Planococcus isolate.

    PubMed

    Sheridan, P P; Brenchley, J E

    2000-06-01

    We isolated a gram-positive, halotolerant psychrophile from a hypersaline pond located on the McMurdo Ice Shelf in Antarctica. A phylogenetic analysis of the 16S rRNA gene sequence of this organism showed that it is a member of the genus Planococcus. This assignment is consistent with the morphology and physiological characteristics of the organism. A gene encoding a beta-galactosidase in this isolate was cloned in an Escherichia coli host. Sequence analysis of this gene placed it in glycosidase family 42 most closely related to an enzyme from Bacillus circulans. Even though an increasing number of family 42 glycosidase sequences are appearing in databases, little information about the biochemical features of these enzymes is available. Therefore, we purified and characterized this enzyme. The purified enzyme did not appear to have any metal requirement, had an optimum pH of 6.5 and an optimum temperature of activity at 42 degrees C, and was irreversibly inactivated within 10 min when it was incubated at 55 degrees C. The enzyme had an apparent K(m) of 4.9 micromol of o-nitrophenyl-beta-D-galactopyranoside, and the V(max) was 467 micromol of o-nitrophenol produced/min/mg of protein at 39 degrees C. Of special interest was the finding that the enzyme remained active at high salt concentrations, which makes it a possible reporter enzyme for halotolerant and halophilic organisms.

  14. Production of cryoprotectant extracellular polysaccharide substances (EPS) by the marine psychrophilic bacterium Colwellia psychrerythraea strain 34H under extreme conditions.

    PubMed

    Marx, Joseph G; Carpenter, Shelly D; Deming, Jody W

    2009-01-01

    Extracellular polysaccharide substances (EPS) play critical roles in microbial ecology, including the colonization of extreme environments in the ocean, from sea ice to the deep sea. After first developing a sugar-free growth medium, we examined the relative effects of temperature, pressure, and salinity on EPS production (on a per cell basis) by the obligately marine and psychrophilic gamma-proteobacterium, Colwellia psychrerythraea strain 34H. Over growth-permissive temperatures of approximately 10 to -4 degrees C, EPS production did not change, but from -8 to -14 degrees C when samples froze, EPS production rose dramatically. Similarly, at growth-permissive hydrostatic pressures of 1-200 atm (1 atm = 101.325 kPa) (at -1 and 8 degrees C), EPS production was unchanged, but at higher pressures of 400 and 600 atm EPS production rose markedly. In salinity tests at 10-100 parts per million (and -1 and 5 degrees C), EPS production increased at the freshest salinity tested. Extreme environmental conditions thus appear to stimulate EPS production by this strain. Furthermore, strain 34H recovered best from deep-freezing to -80 degrees C (not found for Earthly environments) if first supplemented with a preparation of its own EPS, rather than other cryoprotectants like glycerol, suggesting EPS production as both a survival strategy and source of compounds with potentially novel properties for biotechnological and other applications.

  15. Acidification of In-Storage-Psychrophilic-Anaerobic-Digestion (ISPAD) process to reduce ammonia volatilization: Model development and validation.

    PubMed

    Madani-Hosseini, Mahsa; Mulligan, Catherine N; Barrington, Suzelle

    2016-06-01

    In-Storage-Psychrophilic-Anaerobic-Digestion (ISPAD) is an ambient temperature treatment system for wastewaters stored for over 100days under temperate climates, which produces a nitrogen rich digestate susceptible to ammonia (NH3) volatilization. Present acidification techniques reducing NH3 volatilization are not only expensive and with secondary environmental effects, but do not apply to ISPAD relying on batch-to-batch inoculation. The objectives of this study were to identify and validate sequential organic loading (OL) strategies producing imbalances in acidogen and methanogen growth, acidifying ISPAD content one week before emptying to a pH of 6, while also preserving the inoculation potential. This acidification process is challenging as wastewaters often offer a high buffering capacity and ISPAD operational practices foster low microbial populations. A model simulating the ISPAD pH regime was used to optimize 3 different sequential OLs to decrease the ISPAD pH to 6.0. All 3 strategies were compared in terms of biogas production, volatile fatty acid (VFA) concentration, microbial activity, glucose consumption, and pH decrease. Laboratory validation of the model outputs confirmed that a sequential OL of 13kg glucose/m(3) of ISPAD content over 4days could indeed reduce the pH to 6.0. Such OL competes feasibly with present acidification techniques. Nevertheless, more research is required to explain the 3-day lag between the model results and the experimental data. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Structural Investigation of the Oligosaccharide Portion Isolated from the Lipooligosaccharide of the Permafrost Psychrophile Psychrobacter arcticus 273-4.

    PubMed

    Casillo, Angela; Parrilli, Ermenegilda; Filomena, Sannino; Lindner, Buko; Lanzetta, Rosa; Parrilli, Michelangelo; Tutino, Maria Luisa; Corsaro, Maria Michela

    2015-07-22

    Psychrophilic microorganisms have successfully colonized all permanently cold environments from the deep sea to mountain and polar regions. The ability of an organism to survive and grow in cryoenviroments depends on a number of adaptive strategies aimed at maintaining vital cellular functions at subzero temperatures, which include the structural modifications of the membrane. To understand the role of the membrane in the adaptation, it is necessary to characterize the cell-wall components, such as the lipopolysaccharides, that represent the major constituent of the outer membrane. The aim of this study was to investigate the structure of the carbohydrate backbone of the lipooligosaccharide (LOS) isolated from the cold-adapted Psychrobacter arcticus 273-4. The strain, isolated from a 20,000-to-30,000-year-old continuously frozen permafrost in Siberia, was cultivated at 4 °C. The LOS was isolated from dry cells and analyzed by means of chemical methods. In particular, it was degraded either by mild acid hydrolysis or by hydrazinolysis and investigated in detail by (1)H and (13)C NMR spectroscopy and by ESI FT-ICR mass spectrometry. The oligosaccharide was characterized by the substitution of the heptose residue, usually linked to Kdo in the inner core, with a glucose, and for the unusual presence of N-acetylmuramic acid.

  17. Performance of media types in psychrophilic anaerobic treatment of dairy wastewater in attached films packed bed reactors

    SciTech Connect

    Vartak, D.R.; Engler, C.R.; Ricke, S.C.

    1996-12-31

    Retention of microorganisms in anaerobic digesters by providing an attachment medium potentially can increase their productivity at lower operating temperatures. The objective of this work was to investigate the effectiveness of attached-