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Clostridium is an important microbial component in pit mud due to its ability to produce alcohol and short-chain fatty acids. This study presents the characterization and taxonomy of two Gram-stain-positive, strictly anaerobic, rod-shaped mesophilic bacterial strains, designated MT-113 T and MT-5 T , isolated from pit mud in a fermentation cellar used for producing sauce-flavour Chinese baijiu. Phylogenetic analysis based on genome and 16S rRNA gene sequences of strains MT-113 T and MT-5 T indicates their affiliation with the genus Clostridium sensu stricto (Cluster I of the Clostridia), with C. luticellarii FW431 T and C. aromativorans WLY-B-L2 T as the closest related species. The major cellular fatty acids (>10.0%) of both strains are C 14 : 0 and summed feature 1 (iso-C 15 : 1  h and/or C 13 : 0 3-OH). The G+C molar contents of the complete genomes for strains MT-113 T and MT-5 T are 35.84 and 32.74 mol%, respectively. The average nucleotide identity and average amino acid identity values between strains MT-113 T , MT-5 T , C. aromativorans WLY-B-L2 T and C. luticellarii FW431 T range from 79 to 85%. The primary products of glucose fermentation by MT-113 T are acetic, butyric and isovaleric acids, while those of MT-5 T are acetic, isobutyric, butyric and isovaleric acids. Based on their phenotypic, chemotaxonomic and phylogenetic characteristics, strains MT-113 T (=CGMCC 1.18018 T = JCM 36532 T ) and MT-5 T (=CGMCC 1.18016 T = JCM 36530 T ) are proposed as the type strains of two novel species of the genus Clostridium , namely Clostridium lapidicellarium sp. nov. and Clostridium moutaii sp. nov., respectively.

A Gram-stain-positive, strictly anaerobic, endospore-forming and rod-shaped (0.6-0.8×2.7-13.1 µm) bacterium, designated as 5 N-1 T , was isolated from a yellow water sample collected from the manufacturing process of Nongxiangxing baijiu in the Yibin region of Sichuan, PR China. Growth occurred at 15-40 °C (optimum growth at 37 °C), at pH 6.0-9.0 (optimum growth at pH 7.0) and in NaCl concentrations of 0-1 % (w/v) and ethanol concentrations of 0-2 % (v/v). The major fatty acids in strain 5 N-1 T were C 16 : 0 , C 18 : 0 and C 14 : 0 . The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, four unidentified aminophospholipids and one unidentified lipid. Phylogenetic analysis of its 16S rRNA gene sequence indicated that strain 5 N-1 T was most closely related to Clostridium weizhouense YB-6 T (97.70 %) and Clostridium uliginosum DSM 12992 T (97.56 %). The average nucleotide identity and digital DNA‒DNA hybridization values between strain 5 N-1 T and the above two type strains were 80.89 and 80.05 % and 25.80 and 25.30 %, respectively, which were all below the species thresholds. The genome size of strain 5 N-1 T was 3.5 Mbp and the DNA G+C content was 27.5 mol%. Based on the results of phenotypic and genotypic analyses, strain 5 N-1 T represents a novel species of the genus Clostridium , for which the name Clostridium aquiflavi sp. nov. is proposed. The type strain is Clostridium aquiflavi 5 N-1 T (=CICC 24886 T =JCM 35355 T ).

Reductive soil disinfestation (RSD), also known as biological soil disinfestation, is a bioremediation method used to suppress soil-borne plant pathogens by stimulating the activity of indigenous anaerobic bacteria in the soil. An anaerobic bacterial strain (E14 T ) was isolated from an anoxic soil sample subjected to RSD treatment and then comprehensively characterized. Cells of the strain were Gram-stain-positive, curved to sigmoid, and spore-forming rods. Cells were motile with a polar flagellum. Strain E14 T grew in peptone-yeast extract broth, indicating that it utilized proteinous compounds. Strain E14 T was also saccharolytic and produced acetate, isobutyrate, butyrate, isovalerate and gases (H 2 and CO 2 ) as fermentation products. The strain did not decompose any of examined polysaccharides except for starch. The major cellular fatty acids of strain E14 T were iso-C 15:0 and iso-C 15:0 DMA. The closest relative to strain E14 T , based on 16S rRNA gene sequences, was Clostridium thermarum SYSU GA15002 T (96.2 %) in the Clostridiaceae . Whole-genome analysis of strain E14 T showed that its genome was 4.66 Mb long with a genomic DNA G+C content of 32.5 mol%. The average nucleotide identity (ANIb) between strain E14 T and C. thermarum SYSU GA15002 T was 69.0 %. The presence of the genes encoding glycolysis and butyrate production via the acetyl-CoA pathway was confirmed through genome analysis. Based on the obtained phylogenetic, genomic and phenotypic data, we propose that strain E14 T should be assigned to the genus Clostridium in the family Clostridiaceae as Clostridium omnivorum sp. nov. The type strain is E14 T (=NBRC 115133 T =DSM 114974 T ).

A strictly anaerobic, Gram-stain-negative rod-shaped bacterium, designated A1-XYC3 T , was isolated from the faeces of an alpaca ( Lama pacos ). On the basis of the results of a comparative 16S rRNA gene sequence analysis, the isolate was assigned to the genus Clostridium with the highest sequence similarities to Clostridium magnum DSM 2767 T (96.8 %), Clostridium carboxidivorans P7 T (96.3 %) and Clostridium aciditolerans JW/YJL-B3 T (96.1 %). The average nucleotide identity between A1-XYC3 T , C. magnum, C. carboxidivorans and C . aciditolerans was 77.4, 76.1 and 76.6  %, respectively. The predominant components of the cellular fatty acids of A1-XYC3 T were C 14 : 0 , C 16 : 0 and summed feature 10, containing C 18:0 /C 17:0 cyclo. The DNA G+C content was 32.4 mol%. On the basis of biochemical, phylogenetic, genotypic and chemotaxonomic criteria, this isolate represents a novel species within Clostridium sensu stricto for which the name Clostridium tanneri sp. nov. is proposed. The type strain of this species is strain A1-XYC3 T (=CCM 9376 T =NRRL B-65691 T ).

Some species of the genus Clostridium are efficient acetate producers and have been deemed useful for upgrading industrial biogas. An acetogenic, strictly anaerobic, Gram-stain-positive, subterminal endospore-forming bacterium designated strain PL3 T was isolated from peatland soil enrichments with H 2 and CO 2 . Cells of strain PL3 T were 0.8-1.0×4.0-10.0 µm in size and rod-shaped. Growth of strain PL3 T occurred at pH 6.0-7.5 (optimum, pH 7.0), at 20-40 °C (optimum, 30 °C) and with 0-1.5 % (w/v) NaCl (optimum, 0.5%). Biochemical analyses revealed that strain PL3 T metabolized lactose, maltose, raffinose, rhamnose, lactic acid, sorbitol, arabinose and glycerol. Acetic acid was the predominant metabolite under anaerobic respiration with H 2 /CO 2 . The major cellular fatty acids were C 16 : 0 , C 16 : 1 cis 9 and C 17 : 0 cyc. The main polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, aminolipid and aminophospholipid. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain PL3 T belongs to the genus Clostridium with the highest sequence similarity to Clostridium aciditolerans DSM 17425 T (98.6 %) followed by Clostridium nitrophenolicum (97.8 %). The genomic DNA G+C content of strain PL3 T was 31.1 mol%.The genomic in silico DNA-DNA hybridization value between strain PL3 T and C. aciditolerans DSM 17425 T was 25.1 %, with an average nucleotide identity of 80.2 %. Based on phenotypic, chemotaxonomic and phylogenetic differences, strain PL3 T was suggested to represent a novel species of the genus Clostridium , for which the name Clostridium thailandense sp. nov. is proposed. The type strain is PL3 T (=DSM 111812 T =TISTR 2984 T ).

A rod-shaped, Gram-stain-negative, strictly anaerobic, catalase-negative and endospore-forming bacterial strain CSC2 T was isolated from corn silage preserved in Tochigi, Japan. The strain CSC2 T grew at 15-40 °C, at pH 5.0-7.7 and with up to 0.5 % (w/v) NaCl. The main cellular fatty acids were C 14 : 0 , C 16 : 0 and C 16 : 0 dimethyl acetal. The cellular polar lipids detected were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidic acid, lysophosphatidylethanolamine, phosphatidylserine, lysophosphatidylcholine and two unidentified polar lipids. Phylogenetic analysis of the 16S rRNA gene showed that strain CSC2 T was a member of the genus Clostridium and closely related to Clostridium polyendosporum DSM 57272 T (95.6 % gene sequence similarity) and Clostridium fallax ATCC 19400 T (95.3 %). The genomic DNA G+C content of strain CSC2 T was 31.1 mol% (whole genome analysis). The average nucleotide identity based on blast and digital DNA-DNA hybridization values between strain CSC2 T and the type strains of phylogenetically related species were below 71 and 24 %, respectively. On the basis of the genotypic, phenotypic and chemotaxonomic characteristics, it is proposed to designate strain CSC2 T as representing Clostridium zeae sp. nov. The type strain is CSC2 T (=MAFF212476 T =JCM 33766 T =DSM 111242 T ).

