Biodiversity And Polyphasic Taxonomy Of Culturable Halophilic Bacteria And Archaea From Saline-Alkaline Soil In Daqing

Because of the unique physiological structure, hereditary property and metabolic mechanism of halophilic microorganisms, they have attracted widespread attention of scholars at home and abroad. They are a kind of microbiotic resource with value of academic research and utilization. Exploitation of new halophilic microorganisms is beneficial for revealing the mechanisms of salt tolerance, getting new salt tolerance related genes, improving saline lands and so on. Our country with vast territory and long coastline has large saline lands and a lot of salt lake, which provide convenient condition for the isolation and genealogical classification of halophilic microorganisms.In this study, some typical soda salt-alkaline soil samples were collected in Daqing, Heilongjiang Province. Forty seven moderately halophilic bacterial strains and ten extremely halophilic archaeal strains isolated by enrichment culture were collected from surface soil sample (0~15cm). Pure cultures were then obtained by repeated streaking. Phylogenetic analysis based on 16S rDNA gene sequences revealed that moderately halophilic bacterial strains were determined to belong to the genera Alkalibacillus, Bacillus and Halomonas while extremely halophilic archaeal strains belong to the genus Haloterrigena.Strain 15-13T and JX313T were new species candidates of the genus Halomonas and Haloterrigena, respectively. In this paper, we describe the taxonomic status of the two strains by polyphasic technology including morphological, physiological, chemotaxonomic and genotypic analyses. The results showed that the strain 15-13T were moderately halophilic, Gram-negative, aerobic, short rods with several lateral flagella. It could growth at the range of 1~23% (w/v) NaCl (optimum 7%), pH7.0~11.0 (optimum pH9.5) and 20~50oC (optimum 35oC). The major fatty acids (greater than 10%) are C18:1ω7c (60.48%) and C16:0 (13.96%). The DNA G+C content of the type strain is 67.6 mol% (Tm method). Phylogeny of the 16S rRNA gene sequences revealed that the strain 15-13T was a member of the family Halomonadaceae and had a close phylogenetic relationship with the species of the genus Halomonas. The closest phylogenetic neighbour of strain 15-13T was Halomonas pantelleriensis DSM 9661T (98.9% 16S rRNA gene sequence identity). DNA-DNA hybridization percentage between the novel isolated strain and Halomonas pantelleriensis DSM 9661T was 33.8%. Phenotypic features, fatty acid profile and genotypic characterizations of the novel strain supported further its status in the genus Halomonas. On the basis of these data, it suggested that strain 15-13T is a novel species of the genus Halomonas, and the name Halomonas alkalitolerans sp. nov. was proposed. The type strain is 15-13T (=CGMCC 1.9129T =NBRC 106539T).The cells of the extremely halophilic archaeal strain JX313T were coccoid, Gram-negative, non-motile and were not lysised in distilled water. Colonies on agar medium after incubation at 37oC for 7 days turn light red-pigmented. It could growth at the range of NaCl concentrations 10%–saturated solution (w/v) (optimum 17.5%), pH8.0~11.0 (optimum pH10.0) and 20~50oC (optimum 35oC). The DNA G+C content of the type strain is 59.3 mol% (Tm method). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain JX313T was associated with the genera Haloterrigena and Natrinema and is the most closely related to the species Haloterrigena salina JCM 13891T (96.2% sequence similarity) and Haloterrigena hispanica DSM 18328T (96.2%). DNA–DNA hybridization experiments revealed that the hybridization percentage of strain JX313T were less than 50% with related species of the genus Haloterrigena or Natrinema. Furthermore, the cellular polar lipids of strain JX313T, which identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and mannose-2,6-disulfate (1→2)-glucose glycerol diether (S2-DGD), was consistent with the characteristics of polar lipid of genus Haloterrigena. Therefore, phylogenetic analysis, phenotypic assessment and chemotaxonomic data showed that JX313T is a novel species of the genus Haloterrigena, and the name Haloterrigena daqingensis sp. nov. was proposed. The type strain is JX313T (=CGMCC 1.8909T =NBRC 105739T). This description of the genus Haloterrigena was modified based on the taxonomic studies of strain JX313T.