D. G. Zavarzina

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, Russia) sediment samples. The cells of strain Z-0531 are short (1.0–1.5 by 0.3–0.5 µm), 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/l. It can oxidize acetate with such electron acceptors as amorphous Fe(III) hydroxide (AFH), EDTA-Fe(III), anthraquinone-2,6-disulfonate (quinone), Mn(IV), and S0. On medium 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 this strain be assigned to the new genus and species Geoalkalibacter ferrihydriticus gen. nov., sp. nov. (Z-0531T-DSMZ-17813-VKMB-2401).

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).

["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 in the Candidatus status for 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.

Alkaliphilus peptidofermentans sp. nov., a New Alkaliphilic Bacterial Soda Lake Isolate Capable of Peptide Fermentation and Fe(III) Reduction

A novel strain, Z-7036, of anaerobic spore-forming bacteria was isolated from a cellulolytic consortium obtained from the bottom sediments of the low-mineralization soda lake Verkhnee Beloe (Buryatia). The cells of the new strain are short motile gram-positive rods, 1.1–3.0 × 0.25–0.4 μm. The organism is an aerotolerant anaerobe and obligate alkaliphile growing within the pH range of 7.5–9.7 with an optimum at pH 9.1. The strain is mesophilic and halotolerant and grows at NaCl concentrations from 0 to 50 g/l with an optimum at 20 g/l. Carbonates are required. The microorganism ferments peptone, yeast extract, trypticase, tryptone, Bacto Soytone, meat extract, Casamino acids, ornithine, arginine, threonine, and tryptophan. The strain hydrolyzes the bacterial preparations “Gaprin” and “Spirulina”. Acetate and formate are the major fermentation products. The strain reduces amorphous ferric hydroxide (AFH), EDTA-Fe(III), anthraquinone-2,6-disulfonate (quinone), S2O32−, fumarate, and crotonate. Major fatty acids are C16:0, C16:1ω7c, iso-C17, iso-C15, and iso-C17:1. The DNA G+C content is 33.8 ± 0.5 mol %. According to the results of the 16S rRNA gene analysis, strain Z-7036 belongs to the genus Alkaliphilus within the cluster XI of low G+C gram-positive bacteria of the family Clostridiaceae. The novel strain is closely related to A. transvaalensis SAGM1T and A. crotonatoxidans B11-2T (93.3 and 93.9% 16S rRNA sequence identities, respectively). On the basis of the existing genotypic and phenotypic differences, we propose that strain Z-7036 should be classified as a novel species Alkaliphilus peptidofermentans sp. nov.

Description of Anaerobacillus alkalilacustre gen. nov., sp. nov.—Strictly anaerobic diazotrophic bacillus isolated from soda lake and transfer of Bacillus arseniciselenatis, Bacillus macyae, and Bacillus alkalidiazotrophicus to Anaerobacillus as the new combinations A. arseniciselenatis comb. nov., A. macyae comb. nov., and A. alkalidiazotrophicus comb. nov.

An anaerobic, spore-forming bacterium (strain Z-0521) was isolated from the iron-reducing microbial community enriched from sample of bottom sediments from low-mineralized soda lake Khadyn, Tuva upper Yenisey region (Russia). Cells of strain Z-0521 are motile straight Gram-positive rods, 0.7–1.1 (µm in diameter and 3.0–7.0 µm length. It is a mesophilic halotolerante obligate alkaliphilic bacterium with a pH range for growth 8.5–10.7 (optimum at 9.6–9.7). Utilizes carbohydrates. Peptides, organic acids or alcohols are not utilized. In the presence of mannite strain Z-0521 reduces AQDS, arsenate, selenate and selenite. It is capable of N2 fixation and has nitrogenase gene nifH. The dominant cellular fatty acids are C16:0, C16:1w7c and Ca15. The G+C content in the DNA is 36.2 mol %. 16S rRNA gene sequencing identified strain Z-0521 as a member of rRNA group 6 of the genus Bacillus. Its closest relatives are B. alkalidiazotrophicus and B. macyae (98.3 and 98.1% sequence similarity). On the basis of physiological properties and genetic analysis, it is proposed that strain Z-0521T should be assigned to a new species of a new genus, Anaerobacillus alkalilacustre gen. nov., sp. nov. It is also proposed that Bacillus arseniciselenatis, Bacillus macyae and Bacillus alkalidiazotrophicus should be transferred to this new genus, with Anaerobacillus arseniciselenatis (formely Bacillus arseniciselenatis) as the type species.

