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.

Natronogracilivirga saccharolytica gen. nov., sp. nov. and Cyclonatronum proteinivorum gen. nov., sp. nov., haloalkaliphilic organotrophic bacteroidetes from hypersaline soda lakes forming a new family Cyclonatronaceae fam. nov. in the order Balneolales.

Two heterotrophic bacteroidetes strains were isolated as satellites from autotrophic enrichments inoculated with samples from hypersaline soda lakes in southwestern Siberia. Strain Z-1702T is an obligate anaerobic fermentative saccharolytic bacterium from an iron-reducing enrichment culture, while Ca. Cyclonatronum proteinivorum OmegaT is an obligate aerobic proteolytic microorganism from a cyanobacterial enrichment. Cells of isolated bacteria are characterized by highly variable morphology. Both strains are chloride-independent moderate salt-tolerant obligate alkaliphiles and mesophiles. Strain Z-1702T ferments glucose, maltose, fructose, mannose, sorbose, galactose, cellobiose, N-acetyl-glucosamine and alpha-glucans, including starch, glycogen, dextrin, and pullulan. Strain OmegaT is strictly proteolytic utilizing a range of proteins and peptones. The main polar lipid fatty acid in both strains is iso-C15:0, while other major components are various C16 and C17 isomers. According to pairwise sequence alignments using BLAST Gracilimonas was the nearest cultured relative to both strains (<90% of 16S rRNA gene sequence identity). Phylogenetic analysis placed strain Z-1702T and strain OmegaT as two different genera in a deep-branching clade of the new family level within the order Balneolales with genus. Based on physiological characteristics and phylogenetic position of strain Z-1702T it was proposed to represent a novel genus and species Natronogracilivirga saccharolityca gen. nov., sp. nov. (= DSMZ 109061T =JCM 32930T =VKM B 3262T). Furthermore, phylogenetic and phenotypic parameters of N. saccharolityca and C. proteinivorum gen. nov., sp. nov., strain OmegaT (=JCM 31662T, =UNIQEM U979T), make it possible to include them into a new family with a proposed designation Cyclonatronaceae fam. nov..

Metabolic versatility of haloalkaliphilic bacteria from soda lakes belonging to the Alkalispirillum-Alkalilimnicola group.

Four new isolates were obtained from denitrifying enrichments with various electron donors using sediment samples from hypersaline soda lakes. Based on 16S rRNA gene analysis and DNA-DNA hybridization results, they were all identified as members of the Gammaproteobacteria closely associated with the Alkalispirillum-Alkalilimnicola group. Two isolates were obtained from samples enriched with nitrate as electron acceptor and H2 or polysulfide as electron donors, and another two strains were obtained with N2O as the electron acceptor and sulfide or acetate as electron donors. All four new isolates, together with the type strains of the genera Alkalispirillum and Alkalilimnicola originally described as obligate aerobes, were capable of anaerobic growth with acetate using either nitrate or N2O as electron acceptors. Their denitrification pathway, however, was disrupted at the level of nitrite. RuBisCO form I gene was detected and sequenced in the new isolates and in Alkalilimnicola halodurans but not in Alkalispirillum mobile. These data, together with the evidence of Oremland et al. (Appl Environ Microbiol 68:4795-4802, 2002) on the potential of Alkalilimnicola sp. MLHE-1 for autotrophic growth with arsenite as electron donor and nitrate as electron acceptor, demonstrate much higher metabolic diversity of this specific group of haloalkaliphilic Gammaproteobacteria than was originally anticipated.

Desulfonatronum cooperativum sp. nov., a novel hydrogenotrophic, alkaliphilic, sulfate-reducing bacterium, from a syntrophic culture growing on acetate.

A novel alkaliphilic, sulfate-reducing bacterium was isolated from a syntrophic acetate-decomposing community enriched from samples of the soda lake Khadin, Tuva, Russia; the isolate was designated strain Z-7999(T). Cells of strain Z-7999(T) were vibrioid, Gram-negative, 0.4-0.5 x 1.0-2.5 microm and motile by means of a polar flagellum. The temperature range for growth was 15-40 degrees C, with an optimum of 35-38 degrees C. The pH range for growth was 6.7-10.3, with an optimum of pH 8.0-9.0. The NaCl concentration range for growth was 1-80 g l(-1). The novel isolate was obligately anaerobic, was alkaliphilic with a broad pH range and had an obligate requirement for carbonate ions in the growth medium. In the presence of sulfate as electron acceptor, it grew with hydrogen, formate and lactate. It was not able to ferment sugars, organic acids, amino acids or peptides. During growth on formate, strain Z-7999(T) reduced sulfite and thiosulfate to sulfide. It was able to grow lithoheterotrophically with sulfate and formate when acetate was added as a carbon source for biosynthesis of biomass. The G + C content of the genomic DNA of strain Z-7999(T) was 56.5 mol%. Results of comparative 16S rRNA gene sequence analyses revealed that strain Z-7999(T) was part of the delta-Proteobacteria and clustered with other members of the genus Desulfonatronum (similarity values of 95.2 and 95.3 % to Desulfonatronum lacustre and Desulfonatronum thiodismutans, respectively). DNA-DNA hybridization with D. lacustre was 37 %. On the basis of physiological and phylogenetic data, it is proposed that strain Z-7999(T) (= DSM 16749(T) = VKM B-2329(T)) should be placed in the genus Desulfonatronum as a representative of a novel species, Desulfonatronum cooperativum sp. nov.

