BIBLIOGRAFI: CLOSTRIDIUM SP TAHUN 2010 – 2017

1 Basu A, Xin F, Lim TK, Lin Q, Yang KL, He J. (2017) Quantitative proteome profiles help reveal efficient xylose utilization mechanisms in solventogenic Clostridium sp. strain BOH3. Biotechnol Bioeng. 2017 May 5 Abstract Development of sustainable biobutanol production platforms from lignocellulosic materials is impeded by inefficient five carbon sugar uptake by solventogenic bacteria. The recently isolated Clostridium sp. strain BOH3 is particularly advantaged in this regard as it serves as a model organism which can simultaneously utilize both glucose and xylose for high butanol (>15 g/L) production. Strain BOH3 was therefore investigated for its metabolic mechanisms for efficient five carbon sugar uptake using a quantitative proteomics based approach. The proteomics data show that proteins within the CAC1341-1349 operon play a pivotal role for efficient xylose uptake within the cells to produce butanol. Furthermore, up-regulation of key enzymes within the riboflavin synthesis pathway explained that xylose could induce higher riboflavin production capability of the bacteria (e.g., ~80 mg/L from glucose vs ~120 mg/L from xylose). Overall results from the present experimental approach indicated that xylose-fed BOH3 cultures are subjected to high levels of redox stress which coupled with the solvent stress - trigger a sporulation response within the cells earlier than the glucose- fed cultures. The study lays the platform for metabolic engineering strategies in designing organisms for efficient butanol and other value-added chemicals such as riboflavin production. This article is protected by copyright. All rights reserved

2 Ndongo, S. , et.al. (2017) ‘Collinsella phocaeensis’ sp. nov., ‘Clostridium merdae’ sp. nov., ‘Sutterella massiliensis’ sp. nov., ‘Sutturella timonensis’ sp. nov., ‘Enorma phocaeensis’ sp. nov., ‘Mailhella massiliensis’ gen. nov., sp. nov., ‘Mordavella massiliensis’ gen. nov., sp. nov. and ‘Massiliprevotella massiliensis’ gen. nov., sp. nov., 9 new species isolated from fresh stool samples of healthy French patients. New Microbes New Infect.;17: 89-95. Abstract Here we report of summary of the characteristics of ‘Collinsella phocaeensis’ strain Marseille-P3245T sp. nov., ‘Clostridium merdae’ strain Marseille-P2953T, ‘Sutterellamassiliensis’ strainMarseille-P2435T sp. nov., ‘Sutturella timonensis’ strain Marseille-P3282T sp. nov., ‘Enorma phocaeensis’ Marseille- P3242T sp. nov., ‘Mailhella massiliensis’ strain Marseille-P3199T gen. nov., sp. nov., ‘Mordavella massiliensis’ strain Marseille-P3246T sp. nov. And ‘Massiliprevotella massiliensis’ strain Marseille-P2439T sp. nov. isolated from fresh stool samples of healthy French patients.

3 Ruiz-Aguirre A, Polo-López MI, Fernández-Ibáñez P, Zaragoza G. (2017) Integration of Membrane Distillation with solar photo-Fenton for purification of water contaminated with Bacillus sp. and Clostridium sp. spores. Sci Total Environ.;595: 110-118. Abstract Although Membrane Distillation (MD) has been extensively studied for desalination, it has other applications like removing all kinds of solutes from water and concentrating non-volatile substances. MD offers the possibility of producing a clean stream while concentrating valuable compounds from waste streams towards their recovery, or emerging contaminants and pathogens present in wastewater in order to facilitate their chemical elimination. This paper analyses the elimination of bacterial spores from contaminated water with MD and the role ofMD in the subsequent treatment of the concentrate with photo-Fenton process. The experiments were performed at Plataforma Solar de Almería (PSA) using a plate and frame bench module with a Permeate Gap Membrane Distillation (PGMD) configuration. Testswere done for two different kinds of spores in two differentwatermatrixes: distilled water with 3.5 wt% of sea salts contaminated with spores of Bacillus subtilis (B. subtilis) and wastewater after a secondary treatment and still contaminated with Clostridium sp. spores. An analysis of the permeate was performed in all cases to determine its purity, as well as the concentrated stream and its further treatment in order to assess the benefits of usingMD. Results showed a permeate free of spores in all the cases, demonstrating the viability of MD to treat biological contaminated wastewater for further use in agriculture. Moreover, the results obtained after treating the concentrate with photo-Fenton showed a shorter treatment time for the reduction of the spore concentration in the water than that when only photo-Fenton was used.

4 Sakaguchi M, et.al. (2017) Functional dissection of the N-terminal sequence of Clostridium sp. G0005 glucoamylase: identification of components critical for folding the catalytic domain and for constructing the active site structure. Appl Microbiol Biotechnol.;101(6): 2415-2425 Abstract Clostridium sp. G0005 glucoamylase (CGA) is composed of a β-sandwich domain (BD), a linker, and a catalytic domain (CD). In the present study, CGAwas expressed in Escherichia coli as inclusion bodies when the N-terminal region (39 amino acid residues) of the BD was truncated. To further elucidate the role of the N- terminal region of the BD, we constructed N-terminally truncated proteins (Δ19, Δ24, Δ29, and Δ34) and assessed their solubility and activity. Although all evaluated proteins were soluble, their hydrolytic activities toward maltotriose as a substrate varied: Δ19 and Δ24 were almost as active as CGA, but the activity of Δ29 was substantially lower, and Δ34 exhibited little hydrolytic activity. Subsequent truncation analysis of the N- terminal region sequence between residues 25 and 28 revealed that truncation of less than 26 residues did not affect CGA activity, whereas truncation of 26 or more residues resulted in a substantial loss of activity. Based on further site-directed mutagenesis and N-terminal sequence analysis, we concluded that the 26XaaXaaTrp28 sequence of CGA is important in exhibiting CGA activity. These results suggest that the Nterminal region of the BD in bacterial GAs may function not only in folding the protein into the correct structure but also in constructing a competent active site for catalyzing the hydrolytic reaction.

5 Zhao X, Li D, Xu S, Guo Z, Zhang Y, Man L, Jiang B, Hu X. (2017) Clostridium guangxiense sp. nov. and Clostridium neuense sp. nov., two phylogenetically closely related hydrogen-producing species isolated from lake sediment. Int J Syst Evol MicrobiolMar;67(3):710-715. Abstract 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.

6 Afouda P, et.al. (2016) Noncontiguous finished genome sequence and description of Prevotella phocaeensis sp. nov., a new anaerobic species isolated from human gut infected by Clostridium difficile. New Microbes New Infect. 2016;15: 117-127 Abstract Prevotella phocaeensis sp. nov. strain SN19T (= DSM 103364) is a new species isolated from the gut micro- biota of patient with colitis due to Clostridium difficile. Strain SN19T is Gram-negative rod-shaped bacteria, strictly anaerobic, nonmotile and non–endospore forming. The predominance fatty acid is hexadeca- noic acid. Its 16S rRNA showed a 97.70% sequence identity with its phylogenetically closest species, Prevotella oralis. The genome is 2 922 117 bp long and contains 2486 predicted genes including 56 RNA genes.

7 Di Gioia, Diana, et.al. (2016) Lactic acid bacteria as protective cultures in fermented pork meat to prevent Clostridium spp. growth, International Journal of Food Microbiology, Volume 235: 53-59, Abstract: In meat fermented foods, Clostridium spp. growth is kept under control by the addition of nitrite. The growing request of consumers for safer products has led to consider alternative bio-based approaches, the use of protec- tive cultures being one of them. This work is aimed at checking the possibility of using two Lactobacillus spp. strains as protective cultures against Clostridium spp. in pork ground meat for fermented salami preparation. Both Lactobacillus strains displayed anti-clostridia activity in vitro using the spot agar test and after coculturing them in liquid medium with each Clostridium strain. Only one of them, however, namely L. plantarum PCS20, was capable of effectively surviving in ground meat and of performing anti-microbial activity in carnis in a challenge test where meat was inoculated with the Clostridium strain. Therefore, this work pointed out that protective cultures can be a feasible approach for nitrite reduction in fermented meat products.

8 Hosny, M., Set.al. (2016) Description of Clostridium phoceensis sp. nov., a new species within the genus Clostridium, New Microbes and New Infections, Volume 14: 85-92, Abstract: Clostridium phoceensis sp. nov., strain GD3T (= CSUR P1929 = DSM 100334) is the type strain of C. phoceensis sp. nov., a new species within the genus Clostridium. This strain was isolated from the gut microbiota of a 28-year-old healthy French man. C. phoceensis is a Gram-negative, spore-forming, nonmotile, strictly anaerobic bacterium. We describe its complete genome sequence and annotation, together with its phenotypic characteristics.

9 Kamalaskar, Leena, et.al. (2016) Genome sequence and gene expression studies reveal novel hydrogenases mediated hydrogen production by Clostridium biohydrogenum sp. nov., MCM B- 509T, International Journal of Hydrogen Energy, Volume 41, Issue 28: 11990-11999, Abstract: The present study describes Clostridium biohydrogenum sp. nov., MCM B-509T as one of the most efficient hydrogen producing dark fermentative bacteria. MCM B-509 produced 3.35 mol hydrogen per mole of glucose consumed. The draft genome sequence of MCM B-509 provided insights into gene repertoire involved in hydrogen metabolism. Genome analysis revealed the presence of key enzymes such as Fe hydrogenase, NiFe Hydrogenases, NAD dependent hydroxy-butyrate dehydrogenase and D-3 Phosphoglycerate dehydrogenase. Fe-hydrogenases were identified as novel hydrogenases on the basis of low sequence homology with the reference sequences in GenBank database as well as predicted 3D structure. Gene expression studies revealed genes encoding hydrogenases were upregulated and solventogenic pathway down regulated during exponential growth phase. Reverse was observed during stationary growth phase explaining high hydrogen production and decline during respective growth phases. This paper illustrates genetic investigation to explain high hydrogen production potential of C. biohydrogenum sp. nov., MCM B-509T.

10 Kim, Minsun, et.al. (2016) Butyric acid production from softwood hydrolysate by acetate-consuming Clostridium sp. S1 with high butyric acid yield and selectivity, Bioresource Technology, Volume 218: 1208-1214, Abstract: The aim of this work was to study the butyric acid production from softwood hydrolysate by acetate-consuming Clostridium sp. S1. Results showed that Clostridium sp. S1 produced butyric acid by simultaneously utilizing glucose and mannose in softwood hydrolysate and, more remarkably, it consumed acetic acid in hydrolysate. Clostridium sp. S1 utilized each of glucose, mannose, and xylose as well as mixed sugars simultaneously with partially repressed xylose utilization. When softwood (Japanese larch) hydrolysate containing glucose and mannose as the main sugars was used, Clostridium sp. S1 produced 21.17 g/L butyric acid with the yield of 0.47 g/g sugar and the selectivity of 1 (g butyric acid/g total acids) owing to the consumption of acetic acid in hydrolysate. The results demonstrate potential of Clostridium sp. S1 to produce butyric acid selectively and effectively from hydrolysate not only by utilizing mixed sugars simultaneously but also by converting acetic acid to butyric acid.

11 Ransom-Jones E, McDonald JE. (2016) Draft Genome Sequence of Clostridium sp. Strain W14A Isolated from a Cellulose-Degrading Biofilm in a Landfill Leachate Microcosm. Genome Announc., 4(5: e00985-16. Abstract 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.

12 Sakaguchi M, et.al. (2016) Functional dissection of the N-terminal sequence of Clostridium sp. G0005 glucoamylase: identification of components critical for folding the catalytic domain and for constructing the active site structure. Appl Microbiol Biotechnol., Dec 9 Abstract Clostridium sp. G0005 glucoamylase (CGA) is composed of a β-sandwich domain (BD), a linker, and a catalytic domain (CD). In the present study, CGAwas expressed in Escherichia coli as inclusion bodies when the N-terminal region (39 amino acid residues) of the BD was truncated. To further elucidate the role of the N- terminal region of the BD, we constructed N-terminally truncated proteins (Δ19, Δ24, Δ29, and Δ34) and assessed their solubility and activity. Although all evaluated proteins were soluble, their hydrolytic activities toward maltotriose as a substrate varied: Δ19 and Δ24 were almost as active as CGA, but the activity of Δ29 was substantially lower, and Δ34 exhibited little hydrolytic activity. Subsequent truncation analysis of the N- terminal region sequence between residues 25 and 28 revealed that truncation of less than 26 residues did not affect CGA activity, whereas truncation of 26 or more residues resulted in a substantial loss of activity. Based on further site-directed mutagenesis and N-terminal sequence analysis, we concluded that the 26XaaXaaTrp28 sequence of CGA is important in exhibiting CGA activity. These results suggest that the Nterminal region of the BD in bacterial GAs may function not only in folding the protein into the correct structure but also in constructing a competent active site for catalyzing the hydrolytic reaction.

13 Shin, Yeseul et.al. (2016) Clostridium kogasensis sp. nov., a novel member of the genus Clostridium, isolated from soil under a corroded gas pipeline. Anaerobe 39 (2016) 14e18 Abstract Two bacterial strains, YHK0403T 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 5588T (95.8% sequence similarity), followed by Clostridium magnum KCTC 15177T (95.8%), Clostridium drakei KCTC 5440T (95.7%) and Clostridium tyrobutyricum KCTC 5387T (94.9%). The G þ C contents of the isolates were 29.6 mol%. Peptidoglycan in the cell wall was of the A1g 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 C16:1 cis9 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, YHK0403T ¼ KCTC 15258T ¼ JCM 18719T) is proposed.

14 Stiemsma, LT, et.al. (2016) Shifts in Lachnospira and Clostridium sp. in the 3-month stool microbiome are associated with preschool age asthma. Clin Sci, 130(23):2199-2207. Abstract Asthma is a chronic disease of the airways affecting one in ten children in Westernized countries. Recently, our group showed that specific bacterial genera in early life are associated with atopy and wheezing in 1-year-old children. However, little is known about the link between the early life gut microbiome and the diagnosis of asthma in preschool age children. To determine the role of the gut microbiota in preschool age asthma, children up to 4 years of age enrolled in the Canadian Healthy Infant Longitudinal Development (CHILD) study were classified as asthmatic (n=39) or matched healthy controls (n=37). 16S rRNA sequencing and quantitative PCR (qPCR) were used to analyse the composition of the 3-month and 1-year gut microbiome of these children. At 3 months the abundance of the genus, Lachnospira (L), was decreased (P=0.008), whereas the abundance of the species, Clostridium neonatale (C), was increased (P=0.07) in asthmatics. Quartile analysis of stool composition at 3-months revealed a negative association between the ratio of these two bacteria (L/C) and asthma risk by 4 years of age [quartile 1: odds ratio (OR)=15, P=0.02, CI (confidence interval) =1.8–124.7; quartile 2: OR=1.0, ns; quartile 3: OR=0.37, ns]. We conclude that opposing shifts in the relative abundances of Lachnospira and C. neonatale in the first 3 months of life are associated with preschool age asthma, and that the L/C ratio may serve as a potential early life biomarker to predict asthma development.

