[Methanocalculus natronophilus sp. nov., a new alkaliphilic hydrogenotrophic methanogenic archaeon from a soda lake, and proposal of the new family Methanocalculaceae].

A mesophilic hydrogenotrophic methanogenic archaeon, strain Z-7105(T), was isolated from the bottom sediments of a collector in the vicinity of a soda lake Tanatar II (Altai, Russia). The cells were motile, irregular cocci 0.2-1.2 µm in diameter. The organism was an obligate alkaliphile, growing within a pH range from 8.0 to 10.2 with the optimum at pH 9.0-9.5. It was obligately dependent on carbonates, growing at 0.5 to 1.6 M total carbonates with the optimum at 0.7-0.9 M. Sodium ions were also obligately required at concentrations from 0.9 to 3.3 M Na+ (optimum at 1.4-1.9 M). The organism was halotolerant, but Cl- ions were not required. Hydrogen and formate were used as electron donors. Acetate was required for anabolism. The DNA G + C content was 50.2 mol %. According to the results of its 16S rRNA gene sequence analysis, the isolate belonged to the genus Methanocalculus, being the first known alkaliphilic member of this genus. Its similarity to the neutrophilic and halotolerant Methanocalculus species (M. halotolerans, M. taiwanensis, M. pumilus, and M. chunghsingensis) was 98.2-97.1%, which is within the interspecific range for this genus. The level of DNA-DNA hybridization between strain Z-7105(T) and the Methanocalculus type species M. halotolerans DSM 14092(T) was 32%. The genus Methanocalculus, including the new isolate and the previously described species, is distant from other genera of methanogens (< 90% 16S rRNA gene similarity). Based on significant phenotypic differences and the results of phylogenetic analysis, including DNA-DNA hybridization, it is proposed to assign strain Z-7105(T) (= DSM 25006(T), = VKM B-2765(T)) to the new species Methanocalculus natronophilus sp. nov. and to incorporate the genus into the new family Methanocalculaceae fam. nov.

[Geoalkalibacter ferrihydriticus gen. nov., sp. nov., the first alkaliphilic representative of the family Geobacteraceae, isolated from a soda lake].

Investigation of iron reduction in bottom sediments of alkaline soda lakes resulted in the isolation of a new obligately anaerobic iron-reducing bacterium, strain Z-0531, from Lake Khadyn (Tuva Republic, Russia) sediment samples. The cells of strain Z-0531 are short (1.0-1.5 by 0.3-0.5 microm), motile, non-spore-forming, gram-negative rods. The isolate is an obligate alkaliphile, developing in the pH range of 7.8-10.0, with an optimum at pH 8.6. It does not require NaCl but grows at NaCl concentrations of 0-50 g/1l. It can oxidize acetate with such electron acceptors as amorphous Fe(llI) hydroxide (AFH), EDTA-Fe(III), anthraquinone-2,6-disulfonate (quinone), Mn(IV), and S(0). On media with EDTA-Fe(III), the isolate can oxidize, apart from acetate, ethanol, pyruvate, oxalate, arginine, tartrate, lactate, propionate, and serine. H2 is not utilized. The reduced products formed during growth with AFH are siderite or magnetite, depending on the growth conditions. The isolate is incapable of fermenting sugars, peptides, and amino acids. Yeast extract or vitamins are required as growth factors. The organism is capable of dinitrogen fixation and harbors the nifH gene. The DNA G+C content is 55.3 mol %. 16S rRNA analysis places strain Z-0531 into the family Geobacteraceae. Its closest relative (93% similarity) is Desulfuromonas palmitatis. Based on phenotypic distinctions and phylogenetic position, it is proposed that strain Z-0531 be assigned to the new genus and species Geoalkalibacter ferrihydriticus gen. nov., sp. nov.

["Candidatus contubernalis alkalaceticum," an obligately syntrophic alkaliphilic bacterium capable of anaerobic acetate oxidation in a coculture with Desulfonatronum cooperativum].

From the silty sediments of the Khadyn soda lake (Tuva), a binary sulfidogenic bacterial association capable of syntrophic acetate oxidation at pH 10.0 was isolated. An obligately syntrophic, gram-positive, spore-forming alkaliphilic rod-shaped bacterium performs acetate oxidation in a syntrophic association with a hydrogenotrophic, alkaliphilic sulfate-reducing bacterium; the latter organism was previously isolated and characterized as the new species Desulfonatronum cooperativum. Other sulfate-reducing bacteria of the genera Desulfonatronum and Desulfonatronovibrio can also act as the hydrogenotrophic partner. Apart from acetate, the syntrophic culture can oxidize ethanol, propanol, isopropanol, serine, fructose, and isobutyric acid. Selective amplification of 16S rRNA gene fragments of the acetate-utilizing syntrophic component of the binary culture was performed; it was found to cluster with clones of uncultured gram-positive bacteria within the family Syntrophomonadaceae. The acetate-oxidizing bacterium is thus the first representative of this cluster obtained in a laboratory culture. Based on its phylogenetic position, the new acetate-oxidizing syntrophic bacterium is proposed to be assigned, in a Candidate status, to a new genus and species: "Candidatus Contubernalis alkalaceticum."

