Halonatronomonas betaini gen. nov., sp. nov., a haloalkaliphilic isolate from soda lake capable of betaine degradation and proposal of Halarsenatibacteraceae fam. nov. and Halothermotrichaceae fam. nov. within the order Halanaerobiales.

A search for the organisms responsible for anaerobic betaine degradation in soda lakes resulted in isolation of a novel bacterial strain, designated Z-7014T. The cells were Gram-stain-negative, non-endospore-forming rods. Growth occurred at 8-52 °C (optimum 40-45 °C), pH 7.1-10.1 (optimum pH 8.1-8.8) and 1.0-3.5 M Na+ (optimum 1.8 M), i.e. it can be regarded as a haloalkaliphile. The strain utilized a limited range of substrates, mostly peptonaceous but not amino acids, and was able to degrade betaine. Growth on betaine occurred only in the presence of peptonaceous substances which could not be replaced by vitamins. The G + C content of the genomic DNA of strain Z-7014T was 36.1 mol%. The major cellular fatty acids (>5% of the total) were C16:0 DMA, C18: 0 DMA, C16:1ω8, C16:0, C18:1 DMA, C16:1 DMA, C18:1ω9, and C18:0. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain Z-7014T formed a distinct evolutionary lineage in the order Halanaerobiales with the highest similarity to Halarsenitibacter silvermanii SLAS-1T (83.6%), Halothermothrix orenii H168T (85.6%), and Halocella cellulosilytica DSM 7362T (85.6%). AAI and POCP values between strain Z-7014T and type strains of the order Halanaerobiales were 51.7-57.8%, and 33.8-58.3%, respectively. Based on polyphasic results including phylogenomic data, the novel strain could be distinguished from other genera, which suggests that strain Z-7014T represents a novel species of a new genus, for which the name Halonatronomonas betaini gen. nov., sp. nov. is proposed. The type strain is Z-7014T (=KCTC 25237T = VKM B-3506T). On the basis of phylogenomic data, it is also proposed to evolve two novel families Halarsenitibacteraceae fam. nov. and Halothermotrichaceae fam. nov. within the current order Halanaerobiales.

Serinibacter arcticus sp. nov., isolated from a thawing ancient ice wedge.

A novel actinobacterium, strain K3-2T, was isolated in pure culture from a thawing ancient ice wedge at Mammoth Mountain (Eastern Siberia, Russia). Colonies of strain K3-2T were yellowish orange; cells had the fine structure typical of Gram-positive bacteria, were non-motile short rods and were non-spore-forming. Strain K3-2T was mesophilic (optimum growth at 28 °Ð¡), but capable of growing at 4 °Ð¡. The cell-wall peptidoglycan of strain K3-2T contained lysine (the diagnostic diamino acid), glutamic acid, alanine, ornithine, glycine and serine. The polar lipids were phosphatidylglycerol, lysophosphatidylserine, three unidentified phospholipids and glycolipids. The major fatty acids were anteiso-C15 : 0 and C16 : 0. The only menaquinone detected was MK-8(H4). 16S rRNA gene analysis indicated that strain K3-2T belongs to the genus Serinibacter. The closest taxonomically described relatives were Serinibacter salmoneus Kis4-28T and Serinibacter tropicus PS-14-7T, with 97.20 and 97.20 % 16 s rRNA gene sequence similarity, respectively. The average nucleotide identity value of the whole genome sequence between strain K3-2T and S. salmoneus Kis4-28T was 78.9 %. DNA-DNA relatedness values between strain K3-2T and S. salmoneus DSM 21801T (=Kis4-28T) and S. tropicus VKPM Ac 2044T (=PS-14-7T) were 41 and 47 %. Thus, strain K3-2T represents a novel species of the genus Serinibacter for which the name Serinibacter arcticus sp. nov. is proposed. The type strain is K3-2T (DSM 103859T=VKM Ас-2719T).

Methylopila carotae sp. nov., a facultative methylotroph, isolated from a root of Daucus carota L.

