Analysis Of Bacterial Diversity In Yuncheng Salt Lake And Polyphasic Taxonomy Of Four Novel Halophilic Bacteria

Unique physiological characteristics and metabolic mechanisms different from terrestrial microbes are possessed by many halophilic bacteria,which produce special active substances.Therefore,isolation and the study of halophilic mechanism and active substances of halophilic bacteria are of special significance.The Yuncheng salt lake,Shanxi,is one of the three largest sodium sulfate type inland saline lake in the world.High salinity content,as well as the unique salts composition different from both the common saline lakes and the oceans made its microbial diversity and community structure a new research significance.In this paper,the samples of Saline Lake sediments were collected from the different lakes of Yuncheng salt lake,Shanxi.Through the direct spreading method and the enrichment culture and spreading method,the cultured microbes were separated on the normal medium and the oligotrophic medium respectively.200 strains of bacteria were isolated,which were mainly distributed in four phylum of Proteobacteria,Firmicutes,Bacteroides and Actinobacteria,including 29 genera and 58 species.The effects of normal medium and oligotrophic medium,direct spreading and oligotrophic medium spreading on the isolation of culturable microorganism were compared.It was found that in the direct spreading the oligotrophic medium had higher microbial diversity compared with the normal medium,and the dominant species were similar.Compared with oligotrophic spreading direct spreading method had higher microbial diversity,and there were obvious differences in species distribution.3 potential new genera and 13 potential new species were isolated in the separation process.At the same time,the original and enriched samples including each three of mud samples,15 days of enrichment,30 days of enrichment and corresponding water samples which amounts to 12 groups were sequenced by macrogenome.The species and distribution of bacteria in the sample and the succession of microbial diversity during the enrichment process were analyzed.The results showed that Proteobacteria,Firmicutes,Bacteroides and Actinobacteria were the major components in all the samples.The microbial diversity,all measured by both Shannon and simpson index,were compared.Diversity of sample 1 decreased after enrichment,while that of samples 2 and 3 increased after enrichment.After oligotrophic enrichment,the abundance of Firmicutes and Actinobacteria increased significantly,while the Bacteroides decreased slightly and Proteobacteria without significant change.Oligotrophic enrichment is different from previous microbial flora.After enrichment,the abundance of some rare species increased significantly.A total of nine samples were clustered in the original sample and the enriched sample.The clustering of the.original samples were clustered closer,and the enriched samples were clusterd neighborhood.The results showed that oligotrophic enrichment has a significant influence on microbial diversity.Enrichment factor is an important factor affecting the diversity of diversity,and the enrichment process has great influence on Y1 and Y3 samples,and has little influence on Y2 samples.In the process of enrichment,the number of unique OTU decreased obviously after enrichment,indicating that the enrichment process may play a role in the process of culture of bacteria which were rare and difficult to be cultured.Four halophilic bacterial strains were identified by polyphasic study.A Gram-negative,non-motile,rod-shaped,orange-yellow pigmented strain,WDS4A13T,was isolated from a salt mine in Weihai,China.WDS4A13T grows with an optimal PH at 7.0-8.0,33?,with 6%(w/v)NaCl.The polar lipid profile of the novel isolate consisted of diphosphatidylglycerol,phosphatidylethanolamine,glycolipid and an unidentified phospholipid.The predominant menaquinone is MK-6 and anteiso-C15:0,iso-C15:and iso-C16:0 3-OH was the major fatty acids.The DNA G+C content was 35.2 mol%.Phylogenetic analysis based on 16S rRNA gene sequences showed that strain WDS4A13T belonged to the genus Psychroflexus.The levels of similarity between the 16S rRNA gene sequence of strain WDS4A13T and those of the type strains of recognized Psychroflexus species were 92-97%.On the basis of phenotypic,chemotaxonomic and phylogenetic data,strain WDS4A13T represents a novel species within the genus Psychroflexus,for which the name Psychroflexus saliphila sp.nov.is proposed.The type strain is WDS4A13T(=MCCC 1H00134T =KCTC 52043T).A Gram-stain-negative,non-motile,rod-shaped,orange-red pigmented strain,WDS2C27T,was isolated from a marine solar saltern in Weihai,China.WDS2C27T grows with an optimal pH at 7.0-8.0,33?,with 6%(w/v)NaCl.The polar lipid profile of the novel isolate consisted of two unidentified phospholipids(PL1,PL2),diphosphatidylglycerol(DPG),phosphatidylethanolamine(PE)as the major polar lipids.The predominant menaquinone is MK-6 and iso-C15:0,anteiso-C15:0 are the major fatty acids.The DNA G+C content is 35.6 mol%.The levels of similarity between the 16S rRNA gene sequence of strain WDS2C27T and those of the type strains of recognized Psychroflexus species are 91.99-91.13%,of which P.planctonicus,P.aestuariivivens,P.salis shares the similarity of 91.99%,91.40%,91.29%respectively.Phylogenetic analysis based on 16S rRNA gene sequence placed WDS2C27T within the genus Psychroflexus.On the basis of phenotypic,chemotaxonomic and phylogenetic data,strain WDS2C27T represents a novel species,for which the name Psychroflexus halotolerans sp.nov.is proposed.The type strain is WDS2C27T(=MCCC 1H00133T=KCTC 52044T).Strain Y17T was isolated from Yuncheng salt lake,Shanxi province.Aerobic,gram-negative,and endospore forming.Y17T cells were motile,and colonies were colorless and translucent.The optimum growth temperature is 30 °C(growth range 20-40?),optimum salinity 8%(w/v)(salinity growth range 4-20%,w/v),and optimum pH7(pH growth range 6.5-8.5).According to 16SrDNA sequence analysis,Y17T was most similar to the strain of cold Campylobacter(91.1-92.0%).Phylogenetic analysis and physiological and biochemical characteristics indicate that Y17T represents a new genus,which is proposed to be named as Halospirillum yunchengensis.Strain Y22T was isolated from the Yuncheng salt lake,Shanxi province.Aerobic,gram-negative bacilli,and endospore forming.Cells of Y22T were motile,and white colonies were formed.The optimum growth temperature was 30?(range 20-40?),and the optimum salinity was 10%(w/v)(salt).