15 Sun L, Schnürer A. (2016) Draft Genome Sequence of the Cellulolytic Strain Clostridium sp. Bc-iso- 3 Isolated from an Industrial-Scale Anaerobic Digester. Genome Announc.,4(5) : e01188-16. Abstract 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_.

16 Tao Y, Hu X, Zhu X, Jin H, Xu Z, Tang Q, Li X. (2016) Production of Butyrate from Lactate by a Newly Isolated Clostridium sp. BPY5. Appl Biochem Biotechnol.,179(3):361-74. Abstract Lactate-utilizing bacteria play important roles in the production of Chinese strongflavored liquor (CSFL). However, the identity of these bacteria and their lactate-utilizing properties are largely unknown. Here, a lactate-utilizing, butyrate-producing bacterium BPY5 was isolated from an old fermentation pit for CSFL production. The isolate represented a novel species belonging to Clostridium cluster XIVa of family Lachnospiraceae based on phylogenetic analysis using 16S rRNA gene sequences. Strain BPY5 could ferment lactate into butyrate as the major metabolic product. Butyrate was significantly formed at initial lactate concentration from 66 to 104 mM, but substantially declined when initial lactate exceeded 133 mM. At initial lactate concentration of 66 mM, lactate conversion was independent on initial pH from 5.5 to 7.0, but the conversion was completely inhibited when pH dropped below 4.8. Nevertheless, the inhibition on lactate conversion was largely relieved by the addition of acetate, suggesting that exogenous acetate could enhance lactate conversion at low pH condition. Additionally, lactate in CSFL-brewing wastewater was dramatically removed when inoculated with strain BPY5. These results implicate that the isolate may be applied for the industrial production of butyrate or the recovery of butyrate from lactatecontaining wastewater. 17 Youn SH, et.al. (2016) Effective isopropanol-butanol (IB) fermentation with high butanol content using a newly isolated Clostridium sp. A1424. Biotechnol Biofuels, 9:230. Abstract Background: Acetone–butanol–ethanol fermentation has been studied for butanol production. Alternatively, to achieve acetone-free butanol production, use of clostridium strains producing butanol and 1,3-propanediol (1,3- PDO) from glycerol, natural and engineered isopropanol–butanol–ethanol (IBE) producers has been attempted; however, residual 1,3-PDO and acetone, low IBE production by natural IBE producers, and complicated gene modification are limitations. Results: Here, we report an effective isopropanol and butanol (IB) fermentation using a newly isolated Clostridium sp. A1424 capable of producing IB from various substrates with a small residual acetone. Notably, this strain also utilized glycerol and produced butanol and 1,3-PDO. After 46.35 g/L of glucose consumption at pH 5.5-controlled batch fermentation, Clostridium sp. A1424 produced 9.43 g/L of butanol and 13.92 g/L of IB at the productivity of 0.29 and 0.44 g/L/h, respectively, which are the highest values in glucose-based batch fermentations using natural IB producers. More interestingly, using glucose– glycerol mixtures at ratios ranging from 20:2 to 14:8 led to not only acetonefree and 1,3-PDO-free IB fermentation but also enhanced IB production along with a much higher butanol content (butanol/isopropanol ratio of 1.81 with glucose vs. 2.07–6.14 with glucose–glycerol mixture). Furthermore, when the mixture of glucose and crude glycerol at the ratio of 14:8 (total concentration of 35.68 g/L) was used, high butanol/isopropanol ratio (3.44) and butanol titer (9.86 g/L) were achieved with 1.4-fold enhanced butanol yield (0.28 g/g) and productivity (0.41 g/L/h) compared to those with glucose only at pH 5.5. Conclusions: A newly isolated Clostridium sp. A1424 was able to produce butanol and isopropanol from various carbon sources. The productivity and titer of butanol and total alcohol obtained in this study were higher than the previously reported results obtained using other natural IB producers. Use of the mixture of glucose and glycerol was successful to achieve acetone-free, 1,3-PDO-free, and enhanced IB production with higher yield, productivity, and selectivity of butanol compared to those with glucose only, providing great advantages from the perspective of carbon recovery to alcohols. This notable result could be accomplished by isolating an effective IB producer Clostridium sp. A1424 as well as by utilizing glucose–glycerol mixtures.

18 Zhao X, et.al. (2016) Clostridium guangxiense sp. nov. and Clostridium neuense sp. nov., two phylogenetically closely related hydrogen-producing species isolated from lake sediment. Int J Syst Evol Microbiol. Dec 1. Abstract Two novel anaerobic, mesophilic, bio-hydrogen producing bacteria, designated strains ZGM211T and G1T, were isolated from lake sediment. 16S rRNA and ATP synthase beta subunit (atpD) gene sequences phyloge- netic 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 strain ZGM211T and G1T were 40.7 mol% 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.

19 Chiung-Yi, Cheng, (2015) Effects of applied voltage on hydrogen production rate of a single reactor BML with Clostridium sp., Process Safety and Environmental Protection, Volume 98: 383-389, Abstract: This study aimed to explore the influences of single-chamber systems with different applied voltage on bio- hydrogen (H2) production. The reactor used was the bio-electrochemically assisted microbial reactor (BEAMR) membrane-less (BEAMR-membrane-less, BML). The microbial dark fermentative H2 production method was adopted. After the hot screening process and the DNA sequencing, the domesticated dominant microflora was Clostridium sp. This study discussed the influences of the cases with (continuous and intermittent) and without applied voltage separately. The results showed that, the H2 production rate of the case with intermittent applied voltage (117 mL/h g VSS) of 0.24 V was increased of 1.7 folds higher than the without applied voltage (69 mL/h g VSS) and 1.3 folds higher than the case with continuous applied voltage (88.2 mL/h g VSS) of 0.24 V. The produced H2 concentration with intermittent applied voltage was 18.9% (18.6–19.1%) higher than the without applied voltage, while there was no significant difference with continuous applied voltage.

20 Gómez-Torres, Natalia, Sonia Garde, (2015) Impact of Clostridium spp. on cheese characteristics: Microbiology, color, formation of volatile compounds and off-flavors, Food Control, 56: 186-194 Abstract The impact of autochthonous and type-strains of Clostridium tyrobutyricum, Clostridium butyricum, Clostridium beijerinckii and Clostridium sporogenes on spoilage (late blowing defect, LBD), physico-chemical characteristics and volatile profile of cheese has been investigated. Five semi-hard cheeses were produced from ewe milk inoculated with 104 spores/mL of five Clostridium strains and ripened for 60 d. One cheese without clostridial spores served as control. C. tyrobutyricum CECT 4011 and INIA 68 resulted potent cheese spoilers, and caused the appearance of the earliest and greatest symptoms of LBD, affecting cheese pH and color, and leading to accumulation of volatile compounds like butyric, propionic and pentanoic acids and some aldehydes, alcohols and esters associated with cheese rancid and pungent off-odors. Cheeses contaminated with C. beijerinckii INIA 63 and C. sporogenes INIA 71 showed milder and late LBD symptoms, and a volatile profile characterized by higher levels of 2-butanone, 2,3-butanedione and 2-butanol than the rest of cheeses. Despite cheese inoculated with C. butyricum CECT 361 presented a slight blown-pack at the end of ripening, it showed physico-chemical characteristics and a volatile profile similar to control cheese. The first two axes of a principal component analysis (PCA) performed for the 21 significant volatile compounds out of 38, accounting for 91% of the variability between cheeses, separated cheeses made with C. tyrobutyricum CECT 4011 and INIA 68, with severe LBD symptoms, from the rest of cheeses, and also differentiated control cheese and cheese made with C. butyricum CECT 361, from cheeses with milder LBD symptoms made with C. beijerinckii INIA 63 and C. sporogenes INIA 71.

21 Horino H, Ito M, Tonouchi A. (2015) Clostridium oryzae sp. nov., from soil of a Japanese rice field. Int J Syst Evol Microbiol., 65(Pt 3):943-51. Abstract An obligately anaerobic bacterial strain designated KC3T was isolated from a rice straw-degrading culture, for which soil of a Japanese rice field was used as the inoculum. Cells of strain KC3T were determined to be non- cellulolytic, Gram-stain-positive, non-motile, ellipsoidal, spore-forming rods, 0.8–1_4–25 mm. Endospores were formed at a terminal position in elongated cells (12–25 mm, mean 15 mm). The temperature range for growth was 20–50 6C, with an optimum at 37 6C. The pH range for growth was 5.0–7.5, with an optimum at pH 6.0 (slightly acidophilic). Strain KC3T fermented cellobiose to lactate, butyrate, acetate, formate, hydrogen and carbon dioxide. The major cellular fatty acids (.10 %) were C14 : 0, C16 : 0 and C19 : 0 cyclo 11,12 dimethylacetal. The DNA G+C content of strain KC3T was 37.5 mol%. 16S rRNA gene sequence analysis revealed that strain KC3T shared low sequence similarity (,93 %) with type strains of the genus Clostridium sensu stricto (Clostridium rRNA cluster I). Analyses of the DNA gyrase A and ATP synthase beta subunit sequences supported the affiliation of strain KC3T to the genus Clostridium sensu stricto. The evidence presented here indicates that strain KC3T represents a novel species of the genus Clostridium, for which the name Clostridium oryzae sp. nov. is proposed. The type strain of Clostridium oryzae is KC3T (5DSM 28571T5NBRC 110163T)

22 Lanjekar VB, Marathe NP, Shouche YS, Ranade DR. (2015) Clostridium punense sp. nov., an obligate anaerobe isolated from healthy human faeces. Int J Syst Evol Microbiol.,65(12):4749-56.

Abstract An obligately anaerobic, rod-shaped (0.5–1.062.0–10.0 mm), 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 8C (optimum 37 8C). 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 : 1v7c 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 (5DSM 28650T5CCUG 64195T5MCC 2737T).

23 Lee KM,et.al. (2015) Butyric acid production from red algae by a newly isolated Clostridium sp. S1. Biotechnol Lett., 37(9):1837-44. Abstract Objective To produce butyric acid from red algae such as Gelidium amansii in which galactose is a main carbohydrate, microorganisms utilizing galactose and tolerating inhibitors in hydrolysis including levulinic acid and 5-hydroxymethylfurfural (HMF) are required. Results A newly isolated bacterium, Clostridium sp. S1 produced butyric acid not only from galactose as the sole carbon source but also from a mixture of galactose and glucose through simultaneous utilization. Notably, Clostridium sp. S1 produced butyric acid and a small amount of acetic acid with the butyrate:acetate ratio of 45.4:1 and it even converted acetate to butyric acid. Clostridium sp. S1 tolerated 0.5–2 g levulinic acid/l and recovered from HMF inhibition at 0.6–2.5 g/l, resulting in 85–92 % butyric acid concentration of the control culture. When acidpretreated G. amansii hydrolysate was used, Clostridium sp. S1 produced 4.83 g butyric acid/l from 10 g galactose/l and 1 g glucose/l. Conclusion Clostridium sp. S1 produces butyric acid from red algae due to its characteristics in sugar utilization and tolerance to inhibitors, demonstrating its advantage as a red algae-utilizing microorganism

24 Nathani, Neelam M., et.al. (2015) Isolation of chitinolytic Clostridium sp. NCR from Mehsani buffalo rumen, its genomic analysis and potential role in rumen, Genomics Data, Volume 5: 109-111, Abstract: Genomic analysis of Clostridium sp. NCR, an anaerobic Gram positive bacterium which was isolated from rumen fluid of Mehsani breed of buffalo revealed presence of various environmental gene tags (EGTs) involved in pathways for utilizing a wide range of substrates. Here we report the sequence of this rumen isolate, its whole genome sequence has been deposited in DDBJ/EMBL/GenBank under the accession number JQHY00000000. The genome comprises of a 3.62-Mb draft genome with a G + C content of 28.10%, which encodes a total of 3126 proteins. Functional analysis provides information about the microbe's role in maintaining host homeostasis and its fiber degradation potential.

25 O'Neal, Lindsey, et.al. (2015) Clostridium amazonense sp. nov. an obliqately anaerobic bacterium isolated from a remote Amazonian community in Peru, Anaerobe, Volume 35, Part B: 33-37, Abstract: 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 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 NE08VT (DSM 23598T = CCUG 59712T).

26 Patel, Anil Kumar, et.al. (2015) Biohydrogen production from a novel alkalophilic isolate Clostridium sp. IODB-O3, Bioresource Technology, Volume 175: 291-297, Abstract Hydrogen producing bacteria IODB-O3 was isolated from sludge and identified as Clostridium sp. by 16S rDNA gene analysis. In this study, biohydrogen production process was developed using low-cost agro-waste. Maximum H2 was produced at 37 °C and pH 8.5. Maximum H2 yield was obtained 2.54 ± 0.2 mol-H2/mol- reducing sugar from wheat straw pre-hydrolysate (WSPH) and 2.61 ± 0.1 mol-H2/mol-reducing sugar from pre-treated wheat straw enzymatic-hydrolysate (WSEH). The cumulative H2 production (ml/L), 3680 ± 105 and 3270 ± 100, H2 production rate (ml/L/h), 153 ± 5 and 136 ± 5, and specific H2 production (ml/g/h), 511 ± 5 and 681 ± 10 with WSPH and WSEH were obtained, respectively. Biomass pre-treatment via steam explosion generates ample amount of WSPH which remains unutilized for bioethanol production due to non- availability of efficient C5-fermenting microorganisms. This study shows that Clostridium sp. IODB-O3 is capable of utilizing WSPH efficiently for biohydrogen production. This would lead to reduced economic constrain on the overall cellulosic ethanol process and also establish a sustainable biohydrogen production process.

