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

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1 International Journal of Systematic and Evolutionary Microbiology (2014), 64, DOI /ijs Clostridium huakuii sp. nov., an anaerobic, acetogenic bacterium isolated from methanogenic consortia Zhiyong Ruan, 1 Yanwei Wang, 1 Chi Zhang, 2 Jinlong Song, 3 Yi Zhai, 4 Yan Zhuang, 1 Huimin Wang, 1 Xiaorong Chen, 1 Yanting Li, 1 Bingqiang Zhao 1 and Bin Zhao 2 Correspondence Bingqiang Zhao zhaobingqiang@caas.cn Bin Zhao binzhao@mail.hzau.edu.cn 1 Key Laboratory of Microbial Resources (Ministry of Agriculture, China), Institute of Agricultural Resources and Regional Planning, CAAS, Beijing 081, PR China 2 State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan , PR China 3 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 101, PR China 4 Agricultural Engineering Institute, Chongqing Academy of Agricultural Sciences, Chongqing , PR China A Gram-staining-positive, spore-forming, obligately anaerobic, acetogenic bacterium, designated LAM1030 T, was isolated from methanogenic consortia enriched from biogas slurry collected from the large-scale anaerobic digester of Modern Farming Corporation in Hebei Province, China. Cells of strain LAM1030 T were motile, straight or spiral-rod-shaped. Strain LAM1030 T could utilize glucose, fructose, maltose, galactose, lactose, sucrose, cellobiose, mannitol, pyruvate, succinic acid and tryptophan as the sole carbon source. Acetic acid, isovaleric acid and butanoic acid were the main products of glucose fermentation. Sodium sulfite was used as an electron acceptor. Growth of strain LAM1030 T was completely inhibited by the addition of ampicillin, tetracycline, gentamicin or erythromycin at a concentration of 20 mg ml 1. The main polar lipids of strain LAM1030 T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, 11 unknown glycolipids and two unknown phospholipids. No respiratory quinone was detected. The major fatty acids of strain LAM1030 T were C 16 : 0 (21.1 %), C 14 : 0 (10.3 %), summed feature 9 (including C16:0 10- methyl and/or iso-c17:1 v9c) (11.3% ), summed feature 3 (including C16:1 v7c and/or C16:1 v6c) (10.6% ) and iso-c 15 : 0 (6.6 %). Analysis of the 16S rrna gene sequence indicated that strain LAM1030 T belonged to the genus Clostridium and was most closely related to Clostridium subterminale DSM 6970 T, Clostridium thiosulfatireducens DSM T and Clostridium sulfidigenes DSM T, with 97.0, 96.9 and 96.8 % similarity, respectively. The G+C content of the genomic DNA of strain LAM1030 T was 31.2±0.3 mol%. On the basis of its phenotypic, phylogenetic and chemotaxonomic characterization, strain LAM1030 T is suggested to represent a novel species of the genus Clostridium, for which the name Clostridium huakuii sp. nov. is proposed. The type strain is LAM1030 T (5ACCC T 5JCM T ). Methanogenesis is the formation of methane by microbes, which is a key biochemical reaction during the carbon cycle on Earth, and a lot of micro-organisms are involved in the complex biochemical reaction system (Mah et al., 1977; Thauer et al., 2008; Battin et al., 2009; Bastviken et al., 2011). Acetogenic bacteria contribute significantly to the The GenBank/EMBL/DDBJ accession number for the 16S rrna gene sequence of strain LAM1030 T is KC Four supplementary figures and two supplementary tables are available with the online Supplementary Material. turnover of organic matter in methanogenic bioreactors and landfills (McInerney & Bryant, 1981; Ibba & Fynn, 1991; Wiegel, 1994; Barlaz, 1997). Many investigations have been carried out into the isolation and characterization of acetogens (Drake et al., 2008). Fontaine et al. (1942) isolated a novel organism, Clostridium thermoaceticum, which was the first reported acetogen, involved in the conversion of glucose to acetate. At the time of writing, more than acetogenic species have been isolated from various habitats, and the genera Acetobacterium and Clostridium harbour most of the known species. While G 2014 IUMS Printed in Great Britain 4027

