Clostridium Liquoris Sp. Nov., Isolated from a Fermentation Pit Used for The

International Journal of Systematic and Evolutionary Microbiology (2016), 66, 749–754 DOI 10.1099/ijsem.0.000787

Clostridium liquoris sp. nov., isolated from a fermentation pit used for the production of Chinese strong-ﬂavoured liquor Qi Yin,1,2 Yong Tao,1 Xiaoye Zhu,1 Yan Zhou,3 Xiaohong He,1 Lei Cheng,4 Yan Huang4 and Daping Li1

Correspondence 1Key Laboratory of Environmental and Applied Microbiology, Chinese Academy of Sciences & Yong Tao Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu Institute of Biology, [email protected] Chinese Academy of Sciences, Chengdu 610041, PR China Daping Li 2Chengdu Shishi High School, Chengdu 610041, PR China [email protected] 3College of Life Science, Sichuan University, Chengdu 610041, PR China 4Key Laboratory of Development and Application of Rural Renewable Energy, Biogas Institute of Ministry of Agriculture, Chengdu 610041, PR China

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 8C (optimum 37 8C), 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 (5ACCC 00785T5DSM 100320T).

Clostridium is a genus of Gram-positive bacteria belong- as converting bio-waste into organic acids (focusing on ing to the phylum Firmicutes, and contains around 200 acetate, butyrate and caproate) (Mu¨ller, 2003; species (UK-Standards-for-Microbiology-Investigations, 2011; Tracy et al., 2012). There are many reports about pathways Wiegel et al., 2006), of which only a few are pathogenic and metabolism of Clostridium butyricum or Clostridium to humans. A majority of members of the genus Clostri- kluyveri to produce butyrate or caproate in anaerobic fer- dium possess value in commercial applications, such mentation (Cai et al., 2011; Colin et al., 2001; Papoutsakis, 2008; Seedorf et al., 2008). Abbreviation: CSFL, Chinese strong-flavoured liquor. Chinese strong-flavoured liquor (CSFL), also called The GenBank/EMBL/DDBJ accession number for the16S rRNA gene ‘Luzhou-flavor liquor’, accounts for more than 70 % of sequence of strain BEY10T is KC331197. 54 bacterial strains Chinese liquor production every year in China (Zhao (GenBank accession nos 5 KC331155–KC331208) are isolated in et al., 2012). The CSFL quality is to a large degree depen- this study and unpublished. dent on the flavour components (e.g. butyric acid, caproic Two supplementary figures are available with the online Supplementary acid, ethyl caproate and other microbial extracellular fatty Material. acids) produced by microbes in the pit mud of the

