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Sample records for anaerobe clostridium acetobutylicum

  1. Atmospheric vs. anaerobic processing of metabolome samples for the metabolite profiling of a strict anaerobic bacterium, Clostridium acetobutylicum.

    PubMed

    Lee, Sang-Hyun; Kim, Sooah; Kwon, Min-A; Jung, Young Hoon; Shin, Yong-An; Kim, Kyoung Heon

    2014-12-01

    Well-established metabolome sample preparation is a prerequisite for reliable metabolomic data. For metabolome sampling of a Gram-positive strict anaerobe, Clostridium acetobutylicum, fast filtration and metabolite extraction with acetonitrile/methanol/water (2:2:1, v/v) at -20°C under anaerobic conditions has been commonly used. This anaerobic metabolite processing method is laborious and time-consuming since it is conducted in an anaerobic chamber. Also, there have not been any systematic method evaluation and development of metabolome sample preparation for strict anaerobes and Gram-positive bacteria. In this study, metabolome sampling and extraction methods were rigorously evaluated and optimized for C. acetobutylicum by using gas chromatography/time-of-flight mass spectrometry-based metabolomics, in which a total of 116 metabolites were identified. When comparing the atmospheric (i.e., in air) and anaerobic (i.e., in an anaerobic chamber) processing of metabolome sample preparation, there was no significant difference in the quality and quantity of the metabolomic data. For metabolite extraction, pure methanol at -20°C was a better solvent than acetonitrile/methanol/water (2:2:1, v/v/v) at -20°C that is frequently used for C. acetobutylicum, and metabolite profiles were significantly different depending on extraction solvents. This is the first evaluation of metabolite sample preparation under aerobic processing conditions for an anaerobe. This method could be applied conveniently, efficiently, and reliably to metabolome analysis for strict anaerobes in air.

  2. Effect of iron limitation and fur gene inactivation on the transcriptional profile of the strict anaerobe Clostridium acetobutylicum.

    PubMed

    Vasileva, Delyana; Janssen, Holger; Hönicke, Daniel; Ehrenreich, Armin; Bahl, Hubert

    2012-07-01

    Iron is a nutrient of critical importance for the strict anaerobe Clostridium acetobutylicum, as it is involved in numerous basic cellular functions and metabolic pathways. A gene encoding a putative ferric uptake regulator (Fur) has been identified in the genome of C. acetobutylicum. In this work, we inactivated the fur gene by using insertional mutagenesis. The resultant mutant showed a slow-growing phenotype and enhanced sensitivity to oxidative stress, but essentially no dramatic change in its fermentation pattern. A unique feature of its physiology was the overflowing production of riboflavin. To gain further insights into the role of the Fur protein and the mechanisms for establishment of iron balance in C. acetobutylicum, we characterized and compared the gene-expression profile of the fur mutant and the iron-limitation stimulon of the parental strain. Not surprisingly, a repertoire of iron-transport systems was upregulated in both microarray datasets, suggesting that they are regulated by Fur according to the availability of iron. In addition, iron limitation and inactivation of fur affected the expression of several genes involved in energy metabolism. Among them, two genes, encoding a lactate dehydrogenase and a flavodoxin, were highly induced. In order to support the function of the latter, the ribDBAH operon responsible for riboflavin biosynthesis was also upregulated significantly. Furthermore, the iron-starvation response of C. acetobutylicum involved transcriptional modifications that were not detected in the fur mutant, suggesting that there exist additional mechanisms for adaptation to low-iron environments. Collectively, these results demonstrate that the strict anaerobe C. acetobutylicum senses and responds to availability of iron on multiple levels using a sophisticated system, and that Fur plays an important role in this process.

  3. Production of biofuels from pretreated microalgae biomass by anaerobic fermentation with immobilized Clostridium acetobutylicum cells.

    PubMed

    Efremenko, E N; Nikolskaya, A B; Lyagin, I V; Senko, O V; Makhlis, T A; Stepanov, N A; Maslova, O V; Mamedova, F; Varfolomeev, S D

    2012-06-01

    The purpose of this work was to study the possible use of pretreated biomass of various microalgae and cyanobacteria as substrates for acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum cells immobilized into poly(vinyl alcohol) cryogel. To this end, the biochemical composition of photosynthetic microorganisms cultivated under various conditions was studied. The most efficient technique for pretreating microalgal biomass for its subsequent conversion into biofuels appeared to be thermal decomposition at 108 °C. For the first time the maximum productivity of the ABE fermentation in terms of hydrogen (8.5 mmol/L medium/day) was obtained using pretreated biomass of Nannochloropsis sp. Maximum yields of butanol and ethanol were observed with Arthrospira platensis biomass used as the substrate. Immobilized Clostridium cells were demonstrated to be suitable for multiple reuses (for a minimum of five cycles) in ABE fermentation for producing biofuels from pretreated microalgal biomass. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Anaerobic biotransformation of 2,4-dinitrotoluene and 2,6-dinitrotoluene by Clostridium acetobutylicum: A pathway through dihydroxylamino intermediates

    SciTech Connect

    Hughes, J.B.; Wang, C.Y.; Zhang, C.

    1999-04-01

    Experiments were conducted to isolate and identify the intermediates and products of 2,4-dinitrotoluene and 2,6-dinitrotoluene metabolism by Clostridium acetobutylicum. Transformation of both dinitrotoluenes initially resulted in the formation of hydroxylaminonitrotoluenes. Subsequent transformation favored the formation of dihydroxylaminotoluenes, with a limited reduction to aminonitrotoluene isomers. In cell cultures, metabolism beyond the level of dihydroxylaminotoluene was not observed. In cell extracts, where activity could be maintained for periods in excess of those in cell cultures, further transformation yielded aminohydroxylaminotoluenes and eventually diaminotoluenes. These findings further demonstrate the potential for hydroxylamines to be significant intermediates of nitroaromatic transformation under anaerobic fermentative conditions. Interestingly, the rearrangement of dihydroxylaminotoluenes was not observed, as was the case in previous studies of 2,4-dihydroxylamino-6-nitrotoluene metabolism. Dihydroxylaminotoluenes were found to be quite unstable, decomposing rapidly upon exposure to oxygen, complicating the assessment of their fate in remediation processes.

  5. Cellulolytic Activity of Clostridium acetobutylicum.

    PubMed

    Lee, S F; Forsberg, C W; Gibbins, L N

    1985-08-01

    Clostridium acetobutylicum NRRL B527 and ATCC 824 exhibited extracellular and cell-bound endoglucanase and cellobiase activities during growth in a chemically defined medium with cellobiose as the sole source of carbohydrate. For both strains, the endoglucanase was found to be mainly extracellular (70 to 90%) during growth in continuous or batch cultures with the pH maintained at 5.2, whereas the cellobiase was mainly cell associated (60 to 90%). During continuous cultivation of strain B527 with cellobiose as the limiting nutrient, maximum production of the endoglucanase and cellobiase occurred at pH values of 5.2 and 4.8, respectively. In the carbon-limited continuous cultures, strain 824 produced similar levels of endoglucanase, cellobiosidase, and cellobiase activities regardless of the carbon source used. However, in ammonium- or phosphate-limited cultures, with an excess of glucose, only 1/10 of the endoglucanase was produced, and neither cellobiosidase nor cellobiase activities were detectable. A crude extracellular enzyme preparation from strain B527 hydrolyzed carboxymethylcellulose and phosphoric acid-swollen cellulose readily and microcrystalline cellulose (A vicel) to a lesser extent. Glucose accounted for more than 90% of the reducing sugar produced by the hydrolysis of acid-swollen cellulose and Avicel. Strain B527 did not grow in medium with acid-swollen cellulose as the sole source of carbohydrate, although it grew readily on the products obtained by hydrolyzing the cellulose in vitro with a preparation of extracellular cellulase derived from the same organism.

  6. Fermentative hydrogen production in an up-flow anaerobic biofilm reactor inoculated with a co-culture of Clostridium acetobutylicum and Desulfovibrio vulgaris.

    PubMed

    Barca, Cristian; Ranava, David; Bauzan, Marielle; Ferrasse, Jean-Henry; Giudici-Orticoni, Marie-Thérèse; Soric, Audrey

    2016-12-01

    Dark fermentation systems often show low H2 yields and unstable H2 production, as the result of the variability of microbial dynamics and metabolic pathways. Recent batch investigations have demonstrated that an artificial consortium of two anaerobic bacteria, Clostridium acetobutylicum and Desulfovibrio vulgaris Hildenborough, may redirect metabolic fluxes and improve H2 yields. This study aimed at evaluating the scale-up from batch to continuous H2 production in an up-flow anaerobic packed-bed reactor (APBR) continuously fed with a glucose-medium. The effects of various parameters, including void hydraulic retention time (HRTv), pH, and alkalinity, on H2 production performances and metabolic pathways were investigated. The results demonstrated that a stable H2 production was reached after 3-4days of operation. H2 production rates increased significantly with decreasing HRTv from 4 to 2h. Instead, H2 yields remained almost stable despite the change in HRTv, indicating that the decrease in HRTv did not affect the global metabolism. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Annotation of the Clostridium Acetobutylicum Genome

    SciTech Connect

    Daly, M. J.

    2004-06-09

    The genome sequence of the solvent producing bacterium Clostridium acetobutylicum ATCC824, has been determined by the shotgun approach. The genome consists of a 3.94 Mb chromosome and a 192 kb megaplasmid that contains the majority of genes responsible for solvent production. Comparison of C. acetobutylicum to Bacillus subtilis reveals significant local conservation of gene order, which has not been seen in comparisons of other genomes with similar, or, in some cases, closer, phylogenetic proximity. This conservation allows the prediction of many previously undetected operons in both bacteria.

  8. A New Shuttle Plasmid That Stably Replicates in Clostridium acetobutylicum.

    PubMed

    Lee, Sang-Hyun; Kwon, Min-A; Choi, Sunwha; Kim, Sooah; Kim, Jungyeon; Shin, Yong-An; Kim, Kyoung Heon

    2015-10-01

    We have developed a new shuttle plasmid, designated as pLK1-MCS that can replicate in both Clostridium acetobutylicum and Escherichia coli, by combining the pUB110 and pUC19 plasmids. Plasmid pLK1-MCS replicated more stably than previously reported plasmids containing either the pIM13 or the pAMβ1 replicon in the absence of antibiotic selective pressure. The transfer frequency of pLK1-MCS into C. acetobutylicum was similar to the transfer frequency of other shuttle plasmids. We complemented C. acetobutylicum ML1 (that does not produce solvents such as acetone, butanol, and ethanol owing to loss of the megaplasmid pSOL1 harboring the adhE1-ctfAB-adc operon) by introducing pLK1-MCS carrying the adhE1-ctfAB-adc operon into C. acetobutylicum ML1. The transformed cells were able to resume anaerobic solvent production, indicating that the new shuttle plasmid has the potential for practical use in microbial biotechnology.

  9. Effects of butanol on Clostridium acetobutylicum.

    PubMed Central

    Bowles, L K; Ellefson, W L

    1985-01-01

    The internal pH of Clostridium acetobutylicum was determined at various stages during the growth of the organism. Even in the presence of significant quantities of acetic, butyric, and lactic acids, an internal pH of 6.2 was maintained. Experiments using N,N'-dicyclohexylcarbodiimide indicated that a functioning H+-ATPase is necessary for internal pH control. Butanol, one of the end products of the fermentation, had numerous harmful effects on C. acetobutylicum. At a concentration high enough to inhibit growth, butanol destroyed the ability of the cell to maintain internal pH, lowered the intracellular level of ATP, and inhibited glucose uptake. Experiments done at two different external pH values suggested that the butanol-mediated decrease in ATP concentration was independent of the drop in internal pH. Glucose uptake was not affected by arsenate, suggesting that uptake was not ATP dependent. The effects of butanol on C. acetobutylicum are complex, inhibiting several interrelated membrane processes. PMID:2868690

  10. Feasibility of installing and maintaining anaerobiosis using Escherichia coli HD701 as a facultative anaerobe for hydrogen production by Clostridium acetobutylicum ATCC 824 from various carbohydrates.

    PubMed

    Hassan, Sedky H A; Morsy, Fatthy Mohamed

    2015-12-01

    Using Escherichia coli for installing and maintaining anaerobiosis for hydrogen production by Clostridium acetobutylicum ATCC 824 is a cost-effective approach for industrial hydrogen production, as it does not require reducing agents or sparging with inert gases. This study was devoted for investigating the feasibility for installing and maintaining anaerobiosis of hydrogen production by C. acetobutylicum ATCC 824 when using E. coli HD701 utilizable versus non utilizable sugars as a-carbon source. Using E. coli HD701 for installing anaerobiosis showed a comparable hydrogen production yield and efficiency to the use of reducing agents and nitrogen sparging in case of hydrogen production from the E. coli HD701 non utilizable sugars. In contrast, using E. coli HD701 for installing anaerobiosis showed a lower hydrogen production yield and efficiency than the use of reducing agents and nitrogen sparging in case of using glucose as a substrate. This is possibly because E. coli HD701 when using glucose compensate for the substrate, and produce hydrogen with lower efficiency than C. acetobutylicum ATCC 824. These results indicated that the use of E. coli HD701 for installing anaerobiosis would not be economically feasible when using E. coli HD701 utilizable sugars as a carbon source. In contrast, the use of this approach for installing anaerobiosis for hydrogen production from sucrose and starch would have a high potency for industrial applications.

  11. Engineering Clostridium acetobutylicum for alcohol production.

    PubMed

    Hou, Xiaohu; Peng, Wanfeng; Xiong, Lian; Huang, Chao; Chen, Xuefang; Chen, Xinde; Zhang, Weiguo

    2013-06-20

    While Clostridium acetobutylicum has been used for large-scale butanol production (ABE fermentation), its by-product acetone cannot be used as a biofuel. In this study, C. acetobutylicum was engineered for alcohol titers (butanol plus ethanol). The adc gene was inactivated to eliminate acetone production, and glutathione biosynthetic capability was introduced into C. acetobutylicum to improve the strain's robustness by expressing Escherichia coli's gshAB genes in the adc locus. Acetone production was reduced from 2.64±0.22 g/L to 0.15±0.08 g/L in the engineered strain 824adc::gsh, whereas butanol production was increased from 5.17±0.26 g/L to 8.27±0.27 g/L. To further improve the alcohol titers, the metabolic flux in the alcohol biosynthesis pathways was enhanced. Overlapping PCR was used to generate expression cassette EC, which expresses the hbd, thl, crt, and bcd genes, and the Sol operon was amplified to express the adhE and ctfAB genes. Butanol and alcohol production reached 14.86±0.26 g/L and 18.11±0.66 g/L, respectively, in 824adc::gsh Sol-EC. Furthermore, the butanol and alcohol yields were 0.336 g/g and 0.409 g/g, respectively, in 824adc::gsh Sol-EC. This study provided a combined strategy for enhancing alcohol production in C. acetobutylicum. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Hierarchy in pentose sugar metabolism in Clostridium acetobutylicum.

    PubMed

    Aristilde, Ludmilla; Lewis, Ian A; Park, Junyoung O; Rabinowitz, Joshua D

    2015-02-01

    Bacterial metabolism of polysaccharides from plant detritus into acids and solvents is an essential component of the terrestrial carbon cycle. Understanding the underlying metabolic pathways can also contribute to improved production of biofuels. Using a metabolomics approach involving liquid chromatography-mass spectrometry, we investigated the metabolism of mixtures of the cellulosic hexose sugar (glucose) and hemicellulosic pentose sugars (xylose and arabinose) in the anaerobic soil bacterium Clostridium acetobutylicum. Simultaneous feeding of stable isotope-labeled glucose and unlabeled xylose or arabinose revealed that,as expected, glucose was preferentially used as the carbon source. Assimilated pentose sugars accumulated in pentose phosphate pathway (PPP) intermediates with minimal flux into glycolysis. Simultaneous feeding of xylose and arabinose revealed an unexpected hierarchy among the pentose sugars, with arabinose utilized preferentially over xylose. The phosphoketolase pathway (PKP) provides an alternative route of pentose catabolism in C. acetobutylicum that directly converts xylulose-5-phosphate into acetyl-phosphate and glyceraldehyde-3-phosphate, bypassing most of the PPP. When feeding the mixture of pentose sugars, the labeling patterns of lower glycolytic intermediates indicated more flux through the PKP than through the PPP and upper glycolysis, and this was confirmed by quantitative flux modeling. Consistent with direct acetyl-phosphate production from the PKP, growth on the pentose mixture resulted in enhanced acetate excretion. Taken collectively, these findings reveal two hierarchies in clostridial pentose metabolism: xylose is subordinate to arabinose, and the PPP is used less than the PKP.

  13. Hierarchy in Pentose Sugar Metabolism in Clostridium acetobutylicum

    PubMed Central

    Lewis, Ian A.; Park, Junyoung O.

    2014-01-01

    Bacterial metabolism of polysaccharides from plant detritus into acids and solvents is an essential component of the terrestrial carbon cycle. Understanding the underlying metabolic pathways can also contribute to improved production of biofuels. Using a metabolomics approach involving liquid chromatography-mass spectrometry, we investigated the metabolism of mixtures of the cellulosic hexose sugar (glucose) and hemicellulosic pentose sugars (xylose and arabinose) in the anaerobic soil bacterium Clostridium acetobutylicum. Simultaneous feeding of stable isotope-labeled glucose and unlabeled xylose or arabinose revealed that, as expected, glucose was preferentially used as the carbon source. Assimilated pentose sugars accumulated in pentose phosphate pathway (PPP) intermediates with minimal flux into glycolysis. Simultaneous feeding of xylose and arabinose revealed an unexpected hierarchy among the pentose sugars, with arabinose utilized preferentially over xylose. The phosphoketolase pathway (PKP) provides an alternative route of pentose catabolism in C. acetobutylicum that directly converts xylulose-5-phosphate into acetyl-phosphate and glyceraldehyde-3-phosphate, bypassing most of the PPP. When feeding the mixture of pentose sugars, the labeling patterns of lower glycolytic intermediates indicated more flux through the PKP than through the PPP and upper glycolysis, and this was confirmed by quantitative flux modeling. Consistent with direct acetyl-phosphate production from the PKP, growth on the pentose mixture resulted in enhanced acetate excretion. Taken collectively, these findings reveal two hierarchies in clostridial pentose metabolism: xylose is subordinate to arabinose, and the PPP is used less than the PKP. PMID:25527534

  14. Metabolite Analysis of Clostridium acetobutylicum: Fermentation in a Microbial Fuel Cell

    DTIC Science & Technology

    2010-01-01

    Metabolite analysis of Clostridium acetobutylicum : Fermentation in a microbial fuel cell Amethist S. Finch, Timothy D. Mackie, Christian J. Sund...Fermentation products Clostridium acetobutylicum Current generation a b s t r a c t Microbial fuel cells (MFCs) were used to monitor metabolism...changes in Clostridium acetobutylicum fer- mentations. When MFCs were inoculated with C. acetobutylicum , they generated a unique voltage output pattern

  15. Butanol production under microaerobic conditions with a symbiotic system of Clostridium acetobutylicum and Bacillus cereus.

    PubMed

    Wu, Pengfei; Wang, Genyu; Wang, Gehua; Børresen, Børre Tore; Liu, Hongjuan; Zhang, Jianan

    2016-01-14

    One major problem of ABE (acetone, butanol and ethanol) fermentation is high oxygen sensitivity of Clostridium acetobutylicum. Currently, no single strain has been isolated or genetically engineered to produce butanol effectively under aerobic conditions. In our previous work, a symbiotic system TSH06 has been developed successfully by our group, and two strains, C. acetobutylicum TSH1 and Bacillus cereus TSH2, were isolated from TSH06. Compared with single culture, TSH06 showed promotion on cell growth and solvent accumulation under microaerobic conditions. To simulate TSH06, a new symbiotic system was successfully re-constructed by adding living cells of B. cereus TSH2 into C. acetobutylicum TSH1 cultures. During the fermentation process, the function of B. cereus TSH2 was found to deplete oxygen and provide anaerobic environment for C. acetobutylicum TSH1. Furthermore, inoculation ratio of C. acetobutylicum TSH1 and B. cereus TSH2 affected butanol production. In a batch fermentation with optimized inoculation ratio of 5 % C. acetobutylicum TSH1 and 0.5 % B. cereus TSH2, 11.0 g/L butanol and 18.1 g/L ABE were produced under microaerobic static condition. In contrast to the single culture of C. acetobutylicum TSH1, the symbiotic system became more aerotolerant and was able to produce 11.2 g/L butanol in a 5 L bioreactor even with continuous 0.15 L/min air sparging. In addition, qPCR assay demonstrated that the abundance of B. cereus TSH2 increased quickly at first and then decreased sharply to lower than 1 %, whereas C. acetobutylicum TSH1 accounted for more than 99 % of the whole population in solventogenic phase. The characterization of a novel symbiotic system on butanol fermentation was studied. The new symbiotic system re-constructed by co-culture of C. acetobutylicum TSH1 and B. cereus TSH2 showed excellent performance on butanol production under microaerobic conditions. B. cereus TSH2 was a good partner for C. acetobutylicum TSH1 by providing an anaerobic

  16. Single Crossover-Mediated Markerless Genome Engineering in Clostridium acetobutylicum.

    PubMed

    Lee, Sang-Hyun; Kim, Hyun Ju; Shin, Yong-An; Kim, Kyoung Heon; Lee, Sang Jun

    2016-04-28

    A novel genome-engineering tool in Clostridium acetobutylicum was developed based on singlecrossover homologous recombination. A small-sized non-replicable plasmid, pHKO1, was designed for efficient integration into the C. acetobutylicum genome. The integrated pHKO1 plasmid backbone, which included an antibiotic resistance gene, can be excised in vivo by Flp recombinase, leaving a single flippase recognition target sequence in the middle of the targeted gene. Since the pSHL-FLP plasmid, the carrier of the Flp recombinase gene, employed the segregationally unstable pAMβ1 replicon, the plasmid was rapidly cured from the mutant C. acetobutylicum. Consequently, our method makes it easier to engineer C. acetobutylicum.

  17. Ribulokinase and Transcriptional Regulation of Arabinose Metabolism in Clostridium acetobutylicum

    PubMed Central

    Zhang, Lei; Leyn, Semen A.; Gu, Yang; Jiang, Weihong

    2012-01-01

    The transcription factor AraR controls utilization of l-arabinose in Bacillus subtilis. In this study, we combined a comparative genomic reconstruction of AraR regulons in nine Clostridium species with detailed experimental characterization of AraR-mediated regulation in Clostridium acetobutylicum. Based on the reconstructed AraR regulons, a novel ribulokinase, AraK, present in all analyzed Clostridium species was identified, which was a nonorthologous replacement of previously characterized ribulokinases. The predicted function of the araK gene was confirmed by inactivation of the araK gene in C. acetobutylicum and biochemical assays using purified recombinant AraK. In addition to the genes involved in arabinose utilization and arabinoside degradation, extension of the AraR regulon to the pentose phosphate pathway genes in several Clostridium species was revealed. The predicted AraR-binding sites in the C. acetobutylicum genome and the negative effect of l-arabinose on DNA-regulator complex formation were verified by in vitro binding assays. The predicted AraR-controlled genes in C. acetobutylicum were experimentally validated by testing gene expression patterns in both wild-type and araR-inactivated mutant strains during growth in the absence or presence of l-arabinose. PMID:22194461

  18. Ribulokinase and transcriptional regulation of arabinose metabolism in Clostridium acetobutylicum.

    PubMed

    Zhang, Lei; Leyn, Semen A; Gu, Yang; Jiang, Weihong; Rodionov, Dmitry A; Yang, Chen

    2012-03-01

    The transcription factor AraR controls utilization of L-arabinose in Bacillus subtilis. In this study, we combined a comparative genomic reconstruction of AraR regulons in nine Clostridium species with detailed experimental characterization of AraR-mediated regulation in Clostridium acetobutylicum. Based on the reconstructed AraR regulons, a novel ribulokinase, AraK, present in all analyzed Clostridium species was identified, which was a nonorthologous replacement of previously characterized ribulokinases. The predicted function of the araK gene was confirmed by inactivation of the araK gene in C. acetobutylicum and biochemical assays using purified recombinant AraK. In addition to the genes involved in arabinose utilization and arabinoside degradation, extension of the AraR regulon to the pentose phosphate pathway genes in several Clostridium species was revealed. The predicted AraR-binding sites in the C. acetobutylicum genome and the negative effect of L-arabinose on DNA-regulator complex formation were verified by in vitro binding assays. The predicted AraR-controlled genes in C. acetobutylicum were experimentally validated by testing gene expression patterns in both wild-type and araR-inactivated mutant strains during growth in the absence or presence of L-arabinose.

  19. Fermentation of residual glycerol by Clostridium acetobutylicum ATCC 824 in pure and mixed cultures.

    PubMed

    Dams, Rosemeri I; Guilherme, Alexandre A; Vale, Maria S; Nunes, Vanja F; Leitão, Renato C; Santaella, Sandra T

    2016-12-01

    The aim of this research was to estimate the production of hydrogen, organic acids and alcohols by the strain of Clostridium acetobutylicum ATCC 824 using residual glycerol as a carbon source. The experiments were carried out in pure and mixed cultures in batch experiments. Three different sources of inocula for mixed culture were used. Ruminal liquid from goats and sludge collected from two upflow anaerobic sludge blanket reactors treating municipal wastewater and brewery effluent were tested for hydrogen, organic acids and alcohols production with or without C. acetobutylicum ATCC 824. The main detected end-products from the glycerol fermentation were hydrogen, organic acids (acetic, propionic, butyric and caproic) and alcohol (ethanol and 1,3-propanediol - 1,3PD). High hydrogen (0.44 mol H2/mol glycerol consumed) and 1,3PD (0.32 mol 1,3PD/mol glycerol consumed) yields were obtained when the strain C. acetobutylicum ATCC 824 was bioaugmented into the sludge from municipal wastewater using 5 g/L of glycerol. Significant concentrations of n-caproic acid were detected in the ruminal liquid when amended with C. acetobutylicum ATCC 824. The results suggest that glycerol can be used for the generation of H2, 1,3PD and n-caproic acid using C. acetobutylicum ATCC 824 as agent in pure or mixed cultures.

  20. Expression and Characterization of Levansucrase from Clostridium acetobutylicum.

    PubMed

    Gao, Song; Qi, Xianghui; Hart, Darren J; Gao, Herui; An, Yingfeng

    2017-02-01

    The Clostridium acetobutylicum gene Ca-SacB encoding levansucrase was cloned and expressed in Escherichia coli. Ca-SacB is composed of 1287 bp and encodes 428 amino acid residues, which could convert 150 mmol/L sucrose to levan with the liberation of glucose. The optimum pH and temperature of this enzyme for levan formation were pH 6 and 60 °C, respectively. Levansucrase activity of Ca-SacB was completely abolished by 5 mmol/L Ag(+) and Hg(2+). The Km and Vmax values for levansucrase were calculated to be 64 mmol/L and 190 μmol/min/mg, respectively. Interestingly, Ca-SacB was found to have high product specificity, and no fructooligosaccharide was identified in the product, indicating that Ca-SacB may be valuable for industrial production of levan. In addition, Ca-SacB is the first characterized levansucrase isolated from an anaerobic bacterium, which should be valuable for exploring new enzyme resources and deepening the understanding of the catalytic mechanisms of levansucrases.

  1. Genetic and biochemical analysis of solvent formation in Clostridium acetobutylicum

    SciTech Connect

    Bennett, G.N.; Rudolph, F.B.

    1998-05-01

    The anaerobic organism Clostridium acetobutylicum has been used for commercial production of important organic solvents due to its ability to convert a wide variety of crude substrates to acids and alcohols. Current knowledge concerning the molecular genetics, cell regulation and metabolic engineering of this organism is still rather limited. The objectives are to improve the knowledge of the molecular genetics and enzymology of Clostridia in order to make genetic alterations which will more effectively channel cell metabolism toward production of desired products. Two factors that limit butanol production in continuous cultures are: (1) The degeneration of the culture, with an increase in the proportion of cells which are incapable of solvent production. Currently isolated degenerate strains are being evaluated to analyze the molecular mechanism of degeneration to determine if it is due to a genetic loss of solvent related genes, loss of a regulatory element, or an increase in general mutagenesis. Recent studies show two general types of degenerates, one which seems to have lost essential solvent pathway genes and another which has not completely lost all solvent production capability and retains the DNA bearing solvent pathway genes. (2) The production of hydrogen which uses up reducing equivalents in the cell. If the reducing power were more fully directed to the reduction reactions involved in butanol production, the process would be more efficient. The authors have studied oxidation reduction systems related to this process. These studies focus on ferredoxin and rubredoxin and their oxidoreductases.

  2. Reaction engineering studies of acetone-butanol-ethanol fermentation with Clostridium acetobutylicum.

    PubMed

    Schmidt, Michael; Weuster-Botz, Dirk

    2012-05-01

    Acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum has been extensively studied in recent years because the organism is recognized as an excellent butanol producer. A parallel bioreactor system with 48 stirred-tank bioreactors on a 12 mL scale was evaluated for batch cultivations of the strictly anaerobic, butanol-producing C. acetobutylicum ATCC 824. Continuous gassing with nitrogen gas was applied to control anaerobic conditions. Process performances of ABE batch fermentations on a milliliter scale were identical to the liter-scale stirred-tank reactor if reaction conditions were identical on the different scales (e.g., initial medium, pH, temperature, specific evaporation rates, specific power input by the stirrers). The effects of varying initial ammonia concentrations (0.1-4.4 g L(-1) ) were studied in parallel with respect to glucose consumption and butanol production of C. acetobutylicum ATCC 824 as a first application example. The highest butanol yield of 33% (mol mol(-1) ) was observed at initial ammonia concentrations of 0.5 and 1.1 g L(-1) . This is the first report on the successful application of a 48 parallel stirred-tank bioreactor system for reaction engineering studies of strictly anaerobic microorganisms at the milliliter scale. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Stable Escherichia coli-Clostridium acetobutylicum shuttle vector for secretion of murine tumor necrosis factor alpha.

    PubMed

    Theys, J; Nuyts, S; Landuyt, W; Van Mellaert, L; Dillen, C; Böhringer, M; Dürre, P; Lambin, P; Anné, J

    1999-10-01

    Recombinant plasmids were constructed to secrete mouse tumor necrosis factor alpha (mTNF-alpha) from Clostridium acetobutylicum. The shuttle plasmids contained the clostridial endo-beta1, 4-glucanase (eglA) promoter and signal sequence that was fused in frame to the mTNF-alpha cDNA. The construction was first tested in Escherichia coli and then introduced in C. acetobutylicum DSM792 by electroporation. Controls confirmed the presence and stability of the recombinant plasmids in this organism. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an in vitro cytotoxic assay were used to monitor expression and secretion of mTNF-alpha during growth. Significant levels of biologically active mTNF-alpha were measured in both lysates and supernatants. The present report deals with investigations on the elaboration of a gene transfer system for cancer treatment using anaerobic bacteria.

  4. Comparative transcriptomic analysis of Clostridium acetobutylicum biofilm and planktonic cells.

    PubMed

    Liu, Dong; Xu, Jiahui; Wang, Yanyan; Chen, Yong; Shen, Xiaoning; Niu, Huanqing; Guo, Ting; Ying, Hanjie

    2016-01-20

    Biofilm-based immobilization of solventogenic Clostridia has been extensively exploited to overcome traditional bottlenecks in biobutanol production like solvent toxicity and low productivities. However, the molecular basis of solventogenic Clostridia biofilm is rarely explored. Here, for the first time, we report DNA array-based study of Clostridium acetobutylicum biofilm cells to elucidate the transcriptional modulation. Results showed that 16.2% of the C. acetobutylicum genome genes within the biofilm cells were differentially expressed, with most genes being up-regulated. The most dramatic changes occurred with amino acid biosynthesis, with sulfur uptake and cysteine biosynthesis being the most up-regulated and histidine biosynthesis being the most down-regulated in the biofilm cells. It was demonstrated that C. acetobutylicum biofilm cells increased metabolic activities probably by up-regulating iron and sulfur uptake and Fe-S cluster biosynthesis genes as well as glycolysis genes. Furthermore, genes involved in sporulation, granulose formation, extracellular polymer degradation, pentose catabolisms, and various other processes were also notably regulated, indicating that the biofilm mode of growth rendered the cells a distinct phenotype. This study provides valuable insights into the transcriptional regulation in C. acetobutylicum biofilm cells and should be highly useful for understanding and developing the biofilm-based processes. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Purification and Characterization of an Autolysin from Clostridium acetobutylicum

    PubMed Central

    Webster, Jocelyn R.; Reid, Sharon J.; Jones, David T.; Woods, David R.

    1981-01-01

    A proteinaceous substance with antibiotic-like activity, resembling that of a bacteriocin, was isolated from an industrial-scale acetone-butanol fermentation of Clostridium acetobutylicum. The substance, purified by acetone precipitation, diethylaminoethyl cellulose chromatography, and polyacrylamide gel electrophoresis, was characterized as a glycoprotein with a molecular weight of 28,000. The glycoprotein was partially inactivated by certain protease enzymes. It had no effect on deoxyribonucleic acid, ribonucleic acid, or protein synthesis, and it did not result in the loss of intracellular adenosine triphosphate. The glycoprotein lysed sodium dodecyl sulfate-treated cells and cell wall preparations, and therefore it is referred to as an autolysin. The autolysin gene appeared to be chromosomal since plasmid deoxyribonucleic acid was not detected in the C. acetobutylicum strain. PMID:16345710

  6. Cellulose fermentation by a coculture of a mesophilic cellulolytic Clostridium and Clostridium acetobutylicum

    SciTech Connect

    Fond, O.; Petitdemange, E.; Petitdemange, H.; Engasser, J.M.

    1983-01-01

    A coculture of a mesophilic cellulolytic Clostridium with Clostridium acetobutylicum can yield a direct conversion of cellulose into chemicals. In 13 days 30 g/l Solka Floc is degraded and fermented into 14 g/l butyric acid, 4 g/l acetic acid, 3 g/l ethanol, and 1 g/l butanol. A four times higher rate of cellulose hydrolysis than in pure culture of the cellulolytic Clostridium is thus obtained. Fed-batch fermentations of C. acetobutylicum at different glucose feeding rate show that solvents are only produced at a sufficient high rate of glucose supply to the medium. Acids are thus the main products of the coculture because of the limited rate of cellulolysis by the mesophilic strain. 7 references, 5 figures.

  7. Metronidazole activation and isolation of Clostridium acetobutylicum electron transport genes.

    PubMed Central

    Santangelo, J D; Jones, D T; Woods, D R

    1991-01-01

    An Escherichia coli F19 recA, nitrate reductase-deficient mutant was constructed by transposon mutagenesis and shown to be resistant to metronidazole. This mutant was a most suitable host for the isolation of Clostridium acetobutylicum genes on recombinant plasmids, which activated metronidazole and rendered the E. coli F19 strain sensitive to metronidazole. Twenty-five E. coli F19 clones containing different recombinant plasmids were isolated and classified into five groups on the basis of their sensitivity to metronidazole. The clones were tested for nitrate reductase, pyruvate-ferredoxin oxidoreductase, and hydrogenase activities. DNA hybridization and restriction endonuclease mapping revealed that four of the C. acetobutylicum insert DNA fragments on recombinant plasmids were linked in an 11.1-kb chromosomal fragment. DNA sequencing and amino acid homology studies indicated that this DNA fragment contained a flavodoxin gene which encoded a protein of 160 amino acids that activated metronidazole and made the E. coli F19 mutant very sensitive to metronidazole. The flavodoxin and hydrogenase genes which are involved in electron transfer systems were linked on the 11.1-kb DNA fragment from C. acetobutylicum. Images PMID:1991710

  8. Redox-switch regulatory mechanism of thiolase from Clostridium acetobutylicum

    PubMed Central

    Kim, Sangwoo; Jang, Yu-Sin; Ha, Sung-Chul; Ahn, Jae-Woo; Kim, Eun-Jung; Hong Lim, Jae; Cho, Changhee; Shin Ryu, Yong; Kuk Lee, Sung; Lee, Sang Yup; Kim, Kyung-Jin

    2015-01-01

    Thiolase is the first enzyme catalysing the condensation of two acetyl-coenzyme A (CoA) molecules to form acetoacetyl-CoA in a dedicated pathway towards the biosynthesis of n-butanol, an important solvent and biofuel. Here we elucidate the crystal structure of Clostridium acetobutylicum thiolase (CaTHL) in its reduced/oxidized states. CaTHL, unlike those from other aerobic bacteria such as Escherichia coli and Zoogloea ramegera, is regulated by the redox-switch modulation through reversible disulfide bond formation between two catalytic cysteine residues, Cys88 and Cys378. When CaTHL is overexpressed in wild-type C. acetobutylicum, butanol production is reduced due to the disturbance of acidogenic to solventogenic shift. The CaTHLV77Q/N153Y/A286K mutant, which is not able to form disulfide bonds, exhibits higher activity than wild-type CaTHL, and enhances butanol production upon overexpression. On the basis of these results, we suggest that CaTHL functions as a key enzyme in the regulation of the main metabolism of C. acetobutylicum through a redox-switch regulatory mechanism. PMID:26391388

  9. Redox-switch regulatory mechanism of thiolase from Clostridium acetobutylicum.

    PubMed

    Kim, Sangwoo; Jang, Yu-Sin; Ha, Sung-Chul; Ahn, Jae-Woo; Kim, Eun-Jung; Lim, Jae Hong; Cho, Changhee; Ryu, Yong Shin; Lee, Sung Kuk; Lee, Sang Yup; Kim, Kyung-Jin

    2015-09-22

    Thiolase is the first enzyme catalysing the condensation of two acetyl-coenzyme A (CoA) molecules to form acetoacetyl-CoA in a dedicated pathway towards the biosynthesis of n-butanol, an important solvent and biofuel. Here we elucidate the crystal structure of Clostridium acetobutylicum thiolase (CaTHL) in its reduced/oxidized states. CaTHL, unlike those from other aerobic bacteria such as Escherichia coli and Zoogloea ramegera, is regulated by the redox-switch modulation through reversible disulfide bond formation between two catalytic cysteine residues, Cys88 and Cys378. When CaTHL is overexpressed in wild-type C. acetobutylicum, butanol production is reduced due to the disturbance of acidogenic to solventogenic shift. The CaTHL(V77Q/N153Y/A286K) mutant, which is not able to form disulfide bonds, exhibits higher activity than wild-type CaTHL, and enhances butanol production upon overexpression. On the basis of these results, we suggest that CaTHL functions as a key enzyme in the regulation of the main metabolism of C. acetobutylicum through a redox-switch regulatory mechanism.

  10. Cap0037, a Novel Global Regulator of Clostridium acetobutylicum Metabolism.

    PubMed

    Nguyen, Ngoc-Phuong-Thao; Linder, Sonja; Flitsch, Stefanie K; Schiel-Bengelsdorf, Bettina; Dürre, Peter; Soucaille, Philippe

    2016-10-04

    An operon comprising two genes, CA_P0037 and CA_P0036, that encode proteins of unknown function that were previously shown to be highly expressed in acidogenic cells and repressed in solventogenic and alcohologenic cells is located on the pSOL1 megaplasmid of Clostridium acetobutylicum upstream of adhE2 A CA_P0037::int (189/190s) mutant in which an intron was inserted at position 189/190 in the sense strand of CA_P0037 was successfully generated by the Targetron technique. The resultant mutant showed significantly different metabolic flux patterns in acidogenic (producing mainly lactate, butyrate, and butanol) and alcohologenic (producing mainly butyrate, acetate, and lactate) chemostat cultures but not in solventogenic or batch cultures. Transcriptomic investigation of the CA_P0037::int (189/190s) mutant showed that inactivation of CA_P0037 significantly affected the expression of more than 258 genes under acidogenic conditions. Surprisingly, genes belonging to the Fur regulon, involved in iron transport (CA_C1029-CA_C1032), or coding for the main flavodoxin (CA_C0587) were the most significantly expressed genes under all conditions, whereas fur (coding for the ferric uptake regulator) gene expression remained unchanged. Furthermore, most of the genes of the Rex regulon, such as the adhE2 and ldhA genes, and of the PerR regulon, such as rbr3A-rbr3B and dfx, were overexpressed in the mutant. In addition, the whole CA_P0037-CA_P0036 operon was highly expressed under all conditions in the CA_P0037::int (189/190s) mutant, suggesting a self-regulated expression mechanism. Cap0037 was shown to bind to the CA_P0037-CA_P0036 operon, sol operon, and adc promoters, and the binding sites were determined by DNA footprinting. Finally, a putative Cap0037 regulon was generated using a bioinformatic approach. Clostridium acetobutylicum is well-known for its ability to produce solvents, especially n-butanol. Understanding the regulatory network of C. acetobutylicum will be

  11. Pervaporative butanol fermentation by Clostridium acetobutylicum B18

    SciTech Connect

    Geng, Q.; Park, C.H. . Dept. of Agricultural Engineering)

    1994-04-15

    Extractive acetone-butanol-ethanol (ABE) fermentation was carried out successfully using pervaporation and a low-acid-producing Clostridium acetobutylicum B18. A pervaporation module with 0.17 m[sup 2] of surface area was made of silicone membrane of 240 [mu]m thickness. Pervaporation experiments using make-up solutions showed that butanol and acetone fluxes increased linearly with their concentrations in the aqueous phase. Fickian diffusion coefficients were constants for fixed air flow rates, and increased at higher sweep air flow rates. During batch and fed-batch fermentation, pervaporation at an air flow rate of 8 L/min removed butanol and acetone efficiently. Butanol concentration was maintained below 4.5 g/L even though Clostridium acetobutylicum B18 produced butanol steadily. Pervaporation could not remove organic acids efficiently, but organic acids did not accumulate because strain B18 produced little organic acid and recycled added organic acids efficiently. With pervaporation, glucose consumption rate increased compared to without pervaporation, and up to 160 g/L of glucose was consumed during 80 h. Cell growth was not inhibited by possible salt accumulation or oxygen diffusion through the silicone tubing. The culture volume was maintained relatively constant during fed-batch operation because of an offsetting effect of water and product removal by pervaporation and addition of nutrient supplements.

  12. An agr Quorum Sensing System That Regulates Granulose Formation and Sporulation in Clostridium acetobutylicum

    PubMed Central

    Steiner, Elisabeth; Scott, Jamie

    2012-01-01

    The Gram-positive, anaerobic, endospore-forming bacterium Clostridium acetobutylicum has considerable biotechnological potential due to its ability to produce solvents as fermentation products, in particular the biofuel butanol. Its genome contains a putative agr locus, agrBDCA, known in staphylococci to constitute a cyclic peptide-based quorum sensing system. In staphylococci, agrBD is required for the generation of a peptide signal that, upon extracellular accumulation, is sensed by an agrCA-encoded two-component system. Using ClosTron technology, agrB, agrC, and agrA mutants of C. acetobutylicum ATCC 824 were generated and phenotypically characterized. Mutants and wild type displayed similar growth kinetics and no apparent differences in solvent formation under the conditions tested. However, the number of heat-resistant endospores formed by the mutants in liquid culture was reduced by about one order of magnitude. On agar-solidified medium, spore formation was more strongly affected, particularly in agrA and agrC mutants. Similarly, accumulation of the starch-like storage compound granulose was almost undetectable in colonies of agrB, agrA, and agrC mutants. Importantly, these defects could be genetically complemented, demonstrating that they were directly linked to agr inactivation. A diffusible factor produced by agrBD-expressing strains was found to restore granulose and spore formation in the agrB mutant. Furthermore, a synthetic cyclic peptide, designed on the basis of the C. acetobutylicum AgrD sequence, was also capable of complementing the defects of the agrB mutant when added exogenously to the culture. Together, these findings support the hypothesis that agr-dependent quorum sensing is involved in the regulation of sporulation and granulose formation in C. acetobutylicum. PMID:22179241

  13. An agr quorum sensing system that regulates granulose formation and sporulation in Clostridium acetobutylicum.

    PubMed

    Steiner, Elisabeth; Scott, Jamie; Minton, Nigel P; Winzer, Klaus

    2012-02-01

    The Gram-positive, anaerobic, endospore-forming bacterium Clostridium acetobutylicum has considerable biotechnological potential due to its ability to produce solvents as fermentation products, in particular the biofuel butanol. Its genome contains a putative agr locus, agrBDCA, known in staphylococci to constitute a cyclic peptide-based quorum sensing system. In staphylococci, agrBD is required for the generation of a peptide signal that, upon extracellular accumulation, is sensed by an agrCA-encoded two-component system. Using ClosTron technology, agrB, agrC, and agrA mutants of C. acetobutylicum ATCC 824 were generated and phenotypically characterized. Mutants and wild type displayed similar growth kinetics and no apparent differences in solvent formation under the conditions tested. However, the number of heat-resistant endospores formed by the mutants in liquid culture was reduced by about one order of magnitude. On agar-solidified medium, spore formation was more strongly affected, particularly in agrA and agrC mutants. Similarly, accumulation of the starch-like storage compound granulose was almost undetectable in colonies of agrB, agrA, and agrC mutants. Importantly, these defects could be genetically complemented, demonstrating that they were directly linked to agr inactivation. A diffusible factor produced by agrBD-expressing strains was found to restore granulose and spore formation in the agrB mutant. Furthermore, a synthetic cyclic peptide, designed on the basis of the C. acetobutylicum AgrD sequence, was also capable of complementing the defects of the agrB mutant when added exogenously to the culture. Together, these findings support the hypothesis that agr-dependent quorum sensing is involved in the regulation of sporulation and granulose formation in C. acetobutylicum.

  14. In situ hydrogen, acetone, butanol, ethanol and microdiesel production by Clostridium acetobutylicum ATCC 824 from oleaginous fungal biomass.

    PubMed

    Hassan, Elhagag Ahmed; Abd-Alla, Mohamed Hemida; Bagy, Magdy Mohamed Khalil; Morsy, Fatthy Mohamed

    2015-08-01

    An in situ batch fermentation technique was employed for biohydrogen, acetone, butanol, ethanol and microdiesel production from oleaginous fungal biomass using the anaerobic fermentative bacterium Clostridium acetobutylicum ATCC 824. Oleaginous fungal Cunninghamella echinulata biomass which has ability to accumulate up to 71% cellular lipid was used as the substrate carbon source. The maximum cumulative hydrogen by C. acetobutylicum ATCC 824 from crude C. echinulata biomass was 260 ml H2 l(-1), hydrogen production efficiency was 0.32 mol H2 mole(-1) glucose and the hydrogen production rate was 5.2 ml H2 h(-1). Subsequently, the produced acids (acetic and butyric acids) during acidogenesis phase are re-utilized by ABE-producing clostridia and converted into acetone, butanol, and ethanol. The total ABE produced by C. acetobutylicum ATCC 824 during batch fermentation was 3.6 g l(-1) from crude fungal biomass including acetone (1.05 g l(-1)), butanol (2.19 g l(-1)) and ethanol (0.36 g l(-1)). C. acetobutylicum ATCC 824 has ability to produce lipolytic enzymes with a specific activity 5.59 U/mg protein to hydrolyze ester containing substrates. The lipolytic potential of C. acetobutylicum ATCC 824 was used as a biocatalyst for a lipase transesterification process using the produced ethanol from ABE fermentation for microdiesel production. The fatty acid ethyl esters (microdiesel) generated from the lipase transesterification of crude C. echinulata dry mass was analyzed by GC/MS as 15.4% of total FAEEs. The gross energy content of biohydrogen, acetone, butanol, ethanol and biodiesel generated through C. acetobutylicum fermentation from crude C. echinulata dry mass was 3113.14 kJ mol(-1). These results suggest a possibility of integrating biohydrogen, acetone, butanol and ethanol production technology by C. acetobutylicum with microdiesel production from crude C. echinulata dry mass and therefore improve the feasibility and commercialization of bioenergy production

  15. Analysis of Redox Responses During TNT Transformation by Clostridium acetobutylicum ATCC 824 and Mutants Exhibiting Altered Metabolism

    DTIC Science & Technology

    2012-01-01

    relevant for bioremediation studies, and various Clostridium species have been reported to degrade TNT through alternative routes (Ahmad and Hughes 2000...REPORT Analysis of redox responses during TNT transformation by Clostridium acetobutylicum ATCC 824 and mutants exhibiting altered metabolism 14...ABSTRACT 16. SECURITY CLASSIFICATION OF: The transformation of trinitrotoluene (TNT) by several mutant strains of Clostridium acetobutylicum has been

  16. Systems-level metabolic flux profiling elucidates a complete, bifurcated tricarboxylic acid cycle in Clostridium acetobutylicum.

    PubMed

    Amador-Noguez, Daniel; Feng, Xiao-Jiang; Fan, Jing; Roquet, Nathaniel; Rabitz, Herschel; Rabinowitz, Joshua D

    2010-09-01

    Obligatory anaerobic bacteria are major contributors to the overall metabolism of soil and the human gut. The metabolic pathways of these bacteria remain, however, poorly understood. Using isotope tracers, mass spectrometry, and quantitative flux modeling, here we directly map the metabolic pathways of Clostridium acetobutylicum, a soil bacterium whose major fermentation products include the biofuels butanol and hydrogen. While genome annotation suggests the absence of most tricarboxylic acid (TCA) cycle enzymes, our results demonstrate that this bacterium has a complete, albeit bifurcated, TCA cycle; oxaloacetate flows to succinate both through citrate/alpha-ketoglutarate and via malate/fumarate. Our investigations also yielded insights into the pathways utilized for glucose catabolism and amino acid biosynthesis and revealed that the organism's one-carbon metabolism is distinct from that of model microbes, involving reversible pyruvate decarboxylation and the use of pyruvate as the one-carbon donor for biosynthetic reactions. This study represents the first in vivo characterization of the TCA cycle and central metabolism of C. acetobutylicum. Our results establish a role for the full TCA cycle in an obligatory anaerobic organism and demonstrate the importance of complementing genome annotation with isotope tracer studies for determining the metabolic pathways of diverse microbes.

  17. Spontaneous large-scale autolysis in Clostridium acetobutylicum contributes to generation of more spores

    PubMed Central

    Liu, Zhen; Qiao, Kai; Tian, Lei; Zhang, Quan; Liu, Zi-Yong; Li, Fu-Li

    2015-01-01

    Autolysis is a widespread phenomenon in bacteria. In batch fermentation of Clostridium acetobutylicum ATCC 824, there is a spontaneous large-scale autolysis phenomenon with significant decrease of cell density immediately after exponential phase. To unravel the role of autolysis, an autolysin-coding gene, CA_C0554, was disrupted by using ClosTron system to obtain the mutant C. acetobutylicum lyc::int(72). The lower final cell density and faster cell density decrease rate of C. acetobutylicum ATCC 824 than those of C. acetobutylicum lyc::int(72) indicates that CA_C0554 was an important but not the sole autolysin-coding gene responding for the large-scale autolysis. Similar glucose utilization and solvents production but obvious lower cell density of C. acetobutylicum ATCC 824 comparing to C. acetobutylicum lyc::int(72) suggests that lysed C. acetobutylicum ATCC 824 cells were metabolic inactive. On the contrary, the spore density of C. acetobutylicum ATCC 824 is 26.1% higher than that of C. acetobutylicum lyc::int(72) in the final culture broth of batch fermentation. We speculated that spontaneous autolysis of metabolic-inactive cells provided nutrients for the sporulating cells. The present study suggests that one important biological role of spontaneous large-scale autolysis in C. acetobutylicum ATCC 824 batch fermentation is contributing to generation of more spores during sporulation. PMID:26441884

  18. Spontaneous large-scale autolysis in Clostridium acetobutylicum contributes to generation of more spores.

    PubMed

    Liu, Zhen; Qiao, Kai; Tian, Lei; Zhang, Quan; Liu, Zi-Yong; Li, Fu-Li

    2015-01-01

    Autolysis is a widespread phenomenon in bacteria. In batch fermentation of Clostridium acetobutylicum ATCC 824, there is a spontaneous large-scale autolysis phenomenon with significant decrease of cell density immediately after exponential phase. To unravel the role of autolysis, an autolysin-coding gene, CA_C0554, was disrupted by using ClosTron system to obtain the mutant C. acetobutylicum lyc::int(72). The lower final cell density and faster cell density decrease rate of C. acetobutylicum ATCC 824 than those of C. acetobutylicum lyc::int(72) indicates that CA_C0554 was an important but not the sole autolysin-coding gene responding for the large-scale autolysis. Similar glucose utilization and solvents production but obvious lower cell density of C. acetobutylicum ATCC 824 comparing to C. acetobutylicum lyc::int(72) suggests that lysed C. acetobutylicum ATCC 824 cells were metabolic inactive. On the contrary, the spore density of C. acetobutylicum ATCC 824 is 26.1% higher than that of C. acetobutylicum lyc::int(72) in the final culture broth of batch fermentation. We speculated that spontaneous autolysis of metabolic-inactive cells provided nutrients for the sporulating cells. The present study suggests that one important biological role of spontaneous large-scale autolysis in C. acetobutylicum ATCC 824 batch fermentation is contributing to generation of more spores during sporulation.

  19. Systems Biology of Clostridium Acetobutylicum: Sugar Metabolism and TNT Reduction

    NASA Astrophysics Data System (ADS)

    Hurley, Margaret; Sund, Christian; Servinsky, Matthew

    2010-03-01

    Rapid advancements in biotechnology are expected to impact multiple areas of interest to the Army, including decontamination, degradation of toxic chemicals and biofuels. This project is a joint experimental/computational effort to map out the metabolic pathways in Clostridium acetobutylicum, and use this information to develop a systems biology model of this system. This organism has been chosen specifically due to the fact that it has potential application to both biofuel production and nitroaromatic degradation. It is hoped that a systems biology model may provide key information to enhance both of these processes. Details will be presented of a first-generation model of central carbon metabolism in C. Acet., developed upon gene expression data accumulated from bacteria grown on different carbohydrate sources. Additional work will discuss the effect of TNT exposure and potential relevant enhancements of the model.

  20. Elimination of carbon catabolite repression in Clostridium acetobutylicum--a journey toward simultaneous use of xylose and glucose.

    PubMed

    Bruder, Mark; Moo-Young, Murray; Chung, Duane A; Chou, C Perry

    2015-09-01

    The industrial Gram-positive anaerobe Clostridium acetobutylicum is a valued acetone, butanol, and ethanol (ABE) solvent producer that is able to utilize a vast array of carbon sources in fermentation. When glucose is present in the growth medium, however, C. acetobutylicum, like many Gram-positive organisms, exhibits biphasic growth characteristics in which glucose is used preferentially over secondary carbon sources, a phenomenon known as carbon catabolite repression (CCR). The secondary carbon source is only utilized when the supply of glucose is exhausted, resulting in inefficient use of complex carbon sources. As biofuel production is sought from cheap feedstock, attention has turned to lignocellulosic biomass. Growth of C. acetobutylicum on lignocellulose, however, can be limited by CCR. Here, we present a method to relieve the inhibitory effect of CCR and allow simultaneous utilization of the lignocellulosic sugars of glucose and xylose by C. acetobutylicum. First, we utilized an in vivo gene reporter assay to demonstrate that an identified 14-nucleotide catabolite responsive element (CRE) sequence was sufficient to introduce CCR-mediated transcriptional inhibition, while subsequent mutation of the CRE sequence relieved the inhibitory effect. Next, we demonstrated that C. acetobutylicum harboring a CRE-less plasmid-borne xylose and pentose phosphate pathway operon afforded a 7.5-fold increase in xylose utilization in the presence of glucose as compared to a wild-type CRE plasmid-borne operon, effectively overcoming native CCR effects. The methodology presented here should translate to other members of Clostridium that exhibit CCR to enable simultaneous utilization of a vast array of carbon sources.

  1. Comparative genomic and transcriptomic analysis revealed genetic characteristics related to solvent formation and xylose utilization in Clostridium acetobutylicum EA 2018

    PubMed Central

    2011-01-01

    Background Clostridium acetobutylicum, a gram-positive and spore-forming anaerobe, is a major strain for the fermentative production of acetone, butanol and ethanol. But a previously isolated hyper-butanol producing strain C. acetobutylicum EA 2018 does not produce spores and has greater capability of solvent production, especially for butanol, than the type strain C. acetobutylicum ATCC 824. Results Complete genome of C. acetobutylicum EA 2018 was sequenced using Roche 454 pyrosequencing. Genomic comparison with ATCC 824 identified many variations which may contribute to the hyper-butanol producing characteristics in the EA 2018 strain, including a total of 46 deletion sites and 26 insertion sites. In addition, transcriptomic profiling of gene expression in EA 2018 relative to that of ATCC824 revealed expression-level changes of several key genes related to solvent formation. For example, spo0A and adhEII have higher expression level, and most of the acid formation related genes have lower expression level in EA 2018. Interestingly, the results also showed that the variation in CEA_G2622 (CAC2613 in ATCC 824), a putative transcriptional regulator involved in xylose utilization, might accelerate utilization of substrate xylose. Conclusions Comparative analysis of C. acetobutylicum hyper-butanol producing strain EA 2018 and type strain ATCC 824 at both genomic and transcriptomic levels, for the first time, provides molecular-level understanding of non-sporulation, higher solvent production and enhanced xylose utilization in the mutant EA 2018. The information could be valuable for further genetic modification of C. acetobutylicum for more effective butanol production. PMID:21284892

  2. Capturing the response of Clostridium acetobutylicum to chemical stressors using a regulated genome-scale metabolic model

    DOE PAGES

    Dash, Satyakam; Mueller, Thomas J.; Venkataramanan, Keerthi P.; ...

    2014-10-14

    Clostridia are anaerobic Gram-positive Firmicutes containing broad and flexible systems for substrate utilization, which have been used successfully to produce a range of industrial compounds. Clostridium acetobutylicum has been used to produce butanol on an industrial scale through acetone-butanol-ethanol (ABE) fermentation. A genome-scale metabolic (GSM) model is a powerful tool for understanding the metabolic capacities of an organism and developing metabolic engineering strategies for strain development. The integration of stress related specific transcriptomics information with the GSM model provides opportunities for elucidating the focal points of regulation.

  3. Metabolic flexibility of a butyrate pathway mutant of Clostridium acetobutylicum.

    PubMed

    Yoo, Minyeong; Croux, Christian; Meynial-Salles, Isabelle; Soucaille, Philippe

    2017-01-31

    Clostridium acetobutylicum possesses two homologous buk genes, buk (or buk1) and buk2, which encode butyrate kinases involved in the last step of butyrate formation. To investigate the contribution of buk in detail, an in-frame deletion mutant was constructed. However, in all the Δbuk mutants obtained, partial deletions of the upstream ptb gene were observed, and low phosphotransbutyrylase and butyrate kinase activities were measured. This demonstrates that i) buk (CA_C3075) is the key butyrate kinase-encoding gene and that buk2 (CA_C1660) that is poorly transcribed only plays a minor role; and ii) strongly suggests that a Δbuk mutant is not viable if the ptb gene is not also inactivated, probably due to the accumulation of butyryl-phosphate, which might be toxic for the cell. One of the ΔbukΔptb mutants was subjected to quantitative transcriptomic (mRNA molecules/cell) and fluxomic analyses in acidogenic, solventogenic and alcohologenic chemostat cultures. In addition to the low butyrate production, drastic changes in metabolic fluxes were also observed for the mutant: i) under acidogenic conditions, the primary metabolite was butanol and a new metabolite, 2-hydroxy-valerate, was produced ii) under solventogenesis, 58% increased butanol production was obtained compared to the control strain under the same conditions, and a very high yield of butanol formation (0.3gg(-1)) was reached; and iii) under alcohologenesis, the major product was lactate. Furthermore, at the transcriptional level, adhE2, which encodes an aldehyde/alcohol dehydrogenase and is known to be a gene specifically expressed in alcohologenesis, was surprisingly highly expressed in all metabolic states in the mutant. The results presented here not only support the key roles of buk and ptb in butyrate formation but also highlight the metabolic flexibility of C. acetobutylicum in response to genetic alteration of its primary metabolism.

  4. Improving the Clostridium acetobutylicum butanol fermentation by engineering the strain for co-production of riboflavin.

    PubMed

    Cai, Xianpeng; Bennett, George N

    2011-08-01

    Solvent-producing clostridia are well known for their capacity to use a wide variety of renewable biomass and agricultural waste materials for biobutanol production. To investigate the possibility of co-production of a high value chemical during biobutanol production, the Clostridium acetobutylicum riboflavin operon ribGBAH was over-expressed in C. acetobutylicum on Escherichia coli-Clostridium shuttle vector pJIR750. Constructs that either maintained the original C. acetobutylicum translational start codon or modified the start codons of ribG and ribB from TTG to ATG were designed. Riboflavin was successfully produced in both E. coli and C. acetobutylicum using these plasmids, and riboflavin could accumulate up to 27 mg/l in Clostridium culture. Furthermore, the C. acetobutylicum purine pathway was modified by over-expression of the Clostridium purF gene, which encodes the enzyme PRPP amidotransferase. The function of the plasmid pJaF bearing C. acetobutylicum purF was verified by its ability to complement an E. coli purF mutation. However, co-production of riboflavin with biobutanol by use of the purF over-expression plasmid was not improved under the experimental conditions examined. Further rational mutation of the purF gene was conducted by replacement of amino acid codons D302 V and K325Q to make it similar to the feedback-resistant enzymes of other species. However, the co-expression of ribGBAH and purFC in C. acetobutylicum also did not improve riboflavin production. By buffering the culture pH, C. acetobutylicum ATCC 824(pJpGN) could accumulate more than 70 mg/l riboflavin while producing 190 mM butanol in static cultures. Riboflavin production was shown to exert no effect on solvent production at these levels.

  5. Enhancing Butanol Production under the Stress Environments of Co-Culturing Clostridium acetobutylicum/Saccharomyces cerevisiae Integrated with Exogenous Butyrate Addition

    PubMed Central

    Luo, Hongzhen; Ge, Laibing; Zhang, Jingshu; Zhao, Yanli; Ding, Jian; Li, Zhigang; He, Zhenni; Chen, Rui; Shi, Zhongping

    2015-01-01

    In this study, an efficient acetone-butanol-ethanol (ABE) fermentation strategy integrating Clostridium acetobutylicum/Saccharomyces cerevisiae co-culturing system with exogenous butyrate addition, was proposed and experimentally conducted. In solventogenic phase, by adding 0.2 g-DCW/L-broth viable S. cerevisiae cells and 4.0 g/L-broth concentrated butyrate solution into C. acetobutylicum culture broth, final butanol concentration and butanol/acetone ratio in a 7 L anaerobic fermentor reached the highest levels of 15.74 g/L and 2.83 respectively, with the increments of 35% and 43% as compared with those of control. Theoretical and experimental analysis revealed that, the proposed strategy could, 1) extensively induce secretion of amino acids particularly lysine, which are favorable for both C. acetobutylicum survival and butanol synthesis under high butanol concentration environment; 2) enhance the utilization ability of C. acetobutylicum on glucose and over-produce intracellular NADH for butanol synthesis in C. acetobutylicum metabolism simultaneously; 3) direct most of extra consumed glucose into butanol synthesis route. The synergetic actions of effective amino acids assimilation, high rates of substrate consumption and NADH regeneration yielded highest butanol concentration and butanol ratio in C. acetobutylicum under this stress environment. The proposed method supplies an alternative way to improve ABE fermentation performance by traditional fermentation technology. PMID:26489085

  6. Enhancing Butanol Production under the Stress Environments of Co-Culturing Clostridium acetobutylicum/Saccharomyces cerevisiae Integrated with Exogenous Butyrate Addition.

    PubMed

    Luo, Hongzhen; Ge, Laibing; Zhang, Jingshu; Zhao, Yanli; Ding, Jian; Li, Zhigang; He, Zhenni; Chen, Rui; Shi, Zhongping

    2015-01-01

    In this study, an efficient acetone-butanol-ethanol (ABE) fermentation strategy integrating Clostridium acetobutylicum/Saccharomyces cerevisiae co-culturing system with exogenous butyrate addition, was proposed and experimentally conducted. In solventogenic phase, by adding 0.2 g-DCW/L-broth viable S. cerevisiae cells and 4.0 g/L-broth concentrated butyrate solution into C. acetobutylicum culture broth, final butanol concentration and butanol/acetone ratio in a 7 L anaerobic fermentor reached the highest levels of 15.74 g/L and 2.83 respectively, with the increments of 35% and 43% as compared with those of control. Theoretical and experimental analysis revealed that, the proposed strategy could, 1) extensively induce secretion of amino acids particularly lysine, which are favorable for both C. acetobutylicum survival and butanol synthesis under high butanol concentration environment; 2) enhance the utilization ability of C. acetobutylicum on glucose and over-produce intracellular NADH for butanol synthesis in C. acetobutylicum metabolism simultaneously; 3) direct most of extra consumed glucose into butanol synthesis route. The synergetic actions of effective amino acids assimilation, high rates of substrate consumption and NADH regeneration yielded highest butanol concentration and butanol ratio in C. acetobutylicum under this stress environment. The proposed method supplies an alternative way to improve ABE fermentation performance by traditional fermentation technology.

  7. Sequences affecting the regulation of solvent production in Clostridium acetobutylicum.

    PubMed

    Scotcher, Miles C; Huang, Ke-xue; Harrison, Mary L; Rudolph, Frederick B; Bennett, George N

    2003-07-01

    The high solvent phenotype of Clostridium acetobutylicum mutants B and H was complemented by the introduction of a plasmid that contains either an intact or partially-deleted copy of solR, restoring acetone and butanol production to wild-type levels. This demonstrates that the solR open reading frame on pSOLThi is not required to restore solvent levels. The promoter region upstream of alcohol dehydrogense E (adhE) was examined in efforts to identify sites that play major roles in the control of expression. A series of adhE promoter fragments was constructed and the expression of each in acid- and solvent-phases of growth was analyzed using a chloramphenicol acetyl-transferase reporter system. Our results show that a region beyond the 0A box is needed for full induction of the promoter. Additionally, we show that the presence of sequences around a possible processing site designated S2 may have a negative role in the regulation of adhE expression.

  8. Control of butanol formation in Clostridium acetobutylicum by transcriptional activation.

    PubMed

    Thormann, Kai; Feustel, Lothar; Lorenz, Karin; Nakotte, Stephan; Dürre, Peter

    2002-04-01

    The sol operon of Clostridium acetobutylicum is the essential transcription unit for formation of the solvents butanol and acetone. The recent proposal that transcriptional regulation of this operon is controlled by the repressor Orf5/SolR (R. V. Nair, E. M. Green, D. E. Watson, G. N. Bennett, and E. T. Papoutsakis, J. Bacteriol. 181:319-330, 1999) was found to be incorrect. Instead, regulation depends on activation, most probably by the multivalent transcription factor Spo0A. The operon is transcribed from a single promoter. A second signal identified in primer extension studies results from mRNA processing and can be observed only in the natural host, not in a heterologous host. The first structural gene in the operon (adhE, encoding a bifunctional butyraldehyde/butanol dehydrogenase) is translated into two different proteins, the mature AdhE enzyme and the separate butanol dehydrogenase domain. The promoter of the sol operon is preceded by three imperfect repeats and a putative Spo0A-binding motif, which partially overlaps with repeat 3 (R3). Reporter gene analysis performed with the lacZ gene of Thermoanaerobacterium thermosulfurigenes and targeted mutations of the regulatory region revealed that the putative Spo0A-binding motif, R3, and R1 are essential for control. The data obtained also indicate that an additional activator protein is involved.

  9. Microbiological production of acetone-butanol by Clostridium acetobutylicum.

    PubMed

    Abou-Zeid, A A; Fouad, M; Yassein, M

    1978-01-01

    Trials succeeded in raising the efficiencies of the fermentation medium, used in the fermentative production of acetone-butanol by Clostridium acetobutylicum. Egyptian black strap molasses (50.0% sugars) was suitable as carbon source in the fermentation medium, and (NH4)2SO4 was utilized with great success as inorganic nitrogen source. 140.0 g/l black strap molasses (about 7.0% sugars) and 3.0 g/l (NH4)2SO4 were the optimum concentrations for obtaining good yields of acetone and butanol. Molasses and (NH4)2SO4 were preferred because they are cheaper than the other carbon and organic nitrogen sources, used in the fermentative production of acetone-butanol. The percentage increase of the total solvents produced in the fermentation (production medium) was increased by 64.0. The slop (by-product of the acetone-butanol fermentation after distillation) was re-used in the fermentation medium as organic nitrogen source and supported the microorganisms for a good production of acetone and butanol, while when stillage was used in the production medium, the total solvents output was less than that produced in the medium containing slop.

  10. CRISPR-based genome editing and expression control systems in Clostridium acetobutylicum and Clostridium beijerinckii.

    PubMed

    Li, Qi; Chen, Jun; Minton, Nigel P; Zhang, Ying; Wen, Zhiqiang; Liu, Jinle; Yang, Haifeng; Zeng, Zhe; Ren, Xiaodan; Yang, Junjie; Gu, Yang; Jiang, Weihong; Jiang, Yu; Yang, Sheng

    2016-07-01

    Solventogenic clostridia are important industrial microorganisms that produce various chemicals and fuels. Effective genetic tools would facilitate physiological studies aimed both at improving our understanding of metabolism and optimizing solvent productivity through metabolic engineering. Here we have developed an all-in-one, CRISPR-based genome editing plasmid, pNICKclos, that can be used to achieve successive rounds of gene editing in Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii NCIMB 8052 with efficiencies varying from 6.7% to 100% and 18.8% to 100%, respectively. The plasmid specifies the requisite target-specific guide RNA, the gene encoding the Streptococcus pyogenes Cas9 nickase and the genome editing template encompassing the gene-specific homology arms. It can be used to create single target mutants within three days, with a further two days required for the curing of the pNICKclos plasmid ready for a second round of mutagenesis. A S. pyogenes dCas9-mediated gene regulation control system, pdCASclos, was also developed and used in a CRISPRi strategy to successfully repress the expression of spo0A in C. acetobutylicum and C. beijerinckii. The combined application of the established high efficiency CRISPR-Cas9 based genome editing and regulation control systems will greatly accelerate future progress in the understanding and manipulation of metabolism in solventogenic clostridia. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Orf5/SolR: a transcriptional repressor of the sol operon of Clostridium acetobutylicum?

    PubMed

    Thormann, K; Dürre, P

    2001-11-01

    The gene of Orf5 (SolR) of Clostridium acetobutylicum DSM 792 was subcloned and overexpressed in Escherichia coli. The protein was purified with Ni-NTA agarose and used for DNA binding assays. No DNA binding of Orf5 to regions upstream of the sol operon from C. acetobutylicum was observed. Overexpression of Orf5 in C. acetobutylicum led to a change in the organism's pattern of glycosylated exoproteins. The Orf5 protein was localized in the cell membrane fraction and to a small extent in the supernatant medium. Based on these results Orf5 (SolR) appears not to act as a transcriptional repressor in C. acetobutylicum, but instead may be an enzyme involved in glycosylation or deglycosylation.

  12. Predictive modeling in Clostridium acetobutylicum fermentations employing Raman spectroscopy and multivariate data analysis for real-time culture monitoring

    NASA Astrophysics Data System (ADS)

    Zu, Theresah N. K.; Liu, Sanchao; Germane, Katherine L.; Servinsky, Matthew D.; Gerlach, Elliot S.; Mackie, David M.; Sund, Christian J.

    2016-05-01

    The coupling of optical fibers with Raman instrumentation has proven to be effective for real-time monitoring of chemical reactions and fermentations when combined with multivariate statistical data analysis. Raman spectroscopy is relatively fast, with little interference from the water peak present in fermentation media. Medical research has explored this technique for analysis of mammalian cultures for potential diagnosis of some cancers. Other organisms studied via this route include Escherichia coli, Saccharomyces cerevisiae, and some Bacillus sp., though very little work has been performed on Clostridium acetobutylicum cultures. C. acetobutylicum is a gram-positive anaerobic bacterium, which is highly sought after due to its ability to use a broad spectrum of substrates and produce useful byproducts through the well-known Acetone-Butanol-Ethanol (ABE) fermentation. In this work, real-time Raman data was acquired from C. acetobutylicum cultures grown on glucose. Samples were collected concurrently for comparative off-line product analysis. Partial-least squares (PLS) models were built both for agitated cultures and for static cultures from both datasets. Media components and metabolites monitored include glucose, butyric acid, acetic acid, and butanol. Models were cross-validated with independent datasets. Experiments with agitation were more favorable for modeling with goodness of fit (QY) values of 0.99 and goodness of prediction (Q2Y) values of 0.98. Static experiments did not model as well as agitated experiments. Raman results showed the static experiments were chaotic, especially during and shortly after manual sampling.

  13. Direct selection of Clostridium acetobutylicum fermentation mutants by a proton suicide method

    SciTech Connect

    Cueto, P.H.; Mendez, B.S. )

    1990-02-01

    Clostridium acetobutylicum ATCC 10132 mutants altered in acetic acid synthesis or in the shift to solventogenesis were directly selected by a proton suicide method after mutagenic treatment, by using bromide and bromate as selective agents. The mutants were characterized according to their solvent and acid production. On the selection plates they differed in colony phenotype from the parent strain.

  14. Direct Selection of Clostridium acetobutylicum Fermentation Mutants by a Proton Suicide Method.

    PubMed

    Cueto, P H; Méndez, B S

    1990-02-01

    Clostridium acetobutylicum ATCC 10132 mutants altered in acetic acid synthesis or in the shift to solventogenesis were directly selected by a proton suicide method after mutagenic treatment, by using bromide and bromate as selective agents. The mutants were characterized according to their solvent and acid production. On the selection plates they differed in colony phenotype from the parent strain.

  15. Direct Selection of Clostridium acetobutylicum Fermentation Mutants by a Proton Suicide Method

    PubMed Central

    Cueto, Pablo H.; Méndez, Beatriz S.

    1990-01-01

    Clostridium acetobutylicum ATCC 10132 mutants altered in acetic acid synthesis or in the shift to solventogenesis were directly selected by a proton suicide method after mutagenic treatment, by using bromide and bromate as selective agents. The mutants were characterized according to their solvent and acid production. On the selection plates they differed in colony phenotype from the parent strain. PMID:16348133

  16. Antisense RNA Strategies for Metabolic Engineering of Clostridium acetobutylicum

    PubMed Central

    Desai, Ruchir P.; Papoutsakis, Eleftherios T.

    1999-01-01

    We examined the effectiveness of antisense RNA (as RNA) strategies for metabolic engineering of Clostridium acetobutylicum. Strain ATCC 824(pRD4) was developed to produce a 102-nucleotide asRNA with 87% complementarity to the butyrate kinase (BK) gene. Strain ATCC 824(pRD4) exhibited 85 to 90% lower BK and acetate kinase specific activities than the control strain. Strain ATCC 824(pRD4) also exhibited 45 to 50% lower phosphotransbutyrylase (PTB) and phosphotransacetylase specific activities than the control strain. This strain exhibited earlier induction of solventogenesis, which resulted in 50 and 35% higher final concentrations of acetone and butanol, respectively, than the concentrations in the control. Strain ATCC 824(pRD1) was developed to putatively produce a 698-nucleotide asRNA with 96% complementarity to the PTB gene. Strain ATCC 824(pRD1) exhibited 70 and 80% lower PTB and BK activities, respectively, than the control exhibited. It also exhibited 300% higher levels of a lactate dehydrogenase activity than the control exhibited. The growth yields of ATCC 824(pRD1) were 28% less than the growth yields of the control. While the levels of acids were not affected in ATCC 824(pRD1) fermentations, the acetone and butanol concentrations were 96 and 75% lower, respectively, than the concentrations in the control fermentations. The lower level of solvent production by ATCC 824(pRD1) was compensated for by ∼100-fold higher levels of lactate production. The lack of any significant impact on butyrate formation fluxes by the lower PTB and BK levels suggests that butyrate formation fluxes are not controlled by the levels of the butyrate formation enzymes. PMID:10049845

  17. A systems biology approach to investigate the effect of pH-induced gene regulation on solvent production by Clostridium acetobutylicum in continuous culture.

    PubMed

    Haus, Sylvia; Jabbari, Sara; Millat, Thomas; Janssen, Holger; Fischer, Ralf-Jörg; Bahl, Hubert; King, John R; Wolkenhauer, Olaf

    2011-01-19

    Clostridium acetobutylicum is an anaerobic bacterium which is known for its solvent-producing capabilities, namely regarding the bulk chemicals acetone and butanol, the latter being a highly efficient biofuel. For butanol production by C. acetobutylicum to be optimized and exploited on an industrial scale, the effect of pH-induced gene regulation on solvent production by C. acetobutylicum in continuous culture must be understood as fully as possible. We present an ordinary differential equation model combining the metabolic network governing solvent production with regulation at the genetic level of the enzymes required for this process. Parameterizing the model with experimental data from continuous culture, we demonstrate the influence of pH upon fermentation products: at high pH (pH 5.7) acids are the dominant product while at low pH (pH 4.5) this switches to solvents. Through steady-state analyses of the model we focus our investigations on how alteration in gene expression of C. acetobutylicum could be exploited to increase butanol yield in a continuous culture fermentation. Incorporating gene regulation into the model of solvent production by C. acetobutylicum enables an accurate representation of the pH-induced switch to solvent production to be obtained and theoretical investigations of possible synthetic-biology approaches to be pursued. Steady-state analyses suggest that, to increase butanol yield, alterations in the expression of single solvent-associated genes are insufficient; a more complex approach targeting two or more genes is required.

  18. Production of 1,3-propanediol from glycerol by Clostridium acetobutylicum and other Clostridium species

    SciTech Connect

    Forsberg, C.W.

    1987-04-01

    Glycerol was fermented with the production of 1,3-propanediol as the major fermentation product by four strains of Clostridium acetobutylicum, six of C. butylicum, two of C. beijerinckii, one of C. kainantoi, and three of C. butylicum. 1,3-Propanediol was identified by its retention times in gas chromatography and high-pressure liquid chromatography and by its mass spectrum. During growth of C. butylicum B593 in a chemostat culture at pH 6.5, 61% of the glycerol fermented was converted to 1,3-propanediol. When the pH was decreased to 4.9, growth and 1,3-propanediol production were substantially reduced.

