Ekaterina N. Detkova

Culturable diversity of lithotrophic haloalkaliphilic sulfate-reducing bacteria in soda lakes and the description of Desulfonatronum thioautotrophicum sp. nov., Desulfonatronum thiosulfatophilum sp. nov., Desulfonatronovibrio thiodismutans sp. nov., and Desulfonatronovibrio magnus sp. nov.

Soda lake sediments usually contain high concentrations of sulfide indicating active sulfate reduction. Monitoring of sulfate-reducing bacteria (SRB) in soda lakes demonstrated a dominance of two groups of culturable SRB belonging to the order Desulfovibrionales specialized in utilization of inorganic electron donors, such as formate, H2 and thiosulfate. The most interesting physiological trait of the novel haloalkaliphilic SRB isolates was their ability to grow lithotrophically by dismutation of thiosulfate and sulfite. All isolates were obligately alkaliphilic with a pH optimum at 9.5–10 and moderately salt tolerant. Among the fifteen newly isolated strains, four belonged to the genus Desulfonatronum and the others to the genus Desulfonatronovibrio. None of the isolates were closely related to previously described species of these genera. On the basis of phylogenetic, genotypic and phenotypic characterization of the novel soda lake SRB isolates, two novel species each in the genera Desulfonatronum and Desulfonatronovibrio are proposed.

Natroniella acetigena gen. nov. sp. nov., an extremely haloalkaliphilic, homoacetic bacterium : A new member of Haloanaerobiales

Abstract. A new extremely haloalkaliphilic, chemoorganotrophic, homoacetogenic bacterium strain Z-7937T(T-type strain) was isolated from the bottom mud of the soda-depositing Lake Magadi, Kenya. It is an obligately anaerobic, motile, Gram-negative, spore-forming rod growing in the pH range pH 8.1 to 10.7 and optimally in the range pH 9.7 to 10.0 under conditions of high alkalinity caused by saturation with trona. It has an obligate requirement for sodium carbonate and chloride ions. The optimum salt concentration for growth is in the range 12–15% wt/vol, and growth occurs within the range from 10% to 26%. Strain Z-7937T is a mesophile with an optimal temperature for growth of 37°C, and a maximum of 42°C. The G + C content of strain Z-7937T is 31.9 mol%. A limited number of compounds are utilized, including lactate, ethanol, pyruvate, glutamate, and propanol. Acetate is the main end product. 16S rDNA sequence analysis shows strain Z-7937T to be a member of the order Haloanaerobiales and to represent a new branch within the family Halobacteroidaceae. On the basis of its novel physiology and phylogenetic position, we propose strain Z-7937 as a new species of a new genus, Natroniella acetigena gen. nov. sp. nov. The type strain is Z-7937T (= DSM 9952).

Natronoincola histidinovorans gen. nov., sp. nov., A new alkaliphilic acetogenic anaerobe.

Abstract. Two strains, asporogenous Z-7940 and sporogenous Z-7939, of a moderately haloalkaliphilic, obligately anaerobic, fermentative bacteria, motile, with Gram-positive cell wall structure, were isolated from soda deposits in Lake Magadi, Kenya. Both strains are mesophilic and utilize only two amino acids, histidine and glutamate, with formation of acetate and ammonium as the main end products. Strain Z-7939 in addition is able to utilize pyruvate. DNA-DNA homology between strains Z-7940 and Z-7939 was 94%, indicating that in spite of phenotypic differences they belong to the same species. They are true alkaliphiles with a pH range for growth of the type strain Z-7940 from pH 8.0 to pH 10.5, optimum at pH 9.4. Both strains obligately depend on sodium and bicarbonate ions. The optimum salt concentration for growth of the type strain is 8–10% wt/vol and the range from 4% to 16%. The G+C content of strain Z-7940 is 31.9 mol% and the strain Z-7939 is 32.3 mol%. Analysis of 16S rDNA sequence of the type strain shows it to belong to cluster XI of the low G+C Gram-positive bacteria. On the basis of its distinct phylogenetic position and physiological properties, we propose a new genus and new species Natronoincola histidinovorans for these strains. The type strain is Z-7940 (=DSM 11416).