Anaerobic, alkaliphilic, saccharolytic bacterium Alkalibacter saccharofermentans gen. nov., sp. nov. from a soda lake in the Transbaikal region of Russia.

Three strains of new obligately anaerobic alkaliphilic bacteria have been isolated as a saccharolytic component from the cellulolytic community of alkaline Lake Nizhnee Beloe (Transbaikal region, Russia), a lake with low salt concentration. DNA analysis of these strains showed an interspecies level of DNA similarity of 96-100%. Strain Z-79820 was selected for further investigations. Cells were Gram-positive, asporogenous, nonmotile short rods with pointed ends. The strain was a true alkaliphile: growth occurred from pH 7.2 to 10.2 with the optimum at pH 9.0. Strain Z-79820 was halotolerant and could grow in medium with up to 10% (w/v) NaCl, with the optimum between 0 and 4% NaCl. The new isolate obligately depended on Na+ ions in the form of carbonates or chlorides. Total Na+ content needed for optimal growth was 0.46 M Na+, with a wide range from 0.023-0.9 M Na+ at which growth also occurred. The isolate was a mesophile and grew at temperatures from 6 to 50 degrees C (slow growth at 6 and 15 degrees C) with an optimum at 35 degrees C. The organotrophic organism fermented ribose, xylose, glucose, mannose, fructose, sucrose, mannitol, and peptone. The products of glucose fermentation were acetate, ethanol, formate, H2, and CO2. Yeast extract was required for some anabolic needs. The DNA G+C content of the type strain Z-79820 was 42.1 mol%. The new bacterium fell into the 16S rRNA gene cluster XV of the Gram-positive bacteria with low G+C content, where it formed an individual branch. Based on its growth characteristics and genotype traits, we propose the new genus and species named Alkalibacter saccharofermentans with the type strain Z-79820 (=DSM14828), Uniqem-218 (Institute Microbiology, RAS; http://inmi.da.ru).

Anoxynatronum sibiricum gen.nov., sp.nov. alkaliphilic saccharolytic anaerobe from cellulolytic community of Nizhnee Beloe (Transbaikal region).

New alkaliphilic anaerobic fermentative bacteria, strains Z-7981 and Z-7981', with Gram-positive cell walls, were isolated from the cellulolytic community from the soda lake Nizhnee Beloye, south-east of Baikal. Cells were motile rods, which differed in dimensions but, according to 98% DNA/DNA homology, belonged to the same species. Strain Z-7981 was chosen as the type and studied in detail. It did not produce spores and its cells were non-thermoresistant. It was a true alkaliphile with a growth range from pH 7.1 to pH 10.1 and optimal pH for growth at pH 9.1. It was obligately dependent on Na(+) and carbonate ions but not on Cl(-). Growth occurred in media with total sodium content from 0.076 M to 1.27 M Na(+ )with a broad optimum from 0.25 to 0.86 M Na(+). Growth showed an optimum at 35 degrees C, with absence of growth above 46 degrees C. The organism was aerotolerant and was capable of fermentation in non-reducing medium at less than 4.75% O(2) in the gas phase. Strain Z-7981 fermented mono- and disaccharides, sugar alcohols, but only glutamate and cysteine among the amino acids, and the proteinaceous substrates, chitin and dried Spirulina biomass. Fermentation products were acetate and ethanol. Fe(3+) was reduced in a process that yielded no energy. Phylogenetically the new organism belonged to cluster XI of the Gram-positive bacteria with low G+C content and its closest neighboring taxon was Tindallia magadiensis. However, according to its phenotypic and genotypic characters it did not belong to any known genus from this group. We suggest a new genus and species with the name Anoxynatronum sibiricum and strain Z-7981 as its type (=DSM15060).