  19. Pleiotropic functions of catabolite control protein CcpA in Butanol-producing Clostridium acetobutylicum

    PubMed Central

    2012-01-01

    Background Clostridium acetobutylicum has been used to produce butanol in industry. Catabolite control protein A (CcpA), known to mediate carbon catabolite repression (CCR) in low GC gram-positive bacteria, has been identified and characterized in C. acetobutylicum by our previous work (Ren, C. et al. 2010, Metab Eng 12:446–54). To further dissect its regulatory function in C. acetobutylicum, CcpA was investigated using DNA microarray followed by phenotypic, genetic and biochemical validation. Results CcpA controls not only genes in carbon metabolism, but also those genes in solvent production and sporulation of the life cycle in C. acetobutylicum: i) CcpA directly repressed transcription of genes related to transport and metabolism of non-preferred carbon sources such as d-xylose and l-arabinose, and activated expression of genes responsible for d-glucose PTS system; ii) CcpA is involved in positive regulation of the key solventogenic operon sol (adhE1-ctfA-ctfB) and negative regulation of acidogenic gene bukII; and iii) transcriptional alterations were observed for several sporulation-related genes upon ccpA inactivation, which may account for the lower sporulation efficiency in the mutant, suggesting CcpA may be necessary for efficient sporulation of C. acetobutylicum, an important trait adversely affecting the solvent productivity. Conclusions This study provided insights to the pleiotropic functions that CcpA displayed in butanol-producing C. acetobutylicum. The information could be valuable for further dissecting its pleiotropic regulatory mechanism in C. acetobutylicum, and for genetic modification in order to obtain more effective butanol-producing Clostridium strains. PMID:22846451

  20. Pleiotropic functions of catabolite control protein CcpA in Butanol-producing Clostridium acetobutylicum.

    PubMed

    Ren, Cong; Gu, Yang; Wu, Yan; Zhang, Weiwen; Yang, Chen; Yang, Sheng; Jiang, Weihong

    2012-07-30

    Clostridium acetobutylicum has been used to produce butanol in industry. Catabolite control protein A (CcpA), known to mediate carbon catabolite repression (CCR) in low GC gram-positive bacteria, has been identified and characterized in C. acetobutylicum by our previous work (Ren, C. et al. 2010, Metab Eng 12:446-54). To further dissect its regulatory function in C. acetobutylicum, CcpA was investigated using DNA microarray followed by phenotypic, genetic and biochemical validation. CcpA controls not only genes in carbon metabolism, but also those genes in solvent production and sporulation of the life cycle in C. acetobutylicum: i) CcpA directly repressed transcription of genes related to transport and metabolism of non-preferred carbon sources such as d-xylose and l-arabinose, and activated expression of genes responsible for d-glucose PTS system; ii) CcpA is involved in positive regulation of the key solventogenic operon sol (adhE1-ctfA-ctfB) and negative regulation of acidogenic gene bukII; and iii) transcriptional alterations were observed for several sporulation-related genes upon ccpA inactivation, which may account for the lower sporulation efficiency in the mutant, suggesting CcpA may be necessary for efficient sporulation of C. acetobutylicum, an important trait adversely affecting the solvent productivity. This study provided insights to the pleiotropic functions that CcpA displayed in butanol-producing C. acetobutylicum. The information could be valuable for further dissecting its pleiotropic regulatory mechanism in C. acetobutylicum, and for genetic modification in order to obtain more effective butanol-producing Clostridium strains.

  1. pH-induced gene regulation of solvent production by Clostridium acetobutylicum in continuous culture: Parameter estimation and sporulation modelling

    PubMed Central

    Thorn, Graeme J.; King, John R.; Jabbari, Sara

    2013-01-01

    The acetone–butanol (AB) fermentation process in the anaerobic endospore-forming Gram-positive bacterium Clostridium acetobutylicum is useful as a producer of biofuels, particularly butanol. Recent work has concentrated on trying to improve the efficiency of the fermentation method, either through changes in the environmental conditions or by modifying the genome to selectively favour the production of one particular solvent over others. Fermentation of glucose by C. acetobutylicum occurs in two stages: initially the acids acetate and butyrate are produced and excreted and then, as the external pH falls, acetate and butyrate are ingested and further metabolised into the solvents acetone, butanol and ethanol. In order to optimise butanol production, it is important to understand how pH affects the enzyme-controlled reactions in the metabolism process. We adapt an ordinary differential equation model of the metabolic network with regulation at the genetic level for the required enzymes; parametrising the model using experimental data generated from continuous culture, we improve on previous point predictions (S. Haus, S. Jabbari, T. Millat, H. Janssen, R.-J. Fisher, H. Bahl, J. R. King, O. Wolkenhauer, A systems biology approach to investigate the effect of pH-induced gene regulation on solvent production by Clostridium acetobutylicum in continuous culture, BMC Systems Biology 5 (2011)) [1] both by using a different optimisation approach and by computing confidence intervals and correlation coefficients. We find in particular that the parameters are ill-determined from the data and that two separate clusters of parameters appear correlated, reflecting the importance of two metabolic intermediates. We extend the model further to include another aspect of the clostridial survival mechanism, sporulation, and by computation of the Akaike Information Criterion values find that the there is some evidence for the presence of sporulation during the shift. PMID:23201580

  2. pH-induced gene regulation of solvent production by Clostridium acetobutylicum in continuous culture: parameter estimation and sporulation modelling.

    PubMed

    Thorn, Graeme J; King, John R; Jabbari, Sara

    2013-02-01

    The acetone-butanol (AB) fermentation process in the anaerobic endospore-forming Gram-positive bacterium Clostridium acetobutylicum is useful as a producer of biofuels, particularly butanol. Recent work has concentrated on trying to improve the efficiency of the fermentation method, either through changes in the environmental conditions or by modifying the genome to selectively favour the production of one particular solvent over others. Fermentation of glucose by C. acetobutylicum occurs in two stages: initially the acids acetate and butyrate are produced and excreted and then, as the external pH falls, acetate and butyrate are ingested and further metabolised into the solvents acetone, butanol and ethanol. In order to optimise butanol production, it is important to understand how pH affects the enzyme-controlled reactions in the metabolism process. We adapt an ordinary differential equation model of the metabolic network with regulation at the genetic level for the required enzymes; parametrising the model using experimental data generated from continuous culture, we improve on previous point predictions (S. Haus, S. Jabbari, T. Millat, H. Janssen, R.-J. Fisher, H. Bahl, J. R. King, O. Wolkenhauer, A systems biology approach to investigate the effect of pH-induced gene regulation on solvent production by Clostridium acetobutylicum in continuous culture, BMC Systems Biology 5 (2011)) [1] both by using a different optimisation approach and by computing confidence intervals and correlation coefficients. We find in particular that the parameters are ill-determined from the data and that two separate clusters of parameters appear correlated, reflecting the importance of two metabolic intermediates. We extend the model further to include another aspect of the clostridial survival mechanism, sporulation, and by computation of the Akaike Information Criterion values find that the there is some evidence for the presence of sporulation during the shift. Copyright © 2012

  3. Regulation and localization of amylolytic enzymes in Clostridium acetobutylicum ATCC 824

    SciTech Connect

    Annous, B.A.; Blaschek, H.P. )

    1990-08-01

    Amylolytic activity was primarily cell associated when Clostridium acetobutylicum was grown on glucose or maltose and primarily extracellular when grown on dextrin or starch. Total amylolytic activity decreased with increasing glucose concentration. When this microorganism was grown in P2 medium containing starch, the intracellular amylolytic activity was 90% membrane bound and 10% cytoplasmic in nature. The addition of 1% glucose to 2% starch-based P2 medium at different stages of growth indicated that this carbohydrate repressed the synthesis of C. acetobutylicum amylolytic enzymes at the level of transcription.

  4. Workflow for quantitative proteomic analysis of Clostridium acetobutylicum ATCC 824 using iTRAQ tags.

    PubMed

    Hou, Shuyu; Jones, Shawn W; Choe, Leila H; Papoutsakis, Eleftherios T; Lee, Kelvin H

    2013-06-15

    Clostridium acetobutylicum (Cac) is an anaerobic, endospore-forming, Gram-positive bacterium with tremendous promise for use as a biocatalyst for the production of fuels and solvents. Cac proteomic sample preparation for shotgun analysis typically involves a multitude of reagents for harsh lysis conditions and to maintain protein solubility. We describe a protein extraction and preparation method for Cac that is compatible with proteomic shotgun analysis using isobaric labeling approaches. The method is applied to the analysis of Cac grown under butanol stress and labeled using iTRAQ 4-plex reagents. This method relies on the use of calcium carbonate to facilitate lysis by sonication and a commercially available kit to remove detergents prior to labeling. This workflow resulted in the identification and quantitation of 566 unique proteins using ProteinPilot software with a false discovery rate of 0.01% for peptide matches and 0.70% for protein matches. Ninety-five proteins were found to have statistically higher expression levels in butanol-stressed Cac as compared to non-stressed Cac. Sixty-one proteins were found to have statistically lower expression levels in stressed versus non-stressed cells. This method may be applicable to other Gram-positive organisms. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Fermentation of oxidized hexose derivatives by Clostridium acetobutylicum.

    PubMed

    Servinsky, Matthew D; Liu, Sanchao; Gerlach, Elliot S; Germane, Katherine L; Sund, Christian J

    2014-09-18

    Clostridium acetobutylicum fermentations are promising for production of commodity chemicals from heterogeneous biomass due to the wide range of substrates the organism can metabolize. Much work has been done to elucidate the pathways for utilization of aldoses, but little is known about metabolism of more oxidized substrates. Two oxidized hexose derivatives, gluconate and galacturonate, are present in low cost feedstocks, and their metabolism will contribute to overall metabolic output of these substrates. A complete metabolic network for glucose, gluconate, and galacturonate utilization was generated using online databases, previous studies, genomic context, and experimental data. Gluconate appears to be metabolized via the Entner-Doudoroff pathway, and is likely dehydrated to 2-keto-3-deoxy-gluconate before phosphorylation to 2-keto-3-deoxy-6-P-gluconate. Galacturonate appears to be processed via the Ashwell pathway, converging on a common metabolite for gluconate and galacturonate metabolism, 2-keto-3-deoxygluconate. As expected, increasingly oxidized substrates resulted in increasingly oxidized products with galacturonate fermentations being nearly homoacetic. Calculations of expected ATP and reducing equivalent yields and experimental data suggested galacturonate fermentations were reductant limited. Galacturonate fermentation was incomplete, which was not due solely to product inhibition or the inability to utilize low concentrations of galacturonate. Removal of H2 and CO2 by agitation resulted in faster growth, higher cell densities, formation of relatively more oxidized products, and higher product yields for cultures grown on glucose or gluconate. In contrast, cells grown on galacturonate showed reduced growth rates upon agitation, which was likely due to loss in reductant in the form of H2. The growth advantage seen on agitated glucose or gluconate cultures could not be solely attributed to improved ATP economics, thereby indicating other factors are

  6. Cap0037, a Novel Global Regulator of Clostridium acetobutylicum Metabolism

    PubMed Central

    Nguyen, Ngoc-Phuong-Thao; Linder, Sonja; Flitsch, Stefanie K.; Schiel-Bengelsdorf, Bettina; Dürre, Peter

    2016-01-01

    ABSTRACT An operon comprising two genes, CA_P0037 and CA_P0036, that encode proteins of unknown function that were previously shown to be highly expressed in acidogenic cells and repressed in solventogenic and alcohologenic cells is located on the pSOL1 megaplasmid of Clostridium acetobutylicum upstream of adhE2. A CA_P0037::int (189/190s) mutant in which an intron was inserted at position 189/190 in the sense strand of CA_P0037 was successfully generated by the Targetron technique. The resultant mutant showed significantly different metabolic flux patterns in acidogenic (producing mainly lactate, butyrate, and butanol) and alcohologenic (producing mainly butyrate, acetate, and lactate) chemostat cultures but not in solventogenic or batch cultures. Transcriptomic investigation of the CA_P0037::int (189/190s) mutant showed that inactivation of CA_P0037 significantly affected the expression of more than 258 genes under acidogenic conditions. Surprisingly, genes belonging to the Fur regulon, involved in iron transport (CA_C1029-CA_C1032), or coding for the main flavodoxin (CA_C0587) were the most significantly expressed genes under all conditions, whereas fur (coding for the ferric uptake regulator) gene expression remained unchanged. Furthermore, most of the genes of the Rex regulon, such as the adhE2 and ldhA genes, and of the PerR regulon, such as rbr3A-rbr3B and dfx, were overexpressed in the mutant. In addition, the whole CA_P0037-CA_P0036 operon was highly expressed under all conditions in the CA_P0037::int (189/190s) mutant, suggesting a self-regulated expression mechanism. Cap0037 was shown to bind to the CA_P0037-CA_P0036 operon, sol operon, and adc promoters, and the binding sites were determined by DNA footprinting. Finally, a putative Cap0037 regulon was generated using a bioinformatic approach. PMID:27703070

  7. 13C metabolic flux analysis in Clostridium acetobutylicum during growth on L-arabinose

    NASA Astrophysics Data System (ADS)

    Hurley, Margaret; Sund, Christian; Liu, Sanchao; Germane, Katherine; Servinsky, Matthew; Gerlach, Elliot

    2015-03-01

    Clostridium acetobutylicum's metabolic pathways have been studied for decades due to its metabolic diversity and industrial value, yet many details of its metabolism are continuing to emerge. To elucidate the role of xylulose-5-P/fructose-6-P phosphoketolase (XFP), and the recently discovered Pentose Phosphate Pathway (PKP) in C. acetobutylicum, experimental and computational metabolic isotope analysis was performed under growth on glucose, xylose, and arabinose. Results indicate that PKP utilization increased with increasing xylose concentration and this trend was further pronounced during growth on arabinose. This was confirmed by mutation of the gene encoding XFP, which almost completely abolished flux through the PKP during growth on arabinose and resulted in decreased acetate:butyrate ratios. We discuss these experimental and computational results here, and the implications for our understanding of sugar metabolism in C. acetobutylicum.

  8. Biological butanol production from microalgae-based biodiesel residues by Clostridium acetobutylicum.

    PubMed

    Cheng, Hai-Hsuan; Whang, Liang-Ming; Chan, Kun-Chi; Chung, Man-Chien; Wu, Shu-Hsien; Liu, Cheng-Pin; Tien, Shih-Yuan; Chen, Shan-Yuan; Chang, Jo-Shu; Lee, Wen-Jhy

    2015-05-01

    This study conducted batch experiments to evaluate the potential of butanol production from microalgae biodiesel residues by Clostridium acetobutylicum. The results indicated that with 90 g/L of glucose as the sole substrate the highest butanol yield of 0.2 g/g-glucose was found, but the addition of butyrate significantly enhanced the butanol yield. The highest butanol yield of 0.4 g/g-glucose was found with 60 g/L of glucose and 18 g/L of butyrate. Using microalgae biodiesel residues as substrate, C. acetobutylicum produced 3.86 g/L of butanol and achieved butanol yield of 0.13 g/g-carbohydrate via ABE fermentation, but the results indicated that approximately one third of carbohydrate was not utilized by C. acetobutylicum. Biological butanol production from microalgae biodiesel residues can be possible, but further research on fermentation strategies are required to improve production yield.

  9. Comparative shotgun proteomic analysis of Clostridium acetobutylicum from butanol fermentation using glucose and xylose

    SciTech Connect

    Sivagnanam, Kumaran; Raghavan, Vijaya G. S.; Shah, Manesh B; Hettich, Robert {Bob} L; Verberkmoes, Nathan C; Lefsrud, Mark G

    2011-01-01

    Background: Butanol is a second generation biofuel produced by Clostridium acetobutylicum through acetonebutanol- ethanol (ABE) fermentation process. Shotgun proteomics provides a direct approach to study the whole proteome of an organism in depth. This paper focuses on shotgun proteomic profiling of C. acetobutylicum from ABE fermentation using glucose and xylose to understand the functional mechanisms of C. acetobutylicum proteins involved in butanol production. Results: We identified 894 different proteins in C. acetobutylicum from ABE fermentation process by two dimensional - liquid chromatography - tandem mass spectrometry (2D-LC-MS/MS) method. This includes 717 proteins from glucose and 826 proteins from the xylose substrate. A total of 649 proteins were found to be common and 22 significantly differentially expressed proteins were identified between glucose and xylose substrates. Conclusion: Our results demonstrate that flagellar proteins are highly up-regulated with glucose compared to xylose substrate during ABE fermentation. Chemotactic activity was also found to be lost with the xylose substrate due to the absence of CheW and CheV proteins. This is the first report on the shotgun proteomic analysis of C. acetobutylicum ATCC 824 in ABE fermentation between glucose and xylose substrate from a single time data point and the number of proteins identified here is more than any other study performed on this organism up to this report.

  10. Genome Sequence and Comparative Analysis of the Solvent-Producing Bacterium Clostridium acetobutylicum

    PubMed Central

    Nölling, Jörk; Breton, Gary; Omelchenko, Marina V.; Makarova, Kira S.; Zeng, Qiandong; Gibson, Rene; Lee, Hong Mei; Dubois, JoAnn; Qiu, Dayong; Hitti, Joseph; Wolf, Yuri I.; Tatusov, Roman L.; Sabathe, Fabrice; Doucette-Stamm, Lynn; Soucaille, Philippe; Daly, Michael J.; Bennett, George N.; Koonin, Eugene V.; Smith, Douglas R.

    2001-01-01

    The genome sequence of the solvent-producing bacterium Clostridium acetobutylicum ATCC 824 has been determined by the shotgun approach. The genome consists of a 3.94-Mb chromosome and a 192-kb megaplasmid that contains the majority of genes responsible for solvent production. Comparison of C. acetobutylicum to Bacillus subtilis reveals significant local conservation of gene order, which has not been seen in comparisons of other genomes with similar, or, in some cases closer, phylogenetic proximity. This conservation allows the prediction of many previously undetected operons in both bacteria. However, the C. acetobutylicum genome also contains a significant number of predicted operons that are shared with distantly related bacteria and archaea but not with B. subtilis. Phylogenetic analysis is compatible with the dissemination of such operons by horizontal transfer. The enzymes of the solventogenesis pathway and of the cellulosome of C. acetobutylicum comprise a new set of metabolic capacities not previously represented in the collection of complete genomes. These enzymes show a complex pattern of evolutionary affinities, emphasizing the role of lateral gene exchange in the evolution of the unique metabolic profile of the bacterium. Many of the sporulation genes identified in B. subtilis are missing in C. acetobutylicum, which suggests major differences in the sporulation process. Thus, comparative analysis reveals both significant conservation of the genome organization and pronounced differences in many systems that reflect unique adaptive strategies of the two gram-positive bacteria. PMID:11466286

  11. Comparative transcriptome analysis between csrA-disruption Clostridium acetobutylicum and its parent strain.

    PubMed

    Tan, Yang; Liu, Zi-Yong; Liu, Zhen; Zheng, Hua-Jun; Li, Fu-Li

    2015-05-01

    The genome of Clostridium acetobutylicum contains the gene encoding CsrA, a carbon storage regulator. We investigated the function of CsrA in C. acetobutylicum by insertionally inactivating the encoding gene, CA_C2209 using the ClosTron. Disruption of csrA obviously decreases the growth of the organism and reduces the yield of acetone, butanol and ethanol (ABEs). Like the csrA in Escherichia coli, RNA-seq and β-galactosidase analysis revealed that csrA in C. acetobutylicum was closely involved in regulating multiple pathways including flagella assembly, oligopeptide transporting, iron uptake, and central carbon metabolism. It has also been newly demonstrated that csrA in C. acetobutylicum is related to the regulation of pathways involved in the phosphotransferase transporting systems, synthesis of riboflavin, and stage III sporulation. This research represented the first investigation of global regulation by CsrA in the strain belonging to Gram-positive bacteria through transcriptome analysis and provided the important theoretical evidence for improving solvent production by transcriptor engineering in C. acetobutylicum.

  12. Comparative shotgun proteomic analysis of Clostridium acetobutylicum from butanol fermentation using glucose and xylose

    PubMed Central

    2011-01-01

    Background Butanol is a second generation biofuel produced by Clostridium acetobutylicum through acetone-butanol-ethanol (ABE) fermentation process. Shotgun proteomics provides a direct approach to study the whole proteome of an organism in depth. This paper focuses on shotgun proteomic profiling of C. acetobutylicum from ABE fermentation using glucose and xylose to understand the functional mechanisms of C. acetobutylicum proteins involved in butanol production. Results We identified 894 different proteins in C. acetobutylicum from ABE fermentation process by two dimensional - liquid chromatography - tandem mass spectrometry (2D-LC-MS/MS) method. This includes 717 proteins from glucose and 826 proteins from the xylose substrate. A total of 649 proteins were found to be common and 22 significantly differentially expressed proteins were identified between glucose and xylose substrates. Conclusion Our results demonstrate that flagellar proteins are highly up-regulated with glucose compared to xylose substrate during ABE fermentation. Chemotactic activity was also found to be lost with the xylose substrate due to the absence of CheW and CheV proteins. This is the first report on the shotgun proteomic analysis of C. acetobutylicum ATCC 824 in ABE fermentation between glucose and xylose substrate from a single time data point and the number of proteins identified here is more than any other study performed on this organism up to this report. PMID:22008648

  13. Expression of the Klebsiella pneumoniae CG21 acetoin reductase gene in Clostridium acetobutylicum ATCC 824.

    PubMed

    Wardwell, S A; Yang, Y T; Chang, H Y; San, K Y; Rudolph, F B; Bennett, G N

    2001-10-01

    Acetoin reductase catalyzes the production of 2,3-butanediol from acetoin. The gene encoding the acetoin reductase of Klebsiella pneumoniae CG21 was cloned and expressed in Escherichia coli and Clostridium acetobutylicum ATCC 824. The nucleotide sequence of the gene encoding the enzyme was determined to be 768 bp long. Expression of the K. pneumoniae acetoin reductase gene in E. coli revealed that the enzyme has a molecular mass of about 31,000 Da based on sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The K. pneumoniae acetoin reductase gene was cloned into a clostridial/E. coli shuttle vector, and expression of the gene resulted in detectable levels of acetoin reductase activity in both E. coli and C. acetobutylicum. While acetoin, the natural substrate of acetoin reductase, is a typical product of fermentation by C. acetobutylicum, 2,3-butanediol is not. Analysis of culture supernatants by gas chromatography revealed that introduction of the K. pneumoniae acetoin reductase gene into C. acetobutylicum was not sufficient for 2,3-butanediol production even though the cultures were producing acetoin. 2,3-Butanediol was produced by cultures of C. acetobutylicum containing the gene only when commercial acetoin was added.

  14. Redox-responsive repressor Rex modulates alcohol production and oxidative stress tolerance in Clostridium acetobutylicum.

    PubMed

    Zhang, Lei; Nie, Xiaoqun; Ravcheev, Dmitry A; Rodionov, Dmitry A; Sheng, Jia; Gu, Yang; Yang, Sheng; Jiang, Weihong; Yang, Chen

    2014-11-01

    Rex, a transcriptional repressor that modulates its DNA-binding activity in response to NADH/NAD(+) ratio, has recently been found to play a role in the solventogenic shift of Clostridium acetobutylicum. Here, we combined a comparative genomic reconstruction of Rex regulons in 11 diverse clostridial species with detailed experimental characterization of Rex-mediated regulation in C. acetobutylicum. The reconstructed Rex regulons in clostridia included the genes involved in fermentation, hydrogen production, the tricarboxylic acid cycle, NAD biosynthesis, nitrate and sulfite reduction, and CO2/CO fixation. The predicted Rex-binding sites in the genomes of Clostridium spp. were verified by in vitro binding assays with purified Rex protein. Novel members of the C. acetobutylicum Rex regulon were identified and experimentally validated by comparing the transcript levels between the wild-type and rex-inactivated mutant strains. Furthermore, the effects of exposure to methyl viologen or H2O2 on intracellular NADH and NAD(+) concentrations, expression of Rex regulon genes, and physiology of the wild type and rex-inactivated mutant were comparatively analyzed. Our results indicate that Rex responds to NADH/NAD(+) ratio in vivo to regulate gene expression and modulates fermentation product formation and oxidative stress tolerance in C. acetobutylicum. It is suggested that Rex plays an important role in maintaining NADH/NAD(+) homeostasis in clostridia. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  15. Redox-Responsive Repressor Rex Modulates Alcohol Production and Oxidative Stress Tolerance in Clostridium acetobutylicum

    PubMed Central

    Zhang, Lei; Nie, Xiaoqun; Ravcheev, Dmitry A.; Rodionov, Dmitry A.; Sheng, Jia; Gu, Yang; Yang, Sheng; Jiang, Weihong

    2014-01-01

    Rex, a transcriptional repressor that modulates its DNA-binding activity in response to NADH/NAD+ ratio, has recently been found to play a role in the solventogenic shift of Clostridium acetobutylicum. Here, we combined a comparative genomic reconstruction of Rex regulons in 11 diverse clostridial species with detailed experimental characterization of Rex-mediated regulation in C. acetobutylicum. The reconstructed Rex regulons in clostridia included the genes involved in fermentation, hydrogen production, the tricarboxylic acid cycle, NAD biosynthesis, nitrate and sulfite reduction, and CO2/CO fixation. The predicted Rex-binding sites in the genomes of Clostridium spp. were verified by in vitro binding assays with purified Rex protein. Novel members of the C. acetobutylicum Rex regulon were identified and experimentally validated by comparing the transcript levels between the wild-type and rex-inactivated mutant strains. Furthermore, the effects of exposure to methyl viologen or H2O2 on intracellular NADH and NAD+ concentrations, expression of Rex regulon genes, and physiology of the wild type and rex-inactivated mutant were comparatively analyzed. Our results indicate that Rex responds to NADH/NAD+ ratio in vivo to regulate gene expression and modulates fermentation product formation and oxidative stress tolerance in C. acetobutylicum. It is suggested that Rex plays an important role in maintaining NADH/NAD+ homeostasis in clostridia. PMID:25182496

  16. Phosphotransferase activity in Clostridium acetobutylicum from acidogenic and solventogenic phases of growth

    SciTech Connect

    Hutkins, R.W.; Kashket, E.R.

    1986-05-01

    Clostridium acetobutylicum cells, when energized with fructose, transported and phosphorylated the glucose analog 2-deoxyglucose by a phosphoenolpyruvate-dependent phosphotransferase (PT) system. Butanol up to 2% did not inhibit PT activity, although its chaotropic effect on the cell membrane caused cellular phosphoenolpyruvate and the 2-deoxyglucose-6-phosphate to leak out. Cells harvested from the solventogenic phase of batch growth had a significantly lower PT activity than did cells from the acidogenic phase.

  17. Control of Carbon and Electron Flow in Clostridium acetobutylicum Fermentations: Utilization of Carbon Monoxide to Inhibit Hydrogen Production and to Enhance Butanol Yields

    PubMed Central

    Kim, Byung Hong; Bellows, Para; Datta, Rathin; Zeikus, J. G.

    1984-01-01

    Extracts prepared from non-solvent-producing cells of Clostridium acetobutylicum contained methyl viologen-linked hydrogenase activity (20 U/mg of protein at 37°C) but did not display carbon monoxide dehydrogenase activity. CO addition readily inhibited the hydrogenase activity of cell extracts or of viable metabolizing cells. Increasing the partial pressure of CO (2 to 10%) in unshaken anaerobic culture tube headspaces significantly inhibited (90% inhibition at 10% CO) both growth and hydrogen production by C. acetobutylicum. Growth was not sensitive to low partial pressures of CO (i.e., up to 15%) in pH-controlled fermentors (pH 4.5) that were continuously gassed and mixed. CO addition dramatically altered the glucose fermentation balance of C. acetobutylicum by diverting carbon and electrons away from H2, CO2, acetate, and butyrate production and towards production of ethanol and butanol. The butanol concentration was increased from 65 to 106 mM and the butanol productivity (i.e., the ratio of butanol produced/total acids and solvents produced) was increased by 31% when glucose fermentations maintained at pH 4.5 were continuously gassed with 85% N2-15% CO versus N2 alone. The results are discussed in terms of metabolic regulation of C. acetobutylicum saccharide fermentations to achieve maximal butanol or solvent yield. PMID:16346643

  18. A transcriptional study of acidogenic chemostat cells of Clostridium acetobutylicum--solvent stress caused by a transient n-butanol pulse.

    PubMed

    Janssen, Holger; Grimmler, Christina; Ehrenreich, Armin; Bahl, Hubert; Fischer, Ralf-Jörg

    2012-10-31

    The main product of the anaerobic fermentative bacterium Clostridium acetobutylicum is n-butanol, an organic solvent with severe toxic effects on the cells. Therefore, the identification of the molecular factors related to n-butanol stress constitutes a major strategy for furthering the understanding of the biotechnological production of n-butanol, an important industrial biofuel. Previous reports concerning n-butanol stress in C. acetobutylicum dealt exclusively with batch cultures. In this study, for the first time a comprehensive transcriptional analysis of n-butanol-stressed C. acetobutylicum was conducted using stable steady state acidogenic chemostat cultures. A total of 358 differentially expressed genes were significantly affected by n-butanol stress. Similarities, such as the upregulation of general stress genes, and differences in gene expression were compared in detail with earlier DNA microarrays performed in batch cultivation experiments. The main result of this analysis was the observation that genes involved in amino acid and nucleotide biosynthesis, as well as genes for specific transport systems were upregulated by n-butanol. Our results exclude any transcriptional response triggered by exogenous pH changes or solventogenic n-butanol formation. Finally, our data suggest that metabolic flux through the glycerolipid biosynthetic pathway increases, confirming that C. acetobutylicum modifies the cytoplasmic membrane composition in response to n-butanol stress. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Capturing the response of Clostridium acetobutylicum to chemical stressors using a regulated genome-scale metabolic model

    SciTech Connect

    Dash, Satyakam; Mueller, Thomas J.; Venkataramanan, Keerthi P.; Papoutsakis, Eleftherios T.; Maranas, Costas D.

    2014-10-14

    Clostridia are anaerobic Gram-positive Firmicutes containing broad and flexible systems for substrate utilization, which have been used successfully to produce a range of industrial compounds. Clostridium acetobutylicum has been used to produce butanol on an industrial scale through acetone-butanol-ethanol (ABE) fermentation. A genome-scale metabolic (GSM) model is a powerful tool for understanding the metabolic capacities of an organism and developing metabolic engineering strategies for strain development. The integration of stress related specific transcriptomics information with the GSM model provides opportunities for elucidating the focal points of regulation.

  20. Molecular modulation of pleiotropic regulator CcpA for glucose and xylose coutilization by solvent-producing Clostridium acetobutylicum.

    PubMed

    Wu, Yan; Yang, Yunpeng; Ren, Cong; Yang, Chen; Yang, Sheng; Gu, Yang; Jiang, Weihong

    2015-03-01

    Efficient cofermentation of hexose and pentose sugars is essential for ABE (Acetone, Butanol and Ethanol) solvents production from lignocellulosic hydrolysates by Clostridium acetobutylicum, an important industrial microorganism. However, utilization of xylose, the predominant pentose present in lignocellulosic feedstocks, by this anaerobe is limited by CCR (Carbon Catabolite Repression) that is mediated by the catabolite control protein A (CcpA). Here, we reported a novel engineering strategy based on CcpA molecular modulation to overcome the defect. Through CcpA mutagenesis and screening, an amino acid residue, valine 302, was shown to be essential for CcpA-dependent CCR in C. acetobutylicum. When this residue was replaced by asparagine (V302N mutation), CCR could be alleviated and a greatly improved xylose utilization was realized. Transcriptional and DNA binding analysis was then used to elucidate the underlying molecular mechanism. Furthermore, the sol genes (ctfA, ctfB and adhE1) were overexpressed, upon the V302N mutation, to accelerate sugar consumption and solvents formation. The resulting strain (824ccpA-V302N-sol) was capable of using over 90% of the total xylose within 72 h when fermenting a mixture of glucose and xylose (30 g/L glucose and 15 g/L xylose), which was much higher than that (30%) of the control strain 824ccpA-ccpA(C). This is the first report that offered an optimized C. acetobutylicum CcpA with alleviated repression on xylose metabolism, yielding a valuable platform host toward ABE solvents production from lignocellulosic biomass. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  1. Intracellular metabolic changes of Clostridium acetobutylicum and promotion to butanol tolerance during biobutanol fermentation.

    PubMed

    Wang, Yan-Feng; Tian, Juan; Ji, Zhi-Hua; Song, Mao-Yong; Li, Hao

    2016-09-01

    During the fermentation process, Clostridium acetobutylicum cells are often inhibited by the accumulated butanol. However, the mechanism underlying response of C. acetobutylicum to butanol stress remains poorly understood. This study was performed to clarify such mechanism through investigating the butanol stress-associated intracellular biochemical changes at acidogenesis phase (i.e., middle exponential phase) and solventogenesis phase (i.e., early stationary phase) by a gas chromatography-mass spectrometry-based metabolomics strategy. With the aid of partial least-squares-discriminant analysis, a pairwise discrimination between control group and butanol-treated groups was revealed, and 27 metabolites with variable importance in the projection value greater than 1 were identified. Under butanol stress, the glycolysis might be inhibited while TCA cycle might be promoted. Moreover, changes of lipids and fatty acids compositions, amino acid metabolism and osmoregulator concentrations might be the key factors involved in C. acetobutylicum metabolic response to butanol stress. It was suggested that C. acetobutylicum cells might change the levels of long acyl chain saturated fatty acids and branched-chain amino acids to maintain the integrity of cell membrane through adjusting membrane fluidity under butanol stress. The increased level of glycerol was considered to be correlated with osmoregulation and regulating redox balance. In addition, increased levels of some amino acids (i.e., threonine, glycine, alanine, phenylalanine, tyrosine, tryptophan, aspartate and glutamate) might also confer butanol tolerance to C. acetobutylicum. These results highlighted our knowledge about the response or adaptation of C. acetobutylicum to butanol stress, and would contribute to the construction of feasible butanologenic strains with higher butanol tolerance. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Effects of nutritional enrichment on the production of acetone-butanol-ethanol (ABE) by Clostridium acetobutylicum.

    PubMed

    Choi, Sung Jun; Lee, Joungmin; Jang, Yu-Sin; Park, Jin Hwan; Lee, Sang Yup; Kim, In Ho

    2012-12-01

    Clostridium acetobutylicum is an industrially important organism that produces acetone-butanol-ethanol (ABE). The main objective of this study was to characterize the effects of increased cell density on the production of ABE during the phase transition from acidogenesis to solventogenesis in C. acetobutylicum. The increased ABE productivity of C. acetobutylicum was obtained by increasing the cell density using a newly designed medium (designated C. a cetobutylicum medium 1; CAM1). The maximum OD(600) value of C. acetobutylicum ATCC 824 strain obtained with CAM1 was 19.7, which is 1.8 times higher than that obtained with clostridial growth medium (CGM). The overall ABE productivity obtained in the CAM1-fermetation of the ATCC 824 strain was 0.83 g/L/h, which is 1.5 times higher than that (0.55 g/L/h) obtained with CGM. However, the increased productivity obtained with CAM1 did not result in an increase in the final ABE titer, because phase transition occurred at a high titer of acids.

  3. The redox-sensing protein Rex, a transcriptional regulator of solventogenesis in Clostridium acetobutylicum.

    PubMed

    Wietzke, Mandy; Bahl, Hubert

    2012-11-01

    Solventogenic clostridia are characterised by their biphasic fermentative metabolism, and the main final product n-butanol is of particular industrial interest because it can be used as a superior biofuel. During exponential growth, Clostridium acetobutylicum synthesises acetic and butyric acids which are accompanied by the formation of molecular hydrogen and carbon dioxide. During the stationary phase, the solvents acetone, butanol and ethanol are produced. However, the molecular mechanisms of this metabolic switch are largely unknown so far. In this study, in silico, in vitro and in vivo analyses were performed to elucidate the function of the CAC2713-encoded redox-sensing transcriptional repressor Rex and its role in the solventogenic shift of C. acetobutylicum ATCC 824. Electrophoretic mobility shift assays showed that Rex controls the expression of butanol biosynthetic genes as a response to the cellular NADH/NAD(+) ratio. Interestingly, the Rex-negative mutant C. acetobutylicum rex::int(95) produced high amounts of ethanol and butanol, while hydrogen and acetone production were significantly reduced. Both ethanol and butanol (but not acetone) formation started clearly earlier than in the wild type. In addition, the rex mutant showed a de-repression of the bifunctional aldehyde/alcohol dehydrogenase 2 encoded by the adhE2 gene (CAP0035) as demonstrated by increased adhE2 expression as well as high NADH-dependent alcohol dehydrogenase activities. The results presented here clearly indicated that Rex is involved in the redox-dependent solventogenic shift of C. acetobutylicum.

  4. Phosphoketolase Pathway for Xylose Catabolism in Clostridium acetobutylicum Revealed by 13C Metabolic Flux Analysis

    PubMed Central

    Liu, Lixia; Zhang, Lei; Tang, Wei; Gu, Yang; Hua, Qiang; Yang, Sheng; Jiang, Weihong

    2012-01-01

    Solvent-producing clostridia are capable of utilizing pentose sugars, including xylose and arabinose; however, little is known about how pentose sugars are catabolized through the metabolic pathways in clostridia. In this study, we identified the xylose catabolic pathways and quantified their fluxes in Clostridium acetobutylicum based on [1-13C]xylose labeling experiments. The phosphoketolase pathway was found to be active, which contributed up to 40% of the xylose catabolic flux in C. acetobutylicum. The split ratio of the phosphoketolase pathway to the pentose phosphate pathway was markedly increased when the xylose concentration in the culture medium was increased from 10 to 20 g liter−1. To our knowledge, this is the first time that the in vivo activity of the phosphoketolase pathway in clostridia has been revealed. A phosphoketolase from C. acetobutylicum was purified and characterized, and its activity with xylulose-5-P was verified. The phosphoketolase was overexpressed in C. acetobutylicum, which resulted in slightly increased xylose consumption rates during the exponential growth phase and a high level of acetate accumulation. PMID:22865845

  5. Phosphoketolase pathway for xylose catabolism in Clostridium acetobutylicum revealed by 13C metabolic flux analysis.

    PubMed

    Liu, Lixia; Zhang, Lei; Tang, Wei; Gu, Yang; Hua, Qiang; Yang, Sheng; Jiang, Weihong; Yang, Chen

    2012-10-01

    Solvent-producing clostridia are capable of utilizing pentose sugars, including xylose and arabinose; however, little is known about how pentose sugars are catabolized through the metabolic pathways in clostridia. In this study, we identified the xylose catabolic pathways and quantified their fluxes in Clostridium acetobutylicum based on [1-(13)C]xylose labeling experiments. The phosphoketolase pathway was found to be active, which contributed up to 40% of the xylose catabolic flux in C. acetobutylicum. The split ratio of the phosphoketolase pathway to the pentose phosphate pathway was markedly increased when the xylose concentration in the culture medium was increased from 10 to 20 g liter(-1). To our knowledge, this is the first time that the in vivo activity of the phosphoketolase pathway in clostridia has been revealed. A phosphoketolase from C. acetobutylicum was purified and characterized, and its activity with xylulose-5-P was verified. The phosphoketolase was overexpressed in C. acetobutylicum, which resulted in slightly increased xylose consumption rates during the exponential growth phase and a high level of acetate accumulation.

  6. Identification of PTS(Fru) as the major fructose uptake system of Clostridium acetobutylicum.

    PubMed

    Voigt, Christine; Bahl, Hubert; Fischer, Ralf-Jörg

    2014-08-01

    As a member of the saccharolytic clostridia, a variety of different carbohydrates like glucose, fructose, or mannose can be used as carbon and energy source by Clostridium acetobutylicum ATCC 824. Thirteen phosphoenolpyruvate-dependent phosphotransferase systems (PTS) have been identified in C. acetobutylicum, which are likely to be responsible for the uptake of hexoses, hexitols, or disaccharides. Here, we focus on three PTS which are expected to be involved in the uptake of fructose, PTS(Fru), PTS(ManI), and PTS(ManII). To analyze their individual functions, each PTS was inactivated via homologous recombination or insertional mutagenesis. Standardized comparative batch fermentations in a synthetic medium with glucose, fructose, or mannose as sole carbon source identified PTS(Fru) as primary uptake system for fructose, whereas growth with fructose was not impaired in PTS(ManI) and slightly altered in PTS(ManII)-deficient strains of C. acetobutylicum. The inactivation of PTS(ManI) resulted in slower growth on mannose whereas the loss of PTS(ManII) revealed no phenotype during growth on mannose. This is the first time that it has been shown that PTS(Fru) and PTS(ManI) of C. acetobutylicum are directly involved in fructose and mannose uptake, respectively. Moreover, comprehensive comparison of the fermentation products revealed that the loss of PTS(Fru) prevents the solvent shift as no butanol and only basic levels of acetone and ethanol could be determined.

  7. Phosphoketolase flux in Clostridium acetobutylicum during growth on L-arabinose.

    PubMed

    Sund, Christian J; Liu, Sanchao; Germane, Katherine L; Servinsky, Matthew D; Gerlach, Elliot S; Hurley, Margaret M

    2015-02-01

    Clostridium acetobutylicum's metabolic pathways have been studied for decades due to its metabolic diversity and industrial value, yet many details of its metabolism continue to emerge. The flux through the recently discovered pentose phosphoketolase pathway (PKP) in C. acetobutylicum has been determined for growth on xylose but transcriptional analysis indicated the pathway may have a greater contribution to arabinose metabolism. To elucidate the role of xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (XFP), and the PKP in C. acetobutylicum, experimental and computational metabolic isotope analyses were performed under growth conditions of glucose or varying concentrations of xylose and arabinose. A positional bias in labelling between carbons 2 and 4 of butyrate was found and posited to be due to an enzyme isotope effect of the thiolase enzyme. A correction for the positional bias was applied, which resulted in reduction of residual error. Comparisons between model solutions with low residual error indicated flux through each of the two XFP reactions was variable, while the combined flux of the reactions remained relatively constant. PKP utilization increased with increasing xylose concentration and this trend was further pronounced during growth on arabinose. Mutation of the gene encoding XFP almost completely abolished flux through the PKP during growth on arabinose and resulted in decreased acetate/butyrate ratios. Greater flux through the PKP during growth on arabinose when compared with xylose indicated the pathway's primary role in C. acetobutylicum is arabinose metabolism.

  8. Mechanisms of microbial oil recovery by Clostridium acetobutylicum and Bacillus strain JF-2

    SciTech Connect

    Marsh, T.L.; Zhang, X.; Knapp, R.M.; McInerney, M.J.; Sharma, P.K.; Jackson, B.E.

    1995-12-31

    Core displacement experiments at elevated pressures were conducted to determine whether microbial processes are effective under conditions that simulate those found in an actual oil reservoir. The in-situ growth of Clostridium acetobutylicum and Bacillus strain JF-2 resulted in the recovery of residual oil. About 21 and 23% of the residual oil was recovered by C. acetobutylicum and Bacillus strain JF-2, respectively. Flooding cores with cell-free culture fluids of C. acetobutylicum with and without the addition of 50 mM acetone and 100 mM butanol did not result in the recovery of residual oil. Mathematical simulations showed that the amount of gas produced by the clostridial fermentation was not showed that the amount of gas produced by the clostridial fermentation was not sufficient to recover residual oil. Oil recovery by Bacillus strain JF-2 was highly correlated to surfactant production. A biosurfactant-deficient mutant of strain JF-2 was not capable of recovering residual oil. These data show that surfactant production is an important mechanism for microbially enhanced oil recovery. The mechanism for oil recovery by C. acetobutylicum is not understood at this time, but the production of acids, solvents, or gases alone cannot explain the observed increases in oil recovery by this organism.

  9. Biotransformation of furfural and 5-hydroxymethyl furfural (HMF) by Clostridium acetobutylicum ATCC 824 during butanol fermentation.

    PubMed

    Zhang, Yan; Han, Bei; Ezeji, Thaddeus Chukwuemeka

    2012-02-15

    The ability of fermenting microorganisms to tolerate furan aldehyde inhibitors (furfural and 5-hydroxymethyl furfural (HMF)) will enhance efficient bioconversion of lignocellulosic biomass hydrolysates to fuels and chemicals. The effect of furfural and HMF on butanol production by Clostridium acetobutylicum 824 was investigated. Whereas specific growth rates, μ, of C. acetobutylicum in the presence of furfural and HMF were in the range of 15-85% and 23-78%, respectively, of the uninhibited Control, μ increased by 8-15% and 23-38% following exhaustion of furfural and HMF in the bioreactor. Using high performance liquid chromatography and spectrophotometric assays, batch fermentations revealed that furfural and HMF were converted to furfuryl alcohol and 2,5-bis-hydroxymethylfuran, respectively, with specific conversion rates of 2.13g furfural and 0.50g HMF per g (biomass) per hour, by exponentially growing C. acetobutylicum. Biotransformation of these furans to lesser inhibitory compounds by C. acetobutylicum will probably enhance overall fermentation of lignocellulosic hydrolysates to butanol. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Metabolic Engineering of Clostridium acetobutylicum ATCC 824 for Isopropanol-Butanol-Ethanol Fermentation

    PubMed Central

    Lee, Joungmin; Jang, Yu-Sin; Choi, Sung Jun; Im, Jung Ae; Song, Hyohak; Cho, Jung Hee; Seung, Do Young; Papoutsakis, E. Terry; Bennett, George N.

    2012-01-01

    Clostridium acetobutylicum naturally produces acetone as well as butanol and ethanol. Since acetone cannot be used as a biofuel, its production needs to be minimized or suppressed by cell or bioreactor engineering. Thus, there have been attempts to disrupt or inactivate the acetone formation pathway. Here we present another approach, namely, converting acetone to isopropanol by metabolic engineering. Since isopropanol can be used as a fuel additive, the mixture of isopropanol, butanol, and ethanol (IBE) produced by engineered C. acetobutylicum can be directly used as a biofuel. IBE production is achieved by the expression of a primary/secondary alcohol dehydrogenase gene from Clostridium beijerinckii NRRL B-593 (i.e., adhB-593) in C. acetobutylicum ATCC 824. To increase the total alcohol titer, a synthetic acetone operon (act operon; adc-ctfA-ctfB) was constructed and expressed to increase the flux toward isopropanol formation. When this engineering strategy was applied to the PJC4BK strain lacking in the buk gene (encoding butyrate kinase), a significantly higher titer and yield of IBE could be achieved. The resulting PJC4BK(pIPA3-Cm2) strain produced 20.4 g/liter of total alcohol. Fermentation could be prolonged by in situ removal of solvents by gas stripping, and 35.6 g/liter of the IBE mixture could be produced in 45 h. PMID:22210214

  11. Metabolic engineering of Clostridium acetobutylicum ATCC 824 for isopropanol-butanol-ethanol fermentation.

    PubMed

    Lee, Joungmin; Jang, Yu-Sin; Choi, Sung Jun; Im, Jung Ae; Song, Hyohak; Cho, Jung Hee; Seung, Do Young; Papoutsakis, E Terry; Bennett, George N; Lee, Sang Yup

    2012-03-01

    Clostridium acetobutylicum naturally produces acetone as well as butanol and ethanol. Since acetone cannot be used as a biofuel, its production needs to be minimized or suppressed by cell or bioreactor engineering. Thus, there have been attempts to disrupt or inactivate the acetone formation pathway. Here we present another approach, namely, converting acetone to isopropanol by metabolic engineering. Since isopropanol can be used as a fuel additive, the mixture of isopropanol, butanol, and ethanol (IBE) produced by engineered C. acetobutylicum can be directly used as a biofuel. IBE production is achieved by the expression of a primary/secondary alcohol dehydrogenase gene from Clostridium beijerinckii NRRL B-593 (i.e., adh(B-593)) in C. acetobutylicum ATCC 824. To increase the total alcohol titer, a synthetic acetone operon (act operon; adc-ctfA-ctfB) was constructed and expressed to increase the flux toward isopropanol formation. When this engineering strategy was applied to the PJC4BK strain lacking in the buk gene (encoding butyrate kinase), a significantly higher titer and yield of IBE could be achieved. The resulting PJC4BK(pIPA3-Cm2) strain produced 20.4 g/liter of total alcohol. Fermentation could be prolonged by in situ removal of solvents by gas stripping, and 35.6 g/liter of the IBE mixture could be produced in 45 h.

  12. A systems biology approach to investigate the effect of pH-induced gene regulation on solvent production by Clostridium acetobutylicum in continuous culture

    PubMed Central

    2011-01-01

    Background Clostridium acetobutylicum is an anaerobic bacterium which is known for its solvent-producing capabilities, namely regarding the bulk chemicals acetone and butanol, the latter being a highly efficient biofuel. For butanol production by C. acetobutylicum to be optimized and exploited on an industrial scale, the effect of pH-induced gene regulation on solvent production by C. acetobutylicum in continuous culture must be understood as fully as possible. Results We present an ordinary differential equation model combining the metabolic network governing solvent production with regulation at the genetic level of the enzymes required for this process. Parameterizing the model with experimental data from continuous culture, we demonstrate the influence of pH upon fermentation products: at high pH (pH 5.7) acids are the dominant product while at low pH (pH 4.5) this switches to solvents. Through steady-state analyses of the model we focus our investigations on how alteration in gene expression of C. acetobutylicum could be exploited to increase butanol yield in a continuous culture fermentation. Conclusions Incorporating gene regulation into the model of solvent production by C. acetobutylicum enables an accurate representation of the pH-induced switch to solvent production to be obtained and theoretical investigations of possible synthetic-biology approaches to be pursued. Steady-state analyses suggest that, to increase butanol yield, alterations in the expression of single solvent-associated genes are insufficient; a more complex approach targeting two or more genes is required. PMID:21247470

  13. Biobutanol production by a new local isolate of Clostridium acetobutylicum YM1

    NASA Astrophysics Data System (ADS)

    Al-Shorgani, Najeeb Kaid; Tibin, El Mubarak; Kalil, Mohd Sahaid; Yusoff, Wan Mohtar Wan; Hamid, Aidil Abdul

    2014-09-01

    Increasing demand of energy and awareness about environmental pollution has led to increase interest in alternative, clean and renewable energy sources. Biobutanol is considered as the candidate liquid biofuel to replace gasoline. In this study, the capability of a newly isolated strain of local Clostridium acetobutylicum YM1 was tested to produce biobutanol in batch fermentation. Various culture conditions including glucose concentration, initial pH, incubation temperature and inoculum size were investigated for their effects on production of biobutanol using strain YM1. The results showed that the optimal biobutanol production was obtained at glucose concentration 50 g/L, initial pH 6.2, temperature 30°C and inoculum size 10%. These results show that C. acetobutylicum YM1 as a mesophilic bacterium is a potential candidate for biobutanol production.

  14. Identification and Characterization of Two Functionally Unknown Genes Involved in Butanol Tolerance of Clostridium acetobutylicum

    PubMed Central

    Jia, Kaizhi; Zhang, Yanping; Li, Yin

    2012-01-01

    Solvents toxicity is a major limiting factor hampering the cost-effective biotechnological production of chemicals. In Clostridium acetobutylicum, a functionally unknown protein (encoded by SMB_G1518) with a hypothetical alcohol interacting domain was identified. Disruption of SMB_G1518 and/or its downstream gene SMB_G1519 resulted in increased butanol tolerance, while overexpression of SMB_G1518-1519 decreased butanol tolerance. In addition, SMB_G1518-1519 also influences the production of pyruvate:ferredoxin oxidoreductase (PFOR) and flagellar protein hag, the maintenance of cell motility. We conclude that the system of SMB_G1518-1519 protein plays a role in the butanol sensitivity/tolerance phenotype of C. acetobutylicum, and can be considered as potential targets for engineering alcohol tolerance. PMID:22768047

  15. Efficient gene knockdown in Clostridium acetobutylicum by synthetic small regulatory RNAs.

    PubMed

    Cho, Changhee; Lee, Sang Yup

    2017-02-01

    Clostridium is considered a promising microbial host for the production of valuable industrial chemicals. However, Clostridium is notorious for the difficulty of genetic manipulations, and consequently metabolic engineering. Thus, much effort has been exerted to develop novel tools for genetic and metabolic engineering of Clostridium strains. Here, we report the development of a synthetic small regulatory RNA (sRNA)-based system for controlled gene expression in Clostridium acetobutylicum, consisting of a target recognition site, MicC sRNA scaffold, and an RNA chaperone Hfq. To examine the functional operation of sRNA system in C. acetobutylicum, expression control was first examined with the Evoglow fluorescent protein as a model protein. Initially, a C. acetobutylicum protein annotated as Hfq was combined with the synthetic sRNA based on the Escherichia coli MicC scaffold to knockdown Evoglow expression. However, C. acetobutylicum Hfq did not bind to E. coli MicC, while MicC scaffold-based synthetic sRNA itself was able to knockdown the expression of Evoglow. When E. coli hfq gene was introduced, the knockdown efficiency assessed by measuring fluorescence intensity, could be much enhanced. Then, this E. coli MicC scaffold-Hfq system was used to knock down adhE1 gene expression in C. acetobutylicum. Knocking down the adhE1 gene expression using the synthetic sRNA led to a 40% decrease in butanol production (2.5 g/L), compared to that (4.5 g/L) produced by the wild-type strain harboring an empty vector. The sRNA system was further extended to knock down the pta gene expression in the buk mutant C. acetobutylicum strain PJC4BK for enhanced butanol production. The PJC4BK (pPta-Hfq(Eco) ) strain, which has the pta gene expression knocked down, was able to produce 16.9 g/L of butanol, which is higher than that (14.9 g/L) produced by the PJC4BK strain, mainly due to reduced acetic acid production. Fed-batch culture of PJC4BK (pPta-Hfq(Eco) ) strain coupled with

  16. Development of an inducible transposon system for efficient random mutagenesis in Clostridium acetobutylicum

    PubMed Central

    Zhang, Ying; Xu, Shu; Chai, Changsheng; Yang, Sheng; Jiang, Weihong; Minton, Nigel P.; Gu, Yang

    2016-01-01

    Clostridium acetobutylicum is an industrially important Gram-positive organism, which is capable of producing economically important chemicals in the ABE (Acetone, Butanol and Ethanol) fermentation process. Renewed interests in the ABE process necessitate the availability of additional genetics tools to facilitate the derivation of a greater understanding of the underlying metabolic and regulatory control processes in operation through forward genetic strategies. In this study, a xylose inducible, mariner-based, transposon system was developed and shown to allow high-efficient random mutagenesis in the model strain ATCC 824. Of the thiamphenicol resistant colonies obtained, 91.9% were shown to be due to successful transposition of the catP-based mini-transposon element. Phenotypic screening of 200 transposon clones revealed a sporulation-defective clone with an insertion in spo0A, thereby demonstrating that this inducible transposon system can be used for forward genetic studies in C. acetobutylicum. PMID:27001972

  17. Genome analysis of a hyper acetone-butanol-ethanol (ABE) producing Clostridium acetobutylicum BKM19.

    PubMed

    Cho, Changhee; Choe, Donghui; Jang, Yu-Sin; Kim, Kyung-Jin; Kim, Won Jun; Cho, Byung-Kwan; Papoutsakis, E Terry; Bennett, George N; Seung, Do Young; Lee, Sang Yup

    2017-02-01

    Previously the development of a hyper acetone-butanol-ethanol (ABE) producing Clostridium acetobutylicum BKM19 strain capable of producing 30.5% more total solvent by random mutagenesis of its parental strain PJC4BK, which is a buk mutant C. acetobutylicum ATCC 824 strain is reported. Here, BKM19 and PJC4BK strains are re-sequenced by a high-throughput sequencing technique to understand the mutations responsible for enhanced solvent production. In comparison with the C. acetobutylicum PJC4BK, 13 single nucleotide variants (SNVs), one deletion and one back mutation SNV are identified in the C. acetobutylicum BKM19 genome. Except for one SNV found in the megaplasmid, all mutations are found in the chromosome of BKM19. Among them, a mutation in the thlA gene encoding thiolase is further studied with respect to enzyme activity and butanol production. The mutant thiolase (thlA(V5A) ) is showed a 32% higher activity than that of the wild-type thiolase (thlA(WT) ). In batch fermentation, butanol production is increased by 26% and 23% when the thlA(V5A) gene is overexpressed in the wild-type C. acetobutylicum ATCC 824 and in its derivative, the thlA-knockdown TKW-A strain, respectively. Based on structural analysis, the mutation in thiolase does not have a direct effect on the regulatory determinant region (RDR). However, the mutation at the 5(th) residue seems to influence the stability of the RDR, and thus, increases the enzymatic activity and enhances solvent production in the BKM19 strain. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Parallel labeling experiments validate Clostridium acetobutylicum metabolic network model for (13)C metabolic flux analysis.

    PubMed

    Au, Jennifer; Choi, Jungik; Jones, Shawn W; Venkataramanan, Keerthi P; Antoniewicz, Maciek R

    2014-11-01

    In this work, we provide new insights into the metabolism of Clostridium acetobutylicum ATCC 824 obtained using a systematic approach for quantifying fluxes based on parallel labeling experiments and (13)C-metabolic flux analysis ((13)C-MFA). Here, cells were grown in parallel cultures with [1-(13)C]glucose and [U-(13)C]glucose as tracers and (13)C-MFA was used to quantify intracellular metabolic fluxes. Several metabolic network models were compared: an initial model based on current knowledge, and extended network models that included additional reactions that improved the fits of experimental data. While the initial network model did not produce a statistically acceptable fit of (13)C-labeling data, an extended network model with five additional reactions was able to fit all data with 292 redundant measurements. The model was subsequently trimmed to produce a minimal network model of C. acetobutylicum for (13)C-MFA, which could still reproduce all of the experimental data. The flux results provided valuable new insights into the metabolism of C. acetobutylicum. First, we found that TCA cycle was effectively incomplete, as there was no measurable flux between α-ketoglutarate and succinyl-CoA, succinate and fumarate, and malate and oxaloacetate. Second, an active pathway was identified from pyruvate to fumarate via aspartate. Third, we found that isoleucine was produced exclusively through the citramalate synthase pathway in C. acetobutylicum and that CAC3174 was likely responsible for citramalate synthase activity. These model predictions were confirmed in several follow-up tracer experiments. The validated metabolic network model established in this study can be used in future investigations for unbiased (13)C-flux measurements in C. acetobutylicum. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  19. Metabolic engineering of Clostridium acetobutylicum for butyric acid production with high butyric acid selectivity.

    PubMed

    Jang, Yu-Sin; Im, Jung Ae; Choi, So Young; Lee, Jung Im; Lee, Sang Yup

    2014-05-01

    A typical characteristic of the butyric acid-producing Clostridium is coproduction of both butyric and acetic acids. Increasing the butyric acid selectivity important for economical butyric acid production has been rather difficult in clostridia due to their complex metabolic pathways. In this work, Clostridium acetobutylicum was metabolically engineered for highly selective butyric acid production. For this purpose, the second butyrate kinase of C. acetobutylicum encoded by the bukII gene instead of butyrate kinase I encoded by the buk gene was employed. Furthermore, metabolic pathways were engineered to further enhance the NADH-driving force. Batch fermentation of the metabolically engineered C. acetobutylicum strain HCBEKW (pta(-), buk(-), ctfB(-) and adhE1(-)) at pH 6.0 resulted in the production of 32.5g/L of butyric acid with a butyric-to-acetic acid ratio (BA/AA ratio) of 31.3g/g from 83.3g/L of glucose. By further knocking out the hydA gene (encoding hydrogenase) in the HCBEKW strain, the butyric acid titer was not further improved in batch fermentation. However, the BA/AA ratio (28.5g/g) obtained with the HYCBEKW strain (pta(-), buk(-), ctfB(-), adhE1(-) and hydA(-)) was 1.6 times higher than that (18.2g/g) obtained with the HCBEKW strain at pH 5.0, while no improvement was observed at pH 6.0. These results suggested that the buk gene knockout was essential to get a high butyric acid selectivity to acetic acid in C. acetobutylicum. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  20. Altered Electron Flow in Continuous Cultures of Clostridium acetobutylicum Induced by Viologen Dyes

    PubMed Central

    Rao, Govind; Mutharasan, R.

    1987-01-01

    The physiological response of Clostridium acetobutylicum to methyl and benzyl viologen was investigated. Viologen dyes at low concentrations (at levels of parts per million [micrograms per milliliter]) caused significant metabolic shifts. Altered electron flow appeared to direct carbon flow from acid to alcohol production accompanied by decreased hydrogen evolution. Reducing equivalents normally released as free hydrogen were directed toward formation of NADH which, in turn, resulted in increased alcohol production. In addition, it was shown that solvent production can take place at pH 6.3. Contrary to previous reports, butanol production appears to be independent of high levels of acetate-butyrate and glucose. PMID:16347357

  1. Roles of three AbrBs in regulating two-phase Clostridium acetobutylicum fermentation.

    PubMed

    Xue, Qiong; Yang, Yunpeng; Chen, Jun; Chen, Lei; Yang, Sheng; Jiang, Weihong; Gu, Yang

    2016-11-01

    Clostridium acetobutylicum is an important industrial microorganism for n-butanol bioproduction, and its transcription factor AbrB0310 regulates various important cellular processes. However, the roles of two abrB homologues, abrB1941 and abrB3647, have not been determined because they appear inactive during transcription. Here, we performed a detailed investigation into the function of abrB1941 and abrB3647 in C. acetobutylicum. Interestingly, we observed that AbrB3647 exerts an important influence on biphasic fermentation that opposes the influence of AbrB0310, while AbrB1941 might not be essential. When abrB3647 was disrupted using the Targetron system, a greatly improved cellular growth occurred. The following analysis shows that all three AbrBs participated in metabolically regulating acidogenesis, solventogenesis, and a two-phase transition in C. acetobutylicum, but the AbrB0310 and AbrB3647 functions were the most important. Moreover, the target genes subject to AbrB0310 and AbrB3647 regulation closely overlap. Based on these results, we will better understand the roles of the three AbrBs in regulating solventogenic clostridia cell physiology.

  2. Engineering Clostridium acetobutylicum with a histidine kinase knockout for enhanced n-butanol tolerance and production.

    PubMed

    Xu, Mengmeng; Zhao, Jingbo; Yu, Le; Tang, I-Ching; Xue, Chuang; Yang, Shang-Tian

    2015-01-01

    Clostridium acetobutylicum JB200, a mutant strain of C. acetobutylicum ATCC 55025 obtained through strain evolution in a fibrous bed bioreactor, had high butanol tolerance and produced up to ~21 g/L butanol from glucose in batch fermentation, an improvement of ~67 % over the parental strain (~12.6 g/L). Comparative genomic analysis revealed a single-base deletion in the cac3319 gene leading to C-terminal truncation in its encoding histidine kinase (HK) in JB200. To study the effects of cac3319 mutation on cell growth and fermentation, the cac3319 gene in ATCC 55025 was disrupted using the ClosTron group II intron-based gene inactivation system. Compared to ATCC 55025, the cac3319 HK knockout mutant, HKKO, produced 44.4 % more butanol (18.2 ± 1.3 vs. 12.6 ± 0.2 g/L) with a 90 % higher productivity (0.38 ± 0.03 vs. 0.20 ± 0.02 g/L h) due to increased butanol tolerance, confirming, for the first time, that cac3319 plays an important role in regulating solvent production and tolerance in C. acetobutylicum. This work also provides a novel metabolic engineering strategy for generating high-butanol-tolerant and high-butanol-producing strains for industrial applications.

  3. Metabolic engineering of Clostridium acetobutylicum for enhanced production of butyric acid.

    PubMed

    Jang, Yu-Sin; Woo, Hee Moon; Im, Jung Ae; Kim, In Ho; Lee, Sang Yup

    2013-11-01

    Clostridium acetobutylicum has been considered as an attractive platform host for biorefinery due to its metabolic diversity. Considering its capability to overproduce butanol through butyrate, it was thought that butyric acid can also be efficiently produced by this bacterium through metabolic engineering. The pta-ctfB-deficient C. acetobutylicum CEKW, in which genes encoding phosphotransacetylase and CoA-transferase were knocked out, was assessed for its potential as a butyric acid producer in fermentations with four controlled pH values at 5.0, 5.5, 6.0, and 6.4. Butyric acid could be best produced by fermentation of the CEKW at pH 6.0, resulting in the highest titer of 26.6 g/l, which is 6.4 times higher than that obtained with the wild type. However, due to the remaining solventogenic ability of the CEKW, 3.6 g/l solvents were also produced. Thus, the CEKW was further engineered by knocking out the adhE1-encoding aldehyde/alcohol dehydrogenase to prevent solvent production. Batch fermentation of the resulting C. acetobutylicum HCEKW at pH 6.0 showed increased butyric acid production to 30.8 g/l with a ratio of butyric-to-acetic acid (BA/AA) of 6.6 g/g and a productivity of 0.72 g/l/h from 86.9 g/l glucose, while negligible solvent (0.8 g/l ethanol only) was produced. The butyric acid titer, BA/AA ratio, and productivity obtained in this study were the highest values reported for C. acetobutylicum, and the BA/AA ratio and productivity were also comparable to those of native butyric acid producer Clostridium tyrobutyricum. These results suggested that the simultaneous deletion of the pta-ctfB-adhE1 in C. acetobutylicum resulted in metabolic switch from biphasic to acidogenic fermentation, which enhanced butyric acid production.

  4. A Quantitative System-Scale Characterization of the Metabolism of Clostridium acetobutylicum.

    PubMed

    Yoo, Minyeong; Bestel-Corre, Gwenaelle; Croux, Christian; Riviere, Antoine; Meynial-Salles, Isabelle; Soucaille, Philippe

    2015-11-24

    Engineering industrial microorganisms for ambitious applications, for example, the production of second-generation biofuels such as butanol, is impeded by a lack of knowledge of primary metabolism and its regulation. A quantitative system-scale analysis was applied to the biofuel-producing bacterium Clostridium acetobutylicum, a microorganism used for the industrial production of solvent. An improved genome-scale model, iCac967, was first developed based on thorough biochemical characterizations of 15 key metabolic enzymes and on extensive literature analysis to acquire accurate fluxomic data. In parallel, quantitative transcriptomic and proteomic analyses were performed to assess the number of mRNA molecules per cell for all genes under acidogenic, solventogenic, and alcohologenic steady-state conditions as well as the number of cytosolic protein molecules per cell for approximately 700 genes under at least one of the three steady-state conditions. A complete fluxomic, transcriptomic, and proteomic analysis applied to different metabolic states allowed us to better understand the regulation of primary metabolism. Moreover, this analysis enabled the functional characterization of numerous enzymes involved in primary metabolism, including (i) the enzymes involved in the two different butanol pathways and their cofactor specificities, (ii) the primary hydrogenase and its redox partner, (iii) the major butyryl coenzyme A (butyryl-CoA) dehydrogenase, and (iv) the major glyceraldehyde-3-phosphate dehydrogenase. This study provides important information for further metabolic engineering of C. acetobutylicum to develop a commercial process for the production of n-butanol. Currently, there is a resurgence of interest in Clostridium acetobutylicum, the biocatalyst of the historical Weizmann process, to produce n-butanol for use both as a bulk chemical and as a renewable alternative transportation fuel. To develop a commercial process for the production of n-butanol via a

  5. Transformation of heat-treated Clostridium acetobutylicum protoplasts with pUB110 plasmid DNA

    SciTech Connect

    Lin, Y.L.; Blaschek, H.P.

    1984-10-01

    Heat treatment of Clostridium acetobutylicum SA-1 protoplasts at 55/sup 0/C for 15 min before transformation resulted in expression in this microorganism of the kanamycin resistance determinant associated with plasmid pUB110. No heat treatment, or heat treatment at 65 or 44/sup 0/C for various time intervals, resulted in no kanamycin resistance transformants being recovered on selective kanamycin-containing regeneration medium. DNase plate assay indicated that treatment at 55/sup 0/C for 15 min completely inactivated the DNase activity associated with SA-1 protoplasts. Treatment of protoplasts at 65 or 55/sup 0/C for various periods under simulated transformation conditions had an inhibitory effect, although prolonged treatment at 55 or 44/sup 0/C appeared to stimulate DNase activity. Inactivation of protoplast-associated DNase activity by heat treatment at 55/sup 0/C for 15 min correlated with successful expression of kanamycin resistance and suggests that an extremely active, heat-sensitive, protoplast-associated DNase may be a factor in the polyethylene glycol-induced transformation of C. acetobutylicum SA-1 protoplasts. Plasmid pUB110 DNA was isolated from C. acetobutylicum SA-1 kanamycin-resistant (Km/sup r/) transformant cultures by a modification of the procedure used for C. perfringens plasmids. Detection of pUB110 DNA was possible only when diethyl pyrocarbonate was incorporated into isolation protocols to inactivate DNase activity. Restriction studies further verified the presence of pUB110 DNA in C. acetobutylicum SA-1 Km/sup r/ transformants. 36 references, 4 figures, 1 table.

  6. In silico analysis of Clostridium acetobutylicum ATCC 824 metabolic response to an external electron supply.

    PubMed

    Gallardo, Roberto; Acevedo, Alejandro; Quintero, Julián; Paredes, Ivan; Conejeros, Raúl; Aroca, Germán

    2016-02-01

    The biological production of butanol has become an important research field and thanks to genome sequencing and annotation; genome-scale metabolic reconstructions have been developed for several Clostridium species. This work makes use of the iCAC490 model of Clostridium acetobutylicum ATCC 824 to analyze its metabolic capabilities and response to an external electron supply through a constraint-based approach using the Constraint-Based Reconstruction Analysis Toolbox. Several analyses were conducted, which included sensitivity, production envelope, and phenotypic phase planes. The model showed that the use of an external electron supply, which acts as co-reducing agent along with glucose-derived reducing power (electrofermentation), results in an increase in the butanol-specific productivity. However, a proportional increase in the butyrate uptake flux is required. Besides, the uptake of external butyrate leads to the coupling of butanol production and growth, which coincides with results reported in literature. Phenotypic phase planes showed that the reducing capacity becomes more limiting for growth at high butyrate uptake fluxes. An electron uptake flux allows the metabolism to reach the growth optimality line. Although the maximum butanol flux does not coincide with the growth optimality line, a butyrate uptake combined with an electron uptake flux would result in an increased butanol volumetric productivity, being a potential strategy to optimize the production of butanol by C. acetobutylicum ATCC 824.

  7. Phylogenetic and Metabolic Diversity of Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)-transforming Bacteria in Strictly Anaerobic Mixed Cultures Enriched on RDX as Nitrogen Source

    DTIC Science & Technology

    2003-01-01

    crude extract of Clostridium acetobutylicum [33]. Finally, Enterobacteriaceae only exhibited hydroge- nase and RDX-removing activity under anaerobic...accepted 6 August 2003 First published online 6 September 2003 Abstract Five obligate anaerobes that were most closely related to Clostridium ...bifermentans, Clostridium celerecrescens, Clostridium saccharolyticum, Clostridium butyricum and Desulfovibrio desulfuricans by their 16S rRNA genes sequences

  8. Enhancement of butanol tolerance and butanol yield in Clostridium acetobutylicum mutant NT642 obtained by nitrogen ion beam implantation.

    PubMed

    Liu, Xiao-Bo; Gu, Qiu-Ya; Yu, Xiao-Bin; Luo, Wei

    2012-12-01

    As a promising alternative biofuel, biobutanol can be produced through acetone/butanol/ethanol (ABE) fermentation. Currently, ABE fermentation is still a small-scale industry due to its low production and high input cost. Moreover, butanol toxicity to the Clostridium fermentation host limits the accumulation of butanol in the fermentation broth. The wild-type Clostridium acetobutylicum D64 can only produce about 13 g butanol/L and tolerates less than 2% (v/v) butanol. To improve the tolerance of C. acetobutylicum D64 for enhancing the production of butanol, nitrogen ion beam implantation was employed and finally five mutants with enhanced butanol tolerance were obtained. Among these, the most butanol tolerant mutant C. acetobutylicum NT642 can tolerate above 3% (v/v) butanol while the wide-type strain can only withstand 2% (v/v). In batch fermentation, the production of butanol and ABE yield of C. acetobutylicum NT642 was 15.4 g/L and 22.3 g/L, respectively, which were both higher than those of its parental strain and the other mutants using corn or cassava as substrate. Enhancing butanol tolerance is a great precondition for obtaining a hyper-yield producer. Nitrogen ion beam implantation could be a promising biotechnology to improve butanol tolerance and production of the host strain C. acetobutylicum.

  9. Butanol production by immobilised Clostridium acetobutylicum in repeated batch, fed-batch, and continuous modes of fermentation.

    PubMed

    Dolejš, Igor; Krasňan, Vladimír; Stloukal, Radek; Rosenberg, Michal; Rebroš, Martin

    2014-10-01

    Clostridium acetobutylicum immobilised in polyvinylalcohol, lens-shaped hydrogel capsules (LentiKats(®)) was studied for production of butanol and other products of acetone-butanol-ethanol fermentation. After optimising the immobilisation protocol for anaerobic bacteria, continuous, repeated batch, and fed-batch fermentations in repeated batch mode were performed. Using glucose as a substrate, butanol productivity of 0.41 g/L/h and solvent productivity of 0.63 g/L/h were observed at a dilution rate of 0.05 h(-1) during continuous fermentation with a concentrated substrate (60 g/L). Through the process of repeated batch fermentation, the duration of fermentation was reduced from 27.8h (free-cell fermentation) to 3.3h (immobilised cells) with a solvent productivity of 0.77 g/L/h (butanol 0.57 g/L/h). The highest butanol and solvent productivities of 1.21 and 1.91 g/L/h were observed during fed-batch fermentation operated in repeated batch mode with yields of butanol (0.15 g/g) and solvents (0.24 g/g), respectively, produced per gram of glucose. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Enhanced butanol production by Clostridium acetobutylicum NCIMB 13357 grown on date fruit as carbon source in P2 medium.

    PubMed

    Khamaiseh, Emran I; Abdul Hamid, Aidil; Abdeshahian, Peyman; Wan Yusoff, Wan Mohtar; Kalil, Mohd Sahaid

    2014-01-01

    The production of biobutanol was studied by the cultivation of Clostridium acetobutylicum NCIMB 13557 in P2 medium including date fruit as the sole substrate. The effect of P2 medium and the effect of different concentrations of date fruit ranging from 10 to 100 g/L on biobutanol production were investigated. Anaerobic batch culture was carried out at 35 °C incubation temperature and pH 7.0 ± 0.2 for 72 h. Experimental results showed that the lowest yield of biobutanol and acetone-butanol-ethanol (ABE) was 0.32 and 0.35 gram per gram of carbohydrate consumed (g/g), respectively, when an initial date fruit concentration of 10 g/L was utilized. At this fruit date concentration a biobutanol production value of 1.56 g/L was obtained. On the other hand, the maximum yield of biobutanol (0.48 g/g) and ABE (0.63 g/g) was produced at 50 g/L date fruit concentration with a biobutanol production value as high as 11 g/L. However, when a higher initial date fruit concentration was used, biobutanol and ABE production decreased to reach the yield of 0.22 g/g and 0.35 g/g, respectively, where 100 g/L date fruit was used. Similar results also revealed that 10.03 g/L biobutanol was produced using 100 g/L date fruit.

  11. The Issue of Secretion in Heterologous Expression of Clostridium cellulolyticum Cellulase-Encoding Genes in Clostridium acetobutylicum ATCC 824▿

    PubMed Central

    Mingardon, Florence; Chanal, Angélique; Tardif, Chantal; Fierobe, Henri-Pierre

    2011-01-01

    The genes encoding the cellulases Cel5A, Cel8C, Cel9E, Cel48F, Cel9G, and Cel9M from Clostridium cellulolyticum were cloned in the C. acetobutylicum expression vector pSOS952 under the control of a Gram-positive constitutive promoter. The DNA encoding the native leader peptide of the heterologous cellulases was maintained. The transformation of the solventogenic bacterium with the corresponding vectors generated clones in the cases of Cel5A, Cel8C, and Cel9M. Analyses of the recombinant strains indicated that the three cellulases are secreted in an active form to the medium. A large fraction of the secreted cellulases, however, lost the C-terminal dockerin module. In contrast, with the plasmids pSOS952-cel9E, pSOS952-cel48F, and pSOS952-cel9G no colonies were obtained, suggesting that the expression of these genes has an inhibitory effect on growth. The deletion of the DNA encoding the leader peptide of Cel48F in pSOS952-cel48F, however, generated strains of C. acetobutylicum in which mature Cel48F accumulates in the cytoplasm. Thus, the growth inhibition observed when the wild-type cel48F gene is expressed seems related to the secretion of the cellulase. The weakening of the promoter, the coexpression of miniscaffoldin-encoding genes, or the replacement of the native signal sequence of Cel48F by that of secreted heterologous or endogenous proteins failed to generate strains secreting Cel48F. Taken together, our data suggest that a specific chaperone(s) involved in the secretion of the key family 48 cellulase, and probably Cel9G and Cel9E, is missing or insufficiently synthesized in C. acetobutylicum. PMID:21378034

  12. Molecular characterization of an aldehyde/alcohol dehydrogenase gene from Clostridium acetobutylicum ATCC 824.

    PubMed Central

    Nair, R V; Bennett, G N; Papoutsakis, E T

    1994-01-01

    A gene (aad) coding for an aldehyde/alcohol dehydrogenase (AAD) was identified immediately upstream of the previously cloned ctfA (J. W. Cary, D. J. Petersen, E. T. Papoutsakis, and G. N. Bennett, Appl. Environ. Microbiol. 56:1576-1583, 1990) of Clostridium acetobutylicum ATCC 824 and sequenced. The 2,619-bp aad codes for a 96,517-Da protein. Primer extension analysis identified two transcriptional start sites 83 and 243 bp upstream of the aad start codon. The N-terminal section of AAD shows homology to aldehyde dehydrogenases of bacterial, fungal, mammalian, and plant origin, while the C-terminal section shows homology to alcohol dehydrogenases of bacterial (which includes three clostridial alcohol dehydrogenases) and yeast origin. AAD exhibits considerable amino acid homology (56% identity) over its entire sequence to the trifunctional protein encoded by adhE from Escherichia coli. Expression of aad from a plasmid in C. acetobutylicum showed that AAD, which appears as a approximately 96-kDa band in denaturing protein gels, provides elevated activities of NADH-dependent butanol dehydrogenase, NAD-dependent acetaldehyde dehydrogenase and butyraldehyde dehydrogenase, and a small increase in NADH-dependent ethanol dehydrogenase. A 957-bp open reading frame that could potentially encode a 36,704-Da protein was identified upstream of aad. Images PMID:8300540

  13. Integrated, systems metabolic picture of acetone-butanol-ethanol fermentation by Clostridium acetobutylicum.

    PubMed

    Liao, Chen; Seo, Seung-Oh; Celik, Venhar; Liu, Huaiwei; Kong, Wentao; Wang, Yi; Blaschek, Hans; Jin, Yong-Su; Lu, Ting

    2015-07-07

    Microbial metabolism involves complex, system-level processes implemented via the orchestration of metabolic reactions, gene regulation, and environmental cues. One canonical example of such processes is acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum, during which cells convert carbon sources to organic acids that are later reassimilated to produce solvents as a strategy for cellular survival. The complexity and systems nature of the process have been largely underappreciated, rendering challenges in understanding and optimizing solvent production. Here, we present a system-level computational framework for ABE fermentation that combines metabolic reactions, gene regulation, and environmental cues. We developed the framework by decomposing the entire system into three modules, building each module separately, and then assembling them back into an integrated system. During the model construction, a bottom-up approach was used to link molecular events at the single-cell level into the events at the population level. The integrated model was able to successfully reproduce ABE fermentations of the WT C. acetobutylicum (ATCC 824), as well as its mutants, using data obtained from our own experiments and from literature. Furthermore, the model confers successful predictions of the fermentations with various network perturbations across metabolic, genetic, and environmental aspects. From foundation to applications, the framework advances our understanding of complex clostridial metabolism and physiology and also facilitates the development of systems engineering strategies for the production of advanced biofuels.