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.

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)).

Larkinella arboricola sp. nov., a new spiral-shaped bacterium of the phylum Bacteroidetes isolated from the microbial community of decomposing wood.

An aerobic gram-negative bacterial strain Z-0532 with ring-shaped cells forming spirals in the course of growth was isolated from the humified solution produced by spruce wood decomposition. The new isolate was a chemoorganotrophic, mesophilic, moderately acidophilic organism with the temperature range of 6–32°C (optimum at 25–28°C) and pH range from 4.7 to 7.2 (optimum at pH 5.5–6.5). A broad range of substrates was used as carbon and energy sources, including sugars, some organic acids and polyalcohols, and soluble polymeric compounds (gelatin, esculin, starch, xylan, laminarin, dextrin, casein hydrolysate, and Tween-40). According to its physiological and biochemical characteristics, strain Z-0532 is a typical member of the trophic group of oligotrophic bacteria, which utilize the products of wood hydrolysis dissipated by xylotrophic microorganisms. The G+C base content of strain Z-0532 was 52.1 mol %. Sequencing of the 16S rRNA gene of the new isolate revealed 98% similarity to Larkinella insperata LMG 22510T, which is a recently described species of the family Spirosomaceae of the phylum Bacteroidetes. The level of DNA: DNA homology between this species and strain Z-0532 was only 40%. The differences in the phenotypic and genotypic characteristics suggested classification of the isolate obtained from decomposing wood as a new species of the genus Larkinella, Larkinella arboricola sp. nov., with the type strain Z-0532T (=VKM B-2528T = DSM 21851T).

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).

Anaerobic halo- alkaliphilic bacterial community of athalassic, hypersaline Mono lake and Owens Lake in California

The bacterial diversity of microbial extremophiles from the meromictic, hypersaline Mono Lake and a small evaporite pool in Owens Lake of California was studied. In spite of these regions had differing mineral background and different concentrations of NaCl in water they contain the same halo- alkaliphiles anaerobic bacterial community. Three new species of bacteria were detected in this community: primary anaerobe, dissipotrophic saccharolytic spirochete Spirochaeta americana strain AspG1T, primary anaerobe which is proteolytic Tindallia californiensis strain APOT, and secondary anaerobe, hydrogen using Desulfonatronum thiodismutans strain MLF1T, which is sulfate- reducer with chemo-litho-autotrophic metabolism. All of these bacteria are obligate alkaliphiles and dependent upon Na+ ions and CO32- ions in growth mediums. It is interesting that closest relationships for two of these species were isolates from samples of equatorial African soda Magadi lake: Spirochaeta americana AspG1T has 99.4% similarity on 16S rDNA- analyses with Spirochaeta alkalica Z- 7491T, and Tindallia californiensis APOT has 99.1% similarity with Tindallia magadiensis Z-7934T. But result of DNA-DNA- hybridization demonstrated less then 50% similarity between Spirochaeta americana AspG1T and Spirochaeta alkalica Z-7491T. Percent of homology between Tindallia californiensis APOT and Tindallia magadiensis Z-7934T is only 55%. The sulfate-reducer from the alkalic anaerobic community of Magadi lake Desulfonatronovibrio hydrogenovorans Z-7935T was phylogenetically distant from this sulfate-reducer in Mono lake, but genetically closer (99.7% similarity) to the sulfate-reducer, isolated from Central Asian alkalic lake Khadyn in Siberia Desulfonatronum lacustre Z-7951T. The study of key enzymes (hydrogenase and CO- hydrogenase) in Tindallia californiensis APOT and Desulfonatronum thiodismutans MLF1T showed the presence of high activity of both the enzymes in first and only hydrogenase in second isolate. These results indicate that the athalassic, soda Mono and Owens lakes contain an anaerobic alkaliphilic bacterial community with common ancestors of geographically distant regions, but very same in morphological and functional aspects. As suggested Zavarzin, halo- alkaliphiles may represent relics of microbial life from the early formation of the ancient Earth before the Neoproterozoic period.