27 Poehlein A, Bandera A, Horne D, Maier J, Pawlowicz D, Siebert V, Daniel R.(2015) First Insights into the Genome of the N-Methylhydantoin-Degrading Clostridium sp. Strain FS41 (DSM 6877). Genome Announc., 3(2): e00394-15. Abstract Clostridium sp. strain FS41 (DSM 6877) is a strictly anaerobic and Gram-positive spindle-shaped rod. This spore-forming bacterium is able to degrade N-methylhydantoin, with N-carbamoylsarcosine and sarcosine as intermediates. The genome consists of one replicon (6.28 Mb) and harbors 5,735 predicted protein-coding genes.

28 Tushar L, Sasi Jyothsna TS, Sasikala C, Ramana CV. (2015) Draft Genome Sequence of Antimicrobial-Producing Clostridium sp. JC272, Isolated from Marine Sediment. Genome Announc., 3(3): e00650-15. Abstract We announce the draft genome sequence of Clostridium sp. JC272, isolated from a sediment sample collected from marine habitats of Gujarat, India. Clostridium sp. JC272 is an obligate anaerobe and has the ability to produce antimicrobial compounds. The genome sequence indicates the strain’s capability of producing small peptides (microcins), which are potential novel antibiotics.

29 Wang H, Lin H, et.al. (2015) Draft Genome Sequence of Clostridium sp. Ne2, Clostridia from an Enrichment Culture Obtained from Australian Subterranean Termite, Nasutitermes exitiosus. Genome Announc. , 3(2): e00304-15. Abstract 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.

30 Wang, P.P, Bao P, Sun GX. (2015) Identification and catalytic residues of the arsenite methyltransferase from a sulfate-reducing bacterium, Clostridium sp. BXM. FEMS Microbiol Lett., 362(1):1-8. Abstract Arsenic methylation is an important process frequently occurring in anaerobic environments. Anaerobic microorganisms have been implicated as the major contributors for As methylation. However, very little information is available regarding the enzymatic mechanism of As methylation by anaerobes. In this study, one novel sulfate-reducing bacterium isolate, Clostridium sp. BXM, which was isolated from a paddy soil in our laboratory, was demonstrated to have the ability of methylating As. One putative arsenite S-Adenosyl- Methionine methyltransferase (ArsM) gene, CsarsM was cloned from Clostridium sp. BXM. Heterologous expression of CsarsM conferred As resistance and the ability of methylating As to an As-sensitive strain of Escherichia coli. Purified methyltransferase CsArsM catalyzed the formation of methylated products from arsenite, further confirming its function of As methylation. Site-directed mutagenesis studies demonstrated that three conserved cysteine residues at positions 65, 153 and 203 in CsArsM are necessary for arsenite methylation, but only Cysteine 153 and Cysteine 203 are required for the methylation of monomethylarsenic to dimethylarsenic. These results provided the characterization of arsenic methyltransferase from anaerobic sulfate-reducing bacterium. Given that sulfate-reducing bacteria are ubiquitous in various wetlands including paddy soils, enzymatic methylation mediated by these anaerobes is proposed to contribute to the arsenic biogeochemical cycling.

31 Wang Q, Wang CD, Li CH, Li JG, Chen Q, Li YZ. (2015) Clostridium luticellarii sp. nov., isolated from a mud cellar used for producing strong aromatic liquors. Int J Syst Evol Microbiol, 65(12): 4730-3. Abstract A strictly anaerobic, Gram-stain-positive bacterium, designated FW431T, was isolated from a mud cellar used for producing strong aromatic Chinese liquors. The strain was able to produce butanoic acid, an important component of the aroma style of Chinese liquors. Cells of strain FW431T were straight or slightly curved rods with a polar endospore and peritrichous flagella. The major cellular fatty acids (>10 % of the total) were C16 : 0, C18 : 1ω9c and C18 : 0. Biolog assays indicated that the strain preferably metabolizes palatinose, l- fucose, β-hydroxybutyric acid, l-rhamnose and α-ketobutyric acid among 95 carbon sources tested. FW431T was related most closely to Clostridium ljungdahlii DSM 13528T and Clostridium kluyveri DSM 555T based on 16S rRNA gene sequence similarities of 95.0 and 94.2 %, respectively. The DNA G+C content of the genomic DNA was 44.4 mol%. Based on the evidence presented here, FW431T ( = CGMCC 1.5201T = KCTC 15519T) is proposed as the type strain of a novel species, Clostridium luticellarii sp. nov.

32 Yin Q, Tao Y, Zhu X, Zhou Y, He X, Cheng L, Huang Y, Li D. (2015) Clostridium liquoris sp. nov., isolated from a fermentation pit used for the production of Chinese Strong-Flavored Liquor. Int J Syst Evol Microbiol. Nov 23.

Abstract Strain BEY10T was isolated from an old fermentation pit, which had been used for the production of Chinese strong-flavoured liquor for over 20 years. The strain was strictly anaerobic, Gram-stain positive, rod-shaped, non-motile and spore-forming. Strain BEY10T grew at temperatures of 22–47 °C (optimum 37 °C), at pH 5.5– 9.0 (optimum pH 7.5–8.5) and with NaCl concentrations of 0–4 % (w/v) (optimum 0 %). The isolate was able to utilize glucose, mannitol, lactose, xylose, maltose, glycerol, cellobiose and trehalose as carbon sources for growth. The major end-products from glucose fermentation were ethanol and butyric acid. The polar lipids consisted of phosphatidylglycerol, phosphatidylethanolamine, phospholipids, a glycolipid and an aminolipid. The predominant fatty acids (>10 %) were C20 : 0, C18 : 0, C16 : 0, C12 : 0 and C14 : 0. The DNA G+C content was 34.4 mol%. Sequence analysis of the 16S rRNA gene indicated that strain BEY10T belongs to the genus Clostridium in the family Clostridiaceae. The closest phylogenetic neighbour is Clostridium lundense DSM 17049T, showing 97.6 % 16S rRNA gene sequence similarity with strain BEY10T. DNA–DNA relatedness values of strain BEY10T with Clostridium lundense DSM 17049T, Clostridium tetanomorphum DSM 4474T and Clostridium pascui DSM 10365T were 58.8 %, 57.9 % and 42.2 %, respectively. This characterization based on phylogenetic, phenotypic and chemotaxonomic evidence demonstrated that strain BEY10T represents a novel species of the genus Clostridium, for which the name Clostridium liquoris sp. nov. is proposed. The type strain is BEY10T ( = ACCC 00785T = DSM 100320T).

33 Abdeshahian, Peyman, et.al. (2014) The production of biohydrogen by a novel strain Clostridium sp. YM1 in dark fermentation process, International Journal of Hydrogen Energy, Vol. 39: 12524-12531 Abstract In view of increasing attempts for the production of renewable energy, the production of biohydrogen energy by a new mesophilic bacterium Clostridium sp. YM1 was performed for the first time in the dark fermentation. Experimental results showed that the fermentative hydrogen was successfully produced by Clostridium sp. YM1 with the highest cumulative hydrogen volume of 3821 ml/L with a hydrogen yield of 1.7 mol H2/mol glucose consumed. Similar results revealed that optimum incubation temperature and pH value of culture medium were 37 °C and 6.5, respectively. The study of hydrogen production from glucose and xylose revealed that this strain was able to generate higher hydrogen from glucose compared to that from xylose. The profile of volatile fatty acids produced showed that hydrogen generation by Clostridium sp. YM1 was butyrate-type fermentation. Moreover, the findings of this study indicated that an increase in head space of fermentation culture positively enhanced hydrogen production.

34 Goud, R. Kannaiah, Omprakash Sarkar, S. Venkata Mohan, (2014) Regulation of biohydrogen production by heat-shock pretreatment facilitates selective enrichment of Clostridium sp., International Journal of Hydrogen Energy, Volume 39, Issue 14: 7572-7586, Abstract The effect of heat-shock treatment to selectively enrich acidogenic, H2 producing consortia was investigated for inoculum preparation and to control the process operation. Long term operation (520 days) in suspended- batch mode bioreactors illustrated relative efficiency and feasibility of heat-shock treated consortia (15.78 mol/kg CODR) in enhancing H2 production (3.31 mol/kg CODR) when compared to parent (control) consortia. On the contrary, substrate degradation was higher in the control operation (ξCOD, 62.86%; substrate degradation rate (SDR), 1.34 kg CODR/m3-day) compared to heat-shock operation (ξCOD, 52.6%; SDR, 1.10 kg CODR/m3-day). Heat-shock pretreatment has resulted in a marked fermentation pathway shift towards acetic-butyric acid type production. The microbial diversity illustrated dominance in the Clostridia class after applying heat-shock pretreatment. The redox catalytic currents and Tafel analysis strongly support the conclusion of an improved biocatalyst performance after pretreatment with regards to H2 production.

35 Lagier JC, et.al. (2014) Non contiguous-finished genome sequence and description of Clostridium jeddahense sp. nov. Stand Genomic Sci., 9(3):1003-19. Abstract Clostridium jeddahense strain JCDT (= CSUR P693 = DSM 27834) is the type strain of C. jeddahense sp. nov. This strain, whose genome is described here, was isolated from the fecal flora of an obese 24 year-old Saudian male (BMI=52 kg/m2). Clostridium jeddahense strain JCDT is an obligate Gram-positive bacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,613,503 bp long ge-nome (1 chromosome, no plasmid) exhibits a G+C content of 51.95% and contains 3,462 protein-coding and 53 RNA genes, including 4 rRNA genes.

36 Li T, Yan Y, He J. (2014) Reducing cofactors contribute to the increase of butanol production by a wild-type Clostridium sp. strain BOH3. Bioresour Technol, 155:220-8.

Abstract Availability of reducing factors (e.g., NADH and NADPH) plays an important role in improving the efficacy of products conversion in cofactor-dependent production systems. In this study, nicotinic acid (NA), the precursor of NADH and NADPH, was supplemented to the growth medium of a wild-type Clostridium sp. strain BOH3. Results showed that the addition of precursor NA to the medium led to a significant increase in the levels of NADH and NADPH. Meanwhile, a maximal cell growth rate and butanol generation rate were reached by applying a two-stage pH-shift strategy, achieving 18.7 g/L butanol with a yield of 24.6% and a productivity of 0.26 g/L h. The metabolic patterns were shifted towards more reduced metabolites as reflected by higher butanol-to-acetone ratio (11%) and butanol-to-acid ratio (292%). Redistributing metabolic flux to butanol via manipulations of reducing cofactor and pH shift could become an alternative tool to realize metabolic engineering goals.

37 Liu, Chaolan et.al. (2014) Clostridium swellfunianum sp. nov., a novel anaerobic bacterium isolated from the pit mud of Chinese Luzhou-flavor liquor production, Antonie van Leeuwenhoek, Vol. 106: 817-825 Abstract Abstract A novel Gram-positive, strictly anaerobic, spore-forming, rod-shaped bacterium, designated strain S11-3-10T, 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 14823T (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-C15:0, C16:0, C16:0 dma, C14:0, anteiso- C15:0 and iso-C13:0. 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 S11-3-10T (=DSM 27788T = JCM 19606T = CICC 10730T).

38 Marathe, NP, Shetty SA, Lanjekar VB, Rasane MH, Ranade DR, Shouche YS. (2014) Genome sequencing of multidrug resistant novel Clostridium sp. BL8 reveals its potential for pathogenicity. Gut Pathog, 6:30. Abstract Background: The human gut microbiome is important for maintaining the health status of the host. Clostridia are key members of the human gut microbiome, carrying out several important functions in the gut environment. Hence understanding the role of different Clostridium species isolated from human gut is essential. The present study was aimed at investigating the role of novel Clostridium sp. isolate BL8 in human gut using genome sequencing as a tool. Findings: The genome analysis of Clostridium sp. BL8 showed the presence of several adaptive features like bile resistance, presence of sensory and regulatory systems, presence of oxidative stress managing systems and presence of membrane transport systems. The genome of Clostridium sp. BL8 consists of a wide variety of virulence factors like phospholipase C (alpha toxin), hemolysin, aureolysin and exfoliative toxin A, as well as adhesion factors, proteases, Type IV secretion system and antibiotic resistance genes. In vitro antibiotic sensitivity testing showed that Clostridium sp. BL8 was resistant to 11 different tested antibiotics belonging to 6 different classes. The cell cytotoxicity assay confirmed the cytotoxic effect of Clostridium sp. BL8 cells, which killed 40% of the Vero cells after 4 hrs of incubation. Conclusions: Clostridium sp. BL8 has adapted for survival in human gut environment, with presence of different adaptive features. The presence of several virulence factors and cell cytotoxic activity indicate that Clostridium sp. BL8 has a potential to cause infections in humans, however further in vivo studies are necessary to ascertain this fact. Paek J, Lee MH, Kim

39 Mei, Nan, et.al. (2014) Fermentative hydrogen production by a new alkaliphilic Clostridium sp. (strain PROH2) isolated from a shallow submarine hydrothermal chimney in Prony Bay, New Caledonia, International Journal of Hydrogen Energy, Volume 39, Issue 34: 19465-19473, Abstract: The hydrogen-producing strain PROH2 pertaining to the genus Clostridium was successfully isolated from a shallow submarine hydrothermal chimney (Prony Bay, New Caledonia) driven by serpentinization processes. Cell biomass and hydrogen production performances during fermentation by strain PROH2 were studied in a series of batch experiments under various conditions of pH, temperature, NaCl and glucose concentrations. The highest hydrogen yield, 2.71 mol H2/mol glucose, was observed at initial pH 9.5, 37 °C, and glucose concentration 2 g/L, and was comparable to that reported for neutrophilic clostridial species. Hydrogen production by strain PROH2 reached the maximum production rate (0.55 mM-H2/h) at the late exponential phase. Yeast extract was required for growth of strain PROH2 and improved significantly its hydrogen production performances. The isolate could utilize various energy sources including cellobiose, galactose, glucose, maltose, sucrose and trehalose to produce hydrogen. The pattern of end-products of metabolism was also affected by the type of energy sources and culture conditions used. These results indicate that Clostridium sp. strain PROH2 is a good candidate for producing hydrogen under alkaline and mesothermic conditions.