2 Z. Ruan and others studying the microbial community of methanogenic consortia enriched from biogas slurry, a Clostridium-like acetogen, designated LAM1030 T, was isolated. Based on its phenotypic and genotypic characteristics, the new isolate was considered to represent a novel species of the genus Clostridium. Strain LAM1030 T was isolated using a modified Hungate technique (Hungate, 1969). MB medium [10.0 g peptone, 10.0 g yeast extract, 5.0 g glucose, 0.5 g cysteine, 1.0 mg resazurin and 40.0 ml salt solution (DSMZ medium 104), distilled water added to 1 l final volume, ph 7.0) was used for the methanogenic consortia enrichment. MB medium was transferred into serum bottles ( ml) under O 2 -free N 2 and autoclaved at 121 uc for 20 min. Biogas slurry (10 ml) was added into the bottles and incubated anaerobically at 30 uc for 10 days. The Hungate technique (Hungate, 1969; Bryant, 1972) was modified to isolate anaerobes from the enrichment samples using brain heart medium (BD/Difco ). Strains were purified by subculturing onto fresh medium at least twice before being preserved in 25 % (v/v) glycerol at 280 uc for further study. The strain grew well in brain heart infusion medium (BD/ BBL ) at 35 uc. Morphological characteristics of cells from an exponentially growing culture were analysed by using a light microscope (Nikon 80i) and transmission electron microscope (Hitachi 7500) (Ruan, et al., 2014). Standard physiological identifications were carried out according to the methods modified by Cowan & Steel (1965) and Lányi (1987). The optimal physiological conditions for the growth of strain LAM1030 T were investigated in brain heart infusion medium. All tests were conducted independently in duplicate. The strain was incubated in the Hungate tube at temperatures of 10, 15, 20, 25, 30, 35, 40, 45 and 50 uc, ph 5 10 (in 0.5 ph unit increments) and NaCl concentrations of 0 7 % (w/v, in 0.5 % increments) and also 8 % and 10 %. The ph was adjusted to the desired value by using sterile solutions of citric acid/ Na 2 HPO 4 (ph 4.0 to 5.0), MES (ph 5.5 to 6.0), PIPES (ph 6.5 to 7.0), Tricine (ph 7.5 to 8.5), CAPSO (ph 9.0 to 9.5) or CAPS (ph 10.0 to 11.5) added at a final concentration of 30 mm (Ruan et al., 2014). The AN MicroPlate (Biolog) and API 20A kit (biomérieux) were used to test the utilization of various substrates as sole carbon and energy sources according to the manufacturers instructions. API ZYM (biomérieux) was used to test enzyme activities. To test the reduction of electron acceptors, filter-sterilized solutions of sodium nitrite (5 mm), sodium nitrate (20 mm), sodium thiosulfate (20 mm), sodium sulfite (5 mm) or sodium sulfate (20 mm) were added to the basal medium (4 g peptone, 2 g yeast extract, 0.6 g CaCl 2, 0.1 g NH 4 Cl, 0.2 g MgCl 2, 0.1 g KCl, 2 g NaCl, 2.5 g NaHCO 3, 7.2 g HEPES, distilled water added to 1 l, ph 7.0). Elemental sulfur (1 %) and amorphous Fe (III) oxyhydroxide (0.2 %) were added to the basal medium directly before autoclaving (Ramamoorthy et al., 2006). The main products from glucose fermentation in the basal medium were analysed and quantified as described by Steer et al. (2001). Antibiotic sensitivity was tested under different concentrations of ampicillin, tetracycline, gentamicin and erythromycin. Strain LAM1030 T was incubated in the presence of 0, 10, 20, 50,, 200 and 300 mg each antibiotic ml 21. Genomic DNA was extracted and purified by using a TIANamp Bacter DNA kit (Tiangen Biotech) according to the manufacturer s instruction. The 16S rrna gene of strain LAM1030 T was amplified by PCR with the bacterial universal primers 27F (59-AGAGTTTGATCCTGGCTCAG- 39) and 1492R (59-GGTTACCTTGTTACGACTT-39) (Lane, 1991). Purified PCR products of approximately 1.4 kb were sequenced by Life Technologies (Shanghai, China). Sequence similarity analysis and multiple sequence alignment were conducted by using the EzTaxon-e service (Kim et al., 2012) and CLUSTAL X (Thompson et