  14. Integrated, systems metabolic picture of acetone-butanol-ethanol fermentation by Clostridium acetobutylicum

    PubMed Central

    Liao, Chen; Seo, Seung-Oh; Celik, Venhar; Liu, Huaiwei; Kong, Wentao; Wang, Yi; Blaschek, Hans; Jin, Yong-Su; Lu, Ting

    2015-01-01

    Microbial metabolism involves complex, system-level processes implemented via the orchestration of metabolic reactions, gene regulation, and environmental cues. One canonical example of such processes is acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum, during which cells convert carbon sources to organic acids that are later reassimilated to produce solvents as a strategy for cellular survival. The complexity and systems nature of the process have been largely underappreciated, rendering challenges in understanding and optimizing solvent production. Here, we present a system-level computational framework for ABE fermentation that combines metabolic reactions, gene regulation, and environmental cues. We developed the framework by decomposing the entire system into three modules, building each module separately, and then assembling them back into an integrated system. During the model construction, a bottom-up approach was used to link molecular events at the single-cell level into the events at the population level. The integrated model was able to successfully reproduce ABE fermentations of the WT C. acetobutylicum (ATCC 824), as well as its mutants, using data obtained from our own experiments and from literature. Furthermore, the model confers successful predictions of the fermentations with various network perturbations across metabolic, genetic, and environmental aspects. From foundation to applications, the framework advances our understanding of complex clostridial metabolism and physiology and also facilitates the development of systems engineering strategies for the production of advanced biofuels. PMID:26100881

  15. Cell growth behaviors of Clostridium acetobutylicum in a pervaporation membrane bioreactor for butanol fermentation.

    PubMed

    Yao, Peina; Xiao, Zeyi; Chen, Chunyan; Li, Weijia; Deng, Qing

    2016-01-01

    Acetone-butanol-ethanol fermentation using Clostridium acetobutylicum was studied in the continuous and closed-circulating fermentation (CCCF) system. The experiment lasting for 192 H was carried out by integrating fermentation with in situ pervaporation. In the entire process, the cell growth profile took place in the following two phases: the logarithmic phase during early 28 H and the linear phase from 130 to 150 H. This was a unique characteristic compared with the curve of traditional fermentation, and the fitting equations of two growth phases were obtained by Origin software according to the kinetic model of cell growth. Besides, the kinetic parameters that include the butanol yield, maximum specific growth rate, average specific formation rate, and volumetric productivity of butanol were measured as 0.19 g g(-1) , 0.345 H(-1) , 0.134 H(-1) and 0.23 g L(-1)  H(-1) , respectively. The C. acetobutylicum in the CCCF system showed good adaptability and fermentation performance, and the prolonged fermentation period and high production were also the main advantages of CCCF technology. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

  16. High-Level Butanol Production from Cassava Starch by a Newly Isolated Clostridium acetobutylicum.

    PubMed

    Li, Shubo; Guo, Yuan; Lu, Fuzhi; Huang, Jiajian; Pang, Zongwen

    2015-10-01

    A new Clostridium acetobutylicum strain, exhibiting the ability to resist butanol stress and produce butanol, was identified and named GX01. Strain GX01 can use a wide variety of carbohydrates, especially cassava starch, to produce butanol. After the optimization of culture conditions, C. acetobutylicum GX01 could produce 27.3 g/L solvent, including 17.1 g/L butanol, 7.9 g/L acetone, and 2.3 g/L ethanol, from 100 g/L cassava flour and 3 g/L soybean meal. Furthermore, when its acetone-butanol-ethanol (ABE) fermentation was performed in 10- and 30-L bioreactors, the production of total solvent and butanol reached 29.2 and 18.3 g/L, respectively, and 28.8 and 18.8 g/L, respectively. Thus, the high level and stability of butanol production make strain GX01 a promising candidate for ABE fermentation using the low-cost cassava starch.

  17. Enhanced butanol production from cassava with Clostridium acetobutylicum by genome shuffling.

    PubMed

    Li, Shu-Bo; Qian, Yi; Liang, Zheng-Wu; Guo, Yuan; Zhao, Mou-Ming; Pang, Zong-Wen

    2016-04-01

    To obtain strains exhibiting high levels of solvent tolerance and butanol production, wild type strains of Clostridium acetobutylicum butanol-producing strain GX01 and Lactobacillus mucosae butanol-tolerant strain M26 were subjected to mutagenesis combining N-methyl-N-nitro-N-nitrosoguanidine induction with genome shuffling. After four successive rounds of genome shuffling, the C. acetobutylicum shuffled strain GS4-3 showing greater levels of fermentation performances (such as secreting a higher level of amylase, improving the thermal stability, and possessing greater environmental robustness) compared to the wild type strains was isolated. As a result, after optimization of culture conditions, mutant GS4-3 produced 32.6 g/L of total solvent, 20.1 g/L of butanol production, and 0.35 g/L/h of butanol productivity, which were, respectively, increased by 23.5, 23.3, and 40.0 % than the wild-type strain GX01, in a 10 L bioreactor. The enhanced production of butanol and tolerance of solvent of mutant associated with GS4-3 make it promising for acetone/butanol/ethanol fermentation from cassava (Manihot esculenta).

  18. Simultaneous production of butanol and acetoin by metabolically engineered Clostridium acetobutylicum.

    PubMed

    Liu, Dong; Chen, Yong; Ding, Fengying; Guo, Ting; Xie, Jingjing; Zhuang, Wei; Niu, Haunqing; Shi, Xinchi; Zhu, Chenjie; Ying, Hanjie

    2015-01-01

    Biobutanol is a potential fuel substitute and has been receiving increased attention in recent years. However, the economics of biobutanol production have been hampered by a number of bottlenecks such as high cost of raw material and low yield of solvent. Co-production of value-added products is a possible way to improve the economics of biobutanol production. Here, we present metabolic engineering strategies to substitute the major by-product acetone for a value-added product acetoin during butanol fermentation. By overexpressing the α-acetolactate decarboxylase gene alsD in Clostridium acetobutylicum B3, the acetoin yield was markedly increased while acetone formation was reduced. Subsequent disruption of adc gene effectively abolished acetone formation and further increased acetoin yield. After optimization of fermentation conditions, the alsD-overexpressing adc mutant generated butanol (13.8g/L), acetoin (4.3g/L), and ethanol (3.9g/L), but no acetone. Thus, acetone was completely substituted for acetoin, and both mass yield and product value were improved. This study provides valuable insights into the regulation of acetoin synthesis and should be highly useful for the development of acetoin-derived products like 2,3-butanediol and 2-butanol in C. acetobutylicum. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  19. Disruption of the acetate kinase (ack) gene of Clostridium acetobutylicum results in delayed acetate production.

    PubMed

    Kuit, Wouter; Minton, Nigel P; López-Contreras, Ana M; Eggink, Gerrit

    2012-05-01

    In microorganisms, the enzyme acetate kinase (AK) catalyses the formation of ATP from ADP by de-phosphorylation of acetyl phosphate into acetic acid. A mutant strain of Clostridium acetobutylicum lacking acetate kinase activity is expected to have reduced acetate and acetone production compared to the wild type. In this work, a C. acetobutylicum mutant strain with a selectively disrupted ack gene, encoding AK, was constructed and genetically and physiologically characterized. The ack (-) strain showed a reduction in acetate kinase activity of more than 97% compared to the wild type. The fermentation profiles of the ack (-) and wild-type strain were compared using two different fermentation media, CGM and CM1. The latter contains acetate and has a higher iron and magnesium content than CGM. In general, fermentations by the mutant strain showed a clear shift in the timing of peak acetate production relative to butyrate and had increased acid uptake after the onset of solvent formation. Specifically, in acetate containing CM1 medium, acetate production was reduced by more than 80% compared to the wild type under the same conditions, but both strains produced similar final amounts of solvents. Fermentations in CGM showed similar peak acetate and butyrate levels, but increased acetoin (60%), ethanol (63%) and butanol (16%) production and reduced lactate (-50%) formation by the mutant compared to the wild type. These findings are in agreement with the proposed regulatory function of butyryl phosphate as opposed to acetyl phosphate in the metabolic switch of solventogenic clostridia.

  20. Clostridium acetobutylicum Mutants That Produce Butyraldehyde and Altered Quantities of Solvents.

    PubMed

    Rogers, P; Palosaari, N

    1987-12-01

    Spontaneous mutants of Clostridium acetobutylicum NRRL B643 that were resistant to allyl alcohol (AA) were selected and characterized. These mutants contained 10- to 100-fold reduced activities of butanol and ethanol alcohol dehydrogenase. The AA mutants formed two groups and produced no ethanol. Type 1 AA mutants produced significant amounts of a new solvent, butyraldehyde, and contained normal levels of the coenzyme A-dependent butyraldehyde dehydrogenase (BAD). Type 2 AA mutants produced no significant butyraldehyde and lower levels of all solvents, and they contained 45- to 100-fold lower activity levels of BAD. Following ethyl methanesulfonate mutagenesis, low-acid-producing (Acid) mutants were selected and characterized as superinduced solvent producers, yielding more than 99% of theoretical glucose carbon as solvents and only small amounts of acetate and butyrate. Following ethyl methanesulfonate mutagenesis, 13 sporulation-negative (Spo) mutants were characterized; and 3 were found to produce only butyrate and acetate, a minor amount of acetone, and no alcohols. These Spo mutants contained reduced butanol dehydrogenase activity and no BAD enzyme activity. The data support the view that the type 2 AA, the Acid, and the Spo mutants somehow alter normal regulated expression of the solvent pathway in C. acetobutylicum.

  1. Novel substrate specificity of glutathione synthesis enzymes from Streptococcus agalactiae and Clostridium acetobutylicum

    SciTech Connect

    Kino, Kuniki . E-mail: kkino@waseda.jp; Kuratsu, Shoko; Noguchi, Atsushi; Kokubo, Masahiro; Nakazawa, Yuji; Arai, Toshinobu; Yagasaki, Makoto; Kirimura, Kohtaro

    2007-01-12

    Glutathione (GSH) is synthesized by {gamma}-glutamylcysteine synthetase ({gamma}-GCS) and glutathione synthetase (GS) in living organisms. Recently, bifunctional fusion protein, termed {gamma}-GCS-GS catalyzing both {gamma}-GCS and GS reactions from gram-positive firmicutes Streptococcus agalactiae, has been reported. We revealed that in the {gamma}-GCS activity, S. agalactiae {gamma}-GCS-GS had different substrate specificities from those of Escherichia coli {gamma}-GCS. Furthermore, S. agalactiae {gamma}-GCS-GS synthesized several kinds of {gamma}-glutamyltripeptide, {gamma}-Glu-X{sub aa}-Gly, from free three amino acids. In Clostridium acetobutylicum, the genes encoding {gamma}-GCS and putative GS were found to be immediately adjacent by BLAST search, and had amino acid sequence homology with S. agalactiae {gamma}-GCS-GS, respectively. We confirmed that the proteins expressed from each gene showed {gamma}-GCS and GS activity, respectively. C. acetobutylicum GS had broad substrate specificities and synthesized several kinds of {gamma}-glutamyltripeptide, {gamma}-Glu-Cys-X{sub aa}. Whereas the substrate specificities of {gamma}-GCS domain protein and GS domain protein of S. agalactiae {gamma}-GCS-GS were the same as those of S. agalactiae {gamma}-GCS-GS.

  2. A Quantitative System-Scale Characterization of the Metabolism of Clostridium acetobutylicum

    PubMed Central

    Yoo, Minyeong; Bestel-Corre, Gwenaelle; Croux, Christian; Riviere, Antoine; Meynial-Salles, Isabelle

    2015-01-01

    ABSTRACT Engineering industrial microorganisms for ambitious applications, for example, the production of second-generation biofuels such as butanol, is impeded by a lack of knowledge of primary metabolism and its regulation. A quantitative system-scale analysis was applied to the biofuel-producing bacterium Clostridium acetobutylicum, a microorganism used for the industrial production of solvent. An improved genome-scale model, iCac967, was first developed based on thorough biochemical characterizations of 15 key metabolic enzymes and on extensive literature analysis to acquire accurate fluxomic data. In parallel, quantitative transcriptomic and proteomic analyses were performed to assess the number of mRNA molecules per cell for all genes under acidogenic, solventogenic, and alcohologenic steady-state conditions as well as the number of cytosolic protein molecules per cell for approximately 700 genes under at least one of the three steady-state conditions. A complete fluxomic, transcriptomic, and proteomic analysis applied to different metabolic states allowed us to better understand the regulation of primary metabolism. Moreover, this analysis enabled the functional characterization of numerous enzymes involved in primary metabolism, including (i) the enzymes involved in the two different butanol pathways and their cofactor specificities, (ii) the primary hydrogenase and its redox partner, (iii) the major butyryl coenzyme A (butyryl-CoA) dehydrogenase, and (iv) the major glyceraldehyde-3-phosphate dehydrogenase. This study provides important information for further metabolic engineering of C. acetobutylicum to develop a commercial process for the production of n-butanol. PMID:26604256

  3. A two-plasmid inducible CRISPR/Cas9 genome editing tool for Clostridium acetobutylicum.

    PubMed

    Wasels, François; Jean-Marie, Jennifer; Collas, Florent; López-Contreras, Ana M; Lopes Ferreira, Nicolas

    2017-09-01

    CRISPR/Cas-based genetic engineering has revolutionised molecular biology in both eukaryotes and prokaryotes. Several tools dedicated to the genomic transformation of the Clostridium genus of Gram-positive bacteria have been described in the literature; however, the integration of large DNA fragments still remains relatively limited. In this study, a CRISPR/Cas9 genome editing tool using a two-plasmid strategy was developed for the solventogenic strain Clostridium acetobutylicum ATCC 824. Codon-optimised cas9 from Streptococcus pyogenes was placed under the control of an anhydrotetracycline-inducible promoter on one plasmid, while the gRNA expression cassettes and editing templates were located on a second plasmid. Through the sequential introduction of these vectors into the cell, we achieved highly accurate genome modifications, including nucleotide substitution, gene deletion and cassette insertion up to 3.6kb. To demonstrate its potential, this genome editing tool was used to generate a marker-free mutant of ATCC 824 that produced an isopropanol-butanol-ethanol mixture. Whole-genome sequencing confirmed that no off-target modifications were present in the mutants. Such a tool is a prerequisite for efficient metabolic engineering in this solventogenic strain and provides an alternative editing strategy that might be applicable to other Clostridium strains. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  4. Continuous xylose fermentation by Clostridium acetobutylicum--kinetics and energetics issues under acidogenesis conditions.

    PubMed

    Procentese, Alessandra; Raganati, Francesca; Olivieri, Giuseppe; Russo, Maria Elena; Salatino, Piero; Marzocchella, Antonio

    2014-07-01

    The paper reports the assessment of the growth kinetics of Clostridium acetobutylicum DSM 792 adopting xylose as carbon source. Xylose is the fundamental component of hemicellulose hydrolysis, a relevant fraction of lignocellulosic feedstocks for biofuel production. Tests were carried out in a CSTR operated under controlled pH. The effects of acids (acetic and butyric) and solvents (acetone, ethanol and butanol) on the fermentation were investigated. The conversion process was characterized under steady-state conditions in terms of concentration of xylose, cells, acids, and pH. The growth kinetics was expressed by means of a multiple product inhibition and it was able to predict microorganism growth rate under a broad interval of operating conditions, even those typical of solvents production. The mass fractional yield of biomass and products were expressed as a function of the specific growth rate taking into account the Pirt model. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. The mechanism of switching from an acidogenic to butanol-acetone fermentation by Clostridium acetobutylicum

    SciTech Connect

    Rogers, P.

    1992-01-01

    The overall objective of this project is to elucidate the detailed mechanism by which solvent-forming bacteria such as Clostridium acetobutylicum regulate the well-known shift in fermentation pathway between alcohol-acetone and organic acid production. It is desired to eventually isolate and describe: (1) the regulatory genes and protein elements that determine induction of synthesis of the solvent-pathway enzymes; and (2) how this regulation system interacts with the sporulatin induction and development program and with related pathways such as granulse and exopolysaccharide formation in clostridia. A working model forhow clostridial control systems work can be derived from recent research on stress systems in E. coli and sporulation in Bacillus subtilis.

  6. Continuous lactose fermentation by Clostridium acetobutylicum--assessment of acidogenesis kinetics.

    PubMed

    Napoli, Fabio; Olivieri, Giuseppe; Russo, Maria Elena; Marzocchella, Antonio; Salatino, Piero

    2011-01-01

    An assessment of the growth kinetics of acidogenic cells of Clostridium acetobutylicum DSM 792 is reported in the paper. Tests were carried out in a continuous stirred tank reactor under controlled conditions adopting a complex medium supplemented with lactose as carbon source to mimic cheese whey. The effects of acids (acetic and butyric), solvents (acetone, ethanol and butanol) and pH on the growth rate of acidogenic cells were assessed. The conversion process was characterized under steady-state conditions in terms of concentration of lactose, cells, acids, total organic carbon and pH. The growth kinetics was expressed by means of a multiple product inhibition and interacting model including a novel formulation to account for the role of pH. The model has the potential to predict microorganism growth rate under a broad interval of operating conditions, even those typical of solvents production.

  7. Reduction of 2,4,6-trinitrotoluene by Clostridium acetobutylicum through hydroxylamino-nitrotoluene intermediates

    SciTech Connect

    Hughes, J.B.; Wang, C.Y.; Bhadra, R.; Richardson, A.; Bennett, G.N.; Rudolph, F.B.

    1998-03-01

    Studies were conducted to isolate and identify intermediates of 2,4,6-trinitrotoluene (TNT) transformation by Clostridium acetobutylicum and to quantify their concentrations in active whole cell cultures. Only two intermediates of TNT reduction were detected in cell cultures and were identified as 4-hydroxylamino-2,6-dinitrotoluene and 2,4-dihydroxylamino-6-nitrotoluene. Structures were confirmed with {sup 1}H-NMR, {sup 13}C-NMR, and desorption chemical ionization mass spectroscopy. When cells were suspended in a non-growth saline medium, both hydroxylamine forms accumulated. In media capable of supporting cell growth, the 2,4-dihydroxylamino-6-nitrotoluene accumulated with concentrations of 4-hydroxylamino-2,6-dinitrotoluene remaining near detection limits. Studies using purified 2,4-dihydroxylamino-6-nitrotoluene confirmed that its biotransformation rate in active cultures greatly exceeded abiotic decomposition in aqueous medium.

  8. Nutritional factors affecting the ratio of solvents produced by Clostridium acetobutylicum

    SciTech Connect

    Bahl, H.; Gottwald, M.; Kuhn, A.; Rale, V.; Andersch, W.; Gottschalk, G.

    1986-07-01

    Fermentation of whey by Clostridium acetobutylicum yielded butanol and acetone in a ratio of approximately 100:1. This ratio amounted to only 2:1 in synthetic media with glucose, lactose, or glucose plus galactose as substrates. Removal of citrate from whey and addition of minerals resulted in an increase in the amount of acetone produced. Experiments carried out in a chemostat with a low-phosphate synthetic medium revealed that the butanol/acetone ratio could be increased from 2:1 to 3.8:1 by cofermentation of L-lactate and from 2:1 to 8:1 by iron limitation. The performance of the fermentation in a low-iron glucose medium above pH 5.1 yielded L-lactate as the main product. 42 references.

  9. Nutritional Factors Affecting the Ratio of Solvents Produced by Clostridium acetobutylicum

    PubMed Central

    Bahl, H.; Gottwald, M.; Kuhn, A.; Rale, V.; Andersch, W.; Gottschalk, G.

    1986-01-01

    Fermentation of whey by Clostridium acetobutylicum yielded butanol and acetone in a ratio of approximately 100:1. This ratio amounted to only 2:1 in synthetic media with glucose, lactose, or glucose plus galactose as substrates. Removal of citrate from whey and addition of minerals resulted in an increase in the amount of acetone produced. Experiments carried out in a chemostat with a low-phosphate synthetic medium revealed that the butanol/acetone ratio could be increased from 2:1 to 3.8:1 by cofermentation of l-lactate and from 2:1 to 8:1 by iron limitation. The performance of the fermentation in a low-iron glucose medium above pH 5.1 yielded l-lactate as the main product. PMID:16347104

  10. Metabolic engineering of Clostridium acetobutylicum for the enhanced production of isopropanol-butanol-ethanol fuel mixture.

    PubMed

    Jang, Yu-Sin; Malaviya, Alok; Lee, Joungmin; Im, Jung Ae; Lee, Sang Yup; Lee, Julia; Eom, Moon-Ho; Cho, Jung-Hee; Seung, Do Young

    2013-01-01

    Butanol is considered as a superior biofuel, which is conventionally produced by clostridial acetone-butanol-ethanol (ABE) fermentation. Among ABE, only butanol and ethanol can be used as fuel alternatives. Coproduction of acetone thus causes lower yield of fuel alcohols. Thus, this study aimed at developing an improved Clostridium acetobutylicum strain possessing enhanced fuel alcohol production capability. For this, we previously developed a hyper ABE producing BKM19 strain was further engineered to convert acetone into isopropanol. The BKM19 strain was transformed with the plasmid pIPA100 containing the sadh (primary/secondary alcohol dehydrogenase) and hydG (putative electron transfer protein) genes from the Clostridium beijerinckii NRRL B593 cloned under the control of the thiolase promoter. The resulting BKM19 (pIPA100) strain produced 27.9 g/l isopropanol-butanol-ethanol (IBE) as a fuel alcohols with negligible amount of acetone (0.4 g/l) from 97.8 g/l glucose in lab-scale (2 l) batch fermentation. Thus, this metabolically engineered strain was able to produce 99% of total solvent produced as fuel alcohols. The scalability and stability of BKM19 (pIPA100) were evaluated at 200 l pilot-scale fermentation, which showed that the fuel alcohol yield could be improved to 0.37 g/g as compared to 0.29 g/g obtained at lab-scale fermentation, while attaining a similar titer. To the best of our knowledge, this is the highest titer of IBE achieved and the first report on the large scale fermentation of C. acetobutylicum for IBE production. © 2013 American Institute of Chemical Engineers.

  11. Shotgun proteomic monitoring of Clostridium acetobutylicum during stationary phase of butanol fermentation using xylose and comparison with the exponential phase

    SciTech Connect

    Sivagnanam, Kumaran; Raghavan, Vijaya G. S.; Shah, Manesh B; Hettich, Robert {Bob} L; Verberkmoes, Nathan C; Lefsrud, Mark G

    2012-01-01

    Economically viable production of solvents through acetone butanol ethanol (ABE) fermentation requires a detailed understanding of Clostridium acetobutylicum. This study focuses on the proteomic profiling of C. acetobutylicum ATCC 824 from the stationary phase of ABE fermentation using xylose and compares with the exponential growth by shotgun proteomics approach. Comparative proteomic analysis revealed 22.9% of the C. acetobutylicum genome and 18.6% was found to be common in both exponential and stationary phases. The proteomic profile of C. acetobutylicum changed during the ABE fermentation such that 17 proteins were significantly differentially expressed between the two phases. Specifically, the expression of five proteins namely, CAC2873, CAP0164, CAP0165, CAC3298, and CAC1742 involved in the solvent production pathway were found to be significantly lower in the stationary phase compared to the exponential growth. Similarly, the expression of fucose isomerase (CAC2610), xylulose kinase (CAC2612), and a putative uncharacterized protein (CAC2611) involved in the xylose utilization pathway were also significantly lower in the stationary phase. These findings provide an insight into the metabolic behavior of C. acetobutylicum between different phases of ABE fermentation using xylose.

  12. Shotgun proteomic monitoring of Clostridium acetobutylicum during stationary phase of butanol fermentation using xylose and comparison with the exponential phase.

    PubMed

    Sivagnanam, Kumaran; Raghavan, Vijaya G S; Shah, Manesh; Hettich, Robert L; Verberkmoes, Nathan C; Lefsrud, Mark G

    2012-06-01

    Economically viable production of solvents through acetone-butanol-ethanol (ABE) fermentation requires a detailed understanding of Clostridium acetobutylicum. This study focuses on the proteomic profiling of C. acetobutylicum ATCC 824 from the stationary phase of ABE fermentation using xylose and compares with the exponential growth by shotgun proteomics approach. Comparative proteomic analysis revealed 22.9% of the C. acetobutylicum genome and 18.6% was found to be common in both exponential and stationary phases. The proteomic profile of C. acetobutylicum changed during the ABE fermentation such that 17 proteins were significantly differentially expressed between the two phases. Specifically, the expression of five proteins namely, CAC2873, CAP0164, CAP0165, CAC3298, and CAC1742 involved in the solvent production pathway were found to be significantly lower in the stationary phase compared to the exponential growth. Similarly, the expression of fucose isomerase (CAC2610), xylulose kinase (CAC2612), and a putative uncharacterized protein (CAC2611) involved in the xylose utilization pathway were also significantly lower in the stationary phase. These findings provide an insight into the metabolic behavior of C. acetobutylicum between different phases of ABE fermentation using xylose.

  13. Quantitative proteomic analysis of the influence of lignin on biofuel production by Clostridium acetobutylicum ATCC 824.

    PubMed

    Raut, Mahendra P; Couto, Narciso; Pham, Trong K; Evans, Caroline; Noirel, Josselin; Wright, Phillip C

    2016-01-01

    Clostridium acetobutylicum has been a focus of research because of its ability to produce high-value compounds that can be used as biofuels. Lignocellulose is a promising feedstock, but the lignin-cellulose-hemicellulose biomass complex requires chemical pre-treatment to yield fermentable saccharides, including cellulose-derived cellobiose, prior to bioproduction of acetone-butanol-ethanol (ABE) and hydrogen. Fermentation capability is limited by lignin and thus process optimization requires knowledge of lignin inhibition. The effects of lignin on cellular metabolism were evaluated for C. acetobutylicum grown on medium containing either cellobiose only or cellobiose plus lignin. Microscopy, gas chromatography and 8-plex iTRAQ-based quantitative proteomic technologies were applied to interrogate the effect of lignin on cellular morphology, fermentation and the proteome. Our results demonstrate that C. acetobutylicum has reduced performance for solvent production when lignin is present in the medium. Medium supplemented with 1 g L(-1) of lignin led to delay and decreased solvents production (ethanol; 0.47 g L(-1) for cellobiose and 0.27 g L(-1) for cellobiose plus lignin and butanol; 0.13 g L(-1) for cellobiose and 0.04 g L(-1) for cellobiose plus lignin) at 20 and 48 h, respectively, resulting in the accumulation of acetic acid and butyric acid. Of 583 identified proteins (FDR < 1 %), 328 proteins were quantified with at least two unique peptides. Up- or down-regulation of protein expression was determined by comparison of exponential and stationary phases of cellobiose in the presence and absence of lignin. Of relevance, glycolysis and fermentative pathways were mostly down-regulated, during exponential and stationary growth phases in presence of lignin. Moreover, proteins involved in DNA repair, transcription/translation and GTP/ATP-dependent activities were also significantly affected and these changes were associated with altered cell morphology

  14. Engineering Clostridium acetobutylicum for production of kerosene and diesel blendstock precursors.

    PubMed

    Bormann, Sebastian; Baer, Zachary C; Sreekumar, Sanil; Kuchenreuther, Jon M; Dean Toste, F; Blanch, Harvey W; Clark, Douglas S

    2014-09-01

    Processes for the biotechnological production of kerosene and diesel blendstocks are often economically unattractive due to low yields and product titers. Recently, Clostridium acetobutylicum fermentation products acetone, butanol, and ethanol (ABE) were shown to serve as precursors for catalytic upgrading to higher chain-length molecules that can be used as fuel substitutes. To produce suitable kerosene and diesel blendstocks, the butanol:acetone ratio of fermentation products needs to be increased to 2-2.5:1, while ethanol production is minimized. Here we show that the overexpression of selected proteins changes the ratio of ABE products relative to the wild type ATCC 824 strain. Overexpression of the native alcohol/aldehyde dehydrogenase (AAD) has been reported to primarily increase ethanol formation in C. acetobutylicum. We found that overexpression of the AAD(D485G) variant increased ethanol titers by 294%. Catalytic upgrading of the 824(aad(D485G)) ABE products resulted in a blend with nearly 50wt%≤C9 products, which are unsuitable for diesel. To selectively increase butanol production, C. beijerinckii aldehyde dehydrogenase and C. ljungdhalii butanol dehydrogenase were co-expressed (strain designate 824(Cb ald-Cl bdh)), which increased butanol titers by 27% to 16.9gL(-1) while acetone and ethanol titers remained essentially unaffected. The solvent ratio from 824(Cb ald-Cl bdh) resulted in more than 80wt% of catalysis products having a carbon chain length≥C11 which amounts to 9.8gL(-1) of products suitable as kerosene or diesel blendstock based on fermentation volume. To further increase solvent production, we investigated expression of both native and heterologous chaperones in C. acetobutylicum. Expression of a heat shock protein (HSP33) from Bacillus psychrosaccharolyticus increased the total solvent titer by 22%. Co-expression of HSP33 and aldehyde/butanol dehydrogenases further increased ABE formation as well as acetone and butanol yields. HSP33 was

  15. Construction of a restriction-less, marker-less mutant useful for functional genomic and metabolic engineering of the biofuel producer Clostridium acetobutylicum.

    PubMed

    Croux, Christian; Nguyen, Ngoc-Phuong-Thao; Lee, Jieun; Raynaud, Céline; Saint-Prix, Florence; Gonzalez-Pajuelo, Maria; Meynial-Salles, Isabelle; Soucaille, Philippe

    2016-01-01

    Clostridium acetobutylicum is a gram-positive, spore-forming, anaerobic bacterium capable of converting various sugars and polysaccharides into solvents (acetone, butanol, and ethanol). The sequencing of its genome has prompted new approaches to genetic analysis, functional genomics, and metabolic engineering to develop industrial strains for the production of biofuels and bulk chemicals. The method used in this paper to knock-out or knock-in genes in C. acetobutylicum combines the use of an antibiotic-resistance gene for the deletion or replacement of the target gene, the subsequent elimination of the antibiotic-resistance gene with the flippase recombinase system from Saccharomyces cerevisiae, and a C. acetobutylicum strain that lacks upp, which encodes uracil phosphoribosyl-transferase, for subsequent use as a counter-selectable marker. A replicative vector containing (1) a pIMP13 origin of replication from Bacillus subtilis that is functional in Clostridia, (2) a replacement cassette consisting of an antibiotic resistance gene (MLS (R) ) flanked by two FRT sequences, and (3) two sequences homologous to selected regions around target DNA sequence was first constructed. This vector was successfully used to consecutively delete the Cac824I restriction endonuclease encoding gene (CA_C1502) and the upp gene (CA_C2879) in the C. acetobutylicum ATCC824 chromosome. The resulting C. acetobutylicum Δcac1502Δupp strain is marker-less, readily transformable without any previous plasmid methylation and can serve as the host for the "marker-less" genetic exchange system. The third gene, CA_C3535, shown in this study to encode for a type II restriction enzyme (Cac824II) that recognizes the CTGAAG sequence, was deleted using an upp/5-FU counter-selection strategy to improve the efficiency of the method. The restriction-less marker-less strain and the method was successfully used to delete two genes (ctfAB) on the pSOL1 megaplasmid and one gene (ldhA) on the chromosome to get

  16. High-efficient n-butanol production by co-culturing Clostridium acetobutylicum and Saccharomyces cerevisiae integrated with butyrate fermentative supernatant addition.

    PubMed

    Luo, Hongzhen; Zeng, Qingwei; Han, Shuo; Wang, Zhaoyu; Dong, Qing; Bi, Yanhong; Zhao, Yuping

    2017-04-01

    Butanol is not only an important chemical intermediate and solvent in pharmaceutical and cosmetics industries, but also considered as an advanced biofuel. Although species of the natural host Clostridium have been engineered, butanol titers in the anaerobe seem to be limited by its intolerance to butanol less than 13 g/L. Here we aimed to develop a technology for enhancing butanol production by a co-culture system with butyrate fermentative supernatant addition. First, when adding 4.0 g/L butyrate into the acetone-butanol-ethanol (ABE) fermentation broth with single-shot at 24 h, the "acid crash" phenomenon occurred and the ABE fermentation performance deteriorated. Subsequently, we found that adding certain amino acids could effectively enhance butyrate re-assimilation, butanol tolerance and titer (from 11.1 to 14.8 g/L). Additionally, in order to decrease the raw material cost, butyrate fermentative supernatant produced by Clostridium tyrobutyricum was applied to butanol production in the Clostridium acetobutylicum/Saccharomyces cerevisiae co-culture system, instead of adding synthetic butyrate. Final butanol and total ABE concentrations reached higher levels of 16.3 and 24.8 g/L with increments of 46.8 and 37.8%, respectively. These results show that the proposed fermentation strategy has great potential for efficiently butanol production with an economic approach.

  17. Genome shuffling of Clostridium acetobutylicum CICC 8012 for improved production of acetone-butanol-ethanol (ABE).

    PubMed

    Gao, Xiaofeng; Zhao, Hai; Zhang, Guohua; He, Kaize; Jin, Yanling

    2012-08-01

    Genome shuffling was applied to increase ABE production of the strict anaerobe C. acetobutylicum CICC 8012. By using physical and chemical mutagenesis, strains with superior streptomycin sulfate, 2-deoxy-D-glucose and butanol tolerance levels were isolated. These strains were used for genome shuffling. The best performing strain F2-GA was screened after two rounds of genome shuffling. With 55 g glucose/l as carbon source, F2-GA produced 22.21 g ABE/l in 72 h and ABE yield reached 0.42 g/g which was about 34.53 % improvement compared to the wild type. Fermentation parameters and gene expression of several key enzymes in ABE metabolic pathways were varied significantly between F2-GA and the wild type. These results demonstrated the potential use of genome shuffling to microbial breeding which were difficult to deal with traditional methods.

  18. Enhanced butanol production obtained by reinforcing the direct butanol-forming route in Clostridium acetobutylicum.

    PubMed

    Jang, Yu-Sin; Lee, Jin Young; Lee, Joungmin; Park, Jin Hwan; Im, Jung Ae; Eom, Moon-Ho; Lee, Julia; Lee, Sang-Hyun; Song, Hyohak; Cho, Jung-Hee; Seung, Do Young; Lee, Sang Yup

    2012-10-23

    Butanol is an important industrial solvent and advanced biofuel that can be produced by biphasic fermentation by Clostridium acetobutylicum. It has been known that acetate and butyrate first formed during the acidogenic phase are reassimilated to form acetone-butanol-ethanol (cold channel). Butanol can also be formed directly from acetyl-coenzyme A (CoA) through butyryl-CoA (hot channel). However, little is known about the relative contributions of the two butanol-forming pathways. Here we report that the direct butanol-forming pathway is a better channel to optimize for butanol production through metabolic flux and mass balance analyses. Butanol production through the hot channel was maximized by simultaneous disruption of the pta and buk genes, encoding phosphotransacetylase and butyrate kinase, while the adhE1(D485G) gene, encoding a mutated aldehyde/alcohol dehydrogenase, was overexpressed. The ratio of butanol produced through the hot channel to that produced through the cold channel increased from 2.0 in the wild type to 18.8 in the engineered BEKW(pPthlAAD(**)) strain. By reinforcing the direct butanol-forming flux in C. acetobutylicum, 18.9 g/liter of butanol was produced, with a yield of 0.71 mol butanol/mol glucose by batch fermentation, levels which are 160% and 245% higher than those obtained with the wild type. By fed-batch culture of this engineered strain with in situ recovery, 585.3 g of butanol was produced from 1,861.9 g of glucose, with the yield of 0.76 mol butanol/mol glucose and productivity of 1.32 g/liter/h. Studies of two butanol-forming routes and their effects on butanol production in C. acetobutylicum described here will serve as a basis for further metabolic engineering of clostridia aimed toward developing a superior butanol producer. IMPORTANCE Renewable biofuel is one of the answers to solving the energy crisis and climate change problems. Butanol produced naturally by clostridia has superior liquid fuel characteristics and thus has

  19. Expression of Clostridium acetobutylicum ATCC 824 Genes in Escherichia coli for Acetone Production and Acetate Detoxification

    PubMed Central

    Bermejo, Lourdes L.; Welker, Neil E.; Papoutsakis, Eleftherios T.

    1998-01-01

    A synthetic acetone operon (ace4) composed of four Clostridium acetobutylicum ATCC 824 genes (adc, ctfAB, and thl, coding for the acetoacetate decarboxylase, coenzyme A transferase, and thiolase, respectively) under the control of the thl promoter was constructed and was introduced into Escherichia coli on vector pACT. Acetone production demonstrated that ace4 is expressed in E. coli and resulted in the reduction of acetic acid levels in the fermentation broth. Since different E. coli strains vary significantly in their growth characteristics and acetate metabolism, ace4 was expressed in three E. coli strains: ER2275, ATCC 11303, and MC1060. Shake flask cultures of MC1060(pACT) produced ca. 2 mM acetone, while both strains ER2275(pACT) and ATCC 11303(pACT) produced ca. 40 mM acetone. Glucose-fed cultures of strain ATCC 11303(pACT) resulted in a 150% increase in acetone titers compared to those of batch shake flask cultures. External addition of sodium acetate to glucose-fed cultures of ATCC 11303(pACT) resulted in further increased acetone titers. In bioreactor studies, acidic conditions (pH 5.5 versus 6.5) improved acetone production. Despite the substantial acetone evaporation due to aeration and agitation in the bioreactor, 125 to 154 mM acetone accumulated in ATCC 11303(pACT) fermentations. These acetone titers are equal to or higher than those produced by wild-type C. acetobutylicum. This is the first study to demonstrate the ability to use clostridial genes in nonclostridial hosts for solvent production. In addition, acetone-producing E. coli strains may be useful hosts for recombinant protein production in that detrimental acetate accumulation can be avoided. PMID:9501448

  20. Structural Variation in Bacterial Glyoxalase I Enzymes: Investigation of the Metalloenzyme Glyoxalase I from Clostridium acetobutylicum

    SciTech Connect

    Suttisansanee U.; Swaminathan S.; Lau, K.; Lagishetty, S.; Rao, K. N.; Sauder, J. M.; Burley, S. K.; Honek, J. F.

    2011-11-04

    The glyoxalase system catalyzes the conversion of toxic, metabolically produced {alpha}-ketoaldehydes, such as methylglyoxal, into their corresponding nontoxic 2-hydroxycarboxylic acids, leading to detoxification of these cellular metabolites. Previous studies on the first enzyme in the glyoxalase system, glyoxalase I (GlxI), from yeast, protozoa, animals, humans, plants, and Gram-negative bacteria, have suggested two metal activation classes, Zn{sup 2+} and non-Zn{sup 2+} activation. Here, we report a biochemical and structural investigation of the GlxI from Clostridium acetobutylicum, which is the first GlxI enzyme from Gram-positive bacteria that has been fully characterized as to its three-dimensional structure and its detailed metal specificity. It is a Ni{sup 2+}/Co{sup 2+}-activated enzyme, in which the active site geometry forms an octahedral coordination with one metal atom, two water molecules, and four metal-binding ligands, although its inactive Zn{sup 2+}-bound form possesses a trigonal bipyramidal geometry with only one water molecule liganded to the metal center. This enzyme also possesses a unique dimeric molecular structure. Unlike other small homodimeric GlxI where two active sites are located at the dimeric interface, the C. acetobutylicum dimeric GlxI enzyme also forms two active sites but each within single subunits. Interestingly, even though this enzyme possesses a different dimeric structure from previously studied GlxI, its metal activation characteristics are consistent with properties of other GlxI. These findings indicate that metal activation profiles in this class of enzyme hold true across diverse quaternary structure arrangements.

  1. Purification and characterization of the extracellular. alpha. -amylase from Clostridium acetobutylicum ATCC 824

    SciTech Connect

    Paquet, V.; Croux, C.; Goma, G.; Soucaille, P. )

    1991-01-01

    The extracellular {alpha}-amylase (1,4-{alpha}-D-glucanglucanohydrolase; EC 3.2.1.1) from Clostridium acetobutylicum ATCC 824 was purified to homogeneity by anion-exchange chromatography (Mono Q) and gel filtration (Superose 12). The enzyme had an isoelectric point of 4.7 and a molecular weight of 84,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was a monomeric protein, the 19-amino-acid N terminus of which displayed 42% homology with the Bacillus subtilis saccharifying {alpha}-amylase. The amino acid composition of the enzyme showed a high number of acidic and hydrophobic residues and only one cysteine residue per mole. The activity of the {alpha}-amylase was not stimulated by calcium ions (or other metal ions) or inhibited by EDTA, although the enzyme contained seven calcium atoms per molecule. {alpha}-Amylase activity on soluble starch was optimal at pH 5.6 and 45{degree}C. The {alpha}-amylase was stable at an acidic pH but very sensitive to thermal inactivation. It hydrolyzed soluble starch, with a K{sub m} of 3.6 g {center dot} liter{sup {minus}1} and a K{sub cat} of 122 mol of reducing sugars {center dot} s{sup {minus}1} {center dot} mol{sup {minus}1}. The {alpha}-amylase showed greater activity with high-molecular-weight substrates than with low-molecular-weight maltooligosaccharides, hydrolyzed glycogen and pullulan slowly, but did not hydrolyze dextran or cyclodextrins. The major end products of maltohexaose degradation were glucose, maltose, and maltotriose; maltotetraose and maltopentaose were formed as intermediate products. Twenty seven percent of the glucoamylase activity generally detected in the culture supernatant of C. acetobutylicum can be attributed to the {alpha}-amylase.

  2. Enhanced Butanol Production Obtained by Reinforcing the Direct Butanol-Forming Route in Clostridium acetobutylicum

    PubMed Central

    Jang, Yu-Sin; Lee, Jin Young; Lee, Joungmin; Park, Jin Hwan; Im, Jung Ae; Eom, Moon-Ho; Lee, Julia; Lee, Sang-Hyun; Song, Hyohak; Cho, Jung-Hee; Seung, Do Young; Lee, Sang Yup

    2012-01-01

    ABSTRACT Butanol is an important industrial solvent and advanced biofuel that can be produced by biphasic fermentation by Clostridium acetobutylicum. It has been known that acetate and butyrate first formed during the acidogenic phase are reassimilated to form acetone-butanol-ethanol (cold channel). Butanol can also be formed directly from acetyl-coenzyme A (CoA) through butyryl-CoA (hot channel). However, little is known about the relative contributions of the two butanol-forming pathways. Here we report that the direct butanol-forming pathway is a better channel to optimize for butanol production through metabolic flux and mass balance analyses. Butanol production through the hot channel was maximized by simultaneous disruption of the pta and buk genes, encoding phosphotransacetylase and butyrate kinase, while the adhE1D485G gene, encoding a mutated aldehyde/alcohol dehydrogenase, was overexpressed. The ratio of butanol produced through the hot channel to that produced through the cold channel increased from 2.0 in the wild type to 18.8 in the engineered BEKW(pPthlAAD**) strain. By reinforcing the direct butanol-forming flux in C. acetobutylicum, 18.9 g/liter of butanol was produced, with a yield of 0.71 mol butanol/mol glucose by batch fermentation, levels which are 160% and 245% higher than those obtained with the wild type. By fed-batch culture of this engineered strain with in situ recovery, 585.3 g of butanol was produced from 1,861.9 g of glucose, with the yield of 0.76 mol butanol/mol glucose and productivity of 1.32 g/liter/h. Studies of two butanol-forming routes and their effects on butanol production in C. acetobutylicum described here will serve as a basis for further metabolic engineering of clostridia aimed toward developing a superior butanol producer. PMID:23093384

  3. Solvents Production from a Mixture of Glucose and Xylose by Mixed Fermentation of Clostridium acetobutylicum and Saccharomyces cerevisiae.

    PubMed

    Qi, Gao-Xiang; Xiong, Lian; Huang, Chao; Chen, Xue-Fang; Lin, Xiao-Qing; Chen, Xin-De

    2015-10-01

    To overcome the xylose utilization defect in ethanol fermentation by wide-type Saccharomyces cerevisiae and alleviate the carbon catabolite repression (CCR) in acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum, a novel mixed fermentation of S. cerevisiae and C. acetobutylicum was developed. When S. cerevisiae was inoculated 24 h earlier than C. acetobutylicum CH02, a higher solvents yield was achieved with 0.41 g/g, compared to 0.38 g/g in ABE fermentation, and when S. cerevisiae and C. acetobutylicum CH02 were inoculated simultaneously, a higher productivity was achieved with 0.32 g/L/h, compared to 0.15 g/L/h in ABE fermentation. The total solvents yield was improved by the high ethanol yield from glucose. The CCR in mixed fermentation was alleviated when glucose was utilized quickly by S. cerevisiae, and therefore, the productivity was improved. This study suggests that mixed fermentation is an effective solvents production method from a mixture of glucose and xylose.

  4. Combined overexpression of genes involved in pentose phosphate pathway enables enhanced D-xylose utilization by Clostridium acetobutylicum.

    PubMed

    Jin, Lin; Zhang, Hui; Chen, Liwen; Yang, Chen; Yang, Sheng; Jiang, Weihong; Gu, Yang

    2014-03-10

    D-Xylose utilization by Clostridium acetobutylicum, an important industrial microorganism used in ABE (Acetone, Butanol and Ethanol) production, has attracted increasing interests. We demonstrated previously that co-overexpression of genes, encoding d-xylose symporter, D-xylose isomerase and xylulokinase, improved D-xylose utilization by C. acetobutylicum (Xiao, H., et al., 2011. Applied and Environmental Microbiology 77, 7886-7895). Here, we further identified genes involved in PPP (Pentose Phosphate Pathway) in C. acetobutylicum and evaluated their contribution to d-xylose utilization. Among all the candidate genes, the CAC1347, CAC1348, CAC1730 and CAC2880 were validated to encode genes tal, tkl, rpe and rpi, four key genes involved in PPP, respectively. The following combined overexpression of these genes conferred a significantly improved xylose-utilizing ability to the recombinant strain, reaching a solvent titer 42% higher than that of the wild-type strain. This finding offers a useful strategy to optimize d-xylose utilization by C. acetobutylicum. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. d-2,3-Butanediol Production Due to Heterologous Expression of an Acetoin Reductase in Clostridium acetobutylicum ▿ †

    PubMed Central

    Siemerink, Marco A. J.; Kuit, Wouter; López Contreras, Ana M.; Eggink, Gerrit; van der Oost, John; Kengen, Servé W. M.

    2011-01-01

    Acetoin reductase (ACR) catalyzes the conversion of acetoin to 2,3-butanediol. Under certain conditions, Clostridium acetobutylicum ATCC 824 (and strains derived from it) generates both d- and l-stereoisomers of acetoin, but because of the absence of an ACR enzyme, it does not produce 2,3-butanediol. A gene encoding ACR from Clostridium beijerinckii NCIMB 8052 was functionally expressed in C. acetobutylicum under the control of two strong promoters, the constitutive thl promoter and the late exponential adc promoter. Both ACR-overproducing strains were grown in batch cultures, during which 89 to 90% of the natively produced acetoin was converted to 20 to 22 mM d-2,3-butanediol. The addition of a racemic mixture of acetoin led to the production of both d-2,3-butanediol and meso-2,3-butanediol. A metabolic network that is in agreement with the experimental data is proposed. Native 2,3-butanediol production is a first step toward a potential homofermentative 2-butanol-producing strain of C. acetobutylicum. PMID:21335380

  6. D-2,3-butanediol production due to heterologous expression of an acetoin reductase in Clostridium acetobutylicum.

    PubMed

    Siemerink, Marco A J; Kuit, Wouter; López Contreras, Ana M; Eggink, Gerrit; van der Oost, John; Kengen, Servé W M

    2011-04-01

    Acetoin reductase (ACR) catalyzes the conversion of acetoin to 2,3-butanediol. Under certain conditions, Clostridium acetobutylicum ATCC 824 (and strains derived from it) generates both d- and l-stereoisomers of acetoin, but because of the absence of an ACR enzyme, it does not produce 2,3-butanediol. A gene encoding ACR from Clostridium beijerinckii NCIMB 8052 was functionally expressed in C. acetobutylicum under the control of two strong promoters, the constitutive thl promoter and the late exponential adc promoter. Both ACR-overproducing strains were grown in batch cultures, during which 89 to 90% of the natively produced acetoin was converted to 20 to 22 mM d-2,3-butanediol. The addition of a racemic mixture of acetoin led to the production of both d-2,3-butanediol and meso-2,3-butanediol. A metabolic network that is in agreement with the experimental data is proposed. Native 2,3-butanediol production is a first step toward a potential homofermentative 2-butanol-producing strain of C. acetobutylicum.

  7. Development of real-time PCR primer and probe sets for detecting degenerated and non-degenerated forms of the butanol-producing bacterium Clostridium acetobutylicum ATCC 824.

    PubMed

    Lee, Sun-Mi; Cho, Min Ok; Um, Youngsoon; Sang, Byoung-In

    2010-05-01

    Degeneration is one of the limiting factors in butanol fermentation, and it must be monitored and prevented for stable butanol production. In Clostridium acetobutylicum ATCC 824, the most well-known butanol-producing microorganism, degeneration is caused by the loss of the pSOL1 plasmid that carries essential genes involved in solvent production. In this study, we designed two specific primer and probe sets for real-time qPCR (RT-qPCR) detection of C. acetobutylicum ATCC 824 (the C. aceto set) and pSOL1-possessing C. acetobutylicum ATCC 824 (the DGS set). Specific primer and probe sets were designed on the basis of the 16S rDNA sequence and pSOL1 sequence. The number of degenerated C. acetobutylicum could be quantified by subtracting the number of C. acetobutylicum ATCC 824 containing pSOL1 from the total number of C. acetobutylicum ATCC 824. The primer and probe sets permitted the specific detection and quantification of degenerated C. acetobutylicum and total butanol-producing C. acetobutylicum by RT-qPCR.

  8. Expression and nucleotide sequence of the Clostridium acetobutylicum beta-galactosidase gene cloned in Escherichia coli.

    PubMed Central

    Hancock, K R; Rockman, E; Young, C A; Pearce, L; Maddox, I S; Scott, D B

    1991-01-01

    A gene library for Clostridium acetobutylicum NCIB 2951 was constructed in the broad-host-range cosmid pLAFR1, and cosmids containing the beta-galactosidase gene were isolated by direct selection for enzyme activity on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) plates after conjugal transfer of the library to a lac deletion derivative of Escherichia coli. Analysis of various pSUP202 subclones of the lac cosmids on X-Gal plates localized the beta-galactosidase gene to a 5.1-kb EcoRI fragment. Expression of the Clostridium beta-galactosidase gene in E. coli was not subject to glucose repression. By using transposon Tn5 mutagenesis, two gene loci, cbgA (locus I) and cbgR (locus II), were identified as necessary for beta-galactosidase expression in E. coli. DNA sequence analysis of the entire 5.1-kb fragment identified open reading frames of 2,691 and 303 bp, corresponding to locus I and locus II, respectively, and in addition a third truncated open reading frame of 825 bp. The predicted gene product of locus I, CbgA (molecular size, 105 kDa), showed extensive amino acid sequence homology with E. coli LacZ, E. coli EbgA, and Klebsiella pneumoniae LacZ and was in agreement with the size of a polypeptide synthesized in maxicells containing the cloned 5.1-kb fragment. The predicted gene product of locus II, CbgR (molecular size, 11 kDa) shares no significant homology with any other sequence in the current DNA and protein sequence data bases, but Tn5 insertions in this gene prevent the synthesis of CbgA. Complementation experiments indicate that the gene product of cbgR is required in cis with cbgA for expression of beta-galactosidase in E. coli. Images PMID:1850729

  9. Purification and characterization of acidolysin, an acidic metalloprotease produced by Clostridium acetobutylicum ATCC 824.

    PubMed Central

    Croux, C; Paquet, V; Goma, G; Soucaille, P

    1990-01-01

    Acidolysin an extracellular protease produced by Clostridium acetobutylicum ATCC 824 was purified to homogeneity by anion-exchange chromatography with a recovery of 91%. The enzyme was a monomeric protein with a molecular weight of 44,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an acidic isoelectric point of 3.3. Acidolysin was very sensitive to metal-chelating agents and phosphoramidon and was unaffected by sulfhydryl reagents. It was shown to be a calcium- and zinc-containing protease. It exhibited optimal activity against Azocoll at pH 5 and 45 degrees C. It was stable at low pH and heat labile above 50 degrees C. It exhibited specificity toward peptide bonds formed by the amino group of hydrophobic amino acids (isoleucine, leucine, and phenylalanine) and its NH2-terminal amino acid sequence showed a high degree of similarity with that of Bacillus subtilis neutral metalloprotease A. Acidolysin is the first phosphoramidon-sensitive, acidic zinc metalloprotease reported. Images PMID:2082818

  10. Promotion of the Clostridium acetobutylicum ATCC 824 growth and acetone-butanol-ethanol fermentation by flavonoids.

    PubMed

    Wang, Lan; Xia, Menglei; Zhang, Lianhua; Chen, Hongzhang

    2014-07-01

    An unexpected promotion effect of Ginkgo leaf on the growth of Clostridium acetobutylicum ATCC 824 and acetone-butanol-ethanol (ABE) fermentation was investigated. Component analysis of Ginkgo leaf was carried out and flavonoids were determined as the potential key metabolites. Then the flavonoids feeding experiments were carried out. Results showed that addition of only 10 mg/L flavonoids to the fermentation broth can promote butanol and ABE titre up to 14.5 and 17.8 g/L after 5 days of fermentation, that is, 74 and 68% higher than the control. A 2.2-fold biomass also has been achieved. Furthermore, by employing such novel founding, we easily exploited flavonoids from soybean and some agriculture wastes as the wide-distributed and economic feasible ABE fermentation promoter. The mechanism of the above effects was investigated from the perspective of oxidation-reduction potential. This work opens a new way in the efforts to increase the titer of butanol.

  11. Enhanced butanol fermentation using metabolically engineered Clostridium acetobutylicum with ex situ recovery of butanol.

    PubMed

    Lee, Sang-Hyun; Kim, Sooah; Kim, Jung Yeon; Cheong, Nam Yong; Kim, Kyoung Heon

    2016-10-01

    In this study, metabolic target reactions for strain engineering were searched via intracellular coenzyme A (CoA) metabolite analysis. The metabolic reactions catalyzed by thiolase (AtoB) and aldehyde-alcohol dehydrogenase (AdhE1) were considered potential rate-limiting steps. In addition, CoA transferase (CtfAB) was highlighted as being important for the assimilation of organic acids, in order to achieve high butanol production. Based on this quantitative analysis, the BEKW_E1AB-atoB strain was constructed by overexpressing the thl (atoB), adhE1, and ctfAB genes in Clostridium acetobutylicum strain BEKW, which has the phosphotransacetylase (pta) and butyrate kinase (buk) genes knocked out. After 100h of continuous fermentation coupled with adsorptive ex situ butanol recovery, the concentrations found after considering desorption, yield, and productivity for the BEKW_E1AB-atoB strain were 55.7g/L, 0.38g/g, and 2.64g/L/h, respectively. The level of butanol production achieved (2.64g/L/h) represents the highest reported value obtained after adsorptive, long-term fermentation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Effects of H2 and electrochemical reducing power on metabolite production by Clostridium acetobutylicum KCTC1037.

    PubMed

    Jeon, Boyoung; Yi, Junyeong; Park, Doohyun

    2014-01-01

    A conventional fermenter (CF), a single-cathode fermenter (SCF), and a double-cathode fermenter (DCF) were employed to evaluate and compare the effects of H2 and electrochemical reducing power on metabolite production by Clostridium acetobutylicum KCTC1037. The source of the external reducing power for CF was H2, for the SCF was electrochemically reduced neutral red-modified graphite felt electrode (NR-GF), and for the DCF was electrochemically reduced combination of NR-GF and platinum plate electrodes (NR-GF/PtP). The metabolites produced from glucose or CO2 by strain KCTC1037 cultivated in the DCF were butyrate, ethanol, and butanol, but ethanol and butanol were not produced from glucose or CO2 by strain KCTC1037 cultivated in the CF and SCF. It is possible that electrochemically reduced NR-GF/PtP is a more effective source of internal and external reducing power than H2 or NR-GF for strain KCTC1037 to produce metabolites from glucose and CO2. This research might prove useful in developing fermentation technology to actualize direct bioalcohol production of fermentation bacteria from CO2.

  13. Butanol production from hexoses and pentoses by fermentation of Clostridium acetobutylicum.

    PubMed

    Raganati, Francesca; Olivieri, Giuseppe; Götz, Peter; Marzocchella, Antonio; Salatino, Piero

    2015-08-01

    The present paper reports the characterization of ABE (acetone-butanol-ethanol) production by Clostridium acetobutylicum DSM 792 for sugars representative of hydrolysed lignocellulosic biomass (glucose, mannose, arabinose, xylose). The attention was focused on: the selection of an optimal medium for the simultaneous conversion of the investigated sugars; the assessment of interference-synergistic effects during the fermentation of mixtures of the investigated sugars. The synthetic medium was optimised in terms of nutritional factors: the KH2PO4-K2HPO4 concentration was increased up to 5 g/L; the MgSO4 concentration was increased up to 2 g/L; the MnSO4 concentration was increased up to 0.1 g/L; the FeSO4 concentration ranged between 0.002 and 0.01 g/L); the CaCO3 concentration was increased up to 10 g/L. The optimal concentration of the investigated factors was assessed and it varied from one sugar to another. The batch fermentations of a mixture of the four sugars highlighted their synergistic effects. Once set the initial concentration of the sugars (60 g/L), the butanol and solvent concentration increased up to 14.6 and 20.6 g/L, respectively, when the four sugars were present. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Continuous lactose fermentation by Clostridium acetobutylicum--assessment of solventogenic kinetics.

    PubMed

    Procentese, Alessandra; Raganati, Francesca; Olivieri, Giuseppe; Russo, Maria Elena; Salatino, Piero; Marzocchella, Antonio

    2015-03-01

    This work reports the results of a series of tests on the specific butanol production rate by Clostridium acetobutylicum continuous cultures. The tests were carried out using lactose as carbon source to mimic cheese-whey. A continuous stirred tank reactor equipped with a microfiltration unit was used. The dilution rate (D) ranged between 0.02 and 0.15h(-1) and the ratio R of the permeate stream rate to the stream fed to the reactor ranged between 14% and 95%. For each set of D and R values, the continuous cultures were characterized in terms of concentration of cells, acids and solvents. Results were processed to assess the concentration of acidogenic cells, solventogenic cells, spores and the specific butanol production rate. The max butanol productivity was 0.5gL(-1)h(-1) at D=0.1h(-1) and R=95%. The butanol productivity referred to solventogenic cells was expressed as a function of concentration of lactose, acids and butanol. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Immobilization of Clostridium acetobutylicum onto natural textiles and its fermentation properties.

    PubMed

    Zhuang, Wei; Liu, Xiaojing; Yang, Jing; Wu, Jinglan; Zhou, Jingwei; Chen, Yong; Liu, Dong; Ying, Hanjie

    2017-03-01

    Immobilized fermentation has several advantages over traditional suspended fermentation, including simple and continuous operation, improved fermentation performance and reduced cost. Carrier is the most adjustable element among three elements of immobilized fermentation, including carrier, bacteria and environment. In this study, we characterized carrier roughness and surface properties of four types of natural fibres, including linen, cotton, bamboo fibre and silk, to assess their effects on cell immobilization, fermentation performance and stability. Linen with higher specific surface area and roughness could adsorb more bacteria during immobilized fermentation, thereby improving fermentation performance; thus, linen was selected as a suitable carrier and was applied for acetone-butanol-ethanol (ABE) fermentation. To further improve fermentation performance, we also found that microbes of Clostridium acetobutylicum were negatively charged surfaces during fermentation. Therefore, we then modified linen with polyetherimide (PEI) and steric acid (SA) to increase surface positive charge and improve surface property. During ABE fermentation, the adhesion between modified linen and bacteria was increased, adsorption was increased about twofold compared with that of unmodified linen, and butanol productivity was increased 8.16% and 6.80% with PEI- and SA-modified linen as carriers respectively. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  16. Continuous xylose fermentation by Clostridium acetobutylicum--Assessment of solventogenic kinetics.

    PubMed

    Procentese, Alessandra; Raganati, Francesca; Olivieri, Giuseppe; Russo, Maria Elena; Salatino, Piero; Marzocchella, Antonio

    2015-09-01

    This work deals with the specific butanol production rate of Clostridium acetobutylicum using xylose--a relevant fraction of lignocellulosic feedstock for biofuel production--as carbon source. The tests were carried out in a CSTR equipped with a microfiltration unit. The dilution rate (D) ranged between 0.02 and 0.22 h(-1) and the ratio R between the permeate stream rate and the stream fed to the reactor ranged between 14% and 88%. The biomass present in the broth was identified as a heterogeneous cell population consisting of: acidogenic cells, solventogenic cells and spores. The results were processed to assess the concentration of acidogenic cells, solventogenic cells and spores. The specific butanol production rate was also assessed. The max butanol productivity was 1.3 g L(-1) h(-1) at D = 0.17 h(-1) and R = 30%. A comparison between the results reported in a previous work carried out with lactose was made. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Integrated intracellular metabolic profiling and pathway analysis approaches reveal complex metabolic regulation by Clostridium acetobutylicum.

    PubMed

    Liu, Huanhuan; Huang, Di; Wen, Jianping

    2016-02-15

    Clostridium acetobutylicum is one of the most important butanol producing strains. However, environmental stress in the fermentation process usually leads to a lower yield, seriously hampering its industrialization. In order to systematically investigate the key intracellular metabolites that influence the strain growth and butanol production, and find out the critical regulation nodes, an integrated analysis approach has been carried out in this study. Based on the gas chromatography-mass spectrometry technology, the partial least square discriminant analysis and the pathway analysis, 40 metabolic pathways linked with 43 key metabolic nodes were identified. In-depth analysis showed that lots of amino acids metabolism promoted cell growth but exerted slight influence on butanol production, while sugar metabolism was favorable for cell growth but unfavorable for butanol synthesis. Besides, both lysine and succinic acid metabolism generated a complex effect on the whole metabolic network. Dicarboxylate metabolism exerted an indispensable role on cell growth and butanol production. Subsequently, rational feeding strategies were proposed to verify these conclusions and facilitate the butanol biosynthesis. Feeding amino acids, especially glycine and serine, could obviously improve cell growth while yeast extract, citric acid and ethylene glycol could significantly enhance both growth and butanol production. The feeding experiment confirmed that metabolic profiling combined with pathway analysis provided an accurate, reasonable and practical approach to explore the cellular metabolic activity and supplied a basis for improving butanol production. These strategies can also be extended for the production of other important bio-chemical compounds.

  18. Enhanced production of butanol and acetoin by heterologous expression of an acetolactate decarboxylase in Clostridium acetobutylicum.

    PubMed

    Shen, Xiaoning; Liu, Dong; Liu, Jun; Wang, Yanyan; Xu, Jiahui; Yang, Zhengjiao; Guo, Ting; Niu, Huanqing; Ying, Hanjie

    2016-09-01

    Butanol is an important industrial chemical and an attractive transportation fuel. However, the deficiency of reducing equivalents NAD(P)H in butanol fermentation results in a large quantity of oxidation products, which is a major problem limiting the atom economy and economic viability of bio-butanol processes. Here, we integrated the butanol fermentation process with a NADH-generating, acetoin biosynthesis process to improve the butanol production. By overexpressing the α-acetolactate decarboxylase gene alsD from Bacillus subtilis in Clostridium acetobutylicum, acetoin yield was significantly increased at the cost of acetone. After optimization of fermentation conditions, butanol (12.9g/L), acetoin (6.5g/L), and ethanol (1.9g/L) were generated by the recombinant strain, with acetone no more than 1.8g/L. Thus, both mass yield and product value were greatly improved. This study demonstrates that reducing power compensation is effective to improve the atom economy of butanol fermentation, and provides a novel approach to improve the economic viability of bio-butanol production. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Sustainable biobutanol production from pineapple waste by using Clostridium acetobutylicum B 527: Drying kinetics study.

    PubMed

    Khedkar, Manisha A; Nimbalkar, Pranhita R; Gaikwad, Shashank G; Chavan, Prakash V; Bankar, Sandip B

    2017-02-01

    Present investigation explores the use of pineapple peel, a food industry waste, for acetone-butanol-ethanol (ABE) production using Clostridium acetobutylicum B 527. Proximate analysis of pineapple peel shows that it contains 35% cellulose, 19% hemicellulose, and 16% lignin on dry basis. Drying experiments on pineapple peel waste were carried out in the temperature range of 60-120°C and experimental drying data was modeled using moisture diffusion control model to study its effect on ABE production. The production of ABE was further accomplished via acid hydrolysis, detoxification, and fermentation process. Maximum total sugar release obtained by using acid hydrolysis was 97g/L with 95-97% and 10-50% removal of phenolics and acetic acid, respectively during detoxification process. The maximum ABE titer obtained was 5.23g/L with 55.6% substrate consumption when samples dried at 120°C were used as a substrate (after detoxification). Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Secretion and assembly of functional mini-cellulosomes from synthetic chromosomal operons in Clostridium acetobutylicum ATCC 824

    PubMed Central

    2013-01-01

    Background Consolidated bioprocessing (CBP) is reliant on the simultaneous enzyme production, saccharification of biomass, and fermentation of released sugars into valuable products such as butanol. Clostridial species that produce butanol are, however, unable to grow on crystalline cellulose. In contrast, those saccharolytic species that produce predominantly ethanol, such as Clostridium thermocellum and Clostridium cellulolyticum, degrade crystalline cellulose with high efficiency due to their possession of a multienzyme complex termed the cellulosome. This has led to studies directed at endowing butanol-producing species with the genetic potential to produce a cellulosome, albeit by localising the necessary transgenes to unstable autonomous plasmids. Here we have explored the potential of our previously described Allele-Coupled Exchange (ACE) technology for creating strains of the butanol producing species Clostridium acetobutylicum in which the genes encoding the various cellulosome components are stably integrated into the genome. Results We used BioBrick2 (BB2) standardised parts to assemble a range of synthetic genes encoding C. thermocellum cellulosomal scaffoldin proteins (CipA variants) and glycoside hydrolases (GHs, Cel8A, Cel9B, Cel48S and Cel9K) as well as synthetic cellulosomal operons that direct the synthesis of Cel8A, Cel9B and a truncated form of CipA. All synthetic genes and operons were integrated into the C. acetobutylicum genome using the recently developed ACE technology. Heterologous protein expression levels and mini-cellulosome self-assembly were assayed by western blot and native PAGE analysis. Conclusions We demonstrate the successful expression, secretion and self-assembly of cellulosomal subunits by the recombinant C. acetobutylicum strains, providing a platform for the construction of novel cellulosomes. PMID:23962085

  1. Targeted mutagenesis of the Clostridium acetobutylicum acetone-butanol-ethanol fermentation pathway.

    PubMed

    Cooksley, Clare M; Zhang, Ying; Wang, Hengzheng; Redl, Stephanie; Winzer, Klaus; Minton, Nigel P

    2012-11-01

    The production of the chemical solvents acetone and butanol by the bacterium Clostridium acetobutylicum was one of the first large-scale industrial processes to be developed, and in the first part of the last century ranked second in importance only to ethanol production. After a steep decline in its industrial use, there has been a recent resurgence of interest in the acetone-butanol-ethanol (ABE) fermentation process, with a particular emphasis on butanol production. In order to generate strains suitable for efficient use on an industrial scale, metabolic engineering is required to alter the AB ratio in favour of butanol, and eradicate the production of unwanted products of fermentation. Using ClosTron technology, a large-scale targeted mutagenesis in C. acetobutylicum ATCC 824 was carried out, generating a set of 10 mutants, defective in alcohol/aldehyde dehydrogenases 1 and 2 (adhE1, adhE2), butanol dehydrogenases A and B (bdhA, bdhB), phosphotransbutyrylase (ptb), acetate kinase (ack), acetoacetate decarboxylase (adc), CoA transferase (ctfA/ctfB), and a previously uncharacterised putative alcohol dehydrogenase (CAP0059). However, inactivation of the main hydrogenase (hydA) and thiolase (thl) could not be achieved. Constructing such a series of mutants is paramount for the acquisition of information on the mechanism of solvent production in this organism, and the subsequent development of industrial solvent producing strains. Unexpectedly, bdhA and bdhB mutants did not affect solvent production, whereas inactivation of the previously uncharacterised gene CAP0059 resulted in increased acetone, butanol, and ethanol formation. Other mutants showed predicted phenotypes, including a lack of acetone formation (adc, ctfA, and ctfB mutants), an inability to take up acids (ctfA and ctfB mutants), and a much reduced acetate formation (ack mutant). The adhE1 mutant in particular produced very little solvents, demonstrating that this gene was indeed the main contributor to

  2. Acetone-butanol-ethanol production with high productivity using Clostridium acetobutylicum BKM19.

    PubMed

    Jang, Yu-Sin; Malaviya, Alok; Lee, Sang Yup

    2013-06-01

    Conventional acetone-butanol-ethanol (ABE) fermentation is severely limited by low solvent titer and productivities. Thus, this study aims at developing an improved Clostridium acetobutylicum strain possessing enhanced ABE production capability followed by process optimization for high ABE productivity. Random mutagenesis of C. acetobutylicum PJC4BK was performed by screening cells on fluoroacetate plates to isolate a mutant strain, BKM19, which exhibited the total solvent production capability 30.5% higher than the parent strain. The BKM19 produced 32.5 g L(-1) of ABE (17.6 g L(-1) butanol, 10.5 g L(-1) ethanol, and 4.4 g L(-1) acetone) from 85.2 g L(-1) glucose in batch fermentation. A high cell density continuous ABE fermentation of the BKM19 in membrane cell-recycle bioreactor was studied and optimized for improved solvent volumetric productivity. Different dilution rates were examined to find the optimal condition giving highest butanol and ABE productivities. The maximum butanol and ABE productivities of 9.6 and 20.0 g L(-1)  h(-1) , respectively, could be achieved at the dilution rate of 0.85 h(-1) . Further cell recycling experiments were carried out with controlled cell-bleeding at two different bleeding rates. The maximum solvent productivities were obtained when the fermenter was operated at a dilution rate of 0.86 h(-1) with the bleeding rate of 0.04 h(-1) . Under the optimal operational condition, butanol and ABE could be produced with the volumetric productivities of 10.7 and 21.1 g L(-1)  h(-1) , and the yields of 0.17 and 0.34 g g(-1) , respectively. The obtained butanol and ABE volumetric productivities are the highest reported productivities obtained from all known-processes. Copyright © 2013 Wiley Periodicals, Inc.

  3. Structural analysis of Clostridium acetobutylicum ATCC 824 glycoside hydrolase from CAZy family GH105

    SciTech Connect

    Germane, Katherine L.; Servinsky, Matthew D.; Gerlach, Elliot S.; Sund, Christian J.; Hurley, Margaret M.

    2015-07-29

    The crystal structure of the protein product of the C. acetobutylicum ATCC 824 gene CA-C0359 is structurally similar to YteR, an unsaturated rhamnogalacturonyl hydrolase from B. subtilis strain 168. Substrate modeling and electrostatic studies of the active site of the structure of CA-C0359 suggests that the protein can now be considered to be part of CAZy glycoside hydrolase family 105. Clostridium acetobutylicum ATCC 824 gene CA-C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA-C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry (http://scripts.iucr.org/cgi-bin/cr.cgi?rm)) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA-C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate

  4. Increased productivity of Clostridium acetobutylicum fermentation of acetone, butanol, and ethanol by pervaporation through supported ionic liquid membrane.

    PubMed

    Izák, Pavel; Schwarz, Katrin; Ruth, Wolfgang; Bahl, Hubert; Kragl, Udo

    2008-03-01

    Pervaporation proved to be one of the best methods to remove solvents out of a solvent producing Clostridium acetobutylicum culture. By using an ionic liquid (IL)-polydimethylsiloxane (PDMS) ultrafiltration membrane (pore size 60 nm), we could guarantee high stability and selectivity during all measurements carried out at 37 degrees C. Overall solvent productivity of fermentation connected with continuous product removal by pervaporation was 2.34 g l(-1) h(-1). The supported ionic liquid membrane (SILM) was impregnated with 15 wt% of a novel ionic liquid (tetrapropylammonium tetracyano-borate) and 85 wt% of polydimethylsiloxane. Pervaporation, accomplished with the optimized SILM, led to stable and efficient removal of the solvents butan-1-ol and acetone out of a C. acetobutylicum culture. By pervaporation through SILM, we removed more butan-1-ol than C. acetobutylicum was able to produce. Therefore, we added an extra dose of butan-1-ol to run fermentation on limiting values where the bacteria would still be able to survive its lethal concentration (15.82 g/l). After pervaporation was switched off, the bacteria died from high concentration of butan-1-ol, which they produced.

  5. Regulation of nitrogen metabolism, starch utilisation and the beta-hbd-adh1 gene cluster in Clostridium acetobutylicum.

    PubMed

    Woods, D R; Reid, S J

    1995-10-01

    The successful genetic manipulation of Clostridium acetobutylicum for the increased production of solvents will depend on an understanding of gene structure and regulation in the bacterium. The glutamine synthetase (glnA) gene is regulated by antisense RNA, transcribed from a downstream promoter, in the opposite direction to the glnA gene. An open reading frame (ORF) was detected downstream of the glnA gene, which has sequence homology to response regulators with anti-termination activity and may be involved in sensing nitrogen conditions. The expression of the linked beta-hbd, adh1 and fixB genes was investigated throughout the bacterial growth cycle by RNA hybridisation techniques. The adh1 gene was independently expressed as a 2.4-kb transcript which peaked at 12 h, immediately prior to the solventogenic phase. The beta-hbd and fixB genes were transcribed throughout the acidogenic and solventogenic phases. A regulator gene, regA, which complements a Bacillus subtilis ccpA mutant, has been identified and sequenced from C. acetobutylicum P262. The regA gene repressed the degradation of starch by an uncharacterised C. acetobutylicum gene, and may therefore play a role in the utilisation of carbohydrate substrates in this organism.

  6. Structural analysis of Clostridium acetobutylicum ATCC 824 glycoside hydrolase from CAZy family GH105

    PubMed Central

    Germane, Katherine L.; Servinsky, Matthew D.; Gerlach, Elliot S.; Sund, Christian J.; Hurley, Margaret M.

    2015-01-01

    Clostridium acetobutylicum ATCC 824 gene CA_C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA_C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry 1nc5) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA_C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate specificity from that of YteR. PMID:26249707

  7. Effect of zinc supplementation on acetone-butanol-ethanol fermentation by Clostridium acetobutylicum.

    PubMed

    Wu, You-Duo; Xue, Chuang; Chen, Li-Jie; Bai, Feng-Wu

    2013-05-10

    In this article, effect of zinc supplementation on acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum was studied. It was found that when 0.001 g/L ZnSO4·7H2O was supplemented into the medium, solventogenesis was initiated earlier, with 21.0 g/L ABE (12.6 g/L butanol, 6.7 g/L acetone and 1.7 g/L ethanol) produced with a fermentation time of 40 h, compared to 19.4 g/L ABE (11.7 g/L butanol, 6.4 g/L acetone and 1.3g/L ethanol) produced with a fermentation time of 64 h in the control without zinc supplementation, and correspondingly ABE and butanol productivities were increased to 0.53 and 0.32 g/L/h from 0.30 and 0.18 g/L/h, increases of 76.7% and 77.8%, respectively, but their yields were not compromised. The reason for this phenomenon was attributed to rapid acids re-assimilation for more efficient ABE production, which was in accordance with relatively high pH and ORP levels maintained during the fermentation process. The maximum cell density increased by 23.8%, indicating that zinc supplementation stimulated cell growth, and consequently facilitated glucose utilization. However, more zinc supplementation exhibited an inhibitory effect, indicating that zinc supplementation at very low levels such as 0.001 g/L ZnSO4·7H2O will be an economically competitive strategy for improving butanol production.

  8. Global transcriptional changes of Clostridium acetobutylicum cultures with increased butanol:acetone ratios.

    PubMed

    Hönicke, Daniel; Janssen, Holger; Grimmler, Christina; Ehrenreich, Armin; Lütke-Eversloh, Tina

    2012-05-15

    Artificial electron carriers have been widely used to shift the solvent ratio toward butanol in acetone-butanol-ethanol (ABE) fermentation of solventogenic clostridia according to decreased hydrogen production. In this study, first insights on the molecular level were gained to explore the effect of methyl viologen addition to cultures of Clostridium acetobutylicum. Employing batch fermentation in mineral salts medium, the butanol:acetone ratio was successively increased from 2.3 to 12.4 on a 100-ml scale in serum bottles and from 1.4 to 16.5 on a 1300-ml scale in bioreactors, respectively. The latter cultures were used for DNA microarray analyses to provide new information on the transcriptional changes referring to methyl viologen exposure and thus, exhibit gene expression patterns according to the manipulation of the cellular redox balance. Methyl viologen-exposed cultures revealed lower expression levels of the sol operon (CAP0162-0164) and the adjacent adc gene (CAP0165) responsible for solvent formation as well as iron and sulfate transporters and the CAC0105-encoded ferredoxin. On the contrary, genes for riboflavin biosynthesis, for the butyrate/butanol metabolic pathway and genes coding for sugar transport systems were induced. Interestingly, the adhE2-encoded bifunctional NADH-dependent aldhehyde/alcohol-dehydrogenase (CAP0035) was upregulated up to more than 100-fold expression levels as compared to the control culture without methyl viologen addition. The data presented here indicate a transcriptional regulation for decreased acetone biosynthesis and the redox-dependent substitution of adhE1 (CAP0162) by adhE2.

  9. Structural analysis of Clostridium acetobutylicum ATCC 824 glycoside hydrolase from CAZy family GH105.

    PubMed

    Germane, Katherine L; Servinsky, Matthew D; Gerlach, Elliot S; Sund, Christian J; Hurley, Margaret M

    2015-08-01

    Clostridium acetobutylicum ATCC 824 gene CA_C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA_C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry 1nc5) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA_C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate specificity from that of YteR.