40 Nomura, Toshiyuki, et.al. ( 2014) Isolation and characterization of a novel hydrogen-producing strain Clostridium sp. suitable for immobilization, International Journal of Hydrogen Energy, Vol. 39 (3): 1280-1287 Abstract A hydrogen-producing strain of bacteria suitable for immobilization was isolated from anaerobic sludge obtained from a methane fermentation plant. The isolated strain, CFPA-20 was identified as a novel species of the genus Clostridium by phylogenetic analysis of the 16S rRNA sequence. The changes in free energy of interaction of adhesion to polymer resin and self-aggregation were both negative. This indicated that CFPA-20 was thermodynamically favored for immobilization. CFPA-20 grew at a temperature range of 25–37 °C and at a pH range of 4.5–9.0. Immobilization of CFPA-20 on block copolymer polyethylene glycol-b-polypropylene glycol gave a radically improved hydrogen production yield (2.91 mol/mol-glucose) and a maximum hydrogen production rate (568 mL/L-culture/h) compared to the non-immobilized isolate. In addition, the biofilm of CFPA-20 acquired tolerance for volatile fatty acids. Further investigation into this mechanism may ultimately improve the hydrogen production capacity of CFPA-20.

41 Paek, Jayoung , et.al. (2014) Clostridium vulturis sp. nov., isolated from the intestine of the cinereous vulture (Aegypius monachus). Antonie van Leeuwenhoek,106:577–583 Abstract Abstract A Gram-stain positive, strict anaerobe, spore-forming, motile rod-shaped bacterial strain with peritrichous flagella, designated YMB-57T, was isolated from the intestine of a cinereous vulture (Aegypius monachus) in Korea. StrainYMB-57T 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 strainYMB-57T 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 x9c, and C18:1 x9c DMA and the DNA G?C content was determined to be 34.0 mol%. Phenotypic and phylogenetic differences indicate that strain YMB-57T is distinct from other Clostridium species. It is proposed that strain YMB-57T 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-57T (=KCTC 15114T = JCM 17998T).

42 Ruan, Z, et.al. (2014) Clostridium huakuii sp. nov., an anaerobic, acetogenic bacterium isolated from methanogenic consortia. Int J Syst Evol Microbiol., 64:4027-3 Abstract A Gram-staining-positive, spore-forming, obligate anaerobic acetogenic bacterium, designated LAM1030T, 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 LAM1030T 29 were motile, straight or spiral-rod-shaped. Strain LAM1030T could utilize glucose, fructose, maltose, galacatose, lactose, sucrose, cellobiose, ma 1 nnitol, pyruvate, succinic acid, tryptophan as sole carbon source. Acetic acid, isovaleric acid and butanoic acid were the main products of glucose fermentation. Sodium sulfite was used as electron acceptor. Growth of strain LAM1030T was completely inhibited by the addition of 20 μg/ml of ampicillin, tetracycline, gentamicin and erythromycin. The main polar lipids of strain LAM1030T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and eleven unknown glycolipids and two unknown phospholipids. No respiratory quinone was detected. The major fatty acids of strain LAM1030T were C16:0 (21.1%), C14:0 (10.3%) and iso-C15:0 (6.6%). The analysis on the 16S rRNA gene sequence indicated that strain LAM1030T belonged to the genus Clostridium and was most closely related to C. subterminale DSM 6970T, C. thiosulfatireducens DSM 13105T and C. sulfidigenes DSM 18982T, with 97.0%, 96.9% and 96.8% similarity, respectively. The G+C content of the genomic DNA of strain LAM1030T was 31.2±0.3 mol%. On the basis of its phenotypic, phylogenetic and chemotaxonomic characterization, strain LAM1030T was suggested to represent a novel species of the genus Clostridium, for which the name Clostridium huakuii sp. nov. is proposed. The type strain is LAM1030T (=ACCC 00698T = JCM 19186T).

43 Song, ZX, Li XH, Li WW, Bai YX, Fan YT, Hou HW. (2014) Direct bioconversion of raw corn stalk to hydrogen by a new strain Clostridium sp. FS3. Bioresour Technol., 157:91-7 Abstract A new strain FS3 which could achieve an efficient bioconversion of raw corn stalk to hydrogen had been isolated from anaerobic acclimated sludge, and identified as Clostridium butyricum on the basis of a series of physiological and biochemical experiments and 16S rDNA gene sequence. The strain could utilize various carbon sources to produce hydrogen. On the basis of single-factor experiments, the response surface methodology (RSM) was performed to optimize the media for hydrogen production. The maximum hydrogen yield of 92.9 ml/g was observed under the optimal conditions: 20 g/l raw corn stalk, 1.76 g/l NH4HCO3, 0.91 g/l KH2PO4 and 10.4 ml/l nutrient solution. This finding opens a new avenue for direct conversion of raw cellulosic biomass to bio-hydrogen.

44 Thomas, Leya, et.al. (2014) Xylanase and cellulase systems of Clostridium sp.: An insight on molecular approaches for strain improvement, Bioresource Technology, Volume 158: 343-350, Abstract: Bioethanol and biobutanol hold great promise as alternative biofuels, especially for transport sector, because they can be produced from lignocellulosic agro-industrial residues. From techno-economic point of view, the bioprocess for biofuels production should involve minimal processing steps. Consolidated bioprocessing (CBP), which combines various processing steps such as pretreatment, hydrolysis and fermentation in a single bioreactor, could be of great relevance for the production of bioethanol and biobutanol or solvents (acetone, butanol, ethanol), employing clostridia. For CBP, Clostridium holds best promise because it possesses multi- enzyme system involving cellulosome and xylanosome, which comprise several enzymes such as cellulases and xylanases. The aim of this article was to review the recent developments on enzyme systems of clostridia, especially xylanase and cellulase with an effort to analyse the information available on molecular approaches for the improvement of strains with ultimate aim to improve the efficiencies of hydrolysis and fermentation.

45 Tinggang Li, Yu Yan, Jianzhong He, (2014) Reducing cofactors contribute to the increase of butanol production by a wild-type Clostridium sp. strain BOH3, Bioresource Technology, Vol. 155: 220-228 Abstract Availability of reducing factors (e.g., NADH and NADPH) plays an important role in improving the efficacy of products conversion in cofactor-dependent production systems. In this study, nicotinic acid (NA), the precursor of NADH and NADPH, was supplemented to the growth medium of a wild-type Clostridium sp. strain BOH3. Results showed that the addition of precursor NA to the medium led to a significant increase in the levels of NADH and NADPH. Meanwhile, a maximal cell growth rate and butanol generation rate were reached by applying a two-stage pH-shift strategy, achieving 18.7 g/L butanol with a yield of 24.6% and a productivity of 0.26 g/L h. The metabolic patterns were shifted towards more reduced metabolites as reflected by higher butanol-to-acetone ratio (11%) and butanol-to-acid ratio (292%). Redistributing metabolic flux to butanol via manipulations of reducing cofactor and pH shift could become an alternative tool to realize metabolic engineering goals.

46 Wong, YM, Juan JC, Ting A, Wu TY, Gan HM, Austin CM. (2014) Draft Genome Sequence of Clostridium sp. Strain Ade.TY, a New Biohydrogen- and Biochemical-Producing Bacterium Isolated from Landfill Leachate Sludge. Genome Announc., 2(2): e00078-14. Abstract Clostridium sp. strain Ade.TY is potentially a new biohydrogen-producing species isolated from landfill leachate sludge. Here we present the assembly and annotation of its genome, which may provide further insights into its gene interactions for efficient biohydrogen production.

47 Wu YF, Zheng H, Wu QL, Yang H, Liu SJ. (2014) Clostridium algifaecis sp. nov., an anaerobic bacterial species from decomposing algal scum. Int J Syst Evol Microbiol.,64(Pt 11):3844-8. Abstract Two anaerobic bacterial strains, MB9-7T 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 MB9-7T 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 6C, 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 C14 : 0 and C16 : 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 MB9-7T 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 MB9-7T and MB9-9 represent a novel species of the genus Clostridium, with the suggested name Clostridium algifaecis sp. nov. The type strain is MB9-7T (5CGMCC 1.5188T5DSM 28783T).

48 Xin F, Wu YR, He J. (2014) Simultaneous fermentation of glucose and xylose to butanol by Clostridium sp. strain BOH3. Appl Environ Microbiol.,80(15):4771-8. Abstract Cellulose and hemicellulose constitute the major components in sustainable feedstocks, which could be used as substrates for biofuel generation. However, following hydrolysis to monomer sugars, the solventogenic Clostridium will preferentially consume glucose due to transcriptional repression of xylose utilization genes. This is one of the major barriers in making the best of lignocellulosic hydrolysates to produce butanol. In comparison with studies on existing bacteria, this study demonstrates that a newly reported Clostridium sp. strain BOH3 is capable of fermenting 60 g/L of xylose to 14.9 g/L butanol, which is similar to the 14.5 g/L butanol produced from 60 g/L of glucose. More importantly, strain BOH3 consumes glucose and xylose simultaneously, which is shown by its capability for generating 11.7 g/L butanol from a horticultural waste cellulosic hydrolysate containing 39.8 g/L glucose and 20.5 g/L xylose; as well as producing 11.9 g/L butanol from another horticultural waste hemicellulosic hydrolysate containing 58.3 g/L xylose and 5.9 g/L glucose. The high xylose utilization capability in strain BOH3 is attributed to its high xylose-isomerase (0.97 U/mg protein) and xylulokinase activities (1.16 U/mg protein) when compared with low-xylose utilizing solventogenic strains, such as Clostridium sp. strain G117. Interestingly, strain BOH3 was also found to produce riboflavin, 110.5 mg/L from xylose and 76.8 mg/L from glucose during the fermentation process. In summary, Clostridium sp. strain BOH3 is an attractive candidate for application in efficiently converting lignocellulosic hydrolysates to biofuels and other value-added products such as riboflavin.

49 Zhang, C., S.V. Malhotra, A.J. Francis,( 2014) Toxicity of ionic liquids to Clostridium sp. and effects on uranium biosorption, Journal of Hazardous Materials, Volume 264: 246-253, Abstract: As green solvents ionic liquids (ILs) show high potential in nuclear industry for extraction and purification of actinides. However, to date relatively little information has been gained on ILs application in microbial processes, for example biosorption of radionuclides. We investigated the effects of three ILs, 1-butyl-3- methylimidazolium hexafluorophosphate (BMIMPF6), N-ethylpyridinium trifluoroacetate (EtPyCF3COO) and N-ethylpyridinium tetrafluoroborate (EtPyBF4) on the growth and biosorption of uranium by Clostridium sp. The ILs affected the growth of the bacterium as evidenced by decreases in optical density, total gas production, and organic acids production from glucose metabolism. The IC50-48 h of three ILs decreased in the order of BMIMPF6 (8.26 mM) > EtPyBF4 (7.04 mM) > EtPyCF3COO (4.05 mM). Uranium biosorption by the bacterial cells decreased by 75% in the presence of 1% (v/v) BMIMPF6 and by about 90% with 1% (v/v) EtPyBF4 or EtPyCF3COO, in comparison to the control without ILs. The diminished biosorption may be attributed to the membrane damages induced by EtPyBF4 and EtPyCF3COO, which can be visualized by Transmission Electron Microscope (TEM) analysis. Energy-dispersive X-ray spectroscopy (EDS) analysis revealed the accumulation of uranium inside peripheral membrane of the cells exposed to uranium alone or with BMIMPF6, while little or no accumulation was observed in the presence of EtPyBF4 and EtPyCF3COO. These results imply that potential toxicity of ILs towards microorganisms is a particularly important issue in limiting its biotechnological applications.

50 Zhao-Xia, Song, et.al.(2014)Direct bioconversion of raw corn stalk to hydrogen by a new strain Clostridium sp. FS3, Bioresource Technology, Volume 157, April, Pages 91-97, Abstract A new strain FS3 which could achieve an efficient bioconversion of raw corn stalk to hydrogen had been isolated from anaerobic acclimated sludge, and identified as Clostridium butyricum on the basis of a series of physiological and biochemical experiments and 16S rDNA gene sequence. The strain could utilize various carbon sources to produce hydrogen. On the basis of single-factor experiments, the response surface methodology (RSM) was performed to optimize the media for hydrogen production. The maximum hydrogen yield of 92.9 ml/g was observed under the optimal conditions: 20 g/l raw corn stalk, 1.76 g/l NH4HCO3, 0.91 g/l KH2PO4 and 10.4 ml/l nutrient solution. This finding opens a new avenue for direct conversion of raw cellulosic biomass to bio-hydrogen.

51 Berzin V, Tyurin M, Kiriukhin M. (2013) Selective n-butanol production by Clostridium sp. MTButOH1365 during continuous synthesis gas fermentation due to expression of synthetic thiolase, 3-hydroxy butyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase, and NAD-dependent butanol dehydrogenase. Appl Biochem Biotechnol.,169:950-9. Abstract Acetogen Clostridum sp. MT1962 produced 287 mM acetate (p<0.005) and 293 mM ethanol (p<0.005) fermenting synthesis gas blend 60 % CO and 40 %H2 in single-stage continuous fermentation. This strain was metabolically engineered to the biocatalyst Clostridium sp. MTButOH1365. The engineered biocatalyst lost production of ethanol and acetate while initiated the production of 297 mM of n-butanol (p<0.005). The metabolic engineering comprised Cre-lox66/lox71-based elimination of phosphotransacetylase and acetaldehyde dehydrogenase along with integration to chromosome synthetic thiolase, 3-hydroxy butyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase, and NAD-dependent butanol dehydrogenase. This is the first report on elimination of acetate and ethanol production genes and expression of synthetic gene cluster encoding n-butanol biosynthesis pathway in acetogen biocatalyst for selective fuel n-butanol production with no antibiotic support for the introduced genes

52 Jeon, BS, Moon C, Kim BC, Kim H, Um Y, Sang BI. (2013) In situ extractive fermentation for the production of hexanoic acid from galactitol by Clostridium sp. BS-1. Enzyme Microb Technol.,53(3):143-51. Abstract Clostridium sp. BS-1 produces hexanoic acid as a metabolite using galactitol and enhanced hexanoic acid production was obtained by in situ extractive fermentation with Clostridium sp. BS-1 under an optimized medium composition. For medium optimization, five ingredients were selected as variables, and among them yeast extract, tryptone, and sodium butyrate were selected as significant variables according to a fractional factorial experimental design, a steepest ascent experimental design, and a Box–Behnken experimental design. The optimized medium had the following compositions in modified Clostridium acetobutyricum (mCAB) medium: 15.5 g L−1 of yeast extract, 10.13 g L−1 of tryptone, 0.04 g L−1 of FeSO4·7H2O, 0.85 g L−1 of sodium acetate, and 6.47 g L−1 of sodium butyrate. The predicted concentration of hexanoic acid with the optimized medium was 6.98 g L−1, and this was validated experimentally by producing 6.96 g L−1 of hexanoic acid with Clostridium sp. BS-1 under the optimized conditions. In situ extractive fermentation for hexanoic acid removal was then applied in a batch culture system with the optimized medium and 10% (v/v) alamine 336 in oleyl alcohol as an extractive solvent. The pH of the culture in the extractive fermentation was maintained at 5.4–5.6 by an acid balance between production and retrieval by extraction. During a 16 day culture, the hexanoic acid concentration in the solvent increased to 32 g L−1 while it was maintained in a range of 1–2 g L−1 in the medium. The maximum rate of hexanoic acid production was 0.34 g L−1 h−1 in in situ extractive fermentation.