Downloaded from www.microbiologyresearch.org by 000787 G 2015 IUMS Printed in Great Britain 749 IP: 130.132.173.170 On: Mon, 27 Jun 2016 13:30:03 Q. Yin and others fermentation pit (Xu et al., 2010). The genus Clostridium is ranging from 20 to 55 8C, at initial pH values of 4–10 one of the dominant taxa in the pit mud of fermentation (adjusted with 2.5 M NaOH or 2.5 M HCl) and in 0– pits for CSFL production, corresponding to the increase 8 % (w/v) NaCl for 7 days. All experiments were con- of caproic acid concentration and playing an important ducted in triplicate. Cell morphology and size were exam- role in the quality of CSFL (Shi et al., 2010; Tao et al., ined by scanning electron microscope (Quanta200; FEI). 2014). Recently, we isolated 54 bacterial strains (GenBank Samples were coated with Au/Pt before scanning electron accession nos KC331155–KC331208 for 16S rRNA gene microscopy observation. Gram staining was performed by sequences) from the fermentation pit for the production using a Difco Gram stain set and cultures were grown in of CSFL. The most abundant microbes were members of medium A according to standard procedures (Gerhardt the family Clostridiaceae (accounting for 46 % of all iso- et al., 1994). lates), followed by members of the families Eubacteriaceae Genomic DNA was extracted by using the TIANamp Bac- (16.7 %), Lachnospiraceae (13 %) and Lactobacillaceae teria DNA kit (TIANGEN Biotech), and the 16S rRNA gene (11 %). was amplified using universal primers 27F and 1492R In this paper, a novel saccharolytic, butyrate-producing (Lane, 1991). The PCR product was purified using the bacterium, designated BEY10T, was isolated from an old TIANgel Midi Purification kit (TIANGEN Biotech), fermentation pit (located in Mianzhu, Sichuan, China) cloned to the pM18-T vector (TaKaRa) and sequenced which maintained brewing operation for over 20 years using an automated DNA sequencer (ABI 3730; Applied without interruption. Based on the phenotypic and phylo- BioSystems). An almost-complete 16S rRNA gene sequence T genetic characteristics, it is proposed that the isolate rep- (1474 bp) of strain BEY10 was submitted to the GenBank resents a novel species of the genus Clostridium. database, and the identification of phylogenetic neighbours and the calculation of pairwise 16S rRNA gene sequence The anaerobic cultivation technique was used throughout the similarities were achieved using the EzTaxon server course of this study. In brief, pit-mud samples collected from (http://www.ezbiocloud.net/; Kim et al., 2012). Sequences the fermentation pit were inoculated (5 %, v/v) into 100 ml were aligned using CLUSTAL X 1.8 (Thompson et al., serum bottles containing sterilized saline and mixed well. 1997). Phylogenetic trees were reconstructed using the Cultures were then serially diluted with sterilized saline, neighbour-joining (Saitou & Nei, 1987) and maximum- and spread on modified MCM agar (2 %, w/v) plates (the likelihood (Yang, 1997) methods implemented in the pro- 21 isolation medium) containing (l , distilled water): 1.1 g gram MEGA version 5 (Tamura et al., 2011). The topological KH2PO4,0.5g(NH4)2SO4, 1.8 g sodium acetate, 0.5 g Casa- robustness was evaluated by bootstrap analysis (Felsenstein, mino acids, 0.5 g Bacto-peptone, 0.5 g yeast extract, 1.5 g 1985) based on 1000 replicates. KCl, 4.0 g NaHCO3, 0.5 g L-cysteine hydrochloride, 20 ml alcohol, 0.001 g resazurin, 2 ml vitamin solution and 1 ml trace element solution (Atlas, 2010). Plates were incubated for 3–5 days at 30 8C under anaerobic conditions (90 % N2, 5 % CO, and 5 % H2; SHELLAB). Colonies on MCM agar plates were transferred to fresh MCM medium three consecu- tive times to get a pure organism. The liquid medium (denoted medium A) used as a base medium in physiological GL T tests for strain BEY10 , contained the basal salts (Markossian PL et al., 2000) supplemented with 0.1 % (w/v) yeast extract, PL 0.0025 % (w/v) resazurin and 120 mg L-cysteine hydrochlo- PL PL 2 ride l 1; to obtain a solid medium, 2 % agar was added. All PE media had a final pH of 6.8–7.2 after autoclaving. Liquid cul- PG tures were grown with shaking (180 r.p.m.) at 37 8C. The iso- PL late was characterized biochemically using conventional tests as described previously (Tindall et al., 2007) and utilization of various organic substrates as carbon and energy sources was determined by using the API 20A test system (bioMe´rieux) PL AL according to the manufacturer’s instructions. The metabolic PL products from glucose fermentation were determined by an Agilent 1260 Infinity liquid chromatography system (Agilent PL Technologies) equipped with a HPLC column Hi-Plex H (30066.5 mm) and a differential refraction detector (RID).

Growth rates, optimum growth temperature and tolerance to pH and NaCl were determined in medium A containing Fig. 1. Polar lipid proﬁle of the strain BEY10T after two-dimen- 1 % (w/v) yeast extract as the sole carbon and energy sional TLC. PG, phosphatidylglycerol; PE, phosphatidylethanola- source. For these studies, cells were grown at temperatures mine; PL, phospholipids; GL, glycolipid; AL, aminolipid.