al., 1994) respectively. Phylogenetic trees were reconstructed via the neighbour-joining and maximum-parsimony methods with the MEGA4 program package (Tamura et al., 2007). Evolutionary distances were calculated according to the algorithm of Kimura s two-parameter model (Kimura, 1980) for the neighbour-joining method. The DNA G+C content was determined by the thermal denaturation method by using a Beckman DU 800 spectrophotometer (Beckman Coulter); Escherichia coli K12 was used as the reference strain. Chemotaxonomic analyses were performed on strain LAM1030 T and Clostridium thiosulfatireducens DSM T. Cells were harvested in the late exponential phase of growth in brain heart infusion medium at 35 uc. Polar lipids were extracted and separated on silica gel plates (10610 cm, Merck 5554) and further analysed with the method described by Minnikin et al. (1984) and Xu et al. (2011). Sulfuric acid was used to reveal total lipids, ninhydrin for aminolipids, a-naphthol for glycolipids and Zinzadze reagent for phospholipids; the results were analysed as described by Fang et al. (2012). Isoprenoid quinones were extracted from lyophilized cells, which had been cultivated in brain heart infusion medium for 36 h at 35 uc, using chloroform/methanol (2 : 1, v/v), evaporated under vacuum conditions and re-extracted in n-hexane/water (1 : 1, v/v). The crude solution was purified using Sep-Pak Vac silica cartridges (Waters) and subsequently analysed by HPLC (Dionex UltiMate 3000) (Zhu et al., 2012). Analysis of major fatty acids was performed on strain LAM1030 T and C. thiosulfatireducens DSM T. Both strains were incubated in trypticase soy broth (TSB; BD/ Difco ) medium at 35 uc for 36 h. Fatty acid methyl esters were obtained from fresh cells collected from the serum bottles. The identification and quantification of the fatty acid methyl esters as well as the numerical analysis of the fatty acid profiles were carried out by using the Sherlock Microbial Identification System with the standard MIS Library Generation Software (Microbial ID) according to the manufacturer s instruction. The cell morphology of strain LAM1030 T was similar to that of members of the genus Clostridium, endospore-forming, 4028 International Journal of Systematic and Evolutionary Microbiology 64

3 Clostridium huakuii sp. nov. motile, straight or spiral-rod-shaped with a cell size of 0.3 mm to 0.4 mm in width and 1.5 mm to 3.0 mm in length (Fig. S1, available in the online Supplementary Material). Electron micrographs revealed a typical Gram-positive cell wall structure (Fig. S1). The ph and temperature ranges for growth were ph 6.0 to 9.0 (optimum ph ) and 25 to 45 uc (optimum 35 uc), respectively. The strain grew well in medium with 10 g NaCl l 21 and tolerated up to 30 g NaCl l 21. Strain LAM1030 T was able to utilize the following substrates as carbon and energy sources: N-acetyl-Dglucosamine, N-acetyl-b-D-mannosamine, amygdalin, D- arabitol, arbutin, cellobiose, dextrin, i-erythritol, D-fructose, L-fucose, D-galactose, D-galacturonic acid, gentiobiose, D- glucosaminic acid, a-d-glucose, a-d-glucose 1-phosphate, D-glucose 6-phosphate, glycerol, L-a-glycerol phosphate, myo-inositol, a-lactose, lactulose, maltose, maltotriose, D- mannitol, D-mannose, melezitose, melibiose, methyl a-dgalactoside, methyl b-d-galactoside, methyl b-d-glucoside, palatinose, D-raffinose, L-rhamnose, salicin, sucrose, D- trehalose, turanose, pyruvic acid, pyruvic acid methyl ester, succinic acid, inosine, uridine, thymidine-59-monophosphate and uridine-59-monophosphate. Compared with the reference strains, cells of strain LAM 1030 T were positive for the utilization of lactose, D-glucose, D-galactose, D- mannose, cellobiose, maltose, sucrose, D-fructose, succinic acid and D-mannitol, while negative for the utilization of L- alanine, L-glutamate, L-histidine, L-methionine, L-phenylalanine and L-threonine. In API ZYM tests, strain LAM1030 T gave positive reactions for leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, acid