International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1756–1762
DOI 10.1099/ijs.0.060186-0
Hydrogenispora ethanolica gen. nov., sp. nov., an anaerobic carbohydrate-fermenting bacterium from anaerobic sludge Yi Liu,1,2 Jiang-Tao Qiao,1 Xian-Zheng Yuan,1 Rong-Bo Guo1 and Yan-Ling Qiu1 Correspondence Yan-Ling Qiu [email protected]
1
Shandong Industrial Engineering Laboratory of Biogas Production & Utilization, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong Province 266101, PR China
2
University of Chinese Academy of Sciences, Beijing 100049, PR China
An anaerobic, spore-forming, ethanol-hydrogen-coproducing bacterium, designated LX-BT, was isolated from an anaerobic sludge treating herbicide wastewater. Cells of strain LX-BT were nonmotile rods (0.3–0.5¾3.0–18.0 mm). Spores were terminal with a bulged sporangium. Growth occurred at 20–50 6C (optimum 37–45 6C), pH 5.0–8.0 (optimum pH 6.0–7.7) and 0–2.5 % (w/v) NaCl. The strain could grow fermentatively on glucose, maltose, arabinose, fructose, xylose, ribose, galactose, mannose, raffinose, sucrose, pectin, starch, glycerol, fumarate, tryptone and yeast extract. The major end-products of glucose fermentation were acetate, ethanol and hydrogen. Yeast extract was not required but stimulated growth. Nitrate, sulfate, thiosulfate, elemental sulfur, sulfite, anthraquinone-2,6-disulfonate, fumarate and Fe (III) nitrilotriacetate were not used as terminal electron acceptors. The G+C content of the genomic DNA was 56.1 mol%. The major cellular fatty acids were anteiso-C15 : 0, iso-C14 : 0 and C16 : 0. The most abundant polar lipids of strain LX-BT were diphosphatidylglycerol and phosphatidylglycerol. 16S rRNA gene sequence analysis revealed that it belongs to an as-yet-unidentified taxon at the order- or classlevel (OPB54) within the phylum Firmicutes, showing 86.5 % sequence similarity to previously described species of the Desulfotomaculum cluster. The name Hydrogenispora ethanolica gen. nov., sp. nov. is proposed to accommodate strain LX-BT (5DSM 25471T5JCM 18117T5CGMCC 1.5175T) as the type strain.
The phylum Firmicutes is a phylogenetically and phenotypically divergent bacterial phylum, which plays an important role in biodegradation and the carbon cycle in nature (Ludwig et al., 2009). At the time of writing, there are four recognized classes of the phylum Firmicutes: the anaerobic class Clostridia, the obligately or facultatively aerobic class Bacilli, and the classes Erysipelotrichia and Thermolithobacteria (http://www.bacterio.net/; Yutin & Galperin, 2013). Furthermore, many uncultured taxonomic groups exist in the phylum Firmicutes, such as candidate taxa OPB54, which was first detected from Obsidian Pool, a Yellowstone Park hot spring (Hugenholtz et al., 1998). Based on ARB-SILVA taxonomy, OPB54 is assigned to the class Clostridia at the order level (Dunfield et al., 2012) or represents a new class of the phylum Firmicutes (http://arbsilva.de). Environmental clones belonging to OPB54 have The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain LX-BT is AB669474. A supplementary figure is available with the online version of this paper.
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been found in a wide range of habitats, including soils (Brodie et al., 2006; Kanokratana et al., 2011), anaerobic wastewater and waste treatment systems (Tang et al., 2004), the skin microbiome (Kong et al., 2012) and aquatic moss pillars (Nakai et al., 2012). However, no cultivated representatives of this clade have been reported so far. In this study, an ethanol-type hydrogen-producing bacterium (strain LX-BT), the first cultivated strain belonging to the OPB54 cluster of the phylum Firmicutes is described. Strain LX-BT was originally obtained from a mesophilic (35 uC) anaerobic sludge treating herbicide wastewater. The medium used for isolation and cultivation was prepared as described by Sekiguchi et al. (2000). The sludge was washed and transferred into liquid medium supplemented with 50 mg l21 pyrazosulfuron, which was one of the major components of the original herbicide wastewater. After five transfers, the pyrazosulfuron enrichment culture was serially diluted in liquid medium supplemented with glucose (2 mM). Growth and hydrogen production were observed in higher dilution cultures within 2 weeks of incubation. 060186 G 2014 IUMS Printed in Great Britain
Hydrogenispora ethanolica gen. nov., sp. nov.
Cells from the highest dilution were further purified by repeated serial dilutions in glucose medium, then in glucose agar (2 %) roll tubes (Hungate, 1969). Small, dark brown, round colonies were formed after 2 weeks of incubation at 37 uC. The colonies were picked and retransferred to liquid medium supplemented with 2 mM glucose. This roll-tube isolation step involving transfer of single colonies from solid medium to liquid medium was repeated several times, resulting in the isolation of strain LX-BT. Cell morphology was examined under a fluorescent microscope (Olympus BX50F). Transmission electron microscopy (TEM) was performed with a Hitachi H-7000 transmission electron microscope as described by Sekiguchi et al. (2003). Gram staining was performed by the method of Hucker (Doetsch, 1981). Cells of strain LX-BT were non-motile, straight or slightly curved rods, 0.3–0.5 mm in diameter and
(a)
(b)
Fig. 1. Phase-contrast micrograph (a) and thin-section electron micrograph (b) of cells of strain LX-BT grown on glucose. Bulged sporangium indicated by arrow. Bars, 10 mm (a) and 0.5 mm (inset, 0.1 mm) (b). http://ijs.sgmjournals.org
3.0–18.0 mm in length (Fig. 1a). Spores were located terminally and had a bulged sporangium (Fig. 1a). Gram staining was negative, however, electron microscopy demonstrated that strain LX-BT possessed a Gram-positive-type cell wall (Fig. 1b). Physiological characteristics of strain LX-BT were examined as described by Sekiguchi et al. (2000). The temperature range for growth was determined between 15 and 60 uC (5 uC intervals), the pH range was assessed at 37 uC over the range pH 3.5–8.5 (0.5 pH unit intervals) and NaCl tolerance was checked at 0–3.0 % (w/v, 0.5 % intervals). Cell growth was evaluated on the basis of the increase in OD at 400 nm and the production of hydrogen. Unless otherwise indicated, all cultivations were performed at 37 uC in serum vials (20 ml per 50 ml vial) under a gas phase of N2/CO2 (80 : 20, v/v) without shaking. Aerobic growth was tested in a glucose (2 mM) medium under aerobic conditions without reducing agents. Strain LX-BT grew anaerobically on glucose medium at 20–50 uC (optimum, 37–45 uC), at pH 5.0–8.0 (optimum, 6.0–7.7), and with 0–2.5 % (w/v) NaCl, but did not grow at temperatures below 15 uC or above 55 uC after 8 weeks of incubation. The isolate was a strictly anaerobic organism: it could not grow in the presence of oxygen (20 %, v/v, in the gas phase) or after N2/CO2 purging only (without reducing agents). Vigorous growth and hydrogen production were observed with the following substrates (5 mM each unless otherwise