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Polar lipids were extracted using a chloroform/methanol/ constitutes a different clade. Furthermore, the isolate 0.3 % aqueous NaCl mixture (1 : 2 : 0.8, by vol.) (Bligh showed 97.6 %, 96.9 % and 95.3 % 16S rRNA gene sequence & Dyer, 1959) and then were detected by two-dimensional similarity with the type strains of C. lundense, C. tetanomor- silica gel thin-layer chromatography (Tindall et al., 2007). phum and C. pascui respectively, which were the phylogeneti- Strain BEY10T was found to possess a very characteristic cally closest related species. Strain BEY10T showed less than polar lipid pattern of the genus Clostridium, consisting of 95.0 % 16S rRNA gene sequence similarity with other related phosphatidylglycerol, phosphatidylethanolamine, phos- species of the genus Clostridium. pholipids and smaller amounts of a glycolipid and an aminolipid (Fig. 1) (Johnston & Goldﬁne, 1983; Johnston Cells of strain BEY10T were Gram-stain-positive, non-motile, et al., 2010). spore-forming, rod-shaped (0.5–1.0 mm in width and 2.5– For fatty acid analysis, late-exponential-phase cultures of strain 6 mm in length; Fig. S2) and appeared singly or in pairs. The BEY10T, Clostridium lundense DSM 17049T, Clostridium teta- strain formed light yellow colonies with a convex surface nomorphum DSM 4474T and Clostridium pascui DSM 10365T after 72 h at 37 8C. It grew at temperatures of 22–47 8C, were obtained after cultivation in PYG liquid medium at with optimum growth at 37 8C. Growth was observed at 37 8C. Extraction of fatty acid methyl esters and separation pH 5.5–9.0, with optimum growth at pH 7.5–8.5. The isolate by GC were performed by using the Instant FAME method grew optimally in the absence of NaCl but tolerated up to 4 % of the Microbial IdentiﬁcationSystem(MIDI)version6.1 (w/v) NaCl. Physiological and biochemical characteristics of and the RTSBA6 6.10 database (Sasser, 1990). strain BEY10T are compared with the type strains of three closely related species in Table 1. Some characteristics of strain The DNA G+C content was measured by HPLC using an BEY10T are in accordance with characteristics of members Agilent 1200 system (Mesbah et al.,1989).DNA–DNA of the genus Clostridium, whereas others such as negative for hybridization between isolate BEY10T and closely related type indole production and aesculin hydrolysis, and positive for strains were performed as described by De Ley et al. (1970). utilization of some carbon sources including glucose, manni- Phylogenetic analysis based on the neighbour-joining (Fig. 2) tol, lactose, maltose, xylose, glycerol, cellobiose and trehalose and maximum-likelihood algorithms (Fig. S1, available in the were differentiated from the reference strains (Table 1). The online Supplementary Material) revealed that strain BEY10T major fermentation products of strain BEY10T from glucose clustered with members of the genus Clostridium and were ethanol and butyric acid, and a small amount of acetic

0.01 T 100 Clostridium scatologenes ATCC 25775 (M59104) Clostridium carboxidivorans P7T (ACV101000229) 80 Clostridium drakei SL1T (Y18813) T 100 Clostridium pascui DSM 10365 (X96736) Clostridium peptidivorans DSM 12505T (AF156796) DSM 1127T (FR749893) 99 Clostridium malenominatum Clostridium tetani NCTC 279T (X74770) T 81 Clostridium cochlearium ATCC 17787 (M59093) T 75 Clostridium tetanomorphum DSM 4474 (DQ241819) 93 Clostridium lundense DSM 17049T (AY858804) Clostridium liquoris BEY10T (KC331197) 'Clostridium senegalense' JC122 (JF824801) Clostridium sulfidigenes SGB2T (EF199998) T 77 100 Clostridium thiosulfatireducens Lup 21 (AY024332) Clostridium subterminale DSM 6970T (AB294137) 89 Clostridium subterminale ATCC 25774T (L37595) T 100 Clostridium argentinense ATCC 27322 (X68316) Clostridium schirmacherense AP15T (AM114453) A121T (DQ296031) 100 Clostridium tagluense Clostridium algoriphilum 14D1T (AY117755) 77 Clostridium bowmanii DSM 14206T (AJ506120) T 100 Youngiibacter fragilis 232.1 (JF262039) Youngiibacter multivorans DSM 6139T (FR749900)

Fig. 2. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic positions of strain BEY10T, the type strains of other species of the genus Clostridium and representatives of some other related members of the family Clostridiaceae. Bootstrap values .70 % (1000 replicates) are shown at branch nodes. Bar, 1 % sequence divergence.