53 Jinling, Cai, et.al. (2013) Fermentative hydrogen production by a new mesophilic bacterium Clostridium sp. 6A-5 isolated from the sludge of a sugar mill, Renewable Energy, Vol. 59,: 202-209, Abstract The fermentative hydrogen production capability of the newly isolated Clostridium sp. 6A-5 bacterium was studied in a batch cultivation experiment. Various culture conditions (temperature, initial pH, and glucose concentration) were evaluated for their effects on cell growth and hydrogen production (including the yield and rate) of Clostridium sp. 6A-5. Optimal cell growth was observed at 40 °C, initial pH 7.5–8, and glucose concentration 16–26 g/L. The optimal hydrogen yield was obtained at 43 °C, initial pH 8, and glucose concentration 10–16 g/L. Hydrogen began to evolve when cell growth entered the mid-exponential phase and reached the maximum production rate at the late exponential and stationary phases. The maximum hydrogen yield, and rate were 2727 mL/L, and 269.3 mL H2/L h, respectively. These results indicate that Clostridium sp. 6A-5 is a good candidate for mesophilic fermentative hydrogen production.

54 Komonkiat, Itsara, Benjamas Cheirsilp, (2013) Felled oil palm trunk as a renewable source for biobutanol production by Clostridium spp., Bioresource Technology, Volume 146: 200-207, Abstract 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.

55 Monlau, Florian, et.al. (2013) Specific inhibition of biohydrogen-producing Clostridium sp. after dilute-acid pretreatment of sunflower stalks, International Journal of Hydrogen Energy, Vol. 38, Issue 28: 12273-12282, Abstract: Dilute-acid pretreatments are commonly used to solubilize holocelluloses of lignocellulosic materials and represent a promising route to enhance biohydrogen production by dark fermentation. Besides the soluble sugars released, furan derivatives, such as furfural and 5-HMF, as well as phenolic compounds can accumulate in dilute-acid hydrolyzates and that may affect fermentative microbial populations. In this study, biohydrogen production from glucose (5 g VS L−1) in batch tests was investigated in presence of increasing volumes (0% – control, 3.75%, 7.5%, 15% and 35% (v/v)) of dilute acid hydrolyzate generated from sunflower stalks (170 °C, 1 h, 4 g HCl/100 gTS). A sharp decrease of the hydrogen yield was observed from 2.04 mol H2 mol−1 eq. hexose initial in the control to 0 mol H2 mol−1 eq. hexose initial for volumes higher than 15% of added hydrolyzate. Although acetate and butyrate were the main end-products found in the control, ethanol and lactate accumulated accordingly with the increasing addition of hydrolyzate. A clear shift of dominant microbial populations from Clostridium sp. to Sporolactobacillus sp. was concomitantly observed, suggesting a specific inhibition of the biohydrogen-producing bacteria by adding increasing volumes of hydrolyzates.

56 Mu W, Zhang W, Fang D, Zhou L, Jiang B, Zhang T. (2013) Characterization of a D-psicose- producing enzyme, D-psicose 3-epimerase, from Clostridium sp. Biotechnol Lett.,35(9):1481-6. Abstract The gene coding for D-psicose 3-epimerase (DPEase) from Clostridium sp. BNL1100 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by Ni-affinity chromatography. It was a metal- dependent enzyme and required Co2? As optimum cofactor. It displayed catalytic activity maximally at pH 8.0 and 65 _C (as measured over 5 min). The optimum substrate was D-psicose, and the Km, turnover number (kcat), and catalytic efficiency (kcat/Km) for D-psicose were 227 mM, 32,185 min-1, and 141 min-1 mM-1, respectively. At pH 8.0 and 55 _C, 120 g D-psicose l-1 was produced from 500 g D-fructose l-1 after 5 h.

57 Panitz, J.C., et.al. ( 2013) Isolation of a solventogenic Clostridium sp. strain: Fermentation of glycerol to n-butanol, analysis of the bcs operon region and its potential regulatory elements, Systematic and Applied Microbiology, Volume 37 (1): 1-9, Abstract A new solventogenic bacterium, strain GT6, was isolated from standing water sediment. 16S-rRNA gene analysis revealed that GT6 belongs to the heterogeneous Clostridium tetanomorphum group of bacteria exhibiting 99% sequence identity with C. tetanomorphum 4474T. GT6 can utilize a wide range of carbohydrate substrates including glucose, fructose, maltose, xylose and glycerol to produce mainly n-butanol without any acetone. Additional products of GT6 metabolism were ethanol, butyric acid, acetic acid, and trace amounts of 1,3-propanediol. Medium and substrate composition, and culture conditions such as pH and temperature influenced product formation. The major fermentation product from glycerol was n-butanol with a final concentration of up to 11.5 g/L. 3% (v/v) glycerol lead to a total solvent concentration of 14 g/L within 72 h. Growth was not inhibited by glycerol concentrations as high as 15% (v/v).

58 Rosewarne, CP, et.al. (2013) Draft Genome Sequence of Clostridium sp. Maddingley, Isolated from Coal-Seam Gas Formation Water. Genome Announc., 1(1): e00081-12. Abstract Clostridium sp. Maddingley was isolated as an axenic culture from a brown coal-seam formation water sample collected from Victoria, Australia. It lacks the solventogenesis genes found in closely related clostridial strains. Metabolic reconstructions suggest that volatile fatty acids are the main fermentation end products.

59 Singh, Lakhveer, et.al. (2013) Application of polyethylene glycol immobilized Clostridium sp. LS2 for continuous hydrogen production from palm oil mill effluent in upflow anaerobic sludge blanket reactor, Biochemical Engineering Journal, Volume 70: 158-165, Abstract: A novel polyethylene glycol (PEG) gel was fabricated and used as a carrier to immobilize Clostridium sp. LS2 for continuous hydrogen production in an upflow anaerobic sludge blanket (UASB) reactor. Palm oil mill effluent (POME) was used as the substrate carbon source. The optimal amount of PEG-immobilized cells for anaerobic hydrogen production was 12% (w/v) in the UASB reactor. The UASB reactor containing immobilized cells was operated at varying hydraulic retention times (HRT) that ranged from 24 to 6 h at 3.3 g chemical oxygen demand (COD)/L/h organic loading rate (OLR), or at OLRs that ranged from 1.6 to 6.6 at 12 h HRT. The best volumetric hydrogen production rate of 336 mL H2/L/h (or 15.0 mmol/L/h) with a hydrogen yield of 0.35 L H2/g CODremoved was obtained at a HRT of 12 h and an OLR of 5.0 g COD/L/h. The average hydrogen content of biogas and COD reduction were 52% and 62%, respectively. The major soluble metabolites during hydrogen fermentation were butyric acid followed by acetic acid. It is concluded that the PEG-immobilized cell system developed in this work has great potential for continuous hydrogen production from real wastewater (POME) using the UASB reactor.

60 Taghavi, S, Izquierdo JA, van der Lelie D. ( 2013) Complete Genome Sequence of Clostridium sp. Strain DL-VIII, a Novel Solventogenic Clostridium Species Isolated from Anaerobic Sludge. Genome Announc, 1(4): e00605-13. Abstract We report the genome sequence of Clostridium sp. strain DL-VIII, a novel Gram-positive, endospore-forming, solventogenic bacterium isolated from activated anaerobic sludge of a wastewater treatment plant. Aside from a complete sol operon, the 6,477,357-bp genome of DL-VIII reveals genes for several unique enzymes with applications in lignocellulose degradation, including two phenolic acid decarboxylases.

61 Zhang, C., C.J. Dodge, S.V. Malhotra, A.J. Francis, (2013) Bioreduction and precipitation of uranium in ionic liquid aqueous solution by Clostridium sp., Bioresource Technology, 136: 752-756, Abstract: The ionic liquids, 1-butyl-3-methylimidazolium hexafluorophosphate [BMIM][PF6], N- ethylpyridiniumtrifluoroacetate [EtPy][CF3COO] and N-ethylpyridiniumtetrafluoroborate [EtPy][BF4], affected the reduction and precipitation of uranium by Clostridium sp. to a varying degree. Characterization of uranium association with the ionic liquids showed that uranium formed a monodentate complex with the anion BF4− and PF6− of [EtPy][BF4] and [BMIM][PF6], respectively; and a bidentate complex with carboxylate of [EtPy][CF3COO]. Bioreduction of U(VI) was influenced by the type of complex formed: monodentate complexes were readily reduced whereas the bidentate complex of U(VI) with [CF3COO] was recalcitrant. [EtPy][BF4] affected the rate and extent of precipitation of the reduced uranium; at higher concentration the reduced U(IV) remained in the solution phase. The results suggest that by tuning the properties of ionic liquids they may be valuable candidates for uranium biotreatment.

62 Bao, Peng, et.al. (2012) Dechlorination of p,p′-DDTs coupled with sulfate reduction by novel sulfate- reducing bacterium Clostridium sp. BXM, Environmental Pollution, Volume 162: 303-310, Abstract: A novel non-dsrAB (without dissimilatory sulfite reductase genes) sulfate-reducing bacterium (SRB) Clostridium sp. BXM was isolated from a paddy soil. Incubation experiments were then performed to investigate the formation of reduced sulfur compounds (RSC) by Clostridium sp. BXM, and RSC-induced dechlorination of p,p′-DDT in culture medium and soil solution. The RSCs produced were 5.8 mM and 4.5 mM in 28 mM sulfate amended medium and soil solution respectively after 28-day cultivation. The p,p′-DDT dechlorination ratios were 74% and 45.8% for 5.8 mM and 4.5 mM RSCs respectively at 6 h. The metabolites of p,p′-DDT found in the two reaction systems were identified as p,p′-DDD and p,p′-DDE. The dechlorination pathways of p,p′-DDT to p,p′-DDD and p,p′-DDE were proposed, based on mass balance and dechlorination time-courses. The results indicated that RSC-induced natural dechlorination may play an important role in the fate of organochlorines.

63 Berzin V, Kiriukhin M, Tyurin M. (2012) Elimination of acetate production to improve ethanol yield during continuous synthesis gas fermentation by engineered biocatalyst Clostridium sp. MTEtOH550. Appl Biochem Biotechnol.,167(2):338-47. Abstract Acetogen strain Clostridum sp. MT653 produced acetate 273 mM (p<0.005) and ethanol 250 mM (p<0.005) from synthesis gas blend mixture of 64 % CO and 36 %H2. Clostridum sp. MT653 was metabolically engineered to the biocatalyst strain Clostridium sp. MTEtOH550. The biocatalyst increased ethanol yield to 590 mM with no acetate production during single-stage continuous syngas fermentation due to expression of synthetic adh cloned in a multi-copy number expression vector. The acetate production was eliminated by inactivation of the pta gene in Clostridium sp. MTEtOH550. Gene introduction and gene elimination were achieved only using Syngas Biofuels Energy, Inc. Electroporation generator. The electrotransformation efficiencies were 8.0±0.2×106 per microgram of transforming DNA of the expression vector at cell viability ~15 %. The frequency of suicidal vector integration to inactivate pta was ~10−5 per the number of recipient cells. This is the first report on elimination of acetate production and overexpression of synthetic adh gene to engineer acetogen biocatalyst for selective biofuel ethanol production during continuous syngas fermentation.

64 Berzin V, Kiriukhin M, Tyurin M. (2012) Selective production of acetone during continuous synthesis gas fermentation by engineered biocatalyst Clostridium sp. MAceT113. Lett Appl Microbiol., 55(2):149-54. Abstract Aims: To engineer acetogen biocatalyst capable of fermenting synthesis gas blend to acetone as the only liquid carbonaceous product. Methods and Results: The metabolic engineering comprised inactivation of phosphotransa- cetylase via integration of a cassette comprising synthetic genes erm(B), thiolase and HMG-CoA synthase. Acetaldehyde dehydrogenase was inactivated via integration of a cassette consisting of synthetic genes cat, HMGCo A lyase and acetoacetate decarboxylase. The engineered biocatalyst Clostridum sp. MAceT113 lost production of 253 mmol l)1 ethanol and 296 mmol l)1 acetate and started producing 1Æ8 mol l)1 acetone in single-stage continuous syngas fermentation. Conclusions: The acetone concentration in culture broth is economical for bulk manufacture because it is about twenty times of that achieved with known acetone–butanol–ethanol fermentation of sugars. Significance and Impact of the Study: The process shows the opportunity to produce acetone from synthesis gas at concentrations comparable with production of acetone from products of petroleum cracking. This is the first report on elimination of acetate and acetaldehyde production and directing carbon flux from Acetyl-CoA to acetone via a non- naturally occurring in acetogen acetone biosynthesis pathway identified in eukaryotic organisms.