The taxonomic status of the species Clostridium methoxybenzovorans was assessed. The 16S rRNA gene sequence, whole-genome sequence and phenotypic characterizations suggested that the type strain deposited in the American Type Culture Collection ( C. methoxybenzovorans ATCC 700855 T ) is a member of the species Eubacterium callanderi . Hence, C. methoxybenzovorans ATCC 700855 T cannot be used as a reference for taxonomic study. The type strain deposited in the German Collection of Microorganism and Cell Cultures GmbH (DSM 12182 T ) is no longer listed in its online catalogue. Also, both the 16S rRNA gene and the whole-genome sequences of the original strain SR3 T showed high sequence identity with those of Lacrimispora indolis (recently reclassified from Clostridium indolis ) as the most closely related species. Analysis of the two genomes showed average nucleotide identity based on blast and digital DNA-DNA hybridization values of 98.3 and 87.9 %, respectively. Based on these results, C. methoxybenzovorans SR3 T was considered to be a member of L. indolis .

An obligately anaerobic, Gram-stain-positive and spore-forming strain, SNUG30386 T was isolated from a faecal sample of a healthy Korean subject. The strain formed a round ivory-coloured colony and cells were chained rods with tapered ends, approximately 2.0-2.5×0.6-0.8 μm in size. The taxonomic analysis indicated that strain SNUG30386 T was within the family Lachnospiraceae . According to the 16S rRNA gene sequence similarity, the closest species to strain SNUG30386 T was Clostridium symbiosum (95.6 %), followed by Enterocloster asparagiformis (94.8 %), Enterocloster clostridioformis (94.8 %) and Enterocloster lavalensis (94.6 %). The evolutionary tree based on 16S rRNA gene sequences demonstrated that strain SNUG30386 T had split apart at a unique branch point far from other close relatives. Its DNA G+C content was 48.3 mol% calculated from the whole genome sequence. The major cellular fatty acids were C 16 : 0 and C 14 : 0 . Compared to those of the closely related species, strain SNUG30386 T showed distinct biochemical activities such as being unable to utilize most of carbon sources except d-glucose and l-arabinose. As a result, based on its unique phylogenetic clade and taxonomic characteristics, we conclude that strain SNUG30386 T represents a novel species within the genus Clostridium , for which the name Clostridium fessum sp. nov. is proposed. The type strain of the novel species is SNUG30386 T (=KCTC 15633 T = JCM 32258 T ).

An anaerobic, Gram-stain-positive, spore-forming bacterium, designated strain PYR-10 T , was isolated from a mesophilic methanogenic consortium. Cells were 0.7-1.2×6.0-6.3 µm, straight or slightly curved rods, with flagellar motility. Growth was observed in PYG (peptone-yeast glucose) medium at pH 5.5-8.0 (optimum, pH 6.5), 30-55 °C (45 °C) and in NaCl concentrations of 0-15 g l -1 (0 g l -1 ). Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain PYR-10 T belongs to the genus Clostridium . The strain showed 95.4, 93.7, 93.5 and 93.0 % 16S rRNA gene sequence similarity to Clostridium swellfunianum DSM 27788 T , Clostridium pascui DSM 10365 T , Clostridium pasteurianum DSM 525 T and Clostridium punense DSM 28650 T , respectively. The genomic DNA G+C content was 27.7 mol%. The major cellular fatty acids of strain PYR-10 T were iso-C 15 : 0 , C 16 : 0 , C 16 : 0 DMA, anteiso-C 15 : 0 and C 14 : 0 . The main polar lipids were glycolipid, phosphoaminoglycolipid, diphosphatidylglycerol, phosphatidylglycerol, phospholipids, phosphatidylethanolamine and lipids. An unknown menaquinone was detected. 2,6-Diaminopimelic acid was not detected. The whole-cell sugars contained ribose and lower amounts of glucose. Based on the results of phylogenetic, chemotaxonomic and phenotypic analyses, strain PYR-10 T represents a novel species of the genus Clostridium , for which the name Clostridium prolinivorans sp. nov. is proposed. The type strain is strain PYR-10 T (=JCM 33161 T =CCAM 531 T =CGMCC 1.5286 T ).

A novel, Gram-stain-positive, rod-shaped, non-motile, non-spore-forming, obligately anaerobic bacterium, designated strain ZHW00191 T , was isolated from human faeces and characterized by using a polyphasic taxonomic approach. Growth occurred at 25-45 °C (optimum, 37-42 °C), at pH 5.5-10.0 (optimum, pH 6.5-7.0) and with 0-2 % (w/v) NaCl (optimum, 0 %). The end products of glucose fermentation were acetic acid, isobutyric acid and isovaleric acid and a small amount of propionic acid. The dominant cellular fatty acids (>10 %) of strain ZHW00191 T were C 16 : 0 , C 18 : 1 ω 9 с and C 18 : 2 ω6,9 с . Its polar lipid profile comprised diphosphatidylglycerol, phosphatidylglycerol, three unidentified phospholipids and ten unidentified glycolipids. Respiratory quinones were not detected. The cell-wall peptidoglycan contained meso -2,6-diaminopimelic acid, and the whole-cell sugars were ribose and glucose. The genomic DNA G+C content was 32.8 mol%. Analysis of the 16S rRNA gene sequence indicated that ZHW00191 T was most closely related to Clostridium hiranonis TO-931 T (95.3 % similarity). Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) analyses with closely related reference strains indicated that reassociation values were both well below the thresholds of 95-96% and 70 % for species delineation, respectively. Based on phenotypic, chemotaxonomic and genetic studies, a novel genus, Peptacetobacter gen. nov., is proposed. The novel isolate ZHW00191 T (=JCM 33482 T =GDMCC 1.1530 T ) is proposed as the type strain of the type species Peptacetobacter hominis gen. nov., sp. nov. of the proposed new genus. Furthermore, it is proposed that Clostridium hiranonis be transferred to this novel genus, as Peptacetobacter hiranonis comb. nov.

Clostridium diolis shares high similarity based on 16S rRNA gene sequences and fatty acid composition with Clostridium beijerinckii . In this study, the taxonomic status of C. diolis was clarified using genomic and phenotypic approaches. High similarity was detected among C. diolis DSM 15410 T , C. beijerinckii DSM 791 T and NCTC 13035 T , showing average nucleotide identity on blast and in silico DNA-DNA hybridization values over 97 and 85 %, respectively. Results of investigations for substrate utilization and enzyme activity displayed no striking differences between C. diolis DSM 15410 T and C. beijerinckii JCM 1390 T . Based on the results, we propose the reclassification of Clostridium diolis as a later heterotypic synonym of Clostridium beijerinckii . The type strain is ATCC 25752 T (=CIP 104308 T =DSM 791 T =JCM 1390 T =LMG 5716 T =NCTC 13035 T ).

A strictly anaerobic bacterial strain, designated as PI-S10-A1B, was isolated from a sludge sample collected from an industrial effluent dump site at Hyderabad, India. Cells stained Gram-positive and contained terminal endospores. Optimal growth was observed at 30 °C and pH 7.0. It showed negative reactions to catalase and oxidase activities. Phylogenetic analysis of the 16S rRNA gene led to strain PI-S10-A1B T being assigned to the genus Clostridium. It displayed high sequence similarity to species of cluster XIVa including Clostridium amygdalinumBR-10 T (99.84 %), Clostridium saccharolyticum WM1 T (98.93 %) and Clostridium indolis DSM 755 T (98.31 %). It formed a coherent cluster with members of cluster XIVa. Despite high 16S rRNA gene sequence similarity, strain PI-S10-A1B T displayed only 25.3 % identity in DNA-DNA hybridization tests with C. amygdalinum BR-10 T . A draft genome exhibited low values for average nucleotide identity and in silico DNA-DNA hybridization with strains of cluster XIVa. The DNA G+C content was 42.3 mol%. Major lipids were phosphatidylglycerol and diphosphatidylglycerol, with an abundance of phosphoglycolipids. Further, analysis of the draft genome revealed genomic insights against functional aspects. Considering the phenotypic differences and low genomic identity with phylogenetic relatives, strain PI-S10-A1B T is concluded to represent a new species of the genus Clostridium, for which the name Clostridiumindicum sp. nov. is proposed with type strain PI-S10-A1B T (=MTCC 12282 T =DSM 24996 T =JCM 32788 T ).