Fuchsiella alkaliacetigena gen. nov., sp. nov., an alkaliphilic, lithoautotrophic homoacetogen from a soda lake.

The first alkaliphilic obligately anaerobic hydrogenotrophic homoacetogenic bacterium, strain Z-7100(T), was isolated from sediments of the soda-depositing soda lake Tanatar III (Altay, Russia). Cells were thin, flexible rods, motile, Gram-negative and spore-forming. The organism was an obligate alkaliphile, growing at pH 8.5 to 10.5, with optimum growth at pH 8.8-9.3, and it grew in soda brines containing 1.9-4.7 M total Na(+) (optimum at 2.8-3.3 M). It exhibited an obligate dependence upon sodium carbonate but not upon chloride ions with an NaCl range for growth of 0-14% (w/v) and an optimum at 7.0-8.5% (w/v). The isolate was mesophilic and grew at temperatures from 25 to 45 °C, with an optimum at 40 °C. An H(2)+CO(2) mixture, ethanol, pyruvate and lactate were utilized with the formation of acetate as the sole metabolic product. Carbohydrates and amino acids did not support growth. The isolate had a respiratory type of metabolism, reducing NO3(-), SeO(4)(2-) or anthraquinone-2,6-disulfonate (as electron acceptors with ethanol as an electron donor). It was able to grow chemolithotrophically on H(2)+CO(2) in medium supplemented with a vitamin solution only. The major cellular fatty acids were the saturated fatty acids anteiso-C(15), C(14:0) and C(16:0) and the aldehydes C(16), C(14) and anteiso-C(15). The DNA G+C content of the isolate was 32.0 mol%. 16S rRNA gene sequence analysis showed that strain Z-7100(T) is a member of the order Halanaerobiales and represents a new branch within the family Halobacteroidaceae, clustering with the type strain of Selenihalanaerobacter shriftii (92.9% gene sequence similarity). On the basis of its physiological characteristics and phylogenetic position, the isolate is considered to represent a novel species in a new genus within the family Halobacteroidaceae. The name Fuchsiella alkaliacetigena gen. nov., sp. nov. is proposed. The type strain of the type species is Z-7100(T) (=DSM 24880(T)=VKM B-2667(T)).

Natronincola ferrireducens sp. nov., and Natronincola peptidovorans sp. nov., new anaerobic alkaliphilic peptolytic iron-reducing bacteria isolated from soda lakes

Two novel strains of obligately alkaliphilic (pH 7.5–10.2, optimum pH 8.4–8.8) anaerobic spore-forming rod-shaped bacteria, Z-0511 and Z-7031, were isolated from enrichment cultures obtained from the iron-reducing (Lake Khadyn, Tyva) and cellulolytic (Lake Verkhnee Beloe, Buryatia) bacterial communities, respectively. The organisms ferment peptides and do not ferment proteins and amino acids, with the exception of histidine and glutamate utilized by strain Z-0511. The major fermentation products were acetate and propionate for strain Z-0511 and formate and acetate for strain Z-7031, respectively. Carbohydrates and fermentable organic acids could not serve as substrates, except for pyruvate in the case of strain Z-7031. Nitrogen and sulfur compounds were not utilized as electron acceptors by the strains grown on medium with yeast extract. Strain Z-0511 utilized fumarate, crotonate, and EDTA-Fe(III) as electron acceptors. Anthraquinone-2,6-disulfonate (quinone) and Mn(IV) were utilized by both strains, as well as amorphous ferric hydroxide (AFH), which was reduced to iron sesquioxides and magnetite. The presence of AFH stimulated growth; it enhanced the yield of the fermentation products and changed the quantitative ratios of these products. According to a phylogenetic analysis of the 16S rRNA gene sequences and the phenotypic characteristics of the new strains, they were classified as new species of the genus Natronincola, Natronincola ferrireducens sp. nov. Z-0511T (= VKM B-2402, = DSM 18346) and Natronincola peptidovorans sp. nov. Z-7031T (= VKM B-2503, = DSM 18979).

[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-7105T, 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 Clions 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-7105T and the Methanocalculus type species M. halotolerans DSM 14092T 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-7105T (=DSM 25006T, =VKM B-2765T) to the new species Methanocalculus natronophilus sp. nov., and to incorporate the genus into the new family Methanocalculaceae fam. nov.

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).