specified): glucose, maltose, arabinose, fructose, xylose, ribose, galactose, mannose, raffinose, sucrose, pectin (0.5 g l21), starch (0.5 g l21), glycerol, fumarate, tryptone (0.5 %) and yeast extract (0.5 %). Yeast extract was not required but enhanced growth. In syntrophic substrate utilization tests, strain LX-BT was co-cultured with the hydrogenotrophic methanogen Methanospirillum hungatei DSM 864T. None of the following substrates tested supported the growth of strain LX-BT in pure culture or co-culture with M. hungatei DSM 864T (5 mM each unless specified): crotonate, pyruvate, xylan (0.5 %), cellulose, Casamino acids, L-glutamate, serine, glycine, ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, ethylene glycol, citrate, formate, H2/CO2 (80 : 20, v/v, head space) plus acetate, propionate, isobutyrate, butyrate, succinate, malate, benzoate, hydroquinone (2 mM), phenol (2 mM), 4-hydroxybenzoate, 2,5-dihydroxybenzoate or pyrazosulfuron (50 mg l21). In the medium supplemented with glucose (2 mM), the major end-products from glucose were hydrogen, acetate and ethanol (1 mol glucose was converted to approximately 1 mol acetate, 0.86 mol ethanol and 2.52 mol hydrogen, electron recovery 98 %) (Fig. 2). Although the strain produced hydrogen, its growth was not stimulated in co-cultivation with M. hungatei. The ratio of ethanol/acetate from glucose degradation in co-culture was same with that in pure culture. Strain LX-BT did not use any of the following electron acceptors within 4 weeks of incubation with glucose (mM): nitrate (5), sulfate (5), thiosulfate (2), elemental sulfur (5), Fe (III) nitrilotriacetate (2), sulfite (1), fumarate (5) 1757
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Concentration [mmol (l culture)–1]
8 Glucose Hydrogen Acetate Ethanol
6
4
2
0 0
2
4 Time (days)
6
8
Fig. 2. Degradation of glucose by pure culture of strain LX-BT.
or anthraquinone-2,6-disulfonate (AQDS) (2). Sensitivity to antibiotics was tested in glucose medium, containing filter-sterilized antibiotics at 50 mg l21. Growth of strain LX-BT was inhibited by gentamicin, kanamycin, lincomycin and tetracycline, but not ampicillin, chloramphenicol, streptomycin or penicillin. For G+C content determination, DNA was extracted and purified according to the methods of Kamagata & Mikami (1991). The DNA G+C content of strain LX-BT was determined from the melting point (Tm) by thermal denaturation (Mandel et al., 1970) using a Lambda 25 UV-Vis spectrophotometer (Perkin Elmer). DNA from Escherichia coli (Sigma D-2001) was used as a reference for the measurements. The DNA G+C content was 56.1 mol% (SD: ±0.06 mol%). Fatty acids of cells were converted to methyl esters using HCl/methanol and identified by GC-MS (Agilent 7890A gas 140 chromatograph coupled to an Agilent 5975C MSD single quadrupole mass spectrometer) (Hanada et al., 2002). For fatty acid methyl ester and polar lipid analyses, cells were harvested at exponential growth phase from cultures grown anaerobically on 10 mM glucose at 37 uC. The major cellular fatty acids were anteiso-C15 : 0 (58.6 %), iso-C14 : 0 (11.1 %), C16 : 0 (9.6 %), iso-C16 : 0 (6.9 %), iso-C15 : 0 (4.3 %) and C14 : 0 (4.2 %). Polar lipids were extracted and analysed by the methods of Tindall (1990) by using two-dimensional TLC (Merck silica gel 60 F254 plates, layer thickness 0.2 mm, no. 5554). The polar lipid patterns were characterized by the presence of diphosphatidylglycerol, phosphatidylglycerol and other unidentified phospholipids and amino phospholipids (Fig. S1, available in the online Supplementary Material).
polymerase (Perkin Elmer). The PCR primers used in the amplification were the bacterial domain universal primer 8F and the prokaryote universal primer 1490R (Weisburg et al., 1991). The PCR product was sequenced directly on a Beckman CEQ-8000 DNA sequencer using a CEQ DTC quick start kit (Beckman Coulter). The phylogenetic tree based on 16S rRNA gene sequences was constructed by the neighbour-joining method (Saitou & Nei, 1987) implemented in the MEGA5 computer program (Tamura et al., 2007) and the distance matrix was calculated by the Jukes– Cantor model (Jukes & Cantor, 1969). Confidence values of branches in the phylogenetic tree were determined using bootstrap analysis based on 1000 resamplings (Felsenstein, 1985). A total of 1443 base pairs of the 16S rRNA gene from strain LX-BT were sequenced and compared with other related sequences in the databases. Phylogenetic analysis revealed that strain LX-BT is closely affiliated to members of the uncultured OPB54 clade at the order- or class-level based on the ARB-SILVA reference database (http://arb-silva.de). The closest relatives of strain LX-BT based on 16S rRNA gene sequences were environmental clones, such as those from uranium contaminated soil (90– 94 % similarity) (Brodie et al., 2006), and clone AC007 (93 % similarity) from a landfill leachate bioreactor (Burrell et al., 2004). The most closely related cultured species was Clostridium sp. 6-31 (94 % similarity), which was an as-yet-undescribed non-cellulolytic bacterium from biocompost (Sizova et al., 2011). The most closely related described species were Desulfotomaculum hydrothermale (86.5 % similarity), an anaerobic sulfate-reducing bacterium isolated from a hot spring (Haouari et al., 2008), Pelotomaculum propionicicum (86.1 %), a syntrophic propionate-degrading bacterium isolated from a methanogenic sludge (Imachi et al., 2007), and Clostridium straminisolvens (84.9 %), a thermophilic cellulolytic bacterium (Kato et al., 2004) (Fig. 3). Sequence similarity values for the 16S rRNA genes of strain LX-BT and related genera such as Desulfotomaculum (,86.5 %), Pelotomaculum (,86.1 %) and Clostridium (,85 %) in the order Clostridiales of the phylum Firmicutes were in a range 84–86 %, justifying the creation of a new genus and novel species to accommodate the strain from a phylogenetic point of view.
For 16S rRNA gene sequencing, the genomic DNA of strain LX-BT was extracted according to the method of Hiraishi (1992). 16S rRNA genes were amplified by PCR with Taq
Comparative phenotypic traits of strain LX-BT and related genera in the order Clostridiales are shown in Table 1. Strain LX-BT shared some common phenotypic traits with related organisms, such as spore formation and chemoheterotrophic growth. Major differences that distinguished strain LX-BT from other related members of the genera Desulfotomaculum, Pelotomaculum and Clostridium were: (i) morphology, cells of strain LX-BT are long thin rods, forming terminal endospores in a bulged sporangium, while the related organisms were short sausage-shaped rods with central or terminal endospores; (ii) metabolism, strain LX-BT is an ethanol-type fermentation bacterium which could not perform syntrophic substrate oxidation in cooperation with hydrogenotrophic methanogens, or sulfate respiration. However, members of the genus Pelotomaculum
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International Journal of Systematic and Evolutionary Microbiology 64
Hydrogenispora ethanolica gen. nov., sp. nov.