A novel Gram-stain-positive, rod-shaped, obligately anaerobic, non-motile, spore-forming and binary fission encapsulated bacterium, designated strain JN500901 T , was isolated from a mud cellar which has been continuously used for the fermentation of Chinese strong-flavour baijiu for over 100 years. Growth of JN500901 T occurred at pH 4.5-8.0 (optimum, pH 5.0), 20-40 °C (37 °C), 0-2 % (w/v) NaCl and 0-10 % (v/v) ethanol. The Biolog assay revealed that strain JN500901 T metabolized d-fructose, l-fucose, isomaltulose and l-rhamnose among the 95 studied carbon sources. p-Cresol was the predominant volatile metabolite in the fermentation broth of strain JN500901 T incubated in liquid reinforced clostridial medium under anaerobic conditions. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain JN500901 T belongs to Clostridiumsensu stricto, and shared the highest sequence similarity to Clostridiumcarboxidivorans DSM 15243 T (94.2 %), followed by Clostridiumscatologenes DSM 757 T (94.1 %). The dominant cellular fatty acids (>10 %) were C16 : 0 FAME (36.6 %), C19 : 0 cyc 9,10 DMA (19.8 %) and C16 : 1 cis 9 DMA (11.8 %). The complete genome of strain JN500901 T contained a circular chromosome of 2.812 Mb with 2611 genes and 31.0 mol% G+C content. Comparative genome analysis of the strain JN500901 T , Clostridiumcarboxidivorans DSM 15243 T and Clostridiumscatologenes DSM 757 T revealed 74.5 and 74.8 % average nucleotide identity, respectively. Based on the phenotypic, biochemical and phylogenetic analyses presented here, strain JN500901 T is considered to be a novel species of the genus Clostridiumsensustricto, for which the name Clostridium fermenticellae sp. nov. is proposed. The type strain is JN500901 T (=CICC 24501 T =JCM 32827 T ).

Strain YH-tan31 T is a Gram-stain-positive, obligately anaerobic, spore-forming and mesophilic bacterium. The cells are rod-shaped and motile by means of peritrichous flagella. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belongs to the genus Clostridium and is most closely related to Clostridium oryzae DSM 28571 T (97 % sequence identity), followed by Clostridium cellulovorans DSM 3052 T (92.4 %) and Clostridium butyricum KCTC 5387 T (92.1 %). The G+C content of the isolate was 30.9 mol%. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The predominant fatty acids were C14 : 0, C16 : 0, C16 : 0 DMA,and C19 : 0cis 11,12 DMA. The major end products of glucose fermentation were butyrate, acetate and lactate. This isolate represents a new species within the genus Clostridium, for which we propose the name Clostridiumcomposti sp. nov. (type strain, YH-tan31 T ; =KCTC 15630 T =JCM 32793 T ).

A Gram-positive, strictly anaerobic, rod-shaped bacterium, designated YB-7 T , was isolated from activated sludge of an anaerobic baffled reactor pond in Weizhou terminal wastewater treatment plant, Beihai, Guangxi, China. Strain YB-7 T grew at pH 5.0-12.0 (optimum, pH 7.0), 20-45 °C (37 °C) and NaCl concentration of 0-5 % w/v (optimum, 5 %). 16S rRNA gene sequence analysis results showed that strain YB-7 T belonged to the genus Clostridium and it was most closely related to Clostridium tetanomorphum DSM 4474 T (96.9 % similarity). The DNA-DNA relatedness of strain YB-7 T to Clostridium tetanomorphum DSM 4474 T was 47.4 %. The DNA G+C content of strain YB-7 T was determined to be 32.3 mol%, and the predominant cellar fatty acid (>10 %) was C16 : 0. Polar lipids of strain YB-7 T included diphosphatidylglycerol, phosphatidylethylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, two unidentified aminophospholipids, two unidentified phospholipids and unidentified lipids. The results of this study supported the conclusion that strain YB-7 T should be assigned to a new member of the genus Clostridium, for which the name Clostridium beihaiense sp. nov. is proposed. The type strain is YB-7 T (=CICC 24109 T =KCTC 15555 T ).

A strictly anaerobic, Gram-staining-positive, spore-forming rod-shaped bacterium, and designated BXX T , was isolated from cow manure. Colonies on DSMZ medium 311c agar plates were cream, circular, opaque and lustrous. Growth occurred at 20-45 °C with a pH range of 5.0-10.0 and at NaCl concentrations of up to 2 % (w/v). The optimum temperature, pH and NaCl concentration for growth were 30 °C, pH 7 and 1 % (w/v), respectively. The major cellular fatty acids were C16 : 0 (26.8 %), C14 : 0 (22.8 %), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) (16.4 %) and C16 : 1ω9c (10.7 %). The main polar lipids of BXX T were diphosphatidylglycerol, phosphatidylethanolamine, unidentified aminolipids, an unidentified phospholipid and unidentified lipids. Acetate was mainly produced from H2/CO2, H2/CO2/CO (4/3/3, v/v/v), formate, glycerol, 1,2-propanediol, pyruvate, d-fructose and 2-methoxyethanol. BXX T is most closely related to Clostridium thermobutyricumDSM 4928 T , Clostridiumhomopropionicum DSM 5847 T and Clostridium thermopalmarium DSM 5974 T with 16S rRNA gene sequence similarities of 96.9, 96.6 and 96.5 %, respectively. The DNA G+C content of BXX T was 33.7 mol%, which was lower than that of C. thermobutyricum DSM 4928 T (37.0 mol%) and C. thermopalmarium DSM 5974 T (35.7 mol%). In addition, DSM 4928 T and DSM 5974 T are thermophilic members of the genus Clostridium. The absence of C15 : 0 also distinguished BXX T from Clostridium thermobutyricum. On the basis of phylogenetic, phenotypic and chemotaxonomic evidence, the novel isolate represents a novel species within the genus Clostridium, for which the name Clostridium bovifaecis sp. nov is proposed. The type strain of the type species is BXX T (=JCM 32382 T =CGMCC 1.5228 T ).

To clarify the taxonomic position of Eubacterium combesii, the whole genome of its type strain, DSM 20696 T , was sequenced. Comparison of this sequence with known sequences of other bacteria confirmed that E. combesii represented a member of the Clostridium sporogenes/Clostridium botulinum Group I clade. However, the results of phylogenetic analysis also demonstrated that the latter two species did not form the same genetic entity and that E. combesii was in the C. botulinum Group I subclade. Meanwhile, we showed that E. combesii DSM 20696 T did not produce botulinum neurotoxins (BoNTs) and thus should be identified as a strain of C. sporogenes in accordance with the current nomenclature of BoNT-producing clostridia, which is based, in particular, on Opinion 69 issued by the Judicial Commission of the ICSB. However, review of the corresponding Request for an Opinion revealed that it had been based on an erroneous statement. Therefore, we request reconsideration of Opinion 69 and propose to reclassify Eubacterium combesii as a later synonym of Clostridium botulinum. The results of phylogenetic analysis of the other five groups of BoNT-producing clostridia indicated that all the groups were far distant from each other. However, the members of Groups IV-VI are classified as strains of different species, while all members of Groups I-III are designated C. botulinum. Meanwhile, similarly to Group I, Groups II and III are also polyphyletic and appear to consist of two and four species, respectively. These results demonstrate, once again, discrepancies in the nomenclature of BoNT-producing bacteria and corroborate our request for reconsideration of Opinion 69.

A description of an outbreak of necrotizing enterocolitis among neonates, linked to the putative novel species Clostridium neonatale and assignable to the genus Clostridium, was previously reported in brief but that name had never been validly published (Alfa et al. Clin Inf Dis 2002;35:S101-S105). Features of this taxon group and its phylogenetic position with respect to contemporary species in the genus Clostridium were recently reviewed and still found to be unique. Therefore, we provide here a description based on biochemical, chemotaxonomic and antimicrobial susceptibility testing (AST), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, 16S rRNA gene sequencing as well as information obtained by whole genome sequencing (WGS) for strains 99A005 T and 99A006. Those two C. neonatale strains were essentially identical to each other, with genome sizes of 4 658 596-4 705 520 bp and G+C content of 28.4-28.5 mol% (WGS). AST inferred susceptibility to 14 antibiotics. MALDI-TOF spectra were unique and could potentially be used for identification. The type strain is (NML) LCDC 99A005 T [=ATCC BAA-265 T =CCUG 46077 T =St. Boniface Hospital 30686 T ]. While performing this review, we found that the names of 24 validly published species assignable to the genus Clostridium had been omitted from the emended description of the genus (Lawson and Rainey Int J Syst Evol Microbiol 2016;66 :1009-1016). Those species are listed in brief here. Lastly, based on this review, we also propose that Eubacterium budayi, Eubacterium nitritogenes and Eubacterium combesii be transferred to the emended genus Clostridium, as Clostridium budayi comb. nov., Clostridium nitritogenes comb. nov. and Clostridium combesii comb. nov., respectively.