An aerobic facultatively methylotrophic bacterium, designated strain Das4.1T, was isolated from a root of Daucus carota L. The cells of this strain were observed to be Gram-stain negative, asporogenous, non-motile short rods multiplying by binary fission. Strain Das4.1T can utilise methanol, methylamine and a variety of polycarbon compounds as carbon and energy sources. C1-compounds were found to be assimilated via the isocitrate lyase-negative variant of the serine pathway. On medium with 0.5% methanol, growth of strain Das4.1T was observed at pH 5.5-9.0 (optimum, pH 6.0-7.0) and 18-37 °C (optimum, 24-29 °C) and in the presence of 0-2% (w/v) NaCl (optimum, 0.05%). Cells are catalase and oxidase positive and synthesise indole from L-tryptophan. The major fatty acids of methanol-grown cells were identified as C18:1ω7c, C18:0 and 11-methyl-C18:1ω7c. The predominant phospholipids were found to be phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylethanolamine. The major respiratory quinone was identified as Q-10. The DNA G + C content of strain Das4.1T was determined to be 67.3 mol% (Tm). Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that strain Das4.1T belongs to the genus Methylopila and shows high sequence similarity to Methylopila oligotropha 2395AT (98.4%) and Methylopila capsulata IM1T (98.0%). However, the DNA-DNA relatedness of strain Das4.1T with M. oligotropha 2395AT was only 22 ± 3%. Based on genotypic, chemotaxonomic and physiological characterisation, the isolate can be classified as a novel species of the genus Methylopila, for which the name Methylopila carotae sp. nov. is proposed. The type strain is Das4.1T (= VKM B-3244T = CCUG 72399T).

Tundrisphaera lichenicola gen. nov., sp. nov., a psychrotolerant representative of the family Isosphaeraceae from lichen-dominated tundra soils.

Two strains of aerobic, budding, pink-pigmented bacteria, P12T and P515, were isolated from a lichen-dominated peatland and a forested tundra soil of north-western Siberia, respectively. Cells of these isolates were represented by non-motile spheres that occurred singly or were arranged in short chains and aggregates. While growing on solid media, cells of strains P12T and P515 attached to the surface by means of holdfast-like appendages. These isolates were mildly acidophilic (optimum growth at pH 5.5-6.0), psychrotolerant bacteria, which displayed tolerance of low temperatures (4-15 °C), grew optimally at 15-22 °C and did not grow at temperatures above 28 °C. The preferred growth substrates were sugars and some heteropolysaccharides. The major fatty acids were C18 : 1ω9c, C16 : 0 and C14 : 0. Trimethylornithine lipid was the major polar lipid. The only quinone was MK-6, and the G+C content of the DNA was 61.2-62.2 mol%. Strains P12T and P515 possessed identical 16S rRNA gene sequences, which affiliated them with the family Isosphaeraceae, order Planctomycetales, and these displayed the highest similarity (93-94 %) to 16S rRNA gene sequences from members of the genus Singulisphaera. However, the signature fatty acid of species of the genus Singulisphaera, i.e. C18 : 2ω6c,12c, was absent in cells of strains P12T and P515. They also differed from members of the genus Singulisphaera by substrate utilization pattern and a number of physiological characteristics. Based on these data, the novel isolates should be considered as representing a novel genus and species of planctomycetes, for which the name Tundrisphaera lichenicola gen. nov., sp. nov, is proposed. The type strain is P12T (=LMG 29571T=VKM B-3044T).

Fuchsiella ferrireducens sp. nov., a novel haloalkaliphilic, lithoautotrophic homoacetogen capable of iron reduction, and emendation of the description of the genus Fuchsiella.

Two strains of haloalkaliphilic homoacetogenic bacteria capable of iron reduction, Z-7101T and Z-7102, were isolated from soda lake Tanatar III (Altai, Russia). Cells of both strains were flexible, motile, Gram-negative, spore-forming rods. The strains were mesophilic and obligately alkaliphilic: the pH range for growth was 8.5-10.2 (pHopt 9.8). Growth depended on carbonate and chloride ions. The strains were able to grow chemolithoautotrophically on H2+CO2, producing acetate as the only metabolic product. In medium with carbonates as the only potential electron acceptor, the following substrates were utilized for chemo-organotrophic growth: pyruvate, lactate, ethanol, 1-propanol, ethylene glycol and 1-butanol. Strain Z-7101T was able to reduce nitrate, selenate, thiosulfate and anthraquinone 2,6-disulfonate with ethanol as an electron donor. It was also able to reduce synthesized ferrihydrite to siderite with molecular hydrogen or organic compounds, including acetate and formate, as electron donors. It was able to reduce S0 with acetate or formate as electron donors. The DNA G+C content of strain Z-7101T was 34.6 mol%. 16S rRNA gene sequence analysis showed that strains Z-7101T and Z-7102 were members of the order Halanaerobiales and family Halobacteroidaceae, clustering with Fuchsiella alkaliacetigena Z-7100T (98.9-98.4% similarity). DNA-DNA hybridization was 63.0% between strain Z-7101T and F. alkaliacetigena Z-7100T. Based on morphological and physiological differences from F. alkaliacetigena Z-7100T and the results of phylogenetic analysis and DNA-DNA hybridization, it is proposed to assign strains Z-7101T and Z-7102 ( = DSM 26052 = VKM B-2790) to the novel species Fuchsiellaferrireducens sp. nov. The type strain is strain Z-7101T ( = DSM 26031T = VKM B-2766T).