65 Ibnoulkhatib, A., et.al. (2012) Infections ostéoarticulaires post-traumatiques des membres par Clostridium spp., Revue de Chirurgie Orthopédique et Traumatologique, Vol. 98, Issue 6: 626-636, Abstract: Les Clostridium sont des bacilles à gram positif saprophytes telluriques. Ils sont capables de sporulation. Secondairement, ils peuvent se multiplier lorsque les conditions redeviennent favorables. Ces infections peuvent compliquer les fractures ouvertes et les plaies profondes à contamination tellurique.Hypothèse Les infections à Clostridium constituent des évènements particulièrement graves en traumatologie aux résultats incertains malgré un traitement médicochirurgical agressif. Patients et méthode Cette étude rétrospective (12 patients d’âge médian 45 ans) porte sur les infections ostéoarticulaires à Clostridium à la suite d’une fracture ouverte de membre souillée par de la terre et associée à d’importantes lésions des parties molles. Une antibioprophylaxie par amoxicilline-acide clavulanique a été réalisée suivie d’un parage et d’un lavage chirurgical en urgence. L’ostéosynthèse a été réalisée d’emblée chez neuf patients (quatre fixateurs externes et cinq ostéosynthèses internes) et trois fois, différée. Les suites immédiates ont été défavorables dans tous les cas, ce qui a amené à une reprise chirurgicale précoce au délai médian de dix jours (5–25). Résultats La souche de Clostridium était identifiée au délai médian de 14,5 jours (5–160), elle était toujours associée à d’autres germes. Le traitement médicochirurgical de l’infection à Clostridium par débridement, parage et ablation du matériel (quatre fois) et antibiothérapie a abouti à une évolution favorable un fois sans récidive au recul de deux ans et défavorable 11 fois avec retard de consolidation, pseudarthrose septique, troubles cicatriciels et écoulement. Devant cette évolution défavorable, des traitements de « deuxième ligne » ont été réalisés : quatre enclouages centro-médullaires sans greffe osseuse ayant échoué trois fois, deux décortications-greffes ayant conduit à deux échecs, un enclou age avec décortication qui a eu une évolution favorable. Dans quatre cas, une intervention de type membrane induite selon Masquelet a permis la guérison de l’infection. Au recul médian de 24 mois (18– 53), huit infections osseuses étaient guéries et quatre étaient en pseudarthrose septique. Discussion Les infections à Clostridium constituent des infections d’une particulière gravité. Elles sont diagnostiquées tardivement et l’identification des germes est délicate. Le traitement de ces infections est difficile avec une évolution défavorable dans un cas sur trois. Dès lors qu’un Clostridium est identifié dans une infection ostéoarticulaire, la priorité est à la réalisation de gestes chirurgicaux de résection très larges et agressifs car les traitements antibiotiques sont parfois inefficaces in vivo du fait de la résistance des spores. Niveau de preuve IV. Étude rétrospective de cohorte.

66 Ibnoulkhatib, A., et.al. (2012) Post-traumatic bone and/or joint limb infections due to Clostridium spp., Orthopaedics & Traumatology: Surgery & Research, Vol. 98, Issue 6: 696-705, Abstract Clostridium spp. are saprophytic Gram-positive bacteria found in soil and capable of generating endospores. Spore germination occurs when environmental conditions are favorable. Clostridium spp. can cause infections of compound fractures and deep wounds contaminated from soil micro-organisms. Hypothesis Clostridium spp. infections of traffic-related injuries are particularly severe events whose outcome is uncertain even with aggressive medical and surgical treatment. Materials and methods We retrospectively reviewed 12 patients (median age, 45 years) with Clostridium spp. bone and/or joint infections complicating compound limb fractures with soil contamination and extensive soft-tissue damage. Prophylactic amoxicillin-clavulanic acid therapy was administred, followed by emergency surgical wound debridment and lavage. Fracture fixation was performed immediately in nine patients (external in four and internal in five) or at a later time on three patients. The immediate outcome was unfavourable in all 12 cases, requiring early reoperation after a median of 10 days (range, 5–25 days). Results Median time to Clostridium strain identification was 14.5 days (range, 5–160). All infections were polymicrobial. Surgical wound excision, hardware removal (in four cases), and antibiotic therapy produced a favourable outcome in one patient, with no recurrence after 2 years of follow-up; the outcome was unfavourable in 11 cases, with delayed fracture union, septic non-union, impaired healing, and/or chronic sinus tract drainage. Several second-line treatments were used in these 11 patients: intramedullary nailing without bone grafting in four patients, with three failures; decortication and grafting in two patients, with failure in both; nailing with decortication in one patient, who had a good outcome; and the induced membrane procedure described by Masquelet in four patients, all of whom had good outcomes. After a median follow-up of 24 months (range, 18–53 months), the bone infection had subsided in eight patients. The remaining four patients had septic non-union. Discussion Clostridium spp. infections are particularly severe. The diagnosis is delayed and identification of the organism is challenging. The treatment is difficult and results in unfavorable outcomes in one-third of cases. The identification of Clostridium in specimens from an osteoarticular infection indicates a need for extremely extensive and aggressive surgical resection, as spore resistance may impair the in vivo efficacy of antimicrobial agents.

67 Lei Zhao, Guang-li Cao, et.al. (2012) Enhanced bio-hydrogen production by immobilized Clostridium sp. T2 on a new biological carrier, International Journal of Hydrogen Energy, Vol. 37: 162-166, Abstract: Biological mycelia pellets, which are formed spontaneously in the process of Aspergillus niger Y3 fermentation, were explored as carrier for immobilization of Clostridium sp. T2 to improve hydrogen production. Batch fermentation tests showed that optimal dosage and size of mycelia pellets for hydrogen production were 0.350 g 150 ml−1 medium and 1.5 mm. Under these conditions, hydrogen production with immobilized cells on mycelia pellets was further investigated in continuous stirred-tank reactor (CSTR) with hydraulic retention time (HRT) ranging from 12 to 8 h. It obtained that the maximum hydrogen production rate reached 2.76 mmol H2 L−1 h−1 at 10 h HRT, which was 40.8% higher than the carrier-free process, but slightly lower than the counterpart immobilized in sodium alginate with the value of 3.15 mmol H2 L−1 h−1. SEM observation showed that abundant cells were closely adhered to mycelia pellets. The present results indicate the potential of using mycelia pellets as biological carrier for enhancing hydrogen production.

68 Li LL, Taghavi S, Izquierdo JA, van der Lelie D.(2012) Complete genome sequence of Clostridium sp. strain BNL1100, a cellulolytic mesophile isolated from corn stover. J Bacteriol., 194(24):6982-3. Abstract We present the full genome sequence of Clostridium sp. strain BNL1100, a Gram-positive, endospore-forming, lignocellulolytic bacterium isolated from a corn stover enrichment culture. The 4,613,747-bp genome of strain BNL1100 contains 4,025 putative protein-coding genes, of which 103 are glycoside hydrolases, the highest detected number in cluster III clostridia.

69 Ling Jiang, Shuang Li, Yi Hu, Qing Xu, He Huang (2012) Adaptive Evolution for Fast Growth on Glucose and the Effects on the Regulation of Glucose Transport System in Clostridium tyrobutyricum. Biotechnol. Bioeng.;109: 708–718. Abstract Laboratory adaptive evolution of microorganisms offers the possibility of relating acquired mutations to increased fitness of the organism under the conditions used. By combining a fibrous-bed bioreactor, we successfully developed a simple and valuable adaptive evolution strategy in repeated-batch fermentation mode with high initial substrate concentration and evolved Clostridium tyrobutyricum mutant with significantly improved butyric acid volumetric productivity up to 2.25 g/(L h), which is the highest value in batch fermentation reported so far. Further experiments were conducted to pay attention to glucose transport system in consideration of the high glucose consumption rate resulted from evolution. Complete characterization and comparison of the glucose phosphoenolpyruvate (PEP)- dependent phosphotransferase system (PTS) were carried out in the form of toluene-treated cells and cell-free extracts derived from both C. tyrobutyricum wide- type and mutant, while an alternative glucose transport route that requires glucokinase was confirmed by the phenomena of resistance to the glucose analogue 2-deoxyglucose and ATP-dependent glucose phosphorylation. Our results suggest that C. tyrobutyricum mutant is defective in PTS activity and compensates for this defect with enhanced glucokinase activity, resulting in the efficient uptake and consumption of glucose during the whole metabolism.

70 Richter, Hanno et.al. (2012) Prolonged Conversion of n-Butyrate to n-Butanol with Clostridium saccharoperbutylacetonicum in a Two-Stage Continuous Culture with in-situ Product Removal. Biotechnol. Bioeng. 2012;109: 913–921 Abstract n-Butanol was produced continuously in a two-stage fermentor system with integrated product removal from a co-feed of n-butyric acid and glucose. Glucose was always required as a source of ATP and electrons for the conversion of n-butyrate to n-butanol and for biomass growth; for the latter it also served as a carbon source. The first stage generated metabolically active planktonic cells of Clostridium saccharoperbutylacetonicum strain N1-4 that were continuously fed into the second (production) stage; the volumetric ratio of the two fermentors was 1:10. n-Butanol was removed continuously from the second stage via gas stripping. Implementing a two-stage process was observed to dramatically dampen metabolic oscillations (i.e., periodical changes of solventogenic activity). Culture degeneration (i.e., an irreversible loss of solventogenic activity) was avoided by periodical heat shocking and reinoculating stage 1 and by maintaining the concentration of undissociated n-butyric acid in stage 2 at 3.4mMwith a pHauxostat. The system was successfully operated for 42 days during which 93% of the fed n-butyrate was converted to n-butanol at a production rate of 0.39 g/(L_h). The molar yields Yn-butanol/n-butyrate and Yn-butanol/glucose were 2.0, and 0.718, respectively. For the same run, the molar ratio of nbutyrate to glucose consumed was 0.358. The molar yield of carbon in n- butanol produced from carbon in n-butyrate and glucose consumed (Yn-butanol/carbon) was 0.386. These data illustrate that conversion of n-butyrate into n-butanol by solventogenic Clostridium species is feasible and that this can be performed in a continuous system operating for longer than a month. However, our data also demonstrate that a relatively large amount of glucose is required to supply electrons and ATP for this conversion and for cell growth in a continuous culture.

71 Yarlagadda, Venkata Nancharaiah, et.al. (2012) Effect of exogenous electron shuttles on growth and fermentative metabolism in Clostridium sp. BC1, Bioresource Technology, Vol. 108: 295-299, Abstract: In this study, the influence exogenous electron shuttles on the growth and glucose fermentative metabolism of Clostridium sp. BC1 was investigated. Bicarbonate addition to mineral salts (MS) medium accelerated growth and glucose fermentation which shifted acidogenesis (acetic- and butyric-acids) towards solventogenesis (ethanol and butanol). Addition of ferrihydrite, anthraquinone disulfonate, and nicotinamide adenine dinucleotide in bicarbonate to growing culture showed no significant influence on fermentative metabolism. In contrast, methyl viologen (MV) enhanced ethanol- and butanol-production by 28- and 12-fold, respectively with concomitant decrease in hydrogen, acetic- and butyric-acids compared to MS medium. The results show that MV addition affects hydrogenase activity with a significant reduction in hydrogen production and a shift in the direction of electron flow towards enhanced production of ethanol and butanol.

72 Zhang, Yali, Mingrui Yu, and Shang-Tian Yang (2012) Effects of ptb Knockout on Butyric Acid Fermentation by Clostridium tyrobutyricum. Biotechnol. Prog., 28: 52–59, Abstract Clostridium tyrobutyricum ATCC 25755 is an anaerobic, rod-shaped, gram-positive bacterium that produces butyrate, acetate, hydrogen, and carbon dioxide from various saccharides, including glucose and xylose. Phosphotransbutyrylase (PTB) is a key enzyme in the butyric acid synthesis pathway. In this work, effects of ptb knockout by homologous recombination on metabolic flux and product distribution were investigated. When compared with the wild type, the activities of PTB and butyrate kinase in ptb knockout mutant decreased 76 and 42%, respectively; meanwhile, phosphotransacetylase and acetate kinase increased 7 and 29%, respectively. However, ptb knockout did not significantly reduce butyric acid production from glucose or xylose in batch fermentations. Instead, it increased acetic acid and hydrogen production 33.3_53.8% and _11%, respectively. Thus, the ptb knockout did increase the carbon flux toward acetate synthesis, resulting in a significant decrease (28_35% reduction) in the butyrate/acetate ratio in ptb mutant fermentations. In addition, the mutant displayed a higher specific growth rate (0.20 h_1 vs. 0.15 h_1 on glucose and 0.14 h_1 vs. 0.10 h_1 on xylose) and tolerance to butyric acid. Consequently, batch fermentation with the mutant gave higher fermentation rate and productivities (26_48% increase for butyrate, 81_100% increase for acetate, and 38_46% increase for hydrogen). This mutant thus can be used more efficiently than the parental strain in fermentations to produce butyrate, acetate, and hydrogen from glucose and xylose.

73 Chun-Hsiung Hung, et.al. (2011) Interactions between Clostridium sp. and other facultative anaerobes in a self-formed granular sludge hydrogen-producing bioreactor, International Journal of Hydrogen Energy, Volume 36, Issue 14, July, Pages 8704-8711, Abstract: The interactions of Clostridium sp. and other non-hydrogen producing bacteria directly influence anaerobic hydrogen production. In this study, bacteria in a sucrose-feeding hydrogen-producing bioreactor were investigated via 16S rDNA-based analysis. Results showed that Clostridium pasteurianum, Klebsiella sp., and Streptococcus sp. were the predominant microorganisms. The Streptococcus sp. cells were found to localize inside hydrogen-producing granular sludge and were surrounded by clostridia. Significant oxygen consumption was found in the Klebsiella sp. pure culture experiment, in which oxidation reduction potential (ORP) dropped from 100 to −500 mV during the log phase within 2 h. Oxygen consumption by Streptococcus sp. was not significant, and it accumulated EPS under anaerobic conditions. Results suggest that Klebsiella sp. first utilized the oxygen to form anaerobic conditions in this system. Streptococcus sp., on the other hand, produced EPS complexes to strengthen the sludge granule followed by the mass growth of Clostridium sp.

74 Gray, Sean A., et.al. (2011) Synergistic Capture of Clostridium botulinum Type A Neurotoxin by scFv Antibodies to Novel Epitopes. Biotechnol. Bioeng.;108: 2456–2467. Abstract A non-immune library of human single chain fragment variable (scFv) antibodies displayed on Saccharomyces cerevisiae was screened for binding to the Clostridium botulinum neurotoxin serotype A binding domain [BoNT/A (Hc)] with the goal of identifying scFv to novel epitopes. To do this, an antibody-mediated labeling strategy was used in which antigen-binding yeast clones were selected after labeling with previously characterized monoclonal antibodies (MAbs) specific to the Hc. Twenty unique scFv clones were isolated that bound Hc. Of these, 3 also bound to full-length BoNT/A toxin complex with affinities ranging from 5 to 48 nM. Epitope binning showed that the three unique clones recognized at least two epitopes distinct from one another as well as from the detection MAbs. After production in E. coli, scFv were coupled to magnetic particles and tested for their ability to capture BoNT/A holotoxin using an Endopep-MS assay. In this assay, toxin captured by scFv coated magnetic particles was detected by incubation of the complex with a peptide containing a BoNT/A-specific cleavage sequence. Mass spectrometry was used to detect the ratio of intact peptide to cleavage products as evidence for toxin capture. When tested individually, each of the scFv showed a weak positive Endopep-MS result. However, when the particles were coated with all three scFv simultaneously, they exhibited significantly higher Endopep-MS activity, consistent with synergistic binding. These results demonstrate novel approaches toward the isolation and characterization of scFv antibodies specific to unlabeled antigens. They also provide evidence that distinct scFv antibodies can work synergistically to increase the efficiency of antigen capture onto a solid support.