A Gram-stain-variable, strictly anaerobic, rod-shaped, catalase-negative and endospore-forming bacterial strain, designated MJC39 T , was isolated from grass silage preserved in Hokkaido, Japan. Growth occurred at 20-42 °C, pH 5.0-7.0 and NaCl concentrations up to 2 % (w/v). The isolated strain MJC39 T produced butyric acid in peptone yeast extract medium with glucose. The DNA G+C content of strain MJC39 T was 34.4±0.2 mol%. The major cellular fatty acids (>10 %) were C14 : 0, C16 : 0 and summed feature 3 (including C16 : 1ω7c/C16 : 1ω6c). No respiratory quinones were detected. The polar lipids of strain MJC39 T were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, one unidentified lipid, one unidentified aminolipid, two unidentified glycolipids, one unidentified phospholipid, one unidentified aminoglycolipid and one unidentified phosphoaminoglycolipid. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MJC39 T was a member of the genus Clostridium and is closely related to Clostridium tyrobutyricum JCM 11008 T (95.8 % similarity) and Clostridium algifaecis MB9-7 T (95.5 % similarity). Based on the genotypic, phenotypic and chemotaxonomic characteristics, strain MJC39 T represents a novel species of the genus Clostridium, for which the name Clostridium pabulibutyricum sp. nov. is proposed. The type strain is MJC39 T (=JCM 31506 T =DSM 103944 T ).

A strictly anaerobic bacterial strain (SH021 T ) was isolated from a methanogenic reactor. Cells were Gram-stain-positive, motile, straight or slightly curved rods. The optimum temperature for growth was 35 °C, and the optimum pH was 6.1-7.7. The strain was asaccharolytic and utilized amino acids as growth substrates. The strain produced acetate and propionate from l-alanine and l-serine, and propionate and butyrate from l-threonine. Branched-chain amino acids (l-isoleucine, l-leucine and l-valine) were utilized weakly, and isovalerate or isobutyrate was produced. Strain SH021 T utilized pyruvate and lactate, and converted them to acetate and propionate. The genomic DNA G+C content was 38.2 mol%. Compounds related to iso-C15 : 0 were detected as major components in the cellular fatty acids analysis. The diagnostic diamino acid of the cell-wall peptidoglycan was meso-diaminopimelic acid. On the basis of 16S rRNA gene sequences, the most closely related known species were Clostridium propionicum, Clostridium neopropionicum and Clostridium lactatifermentans in cluster XIVb of the class Clostridia. Based on the phylogenetic and phenotypic data, Anaerotignum aminivorans gen. nov., sp. nov. is proposed to accommodate strain SH021 T (=JCM 31556 T =DSM 103575 T ). For the three related species of the genus Clostridium, Anaerotignum propionicum comb. nov. (type strain DSM 1682 T =JCM 1430 T =ATCC 25522 T =CCUG 9280 T =NCIMB 10656 T =VPI 5303 T ), Anaerotignum neopropionicum comb. nov. (type strain X4 T =DSM 3847 T =KCTC 15564 T ) and Anaerotignum lactatifermentans comb. nov. (type strain G17 T =DSM 14214 T =LMG 20954 T ) are proposed with emended descriptions of these species.

Several strictly anaerobic bacteria that are Gram-stain-positive have the ability to use uric acid as the sole source of carbon and energy. The phylogeny of three such species, Clostridium acidurici, Clostridium purinilyticum, and Eubacterium angustum, members of the Clostridium cluster XII that ferment purines, but not most amino acids or carbohydrates, has been re-examined, taking advantage of their recently sequenced genomes. Phylogenetic analyses, based on 16S rRNA gene sequences, protein sequences of RpoB and GyrB, and on a concatenated alignment of 50 ribosomal proteins, revealed tight clustering of C. acidurici and C. purinilyticum. Eubacterium angustum showed consistent association with C. acidurici and C. purinilyticum , but differed from these two in terms of the genome size, G+C content of its chromosomal DNA and its inability to form spores. We propose reassigning C. acidurici and C. purinilyticum to the novel genus Gottschalkia as Gottschalkia acidurici gen. nov. comb. nov. (the type species of the genus) and Gottschalkia purinilytica comb. nov., respectively. Eubacterium angustum is proposed to be reclassified as Andreesenia angusta gen. nov. comb. nov. Furthermore, based on the phylogenetic data and similar metabolic properties, we propose assigning genera Gottschalkia and Andreesenia to the novel family Gottschalkiaceae. Metagenomic sequencing data indicate the widespread distibution of organisms falling within the radiation of the proposed family Gottschalkiaceae in terrestrial and aquatic habitats from upstate New York to Antarctica, most likely due to their ability to metabolize avian-produced uric acid.

Obligately anaerobic, Gram-stain-positive, spore-forming bacteria indistinguishable by pulsed-field gel electrophoresis were isolated from non-dairy protein shakes in bloated bottles. One of the isolates, strain IEH 97212T, was selected for further study. The strain was closely related to Clostridium sporogenes and Clostridium botulinum Group 1 based on 16S rRNA gene sequence similarities. Phylogenetic analysis also showed that strain IEH 97212T and strain PE (=DSM 18688), a bacterium isolated from solfataric mud, had identical 16S rRNA gene sequences. Strains IEH 97 212T and DSM 18 688 were relatively more thermophilic (temperature range for growth: 30-55 °C) and less halotolerant [growth range: 0-2.5 % (w/v) NaCl] than C. sporogenes and C. botulinum. They were negative for catalase, oxidase, urease and l-pyrrolidonyl-arylamidase and did not produce indole. The strains produced acid from d-glucose, maltose and trehalose, and hydrolysed gelatin, but did not hydrolyse aesculin. The end-products of growth included acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, isocaproic acid, phenylpropionic acid, 2-piperidinone, 2-pyrrolidinone and gas(es). The predominant fatty acids were C14 : 0, C16 : 0 and C18 : 1ω9c. The genomic DNA G+C content of strains IEH 97212T and DSM 18688 was 26.9 and 26.7 mol%, respectively. According to the digital DNA-DNA hybridization data, the relatedness of these strains was 98.4 %, while they showed only 35.7-36.0 % relatedness to C. sporogenes. Based on the results of this polyphasic study, these strains represent a novel species, for which the name Clostridium tepidum sp. nov. is proposed, with the type strain IEH 97212T (=NRRL B-65463T=DSM 104389T).

Two bacterial strains, designated EGH7T and TSAH33, were isolated from human faeces and characterized by using a polyphasic taxonomic approach that included analysis of morphology, phenotypic and biochemical features, cellular fatty acid profiles and phylogenetic position based on 16S rRNA and hsp60 gene sequence analyses. The results of 16S rRNA gene sequence analysis indicated that these strains represented members of the family Lachnospiraceae and formed a monophyletic cluster near Eubacterium contortum JCM 6483T (95 % sequence similarity), Ruminococcus gnavus JCM 6515T (95 %), Clostridium oroticum JCM 1429T (95 %), Eubacterium fissicatena JCM 31501T (95 %) and Clostridium nexile JCM 31500T (94 %). The results of a hsp60 gene sequence analysis supported the phylogenetic tree based on the 16S rRNA gene sequence, with a sequence similarity value of between 77.9 and 84.8 % to the five strains listed above. The novel strains were obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-stain-positive cocco-bacilli. The strains formed characteristic umbilicated colonies on EG agar plates. The major cellular fatty acids were C18 : 1ω9c, C16 : 0 and C18 : 1ω9c dimethyl acetal (DMA). EGH7T and TSAH33 have DNA G+C contents of 46.9 and 45.5 mol%, respectively. On the basis of these data, strains EGH7T and TSAH33 represent a novel species of a novel genus, for which the name Faecalimonas umbilicata gen. nov., sp. nov. is proposed. The type strain of F. umbilicata is EGH7T (=JCM 30896T=DSM 103426T).

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

In 1994, analyses of clostridial 16S rRNA gene sequences led to the assignment of 18 species to Clostridium cluster XI, separating them from Clostridium sensu stricto (Clostridium cluster I). Subsequently, most cluster XI species have been assigned to the family Peptostreptococcaceae with some species being reassigned to new genera. However, several misclassified Clostridium species remained, creating a taxonomic conundrum and confusion regarding their status. Here, we have re-examined the phylogeny of cluster XI species by comparing the 16S rRNA gene-based trees with protein- and genome-based trees, where available. The resulting phylogeny of the Peptostreptococcaceae was consistent with the recent proposals on creating seven new genera within this family. This analysis also revealed a tight clustering of Clostridium litorale and Eubacterium acidaminophilum. Based on these data, we propose reassigning these two organisms to the new genus Peptoclostridium as Peptoclostridium litorale gen. nov. comb. nov. (the type species of the genus) and Peptoclostridium acidaminophilum comb. nov., respectively. As correctly noted in the original publications, the genera Acetoanaerobium and Proteocatella also fall within cluster XI, and can be assigned to the Peptostreptococcaceae. Clostridium sticklandii, which falls within radiation of genus Acetoanaerobium, is proposed to be reclassified as Acetoanaerobium sticklandii comb. nov. The remaining misnamed members of the Peptostreptococcaceae, [Clostridium] hiranonis, [Clostridium] paradoxum and [Clostridium] thermoalcaliphilum, still remain to be properly classified.

