Natronospira proteinivora gen. nov., sp. nov, an extremely salttolerant, alkaliphilic gammaproteobacterium from hypersaline soda lakes

Brine samples from Kulunda Steppe soda lakes (Altai, Russia) were inoculated into a hypersaline alkaline mineral medium with β-keratin (chicken feather) as a substrate. The micro-organisms dominating the enrichment culture were isolated by limiting serial dilution on the same medium with casein as a substrate. The cells of strain BSker1T were motile, curved rods. The strain was an obligately aerobic heterotroph utilizing proteins and peptides as growth substrates. The isolate was an obligate alkaliphile with a pH range for growth from pH 8.5 to 10.25 (optimum at pH 9.5), and it was extremely salt tolerant, growing with between 1 and 4.5 M total Na+ (optimally at 2–2.5 M). BSker1T had a unique composition of polar lipid fatty acids, dominated by two C17 species. The membrane polar lipids included multiple unidentified phospholipids and two aminolipids. According to phylogenetic analysis of the 16S rRNA gene sequence, the isolate forms a novel branch within the family Ectothiorhodospiraceae (class Gammaproteobacteria) with the highest sequence similarity to the members of this family being 91 %. On the basis of distinct phenotypic and genotypic properties, strain BSker1T (=JCM 31341T=UNIQEM U1008T) is proposed to be classified as a representative of a novel genus and species, Natronospira proteinivora gen. nov., sp. nov.Accepted Author ManuscriptBT/Environmental Biotechnolog

Selective enrichment on a wide polylsaccharide spectrum allowed isolation of novel metabolic and taxonomic groups of haloarchaea from hypersaline lakes

Extremely halophilic archaea (haloarchaea) of the class Halobacteria isa dominant group of aerobic heterotrophic prokaryotic communities insalt-saturated habitats, such as salt lakes and solar salterns. Most of thepure cultures of haloarchaea were enriched, isolated, and cultivated onrich soluble substrates such as amino acids, peptides or simple sugars.So far, the evidences on the capability of haloarchaea to use differentpolysaccharides as growth substrates remained scarce. However, it isbecoming increasingly obvious that these archaea can also actively participatein mineralization of complex biopolymers, in particular cellulose and chitin–two dominant biomass polysaccharides on the planet. Here we used anarray of commercially available homo- and heteropolysaccharides to enrichhydrolytic haloarchaea from hypersaline salt lakes with neutral pH and fromalkaline soda lakes. This resulted in isolation of a range of halo- and natronoarchaea, respectively, belonging to already described taxa as well as several new genus-level lineages. In some cases, the isolates enriched with different polysaccharides happened to be closely related, thus representing generalistic ecotype, while the others were narrow specialists. In general, soda lakes yielded a broader range of polysaccharide-utilizing specialists in comparison to neutral salt lakes. The results demonstrated a significant diversity of halo(natrono)archaea with a previously unrecognized potential for utilizationof a broad range of natural polysaccharides in hypersaline habitats.BT/Environmental Biotechnolog

Diversity of cultivated aerobic poly-hydrolytic bacteria in saline alkaline soils

Alkaline saline soils, known also as ''soda solonchaks'', represent a natural soda habitat which differs from soda lake sediments by higher aeration and lower humidity. The microbiology of soda soils, in contrast to the more intensively studied soda lakes, remains poorly explored. In this work we investigate the diversity of culturable aerobic haloalkalitolerant bacteria with various hydrolytic activities from soda soils at different locations in Central Asia, Africa, and North America. In total, 179 pure cultures were obtained by using media with various polymers at pH 10 and 0.6 M total Na+. According to the 16S rRNA gene sequence analysis, most of the isolates belonged to Firmicutes and Actinobacteria. Most isolates possessed multiple hydrolytic activities, including endoglucanase, xylanase, amylase and protease. The pH profiling of selected representatives of actinobacteria and endospore-forming bacteria showed, that the former were facultative alkaliphiles, while the latter were mostly obligate alkaliphiles. The hydrolases of selected representatives from both groups were active at a broad pH range from six to 11. Overall, this work demonstrates the presence of a rich hydrolytic bacterial community in soda soils which might be explored further for production of haloalkalistable hydrolases.BT/Environmental Biotechnolog

Natronotalea proteinilytica gen. nov., sp. nov. and Longimonas haloalkaliphila sp. nov., extremely haloalkaliphilic members of the phylum Rhodothermaeota from hypersaline alkaline lakes