  10. Green synthesized iron oxide nanoparticles effect on fermentative hydrogen production by Clostridium acetobutylicum.

    PubMed

    Mohanraj, Sundaresan; Kodhaiyolii, Shanmugam; Rengasamy, Mookan; Pugalenthi, Velan

    2014-05-01

    A green synthesis of iron oxide nanoparticles (FeNPs) was developed using Murraya koenigii leaf extract as reducing and stabilizing agent. UV-vis spectra show that the absorption band centred at a wavelength of 277 nm which corresponds to the surface plasmon resonances of synthesized FeNPs. Fourier transform infrared spectroscopy spectrum exhibits that the characteristic band at 580 cm(-1) is assigned to Fe-O of γ-Fe2O3. Transmission electron microscopy image confirms that the spherical with irregular shaped aggregates and average size of nanoparticles was found to be ∼59 nm. The effect of synthesized FeNPs on fermentative hydrogen production was evaluated from glucose by Clostridium acetobutylicum NCIM 2337. The hydrogen yield in control experiment was obtained as 1.74 ± 0.08 mol H2/mol glucose whereas the highest hydrogen yield in FeNPs supplemented experiment was achieved as 2.33 ± 0.09 mol H2/mol glucose at 175 mg/L of FeNPs. In addition, the hydrogen content and hydrogen production rate were also increased from 34 ± 0.8 to 52 ± 0.8 % and 23 to 25.3 mL/h, respectively. The effect of FeNPs was compared with supplementation of FeSO4 on fermentative process. The supplementation of FeNPs enhanced the hydrogen production in comparison with control and FeSO4. The supplementation of FeNPs led to the change of the metabolic pathway towards high hydrogen production due to the enhancement of ferredoxin activity. The fermentation type was shifted from butyrate to acetate/butyrate fermentation type at the addition of FeNPs.

  11. Biobutanol production in a Clostridium acetobutylicum biofilm reactor integrated with simultaneous product recovery by adsorption

    PubMed Central

    2014-01-01

    Background Clostridium acetobutylicum can propagate on fibrous matrices and form biofilms that have improved butanol tolerance and a high fermentation rate and can be repeatedly used. Previously, a novel macroporous resin, KA-I, was synthesized in our laboratory and was demonstrated to be a good adsorbent with high selectivity and capacity for butanol recovery from a model solution. Based on these results, we aimed to develop a process integrating a biofilm reactor with simultaneous product recovery using the KA-I resin to maximize the production efficiency of biobutanol. Results KA-I showed great affinity for butanol and butyrate and could selectively enhance acetoin production at the expense of acetone during the fermentation. The biofilm reactor exhibited high productivity with considerably low broth turbidity during repeated batch fermentations. By maintaining the butanol level above 6.5 g/L in the biofilm reactor, butyrate adsorption by the KA-I resin was effectively reduced. Co-adsorption of acetone by the resin improved the fermentation performance. By redox modulation with methyl viologen (MV), the butanol-acetone ratio and the total product yield increased. An equivalent solvent titer of 96.5 to 130.7 g/L was achieved with a productivity of 1.0 to 1.5 g · L-1 · h-1. The solvent concentration and productivity increased by 4 to 6-fold and 3 to 5-fold, respectively, compared to traditional batch fermentation using planktonic culture. Conclusions Compared to the conventional process, the integrated process dramatically improved the productivity and reduced the energy consumption as well as water usage in biobutanol production. While genetic engineering focuses on strain improvement to enhance butanol production, process development can fully exploit the productivity of a strain and maximize the production efficiency. PMID:24401161

  12. The influence of the pentose's pathway of the Clostridium Acetobutylicum on the production of butanol: Insights from mathematical modeling

    NASA Astrophysics Data System (ADS)

    Soares, Rafael Ferreira; da Silva, Fabrício Alves Barbosa; Guimarães, Ana Carolina Ramos; Caffarena, Ernesto Raul

    2016-12-01

    This work is a pilot study for further analysis of the organism Trypanosoma cruzi (T. cruzi) and the influences of the Pentose's Pathway on the parasite Clostridium acetobutylicum, already cataloged in the database of OptFlux program. We used the approach parcimonius Flux Balance Analysis (pFBA) to simulate the wild type organism and the mutant with an inhibition of the R_01056 reaction in pentose's pathway. Results showed a reduction of approximately 1/3 of the biomass and 2/3 of the butanol production. This reduction shows the direct influence of the Pentose's Pathway on the primary production of metabolites and the biomass generation from the Clostridium metabolites. This information prompted us to build in the future an SBML parameter file to represent the flow of T.cruzi pathways, which will be essential for the development of new drugs against.

  13. Recent advances and state-of-the-art strategies in strain and process engineering for biobutanol production by Clostridium acetobutylicum.

    PubMed

    Xue, Chuang; Zhao, Jingbo; Chen, Lijie; Yang, Shang-Tian; Bai, Fengwu

    Butanol as an advanced biofuel has gained great attention due to its environmental benefits and superior properties compared to ethanol. However, the cost of biobutanol production via conventional acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum is not economically competitive, which has hampered its industrial application. The strain performance and downstream process greatly impact the economics of biobutanol production. Although various engineered strains with carefully orchestrated metabolic and sporulation-specific pathways have been developed, none of them is ideal for industrial biobutanol production. For further strain improvement, it is necessary to develop advanced genome editing tools and a deep understanding of cellular functioning of genes in metabolic and regulatory pathways. Processes with integrated product recovery can increase fermentation productivity by continuously removing inhibitory products while generating butanol (ABE) in a concentrated solution. In this review, we provide an overview of recent advances in C. acetobutylicum strain engineering and process development focusing on in situ product recovery. With deep understanding of systematic cellular bioinformatics, the exploration of state-of-the-art genome editing tools such as CRISPR-Cas for targeted gene knock-out and knock-in would play a vital role in Clostridium cell engineering for biobutanol production. Developing advanced hybrid separation processes for in situ butanol recovery, which will be discussed with a detailed comparison of advantages and disadvantages of various recovery techniques, is also imperative to the economical development of biobutanol. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Development of an anhydrotetracycline-inducible gene expression system for solvent-producing Clostridium acetobutylicum: A useful tool for strain engineering.

    PubMed

    Dong, Hongjun; Tao, Wenwen; Zhang, Yanping; Li, Yin

    2012-01-01

    Clostridium acetobutylicum is an important solvent (acetone-butanol-ethanol) producing bacterium. However, a stringent, effective, and convenient-to-use inducible gene expression system that can be used for regulating the gene expression strength in C. acetobutylicum is currently not available. Here, we report an anhydrotetracycline-inducible gene expression system for solvent-producing bacterium C. acetobutylicum. This system consists of a functional chloramphenicol acetyltransferase gene promoter containing tet operators (tetO), Pthl promoter (thiolase gene promoter from C. acetobutylicum) controlling TetR repressor expression cassette, and the chemical inducer anhydrotetracycline (aTc). The optimized system, designated as pGusA2-2tetO1, allows gene regulation in an inducer aTc concentration-dependent way, with an inducibility of over two orders of magnitude. The stringency of TetR repression supports the introduction of the genes encoding counterselective marker into C. acetobutylicum, which can be used to increase the mutant screening efficiency. This aTc-inducible gene expression system will thus increase the genetic manipulation capability for engineering C. acetobutylicum. Copyright © 2011 Elsevier Inc. All rights reserved.

  15. Transcription factors and genetic circuits orchestrating the complex, multilayered response of Clostridium acetobutylicum to butanol and butyrate stress

    PubMed Central

    2013-01-01

    Background Organisms of the genus Clostridium are Gram-positive endospore formers of great importance to the carbon cycle, human normo- and pathophysiology, but also in biofuel and biorefinery applications. Exposure of Clostridium organisms to chemical and in particular toxic metabolite stress is ubiquitous in both natural (such as in the human microbiome) and engineered environments, engaging both the general stress response as well as specialized programs. Yet, despite its fundamental and applied significance, it remains largely unexplored at the systems level. Results We generated a total of 96 individual sets of microarray data examining the transcriptional changes in C. acetobutylicum, a model Clostridium organism, in response to three levels of chemical stress from the native metabolites, butanol and butyrate. We identified 164 significantly differentially expressed transcriptional regulators and detailed the cellular programs associated with general and stressor-specific responses, many previously unexplored. Pattern-based, comparative genomic analyses enabled us, for the first time, to construct a detailed picture of the genetic circuitry underlying the stress response. Notably, a list of the regulons and DNA binding motifs of the stress-related transcription factors were identified: two heat-shock response regulators, HrcA and CtsR; the SOS response regulator LexA; the redox sensor Rex; and the peroxide sensor PerR. Moreover, several transcriptional regulators controlling stress-responsive amino acid and purine metabolism and their regulons were also identified, including ArgR (arginine biosynthesis and catabolism regulator), HisR (histidine biosynthesis regulator), CymR (cysteine metabolism repressor) and PurR (purine metabolism repressor). Conclusions Using an exceptionally large set of temporal transcriptional data and regulon analyses, we successfully built a STRING-based stress response network model integrating important players for the general and

  16. Transcription factors and genetic circuits orchestrating the complex, multilayered response of Clostridium acetobutylicum to butanol and butyrate stress.

    PubMed

    Wang, Qinghua; Venkataramanan, Keerthi Prasad; Huang, Hongzhan; Papoutsakis, Eleftherios T; Wu, Cathy H

    2013-11-06

    Organisms of the genus Clostridium are Gram-positive endospore formers of great importance to the carbon cycle, human normo- and pathophysiology, but also in biofuel and biorefinery applications. Exposure of Clostridium organisms to chemical and in particular toxic metabolite stress is ubiquitous in both natural (such as in the human microbiome) and engineered environments, engaging both the general stress response as well as specialized programs. Yet, despite its fundamental and applied significance, it remains largely unexplored at the systems level. We generated a total of 96 individual sets of microarray data examining the transcriptional changes in C. acetobutylicum, a model Clostridium organism, in response to three levels of chemical stress from the native metabolites, butanol and butyrate. We identified 164 significantly differentially expressed transcriptional regulators and detailed the cellular programs associated with general and stressor-specific responses, many previously unexplored. Pattern-based, comparative genomic analyses enabled us, for the first time, to construct a detailed picture of the genetic circuitry underlying the stress response. Notably, a list of the regulons and DNA binding motifs of the stress-related transcription factors were identified: two heat-shock response regulators, HrcA and CtsR; the SOS response regulator LexA; the redox sensor Rex; and the peroxide sensor PerR. Moreover, several transcriptional regulators controlling stress-responsive amino acid and purine metabolism and their regulons were also identified, including ArgR (arginine biosynthesis and catabolism regulator), HisR (histidine biosynthesis regulator), CymR (cysteine metabolism repressor) and PurR (purine metabolism repressor). Using an exceptionally large set of temporal transcriptional data and regulon analyses, we successfully built a STRING-based stress response network model integrating important players for the general and specialized metabolite stress

  17. Proteomic analyses of the phase transition from acidogenesis to solventogenesis using solventogenic and non-solventogenic Clostridium acetobutylicum strains.

    PubMed

    Jang, Yu-Sin; Han, Mee-Jung; Lee, Joungmin; Im, Jung Ae; Lee, Yu Hyun; Papoutsakis, Eleftherios Terry; Bennett, George; Lee, Sang Yup

    2014-06-01

    The fermentation carried out by the solvent-producing bacterium, Clostridium acetobutylicum, is characterized by two distinct phases: acidogenic and solventogenic phases. Understanding the cellular physiological changes occurring during the phase transition in clostridial fermentation is important for the enhanced production of solvents. To identify protein changes upon entry to stationary phase where solvents are typically produced, we herein analyzed the proteomic profiles of the parental wild type C. acetobutylicum strains, ATCC 824, the non-solventogenic strain, M5 that has lost the solventogenic megaplasmid pSOL1, and the synthetic simplified alcohol forming strain, M5 (pIMP1E1AB) expressing plasmid-based CoA-transferase (CtfAB) and aldehyde/alcohol dehydrogenase (AdhE1). A total of 68 protein spots, corresponding to 56 unique proteins, were unambiguously identified as being differentially present after the phase transitions in the three C. acetobutylicum strains. In addition to changes in proteins known to be involved in solventogenesis (AdhE1 and CtfB), we identified significant alterations in enzymes involved in sugar transport and metabolism, fermentative pathway, heat shock proteins, translation, and amino acid biosynthesis upon entry into the stationary phase. Of these, four increased proteins (AdhE1, CAC0233, CtfB and phosphocarrier protein HPr) and six decreased proteins (butyrate kinase, ferredoxin:pyruvate oxidoreductase, phenylalanyl-tRNA synthetase, adenylosuccinate synthase, pyruvate kinase and valyl-tRNA synthetase) showed similar patterns in the two strains capable of butanol formation. Interestingly, significant changes of several proteins by post-translational modifications were observed in the solventogenic phase. The proteomic data from this study will improve our understanding on how cell physiology is affected through protein levels patterns in clostridia.

  18. Chemostat cultivation and transcriptional analyses of Clostridium acetobutylicum mutants with defects in the acid and acetone biosynthetic pathways.

    PubMed

    Hönicke, Daniel; Lütke-Eversloh, Tina; Liu, Ziyong; Lehmann, Dörte; Liebl, Wolfgang; Ehrenreich, Armin

    2014-12-01

    Clostridium acetobutylicum is a model organism for the biotechnologically important acetone-butanol-ethanol (ABE) fermentation. With the objective to rationally develop strains with improved butanol production, detailed insights into the physiological and genetic mechanisms of solvent production are required. Therefore, pH-controlled phosphate-limited chemostat cultivation and DNA microarray technology were employed for an in-depth analysis of knockout mutants with defects in the central fermentative metabolism. The set of studied mutants included strains with inactivated phosphotransacetylase (pta), phosphotransbutyrylase (ptb), and acetoacetate decarboxylase (adc) encoding genes, as well as an adc/pta double knockout mutant. A comprehensive physiological characterization of the mutants was performed by continuous cultivation, allowing for a well-defined separation of acidogenic and solventogenic growth, combined with the advantage of the high reproducibility of steady-state conditions. The ptb-negative strain C. acetobutylicum ptb::int(87) exhibited the most striking metabolite profile: Sizable amounts of butanol (29 ± 1.3 mM) were already produced during acidogenic growth. The product patterns of the mutants as well as accompanying transcriptomic data are presented and discussed.

  19. Modifying the product pattern of Clostridium acetobutylicum: physiological effects of disrupting the acetate and acetone formation pathways.

    PubMed

    Lehmann, Dörte; Hönicke, Daniel; Ehrenreich, Armin; Schmidt, Michael; Weuster-Botz, Dirk; Bahl, Hubert; Lütke-Eversloh, Tina

    2012-05-01

    Clostridial acetone-butanol-ethanol (ABE) fermentation is a natural source for microbial n-butanol production and regained much interest in academia and industry in the past years. Due to the difficult genetic accessibility of Clostridium acetobutylicum and other solventogenic clostridia, successful metabolic engineering approaches are still rare. In this study, a set of five knock-out mutants with defects in the central fermentative metabolism were generated using the ClosTron technology, including the construction of targeted double knock-out mutants of C. acetobtuylicum ATCC 824. While disruption of the acetate biosynthetic pathway had no significant impact on the metabolite distribution, mutants with defects in the acetone pathway, including both acetoacetate decarboxylase (Adc)-negative and acetoacetyl-CoA:acyl-CoA transferase (CtfAB)-negative mutants, exhibited high amounts of acetate in the fermentation broth. Distinct butyrate increase and decrease patterns during the course of fermentations provided experimental evidence that butyrate, but not acetate, is re-assimilated via an Adc/CtfAB-independent pathway in C. acetobutylicum. Interestingly, combining the adc and ctfA mutations with a knock-out of the phosphotransacetylase (Pta)-encoding gene, acetate production was drastically reduced, resulting in an increased flux towards butyrate. Except for the Pta-negative single mutant, all mutants exhibited a significantly reduced solvent production.

  20. Multiple orphan histidine kinases interact directly with Spo0A to control the initiation of endospore formation in Clostridium acetobutylicum

    PubMed Central

    Steiner, Elisabeth; Dago, Angel E.; Young, Danielle I.; Heap, John T.; Minton, Nigel P.; Hoch, James A.

    2011-01-01

    The phosphorylated Spo0A transcription factor controls the initiation of endospore formation in Clostridium acetobutylicum, but genes encoding key phosphorelay components, Spo0F and Spo0B, are missing in the genome. We hypothesized that the five orphan histidine kinases of C. acetobutylicum interact directly with Spo0A to control its phosphorylation state. Sequential targeted gene disruption and gene expression profiling provided evidence for two pathways for Spo0A activation, one dependent on a histidine kinase encoded by cac0323, the other on both histidine kinases encoded by cac0903 and cac3319. Purified Cac0903 and Cac3319 kinases autophosphorylated and transferred phosphoryl groups to Spo0A in vitro, confirming their role in Spo0A activation in vivo. A cac0437 mutant hyper-sporulated, suggesting that Cac0437 is a modulator that prevents sporulation and maintains cellular Spo0A~P homeostasis during growth. Accordingly, Cac0437 has apparently lost the ability to autophosphorylate in vitro; instead it catalyses the ATP-dependent dephosphorylation of Spo0A~P releasing inorganic phosphate. Direct phosphorylation of Spo0A by histidine kinases and dephosphorylation by kinase-like proteins may be a common feature of the clostridia that may represent the ancestral state before the great oxygen event some 2.4 billion years ago, after which additional phosphorelay proteins were recruited in the evolutionary lineage that led to the bacilli. PMID:21401736

  1. Synergistic effect of calcium and zinc on glucose/xylose utilization and butanol tolerance of Clostridium acetobutylicum.

    PubMed

    Wu, Youduo; Xue, Chuang; Chen, Lijie; Yuan, Wenjie; Bai, Fengwu

    2016-03-01

    Biobutanol outperforms bioethanol as an advanced biofuel, but is not economically competitive in terms of its titer, yield and productivity associated with feedstocks and energy cost. In this work, the synergistic effect of calcium and zinc was investigated in the acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum using glucose, xylose and glucose/xylose mixtures as carbon source(s). Significant improvements associated with enhanced glucose/xylose utilization, cell growth, acids re-assimilation and butanol biosynthesis were achieved. Especially, the maximum butanol and ABE production of 16.1 and 25.9 g L(-1) were achieved from 69.3 g L(-1) glucose with butanol/ABE productivities of 0.40 and 0.65 g L(-1) h(-1) compared to those of 11.7 and 19.4 g/L with 0.18 and 0.30 g L(-1) h(-1) obtained in the control respectively without any supplement. More importantly, zinc was significantly involved in the butanol tolerance based on the improved xylose utilization under various butanol-shock conditions (2, 4, 6, 8 and 10 g L(-1) butanol). Under the same conditions, calcium and zinc co-supplementation led to the best xylose utilization and butanol production. These results suggested that calcium and zinc could play synergistic roles improving ABE fermentation by C. acetobutylicum. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Production of an acetone-butanol-ethanol mixture from Clostridium acetobutylicum and its conversion to high-value biofuels.

    PubMed

    Sreekumar, Sanil; Baer, Zachary C; Pazhamalai, Anbarasan; Gunbas, Gorkem; Grippo, Adam; Blanch, Harvey W; Clark, Douglas S; Toste, F Dean

    2015-03-01

    Clostridium acetobutylicum is a bacterial species that ferments sugar to a mixture of organic solvents (acetone, butanol and ethanol). This protocol delineates a methodology to combine solventogenic clostridial fermentation and chemical catalysis via extractive fermentation for the production of biofuel blendstocks. Extractive fermentation of C. acetobutylicum is operated in fed-batch mode with a concentrated feed solution (500 grams per liter glucose and 50 grams per liter yeast extract) for 60 h, producing in excess of 40 g of solvents (acetone, butanol and ethanol) between the completely immiscible extractant and aqueous phases of the bioreactor. After distillation of the extractant phase, the acetone, butanol and ethanol mixture is upgraded to long-chain ketones over a palladium-hydrotalcite (Pd-HT) catalyst. This reaction is generally carried out in batch with a high-pressure Q-tube for 20 h at 250 °C. Following this protocol enables the production of ∼0.5 g of high-value biofuel precursors from a 1.7-g portion of fermentation solvents.

  3. Metabolite labelling reveals hierarchies in Clostridium acetobutylicum that selectively channel carbons from sugar mixtures towards biofuel precursors.

    PubMed

    Aristilde, Ludmilla

    2017-01-01

    Clostridial fermentation of cellulose and hemicellulose relies on the cellular physiology controlling the metabolism of the cellulosic hexose sugar (glucose) with respect to the hemicellulosic pentose sugars (xylose and arabinose) and the hemicellulosic hexose sugars (galactose and mannose). Here, liquid chromatography-mass spectrometry and stable isotope tracers in Clostridium acetobutylicum were applied to investigate the metabolic hierarchy of glucose relative to the different hemicellulosic sugars towards two important biofuel precursors, acetyl-coenzyme A and butyryl-coenzyme A. The findings revealed constitutive metabolic hierarchies in C. acetobutylicum that facilitate (i) selective investment of hemicellulosic pentoses towards ribonucleotide biosynthesis without substantial investment into biofuel production and (ii) selective contribution of hemicellulosic hexoses through the glycolytic pathway towards biofuel precursors. Long-term isotopic enrichment demonstrated incorporation of both pentose sugars into pentose-phosphates and ribonucleotides in the presence of glucose. Kinetic labelling data, however, showed that xylose was not routed towards the biofuel precursors but there was minor contribution from arabinose. Glucose hierarchy over the hemicellulosic hexoses was substrate-dependent. Kinetic labelling of hexose-phosphates and triose-phosphates indicated that mannose was assimilated but not galactose. Labelling of both biofuel precursors confirmed this metabolic preference. These results highlight important metabolic considerations in the accounting of clostridial mixed-sugar utilization. © 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  4. Transcriptional analysis of micronutrient zinc-associated response for enhanced carbohydrate utilization and earlier solventogenesis in Clostridium acetobutylicum.

    PubMed

    Wu, You-Duo; Xue, Chuang; Chen, Li-Jie; Wan, Hui-Hui; Bai, Feng-Wu

    2015-11-20

    The micronutrient zinc plays vital roles in ABE fermentation by Clostridium acetobutylicum. In order to elucidate the zinc-associated response for enhanced glucose utilization and earlier solventogenesis, transcriptional analysis was performed on cells grown in glucose medium at the exponential growth phase of 16 h without/with supplementary zinc. Correspondingly, the gene glcG (CAC0570) encoding a glucose-specific PTS was significantly upregulated accompanied with the other two genes CAC1353 and CAC1354 for glucose transport in the presence of zinc. Additionally, genes involved in the metabolisms of six other carbohydrates (maltose, cellobiose, fructose, mannose, xylose and arabinose) were differentially expressed, indicating that the regulatory effect of micronutrient zinc is carbohydrate-specific with respects to the improved/inhibited carbohydrate utilization. More importantly, multiple genes responsible for glycolysis (glcK and pykA), acidogenesis (thlA, crt, etfA, etfB and bcd) and solventogenesis (ctfB and bdhA) of C. acetobutylicum prominently responded to the supplementary zinc at differential expression levels. Comparative analysis of intracellular metabolites revealed that the branch node intermediates such as acetyl-CoA, acetoacetyl-CoA, butyl-CoA, and reducing power NADH remained relatively lower whereas more ATP was generated due to enhanced glycolysis pathway and earlier initiation of solventogenesis, suggesting that the micronutrient zinc-associated response for the selected intracellular metabolisms is significantly pleiotropic.

  5. Carbon 13-Metabolic Flux Analysis derived constraint-based metabolic modelling of Clostridium acetobutylicum in stressed chemostat conditions.

    PubMed

    Wallenius, Janne; Maaheimo, Hannu; Eerikäinen, Tero

    2016-11-01

    The metabolism of butanol producing bacteria Clostridium acetobutylicum was studied in chemostat with glucose limited conditions, butanol stimulus, and as a reference cultivation. COnstraint-Based Reconstruction and Analysis (COBRA) was applied using additional constraints from (13)C Metabolic Flux Analysis ((13)C-MFA) and experimental measurement results. A model consisting of 451 metabolites and 604 reactions was utilized in flux balance analysis (FBA). The stringency of the flux spaces considering different optimization objectives, i.e. growth rate maximization, ATP maintenance, and NADH/NADPH formation, for flux variance analysis (FVA) was studied in the different modelled conditions. Also a previously uncharacterized exopolysaccharide (EPS) produced by C. acetobutylicum was characterized on monosaccharide level. The major monosaccharide components of the EPS were 40n-% rhamnose, 34n-% glucose, 13n-% mannose, 10n-% galactose, and 2n-% arabinose. The EPS was studied to have butanol adsorbing property, 70(butanol)mg(EPS)g(-1) at 37°C. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. The Two-Component System PhoPR of Clostridium acetobutylicum Is Involved in Phosphate-Dependent Gene Regulation ▿

    PubMed Central

    Fiedler, Tomas; Mix, Maren; Meyer, Uta; Mikkat, Stefan; Glocker, Michael O.; Bahl, Hubert; Fischer, Ralf-Jörg

    2008-01-01

    The phoPR gene locus of Clostridium acetobutylicum ATCC 824 comprises two genes, phoP and phoR. Deduced proteins are predicted to represent a response regulator and sensor kinase of a phosphate-dependent two-component regulatory system. We analyzed the expression patterns of phoPR in Pi-limited chemostat cultures and in response to Pi pulses. A basic transcription level under high-phosphate conditions was shown, and a significant increase in mRNA transcript levels was found when external Pi concentrations dropped below 0.3 mM. In two-dimensional gel electrophoresis experiments, a 2.5-fold increase in PhoP was observed under Pi-limiting growth conditions compared to growth with an excess of Pi. At least three different transcription start points for phoP were determined by primer extension analyses. Proteins PhoP and an N-terminally truncated *PhoR were individually expressed heterologously in Escherichia coli and purified. Autophosphorylation of *PhoR and phosphorylation of PhoP were shown in vitro. Electromobility shift assays proved that there was a specific binding of PhoP to the promoter region of the phosphate-regulated pst operon of C. acetobutylicum. PMID:18689481

  7. Transcriptional analysis of micronutrient zinc-associated response for enhanced carbohydrate utilization and earlier solventogenesis in Clostridium acetobutylicum

    PubMed Central

    Wu, You-Duo; Xue, Chuang; Chen, Li-Jie; Wan, Hui-Hui; Bai, Feng-Wu

    2015-01-01

    The micronutrient zinc plays vital roles in ABE fermentation by Clostridium acetobutylicum. In order to elucidate the zinc-associated response for enhanced glucose utilization and earlier solventogenesis, transcriptional analysis was performed on cells grown in glucose medium at the exponential growth phase of 16 h without/with supplementary zinc. Correspondingly, the gene glcG (CAC0570) encoding a glucose-specific PTS was significantly upregulated accompanied with the other two genes CAC1353 and CAC1354 for glucose transport in the presence of zinc. Additionally, genes involved in the metabolisms of six other carbohydrates (maltose, cellobiose, fructose, mannose, xylose and arabinose) were differentially expressed, indicating that the regulatory effect of micronutrient zinc is carbohydrate-specific with respects to the improved/inhibited carbohydrate utilization. More importantly, multiple genes responsible for glycolysis (glcK and pykA), acidogenesis (thlA, crt, etfA, etfB and bcd) and solventogenesis (ctfB and bdhA) of C. acetobutylicum prominently responded to the supplementary zinc at differential expression levels. Comparative analysis of intracellular metabolites revealed that the branch node intermediates such as acetyl-CoA, acetoacetyl-CoA, butyl-CoA, and reducing power NADH remained relatively lower whereas more ATP was generated due to enhanced glycolysis pathway and earlier initiation of solventogenesis, suggesting that the micronutrient zinc-associated response for the selected intracellular metabolisms is significantly pleiotropic. PMID:26586044

  8. Stable and enhanced gene expression in Clostridium acetobutylicum using synthetic untranslated regions with a stem-loop.

    PubMed

    Lee, Joungmin; Jang, Yu-Sin; Papoutsakis, Eleftherios T; Lee, Sang Yup

    2016-07-20

    Gene overexpression is one of the most basic strategies in metabolic engineering, but the factors determining gene expression levels have been poorly studied in Clostridium species. In this study, we found that a short single-stranded 5' untranslated region (UTR) sequence led to decreased gene expression in Clostridium acetobutylicum. Using an in vitro enzyme assay and reverse transcription-quantitative PCR, we found that addition of a small stem-loop at the 5' end of mRNA increased mRNA levels and thereby protein expression levels up to 4.6-fold, possibly protecting mRNA from exonuclease attack. Gene-expression levels were apparently independent of the stability of the added stem-loop; the existence of a stem-loop itself appears to be more important. Our results indicate that efficient expression cassettes can be designed by taking the 5' UTR into consideration, as the expression levels can vary even though the same promoter and RBS are used. These findings will be useful for developing a more reliable gene expression system for metabolic engineering of Clostridium strains. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Fermentation characterization and flux analysis of recombinant strains of Clostridium acetobutylicum with an inactivated solR gene.

    PubMed

    Harris, L M; Blank, L; Desai, R P; Welker, N E; Papoutsakis, E T

    2001-11-01

    The effect of solR inactivation on the metabolism of Clostridium acetobutylicum was examined using fermentation characterization and metabolic flux analysis. The solR-inactivated strain (SolRH) of this study had a higher rate of glucose utilization and produced higher solvent concentrations (by 25%, 14%, and 81%, respectively, for butanol, acetone, and ethanol) compared to the wild type. Strain SolRH(pTAAD), carrying a plasmid-encoded copy of the bifunctional alcohol/aldehyde dehydrogenase gene (aad) used in butanol production, produced even higher concentrations of solvents (by 21%, 45%, and 62%, respectively, for butanol, acetone, and ethanol) than strain SolRH. Clarithromycin used for strain SolRH maintenance during SolRH(pTAAD) fermentations did not alter product formation; however, tetracycline used for pTAAD maintenance resulted in 90% lower solvent production.

  10. Assessment of morphological changes of Clostridium acetobutylicum by flow cytometry during acetone/butanol/ethanol extractive fermentation.

    PubMed

    González-Peñas, Helena; Lu-Chau, Thelmo Alejandro; Moreira, Maria Teresa; Lema, Juan Manuel

    2015-03-01

    Acetone/butanol/ethanol (ABE) fermentation by Clostridium acetobutylicum was investigated in extractive fed-batch experiments. In conventional fermentations, metabolic activity ceases when a critical threshold products concentration is reached (~21.6 g solvents l(-1)). Solvents production was increased up to 36.6 and 37.2 g l(-1), respectively, using 2-butyl-1-octanol (aqueous to organic ratio: 1:0.25 v/v) and pomace olive oil (1:1 v/v) as extraction solvents. The morphological changes of different cell types were monitored and quantified using flow cytometry. Butanol production in extractive fermentations with pomace olive oil was achieved mainly by vegetative cells, whereas the percentage of sporulating cells was lower than 10%.

  11. Effect of pH and lactose concentration on solvent production from whey permeate using Clostridium acetobutylicum

    SciTech Connect

    Ennis, B.M.; Maddox, I.S.

    1987-02-20

    A study was performed to optimize the production of solvents from whey permeate in batch fermentation using Clostridium acetobutylicum P262. Fermentations performed at relatively low pH values resulted in high solvent yields and productivities, but lactose utilization was incomplete. At higher pH values, lactose-utilization was improved but acid production dominated over solvent production. When operating at the higher pH values, an increase in the initial lactose concentration of the whey permeate resulted in lower rates of lactose utilization, and this was accompanied by increased solvent production and decreased acid production. Analysis of data from several experiments revealed a strong inverse relationship between solvent yield and lactose utilization rate. Thus, conditions which minimize the lactose utilization rate such as low culture pH values or high initial lactose concentrations, favor solventogenesis at the expense of acid production. 12 references.

  12. Improving cellular robustness and butanol titers of Clostridium acetobutylicum ATCC824 by introducing heat shock proteins from an extremophilic bacterium.

    PubMed

    Liao, Zhengping; Zhang, Yanan; Luo, Sheng; Suo, Yukai; Zhang, Shaozhi; Wang, Jufang

    2017-06-20

    In recent years, increasing concerns over environment, energy and climate have renewed interest in biotechnological production of butanol. However, growth inhibition by fermentation products and inhibitory components from raw biomass has hindered the development of acetone-butanol-ethanol (ABE) fermentation. Improving the cellular robustness of Clostridium acetobutylicum is of great importance for efficient ABE production. In this study, we attempted to improve the robustness and butanol titers of C. acetobutylicum ATCC824 by overexpressing GroESL and DnaK from the extremely radioresistant bacterium Deinococcus wulumuqiensis R12 and from C. acetobutylicum ATCC824 itself. Three recombinant strains were obtained and designated 824(dnaK R12), 824(groESL R12) and 824(groESL824). These three recombinants were found to have significantly improved tolerances to stresses including butanol, furfural, oxidation and acid. Meanwhile, the butanol titers increased to 13.0g/L, 11.2g/L and 10.7g/L, which were 49.4%, 28.7% and 23.0% higher than that from the wild-type strain (8.7g/L), respectively. For 824(dnaK R12), the production of acetic and butyric acids decreased by 97.1% (1.4g/L vs. 0.04g/L) and 100% (0.3g/L vs. 0g/L), respectively, compared with the wild-type strain. Overexpressing GroESL and DnaK from D. wulumuqiensis R12 also resulted in better growth and ABE production than the wild-type strain on fermentation in the presence of 2.5g/L furfural. Strain 824(groESL R12) was superior to 824(groESL 824) in diverse types of stress-tolerance and butanol titer, indicating that GroESL from the extremophilic bacterium could perform its function more efficiently in the heterologous host than native GroESL. Our study provides evidence that extremophilic bacteria can be excellent resources for engineering C. acetobutylicum to improve its robustness and butanol titer. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Small acid-soluble spore proteins of Clostridium acetobutylicum are able to protect DNA in vitro and are specifically cleaved by germination protease GPR and spore protease YyaC.

    PubMed

    Wetzel, Daniela; Fischer, Ralf-Jörg

    2015-11-01

    Small acid-soluble proteins (SASPs) play an important role in protection of DNA in dormant bacterial endospores against damage by heat, UV radiation or enzymic degradation. In the genome of the strict anaerobe Clostridium acetobutylicum, five genes encoding SASPs have been annotated and here a further sixth candidate is suggested. The ssp genes are expressed in parallel dependent upon Spo0A, a master regulator of sporulation. Analysis of the transcription start points revealed a σG or a σF consensus promoter upstream of each ssp gene, confirming a forespore-specific gene expression. SASPs were termed SspA (Cac2365), SspB (Cac1522), SspD (Cac1620), SspF (Cac2372), SspH (Cac1663) and Tlp (Cac1487). Here it is shown that with the exception of Tlp, every purified recombinant SASP is able to bind DNA in vitro thereby protecting it against enzymic degradation by DNase I. Moreover, SspB and SspD were specifically cleaved by the two germination-specific proteases GPR (Cac1275) and YyaC (Cac2857), which were overexpressed in Escherichia coli and activated by an autocleavage reaction. Thus, for the first time to our knowledge, GPR-like activity and SASP specificity could be demonstrated for a YyaC-like protein. Collectively, the results assign SspA, SspB, SspD, SspF and SspH of C. acetobutylicum as members of α/β-type SASPs, whereas Tlp seems to be a non-DNA-binding spore protein of unknown function. In acetic acid-extracted proteins of dormant spores of C. acetobutylicum, SspA was identified almost exclusively, indicating its dominant biological role as a major α/β-type SASP in vivo.

  14. Inactivation of σF in Clostridium acetobutylicum ATCC 824 Blocks Sporulation Prior to Asymmetric Division and Abolishes σE and σG Protein Expression but Does Not Block Solvent Formation ▿ †

    PubMed Central

    Jones, Shawn W.; Tracy, Bryan P.; Gaida, Stefan M.; Papoutsakis, Eleftherios T.

    2011-01-01

    Clostridium acetobutylicum is both a model organism for the understanding of sporulation in solventogenic clostridia and its relationship to solvent formation and an industrial organism for anaerobic acetone-butanol-ethanol (ABE) fermentation. How solvent production is coupled to endospore formation—both stationary-phase events—remains incompletely understood at the molecular level. Specifically, it is unclear how sporulation-specific sigma factors affect solvent formation. Here the sigF gene in C. acetobutylicum was successfully disrupted and silenced. Not only σF but also the sigma factors σE and σG were not detected in the sigF mutant (FKO1), and differentiation was stopped prior to asymmetric division. Since plasmid expression of the spoIIA operon (spoIIAA-spoIIAB-sigF) failed to complement FKO1, the operon was integrated into the FKO1 chromosome to generate strain FKO1-C. In FKO1-C, σF expression was restored along with sporulation and σE and σG protein expression. Quantitative reverse transcription-PCR (RT-PCR) analysis of a select set of genes (csfB, gpr, spoIIP, sigG, lonB, and spoIIR) that could be controlled by σF, based on the Bacillus subtilis model, indicated that sigG may be under the control of σF, but spoIIR, an important activator of σE in B. subtilis, is not, and neither are the rest of the genes investigated. FKO1 produced solvents at a level similar to that of the parent strain, but solvent levels were dependent on the physiological state of the inoculum. Finally, the complementation strain FKO1-C is the first reported instance of purposeful integration of multiple functional genes into a clostridial chromosome—here, the C. acetobutylicum chromosome—with the aim of altering cell metabolism and differentiation. PMID:21421765

  15. Effect of cocultures on the production of butanol by Clostridium sp. [C. butyricum, C. pasteurianum, C. butylicum, and C. acetobutylicum

    SciTech Connect

    Bergstrom, S.L.; Foutch, G.L.

    1983-01-01

    One of the problems with the production of butanol by fermentation is that a mixture of other solvents, primarily acetone and ethanol, are also produced. The ratio of butanol to acetone to ethanol is approximately 6:3:1. If the metabolism of the Clostridium can be shifted away from acetone and ethanol, then a higher percentage of the sugar carbon can be converted to butanol. This study examines the effects that cocultures of Clostridium have on the yield of butanol from glucose. C. butyricum and C. pasteurianum produced high concentrations of butyric acid from these sugars. C. butylicum and C. acetobutylicum are capable of utilizing this butyric acid for energy, resulting in butanol production. By using combinations of these strains in coculture a higher initial conversion to butyric acid would be expected followed by a higher overall yield of butanol. This coculture would not be expected to have an increased tolerance for butanol in the fermentation broth. 6 references, 1 figure, 3 tables.

  16. Isolation of Clostridium tetani from anaerobic empyema.

    PubMed

    Mayall, B C; Snashall, E A; Peel, M M

    1998-11-01

    We report the isolation of Clostridium tetani (along with Fusobacterium mortiferum) from empyema pus. The patient, a 68 year old retired farmer from rural NSW, had recently undergone cholecystectomy, had heart failure and developed an empyema. He improved after drainage of the empyema and penicillin therapy, but died suddenly during convalescence.

  17. An improved kinetic model for the acetone-butanol-ethanol pathway of Clostridium acetobutylicum and model-based perturbation analysis

    PubMed Central

    2011-01-01

    Background Comprehensive kinetic models of microbial metabolism can enhance the understanding of system dynamics and regulatory mechanisms, which is helpful in optimizing microbial production of industrial chemicals. Clostridium acetobutylicum produces solvents (acetone-butanol–ethanol, ABE) through the ABE pathway. To systematically assess the potential of increased production of solvents, kinetic modeling has been applied to analyze the dynamics of this pathway and make predictive simulations. Up to date, only one kinetic model for C. acetobutylicum supported by experiment has been reported as far as we know. But this model did not integrate the metabolic regulatory effects of transcriptional control and other complex factors. It also left out the information of some key intermediates (e.g. butyryl-phosphate). Results We have developed an improved kinetic model featured with the incorporation of butyryl-phosphate, inclusion of net effects of complex metabolic regulations, and quantification of endogenous enzyme activity variations caused by these regulations. The simulation results of our model are more consistent with published experimental data than the previous model, especially in terms of reflecting the kinetics of butyryl-phosphate and butyrate. Through parameter perturbation analysis, it was found that butyrate kinase has large and positive influence on butanol production while CoA transferase has negative effect on butanol production, suggesting that butyrate kinase has more efficiency in converting butyrate to butanol than CoA transferase. Conclusions Our improved kinetic model of the ABE process has more capacity in approaching real circumstances, providing much more insight in the regulatory mechanisms and potential key points for optimization of solvent productions. Moreover, the modeling strategy can be extended to other biological processes. PMID:21689471

  18. A transcriptional study of acidogenic chemostat cells of Clostridium acetobutylicum--cellular behavior in adaptation to n-butanol.

    PubMed

    Schwarz, Katrin M; Kuit, Wouter; Grimmler, Christina; Ehrenreich, Armin; Kengen, Servé W M

    2012-10-31

    To gain more insight into the butanol stress response of Clostridium acetobutylicum the transcriptional response of a steady state acidogenic culture to different levels of n-butanol (0.25-1%) was investigated. No effect was observed on the fermentation pattern and expression of typical solvent genes (aad, ctfA/B, adc, bdhA/B, ptb, buk). Elevated levels of butanol mainly affected class I heat-shock genes (hrcA, grpE, dnaK, dnaJ, groES, groEL, hsp90), which were upregulated in a dose- and time-dependent manner, and genes encoding proteins involved in the membrane composition (fab and fad or glycerophospholipid related genes) and various ABC-transporters of unknown specificity. Interestingly, fab and fad genes were embedded in a large, entirely repressed cluster (CAC1988-CAC2019), which inter alia encoded an iron-specific ABC-transporter and molybdenum-cofactor synthesis proteins. Of the glycerophospholipid metabolism, the glycerol-3-phosphate dehydrogenase (glpA) gene was highly upregulated, whereas a glycerophosphodiester ABC-transporter (ugpAEBC) and a phosphodiesterase (ugpC) were repressed. On the megaplasmid, only a few genes showed differential expression, e.g. a rare lipoprotein (CAP0058, repressed) and a membrane protein (CAP0102, upregulated) gene. Observed transcriptional responses suggest that C. acetobutylicum reacts to butanol stress by induction of the general stress response and changing its cell envelope and transporter composition, but leaving the central catabolism unaffected. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Elucidating the contributions of multiple aldehyde/alcohol dehydrogenases to butanol and ethanol production in Clostridium acetobutylicum

    PubMed Central

    Dai, Zongjie; Dong, Hongjun; Zhang, Yanping; Li, Yin

    2016-01-01

    Ethanol and butanol biosynthesis in Clostridium acetobutylicum share common aldehyde/alcohol dehydrogenases. However, little is known about the relative contributions of these multiple dehydrogenases to ethanol and butanol production respectively. The contributions of six aldehyde/alcohol dehydrogenases of C. acetobutylicum on butanol and ethanol production were evaluated through inactivation of the corresponding genes respectively. For butanol production, the relative contributions from these enzymes were: AdhE1 > BdhB > BdhA ≈ YqhD > SMB_P058 > AdhE2. For ethanol production, the contributions were: AdhE1 > BdhB > YqhD > SMB_P058 > AdhE2 > BdhA. AdhE1 and BdhB are two essential enzymes for butanol and ethanol production. AdhE1 was relatively specific for butanol production over ethanol, while BdhB, YqhD, and SMB_P058 favor ethanol production over butanol. Butanol synthesis was increased in the adhE2 mutant, which had a higher butanol/ethanol ratio (8.15:1) compared with wild type strain (6.65:1). Both the SMB_P058 mutant and yqhD mutant produced less ethanol without loss of butanol formation, which led to higher butanol/ethanol ratio, 10.12:1 and 10.17:1, respectively. To engineer a more efficient butanol-producing strain, adhE1 could be overexpressed, furthermore, adhE2, SMB_P058, yqhD are promising gene inactivation targets. This work provides useful information guiding future strain improvement for butanol production. PMID:27321949

  20. Coenzyme A-transferase-independent butyrate re-assimilation in Clostridium acetobutylicum-evidence from a mathematical model.

    PubMed

    Millat, Thomas; Voigt, Christine; Janssen, Holger; Cooksley, Clare M; Winzer, Klaus; Minton, Nigel P; Bahl, Hubert; Fischer, Ralf-Jörg; Wolkenhauer, Olaf

    2014-11-01

    The hetero-dimeric CoA-transferase CtfA/B is believed to be crucial for the metabolic transition from acidogenesis to solventogenesis in Clostridium acetobutylicum as part of the industrial-relevant acetone-butanol-ethanol (ABE) fermentation. Here, the enzyme is assumed to mediate re-assimilation of acetate and butyrate during a pH-induced metabolic shift and to faciliate the first step of acetone formation from acetoacetyl-CoA. However, recent investigations using phosphate-limited continuous cultures have questioned this common dogma. To address the emerging experimental discrepancies, we investigated the mutant strain Cac-ctfA398s::CT using chemostat cultures. As a consequence of this mutation, the cells are unable to express functional ctfA and are thus lacking CoA-transferase activity. A mathematical model of the pH-induced metabolic shift, which was recently developed for the wild type, is used to analyse the observed behaviour of the mutant strain with a focus on re-assimilation activities for the two produced acids. Our theoretical analysis reveals that the ctfA mutant still re-assimilates butyrate, but not acetate. Based upon this finding, we conclude that C. acetobutylicum possesses a CoA-tranferase-independent butyrate uptake mechanism that is activated by decreasing pH levels. Furthermore, we observe that butanol formation is not inhibited under our experimental conditions, as suggested by previous batch culture experiments. In concordance with recent batch experiments, acetone formation is abolished in chemostat cultures using the ctfa mutant.

  1. An improved kinetic model for the acetone-butanol-ethanol pathway of Clostridium acetobutylicum and model-based perturbation analysis.

    PubMed

    Li, Ru-Dong; Li, Yuan-Yuan; Lu, Ling-Yi; Ren, Cong; Li, Yi-Xue; Liu, Lei

    2011-06-20

    Comprehensive kinetic models of microbial metabolism can enhance the understanding of system dynamics and regulatory mechanisms, which is helpful in optimizing microbial production of industrial chemicals. Clostridium acetobutylicum produces solvents (acetone-butanol-ethanol, ABE) through the ABE pathway. To systematically assess the potential of increased production of solvents, kinetic modeling has been applied to analyze the dynamics of this pathway and make predictive simulations. Up to date, only one kinetic model for C. acetobutylicum supported by experiment has been reported as far as we know. But this model did not integrate the metabolic regulatory effects of transcriptional control and other complex factors. It also left out the information of some key intermediates (e.g. butyryl-phosphate). We have developed an improved kinetic model featured with the incorporation of butyryl-phosphate, inclusion of net effects of complex metabolic regulations, and quantification of endogenous enzyme activity variations caused by these regulations. The simulation results of our model are more consistent with published experimental data than the previous model, especially in terms of reflecting the kinetics of butyryl-phosphate and butyrate. Through parameter perturbation analysis, it was found that butyrate kinase has large and positive influence on butanol production while CoA transferase has negative effect on butanol production, suggesting that butyrate kinase has more efficiency in converting butyrate to butanol than CoA transferase. Our improved kinetic model of the ABE process has more capacity in approaching real circumstances, providing much more insight in the regulatory mechanisms and potential key points for optimization of solvent productions. Moreover, the modeling strategy can be extended to other biological processes.

  2. Transcriptional Analysis of spo0A Overexpression in Clostridium acetobutylicum and Its Effect on the Cell's Response to Butanol Stress

    PubMed Central

    Alsaker, Keith V.; Spitzer, Thomas R.; Papoutsakis, Eleftherios T.

    2004-01-01

    Spo0A is the regulator of stationary-phase events and is required for transcription of solvent formation genes in Clostridium acetobutylicum. In order to elucidate the role of spo0A in differentiation, we performed transcriptional analysis of 824(pMSPOA) (a spo0A-overexpressing C. acetobutylicum strain with enhanced sporulation) against a plasmid control strain. DNA microarray data were contrasted to data from a spo0A knockout strain (SKO1) that neither sporulates nor produces solvents. Transcripts of fatty acid metabolism genes, motility and chemotaxis genes, heat shock protein genes, and genes encoding the Fts family of cell division proteins were differentially expressed in the two strains, suggesting that these genes play roles in sporulation and the solvent stress response. 824(pMSPOA) alone showed significant downregulation of many glycolytic genes in stationary phase, which is consistent with metabolic flux analysis data. Surprisingly, spo0A overexpression resulted in only nominal transcriptional changes of regulatory genes (abrB and sigF) whose expression was significantly altered in SKO1. Overexpression of spo0A imparted increased tolerance and prolonged metabolism in response to butanol stress. While most of the differentially expressed genes appear to be part of a general stress response (similar to patterns in two plasmid control strains and a groESL-overexpressing strain), several genes were expressed at higher levels at early time points after butanol challenge only in 824(pMSPOA). Most of these genes were related to butyryl coenzyme A and butyrate formation and/or assimilation, but they also included the cell division gene ftsX, the gyrase subunit-encoding genes gyrB and gyrA, DNA synthesis and repair genes, and fatty acid synthesis genes, all of which might play a role in the immediate butanol stress response, and thus in enhanced butanol tolerance. PMID:15028679

  3. Production of a functional cell wall-anchored minicellulosome by recombinant Clostridium acetobutylicum ATCC 824.

    PubMed

    Willson, Benjamin J; Kovács, Katalin; Wilding-Steele, Tom; Markus, Robert; Winzer, Klaus; Minton, Nigel P

    2016-01-01

    The use of fossil fuels is no longer tenable. Not only are they a finite resource, their use is damaging the environment through pollution and global warming. Alternative, environmentally friendly, renewable sources of chemicals and fuels are required. To date, the focus has been on using lignocellulose as a feedstock for microbial fermentation. However, its recalcitrance to deconstruction is making the development of economic processes extremely challenging. One solution is the generation of an organism suitable for use in consolidated bioprocessing (CBP), i.e. one able to both hydrolyse lignocellulose and ferment the released sugars, and this represents an important goal for synthetic biology. We aim to use synthetic biology to develop the solventogenic bacterium C. acetobutylicum as a CBP organism through the introduction of a cellulosome, a complex of cellulolytic enzymes bound to a scaffold protein called a scaffoldin. In previous work, we were able to demonstrate the in vivo production of a C. thermocellum-derived minicellulosome by recombinant strains of C. acetobutylicum, and aim to develop on this success, addressing potential issues with the previous strategy. The genes for the cellulosomal enzymes Cel9G, Cel48F, and Xyn10A from C. cellulolyticum were integrated into the C. acetobutylicum genome using Allele-Coupled Exchange (ACE) technology, along with a miniscaffoldin derived from C. cellulolyticum CipC. The possibility of anchoring the recombinant cellulosome to the cell surface using the native sortase system was assessed, and the cellulolytic properties of the recombinant strains were assayed via plate growth, batch fermentation and sugar release assays. We have been able to demonstrate the synthesis and in vivo assembly of a four-component minicellulosome by recombinant C. acetobutylicum strains. Furthermore, we have been able to anchor a minicellulosome to the C. acetobutylicum cell wall by the use of the native sortase system. The recombinant

  4. The metabolic network of Clostridium acetobutylicum: Comparison of the approximate Bayesian computation via sequential Monte Carlo (ABC-SMC) and profile likelihood estimation (PLE) methods for determinability analysis.

    PubMed

    Thorn, Graeme J; King, John R

    2016-01-01

    The Gram-positive bacterium Clostridium acetobutylicum is an anaerobic endospore-forming species which produces acetone, butanol and ethanol via the acetone-butanol (AB) fermentation process, leading to biofuels including butanol. In previous work we looked to estimate the parameters in an ordinary differential equation model of the glucose metabolism network using data from pH-controlled continuous culture experiments. Here we combine two approaches, namely the approximate Bayesian computation via an existing sequential Monte Carlo (ABC-SMC) method (to compute credible intervals for the parameters), and the profile likelihood estimation (PLE) (to improve the calculation of confidence intervals for the same parameters), the parameters in both cases being derived from experimental data from forward shift experiments. We also apply the ABC-SMC method to investigate which of the models introduced previously (one non-sporulation and four sporulation models) have the greatest strength of evidence. We find that the joint approximate posterior distribution of the parameters determines the same parameters as previously, including all of the basal and increased enzyme production rates and enzyme reaction activity parameters, as well as the Michaelis-Menten kinetic parameters for glucose ingestion, while other parameters are not as well-determined, particularly those connected with the internal metabolites acetyl-CoA, acetoacetyl-CoA and butyryl-CoA. We also find that the approximate posterior is strongly non-Gaussian, indicating that our previous assumption of elliptical contours of the distribution is not valid, which has the effect of reducing the numbers of pairs of parameters that are (linearly) correlated with each other. Calculations of confidence intervals using the PLE method back this up. Finally, we find that all five of our models are equally likely, given the data available at present. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Crystal structure of Clostridium acetobutylicum Aspartate kinase (CaAK): An important allosteric enzyme for amino acids production.

    PubMed

    Manjasetty, Babu A; Chance, Mark R; Burley, Stephen K; Panjikar, Santosh; Almo, Steven C

    2014-09-01

    Aspartate kinase (AK) is an enzyme which is tightly regulated through feedback control and responsible for the synthesis of 4-phospho-L-aspartate from L-aspartate. This intermediate step is at an important branch point where one path leads to the synthesis of lysine and the other to threonine, methionine and isoleucine. Concerted feedback inhibition of AK is mediated by threonine and lysine and varies between the species. The crystal structure of biotechnologically important Clostridium acetobutylicum aspartate kinase (CaAK; E.C. 2.7.2.4; Mw=48,030Da; 437aa; SwissProt: Q97MC0) has been determined to 3Å resolution. CaAK acquires a protein fold similar to the other known structures of AKs despite the low sequence identity (<30%). It is composed of two domains: an N-terminal catalytic domain (kinase) domain and a C-terminal regulatory domain further comprised of two small domains belonging to the ACT domain family. Pairwise comparison of 12 molecules in the asymmetric unit helped to identify the bending regions which are in the vicinity of ATP binding site involved in domain movements between the catalytic and regulatory domains. All 12 CaAK molecules adopt fully open T-state conformation leading to the formation of three tetramers unique among other similar AK structures. On the basis of comparative structural analysis, we discuss tetramer formation based on the large conformational changes in the catalytic domain associated with the lysine binding at the regulatory domains. The structure described herein is homologous to a target in wide-spread pathogenic (toxin producing) bacteria such as Clostridium tetani (64% sequence identity) suggesting the potential of the structure solved here to be applied for modeling drug interactions. CaAK structure may serve as a guide to better understand and engineer lysine biosynthesis for the biotechnology industry.

  6. Crystal structure of Clostridium acetobutylicum Aspartate kinase (CaAK): An important allosteric enzyme for amino acids production

    PubMed Central

    Manjasetty, Babu A.; Chance, Mark R.; Burley, Stephen K.; Panjikar, Santosh; Almo, Steven C.

    2014-01-01

    Aspartate kinase (AK) is an enzyme which is tightly regulated through feedback control and responsible for the synthesis of 4-phospho-L-aspartate from L-aspartate. This intermediate step is at an important branch point where one path leads to the synthesis of lysine and the other to threonine, methionine and isoleucine. Concerted feedback inhibition of AK is mediated by threonine and lysine and varies between the species. The crystal structure of biotechnologically important Clostridium acetobutylicum aspartate kinase (CaAK; E.C. 2.7.2.4; Mw=48,030Da; 437aa; SwissProt: Q97MC0) has been determined to 3Å resolution. CaAK acquires a protein fold similar to the other known structures of AKs despite the low sequence identity (<30%). It is composed of two domains: an N-terminal catalytic domain (kinase) domain and a C-terminal regulatory domain further comprised of two small domains belonging to the ACT domain family. Pairwise comparison of 12 molecules in the asymmetric unit helped to identify the bending regions which are in the vicinity of ATP binding site involved in domain movements between the catalytic and regulatory domains. All 12 CaAK molecules adopt fully open T-state conformation leading to the formation of three tetramers unique among other similar AK structures. On the basis of comparative structural analysis, we discuss tetramer formation based on the large conformational changes in the catalytic domain associated with the lysine binding at the regulatory domains. The structure described herein is homologous to a target in wide-spread pathogenic (toxin producing) bacteria such as Clostridium tetani (64% sequence identity) suggesting the potential of the structure solved here to be applied for modeling drug interactions. CaAK structure may serve as a guide to better understand and engineer lysine biosynthesis for the biotechnology industry. PMID:25170437

  7. Integrated bioprocess for long-term continuous cultivation of Clostridium acetobutylicum coupled to pervaporation with PDMS composite membranes.

    PubMed

    Van Hecke, Wouter; Vandezande, Pieter; Claes, Stan; Vangeel, Silvia; Beckers, Herman; Diels, Ludo; De Wever, Heleen

    2012-05-01

    A continuous cultivation of Clostridium acetobutylicum ATCC 824 is described using a two-stage design to mimic the two phases of batch culture growth of the organism. A hydrophobic pervaporation unit was coupled to the second fermentor containing the highest solvent titers. This in situ product recovery technology efficiently decreased butanol toxicity in the fermentor while the permeate was enriched to 57-195 g L(-1) total solvents depending on the solvent concentrations in the fermentor. By the alleviation of product inhibition, the glucose concentration could be increased from 60 to 126 g L(-1) while the productivity increased concomitantly from 0.13 to 0.30 g L(-1)h(-1). The continuous fermentation was conducted for 1172 h during which the pervaporation was coupled to the second fermentor for 475 h with an average flux of 367 g m(-2)h(-1). The energy consumption was calculated for a 2 wt.% n-butanol fermentation broth and compared with the conventional process.

  8. A Standard Operating Procedure (SOP) for the preparation of intra- and extracellular proteins of Clostridium acetobutylicum for proteome analysis.

    PubMed

    Schwarz, Katrin; Fiedler, Tomas; Fischer, Ralf-Jörg; Bahl, Hubert

    2007-02-01

    We report on the development of a Standard Operating Procedure (SOP) for extraction and handling of intra- and extracellular protein fractions of Clostridium acetobutylicum ATCC 824 for reproducible high quality two-dimensional gel electrophoresis (2-DE) analyses. Standardized cells from a phosphate-limited chemostat were used to evaluate different protein preparation methods. For the preparation of the secretome, a dialysis/ultrafiltration procedure resulted in higher protein yields and proved to be more reliable compared to different precipitation methods using TCA, DOC-TCA, acetone, and PEG 6000. Sonication was found to be the most efficient method among different tested techniques of cell disruption for the analysis of the intracellular proteome. Furthermore, the effect of protease inhibitors and sample storage conditions were tested for both intra- and extracellular protein samples. Significant changes in the protein pattern were observed depending on the addition of protease inhibitors. 2-DE gels with a pH gradient from 4 to 7 prepared according to the developed SOP contained at least 736 intracellular and 324 extracellular protein spots.

  9. Continuous two stage acetone-butanol-ethanol fermentation with integrated solvent removal using Clostridium acetobutylicum B 5313.

    PubMed

    Bankar, Sandip B; Survase, Shrikant A; Singhal, Rekha S; Granström, Tom

    2012-02-01

    The objective of this study was to optimize continuous acetone-butanol-ethanol (ABE) fermentation using a two stage chemostat system integrated with liquid-liquid extraction of solvents produced in the first stage. This minimized end product inhibition by butanol and subsequently enhanced glucose utilization and solvent production in continuous cultures of Clostridium acetobutylicum B 5313. During continuous two-stage ABE fermentation, sugarcane bagasse was used as the cell holding material for the both stages and liquid-liquid extraction was performed using an oleyl alcohol and decanol mixture. An overall solvent production of 25.32g/L (acetone 5.93g/L, butanol 16.90g/L and ethanol 2.48g/L) was observed as compared to 15.98g/L in the single stage chemostat with highest solvent productivity and solvent yield of 2.5g/Lh and of 0.35g/g, respectively. Maximum glucose utilization (83.21%) at a dilution rate of 0.051/h was observed as compared to 54.38% in the single stage chemostat.

  10. Efficient acetone-butanol-ethanol production (ABE) by Clostridium acetobutylicum XY16 immobilized on chemically modified sugarcane bagasse.

    PubMed

    Kong, Xiangping; He, Aiyong; Zhao, Jie; Wu, Hao; Jiang, Min

    2015-07-01

    Sugarcane bagasse was chemically modified by polyethylenimine (PEI) and glutaraldehyde (GA) and then used as a support to immobilize Clostridium acetobutylicum XY16 in the process of butanol production. Compared with batch fermentation using unmodified sugarcane bagasse, 22.3 g/L total solvents were produced by cells immobilized on 4 g/L PEI treated sugarcane bagasse with high solvent productivity of 0.62 g/(L h) and glucose consumption rate of 1.67 g/(L h). Improvement of 14, 43, and 37 % in total solvent titer, solvent productivity and glucose consumption rate was observed, respectively. Enhanced solvent production of 25.14 g/L was obtained when using a high concentration of glucose of 80 g/L. Continuous fermentation was studied using PEI/GA modified sugarcane bagasse as immobilization support with a range of dilution which rates from 0.2 to 2.5 to find an optimal condition. The maximum solvent productivity of 11.32 g/(L h) was obtained at a high dilution rate of 2.0 h(-1).

  11. Enhanced butanol production by solvent tolerance Clostridium acetobutylicum SE25 from cassava flour in a fibrous bed bioreactor.

    PubMed

    Li, Han-Guang; Ma, Xing-Xing; Zhang, Qing-Hua; Luo, Wei; Wu, Ya-Qing; Li, Xun-Hang

    2016-12-01

    To enhance the butanol productivity and reduce the material cost, acetone, butanol, and ethanol fermentation by Clostridium acetobutylicum SE25 was investigated using batch, repeated-batch and continuous cultures in a fibrous bed bioreactor, where cassava flour was used as the substrate. With periodical nutrient supplementation, stable butanol production was maintained for about 360h in a 6-cycle repeated-batch fermentation with an average butanol productivity of 0.28g/L/h and butanol yield of 0.32g/g-starch. In addition, the highest butanol productivity of 0.63g/L/h and butanol yield of 0.36g/g-starch were achieved when the dilution rate were investigated in continuous production of acetone, butanol, and ethanol using a fibrous bed bioreactor, which were 231.6% and 28.6% higher than those of the free-cell fermentation. On the other hand, this study also successfully comfirmed that the biofilm can provide an effective protection for the microbial cells which are growing in stressful environment. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Sequence of the lyc gene encoding the autolytic lysozyme of Clostridium acetobutylicum ATCC824: comparison with other lytic enzymes.

    PubMed

    Croux, C; García, J L

    1991-07-31

    The lyc gene, encoding an autolytic lysozyme from Clostridium acetobutylicum ATCC824, has been cloned. The nucleotide sequence of the lyc gene has been determined and found to encode a protein of 324 amino acids (aa) with a deduced Mr of 34,939. The lyc gene is preceded by two open reading frames with unknown functions, suggesting that this gene is part of an operon. Comparison between the deduced aa sequence of the lyc gene and the directly determined N-terminal sequence of the extracellular clostridial lysozyme suggests that the enzyme is synthesized without a cleavable signal peptide. Moreover, the comparative analyses between the clostridial lysozyme and other known cell-wall lytic enzymes revealed a significant similarity with the N-terminal portion of the lysozymes of Streptomyces globisporus, the fungus Chalaropsis, the Lactobacillus bulgaricus bacteriophage mv1, and the Streptococcus pneumoniae bacteriophages of the Cp family (CPL lysozymes). In addition, the analyses showed that the C-terminal half of the clostridial lysozyme was homologous to the N-terminal domain of the muramoyl-pentapeptide-carboxypeptidase of Streptomyces albus, suggesting a role in substrate binding. The existence of five putative repeated motifs in the C-terminal region of the autolytic lysozyme suggests that this region could play a role in the recognition of the polymeric substrate.

  13. The mechanism of switching from an acidogenic to butanol-acetone fermentation by Clostridium acetobutylicum. Technical progress report, July 1990--June 1993

    SciTech Connect

    Rogers, P.

    1994-11-01

    The overall objective of this project was to elucidate the detailed mechanism by which solvent-forming bacteria such as Clostridium acetobutylicum regulate the well-known shift in fermentation pathway between alcohol-acetone and organic acid production. We eventually want to isolate and describe: (1) the regulatory genes and protein elements that determine induction of synthesis of the solvent-pathway enzymes; and (2) how this regulation system interacts with the sporulation induction and development program and with related pathways such as granulose and exopolysaccharide formation in clostridia. A working model for how clostridial control systems work can be derived from recent research on stress systems in E. coli and sporulation in Bacillus subtilis. This research was centered upon the technique of employing transposable elements that create gene fusions and mutations due to insertion in the chromosome of gram positive bacteria. Our approach was based on recent demonstration in our laboratory and by others of transconjugation of Tn916 into C. acetobutylicum and its insertion into the chromosome. A panel of strains with Tn916 inserts that are also solvent-negative and/or asporogenic were used to identify specific regulatory genes. A second approach was based upon electroporative transformation of plasmid PTV1 DNA carrying transposon Tn917 into C. acetobutylicum. Insertion of Tn917 lac to report activity of genes and functions in vegetative and stationary or slow-growing cells will be investigated.

  14. Enhancing acetone biosynthesis and acetone-butanol-ethanol fermentation performance by co-culturing Clostridium acetobutylicum/Saccharomyces cerevisiae integrated with exogenous acetate addition.

    PubMed

    Luo, Hongzhen; Ge, Laibing; Zhang, Jingshu; Ding, Jian; Chen, Rui; Shi, Zhongping

    2016-01-01

    Acetone is the major by-product in ABE fermentations, most researches focused on increasing butanol/acetone ratio by decreasing acetone biosynthesis. However, economics of ABE fermentation industry strongly relies on evaluating acetone as a valuable platform chemical. Therefore, a novel ABE fermentation strategy focusing on bio-acetone production by co-culturing Clostridium acetobutylicum/Saccharomyces cerevisiae with exogenous acetate addition was proposed. Experimental and theoretical analysis revealed the strategy could, enhance C. acetobutylicum survival oriented amino acids assimilation in the cells; control NADH regeneration rate at moderately lower level to enhance acetone synthesis but without sacrificing butanol production; enhance the utilization ability of C. acetobutylicum on glucose and direct most of extra consumed glucose into acetone/butanol synthesis routes. By implementing the strategy using synthetic or acetate fermentative supernatant, acetone concentrations increased to 8.27-8.55g/L from 5.86g/L of the control, while butanol concentrations also elevated to the higher levels of 13.91-14.23g/L from 11.63g/L simultaneously.

  15. Synergistic dark and photo-fermentation continuous system for hydrogen production from molasses by Clostridium acetobutylicum ATCC 824 and Rhodobacter capsulatus DSM 1710.

    PubMed

    Morsy, Fatthy Mohamed

    2017-04-01

    This study investigated synergistic dark and photo-fermentation using continuous fermentation system (CFS). The system relies on connecting several fermenters from bottom of one to top culture level of the next in a manner that allows for delaying movement of the substrate and thus for its full consumption. While H2 was collected, CFS allowed for moving liquid byproducts toward the outlet and hence continuous productivity. CFS could be efficiently used for: (1) Continuous dark and photo-fermentation H2 production by Clostridium acetobutylicum and Rhodobacter capsulatus producing 5.65moleH2mole(-1) hexose; (2) Continuous dark-fermentation synergistic H2, acetone, butanol and ethanol (ABE) production by C. acetobutylicum which produced per mole hexose, 2.43mol H2 along with 73.08g ABE (3) Continuous H2 and methane production by C. acetobutylicum and bacterial sludge producing, per mole hexose, 1.64mol pure H2 and 2.56mol CH4 mixed with 0.37mol H2·The hydraulic retention time (HRT) for whole system was short where organic acids produced in dark-fermentation in first fermenter were synergistically utilized for H2 production by R. capsulatus in subsequent fermenters. CFS is suitable for fast-digestible sugars but not lignocelluloses or other hard-digestible organics, requiring prolonged HRT, unless such polymeric organics were hydrolyzed prior to fermentation.

  16. Simultaneous production of isopropanol, butanol, ethanol and 2,3-butanediol by Clostridium acetobutylicum ATCC 824 engineered strains

    PubMed Central

    2012-01-01

    Isopropanol represents a widely-used commercial alcohol which is currently produced from petroleum. In nature, isopropanol is excreted by some strains of Clostridium beijerinckii, simultaneously with butanol and ethanol during the isopropanol butanol ethanol (IBE) fermentation. In order to increase isopropanol production, the gene encoding the secondary-alcohol dehydrogenase enzyme from C. beijerinckii NRRL B593 (adh) which catalyzes the reduction of acetone to isopropanol, was cloned into the acetone, butanol and ethanol (ABE)-producing strain C. acetobutylicum ATCC 824. The transformants showed high capacity for conversion of acetone into isopropanol (> 95%). To increase isopropanol production levels in ATCC 824, polycistronic transcription units containing, in addition to the adh gene, homologous genes of the acetoacetate decarboxylase (adc), and/or the acetoacetyl-CoA:acetate/butyrate:CoA transferase subunits A and B (ctfA and ctfB) were constructed and introduced into the wild-type strain. Combined overexpression of the ctfA and ctfB genes resulted in enhanced solvent production. In non-pH-controlled batch cultures, the total solvents excreted by the transformant overexpressing the adh, ctfA, ctfB and adc genes were 24.4 g/L IBE (including 8.8 g/L isopropanol), while the control strain harbouring an empty plasmid produced only 20.2 g/L ABE (including 7.6 g/L acetone). The overexpression of the adc gene had limited effect on IBE production. Interestingly, all transformants with the adh gene converted acetoin (a minor fermentation product) into 2,3-butanediol, highlighting the wide metabolic versatility of solvent-producing Clostridia. PMID:22909015

  17. Modelling the role of CtfA/B in reverse shift continuous culture experiments of Clostridium acetobutylicum.

    PubMed

    Thorn, Graeme J; King, John R

    2016-06-01

    In continuous phosphate-limited conditions, under pH control from high pH (pH ≳ 5.2) to low pH (pH ≲ 5.2), the metabolism of the Gram-positive bacterium Clostridium acetobutylicum,switches from acid to solvent production. Three main enzymes are responsible for the shift, acetoacetate decarboxylase (Adc), alcohol dehydrogenase (AdhE1/2) and a CoA-transferase (CtfA/B), which are produced in increased quantities during solventogenesis. A two-population model, Millat et al. (2013) and fitted to such 'forward'-shift data, can explain this, as well as observed changes in optical density immediately following the shift: an acidogenic subpopulation is washed out and a solventogenic subpopulation grows in its place, each with distinct physiologies and proteomes. We fit this model to a 'reverse'-shift experiment, where the pH is increased from solventogenic to acidogenic conditions. We find corresponding changes in reaction rates, with AdhE1 and Adc production falling, as in the 'forward' experiments; however, for CtfA/B, the best fit surprisingly arises from the same level of production in both conditions. We propose experiments that would test whether this is a model artefact or accurately reflects cultures shifted in this reverse direction, and, if true, may suggest that over-expressing CtfA/B in both solventogenic and acidogenic conditions could improve the efficiency of fermentation. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Clostridium acetobutylicum 8-Oxoguanine DNA Glycosylase (Ogg) Differs from Eukaryotic Oggs with Respect to Opposite Base Discrimination†

    PubMed Central

    Robey-Bond, Susan M.; Barrantes-Reynolds, Ramiro; Bond, Jeffrey P.; Wallace, Susan S.; Bandaru, Viswanath

    2008-01-01

    During repair of damaged DNA, the oxidized base 8-oxoguanine (8-oxoG) is removed by 8-oxoguanine—DNA glycosylase (Ogg) in eukaryotes and most archaea, whereas in most bacteria it is removed by formamidopyrimidine—DNA glycosylase (Fpg). We report the first characterization of a bacterial Ogg, Clostridium acetobutylicum Ogg (CacOgg). Like human OGG1 and Escherichia coli Fpg (EcoFpg), CacOgg excised 8-oxoguanine. However, unlike hOGG1 and EcoFpg, CacOgg showed little preference for the base opposite the damage during base excision and removed 8-oxoguanine from single-stranded DNA. Thus, our results showed unambiguous qualitative functional differences in vitro between CacOgg and both hOGG1 and EcoFpg. CacOgg differs in sequence from the eukaryotic enzymes at two sequence positions, M132 and F179, which align with amino acids (R154 and Y203) in human OGG1 (hOGG1) found to be involved in opposite base interaction. To address the sequence basis for functional differences with respect to opposite base interactions, we prepared three CacOgg variants, M132R, F179Y, and M132R/F179Y. All three variants showed a substantial increase in specificity for 8-oxoG·C relative to 8-oxoG·A. While we were unable to definitively associate these qualitative functional differences with differences in selective pressure between eukaryotes, Clostridia, and other bacteria, our results are consistent with the idea that evolution of Ogg function is based on kinetic control of repair. PMID:18578506

  19. Structural Characterization of Clostridium acetobutylicum 8-Oxoguanine DNA Glycosylase in Its Apo Form and in Complex with 8-Oxodeoxyguanosine

    SciTech Connect

    Faucher, Frédérick; Robey-Bond, Susan M.; Wallace, Susan S.; Doublié, Sylvie

    2009-06-30

    DNA is subject to a multitude of oxidative damages generated by oxidizing agents from metabolism and exogenous sources and by ionizing radiation. Guanine is particularly vulnerable to oxidation, and the most common oxidative product 8-oxoguanine (8-oxoG) is the most prevalent lesion observed in DNA molecules. 8-OxoG can form a normal Watson-Crick pair with cytosine (8-oxoG:C), but it can also form a stable Hoogsteen pair with adenine (8-oxoG:A), leading to a G:C {yields} T:A transversion after replication. Fortunately, 8-oxoG is recognized and excised by either of two DNA glycosylases of the base excision repair pathway: formamidopyrimidine-DNA glycosylase and 8-oxoguanine DNA glycosylase (Ogg). While Clostridium acetobutylicum Ogg (CacOgg) DNA glycosylase can specifically recognize and remove 8-oxoG, it displays little preference for the base opposite the lesion, which is unusual for a member of the Ogg1 family. This work describes the crystal structures of CacOgg in its apo form and in complex with 8-oxo-2'-deoxyguanosine. A structural comparison between the apo form and the liganded form of the enzyme reveals a structural reorganization of the C-terminal domain upon binding of 8-oxoG, similar to that reported for human OGG1. A structural comparison of CacOgg with human OGG1, in complex with 8-oxoG containing DNA, provides a structural rationale for the lack of opposite base specificity displayed by CacOgg.

  20. Introducing a single secondary alcohol dehydrogenase into butanol-tolerant Clostridium acetobutylicum Rh8 switches ABE fermentation to high level IBE fermentation

    PubMed Central

    2012-01-01

    Background Previously we have developed a butanol tolerant mutant of Clostridium acetobutylicum Rh8, from the wild type strain DSM 1731. Strain Rh8 can tolerate up to 19 g/L butanol, with solvent titer improved accordingly, thus exhibiting industrial application potential. To test if strain Rh8 can be used for production of high level mixed alcohols, a single secondary alcohol dehydrogenase from Clostridium beijerinckii NRRL B593 was overexpressed in strain Rh8 under the control of thl promoter. Results The heterogenous gene sADH was functionally expressed in C. acetobutylicum Rh8. This simple, one-step engineering approach switched the traditional ABE (acetone-butanol-ethanol) fermentation to IBE (isopropanol-butanol-ethanol) fermentation. The total alcohol titer reached 23.88 g/l (7.6 g/l isopropanol, 15 g/l butanol, and 1.28 g/l ethanol) with a yield to glucose of 31.42%. The acid (butyrate and acetate) assimilation rate in isopropanol producing strain Rh8(psADH) was increased. Conclusions The improved butanol tolerance and the enhanced solvent biosynthesis machinery in strain Rh8 is beneficial for production of high concentration of mixed alcohols. Strain Rh8 can thus be considered as a good host for further engineering of solvent/alcohol production. PMID:22742819

  1. Heterologous Expression of the Clostridium carboxidivorans CO Dehydrogenase Alone or Together with the Acetyl Coenzyme A Synthase Enables both Reduction of CO2 and Oxidation of CO by Clostridium acetobutylicum.

    PubMed

    Carlson, Ellinor D; Papoutsakis, Eleftherios T

    2017-08-15

    With recent advances in synthetic biology, CO2 could be utilized as a carbon feedstock by native or engineered organisms, assuming the availability of electrons. Two key enzymes used in autotrophic CO2 fixation are the CO dehydrogenase (CODH) and acetyl coenzyme A (acetyl-CoA) synthase (ACS), which form a bifunctional heterotetrameric complex. The CODH/ACS complex can reversibly catalyze CO2 to CO, effectively enabling a biological water-gas shift reaction at ambient temperatures and pressures. The CODH/ACS complex is part of the Wood-Ljungdahl pathway (WLP) used by acetogens to fix CO2, and it has been well characterized in native hosts. So far, only a few recombinant CODH/ACS complexes have been expressed in heterologous hosts, none of which demonstrated in vivo CO2 reduction. Here, functional expression of the Clostridium carboxidivorans CODH/ACS complex is demonstrated in the solventogen Clostridium acetobutylicum, which was engineered to express CODH alone or together with the ACS. Both strains exhibited CO2 reduction and CO oxidation activities. The CODH reactions were interrogated using isotopic labeling, thus verifying that CO was a direct product of CO2 reduction, and vice versa. CODH apparently uses a native C. acetobutylicum ferredoxin as an electron carrier for CO2 reduction. Heterologous CODH activity depended on actively growing cells and required the addition of nickel, which is inserted into CODH without the need to express the native Ni insertase protein. Increasing CO concentrations in the gas phase inhibited CODH activity and altered the metabolite profile of the CODH-expressing cells. This work provides the foundation for engineering a complete and functional WLP in nonnative host organisms.IMPORTANCE Functional expression of CO dehydrogenase (CODH) from Clostridium carboxidivorans was demonstrated in C. acetobutylicum, which is natively incapable of CO2 fixation. The expression of CODH, alone or together with the C. carboxidivorans acetyl

  2. Mutant strain of C. acetobutylicum and process for making butanol

    DOEpatents

    Jain, Mahendra K.; Beacom, Daniel; Datta, Rathin

    1993-01-01

    A biologically pure asporogenic mutant of Clostridium acetobutylicum is produced by growing sporogenic C. acetobutylicum ATCC 4259 and treating the parent strain with ethane methane sulfonate. The mutant which as been designated C. acetobutylicum ATCC 55025 is useful in an improved ABE fermentation process, and produces high concentrations of butanol and total solvents.