In a recent publication that attempts to deal with the growing problem of taxa being added to the genus Clostridium that are outside of Clostridium (16S rRNA) group I, a solution is proposed that seeks to limit the genus Clostridium Prazmowski 1880 (Approved Lists 1980) to a small number of species 'related' to the type species, Clostridium butyricum Prazmowski 1880 (Approved Lists 1980). It has been proposed that this genus should also include members of the genus Sarcina Goodsir 1842 (Approved Lists 1980), Sarcinamaxima Lindner 1888 (Approved Lists 1980) and Sarcinaventriculi Goodsir 1842 (Approved Lists 1980), the latter being the nomenclatural type of the genus Sarcina Goodsir 1842 (Approved Lists 1980). In making proposals to treat the genus name Sarcina Goodsir 1842 (Approved Lists 1980) as a synonym of ClostridiumPrazmowski 1880 (Approved Lists 1980), reference is made to the wording of the International Code of Nomenclature of Bacteria. However, while that wording is factually correct, other parts of the Code are relevant to this issue and clearly indicate that the proposed course of action is not sanctioned by texts that have not been directly made reference to. Rather than avoiding confusion it has been contributed to, and it is necessary to document where the problems lie.

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

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

An obligately anaerobic bacterial strain designated KC3(T) was isolated from a rice straw-degrading culture, for which soil of a Japanese rice field was used as the inoculum. Cells of strain KC3(T) were determined to be non-cellulolytic, Gram-stain-positive, non-motile, ellipsoidal, spore-forming rods, 0.8-1×4-25 µm. Endospores were formed at a terminal position in elongated cells (12-25 µm, mean 15 µm). The temperature range for growth was 20-50 °C, with an optimum at 37 °C. The pH range for growth was 5.0-7.5, with an optimum at pH 6.0 (slightly acidophilic). Strain KC3(T) fermented cellobiose to lactate, butyrate, acetate, formate, hydrogen and carbon dioxide. The major cellular fatty acids (>10 %) were C14 : 0, C16 : 0 and C19 : 0 cyclo 11,12 dimethylacetal. The DNA G+C content of strain KC3(T) was 37.5 mol%. 16S rRNA gene sequence analysis revealed that strain KC3(T) shared low sequence similarity (<93 %) with type strains of the genus Clostridium sensu stricto (Clostridium rRNA cluster I). Analyses of the DNA gyrase A and ATP synthase beta subunit sequences supported the affiliation of strain KC3(T) to the genus Clostridium sensu stricto. The evidence presented here indicates that strain KC3(T) represents a novel species of the genus Clostridium, for which the name Clostridium oryzae sp. nov. is proposed. The type strain of Clostridium oryzae is KC3(T) ( = DSM 28571(T) = NBRC 110163(T))., (© 2015 IUMS.)

A Gram-staining-positive, spore-forming, obligately anaerobic, acetogenic bacterium, designated LAM1030(T), was isolated from methanogenic consortia enriched from biogas slurry collected from the large-scale anaerobic digester of Modern Farming Corporation in Hebei Province, China. Cells of strain LAM1030(T) were motile, straight or spiral-rod-shaped. Strain LAM1030(T) could utilize glucose, fructose, maltose, galactose, lactose, sucrose, cellobiose, mannitol, pyruvate, succinic acid and tryptophan as the sole carbon source. Acetic acid, isovaleric acid and butanoic acid were the main products of glucose fermentation. Sodium sulfite was used as an electron acceptor. Growth of strain LAM1030(T) was completely inhibited by the addition of ampicillin, tetracycline, gentamicin or erythromycin at a concentration of 20 µg ml(-1). The main polar lipids of strain LAM1030(T) were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, 11 unknown glycolipids and two unknown phospholipids. No respiratory quinone was detected. The major fatty acids of strain LAM1030(T) were C16 : 0 (21.1 %), C14 : 0 (10.3 %), summed feature 9 (including C16:0 10-methyl and/or iso-C17:1 ω9c) (11.3% ), summed feature 3 (including C16:1 ω7c and/or C16:1 ω6c) (10.6% ) and iso-C15 : 0 (6.6 %). Analysis of the 16S rRNA gene sequence indicated that strain LAM1030(T) belonged to the genus Clostridium and was most closely related to Clostridium subterminale DSM 6970(T), Clostridium thiosulfatireducens DSM 13105(T) and Clostridium sulfidigenes DSM 18982(T), with 97.0, 96.9 and 96.8 % similarity, respectively. The G+C content of the genomic DNA of strain LAM1030(T) was 31.2±0.3 mol%. On the basis of its phenotypic, phylogenetic and chemotaxonomic characterization, strain LAM1030(T) is suggested to represent a novel species of the genus Clostridium, for which the name Clostridium huakuii sp. nov. is proposed. The type strain is LAM1030(T) ( = ACCC 00698(T) = JCM 19186(T))., (© 2014 IUMS.)

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

A novel anaerobic, mesophilic, hydrogen-producing bacterium, designated strain M2/40(T), was isolated from a mesophilic, two-phase, laboratory-scale biogas reactor fed continuously with maize silage supplemented with 5% wheat straw. 16S rRNA gene sequence comparison revealed an affiliation to the genus Clostridium sensu stricto (cluster I of the clostridia), with Clostridium cellulovorans as the closest characterized species, showing 93.8% sequence similarity to the type strain. Cells of strain M2/40(T) were rods to elongated filamentous rods that showed variable Gram staining. Optimal growth occurred at 35 °C and at pH 7. Grown on glucose, the main fermentation products were H2, CO2, formate, lactate and propionate. The DNA G+C content was 29.6 mol%. The major fatty acids (>10 %) were C(16 : 0), summed feature 10 (C(18 : 1)ω11c/ω9t/ω6t and/or unknown ECL 17.834) and C(18 : 1)ω11c dimethylacetal. Based on phenotypic, chemotaxonomic and phylogenetic differences, strain M2/40(T) represents a novel species within the genus Clostridium, for which we propose the name Clostridium bornimense sp. nov. The type strain is M2/40(T) ( = DSM 25664(T) = CECT 8097(T))., (© 2014 IUMS.)

A Cr(VI)-resistant, Gram-positive, spore-forming, obligate anaerobe, designated GCAF-1(T), was isolated from chromium-contaminated soil by its ability to reduce Cr(VI) in low concentrations. Mixed acid fermentation during growth on glucose resulted in accumulation of acetate, butyrate, formate and lactate. Morphological studies indicated the presence of peritrichous flagella, pili and an S-layer. The major cellular fatty acids (>5 %) were C(16 : 0), C(14 : 0), summed feature 3 (comprising iso-C(15 : 0) 2-OH and/or C(16 : 1)ω7c), C(18 : 1)ω7c, C(16 : 1)ω9c, summed feature 4 (comprising iso-C(17 : 1) I and/or anteiso-C(17 : 1) B) and C(18 : 1)ω9c. The DNA G+C content of strain GCAF-1(T) was 30.7 mol%. Phylogenetic interference indicated that strain GCAF-1(T) clustered with group I of the genus Clostridium. Of strains within this cluster, strain GCAF-1(T) shared the highest 16S rRNA gene sequence similarities (98.1-98.9 %) with Clostridium beijerinckii DSM 791(T), C. saccharobutylicum NCP 262(T), C. saccharoperbutylacetonicum N1-4(T), C. puniceum DSM 2619(T) and C. roseum DSM 51(T). However, strain GCAF-1(T) could be clearly distinguished from its closest phylogenetic neighbours by low levels of DNA-DNA relatedness (<50 %) and some phenotypic features. Based on the evidence presented here, strain GCAF-1(T) ( = DSM 23318(T) = KCTC 5935(T)) represents a novel species of the genus Clostridium, for which the name Clostridium chromiireducens sp. nov. is proposed.