Pelotomaculum propionicicum DSM 15578T (AB154390) Cryptanaerobacter phenolicus LR7.2T (AY327251) Rice field soil, Pelotomaculum sp. FP rrnB (AB159558) 76 100 Pelotomaculum schinkii DSM 15200T (X91170) 0.02 Pelotomaculum isophthalicicum JIT (AB232785) 100 Pelotomaculum terephthalicicum DSM 16121T (AB091323) 100 Pelotomaculum thermopropionicum SIT (NR_040840) Desulfotomaculum aeronauticum DSM 10349T (X98407) 94 96 ‘Desulfotomaculum reducens’ MI-1 (U95951) 100 Desulfotomaculum putei TH-11T (AF053929) 95 96 Desulfotomaculum hydrothermale DSM 18033T (EF081293) 50 Desulfotomaculum carboxydivorans DSM 14880T (AY961415) 93 Desulfotomaculum acetoxidans DSM 771T (Y11566) 47 Sporotomaculum hydroxybenzoicum DSM 5475T (Y14845) Desulfotomaculum sapomandens DSM 3223T (AF168365) 100 34 Desulfotomaculum thermoacetoxidans DSM 5813T (Y11573) Desulfotomaculum australicum AB33T (M96665) 100 Desulfotomaculum kuznetsovii DSM 6115T (NR_026410) 99 Thermolithobacter ferrireducens JW/KA-2T (NR_041802) New order/class Peat swamp forest soil, uncultured bacterium PW185 (GQ402678) 52 OPB54 72 Skin popliteal fossa, uncultured bacterium ncd557h04c1 (JF049253) 100 Uranium-contaminated soil, uncultured bacterium AKAU3574 (DQ125579) 72 Aquatic moss pillars, uncultured bacterium MPB2-134 (AB630810) 23 Clostridium sp. 6-31 (FJ808611) 68 Methanogenic landfill leachate bioreactor, uncultured Clostridium sp. AC007 (AY330129) 93 39 Thermophilic anaerobic soild waste bioreactor, uncultured bacterium HAW-R60-B-745d-BD (FN436105) 100 Hydrogenispora ethanolica LX-BT (AB669474) 56 Rice paddy soil, uncultured bacterium TSBAR001_M17 (AB486189) Thermophilic anaerobic solid waste bioreactor, uncultured bacterium B55_K_B_D09 (DQ887958) 43 Skin popliteal fossa, uncultured bacterium ncd1975b01c1 (JF172030) 92 Leachate sediment, uncultured Clostridium sp. De150 (HQ183805) 100 98 Pit mud, uncultured bacterium b19-147 (JX576081) 70 Thermophilic anaerobic municipal solid waste digester, uncultured bacterium MBA05 (AB114315) 68 Clostridium straminisolvens DSM 16021T (AB125279) Caldanaerobius fijiensis DSM 17918T (EF507903) 100 100 Caldanaerobius zeae mel2T (U75933) 36 Thermoanaerobacterium thermosaccharolyticum D120-70 (AF247003) Caldicellulosiruptor saccharolyticus DSM 8903T (NR_074845) 21 Caldicellulosiruptor bescii DSM 6725T (L09180) 100 ‘Tepidanaerobacter acetatoxydans’ DSM 21804 (NR_074537) Thermoanaerobacter sulfurophilus L-64T (Y16940) 99 77 Caldanaerobacter subterraneus SL9T (AY216597) 13 63 Thermanaeromonas toyohensis DSM 14490T (NR_024777) Moorella thermoacetica AMP (AY884087) Syntrophomonas bryantii DSM 3014T (HE654006) 100 37 Thermosyntropha lipolytica JW/VS-265T (X99980) Carboxydothermus hydrogenoformans Z-2901T (NC_007503) 44 Syntrophaceticus schinkii Sp3T (EU386162) 84 Thermacetogenium phaeum DSM 12270T (NC_018870) 100 Thermaerobacter litoralis KW1T (NR_043281) 100 Thermaerobacter subterraneus ir-3 (EU214630) Symbiobacterium thermophilum IAM 14863T (NC_006177) 74 ‘Caldinitratiruptor microaerophilus’ CA62N (GQ405534) 99 63 94
62
100
Sulfobacillus thermotolerans Kuch1T (JX966410) Sulfobacillus thermosulfidooxidans DSM 9293T (NR_040945) Anaerolinea thermolimosa DSM 16554T (NR_040970)
Fig. 3. Phylogenetic relationships among strain LX-BT and representatives within the bacterial phylum Firmicutes. The 16S rRNA gene sequence of Anaerolinea thermolimosa DSM 16554T was used as an out-group. The tree was calculated on the basis of a distance matrix analysis of 16S rRNA gene sequences (neighbour-joining tree). Bar, 0.02 nucleotide changes per sequence position. Numbers at nodes show bootstrap values (%) obtained with 1000 resampling analyses.