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Table 1. Differential features of isolate BEY10T and type strains of the phylogenetically closest related species of the genus Clostridium

Strains: 1, BEY10T;2,C. lundense DSM 17049T;3,C. tetanomorphum DSM 4474T;4C. pascui DSM 10365T. Data (colony morphology, cell morphology and DNA G+C content) for the reference strain were obtained from references (Cirne et al., 2006; Wilde et al., 1997; Wilde et al., 1989) or generated in this work. +, Positive; 2, negative; W, weakly positive; ND, not detected.

Characteristic 1 2 3 4

Colony morphology Form Convex Convex Flat Convex Diameter (mm) 1 1 2–5 1–2 Pigmentation Light yellow Cream Grey White–grey Cell morphology Shape Rods Rods Slender rods Straight rods Size (mm) 0.5–1.062.5–6 0.5662.8–4.5 0.7–1.063.0–10.0 0.75–1.063.2–8 DNA G+C content (mol%) 34.4 31.2 28 27 Hydrolysis of aesculin 2 ++ + Utilization of: Glucose + 2 + 2 Mannitol + 22 2 Lactose + 2 + 2 Maltose + 22 2 Xylose + 2 + 2 Glycerol + 22 2 Cellobiose + 22 2 Mannose W 22 2 Melezitose W 22 2 Rafﬁnose W 22 2 Sorbitol W 2 W 2 Rhamnose W 22 2 Trehalose + 22 2 Fermentation products from glucose* E,B,a,l ND ND ND

*Fermentation products: a, acetate; b, butyrate; e, ethanol; l, lactose. Upper- and lower-case letters indicate major and minor fermentation products, respectively.

acid and lactic acid (Table 1). These compounds play C18 : 0 (13.1 %), C12 : 0 (13.0 %) and C14 : 0 (10.8 %). important roles in the flavour of Chinese liquor (Liu et al., Detailed fatty acid profiles of strain BEY10T and the type 2014). strains of three closely related species of the genus Clostri- dium are shown in Table 2. All the strains were tested under The genus Clostridium exhibits a wide range of phenotypic the same growth conditions. As shown in Table 2, C characteristics (Van Dyke & McCarthy, 2002). To our 16 : 0 and C were the major fatty acids of three closely related knowledge, the fatty acid types mainly constitute C , 14 : 0 16 : 0 species of the genus Clostridium, but high amounts of C or C , including in the species C. lundense, C. 16 : 1 14 : 0 C and C in the isolate showed it was unique and tetanomorphum, C. pascui, C. tagluense, C. algoriphilum, 20 : 0 18 : 0 distinct from other closely related strains. C. bowmanii, C. frigoris, C. lacusfryxellense, C. psychrophilum, C. estertheticum subsp. laramiense and C. estertheticum The DNA G+C content of strain BEY10T was 34.4 mol%, subsp. estertheticum (Shcherbakova et al., 2005; Spring slightly higher than that of C. lundense DSM 17049T (Cirne et al., 2003; Suetin et al., 2009). According to phospholipid et al., 2006), C. tetanomorphum DSM 4474T (Wilde et al., T fatty acid (PLFA) analysis in CSFL, C18 : 0 and some fatty 1989) and C. pascui DSM 10365 (Wilde et al., 1997) acids with more carbons appeared in the maturing pit (Table 1). The DNA–DNA hybridization values between mud, which means the distinctive fatty acids may affect strain BEY10T and the closest three type strains, C. lundense the fat scent of CSFL (Zhao, et al., 2012). However, little DSM 17049T, C. tetanomorphum DSM 4474T and C. pascui has been reported about species of the genus Clostridium DSM 10365T, were 58.8 %, 57.9 % and 42.4 %, respect- containing eighteen or more carbon fatty acids in Chinese ively. These values are significantly below the threshold pit mud. In this study, the dominant fatty acids (.10 %) value of 70 % recommended by Wayne et al. (1987) for T of strain BEY10 were C20 : 0 (29.5 %), C16 : 0 (17.7 %), assignment of strains to the same species. Downloaded from www.microbiologyresearch.org by 752 International Journal of Systematic and Evolutionary Microbiology 66 IP: 130.132.173.170 On: Mon, 27 Jun 2016 13:30:03 Clostridium liquoris sp. nov.