Planctomicrobium piriforme gen. nov., sp. nov., a stalked planctomycete from a littoral wetland of a boreal lake.

An aerobic, budding, non-pigmented and rosette-forming bacterium was isolated from a littoral wetland of a boreal lake located in Valaam Island, northern Russia, and designated strain P3(T). Ellipsoidal to pear-shaped cells of this bacterium were covered with crateriform pits and possessed stalks suggesting a planctomycete morphotype. 16S rRNA gene sequence analysis confirmed that strain P3(T) was a member of the order Planctomycetales and belonged to a phylogenetic lineage defined by the genus Planctomyces , with 89 and 86% sequence similarity to Planctomyces brasiliensis and Planctomyces maris , respectively. Strain P3(T) was a mildly acidophilic, mesophilic organism capable of growth at pH values between pH 4.2 and 7.1 (with an optimum at pH 6.0-6.5) and at temperatures between 10 and 30 °C (optimum at 20-28 °C). Most sugars, a number of polysaccharides and several organic acids were the preferred growth substrates. Compared with Planctomyces brasiliensis and Planctomyces maris , which require NaCl for growth, strain P3(T) was salt-sensitive and did not develop at NaCl concentrations above 0.5% (w/v). The major fatty acids were C16 : 0 and C16 : 1ω7c; the cells also contained significant amounts of C18 : 1ω7c and C18 : 0. The major intact polar lipids were diacylglycerol-O-(N,N,N-trimethyl)homoserine (DGTS) lipids; the major neutral lipids were long-chain 1,(ω-1)-diols and C31 : 9 hydrocarbon. The quinone was MK-6, and the G+C content of the DNA was 59.0 mol%. Strain P3(T) differed from Planctomyces brasiliensis and Planctomyces maris by cell morphology, substrate utilization pattern and a number of physiological characteristics. Based on these data, the novel isolate should be considered as representing a novel genus and species of planctomycetes, for which the name Planctomicrobium piriforme gen. nov., sp. nov., is proposed. The type strain is P3(T) ( =DSM 26348(T) =VKM B-2887(T)).

Methanospirillum stamsii sp. nov., a psychrotolerant, hydrogenotrophic, methanogenic archaeon isolated from an anaerobic expanded granular sludge bed bioreactor operated at low temperature.

A psychrotolerant hydrogenotrophic methanogen, strain Pt1, was isolated from a syntrophic propionate-oxidizing methanogenic consortium obtained from granulated biomass of a two-stage low-temperature (3-8 °C) anaerobic expanded granular sludge bed (EGSB) bioreactor, fed with a mixture of volatile fatty acids (VFAs) (acetate, propionate and butyrate). The strain was strictly anaerobic, and cells were curved rods, 0.4-0.5×7.5-25 µm, that sometimes formed wavy filaments from 25 to several hundred micrometres in length. Cells stained Gram-negative and were non-sporulating. They were gently motile by means of tufted flagella. The strain grew at 5-37 °C (optimum at 20-30 °C), at pH 6.0-10 (optimum 7.0-7.5) and with 0-0.3 M NaCl (optimum 0 M NaCl). Growth and methane production was found with H2/CO2 and very weak growth with formate. Acetate and yeast extract stimulated growth, but were not essential. The G+C content of the DNA of strain Pt1 was 40 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Pt1 was a member of the genus Methanospirillum and showed 97.5 % sequence similarity to Methanospirillum hungatei JF1(T) and 94 % sequence similarity to Methanospirillum lacunae Ki8-1(T). DNA-DNA hybridization of strain Pt1 with Methanospirillum hungatei JF1(T) revealed 39 % relatedness. On the basis of its phenotypic characteristics and phylogenetic position, strain Pt1 is a representative of a novel species of the genus Methanospirillum, for which the name Methanospirillum stamsii sp. nov. is proposed. The type strain is Pt1(T) ( = DSM 26304(T) = VKM B-2808(T)).

