Genetic Diversity And Enzymology Properties Of Culturable Halophiles In Daishan Solar Saltern

This study was aimed at investigating the diversity of culturable halophiles in Daishan Saltworks, a hypersaline environment, and discovering new strains with enzyme-producing activities. A total of181strains were isolated from soil and saline water collected from the Daishan Saltworks by culture-dependent method based on selective media. Specific primers were used to amplify16S rRNA gene of bacteria and archaea. The operation taxonomy units (OTUs) were determined by ARDRA method and the representative strain of every OTU was sequenced. The phylogenetic position of all the isolated strains was determined by16S rRNA sequencing. The results showed the isolated181strains displayed21operational taxonomic units (OTUs), of which12OTUs belonged to halophilic bacteria and the others were haloarchaea. Phylogenetic analysis indicated that there were7genera presented among the halophilic bacteria group, include Halomonas, Pseudomonas, Marinobacte, Microbulbifer, Idiomarina, Halobacillus and Piscibacillus, the dominant halophilic strains were Halomonas, which accounted for46.77%in halophilic bacteria. There were4genera among the haloarchaea group, include Haloarcula, Halorubrum, Haloferax, Haloterrigena, the dominant halophilic strains were Haloarcula, which accounted for49.12%in haloarchaea respectively. All the results indicated an abundant diversity of halophiles in Daishan Saltworks.Amylase, protease, lipase and cellulase from181strains were screened. The result showed that39strains have amylase activity,20strains have protease activity, and39strains have lipase activity. Additionally, there are3strains can produce three enzymes, besides18strains produce amylase and lipase,7strains produce amylase and protease,5strains produce lipase and protease. Most of enzymes producing strains belong to Halomonas, then Haloferax and Haloarcula. It was indicated that there are abundant resource of halophilic microorganism and enzyme in Daishan Saltworks.On the basis of the hamology and phylogenetic analysis of16S rDNA, it was considered that DSFS111which can produce amylase and lipase was related to the genera Haloarcula. Colonies on agar plates containing25%(w/v) total salts were red, smooth-faced, elevated and round, diameter3mm after a week. This microbe could grow in medium containing18-35%NaCl, optimal salinity at25%, the tolerance temperature between25℃to50℃, and optimal temperature at37℃, pH at6.0-8.0while optimally at7. Cells of the strain are round and Gram-negative, no spores and containing lipid particles.Enzyme characteristics of DSFD111were investigated in this study. The results shown that the amylase activity was in20℃to60℃range, with the highest activity at37℃, and in pH6to8range, with the strongest activity pH in7. The amylase appears good thermal stability between37℃to50℃and pH stability between pH6-8.Moreover, the condition of producing enzyme was detected, the result shown that the activity of amylase was positive correlated with the growing status. Single factor experiment proved that the optimum fermenting condition of Haloarcula sp. DSFD111producting amylase is:25%concentration of NaCl,1%soluble starch as carbon source,1%bacterial peptone as nitrogen source, pH7.0,37℃shaking culture for60-90h. This provided a favorable scientific basis in the the Soltworks environment to seek high activity amylase strains.It was indicated that there are abundant resource of halophilic microorganism and enzyme in Daishan Solar Saltern.