  3. Transcription of the pst Operon of Clostridium acetobutylicum Is Dependent on Phosphate Concentration and pH

    PubMed Central

    Fischer, Ralf-Jörg; Oehmcke, Sonja; Meyer, Uta; Mix, Maren; Schwarz, Katrin; Fiedler, Tomas; Bahl, Hubert

    2006-01-01

    The pst operon of Clostridium acetobutylicum ATCC 824 comprises five genes, pstS, pstC, pstA, pstB, and phoU, and shows a gene architecture identical to that of Escherichia coli. Deduced proteins are predicted to represent a high-affinity phosphate-specific ABC (ATP-binding cassette) transport system (Pst) and a protein homologous to PhoU, a negative phosphate regulon regulator. We analyzed the expression patterns of the pst operon in Pi-limited chemostat cultures during acid production at pH 5.8 or solvent production at pH 4.5 and in response to Pi pulses. Specific mRNA transcripts were found only when external Pi concentrations had dropped below 0.2 mM. Two specific transcripts were detected, a 4.7-kb polycistronic mRNA spanning the whole operon and a quantitatively dominating 1.2-kb mRNA representing the first gene, pstS. The mRNA levels clearly differed depending on the external pH. The amounts of the full-length mRNA detected were about two times higher at pH 5.8 than at pH 4.5. The level of pstS mRNA increased by a factor of at least 8 at pH 5.8 compared to pH 4.5 results. Primer extension experiments revealed only one putative transcription start point 80 nucleotides upstream of pstS. Thus, additional regulatory sites are proposed in the promoter region, integrating two different extracellular signals, namely, depletion of inorganic phosphate and the pH of the environment. After phosphate pulses were applied to a phosphate-limited chemostat we observed faster phosphate consumption at pH 5.8 than at pH 4.5, although higher optical densities were recorded at pH 4.5. PMID:16855236

  4. Northern, morphological, and fermentation analysis of spo0A inactivation and overexpression in Clostridium acetobutylicum ATCC 824.

    PubMed

    Harris, Latonia M; Welker, Neil E; Papoutsakis, Eleftherios T

    2002-07-01

    The Clostridium acetobutylicum ATCC 824 spo0A gene was cloned, and two recombinant strains were generated, an spo0A inactivation strain (SKO1) and an spo0A overexpression strain [824(pMPSOA)]. SKO1 was developed by targeted gene inactivation with a replicative plasmid capable of double-crossover chromosomal integration--a technique never used before with solventogenic clostridia. SKO1 was severely deficient in solvent formation: it produced only 2 mM acetone and 13 mM butanol, compared to the 92 mM acetone and 172 mM butanol produced by the parental strain. After 72 h of growth on solid media, SKO1 formed long filaments of rod-shaped cells that failed to septate. SKO1 cells never achieved the swollen clostridial form typical of the parental strain and did not form endospores. No spo0A transcripts were detected in SKO1, while transcription of two solvent formation operons (aad-ctfA-ctfB and adc; both containing 0A boxes in their promoter regions) was limited. Strain 824(pMSPOA) produced higher butanol concentrations than the control strain [824(pIMP1)] and dramatically elevated spo0A transcript levels and displayed a bimodal pattern of spo0A transcription similar to that of B. subtilis. Microscopic studies indicated that sporulation was both enhanced and accelerated due to spo0A overexpression compared to that of both the 824(pIMP1) and parental strains. Consistent with that, expression of the key solvent formation genes (aad-ctfA-ctfB and adc) and three sporulation-specific genes (spoIIGA, sigE, and sigG) was observed earlier in strain 824(pMSPOA) than in the plasmid control. These data support the hypothesis that Spo0A is a transcriptional regulator that positively controls sporulation and solvent production. Its effect on solvent formation is a balancing act in regulating sporulation versus solvent gene expression: its overexpression apparently tips the balance in favor of accelerated and enhanced sporulation at the expense of overall solvent production.

  5. Cloning and expression of clostridium acetobutylicum ATCC 824 acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase in Escherichia coli

    SciTech Connect

    Cary, J.W.; Petersen, D.J.; Bennett, G.N. ); Papoutsakis, E.T. )

    1990-06-01

    Coenzyme A (CoA)-transferase (acetoacetyl-CoA:acetate/butyrate:CoA-transferase (butyrate-acetoacetate CoA-transferase) (EC 2.8.3.9)) of Clostridium acetobutylicum ATCC 824 is an important enzyme in the metabolic shift between the acid-producing and solvent-forming states of this organism. The genes encoding the two subunits of this enzyme have been cloned and subsequent subcloning experiments established the position of the structural genes for CoA-transferase. Complementation of Escherichia coli ato mutants with the recombinant plasmid pCoAT4 (pUC19 carrying a 1.8-kilobase insert of C. acetobutylicum DNA encoding CoA-transferase activity) enabled the transformants to grow on butyrate as a sole carbon source. Despite the ability of CoA-transferase to complement the ato defect in E. coli mutants, Southern blot and Western blot (immunoblot) analyses showed showed that neither the C. acetobutylicum genes encoding CoA-transferase nor the enzyme itself shared any apparent homology with its E. coli counterpart. Polypeptides of M{sub r} of the purified CoA-transferase subunits were observed by Western blot and maxicell analysis of whole-cell extracts of E.coli harboring pCoAT4. The proximity and orientation of the genes suggest that the genes encoding the two subunits of CoA-transferase may form an operon similar to that found in E. coli. In the plasmid, however, transcription appears to be primarily from the lac promoter of the vector.

  6. Integrative modelling of pH-dependent enzyme activity and transcriptomic regulation of the acetone-butanol-ethanol fermentation of Clostridium acetobutylicum in continuous culture.

    PubMed

    Millat, Thomas; Janssen, Holger; Bahl, Hubert; Fischer, Ralf-Jörg; Wolkenhauer, Olaf

    2013-09-01

    In a continuous culture under phosphate limitation the metabolism of Clostridium acetobutylicum depends on the external pH level. By comparing seven steady-state conditions between pH 5.7 and pH 4.5 we show that the switch from acidogenesis to solventogenesis occurs between pH 5.3 and pH 5.0 with an intermediate state at pH 5.1. Here, an integrative study is presented investigating how a changing external pH level affects the clostridial acetone-butanol-ethanol (ABE) fermentation pathway. This is of particular interest as the biotechnological production of n-butanol as biofuel has recently returned into the focus of industrial applications. One prerequisite is the furthering of the knowledge of the factors determining the solvent production and their integrative regulations. We have mathematically analysed the influence of pH-dependent specific enzyme activities of branch points of the metabolism on the product formation. This kinetic regulation was compared with transcriptomic regulation regarding gene transcription and the proteomic profile. Furthermore, both regulatory mechanisms were combined yielding a detailed projection of their individual and joint effects on the product formation. The resulting model represents an important platform for future developments of industrial butanol production based on C. acetobutylicum.

  7. Regulation of the sol locus genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824 by a putative transcriptional repressor.

    PubMed

    Nair, R V; Green, E M; Watson, D E; Bennett, G N; Papoutsakis, E T

    1999-01-01

    A gene (orf1, now designated solR) previously identified upstream of the aldehyde/alcohol dehydrogenase gene aad (R. V. Nair, G. N. Bennett, and E. T. Papoutsakis, J. Bacteriol. 176:871-885, 1994) was found to encode a repressor of the sol locus (aad, ctfA, ctfB and adc) genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824. Primer extension analysis identified a transcriptional start site 35 bp upstream of the solR start codon. Amino acid comparisons of SolR identified a potential helix-turn-helix DNA-binding motif in the C-terminal half towards the center of the protein, suggesting a regulatory role. Overexpression of SolR in strain ATCC 824(pCO1) resulted in a solvent-negative phenotype owing to its deleterious effect on the transcription of the sol locus genes. Inactivation of solR in C. acetobutylicum via homologous recombination yielded mutants B and H (ATCC 824 solR::pO1X) which exhibited deregulated solvent production characterized by increased flux towards butanol and acetone formation, earlier induction of aad, lower overall acid production, markedly improved yields of solvents on glucose, a prolonged solvent production phase, and increased biomass accumulation compared to those of the wild-type strain.

  8. Integrative modelling of pH-dependent enzyme activity and transcriptomic regulation of the acetone–butanol–ethanol fermentation of Clostridium acetobutylicum in continuous culture

    PubMed Central

    Millat, Thomas; Janssen, Holger; Bahl, Hubert; Fischer, Ralf-Jörg; Wolkenhauer, Olaf

    2013-01-01

    Summary In a continuous culture under phosphate limitation the metabolism of Clostridium acetobutylicum depends on the external pH level. By comparing seven steady-state conditions between pH 5.7 and pH 4.5 we show that the switch from acidogenesis to solventogenesis occurs between pH 5.3 and pH 5.0 with an intermediate state at pH 5.1. Here, an integrative study is presented investigating how a changing external pH level affects the clostridial acetone–butanol–ethanol (ABE) fermentation pathway. This is of particular interest as the biotechnological production of n-butanol as biofuel has recently returned into the focus of industrial applications. One prerequisite is the furthering of the knowledge of the factors determining the solvent production and their integrative regulations. We have mathematically analysed the influence of pH-dependent specific enzyme activities of branch points of the metabolism on the product formation. This kinetic regulation was compared with transcriptomic regulation regarding gene transcription and the proteomic profile. Furthermore, both regulatory mechanisms were combined yielding a detailed projection of their individual and joint effects on the product formation. The resulting model represents an important platform for future developments of industrial butanol production based on C. acetobutylicum. PMID:23332010

  9. Regulation of the sol Locus Genes for Butanol and Acetone Formation in Clostridium acetobutylicum ATCC 824 by a Putative Transcriptional Repressor

    PubMed Central

    Nair, Ramesh V.; Green, Edward M.; Watson, David E.; Bennett, George N.; Papoutsakis, Eleftherios T.

    1999-01-01

    A gene (orf1, now designated solR) previously identified upstream of the aldehyde/alcohol dehydrogenase gene aad (R. V. Nair, G. N. Bennett, and E. T. Papoutsakis, J. Bacteriol. 176:871–885, 1994) was found to encode a repressor of the sol locus (aad, ctfA, ctfB and adc) genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824. Primer extension analysis identified a transcriptional start site 35 bp upstream of the solR start codon. Amino acid comparisons of SolR identified a potential helix-turn-helix DNA-binding motif in the C-terminal half towards the center of the protein, suggesting a regulatory role. Overexpression of SolR in strain ATCC 824(pCO1) resulted in a solvent-negative phenotype owing to its deleterious effect on the transcription of the sol locus genes. Inactivation of solR in C. acetobutylicum via homologous recombination yielded mutants B and H (ATCC 824 solR::pO1X) which exhibited deregulated solvent production characterized by increased flux towards butanol and acetone formation, earlier induction of aad, lower overall acid production, markedly improved yields of solvents on glucose, a prolonged solvent production phase, and increased biomass accumulation compared to those of the wild-type strain. PMID:9864345

  10. Metabolic engineering of Clostridium acetobutylicum ATCC 824 for the high-yield production of a biofuel composed of an isopropanol/butanol/ethanol mixture.

    PubMed

    Dusséaux, Simon; Croux, Christian; Soucaille, Philippe; Meynial-Salles, Isabelle

    2013-07-01

    Clostridium acetobutylicum was metabolically engineered to produce a biofuel consisting of an isopropanol/butanol/ethanol mixture. For this purpose, different synthetic isopropanol operons were constructed and introduced on plasmids in a butyrate minus mutant strain (C. acetobutylicum ATCC 824 Δcac15ΔuppΔbuk). The best strain expressing the isopropanol operon from the thl promoter was selected from batch experiments at pH 5. By further optimizing the pH of the culture, a biofuel mixture with almost no by-products was produced at a titer, a yield and productivity never reached before, opening the opportunities to develop an industrial process for alternative biofuels with Clostridial species. Furthermore, by performing in vivo and in vitro flux analysis of the synthetic isopropanol pathway, this flux was identified to be limited by the [acetate](int) and the high Km of CoA-transferase for acetate. Decreasing the Km of this enzyme using a protein engineering approach would be a good target for improving isopropanol production and avoiding acetate accumulation in the culture medium. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Comparative genomic and proteomic analyses of Clostridium acetobutylicum Rh8 and its parent strain DSM 1731 revealed new understandings on butanol tolerance.

    PubMed

    Bao, Guanhui; Dong, Hongjun; Zhu, Yan; Mao, Shaoming; Zhang, Tianrui; Zhang, Yanping; Chen, Zugen; Li, Yin

    2014-08-08

    Clostridium acetobutylicum strain Rh8 is a butanol-tolerant mutant which can tolerate up to 19g/L butanol, 46% higher than that of its parent strain DSM 1731. We previously performed comparative cytoplasm- and membrane-proteomic analyses to understand the mechanism underlying the improved butanol tolerance of strain Rh8. In this work, we further extended this comparison to the genomic level. Compared with the genome of the parent strain DSM 1731, two insertion sites, four deletion sites, and 67 single nucleotide variations (SNVs) are distributed throughout the genome of strain Rh8. Among the 67 SNVs, 16 SNVs are located in the predicted promoters and intergenic regions; while 29 SNVs are located in the coding sequence, affecting a total of 21 proteins involved in transport, cell structure, DNA replication, and protein translation. The remaining 22 SNVs are located in the ribosomal genes, affecting a total of 12 rRNA genes in different operons. Analysis of previous comparative proteomic data indicated that none of the differentially expressed proteins have mutations in its corresponding genes. Rchange Algorithms analysis indicated that the mutations occurred in the ribosomal genes might change the ribosome RNA thermodynamic characteristics, thus affect the translation strength of these proteins. Take together, the improved butanol tolerance of C. acetobutylicum strain Rh8 might be acquired through regulating the translational process to achieve different expression strength of genes involved in butanol tolerance. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Combination of RNA sequencing and metabolite data to elucidate improved toxic compound tolerance and butanol fermentation of Clostridium acetobutylicum from wheat straw hydrolysate by supplying sodium sulfide.

    PubMed

    Jin, Yanling; Fang, Yang; Huang, Mengjun; Sun, Jiaolong; Huang, Yuhong; Gao, Xiaofeng; Li, Renqiang; He, Kaize; Zhao, Hai

    2015-12-01

    Sodium sulfide (SS) was added to the non-detoxified wheat straw hydrolysate for ABE fermentation by Clostridium acetobutylicum CICC8012. Biochemical measurements demonstrated that supplementation with SS promoted earlier and enhanced conversion of acid to ABE and led to a 27.48% improvement in sugar consumption, a 20.48% improvement in the sugar-based ABE yield, a 47.63% improvement in the butanol titer, and a 53.50% improvement in the ABE concentration. The response of C. acetobutylicum CICC8012 at the mRNA level was examined by a transcriptional analysis performed with RNA sequencing. The expression of genes involved in the membrane transport of carbohydrates, glycolysis, and ABE formation increased following SS-supplemented fermentation, whereas the expression of genes encoding enzymes involved in acid formation decreased, which indicates that supplemental SS affected the central fermentative pathway, down-regulated the metabolic flux toward the acid formation branches, and up-regulated the metabolic flux toward the ABE formation branches. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Cloning, expression, and purification of glutamine synthetase from Clostridum acetobutylicum

    SciTech Connect

    Usdin, K.P.; Zappe, H.; Jones, D.T.; Woods, D.R.

    1986-09-01

    A glutamine synthetase (GS) gene, glnA, from the gram-positive obligate anaerobe Clostridium acetobutylicum was cloned on recombinant plasmid pHZ200 and enabled Escherichia coli glnA deletion mutants to utilize (NH/sub 4/)/sub 2/ as a sole source of nitrogen. The cloned C. acetobutylicum gene was expressed from a regulatory region contained within the cloned DNA fragment. glnA expression was subject to nitrogen regulation in E. coli. This cloned glnA DNA did not enable an E. coli glnA ntrB ntrC deletion mutant to utilize arginine or low levels of glutamine as sole nitrogen sources, and failed to activate histidase activity in this strain which contained the Klebsiella aerogenes hut operon. The GS produced by pHZ200 was purified and had an apparent subunit molecular weight of approximately 59,000. There was no DNA or protein homology between the cloned C. acetobutylicum glnA gene and GS and the corresponding gene and GS from E. coli. The C. acetobutylicum GS was inhibited by Mg/sup 2 +/ in the ..gamma..-glutamyl transferase assay, but there was no evidence that the GS was adenylylated.

  14. The mechanism of switching from an acidogenic to butanol-acetone fermentation by Clostridium acetobutylicum. Technical progress report, July 1990--December 1992

    SciTech Connect

    Rogers, P.

    1992-12-31

    The overall objective of this project is to elucidate the detailed mechanism by which solvent-forming bacteria such as Clostridium acetobutylicum regulate the well-known shift in fermentation pathway between alcohol-acetone and organic acid production. It is desired to eventually isolate and describe: (1) the regulatory genes and protein elements that determine induction of synthesis of the solvent-pathway enzymes; and (2) how this regulation system interacts with the sporulatin induction and development program and with related pathways such as granulse and exopolysaccharide formation in clostridia. A working model forhow clostridial control systems work can be derived from recent research on stress systems in E. coli and sporulation in Bacillus subtilis.

  15. Clostridium thermoalcaliphilum sp. nov., an anaerobic and thermotolerant facultative alkaliphile

    SciTech Connect

    Youhong Li; Engle, M.; Wiegel, J.

    1994-01-01

    An anaerobic and thermophilic alkaliphile, strain JW/YL23-2{sub T} (T = type strain), was isolated from sewage sludge obtained from a sewage plant in Atlanta, Ga. at pH 10.1 and 50{degrees}C, the doubling time of this strain was 19 min. Strain JW/YL23-2{sub T}, a motile rod-shaped bacterium with 2 to 12 peritrichous flagella, exhibited a negative Gram stain reaction but was gram-type positive as judged by the polymyxin B test. No heat-stable (85{degrees}C, 15 min) endospores were detected. At 50{degrees}C, growth occurred at pH values ranging from 7.0 to 11.0; the optimum pH was 9.6 to 10.1. The temperature range for growth ranged from 27 to 57.5{degrees}C; the optimum temperature was 48 to 51{degrees}C (pH 10.1). Dissimilatory sulfate reduction was not detected. The organism utilized glucose, fructose, sucrose, maltose, cellobiose, and Casamino Acids. The DNA G+C content was 32 mol% (as determined by chemical analysis). A 16S rRNA sequence analysis revealed a 2% inferred evolutionary distance to Clostridium paradoxum. However, the cell wall type of strain JW/YL23-2{sup T} was A4{beta} (L-Orn-D-Asp), while that of C. paradoxum was Al{sub {tau}} (m-diaminopimelic acid direct). On the basis of the alkaline pH values and high temperatures for optimal growth, the inability to form spores, and other characteristics different from C. paradoxum characteristics, strain JW/YL-23-2 was placed in a new species, Clostridium thermoalcaliphilum; JW/YL23-2 (= DSM 7309) is the type strain of this new species.

  16. Clostridium thermoalcaliphilum sp. nov., an anaerobic and thermotolerant facultative alkaliphile.

    PubMed

    Li, Y; Engle, M; Weiss, N; Mandelco, L; Wiegel, J

    1994-01-01

    An anaerobic and thermophilic alkaliphile, strain JW/YL23-2T (T = type strain), was isolated from sewage sludge obtained from a sewage plant in Atlanta, Ga. At pH 10.1 and 50 degrees C, the doubling time of this strain was 19 min. Strain JW/YL23-2T, a motile rod-shaped bacterium with 2 to 12 peritrichous flagella, exhibited a negative Gram stain reaction but was gram-type positive as judged by the polymyxin B test. No heat-stable (85 degrees C, 15 min) endospores were detected. At 50 degrees C, growth occurred at pH values ranging from 7.0 to 11.0; the optimum pH was 9.6 to 10.1. The temperature range for growth ranged from 27 to 57.5 degrees C; the optimum temperature was 48 to 51 degrees C (pH 10.1). Dissimilatory sulfate reduction was not detected. The organism utilized glucose, fructose, sucrose, maltose, cellobiose, and Casamino Acids. The DNA G+C content was 32 mol% (as determined by chemical analysis). A 16S rRNA sequence analysis revealed a 2% inferred evolutionary distance to Clostridium paradoxum. However, the cell wall type of strain JW/YL23-2T was A4 beta (L-Orn-D-Asp), while that of C. paradoxum was A1 tau (m-diaminopimelic acid direct). On the basis of the alkaline pH values and high temperatures for optimal growth, the inability to form spores, and other characteristics different from C. paradoxum characteristics, strain JW/YL-23-2 was placed in a new species, Clostridium thermoalcaliphilum; JW/YL23-2 (= DSM 7309) is the type strain of this new species.

  17. Acetone, isopropanol, and butanol production by Clostridium beijerinckii (syn. Clostridium butylicum) and Clostridium aurantibutyricum

    SciTech Connect

    George, H.A.; Johnson, J.L.; Moore, W.E.C.; Holdeman, L.V.; Chen, J.S.

    1983-03-01

    Thirty-four strains representing 15 species of anaerobic bacteria were screened for acetone, isopropanol, and n-butanol (solvent) production. Under our culture conditions, several strains of Clostridium beijerinckii and C. aurantibutyricum produced at least 40 mM n-butanol (C. acetobutylicum strains produced up to 41 mM n-butanol under similar conditions). Both solvent-producing and non-solvent-producing strains of C. beijerinckii have high DNA homology with a reference strain of C. beijerinckii. Strains labeled ''Clostridium butylicum'' are phenotypically similar to C. beijerinckii and showed at least 78% DNA homology to a reference strain of C. beijerinckii. Therefore, these ''C. butylicum'' strains are members of C. beijerinckii. An earlier DNA homology study has shown that C. beijerinckii, C. aurantibutyricum, and C. acetobutylicum are distinct species.

  18. Common Mesophilic Anaerobes, Including Clostridium botulinum and Clostridium tetani, in 21 Soil Specimens

    PubMed Central

    Smith, Louis Ds.

    1975-01-01

    A relatively rich medium was markedly superior to a dilute medium for the isolation of anaerobic bacteria from soil. The obligate anaerobes isolated from 21 soil samples were all clostridia and the counts ranged from 2.7 × 102 to 3.3 × 106 per g. The organisms most frequently isolated were Clostridium subterminale, C. sordellii, C. sporogenes, C. indolis, C. bifermentans, C. mangenoti, and C. perfringens. Seventeen other species were also recognized but almost one-third of the isolates could not be identified with any known species of Clostridum. C. botulinum type A was demonstrated in six soil samples, and type B in one. These soils were neutral to alkaline in reaction (average pH 7.9) and low in organic matter content (1.4%). The association of C. botulinum types A and B with neutral to alkaline soils was statistically significant (P = 0.001) as was their association with soils low in organic matter (P = 0.005). C. botulinum types E and F were found in one soil sample, pH 4.5, with organic matter 13.7%. C. tetani was isolated from two soil samples, both of intermediate pH value and higher than average organic matter content. PMID:238468

  19. Comparative genomic and proteomic analyses of Clostridium acetobutylicum Rh8 and its parent strain DSM 1731 revealed new understandings on butanol tolerance

    SciTech Connect

    Bao, Guanhui; Dong, Hongjun; Zhu, Yan; Mao, Shaoming; Zhang, Tianrui; Zhang, Yanping; Chen, Zugen; Li, Yin

    2014-08-08

    Highlights: • Genomes of a butanol tolerant strain and its parent strain were deciphered. • Comparative genomic and proteomic was applied to understand butanol tolerance. • None differentially expressed proteins have mutations in its corresponding genes. • Mutations in ribosome might be responsible for the global difference of proteomics. - Abstract: Clostridium acetobutylicum strain Rh8 is a butanol-tolerant mutant which can tolerate up to 19 g/L butanol, 46% higher than that of its parent strain DSM 1731. We previously performed comparative cytoplasm- and membrane-proteomic analyses to understand the mechanism underlying the improved butanol tolerance of strain Rh8. In this work, we further extended this comparison to the genomic level. Compared with the genome of the parent strain DSM 1731, two insertion sites, four deletion sites, and 67 single nucleotide variations (SNVs) are distributed throughout the genome of strain Rh8. Among the 67 SNVs, 16 SNVs are located in the predicted promoters and intergenic regions; while 29 SNVs are located in the coding sequence, affecting a total of 21 proteins involved in transport, cell structure, DNA replication, and protein translation. The remaining 22 SNVs are located in the ribosomal genes, affecting a total of 12 rRNA genes in different operons. Analysis of previous comparative proteomic data indicated that none of the differentially expressed proteins have mutations in its corresponding genes. Rchange Algorithms analysis indicated that the mutations occurred in the ribosomal genes might change the ribosome RNA thermodynamic characteristics, thus affect the translation strength of these proteins. Take together, the improved butanol tolerance of C. acetobutylicum strain Rh8 might be acquired through regulating the translational process to achieve different expression strength of genes involved in butanol tolerance.

  20. Molecular Characterization and Transcriptional Analysis of adhE2, the Gene Encoding the NADH-Dependent Aldehyde/Alcohol Dehydrogenase Responsible for Butanol Production in Alcohologenic Cultures of Clostridium acetobutylicum ATCC 824

    PubMed Central

    Fontaine, Lisa; Meynial-Salles, Isabelle; Girbal, Laurence; Yang, Xinghong; Croux, Christian; Soucaille, Philippe

    2002-01-01

    The adhE2 gene of Clostridium acetobutylicum ATCC 824, coding for an aldehyde/alcohol dehydrogenase (AADH), was characterized from molecular and biochemical points of view. The 2,577-bp adhE2 codes for a 94.4-kDa protein. adhE2 is expressed, as a monocistronic operon, in alcohologenic cultures and not in solventogenic cultures. Primer extension analysis identified two transcriptional start sites 160 and 215 bp upstream of the adhE2 start codon. The expression of adhE2 from a plasmid in the DG1 mutant of C. acetobutylicum, a mutant cured of the pSOL1 megaplasmid, restored butanol production and provided elevated activities of NADH-dependent butyraldehyde and butanol dehydrogenases. The recombinant AdhE2 protein expressed in E. coli as a Strep-tag fusion protein and purified to homogeneity also demonstrated NADH-dependent butyraldehyde and butanol dehydrogenase activities. This is the second AADH identified in C. acetobutylicum ATCC 824, and to our knowledge this is the first example of a bacterium with two AADHs. It is noteworthy that the two corresponding genes, adhE and adhE2, are carried by the pSOL1 megaplasmid of C. acetobutylicum ATCC 824. PMID:11790753

  1. Butyric acid from anaerobic fermentation of lignocellulosic biomass hydrolysates by Clostridium sp. strain RPT-4213

    USDA-ARS?s Scientific Manuscript database

    A novel Clostridium sp. strain RPT-4213 was found producing butyrate under strict anaerobic conditions. This strain produced 9.47 g L-1 butyric acid from MRS media (0.48 g/g glucose). RPT-4213 was also used to ferment dilute acid pretreated hydrolysates including wheat straw (WSH), corn fiber (CFH...

  2. Butyric acid from anaerobic fermentation of lignocellulosic biomass hydrolysates by Clostridium tyrobutyricum strain RPT-4213

    USDA-ARS?s Scientific Manuscript database

    A newly isolated Clostridium sp. strain RPT-4213 was found to produce butyrate under anaerobic conditions. Fermentations using Lactobacilli MRS Broth produced 9.47 g L-1 butyric acid from glucose (0.48 g/g glucose). However, the strain was not capable of utilizing five carbon sugars. To assess the a...

  3. Direct fermentation of gelatinized cassava starch to acetone, butanol, and ethanol using Clostridium acetobutylicum mutant obtained by atmospheric and room temperature plasma.

    PubMed

    Li, Han-guang; Luo, Wei; Wang, Qiang; Yu, Xiao-bin

    2014-04-01

    The mutant strain designated as ART18, obtained from the wild-type strain Clostridium acetobutylicum PW12 treated by atmospheric and room temperature plasma, showed higher solvent tolerance and butanol production than that of the wild-type strain. The production of butanol was 11.3 ± 0.5 g/L, 31 % higher than that of the wild-type strain when it was used for acetone, butanol, and ethanol fermentation in P2 medium. Furthermore, the effects of cassava flour concentration, pH regulators, and vitamins on the ABE production were also investigated. The highest butanol production of 15.8 ± 0.8 g/L and butanol yield (0.31 g/g) were achieved after the above factors were optimized. When acetone, butanol, and ethanol fermentation by ART18 was carried out in a 15-L bioreactor, the butanol production, the productivity of butanol, and the total solvent were 16.3 ± 0.9, 0.19, and 0.28 g/L(/)h, respectively. These results indicate that ART18 is a promising industrial producer in ABE fermentation.

  4. Acetone-butanol-ethanol (ABE) fermentation using Clostridium acetobutylicum XY16 and in situ recovery by PDMS/ceramic composite membrane.

    PubMed

    Wu, Hao; Chen, Xiao-Peng; Liu, Gong-Ping; Jiang, Min; Guo, Ting; Jin, Wan-Qin; Wei, Ping; Zhu, Da-Wei

    2012-09-01

    PDMS/ceramic composite membrane was directly integrated with acetone-butanol-ethanol (ABE) fermentation using Clostridium acetobutylicum XY16 at 37 °C and in situ removing ABE from fermentation broth. The membrane was integrated with batch fermentation, and approximately 46 % solvent was extracted. The solvent in permeates was 118 g/L, and solvent productivity was 0.303 g/(L/h), which was approximately 33 % higher compared with the batch fermentation without in situ recovery. The fed-batch fermentation with in situ recovery by pervaporation continued for more than 200 h, 61 % solvent was extracted, and the solvent in penetration was 96.2 g/L. The total flux ranged from 0.338 to 0.847 kg/(m(2)/h) and the separation factor of butanol ranged from 5.1 to 27.1 in this process. The membrane was fouled by the active fermentation broth, nevertheless the separation performances were partially recovered by offline membrane cleaning, and the solvent productivity was increased to 0.252 g/(L/h), which was 19 % higher compared with that in situ recovery process without membrane cleaning.

  5. Stable high-titer n-butanol production from sucrose and sugarcane juice by Clostridium acetobutylicum JB200 in repeated batch fermentations.

    PubMed

    Jiang, Wenyan; Zhao, Jingbo; Wang, Zhongqiang; Yang, Shang-Tian

    2014-07-01

    The production of n-butanol, a widely used industrial chemical and promising transportation fuel, from abundant, low-cost substrates, such as sugarcane juice, in acetone-butanol-ethanol (ABE) fermentation was studied with Clostridium acetobutylicum JB200, a mutant with high butanol tolerance and capable of producing high-titer (>20 g/L) n-butanol from glucose. Although JB200 is a favorable host for industrial bio-butanol production, its fermentation performance with sucrose and sugarcane juice as substrates has not been well studied. In this study, the long-term n-butanol production from sucrose by JB200 was evaluated with cells immobilized in a fibrous-bed bioreactor (FBB), showing stable performance with high titer (16-20 g/L), yield (∼ 0.21 g/g sucrose) and productivity (∼ 0.32 g/Lh) for 16 consecutive batches over 800 h. Sugarcane thick juice as low-cost substrate was then tested in 3 consecutive batches, which gave similar n-butanol production, demonstrating that JB200 is a robust and promising strain for industrial ABE fermentation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. PTS regulation domain-containing transcriptional activator CelR and sigma factor σ(54) control cellobiose utilization in Clostridium acetobutylicum.

    PubMed

    Nie, Xiaoqun; Yang, Bin; Zhang, Lei; Gu, Yang; Yang, Sheng; Jiang, Weihong; Yang, Chen

    2016-04-01

    The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulation domain (PRD)-containing enhancer binding proteins (EBPs) are an important class of σ(54) -interacting transcriptional activators. Although PRD-containing EBPs are present in many Firmicutes, most of their regulatory functions remain unclear. In this study, the transcriptional regulons of about 50 PRD-containing EBPs in diverse Firmicutes species are reconstructed by using a comparative genomic approach, which contain the genes associated with utilization of β-glucosides, fructose/levan, mannose/glucose, pentitols, and glucosamine/fructosamine. We then present experimental evidence that the cel operon involved in cellobiose utilization is directly regulated by CelR and σ(54) (SigL) in Clostridium acetobutylicum. The predicted three CelR-binding sites and σ(54) promoter elements upstream of the cel operon are verified by in vitro binding assays. We show that CelR has an ATPase activity, which is strongly stimulated by the presence of DNA containing the CelR-binding sites. Moreover, mutations in any one of the three CelR-binding sites significantly decreased the cel promoter activity probably due to the need for all three DNA sites for maximal ATPase activity of CelR. It is suggested that CelR is regulated by PTS-mediated phosphorylation at His-551 and His-829, which exerts a positive effect and an inhibitory effect, respectively, on the CelR activity. © 2015 John Wiley & Sons Ltd.

  7. Clostridium amazonense sp. nov. an obliqately anaerobic bacterium isolated from a remote Amazonian community in Peru

    PubMed Central

    O’Neal, Lindsey; Obregón-Tito, Alexandra J.; Tito, Raul Y.; Ozga, Andrew T.; Polo, Susan I.; Lewis, Cecil M.; Lawson, Paul A.

    2015-01-01

    A strictly anaerobic Gram-stain positive, spore-forming, rod-shaped bacterium designated NE08VT, was isolated from a fecal sample of an individual residing in a remote Amazonian community in Peru. Phylogenetic analysis based on the 16S rRNA gene sequence showed the organism belonged to the genus Clostridium and is most closely related to Clostridium vulturis (97.4% sequence similarity) and was further characterized using biochemical and chemotaxonomic methods. The major cellular fatty acids were anteiso C13:0 and C16:0 with a genomic DNA G + C content of 31.6 mol%. Fermentation products during growth on glucose were acetate and butyrate. Based on phylogenetic, phenotypic and chemotaxonomic information, strain NE08V was identified as representing a novel species of the genus Clostridium, for which the name Clostridium amazonense sp. nov. is proposed. The type strain is NE08VT (DSM 23598T = CCUG 59712T). PMID:26123611

  8. σK of Clostridium acetobutylicum is the first known sporulation-specific sigma factor with two developmentally separated roles, one early and one late in sporulation.

    PubMed

    Al-Hinai, Mohab A; Jones, Shawn W; Papoutsakis, Eleftherios T

    2014-01-01

    Sporulation in the model endospore-forming organism Bacillus subtilis proceeds via the sequential and stage-specific activation of the sporulation-specific sigma factors, σ(H) (early), σ(F), σ(E), σ(G), and σ(K) (late). Here we show that the Clostridium acetobutylicum σ(K) acts both early, prior to Spo0A expression, and late, past σ(G) activation, thus departing from the B. subtilis model. The C. acetobutylicum sigK deletion (ΔsigK) mutant was unable to sporulate, and solventogenesis, the characteristic stationary-phase phenomenon for this organism, was severely diminished. Transmission electron microscopy demonstrated that the ΔsigK mutant does not develop an asymmetric septum and produces no granulose. Complementation of sigK restored sporulation and solventogenesis to wild-type levels. Spo0A and σ(G) proteins were not detectable by Western analysis, while σ(F) protein levels were significantly reduced in the ΔsigK mutant. spo0A, sigF, sigE, sigG, spoIIE, and adhE1 transcript levels were all downregulated in the ΔsigK mutant, while those of the sigH transcript were unaffected during the exponential and transitional phases of culture. These data show that σ(K) is necessary for sporulation prior to spo0A expression. Plasmid-based expression of spo0A in the ΔsigK mutant from a nonnative promoter restored solventogenesis and the production of Spo0A, σ(F), σ(E), and σ(G), but not sporulation, which was blocked past the σ(G) stage of development, thus demonstrating that σ(K) is also necessary in late sporulation. sigK is expressed very early at low levels in exponential phase but is strongly upregulated during the middle to late stationary phase. This is the first sporulation-specific sigma factor shown to have two developmentally separated roles.

  9. σK of Clostridium acetobutylicum Is the First Known Sporulation-Specific Sigma Factor with Two Developmentally Separated Roles, One Early and One Late in Sporulation

    PubMed Central

    Al-Hinai, Mohab A.; Jones, Shawn W.

    2014-01-01

    Sporulation in the model endospore-forming organism Bacillus subtilis proceeds via the sequential and stage-specific activation of the sporulation-specific sigma factors, σH (early), σF, σE, σG, and σK (late). Here we show that the Clostridium acetobutylicum σK acts both early, prior to Spo0A expression, and late, past σG activation, thus departing from the B. subtilis model. The C. acetobutylicum sigK deletion (ΔsigK) mutant was unable to sporulate, and solventogenesis, the characteristic stationary-phase phenomenon for this organism, was severely diminished. Transmission electron microscopy demonstrated that the ΔsigK mutant does not develop an asymmetric septum and produces no granulose. Complementation of sigK restored sporulation and solventogenesis to wild-type levels. Spo0A and σG proteins were not detectable by Western analysis, while σF protein levels were significantly reduced in the ΔsigK mutant. spo0A, sigF, sigE, sigG, spoIIE, and adhE1 transcript levels were all downregulated in the ΔsigK mutant, while those of the sigH transcript were unaffected during the exponential and transitional phases of culture. These data show that σK is necessary for sporulation prior to spo0A expression. Plasmid-based expression of spo0A in the ΔsigK mutant from a nonnative promoter restored solventogenesis and the production of Spo0A, σF, σE, and σG, but not sporulation, which was blocked past the σG stage of development, thus demonstrating that σK is also necessary in late sporulation. sigK is expressed very early at low levels in exponential phase but is strongly upregulated during the middle to late stationary phase. This is the first sporulation-specific sigma factor shown to have two developmentally separated roles. PMID:24187083

  10. Clostridium herbivorans sp. nov., a cellulolytic anaerobe from the pig intestine.

    PubMed

    Varel, V H; Tanner, R S; Woese, C R

    1995-07-01

    A new cellulolytic anaerobic clostridium was isolated from the intestinal tract of pigs. The single isolate was a gram-positive, motile rod, formed terminal to subterminal swollen sporangia, and required a fermentable carbohydrate for growth. Cellulose, cellobiose, maltose, starch, and glycogen supported growth, but glucose and fructose did not. The major end products from the fermentation of cellobiose were butyrate and formate; minor amounts of hydrogen and ethanol were also formed. Ruminal fluid (15%) or yeast extract (1%) was required for good growth. The optimum temperature for growth was 39 to 42 degrees C, and the optimum pH was 6.8 to 7.2. Cell lysis occurred rapidly once stationary growth was reached. A 16S rRNA sequence analysis showed that the strain was related to a group of gram-positive anaerobes that includes Clostridium oroticum and the cellulolytic species Clostridium polysaccharolyticum and Clostridium populeti. The DNA base composition of the isolate is 38 mol% G + C. We propose the name Clostridium herbivorans for this organism; strain 54408 (= ATCC 49925) is the type strain.

  11. ClosTron-mediated engineering of Clostridium.

    PubMed

    Kuehne, Sarah A; Heap, John T; Cooksley, Clare M; Cartman, Stephen T; Minton, Nigel P

    2011-01-01

    The genus Clostridium is a diverse assemblage of Gram positive, anaerobic, endospore-forming bacteria. Whilst certain species have achieved notoriety as important animal and human pathogens (e.g. Clostridium difficile, Clostridium botulinum, Clostridium tetani, and Clostridium perfringens), the vast majority of the genus are entirely benign, and are able to undertake all manner of useful biotransformations. Prominent amongst them are those species able to produce the biofuels, butanol and ethanol from biomass-derived residues, such as Clostridium acetobutylicum, Clostridium beijerinkii, Clostridium thermocellum, and Clostridium phytofermentans. The prominence of the genus in disease and biotechnology has led to the need for more effective means of genetic modification. The historical absence of methods based on conventional strategies for "knock-in" and "knock-out" in Clostridium has led to the adoption of recombination-independent procedures, typified by ClosTron technology. The ClosTron uses a retargeted group II intron and a retro-transposition-activated marker to selectively insert DNA into defined sites within the genome, to bring about gene inactivation and/or cargo DNA delivery. The procedure is extremely efficient, rapid, and requires minimal effort by the operator.

  12. Anaerobic decomposition of humic substances by Clostridium from the deep subsurface.

    PubMed

    Ueno, Akio; Shimizu, Satoru; Tamamura, Shuji; Okuyama, Hidetoshi; Naganuma, Takeshi; Kaneko, Katsuhiko

    2016-01-08

    Decomposition of humic substances (HSs) is a slow and cryptic but non-negligible component of carbon cycling in sediments. Aerobic decomposition of HSs by microorganisms in the surface environment has been well documented; however, the mechanism of anaerobic microbial decomposition of HSs is not completely understood. Moreover, no microorganisms capable of anaerobic decomposition of HSs have been isolated. Here, we report the anaerobic decomposition of humic acids (HAs) by the anaerobic bacterium Clostridium sp. HSAI-1 isolated from the deep terrestrial subsurface. The use of (14)C-labelled polycatechol as an HA analogue demonstrated that the bacterium decomposed this substance up to 7.4% over 14 days. The decomposition of commercial and natural HAs by the bacterium yielded lower molecular mass fractions, as determined using high-performance size-exclusion chromatography. Fourier transform infrared spectroscopy revealed the removal of carboxyl groups and polysaccharide-related substances, as well as the generation of aliphatic components, amide and aromatic groups. Therefore, our results suggest that Clostridium sp. HSAI-1 anaerobically decomposes and transforms HSs. This study improves our understanding of the anaerobic decomposition of HSs in the hidden carbon cycling in the Earth's subsurface.

  13. Anaerobic decomposition of humic substances by Clostridium from the deep subsurface

    PubMed Central

    Ueno, Akio; Shimizu, Satoru; Tamamura, Shuji; Okuyama, Hidetoshi; Naganuma, Takeshi; Kaneko, Katsuhiko

    2016-01-01

    Decomposition of humic substances (HSs) is a slow and cryptic but non-negligible component of carbon cycling in sediments. Aerobic decomposition of HSs by microorganisms in the surface environment has been well documented; however, the mechanism of anaerobic microbial decomposition of HSs is not completely understood. Moreover, no microorganisms capable of anaerobic decomposition of HSs have been isolated. Here, we report the anaerobic decomposition of humic acids (HAs) by the anaerobic bacterium Clostridium sp. HSAI-1 isolated from the deep terrestrial subsurface. The use of 14C-labelled polycatechol as an HA analogue demonstrated that the bacterium decomposed this substance up to 7.4% over 14 days. The decomposition of commercial and natural HAs by the bacterium yielded lower molecular mass fractions, as determined using high-performance size-exclusion chromatography. Fourier transform infrared spectroscopy revealed the removal of carboxyl groups and polysaccharide-related substances, as well as the generation of aliphatic components, amide and aromatic groups. Therefore, our results suggest that Clostridium sp. HSAI-1 anaerobically decomposes and transforms HSs. This study improves our understanding of the anaerobic decomposition of HSs in the hidden carbon cycling in the Earth’s subsurface. PMID:26743007

  14. Anaerobic decomposition of humic substances by Clostridium from the deep subsurface

    NASA Astrophysics Data System (ADS)

    Ueno, Akio; Shimizu, Satoru; Tamamura, Shuji; Okuyama, Hidetoshi; Naganuma, Takeshi; Kaneko, Katsuhiko

    2016-01-01

    Decomposition of humic substances (HSs) is a slow and cryptic but non-negligible component of carbon cycling in sediments. Aerobic decomposition of HSs by microorganisms in the surface environment has been well documented; however, the mechanism of anaerobic microbial decomposition of HSs is not completely understood. Moreover, no microorganisms capable of anaerobic decomposition of HSs have been isolated. Here, we report the anaerobic decomposition of humic acids (HAs) by the anaerobic bacterium Clostridium sp. HSAI-1 isolated from the deep terrestrial subsurface. The use of 14C-labelled polycatechol as an HA analogue demonstrated that the bacterium decomposed this substance up to 7.4% over 14 days. The decomposition of commercial and natural HAs by the bacterium yielded lower molecular mass fractions, as determined using high-performance size-exclusion chromatography. Fourier transform infrared spectroscopy revealed the removal of carboxyl groups and polysaccharide-related substances, as well as the generation of aliphatic components, amide and aromatic groups. Therefore, our results suggest that Clostridium sp. HSAI-1 anaerobically decomposes and transforms HSs. This study improves our understanding of the anaerobic decomposition of HSs in the hidden carbon cycling in the Earth’s subsurface.

  15. Calorimetric studies of the growth of anaerobic microbes.

    PubMed

    Miyake, Hideo; Maeda, Yukiko; Ishikawa, Takashi; Tanaka, Akiyoshi

    2016-09-01

    This article aims to validate the use of calorimetry to measure the growth of anaerobic microbes. It has been difficult to monitor the growth of strict anaerobes while maintaining optimal growth conditions. Traditionally, optical density and ATP concentration are usually used as measures of the growth of anaerobic microbes. However, to take these measurements it is necessary to extract an aliquot of the culture, which can be difficult while maintaining anaerobic conditions. In this study, calorimetry was used to continuously and nondestructively measure the heat generated by the growth of anaerobic microbes as a function of time. Clostridium acetobutylicum, Clostridium beijerinckii, and Clostridium cellulovorans were used as representative anaerobic microbes. Using a multiplex isothermal calorimeter, we observed that peak time (tp) of C. acetobutylicum heat evolution increased as the inoculation rate decreased. This strong correlation between the inoculation rate and tp showed that it was possible to measure the growth rate of anaerobic microbes by calorimetry. Overall, our results showed that there is a very good correlation between heat evolution and optical density/ATP concentration, validating the use of the method. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  16. High-titer n-butanol production by clostridium acetobutylicum JB200 in fed-batch fermentation with intermittent gas stripping.

    PubMed

    Xue, Chuang; Zhao, Jingbo; Lu, Congcong; Yang, Shang-Tian; Bai, Fengwu; Tang, I-Ching

    2012-11-01

    Acetone-butanol-ethanol (ABE) fermentation with a hyper-butanol producing Clostridium acetobutylicum JB200 was studied for its potential to produce a high titer of butanol that can be readily recovered with gas stripping. In batch fermentation without gas stripping, a final butanol concentration of 19.1 g/L was produced from 86.4 g/L glucose consumed in 78 h, and butanol productivity and yield were 0.24 g/L h and 0.21 g/g, respectively. In contrast, when gas stripping was applied intermittently in fed-batch fermentation, 172 g/L ABE (113.3 g/L butanol, 49.2 g/L acetone, 9.7 g/L ethanol) were produced from 474.9 g/L glucose in six feeding cycles over 326 h. The overall productivity and yield were 0.53 g/L h and 0.36 g/g for ABE and 0.35 g/L h and 0.24 g/g for butanol, respectively. The higher productivity was attributed to the reduced butanol concentration in the fermentation broth by gas stripping that alleviated butanol inhibition, whereas the increased butanol yield could be attributed to the reduced acids accumulation as most acids produced in acidogenesis were reassimilated by cells for ABE production. The intermittent gas stripping produced a highly concentrated condensate containing 195.9 g/L ABE or 150.5 g/L butanol that far exceeded butanol solubility in water. After liquid-liquid demixing or phase separation, a final product containing ~610 g/L butanol, ~40 g/L acetone, ~10 g/L ethanol, and no acids was obtained. Compared to conventional ABE fermentation, the fed-batch fermentation with intermittent gas stripping has the potential to reduce at least 90% of energy consumption and water usage in n-butanol production from glucose. Copyright © 2012 Wiley Periodicals, Inc.

  17. Genome-Scale Model for Clostridium acetobutylicum: Part II. Development of Specific Proton Flux States and Numerically Determined Sub-Systems

    PubMed Central

    Senger, Ryan S.; Papoutsakis, Eleftherios T.

    2009-01-01

    A regulated genome-scale model for Clostridium acetobutylicum ATCC 824 was developed based on its metabolic network reconstruction. To aid model convergence and limit the number of flux-vector possible solutions (the size of the phenotypic solution space), modeling strategies were developed to impose a new type of constraint at the endo–exo-metabolome interface. This constraint is termed the specific proton flux state, and its use enabled accurate prediction of the extracellular medium pH during vegetative growth of batch cultures. The specific proton flux refers to the influx or efflux of free protons (per unit biomass) across the cell membrane. A specific proton flux state encompasses a defined range of specific proton fluxes and includes all metabolic flux distributions resulting in a specific proton flux within this range. Effective simulation of time-course batch fermentation required the use of independent flux balance solutions from an optimum set of specific proton flux states. Using a real-coded genetic algorithm to optimize temporal bounds of specific proton flux states, we show that six separate specific proton flux states are required to model vegetative-growth metabolism and accurately predict the extracellular medium pH. Further, we define the apparent proton flux stoichiometry per weak acids efflux and show that this value decreases from ~3.5 mol of protons secreted per mole of weak acids at the start of the culture to ~0 at the end of vegetative growth. Calculations revealed that when specific weak acids production is maximized in vegetative growth, the net proton exchange between the cell and environment occurs primarily through weak acids efflux (apparent proton flux stoichiometry is 1). However, proton efflux through cation channels during the early stages of acidogenesis was found to be significant. We have also developed the concept of numerically determined sub-systems of genome-scale metabolic networks here as a sub-network with a one

  18. Clostridium amazonense sp. nov. an obliqately anaerobic bacterium isolated from a remote Amazonian community in Peru.

    PubMed

    O'Neal, Lindsey; Obregón-Tito, Alexandra J; Tito, Raul Y; Ozga, Andrew T; Polo, Susan I; Lewis, Cecil M; Lawson, Paul A

    2015-10-01

    A strictly anaerobic Gram-stain positive, spore-forming, rod-shaped bacterium designated NE08V(T), was isolated from a fecal sample of an individual residing in a remote Amazonian community in Peru. Phylogenetic analysis based on the 16S rRNA gene sequence showed the organism belonged to the genus Clostridium and is most closely related to Clostridium vulturis (97.4% sequence similarity) and was further characterized using biochemical and chemotaxonomic methods. The major cellular fatty acids were anteiso C13:0 and C16:0 with a genomic DNA G + C content of 31.6 mol%. Fermentation products during growth with PYG were acetate and butyrate. Based on phylogenetic, phenotypic and chemotaxonomic information, strain NE08V was identified as representing a novel species of the genus Clostridium, for which the name Clostridium amazonense sp. nov. is proposed. The type strain is NE08V(T) (DSM 23598(T) = CCUG 59712(T)). Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. An Esterase from Anaerobic Clostridium hathewayi Can Hydrolyze Aliphatic-Aromatic Polyesters.

    PubMed

    Perz, Veronika; Hromic, Altijana; Baumschlager, Armin; Steinkellner, Georg; Pavkov-Keller, Tea; Gruber, Karl; Bleymaier, Klaus; Zitzenbacher, Sabine; Zankel, Armin; Mayrhofer, Claudia; Sinkel, Carsten; Kueper, Ulf; Schlegel, Katharina; Ribitsch, Doris; Guebitz, Georg M

    2016-03-15

    Recently, a variety of biodegradable polymers have been developed as alternatives to recalcitrant materials. Although many studies on polyester biodegradability have focused on aerobic environments, there is much less known on biodegradation of polyesters in natural and artificial anaerobic habitats. Consequently, the potential of anaerobic biogas sludge to hydrolyze the synthetic compostable polyester PBAT (poly(butylene adipate-co-butylene terephthalate) was evaluated in this study. On the basis of reverse-phase high-performance liquid chromatography (RP-HPLC) analysis, accumulation of terephthalic acid (Ta) was observed in all anaerobic batches within the first 14 days. Thereafter, a decline of Ta was observed, which occurred presumably due to consumption by the microbial population. The esterase Chath_Est1 from the anaerobic risk 1 strain Clostridium hathewayi DSM-13479 was found to hydrolyze PBAT. Detailed characterization of this esterase including elucidation of the crystal structure was performed. The crystal structure indicates that Chath_Est1 belongs to the α/β-hydrolases family. This study gives a clear hint that also micro-organisms in anaerobic habitats can degrade manmade PBAT.

  20. A quantitative metabolomics study of high sodium response in Clostridium acetobutylicum ATCC 824 acetone-butanol-ethanol (ABE) fermentation

    PubMed Central

    Zhao, Xinhe; Condruz, Stefan; Chen, Jingkui; Jolicoeur, Mario

    2016-01-01

    Hemicellulose hydrolysates, sugar-rich feedstocks used in biobutanol refinery, are normally obtained by adding sodium hydroxide in the hydrolyze process. However, the resulting high sodium concentration in the hydrolysate inhibits ABE (acetone-butanol-ethanol) fermentation, and thus limits the use of these low-cost feedstocks. We have thus studied the effect of high sodium on the metabolic behavior of Clostridium acetobutyricum ATCC 824, with xylose as the carbon source. At a threshold sodium concentration of 200 mM, a decrease of the maximum cell dry weight (−19.50 ± 0.85%) and of ABE yield (−35.14 ± 3.50% acetone, −33.37 ± 0.74% butanol, −22.95 ± 1.81% ethanol) were observed compared to control culture. However, solvents specific productivities were not affected by supplementing sodium. The main effects of high sodium on cell metabolism were observed in acidogenesis, during which we observed the accumulation of ATP and NADH, and the inhibition of the pentose phosphate (PPP) and the glycolytic pathways with up to 80.73 ± 1.47% and 68.84 ± 3.42% decrease of the associated metabolic intermediates, respectively. However, the NADP+-to-NADPH ratio was constant for the whole culture duration, a phenomenon explaining the robustness of solvents specific productivities. Therefore, high sodium, which inhibited biomass growth through coordinated metabolic effects, interestingly triggered cell robustness on solvents specific productivity. PMID:27321153

  1. Clostridium huakuii sp. nov., an anaerobic, acetogenic bacterium isolated from methanogenic consortia.

    PubMed

    Ruan, Zhiyong; Wang, Yanwei; Zhang, Chi; Song, Jinlong; Zhai, Yi; Zhuang, Yan; Wang, Huimin; Chen, Xiaorong; Li, Yanting; Zhao, Bingqiang; Zhao, Bin

    2014-12-01

    A Gram-staining-positive, spore-forming, obligately anaerobic, acetogenic bacterium, designated LAM1030(T), was isolated from methanogenic consortia enriched from biogas slurry collected from the large-scale anaerobic digester of Modern Farming Corporation in Hebei Province, China. Cells of strain LAM1030(T) were motile, straight or spiral-rod-shaped. Strain LAM1030(T) could utilize glucose, fructose, maltose, galactose, lactose, sucrose, cellobiose, mannitol, pyruvate, succinic acid and tryptophan as the sole carbon source. Acetic acid, isovaleric acid and butanoic acid were the main products of glucose fermentation. Sodium sulfite was used as an electron acceptor. Growth of strain LAM1030(T) was completely inhibited by the addition of ampicillin, tetracycline, gentamicin or erythromycin at a concentration of 20 µg ml(-1). The main polar lipids of strain LAM1030(T) were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, 11 unknown glycolipids and two unknown phospholipids. No respiratory quinone was detected. The major fatty acids of strain LAM1030(T) were C16 : 0 (21.1 %), C14 : 0 (10.3 %), summed feature 9 (including C16:0 10-methyl and/or iso-C17:1 ω9c) (11.3% ), summed feature 3 (including C16:1 ω7c and/or C16:1 ω6c) (10.6% ) and iso-C15 : 0 (6.6 %). Analysis of the 16S rRNA gene sequence indicated that strain LAM1030(T) belonged to the genus Clostridium and was most closely related to Clostridium subterminale DSM 6970(T), Clostridium thiosulfatireducens DSM 13105(T) and Clostridium sulfidigenes DSM 18982(T), with 97.0, 96.9 and 96.8 % similarity, respectively. The G+C content of the genomic DNA of strain LAM1030(T) was 31.2±0.3 mol%. On the basis of its phenotypic, phylogenetic and chemotaxonomic characterization, strain LAM1030(T) is suggested to represent a novel species of the genus Clostridium, for which the name Clostridium huakuii sp. nov. is proposed. The type strain is LAM1030(T) ( = ACCC 00698(T

  2. Clostridium algifaecis sp. nov., an anaerobic bacterial species from decomposing algal scum.

    PubMed

    Wu, Yu-Fan; Zheng, Hui; Wu, Qing-Long; Yang, Hong; Liu, Shuang-Jiang

    2014-11-01

    Two anaerobic bacterial strains, MB9-7(T) and MB9-9, were isolated from decomposing algal scum and were characterized using a polyphasic approach. Phylogenetic analysis of 16S rRNA gene sequences showed that strains MB9-7(T) and MB9-9 are closely related to each other (99.7% similarity) and they are also closely related to Clostridium tyrobutyricum (96.5%). The two strains were Gram-stain positive and rod-shaped. Growth occurred at 20-45 °C, at pH 4.0-8.0 and at NaCl concentrations of up to 2% (w/v). Acid was produced from glucose, xylose and mannose. Products of fermentation in PYG medium were mainly butyrate, acetate, carbon dioxide and hydrogen. The predominant cellular fatty acids were C(14:0) and C(16:0). The cellular polar lipids comprised phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, two glycolipids, one phospholipid, one aminophospholipid and two aminolipids. The DNA G+C contents of strain MB9-7(T) and MB9-9 were 27.9 and 28.7 mol%, respectively. These results support the assignment of the new isolates to the genus Clostridium and also distinguish them from other species of the genus Clostridium. Hence, it is proposed that strains MB9-7(T) and MB9-9 represent a novel species of the genus Clostridium, with the suggested name Clostridium algifaecis sp. nov. The type strain is MB9-7(T) ( =CGMCC 1.5188(T) =DSM 28783(T)). © 2014 IUMS.

  3. Clostridium aldrichii sp. nov., a cellulolytic mesophile inhabiting a wood-fermenting anaerobic digester.

    PubMed

    Yang, J C; Chynoweth, D P; Williams, D S; Li, A

    1990-07-01

    An anaerobic, mesophilic, spore-forming, cellulolytic bacterium was repeatedly isolated from a wood-fermenting anaerobic digester. Cells of this organism were gram-positive rods, motile with a bundle of polar flagella, and formed subterminal oblong spores. The colonies in agar had an irregular shape with many platelike structures and were greyish white. Cellulose, xylan, and cellobiose served as substrates for growth. Acetate, propionate, butyrate, isobutyrate, isovalerate, lactate, succinate, H2, and CO2 were products of cellobiose fermentation. The optimal temperature and pH for growth were 35 degrees C and 7, respectively. The DNA composition was 40 mol% G + C. The name Clostridium aldrichii sp. nov. is proposed. The type strain is P-1 (= OGI 112, = ATCC 49358).

  4. Clostridium punense sp. nov., an obligate anaerobe isolated from healthy human faeces.

    PubMed

    Lanjekar, Vikram Bholanath; Marathe, Nachiket Prakash; Shouche, Yogesh Shreepad; Ranade, Dilip Ramchandra

    2015-12-01

    An obligately anaerobic, rod-shaped (0.5-1.0 × 2.0-10.0 μm), Gram-stain-positive bacterium, occurring mainly singly or in pairs, and designated BLPYG-8T, was isolated from faeces of a healthy human volunteer aged 56 years. Cells were non-motile. Oval, terminal spores were formed that swell the cells. The strain was affiliated with the genus Clostridium sensu stricto (Clostridium rRNA cluster I) as revealed by 16S rRNA gene sequence analysis. Strain BLPYG-8T showed 97.3 to 97.4 % 16S rRNA gene sequence similarity with Clostridium sulfidigenes DSM 18982T, Clostridium subterminale DSM 6970T and Clostridium thiosulfatireducens DSM 13105T. DNA-DNA hybridization and phenotypic analysis showed that the strain was distinct from its closest relatives, C. sulfidigenes DSM 18982T, C. subterminale DSM 6970T, C. thiosulfatireducens DSM 13105T with 54.2, 53.9 and 53.3 % DNA-DNA relatedness, respectively. Strain BLPYG-8T grew in PYG broth at temperatures between 20 and 40 °C (optimum 37 °C). The strain utilized a range of amino acids as well as carbohydrates as a source of carbon and energy. Glucose fermentation resulted in the formation of volatile fatty acids mainly acetic acid, n-butyric acid and organic acids such as succinic and lactic acid. The DNA G+C content of strain BLPYG-8T was 44.1 mol%. The major fatty acids (>10 %) were C14 : 0, iso-C15 : 0, C16 : 1ω7c and C16 : 0. Phylogenetic analysis and specific phenotypic characteristics and/or DNA G+C content differentiated the strain from its closest relatives. On the basis of these data, strain BLPYG-8T represents a novel species of the genus Clostridium, for which the name Clostridium punense sp. nov. is proposed. The type strain is BLPYG-8T ( = DSM 28650T = CCUG 64195T = MCC 2737T).

  5. Physical Characterization of Clostridium Botulinum Neurotoxin Genes

    DTIC Science & Technology

    1993-10-01

    Clostridium sporogenes 52 2.3 An Expression System for Clostridium acetobutylicum 58 2.4 Attempted Expression of BoNT/A Hc-encoding Fragments 74 2.5 Status... Clostridium / E. coli shuttle vector pMTL500ET 56 15. The C. acetobutylicum expression vector pMTL500F 59 16. Inducible expression of a cat gene using...expression system, developed in this laboratory independently of this contract, for Clostridium acetobutylicum . Although the promoter in question (fac) is

  6. Physical Characterization of Clostridium Botulinum Neurotoxin Genes

    DTIC Science & Technology

    1992-02-17

    Attention was switched to employing Clostridium acetobutylicum NCIB 8052 as the recombinant host and efforts focused on obtaining regulated...Transfer in Clostridium sporogenes 41 2.3 An Expression System for Clostridium acetobutylicum 47 CONCLUSIONS 57- 58 REFERENCES 59-64 CONTENTS Page Number...A[lac-pro] supE thi hsdDS/ F’- traD36 proA+ Br lacP lacZAM15) and 168 trpC, respectively. The Clostridium acetobutylicum strain employed was NCIB

  7. The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features

    PubMed Central

    Seedorf, Henning; Fricke, W. Florian; Veith, Birgit; Brüggemann, Holger; Liesegang, Heiko; Strittmatter, Axel; Miethke, Marcus; Buckel, Wolfgang; Hinderberger, Julia; Li, Fuli; Hagemeier, Christoph; Thauer, Rudolf K.; Gottschalk, Gerhard

    2008-01-01

    Clostridium kluyveri is unique among the clostridia; it grows anaerobically on ethanol and acetate as sole energy sources. Fermentation products are butyrate, caproate, and H2. We report here the genome sequence of C. kluyveri, which revealed new insights into the metabolic capabilities of this well studied organism. A membrane-bound energy-converting NADH:ferredoxin oxidoreductase (RnfCDGEAB) and a cytoplasmic butyryl-CoA dehydrogenase complex (Bcd/EtfAB) coupling the reduction of crotonyl-CoA to butyryl-CoA with the reduction of ferredoxin represent a new energy-conserving module in anaerobes. The genes for NAD-dependent ethanol dehydrogenase and NAD(P)-dependent acetaldehyde dehydrogenase are located next to genes for microcompartment proteins, suggesting that the two enzymes, which are isolated together in a macromolecular complex, form a carboxysome-like structure. Unique for a strict anaerobe, C. kluyveri harbors three sets of genes predicted to encode for polyketide/nonribosomal peptide synthetase hybrides and one set for a nonribosomal peptide synthetase. The latter is predicted to catalyze the synthesis of a new siderophore, which is formed under iron-deficient growth conditions. PMID:18218779

  8. Improvement of the butanol production selectivity and butanol to acetone ratio (B:A) by addition of electron carriers in the batch culture of a new local isolate of Clostridium acetobutylicum YM1.

    PubMed

    Nasser Al-Shorgani, Najeeb Kaid; Kalil, Mohd Sahaid; Wan Yusoff, Wan Mohtar; Shukor, Hafiza; Hamid, Aidil Abdul

    2015-12-01

    Improvement in the butanol production selectivity or enhanced butanol:acetone ratio (B:A) is desirable in acetone-butanol-ethanol (ABE) fermentation by Clostridium strains. In this study, artificial electron carriers were added to the fermentation medium of a new isolate of Clostridium acetobutylicum YM1 in order to improve the butanol yield and B:A ratio. The results revealed that medium supplementation with electron carriers changed the metabolism flux of electron and carbon in ABE fermentation by YM1. A decrease in acetone production, which subsequently improved the B:A ratio, was observed. Further improvement in the butanol production and B:A ratios were obtained when the fermentation medium was supplemented with butyric acid. The maximum butanol production (18.20 ± 1.38 g/L) was gained when a combination of methyl red and butyric acid was added. Although the addition of benzyl viologen (0.1 mM) and butyric acid resulted in high a B:A ratio of 16:1 (800% increment compared with the conventional 2:1 ratio), the addition of benzyl viologen to the culture after 4 h resulted in the production of 18.05 g/L butanol. Manipulating the metabolic flux to butanol through the addition of electron carriers could become an alternative strategy to achieve higher butanol productivity and improve the B:A ratio.

  9. Isolation and characterization of an anaerobic, cellulolytic bacterium, Clostridium cellulovorans sp. nov

    SciTech Connect

    Sleat, R.; Mah, R.A.; Robinson, R.

    1984-07-01

    A new anaerobic, mesophilic, spore-forming cellulolytic bacterium is described. Cellulose is cleared within 24 to 48 h around colonies formed in cellulose agar roll tubes. Cells stain gram negative and are nonmotile rods which form oblong spores either centrally or subterminally in a clostridial swelling. Colonies are irregular with an opaque edge and a center devoid of both vegetative cells and spores. Cellulose, xylan, pectin, cellubiose, glucose, maltose, galactose, sucrose, lactose, and mannose serve as substrates for growth. H/sub 2/, CO/sub 2/, acetate, butyrate, formate, and lactate are produced during fermentation of cellulose or cellobiose. The temperature and pH for optimum growth are 37/sup 0/C and 7.0, respectively. The DNA composition is 26 to 27 mol% guanine plus cytosine. This bacterium resembles Clostridium lochhheadii in morphological and some biochemical characteristics but is not identical to it. The name Clostridium cellulovorans sp. nov. is proposed. The type strain is 743B (ATCC 35296).

  10. Mechanism of Aminoglycoside Antibiotic Resistance in Anaerobic Bacteria: Clostridium perfringens and Bacteroides fragilis

    PubMed Central

    Bryan, L. E.; Kowand, S. K.; Van Den Elzen, H. M.

    1979-01-01

    Cell-free amino acid incorporation using ribosomes from strains of either Clostridium perfringens or Bacteroides fragilis was shown to be susceptible to inhibition by streptomycin and gentamicin. Ribosomes bound dihydrostreptomycin as effectively as ribosomes from Escherichia coli. No inactivation of streptomycin or gentamicin was detected by cell extracts of either anaerobic bacterial species. B. fragilis, grown without added hemin, menadione, and fumarate, and C. perfringens did not show any time-dependent accumulation of dihydrostreptomycin or gentamicin at concentrations tested. Decreased resistance to aminoglycosides and time-dependent uptake of dihydrostreptomycin at 500 μg/ml was observed with B. fragilis grown with hemin, menadione, and fumarate. With the last additions, cytochrome b was detected by cytochrome spectra of B. fragilis. These results demonstrate that anaerobic bacteria unable to carry out oxygen- or nitrate-dependent electron transport are resistant to streptomycin and gentamicin because of failure to transport aminoglycosides. The induction of fumarate-dependent electron transport in B. fragilis is associated with some aminoglycoside transport that is of poor efficiency relative to bacteria with electron transport to oxygen or nitrate. PMID:218500

  11. Investigation of the relation between anaerobic bacteria genus clostridium and late-onset autism etiology in children.

    PubMed

    Keşli, Recep; Gökçen, Cem; Buluğ, Ufuk; Terzi, Yüksel

    2014-01-01

    The aim of this study was to investigate the relation between the etiology of late-onset childhood autism and anaerobic bacteria. Thirty children diagnosed with autistic disorder and control group have been included in the study. 3-(3-hydroxy phenyl)-3-hydroxypropionic acid (HPHPA) excretion rates which is a metabolic product of the genus Clostridium, were measured via mass spectrometry-gas chromatography (MS-GC) method from urine samples. When the assayed average HPHPA values compared with each group, a statistically significant difference was found (p < 0.05). Data obtained from this study support the existence of a significant correlation between autism etiology and anaerobic bacteria.

  12. Trophic links between the acetogen Clostridium glycolicum KHa and the fermentative anaerobe Bacteroides xylanolyticus KHb, isolated from Hawaiian forest soil.

    PubMed

    Hunger, Sindy; Gössner, Anita S; Drake, Harold L

    2011-09-01

    Isolate KH was obtained from Hawaiian forest soil and found to be composed of two functionally linked anaerobes, KHa and KHb. Gene analyses (16S rRNA, fhs, cooS) identified KHa as an acetogenic strain of Clostridium glycolicum and KHb as Bacteroides xylanolyticus. KHb fermented xylan and other saccharides that KHa could not utilize and formed products (e.g., ethanol and H(2)) that supported the acetogenic growth of KHa.

  13. Butanol production employing fed-batch fermentation by Clostridium acetobutylicum GX01 using alkali-pretreated sugarcane bagasse hydrolysed by enzymes from Thermoascus aurantiacus QS 7-2-4.

    PubMed

    Pang, Zong-Wen; Lu, Wei; Zhang, Hui; Liang, Zheng-Wu; Liang, Jing-Juan; Du, Liang-Wei; Duan, Cheng-Jie; Feng, Jia-Xun

    2016-07-01

    Sugarcane bagasse (SB) is a potential feedstock for butanol production. However, biological production of butanol from SB is less economically viable. In this study, evaluation of eight pretreatments on SB showed that alkali pretreatment efficiently removed lignin from SB while retaining the intact native structure of the released microfibrils. In total, 99% of cellulose and 100% of hemicellulose in alkali-pretreated SB were hydrolysed by enzymes from Thermoascus aurantiacus. The hydrolysate was used to produce butanol in a fed-batch fermentation by Clostridium acetobutylicum. At 60h, 14.17 and 21.11gL(-1) of butanol and acetone-butanol-ethanol (ABE) were produced from 68.89gL(-1) of total sugars, respectively, yielding 0.22 and 0.33gg(-1) of sugars. The maximum yield of butanol and ABE reached 15.4g and 22.9g per 100g raw SB, respectively. This established process may have potential application for butanol production from SB.

  14. Reductive Disslocation of Pu(IV) by Clostridium sp. Under Anaerobic Conditions

    SciTech Connect

    Francis,A.; Dodge, C.; Gillow, J.

    2008-01-01

    An anaerobic, gram positive, spore-forming bacterium Clostridium sp., common in soils and wastes, capable of reduction of Fe(III) to Fe(II), Mn(IV) to Mn(II), Tc(VII) to Tc(IV), and U(VI) to U(IV), reduced Pu(IV) to Pu(III). Addition of 242Pu (IV)-nitrate to the bacterial growth medium at pH 6.4 resulted in the precipitation of Pu as amorphous Pu(OH)4 due to hydrolysis and polymerization reactions. The Pu (1 x 10-5 M) had no effect upon growth of the bacterium as evidenced by glucose consumption; carbon dioxide and hydrogen production; a decrease in pH of the medium from 6.4 to 3.0 due to production of acetic and butyric acids from glucose fermentation; and a change in the Eh of the culture medium from +50 to -180 mV. Commensurate with bacterial growth, Pu was rapidly solubilized as evidenced by an increase in Pu concentration in solution which passed through a 0.03 {mu}m filtration. Selective solvent extraction of the culture by thenoyltrifluoroacetone (TTA) indicated the presence of a reduced Pu species in the soluble fraction. X-ray absorption near edge spectroscopic (XANES) analysis of Pu in the culture sample at the Pu LIII absorption edge (18.054 keV) showed a shift of -3 eV compared to a Pu(IV) standard indicating reduction of Pu(IV) to Pu(III). These results suggest that, although Pu generally exists as insoluble Pu(IV) in the environment, under appropriate conditions, anaerobic microbial activity could affect the long-term stability and mobility of Pu by its reductive dissolution.

  15. Facultative Anaerobe Caldibacillus debilis GB1: Characterization and Use in a Designed Aerotolerant, Cellulose-Degrading Coculture with Clostridium thermocellum.

    PubMed

    Wushke, Scott; Levin, David B; Cicek, Nazim; Sparling, Richard

    2015-08-15

    Development of a designed coculture that can achieve aerotolerant ethanogenic biofuel production from cellulose can reduce the costs of maintaining anaerobic conditions during industrial consolidated bioprocessing (CBP). To this end, a strain of Caldibacillus debilis isolated from an air-tolerant cellulolytic consortium which included a Clostridium thermocellum strain was characterized and compared with the C. debilis type strain. Characterization of isolate C. debilis GB1 and comparisons with the type strain of C. debilis revealed significant physiological differences, including (i) the absence of anaerobic metabolism in the type strain and (ii) different end product synthesis profiles under the experimental conditions used. The designed cocultures displayed unique responses to oxidative conditions, including an increase in lactate production. We show here that when the two species were cultured together, the noncellulolytic facultative anaerobe C. debilis GB1 provided respiratory protection for C. thermocellum, allowing the synergistic utilization of cellulose even under an aerobic atmosphere.

  16. Effect of substrate loading on hydrogen production during anaerobic fermentation by Clostridium thermocellum 27405.

    PubMed

    Islam, Rumana; Cicek, Nazim; Sparling, Richard; Levin, David

    2006-09-01

    We have investigated hydrogen (H2) production by the cellulose-degrading anaerobic bacterium, Clostridium thermocellum. In the following experiments, batch-fermentations were carried out with cellobiose at three different substrate concentrations to observe the effects of carbon-limited or carbon-excess conditions on the carbon flow, H2-production, and synthesis of other fermentation end products, such as ethanol and organic acids. Rates of cell growth were unaffected by different substrate concentrations. H2, carbon dioxide (CO2), acetate, and ethanol were the main products of fermentation. Other significant end products detected were formate and lactate. In cultures where cell growth was severely limited due to low initial substrate concentrations, hydrogen yields of 1 mol H2/mol of glucose were obtained. In the cultures where growth ceased due to carbon depletion, lactate and formate represented a small fraction of the total end products produced, which consisted mainly of H2, CO2, acetate, and ethanol throughout growth. In cultures with high initial substrate concentrations, cellobiose consumption was incomplete and cell growth was limited by factors other than carbon availability. H2-production continued even in stationary phase and H2/CO2 ratios were consistently greater than 1 with a maximum of 1.2 at the stationary phase. A maximum specific H2 production rate of 14.6 mmol g dry cell(-1) h(-1) was observed. As cells entered stationary phase, extracellular pyruvate production was observed in high substrate concentration cultures and lactate became a major end product.

  17. Inactivation of Clostridium difficile in sewage sludge by anaerobic thermophilic digestion.

    PubMed

    Xu, Changyun; Salsali, Hamidreza; Weese, Scott; Warriner, Keith

    2016-01-01

    There has been an increase in community-associated Clostridium difficile infections with biosolids derived from wastewater treatment being identified as one potential source. The current study evaluated the efficacy of thermophilic digestion in decreasing levels of C. difficile ribotype 078 associated with sewage sludge. Five isolates of C. difficile 078 were introduced (final density of 5 log CFU/g) into digested sludge and subjected to anaerobic digestion at mesophilic (36 or 42 °C) or thermophilic (55 °C) temperatures for up to 60 days. It was found that mesophilic digestion at 36 °C did not result in a significant reduction in C. difficile spore levels. In contrast, thermophilic sludge digestion reduced endospore levels at a rate of 0.19-2.68 log CFU/day, depending on the strain tested. The mechanism of lethality was indirect - by stimulating germination then inactivating the resultant vegetative cells. Acidification of sludge by adding acetic acid (6 g/L) inhibited the germination of spores regardless of the sludge digestion temperature. In conclusion, thermophilic digestion can be applied to reduce C. difficile in biosolids, thereby reducing the environmental burden of the enteric pathogen.

  18. Isolation and characterization of a moderately thermophilic anaerobic alkaliphile, Clostridium paradoxum sp. nov.

    SciTech Connect

    Youhong Li; Wiegel, J.; Mandelco, L.

    1993-07-01

    Alkaliphilic, moderately thermophilic anaerobic bacteria able to grow above pH 10.5 and 55{degrees}C were isolated from various sewage plants in the United States. The strains were motile with two to six peritrichous flagella and formed round to slightly oval terminal spores in terminally distended and slightly enlarged cells. Sporulated cells remained motile. The pH range for growth was between 7.0 and 11.1, with an optimum of around 10.1. At pH 10.1 the temperature range for growth was between 30 and 63{degrees}C, with an optimum of 56{degrees}C. The shortest observed doubling time (glucose) was around 16 min at 56{degrees}C and pH 10.1. No dissimilatory sulfate reduction was detected. The organism utilized glucose, fructose, sucrose, maltose, and pyruvate but required yeast extract or tryptone for growth. Optimal NaCl concentrations for growth were between 50 and 200 mM. The guanine-plus-cytosine content was 30.0 {+-} 0.10 mol%. On the basis of unique properties and 16S rRNA analysis, the strains are placed in a new species, Clostridium paradoxum, referring to the unusual retainment of motility by sporulated cells. Strain JW-YL-7 (DSM 7308) is designated as the type strain.

  19. Extending CRISPR-Cas9 Technology from Genome Editing to Transcriptional Engineering in the Genus Clostridium

    PubMed Central

    Bruder, Mark R.; Pyne, Michael E.; Moo-Young, Murray

    2016-01-01

    ABSTRACT The discovery and exploitation of the prokaryotic adaptive immunity system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins have revolutionized genetic engineering. CRISPR-Cas tools have enabled extensive genome editing as well as efficient modulation of the transcriptional program in a multitude of organisms. Progress in the development of genetic engineering tools for the genus Clostridium has lagged behind that of many other prokaryotes, presenting the CRISPR-Cas technology an opportunity to resolve a long-existing issue. Here, we applied the Streptococcus pyogenes type II CRISPR-Cas9 (SpCRISPR-Cas9) system for genome editing in Clostridium acetobutylicum DSM792. We further explored the utility of the SpCRISPR-Cas9 machinery for gene-specific transcriptional repression. For proof-of-concept demonstration, a plasmid-encoded fluorescent protein gene was used for transcriptional repression in C. acetobutylicum. Subsequently, we targeted the carbon catabolite repression (CCR) system of C. acetobutylicum through transcriptional repression of the hprK gene encoding HPr kinase/phosphorylase, leading to the coutilization of glucose and xylose, which are two abundant carbon sources from lignocellulosic feedstocks. Similar approaches based on SpCRISPR-Cas9 for genome editing and transcriptional repression were also demonstrated in Clostridium pasteurianum ATCC 6013. As such, this work lays a foundation for the derivation of clostridial strains for industrial purposes. IMPORTANCE After recognizing the industrial potential of Clostridium for decades, methods for the genetic manipulation of these anaerobic bacteria are still underdeveloped. This study reports the implementation of CRISPR-Cas technology for genome editing and transcriptional regulation in Clostridium acetobutylicum, which is arguably the most common industrial clostridial strain. The developed genetic tools enable simpler, more reliable

  20. The Selenoproteome of Clostridium sp. OhILAs: Characterization of Anaerobic Bacterial Selenoprotein Methionine Sulfoxide Reductase A

    PubMed Central

    Kim, Hwa-Young; Zhang, Yan; Lee, Byung Cheon; Kim, Jae-Ryong; Gladyshev, Vadim N.