could perform syntrophic substrate oxidation with hydrogenotrophic methanogens (Imachi et al., 2002, 2007; Qiu et al., 2006), and members of the genus Desulfotomaculum could perform sulfate respiration (Haouari et al., 2008; Liu et al., 1997; Parshina et al., 2005). Strain LX-BT showed similar fermentative metabolism to C. straminisolvens in the family Clostridiaceae, however, their substrate spectrums for growth were different. For example, strain LX-BT was able to grow on various carbohydrates and proteinaceous compounds, whereas C. straminisolvens is a thermophilic cellulolytic bacterium, which can only use cellulose and cellobiose (Kato et al., 2004). http://ijs.sgmjournals.org
Strain LX-BT is the first cultivable isolate accommodated in the OPB54 lineage. On the basis of phylogenetic, genetic and physiological properties, the strain represents a novel species of a novel genus in the phylum Firmicutes. The name Hydrogenispora ethanolica is proposed. Description of Hydrogenispora gen. nov. Hydrogenispora [Hy.dro.ge.ni.spo9ra. N.L. n. hydrogenum (from Gr. n. hudoˆr water; and Gr. v. gennaio to produce) hydrogen; Gr. n. spora a seed, and, in biology, a spore; Hydrogenispora a spore-forming, hydrogen-producing bacterium]. 1759
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Table 1. Characteristics of strain LX-BT and members of the related genera Desulfotomaculum, Pelotomaculum and Clostridium within the phylum Firmicutes Strains: 1, LX-BT; 2, Desulfotomaculum hydrothermale DSM 18033T; 3, Desulfotomaculum putei DSM 12395T; 4, Desulfotomaculum carboxydivorans DSM 14480T; 5, Pelotomaculum thermopropionicum DSM 13744T; 6, Pelotomaculum terephthalicicum DSM 16121T; 7, Clostridium straminisolvens DSM 16021T. +, Positive; 2, negative; ND, no data available. Characteristic
OPB54
Order Clostridiales Family Peptococcaceae
1
2
3
4
Family Clostridiaceae 5
6
7
International Journal of Systematic and Evolutionary Microbiology 64
Similarity (%) with strain LX-BT Cell size (mm) Motility Spore location Optimum temperature (uC) for growth (range) Optimum pH for growth (range) NaCl tolerance (%, w/v) DNA G+C content (mol%) Major cellular fatty acids Substrate utilization Carbohydrates Glycerol Fumarate Pyruvate Lactate Metabolism
– 86.5 85.6 85.3 86.1 0.3–0.563.0–18.0 1.063.0–6.0 1.0–1.162.0–5.0 0.5–1.565.0–15.0 0.7–0.861.7–2.8 2 + + + 2 Terminal Subterminal to terminal Paracentral Subterminal or terminal Central 37–45 (20–50) 55 (40–60) 64 (40–65) 55 (30–68) 55 (45–65)
84.9 0.8–1.062.0–3.0 + Central 37 (30–40)
84.9 0.5–1.063.0–8.0 2 Subterminal 50–55 (50–60)
6.0–7.7 (5.0–8.0) 0–2.5 56.1 anteiso-C15 : 0
7.1 (5.8–8.2) 0–1.5 46.8
7.0 (6.5–7.5) 0–1.5 53.6 iso-C15 : 0, C14 : 0
7.5 (6.0–8.5)
+ + + 2 2 Fermentative
Electron acceptors Habitat
2 Anaerobic sludge
2 + 2 + + Fermentative, respiratory Sulfate Hot spring
This study
Haouari et al. (2008)
Reference
ND
7.5 (6.0–7.9) 0–2.0 47.1 C16 : 0, iso-C15 : 0
6.8–7.2 (6.0–8.0) 0–1.7 46.9 C16 : 0, iso-C16 : 0
7.0 (6.5–8.0) 0–0.4 52.8 iso-C15 : 0
Only fructose
+ 2 2 + + Fermentative, respiratory Sulfate Anaerobic sludge
2 2 + + + Fermentative, syntrophic Fumarate Anaerobic sludge
ND ND
+ 2 Fermentative, respiratory Sulfate Terrestrial subsurface Liu et al. (1997)
ND
41.3 ND
2 Only cellulose, cellobiose 2 ND 2 ND 2 ND 2 ND Fermentative, Fermentative syntrophic ND 2 Anaerobic sludge Rice straw
Parshina et al. (2005) Imachi et al. (2002) Qiu et al. (2006)
Kato et al. (2004)
Hydrogenispora ethanolica gen. nov., sp. nov.
Strictly anaerobic, non-motile, spore-forming rods. Ferment various sugars but not amino acids or aromatic compounds. Oxygen, nitrate, sulfate, thiosulfate, elemental sulfur, sulfite, anthraquinone-2,6-disulfonate, fumarate and Fe (III) nitrilotriacetate do not serve as electron acceptors for growth. The type species is Hydrogenispora ethanolica.
Shows the following characteristics in addition to those given for the genus. Cells are rods, 0.3–0.5 mm in diameter and 3.0–18.0 mm in length. Produces terminal endospores in bulged sporangium. Surface colonies on agar are dark brown and round after cultivation at 37 uC for 1–2 weeks. Grows at 20–50 uC (optimum 37–45 uC), pH 5.0–8.0 (optimum pH 6.0–7.7) and with 0–2.5 % (w/v) NaCl. Yeast extract is not required but stimulates growth. Ferments glucose, maltose, arabinose, fructose, xylose, ribose, sucrose, galactose, mannose, raffinose, pectin, starch, glycerol, fumarate, tryptone and yeast extract in pure culture. The major end-products from glucose are acetate, ethanol and hydrogen. Sensitive to gentamicin, kanamycin, lincomycin and tetracycline, but resistant to ampicillin, chloramphenicol, streptomycin and penicillin. The main fatty acids are anteiso-C15 : 0, iso-C14 : 0 and C16 : 0. The most abundant polar lipids are diphosphatidylglycerol and phosphatidylglycerol. T
The type strain is LX-B (5DSM 25471 5JCM 18117 5 CGMCC 1.5175T), isolated from a mesophilic anaerobic sludge treating herbicide wastewater. The DNA G+C content of the type strain is 56.1 mol%.
Acknowledgements We thank Dr Yuji Sekiguchi of the Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) for help with phylogenetic analysis, and Fei Li and Xiaofei Yan for transmission electron microscopy. This work was supported by the Taishan Scholar Program of Shandong Province, the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2YW-G-052, KG2D-EW-304-1), the National Natural Science Foundation of China (51078344) and the National Key Technology R&D Program (2013BAD22B00).
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philic, aerotolerant and cellulolytic bacterium isolated from a cellulose-degrading bacterial community. Int J Syst Evol Microbiol 54, 2043–2047. Kong, H. H., Oh, J., Deming, C., Conlan, S., Grice, E. A., Beatson, M. A., Nomicos, E., Polley, E. C., Komarow, H. D. & other authors (2012). Temporal shifts in the skin microbiome associated with
disease flares and treatment in children with atopic dermatitis. Genome Res 22, 850–859. Liu, Y. T., Karnauchow, T. M., Jarrell, K. F., Balkwill, D. L., Drake, G. R., Ringelberg, D., Clarno, R. & Boone, D. R. (1997). Description of two
new thermophilic Desulfotomaculum spp., Desulfotomaculum putei sp. 1761
Y. Liu and others nov, from a deep terrestrial subsurface, and Desulfotomaculum luciae sp. nov, from a hot spring. Int J Syst Bacteriol 47, 615–621.
Sekiguchi, Y., Kamagata, Y., Nakamura, K., Ohashi, A. & Harada, H. (2000). Syntrophothermus lipocalidus gen. nov., sp. nov., a novel
Ludwig, W., Schleifer, K.-H. & Whitman, W. B. (2009). Revised road
thermophilic, syntrophic, fatty-acid-oxidizing anaerobe which utilizes isobutyrate. Int J Syst Evol Microbiol 50, 771–779.
map to the phylum Firmicutes. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol 3, pp. 1–13. Edited by P. De Vos, G. M. Garrity, D. Jones, N. R. Krieg, W. Ludwig, F. A. Rainey, K.-H. Schleifer & W. B. Whitman. New York: Springer. Mandel, M., Igambi, L., Bergendahl, J., Dodson, M. L., Jr & Scheltgen, E. (1970). Correlation of melting temperature and cesium chloride buoyant
density of bacterial deoxyribonucleic acid. J Bacteriol 101, 333–338. Nakai, R., Abe, T., Baba, T., Imura, S., Kagoshima, H., Kanda, H., Kanekiyo, A., Kohara, Y., Koi, A. & other authors (2012). Microflorae
of aquatic moss pillars in a freshwater lake, East Antarctica, based on fatty acid and 16S rRNA gene analyses. Polar Biol 35, 425–433. Parshina, S. N., Sipma, J., Nakashimada, Y., Henstra, A. M., Smidt, H., Lysenko, A. M., Lens, P. N. L., Lettinga, G. & Stams, A. J. M. (2005).