Thermovenabulum ferriorganovorum gen. nov., sp. nov., a novel thermophilic, anaerobic, endospore-forming bacterium.

A thermophilic, anaerobic, spore-forming bacterium (strain Z-9801T) was isolated from a terrestrial hydrothermal source in the Uzon caldera on the Kamchatka peninsula. Cells of strain Z-9801T were straight, sometimes branched rods, 0.5-0.6 microm in diameter and 1.5-7.0 microm in length, with peritrichous flagella. The temperature range for growth was 45-76 degrees C, with an optimum at 63-65 degrees C. The pH range for growth was 4.8-8.2, with an optimum at 6.7-6.9. The substrates utilized by strain Z-9801T included peptone, yeast extract, beef extract, Casamino acids, starch, pyruvate, melibiose, sucrose, fructose, maltose, xylose and ribose. The fermentation products from melibiose were ethanol, acetate, H2 and CO2. Strain Z-9801T used H2 in the presence of Fe(III) and an organic electron donor. Strain Z-9801T reduced Fe(III), Mn(IV), nitrate, fumarate, sulfite, thiosulfate, elemental sulfur and 9,10-anthraquinone 2,6-disulfonate. The G+C content of strain Z-9801T DNA was 36 mol%. 16S rDNA sequence analysis revealed that the isolated organism forms a separate branch within the Bacillus/Clostridium group. On the basis of physiological properties and phylogenetic analysis, it is proposed that strain Z-9801T (= DSM 14006T = UNIQEM 210T) should be assigned to a novel species of a new genus, Thermovenabulum ferriorganovorum gen. nov., sp. nov.

Thermanaerovibrio velox sp. nov., a new anaerobic, thermophilic, organotrophic bacterium that reduces elemental sulfur, and emended description of the genus Thermanaerovibrio.

A moderately thermophilic, organotrophic bacterium with vibrioid cells was isolated from a sample of a cyanobacterial mat from caldera Uzon, Kamchatka, Russia, and designated strain Z-9701T. Cells of strain Z-9701T were curved, Gram-negative rods, 0.5-0.7 x 2.5-5.0 microm in size, with tapering ends and with fast, wavy movement by means of lateral flagella located on the concave side of the cell. Colonies were small, white, irregular or round, 0.2 mm in diameter, and with even edges. Strain Z-9701T was an obligate anaerobe with a temperature optimum at 60-65 degrees C and a pH optimum at 7.3. It fermented glucose, fructose, mannose, N-acetyl-D-glucosamine, adonite, arginine, serine, peptone, yeast extract and Casamino acids. The fermentation products formed during growth on glucose were acetate, lactate, H2, CO2 and ethanol. Strain Z-9701T reduced elemental sulfur to H2S during organotrophic growth with glucose or peptides as energy and carbon sources. In the presence of S0, strain Z-9701T was capable of lithotrophic growth with molecular hydrogen as energy substrate and 0.1 g yeast extract l(-1) as carbon source. Sulfate, thiosulfate, nitrate, Fe(III) and sulfite were not reduced and did not stimulate growth. The G+C content of strain Z-9701T DNA was 54.6 mol%. The results of 16S rDNA sequence analyses revealed that strain Z-9701T belongs to the cluster within the Clostridium group formed by Thermanaerovibrio acidaminovorans, Dethiosulfovibrio peptidovorans, Anaerobaculum thermoterrenum and Aminobacterium colombiense, but the level of sequence similarity with the members of this cluster was not very high (87.6-92.2%). Among these organisms, Thermanaerovibrio acidaminovorans is phenotypically close to strain Z-9701T. However, the two organisms showed a relatively low level of similarity of their 16S rRNA sequences (92.2%) and of DNA-DNA hybridization (15 +/- 1%). Nevertheless, on the basis of the similar morphology and physiology of the new isolate and Thermanaerovibrio acidaminovorans, strain Z-9701T was placed in the genus Thermanaerovibrio and a new species, Thermanaerovibrio velox, proposed for it. The type strain is Z-9701T (= DSM 12556T).