An obligately anaerobic, Gram-positive, spore-forming bacterial strain, designated SL206(T), was isolated from pear orchard soils. Strain SL206(T) cells were straight or slightly curved rods, with motility by peritrichate flagella. Cell walls contained meso-diaminopimelic acid; wall sugars were glucose, rhamnose and mannose. The major fatty acids were C(16 : 0), C(18 : 1)omega9c and summed feature 10 (containing C(18 : 1)omega11c/9t/6t). API 20A reactions were negative for oxidase, catalase and acid production from l-rhamnose, sucrose, trehalose, d-xylose, melezitose, salicin and d-sorbitol, and positive for acid production from d-glucose, sucrose, maltose, d-mannose and raffinose. Glucose was fermented to acetate, butyrate, CO(2), H(2) and ethanol in culture. The G+C content of the genomic DNA was 31.1 mol%. Based on comparative 16S rRNA gene sequence analysis, the isolate belonged to the genus Clostridium and formed a clade with Clostridium pasteurianum. The species most closely related to strain SL206(T) were C. pasteurianum (98.6 % similarity) and Clostridium acidisoli (97.8 % similarity). In DNA-DNA relatedness studies, the isolate had 59.5 % relatedness with C. pasteurianum and thus represented a unique species. On the basis of these studies, strain SL206(T) (=KCTC 5449(T) =JCM 14858(T)) is proposed to represent the type strain of a novel species, Clostridium arbusti sp. nov.

An obligate anaerobic, Gram-staining-negative, mesophilic, cellulolytic bacterium, strain H1(T), was isolated from the rumen content of yak. Cells were straight to slightly curved rods, 0.8-1.0 x 3.0-4.0 microm in size, non-motile and encapsulated with mucous materials. Elliptical and terminal spores that swelled the cells were produced occasionally. The strain grew at 25-45 degrees C (optimum, 38 degrees C) and pH 6.0-7.8 (optimum, pH 6.7). Cellulose, cellobiose, xylan, xylose and maltose were used as carbon and energy sources, but not glucose. Products from cellulose and cellobiose fermentation were formic acid, acetic acid, carbon dioxide and trace amounts of ethanol, lactic acid and succinic acid. The genomic DNA G+C content was 33.7+/-1.2 mol%. The predominant fatty acids were C(16 : 0) (27.1 %), C(14 : 0) (9.2 %) and iso-C( 16 : 0) (6.4%). Based on the 16S rRNA gene sequence analysis, strain H1(T) was affiliated to the clostridial rRNA cluster XIVb and showed the highest 16S rRNA gene sequence similarity to Clostridium lentocellum DSM 5427(T) (96.0 %). These two strains formed a distinct lineage of the family 'Lachnospiraceae '. Based on data from this polyphasic taxonomic study, a new genus, Cellulosilyticum gen. nov., is proposed. Cellulosilyticum ruminicola sp. nov. is proposed for strain H1(T). The type strain of Cellulosilyticum ruminicola sp. nov. is strain H1(T) (=CGMCC 1.5065(T)=JCM 14822(T)). Clostridium lentocellum was reclassified in the new genus as Cellulosilyticum lentocellum comb. nov. (type strain RHM5(T)=ATCC 49066( T)=DSM 5427(T)=NCIMB 11756(T)).

We isolated several strains from various clinical samples (five samples of blood, four of intra-abdominal pus and one of infected soft tissue) that were anaerobic, motile or non-motile and Gram-positive rods. Some of the strains formed spores. Phylogenetic analysis of the 16S rRNA gene sequence showed that these organisms could be placed within clostridial cluster IV as defined by Collins et al. [(1994). Int J Syst Bacteriol 44, 812-826] and shared more than 99 % sequence similarity with Clostridium orbiscindens DSM 6740(T) and Eubacterium plautii DSM 4000(T). Together, they formed a distinct cluster, with Bacteroides capillosus ATCC 29799(T) branching off from this line of descent with sequence similarities of 97.1-97.4 %. The next nearest neighbours of these organisms were Clostridium viride, Oscillibacter valericigenes, Papillibacter cinnamivorans and Sporobacter termitidis, with sequence similarities to the respective type strains of 93.1-93.4, 91.2-91.4, 89.8-90 and 88.7-89.3 %. On the basis of biochemical properties, phylogenetic position, DNA G+C content and DNA-DNA hybridization, it is proposed to unify Clostridium orbiscindens and Eubacterium plautii in a new genus as Flavonifractor plautii gen. nov., comb. nov., with the type strain Prévot S1(T) (=ATCC 29863(T) =VPI 0310(T) =DSM 4000(T)), and to reassign Bacteroides capillosus to Pseudoflavonifractor capillosus gen. nov., comb. nov., with the type strain CCUG 15402A(T) (=ATCC 29799(T) =VPI R2-29-1(T)).

Four hydrogen-producing, aerotolerant, anaerobic bacterial strains isolated from chlorinated solvent-contaminated groundwater were characterized using a polyphasic approach. Three of the strains, designated BL-18, BL-19 and BL-20(T), were found to be identical in 16S rRNA gene sequences and in phenotypic properties. Cells of these strains are Gram-positive-staining, spore-forming, motile rods with peritrichous flagella. Growth occurred at 15-40 degrees C, pH 5.0-10.0 and at NaCl concentrations up to 5 % (w/v). Acid was produced in fermentation of cellobiose, fructose, galactose (weak), glucose, maltose and salicin. Products of fermentation in PYG medium were acetate, butyrate, ethanol, formate, carbon dioxide and hydrogen. Dominant cellular fatty acids when grown in PYG medium were C(13 : 0) iso, C(16 : 0), C(13 : 0) anteiso, C(15 : 0) iso and C(15 : 0) anteiso. The genomic DNA G+C content was 30.4 mol%. These isolates can be differentiated from their closest phylogenetic relative, the cluster I Clostridium species Clostridium frigidicarnis (97.2 % similar to the type strain in 16S rRNA gene sequence), on the basis of phenotypic and chemotaxonomic properties. The other strain characterized in this study, BL-28(T), was Gram-positive-staining with spore-forming, rod-shaped cells. Growth occurred at 15-46 degrees C, pH 6.0-8.5 and at NaCl concentrations up to 3 % (w/v). Acid was produced from cellobiose, dextran, fructose (weak), glucose, maltose, salicin and trehalose. End products of PYG fermentation included acetate, butyrate, pyruvate, carbon dioxide and hydrogen. Dominant cellular fatty acids from cells grown in PYG medium at 30 degrees C were C(14 : 0), C(14 : 0) dimethyl aldehyde, C(16 : 0) and C(12 : 0). The DNA G+C content was 28.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain BL-28(T) falls within cluster I of the genus Clostridium, but with

A novel strictly anaerobic, endospore-forming, rod-shaped, Gram-positive bacterium, designated strain SGB2(T), was isolated from a mixed culture from a pond sediment during screening for sulfate-reducing bacteria capable of degrading cyanophycin (CGP). In this study, the taxonomic characterization of this mesophilic, proteolytic Clostridium isolate and the role which it, and its phylogenetic relatives, may play in peptide degradation and in the sulfur cycle are reported. Strain SGB2(T) was a commensal strain, utilizing CGP degradation products produced by other micro-organisms. Cells were motile until sporulation, forming oval, terminal spores that swell the cells. It showed optimum growth at 34 degrees C, pH 6.6 and in the absence of NaCl. Strain SGB2(T) utilized proteinaceous compounds such as peptone, Casamino acids, gelatin and trypticase soy, in addition to several amino acids and pyruvate. Utilization of many of these compounds was enhanced in the presence of thiosulfate. The isolate was unable to use any of the carbohydrates or alcohols investigated or CGP as carbon and energy sources. Thiosulfate and elemental sulfur were used as terminal electron acceptors. Phylogenetic analysis revealed that strain SGB2(T) belongs to the low-G+C-containing Clostridiales group. It exhibited 99 % 16S rRNA gene sequence similarity to its closest relatives Clostridium thiosulfatireducens Lup21(T) and Clostridium subterminale DSM 6970(T). DNA-DNA hybridization values with these two strains were 39.4 and 42.1 %, respectively. Based on phenotypic, genotypic and phylogenetic characteristics, we conclude that the isolate represents a novel species of the genus Clostridium, Clostridium sulfidigenes sp. nov. The type strain is SGB2(T) (=DSM 18982(T) =ATCC BAA-1538(T)).

Two novel anaerobic, moderately thermophilic and cellulose-/cellobiose-digesting bacteria, EBR45(T) and EBR596(T), were isolated from anaerobic sludge of a cellulose-degrading methanogenic bioreactor. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains belonged to cluster III within the low-G+C-content Gram-positive bacteria. The close relatives of EBR45(T) were Clostridium straminisolvens DSM 16021(T) (sequence identity, 94.6 %) and Clostridium thermocellum DSM 1237(T) (93.4 %). The closest relative of EBR596(T) was Clostridium stercorarium DSM 8532(T) (95.9 %). Both isolates were rod-shaped sporulators, growing optimally at 60 degrees C. EBR45(T) was Gram-staining-reaction-variable and non-motile, formed bright-yellow colonies on solid media, and grew on a relatively narrow range of carbohydrates including cellulose and cellobiose. EBR596(T) was Gram-staining-reaction-negative and motile, formed glossy white colonies and grew on cellobiose and various carbohydrates except cellulose. Major fatty acid compositions were 16 : 0 iso, 16 : 0 and 16 : 0 dimethylacetal (strain EBR45(T)) and 15 : 0 iso, 16 : 0 iso, 15 : 0 anteiso and 17 : 0 anteiso (strain EBR596(T)). The DNA G+C contents were 36.9 mol% (EBR45(T)) and 51.1 mol% (EBR596(T)). Based on the phenotypic and phylogenetic data and genomic distinctiveness, strains EBR45(T) and EBR596(T) represent two novel species, for which the names Clostridium clariflavum sp. nov. (type strain EBR45(T) =DSM 19732(T) =NBRC 101661(T)) and Clostridium caenicola sp. nov. (type strain EBR596(T) =DSM 19027(T) =NBRC 102590(T)) are proposed.