Desulfotomaculum carboxydivorans sp. nov., a novel sulfate-reducing bacterium capable of growth at 100 % CO. Int J Syst Evol Microbiol 55, 2159–2165. Qiu, Y. L., Sekiguchi, Y., Hanada, S., Imachi, H., Tseng, I. C., Cheng, S. S., Ohashi, A., Harada, H. & Kamagata, Y. (2006). Pelotomaculum
terephthalicum sp. nov. and Pelotomaculum isophthalicum sp. nov.: two anaerobic bacteria that degrade phthalate isomers in syntrophic association with hydrogenotrophic methanogens. Arch Microbiol 185, 172–182.
Sekiguchi, Y., Yamada, T., Hanada, S., Ohashi, A., Harada, H. & Kamagata, Y. (2003). Anaerolinea thermophila gen. nov., sp. nov. and
Caldilinea aerophila gen. nov., sp. nov., novel filamentous thermophiles that represent a previously uncultured lineage of the domain Bacteria at the subphylum level. Int J Syst Evol Microbiol 53, 1843– 1851. Sizova, M. V., Izquierdo, J. A., Panikov, N. S. & Lynd, L. R. (2011).
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DOI 10.1099/ijs.0.060186-0
Hydrogenispora ethanolica gen. nov., sp. nov., an anaerobic carbohydrate-fermenting bacterium from anaerobic sludge Yi Liu,1,2 Jiang-Tao Qiao,1 Xian-Zheng Yuan,1 Rong-Bo Guo1 and Yan-Ling Qiu1 Correspondence Yan-Ling Qiu [email protected]
1
Shandong Industrial Engineering Laboratory of Biogas Production & Utilization, Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong Province 266101, PR China
2
University of Chinese Academy of Sciences, Beijing 100049, PR China
An anaerobic, spore-forming, ethanol-hydrogen-coproducing bacterium, designated LX-BT, was isolated from an anaerobic sludge treating herbicide wastewater. Cells of strain LX-BT were nonmotile rods (0.3–0.5¾3.0–18.0 mm). Spores were terminal with a bulged sporangium. Growth occurred at 20–50 6C (optimum 37–45 6C), pH 5.0–8.0 (optimum pH 6.0–7.7) and 0–2.5 % (w/v) NaCl. The strain could grow fermentatively on glucose, maltose, arabinose, fructose, xylose, ribose, galactose, mannose, raffinose, sucrose, pectin, starch, glycerol, fumarate, tryptone and yeast extract. The major end-products of glucose fermentation were acetate, ethanol and hydrogen. Yeast extract was not required but stimulated growth. Nitrate, sulfate, thiosulfate, elemental sulfur, sulfite, anthraquinone-2,6-disulfonate, fumarate and Fe (III) nitrilotriacetate were not used as terminal electron acceptors. The G+C content of the genomic DNA was 56.1 mol%. The major cellular fatty acids were anteiso-C15 : 0, iso-C14 : 0 and C16 : 0. The most abundant polar lipids of strain LX-BT were diphosphatidylglycerol and phosphatidylglycerol. 16S rRNA gene sequence analysis revealed that it belongs to an as-yet-unidentified taxon at the order- or classlevel (OPB54) within the phylum Firmicutes, showing 86.5 % sequence similarity to previously described species of the Desulfotomaculum cluster. The name Hydrogenispora ethanolica gen. nov., sp. nov. is proposed to accommodate strain LX-BT (5DSM 25471T5JCM 18117T5CGMCC 1.5175T) as the type strain.
The phylum Firmicutes is a phylogenetically and phenotypically divergent bacterial phylum, which plays an important role in biodegradation and the carbon cycle in nature (Ludwig et al., 2009). At the time of writing, there are four recognized classes of the phylum Firmicutes: the anaerobic class Clostridia, the obligately or facultatively aerobic class Bacilli, and the classes Erysipelotrichia and Thermolithobacteria (http://www.bacterio.net/; Yutin & Galperin, 2013). Furthermore, many uncultured taxonomic groups exist in the phylum Firmicutes, such as candidate taxa OPB54, which was first detected from Obsidian Pool, a Yellowstone Park hot spring (Hugenholtz et al., 1998). Based on ARB-SILVA taxonomy, OPB54 is assigned to the class Clostridia at the order level (Dunfield et al., 2012) or represents a new class of the phylum Firmicutes (http://arbsilva.de). Environmental clones belonging to OPB54 have The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain LX-BT is AB669474. A supplementary figure is available with the online version of this paper.
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been found in a wide range of habitats, including soils (Brodie et al., 2006; Kanokratana et al., 2011), anaerobic wastewater and waste treatment systems (Tang et al., 2004), the skin microbiome (Kong et al., 2012) and aquatic moss pillars (Nakai et al., 2012). However, no cultivated representatives of this clade have been reported so far. In this study, an ethanol-type hydrogen-producing bacterium (strain LX-BT), the first cultivated strain belonging to the OPB54 cluster of the phylum Firmicutes is described. Strain LX-BT was originally obtained from a mesophilic (35 uC) anaerobic sludge treating herbicide wastewater. The medium used for isolation and cultivation was prepared as described by Sekiguchi et al. (2000). The sludge was washed and transferred into liquid medium supplemented with 50 mg l21 pyrazosulfuron, which was one of the major components of the original herbicide wastewater. After five transfers, the pyrazosulfuron enrichment culture was serially diluted in liquid medium supplemented with glucose (2 mM). Growth and hydrogen production were observed in higher dilution cultures within 2 weeks of incubation. 060186 G 2014 IUMS Printed in Great Britain
Hydrogenispora ethanolica gen. nov., sp. nov.