Two proteolytic bacterial strains, BSker2Tand BSker3T, were enriched from sediments of hypersaline alkaline lakes in Kulunda Steppe (Altai, Russia) with chicken feathers as substrate, followed by pure culture isolation on hypersaline alkaline media with casein. The cells were non-motile, filamentous, flexible rods. The isolates were obligately aerobic heterotrophs utilizing proteins and peptides as growth substrates. Both were obligate alkaliphiles, but differed in their pH optimum for growth: pH 9.5-9.8 for Bsker2Tand pH 8.5-8.8 for BSker3T. The salt range for growth of both isolates was between 2 and 4.5 M total Na+ with an optimum at 2.5-3 M. No organic osmolytes were detected in cells of BSker2T, but they accumulated high intracellular concentrations of K+. The polar lipid fatty acids were dominated by unsaturated C16 and C18 species. The 16S rRNA gene phylogeny indicated that both strains belong to the recently proposed phylum Rhodothermaeota. BSker2Tforms a novel genus-level branch, while BSker3Trepresents a novel species-level member in the genus Longimonas. On the basis of distinct phenotypic and genotypic properties, strain BSker2T(=JCM 31342T=UNIQEM U1009T) is proposed to be classified as a representative of a novel genus and species, Natronotalea proteinilyticagen. nov., sp. nov., and strain BSker3T(=JCM 31343T=UNIQEM U1010T) as a representative of a novel species, Longimonas haloalkaliphila sp. nov.Accepted Author ManuscriptBT/Environmental Biotechnolog

Natronosporangium hydrolyticum gen. nov., sp. nov., a haloalkaliphilic polyhydrolytic actinobacterium from a soda solonchak soil in Central Asia

During a cultural diversity survey on hydrolytic bacteria in saline alkaline soils, a hydrolytic actinobacterium strain ACPA39T was enriched and isolated in pure culture from a soda solonchak soil in southwestern Siberia. It forms a substrate mycelium with rod-shaped sporangia containing 1–3 exospores. The isolate is obligately alkaliphilic, growing at pH 7.5–10.3 (optimum at 8.5–9.0) and moderately halophilic, tolerating up to 3 M total Na+ in the form of sodium carbonates. It is an obligately aerobic, organoheteroterophic, saccharolytic bacterium, utilizing various sugars and alpha/beta-glucans as growth substrates. According to the 16S rRNA gene-based phylogenetic analysis, strain ACPA39T forms a distinct branch within the family Micromonosporaceae, with the sequence identities below 94.5% with type strains of other genera. This is confirmed by phylogenomic analysis based on the 120 conserved single copy protein-based markers and genomic indexes (ANI, AAI). The cell-wall of ACPA39T contained meso-DAP, glycine, glutamic acid and alanine in a equimolar ratio, characteristic of the peptidoglycan type A1γ'. The whole-cell sugars include galactose and xylose. The major menaquinone is MK-10(H4). The identified polar lipids consist of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. The polar lipid fatty acids were dominated by anteiso-C17:0, iso-C16:0, iso-C17:0, 10 Me-C18:0 and C18:1ω9. Based on the distinct phylogeny, the chemotaxonomy features and unique phenotypic properties, strain ACPA39T (DSM 106523T = VKM 2772T) is classified into a new genus and species in the family Micromonosporaceae for which the name Natronosporangium hydrolitycum gen. nov., sp. nov. is proposed.BT/Environmental Biotechnolog

Halococcoides cellulosivorans gen. nov., sp. nov., an extremely halophilic cellulose-utilizing haloarchaeon from hypersaline lakes

An extremely halophilic euryarchaeon, strain HArcel1T, was enriched and isolated in pure culture from the surface brines and sediments of hypersaline athalassic lakes in the Kulunda Steppe (Altai region, Russia) using amorphous cellulose as the growth substrate. The colonies of HArcel1T are pale-orange, and form large zones of cellulose hydrolysis around them. The cells are non-motile cocci of variable size with a thin monolayer cell wall. The isolate is an obligate aerobic heterotroph capable of growth with only three substrates: various forms of insoluble cellulose, xylan and cellobiose. Strain HArcel1T is an extremely halophilic neutrophile, growing within the salinity range from 2.5 to 5 M NaCl (optimum at 3.5-4 M). The core archaeal lipids are dominated by C20-C20 and C25-C20 dialkyl glycerol ethers, in approximately 6:1 proportion. The 16S rRNA and rpoB' gene analysis indicated that HArcel1T forms a separate lineage within the family Haloarculaceae, order Halobacteriales, with the genera Halorhabdus and Halopricus as closest relatives. On the basis of the unique phenotypic properties and distinct phylogeny of the 16S rRNA and rpoB' genes, it is suggested that strain HArcel1T is classified into a new genus and species Halococcoides cellulosivorans gen. nov., sp. nov. (JCM 31941T=UNIQEM U975T).Accepted Author ManuscriptBT/Environmental Biotechnolog