A strictly anaerobic, Gram-positive, psychrotolerant, endospore-forming bacterium (strain A121(T)) was isolated from a permafrost sample collected in the Canadian High Arctic. Phylogenetic analysis of the 16S rRNA gene sequence of strain A121(T) showed its affiliation with the group of psychrophilic and psychrotolerant members of cluster I of the genus Clostridium, Clostridium bowmanii DSM 14206(T) being the closest relative (sequence similarity 98.5 %). Levels of DNA-DNA relatedness between strain A121(T) and the type strains of phylogenetically related species ranged from 33 to 52 %. Strain A121(T) grew in PY broth at temperatures between 4 and 28 degrees C (optimum 15-20 degrees C), at pH 6.0-8.0 (optimum pH 6.5-7.2) and in NaCl concentrations of 0-10.0 % (optimum 0-2.0 %). The strain utilized a narrow range of carbohydrates as sources of carbon and energy, including glucose, fructose, trehalose, maltose and starch; it also hydrolysed gelatin. Predominant fatty acids were C(16 : 1) cis9, C(16 : 1) cis9 DMA, C(16 : 0) and C(14 : 0). The DNA G+C content was 31.5 mol%. On the basis of its overall genotypic and phenotypic characteristics, strain A121(T) is classified within a novel species of the genus Clostridium, Clostridium tagluense sp. nov. The type strain is A121(T) (=VKM B-2369(T) =DSM 17763(T)).

A strictly anaerobic, mesophilic, cellulolytic bacterial strain, designated CDT-1(T), was isolated from rice-straw residue from a methanogenic reactor treating waste from cattle farms. The isolation was performed using enrichment culture with filter paper as a substrate. Cells stained Gram-negative, but reacted Gram-positively in the KOH test. Cells were slightly curved rods and were motile by means of peritrichous flagella. The strain produced yellow pigment when grown on filter-paper fragments. Although spore formation was not confirmed microscopically, thermotolerant cells were produced when the strain was grown on filter paper. The optimum temperature for growth was 33 degrees C and the optimum pH was 7.4. Oxidase, catalase and nitrate-reducing activities were absent. The strain utilized xylose, fructose, glucose, cellobiose, xylooligosaccharide, cellulose (filter-paper fragments and ball-milled filter paper) and xylan. The major fermentation products were acetate, ethanol, H(2) and CO(2). The major cellular fatty acids were iso-C(15 : 0), iso-C(14 : 0) and C(16 : 0) DMA. The cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. The genomic DNA G+C content was 40.7 mol%. On the basis of 16S rRNA gene sequence similarities, strain CDT-1(T) could be placed in cluster III of the genus Clostridium, being closely related to type strains of Clostridium hungatei (96.6 % sequence similarity), Clostridium termitidis (96.2 %) and Clostridium papyrosolvens (96.1 %). On the basis of the cellular, physiological and phylogenetic differences between CDT-1(T) and its close relatives, this strain represents a novel species of the genus Clostridium, for which the name Clostridium sufflavum sp. nov. is proposed. The type strain is CDT-1(T) (=JCM 14807(T)=DSM 19573(T)).

Two vancomycin-resistant, strictly anaerobic, Gram-positive, rod-shaped, spore-forming organisms (strains CCRI-9842(T) and CCRI-9929) isolated from human faecal specimens in Québec, Canada, and Australia were characterized using phenotypic, biochemical and molecular taxonomic methods. Pairwise analysis of the 16S rRNA gene sequences showed that both strains were closely related to each other genetically (displaying 99.2 % sequence similarity) and represented a previously unknown subline within the Clostridium coccoides rRNA group of organisms (rRNA cluster XIVa of the genus Clostridium). Strains CCRI-9842(T) and CCRI-9929 used carbohydrates as fermentable substrates, producing acetic acid as the major product of glucose metabolism. The novel strains were most closely related to Clostridium asparagiforme, Clostridium bolteae and Clostridium clostridioforme, but morphological, biochemical and phylogenetic studies demonstrated that they represent a previously unidentified species of the genus Clostridium. This was confirmed by the unique cellular fatty acid composition of strains CCRI-9842(T) and CCRI-9929. Therefore, on the basis of data from the polyphasic taxonomic analysis, it is proposed that strains CCRI-9842(T) and CCRI-9929 represent a novel species of the genus Clostridium, for which the name Clostridium lavalense sp. nov. is proposed. The type strain is CCRI-9842(T) (=CCUG 54291(T)=JCM 14986(T)=NML 03-A-015(T)).

The species of Clostridium comprise a very heterogeneous assemblage of bacteria that do not form a phylogenetically coherent group. It has been proposed previously that only a subset of the species of Clostridium that form a distinct cluster in the 16S rRNA tree (cluster I) should be regarded as the true representatives of the genus Clostridium (i.e. Clostridium sensu stricto). However, this cluster is presently defined only in phylogenetic terms, and no biochemical, molecular or phenotypic characteristic is known that is unique to species from this cluster. We report here phylogenomic and comparative analyses based on sequenced clostridial genomes in an attempt to bridge this gap and to clarify the evolutionary relationships among species of clostridia. In phylogenetic trees for species of clostridia based on concatenated sequences for 37 highly conserved proteins, the species of Clostridium cluster I formed a strongly supported clade that was separated from all other clostridia by a long branch. Several other Clostridium species that are not part of this cluster grouped reliably with other species of clostridia in a number of well-resolved clades. Our comparative genomic analyses have identified three conserved indels in three highly conserved proteins (a 4 aa insert in DNA gyrase A, a 1 aa deletion in ATP synthase beta subunit and a 1 aa insert in ribosomal protein S2) that are unique to the species of Clostridium cluster I and are not found in any other bacteria. blastp searches on various proteins in the genomes of Clostridium tetani E88 and Clostridium perfringens SM101 have also identified more than 10 proteins that are found uniquely in the cluster I species. These results provide evidence that the species of Clostridium cluster I not only are phylogenetically distinct but also share many unique molecular characteristics. These newly identified molecular markers provide useful tools to define and circumscribe the genus Clostridium sensu stricto in more definitive terms. We have also identified a 7-9 aa conserved insert in the enzyme phosphoglycerate dehydrogenase that is uniquely found in the Clostridium thermocellum, Thermoanaerobacter pseudethanolicus, Thermoanaerobacter tengcogensis and Caldicellulosiruptor saccharolyticus homologues, and is absent from all other bacteria. These species form a well-defined clade in the phylogenetic trees and this indel provides a potential molecular marker for this clostridial cluster.

It is proposed that Clostridium proteoclasticum be reclassified as Butyrivibrio proteoclasticus comb. nov. on the basis of phylogenetic position, DNA G+C content and physiological traits. Phylogenetic analyses based on 16S rRNA gene sequences from an extensive range of taxa within clostridial rRNA subcluster XIVa grouped C. proteoclasticum together with isolates of the genus Butyrivibrio, though this species was genetically distinct from the extant Butyrivibrio species examined. The DNA G+C content of C. proteoclasticum was originally erroneously reported as 28 mol%. However the genome sequence of the type strain of C. proteoclasticum, strain B316(T), and HPLC analysis estimate the DNA G+C content as 40 mol%, which is within the range reported for strains of Butyrivibrio. C. proteoclasticum was distinguishable from other species of the genus Butyrivibrio as the 16S rRNA gene from strain B316(T) shared less than 97 % sequence similarity with sequences from the type strains of Butyrivibrio species. C. proteoclasticum was also able to convert linoleic acid to stearic acid, in contrast to other species of Butyrivibrio. Physiological characteristics, including carbon source utilization, volatile fatty acid production and proteinase activities, were assessed for a panel of representative strains of the genera Butyrivibrio and Pseudobutyrivibrio and C. proteoclasticum. These data, together with the phylogenetic analyses, support the reclassification of Clostridium proteoclasticum as a separate species within the genus Butyrivibrio, Butyrivibrio proteoclasticus comb. nov. (type strain B316(T)=ATCC 51982(T)=DSM 14932(T)).