Cells from the highest dilution were further purified by repeated serial dilutions in glucose medium, then in glucose agar (2 %) roll tubes (Hungate, 1969). Small, dark brown, round colonies were formed after 2 weeks of incubation at 37 uC. The colonies were picked and retransferred to liquid medium supplemented with 2 mM glucose. This roll-tube isolation step involving transfer of single colonies from solid medium to liquid medium was repeated several times, resulting in the isolation of strain LX-BT. Cell morphology was examined under a fluorescent microscope (Olympus BX50F). Transmission electron microscopy (TEM) was performed with a Hitachi H-7000 transmission electron microscope as described by Sekiguchi et al. (2003). Gram staining was performed by the method of Hucker (Doetsch, 1981). Cells of strain LX-BT were non-motile, straight or slightly curved rods, 0.3–0.5 mm in diameter and
(a)
(b)
Fig. 1. Phase-contrast micrograph (a) and thin-section electron micrograph (b) of cells of strain LX-BT grown on glucose. Bulged sporangium indicated by arrow. Bars, 10 mm (a) and 0.5 mm (inset, 0.1 mm) (b). http://ijs.sgmjournals.org
3.0–18.0 mm in length (Fig. 1a). Spores were located terminally and had a bulged sporangium (Fig. 1a). Gram staining was negative, however, electron microscopy demonstrated that strain LX-BT possessed a Gram-positive-type cell wall (Fig. 1b). Physiological characteristics of strain LX-BT were examined as described by Sekiguchi et al. (2000). The temperature range for growth was determined between 15 and 60 uC (5 uC intervals), the pH range was assessed at 37 uC over the range pH 3.5–8.5 (0.5 pH unit intervals) and NaCl tolerance was checked at 0–3.0 % (w/v, 0.5 % intervals). Cell growth was evaluated on the basis of the increase in OD at 400 nm and the production of hydrogen. Unless otherwise indicated, all cultivations were performed at 37 uC in serum vials (20 ml per 50 ml vial) under a gas phase of N2/CO2 (80 : 20, v/v) without shaking. Aerobic growth was tested in a glucose (2 mM) medium under aerobic conditions without reducing agents. Strain LX-BT grew anaerobically on glucose medium at 20–50 uC (optimum, 37–45 uC), at pH 5.0–8.0 (optimum, 6.0–7.7), and with 0–2.5 % (w/v) NaCl, but did not grow at temperatures below 15 uC or above 55 uC after 8 weeks of incubation. The isolate was a strictly anaerobic organism: it could not grow in the presence of oxygen (20 %, v/v, in the gas phase) or after N2/CO2 purging only (without reducing agents). Vigorous growth and hydrogen production were observed with the following substrates (5 mM each unless otherwise specified): glucose, maltose, arabinose, fructose, xylose, ribose, galactose, mannose, raffinose, sucrose, pectin (0.5 g l21), starch (0.5 g l21), glycerol, fumarate, tryptone (0.5 %) and yeast extract (0.5 %). Yeast extract was not required but enhanced growth. In syntrophic substrate utilization tests, strain LX-BT was co-cultured with the hydrogenotrophic methanogen Methanospirillum hungatei DSM 864T. None of the following substrates tested supported the growth of strain LX-BT in pure culture or co-culture with M. hungatei DSM 864T (5 mM each unless specified): crotonate, pyruvate, xylan (0.5 %), cellulose, Casamino acids, L-glutamate, serine, glycine, ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, ethylene glycol, citrate, formate, H2/CO2 (80 : 20, v/v, head space) plus acetate, propionate, isobutyrate, butyrate, succinate, malate, benzoate, hydroquinone (2 mM), phenol (2 mM), 4-hydroxybenzoate, 2,5-dihydroxybenzoate or pyrazosulfuron (50 mg l21). In the medium supplemented with glucose (2 mM), the major end-products from glucose were hydrogen, acetate and ethanol (1 mol glucose was converted to approximately 1 mol acetate, 0.86 mol ethanol and 2.52 mol hydrogen, electron recovery 98 %) (Fig. 2). Although the strain produced hydrogen, its growth was not stimulated in co-cultivation with M. hungatei. The ratio of ethanol/acetate from glucose degradation in co-culture was same with that in pure culture. Strain LX-BT did not use any of the following electron acceptors within 4 weeks of incubation with glucose (mM): nitrate (5), sulfate (5), thiosulfate (2), elemental sulfur (5), Fe (III) nitrilotriacetate (2), sulfite (1), fumarate (5) 1757
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Concentration [mmol (l culture)–1]
8 Glucose Hydrogen Acetate Ethanol
6
4
2
0 0
2
4 Time (days)
6
8
Fig. 2. Degradation of glucose by pure culture of strain LX-BT.
or anthraquinone-2,6-disulfonate (AQDS) (2). Sensitivity to antibiotics was tested in glucose medium, containing filter-sterilized antibiotics at 50 mg l21. Growth of strain LX-BT was inhibited by gentamicin, kanamycin, lincomycin and tetracycline, but not ampicillin, chloramphenicol, streptomycin or penicillin. For G+C content determination, DNA was extracted and purified according to the methods of Kamagata & Mikami (1991). The DNA G+C content of strain LX-BT was determined from the melting point (Tm) by thermal denaturation (Mandel et al., 1970) using a Lambda 25 UV-Vis spectrophotometer (Perkin Elmer). DNA from Escherichia coli (Sigma D-2001) was used as a reference for the measurements. The DNA G+C content was 56.1 mol% (SD: ±0.06 mol%). Fatty acids of cells were converted to methyl esters using HCl/methanol and identified by GC-MS (Agilent 7890A gas 140 chromatograph coupled to an Agilent 5975C MSD single quadrupole mass spectrometer) (Hanada et al., 2002). For fatty acid methyl ester and polar lipid analyses, cells were harvested at exponential growth phase from cultures grown anaerobically on 10 mM glucose at 37 uC. The major cellular fatty acids were anteiso-C15 : 0 (58.6 %), iso-C14 : 0 (11.1 %), C16 : 0 (9.6 %), iso-C16 : 0 (6.9 %), iso-C15 : 0 (4.3 %) and C14 : 0 (4.2 %). Polar lipids were extracted and analysed by the methods of Tindall (1990) by using two-dimensional TLC (Merck silica gel 60 F254 plates, layer thickness 0.2 mm, no. 5554). The polar lipid patterns were characterized by the presence of diphosphatidylglycerol, phosphatidylglycerol and other unidentified phospholipids and amino phospholipids (Fig. S1, available in the online Supplementary Material).
polymerase (Perkin Elmer). The PCR primers used in the amplification were the bacterial domain universal primer 8F and the prokaryote universal primer 1490R (Weisburg et al., 1991). The PCR product was sequenced directly on a Beckman CEQ-8000 DNA sequencer using a CEQ DTC quick start kit (Beckman Coulter). The phylogenetic tree based on 16S rRNA gene sequences was constructed by the neighbour-joining method (Saitou & Nei, 1987) implemented in the MEGA5 computer program (Tamura et al., 2007) and the distance matrix was calculated by the Jukes– Cantor model (Jukes & Cantor, 1969). Confidence values of branches in the phylogenetic tree were determined using bootstrap analysis based on 1000 resamplings (Felsenstein, 1985). A total of 1443 base pairs of the 16S rRNA gene from strain LX-BT were sequenced and compared with other related sequences in the databases. Phylogenetic analysis revealed that strain LX-BT is closely affiliated to members of the uncultured OPB54 clade at the order- or class-level based on the ARB-SILVA reference database (http://arb-silva.de). The closest relatives of strain LX-BT based on 16S rRNA gene sequences were environmental clones, such as those from uranium contaminated soil (90– 94 % similarity) (Brodie et al., 2006), and clone AC007 (93 % similarity) from a landfill leachate bioreactor (Burrell et al., 2004). The most closely related cultured species was Clostridium sp. 6-31 (94 % similarity), which was an as-yet-undescribed non-cellulolytic bacterium from biocompost (Sizova et al., 2011). The most closely related described species were Desulfotomaculum hydrothermale (86.5 % similarity), an anaerobic sulfate-reducing bacterium isolated from a hot spring (Haouari et al., 2008), Pelotomaculum propionicicum (86.1 %), a syntrophic propionate-degrading bacterium isolated from a methanogenic sludge (Imachi et al., 2007), and Clostridium straminisolvens (84.9 %), a thermophilic cellulolytic bacterium (Kato et al., 2004) (Fig. 3). Sequence similarity values for the 16S rRNA genes of strain LX-BT and related genera such as Desulfotomaculum (,86.5 %), Pelotomaculum (,86.1 %) and Clostridium (,85 %) in the order Clostridiales of the phylum Firmicutes were in a range 84–86 %, justifying the creation of a new genus and novel species to accommodate the strain from a phylogenetic point of view.