75 Hao Wang, Sanjay V. Malhotra, Arokiasamy J. Francis, (2011) Toxicity of various anions associated with methoxyethyl methyl imidazolium-based ionic liquids on Clostridium sp., Chemosphere, Vol. 82, Issue 11:1597-1603, Abstract: We investigated the effects on the growth of the anaerobic bacterium, Clostridium sp., of the ionic liquid, 1- methoxyethyl-3-methyl imidazolium [MOEMIM]+, derived from imidazolium cation and paired with one of a variety of counter-ions, viz., tetrafluoroborate [ BF 4 ] - , hexafluorophosphate [ PF 6 ] - , trifluoroacetate [ CF 3 COO ] - , bis(trifluoromethane)sulfonamide [ Tf 2 N ] - , methane sulfonate [OMS], and 1-butyl-3-methyl imidazolium tetrafluoroborate [BMIM][BF4]. These anions, in association with [MOEMIM]+ lowered the growth rate of the bacterium, showing the following trend: [ Tf 2 N ] - ≧ [ PF 6 ] - > [ BF 4 ] - > [ CF 3 COO ] - > [OMS]−. Anions incorporating fluorine were more toxic than those without it, and their toxicity rose with an increase in the number of fluorine atoms. Also, [MOEMIM]+ [ BF 4 ] - was less toxic than [BMIM]+ [ BF 4 ] - , probably due to the presence of a methoxyethyl functional group integrated in the cation side chain.

76 Ho KL, Lee DJ. (2011) Harvesting biohydrogen from cellobiose from sulfide or nitrite-containing wastewaters using Clostridium sp. R1. Bioresour Technol.,102(18):8547-9. Abstract Harvesting biohydrogen from inhibiting wastewaters is of practical interest since the toxicity of compounds in a wastewater stream commonly prevents the bioenergy content being recovered. The isolated Clostridium sp. R1 is utilized to degrade cellobiose in sulfide or nitrite-containing medium for biohydrogen production. The strain can effectively degrade cellobiose free of severe inhibitory effects at up to 200 mg l_1 sulfide or to 5 mg l_1 nitrite, yielding hydrogen at >2.0 mol H2 mol_1 cellobiose. Principal metabolites of cellobiose fermentation are acetate and butyrate, with the concentration of the former increases with increasing sulfide and nitrite concentrations. The isolated strain can yield hydrogen from cellobiose in sulfide-laden wastewaters. However, the present of nitrite significantly limit the efficiency of the biohydrogen harvesting process.

77 Kuo-Ling ,Ho, Duu-Jong Lee (2011) Harvesting biohydrogen from cellobiose from sulfide or nitrite- containing wastewaters using Clostridium sp. R1, Bioresource Technology, Vol. 102 : 8547-8549 Abstract Harvesting biohydrogen from inhibiting wastewaters is of practical interest since the toxicity of compounds in a wastewater stream commonly prevents the bioenergy content being recovered. The isolated Clostridium sp. R1 is utilized to degrade cellobiose in sulfide or nitrite-containing medium for biohydrogen production. The strain can effectively degrade cellobiose free of severe inhibitory effects at up to 200 mg l−1 sulfide or to 5 mg l−1 nitrite, yielding hydrogen at >2.0 mol H2 mol−1 cellobiose. Principal metabolites of cellobiose fermentation are acetate and butyrate, with the concentration of the former increases with increasing sulfide and nitrite concentrations. The isolated strain can yield hydrogen from cellobiose in sulfide-laden wastewaters. However, the present of nitrite significantly limit the efficiency of the biohydrogen harvesting process. 78 Ling Jiang, (2011) Enhanced Butyric Acid Tolerance and Bioproduction by Clostridium tyrobutyricum Immobilized in a Fibrous Bed Bioreactor. Biotechnol. Bioeng. ,108: 31–40. Abstract Repeated fed-batch fermentation of glucose by Clostridium tyrobutyricum immobilized in a fibrous bed bioreactor (FBB) was successfully employed to produce butyric acid at a high final concentration as well as to adapt a butyric-acid-tolerant strain. At the end of the eighth fedbatch fermentation, the butyric acid concentration reached 86.9_2.17 g/L, which to our knowledge is the highest butyric acid concentration ever produced in the traditional fermentation process. To understand the mechanism and factors contributing to the improved butyric acid production and enhanced acid tolerance, adapted strains were harvested from the FBB and characterized for their physiological properties, including specific growth rate, acid-forming enzymes, intracellular pH, membrane-bound ATPase and cell morphology. Compared with the original culture used to seed the bioreactor, the adapted culture showed significantly reduced inhibition effects of butyric acid on specific growth rate, cellular activities of butyric-acid-forming enzyme phosphotransbutyrylase (PTB) and ATPase, together with elevated intracellular pH, and elongated rod morphology.

79 Lo YC, Huang CY, Cheng CL, Lin CY, Chang JS. (2011) Characterization of cellulolytic enzymes and bioH2 production from anaerobic thermophilic Clostridium sp. TCW1. Bioresour Technol.,102(18): 8384-92. Abstract A thermophilic anaerobic bacterium Clostridium sp. TCW1 was isolated from dairy cow dung and was used to produce hydrogen from cellulosic feedstock. Extracellular cellulolytic enzymes produced from TCW1 strain were identified as endoglucanases (45, 53 and 70 kDa), exoglucanase (70 kDa), xylanases (53 and 60 kDa), and b-glucosidase (45 kDa). The endoglucanase and xylanase were more abundant. The optimal conditions for H2 production and enzyme production of the TCW1 strain were the same (60 _C, initial pH 7, agitation rate of 200 rpm). Ten cellulosic feedstock, including pure or natural cellulosic materials, were used as feedstock for hydrogen production by Clostridium strain TCW1 under optimal culture conditions. Using filter paper at 5.0 g/L resulted in the most effective hydrogen production performance, achieving a H2 production rate and yield of 57.7 ml/h/L and 2.03 mol H2/mol hexose, respectively. Production of cellulolytic enzyme activities was positively correlated with the efficiency of dark-H2 fermentation.

80 Mingjie Jin, Venkatesh Balan, Christa Gunawan, Bruce E. Dale (2011) Consolidated Bioprocessing (CBP) Performance of Clostridium phytofermentans on AFEX-Treated Corn Stover for Ethanol Production. Biotechnol. Bioeng. 2011;108: 1290–1297 Abstract Consolidated bioprocessing (CBP) is believed to be a potentially cost-efficient and commercially viable way to produce cellulosic biofuels. In this study, we have evaluated the performance of the CBP organism Clostridium phytofermentans (ATCC 700394) on AFEX-treated corn stover (AFEX-CS). Fermentation conditions including temperature, inoculation size, nutrients, and initial pH were investigated. At optimal conditions with 0.5% (w/w) glucan loading of AFEX-CS, C. phytofermentans hydrolyzed 76% of glucan and 88.6% of xylan in 10 days. These values reached 87% and 102% of those obtained by simultaneous saccharification and co- fermentation (SSCF) using commercial enzymes and S. cerevisiae 424A. Ethanol titer for CBP was found to be 2.8 g/L which was 71.8% of that yielded by SSCF (3.9 g/L). Decomposition products from AFEX-CS helped to increase ethanol yield somewhat during CBP. Particle size played a crucial role in the enhancement of sugar conversion by CBP.

81 Nancharaiah, Y. Venkata, A.J. Francis (2011) Alkyl-methylimidazolium ionic liquids affect the growth and fermentative metabolism of Clostridium sp., Bioresource Techn., Vol. 102: 6573-6578, Abstract In this study, the effect of ionic liquids, 1-ethyl-3-methylimidazolium acetate [EMIM][Ac], 1-ethyl-3- methylimidazolium diethylphosphate [EMIM][DEP], and 1-methyl-3-methylimidazolium dimethylphosphate [MMIM][DMP] on the growth and glucose fermentation of Clostridium sp. was investigated. Among the three ionic liquids tested, [MMIM][DMP] was found to be least toxic. Growth of Clostridium sp. was not inhibited up to 2.5, 4 and 4 g L−1 of [EMIM][Ac], [EMIM][DEP] and [MMIM][DMP], respectively. [EMIM][Ac] at <2.5 g L−1, showed hormetic effect and stimulated the growth and fermentation by modulating medium pH. Total organic acid production increased in the presence of 2.5 and 2 g L−1 of [EMIM][Ac] and [MMIM][DMP]. Ionic liquids had no significant influence on alcohol production at <2.5 g L−1. Total gas production was affected by ILs at ⩾2.5 g L−1 and varied with type of methylimidazolium IL. Overall, the results show that the growth and fermentative metabolism of Clostridium sp. is not impacted by ILs at concentrations below 2.5 g L−1.

82 Ponthein, Watchara, Benjamas Cheirsilp (2011) Development of Acetone Butanol Ethanol (ABE) Production from Palm Pressed Fiber by Mixed Culture of Clostridium sp. and Bacillus sp., Energy Procedia, Volume 9: 459-467, Abstract: The palm pressed fiber (PPF) was treated either with NaOH 10% (w/v) and boiling for 15 min or H2SO4 0.5% (v/v) and autoclaved at 121 °C for 60 min. The PPF residues after pretreatment with NaOH, H2SO4 and both were 63.28, 62.20 and 47.00% (w/w), respectively. The pretreatment with both NaOH and H2SO4 reduced significantly lignin content and the highest cellulose content of 76.32% (w/w) was obtained. The ABE production using pretreated PPF as a carbon source was attempted in a single culture of Clostridium acetobutylicum DSM 1713 and a mixed culture of C. acetobutylicum DSM 1713 with Bacillus cellulolyticus JCM 9156 with and without addition of cellulase. It was found that the single and mixed cultures with the addition of 30 U cellulase gave the ABE production of 3.97 and 3.95 g/L, respectively, at 144 h. While the cultures without cellulase produced much less ABE (0.25-0.49 g/L). Although, the mixed culture did not improve ABE production, it could reduce the use of reducing agent and nitrogen gas to ensure the anaerobic condition. The optimum condition for ABE production by the mixed culture using pretreated PPF was 5.0 g/L PPF and 9.0 g/ L isolated soy protein (ISP), at pH 6.5, 37 °C, 60 rpm with the addition of 30 U cellulase. At this condition, 4.95 g/L of ABE was obtained at 144 h.

83 Ramachandran U, Wrana N, Cicek N, Sparling R, Levin DB. (2011) Isolation and characterization of a hydrogen- and ethanol-producing Clostridium sp. Strain URNW. Can J Microbiol., 57(3): 236-43. Abstract: Identification, characterization, and end-product synthesis patterns were analyzed in a newly identified mesophilic, anaerobic Clostridium sp. strain URNW, capable of producing hydrogen (H2) and ethanol. Metabolic profiling was used to characterize putative end-product synthesis pathways of the Clostridium sp. strain URNW, which was found to grow on cellobiose; on hexose sugars, such as glucose, sucrose, and mannose; and on sugar alcohols, like mannitol and sorbitol. When grown in batch cultures on 2 g cellobiose_L– 1, Clostridium sp. strain URNW showed a cell generation time of 1.5 h, and the major end-products were H2, formate, carbon dioxide (CO2), lactate, butyrate, acetate, pyruvate, and ethanol. The total volumetric H2 production was 14.2 mmol_(L culture)–1 and the total production of ethanol was 0.4 mmol_ (L culture)–1. The maximum yield of H2 was 1.3 mol_(mol glucose equivalent)–1 at a carbon recovery of 94%. The specific production rates of H2, CO2, and ethanol were 0.45, 0.13, and 0.003 mol_h-1-(g dry cell mass)–1, respectively. BLAST analyses of 16S rDNA and chaperonin 60 (cpn60) sequences from Clostridium sp. strain URNW revealed a 98% nucleotide sequence identity with the 16S rDNA and cpn60 sequences from Clostridium intestinale ATCC 49213. Phylogenetic analyses placed Clostridium sp. strain URNW within the butyrate- synthesizing clostridia.

84 Tran, Giang Hai, et.al. (2011) Probing the Active Site of Cellodextrin Phosphorylase from Clostridium stercorarium: Kinetic Characterization, Ligand Docking, and Site-Directed Mutagenesis. Engineers Biotechnol. Prog., 27: 326–332, Abstract Cellodextrin phosphorylase from Clostridium stercorarium has been recombinantly expressed in Escherichia coli for the first time. Kinetic characterization of the purified enzyme has revealed that aryl and alkyl b- glucosides can be efficiently glycosylated, an activity that has not yet been described for this enzyme class. To obtain a better understanding of the factors that determine the enzyme’s specificity, homology modeling and ligand docking were applied. Residue W168 has been found to form a hydrophobic stacking interaction with the substrate in subsite þ2, and its importance has been examined by means of site-directed mutagenesis. The mutant W168A retains about half of its catalytic activity, indicating that other residues also contribute to the binding affinity of subsite þ2. Finally, residue D474 has been identified as the catalytic acid, interacting with the glycosidic oxygen between subsites _1 and þ1. Mutating this residue results in complete loss of activity. These results, for the first time, provide an insight in the enzyme–substrate interactions that determine the activity and specificity of cellodextrin phosphorylases. V

85 Wang H, Malhotra SV, Francis AJ. (2011) Toxicity of various anions associated with methoxyethyl methyl imidazolium-based ionic liquids on Clostridium sp. Chemosphere,82(11):1597-603. Abstract We investigated the effects on the growth of the anaerobic bacterium, Clostridium sp., of the ionic liquid, 1-methoxyethyl-3-methyl imidazolium [MOEMIM]+, derived from imidazolium cation and paired with one of a variety of counter-ions, viz., tetrafluoroborate ½BF4_, hexafluorophosphate ½PF6_, trifluoroacetate ½CF3COO_, bis(trifluoromethane)sulfonamide ½Tf2N_, methane sulfonate [OMS], and 1-butyl-3-methyl imidazolium tetrafluoroborate [BMIM][BF4]. These anions, in association with [MOEMIM]+ lowered the growth rate of the bacterium, showing the following trend: ½Tf2N_ = ½PF6_ > ½BF4_ > ½CF3COO_ > [OMS]_. Anions incorporating fluorine were more toxic than those without it, and their toxicity rose with an increase in the number of fluorine atoms. Also, [MOEMIM]+ ½BF4_ was less toxic than [BMIM]+ ½BF4_, probably due to the presence of a methoxyethyl functional group integrated in the cation side chain.