    2008-01-01

    SUMMARY Selenocysteine (Sec) is incorporated into proteins in response to UGA codons. This residue is frequently found at the catalytic sites of oxidoreductases. In the present study, we characterized the selenoproteome of an anaerobic bacterium, Clostridium sp. (also known as Alkaliphilus oremlandii) OhILA, and identified 13 selenoprotein genes, 5 of which have not been previously described. One of the detected selenoproteins was methionine sulfoxide reductase A (MsrA), an antioxidant enzyme that repairs oxidatively damaged methionines in a stereospecific manner. To date, little is known about MsrA from anaerobes. We characterized this selenoprotein MsrA which had a single Sec residue at the catalytic site but no cysteine (Cys) residues in the protein sequence. Its SECIS (Sec insertion sequence) element did not resemble those in Escherichia coli. Although with low translational efficiency, the expression of the Clostridium selenoprotein msrA gene in E. coli could be demonstrated by 75Se metabolic labeling, immunoblot analyses, and enzyme assays, indicating that its SECIS element was recognized by the E. coli Sec insertion machinery. We found that the Sec-containing MsrA exhibited at least a 20-fold higher activity than its Cys mutant form, indicating a critical role of Sec in the catalytic activity of the enzyme. Furthermore, our data revealed that the Clostridium MsrA was inefficiently reducible by thioredoxin, which is a typical reducing agent for MsrA, suggesting the use of alternative electron donors in this anaerobic bacterium that directly act on the selenenic acid intermediate and do not require resolving Cys residues. PMID:18767149

  1. Selective medium for isolation of Clostridium butyricum from human feces.

    PubMed Central

    Popoff, M R

    1984-01-01

    A selective medium, Clostridium butyricum isolation medium (BIM), is described for the isolation of C. butyricum from human feces. The BIM is a synthetic minimal medium and contains trimethoprim (16 micrograms/ml), D-cycloserine (10 micrograms/ml), and polymyxin B sulfate (20 micrograms/ml) as selective inhibitory agents. Qualitative tests indicated that C. butyricum and other butyric acid-producing clostridia grew on BIM, Clostridium sphenoides and Bacillus cereus produced small colonies, and other clostridia and other obligate anaerobic or facultatively anerobic bacteria were inhibited. Quantitative recovery of C. butyricum from cultures or seeded fecal samples was comparable with BIM and with complex medium, but the quantitative recovery of the other butyric acid-producing clostridia tested (C. beijerinckii, C. acetobutylicum) was lower with BIM than with complex medium. The BIM should aid the rapid isolation of C. butyricum from fecal samples and should be useful for bacteriological investigation of neonatal necrotizing enterocolitis. PMID:6490827

  2. Isolation and characterization of an active mannanase-producing anaerobic bacterium, Clostridium tertium KT-5A, from lotus soil.

    PubMed

    Kataoka, N; Tokiwa, Y

    1998-03-01

    Of 10 strains of mannanase-producing anaerobic bacteria isolated from soils and methanogenic sludges, Clostridium tertium KT-5A, which was isolated from lotus soil, produced high amounts of extracellular beta-1,4-mannanase. The isolate was an aerotolerant anaerobe without quinon systems; the cell growth cultivated with no addition of reducing agents was also stable. High yields of mannanase were obtained by inducing enzyme production with galactomannan guar gum and beef extract/peptone as carbon and nitrogen sources, respectively. Fermentation end products on galactomannan fermentation were formate, acetate, lactate, butyrate, carbon dioxide and hydrogen. The extracellular mannanase displayed high activity on galactomannans of locust bean gum galactose/mannose (G/M) ratio 1:4 and spino gum (G/M 1:3), but weak activity on guar gum galactomannan (G/M 1:2) and konjac glucomannan. As far as is known, this is the first report on the isolation of an active mannanase-producing anaerobic bacterium from natural environments.

  3. Ecophysiological adaptations of anaerobic bacteria to low pH: analysis of anaerobic digestion in acidic bog sediments. [Lactobacillus; Clostridium; Sarcina ventriculi

    SciTech Connect

    Goodwin, S.; Zeikus, G.J.

    1987-01-01

    The dynamics of anaerobic digestion were examined in the low-pH sediments of Crystal Bog in Wisconsin. The sediments (pH 4.9) contained 71% organic matter and the following concentrations of dissolved gases (micromoles per liter):CO/sub 2/, 1140; CH/sub 4/, 490; and H/sub 2/, 0.01. The rate of methane production was 6.2 ..mu..mol/liter of sediment per h, which is slower than eutrophic, neutral sediments. Microbial metabolic processes displayed the following pH optima: hydrolysis reactions, between 4.2 and 5.6; aceticlastic methanogenesis, 5.2; and hydrogen-consuming reactions, 5.6. The turnover rate constants for key intermediary metabolites were (h/sup -1/): glucose, 1.10; lactate, 0.277; acetate, 0.118; and ethanol, 0.089. The populations of anaerobes were low, with hydrolytic groups (10/sup 6//ml) several orders of magnitude higher than methanogens (10/sup 2//ml). The addition of carbon electron donors to the sediment resulted in the accumulation of hydrogen, whereas the addition of hydrogen resulted in the accumulation of fatty acids and the inhibition of hydrogen-producing acetogenic reactions. Strains of Lactobacillus, Clostridium, and Sarcina ventriculi were isolated from the bog, and their physiological attributes were characterized in relation to hydrolytic process functions in the sediments. The present studies provide evidence that the pH present in the bog sediments alter anaerobic digestion processes s, that total biocatalytic activity is lower bu the general carbon and electron flow pathways are similar to those of neutral anoxic sediments.

  4. Sensitive and selective culture medium for detection of environmental Clostridium difficile isolates without requirement for anaerobic culture conditions.

    PubMed

    Cadnum, Jennifer L; Hurless, Kelly N; Deshpande, Abhishek; Nerandzic, Michelle M; Kundrapu, Sirisha; Donskey, Curtis J

    2014-09-01

    Effective and easy-to-use methods for detecting Clostridium difficile spore contamination would be useful for identifying environmental reservoirs and monitoring the effectiveness of room disinfection. Culture-based detection methods are sensitive for detecting C. difficile, but their utility is limited due to the requirement of anaerobic culture conditions and microbiological expertise. We developed a low-cost selective broth medium containing thioglycolic acid and l-cystine, termed C. difficile brucella broth with thioglycolic acid and l-cystine (CDBB-TC), for the detection of C. difficile from environmental specimens under aerobic culture conditions. The sensitivity and specificity of CDBB-TC (under aerobic culture conditions) were compared to those of CDBB (under anaerobic culture conditions) for the recovery of C. difficile from swabs collected from hospital room surfaces. CDBB-TC was significantly more sensitive than CDBB for recovering environmental C. difficile (36/41 [88%] versus 21/41 [51%], respectively; P = 0.006). C. difficile latex agglutination, an enzyme immunoassay for toxins A and B or glutamate dehydrogenase, and a PCR for toxin B genes were all effective as confirmatory tests. For 477 total environmental cultures, the specificity of CDBB-TC versus that of CDBB based upon false-positive yellow-color development of the medium without recovery of C. difficile was 100% (0 false-positive results) versus 96% (18 false-positive results), respectively. False-positive cultures for CDBB were attributable to the growth of anaerobic non-C. difficile organisms that did not grow in CDBB-TC. Our results suggest that CDBB-TC provides a sensitive and selective medium for the recovery of C. difficile organisms from environmental samples, without the need for anaerobic culture conditions. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  5. Assessing Methanobrevibacter smithii and Clostridium difficile as not conventional faecal indicators in effluents of a wastewater treatment plant integrated with sludge anaerobic digestion.

    PubMed

    Romanazzi, Valeria; Bonetta, Silvia; Fornasero, Stefania; De Ceglia, Margherita; Gilli, Giorgio; Traversi, Deborah

    2016-12-15

    Wastewater treatment plants (WWTP) are an important source of surface water contamination by enteric pathogens, affecting the role of environmental water as a microbial reservoir. We describe the release to the environment of certain anaerobes of human and environmental concern. The work was focused on emerging microbial targets. They are tracing, by RT-qPCR, on WWTP effluents, both liquid and solid, when an anaerobic digestion step is included. The focus is placed on Clostridium spp. with the specific quantification of Clostridium perfringens, as typical bioindicator, and Clostridium difficile, as emerging pathogen not only confined into nosocomial infection. Moreover methanogens were quantified for their involvement in the anaerobic digestion, and in particular on Methanobrevibacter smithii as major methanogenic component of the human gut microbiome and as not conventional faecal indicator. In the water samples, a reduction, statistically significant, in all microbial targets was observed (p < 0.01), 2 log for the total bacteria, 1.4 log for the Clostridium spp. and M. smithii, 1 log for total methanogens, C. perfringens and C. difficile. The AD process contribute to a significant change in microbial levels into the sludge for total bacteria and total methanogens (p < 0.01), both when the input sludge are primary and secondary, while for the presence of Clostridium spp. and C. difficile there was not a significant change. The produced data are innovative showing which is the diffusion of such anaerobic microorganisms throughout the WWTP and opening a discussion on the implementation of possible techniques for a more efficient microbial removal from effluents, particularly bio-solids, to reduce the potential release of pathogens into the environment. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Ferredoxin and Formyltetrahydrofolate Synthetase: Comparative Studies with Clostridium acidiurici, Clostridium cylindrosporum, and Newly Isolated Anaerobic Uric Acid-Fermenting Strains

    PubMed Central

    Champion, Alexander B.; Rabinowitz, Jesse C.

    1977-01-01

    Six strains of Clostridium acidiurici and three strains of C. cylindrosporum were isolated from soil samples by enrichment culture with uric acid as the source of carbon, nitrogen, and energy. The newly isolated strains were characterized by their spore morphology and the amounts of glycine and formate formed by the fermentation of uric acid. The strains were easily identified as belonging to one species or the other on the basis of spore morphology and formate production. The crystal properties and spectra of the native ferredoxins of all the strains isolated and the amino acid composition and partial carboxy-terminal sequence of all their apoferredoxins were determined. All the ferredoxins were tested for cross-reactivity with antiserum to C. acidiurici ferredoxin by microcomplement fixation. Five of the six C. acidiurici strains, which had ferredoxins with amino acid compositions identical to that from C. acidiurici, also showed immunological identity (immunological distance = 0.0). These results suggest sequence identity. The one strain with a different amino acid composition failed to show complete cross-reactivity. Two of the three C. cylindrosporum strains have ferredoxin amino acid compositions identical to that from C. cylindrosporum. The third strain had a minimum of five differences in sequence. All C. cylindrosporum strains had ferredoxins that differed considerably from C. acidiurici strains (minimum of eight to nine differences), and none of these ferredoxins cross-reacted with antisera to C. acidiurici ferredoxin. Antisera were prepared to formyltetrahydrofolate synthetase from C. acidiurici and C. cylindrosporum, and all possible comparisons were made by using immunodiffusion and microcomplement fixation. There is more intraspecies variation in the synthetases than in the ferredoxins; however, the results suggest considerable interspecies differences in both proteins. These results suggest a low degree of genomic relatedness between the two species

  7. Genetic and Biochemical Analysis of Anaerobic TNT Biodegradation

    DTIC Science & Technology

    2004-08-04

    using cultures of Clostridium acetobutylicum ATCC824. The studies included (1) the characterization of products of TNT degradation, (2) investigation...describes cloning and expression of two nitroreductases from C. acetobutylicum . TNT transformation of about the same rate was found in strain M5, a mutant...of C. acetobutylicum lacking the large plasmid which bears the solvent-producing genes. This confirms the finding that TNT degradation is

  8. Clostridium swellfunianum sp. nov., a novel anaerobic bacterium isolated from the pit mud of Chinese Luzhou-flavor liquor production.

    PubMed

    Liu, Chaolan; Huang, Dan; Liu, Laiyan; Zhang, Jin; Deng, Yu; Chen, Ling; Zhang, Wenxue; Wu, Zhengyun; Fan, Ao; Lai, Dengyi; Dai, Lirong

    2014-10-01

    A novel Gram-positive, strictly anaerobic, spore-forming, rod-shaped bacterium, designated strain S11-3-10(T), was isolated from the pit mud used for Chinese Luzhou-flavor liquor production. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that the strain formed a monophyletic clade with the closely related type strains of Clostridium cluster I and was most closely related to Clostridium amylolyticum JCM 14823(T) (94.38%). The temperature, pH, and NaCl range for growth was determined to be 20-45 °C (optimum 37 °C), 4.0-10.0 (optimum pH 7.3), and 0-3.0% (w/v), respectively. The strain was able to tolerate up to 7.5 % (v/v) ethanol. Yeast extract or peptone was found to be required for growth. Acids were found to be produced from glucose, mannose and trehalose. The major end products from glucose fermentation were identified as ethanol, acetate and hydrogen. The polar lipids were found to consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and unidentified phospholipids and polar lipids. The major fatty acids (>5%) were identified as iso-C(15:0), C(16:0), C(16:0)dma, C(14:0), anteiso-C(15:0) and iso-C(13:0). No respiratory quinone was detected. The diamino acid in the cell wall peptidoglycan was identified as meso-diaminopimelic acid and the whole-cell sugars were found to include galactose and glucose as major components. The DNA G+C content was determined to be 36.4 mol%. Based on the phylogenetic, chemotaxonomic and phenotypic evidence, the isolate is considered to represent a novel species of the genus Clostridium for which the name Clostridium swellfunianum sp. nov. is proposed. The type strain is S11-3-10(T) (=DSM 27788(T) = JCM 19606(T) = CICC 10730(T)).

  9. Deciphering Clostridium tyrobutyricum Metabolism Based on the Whole-Genome Sequence and Proteome Analyses

    PubMed Central

    Lee, Joungmin; Jang, Yu-Sin; Han, Mee-Jung; Kim, Jin Young

    2016-01-01

    ABSTRACT Clostridium tyrobutyricum is a Gram-positive anaerobic bacterium that efficiently produces butyric acid and is considered a promising host for anaerobic production of bulk chemicals. Due to limited knowledge on the genetic and metabolic characteristics of this strain, however, little progress has been made in metabolic engineering of this strain. Here we report the complete genome sequence of C. tyrobutyricum KCTC 5387 (ATCC 25755), which consists of a 3.07-Mbp chromosome and a 63-kbp plasmid. The results of genomic analyses suggested that C. tyrobutyricum produces butyrate from butyryl-coenzyme A (butyryl-CoA) through acetate reassimilation by CoA transferase, differently from Clostridium acetobutylicum, which uses the phosphotransbutyrylase-butyrate kinase pathway; this was validated by reverse transcription-PCR (RT-PCR) of related genes, protein expression levels, in vitro CoA transferase assay, and fed-batch fermentation. In addition, the changes in protein expression levels during the course of batch fermentations on glucose were examined by shotgun proteomics. Unlike C. acetobutylicum, the expression levels of proteins involved in glycolytic and fermentative pathways in C. tyrobutyricum did not decrease even at the stationary phase. Proteins related to energy conservation mechanisms, including Rnf complex, NfnAB, and pyruvate-phosphate dikinase that are absent in C. acetobutylicum, were identified. Such features explain why this organism can produce butyric acid to a much higher titer and better tolerate toxic metabolites. This study presenting the complete genome sequence, global protein expression profiles, and genome-based metabolic characteristics during the batch fermentation of C. tyrobutyricum will be valuable in designing strategies for metabolic engineering of this strain. PMID:27302759

  10. Clostridium guangxiense sp. nov. and Clostridium neuense sp. nov., two phylogenetically closely related hydrogen-producing species isolated from lake sediment.

    PubMed

    Zhao, Xin; Li, Danyang; Xu, Shuhong; Guo, Zhanghao; Zhang, Yan; Man, Lin; Jiang, Binhui; Hu, Xiaomin

    2017-03-01

    Two novel anaerobic, mesophilic, biohydrogen-producing bacteria, designated strains ZGM211T and G1T, were isolated from lake sediment. 16S rRNA and ATP synthase beta subunit (atpD) gene sequences and phylogenetic analysis of strains ZGM211T and G1T revealed an affiliation to the genus Clostridium sensu stricto (cluster I of the clostridia), with Clostridium acetobutylicum as the closest characterized species, showing the same sequence similarity of 96.4 % to the type strain (98.9 % between the two isolates). Cells of the two strains were rod shaped. Growth occurred at 20-45 °C, pH 4.0-8.0 and NaCl concentrations up to 2 % (w/v). Grown on glucose, the main fermentation products were H2, CO2, acetate and butyrate. The major fatty acids were C14 : 0 and C16 : 0. The DNA G+C contents of strains ZGM211T and G1T were 40.7 and 41.5 mol%, respectively. Based on phenotypic, chemotaxonomic and phylogenetic differences, strains ZGM211T (=CICC 24070T=BCRC 80950T) and G1T (=CICC 24069T=BCRC 80949T) are proposed as the type strains of novel species of the genus Clostridium with the names Clostridium guangxiense sp. nov. and Clostridium neuense sp. nov., respectively.

  11. Anaerobic

    MedlinePlus

    ... more prolonged exercise like walking or jogging. Anaerobic reactions are faster. We need them during shorter, more intense activities like sprinting. Anaerobic exercise leads to a buildup of lactic acid in our tissues. We need oxygen to remove ...

  12. Co-utilization of glycerol and lignocellulosic hydrolysates enhances anaerobic 1,3-propanediol production by Clostridium diolis

    PubMed Central

    Xin, Bo; Wang, Yu; Tao, Fei; Li, Lixiang; Ma, Cuiqing; Xu, Ping

    2016-01-01

    Anaerobic fermentation using lignocellulosic hydrolysates as co-substrates is an economically attractive method to enhance 1,3-propanediol (1,3-PD) production by increasing the conversion yield from glycerol. Lignocellulosic hydrolysates contain the mixed sugars that are primarily glucose, xylose, and arabinose. Therefore, these three individual sugars were used, separately, as co-substrates with glycerol, in 1,3-PD production by a Clostridium diolis strain DSM 15410, resulting in an 18%–28% increase in the 1,3-PD yield. Co-fermentation of the mixed sugars and glycerol obtained a higher intracellular NADH/NAD+ ratio and increased the 1,3-PD yield by 22% relative to fermentation of glycerol alone. Thereafter, two kinds of lignocellulosic hydrolysates, corn stover hydrolysate and corncob molasses, were individually co-fermented with glycerol. The maximum 1,3-PD yield from glycerol reached 0.85 mol/mol. Fed-batch co-fermentation was also performed, improving the 1,3-PD yield (from 0.62 mol/mol to 0.82 mol/mol). These results demonstrate that the co-fermentation strategy is an efficient and economical way to produce 1,3-PD from glycerol. PMID:26750307

  13. Co-utilization of glycerol and lignocellulosic hydrolysates enhances anaerobic 1,3-propanediol production by Clostridium diolis.

    PubMed

    Xin, Bo; Wang, Yu; Tao, Fei; Li, Lixiang; Ma, Cuiqing; Xu, Ping

    2016-01-11

    Anaerobic fermentation using lignocellulosic hydrolysates as co-substrates is an economically attractive method to enhance 1,3-propanediol (1,3-PD) production by increasing the conversion yield from glycerol. Lignocellulosic hydrolysates contain the mixed sugars that are primarily glucose, xylose, and arabinose. Therefore, these three individual sugars were used, separately, as co-substrates with glycerol, in 1,3-PD production by a Clostridium diolis strain DSM 15410, resulting in an 18%-28% increase in the 1,3-PD yield. Co-fermentation of the mixed sugars and glycerol obtained a higher intracellular NADH/NAD(+) ratio and increased the 1,3-PD yield by 22% relative to fermentation of glycerol alone. Thereafter, two kinds of lignocellulosic hydrolysates, corn stover hydrolysate and corncob molasses, were individually co-fermented with glycerol. The maximum 1,3-PD yield from glycerol reached 0.85 mol/mol. Fed-batch co-fermentation was also performed, improving the 1,3-PD yield (from 0.62 mol/mol to 0.82 mol/mol). These results demonstrate that the co-fermentation strategy is an efficient and economical way to produce 1,3-PD from glycerol.

  14. Complete dechlorination of tetrachloroethylene by use of an anaerobic Clostridium bifermentans DPH-1 and zero-valent iron.

    PubMed

    Chang, Y C; Kikuchi, S; Kawauchi, N; Sato, T; Takamizawa, K

    2008-04-01

    A laboratory test was conducted to examine the combined effect of an anaerobic Clostridium bifermentans DPH-1 and addition of zero-valent iron (Fe0) on the reductive dechlorination of tetrachloroethylene (PCE). In addition, the dechlorination of cis-1,2-dichloroethylene (cDCE) produced from PCE was examined using Fe0. The cDCE produced was completely dechlorinated to non-toxic end products, mostly, ethylene by a subsequent chemical reductive process. Production of ethylene was dramatically increased with increase of initial cDCE concentration in the range of 10.3 microM to 928 microM (1.0-90 mg l(-1)) and the velocity constant was calculated to be 0.38 day(-1). On the other hand, the combined use of strain DPH-1 and Fe0 showed the most significant effect on the initial PCE dechlorination, but cohesion of Fe0 was found to inhibit the dechlorination rate of PCE. It is thought that phosphoric acid iron contained in a medium forms film on the surface of iron particle, so oxidation of iron is inhibited.

  15. Laboratory identification of anaerobic bacteria isolated on Clostridium difficile selective medium.

    PubMed

    Rodriguez, Cristina; Warszawski, Nathalie; Korsak, Nicolas; Taminiau, Bernard; Van Broeck, Johan; Delmée, Michel; Daube, Georges

    2016-06-01

    Despite increasing interest in the bacterium, the methodology for Clostridium difficile recovery has not yet been standardized. Cycloserine-cefoxitin fructose taurocholate (CCFT) has historically been the most used medium for C. difficile isolation from human, animal, environmental, and food samples, and presumptive identification is usually based on colony morphologies. However, CCFT is not totally selective. This study describes the recovery of 24 bacteria species belonging to 10 different genera other than C. difficile, present in the environment and foods of a retirement establishment that were not inhibited in the C. difficile selective medium. These findings provide insight for further environmental and food studies as well as for the isolation of C. difficile on supplemented CCFT.

  16. Clostridium difficile virulence factors: Insights into an anaerobic spore-forming pathogen

    PubMed Central

    Awad, Milena M; Johanesen, Priscilla A; Carter, Glen P; Rose, Edward; Lyras, Dena

    2014-01-01

    The worldwide emergence of epidemic strains of Clostridium difficile linked to increased disease severity and mortality has resulted in greater research efforts toward determining the virulence factors and pathogenesis mechanisms used by this organism to cause disease. C. difficile is an opportunist pathogen that employs many factors to infect and damage the host, often with devastating consequences. This review will focus on the role of the 2 major virulence factors, toxin A (TcdA) and toxin B (TcdB), as well as the role of other putative virulence factors, such as binary toxin, in C. difficile-mediated infection. Consideration is given to the importance of spores in both the initiation of disease and disease recurrence and also to the role that surface proteins play in host interactions. PMID:25483328

  17. ANAEROBIC TRANSFORMATION OF 2, 4 AND 2, 2- DINITROTOLUENES BY CLOSTRIDIUM ACETOBUTYLICUM: A PATHWAY THROUGH DIHDROXYALMINO- INTERMEDIATES. (R825513C006)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  18. ANAEROBIC TRANSFORMATION OF 2, 4 AND 2, 2- DINITROTOLUENES BY CLOSTRIDIUM ACETOBUTYLICUM: A PATHWAY THROUGH DIHDROXYALMINO- INTERMEDIATES. (R825513C006)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  19. Wukongibacter baidiensis gen. nov., sp. nov., an anaerobic bacterium isolated from hydrothermal sulfides, and proposal for the reclassification of the closely related Clostridium halophilum and Clostridium caminithermale within Maledivibacter gen. nov. and Paramaledivibacter gen. nov., respectively.

    PubMed

    Li, Guangyu; Zeng, Xiang; Liu, Xiupian; Zhang, Xiaobo; Shao, Zongze

    2016-11-01

    An anaerobic, Gram-stain-positive, spore-forming bacterium, designated DY30321T, was isolated from a sample of mixed hydrothermal sulfides collected during cruise DY30 of R/V Da Yang Yi Hao. Cells of strain DY30321T were rod-shaped with rounded ends, and were not motile. Strain DY30321T grew optimally at pH 8.0, at 30 °C and at a salinity (sea salts) of 30-40 g l-1. The principal fatty acids of strain DY30321T were C14 : 0 and summed feature 1 (comprising iso H-C15 : 1/C13 : 0 3-OH). The predominant polar lipids of strain DY30321T were diphosphatidylglycerol, phosphatidylcholine and phosphatidylethanolamine. No respiratory quinone was detected. The G+C content of the genomic DNA of strain DY30321T was 33.4 mol%. Phylogenetically, strain DY30321T branched within the family Peptostreptococcaceae, with (misclassified) Clostridium halophilum M1T being its closest phylogenetic relative (94.6 % 16S rRNA gene sequence similarity), followed by (misclassified) Clostridium caminithermale DVird3T (92.1 %). These strains showed very low 16S rRNA gene sequence similarity (<84 %) to Clostrdium butyricum ATCC 19398T, the type species of the genus Clostridium sensu stricto. On the basis of its phenotypic, phylogenetic and chemotaxonomic characteristics, strain DY30321T (=KCTC 15549T=MCCC 1A01532T) is considered as the type strain of a novel species of a new genus in the family Peptostreptococcaceae, for which the name Wukongibacterbaidiensis gen. nov., sp. nov. is proposed. Maledivibacter gen. nov. is proposed to accommodate Clostridium halophilum as Maledivibacter halophilus comb. nov. (type species of the genus), and Paramaledivibacter gen. nov. to accommodate Clostridium caminithermale as Paramaledivibacter caminithermalis comb. nov. (type species of the genus).

  20. Physiology and Sporulation in Clostridium.

    PubMed

    Dürre, Peter

    2014-08-01

    Clostridia are Gram-positive, anaerobic, endospore-forming bacteria, incapable of dissimilatory sulfate reduction. Comprising approximately 180 species, the genus Clostridium is one of the largest bacterial genera. Physiology is mostly devoted to acid production. Numerous pathways are known, such as the homoacetate fermentation by acetogens, the propionate fermentation by Clostridium propionicum, and the butyrate/butanol fermentation by C. acetobutylicum, a well-known solvent producer. Clostridia degrade sugars, alcohols, amino acids, purines, pyrimidines, and polymers such as starch and cellulose. Energy conservation can be performed by substrate-level phosphorylation as well as by the generation of ion gradients. Endospore formation resembles the mechanism elucidated in Bacillus. Morphology, contents, and properties of spores are very similar to bacilli endospores. Sporulating clostridia usually form swollen mother cells and accumulate the storage substance granulose. However, clostridial sporulation differs by not employing the so-called phosphorelay. Initiation starts by direct phosphorylation of the master regulator Spo0A. The cascade of sporulation-specific sigma factors is again identical to what is known from Bacillus. The onset of sporulation is coupled in some species to either solvent (acetone, butanol) or toxin (e.g., C. perfringens enterotoxin) formation. The germination of spores is often induced by various amino acids, often in combination with phosphate and sodium ions. In medical applications, C. butyricum spores are used as a C. difficile prophylaxis and as treatment against diarrhea. Recombinant spores are currently under investigation and testing as antitumor agents, because they germinate only in hypoxic tissues (i.e., tumor tissue), allowing precise targeting and direct killing of tumor cells.

  1. Uncoupler-Resistant Glucose Uptake by the Thermophilic Glycolytic Anaerobe Thermoanaerobacter thermosulfuricus (Clostridium thermohydrosulfuricum)

    PubMed Central

    Cook, Gregory M.; Janssen, Peter H.; Morgan, Hugh W.

    1993-01-01

    The transport of glucose across the bacterial cell membrane of Thermoanaerobacter thermosulfuricus (Clostridium thermohydrosulfuricum) Rt8.B1 was governed by a permease which did not catalyze concomitant substrate transport and phosphorylation and thus was not a phosphoenolpyruvate-dependent phosphotransferase. Glucose uptake was carrier mediated, could not be driven by an artificial membrane potential (Δψ) in the presence or absence of sodium, and was not sensitive to inhibitors which dissipate the proton motive force (Δp; tetrachlorosalicylanilide, N,N-dicyclohexylcarboiimide, and 2,4-dinitrophenol), and no uptake of the nonmetabolizable analog 2-deoxyglucose could be demonstrated. The glucokinase apparent Km for glucose (0.21 mM) was similar to the Kt (affinity constant) for glucose uptake (0.15 mM), suggesting that glucokinase controls the rate of glucose uptake. Inhibitors of ATP synthesis (iodoacetate and sodium fluoride) also inhibited glucose uptake, and this effect was due to a reduction in the level of ATP available to glucokinase for glucose phosphorylation. These results indicated that T. thermosulfuricus Rt8.B1 lacks a concentrative uptake system for glucose and that uptake is via facilitated diffusion, followed by ATP-dependent phosphorylation by glucokinase. In T. thermosulfuricus Rt8.B1, glucose is metabolized by the Embden-Meyerhof-Parnas pathway, which yields 2 mol of ATP (G. M. Cook, unpublished data). Since only 1 mol of ATP is used to transport 1 mol of glucose, the energetics of this system are therefore similar to those found in bacteria which possess a phosphotransferase. PMID:16349043

  2. Transcriptional Analysis of Clostridium beijerinckii NCIMB 8052 and the Hyper-Butanol-Producing Mutant BA101 during the Shift from Acidogenesis to Solventogenesis▿ †

    PubMed Central

    Shi, Zhen; Blaschek, Hans P.

    2008-01-01

    Clostridium beijerinckii is an anaerobic bacterium used for the fermentative production of acetone and butanol. The recent availability of genomic sequence information for C. beijerinckii NCIMB 8052 has allowed for an examination of gene expression during the shift from acidogenesis to solventogenesis over the time course of a batch fermentation using a ca. 500-gene set DNA microarray. The microarray was constructed using a collection of genes which are orthologs of members of gene families previously found to be important to the physiology of C. acetobutylicum ATCC 824. Similar to the onset of solventogenesis in C. acetobutylicum 824, the onset of solventogenesis in C. beijerinckii 8052 was concurrent with the initiation of sporulation. However, forespores and endospores developed more rapidly in C. beijerinckii 8052 than in C. acetobutylicum 824, consistent with the accelerated expression of the sigE- and sigG-regulated genes in C. beijerinckii 8052. The comparison of gene expression patterns and morphological changes in C. beijerinckii 8052 and the hyper-butanol-producing C. beijerinckii strain BA101 indicated that BA101 was less efficient in sporulation and phosphotransferase system-mediated sugar transport than 8052 but that it exhibited elevated expression of several primary metabolic genes and chemotaxis/motility genes. PMID:18849451

  3. The genome sequence of Clostridium tetani, the causative agent of tetanus disease.

    PubMed

    Bruggemann, Holger; Baumer, Sebastian; Fricke, Wolfgang Florian; Wiezer, Arnim; Liesegang, Heiko; Decker, Iwona; Herzberg, Christina; Martinez-Arias, Rosa; Merkl, Rainer; Henne, Anke; Gottschalk, Gerhard

    2003-02-04

    Tetanus disease is one of the most dramatic and globally prevalent diseases of humans and vertebrate animals, and has been reported for over 24 centuries. The manifestation of the disease, spastic paralysis, is caused by the second most poisonous substance known, the tetanus toxin, with a human lethal dose of approximately 1 ng/kg. Fortunately, this disease is successfully controlled through immunization with tetanus toxoid; nevertheless, according to the World Health Organization, an estimated 400,000 cases still occur each year, mainly of neonatal tetanus. The causative agent of tetanus disease is Clostridium tetani, an anaerobic spore-forming bacterium, whose natural habitat is soil, dust, and intestinal tracts of various animals. Here we report the complete genome sequence of toxigenic C. tetani E88, a variant of strain Massachusetts. The genome consists of a 2,799,250-bp chromosome encoding 2,372 ORFs. The tetanus toxin and a collagenase are encoded on a 74,082-bp plasmid, containing 61 ORFs. Additional virulence-related factors could be identified, such as an array of surface-layer and adhesion proteins (35 ORFs), some of them unique to C. tetani. Comparative genomics with the genomes of Clostridium perfringens, the causative agent of gas gangrene, and Clostridium acetobutylicum, a nonpathogenic solvent producer, revealed a remarkable capacity of C. tetani: The organism can rely on an extensive sodium ion bioenergetics. Additional candidate genes involved in the establishment and maintenance of a pathogenic lifestyle of C. tetani are presented.

  4. The genome sequence of Clostridium tetani, the causative agent of tetanus disease

    PubMed Central

    Brüggemann, Holger; Bäumer, Sebastian; Fricke, Wolfgang Florian; Wiezer, Arnim; Liesegang, Heiko; Decker, Iwona; Herzberg, Christina; Martínez-Arias, Rosa; Merkl, Rainer; Henne, Anke; Gottschalk, Gerhard

    2003-01-01

    Tetanus disease is one of the most dramatic and globally prevalent diseases of humans and vertebrate animals, and has been reported for over 24 centuries. The manifestation of the disease, spastic paralysis, is caused by the second most poisonous substance known, the tetanus toxin, with a human lethal dose of ≈1 ng/kg. Fortunately, this disease is successfully controlled through immunization with tetanus toxoid; nevertheless, according to the World Health Organization, an estimated 400,000 cases still occur each year, mainly of neonatal tetanus. The causative agent of tetanus disease is Clostridium tetani, an anaerobic spore-forming bacterium, whose natural habitat is soil, dust, and intestinal tracts of various animals. Here we report the complete genome sequence of toxigenic C. tetani E88, a variant of strain Massachusetts. The genome consists of a 2,799,250-bp chromosome encoding 2,372 ORFs. The tetanus toxin and a collagenase are encoded on a 74,082-bp plasmid, containing 61 ORFs. Additional virulence-related factors could be identified, such as an array of surface-layer and adhesion proteins (35 ORFs), some of them unique to C. tetani. Comparative genomics with the genomes of Clostridium perfringens, the causative agent of gas gangrene, and Clostridium acetobutylicum, a nonpathogenic solvent producer, revealed a remarkable capacity of C. tetani: The organism can rely on an extensive sodium ion bioenergetics. Additional candidate genes involved in the establishment and maintenance of a pathogenic lifestyle of C. tetani are presented. PMID:12552129

  5. Evaluation of hydrogen production by clostridium strains on beet molasses.

    PubMed

    Avci, Ayşe; Kiliç, Nur Koçberber; Dönmez, Gönöl; Dönmez, Sedat

    2014-01-01

    Clostridium acetobutylicum DSM 792, C. acetobutylicum DSM 1731 and two newly isolated bacteria defined as the members of genus Clostridium - based on the 16S rRNA analysis and biochemical traits - were characterized with regard to their hydrogen production in media containing increasing beet molasses concentrations. The highest hydrogen yield was observed for C. acetobutylicum DSM 792 with a yield of 2.8 mol H2 mol-1 hexose in medium including 60 g L-1 molasses. This bacterium also produced the maximum amount of hydrogen (5908.8 mL L-1) at the same molasses concentration. A slightly lower hydrogen yield was measured for C. acetobutylicum DSM 1731 (2.5 mol H2 mol-1 hexose) when grown on 40 g L-1 molasses. The new isolates Clostridium roseum C and Clostridium saccharoperbutylacetonicum PF produced hydrogen with yields of 2.0 mol H2 mol-1 hexose at 40 and 60 g L-1 molasses and 2.1 mol H2 mol-1 hexose at 40 gL-1 molasses, respectively.

  6. Structural characterization of the polar lipids of Clostridium novyi NT. Further evidence for a novel anaerobic biosynthetic pathway to plasmalogens.

    PubMed

    Guan, Ziqiang; Johnston, Norah C; Aygun-Sunar, Semra; Daldal, Fevzi; Raetz, Christian R H; Goldfine, Howard

    2011-03-01

    A study of the polar lipids of Clostridium novyi NT has revealed the presence of phosphatidylethanolamine (PE) and cardiolipin as major phospholipids with smaller amounts of phosphatidylglycerol (PG), lysyl-PG and alanyl-PG. Other minor phospholipids included phosphatidic acid, CDP-diacylglycerol, phosphatidylserine (PS) and phosphatidylthreonine (PT). PE, PG and amino acyl PG were present in both the diacyl and alk-1'-enyl acyl (plasmalogen) forms and cardiolipin plasmalogens were found to contain one or two alk-1'-enyl chains. In contrast, the precursor lipids phosphatidic acid, CDP-diacylglycerol and PS were present almost exclusively as diacyl phospholipids. These findings are consistent with the hypothesis that plasmalogens are formed from diacylated phospholipids at a late stage of phospholipid formation in Clostridium species. This novel pathway contrasts with the route in animals in which a saturated ether bond is formed at an early stage of plasmalogen biosynthesis and the alk-1-enyl bond is formed by an aerobic mechanism. Copyright © 2010 Elsevier B.V. All rights reserved.

  7. Structural Characterization of the Polar Lipids of Clostridium novyi NT. Further Evidence for a Novel Anaerobic Biosynthetic Pathway to Plasmalogens

    PubMed Central

    Guan, Ziqiang; Johnston, Norah C.; Aygun-Sunar, Semra; Daldal, Fevzi; Raetz, Christian R. H.; Goldfine, Howard

    2011-01-01

    A study of the polar lipids of Clostridium novyi NT has revealed the presence of phosphatidylethanolamine (PE) and cardiolipin as major phospholipids with smaller amounts of phosphatidylglycerol (PG), lysyl-PG and alanyl-PG. Other minor phospholipids included phosphatidic acid, CDP-diacylglycerol, phosphatidylserine (PS) and phosphatidylthreonine (PT). PE, PG and amino acyl PG were present in both the diacyl and alk-1’-enyl acyl (plasmalogen) forms and cardiolipin plasmalogens were found to contain one or two alk-1’-enyl chains. In contrast, the precursor lipids phosphatidic acid, CDP-diacylglycerol and PS were present almost exclusively as diacyl phospholipids. These findings are consistent with the hypothesis that plasmalogens are formed from diacylated phospholipids at a late stage of phospholipid formation in Clostridium species. This novel pathway contrasts with the route in animals in which a saturated ether bond is formed at an early stage of plasmalogen biosynthesis and the alk-1-enyl bond is formed by an aerobic mechanism. PMID:21195206

  8. MRI-Monitored Intra-Tumoral Injection of Iron-Oxide Labeled Clostridium novyi-NT Anaerobes in Pancreatic Carcinoma Mouse Model

    PubMed Central

    Zheng, Linfeng; Zhang, Zhuoli; Khazaie, Khashayarsha; Saha, Saurabh; Lewandowski, Robert J.; Zhang, Guixiang; Larson, Andrew C.

    2014-01-01

    Objectives To validate the feasibility of labeling Clostridium novyi-NT (C.novyi-NT) anaerobes with iron-oxide nanoparticles for magnetic resonance imaging (MRI) and demonstrate the potential to use MRI to visualize intra-tumoral delivery of these iron-oxide labeled C.novyi-NT during percutaneous injection procedures. Materials and Methods All studies were approved by IACUC. C.novyi-NT were labeled with hybrid iron-oxide Texas red nanoparticles. Growth of labeled and control samples were evaluated with optical density. Labeling was confirmed with confocal fluorescence and transmission electron microscopy (TEM). MRI were performed using a 7 Tesla scanner with T2*-weighted (T2*W) sequence. Contrast-to-noise ratio (CNR) measurements were performed for phantoms and signal-to-noise ratio (SNR) measurements performed in C57BL/6 mice (n = 12) with Panc02 xenografts before and after percutaneous injection of iron-oxide labeled C.novyi-NT. MRI was repeated 3 and 7 days post-injection. Hematoxylin-eosin (HE), Prussian blue and Gram staining of tumor specimens were performed for confirmation of intra-tumoral delivery. Results Iron-oxide labeling had no influence upon C.novyi-NT growth. The signal intensity (SI) within T2*W images was significantly decreased for iron-oxide labeled C.novyi-NT phantoms compared to unlabeled controls. Under confocal fluorescence microscopy, the iron-oxide labeled C.novyi-NT exhibited a uniform red fluorescence consistent with observed regions of DAPI staining and overall labeling efficiency was 100% (all DAPI stained C.novyi-NT exhibited red fluorescence). Within TEM images, a large number iron granules were observed within the iron-oxide labeled C.novyi-NT; these were not observed within unlabeled controls. Intra-procedural MRI measurements permitted in vivo visualization of the intra-tumoral distribution of iron-oxide labeled C.novyi-NT following percutaneous injection (depicted as punctate regions of SI reductions within T2*-weighted

  9. MRI-monitored intra-tumoral injection of iron-oxide labeled Clostridium novyi-NT anaerobes in pancreatic carcinoma mouse model.

    PubMed

    Zheng, Linfeng; Zhang, Zhuoli; Khazaie, Khashayarsha; Saha, Saurabh; Lewandowski, Robert J; Zhang, Guixiang; Larson, Andrew C

    2014-01-01

    To validate the feasibility of labeling Clostridium novyi-NT (C.novyi-NT) anaerobes with iron-oxide nanoparticles for magnetic resonance imaging (MRI) and demonstrate the potential to use MRI to visualize intra-tumoral delivery of these iron-oxide labeled C.novyi-NT during percutaneous injection procedures. All studies were approved by IACUC. C.novyi-NT were labeled with hybrid iron-oxide Texas red nanoparticles. Growth of labeled and control samples were evaluated with optical density. Labeling was confirmed with confocal fluorescence and transmission electron microscopy (TEM). MRI were performed using a 7 Tesla scanner with T2*-weighted (T2*W) sequence. Contrast-to-noise ratio (CNR) measurements were performed for phantoms and signal-to-noise ratio (SNR) measurements performed in C57BL/6 mice (n = 12) with Panc02 xenografts before and after percutaneous injection of iron-oxide labeled C.novyi-NT. MRI was repeated 3 and 7 days post-injection. Hematoxylin-eosin (HE), Prussian blue and Gram staining of tumor specimens were performed for confirmation of intra-tumoral delivery. Iron-oxide labeling had no influence upon C.novyi-NT growth. The signal intensity (SI) within T2*W images was significantly decreased for iron-oxide labeled C.novyi-NT phantoms compared to unlabeled controls. Under confocal fluorescence microscopy, the iron-oxide labeled C.novyi-NT exhibited a uniform red fluorescence consistent with observed regions of DAPI staining and overall labeling efficiency was 100% (all DAPI stained C.novyi-NT exhibited red fluorescence). Within TEM images, a large number iron granules were observed within the iron-oxide labeled C.novyi-NT; these were not observed within unlabeled controls. Intra-procedural MRI measurements permitted in vivo visualization of the intra-tumoral distribution of iron-oxide labeled C.novyi-NT following percutaneous injection (depicted as punctate regions of SI reductions within T2*-weighted images); tumor SNR decreased significantly

  10. Metabolic properties of Eubacterium pyruvativorans, a ruminal 'hyper-ammonia-producing' anaerobe with metabolic properties analogous to those of Clostridium kluyveri.

    PubMed

    Wallace, R John; Chaudhary, Lal C; Miyagawa, Eiichi; McKain, N; Walker, Nicola D

    2004-09-01

    Eubacterium pyruvativorans I-6(T) is a non-saccharolytic, amino-acid-fermenting anaerobe from the rumen, isolated by its ability to grow on pancreatic casein hydrolysate (PCH) as sole C source. This study investigated its metabolic properties and its likely ecological niche. Additional growth was supported by pyruvate, vinyl acetate, and, to a lesser extent, lactate and crotonate, and also by a mixture of amino acids (alanine, glycine, serine and threonine) predicted to be catabolized to pyruvate. No single amino acid supported growth, and peptides were required for growth on amino acids. Alanine, followed by leucine, serine and proline, were used most extensively during growth, but only alanine and asparate were extensively modified before incorporation. Growth on PCH, but not on pyruvate, was increased by the addition of acetate, propionate and butyrate. l-Lactate was fermented incompletely, mainly to acetate, but no lactate-C was incorporated. Propionate and butyrate were utilized during growth, forming valerate and caproate, respectively. Labelling experiments suggested a metabolic pattern where two C atoms of butyrate, valerate and caproate were derived from amino acids, with the others being formed from acetate, propionate and butyrate. The metabolic strategy of E. pyruvativorans therefore resembles that of Clostridium kluyveri, which ferments ethanol only when the reaction is coupled to acetate, propionate or butyrate utilization. The fermentative niche of E. pyruvativorans appears to be to scavenge amino acids, lactate and possibly other metabolites in order to generate ATP via acetate formation, using volatile fatty acid elongation with C(2) units derived from other substrates to dispose of reducing equivalents.

  11. The VirS/VirR two-component system regulates the anaerobic cytotoxicity, intestinal pathogenicity, and enterotoxemic lethality of Clostridium perfringens type C isolate CN3685.

    PubMed

    Ma, Menglin; Vidal, Jorge; Saputo, Juliann; McClane, Bruce A; Uzal, Francisco

    2011-01-25

    Clostridium perfringens vegetative cells cause both histotoxic infections (e.g., gas gangrene) and diseases originating in the intestines (e.g., hemorrhagic necrotizing enteritis or lethal enterotoxemia). Despite their medical and veterinary importance, the molecular pathogenicity of C. perfringens vegetative cells causing diseases of intestinal origin remains poorly understood. However, C. perfringens beta toxin (CPB) was recently shown to be important when vegetative cells of C. perfringens type C strain CN3685 induce hemorrhagic necrotizing enteritis and lethal enterotoxemia. Additionally, the VirS/VirR two-component regulatory system was found to control CPB production by CN3685 vegetative cells during aerobic infection of cultured enterocyte-like Caco-2 cells. Using an isogenic virR null mutant, the current study now reports that the VirS/VirR system also regulates CN3685 cytotoxicity during infection of Caco-2 cells under anaerobic conditions, as found in the intestines. More importantly, the virR mutant lost the ability to cause hemorrhagic necrotic enteritis in rabbit small intestinal loops. Western blot analyses demonstrated that the VirS/VirR system mediates necrotizing enteritis, at least in part, by controlling in vivo CPB production. In addition, vegetative cells of the isogenic virR null mutant were, relative to wild-type vegetative cells, strongly attenuated in their lethality in a mouse enterotoxemia model. Collectively, these results identify the first regulator of in vivo pathogenicity for C. perfringens vegetative cells causing disease originating in the complex intestinal environment. Since VirS/VirR also mediates histotoxic infections, this two-component regulatory system now assumes a global role in regulating a spectrum of infections caused by C. perfringens vegetative cells.

  12. The VirS/VirR Two-Component System Regulates the Anaerobic Cytotoxicity, Intestinal Pathogenicity, and Enterotoxemic Lethality of Clostridium perfringens Type C Isolate CN3685

    PubMed Central

    Ma, Menglin; Vidal, Jorge; Saputo, Juliann; McClane, Bruce A.; Uzal, Francisco

    2011-01-01

    Clostridium perfringens vegetative cells cause both histotoxic infections (e.g., gas gangrene) and diseases originating in the intestines (e.g., hemorrhagic necrotizing enteritis or lethal enterotoxemia). Despite their medical and veterinary importance, the molecular pathogenicity of C. perfringens vegetative cells causing diseases of intestinal origin remains poorly understood. However, C. perfringens beta toxin (CPB) was recently shown to be important when vegetative cells of C. perfringens type C strain CN3685 induce hemorrhagic necrotizing enteritis and lethal enterotoxemia. Additionally, the VirS/VirR two-component regulatory system was found to control CPB production by CN3685 vegetative cells during aerobic infection of cultured enterocyte-like Caco-2 cells. Using an isogenic virR null mutant, the current study now reports that the VirS/VirR system also regulates CN3685 cytotoxicity during infection of Caco-2 cells under anaerobic conditions, as found in the intestines. More importantly, the virR mutant lost the ability to cause hemorrhagic necrotic enteritis in rabbit small intestinal loops. Western blot analyses demonstrated that the VirS/VirR system mediates necrotizing enteritis, at least in part, by controlling in vivo CPB production. In addition, vegetative cells of the isogenic virR null mutant were, relative to wild-type vegetative cells, strongly attenuated in their lethality in a mouse enterotoxemia model. Collectively, these results identify the first regulator of in vivo pathogenicity for C. perfringens vegetative cells causing disease originating in the complex intestinal environment. Since VirS/VirR also mediates histotoxic infections, this two-component regulatory system now assumes a global role in regulating a spectrum of infections caused by C. perfringens vegetative cells. PMID:21264065

  13. Clostridium geopurificans strain MJ1 sp. nov., a strictly anaerobic bacterium that grows via fermentation and reduces the cyclic nitramine explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX).

    PubMed

    Kwon, Man Jae; Wei, Na; Millerick, Kayleigh; Popovic, Jovan; Finneran, Kevin

    2014-06-01

    A fermentative, non-spore forming, motile, rod-shaped bacterium, designated strain MJ1(T), was isolated from an RDX contaminated aquifer at a live-fire training site in Northwest NJ, United States. On the basis of 16S rRNA gene sequencing and DNA base composition, strain MJ1(T) was assigned to the Firmicutes. The DNA G+C content was 42.8 mol%. Fermentative growth was supported by glucose and citrate in a defined basal medium. The bacterium is a strict anaerobe that grows between at pH 6.0 and pH 8.0 and 18 and 37 °C. The culture did not grow with hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) as the electron acceptor or mineralize RDX under these conditions. However, MJ1(T) transformed RDX into MNX, methylenedinitramine, formaldehyde, formate, ammonium, nitrous oxide, and nitrate. The nearest phylogenetic relative with a validly published name was Desulfotomaculum guttoideum (95 % similarity). However, MJ1(T) was also related to Clostridium celerecrescens DSM 5628 (95 %), Clostridium indolis DSM 755 (94 %), and Clostridium sphenoides DSM 632 (94 %). DNA:DNA hybridization with these strains was between 6.7 and 58.7 percent. The dominant cellular fatty acids (greater than 5 % of the total, which was 99.0 % recovery) were 16:0 fatty acid methyl ester (FAME) (32.12 %), 18:1cis 11 dimethyl acetal (DMA) (16.47 %), 16:1cis 9 DMA (10.28 %), 16:1cis 9 FAME (8.10 %), and 18:1cis 9 DMA (5.36 %). On the basis of morphological, physiological, and phylogenetic data, Clostridium geopurificans is proposed as a new species in genus Clostridium, with strain MJ1(T) as the type strain.

  14. Scale-up of anaerobic 1,3-propanediol production by Clostridium butyricum DSP1 from crude glycerol

    PubMed Central

    2014-01-01

    Background As the production of biofuels from raw materials continuously increases, optimization of production processes is necessary. A very important issue is the development of wasteless methods of biodiesel production. One way of utilization of glycerol generated in biodiesel production is its microbial conversion to 1,3-PD (1,3-propanediol). Results The study investigated the scale-up of 1,3-PD synthesis from crude glycerol by Clostridium butyricum. Batch fermentations were carried out in 6.6 L, 42 L and 150 L bioreactors. It was observed that cultivation of C. butyricum on a pilot scale did not decrease the efficiency of 1,3-PD production. The highest concentrations of 1,3-PD, 37 g/L for batch fermentation and 71 g/L for fed-batch fermentation, were obtained in the 6.6 L bioreactor. The kinetic parameters of 1,3-PD synthesis from crude glycerol established for batch fermentation were similar regarding all three bioreactor capacities. During fed-batch fermentation, the concentration of 1,3-PD in the 150 L bioreactor was lower and the substrate was not completely utilized. That suggested the presence of multifunctional environmental stresses in the 150 L bioreactor, which was confirmed by protein analysis. Conclusion The values of effectivity parameters for 1,3-PD synthesis in batch fermentations carried out in 6.6 L, 42 L and 150 L bioreactors were similar. The parameters obtained during fed-batch fermentations in the 150 L bioreactor differed in the rate and percentage of substrate utilization. The analysis of cell proteins demonstrated that a number of multifunctional stresses occurred during fed-batch fermentations in the 150 L bioreactor, which suggests the possibility of identifying the key stages in the biochemical process where inhibition of 1,3-PD synthesis pathways can be observed. PMID:24555775

  15. FT-IR spectroscopic analysis for studying Clostridium cell response to conversion of enzymatically hydrolyzed hay

    NASA Astrophysics Data System (ADS)

    Grube, Mara; Gavare, Marita; Nescerecka, Alina; Tihomirova, Kristina; Mezule, Linda; Juhna, Talis

    2013-07-01

    Grass hay is one of assailable cellulose containing non-food agricultural wastes that can be used as a carbohydrate source by microorganisms producing biofuels. In this study three Clostridium strains Clostridium acetobutylicum, Clostridium beijerinckii and Clostridium tetanomorphum, capable of producing acetone, butanol and ethanol (ABE) were adapted to convert enzymatically hydrolyzed hay used as a growth media additive. The results of growth curves, substrate degradation kinetics and FT-IR analyses of bacterial biomass macromolecular composition showed diverse strain-specific cell response to the growth medium composition.

  16. Metabolism of the /sup 18/O-methoxy substituent of 3-methoxybenzoic acid and other unlabeled methoxybenzoic acids by anaerobic bacteria. [Eubacterium limosum; Acetobacterium woodil; Syntrophococcus; Clostridium; Desulfotomaculum; Enterobacter

    SciTech Connect

    DeWeerd, J.A.; Saxena, A.; Nagle, D.P. Jr.; Sulflita, J.M.

    1988-05-01

    The mechanism of the bioconversion of methoxylated benzoic acids to the hydroxylated derivatives was investigated with a model substrate and cultures of one anaerobic consortium, eight strict anaerobic bacteria, and one facultative anaerobic microorganism. We found that a haloaromatic dehalogenating consortium, a dehalogenating isolate from that consortium, Eubacterium, limosum, and a strain of Acetobacterium woodii metabolized 3-(methoxy-/sup 18/O)methoxybenzoic acid (3-anisic acid) to 3-(hydroxy-/sup 18/O)hydroxybenzoic acid stoichiometrically at rates of 1.5, 3.2, 52.4, and 36.7 nmol/min per mg of protein, respectively. A different strain of Acetobacterium and strains of Syntrophococcus, Clostridium Desulfotomaculum, Enterobacter, and an anaerobic bacterium, strain TH-001, were unable to transform this compound. The O-demethylating ability of E. limosum was induced only with appropriate methoxylated benzoates but not with D-glucose, lactate, isoleucine, or methanol. Cross-acclimation and growth experiments with E. limosum showed a rate of metabolism that was an order of magnitude slower and showed no growth with either 4-methoxysalicylic acid (2-hydroxy-4-methoxybenzoic acid) or 4-anisic acid (4-methoxybenzoic acid) when adapted to 3-anisic acid. However, A. woodii NZva-16 showed slower rates and no growth with 3- or 4-methoxysalicylic acid when adapted to 3-anisic acid in similar experiments.

  17. Engineering Clostridium Strain to Accept Unmethylated DNA

    PubMed Central

    Dong, Hongjun; Zhang, Yanping; Dai, Zongjie; Li, Yin

    2010-01-01

    It is difficult to genetically manipulate the medically and biotechnologically important genus Clostridium due to the existence of the restriction and modification (RM) systems. We identified and engineered the RM system of a model clostridial species, C. acetobutylicum, with the aim to allow the host to accept the unmethylated DNA efficiently. A gene CAC1502 putatively encoding the type II restriction endonuclease Cac824I was identified from the genome of C. acetobutylicum DSM1731, and disrupted using the ClosTron system based on group II intron insertion. The resulting strain SMB009 lost the type II restriction endonuclease activity, and can be transformed with unmethylated DNA as efficiently as with methylated DNA. The strategy reported here makes it easy to genetically modify the clostridial species using unmethylated DNA, which will help to advance the understanding of the clostridial physiology from the molecular level. PMID:20161730

  18. Comparative In Vitro Activities of SMT19969, a New Antimicrobial Agent, against Clostridium difficile and 350 Gram-Positive and Gram-Negative Aerobic and Anaerobic Intestinal Flora Isolates

    PubMed Central

    Citron, Diane M.; Tyrrell, Kerin L.; Merriam, C. Vreni

    2013-01-01

    The comparative in vitro activity of SMT19969, a novel, narrow-spectrum, nonabsorbable agent, was studied against 50 ribotype-defined Clostridium difficile strains, 174 Gram-positive and 136 Gram-negative intestinal anaerobes, and 40 Gram-positive aerobes. SMT19969 was one dilution more active against C. difficile isolates (MIC range, 0.125 to 0.5 μg/ml; MIC90, 0.25 μg/ml), including ribotype 027 strains, than fidaxomicin (range, 0.06 to 1 μg/ml; MIC90, 0.5 μg/ml) and two to six dilutions lower than either vancomycin or metronidazole. SMT19969 and fidaxomicin were generally less active against Gram-negative anaerobes, especially the Bacteroides fragilis group species, than vancomycin and metronidazole, suggesting that SMT19969 has a lesser impact on the normal intestinal microbiota that maintain colonization resistance. SMT19969 showed limited activity against other Gram-positive anaerobes, including Bifidobacteria species, Eggerthella lenta, Finegoldia magna, and Peptostreptococcus anaerobius, with MIC90s of >512, >512, 64, and 64 μg/ml, respectively. Clostridium species showed various levels of susceptibility, with C. innocuum being susceptible (MIC90, 1 μg/ml) and C. ramosum and C. perfringens being nonsusceptible (MIC90, >512 μg/ml). Activity against Lactobacillus spp. (range, 0.06 to >512 μg/ml; MIC90, >512 μg/ml) was comparable to that of fidaxomicin and varied by species and strain. Gram-positive aerobic cocci (Staphylococcus aureus, Enterococcus faecalis, E. faecium, and streptococci) showed high SMT19969 MIC90 values (128 to >512 μg/ml). PMID:23877700

  19. Butanol Production from Crystalline Cellulose by Cocultured Clostridium thermocellum and Clostridium saccharoperbutylacetonicum N1-4 ▿

    PubMed Central

    Nakayama, Shunichi; Kiyoshi, Keiji; Kadokura, Toshimori; Nakazato, Atsumi

    2011-01-01

    We investigated butanol production from crystalline cellulose by cocultured cellulolytic Clostridium thermocellum and the butanol-producing strain, Clostridium saccharoperbutylacetonicum (strain N1-4). Butanol was produced from Avicel cellulose after it was incubated with C. thermocellum for at least 24 h at 60°C before the addition of strain N1-4. Butanol produced by strain N1-4 on 4% Avicel cellulose peaked (7.9 g/liter) after 9 days of incubation at 30°C, and acetone was undetectable in this coculture system. Less butanol was produced by cocultured Clostridium acetobutylicum and Clostridium beijerinckii than by strain N1-4, indicating that strain N1-4 was the optimal strain for producing butanol from crystalline cellulose in this coculture system. PMID:21764954

  20. Anaerobic thermophilic culture system

    DOEpatents

    Ljungdahl, Lars G.; Wiegel, Jurgen K. W.

    1981-01-01

    A mixed culture system of the newly discovered microorganism Thermoanaerobacter ethanolicus ATCC31550 and the microorganism Clostridium thermocellum ATCC31549 is described. In a mixed nutrient culture medium that contains cellulose, these microorganisms have been coupled and cultivated to efficiently ferment cellulose to produce recoverable quantities of ethanol under anaerobic, thermophilic conditions.

  1. Draft Genome Sequence of the Cellulolytic Strain Clostridium sp. Bc-iso-3 Isolated from an Industrial-Scale Anaerobic Digester

    PubMed Central

    2016-01-01

    Clostridium sp. Bc-iso-3 is a cellulolytic strain isolated from a Swedish industrial-scale biogas digester. Here, we present the draft genome sequence of this strain, which consists of four contigs with a total length of 4,327,139 bp and an average coverage of 312.97×. PMID:27789641

  2. The Purine-Utilizing Bacterium Clostridium acidurici 9a: A Genome-Guided Metabolic Reconsideration

    PubMed Central

    Hartwich, Katrin; Poehlein, Anja; Daniel, Rolf

    2012-01-01

    Clostridium acidurici is an anaerobic, homoacetogenic bacterium, which is able to use purines such as uric acid as sole carbon, nitrogen, and energy source. Together with the two other known purinolytic clostridia C. cylindrosporum and C. purinilyticum, C. acidurici serves as a model organism for investigation of purine fermentation. Here, we present the first complete sequence and analysis of a genome derived from a purinolytic Clostridium. The genome of C. acidurici 9a consists of one chromosome (3,105,335 bp) and one small circular plasmid (2,913 bp). The lack of candidate genes encoding glycine reductase indicates that C. acidurici 9a uses the energetically less favorable glycine-serine-pyruvate pathway for glycine degradation. In accordance with the specialized lifestyle and the corresponding narrow substrate spectrum of C. acidurici 9a, the number of genes involved in carbohydrate transport and metabolism is significantly lower than in other clostridia such as C. acetobutylicum, C. saccharolyticum, and C. beijerinckii. The only amino acid that can be degraded by C. acidurici is glycine but growth on glycine only occurs in the presence of a fermentable purine. Nevertheless, the addition of glycine resulted in increased transcription levels of genes encoding enzymes involved in the glycine-serine-pyruvate pathway such as serine hydroxymethyltransferase and acetate kinase, whereas the transcription levels of formate dehydrogenase-encoding genes decreased. Sugars could not be utilized by C. acidurici but the full genetic repertoire for glycolysis was detected. In addition, genes encoding enzymes that mediate resistance against several antimicrobials and metals were identified. High resistance of C. acidurici towards bacitracin, acriflavine and azaleucine was experimentally confirmed. PMID:23240052

  3. Recent advances in germination of Clostridium spores.

    PubMed

    Olguín-Araneda, Valeria; Banawas, Saeed; Sarker, Mahfuzur R; Paredes-Sabja, Daniel

    2015-05-01

    Members of Clostridium genus are a diverse group of anaerobic spore-formers that includes several pathogenic species. Their anaerobic requirement enhances the importance of the dormant spore morphotype during infection, persistence and transmission. Bacterial spores are metabolically inactive and may survive for long times in the environment and germinate in presence of nutrients termed germinants. Recent progress with spores of several Clostridium species has identified the germinant receptors (GRs) involved in nutrient germinant recognition and initiation of spore germination. Signal transduction from GRs to the downstream effectors remains poorly understood but involves the release of dipicolinic acid. Two mechanistically different cortex hydrolytic machineries are present in Clostridium spores. Recent studies have also shed light into novel biological events that occur during spore formation (accumulation of transcriptional units) and transcription during early spore outgrowth. In summary, this review will cover all of the recent advances in Clostridium spore germination. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  4. An unexpected negative influence of light intensity on hydrogen production by dark fermentative bacteria Clostridium beijerinckii.

    PubMed

    Zagrodnik, R; Laniecki, M

    2016-01-01

    The role of light intensity on biohydrogen production from glucose by Clostridium beijerinckii, Clostridium acetobutylicum, and Rhodobacter sphaeroides was studied to evaluate the performance and possible application in co-culture fermentation system. The applied source of light had spectrum similar to the solar radiation. The influence of light intensity on hydrogen production in dark process by C. acetobutylicum was negligible. In contrast, dark fermentation by C. beijerinckii bacteria showed a significant decrease (83%) in produced hydrogen at light intensity of 540W/m(2). Here, the redirection of metabolism from acetic and butyric acid formation towards lactic acid was observed. This not yet reported effect was probably caused by irradiation of these bacteria by light within UVA range, which is an important component of the solar radiation. The excessive illumination with light of intensity higher than 200W/m(2) resulted in decrease in hydrogen production with photofermentative bacteria as well.

  5. The Clostridium Sporulation Programs: Diversity and Preservation of Endospore Differentiation

    PubMed Central

    Al-Hinai, Mohab A.; Jones, Shawn W.

    2015-01-01

    SUMMARY Bacillus and Clostridium organisms initiate the sporulation process when unfavorable conditions are detected. The sporulation process is a carefully orchestrated cascade of events at both the transcriptional and posttranslational levels involving a multitude of sigma factors, transcription factors, proteases, and phosphatases. Like Bacillus genomes, sequenced Clostridium genomes contain genes for all major sporulation-specific transcription and sigma factors (spo0A, sigH, sigF, sigE, sigG, and sigK) that orchestrate the sporulation program. However, recent studies have shown that there are substantial differences in the sporulation programs between the two genera as well as among different Clostridium species. First, in the absence of a Bacillus-like phosphorelay system, activation of Spo0A in Clostridium organisms is carried out by a number of orphan histidine kinases. Second, downstream of Spo0A, the transcriptional and posttranslational regulation of the canonical set of four sporulation-specific sigma factors (σF, σE, σG, and σK) display different patterns, not only compared to Bacillus but also among Clostridium organisms. Finally, recent studies demonstrated that σK, the last sigma factor to be activated according to the Bacillus subtilis model, is involved in the very early stages of sporulation in Clostridium acetobutylicum, C. perfringens, and C. botulinum as well as in the very late stages of spore maturation in C. acetobutylicum. Despite profound differences in initiation, propagation, and orchestration of expression of spore morphogenetic components, these findings demonstrate not only the robustness of the endospore sporulation program but also the plasticity of the program to generate different complex phenotypes, some apparently regulated at the epigenetic level. PMID:25631287

  6. The Clostridium sporulation programs: diversity and preservation of endospore differentiation.

    PubMed

    Al-Hinai, Mohab A; Jones, Shawn W; Papoutsakis, Eleftherios T

    2015-03-01

    Bacillus and Clostridium organisms initiate the sporulation process when unfavorable conditions are detected. The sporulation process is a carefully orchestrated cascade of events at both the transcriptional and posttranslational levels involving a multitude of sigma factors, transcription factors, proteases, and phosphatases. Like Bacillus genomes, sequenced Clostridium genomes contain genes for all major sporulation-specific transcription and sigma factors (spo0A, sigH, sigF, sigE, sigG, and sigK) that orchestrate the sporulation program. However, recent studies have shown that there are substantial differences in the sporulation programs between the two genera as well as among different Clostridium species. First, in the absence of a Bacillus-like phosphorelay system, activation of Spo0A in Clostridium organisms is carried out by a number of orphan histidine kinases. Second, downstream of Spo0A, the transcriptional and posttranslational regulation of the canonical set of four sporulation-specific sigma factors (σ(F), σ(E), σ(G), and σ(K)) display different patterns, not only compared to Bacillus but also among Clostridium organisms. Finally, recent studies demonstrated that σ(K), the last sigma factor to be activated according to the Bacillus subtilis model, is involved in the very early stages of sporulation in Clostridium acetobutylicum, C. perfringens, and C. botulinum as well as in the very late stages of spore maturation in C. acetobutylicum. Despite profound differences in initiation, propagation, and orchestration of expression of spore morphogenetic components, these findings demonstrate not only the robustness of the endospore sporulation program but also the plasticity of the program to generate different complex phenotypes, some apparently regulated at the epigenetic level. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. Genome Sequence of Clostridium tunisiense TJ, Isolated from Drain Sediment from a Pesticide Factory

    PubMed Central

    Sun, Lili; Wang, Yu; Yu, Chunyan; Zhao, Yongqin

    2012-01-01

    Clostridium tunisiense is a Gram-positive, obligate anaerobe that was first isolated in an anaerobic evironment under eutrophication. Here we report the first genome sequence of the Clostridium tunisiense TJ isolated from drain sediment of a pesticide factory in Tianjin, China. The genome is of great importance for both basic and application research. PMID:23209212

  8. Genome sequence of Clostridium tunisiense TJ, isolated from drain sediment from a pesticide factory.

    PubMed

    Sun, Lili; Wang, Yu; Yu, Chunyan; Zhao, Yongqin; Gan, Yinbo

    2012-12-01

    Clostridium tunisiense is a Gram-positive, obligate anaerobe that was first isolated in an anaerobic environment under eutrophication. Here we report the first genome sequence of the Clostridium tunisiense TJ isolated from drain sediment of a pesticide factory in Tianjin, China. The genome is of great importance for both basic and application research.

  9. A roadmap for gene system development in Clostridium.

    PubMed

    Minton, Nigel P; Ehsaan, Muhammad; Humphreys, Christopher M; Little, Gareth T; Baker, Jonathan; Henstra, Anne M; Liew, Fungmin; Kelly, Michelle L; Sheng, Lili; Schwarz, Katrin; Zhang, Ying

    2016-10-01

    Clostridium species are both heroes and villains. Some cause serious human and animal diseases, those present in the gut microbiota generally contribute to health and wellbeing, while others represent useful industrial chassis for the production of chemicals and fuels. To understand, counter or exploit, there is a fundamental requirement for effective systems that may be used for directed or random genome modifications. We have formulated a simple roadmap whereby the necessary gene systems maybe developed and deployed. At its heart is the use of 'pseudo-suicide' vectors and the creation of a pyrE mutant (a uracil auxotroph), initially aided by ClosTron technology, but ultimately made using a special form of allelic exchange termed ACE (Allele-Coupled Exchange). All mutants, regardless of the mutagen employed, are made in this host. This is because through the use of ACE vectors, mutants can be rapidly complemented concomitant with correction of the pyrE allele and restoration of uracil prototrophy. This avoids the phenotypic effects frequently observed with high copy number plasmids and dispenses with the need to add antibiotic to ensure plasmid retention. Once available, the pyrE host may be used to stably insert all manner of application specific modules. Examples include, a sigma factor to allow deployment of a mariner transposon, hydrolases involved in biomass deconstruction and therapeutic genes in cancer delivery vehicles. To date, provided DNA transfer is obtained, we have not encountered any clostridial species where this technology cannot be applied. These include, Clostridium difficile, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium botulinum, Clostridium perfringens, Clostridium sporogenes, Clostridium pasteurianum, Clostridium ljungdahlii, Clostridium autoethanogenum and even Geobacillus thermoglucosidasius. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Small RNAs in the genus Clostridium.

    PubMed

    Chen, Yili; Indurthi, Dinesh C; Jones, Shawn W; Papoutsakis, Eleftherios T

    2011-01-25

    The genus Clostridium includes major human pathogens and species important to cellulose degradation, the carbon cycle, and biotechnology. Small RNAs (sRNAs) are emerging as crucial regulatory molecules in all organisms, but they have not been investigated in clostridia. Research on sRNAs in clostridia is hindered by the absence of a systematic method to identify sRNA candidates, thus delegating clostridial sRNA research to a hit-and-miss process. Thus, we wanted to develop a method to identify potential sRNAs in the Clostridium genus to open up the field of sRNA research in clostridia. Using comparative genomics analyses combined with predictions of rho-independent terminators and promoters, we predicted sRNAs in 21 clostridial genomes: Clostridium acetobutylicum, C. beijerinckii, C. botulinum (eight strains), C. cellulolyticum, C. difficile, C. kluyveri (two strains), C. novyi, C. perfringens (three strains), C. phytofermentans, C. tetani, and C. thermocellum. Although more than one-third of predicted sRNAs have Shine-Dalgarno (SD) sequences, only one-sixth have a start codon downstream of SD sequences; thus, most of the predicted sRNAs are noncoding RNAs. Quantitative reverse transcription-PCR (Q-RT-PCR) and Northern analysis were employed to test the presence of a randomly chosen set of sRNAs in C. acetobutylicum and several C. botulinum strains, leading to the confirmation of a large fraction of the tested sRNAs. We identified a conserved, novel sRNA which, together with the downstream gene coding for an ATP-binding cassette (ABC) transporter gene, responds to the antibiotic clindamycin. The number of predicted sRNAs correlated with the physiological function of the species (high for pathogens, low for cellulolytic, and intermediate for solventogenic), but not with 16S rRNA-based phylogeny.

  11. Influence of high gas production during thermophilic anaerobic digestion in pilot-scale and lab-scale reactors on survival of the thermotolerant pathogens Clostridium perfringens and Campylobacter jejuni in piggery wastewater.

    PubMed

    Skillman, L C; Bajsa, O; Ho, L; Santhanam, B; Kumar, M; Ho, G

    2009-07-01

    Safe reuse of animal wastes to capture energy and nutrients, through anaerobic digestion processes, is becoming an increasingly desirable solution to environmental pollution. Pathogen decay is the most important safety consideration and is in general, improved at elevated temperatures and longer hydraulic residence times. During routine sampling to assess pathogen decay in thermophilic digestion, an inversely proportional relationship between levels of Clostridium perfringens and gas production was observed. Further samples were collected from pilot-scale, bench-scale thermophilic reactors and batch scale vials to assess whether gas production (predominantly methane) could be a useful indicator of decay of the thermotolerant pathogens C. perfringens and Campylobacter jejuni. Pathogen levels did appear to be lower where gas production and levels of methanogens were higher. This was evident at each operating temperature (50, 57, 65 degrees C) in the pilot-scale thermophilic digesters, although higher temperatures also reduced the numbers of pathogens detected. When methane production was higher, either when feed rate was increased, or pH was lowered from 8.2 (piggery wastewater) to 6.5, lower numbers of pathogens were detected. Although a number of related factors are known to influence the amount and rate of methane production, it may be a useful indicator of the removal of the pathogens C. perfringens and C. jejuni.

  12. Antipathogenic activity of probiotics against Salmonella Typhimurium and Clostridium difficile in anaerobic batch culture systems: is it due to synergies in probiotic mixtures or the specificity of single strains?

    PubMed

    Tejero-Sariñena, Sandra; Barlow, Janine; Costabile, Adele; Gibson, Glenn R; Rowland, Ian

    2013-12-01

    Probiotics are currently being investigated for prevention of infections caused by enteric pathogens. The aim of this in vitro study was to evaluate the influence of three single probiotics: Lactobacillus casei NCIMB 30185 (PXN 37), Lactobacillus acidophilus NCIMB 30184 (PXN 35), Bifidobacterium breve NCIMB 30180 (PXN 25) and a probiotic mixture containing the above strains plus twelve other strains belonging to the Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus and Bacillus genera on the survival of Salmonella Typhimurium and Clostridium difficile using pH-controlled anaerobic batch cultures containing mixed faecal bacteria. Changes in relevant bacterial groups and effects of probiotic addition on survival of the two pathogens were assessed over 24 h. Quantitative analysis of bacterial populations revealed that there was a significant increase in lactobacilli and/or bifidobacteria numbers, depending on probiotic addition, compared with the control (no added probiotic). There was also a significant reduction in S. Typhimurium and C. difficile numbers in the presence of certain probiotics compared with controls. Of the probiotic treatments, two single strains namely L. casei NCIMB 30185 (PXN 37), and B. breve NCIMB 30180 (PXN 25) were the most potent in reducing the numbers of S. Typhimurium and C. difficile. In addition, the supplementation with probiotics into the systems influenced some fermentations parameters. Acetate was found in the largest concentrations in all vessels and lactate and formate were generally detected in higher amounts in vessels with probiotic addition compared to controls.

  13. Complete Genome Sequence of Clostridium clariflavum DSM 19732

    SciTech Connect

    Goodwin, Lynne A.; Davenport, Karen W.; Teshima, Hazuki; Bruce, David; Detter, J. Chris; Tapia, Roxanne; Han, Cliff; Land, Miriam L; Hauser, Loren John; Jeffries, Cynthia; Han, James; Pitluck, Sam; Nolan, Matt; Chen, Amy; Huntemann, Marcel; Mavromatis, K; Mikhailova, Natalia; Liolios, Konstantinos; Woyke, Tanja; Lynd, Lee R

    2012-01-01

    Clostridium clariflavum is a Cluster III Clostridium within the family Clostridiaceae isolated from thermophilic anaerobic sludge (Shiratori et al, 2009). This species is of interest because of its similarity to the model cellulolytic organism Clostridium thermocellum and for the ability of environmental isolates to break down cellulose and hemicellulose. Here we describe features of the 4,897,678 bp long genome and its annotation, consisting of 4,131 proteincoding and 98 RNA genes, for the type strain DSM 19732.

  14. Techniques for controlling variability in gram staining of obligate anaerobes.

    PubMed Central

    Johnson, M J; Thatcher, E; Cox, M E

    1995-01-01

    Identification of anaerobes recovered from clinical samples is complicated by the fact that certain gram-positive anaerobes routinely stain gram negative; Peptostreptococcus asaccharolyticus, Eubacterium plautii, Clostridium ramosum, Clostridium symbiosum, and Clostridium clostridiiforme are among the nonconformists with regard to conventional Gram-staining procedures. Accurate Gram staining of American Type Culture Collection strains of these anaerobic bacteria is possible by implementing fixing and staining techniques within a gloveless anaerobic chamber. Under anaerobic conditions, gram-positive staining occurred in all test organisms with "quick" fixing techniques with both absolute methanol and formalin. The results support the hypothesis that, when anaerobic bacteria are exposed to oxygen, a breakdown of the physical integrity of the cell wall occurs, introducing Gram stain variability in gram-positive anaerobes. PMID:7538512

  15. Extending CRISPR-Cas9 Technology from Genome Editing to Transcriptional Engineering in the Genus Clostridium.

    PubMed

    Bruder, Mark R; Pyne, Michael E; Moo-Young, Murray; Chung, Duane A; Chou, C Perry

    2016-10-15

    The discovery and exploitation of the prokaryotic adaptive immunity system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins have revolutionized genetic engineering. CRISPR-Cas tools have enabled extensive genome editing as well as efficient modulation of the transcriptional program in a multitude of organisms. Progress in the development of genetic engineering tools for the genus Clostridium has lagged behind that of many other prokaryotes, presenting the CRISPR-Cas technology an opportunity to resolve a long-existing issue. Here, we applied the Streptococcus pyogenes type II CRISPR-Cas9 (SpCRISPR-Cas9) system for genome editing in Clostridium acetobutylicum DSM792. We further explored the utility of the SpCRISPR-Cas9 machinery for gene-specific transcriptional repression. For proof-of-concept demonstration, a plasmid-encoded fluorescent protein gene was used for transcriptional repression in C. acetobutylicum Subsequently, we targeted the carbon catabolite repression (CCR) system of C. acetobutylicum through transcriptional repression of the hprK gene encoding HPr kinase/phosphorylase, leading to the coutilization of glucose and xylose, which are two abundant carbon sources from lignocellulosic feedstocks. Similar approaches based on SpCRISPR-Cas9 for genome editing and transcriptional repression were also demonstrated in Clostridium pasteurianum ATCC 6013. As such, this work lays a foundation for the derivation of clostridial strains for industrial purposes.

  16. Cloning and expression of the first anaerobic toxin gene from Clostridium bifermentans subsp. malaysia, encoding a new mosquitocidal protein with homologies to Bacillus thuringiensis delta-endotoxins.