phosphatase, naphthol-as-bi-phosphohydrolase, a-glucosidase and a- mannosidase, while negative reactions were obtained for alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), a-chymotrypsin, b-galactosidase, b-glucuronidase, b-glucosidase, N-acetyl-b-glucosaminidase and a- fucosidase. A comparison of the physiological characteristics between strain LAM1030 T and relative reference strains is shown in Tables 1 and S1. Acetic acid (86.1 %), isovaleric acid (12.2 %) and butanoic acid (1.7 %) were the main endproducts of glucose fermentation. Sodium sulfite was used as an electron acceptor. Growth of strain LAM1030 T was completely inhibited by the addition of ampicillin, tetracycline, gentamicin or erythromycin at a concentration of 20 mg ml 21. Table 1. Differential phenotypic, physiological and genotypic characteristics of strain LAM1030 T and its relatives Strains: 1, LAM1030 T ;2,Clostridium subterminale DSM 6970 T (data from Thabet et al., 2004; Sallam & Steinbüchel, 2009) 3, C. thiosulfatireducens DSM T (this study and Hernández-Eugenio et al., 2002); 4, Clostridium sulfidigenes DSM T (Sallam & Steinbüchel, 2009). +, Positive; 2, negative. Characteristic Source Biogas slurry Human infections Cheese factory Pond sediment wastewater Cell width (mm) Cell length (mm) ph for growth Range Optimum Temperature for growth Range Optimum NaCl concentration for growth Range (g l 21 ) Optimum (g l 21 ) DNA G+C content (mol%) 31.2±0.3* 28D 33.3±0.3* 32.3D Thiosulfate reduction Sulfite reduction Utilization of: L-Alanine L-Glutamate L-Histidine L-Methionine L-Phenylalanine L-Threonine DL-Tryptophan *DNA G+C content was determined by the T m method. DDNA G+C content was determined by HPLC

4 Z. Ruan and others Clostridium estertheticum NCIMB T (X68181) Clostridium lacusfryxellense DSM T (AJ506118) Clostridium frigoris DSM T (AJ506117) Clostridum algoriphilum 14D1 T (AY117755) Clostridum schirmacherense AP15 T (AM114453) Clostridum tagluense A121 T (DQ296031) Clostridium psychrophilum A-1/C-an/I T (AJ297443) Clostridium pascui DSM T (X96736) Clostridium tetanomorphum DSM 4474 T (DQ241819) Clostridium lundense DSM T (AY858804) Clostridum bowmanii DSM T (AJ506120) Clostridium argentinense ATCC T (X68316) Clostridium huakuii LAM1030 T (KC967412) 60 Clostridium senegalense JC122 (JF824801) Clostridum subterminale DSM 6970 T (AB294137) Clostridum thiosulfatireducens DSM T (AY024322) Clostridum sulfidigenes DSM T (EF199998) Fig. 1. Neighbour-joining phylogenetic tree based on a comparison of the 16S rrna gene sequences of strain LAM1030 T and its closest relatives. Bootstrap values (percentages) based on X resamplings are shown at nodes. Bar, 0.05 substitutions per nucleotide position. An almost complete 16S rrna gene sequence (1394 nt) was obtained from strain LAM1030 T. The results of 16S rrna gene sequence analysis indicated that strain LAM1030 T belonged to the genus Clostridium and was most closely related to Clostridium subterminale DSM 6970 T, C. thiosulfatireducens DSM T and Clostridium sulfidigenes DSM T, with 97.0, 96.9 and 96.8 % similarity, respectively (Fig. 1). The topologies of the phylogenetic tree built by using the maximum-parsimony method supported the result that strain LAM1030 T formed a stable clade with these three species (Fig. S2). The G+C content of the genomic DNA of strain LAM1030 T was 31.2±0.3 mol% as determined by the T m method, which was lower than those of C. thiosulfatireducens DSM T and C. sulfidigenes DSM T (33.3 and 32.3 mol%, respectively), but higher than that of C. subterminale DSM 6970 T (28 mol%). The low DNA G+C content was in the range of DNA G+C content of the genus Clostridium. The main polar lipids of strain LAM1030 T were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), 11 unknown glycolipids and two unknown phospholipids. Although the main polar lipids of strain LAM1030 T and C. thiosulfatireducens DSM T were similar, differences existed in their content and other minor components (Figs S3 and S4). No