Table 2. Cellular fatty acid contents (%) of isolate BEY10T Mianzhu, Sichuan, China. The DNA G+C content of the and type strains of phylogenetically closest related species of type strain is 34.4 mol%. Clostridium

Strains: 1, BEY10T;2,C. lundense DSM 17049T;3,C. tetanomorphum Acknowledgements T T DSM 4474 ;4C. pascui DSM 10365 . All data are from this study. TR, This work was supported by the Natural Science Foundation of China Trace (,1 %); 2, not detected. Fatty acids amounting to ,1%of (nos 31270531 and 31470020), and the Open-foundation project of the total fatty acids in all strains are not shown. Key Laboratory of Environmental and Applied Microbiology, Chinese Academy of Sciences (no. KLCAS-2013). Fatty acid 1234

C11 : 0 2 2.0 22 iso-C11 : 0 3-OH 1.5 2.3 1.7 5.3 References C12 : 0 13.0 1.9 2 3.6 Atlas, R. M. (2010). Handbook of Microbiological Media, 4th edn. Boca iso-C12 : 0 3-OH 2 1.1 22 Raton, FL: CRC Press. C13 : 1 2 3.8 1.1 2 C14 : 0 10.8 43.4 19.5 22.6 Bligh, E. G. & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37, 911–917. C16 : 0 17.7 19.4 54.3 27.4 anteiso-C16 : 0 TR 221.9 Cai, G., Jin, B., Saint, C. & Monis, P. (2011). Genetic manipulation of C16 : 1v9c TR 7.1 TR 4.8 butyrate formation pathways in Clostridium butyricum. J Biotechnol 155, 269–274. anteiso-C17 : 0 1.3 1.5 2.3 5.0 iso-C17 : 1v5c 2.3 TR TR TR Cirne, D. G., Delgado, O. D., Marichamy, S. & Mattiasson, B. (2006). C18 : 0 13.1 3.1 3.4 8.1 Clostridium lundense sp. nov., a novel anaerobic lipolytic bacterium isolated from bovine rumen. Int J Syst Evol Microbiol 56, 625–628. C18 : 1v9c 1.3 223.3 C19 : 0 1.0 222 Colin, T., Bories, A., Lavigne, C. & Moulin, G. (2001). Effects of acetate iso-C19 : 1 1.6 222 and butyrate during glycerol fermentation by Clostridium butyricum. Curr Microbiol 43, 238–243. C20 : 0 29.5 222 C20 : 1v7c 4.0 222 De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative C20 : 1v9c 1.3 222 measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142. Felsenstein, J. (1985). Confidence limits on phylogenies: an approach Based on the analysis of morphological, phylogenetic and using the bootstrap. Evolution 39, 783–791. T physiological characteristics, strain BEY10 represents a Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (1994). novel species of the genus Clostridium, for which the Methods for General and Molecular Bacteriology. Washington, DC: name Clostridium liquoris sp. nov. is proposed. American Society for Microbiology. Johnston, N. C. & Goldfine, H. (1983). Lipid composition in the classification of the butyric acid-producing clostridia. J Gen Description of Clostridium liquoris sp. nov. Microbiol 129, 1075–1081. Clostridium liquoris (li9quo.ris. L. gen. n. liquoris of a Johnston, N. C., Aygun-Sunar, S., Guan, Z., Ribeiro, A. A., Daldal, F., Raetz, C. R. H. & Goldfine, H. (2010). liquor, relating to the fermentation pit of Chinese liquor, A phosphoethanolamine- modified glycosyl diradylglycerol in the polar lipids of Clostridium where the type strain is isolated). tetani. J Lipid Res 51, 1953–1961. Cells are Gram-stain-positive, strictly anaerobic, rod- Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., shaped, 0.5–1.0|2.5–6 mm, non-motile and spore-form- Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that ing. Colonies are light yellow with a circular convex surface 62 after 72 h at 37 uC when grown on peptone/yeast medium. represent uncultured species. Int J Syst Evol Microbiol , 716–721. Growth occurs at 22–47 uC (optimum 37 uC), in 0–4 % (w/ Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid v) NaCl (optimum 0 %) and at pH 5.5–9.0 (optimum Techniques in Bacterial Systematics, pp. 115–175. Edited by E. Stackebrandt & M. Goodfellow. Chichester: Wiley. pH 7.5–8.5). Tests for catalase and indole production are Liu, C., Huang, D., Liu, L., Zhang, J., Deng, Y., Chen, L., Zhang, W., Wu, negative. Gelatin and aesculin are not hydrolysed. Utilizes Z., Fan, A. & other authors (2014). Clostridium swellfunianum sp. glucose, mannitol, lactose, xylose, maltose, glycerol, cello- nov., a novel anaerobic bacterium isolated from the pit mud of biose and trehalose as sole carbon sources; mannose, mele- Chinese Luzhou-flavor liquor production. Antonie van Leeuwenhoek zitose, raffinose, sorbitol and rhamnose are weakly utilized. 106, 817–825. Ethanol and butyric acid are the major fermentation pro- Markossian, S., Becker, P., Ma¨ rkl, H. & Antranikian, G. (2000). ducts from glucose. The polar lipids consist of phosphati- Isolation and characterization of lipid-degrading Bacillus dylglycerol, phosphatidylethanolamine, phospholipids, a thermoleovorans IHI-91 from an icelandic hot spring. Extremophiles glycolipid and an aminolipid. The major fatty acids are 4, 365–371. C20 : 0,C16 : 0,C18 : 0,C12 : 0 and C14 : 0. Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by T T T The type strain BEY10 (5 ACCC 00785 5 DSM 100320 ) high-performance liquid chromatography. Int J Syst Bacteriol 39, was isolated from an old fermentation pit located in 159–167.