    PubMed Central

    Barloy, F; Delécluse, A; Nicolas, L; Lecadet, M M

    1996-01-01

    A gene (cbm71) encoding a 71,128-Da mosquitocidal protein (Cbm71) was obtained by screening a size-fractionated XbaI digest of total genomic DNA from Clostridium bifermentans subsp. malaysia CH18 with two gene-specific oligonucleotide probes. The sequence of the Cbm71 protein, as deduced from the sequence of cbm71, corresponds to that of the 66-kDa protein previously described as one of the mosquitocidal components of C. bifermentans subsp. malaysia. Cbm71 shows limited similarities with Bacillus thuringiensis delta-endotoxins, especially in the four first conserved blocks. However, Cbm71 was not immunologically related to any of the Cry toxins and thus belongs to a novel class of mosquitocidal protein. The cbm71 gene was expressed in a nontoxic strain of B. thuringiensis, and Cbm71 was produced during sporulation and secreted to the supernatant of culture. Trichloroacetic-precipitated supernatant preparations were toxic for mosquito larvae of the species Aedes aegypti, Culex pipiens, and Anopheles stephensi. PMID:8655486

  17. Complementation of a Clostridium perfringens spo0A mutant with wild-type spo0A from other Clostridium species.

    PubMed

    Huang, I-Hsiu; Sarker, Mahfuzur R

    2006-09-01

    To evaluate whether C. perfringens can be used as a model organism for studying the sporulation process in other clostridia, C. perfringens spo0A mutant IH101 was complemented with wild-type spo0A from four different Clostridium species. Wild-type spo0A from C. acetobutylicum or C. tetani, but not from C. botulinum or C. difficile, restored sporulation and enterotoxin production in IH101. The ability of spo0A from C. botulinum or C. difficile to complement the lack of spore formation in IH101 might be due, at least in part, to the low levels of spo0A transcription and Spo0A production.

  18. NanR, a Transcriptional Regulator That Binds to the Promoters of Genes Involved in Sialic Acid Metabolism in the Anaerobic Pathogen Clostridium perfringens.

    PubMed

    Therit, Blair; Cheung, Jackie K; Rood, Julian I; Melville, Stephen B

    2015-01-01

    Among many other virulence factors, Clostridium perfringens produces three sialidases NanH, NanI and NanJ. NanH lacks a secretion signal peptide and is predicted to be an intracellular enzyme, while NanI and NanJ are secreted. Previously, we had identified part of an operon encoding NanE (epimerase) and NanA (sialic acid lyase) enzymes. Further analysis of the entire operon suggests that it encodes a complete pathway for the transport and metabolism of sialic acid along with a putative transcriptional regulator, NanR. The addition of 30 mM N-acetyl neuraminic acid (Neu5Ac) to a semi-defined medium significantly enhanced the growth yield of strain 13, suggesting that Neu5Ac can be used as a nutrient. C. perfringens strain 13 lacks a nanH gene, but has NanI- and NanJ-encoding genes. Analysis of nanI, nanJ, and nanInanJ mutants constructed by homologous recombination revealed that the expression of the major sialidase, NanI, was induced by the addition of Neu5Ac to the medium, and that in separate experiments, the same was true of a nanI-gusA transcriptional fusion. For the nanI and nanJ genes, primer extension identified three and two putative transcription start sites, respectively. Gel mobility shift assays using purified NanR and DNA from the promoter regions of the nanI and nanE genes showed high affinity, specific binding by NanR. We propose that NanR is a global regulator of sialic acid-associated genes and that it responds, in a positive feedback loop, to the concentration of sialic acid in the cell.

  19. NanR, a Transcriptional Regulator That Binds to the Promoters of Genes Involved in Sialic Acid Metabolism in the Anaerobic Pathogen Clostridium perfringens

    PubMed Central

    Therit, Blair; Cheung, Jackie K.; Rood, Julian I.; Melville, Stephen B.

    2015-01-01

    Among many other virulence factors, Clostridium perfringens produces three sialidases NanH, NanI and NanJ. NanH lacks a secretion signal peptide and is predicted to be an intracellular enzyme, while NanI and NanJ are secreted. Previously, we had identified part of an operon encoding NanE (epimerase) and NanA (sialic acid lyase) enzymes. Further analysis of the entire operon suggests that it encodes a complete pathway for the transport and metabolism of sialic acid along with a putative transcriptional regulator, NanR. The addition of 30 mM N-acetyl neuraminic acid (Neu5Ac) to a semi-defined medium significantly enhanced the growth yield of strain 13, suggesting that Neu5Ac can be used as a nutrient. C. perfringens strain 13 lacks a nanH gene, but has NanI- and NanJ-encoding genes. Analysis of nanI, nanJ, and nanInanJ mutants constructed by homologous recombination revealed that the expression of the major sialidase, NanI, was induced by the addition of Neu5Ac to the medium, and that in separate experiments, the same was true of a nanI-gusA transcriptional fusion. For the nanI and nanJ genes, primer extension identified three and two putative transcription start sites, respectively. Gel mobility shift assays using purified NanR and DNA from the promoter regions of the nanI and nanE genes showed high affinity, specific binding by NanR. We propose that NanR is a global regulator of sialic acid-associated genes and that it responds, in a positive feedback loop, to the concentration of sialic acid in the cell. PMID:26197388

  20. First Report of Clostridium lavalense Isolated in Human Blood Cultures.

    PubMed

    Garceau, Richard; Bourque, Christine; Thibault, Louise; Côté, Jean-Charles; Longtin, Jean; Domingo, Marc-Christian

    2016-01-01

    An 88-year-old man was admitted to the hospital with worsening malaise, fever, and weakness. Anaerobic blood culture bottles revealed the presence of an anaerobic, Gram-positive sporulated bacillus. Empirical antibiotherapy with intravenous piperacillin-tazobactam was initiated. The patient defervesced after four days and was switched to oral amoxicillin on his 6th day of antibiotic therapy and later discharged from the hospital. Four months later, he had recovered. The bacterium was initially identified as Clostridium butyricum using anaerobic manual identification panel. 16S rRNA gene sequence and phylogenetic analysis showed the bacterium to be Clostridium lavalense, a recently described species with no previously published case of isolation in human diagnostic samples so far. This is the first report of Clostridium lavalense isolation from human blood cultures. Further studies are needed in order to elucidate the role of Clostridium lavalense in human disease and its virulence factors.

  1. First Report of Clostridium lavalense Isolated in Human Blood Cultures

    PubMed Central

    Bourque, Christine; Thibault, Louise; Côté, Jean-Charles; Domingo, Marc-Christian

    2016-01-01

    An 88-year-old man was admitted to the hospital with worsening malaise, fever, and weakness. Anaerobic blood culture bottles revealed the presence of an anaerobic, Gram-positive sporulated bacillus. Empirical antibiotherapy with intravenous piperacillin-tazobactam was initiated. The patient defervesced after four days and was switched to oral amoxicillin on his 6th day of antibiotic therapy and later discharged from the hospital. Four months later, he had recovered. The bacterium was initially identified as Clostridium butyricum using anaerobic manual identification panel. 16S rRNA gene sequence and phylogenetic analysis showed the bacterium to be Clostridium lavalense, a recently described species with no previously published case of isolation in human diagnostic samples so far. This is the first report of Clostridium lavalense isolation from human blood cultures. Further studies are needed in order to elucidate the role of Clostridium lavalense in human disease and its virulence factors. PMID:27478446

  2. Biotechnological potential of Clostridium butyricum bacteria

    PubMed Central

    Szymanowska-Powałowska, Daria; Orczyk, Dorota; Leja, Katarzyna

    2014-01-01

    In response to demand from industry for microorganisms with auspicious biotechnological potential, a worldwide interest has developed in bacteria and fungi isolation. Microorganisms of interesting metabolic properties include non-pathogenic bacteria of the genus Clostridium, particularly C. acetobutylicum, C. butyricum and C. pasteurianum. A well-known property of C. butyricum is their ability to produce butyric acid, as well as effectively convert glycerol to 1,3-propanediol (38.2 g/L). A conversion rate of 0.66 mol 1,3-propanediol/mol of glycerol has been obtained. Results of the studies described in the present paper broaden our knowledge of characteristic features of C. butyricum specific isolates in terms of their phylogenetic affiliation, fermentation capacity and antibacterial properties. PMID:25477923

  3. Physiology, biochemistry, and genetics of a pure culture of an obligatory anaerobic bacterium that utilizes 2,4,-6-trinitrotoluene (TNT) and biodegradation of RDX by pure cultures of obligatory anaerobic bacteria of the genus clostridium. Final report, 1 September 1993-31 August 1996

    SciTech Connect

    Crawford, R.L.; Crawford, D.L.

    1996-09-01

    In work supported by the US AFOSR (grant F49620-94-1-0306) we are conducting detailed biochemical and genetic studies of three strains of Clostridium bifernientans, obligatory anaerobic bacteria that appear to completely degrade a variety of nitroaromatic compounds, including 2,4,6-trinitrotoluene (TNT). We are determining the optimal physiological conditions for the degradative activities of C. bifermentans strains; and identifying and characterizing enzymes and genes involved in the biotransformation of nitroaromatic compounds by C. bifermentans. In our AASERT supplemental grant(AFOSR-93-1-O464) we expanded these goals to the explosive RDX (1,3,5-triaza-1, 3,5-trinitrocyclohexane). The AASERT grant funded two graduate students, who characterized the ability of C. bifermentans to degrade RDX (Regan, K. N., and R.L. Crawford, 1994. Biotechnol. Kett. 16: 1081- 1086), and prepared both genomic and plasmid DNA libraries from C. bifermentans. This genetic work will accelerate our progress toward our goal of characterizing the genetics of TNT/RDx degradation by our clostridia (K. Diedrich, M.S. thesis, University of Idaho; in preparation).

  4. Coculture Production of Butanol by Clostridium Bacteria

    NASA Technical Reports Server (NTRS)

    Bergstrom, S. L.; Foutch, G. L.

    1985-01-01

    Production of butanol by anaerobic fermentation of sugars enhanced by use of two Clostridium species, one of which feeds on metabolic product of other. Renewed interest in fermentation process for making butanol stimulated by potential use of butanol as surfactant in enhanced oil recovery. Butanol also used as fuel or as chemical feedstock and currently produced synthetically from petroleum.

  5. Coculture Production of Butanol by Clostridium Bacteria

    NASA Technical Reports Server (NTRS)

    Bergstrom, S. L.; Foutch, G. L.

    1985-01-01

    Production of butanol by anaerobic fermentation of sugars enhanced by use of two Clostridium species, one of which feeds on metabolic product of other. Renewed interest in fermentation process for making butanol stimulated by potential use of butanol as surfactant in enhanced oil recovery. Butanol also used as fuel or as chemical feedstock and currently produced synthetically from petroleum.

  6. Comparative Analysis of Clostridium perfringens Bacteriophage

    USDA-ARS?s Scientific Manuscript database

    Background: Clostridium perfringens are Gram-positive bacteria that are a major bacterial cause of food-borne disease and gas gangrene among humans. These anaerobic bacteria are also the presumptive etiologic agent of necrotic enteritis among chickens. Pathogenesis and symptoms of a necrotic enterit...

  7. Clostridium difficile

    MedlinePlus

    ... 18-21yrs. Healthy Living Healthy Living Healthy Living Nutrition Fitness Sports Oral Health Emotional Wellness Growing Healthy Sleep Safety & ... Head Neck & Nervous System Heart Infections Learning Disabilities Obesity Orthopedic Prevention ... Children > Health Issues > Conditions > Abdominal > Clostridium difficile Health Issues ...

  8. Biofilm-growing intestinal anaerobic bacteria.

    PubMed

    Donelli, Gianfranco; Vuotto, Claudia; Cardines, Rita; Mastrantonio, Paola

    2012-07-01

    Sessile growth of anaerobic bacteria from the human intestinal tract has been poorly investigated, so far. We recently reported data on the close association existing between biliary stent clogging and polymicrobial biofilm development in its lumen. By exploiting the explanted stents as a rich source of anaerobic bacterial strains belonging to the genera Bacteroides, Clostridium, Fusobacterium, Finegoldia, Prevotella, and Veillonella, the present study focused on their ability to adhere, to grow in sessile mode and to form in vitro mono- or dual-species biofilms. Experiments on dual-species biofilm formation were planned on the basis of the anaerobic strains isolated from each clogged biliary stent, by selecting those in which a couple of anaerobic strains belonging to different species contributed to the polymicrobial biofilm development. Then, strains were investigated by field emission scanning electron microscopy and confocal laser scanning microscopy to reveal if they are able to grow as mono- and/or dual-species biofilms. As far as we know, this is the first report on the ability to adhere and form mono/dual-species biofilms exhibited by strains belonging to the species Bacteroides oralis, Clostridium difficile, Clostridium baratii, Clostridium fallax, Clostridium bifermentans, Finegoldia magna, and Fusobacterium necrophorum. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  9. Clostridium difficile Infection

    MedlinePlus

    ... Schedules Nutrient Shortfall Questionnaire Home Diseases and Conditions Clostridium difficile (C. diff.) Infection Clostridium difficile (C. diff.) Infection Condition Family HealthSeniors Share ...

  10. First isolation of Clostridium indolis in a patient with chronic osteitis: a case report and literature review of human infections related to Clostridium saccharolyticum group species.

    PubMed

    Lotte, Romain; Lotte, Laurène; Bouvet, Philippe; Degand, Nicolas; Bal, Antonin; Carles, Michel; de Dompsure, Regis Bernard; Popoff, Michel-Robert; Ruimy, Raymond

    2016-12-01

    Clostridium indolis is an anaerobic spore-forming Gram-positive bacillus belonging to the Clostridium saccharolyticum group. Its clinical significance in human remains poorly known. We describe the first case of osteitis related to C. indolis, identified by MALDI-TOF mass spectrometry and provide a literature review of human infections related to C. saccharolyticum group species.

  11. Small RNAs in the Genus Clostridium

    PubMed Central

    Chen, Yili; Indurthi, Dinesh C.; Jones, Shawn W.; Papoutsakis, Eleftherios T.

    2011-01-01

    The genus Clostridium includes major human pathogens and species important to cellulose degradation, the carbon cycle, and biotechnology. Small RNAs (sRNAs) are emerging as crucial regulatory molecules in all organisms, but they have not been investigated in clostridia. Research on sRNAs in clostridia is hindered by the absence of a systematic method to identify sRNA candidates, thus delegating clostridial sRNA research to a hit-and-miss process. Thus, we wanted to develop a method to identify potential sRNAs in the Clostridium genus to open up the field of sRNA research in clostridia. Using comparative genomics analyses combined with predictions of rho-independent terminators and promoters, we predicted sRNAs in 21 clostridial genomes: Clostridium acetobutylicum, C. beijerinckii, C. botulinum (eight strains), C. cellulolyticum, C. difficile, C. kluyveri (two strains), C. novyi, C. perfringens (three strains), C. phytofermentans, C. tetani, and C. thermocellum. Although more than one-third of predicted sRNAs have Shine-Dalgarno (SD) sequences, only one-sixth have a start codon downstream of SD sequences; thus, most of the predicted sRNAs are noncoding RNAs. Quantitative reverse transcription-PCR (Q-RT-PCR) and Northern analysis were employed to test the presence of a randomly chosen set of sRNAs in C. acetobutylicum and several C. botulinum strains, leading to the confirmation of a large fraction of the tested sRNAs. We identified a conserved, novel sRNA which, together with the downstream gene coding for an ATP-binding cassette (ABC) transporter gene, responds to the antibiotic clindamycin. The number of predicted sRNAs correlated with the physiological function of the species (high for pathogens, low for cellulolytic, and intermediate for solventogenic), but not with 16S rRNA-based phylogeny. PMID:21264064

  12. Draft Genome Sequence of the Cellulolytic and Xylanolytic Thermophile Clostridium clariflavum Strain 4-2a.

    PubMed

    Rooney, Elise A; Rowe, Kenneth T; Guseva, Anna; Huntemann, Marcel; Han, James K; Chen, Amy; Kyrpides, Nikos C; Mavromatis, Konstantinos; Markowitz, Victor M; Palaniappan, Krishna; Ivanova, Natalia; Pati, Amrita; Liolios, Konstantinos; Nordberg, Henrik P; Cantor, Michael N; Hua, Susan X; Shapiro, Nicole; Woyke, Tanja; Lynd, Lee R; Izquierdo, Javier A

    2015-07-23

    Clostridium clariflavum strain 4-2a, a novel strain isolated from a thermophilic biocompost pile, has demonstrated an extensive capability to utilize both cellulose and hemicellulose under thermophilic anaerobic conditions. Here, we report the draft genome of this strain.

  13. Metabolic control of Clostridium thermocellum via selective inhibition and compensatory product formation

    USDA-ARS?s Scientific Manuscript database

    Clostridium thermocellum is a thermophilic, anaerobic bacterium that catabolizes recalcitrant plant fibers such as cellulose. Cellulose is depolymerized by an extracellular, membrane-associated enzyme system, and the sugars are then transported across the cell membrane for fermentation. C. thermoc...

  14. Identification, distribution, and toxigenicity of obligate anaerobes in polluted waters.

    PubMed Central

    Daily, O P; Joseph, S W; Gillmore, J D; Colwell, R R; Seidler, R J

    1981-01-01

    A seasonal occurrence of obligately anaerobic bacteria, predominantly of the genera Bacteroides and Clostridium, in a polluted water site has been observed. The number of anaerobes varied from 1.8 X 10(3) cells/ml in the warmer months to 10 cells/ml in winter. Several isolates were toxigenic, indicating a potential human health hazard. PMID:7235706

  15. Relationship of Bacteriophages to the Toxigenicity of Clostridium botulinum and Closely Related Organisms

    DTIC Science & Technology

    1981-01-01

    CLOSTRIDIUM NOVYI TYPE A BY BACTERIOPHAGES C. botulinum and C. novyi are pathogenic anaerobes that are characterized by their ability to produce powerful...A-., Ij7L.oI..I-C.C= RELATIONSHIP OF BACTERIOPHAGES TO THE TOXIGENICITY OF CLOSTRIDIUM BOTULINUM AND CLOSELY RELATED ORGANISMS M. W. Eklund F. T...specific neurotoxins, the species Clostridium botulinum is divided into types A through G. Even though the different toxin types represent a heterogenous

  16. Two Serious Cases of Infection with Clostridium celatum after 40 Years in Hiding?

    PubMed Central

    Hoegh, Silje Vermedal; Holt, Hanne Marie; Justesen, Ulrik Stenz

    2015-01-01

    Clostridium celatum [ce.la'tum. L. adj. celatum hidden] has been known since 1974, when it was isolated from human feces. In 40 years, no association with human infection has been reported. In this work, we present two serious cases of infection with the anaerobic Gram-positive rod Clostridium celatum. PMID:26560535

  17. Two Serious Cases of Infection with Clostridium celatum after 40 Years in Hiding?

    PubMed

    Agergaard, Charlotte Nielsen; Hoegh, Silje Vermedal; Holt, Hanne Marie; Justesen, Ulrik Stenz

    2016-01-01

    Clostridium celatum [ce.la'tum. L. adj. celatum hidden] has been known since 1974, when it was isolated from human feces. In 40 years, no association with human infection has been reported. In this work, we present two serious cases of infection with the anaerobic Gram-positive rod Clostridium celatum. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  18. Clostridium novyi, sordellii, and tetani: mechanisms of disease.

    PubMed

    Aronoff, David M

    2013-12-01

    Clostridia represent a diverse group of spore-forming gram positive anaerobes that include several pathogenic species. In general, diseases caused by clostridia are a result of intoxication of the infected host. Thus, clostridial toxins have been targeted for diagnostic, therapeutic, and preventive strategies against infection. Studying the mechanisms of action of clostridial toxins has not only shed light on the pathogenesis of infection but has provided important new insights into cell biology and immunology. A primary purpose of this manuscript is to provide a succinct review on the mechanisms of disease caused by intoxication by the pathogens Clostridium tetani, Clostridium novyi, and Clostridium sordellii.

  19. Clostridium difficile: from obscurity to superbug.

    PubMed

    Brazier, J S

    2008-01-01

    According to the UK media and popular press, Clostridium difficile is now a fully fledged member of that notorious but ill-defined group of microorganisms portrayed to the general public as superbugs. Following the trail blazed by methicillin-resistant Staphylococcus aureus (MRSA), C. difficile has made the transition from being an obscure anaerobic bacterium, mainly of interest to specialist anaerobic microbiologists, to that of an infamous superbug responsible for outbreaks of hospital-acquired infection that commonly result in serious disease and death. This review tracks the rise in scientific knowledge and public awareness of this organism.

  20. Genetic Analysis of Nitroaromatic Degradation by Clostridium

    DTIC Science & Technology

    2013-07-30

    Phenazine, a molecule produced by some soil bacteria was found to have a significant effect on metabolite pattern in two clostridium test strains...found certain other redox active dyes (some naturally occurring in soil environments) also generate this change in acid profile of anaerobic fermenters ...Insulation of a synthetic hydrogen metabolism circuit in bacteria . J Biol Eng. 2010 Feb 25;4:3. Akhtar MK, Jones PR. Engineering of a synthetic hydF

  1. Acetone-butanol-ethanol production from substandard and surplus dates by Egyptian native Clostridium strains.

    PubMed

    Abd-Alla, Mohamed Hemida; Zohri, Abdel-Naser Ahmed; El-Enany, Abdel-Wahab Elsadek; Ali, Shimaa Mohamed

    2015-04-01

    One hundred and seven mesophilic isolates of Clostridium were isolated from agricultural soils cultivated with different plants in Assuit Governorate, Egypt. Eighty isolates (out of 107) showed the ability to produce ABE (Acetone, butanol and ethanol) on T6 medium ranging from 0.036 to 31.89 g/L. The highest numbers of ABE producing isolates were obtained from soil samples of potato contributing 27 isolates, followed by 18 isolates from wheat and 10 isolates from onion. On the other hand, there were three native isolates that produced ABE more than those produced by the reference isolate Clostridium acetobutylicum ATCC 824 (11.543 g/L). The three isolates were identified based on phenotypic and gene encoding 16S rRNA as Clostridium beijerinckii ASU10 (KF372577), Clostridium chauvoei ASU55 (KF372580) and Clostridium roseum ASU58 (KF372581). The highest ABE level from substandard and surplus dates was produced by C. beijerinckii ASU10 (24.07 g/L) comprising butanol 67.15% (16.16 g/L), acetone 30.73% (7.4 g/L) and ethanol 2.12% (0.51 g/L), while C. roseum ASU58 and C. chauvoei ASU55 produced ABE contributing 20.20 and 13.79 g/L, respectively. ABE production by C. acetobutylicum ATCC 824 was 15.01 g/L. This study proved that the native strains C. beijerinckii ASU10 and C. roseum ASU58 have high competitive efficacy on ABE production from economical substrate as substandard and surplus date fruits. Additionally, using this substrate without any nutritional components is considered to be a commercial substrate for desired ABE production.

  2. Anaerobic bacteria

    MedlinePlus

    Anaerobic bacteria are bacteria that do not live or grow when oxygen is present. In humans, these bacteria ... Goldstein EJ. Diseases caused by non-spore forming anaerobic bacteria. In: Goldman L, Schafer AI, eds. Goldman's Cecil ...

  3. Clostridium difficile

    PubMed Central

    Curry, Scott R.

    2017-01-01

    SYNOPSIS Clostridium difficile infections (CDI) have emerged as one of the principal threats to the health of hospitalized and immunocompromised patients. Nucleic acid testing for C. difficile toxin genes has eclipsed traditional clinical diagnostics for CDI in sensitivity and is now widespread in clinical use, but preliminary evidence suggests that this may have come at a cost of substantially reduced positive predictive value. The importance of C. difficile colonization is increasingly recognized not only as a source for false positive clinical testing but also as a source of new infections within hospitals and other healthcare environments. In the last five years, several new treatment strategies that capitalize on the increasing understanding of the altered microbiome and host defenses in CDI patients have completed clinical trials, including fecal microbiota transplantation (FMT). This article highlights the changing epidemiology, laboratory diagnostics, pathogenesis, and treatment of CDI. PMID:20513554

  4. Collagenase Clostridium Histolyticum Injection

    MedlinePlus

    Collagenase Clostridium histolyticum injection is used to treat Dupuytren's contracture (a painless thickening and tightening of tissue [cord] beneath ... of tissue can be felt upon examination. Collagenase Clostridium histolyticum injection is also used to treat Peyronie's ...

  5. The Role of Microorganisms in Marine Corrosion

    DTIC Science & Technology

    1990-11-30

    palladium surface. Species of Clostridium produced high concentrations of hydrogen. However, hydrogen production by Desulfovibrio sulfuricans was...biofilms. The permeation characteristics of Clostridium reflect metabolic processes which differ from other anaerobic bacteria. For example, Clostridium ... acetobutylicum displayed a rapid decrease in hydrogen production in the biofilm, reflecting a movement to hydrogen utilization. In the case of

  6. Ceftolozane-Tazobactam Activity against Phylogenetically Diverse Clostridium difficile Strains

    PubMed Central

    Gonzalez, Mark D.; Wallace, Meghan A.; Hink, Tiffany; Dubberke, Erik R.

    2015-01-01

    Ceftolozane-tazobactam (C/T) is approved for the treatment of complicated intra-abdominal and urinary tract infections and has varied activity against anaerobic bacteria. Here, we evaluate the activity of C/T against a phylogenetically diverse collection of Clostridium difficile isolates and report uniformly high MICs (≥256 μg/ml) to C/T. PMID:26282409

  7. Complementation of a Clostridium perfringens spo0A Mutant with Wild-Type spo0A from Other Clostridium Species

    PubMed Central

    Huang, I-Hsiu; Sarker, Mahfuzur R.

    2006-01-01

    To evaluate whether C. perfringens can be used as a model organism for studying the sporulation process in other clostridia, C. perfringens spo0A mutant IH101 was complemented with wild-type spo0A from four different Clostridium species. Wild-type spo0A from C. acetobutylicum or C. tetani, but not from C. botulinum or C. difficile, restored sporulation and enterotoxin production in IH101. The ability of spo0A from C. botulinum or C. difficile to complement the lack of spore formation in IH101 might be due, at least in part, to the low levels of spo0A transcription and Spo0A production. PMID:16957268

  8. Degradation of PCE by Two Kinds of Anaerobic Bacteria

    NASA Astrophysics Data System (ADS)

    Kim, Eun-Sook; Takaoka, Hidemitsu; Takamizawa, Kazuhiro

    Anaerobic decomposition of PCE was examined using Clostridium bifermentans DPH-1 with degradation ability of PCE to cis-1,2-dichloroethylene (DCE) and cis-DCE decomposing bacterium, Clostridium sp. strain KYT-1. In the serial 2 step reactions, it was demonstrated that PCE was degraded completely by Clostridium bifermentans DPH-1 in the first reaction and 39% of cis-DCE, byproduct of PCE degrading was eliminated by Clostridium sp. strain KYT-1 in the second reaction with glucose provided as carbon source. On the other hand, in the mixed culture of anaerobic bacteria, PCE was not eliminated perfectly and remained as much as 33% of initial concentration of PCE. But the accumulation of cis-DCE and VC as intermediate metabolites of PCE degradation was not shown.

  9. Draft Genome Sequence of Clostridium sp. Ne2, Clostridia from an Enrichment Culture Obtained from Australian Subterranean Termite, Nasutitermes exitiosus.

    PubMed

    Wang, Han; Lin, Hai; Tran-Dinh, Nai; Li, Dongmei; Greenfield, Paul; Midgley, David J

    2015-04-23

    The draft genome sequence of Clostridium sp. Ne2 was reconstructed from a metagenome of a hydrogenogenic microbial consortium. The organism is most closely related to Clostridium magnum and is a strict anaerobe that is predicted to ferment a range of simple sugars. Copyright © 2015 Wang et al.

  10. Draft Genome Sequence of Clostridium sp. Ne2, Clostridia from an Enrichment Culture Obtained from Australian Subterranean Termite, Nasutitermes exitiosus

    PubMed Central

    Lin, Hai; Tran-Dinh, Nai; Li, Dongmei; Greenfield, Paul; Midgley, David J.

    2015-01-01

    The draft genome sequence of Clostridium sp. Ne2 was reconstructed from a metagenome of a hydrogenogenic microbial consortium. The organism is most closely related to Clostridium magnum and is a strict anaerobe that is predicted to ferment a range of simple sugars. PMID:25908129

  11. Characterization of Clostridium sp. RKD producing botulinum-like neurotoxin.

    PubMed

    Dixit, Aparna; Dhaked, Ram Kumar; Alam, Syed Imteyaz; Singh, Lokendra

    2005-07-01

    A Gram positive, motile, rod-shaped, strictly anaerobic bacterium isolated from intestine of decaying fish was identified as Clostridium sp. RKD and produced a botulinum type B-like neurotoxin as suggested by mouse bioassay and protection with anti botulinum antibodies. The neurotoxicity was functionally characterized by the phrenic nerve hemi-diaphragm assay. Phylogenetic analysis based on 16S rDNA sequence, placed it at a different position from the reported strains of Clostridium botulinum. The strain exhibited differences from both Clostridium botulinum and Clostridium tetani with respect to morphological, biochemical and chemotaxonomic characteristics. Botulinum group specific and serotype specific primers amplified the DNA fragments of 260 and 727 bp, respectively, indicating presence of botulinum type 'B' toxin gene. Sequence of nearly 700 bp amplified using primers specific for botulinum neurotoxin type B gene, did not show any significant match in the database when subjected to BLAST search.

  12. Variability in DPA and Calcium Content in the Spores of Clostridium Species.

    PubMed

    Jamroskovic, Jan; Chromikova, Zuzana; List, Cornelia; Bartova, Barbora; Barak, Imrich; Bernier-Latmani, Rizlan

    2016-01-01

    Spores of a number of clostridial species, and their resistance to thermal treatment is a major concern for the food industry. Spore resistance to wet heat is related to the level of spore hydration, which is inversely correlated with the content of calcium and dipicolinic acid (DPA) in the spore core. It is widely believed that the accumulation of DPA and calcium in the spore core is a fundamental component of the sporulation process for all endospore forming species. We have noticed heterogeneity in the heat resistance capacity and overall DPA/calcium content among the spores of several species belonging to Clostridium sensu stricto group: two C. acetobutylicum strains (DSM 792 and 1731), two C. beijerinckii strains (DSM 791 and NCIMB 8052), and a C. collagenovorans strain (DSM 3089). A C. beijerinckii strain (DSM 791) and a C. acetobutylicum strain (DSM 792) display low Ca and DPA levels. In addition, these two species, with the lowest average Ca/DPA content amongst the strains considered, also exhibit minimal heat resistance. There appears to be no correlation between the Ca/DPA content and the phylogenetic distribution of the C. acetobutylicum and C. beijerinckii species based either on the 16S rRNA or the spoVA gene. This finding suggests that a subset of Clostridium sensu stricto species produce spores with low resistance to wet heat. Additionally, analysis of individual spores using STEM-EDS and STXM revealed that DPA and calcium levels can also vary amongst individual spores in a single spore population.

  13. Variability in DPA and Calcium Content in the Spores of Clostridium Species

    PubMed Central

    Jamroskovic, Jan; Chromikova, Zuzana; List, Cornelia; Bartova, Barbora; Barak, Imrich; Bernier-Latmani, Rizlan

    2016-01-01

    Spores of a number of clostridial species, and their resistance to thermal treatment is a major concern for the food industry. Spore resistance to wet heat is related to the level of spore hydration, which is inversely correlated with the content of calcium and dipicolinic acid (DPA) in the spore core. It is widely believed that the accumulation of DPA and calcium in the spore core is a fundamental component of the sporulation process for all endospore forming species. We have noticed heterogeneity in the heat resistance capacity and overall DPA/calcium content among the spores of several species belonging to Clostridium sensu stricto group: two C. acetobutylicum strains (DSM 792 and 1731), two C. beijerinckii strains (DSM 791 and NCIMB 8052), and a C. collagenovorans strain (DSM 3089). A C. beijerinckii strain (DSM 791) and a C. acetobutylicum strain (DSM 792) display low Ca and DPA levels. In addition, these two species, with the lowest average Ca/DPA content amongst the strains considered, also exhibit minimal heat resistance. There appears to be no correlation between the Ca/DPA content and the phylogenetic distribution of the C. acetobutylicum and C. beijerinckii species based either on the 16S rRNA or the spoVA gene. This finding suggests that a subset of Clostridium sensu stricto species produce spores with low resistance to wet heat. Additionally, analysis of individual spores using STEM-EDS and STXM revealed that DPA and calcium levels can also vary amongst individual spores in a single spore population. PMID:27891119

  14. The acetate kinase of Clostridum acetobutylicum strain P262.

    PubMed

    Diez-Gonzalez, F; Russell, J B; Hunter, J B

    1996-12-01

    Clostridum acetobutylicum strain P262 fermented glucose, pyruvate, or lactate, and the butyrate production was substrate-dependent. Differences in butyrate yield could not be explained by changes in butyrate kinase activities, but the butyrate production was inversely related to acetate kinase activity. The acetate kinase had a pH optimum of 8.0, a Km for acetate of 160 mM, and a kcat of 16, 800 min-1. The enyzme had a native molecular mass of 78 kDa; the size of 42 kDa on SDS-PAGE indicated that the acetate kinase of strain P262 was a homodimer.

  15. Characterization of an acetoin reductase/2,3-butanediol dehydrogenase from Clostridium ljungdahlii DSM 13528.

    PubMed

    Tan, Yang; Liu, Zi-Yong; Liu, Zhen; Li, Fu-Li

    2015-11-01

    Acetoin reductase catalyzes the formation of 2,3-butanediol from acetoin. In Clostridium ljungdahlii DSM 13528, the gene CLJU_c23220 encoding the putative Zn(2+)-dependent alcohol dehydrogenase was cloned and expressed in Escherichia coli. The recombinant enzyme, CLAR, can catalyze the conversion of acetoin to 2,3-butanediol with NADPH as the cofactor. Furthermore, the gene CLJU_c23220 was introduced into Clostridium acetobutylicum ATCC 824 and the transformant was conferred the capacity of 2,3-butanediol production. In batch fermentation the transformant produced up to 3.1g/L of 2,3-butanediol, as well as acetone, butanol and ethanol (ABE, 17.8 g/L) in amounts similar to those produced by the wild type strain. This study provides conclusive evidence at the protein level that CLJU_c23220 is the key gene responsible for the conversion of acetoin to 2,3-butanediol in C. ljungdahlii DSM 13528. Moreover, the C. acetobutylicum ATCC 824 was modified via one-step metabolic engineering to produce 2,3-butanediol without influencing the ABE production.

  16. Molecular characterization of two Clostridium acetobutylicum ATCC 824 butanol dehydrogenase isozyme genes.

    PubMed Central

    Walter, K A; Bennett, G N; Papoutsakis, E T

    1992-01-01

    A 4-kb segment of DNA containing two previously cloned butanol dehydrogenase (BDH) isozyme genes (D. Petersen, R. Welch, F. Rudolph, and G. Bennett, J. Bacteriol. 173:1831-1834, 1991) was sequenced. Two complete open reading frames (ORFs) were identified (bdhA and bdhB), along with a third truncated ORF (ORF1). The translation products of bdhA and bdhB corresponded to the N-terminal sequences of the purified BDH I and BDH II proteins, respectively. The two isozymes had a high amino acid identity (73%) and showed homology to a newly described class of alcohol dehydrogenases. Northern blots revealed that bdhA and bdhB did not form an operon. Primer extension experiments located single transcriptional start sites 37 and 58 bp upstream of the start codons of bdhA and bdhB, respectively. The -10 and -35 promoter regions for these genes were almost identical. bdhA and bdhB were found to be induced or derepressed immediately prior to significant butanol production in controlled pH 5.0 batch fermentations. Images PMID:1385386

  17. Discovery of External Modulators of the Fe-Fe Hydrogenase Enzyme in Clostridium acetobutylicum

    DTIC Science & Technology

    2015-02-01

    molecular weight:  Daltons) that can be potentially experimentally tested to reduce HydA activity in bacteria . Computational 3 prescreening...and Marburg viruses6–9 and an ATP (adenosine triphosphate)–powered secretion system in plague bacteria .10 Two million commercially available...established to test the effects that potential HydA inhibitors may have on gas production in C. acet. fermentations . Typically, gas production is

  18. Recent progress on industrial fermentative production of acetone-butanol-ethanol by Clostridium acetobutylicum in China.

    PubMed

    Ni, Ye; Sun, Zhihao

    2009-06-01

    China is one of the few countries, which maintained the fermentative acetone-butanol-ethanol (ABE) production for several decades. Until the end of the last century, the ABE fermentation from grain was operated in a few industrial scale plants. Due to the strong competition from the petrochemical industries, the fermentative ABE production lost its position in the 1990s, when all the solvent fermentation plants in China were closed. Under the current circumstances of concern about energy limitations and environmental pollution, new opportunities have emerged for the traditional ABE fermentation industry since it could again be potentially competitive with chemical synthesis. From 2006, several ABE fermentation plants in China have resumed production. The total solvent (acetone, butanol, and ethanol) production capacity from ten plants reached 210,000 tons, and the total solvent production is expected to be extended to 1,000,000 tons (based on the available data as of Sept. 2008). This article reviews current work in strain development, the continuous fermentation process, solvent recovery, and economic evaluation of ABE process in China. Challenges for an economically competitive ABE process in the future are also discussed.

  19. Biobutanol production by Clostridium acetobutylicum using xylose recovered from birch Kraft black liquor.

    PubMed

    Kudahettige-Nilsson, Rasika L; Helmerius, Jonas; Nilsson, Robert T; Sjöblom, Magnus; Hodge, David B; Rova, Ulrika

    2015-01-01

    Acetone-butanol-ethanol (ABE) fermentation was studied using acid-hydrolyzed xylan recovered from hardwood Kraft black liquor by CO2 acidification as the only carbon source. Detoxification of hydrolyzate using activated carbon was conducted to evaluate the impact of inhibitor removal and fermentation. Xylose hydrolysis yields as high as 18.4% were demonstrated at the highest severity hydrolysis condition. Detoxification using active carbon was effective for removal of both phenolics (76-81%) and HMF (38-52%). Batch fermentation of the hydrolyzate and semi-defined P2 media resulted in a total solvent yield of 0.12-0.13g/g and 0.34g/g, corresponding to a butanol concentration of 1.8-2.1g/L and 7.3g/L respectively. This work is the first study of a process for the production of a biologically-derived biofuel from hemicelluloses solubilized during Kraft pulping and demonstrates the feasibility of utilizing xylan recovered directly from industrial Kraft pulping liquors as a feedstock for biological production of biofuels such as butanol.

  20. TRANSFORMATION OF TNT AND RELATED NITROAROMATIC COMPOUNDS BY CLOSTRIDIUM ACETOBUTYLICUM. (R825513C006)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  1. TRANSFORMATION OF TNT AND RELATED NITROAROMATIC COMPOUNDS BY CLOSTRIDIUM ACETOBUTYLICUM. (R825513C006)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  2. Alternative non-chromatographic method for alcohols determination in Clostridium acetobutylicum fermentations.

    PubMed

    Noriega-Medrano, Laura J; Vega-Estrada, Jesús; Ortega-López, Jaime; Ruiz-Medrano, Roberto; Cristiani-Urbina, Eliseo; Montes-Horcasitas, Maria Del Carmen

    2016-07-01

    An economic, simple, quantitative, and non-chromatographic method for the determination of alcohols using microdiffusion principle has been adapted and validated for acetone-butanol-ethanol (ABE) fermentation samples. This method, based on alcohols oxidation using potassium dichromate in acid medium, and detection by spectrophotometry, was evaluated varying, both, temperature (35°C, 45°C, and 55°C) and reaction time (0 to 125min). With a sample analysis time of 90min at 45°C, a limit of detection (LOD), and a limit of quantification (LOQ) of 0.10, and 0.40g/L, respectively. The proposed method has been successfully applied to determine butanol and ethanol concentrations in ABE fermentation samples with the advantage that multiple samples can be analyzed simultaneously. The measurements obtained with the proposed method were in good agreement with those obtained with the Gas Chromatography Method (GCM). This proposed method is useful for routine analysis of alcohols and screening samples in laboratories and industries.

  3. Anaerobic Infections

    MedlinePlus

    ... doses of antibiotics taken by mouth for months. Bacteroides and Prevotella infections. Bacterial organisms from species called Bacteroides and Prevotella are anaerobic. They are common organisms ...

  4. New Insight into Sugarcane Industry Waste Utilization (Press Mud) for Cleaner Biobutanol Production by Using C. acetobutylicum NRRL B-527.

    PubMed

    Nimbalkar, Pranhita R; Khedkar, Manisha A; Gaikwad, Shashank G; Chavan, Prakash V; Bankar, Sandip B

    2017-05-05

    In the present study, press mud, a sugar industry waste, was explored for biobutanol production to strengthen agricultural economy. The fermentative production of biobutanol was investigated via series of steps, viz. characterization, drying, acid hydrolysis, detoxification, and fermentation. Press mud contains an adequate amount of cellulose (22.3%) and hemicellulose (21.67%) on dry basis, and hence, it can be utilized for further acetone-butanol-ethanol (ABE) production. Drying experiments were conducted in the temperature range of 60-120 °C to circumvent microbial spoilage and enhance storability of press mud. Furthermore, acidic pretreatment variables, viz. sulfuric acid concentration, solid to liquid ratio, and time, were optimized using response surface methodology. The corresponding values were found to be 1.5% (v/v), 1:5 g/mL, and 15 min, respectively. In addition, detoxification studies were also conducted using activated charcoal, which removed almost 93-97% phenolics and around 98% furans, which are toxic to microorganisms during fermentation. Finally, the batch fermentation of detoxified press mud slurry (the sample dried at 100 °C and pretreated) using Clostridium acetobutylicum NRRL B-527 resulted in a higher butanol production of 4.43 g/L with a total ABE of 6.69 g/L.

  5. Biofilm formation by Clostridium difficile

    PubMed Central

    Dapa, Tanja; Unnikrishnan, Meera

    2013-01-01

    Clostridium difficile infection (CDI) is a major healthcare-associated disease worldwide. Recurring infections and increasing antibiotic resistance have complicated treatment of CDI. While C. difficile spores are important for transmission and persistence of CDI, other factors such as gut colonization and formation of bacterial communities in the gut may also contribute to pathogenesis and persistence, but have not been well investigated. Recently, we reported that important clinical C. difficile strains are able to form composite biofilms in vitro. C. difficile biofilm formation is a complex process, modulated by several different factors, including cell surface components and regulators. We also reported that bacteria within biofilms are more resistant to high concentrations of vancomycin, the antibiotic of choice for treatment of CDI. Here we summarize our recent findings and discuss the implications of biofilm formation by this anaerobic gut pathogen in disease pathogenesis and treatment. PMID:23892245

  6. Description of Anaerobacterium chartisolvens gen. nov., sp. nov., an obligately anaerobic bacterium from Clostridium rRNA cluster III isolated from soil of a Japanese rice field, and reclassification of Bacteroides cellulosolvens Murray et al. 1984 as Pseudobacteroides cellulosolvens gen. nov., comb. nov.

    PubMed

    Horino, Haruka; Fujita, Takashi; Tonouchi, Akio

    2014-04-01

    An obligately anaerobic bacterial strain designated T-1-35(T) was isolated as a dominant cultivable cellulose-degrading bacterium from soil of a Japanese rice field as an anaerobic filter-paper degrader. Cells of strain T-1-35(T) stained Gram-positive and were non-spore-forming rods with rounded ends, 0.8-1.0×3.5-15.0 µm, and motile by means of two to four polar flagella. Cells of strain T-1-35(T) exhibited pleomorphism: in aged cultures (over 90 days of incubation), almost all cells were irregularly shaped. Although no spore formation was observed, cells tolerated high temperatures, up to 90 °C for 10 min. The temperature range for growth was 15-40 °C, with an optimum at 35 °C. The pH range for growth was 5.5-9.0, with an optimum at pH 8.0-8.5 (slightly alkaliphilic). Strain T-1-35(T) fermented some carbohydrates to produce ethanol and lactate as the major products. Major cellular fatty acids were iso-C16 : 0 and iso-C13 : 0 3-OH. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain T-1-35(T) belonged to Clostridium rRNA cluster III. The closest relative of strain T-1-35(T) was Bacteroides cellulosolvens WM2(T), with 16S rRNA gene sequence similarity of 93.4 %. Phenotypic, physiological and molecular genetic methods demonstrated that strain T-1-35(T) was distinct from its phylogenetic relatives (members of Clostridium rRNA cluster III) because it predominantly produced ethanol, iso-C13 : 0 3-OH was a major cellular fatty acid and it always exhibited pleomorphism. On the basis of the results of a polyphasic taxonomic study, strain T-1-35(T) is considered to represent a novel genus and species, Anaerobacterium chartisolvens gen. nov., sp. nov. The type strain of Anaerobacterium chartisolvens is T-1-35(T) ( = DSM 27016(T) = NBRC 109520(T)). In addition, from the results of our phylogenetic analysis and its phenotypic features, the species Bacteroides cellulosolvens Murray et al. 1984 is proposed to be reclassified

  7. Clostridium phytofermentans sp. nov., a cellulolytic mesophile from forest soil.

    PubMed

    Warnick, Thomas A; Methé, Barbara A; Leschine, Susan B

    2002-07-01

    An obligately anaerobic, mesophilic, cellulolytic bacterium, strain ISDgT, was isolated from forest soil. Cells of this isolate stained Gram-negative, despite possessing a Gram-positive cell-wall ultrastructure, and were motile, straight rods that formed spherical terminal spores that swelled the sporangium. Cellulose, pectin, polygalacturonic acid, starch, xylan, arabinose, cellobiose, fructose, galactose, gentiobiose, glucose, lactose, maltose, mannose, ribose and xylose supported growth. The major end products of fermentation were ethanol, acetate, CO2 and H2; formate and lactate were minor products. The optimum temperature for growth was 35-37 degrees C. Phylogenetic analyses based on 16S rRNA sequence comparisons showed that strain ISDgT was related to a group of anaerobes that included Clostridium herbivorans, Clostridium polysaccharolyticum and Clostridium populeti. The G+C content of this strain was 35.9 mol%. On the basis of numerous genotypic and phenotypic differences between strain ISDgT and its close relatives, strain ISDgT is proposed as a novel species in the genus Clostridium, for which the name Clostridium phytofermentans sp. nov. is proposed. The type strain is ISDgT (= ATCC 700394T).

  8. Autism and Clostridium tetani.

    PubMed

    Bolte, E R

    1998-08-01

    Autism is a severe developmental disability believed to have multiple etiologies. This paper outlines the possibility of a subacute, chronic tetanus infection of the intestinal tract as the underlying cause for symptoms of autism observed in some individuals. A significant percentage of individuals with autism have a history of extensive antibiotic use. Oral antibiotics significantly disrupt protective intestinal microbiota, creating a favorable environment for colonization by opportunistic pathogens. Clostridium tetani is an ubiquitous anaerobic bacillus that produces a potent neurotoxin. Intestinal colonization by C. tetani, and subsequent neurotoxin release, have been demonstrated in laboratory animals which were fed vegetative cells. The vagus nerve is capable of transporting tetanus neurotoxin (TeNT) and provides a route of ascent from the intestinal tract to the CNS. This route bypasses TeNT's normal preferential binding sites in the spinal cord, and therefore the symptoms of a typical tetanus infection are not evident. Once in the brain, TeNT disrupts the release of neurotransmitters by the proteolytic cleavage of synaptobrevin, a synaptic vesicle membrane protein. This inhibition of neurotransmitter release would explain a wide variety of behavioral deficits apparent in autism. Lab animals injected in the brain with TeNT have exhibited many of these behaviors. Some children with autism have also shown a significant reduction in stereotyped behaviors when treated with antimicrobials effective against intestinal clostridia. When viewed as sequelae to a subacute, chronic tetanus infection, many of the puzzling abnormalities of autism have a logical basis. A review of atypical tetanus cases, and strategies to test the validity of this paper's hypothesis, are included.

  9. Biofilms of Clostridium species.

    PubMed

    Pantaléon, Véronique; Bouttier, Sylvie; Soavelomandroso, Anna Philibertine; Janoir, Claire; Candela, Thomas

    2014-12-01

    The biofilm is a microbial community embedded in a synthesized matrix and is the main bacterial way of life. A biofilm adheres on surfaces or is found on interfaces. It protects bacteria from the environment, toxic molecules and may have a role in virulence. Clostridium species are spread throughout both environments and hosts, but their biofilms have not been extensively described in comparison with other bacterial species. In this review we describe all biofilms formed by Clostridium species during both industrial processes and in mammals where biofilms may be formed either during infections or associated to microbiota in the gut. We have specifically focussed on Clostridium difficile and Clostridium perfringens biofilms, which have been studied in vitro. Regulatory processes including sporulation and germination highlight how these Clostridium species live in biofilms. Furthermore, biofilms may have a role in the survival and spreading of Clostridium species.

  10. Microbial Gas Production Used to Achieve Autonomous Buoyancy Control

    DTIC Science & Technology

    2010-09-01

    counter weight. The microbe used is cultured in the Erlenmeyer flask. Clostridium acetobutylicum for micro1bial ballast In order to utilize microbial...34 20 0 --100% 0 20 40 60 80 Time(hr) Figure 6. Gas production by different S()lid-phase agar cultures of C. acetobutvlicum. Clostridium ... acetobutylicum as our model microbe; a gram-positive anaerobic bacterium known for its ability to produce hydrogen gas 4,5 To determine if there

  11. Septic arthritis due to Clostridium ramosum.

    PubMed

    García-Jiménez, Antonio; Prim, Núria; Crusi, Xavier; Benito, Natividad

    2016-04-01

    Clostridium species are anaerobic bacilli that are rarely reported as etiologic agents of infectious arthritis. Previous cases of arthritis caused by Clostridium ramosum have not been reported. We describe the first 2 cases of C. ramosum arthritis. We reviewed the etiology of arthritis in our hospital during the previous 15 years. Both patients had underlying immunocompromising conditions and their infections involved a joint with preexisting disease: patient 1 had rheumatic arthritis and a prosthetic joint; patient 2, chronic renal failure on dialysis and hip osteoarthritis. The infection was hematogenously acquired and the course was indolent but destructive in both the cases. Management included open arthrotomy and resection arthroplasty. The infection had a persisting and relapsing course, and prolonged antibiotic treatment was required. In the literature review, we found 55 previous cases of arthritis caused by Clostridium species between 1966 and 2014; Clostridium perfringens was the most common infecting species; the infection was traumatically acquired in most of the cases. A total of 15 patients have been described with infections caused by C. ramosum; none had septic arthritis. The majority were elderly or immunocompromised adults. Proper collection, transportation and processing of clinical specimens is essential for diagnosing clostridial infections. More information about the best management of clostridial arthritis are needed. We describe the first 2 cases of septic arthritis caused by C. ramosum. They shared several pathogenic and clinical features. The possibility of anaerobic arthritis should always be considered when collecting diagnostic specimens. An increasing number of clostridial arthritis cases are likely to be diagnosed in future years. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Purification of Clostridium toxoids.

    PubMed

    Buchowicz, I; Hay, M; Schiller, B; Korbecki, M; Sochańska, R

    1977-01-01

    A two-step fractionation procedure was applied for purification and concentration of the individual Clostridium toxoids. The toxoids were precipitated with hydrochloric acid in the presence of sodium sextametaphosphate, then antigenic fractions were separated from inactive contaminants by Sephadex G-75 filtration. Specific activity of the preparations thus obtained, as determined by Mancini radial immunodiffusion, was 150--565 binding units per mg of protein nitrogen for Clostridium perfringens toxoid, 204--352 binding units for Clostridium oedematiens toxoid and 26.6 -- 51.2 binding units for Clostridium septicum toxoid.

  13. A Universal Mariner Transposon System for Forward Genetic Studies in the Genus Clostridium

    PubMed Central

    Zhang, Ying; Grosse-Honebrink, Alexander; Minton, Nigel P.

    2015-01-01

    DNA transposons represent an essential tool in the armoury of the molecular microbiologist. We previously developed a catP-based mini transposon system for Clostridium difficile in which the expression of the transposase gene was dependent on a sigma factor unique to C. difficile, TcdR. Here we have shown that the host range of the transposon is easily extended through the rapid chromosomal insertion of the tcdR gene at the pyrE locus of the intended clostridial target using Allele-Coupled Exchange (ACE). To increase the effectiveness of the system, a novel replicon conditional for plasmid maintenance was developed, which no longer supports the effective retention of the transposon delivery vehicle in the presence of the inducer isopropyl β-D-1-thiogalactopyranoside (IPTG). As a consequence, those thiamphenicol resistant colonies that arise in clostridial recipients, following plating on agar medium supplemented with IPTG, are almost exclusively due to insertion of the mini transposon into the genome. The system has been exemplified in both Clostridium acetobutylicum and Clostridium sporogenes, where transposon insertion has been shown to be entirely random. Moreover, appropriate screening of both libraries resulted in the isolation of auxotrophic mutants as well as cells deficient in spore formation/germination. This strategy is capable of being implemented in any Clostridium species. PMID:25836262

  14. A universal mariner transposon system for forward genetic studies in the genus Clostridium.

    PubMed

    Zhang, Ying; Grosse-Honebrink, Alexander; Minton, Nigel P

    2015-01-01

    DNA transposons represent an essential tool in the armoury of the molecular microbiologist. We previously developed a catP-based mini transposon system for Clostridium difficile in which the expression of the transposase gene was dependent on a sigma factor unique to C. difficile, TcdR. Here we have shown that the host range of the transposon is easily extended through the rapid chromosomal insertion of the tcdR gene at the pyrE locus of the intended clostridial target using Allele-Coupled Exchange (ACE). To increase the effectiveness of the system, a novel replicon conditional for plasmid maintenance was developed, which no longer supports the effective retention of the transposon delivery vehicle in the presence of the inducer isopropyl β-D-1-thiogalactopyranoside (IPTG). As a consequence, those thiamphenicol resistant colonies that arise in clostridial recipients, following plating on agar medium supplemented with IPTG, are almost exclusively due to insertion of the mini transposon into the genome. The system has been exemplified in both Clostridium acetobutylicum and Clostridium sporogenes, where transposon insertion has been shown to be entirely random. Moreover, appropriate screening of both libraries resulted in the isolation of auxotrophic mutants as well as cells deficient in spore formation/germination. This strategy is capable of being implemented in any Clostridium species.

  15. Regulation of Toxin Production in Clostridium perfringens

    PubMed Central

    Ohtani, Kaori; Shimizu, Tohru

    2016-01-01

    The Gram-positive anaerobic bacterium Clostridium perfringens is widely distributed in nature, especially in soil and the gastrointestinal tracts of humans and animals. C. perfringens causes gas gangrene and food poisoning, and it produces extracellular enzymes and toxins that are thought to act synergistically and contribute to its pathogenesis. A complicated regulatory network of toxin genes has been reported that includes a two-component system for regulatory RNA and cell-cell communication. It is necessary to clarify the global regulatory system of these genes in order to understand and treat the virulence of C. perfringens. We summarize the existing knowledge about the regulatory mechanisms here. PMID:27399773

  16. Metabolic engineering of Clostridium tyrobutyricum for n-butanol production from maltose and soluble starch by overexpressing α-glucosidase.

    PubMed

    Yu, Le; Xu, Mengmeng; Tang, I-Ching; Yang, Shang-Tian

    2015-07-01

    Clostridium tyrobutyricum does not have the enzymes needed for using maltose or starch. Two extracellular α-glucosidases encoded by agluI and agluII from Clostridium acetobutylicum ATCC 824 catalyzing the hydrolysis of α-1,4-glycosidic bonds in maltose and starch from the non-reducing end were cloned and expressed in C. tyrobutyricum (Δack, adhE2), and their effects on n-butanol production from maltose and soluble starch in batch fermentations were studied. Compared to the parental strain grown on glucose, mutants expressing agluI showed robust activity in breaking down maltose and produced more butanol (17.2 vs. 9.5 g/L) with a higher butanol yield (0.20 vs. 0.10 g/g) and productivity (0.29 vs. 0.16 g/L h). The mutant was also able to use soluble starch as substrate, although at a slower rate compared to maltose. Compared to C. acetobutylicum ATCC 824, the mutant produced more butanol from maltose (17.2 vs. 11.2 g/L) and soluble starch (16.2 vs. 8.8 g/L) in batch fermentations. The mutant was stable in batch fermentation without adding antibiotics, achieving a high butanol productivity of 0.40 g/L h. This mutant strain thus can be used in industrial production of n-butanol from maltose and soluble starch.

  17. Clostridium difficile spore biology: sporulation, germination, and spore structural proteins

    PubMed Central

    Paredes-Sabja, Daniel; Shen, Aimee; Sorg, Joseph A.

    2014-01-01

    Clostridium difficile is a Gram-positive, spore-forming obligate anaerobe and a major nosocomial pathogen of world-wide concern. Due to its strict anaerobic requirements, the infectious and transmissible morphotype is the dormant spore. In susceptible patients, C. difficile spores germinate in the colon to form the vegetative cells that initiate Clostridium difficile infections (CDI). During CDI, C. difficile induces a sporulation pathway that produces more spores; these spores are responsible for the persistence of C. difficile in patients and horizontal transmission between hospitalized patients. While important to the C. difficile lifecycle, the C. difficile spore proteome is poorly conserved when compared to members of the Bacillus genus. Further, recent studies have revealed significant differences between C. difficile and B. subtilis at the level of sporulation, germination and spore coat and exosporium morphogenesis. In this review, the regulation of the sporulation and germination pathways and the morphogenesis of the spore coat and exosporium will be discussed. PMID:24814671

  18. Isolation and Identification of Psychrophilic Species of Clostridium from Milk

    PubMed Central

    Bhadsavle, C. H.; Shehata, T. E.; Collins, E. B.

    1972-01-01

    Four of 48 raw milk samples contained catalase-negative, gram-positive, motile, sporeforming, rod-shaped bacteria that grew optimally at 22 to 30 C and slowly at low temperatures. Isolates from two samples had a minimal growth temperature of 4 C, were anaerobic, and had characteristics similar to Clostridium hastiforme; those from the other two samples had a minimal growth temperature of 0 ± 1 C, were anaerobic, aerotolerant, and had characteristics similar to C. carnis. Specific growth rates, doubling times, ability to grow in pasteurized milk stored in commercial cartons, and resistance of spores to heating were determined for one strain of C. hastiforme. PMID:4565634

  19. Splenic abscess with Clostridium novyi bacteraemia and sepsis.

    PubMed

    Vleminckx, W G; Diltoer, M W; Spapen, H D; Pierard, D; De Mey, J; Delvaux, G R; Huyghens, L P

    1997-03-01

    Splenic abscess is an uncommon entity and usually results in the death of the patient when left undiagnosed. A case is presented where bacteraemia with an anaerobic Gram-positive bacillus was associated with splenic abscess. Despite treatment with splenectomy and antibiotics the patient developed a multiple organ dysfunction syndrome (MODS) and died. Of particular interest was the isolation of Clostridium novyi type A from the blood in a patient without gas gangrene but with splenic suppuration.

  20. Clostridium Difficile Infections

    MedlinePlus

    Clostridium difficile (C. difficile) is a bacterium that causes diarrhea and more serious intestinal conditions such as colitis. Symptoms include Watery ... Nausea Abdominal pain or tenderness You might get C. difficile disease if you have an illness that ...

  1. Clostridium tepidum sp. nov., a close relative of Clostridium sporogenes and Clostridium botulinum Group I.

    PubMed

    Dobritsa, Anatoly P; Kutumbaka, Kirthi K; Werner, Kirsten; Wiedmann, Martin; Asmus, Aaron; Samadpour, Mansour

    2017-07-01

    Obligately anaerobic, Gram-stain-positive, spore-forming bacteria indistinguishable by pulsed-field gel electrophoresis were isolated from non-dairy protein shakes in bloated bottles. One of the isolates, strain IEH 97212T, was selected for further study. The strain was closely related to Clostridium sporogenes and Clostridium botulinum Group 1 based on 16S rRNA gene sequence similarities. Phylogenetic analysis also showed that strain IEH 97212T and strain PE (=DSM 18688), a bacterium isolated from solfataric mud, had identical 16S rRNA gene sequences. Strains IEH 97 212T and DSM 18 688 were relatively more thermophilic (temperature range for growth: 30-55 °C) and less halotolerant [growth range: 0-2.5 % (w/v) NaCl] than C. sporogenes and C. botulinum. They were negative for catalase, oxidase, urease and l-pyrrolidonyl-arylamidase and did not produce indole. The strains produced acid from d-glucose, maltose and trehalose, and hydrolysed gelatin, but did not hydrolyse aesculin. The end-products of growth included acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, isocaproic acid, phenylpropionic acid, 2-piperidinone, 2-pyrrolidinone and gas(es). The predominant fatty acids were C14 : 0, C16 : 0 and C18 : 1ω9c. The genomic DNA G+C content of strains IEH 97212T and DSM 18688 was 26.9 and 26.7 mol%, respectively. According to the digital DNA-DNA hybridization data, the relatedness of these strains was 98.4 %, while they showed only 35.7-36.0 % relatedness to C. sporogenes. Based on the results of this polyphasic study, these strains represent a novel species, for which the name Clostridium tepidum sp. nov. is proposed, with the type strain IEH 97212T (=NRRL B-65463T=DSM 104389T).

  2. Clostridium lavalense sp. nov., a glycopeptide-resistant species isolated from human faeces.

    PubMed

    Domingo, M-C; Huletsky, A; Boissinot, M; Hélie, M-C; Bernal, A; Bernard, K A; Grayson, M L; Picard, F J; Bergeron, M G

    2009-03-01

    Two vancomycin-resistant, strictly anaerobic, Gram-positive, rod-shaped, spore-forming organisms (strains CCRI-9842(T) and CCRI-9929) isolated from human faecal specimens in Québec, Canada, and Australia were characterized using phenotypic, biochemical and molecular taxonomic methods. Pairwise analysis of the 16S rRNA gene sequences showed that both strains were closely related to each other genetically (displaying 99.2 % sequence similarity) and represented a previously unknown subline within the Clostridium coccoides rRNA group of organisms (rRNA cluster XIVa of the genus Clostridium). Strains CCRI-9842(T) and CCRI-9929 used carbohydrates as fermentable substrates, producing acetic acid as the major product of glucose metabolism. The novel strains were most closely related to Clostridium asparagiforme, Clostridium bolteae and Clostridium clostridioforme, but morphological, biochemical and phylogenetic studies demonstrated that they represent a previously unidentified species of the genus Clostridium. This was confirmed by the unique cellular fatty acid composition of strains CCRI-9842(T) and CCRI-9929. Therefore, on the basis of data from the polyphasic taxonomic analysis, it is proposed that strains CCRI-9842(T) and CCRI-9929 represent a novel species of the genus Clostridium, for which the name Clostridium lavalense sp. nov. is proposed. The type strain is CCRI-9842(T) (=CCUG 54291(T)=JCM 14986(T)=NML 03-A-015(T)).

  3. Cryptic polyketide synthase genes in non-pathogenic Clostridium SPP.

    PubMed

    Behnken, Swantje; Hertweck, Christian

    2012-01-01

    Modular type I polyketide synthases (PKS) produce a vast array of bacterial metabolites with highly diverse biological functions. Notably, all known polyketides were isolated from aerobic bacteria, and yet no example has been reported for strict anaerobes. In this study we explored the diversity and distribution of PKS genes in the genus Clostridium. In addition to comparative genomic analyses combined with predictions of modular type I polyketide synthase (PKS) gene clusters in sequenced genomes of Clostridium spp., a representative selection of other species inhabiting a variety of ecological niches was investigated by PCR screening for PKS genes. Our data reveal that all studied pathogenic Clostridium spp. are devoid of putative PKS genes. In stark contrast, cryptic PKS genes are widespread in genomes of non-pathogenic Clostridium species. According to phylogenetic analyses, the Clostridium PKS genes have unusual and diverse origins. However, reverse transcription quantitative PCR demonstrates that these genes are silent under standard cultivation conditions, explaining why the related metabolites have been overlooked until now. This study presents clostridia as a putative source for novel bioactive polyketides.

  4. Cryptic Polyketide Synthase Genes in Non-Pathogenic Clostridium SPP

    PubMed Central

    Behnken, Swantje; Hertweck, Christian

    2012-01-01

    Modular type I polyketide synthases (PKS) produce a vast array of bacterial metabolites with highly diverse biological functions. Notably, all known polyketides were isolated from aerobic bacteria, and yet no example has been reported for strict anaerobes. In this study we explored the diversity and distribution of PKS genes in the genus Clostridium. In addition to comparative genomic analyses combined with predictions of modular type I polyketide synthase (PKS) gene clusters in sequenced genomes of Clostridium spp., a representative selection of other species inhabiting a variety of ecological niches was investigated by PCR screening for PKS genes. Our data reveal that all studied pathogenic Clostridium spp. are devoid of putative PKS genes. In stark contrast, cryptic PKS genes are widespread in genomes of non-pathogenic Clostridium species. According to phylogenetic analyses, the Clostridium PKS genes have unusual and diverse origins. However, reverse transcription quantitative PCR demonstrates that these genes are silent under standard cultivation conditions, explaining why the related metabolites have been overlooked until now. This study presents clostridia as a putative source for novel bioactive polyketides. PMID:22235310

  5. Characterization of a butanol-acetone-producing Clostridium strain and identification of its solventogenic genes.

    PubMed

    Chua, Teck Khiang; Liang, Da-Wei; Qi, Chao; Yang, Kun-Lin; He, Jianzhong

    2013-05-01

    A unique Clostridium species strain G117 was obtained in this study to be capable of producing dominant butanol from glucose. Butanol of 13.50 g/L was produced when culture G117 was fed with 60 g/L glucose, which is ~20% higher than previously reported butanol production by wild-type Clostridium acetobutylicum ATCC 824 under similar conditions. Strain G117 also distinguishes itself by generating negligible amount of ethanol, but producing butanol and acetone as biosolvent end-products. A butanol dehydrogenase gene (bdh gene) was identified in strain G117, which demonstrated a ~200-fold increase in transcription level measured by quantitative real-time PCR after 10h of culture growth. The high transcription suggests that this bdh gene could be a putative gene involved in butanol production. In all, Clostridium sp. strain G117 serves as a potential candidate for industrial biobutanol production while the absence of ethanol ensures an economic-efficient separation and purification of butanol.

  6. Felled oil palm trunk as a renewable source for biobutanol production by Clostridium spp.

    PubMed

    Komonkiat, Itsara; Cheirsilp, Benjamas

    2013-10-01

    This study aimed to convert felled oil palm trunk to biobutanol by Clostridium spp. For efficient utilization of oil palm trunk, it was separated into sap and trunk fiber. The sap was used directly while the trunk fiber was hydrolyzed to fermentable sugars before use. Among five clostridia strains screened, Clostridium acetobutylicum DSM 1731 was the most suitable strain for butanol production from the sap without any supplementation of nutrients. It produced the highest amount of butanol (14.4 g/L) from the sap (sugar concentration of 50 g/L) with butanol yield of 0.35 g/g. When hydrolysate from the trunk fiber was used as an alternative carbon source (sugar concentration of 30 g/L), of the strains tested Clostridium beijerinckii TISTR 1461 produced the highest amount of butanol (10.0 g/L) with butanol yield of 0.41 g/g. The results presented herein suggest that oil palm trunk is a promising renewable substrate for biobutanol production. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Metabolic engineering of Clostridium tyrobutyricum for n-butanol production: effects of CoA transferase.

    PubMed

    Yu, Le; Zhao, Jingbo; Xu, Mengmeng; Dong, Jie; Varghese, Saju; Yu, Mingrui; Tang, I-Ching; Yang, Shang-Tian

    2015-06-01

    The overexpression of CoA transferase (ctfAB), which catalyzes the reaction: acetate/butyrate + acetoacetyl-CoA → acetyl/butyryl-CoA + acetoacetate, was studied for its effects on acid reassimilation and butanol biosynthesis in Clostridium tyrobutyricum (Δack, adhE2). The plasmid pMTL007 was used to co-express adhE2 and ctfAB from Clostridium acetobutylicum ATCC 824. In addition, the sol operon containing ctfAB, adc (acetoacetate decarboxylase), and ald (aldehyde dehydrogenase) was also cloned from Clostridium beijerinckii NCIMB 8052 and expressed in C. tyrobutyricum (Δack, adhE2). Mutants expressing these genes were evaluated for their ability to produce butanol from glucose in batch fermentations at pH 5.0 and 6.0. Compared to C. tyrobutyricum (Δack, adhE2) without expressing ctfAB, all mutants with ctfAB overexpression produced more butanol, with butanol yield increased to 0.22 - 0.26 g/g (vs. 0.10 - 0.13 g/g) and productivity to 0.35 g/l h (vs. 0.13 g/l h) because of the reduced acetate and butyrate production. The expression of ctfAB also resulted in acetone production from acetoacetate through a non-enzymatic decarboxylation.

  8. Regulation of toxin and bacteriocin gene expression in Clostridium by interchangeable RNA polymerase sigma factors.

    PubMed

    Dupuy, Bruno; Raffestin, Stéphanie; Matamouros, Susana; Mani, Nagraj; Popoff, Michel R; Sonenshein, Abraham L

    2006-05-01

    The production of major extracellular toxins by pathogenic strains of Clostridium botulinum, Clostridium tetani and Clostridium difficile, and a bacteriocin by Clostridium perfringens is dependent on a related group of RNA polymerase sigma-factors. These sigma-factors (BotR, TetR, TcdR and UviA) were shown to be sufficiently similar that they could substitute for one another in in vitro DNA binding and run-off transcription experiments. In cells, however, the sigma-factors fell into two subclasses. BotR and TetR were able to direct transcription of their target genes in a fully reciprocal manner. Similarly, UviA and TcdR were fully interchangeable. Neither BotR nor TetR could substitute for UviA or TcdR, however, and neither UviA nor TcdR could direct transcription of the natural targets of BotR or TetR. The extent of functional interchangeability of the sigma-factors was attributed to the strong conservation of their subregion 4.2 sequences and the conserved -35 sequences of their target promoters, while restrictions on interchangeability were attributed to variations in their subregion 2.4 sequences and the target site -10 sequences. The four sigma-factors have been assigned to group 5 of the sigma(70) family and seem to have arisen from a common ancestral protein that may have co-evolved with the genes whose transcription they direct. A fifth Clostridiumsigma-factor, sigma(Y) of Clostridium acetobutylicum, resembles the TcdR family, but was not functionally interchangeable with members of this family.

  9. Clostridium kogasensis sp. nov., a novel member of the genus Clostridium, isolated from soil under a corroded gas pipeline.

    PubMed

    Shin, Yeseul; Kang, Seok-Seong; Paek, Jayoung; Jin, Tae Eun; Song, Hong Seok; Kim, Hongik; Park, Hee-Moon; Chang, Young-Hyo

    2016-06-01

    Two bacterial strains, YHK0403(T) and YHK0508, isolated from soil under a corroded gas pipe line, were revealed as Gram-negative, obligately anaerobic, spore-forming and mesophilic bacteria. The cells were rod-shaped and motile by means of peritrichous flagella. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolates were members of the genus Clostridium and were the most closely related to Clostridium scatologenes KCTC 5588(T) (95.8% sequence similarity), followed by Clostridium magnum KCTC 15177(T) (95.8%), Clostridium drakei KCTC 5440(T) (95.7%) and Clostridium tyrobutyricum KCTC 5387(T) (94.9%). The G + C contents of the isolates were 29.6 mol%. Peptidoglycan in the cell wall was of the A1γ type with meso-diaminopimelic acid. The major polar lipid was diphosphatidylglycerol (DPG), and other minor lipids were revealed as phosphatidylglycerol (PG), phosphatidylethanolamine (PE), two unknown glycolipids (GL1 and GL2), an unknown aminoglycolipid (NGL), two unknown aminophospholipids (PN1 and PN2) and four unknown phospholipids (PL1 to PL4). Predominant fatty acids were C16:0 and C16:1cis9 DMA. The major end products from glucose fermentation were identified as butyrate (12.2 mmol) and acetate (9.8 mmol). Collectively, the results from a wide range of phenotypic tests, chemotaxonomic tests, and phylogenetic analysis indicated that the two isolates represent novel species of the genus Clostridium, for which the name Clostridium kogasensis sp. nov. (type strain, YHK0403(T) = KCTC 15258(T) = JCM 18719(T)) is proposed. Copyright © 2016. Published by Elsevier Ltd.

  10. An active principle of Nigella sativa L., thymoquinone, showing significant antimicrobial activity against anaerobic bacteria

    PubMed Central

    Randhawa, Mohammad Akram; Alenazy, Awwad Khalaf; Alrowaili, Majed Gorayan; Basha, Jamith

    2017-01-01

    Aim/Background: Thymoquinone (TQ) is the major active principle of Nigella sativa seed (black seed) and is known to control many fungi, bacteria, and some viruses. However, the activity of TQ against anaerobic bacteria is not well demonstrated. Anaerobic bacteria can cause severe infections, including diarrhea, aspiration pneumonia, and brain abscess, particularly in immunodeficient individuals. The present study aimed to investigate the in vitro antimicrobial activity of TQ against some anaerobic pathogens in comparison to metronidazole. Methods: Standard, ATCC, strains of four anaerobic bacteria (Clostridium difficile, Clostridium perfringens, Bacteroides fragilis, and Bacteroides thetaiotaomicron), were initially isolated on special Brucella agar base (with hemin and vitamin K). Then, minimum inhibitory concentrations (MICs) of TQ and metronidazole were determined against these anaerobes when grown in Brucella agar, using serial agar dilution method according to the recommended guidelines for anaerobic organisms instructed by the Clinical and Laboratory Standards Institute. Results: TQ showed a significant antimicrobial activity against anaerobic bacteria although much weaker than metronidazole. MICs of TQ and metronidazole against various anaerobic human pathogens tested were found to be between 10-160 mg/L and 0.19-6.25 mg/L, respectively. Conclusions: TQ controlled the anaerobic human pathogenic bacteria, which supports the use of N. sativa in the treatment of diarrhea in folk medicine. Further investigations are in need for determination of the synergistic effect of TQ in combination with metronidazole and the activity of derivatives of TQ against anaerobic infections. PMID:28163966

  11. Near complete genome sequence of Clostridium paradoxum strain JW-YL-7

    SciTech Connect

    Lancaster, Andrew; Utturkar, Sagar M.; Poole, Farris; Klingeman, Dawn Marie; Elias, Dwayne A.; Adams, Michael W. W.; Brown, Steven D.

    2016-05-05

    Clostridium paradoxum strain JW-YL-7 is a moderately thermophilic anaerobic alkaliphile isolated from the municipal sewage treatment plant in Athens, GA. We report the near-complete genome sequence of C. paradoxum strain JW-YL-7 obtained by using PacBio DNA sequencing and Pilon for sequence assembly refinement with Illumina data.

  12. Near complete genome sequence of Clostridium paradoxum strain JW-YL-7

    DOE PAGES

    Lancaster, Andrew; Utturkar, Sagar M.; Poole, Farris; ...

    2016-05-05

    Clostridium paradoxum strain JW-YL-7 is a moderately thermophilic anaerobic alkaliphile isolated from the municipal sewage treatment plant in Athens, GA. We report the near-complete genome sequence of C. paradoxum strain JW-YL-7 obtained by using PacBio DNA sequencing and Pilon for sequence assembly refinement with Illumina data.

  13. Complete Genome Sequence of the Cellulolytic Thermophile Clostridium thermocellum DSM1313

    SciTech Connect

    Feinberg, Lawrence F; Foden, Justine; Barrett, Trisha; Davenport, Karen W.; Bruce, David; Detter, J. Chris; Tapia, Roxanne; Han, Cliff; Lapidus, Alla L.; Lucas, Susan; Cheng, Jan-Fang; Pitluck, Sam; Woyke, Tanja; Ivanova, N; Mikhailova, Natalia; Land, Miriam L; Hauser, Loren John; Argyros, Aaron; Goodwin, Lynne A.; Hogsett, David; Caiazza, Nicky

    2011-01-01

    Clostridium thermocellum DSM1313 is a thermophilic, anaerobic bacterium with some of the highest rates of cellulose hydrolysis reported. The complete genome sequence reveals a suite of carbohydrate-active enzymes and demonstrates a level of diversity at the species level distinguishing it from the type strain ATCC27405.

  14. Draft genome sequences of clostridium strains native to Colombia with the potential to produce solvents.

    PubMed

    Rosas-Morales, Juan Pablo; Perez-Mancilla, Ximena; López-Kleine, Liliana; Montoya Castaño, Dolly; Riaño-Pachón, Diego Mauricio

    2015-05-21

    Genomes from four Clostridium sp. strains considered to be mesophilic anaerobic bacteria, isolated from crop soil in Colombia, with a strong potential to produce alcohols like 1,3-propanediol, were analyzed. We present the draft genome of these strains, which will be useful for developing genetic engineering strategies.

  15. Characterization of anti-Clostridium perfringens bacteriophages isolated on poultry farms in Central Russia

    USDA-ARS?s Scientific Manuscript database

    Clostridium perfringens is a main food-borne pathogen causing human diseases. Besides, these Gram-positive anaerobes are responsible for the development of avian necrotic enteritidis, the wide-spread disease in countries engaged in the poultry breeding. For minimization followed by complete exclu...

  16. Draft Genome Sequences of Clostridium Strains Native to Colombia with the Potential To Produce Solvents

    PubMed Central

    Rosas-Morales, Juan Pablo; Perez-Mancilla, Ximena; López-Kleine, Liliana

    2015-01-01

    Genomes from four Clostridium sp. strains considered to be mesophilic anaerobic bacteria, isolated from crop soil in Colombia, with a strong potential to produce alcohols like 1,3-propanediol, were analyzed. We present the draft genome of these strains, which will be useful for developing genetic engineering strategies. PMID:25999575

  17. First Insights into the Draft Genome of Clostridium colicanis DSM 13634, Isolated from Canine Feces

    PubMed Central

    Poehlein, Anja; Schilling, Tobias; Bhaskar Sathya Narayanan, Udhaya

    2016-01-01

    Clostridium colicanis DSM 13634 is a strictly anaerobic, rod-shaped, and spore-forming bacterium. It produces acids from common sugars such as glucose and fructose. The draft genome consists of one chromosome (2.6 Mbp) and contains 2,159 predicted protein-encoding genes. PMID:27198021

  18. Draft Genome Sequence of the Cellulolytic and Xylanolytic Thermophile Clostridium clariflavum Strain 4-2a

    PubMed Central

    Rooney, Elise A.; Rowe, Kenneth T.; Guseva, Anna; Huntemann, Marcel; Han, James K.; Chen, Amy; Kyrpides, Nikos C.; Mavromatis, Konstantinos; Markowitz, Victor M.; Palaniappan, Krishna; Ivanova, Natalia; Pati, Amrita; Liolios, Konstantinos; Nordberg, Henrik P.; Cantor, Michael N.; Hua, Susan X.; Shapiro, Nicole; Woyke, Tanja; Lynd, Lee R.