Methylomonas paludis sp. nov., the first acid-tolerant member of the genus Methylomonas, from an acidic wetland.

An aerobic methanotrophic bacterium was isolated from an acidic (pH 3.9) Sphagnum peat bog in north-eastern Russia and designated strain MG30(T). Cells of this strain were Gram-negative, pale pink-pigmented, non-motile, thick rods that were covered by large polysaccharide capsules and contained an intracytoplasmic membrane system typical of type I methanotrophs. They possessed a particulate methane monooxygenase enzyme (pMMO) and utilized only methane and methanol. Carbon was assimilated via the ribulose-monophosphate pathway; nitrogen was fixed via an oxygen-sensitive nitrogenase. Strain MG30(T) was able to grow at a pH range of 3.8-7.3 (optimum pH 5.8-6.4) and at temperatures between 8 and 30 °C (optimum 20-25 °C). The major cellular fatty acids were C16:1ω5t, C16:1ω8c, C16:1ω7c and C14:0; the DNA G+C content was 48.5 mol%. The isolate belongs to the family Methylococcaceae of the class Gammaproteobacteria and displayed 94.7-96.9% 16S rRNA gene sequence similarity to members of the genus Methylomonas. However, strain MG30(T) differed from all taxonomically characterized members of this genus by the absence of motility, the ability to grow in acidic conditions and low DNA G+C content. Therefore, we propose to classify this strain as representing a novel, acid-tolerant species of the genus Methylomonas, Methylomonas paludis sp. nov. Strain MG30(T) (=DSM 24973(T)=VKM B-2745(T)) is the type strain.

Singulisphaera rosea sp. nov., a planctomycete from acidic Sphagnum peat, and emended description of the genus Singulisphaera.

An aerobic, pink-pigmented, budding bacterium, designated strain S26(T), was isolated from an acidic Sphagnum peat bog of north-western Russia. Cells were non-motile and spherical, occurring singly, in pairs or in short chains, and were able to attach to surfaces by means of a holdfast material. Strain S26(T) was a moderately acidophilic, mesophilic organism capable of growth at pH 3.2-7.1 (optimum at pH 4.8-5.0) and at 4-33 °C (optimum at 20-26 °C). Most sugars, several organic acids and polyalcohols were the preferred growth substrates. The major fatty acids were C(16:0), C(18:1)ω9c and C(18:2)ω6c,12c. The major neutral lipids were n-C(31:9) hydrocarbon and squalene; the polar lipids were phosphatidylglycerol, phosphatidylcholine and components with an unknown structure. The DNA G+C content of strain S26(T) was 62.2 mol%. 16S rRNA gene sequence analysis showed that strain S26(T) is a member of the order Planctomycetales. Among taxonomically characterized representatives of this order, highest levels of 16S rRNA gene sequence similarity (95.1-95.2%) were observed with strains of the non-filamentous, peat-inhabiting planctomycete Singulisphaera acidiphila. Strain S26(T) could be differentiated from Singulisphaera acidiphila based on pigmentation, significant differences in substrate utilization patterns, greater tolerance of acidic conditions and the presence of C(16:1)ω9c. Based on the data presented, strain S26(T) is considered to represent a novel species of the genus Singulisphaera, for which the name Singulisphaera rosea sp. nov. is proposed; the type strain is S26(T) (=DSM 23044(T)=VKM B-2599(T)).

Sulfur-dependent respiration under extremely haloalkaline conditions in soda lake 'acetogens' and the description of Natroniella sulfidigena sp. nov.

Microbial sulfidogenesis is the main dissimilatory anaerobic process in anoxic sediments of extremely haloalkaline soda lakes. In soda lakes with a salinity >2 M of the total Na(+) sulfate reduction is depressed, while thiosulfate- and sulfur-dependent sulfidogenesis may still be very active. Anaerobic enrichments at pH 10 and a salinity of 2-4 M total Na(+) from sediments of hypersaline soda lakes with thiosulfate and elemental sulfur as electron acceptors and simple nonfermentable electron donors resulted in the isolation of two groups of haloalkaliphilic bacteria capable of dissimilatory sulfidogenesis. Both were closely related to obligately heterotrophic fermentative homoacetogens from soda lakes. The salt-tolerant alkaliphilic thiosulfate-reducing isolates were identified as representatives of Tindallia magadiensis, while the extremely natronophilic obligate sulfur/polysulfide-respiring strains belonged to the genus Natroniella and are proposed here as a novel species Natroniella sulfidigena. Despite the close phylogenetic relation to Natroniella acetigena, it drastically differed from the type strain phenotypically (chemolithoautotrophic and acetate-dependent sulfur respiration, absence of acetate as the final metabolic product). Apparently, in the absence of specialized respiratory sulfidogens, primarily fermentative bacteria that are well adapted to extreme salinity may take over an uncharacteristic ecological function. This finding, once again, exemplifies the importance of isolation and phenotypic investigation of pure cultures.