For 16S rRNA gene sequencing, the genomic DNA of strain LX-BT was extracted according to the method of Hiraishi (1992). 16S rRNA genes were amplified by PCR with Taq
Comparative phenotypic traits of strain LX-BT and related genera in the order Clostridiales are shown in Table 1. Strain LX-BT shared some common phenotypic traits with related organisms, such as spore formation and chemoheterotrophic growth. Major differences that distinguished strain LX-BT from other related members of the genera Desulfotomaculum, Pelotomaculum and Clostridium were: (i) morphology, cells of strain LX-BT are long thin rods, forming terminal endospores in a bulged sporangium, while the related organisms were short sausage-shaped rods with central or terminal endospores; (ii) metabolism, strain LX-BT is an ethanol-type fermentation bacterium which could not perform syntrophic substrate oxidation in cooperation with hydrogenotrophic methanogens, or sulfate respiration. However, members of the genus Pelotomaculum
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International Journal of Systematic and Evolutionary Microbiology 64
Hydrogenispora ethanolica gen. nov., sp. nov.
Pelotomaculum propionicicum DSM 15578T (AB154390) Cryptanaerobacter phenolicus LR7.2T (AY327251) Rice field soil, Pelotomaculum sp. FP rrnB (AB159558) 76 100 Pelotomaculum schinkii DSM 15200T (X91170) 0.02 Pelotomaculum isophthalicicum JIT (AB232785) 100 Pelotomaculum terephthalicicum DSM 16121T (AB091323) 100 Pelotomaculum thermopropionicum SIT (NR_040840) Desulfotomaculum aeronauticum DSM 10349T (X98407) 94 96 ‘Desulfotomaculum reducens’ MI-1 (U95951) 100 Desulfotomaculum putei TH-11T (AF053929) 95 96 Desulfotomaculum hydrothermale DSM 18033T (EF081293) 50 Desulfotomaculum carboxydivorans DSM 14880T (AY961415) 93 Desulfotomaculum acetoxidans DSM 771T (Y11566) 47 Sporotomaculum hydroxybenzoicum DSM 5475T (Y14845) Desulfotomaculum sapomandens DSM 3223T (AF168365) 100 34 Desulfotomaculum thermoacetoxidans DSM 5813T (Y11573) Desulfotomaculum australicum AB33T (M96665) 100 Desulfotomaculum kuznetsovii DSM 6115T (NR_026410) 99 Thermolithobacter ferrireducens JW/KA-2T (NR_041802) New order/class Peat swamp forest soil, uncultured bacterium PW185 (GQ402678) 52 OPB54 72 Skin popliteal fossa, uncultured bacterium ncd557h04c1 (JF049253) 100 Uranium-contaminated soil, uncultured bacterium AKAU3574 (DQ125579) 72 Aquatic moss pillars, uncultured bacterium MPB2-134 (AB630810) 23 Clostridium sp. 6-31 (FJ808611) 68 Methanogenic landfill leachate bioreactor, uncultured Clostridium sp. AC007 (AY330129) 93 39 Thermophilic anaerobic soild waste bioreactor, uncultured bacterium HAW-R60-B-745d-BD (FN436105) 100 Hydrogenispora ethanolica LX-BT (AB669474) 56 Rice paddy soil, uncultured bacterium TSBAR001_M17 (AB486189) Thermophilic anaerobic solid waste bioreactor, uncultured bacterium B55_K_B_D09 (DQ887958) 43 Skin popliteal fossa, uncultured bacterium ncd1975b01c1 (JF172030) 92 Leachate sediment, uncultured Clostridium sp. De150 (HQ183805) 100 98 Pit mud, uncultured bacterium b19-147 (JX576081) 70 Thermophilic anaerobic municipal solid waste digester, uncultured bacterium MBA05 (AB114315) 68 Clostridium straminisolvens DSM 16021T (AB125279) Caldanaerobius fijiensis DSM 17918T (EF507903) 100 100 Caldanaerobius zeae mel2T (U75933) 36 Thermoanaerobacterium thermosaccharolyticum D120-70 (AF247003) Caldicellulosiruptor saccharolyticus DSM 8903T (NR_074845) 21 Caldicellulosiruptor bescii DSM 6725T (L09180) 100 ‘Tepidanaerobacter acetatoxydans’ DSM 21804 (NR_074537) Thermoanaerobacter sulfurophilus L-64T (Y16940) 99 77 Caldanaerobacter subterraneus SL9T (AY216597) 13 63 Thermanaeromonas toyohensis DSM 14490T (NR_024777) Moorella thermoacetica AMP (AY884087) Syntrophomonas bryantii DSM 3014T (HE654006) 100 37 Thermosyntropha lipolytica JW/VS-265T (X99980) Carboxydothermus hydrogenoformans Z-2901T (NC_007503) 44 Syntrophaceticus schinkii Sp3T (EU386162) 84 Thermacetogenium phaeum DSM 12270T (NC_018870) 100 Thermaerobacter litoralis KW1T (NR_043281) 100 Thermaerobacter subterraneus ir-3 (EU214630) Symbiobacterium thermophilum IAM 14863T (NC_006177) 74 ‘Caldinitratiruptor microaerophilus’ CA62N (GQ405534) 99 63 94
62
100
Sulfobacillus thermotolerans Kuch1T (JX966410) Sulfobacillus thermosulfidooxidans DSM 9293T (NR_040945) Anaerolinea thermolimosa DSM 16554T (NR_040970)
Fig. 3. Phylogenetic relationships among strain LX-BT and representatives within the bacterial phylum Firmicutes. The 16S rRNA gene sequence of Anaerolinea thermolimosa DSM 16554T was used as an out-group. The tree was calculated on the basis of a distance matrix analysis of 16S rRNA gene sequences (neighbour-joining tree). Bar, 0.02 nucleotide changes per sequence position. Numbers at nodes show bootstrap values (%) obtained with 1000 resampling analyses.