Downloaded from www.microbiologyresearch.org by http://ijs.microbiologyresearch.org 753 IP: 130.132.173.170 On: Mon, 27 Jun 2016 13:30:03 Q. Yin and others

Mu¨ ller, V. (2003). Energy conservation in acetogenic bacteria. Appl Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Environ Microbiol 69, 6345–6353. Higgins, D. G. (1997). The CLUSTAL_Xwindows interface: flexible Papoutsakis, E. T. (2008). Engineering solventogenic clostridia. Curr strategies for multiple sequence alignment aided by quality analysis 25 Opin Biotechnol 19, 420–429. tools. Nucleic Acids Res , 4876–4882. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method Tindall, B. J., Sikorski, J., Smibert, R. A. & Krieg, N. R. (2007). for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425. Phenotypic characterization and the principles of comparative systematics. In Methods for General and Molecular Microbiology, pp. Sasser, M. (1990). Identification of bacteria by gas chromatography 330–393. Edited by C. A. Reddy, T. J. Beveridge, J. A. Breznak, of cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI G. A. Marzluf, T. M. Schmidt & L. R. Snyder. Washington, DC: Inc. American Society for Microbiology. Seedorf, H., Fricke, W. F., Veith, B., Bru¨ ggemann, H., Liesegang, H., Tracy, B. P., Jones, S. W., Fast, A. G., Indurthi, D. C. & Papoutsakis, Strittmatter, A., Miethke, M., Buckel, W., Hinderberger, J. & other E. T. (2012). Clostridia: the importance of their exceptional substrate authors (2008). The genome of Clostridium kluyveri, a strict and metabolite diversity for biofuel and biorefinery applications. Curr anaerobe with unique metabolic features. Proc Natl Acad Sci U S A Opin Biotechnol 23, 364–381. 105, 2128–2133. UK-Standards-for-Microbiology-Investigations (2011). Identification Shcherbakova, V. A., Chuvilskaya, N. A., Rivkina, E. M., of Clostridium species. In: https://www.gov.uk/government/uploads/ Pecheritsyna, S. A., Laurinavichius, K. S., Suzina, N. E., Osipov, system/uploads/attachment_data/file/400746/ID_8i4.pdf G. A., Lysenko, A. M., Gilichinsky, D. A. & Akimenko, V. K. (2005). Novel psychrophilic anaerobic spore-forming bacterium from the Van Dyke, M. I. & McCarthy, A. J. (2002). Molecular biological overcooled water brine in permafrost: description Clostridium detection and characterization of Clostridium populations in 68 algoriphilum sp. nov. Extremophiles 9, 239–246. municipal landfill sites. Appl Environ Microbiol , 2049–2053. Shi, S., Zhang, W. X., Deng, Y., Zhong, F. D. & Hu, F. (2010). Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, Construction of microbial fingerprint in Chinese liquor production O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & by DGGE technique. China Brewing 3, 118–120 (In Chinese). other authors (1987). International Committee on Systematic Spring, S., Merkhoffer, B., Weiss, N., Kroppenstedt, R. M., Hippe, H. Bacteriology. Report of the ad hoc committee on reconciliation & Stackebrandt, E. (2003). Characterization of novel psychrophilic of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464. clostridia from an Antarctic microbial mat: description of Wiegel, J., Tanner, R. & Rainey, F. (2006). An Introduction to the Clostridium frigoris sp. nov., Clostridium lacusfryxellense sp. nov., Family Clostridiaceae.InThe Prokaryotes, 3rd edn., pp. 654–678. Clostridium bowmanii sp. nov. and Clostridium psychrophilum sp. Edited by M. Dworkin, S. Falkow, E. Rosenberg, K.-H. Schleifer & nov. and reclassification of Clostridium laramiense as Clostridium E. Stackebrandt. New York: Springer. estertheticum subsp. laramiense subsp. nov. Int J Syst Evol Microbiol Wilde, E., Hippe, H., Tosunoglu, N., Schallehn, G., Herwig, K. & 53, 1019–1029. Gottschalk, G. (1989). Clostridium tetanomorphum sp. nov., nom. Suetin, S. V., Shcherbakova, V. A., Chuvilskaya, N. A., Rivkina, E. M., rev. Int J Syst Bacteriol 39, 127–134. Suzina, N. E., Lysenko, A. M. & Gilichinsky, D. A. (2009). Wilde, E., Collins, M. D. & Hippe, H. (1997). Clostridium pascui sp. Clostridium tagluense sp. nov., a psychrotolerant, anaerobic, spore- nov., a new glutamate-fermenting sporeformer from a pasture in forming bacterium from permafrost. Int J Syst Evol Microbiol 59, Pakistan. Int J Syst Bacteriol 47, 164–170. 1421–1426. Xu, Y., Wang, D., Fan, W., Mu, X. & Chen, J. (2010). Traditional Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, Chinese Biotechnology. In Biotechnology in China, pp. 189–233. S. (2011). MEGA5: molecular evolutionary genetics analysis using Edited by G. T. Tsao, P. Ouyang & J. Chen. Berlin: Springer. maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739. Yang, Z. (1997). paml: a program package for phylogenetic analysis by maximum likelihood. Comput Appl Biosci 13, 555–556. Tao, Y., Li, J., Rui, J., Xu, Z., Zhou, Y., Hu, X., Wang, X., Liu, M., Li, D. & Li, X. (2014). Prokaryotic communities in pit mud from different- Zhao, J.-S., Zheng, J., Zhou, R.-Q. & Shi, B. (2012). Microbial aged cellars used for the production of Chinese strong-flavored community structure of pit mud in a Chinese strong aromatic liquor. Appl Environ Microbiol 80, 2254–2260. liquor fermentation pit. J Inst Brew 118, 356–360.

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