Acidisoma tundrae gen. nov., sp. nov. and Acidisoma sibiricum sp. nov., two acidophilic, psychrotolerant members of the Alphaproteobacteria from acidic northern wetlands.

Three obligately aerobic, heterotrophic bacteria, designated strains WM1T, TPB606T and TPB621, were isolated from acidic Sphagnum-dominated tundra and Siberian wetlands in Russia. Cells of these isolates were Gram-negative, non-motile coccobacilli that occurred singly, in pairs or in chains, and were covered by large capsules. The novel strains were moderately acidophilic and psychrotolerant organisms capable of growth at pH 3.0-7.6 and 2-30 degrees C. Cells contained numerous intracellular poly-beta-hydroxybutyrate granules (3-4 per cell). The major cellular fatty acid was cyclo C19:0omega8c and the predominant quinone was Q-10. Strains TPB606T and TPB621, isolated from Siberian wetland, possessed almost identical 16S rRNA gene sequences and shared 97.2% sequence similarity with tundra strain WM1T. The three strains were shown to belong to the Alphaproteobacteria, but were related only distantly to the type strains of acidophilic bacteria Acidisphaera rubrifaciens (93.4-94.3% 16S rRNA gene sequence similarity), Rhodopila globiformis (92.2-93.3%), and members of the genera Acidiphilium (91.3-93%) and Acidocella (91.8-92.4%). The DNA G+C contents of the novel strains were 60.5-61.9 mol%. The low levels of DNA-DNA relatedness (37%) and a number of phenotypic differences between the Siberian strains TPB606T and TPB621 and the tundra strain WM1T indicated that they represent two separate species. As the three isolates are clearly distinct from all recognized acidophilic members of the Alphaproteobacteria, they are considered to represent two novel species of a new genus, for which the names Acidisoma tundrae gen. nov., sp. nov. and Acidisoma sibiricum sp. nov. are proposed. The type strain of Acidisoma sibiricum is TPB606T (=DSM 21000T=VKM B-2487T) and the type strain of Acidisoma tundrae is WM1T (=DSM 19999T=VKM B-2488T).

Tindallia californiensis sp. nov., a new anaerobic, haloalkaliphilic, spore-forming acetogen isolated from Mono Lake in California.

A novel extremely haloalkaliphilic, strictly anaerobic, acetogenic bacterium strain APO was isolated from sediments of the athalassic, meromictic, alkaline Mono Lake in California. The Gram-positive, spore-forming, slightly curved rods with sizes 0.55-0.7x1.7-3.0 microm were motile by a single laterally attached flagellum. Strain APO was mesophilic (range 10-48 degrees C, optimum of 37 degrees C); halophilic (NaCl range 1-20% (w/v) with optimum of 3-5% (w/v), and alkaliphilic (pH range 8.0-10.5, optimum 9.5). The novel isolate required sodium ions in the medium. Strain APO was an organotroph with a fermentative type of metabolism and used the substrates peptone, bacto-tryptone, casamino acid, yeast extract, l-serine, l-lysine, l-histidine, l-arginine, and pyruvate. The new isolate performed the Stickland reaction with the following amino acid pairs: proline + alanine, glycine + alanine, and tryptophan + valine. The main end product of growth was acetate. High activity of CO dehydrogenase and hydrogenase indicated the presence of a homoacetogenic, non-cycling acetyl-CoA pathway. Strain APO was resistant to kanamycin but sensitive to chloramphenicol, tetracycline, and gentamycin. The G+C content of the genomic DNA was 44.4 mol% (by HPLC method). The sequence of the 16S rRNA gene of strain APO possessed 98.2% similarity with the sequence from Tindallia magadiensis Z-7934, but the DNA-DNA hybridization value between these organisms was only 55%. On the basis of these physiological and molecular properties, strain APO is proposed to be a novel species of the genus Tindallia with the name Tindallia californiensis sp. nov., (type strain APO = ATCC BAA-393 = DSM 14871).