could perform syntrophic substrate oxidation with hydrogenotrophic methanogens (Imachi et al., 2002, 2007; Qiu et al., 2006), and members of the genus Desulfotomaculum could perform sulfate respiration (Haouari et al., 2008; Liu et al., 1997; Parshina et al., 2005). Strain LX-BT showed similar fermentative metabolism to C. straminisolvens in the family Clostridiaceae, however, their substrate spectrums for growth were different. For example, strain LX-BT was able to grow on various carbohydrates and proteinaceous compounds, whereas C. straminisolvens is a thermophilic cellulolytic bacterium, which can only use cellulose and cellobiose (Kato et al., 2004). http://ijs.sgmjournals.org
Strain LX-BT is the first cultivable isolate accommodated in the OPB54 lineage. On the basis of phylogenetic, genetic and physiological properties, the strain represents a novel species of a novel genus in the phylum Firmicutes. The name Hydrogenispora ethanolica is proposed. Description of Hydrogenispora gen. nov. Hydrogenispora [Hy.dro.ge.ni.spo9ra. N.L. n. hydrogenum (from Gr. n. hudoˆr water; and Gr. v. gennaio to produce) hydrogen; Gr. n. spora a seed, and, in biology, a spore; Hydrogenispora a spore-forming, hydrogen-producing bacterium]. 1759
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Table 1. Characteristics of strain LX-BT and members of the related genera Desulfotomaculum, Pelotomaculum and Clostridium within the phylum Firmicutes Strains: 1, LX-BT; 2, Desulfotomaculum hydrothermale DSM 18033T; 3, Desulfotomaculum putei DSM 12395T; 4, Desulfotomaculum carboxydivorans DSM 14480T; 5, Pelotomaculum thermopropionicum DSM 13744T; 6, Pelotomaculum terephthalicicum DSM 16121T; 7, Clostridium straminisolvens DSM 16021T. +, Positive; 2, negative; ND, no data available. Characteristic
OPB54
Order Clostridiales Family Peptococcaceae
1
2
3
4
Family Clostridiaceae 5
6
7
International Journal of Systematic and Evolutionary Microbiology 64
Similarity (%) with strain LX-BT Cell size (mm) Motility Spore location Optimum temperature (uC) for growth (range) Optimum pH for growth (range) NaCl tolerance (%, w/v) DNA G+C content (mol%) Major cellular fatty acids Substrate utilization Carbohydrates Glycerol Fumarate Pyruvate Lactate Metabolism
– 86.5 85.6 85.3 86.1 0.3–0.563.0–18.0 1.063.0–6.0 1.0–1.162.0–5.0 0.5–1.565.0–15.0 0.7–0.861.7–2.8 2 + + + 2 Terminal Subterminal to terminal Paracentral Subterminal or terminal Central 37–45 (20–50) 55 (40–60) 64 (40–65) 55 (30–68) 55 (45–65)
84.9 0.8–1.062.0–3.0 + Central 37 (30–40)
84.9 0.5–1.063.0–8.0 2 Subterminal 50–55 (50–60)
6.0–7.7 (5.0–8.0) 0–2.5 56.1 anteiso-C15 : 0
7.1 (5.8–8.2) 0–1.5 46.8
7.0 (6.5–7.5) 0–1.5 53.6 iso-C15 : 0, C14 : 0
7.5 (6.0–8.5)
+ + + 2 2 Fermentative
Electron acceptors Habitat
2 Anaerobic sludge
2 + 2 + + Fermentative, respiratory Sulfate Hot spring
This study
Haouari et al. (2008)
Reference
ND
7.5 (6.0–7.9) 0–2.0 47.1 C16 : 0, iso-C15 : 0
6.8–7.2 (6.0–8.0) 0–1.7 46.9 C16 : 0, iso-C16 : 0
7.0 (6.5–8.0) 0–0.4 52.8 iso-C15 : 0
Only fructose
+ 2 2 + + Fermentative, respiratory Sulfate Anaerobic sludge
2 2 + + + Fermentative, syntrophic Fumarate Anaerobic sludge
ND ND
+ 2 Fermentative, respiratory Sulfate Terrestrial subsurface Liu et al. (1997)
ND
41.3 ND
2 Only cellulose, cellobiose 2 ND 2 ND 2 ND 2 ND Fermentative, Fermentative syntrophic ND 2 Anaerobic sludge Rice straw
Parshina et al. (2005) Imachi et al. (2002) Qiu et al. (2006)
Kato et al. (2004)
Hydrogenispora ethanolica gen. nov., sp. nov.
Strictly anaerobic, non-motile, spore-forming rods. Ferment various sugars but not amino acids or aromatic compounds. Oxygen, nitrate, sulfate, thiosulfate, elemental sulfur, sulfite, anthraquinone-2,6-disulfonate, fumarate and Fe (III) nitrilotriacetate do not serve as electron acceptors for growth. The type species is Hydrogenispora ethanolica.
Shows the following characteristics in addition to those given for the genus. Cells are rods, 0.3–0.5 mm in diameter and 3.0–18.0 mm in length. Produces terminal endospores in bulged sporangium. Surface colonies on agar are dark brown and round after cultivation at 37 uC for 1–2 weeks. Grows at 20–50 uC (optimum 37–45 uC), pH 5.0–8.0 (optimum pH 6.0–7.7) and with 0–2.5 % (w/v) NaCl. Yeast extract is not required but stimulates growth. Ferments glucose, maltose, arabinose, fructose, xylose, ribose, sucrose, galactose, mannose, raffinose, pectin, starch, glycerol, fumarate, tryptone and yeast extract in pure culture. The major end-products from glucose are acetate, ethanol and hydrogen. Sensitive to gentamicin, kanamycin, lincomycin and tetracycline, but resistant to ampicillin, chloramphenicol, streptomycin and penicillin. The main fatty acids are anteiso-C15 : 0, iso-C14 : 0 and C16 : 0. The most abundant polar lipids are diphosphatidylglycerol and phosphatidylglycerol. T
The type strain is LX-B (5DSM 25471 5JCM 18117 5 CGMCC 1.5175T), isolated from a mesophilic anaerobic sludge treating herbicide wastewater. The DNA G+C content of the type strain is 56.1 mol%.
Acknowledgements We thank Dr Yuji Sekiguchi of the Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) for help with phylogenetic analysis, and Fei Li and Xiaofei Yan for transmission electron microscopy. This work was supported by the Taishan Scholar Program of Shandong Province, the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2YW-G-052, KG2D-EW-304-1), the National Natural Science Foundation of China (51078344) and the National Key Technology R&D Program (2013BAD22B00).
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Hydrogenispora ethanolica (e.tha.no9li.ca. N.L. n. ethanol ethanol; L. fem. suff. -ica suffix used with the sense of pertaining to; N.L. fem. adj. ethanolica belonging to ethanol, referring to ethanol, which is produced by the species).
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