86 Yung-Chung, et.al. (2011) Characterization of cellulolytic enzymes and bioH2 production from anaerobic thermophilic Clostridium sp. TCW1, Bioresource Technology, Vol. 102: 8384-8392 Abstract A thermophilic anaerobic bacterium Clostridium sp. TCW1 was isolated from dairy cow dung and was used to produce hydrogen from cellulosic feedstock. Extracellular cellulolytic enzymes produced from TCW1 strain were identified as endoglucanases (45, 53 and 70 kDa), exoglucanase (70 kDa), xylanases (53 and 60 kDa), and β-glucosidase (45 kDa). The endoglucanase and xylanase were more abundant. The optimal conditions for H2 production and enzyme production of the TCW1 strain were the same (60 °C, initial pH 7, agitation rate of 200 rpm). Ten cellulosic feedstock, including pure or natural cellulosic materials, were used as feedstock for hydrogen production by Clostridium strain TCW1 under optimal culture conditions. Using filter paper at 5.0 g/L resulted in the most effective hydrogen production performance, achieving a H2 production rate and yield of 57.7 ml/h/L and 2.03 mol H2/mol hexose, respectively. Production of cellulolytic enzyme activities was positively correlated with the efficiency of dark-H2 fermentation.

87 Akila G, Chandra TS. (2010) Stimulation of biomethanation by Clostridium sp. PXYL1 in coculture with a Methanosarcina strain PMET1 at psychrophilic temperatures. J Appl Microbiol., 108: 204-13. Abstract Aim: Bioaugumentation of low temperature biogas production was attempted by addition of cold-adapted Clostridium and a methanogen. Methods and Results: A psychrotrophic xylanolytic acetogenic strain Clostridium sp. PXYL1 growing optimally at 20_C and pH 5Æ3 and a Methanosarcina strain, PMET1, growing optimally on acetate and producing methane at 15_C were isolated from a cattle manure digester. Anaerobic conversion of xylose at 15_C with the coculture of the two strains was performed, and batch culture methane production characteristics indicated that methanogenesis occurred via acetate through ‘acetoclastic’ pathway. Stimulation studies were also undertaken to evaluate the effect of exogenous addition of the coculture on biogas yields at 15_C. Addition of 3 ml of PXYL1 at the rate of 12 ・ 102 CFU ml)1 increased the biogas 1Æ7-fold (33 l per kg cowdung) when compared to control (19Æ3 l per kg cowdung) as well as increased the volatile fatty acid (VFA) levels to 3210 mg l)1 when compared to 1140 mg l)1 in controls. Exogenous of addition of 10 ml PMET1 inoculum at the rate of 6Æ8 ± 102 CFU ml)1 in addition to PXYL1 served to further improve the biogas yields to 46 l kg)1 as well as significantly brought down the VFA levels to 1350 mg l)1. Conclusions: Our results suggest that the rate-limiting methanogenic step at low temperatures could be overcome and that biogas yields improved by manipulating the population of the acetoclastic methanogens. Significance and Impact of the Study: Stimulation of biomethanation at low temperature by coculture.

88 Chatzifragkou, Afroditi, et.al. (2010) Effect of Biodiesel-Derived Waste Glycerol Impurities on Biomass and 1,3-Propanediol Production of Clostridium butyricum VPI 1718. Biotechnol. Bioeng. 2010;107: 76–84. Abstract Aim of the present study was to assess and evaluate the impact of various kinds of impurities of biodiesel- derived raw glycerol feedstock, upon microbial growth and 1,3-propanediol (1,3-PDO) production by Clostridium butyricum. Preliminary trials in 200-mL anaerobic bottles revealed that the presence of NaCl at a concentration of 4.5% (w/w of glycerol) in growth medium imposed an evident inhibitory effect, in contrast with phosphoric salts. However, the application of NaCl at elevated quantities during batch bioreactor experiments [up to 30% (w/w of glycerol)], did neither affect the microbial growth, nor the 1,3-PDO production. Moreover, when oleic acid was added into the growth medium at 2% (w/w of glycerol), a total preclusion of the strain was observed. In order to further investigate whether the nature of oleic acid itself or the presence of the double bond induced the inhibitory phenomenon, stearic acid was added into the medium at the same concentration (2%, w/w, of glycerol). Indeed, no inhibitory effect was observed in the fermentor, suggesting that the presence of the double bond may play a key role in the growth behavior of the microorganism. Finally, methanol effect was tested in batch and continuous bioreactor operations. Interestingly enough, the alcohol addition did not affect the microbial bioconversion of glycerol into 1,3- PDO, even when imposed at relatively high concentrations (10%, w/w, of glycerol) in batch-bioreactor operations. In continuous experiments, methanol was added when steady state had been achieved, and although in one case high concentration was added into the chemostat (5 g/L), the system re-obtained a steady state without indications of negative effect upon biomass production due to the alcohol.

89 Jeon, Byoung Seung, Kim BC, Um Y, Sang BI (2010) Production of hexanoic acid from D-galactitol by a newly isolated Clostridium sp. BS-1. Appl Microbiol Biotechnol., 88(5):1161-7. Abstract In a study screening anaerobic microbes utilizing D-galactitol as a fermentable carbon source, four bacterial strains were isolated from an enrichment culture producing H2, ethanol, butanol, acetic acid, butyric acid, and hexanoic acid. Among these isolates, strain BS-1 produced hexanoic acid as a major metabolic product of anaerobic fermentation with D-galactitol. Strain BS-1 belonged to the genus Clostridium based on phylogenetic analysis using 16S rRNA gene sequences, and the most closely related strain was Clostridium sporosphaeroides DSM 1294T, with 94.4% 16S rRNA gene similarity. In batch cultures, Clostridium sp. BS-1 produced 550±31 mLL−1 of H2, 0.36±0.01 gL−1 of acetic acid, 0.44±0.01 gL−1 of butyric acid, and 0.98±0.03 gL−1 of hexanoic acid in a 4-day cultivation. The production of hexanoic acid increased to 1.22 and 1.73 gL−1 with the addition of 1.5 gL−1 of sodium acetate and 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), respectively. Especially when 1.5 gL−1 of sodium acetate and 100 mM MES were added simultaneously, the production of hexanoic acid increased up to 2.99 gL−1. Without adding sodium acetate, 2.75 gL−1 of hexanoic acid production from D-galactitol was achieved using a coculture of Clostridium sp. BS-1 and one of the isolates, Clostridium sp. BS-7, in the presence of 100 mM MES. In addition, volatile fatty acid (VFA) production by Clostridium sp. BS- 1 from D-galactitol and D-glucose was enhanced when a more reduced culture redox potential (CRP) was applied via addition of Na2S·9H2O.

90 Ji-Fei Xu, et.al. (2010) Cell growth and hydrogen production on the mixture of xylose and glucose using a novel strain of Clostridium sp. HR-1 isolated from cow dung compost, International Journal of Hydrogen Energy, Volume 35, Issue 24: 13467-13474, Abstract: A novel mesophilic hydrogen-producing bacterium was isolated from cow dung compost and designated as Clostridium sp. HR-1 by 16S rRNA gene sequence. The optimum condition for hydrogen production by strain HR-1 was pH of 6.5, temperature of 37 °C and yeast extract as nitrogen sources. The strain HR-1 has the ability to utilize kinds of hexose and pentose as carbon sources for growth and H2 production. Cell growth and hydrogen productivity were investigated for batch fermentation on media containing different ratios of xylose and glucose. Glucose was the preferred substrate in the glucose and xylose mixtures. The high glucose fraction had higher cell biomass production rate. The rate of glucose consumption was higher than xylose consumption, and remained essentially constant independent of xylose content of the mixture. The rate of xylose utilization was decreased with increasing of the glucose fraction. The average H2 yield and specific H2 production rates with xylose and glucose are 1.63 mol-H2/mol xylose and 11.14-H2 mmol/h g-cdw, and 2.02 mol-H2/mol- glucose and 9.37 mmol-H2/h g-cdw, respectively. Using the same initial substrate concentration, the maximum average H2 yield and specific H2 production rates with the mixtures of 9 g/l xylose and 3 g/l glucose was 2.01 mol-H2/mol-mixed sugar and 12.56 mmol-H2/h g-cdw, respectively. During the fermentation, the main soluble microbial products were ethanol and acetate which showed trends with the different ratios of xylose and glucose.

91 Kuo-Ling Ho, Yu-You Chen, Duu-Jong Lee (2010) Biohydrogen production from cellobiose in phenol and cresol-containing medium using Clostridium sp. R1, International Journal of Hydrogen Energy, Vol. 35 (19): 10239-10244, Abstract: Cellobiose fermentation in batch test using an isolated strain, Clostridium sp. R1, was investigated. The Clostridium sp. R1 achieved a maximum hydrogen yield of 3.5 mol H2 mol−1 cellobiose at pH 6 and 30 °C, higher than most yields reported in literature. This strain can generate hydrogen from a number of carbohy- drates, including galactose, glucose, mannose, maltose, sucrose, and starch. This strain can also convert cellobiose to hydrogen in the presence of toxic phenol or cresol. The inhibition effects of phenolic compounds on strain R1 activity followed phenol > p-cresol > o-cresol > m-cresol. Co-culturing with another strain, Clostridium butyricum, can co-degrade some of the phenol as substrates. The new isolated strain can yield hydrogen from phenol-containing wastewaters.

92 Lincke, Thorger, et.al. (2010) Closthioamide: An Unprecedented Polythioamide Antibiotic from the Strictly Anaerobic Bacterium Clostridium cellulolyticum. Angew. Chem., 122, 2055 –2057 Abstract Bacteria belonging to the genus Clostridium are among the most prominent microorganisms that lead an obligate anaerobic lifestyle. Clostridia occur in gastrointestinal tracts and are ubiquitously distributed in soil and sediments, rapidly decaying organic matter.[1] Owing to their potent catabolic properties, interest in these organisms has grown rapidly over the past few years. Not only are clostridia routinely employed to degrade anthropogenic cellulosic waste products, they have also been increasingly exploited to meet the need for renewable chemicals and biofuels.[2] Although various pathogenic species produce the most powerful neurotoxins known to mankind, the tetanus and botulinum toxins, respectively,[3] no secondary metabolites have yet been isolated from these or any other strictly anaerobic bacteria. However, bioinformatics analysis (“mining”) of the recently sequenced genomes of Clostridium spp., for example, Clostridium kluyveri[4] and Clostridium cellulolyticum, indicated that these bacteria harbor genes for the biosynthesis of secondary metabolites. Since the encoded cryptic natural products have been overlooked so far, it appears the biosynthesis genes remain dormant under standard laboratory conditions and are only triggered in the presence of particular stimuli. Herein we report the discovery of the first secondary metabolite, a hitherto fully unprecedented type of polythioamide, from a strictly anaerobic bacterium, Clostridium cellulolyticum. C. cellulolyticum is an anaerobic nonruminal Gram-positive bacterium that was isolated from decayed grass compost and is an important industrial strain as a result of its ability to degrade crystalline cellulose.[5] Yet, no secondary metabolite has been reported from this well-known model organism. To investigate secondary-metabolite production, we cultivated a strain of C. cellulolyticum, DSM 5812 (=ATCC 35319), in 1 L fermenters with pH control by using complex media. However, under these standard growth conditions as reported for this strain, no secondary-metabolite formation was observed. Therefore, we sought to induce natural product biosynthesis by applying external triggers.

93 Keith V. Alsaker, et.al. (2010) Metabolite Stress and Tolerance in the Production of Biofuels and Chemicals: Gene-Expression-Based Systems Analysis of Butanol, Butyrate, and Acetate Stresses in the Anaerobe Clostridium acetobutylicum. Biotechnol. Bioeng.;105: 1131–1147 Abstract Metabolite accumulation has pleiotropic, toxic, or beneficial effects on cell physiology, but such effects are not well understood at the molecular level. Cells respond and adapt to metabolite stress by mechanisms largely unexplored, especially in the context of multiple and simultaneous stresses. Solventogenic and related clostridia have an inherent advantage for production of biofuels and chemicals directly from cellulosic material and other complex carbohydrates, but issues of product/metabolite tolerance and related culture productivities remain. Using DNA microarray-based gene expression analysis, the transcriptional- stress responses of Clostridium acetobutylicum to fermentation acids acetate and butyrate and the solvent product butanol were analyzed and compared in the context of cell physiology. Ontological analysis demonstrated that stress by all three metabolites resulted in upregulation of genes related to post-translational modifications and chaperone activity, and downregulation of the translationmachinery genes. Motility genes were downregulated by acetate-stress only. The general metabolite stress included upregulation of numerous stress genes (dnaK, groES, groEL, hsp90, hsp18, clpC, and htrA), the solventogenic operon aad-ctfA-ctfB, and other solventogenic genes. Acetate stress downregulated expression of the butyryl-CoA- and butyrate- formation genes, while butyrate stress downregulated expression of acetate-formation genes. Pyrimidine-biosynthesis genes were downregulated by most stresses, but purine-biosynthesis genes were upregulated by acetate and butyrate, possibly for thiamine and histidine biosynthesis. Methionine-biosynthesis genes were upregulated by acetate stress, indicating a possibly conserved stress response mechanism also observed in Escherichia coli. Nitrogen-fixation gene expression was upregulated by acetate stress. Butyrate stress upregulated many iron-metabolism genes, riboflavin-biosynthesis genes, and several genes related to cellular repair from oxidative stress, such as perR and superoxide dismutases. Butanol stress upregulated the glycerol metabolism genes glpA and glpF. Surprisingly, metabolite stress had no apparent effect on the expression of the sporulation-cascade genes. It is argued that the list of upregulated genes in response to the three metabolite stresses includes several genes whose overexpression would likely impart tolerance, thus making the information generated in this study, a valuable source for the development of tolerant recombinant strains.