respiratory quinone was detected in strain LAM1030 T. The fatty acid profile of strain LAM1030 T was similar to that of C. thiosulfatireducens DSM T. The major fatty acids of strain LAM1030 T were C 16 : 0 (21.1 %), C 14 : 0 (10.3 %), summed feature 9 (including C16:0 10-methyl and/or iso- C17:1 v9c) (11.3% ), summed feature 3 (including C16:1 v7c and/or C16:1 v6c) (10.6% ) and iso-c 15 : 0 (6.6 %); detailed fatty acid compositions of strain LAM1030 T and C. thiosulfatireducens DSM T are shown in Table S2. On the basis of phenotypic, phylogenetic and chemotaxonomic characteristics, strain LAM1030 T is considered to represent a novel species of the genus Clostridium, for which the name Clostridium huakuii sp. nov. is proposed. Description of Clostridium huakuii sp. nov. Clostridium huakuii (hua.ku9i.i. N.L. gen. n. huakuii of Hua-Kui, named in honour of the Chinese microbiologist Hua-Kui Chen for his contributions to soil microbiology in China). Cells of strain LAM1030 T are anaerobic, motile, Gramstaining-positive, spore-forming, straight or spiral-rodshaped with a cell size of 0.3 to 0.4 mm61.5 to 3.0 mm. The ph and temperature ranges for growth are ph 6.0 to 9.0 (optimum ph ) and 25 to 45 uc (optimum 35 uc), respectively. Grows well in medium with 10 g NaCl l 21 and tolerates up to 30 g NaCl l 21. Able to utilize N-acetyl- D-glucosamine, N-acetyl-b-D-mannosamine, amygdalin, D-arabitol, arbutin, cellobiose, dextrin, i-erythritol, D- fructose, L-fucose, D-galactose, D-galacturonic acid, gentiobiose, D-glucosaminic acid, a-d-glucose, a-d-glucose 1-phosphate, D-glucose 6-phosphate, glycerol, L-a-glycerol phosphate, myo-inositol, a- lactose, lactulose, maltose, maltotriose, D-mannitol, D-mannose, melezitose, melibiose, methyl a-d-galactoside, methyl b-d-galactoside, methyl b-d-glucoside, palatinose, D-raffinose, L-rhamnose, salicin, sucrose, D-trehalose, turanose, pyruvic acid, pyruvic acid methyl ester, succinic acid, inosine, uridine, thymidine-59-monophosphate and uridine-59-monophosphate, 4030 International Journal of Systematic and Evolutionary Microbiology 64

5 Clostridium huakuii sp. nov. but unable to utilize L-alanine, L-glutamate, L-histidine, L- methionine, L-phenylalanine or L-threonine as carbon and energy sources. Positive reactions for leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, acid phosphatase, naphthol-as-bi-phosphohydrolase, a-glucosidase and a-mannosidase in the API ZYM system. The main products of glucose fermentation are acetic acid, isovaleric acid and butanoic acid. Sodium sulfite can be used as an electron acceptor. Growth is completely inhibited by the addition of ampicillin, tetracycline, gentamicin or erythromycin at 20 mg ml 21.C 16 : 0,C 14 : 0, summed feature 9 (including C16:0 10-methyl and/or iso- C17:1 v9c) (11.3% ), summed feature 3 (including C16:1 v7c and/or C16:1 v6c) and iso-c 15 : 0 are the major fatty acids. The main polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, 11 unknown glycolipids and two unknown phospholipids. No respiratory quinone is detected. The type strain is LAM1030 T (5ACCC T 5JCM T ), which is isolated from methanogenic consortia enriched from biogas slurry collected from the large-scale anaerobic digester of Modern Farming Corporation in Hebei Province, China. The genomic DNA G+C content of the type strain is 31.2±0.3 mol% as determined by the T m method. Acknowledgements We thank Professor Min Wu from Zhejiang University greatly for his technical assistance. This work was supported by National Nonprofit Institute Research Grant of the Chinese Academy of Agricultural Sciences (CAAS; no ), National Key Technology R&D Program of China (nos 2013BAD05B04F02 and 2011BAD11B05), National Natural Science Foundation of China (NSFC; no ), Foundation of the Key Laboratory of Development and Application of Rural Renewable Energy (MOA, China) (no ) and Science Foundation of Modern Farming Group (no. MF20518). References Barlaz, M. A. (1997). 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