A Gram-stain-positive, strictly anaerobic, mesophilic, amylolytic, rod-shaped bacterium, designated strain SW408(T), was isolated from a laboratory-scale H(2)-producing upflow anaerobic sludge blanket reactor. The strain grew at 24-45 degrees C (no growth at or below 22 degrees C or at or above 47 degrees C), with optimum growth at 37 degrees C. The pH range for growth was 4.0-9.0 (no growth at or below pH 3.6 or at or above pH 9.3), with optimum growth at pH 7.0. Starch, cellobiose, glucose, fructose, galactose, lactose, maltose, mannose, ribose and sucrose supported growth. The major end products from glucose fermentation were ethanol, acetate, hydrogen and carbon dioxide. Abundant H(2) was produced from starch fermentation. The DNA G+C content was 33.1 mol% (T(m) method). Phylogenetic analysis based on 16S rRNA gene sequence analysis showed that the bacterium represents a previously unrecognized species within Clostridium rRNA cluster I and is most closely related to the type strain of Clostridium frigidicarnis (94.9% similarity). On the basis of phenotypic, genotypic and phylogenetic characteristics, strain SW408(T) was identified as a representative of a novel species of the genus Clostridium, for which the name Clostridium amylolyticum sp. nov. is proposed. The type strain is SW408(T) (=JCM 14823(T)=AS 1.5069(T)=CGMCC 1.5069(T)).

Phenotypic and phylogenetic studies were performed on 15 isolates of an unidentified Gram-positive, anaerobic, non-sporulating coccobacillus-shaped bacterium isolated from human faeces. The novel organisms were catalase-negative, indole-negative and produced acetate and succinate as end products of metabolism. Comparative 16S rRNA gene sequencing demonstrated that the 15 isolates were highly related to each other and formed a hitherto unknown subline within the clostridial rRNA cluster XIVa. The novel isolates formed a robust phylogenetic group with a number of organisms which included Clostridium coccoides, Ruminococcus luti, Ruminococcus obeum and a number of other misclassified ruminococci. On the basis of these studies, a novel genus, Blautia gen. nov., is proposed. It is suggested that Clostridium coccoides, Ruminococcus hansenii, Ruminococcus hydrogenotrophicus, Ruminococcus luti, Ruminococcus productus, and Ruminococcus schinkii are transferred to this genus as Blautia coccoides gen. nov., comb. nov., Blautia hansenii comb. nov., Blautia hydrogenotrophica comb. nov., Blautia luti comb. nov., Blautia producta comb. nov. and Blautia schinkii comb. nov. One of the new isolates, the hitherto unknown coccus-shaped bacterial strain WAL 14507T (=ATCC BAA-1564T=DSM 19850T) is proposed as representing the type strain of a novel species, Blautia wexlerae sp. nov.

A moderately thermophilic, anaerobic bacterium (strain SG 508T) was isolated from a hydrothermal vent chimney located at 1 degrees N on the East Pacific Rise at a depth of 2650 m. Cells of strain SG 508T were straight to slightly curved rods, 0.4-0.6 microm in diameter and 2.0-3.0 microm in length. Spore formation was observed only below pH 5.5. The temperature range for growth was 22-60 degrees C, with optimum growth at 50 degrees C. The pH range for growth was 4.0-8.5, with optimum growth at pH 6.0-6.8. Growth of strain SG 508T was observed at NaCl concentrations ranging from 1.0 to 6.0 % (w/v), with optimum growth at 2.5 % (w/v). Substrates utilized by strain SG 508T included casein, peptone, tryptone, yeast extract, beef extract, starch, maltose and glucose. The products of glucose fermentation were ethanol, acetate, H2, formate and CO2. Strain SG 508T was able to reduce elemental sulfur to hydrogen sulfide. The DNA G+C content of strain SG 508T was 30.9 mol%. 16S rRNA gene sequence analysis revealed that the isolated organism belonged to cluster I of the genus Clostridium. On the basis of its physiological properties and data from phylogenetic analyses, strain SG 508T is considered to represent a novel species of the genus Clostridium, for which the name Clostridium tepidiprofundi sp. nov. is proposed. The type strain is SG 508T (=DSM 19306T =VKM B-2459T).

A mesophilic, strictly anaerobic bacterium, strain Sjm18-20(T), was isolated from the alimentary canal of a Japanese corbicula clam. Cells of strain Sjm18-20(T) were Gram-negative, non-sporulating, straight to slightly curved rods, 2.5-6.0 microm long, and were motile with oscillatory movements by means of peritrichous flagella. Cells elongated to 30 microm after prolonged cultivation. Optimum growth was observed at 30 degrees C and pH 6.0-6.5. Growth occurred below 4.0 % (w/v) NaCl. Strain Sjm18-20(T) produced acid from D-glucose and a few pentoses such as L-arabinose, D-ribose and D-xylose. n-Valeric acid was the major end product from glucose. The genomic DNA G+C content of strain Sjm18-20(T) was 52.9 mol%. Phylogenetic analysis based on the 16S rRNA gene revealed that strain Sjm18-20(T) could be accommodated in clostridial cluster IV of the low-G+C-content Gram-positive bacteria and that the closest neighbour of this organism (92.6-92.9 % similarity) was the cloned 16S rRNA gene sequence of a not-yet cultured bacterium, thought to represent Oscillospira guilliermondii. The nearest cultivated neighbours of strain Sjm18-20(T) were Clostridium orbiscindens DSM 6740(T) and Clostridium viride T2-7(T), with sequence similarities of 91.3 and 89.1 %, respectively. On the basis of phenotypic features and phylogenetic position, it is proposed that this isolate represents a novel species in a new genus, Oscillibacter valericigenes gen. nov., sp. nov. The type strain of Oscillibacter valericigenes is Sjm18-20(T) (=NBRC 101213(T) =DSM 18026(T)).

An obligate anaerobic, mesophilic, motile and endospore-forming bacterium, designated 1D(T), was isolated from a subsurface soil sample. The young culture of strain 1D(T) was Gram-positive and formed oval spores that were central in position. Based on the biochemical, chemotaxonomic and physiological data, strain 1D(T) appears to be a member of the genus Clostridium. Strain 1D(T) was found to be capable of degrading p-nitrophenol (pNP) at a concentration of 0.5 mM under anaerobic conditions as revealed by HPLC analysis. The major fatty acids were C(16 : 0 )(28.02 %), iso-C(17 : 1) I/anteiso B (23.05 %) and C(14 : 0) (10.02 %). The major polar lipid content was diphosphatidylglycerol. Strain 1D(T) showed highest 16S rRNA gene sequence similarity to Clostridium aciditolerans JW/YJL-B3(T) (98.2 %) and similarity was less for Clostridium scatologenes ATCC 25775(T) (95.1 %), Clostridium drakei SL1(T) (95.0 %) and Clostridium carboxidivorans P7(T) (95.0 %). Phylogenetic analysis showed that it formed a coherent cluster with the species belonging to cluster I of the genus Clostridium. The DNA G+C content was 35.5 mol%. DNA-DNA hybridization analysis indicated a mean value of 36.4 % between strain 1D(T) and its closest relative C. aciditolerans. Several phenotypic differences from the closely related species were also revealed. On the basis of the polyphasic characteristics, strain 1D(T) represents a novel species of the genus Clostridium, for which the name Clostridium nitrophenolicum sp. nov. is proposed. The type strain is 1D(T) (=MTCC 7832(T)=JCM 14030(T)).

Strain ILE-2(T) was isolated from an upflow anaerobic sludge bed reactor treating brewery wastewater. The motile, non-sporulating, slightly curved cells (2-4 x 0.1 microm) stained Gram-negative and grew optimally at 42 degrees C and pH 7.1 with 0.5 % NaCl. The strain required yeast extract for growth and fermented Casamino acids, peptone, isoleucine, arginine, lysine, alanine, valine, glutamate, histidine, glutamine, methionine, malate, fumarate, glycerol and pyruvate to acetate, propionate and minor amounts of branched-chain fatty acids. Carbohydrates, formate, acetate, propionate, butyrate, isovalerate, methanol, ethanol, 1-propanol, butanol, lactate, succinate, starch, casein, gelatin, xylan and a number of other amino acids were not utilized. The DNA G+C content of strain ILE-2(T) was 52.7 mol%. 16S rRNA gene sequence analysis revealed that ILE-2(T) was distantly related to members of the genera Aminobacterium (83 % similarity) and Aminomonas (85 % similarity) in the family Syntrophomonadaceae, order Clostridiales, phylum Firmicutes. On the basis of the results of our polyphasic analysis, strain ILE-2(T) represents a novel species and genus within the family Syntrophomonadaceae, for which the name Aminiphilus circumscriptus gen. nov., sp. nov. is proposed. The type strain of Aminiphilus circumscriptus is ILE-2(T) (=DSM 16581(T) =JCM 14039(T)).