    2015-01-01

    Clostridium clariflavum strain 4-2a, a novel strain isolated from a thermophilic biocompost pile, has demonstrated an extensive capability to utilize both cellulose and hemicellulose under thermophilic anaerobic conditions. Here, we report the draft genome of this strain. PMID:26205857

  19. Genome Sequence of the Solvent-Producing Bacterium Clostridium carboxidivorans Strain P7T▿

    PubMed Central

    Paul, Debarati; Austin, Frank W.; Arick, Tony; Bridges, Susan M.; Burgess, Shane C.; Dandass, Yoginder S.; Lawrence, Mark L.

    2010-01-01

    Clostridium carboxidivorans strain P7T is a strictly anaerobic acetogenic bacterium that produces acetate, ethanol, butanol, and butyrate. The C. carboxidivorans genome contains all the genes for the carbonyl branch of the Wood-Ljungdahl pathway for CO2 fixation, and it encodes enzymes for conversion of acetyl coenzyme A into butanol and butyrate. PMID:20729368

  20. Molecular Characterization of Podoviridae Bacteriophages Virulent for Clostridium perfringens and Comparison of Their Predicted Lytic Proteins

    USDA-ARS?s Scientific Manuscript database

    Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that plays a significant role in human food-borne disease as well as non-food-borne human, animal and poultry diseases. There has been a resurgent interest in the use of bacteriophages or their gene products to control ba...

  1. Fatal clostridial enterotoxemia (Clostridium glycolicum) in an ornate Nile monitor (Varanus ornatus).

    PubMed

    Bertelsen, Mads F; Weese, J Scott

    2006-03-01

    Enterotoxemia caused by Clostridium glycolicum was identified as the cause of circulatory collapse and death in a female, 3-yr-old, captive-bred ornate Nile monitor (Varanus ornatus). Anaerobic culture of fecal samples from 12 other monitor lizards from the collection failed to demonstrate the presence of this pathogen.

  2. Wood adhesives prepared from lucerne fiber fermentation residues of Ruminococcus albus and Clostridium thermocellum

    Treesearch

    P. J. Weimer; R. G. Koegel; Linda F. Lorenz; Charles R. Frihart; William R. Kenealy

    2005-01-01

    Fermentation residues (consisting of incompletely fermented fiber, adherent bacterial cells, and a glycocalyx material that enhanced bacterial adherence) were obtained by growing the anaerobic cellulolytic bacteria Ruminococcus albus 7 or Clostridium thermocellum ATCC 27405 on a fibrous fraction derived from lucerne (Medicago sativa L.). The dried residue was able to...

  3. Endocarditis caused by anaerobic bacteria.

    PubMed

    Kestler, M; Muñoz, P; Marín, M; Goenaga, M A; Idígoras Viedma, P; de Alarcón, A; Lepe, J A; Sousa Regueiro, D; Bravo-Ferrer, J M; Pajarón, M; Costas, C; García-López, M V; Hidalgo-Tenorio, C; Moreno, M; Bouza, E

    2017-04-05

    Infective endocarditis (IE) caused by anaerobic bacteria is a rare and poorly characterized disease. Most data reported in the literature are from case reports [1-3]. Therefore, we assessed the situation of anaerobic IE (AIE) in Spain using the database of the Spanish Collaboration on Endocarditis (GAMES). We performed a prospective study from 2008 to 2016 in 26 Spanish centers. We included 2491 consecutive cases of definite IE (Duke criteria). Anaerobic bacteria caused 22 cases (0.9%) of definite IE. Median age was 66 years (IQR, 56-73), and 19 (86.4%) patients were men. Most patients (14 [63.6%]) had prosthetic valve IE and all episodes were left-sided: aortic valves, 12 (54.5%); and mitral valves, 8 (36.4%). The most common pathogens were Propionibacterium acnes (14 [63.6%]), Lactobacillus spp (3 [13.63%]), and Clostridium spp. (2 [9.0%]), and the infection was mainly odontogenic. Fifteen of the 22 patients (68.2%) underwent cardiac surgery. Mortality was 18.2% during admission and 5.5% after 1 year of follow-up. When patients with AIE were compared with the rest of the cohort, we found that although those with AIE had a similar age and Charlson comorbidity index, they were more likely to have community-acquired IE (86.4% vs. 60.9%, p = 0.01), have undergone cardiac surgery (68.2% vs 48.7% p = 0.06), and have had lower mortality rates during admission (18.2% vs. 27.3%). IE due to anaerobic bacteria is an uncommon disease that affects mainly prosthetic valves and frequently requires surgery. Otherwise, there are no major differences between AIE and IE caused by other microorganisms. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Microbiology and physiology of anaerobic fermentations of cellulose. Progress report, September 1982-March 1983

    SciTech Connect

    Peck, H.D. Jr.; Ljungdahl, L.G.

    1983-01-01

    Research progress for the period September 1982-March 1983 is reported. The cellulose enzyme system of the anaerobic thermophile Clostridium thermocellum is being studied. Mutants have been obtained from thermoanaerobacter ethanolicus which produce higher amounts of ethanol than does the wild type. Clostridium thermoautotrophicum has been studied with respect to the pathway of acetate synthesis from CO/sub 2/. Progress has been achieved on properties of the NADP-dependent formate dehydrogenase and the CO dehydrogenase of Clostridium thermoaceticum. The CO dehydrogenase of Acetobacterium woodii has been isolated. Research has continued on the bioenergetics of dissimilatory sulfate reduction in sulfate reducing and methanogenic bacteria.

  5. Occurrence of anaerobic bacteria in diseases of the dog and cat.

    PubMed

    Berg, J N; Fales, W H; Scanlan, C M

    1979-06-01

    A survey for anaerobic bacteria was conducted in 314 clinical specimens from dogs and cats. A total of 187 anaerobic isolates in pure and mixed culture were isolated from 111 of the specimens that contained anaerobic bacteria. Common isolated included Actinomyces (9.1%), Clostridium perfringens (19.3%), other Clostridium spp (11.2%), Peptostreptococcus anaerobius (7.5%), Bacteroides melaninogenicus (13.4%), other Bacteroides spp (17.6%), and Fusobacterium necrophorum (5.3%). Anaerobic bacteria were involved in serious lesions that often were life threatening to the animals. Antibiotic susceptibility data indicated that the lincomycin family, the penicillin family, chloramphenicol, and cephaloridine are preferred drugs for treatment of anaerobic infections. Data from the survey were used in formulation of a table to aid practitioners in clinical diagnosis of disease caused by anaerobes. Clostridium perfringens was isolated in large numbers from five of six dogs with a clinical diagnosis of canine hemorrhagic gastroenteritis and from one cat with hemorrhagic diarrhea. Experimental infections were induced in rats, using caine feces as inoculum. Induced lesions contained aerobic and anaerobic bacteria similar to those bacteria isolated in the clinical survey, indicating that feces may serve as a major source of these bacteria in clinical infections of the dog.

  6. Clostridium chauvoei in hens.

    PubMed

    Prukner-Radovcic, E; Milakovic-Novak, L; Ivesa-Petricevic, S; Grgic, N

    1995-03-01

    The bacterium Clostridium chauvoei causes disease in certain animals, most frequently in cattle and sheep. It occurs rarely in pigs, while equines and poultry appear to be resistant to infection. Two cases are presented in which C. chauvoei was isolated from disease of complex aetiology in hens. In Case I, 15-week-old light hybrid chickens were affected with chronic respiratory disease, coccidiosis, ascariasis and inflammation of the skin on the head, with necrosis of the comb. Growth was uneven and mortality reached 24%. Clostridium chauvoei was isolated from two of three combs examined. In Case II a flock of broiler breeders aged 11 weeks developed coccidiosis and, owing to disease or death, 60% were excluded from production. Clostridium chauvoei was isolated from all of 10 livers examined. These results demonstrate that C. chauvoei can infect chickens and that its possible role as a pathogen under certain circumstances should be further investigated.

  7. Formation of involatile methylantimony species by Clostridium spp.

    PubMed

    Smith, L M; Craig, P J; Jenkins, R O

    2002-04-01

    Trimethylantimony was detected by gas chromatography-mass spectrometry (GC-MS) in the headspace of a soil enrichment culture designed to promote growth of clostridia. Clostridial isolates from the soil enrichment culture were shown to biomethylate inorganic antimony in monseptic culture, using hydride generation-gas chromatographyatomic absorption spectrometry (HG-GC-AAS). GC-MS profiles of headspace gases from soil enrichment cultures shown to generate trimethylantimony, were used to select characterised Clostridium spp for assessment of antimony biomethylation capability. Involatile methylantimony species (up to 21 microg Sb dm(-3)) were detected by HG-GC-AAS in the medium of monoseptic cultures of C. acetobutylicum, C. butyricum and C. cochlearium. The relative quantities of involatile mono-, di- and trimethylantimony species produced over the course of a 28-day cultivation period is consistent with trimethylantimony oxide being a final product of antimony biomethylation by these bacteria, with mono- and dimethylantimony species appearing transiently in the cultures as intermediates of an antimony biomethylation pathway. Clostridia may be the principal agents of antimony biomethylation in methanogenic environments and could give rise to methylated forms of antimony in both the aqueous and gaseous phases.

  8. Clostridium vulturis sp. nov., isolated from the intestine of the cinereous vulture (Aegypius monachus).

    PubMed

    Paek, Jayoung; Lee, Mi-Hwa; Kim, Byung-Chun; Sang, Byoung-In; Paek, Woon Kee; Jin, Tae-Eun; Shin, Yeseul; Park, In-Soon; Chang, Young-Hyo

    2014-09-01

    A Gram-stain positive, strict anaerobe, spore-forming, motile rod-shaped bacterial strain with peritrichous flagella, designated YMB-57(T), was isolated from the intestine of a cinereous vulture (Aegypius monachus) in Korea. Strain YMB-57(T) was found to show optimal growth at 37 °C, pH 7.5 and 1.0 % (w/v) NaCl. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain YMB-57(T) belongs to the genus Clostridium and is most closely related to the type strains of Clostridium subterminale (96.9 % sequence similarity), Clostridium thiosulfatireducens (96.7 %) and Clostridium sulfidigenes (96.6 %). The main fermentation end-products identified following growth in PYG medium were acetate, butyrate, ethanol, propanol, carbon dioxide and hydrogen. Peptone was converted to ethanol, and butanol, whereas glucose was fermented to ethanol. The major cellular fatty acids were identified as C16:0, C18:1 ω9c, and C18:1 ω9c DMA and the DNA G+C content was determined to be 34.0 mol%. Phenotypic and phylogenetic differences indicate that strain YMB-57(T) is distinct from other Clostridium species. It is proposed that strain YMB-57(T) be classified as the type strain of a novel species of the genus Clostridium, with the name Clostridium vulturis sp. nov. The type strain is YMB-57(T) (=KCTC 15114(T) = JCM 17998(T)).

  9. Anaerobic Digestion.

    PubMed

    Liebetrau, Jan; Sträuber, Heike; Kretzschmar, Jörg; Denysenko, Velina; Nelles, Michael

    2017-04-09

    The term anaerobic digestion usually refers to the microbial conversion of organic material to biogas, which mainly consists of methane and carbon dioxide. The technical application of the naturally-occurring process is used to provide a renewable energy carrier and - as the substrate is often waste material - to reduce the organic matter content of the substrate prior to disposal.Applications can be found in sewage sludge treatment, the treatment of industrial and municipal solid wastes and wastewaters (including landfill gas utilization), and the conversion of agricultural residues and energy crops.For biorefinery concepts, the anaerobic digestion (AD) process is, on the one hand, an option to treat organic residues from other production processes. Concomitant effects are the reduction of organic carbon within the treated substance, the conversion of nitrogen and sulfur components, and the production of an energy-rich gas - the biogas. On the other hand, the multistep conversion of complex organic material offers the possibility of interrupting the conversion chain and locking out intermediates for utilization as basic material within the chemical industry.

  10. Bacterial ecology of abattoir wastewater treated by an anaerobic digestor

    PubMed Central

    Jabari, Linda; Gannoun, Hana; Khelifi, Eltaief; Cayol, Jean-Luc; Godon, Jean-Jacques; Hamdi, Moktar; Fardeau, Marie-Laure

    2016-01-01

    Wastewater from an anaerobic treatment plant at a slaughterhouse was analysed to determine the bacterial biodiversity present. Molecular analysis of the anaerobic sludge obtained from the treatment plant showed significant diversity, as 27 different phyla were identified. Firmicutes, Proteobacteria, Bacteroidetes, Thermotogae, Euryarchaeota (methanogens), and msbl6 (candidate division) were the dominant phyla of the anaerobic treatment plant and represented 21.7%, 18.5%, 11.5%, 9.4%, 8.9%, and 8.8% of the total bacteria identified, respectively. The dominant bacteria isolated were Clostridium, Bacteroides, Desulfobulbus, Desulfomicrobium, Desulfovibrio and Desulfotomaculum. Our results revealed the presence of new species, genera and families of microorganisms. The most interesting strains were characterised. Three new bacteria involved in anaerobic digestion of abattoir wastewater were published. PMID:26887229

  11. Bacteriophages of Clostridium perfringens

    USDA-ARS?s Scientific Manuscript database

    The specific aims of the book chapter are to: (1) Briefly review the nomenclature of bacteriophages and how these agents are classified. (2) Discuss the problems associated with addition/removal of antibiotics in commercial animal feeds. (3) Provide a brief overview of Clostridium perfringens biolog...

  12. Clostridium tetani bacteraemia.

    PubMed

    Hallit, Rabih Riad; Afridi, Muhammad; Sison, Raymund; Salem, Elie; Boghossian, Jack; Slim, Jihad

    2013-01-01

    Tetanus is a neuromuscular disease in which Clostridium tetani exotoxin (tetanospasmin) produces muscle spasms, incapacitating its host. To our knowledge, C. tetani bacteraemia has never been reported in the literature. The ideal management of this entity remains unresolved given that there is no literature to guide the therapy.

  13. Anaerobic thermophiles.

    PubMed

    Canganella, Francesco; Wiegel, Juergen

    2014-02-26

    The term "extremophile" was introduced to describe any organism capable of living and growing under extreme conditions. With the further development of studies on microbial ecology and taxonomy, a variety of "extreme" environments have been found and an increasing number of extremophiles are being described. Extremophiles have also been investigated as far as regarding the search for life on other planets and even evaluating the hypothesis that life on Earth originally came from space. The first extreme environments to be largely investigated were those characterized by elevated temperatures. The naturally "hot environments" on Earth range from solar heated surface soils and water with temperatures up to 65 °C, subterranean sites such as oil reserves and terrestrial geothermal with temperatures ranging from slightly above ambient to above 100 °C, to submarine hydrothermal systems with temperatures exceeding 300 °C. There are also human-made environments with elevated temperatures such as compost piles, slag heaps, industrial processes and water heaters. Thermophilic anaerobic microorganisms have been known for a long time, but scientists have often resisted the belief that some organisms do not only survive at high temperatures, but actually thrive under those hot conditions. They are perhaps one of the most interesting varieties of extremophilic organisms. These microorganisms can thrive at temperatures over 50 °C and, based on their optimal temperature, anaerobic thermophiles can be subdivided into three main groups: thermophiles with an optimal temperature between 50 °C and 64 °C and a maximum at 70 °C, extreme thermophiles with an optimal temperature between 65 °C and 80 °C, and finally hyperthermophiles with an optimal temperature above 80 °C and a maximum above 90 °C. The finding of novel extremely thermophilic and hyperthermophilic anaerobic bacteria in recent years, and the fact that a large fraction of them belong to the Archaea has definitely

  14. Anaerobic Thermophiles

    PubMed Central

    Canganella, Francesco; Wiegel, Juergen

    2014-01-01

    The term “extremophile” was introduced to describe any organism capable of living and growing under extreme conditions. With the further development of studies on microbial ecology and taxonomy, a variety of “extreme” environments have been found and an increasing number of extremophiles are being described. Extremophiles have also been investigated as far as regarding the search for life on other planets and even evaluating the hypothesis that life on Earth originally came from space. The first extreme environments to be largely investigated were those characterized by elevated temperatures. The naturally “hot environments” on Earth range from solar heated surface soils and water with temperatures up to 65 °C, subterranean sites such as oil reserves and terrestrial geothermal with temperatures ranging from slightly above ambient to above 100 °C, to submarine hydrothermal systems with temperatures exceeding 300 °C. There are also human-made environments with elevated temperatures such as compost piles, slag heaps, industrial processes and water heaters. Thermophilic anaerobic microorganisms have been known for a long time, but scientists have often resisted the belief that some organisms do not only survive at high temperatures, but actually thrive under those hot conditions. They are perhaps one of the most interesting varieties of extremophilic organisms. These microorganisms can thrive at temperatures over 50 °C and, based on their optimal temperature, anaerobic thermophiles can be subdivided into three main groups: thermophiles with an optimal temperature between 50 °C and 64 °C and a maximum at 70 °C, extreme thermophiles with an optimal temperature between 65 °C and 80 °C, and finally hyperthermophiles with an optimal temperature above 80 °C and a maximum above 90 °C. The finding of novel extremely thermophilic and hyperthermophilic anaerobic bacteria in recent years, and the fact that a large fraction of them belong to the Archaea has

  15. Toxin Plasmids of Clostridium perfringens

    PubMed Central

    Li, Jihong; Adams, Vicki; Bannam, Trudi L.; Miyamoto, Kazuaki; Garcia, Jorge P.; Uzal, Francisco A.; Rood, Julian I.

    2013-01-01

    SUMMARY In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract. PMID:23699255

  16. Treatment of RDX & HMX Plumes Using Mulch Biowalls

    DTIC Science & Technology

    2008-08-01

    by Clostridium acetobutylicum cell-free extract. Chemosphere, 2002. 50: p. 665-671. August 2008 ESTCP ER-0426 64 Final Technical...ER-0426 61 Final Technical Report 7. REFERENCES 1. Ahmad, F. and J.B. Hughes, Anaerobic Transformation of TNT by Clostridium , In...K.M. and R.L. Crawford, Characterization of Clostridium bifermentans and its biotransformation of 2,4,6-trinitrotoluene and 1,3,5-triaza-1,3,5

  17. Formic Acid Triggers the “Acid Crash” of Acetone-Butanol-Ethanol Fermentation by Clostridium acetobutylicum▿

    PubMed Central

    Wang, Shaohua; Zhang, Yanping; Dong, Hongjun; Mao, Shaoming; Zhu, Yan; Wang, Runjiang; Luan, Guodong; Li, Yin

    2011-01-01

    Solvent production by Clostridium acetobutylicum collapses when cells are grown in pH-uncontrolled glucose medium, the so-called “acid crash” phenomenon. It is generally accepted that the fast accumulation of acetic acid and butyric acid triggers the acid crash. We found that addition of 1 mM formic acid into corn mash medium could trigger acid crash, suggesting that formic acid might be related to acid crash. When it was grown in pH-uncontrolled glucose medium or glucose-rich medium, C. acetobutylicum DSM 1731 containing the empty plasmid pIMP1 failed to produce solvents and was found to accumulate 0.5 to 1.24 mM formic acid intracellularly. In contrast, recombinant strain DSM 1731 with formate dehydrogenase activity did not accumulate formic acid intracellularly and could produce solvent as usual. We therefore conclude that the accumulation of formic acid, rather than acetic acid and butyric acid, is responsible for the acid crash of acetone-butanol-ethanol fermentation. PMID:21216898

  18. Novel and neglected issues of acetone-butanol-ethanol (ABE) fermentation by clostridia: Clostridium metabolic diversity, tools for process mapping and continuous fermentation systems.

    PubMed

    Patakova, Petra; Linhova, Michaela; Rychtera, Mojmir; Paulova, Leona; Melzoch, Karel

    2013-01-01

    This review emphasises the fact that studies of acetone-butanol-ethanol (ABE) fermentation by solventogenic clostridia cannot be limited to research on the strain Clostridium acetobutylicum ATCC 824. Various 1-butanol producing species of the genus Clostridium, which differ in their patterns of product formation and abilities to ferment particular carbohydrates or glycerol, are described. Special attention is devoted to species and strains that do not produce acetone naturally and to the utilisation of lactose, inulin, glycerol and mixtures of pentose and hexose carbohydrates. Furthermore, process-mapping tools based on different principles, including flow cytometry, DNA microarray analysis, mass spectrometry, Raman microscopy, FT-IR spectroscopy and anisotropy of electrical polarisability, which might facilitate fermentation control and a deeper understanding of ABE fermentation, are introduced. At present, the methods with the greatest potential are flow cytometry and transcriptome analysis. Flow cytometry can be used to visualise and capture cells within clostridial populations as they progress through the normal cell cycle, in which symmetric and asymmetric cell division phases alternate. Cell viability of a population of Clostridium pasteurianum NRRL B-598 was determined by flow cytometry. Transcriptome analysis has been used in various studies including the detection of genes expressed in solventogenic phase, at sporulation, in the stress response, to compare expression patterns of different strains or parent and mutant strains, for studies of catabolite repression, and for the detection of genes involved in the transport and metabolism of 11 different carbohydrates. Interestingly, the results of transcriptome analysis also challenge our earlier understanding of the role of the Spo0A regulator in initiation of solventogenesis in C. acetobutylicum ATCC 824. Lastly, the review describes other significant recent discoveries, including the deleterious effects of

  19. Cultivation of Anaerobic and Facultatively Anaerobic Bacteria from Spacecraft-Associated Clean Rooms▿

    PubMed Central

    Stieglmeier, Michaela; Wirth, Reinhard; Kminek, Gerhard; Moissl-Eichinger, Christine

    2009-01-01

    In the course of this biodiversity study, the cultivable microbial community of European spacecraft-associated clean rooms and the Herschel Space Observatory located therein were analyzed during routine assembly operations. Here, we focused on microorganisms capable of growing without oxygen. Anaerobes play a significant role in planetary protection considerations since extraterrestrial environments like Mars probably do not provide enough oxygen for fully aerobic microbial growth. A broad assortment of anaerobic media was used in our cultivation strategies, which focused on microorganisms with special metabolic skills. The majority of the isolated strains grew on anaerobic, complex, nutrient-rich media. Autotrophic microorganisms or microbes capable of fixing nitrogen were also cultivated. A broad range of facultatively anaerobic bacteria was detected during this study and also, for the first time, some strictly anaerobic bacteria (Clostridium and Propionibacterium) were isolated from spacecraft-associated clean rooms. The multiassay cultivation approach was the basis for the detection of several bacteria that had not been cultivated from these special environments before and also led to the discovery of two novel microbial species of Pseudomonas and Paenibacillus. PMID:19363082

  20. Toxigenicity of Clostridium histolyticum

    PubMed Central

    Nishida, Shoki; Imaizumi, Masaaki

    1966-01-01

    Nishida, Shoki (Kanazawa University, Kanazawa, Japan), and Masaaki Imaizumi. Toxigenicity of Clostridium histolyticum. J. Bacteriol. 91:477–483. 1966.—From 234 soil samples, 21 strains of Clostridium histolyticum of different levels of α-toxigenicity were isolated by a new method specially designed for the isolation of this species. The α-toxigenicity of freshly isolated strains and of stock strains was closely associated with the potentiality for sporulation, growth, and smooth-colony formation. The presence of sugars, particularly xylose and arabinose, was inhibitory for growth. A few controversies on the biological properties of this species seem to be due to disregard for the growth-inhibiting effects of these sugars. PMID:5935337

  1. Genome sequence of Clostridium sporogenes DSM 795(T), an amino acid-degrading, nontoxic surrogate of neurotoxin-producing Clostridium botulinum.

    PubMed

    Poehlein, Anja; Riegel, Karin; König, Sandra M; Leimbach, Andreas; Daniel, Rolf; Dürre, Peter

    2015-01-01

    Clostridium sporogenes DSM 795 is the type strain of the species Clostridium sporogenes, first described by Metchnikoff in 1908. It is a Gram-positive, rod-shaped, anaerobic bacterium isolated from human faeces and belongs to the proteolytic branch of clostridia. C. sporogenes attracts special interest because of its potential use in a bacterial therapy for certain cancer types. Genome sequencing and annotation revealed several gene clusters coding for proteins involved in anaerobic degradation of amino acids, such as glycine and betaine via Stickland reaction. Genome comparison showed that C. sporogenes is closely related to C. botulinum. The genome of C. sporogenes DSM 795 consists of a circular chromosome of 4.1 Mb with an overall GC content of 27.81 mol% harboring 3,744 protein-coding genes, and 80 RNAs.

  2. Degradation of natural and synthetic polyesters under anaerobic conditions.

    PubMed

    Abou-Zeid, D M; Müller, R J; Deckwer, W D

    2001-03-30

    Often, degradability under anaerobic conditions is desirable for plastics claimed to be biodegradable, e.g. in anaerobic biowaste treatment plants, landfills and in natural anaerobic sediments. The biodegradation of the natural polyesters poly(beta-hydroxybutyrate) (PHB), poly(beta-hydroxybutyrate-co-11.6%-beta-hydroxyvalerate) (PHBV) and the synthetic polyester poly(epsilon-caprolactone) (PCL) was studied in two anaerobic sludges and individual polyester degrading anaerobic strains were isolated, characterized and used for degradation experiments under controlled laboratory conditions. Incubation of PHB and PHBV films in two anaerobic sludges exhibited significant degradation in a time scale of 6-10 weeks monitored by weight loss and biogas formation. In contrast to aerobic conditions, PHB was degraded anaerobically more rapidly than the copolyester PHBV, when tested with either mixed cultures or a single strained isolate. PCL tends to degrade slower than the natural polyesters PHB and PHBV. Four PHB and PCL degrading isolates were taxonomically identified and are obviously new species belonging to the genus Clostridium group I. The depolymerizing enzyme systems of PHB and PCL degrading isolates are supposed to be different. Using one isolated strain in an optimized laboratory degradation test with PHB powder, the degradation time was drastically reduced compared to the degradation in sludges (2 days vs. 6-10 weeks).

  3. Butanol production from cane molasses by Clostridium saccharobutylicum DSM 13864: batch and semicontinuous fermentation.

    PubMed

    Ni, Ye; Wang, Yun; Sun, Zhihao

    2012-04-01

    Clostridium acetobutylicum strains used in most Chinese ABE (acetone-butanol-ethanol) plants favorably ferment starchy materials like corn, cassava, etc., rather than sugar materials. This is one major problem of ABE industry in China and significantly limits the exploitation of cheap waste sugar materials. In this work, cane molasses were utilized as substrate in ABE production by Clostridium saccharobutylicum DSM 13864. Under optimum conditions, total solvent of 19.80 g/L (13.40 g/L butanol) was reached after 72 h of fermentation in an Erlenmeyer flask. In a 5-L bioreactor, total solvent of 17.88 g/L was attained after 36 h of fermentation, and the productivity and yield were 0.50 g/L/h and 0.33 g ABE/g sugar consumption, respectively. To further enhance the productivity, a two-stage semicontinuous fermentation process was steadily operated for over 8 days (205 h, 26 cycles) with average productivity (stage II) of 1.05 g/L/h and cell concentration (stage I) of 7.43 OD(660), respectively. The average batch fermentation time (stage I and II) was reduced to 21-25 h with average solvent of 15.27 g/L. This study provides valuable process data for the development of industrial ABE fermentation process using cane molasses as substrate.

  4. Peptoniphilus methioninivorax sp. nov., A Novel Gram-Positive Anaerobic Coccus Isolated from Retail Ground Beef

    USDA-ARS?s Scientific Manuscript database

    Strain NRRL B-23883 was isolated from retail ground beef as part of a study on the genetic diversity of Clostridium perfringens. The strain was found to be a strictly anaerobic, gram-type positive coccus that was able to utilize peptone as a sole carbon source. Subsequent to sequencing the 16S rib...

  5. Phosphine generation by mixed- and monoseptic-cultures of anaerobic bacteria.

    PubMed

    Jenkins, R O; Morris, T A; Craig, P J; Ritchie, A W; Ostah, N

    2000-04-24

    A microbial basis for bioreductive generation of phosphine is proposed, which could account at least in part for the presence of this toxic gas in natural anaerobic environments and in sewage and landfill gases. Phosphine generation under anaerobic growth conditions was dependent upon both the culture inoculum source (animal faeces) and enrichment culture conditions. Phosphine was detected in headspace gases from mixed cultures under conditions promoting fermentative growth of mixed acid and butyric acid bacteria, either in the presence or absence of methane generation. Monoseptic cultures of certain mixed acid fermentors (Escherichia coli, Salmonella gallinarum, and Salmonella arizonae) and solvent fermentors (Clostridium sporogenes, Clostridium acetobutyricum and Clostridium cochliarium) also generated phosphine. Such fermentative bacteria participate in the multi-stage process of methanogenesis in nature. Generation of phosphine by these bacteria, rather than by methanoarchaea themselves, could explain the apparent correlation between methanogenesis and the formation of phosphine in nature.

  6. [Characteristics of Clostridium tetani and laboratory diagnosis of tetanus].

    PubMed

    Smietańska, Karolina; Rokosz-Chudziak, Natalia; Rastawicki, Waldemar

    2013-01-01

    The causative agent of tetanus is the obligate anaerobic bacterium--Clostridium tetani. These bacteria form endospores that are able to survive long periods of exposure to air and other adverse environmental conditions. Infection generally occurs through wound contamination. We can distinguish several forms of tetanus: generalized, local and neonatal. Diagnosis of tetanus is based primarily on the patient's clinical symptoms (muscle cramps, painful back muscle spasms, generalized contractions of the arcuate curvature of the body) as well as on microbiological diagnosis. This article is a brief review of C. tetani and diagnosis of infections caused by these organisms in humans.

  7. The prospect for vaccines to prevent Clostridium difficile infection.

    PubMed

    Ghose, Chandrabali; Kelly, Ciarán P

    2015-03-01

    Clostridium difficile is a spore-forming anaerobic gram-positive organism that is the leading cause of antibiotic-associated nosocomial infectious diarrhea in the Western world. This article describes the evolving epidemiology of C difficile infection (CDI) in the twenty-first century, evaluates the importance of vaccines against the disease, and defines the roles of both innate and adaptive host immune responses in CDI. The effects of passive immunotherapy and active vaccination against CDI in both humans and animals are also discussed.

  8. Molecular Characterization of Podoviral Bacteriophages Virulent for Clostridium perfringens and Their Comparison with Members of the Picovirinae

    USDA-ARS?s Scientific Manuscript database

    Clostridium perfringens is a Gram positive, spore-forming anaerobic bacterium that plays a significant role in human food-borne disease as well as non-food-borne human, animal, and poultry diseases. There has been a resurgent interest in the use of bacteriophages or their gene products to control b...

  9. Fatal myocarditis due to Clostridium novyi type B in a previously healthy woman: case report and literature review.

    PubMed

    Ma, Marek; Boyd, J Todd; Trinh, Hien T; Coombs, Jeremy W; Fermann, Gregory J

    2007-01-01

    Clostridium novyi is a Gram-positive anaerobe, which is commonly a pathogen of domestic and wild animals. Disease in humans typically presents as myonecrosis. C. novyi has not previously been reported as a cause of myocarditis. We report a fatal case with infection of the myocardium by C. novyi type B.

  10. Myonecrosis by Clostridium septicum in a dog, diagnosed by a new multiplex-PCR.

    PubMed

    Ribeiro, Márcio Garcia; Silva, Rodrigo Otávio Silveira; Pires, Prhiscylla Sadanã; Martinho, Anna Paula Vitirito; Lucas, Thays Mizuki; Teixeira, Ana Izabel Passarela; Paes, Antonio Carlos; Barros, Claudenice Batista; Lobato, Francisco Carlos Faria

    2012-10-01

    Clostridial myositis is an acute, generally fatal toxemia that is considered to be rare in pet animals. The present report describes an unusual canine clostridial myositis that was diagnosed by a new multiplex-PCR (mPCR) designed for simultaneous identification of Clostridium sordellii, Clostridium septicum, Clostridium perfringens type A, Clostridium chauvoei, and Clostridium novyi type A. A ten-month-old male Rottweiler dog, that had displayed lameness and swelling of the left limb for 12 h, was admitted to a veterinary hospital. The animal was weak, dyspneic and hyperthermic, and a clinical examination indicated the presence of gas and edema in the limb. Despite emergency treatment, the animal died in only a few minutes. Samples of muscular tissue from the necrotic area were aseptically collected and plated onto defibrinated sheep blood agar (5%) in anaerobic conditions. Colonies suggestive of Clostridium spp. were submitted to testing by multiplex-PCR. Impression smears of the tissues, visualized with Gram and also with panoptic stains, revealed long rod-shaped organisms, and specimens also tested positive using the fluorescent antibody technique (FAT). The FAT and mPCR tests enabled a diagnosis of C. septicum myonecrosis in the dog.

  11. REDUCTION OF 2,4,6-TRINITROTOLUENE BY CLOSTRIDIUM ACETOBUTYLICUM THROUGH HYDROXYLAMINO-NITROTOLUENE INTERMEDIATES. (R825513C006)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  12. Genetic and biochemical analysis of solvent formation in Clostridium acetobutylicum. Progress report, September 1, 1992--July 31, 1996

    SciTech Connect

    Bennett, G.N.; Rudolph, F.B.

    1997-01-01

    Several degenerate strains were isolated and characterized by sporulation, motility and growth properties. Cell appearance and colony morphology were also recorded. Enzymatic assays revealed reduced butyraldehyde dehydrogenase and Co-A transferase enzyme activities in the degenerates. DNA analysis revealed that in complete degenerate strains the genes of the solvent locus were absent. Gyrase inhibitors slightly reduced the growth rate and decreased acetone formation preferentially. In an effort to analyze the role of sporulation sigma factors in solvent gene expression, recombination experiments were conducted and led to strains with increased solvent production. Analysis of redox systems has resulted in the sequence analysis of a cluster encoding formyl transferase proteins and an oxidoreductase-like gene. The genes for the two subunits of an apparent electron transfer flavoprotein were sequenced and suggest this factor acts to carry electrons to the butyryl-CoA dehydrogenase. The genes encoding the Fo subunits of the membrane ATPase have been sequenced.

  13. CaCO3 supplementation alleviates the inhibition of formic acid on acetone/butanol/ethanol fermentation by Clostridium acetobutylicum.

    PubMed

    Qi, Gaoxiang; Xiong, Lian; Lin, Xiaoqing; Huang, Chao; Li, Hailong; Chen, Xuefang; Chen, Xinde

    2017-01-01

    To investigate the inhibiting effect of formic acid on acetone/butanol/ethanol (ABE) fermentation and explain the mechanism of the alleviation in the inhibiting effect under CaCO3 supplementation condition. From the medium containing 50 g sugars l(-1) and 0.5 g formic acid l(-1), only 0.75 g ABE l(-1) was produced when pH was adjusted by KOH and fermentation ended prematurely before the transformation from acidogenesis to solventogenesis. In contrast, 11.4 g ABE l(-1) was produced when pH was adjusted by 4 g CaCO3 l(-1). The beneficial effect can be ascribed to the buffering capacity of CaCO3. Comparative analysis results showed that the undissociated formic acid concentration and acid production coupled with ATP and NADH was affected by the pH buffering capacity of CaCO3. Four millimole undissociated formic acid was the threshold at which the transformation to solventogenesis occurred. The inhibiting effect of formic acid on ABE fermentation can be alleviated by CaCO3 supplementation due to its buffering capacity.

  14. Impact of sweet sorghum cuticular waxes (SSCW) on acetone-butanol-ethanol fermentation using Clostridium acetobutylicum ABE1201.

    PubMed

    Cai, Di; Chang, Zhen; Wang, Chengyu; Ren, Wenqiang; Wang, Zheng; Qin, Peiyong; Tan, Tianwei

    2013-12-01

    The effect of cuticular waxes of sweet sorghum stem on acetone-butanol-ethanol (ABE) fermentation process was investigated. About 22.9% of butanol and 25.4% of ABE were decreased with fermentation period extended when SSCW was added. The inhibition of SSCW militate against both acidogenesis and solventogenesis phase, which were inconsistent with the inhibition of lignocellulose hydrolysate. Further studies on the composition of SSCW were performed. Regulations of inhibition with different carbon chain length of main compositions of SSCW on ABE fermentation were also investigated. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. REDUCTION OF 2,4,6-TRINITROTOLUENE BY CLOSTRIDIUM ACETOBUTYLICUM THROUGH HYDROXYLAMINO-NITROTOLUENE INTERMEDIATES. (R825513C006)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  16. Clostridium difficile infection: molecular pathogenesis and novel therapeutics

    PubMed Central

    Rineh, Ardeshir; Kelso, Michael J; Vatansever, Fatma; Tegos, George P; Hamblin, Michael R

    2015-01-01

    The Gram-positive anaerobic bacterium Clostridium difficile produces toxins A and B, which can cause a spectrum of diseases from pseudomembranous colitis to C. difficile-associated diarrhea. A limited number of C. difficile strains also produce a binary toxin that exhibits ADP ribosyltransferase activity. Here, the structure and the mechanism of action of these toxins as well as their role in disease are reviewed. Nosocomial C. difficile infection is often contracted in hospital when patients treated with antibiotics suffer a disturbance in normal gut microflora. C. difficile spores can persist on dry, inanimate surface for months. Metronidazole and oral vancomycin are clinically used for treatment of C. difficile infection but clinical failure and concern about promotion of resistance are motivating the search for novel non-antibiotic therapeutics. Methods for controlling both toxins and spores, replacing gut microflora by probiotics or fecal transplant, and killing bacteria in the anaerobic gut by photodynamic therapy are discussed. PMID:24410618

  17. Prevalence of Clostridium species and behaviour of Clostridium botulinum in gnocchi, a REPFED of italian origin.

    PubMed

    Del Torre, M; Stecchini, M L; Braconnier, A; Peck, M W

    2004-11-01

    Sales and consumption of refrigerated processed foods of extended durability (REPFEDs) have increased many-fold in Europe over the last 10 years. The safety and quality of these convenient ready-to-eat foods relies on a combination of mild heat treatment and refrigerated storage, sometimes in combination with other hurdles such as mild preservative factors. The major hazard to the microbiological safety of these foods is Clostridium botulinum. This paper reports on the prevalence and behaviour of proteolytic C. botulinum and non-proteolytic C. botulinum in gnocchi, a potato-based REPFED of Italian origin. Attempts to isolate proteolytic C. botulinum and non-proteolytic C. botulinum from gnocchi and its ingredients were unsuccessful. Based on assessment of the adequacy of the methods used, it was estimated that for proteolytic C. botulinum there was < 25 spores/kg of gnocchi and < 70 spores/kg of ingredients. The total anaerobic microbial load of gnocchi and its ingredients was low, with an estimated 1 MPN/g in processed gnocchi. Most of the anaerobic flora was facultatively anaerobic. A few obligately anaerobic bacteria were isolated from gnocchi and its ingredients and belonged to different Clostridium species. The protection factor, number of decimal reductions in the probability of toxigenesis from a single spore, was determined for eight different gnocchi formulations by challenge test studies. For all gnocchi stored at 8 degrees C (as recommended by the manufacturer) or 12 degrees C (mild temperature abuse), growth and toxin production were not detected in 75 days. The protection factor was >4.2 for proteolytic C. botulinum, and >6.2 for non-proteolytic C. botulinum. When inoculated packs were stored at 20 degrees C (severe temperature abuse), toxin production in 75 days was prevented by the inclusion of 0.09% (w/w) sorbic acid (protection factors as above), however in the absence of sorbic acid the packs became toxic before the end of the intended shelf

  18. Livestock Anaerobic Digester Database

    EPA Pesticide Factsheets

    The Anaerobic Digester Database provides basic information about anaerobic digesters on livestock farms in the United States, organized in Excel spreadsheets. It includes projects that are under construction, operating, or shut down.

  19. My Lifelong Passion for Biochemistry and Anaerobic Microorganisms.

    PubMed

    Thauer, Rudolf Kurt

    2015-01-01

    Early parental influence led me first to medical school, but after developing a passion for biochemistry and sensing the need for a deeper foundation, I changed to chemistry. During breaks between semesters, I worked in various biochemistry labs to acquire a feeling for the different areas of investigation. The scientific puzzle that fascinated me most was the metabolism of the anaerobic bacterium Clostridium kluyveri, which I took on in 1965 in Karl Decker's lab in Freiburg, Germany. I quickly realized that little was known about the biochemistry of strict anaerobes such as clostridia, methanogens, acetogens, and sulfate-reducing bacteria and that these were ideal model organisms to study fundamental questions of energy conservation, CO2 fixation, and the evolution of metabolic pathways. My passion for anaerobes was born then and is unabated even after 50 years of study.

  20. Microbiology and physiology of anaerobic fermentations of cellulose

    SciTech Connect

    Wiegel, J.

    1991-05-01

    The biochemistry and physiology of four major groups of anaerobic bacteria involved in the conversion of cellulose to methane or chemical feedstocks are examined. Aspects of metabolism which are relevant to the interactions and bioenergetics of consortia are being studied. Properties of the cellulolytic enzyme cluster of Clostridium thermocellum are investigated. Five different hydrogenases have been characterized in detail from anaerobic bacteria. Genes for different hydrogenases are being cloned and sequenced to determine their structural relationships. The role of metal clusters in activation of H{sub 2} is being investigated, as is the structure and role of metal clusters in formate metabolism. The function of formate in the total synthesis of acetate from CO{sub 2} and the role of this primary in anaerobes will be examined as well. Finally, these enzyme studies will be performed on thermophilic bacteria and new, pertinent species will be isolated. 50 refs., 3 figs., 1 tab.

  1. Clostridium difficile Infection

    PubMed Central

    Heinlen, Latisha; Ballard, Jimmy D.

    2010-01-01

    Clostridium difficile is the leading cause of hospital-acquired diarrhea in Europe and North America and is a serious re-emerging pathogen. Recent outbreaks have led to increasing morbidity and mortality and have been associated with a new strain (BI/NAP1/027) of C. difficile that produces more toxin than historical strains. With the increasing incidence of C. difficile infection, clinicians have also seen a change in the epidemiology with increased infections in previously low-risk populations. This chapter highlights the current knowledge on C. difficile virulence, human disease, epidemic outbreaks, and optimal treatment strategies. PMID:20697257

  2. Ultrastructure and extreme heat resistance of spores from thermophilic Clostridium species.

    PubMed Central

    Hyun, H H; Zeikus, J G; Longin, R; Millet, J; Ryter, A

    1983-01-01

    The heat resistance and ultrastructural features of spore suspensions prepared from Clostridium thermocellum LQRI, Clostridium thermosulfurogenes 4B, and Clostridium thermohydrosulfuricum 39E were compared as a function of decimal reduction time. The decimal reduction times at 121 degrees C for strains LQRI, 4B, and 39E were 0.5, 2.5, and 11 min. The higher degree of spore heat resistance was associated with a spore architecture displaying a thicker cortex layer. Heat resistance of these spores was proportional to the ratio of spore cortex volume to cytoplasmic volume. These ratios for spores of strains LQRI, 4B, and 39E were 1.4, 1.6, and 6.6, respectively. The extreme heat resistance and autoclavable nature of C. thermohydrosulfuricum spores under routine sterilization procedures is suggested as a common cause of laboratory contamination with pure cultures of thermophilic, saccharide-fermenting anaerobes. Images PMID:6643392

  3. [Toxins of Clostridium perfringens as a natural and bioterroristic threats].

    PubMed

    Omernik, Andrzej; Płusa, Tadeusz

    2015-09-01

    Clostridium perfringens is absolutely anaerobic rod-shaped, sporeforming bacterium. The morbidity is connected with producing toxins. Depending on the type of toxin produced Clostridium perfringens can be divided into five serotypes:A-E. Under natural conditions, this bacterium is responsible for local outbreaks of food poisoning associated with eating contaminated food which which was improperly heat treated. Some countries with lower economic level are endemic foci of necrotizing enteritis caused by Clostridium perfringens. The bacterium is also a major cause of gas gangrene. It is a disease, associated with wound infection, with potentially fatal prognosis in the case of treatment's delays. In the absence of early radical surgery, antibiotic therapy and (if available) hyperbaric treatment leads to the spread of toxins in the body causing shock, coma and death. Due to the force of produced toxins is a pathogen that poses a substrate for the production of biological weapons. It could potentially be used to induce outbreaks of food poisoning and by missiles contamination by spore lead to increased morbidity of gas gangrene in injured soldiers. C. perfringens types B and D produce epsilon toxin considered to be the third most powerful bacterial toxin. Because of the ability to disperse the toxin as an aerosol and a lack of methods of treatment and prevention of poisoning possible factors it is a potential tool for bioterrorism It is advisable to continue research into vaccines and treatments for poisoning toxins of C. perfringens. © 2015 MEDPRESS.

  4. Quinolone activity against anaerobes.

    PubMed

    Appelbaum, P C

    1999-01-01

    The first generation of fluoroquinolones such as ciprofloxacin and ofloxacin are inactive against most anaerobic bacteria. However, some broad-spectrum quinolones, which have recently become clinically available or are under active development, have significant antianaerobic activity. This review summarises the in vitro activity of currently available, as well as experimental, quinolones against clinically significant anaerobic bacteria. Quinolones with low activity against anaerobes include ciprofloxacin, ofloxacin, levofloxacin, fleroxacin, pefloxacin, enoxacin and lomefloxacin. Compounds with intermediate antianaerobic activity include sparfloxacin and grepafloxacin. Trovafloxacin, gatifloxacin and moxifloxacin yield low MICs against most groups of anaerobes. Quinolones with the greatest in vitro activity against anaerobes include clinafloxacin and sitafloxacin (DU-6859a).

  5. Clostridium difficile spore biology: sporulation, germination, and spore structural proteins.

    PubMed

    Paredes-Sabja, Daniel; Shen, Aimee; Sorg, Joseph A

    2014-07-01

    Clostridium difficile is a Gram-positive, spore-forming obligate anaerobe and a major nosocomial pathogen of worldwide concern. Owing to its strict anaerobic requirements, the infectious and transmissible morphotype is the dormant spore. In susceptible patients, C. difficile spores germinate in the colon to form the vegetative cells that initiate Clostridium difficile infections (CDI). During CDI, C. difficile induces a sporulation pathway that produces more spores; these spores are responsible for the persistence of C. difficile in patients and horizontal transmission between hospitalized patients. Although important to the C. difficile lifecycle, the C. difficile spore proteome is poorly conserved when compared to members of the Bacillus genus. Further, recent studies have revealed significant differences between C. difficile and Bacillus subtilis at the level of sporulation, germination, and spore coat and exosporium morphogenesis. In this review, the regulation of the sporulation and germination pathways and the morphogenesis of the spore coat and exosporium will be discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Clostridium tertium bacteremia in a patient with glyphosate ingestion.

    PubMed

    You, Myung-Jo; Shin, Gee-Wook; Lee, Chang-Seop

    2015-01-06

    Clostridium tertium is distributed in the soil and in animal and human gastrointestinal tracts. C. tertium has been isolated from patients with blood diseases, immune disorders, and abdominal surgeries. Glyphosate is toxic, causing cause eye and skin irritation, gastrointestinal pain, and vomiting. Ingestion of herbicides modifies the gastrointestinal environment, which stresses the living organisms. However, there has been little attention to cases of bacteremia in patients recovering from suicide attempt by ingesting herbicide. Clostridium tertium was identified in a 44-year-old female who attempted suicide by glyphosate (a herbicide) ingestion. The 16S rRNA sequences from all colonies were 99% identical with that of C. tertium (AB618789) found on a BLAST search of the NCBI database. The bacterium was cultured on TSA under aerobic and anaerobic conditions. Antimicrobial susceptibility tests performed under both aerobic and anaerobic conditions showed that the bacterium was susceptible to penicillin, a combination of β-lactamase inhibitor and piperacillin or amoxicillin, and first- and second- generation cephalosporins. However, it was resistant to third- and fourth-generation cephalosporins. Glyphosate herbicide might be a predisposing factor responsible for the pathogenesis of C. tertium. The results highlight the need for careful diagnosis and selection of antibiotics in the treatment of this organism.

  7. Rapid Confirmation of Clostridium perfringens by Using Chromogenic and Fluorogenic Substrates

    PubMed Central

    Adcock, Philip W.; Saint, Christopher P.

    2001-01-01

    The use of 4-methylumbelliferyl phosphate (MUP) and ortho-nitrophenyl-β-d-galactopyranoside (ONPG) for the identification of Clostridium perfringens was investigated. A liquid assay containing both MUP and ONPG was a highly specific alternative method for C. perfringens confirmation, reducing incubation time from 48 to only 4 h. The assay solution is easy to prepare, does not require anaerobic conditions for use, and has an extended shelf life. PMID:11526053

  8. Clostridium saccharogumia sp. nov. and Lactonifactor longoviformis gen. nov., sp. nov., two novel human faecal bacteria involved in the conversion of the dietary phytoestrogen secoisolariciresinol diglucoside.

    PubMed

    Clavel, Thomas; Lippman, Ramona; Gavini, Françoise; Doré, Joël; Blaut, Michael

    2007-01-01

    Two anaerobic bacteria involved in the conversion of the plant lignan secoisolariciresinol diglucoside were isolated from faeces of a healthy male adult. The first isolate, strain SDG-Mt85-3Db, was a mesophilic strictly anaerobic Gram-positive helically coiled rod. Based on 16S r RNA gene sequence analysis, its nearest relatives were Clostridium cocleatum (96.7% similarity) and Clostridium ramosum (96.6%). In contrast to these species, the isolate was devoid of alpha-galactosidase and -glucosidase and did not grow on maltose, melibiose, raffinose, rhamnose and trehalose. The hypothesis that strain SDG-Mt85-3Db represents a new bacterial species of the Clostridium cluster XVIII was confirmed by DNA-DNA hybridisation experiments. The G+C content of DNA of strain SDG-Mt85-3Db (30.7+/-0.8 mol%) was comparable with that of Clostridium butyricum, the type species of the genus Clostridium. The name Clostridium saccharogumia is proposed for strain SDG-Mt85-3Db (=DSM 17460T=CCUG 51486T). The second isolate, strain ED-Mt61/PYG-s6, was a mesophilic strictly anaerobic Gram-positive regular rod. Based on 16S rRNA gene sequence analysis, its nearest relatives were Clostridium amygdalinum (93.3%), Clostridium saccharolyticum (93.1%) and Ruminococcus productus (93.0%). The isolate differed from these species in its ability to dehydrogenate enterodiol. It also possessed alpha-arabinosidase and -galactosidase and had a higher G+C content of DNA (48.0 mol%). According to these findings, it is proposed to create a novel genus, Lactonifactor, and a novel species, Lactonifactor longoviformis, to accommodate strain ED-Mt61/PYG-s6. The type strain is DSM 17459T (=CCUG 51487T).

  9. Quantitative real-time PCR assay for Clostridium septicum in poultry gangrenous dermatitis associated samples.

    PubMed

    Neumann, A P; Dunham, S M; Rehberger, T G; Siragusa, G R

    2010-08-01

    Clostridium septicum is a spore-forming anaerobe frequently implicated in cases of gangrenous dermatitis (GD) and other spontaneously occurring myonecrotic infections of poultry. Although C. septicum is readily cultured from diseased tissues it can be difficult to enumerate due to its tendency to swarm over the surface of agar plates. In this study a quantitative real-time PCR assay was developed in order to more accurately measure the levels of C. septicum in healthy as well as GD associated poultry samples. The assay was specifically designed to target the C. septicum alpha toxin gene, csa, which is, to our knowledge, carried by all strains of C. septicum and has been shown to be essential for virulence. Genomic DNAs from a diverse collection of bacterial species, including closely related Clostridium chauvoei, Clostridium carnis, Clostridium tertium as well as several strains of Clostridium perfringens, all failed to produce a positive reaction. An approximate reproducible limit of detection in spiked extracts of at least 10(3) cfu/g of C. septicum was observed for a variety of different sample types. C. septicum levels in broiler chicken field samples estimated from the results of qPCR were statistically correlated to culture based enumerations obtained from those same tissues.

  10. Clostridium polynesiense sp. nov., a new member of the human gut microbiota in French Polynesia.

    PubMed

    Sankar, Senthil Alias; Rathored, Jaishriram; Metidji, Sarah; Lagier, Jean-Christophe; Khelaifia, Saber; Labas, Noemie; Musso, Didier; Raoult, Didier; Fournier, Pierre-Edouard

    2015-12-01

    Strain MS1, a Gram-positive, obligately anaerobic, motile and spore-forming rod belonging to the Clostridium genus, was isolated from the feces of a healthy Polynesian male living in French Polynesia. The temperature range for growth was 30-45 °C. We sequenced its complete genome and studied its phenotypic characteristics. The 3,560,738-bp long genome (one chromosome, no plasmid, G + C content 34%) contained 3535 protein-coding and 70 RNA genes. Strain MS1 exhibited a 98.24% 16S rRNA similarity with Clostridium amylolyticum, the phylogenetically closest species. When compared with other Clostridium species with standing in nomenclature, it had an average genomic similarity of 68.8-70%, a unique MALDI-TOF spectrum, and differed in nitrate reduction, motility and L-arabinose and D-lactose metabolism with most of the closest species. Therefore, strain MS1 is sufficiently distinct from type strains of the genus Clostridium to represent a novel species within this genus, for which the name Clostridium polynesiense sp. nov. is proposed. The type strain of C. polynesiense is MS1(T) (= CSUR P630 = DSM 27072). Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Vaccines against Clostridium difficile

    PubMed Central

    Leuzzi, Rosanna; Adamo, Roberto; Scarselli, Maria

    2014-01-01

    Clostridium difficile infection (CDI) is recognized as a major cause of nosocomial diseases ranging from antibiotic related diarrhea to fulminant colitis. Emergence during the last 2 decades of C. difficile strains associated with high incidence, severity and lethal outcomes has increased the challenges for CDI treatment. A limited number of drugs have proven to be effective against CDI and concerns about antibiotic resistance as well as recurring disease solicited the search for novel therapeutic strategies. Active vaccination provides the attractive opportunity to prevent CDI, and intense research in recent years led to development of experimental vaccines, 3 of which are currently under clinical evaluation. This review summarizes recent achievements and remaining challenges in the field of C. difficile vaccines, and discusses future perspectives in view of newly-identified candidate antigens. PMID:24637887

  12. Vaccines against Clostridium difficile.

    PubMed

    Leuzzi, Rosanna; Adamo, Roberto; Scarselli, Maria

    2014-01-01

    Clostridium difficile infection (CDI) is recognized as a major cause of nosocomial diseases ranging from antibiotic related diarrhea to fulminant colitis. Emergence during the last 2 decades of C. difficile strains associated with high incidence, severity and lethal outcomes has increased the challenges for CDI treatment. A limited number of drugs have proven to be effective against CDI and concerns about antibiotic resistance as well as recurring disease solicited the search for novel therapeutic strategies. Active vaccination provides the attractive opportunity to prevent CDI, and intense research in recent years led to development of experimental vaccines, 3 of which are currently under clinical evaluation. This review summarizes recent achievements and remaining challenges in the field of C. difficile vaccines, and discusses future perspectives in view of newly-identified candidate antigens.

  13. [Clostridium difficile enteritis].

    PubMed

    Ramos Martínez, Antonio; Romero Pizarro, Yolanda; Martínez Arrieta, Félix; Balandín Moreno, Bárbara; Múñez Rubio, Elena; Cuiñas León, Karina; Sánchez Romero, Isabel; Cantos López de Ibargüen, Blanca; Asensio Vegas, Angel

    2011-10-01

    Clostridium difficile infection of the small intestine is infrequent. We present the first case of C. difficile enteritis (CDE) diagnosed in Spain and provide a review of the literature. A 30-year-old man underwent surgery for recurrence of a retroperitoneal germ cell tumor. Seven days later the patient developed vomiting, diarrhea and, finally, intestinal obstruction due to pseudomembranes caused by CDE. Only 57 cases of CDE have been reported in the literature. The mean age was 52±17 years with a range of 18 to 86 years. Twenty-nine patients (50%) had inflammatory bowel disease. Forty-seven (81%) had a history of colon or small intestine surgery. Mortality was higher in older patients and in those without inflammatory bowel disease. CDE is characterized by high severity and mortality. 2011 Elsevier España, S.L. All rights reserved.

  14. Clostridium difficile infection.

    PubMed

    Alcalá Hernández, Luis; Reigadas Ramírez, Elena; Bouza Santiago, Emilio

    2017-05-23

    Clostridium difficile infection (CDI) is the main cause of nosocomial diarrhea in industrialized countries and the source of a growing number of cases of diarrhea in the community. The outbreak of the hypervirulent strain belonging to ribotype 027 has increased the incidence and severity of CDI in some countries. Although CDI usually courses as a mild diarrhea it can lead to severe forms such as toxic megacolon or septic shock. One of every 2 episodes of CDI is not diagnosed in Spanish hospitals due to a lack of clinical suspicion or the use of insensitive diagnostic methods. The diagnostic techniques of choice are algorithms based on the detection of glutamate dehydrogenase and molecular detection of the genes of the toxins with or without the direct detection of the toxins. The recommended treatment for CDI depends on the type of infection and the characteristics of the patient. Copyright © 2017 Elsevier España, S.L.U. All rights reserved.

  15. Comparative genomic analysis of dha regulon and related genes for anaerobic glycerol metabolism in bacteria.

    PubMed

    Sun, Jibin; van den Heuvel, Joop; Soucaille, Philippe; Qu, Yinbo; Zeng, An-Ping

    2003-01-01

    The dihydroxyacetone (dha) regulon of bacteria encodes genes for the anaerobic metabolism of glycerol. In this work, genomic data are used to analyze and compare the dha regulon and related genes in different organisms in silico with respect to gene organization, sequence similarity, and possible functions. Database searches showed that among the organisms, the genomes of which have been sequenced so far, only two, i.e., Klebsiella pneumoniae MGH 78578 and Clostridium perfringens contain a complete dha regulon bearing all known enzymes. The components and their organization in the dha regulon of these two organisms differ considerably from each other and also from the previously partially sequenced dha regulons in Citrobacter freundii, Clostridium pasteurianum, and Clostridium butyricum. Unlike all of the other organisms, genes for the oxidative and reductive pathways of anaerobic glycerol metabolism in C. perfringens are located in two separate organization units on the chromosome. Comparisons of deduced protein sequences of genes with similar functions showed that the dha regulon components in K. pneumoniae and C. freundii have high similarities (80-95%) but lower similarities to those of the Clostridium species (30-80%). Interestingly, the protein sequence similarities among the dha genes of the Clostridium species are in many cases even lower than those between the Clostridium species and K. pneumoniae or C. freundii, suggesting two different types of dha regulon in the Clostridium species studied. The in silico reconstruction and comparison of dha regulons revealed several new genes in the microorganisms studied. In particular, a novel dha kinase that is phosphoenolpyruvate-dependent is identified and experimentally confirmed for K. pneumoniae in addition to the known ATP-dependent dha kinase. This finding gives new insights into the regulation of glycerol metabolism in K. pneumoniae and explains some hitherto not well understood experimental observations.

  16. The effect of rumen chitinolytic bacteria on cellulolytic anaerobic fungi.

    PubMed

    Kopecný, J; Hodrová, B; Stewart, C S

    1996-09-01

    The polycentric anaerobic fungus Orpinomyces joyonii A4 was cultivated on microcrystalline cellulose alone and in association with the rumen chitinolytic bacterium Clostridium sp. strain ChK5, which shows strong phenotypic similarity to Clostridium tertium. The presence of strain ChK5 significantly depressed the solubilization of microcrystalline cellulose, the production of short-chain fatty acids (SCFA) and the release of endoglucanase by the fungus. Co-culture of the monocentric anaerobic fungus Neocallimastix frontalis strain RE1, Neocallimastix sp. strain G-1 and Caecomyces sp. strain SC2 with strain ChK5 also resulted in depressed fungal cellulolysis. Cell-free supernatant fluids from strain ChK5 inhibited the release of reducing sugars from carboxymethylcellulose by cell-free supernatant fluids from O. joyonii strain A4. Strain 007 of the cellulolytic anaerobe Ruminococcus flavefaciens was also shown to produce small amounts of soluble products upon incubation with colloidal chitin. Mixtures of culture supernates from this bacterium and from O. joyonii strain A4 showed cellulase activity that was less than that of the component cultures. It is suggested that the ability of some rumen bacteria to hydrolyse or transform chitin may be an important factor in the interactions between bacteria and fungi in the rumen.

  17. Novel System for Efficient Isolation of Clostridium Double-Crossover Allelic Exchange Mutants Enabling Markerless Chromosomal Gene Deletions and DNA Integration

    PubMed Central

    Al-Hinai, Mohab A.; Fast, Alan G.

    2012-01-01

    Isolation of Clostridium mutants based on gene replacement via allelic exchange remains a major limitation for this important genus. Use of a heterologous counterselection marker can facilitate the identification of the generally rare allelic exchange events. We report on the development of an inducible counterselection marker and describe its utility and broad potential in quickly and efficiently generating markerless DNA deletions and integrations at any genomic locus without the need for auxotrophic mutants or the use of the mobile group II introns. This system is based on a codon-optimized mazF toxin gene from Escherichia coli under the control of a lactose-inducible promoter from Clostridium perfringens. This system is potentially applicable to almost all members of the genus Clostridium due to their similarly low genomic GC content and comparable codon usage. We isolated all allelic-exchange-based gene deletions (ca_p0167, sigF, and sigK) or disruptions (ca_p0157 and sigF) we attempted and integrated a 3.6-kb heterologous DNA sequence (made up of a Clostridium ljungdahlii 2.1-kb formate dehydrogenase [fdh] gene plus a FLP recombination target [FRT]-flanked thiamphenicol resistance marker) into the Clostridium acetobutylicum chromosome. Furthermore, we report on the development of a plasmid system with inducible segregational instability, thus enabling efficient deployment of the FLP-FRT system to generate markerless deletion or integration mutants. This enabled expeditious deletion of the thiamphenicol resistance marker from the fdh integrant strain as well as the sigK deletion strain. More generally, our system can potentially be applied to other organisms with underdeveloped genetic tools. PMID:22983967

  18. [Antimicrobial susceptibility testing of anaerobic bacteria].

    PubMed

    García-Sánchez, José E; García-Sánchez, Enrique; García-García, María Inmaculada

    2014-02-01

    The anaerobic bacteria resistance to antibiotics is increasing, and even has appeared against the most active of those, like metronidazol and carbapenems. This fact forces to make and periodical sensibility tests -at least in the most aggressive and virulent species, in cases that they are isolated from life locations and in the absence of therapeutic response- to check the local sensibility and to establish suitable empiric therapies, all based on multicentric studies carried out in order to this or well to check the activity of new antibiotics. For the laboratory routine, the easiest sensibility method is the E-test/MIC evaluator. Another alternative is microdilution, that's only normalized for Bacteroides. There are preliminary facts that allow the use of disc diffusion method in some species of Bacteroides and Clostridium. For the temporal and multicentric studies, the procedure is dilution in agar plate, the reference method. Copyright © 2014 Elsevier España, S.L. All rights reserved.

  19. Anaerobes as Sources of Bioactive Compounds and Health Promoting Tools.

    PubMed

    Mamo, Gashaw

    Aerobic microorganisms have been sources of medicinal agents for several decades and an impressive variety of drugs have been isolated from their cultures, studied and formulated to treat or prevent diseases. On the other hand, anaerobes, which are believed to be the oldest life forms on earth and evolved remarkably diverse physiological functions, have largely been neglected as sources of bioactive compounds. However, results obtained from the limited research done so far show that anaerobes are capable of producing a range of interesting bioactive compounds that can promote human health. In fact, some of these bioactive compounds are found to be novel in their structure and/or mode of action.Anaerobes play health-promoting roles through their bioactive products as well as application of whole cells. The bioactive compounds produced by these microorganisms include antimicrobial agents and substances such as immunomodulators and vitamins. Bacteriocins produced by anaerobes have been in use as preservatives for about 40 years. Because these substances are effective at low concentrations, encounter relatively less resistance from bacteria and are safe to use, there is a growing interest in these antimicrobial agents. Moreover, several antibiotics have been reported from the cultures of anaerobes. Closthioamide and andrimid produced by Clostridium cellulolyticum and Pantoea agglomerans, respectively, are examples of novel antibiotics of anaerobe origin. The discovery of such novel bioactive compounds is expected to encourage further studies which can potentially lead to tapping of the antibiotic production potential of this fascinating group of microorganisms.Anaerobes are widely used in preparation of fermented foods and beverages. During the fermentation processes, these organisms produce a number of bioactive compounds including anticancer, antihypertensive and antioxidant substances. The well-known health promoting effect of fermented food is mostly due to these

  20. Tips to Prevent Illness from Clostridium Perfringens

    MedlinePlus

    ... C. perfringens Recommend on Facebook Tweet Share Compartir Clostridium perfringens ( C. perfringens ) is one of the most common ... gov More Information More Information Learn more about Clostridium perfringens Find out safe minimum cooking temperatures for foods ...

  1. Sequential degradation of chlorophenols in anaerobic freshwater sediments

    SciTech Connect

    Zhang, X.

    1993-01-01

    Anaerobic degradation of 2,4-dichlorophenol and 3-chloro-4-hydroxybenzoate in the freshwater sediment samples was investigated. Studies of the enrichment cultures and a pure culture, adaptation times, correlation of substrate degradation and product accumulation, maximal observed transformation rates, temperature and pH ranges for the transformation provided the bases for the proposed sequential pathway for degradation of 2,4-dichlorophenol. At least six different bacterial species were required to catalyze following reactions: (1) the dechlorination of 2,4-dichlorophenol; (2) the dechlorination of 4-chlorophenol; (3) the para-carboxylation of phenol; (4) the reductive dehydroxylation of para-hydroxybenzoate; (5) the degradation of benzoate to acetate, H[sub 2] and CO[sub 2]; and (6) the conversion of H[sub 2]/CO[sub 2] and acetate to methane. The rate limiting reaction in the pathway was the dechlorination of 4-chlorophenol. A new species, Clostridium [open quote]hydroxybenzoicum[close quote], isolated from the enrichment, catalyzed the carboxylation of phenol at the para-position to 4-hydroxybenzoate by a reversible decarboxylation/carboxylation enzyme. 3,4-Dihydroxybenzoate was decarboxylated by a second enzyme in this organism. The activities were biotin and ATP independent. The bacterium, in a pure culture, did not benefit from the decarboxylation reaction but apparently it benefited in the phenol-degrading enrichment culture. Of 46 strains (42 species) tested, only three exhibited hydroxybenzoate decarboxylation activities:Clostridium thermoaceticum, Clostridium thermoautotrophicum,Clostridium scatologenes. The history of the sediment determined the first step in the anaerobic degradation.

  2. Action of nitroheterocyclic drugs against Clostridium difficile

    PubMed Central

    Kumar, Manish; Adhikari, Sudip; Hurdle, Julian G.

    2014-01-01

    The nitroheterocyclic classes of drugs have a long history of use in treating anaerobic infections, as exemplified by metronidazole as a first-line treatment for mild-to-moderate Clostridium difficile infection (CDI). Since direct comparisons of the three major classes of nitroheterocyclic drugs (i.e. nitroimidazole, nitazoxanide and nitrofurans) and nitrosating agents against C. difficile are under-examined, in this study their actions against C. difficile were compared. Results show that whilst transient resistance occurs to metronidazole and nitazoxanide, stable resistance arises to nitrofurans upon serial passage. All compounds killed C. difficile at high concentrations in addition to the host defence nitrosating agent S-nitrosoglutathione (GSNO). This suggests that GSNO killing of C. difficile contributes to its efficacy in murine CDI. Although nitric oxide production could not be detected for the nitroheterocyclic drugs, the cellular response to metronidazole and nitrofurans has some overlap with the response to GSNO, causing significant upregulation of the hybrid-cluster protein Hcp that responds to nitrosative stress. These findings provide new insights into the action of nitroheterocyclic drugs against C. difficile. PMID:25129314

  3. Perfringolysin O: The Underrated Clostridium perfringens Toxin?

    PubMed Central

    Verherstraeten, Stefanie; Goossens, Evy; Valgaeren, Bonnie; Pardon, Bart; Timbermont, Leen; Haesebrouck, Freddy; Ducatelle, Richard; Deprez, Piet; Wade, Kristin R.; Tweten, Rodney; Van Immerseel, Filip

    2015-01-01

    The anaerobic bacterium Clostridium perfringens expresses multiple toxins that promote disease development in both humans and animals. One such toxin is perfringolysin O (PFO, classically referred to as θ toxin), a pore-forming cholesterol-dependent cytolysin (CDC). PFO is secreted as a water-soluble monomer that recognizes and binds membranes via cholesterol. Membrane-bound monomers undergo structural changes that culminate in the formation of an oligomerized prepore complex on the membrane surface. The prepore then undergoes conversion into the bilayer-spanning pore measuring approximately 250–300 Å in diameter. PFO is expressed in nearly all identified C. perfringens strains and harbors interesting traits that suggest a potential undefined role for PFO in disease development. Research has demonstrated a role for PFO in gas gangrene progression and bovine necrohemorrhagic enteritis, but there is limited data available to determine if PFO also functions in additional disease presentations caused by C. perfringens. This review summarizes the known structural and functional characteristics of PFO, while highlighting recent insights into the potential contributions of PFO to disease pathogenesis. PMID:26008232

  4. Draft Genome Sequence of Clostridium sp. Strain W14A Isolated from a Cellulose-Degrading Biofilm in a Landfill Leachate Microcosm

    PubMed Central

    2016-01-01

    Here, we report the draft genome of Clostridium sp. strain W14A, isolated from the anaerobic, cellulolytic biofilm of a cotton string sample incubated in a landfill leachate microcosm. The draft genome comprises 131 contigs, 3,823,510 bp, 51.5% G+C content, and 4,119 predicted coding domain sequences. PMID:27660778

  5. Characterization of Bacteriophages Virulent for Clostridium perfringens and Identification of Phage Lytic Enzymes as Alternatives to Antibiotics for Potential Control of the Bacterium.

    USDA-ARS?s Scientific Manuscript database

    Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that plays a significant role in human food-borne disease as well as non-food-borne human, animal, and poultry diseases. There has been a resurgent interest in the use of bacteriophages or their gene products to control b...

  6. Characterization of Bacteriophages Virulent for Clostridium perfringens and Identification of Phage Lytic Enzymes as Alternatives to Antibiotics for Potential Control of the Bacterium

    USDA-ARS?s Scientific Manuscript database

    Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that plays a significant role in human food-borne disease as well as non-food-borne human, animal, and poultry diseases. There has been a resurgent interest in the use of bacteriophages or their gene products to control b...

  7. Expression of a Clostridium perfringens genome-encoded putative N-acetylmuramoyl-L-alanine amidase as a potential antimicrobial to control the bacterium

    USDA-ARS?s Scientific Manuscript database

    Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that plays a substantial role in non-foodborne human, animal and avian diseases as well as human foodborne disease. Previously discovered C. perfringens bacteriophage lytic enzyme amino acid sequences were utilized to iden...

  8. Draft Genome Sequences for Clostridium thermocellum Wild-Type Strain YS and Derived Cellulose Adhesion-Defective Mutant Strain AD2

    SciTech Connect

    Brown, Steven D; Lamed, Raphael; Morag, Ely; Borovok, Ilya; Shoham, Yuval; Klingeman, Dawn Marie; Johnson, Courtney M; Yang, Zamin; Land, Miriam L; Utturkar, Sagar M; Keller, Martin; Bayer, Edward A

    2012-01-01

    Clostridium thermocellum wild-type strain YS is an anaerobic, thermophilic, cellulolytic bacterium capable of directly converting cellulosic substrates into ethanol. Strain YS and a derived cellulose adhesion-defective mutant strain AD2 played pivotal roles in describing the original cellulosome concept. We present their draft genome sequences.

  9. Characterization of bacteriophages virulent for Clostridium perfringens and identification of phage lytic enzymes as alternatives to antibiotics for potential control of the bacterium

    USDA-ARS?s Scientific Manuscript database

    There has been a resurgent interest in the use of bacteriophages or their gene products to control bacterial pathogens as alternatives to currently utilized antibiotics. Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that plays a significant role in human food-borne di...

  10. Characterization of bacteriophages virulent for Clostridium perfringens and identification of phage lytic enzymes as alternatives to antibiotics for potential control of the bacterium

    USDA-ARS?s Scientific Manuscript database

    There has been a resurgent interest in the use of bacteriophages or their gene products to control bacterial pathogens as alternatives to currently utilized antibiotics. Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that plays a significant role in human food-borne d...

  11. Proposal to restrict the genus Clostridium (Prazmowski) to Clostridium butyricum and related species.

    PubMed

    Lawson, Paul A; Rainey, Fred A

    2015-12-07

    The genus Clostridium as presently constituted is phylogenetically and phenotypically incoherent. Polyphasic taxonomic data indicate that the genus comprises a collection of very heterogeneous species. Numerous phylogenetic studies, principally based on sequencing of the 16S rRNA gene indicate that the genus Clostridium should be restricted to Clostridium cluster I as Clostridium sensu stricto. Despite these findings, authors continue to add new species to the genus Clostridium that do not fall within the radiation of cluster I and the type species C. butryicum thus perpetuating the confusion associated with the taxonomy of this group. Here we formally propose that members of the Clostridium (Prazmowski) be restricted to the type species Clostridium butyricum and cluster I species. Eubacterium moniliforme, Eubacterium tarantellae, Sarcina maxima, and Sarcina ventriculi should be transferred to the genus Clostridium as Clostridium moniliforme comb. nov., Clostridium tarantellae comb. nov., Clostridium maximum comb. nov., and Clostridium ventriculi comb. nov. A novel genus Hathewaya gen. nov.is proposed for the species Clostridium histolyticum, Clostridium limosum and Clostridium proteolyticum as Hathewaya histolytica gen. nov. com. nov., Hathewaya limosa com. nov. and Hathewaya proteolytica comb. nov. The type species of Hathewaya is Hathewaya histolytica.

  12. Isolation and identification of Clostridium perfringens in the venom and fangs of Loxosceles intermedia (brown spider): enhancement of the dermonecrotic lesion in loxoscelism.

    PubMed

    Monteiro, Cristina Leise B; Rubel, Rosália; Cogo, Laura Lúcia; Mangili, Oldemir C; Gremski, Waldemiro; Veiga, Silvio S

    2002-04-01

    Loxoscelism or the envenoming by the brown spiders (Loxosceles genus spiders), may produce extensive dermonecrosis and hemorrhage at the bite site and, eventually, systemic reactions that may be lethal. Isolation and identification of many different bacteria, among them Clostridium perfringens, of great medical importance due to its involvement in dermonecrotizing and systemic conditions, was carried out from the venomous apparatus (fangs and venom) of spiders obtained directly from nature, through microbiological cultures in aerobic and anaerobic conditions. Working with Loxosceles intermedia venom (alone) and with the venom conjugated with Clostridium perfringens using rabbits as experimental models for dermonecrosis, allowed for the observation that venom and anaerobic bacteria conjugated resulted in a striking increase of the dermonecrotic picture when compared to venom alone, suggesting a role for Clostridium perfringens in the severe dermonecrotic picture of these patients and opening the possibility for the association of antibiotic therapy in treating loxoscelism.

  13. Paraclostridium benzoelyticum gen. nov. sp. nov., isolated from marine sediment and reclassification of Clostridium bifermentans as Paraclostridium bifermentans comb. nov. Proposal of a new genus Paeniclostridium gen. nov. to accommodate Clostridium sordellii and Clostridium ghonii.

    PubMed

    T S, Sasi Jyothsna; L, Tushar; Ch, Sasikala; Ch V, Ramana

    2016-01-05

    Twenty three rod shaped, endospore forming, Gram-stain-positive, obligately anaerobic bacteria were isolated from different marine sediment samples of Gujarat. All the twenty three strains have 16S rRNA gene sequence similarity of ~100%. Strain JC272T was designated as the type strain and has sequence similarity with Clostridium bifermentans ATCC638T (99.8%), Clostridium ghonii JCM1400T (98.0%), Clostridium sordellii ATCC9714T (97.9%) and other members of the genus Clostridium (<96.4%). C16:0, C18:0, C17:0, C16:1ω9C and iso-C16:0 are the major (>5%) fatty acids. Strain JC272T contains diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and unidentified amino lipids (AL1&AL2). However, genome based analysis of ANI and in silico DDH of strain JC272T with C. bifermentans ATCC 638T yielded values of 94.35% and 58.5+2.8%, respectively. G+C mol% of strain JC272T was 28.3%. Strain JC272T together with C. bifermentans fall outside Clostridium rRNA cluster I considered as Clostridium senso stricto. Based on ANI value, in-silico DDH, distinct morphological and physiological differences from the previously described taxa, we propose strain JC272T as a representative of a new genus and species in the family Clostridiaceae, for which the name Paraclostridium benzoelyticum gen. nov., sp. nov. is proposed. Type strain is JC272T (=KCTC15476T =LMG28745T). It is also proposed to transfer C. bifermentans to this new genus, as Paraclostridium bifermentans comb. nov. (type strain is ATCC638T =DSM14991T =JCM1386T). We also propose the genus Paeniclostridium gen. nov. to accommodate Clostridium sordellii and Clostridium ghonii as Paeniclostridium sordellii comb. nov. (type strain is ATCC9714T =LMG15708T =JCM3814T) and Paeniclostridium ghonii comb. nov. (type strain is ATCC25757T = DSM15049T =JCM1400T).

  14. Clostridiumm ljungdahlii, an anaerobic ethanol and acetate producing microorganism

    DOEpatents

    Gaddy, J.L.; Clausen, E.C.

    1992-12-22

    A newly discovered microorganism was isolated in a biologically pure culture and designated Clostridium ljungdahlii, having the identifying characteristics of ATCC No. 49587. Cultured in an aqueous nutrient medium under anaerobic conditions, this microorganism is capable of producing ethanol and acetate from CO and H[sub 2]O and/or CO[sub 2] and H[sub 2] in synthesis gas. Under optimal growth conditions, the microorganism produces acetate in preference to ethanol. Conversely, under non-growth conditions, ethanol production is favored over acetate. 3 figs.

  15. Clostridiumm ljungdahlii, an anaerobic ethanol and acetate producing microorganism

    DOEpatents

    Gaddy, James L.; Clausen, Edgar C.

    1992-01-01

    A newly discovered microorganism was isolated in a biologically pure culture and designated Clostridium ljungdahlii, having the identifying characteristics of ATCC No. 49587. Cultured in an aqueous nutrient medium under anaerobic conditions, this microorganism is capable of producing ethanol and acetate from CO and H.sub.2 O and/or CO.sub.2 and H.sub.2 in synthesis gas. Under optimal growth conditions, the microorganism produces acetate in preference to ethanol. Conversely, under non-growth conditions, ethanol production is