Analysis And Exoenzme Charateristics Of Halophilic And Halotolerant Bacteria Isolated From Sandy Soil From The Tianmo Desert In Zhangjiakou City,Hebei Province,China

In this study,to investigate the diversity and exoenzyme characteristics of halophilic and halotolerant bacteria in sandy soil samples collected from the Tianmo Desert in Zhangjiakou City,Hebei Province,PR China?40°17?30??–40°17?46??N,115°41?02??–115°41?13??E;574⁵69ASL?,bacteria strains were isolated and purified using culture-dependent method.The phylogenetic diversity of the isolates was investigated by using16S rRNA gene sequence analysis,and the exoenzyme characteristics was studied using some conventional methods,and the taxonomic position of some strains representing potential new taxa/species was investigated by means of the polyphasic taxonomic approach.We isolated 264 halophilic/halotolerant bacteria from the samples on 4 media?NA,MA,MH,JCM611?supplemented with 10,15 and 20%?w/v?NaCl.On the basis of some morphological?colony,cell?and physiological?salt tolerance?characteristics,76representative strains were selected to perform the phylogenetic analysis and exoenzyme observation.The phylogenetic analysis result showed that,these 76 halophilic and halotolerant strains belonged 33 species,being members of 15 genera?Streptomyces,Kocuria,Citricoccus,Brachybacterium,Dietzia,Virgibacillus,Thalassobacillus,Terribacillus,Piscibacillus,Oceanobacillus,Lentibacillus,Halobacillus,Gracilibacillus,Bacillus,Jeotgalibacillus?of 6 families?Streptomycetaceae,Micrococcaceae,Dietziaceae,Dermabacteraceae,Planococcaceae,Bacillaceae?in 2 phyla?Actinobacteria,Firmicutes?.The most abundant strains were within the phylum Firmicutes?69 strains,90.8%?,and the most dominant family was Bacillaceae?67,88.1%?,and the the most dominant genus was Bacillus?30,39.4%?.A smoll amount of isolates?7 strains?belonged to the phylum Actinobacteria.There were obvious genetic divergences between most isolates and their closestly related type strains?sequence similarities,2 strains,100%;other strains,97.28%to 99.93%?.At least 6 strains?JSM 1684010,JSM 1684019,JSM1685063,JSM 1685098,JSM 1685112,JSM 1685114?should be members of 5 potential novel species of 4 recognized genera?Bacillus,Oceanobacillus,Virgibacillus,Lentibacillus?.The results of exoenzyme observation showed that,73?96.1%?out of the 76 strains tested showed one or more exoenzyme-acitvities of the 4 enzyme tested?amylase,gelatinase,urease,esterase?,and 34 strains?44.7%?showed amylase-positive,24?31.6%?gelatinase-positive,43?56.6%?esterase-positive,and 57?75%?urease-positive.And 15strains showed only one exoenzyme-activity,and 34?44.7%?,21?27.6%?and 3 strains?3.9%?showed 2,3 and 4 exoenzyme-activities,respectively.The results also showed that there were 6 strains?JSM 1684012,JSM 1684041,JSM 1684067,JSM 1685081,JSM 1685082,JSM 1685098?exhibited relatively strong amylase-activity,and 3 strains?JSM 1684007,JSM 1684012,JSM 1684071?exhibited relatively strong gelatinase-activity,and 5 strains?JSM 1684003,JSM 1684086,JSM 1684093,JSM 1684099,JSM1685085?exhibited relatively strong urease-activity,and 8 strains?JSM 1684098,JSM1684121,JSM 1684137,JSM 1685063,JSM 1685072,JSM 1685081,JSM 1685099,JSM 1685003?exhibited relatively strong esterase-activity.To investigate the taxonomic position of strain JSM 1685098,the polyphasic taxonomic approach was performed.The results showed that the strain JSM 1685098 was slightly halophilic,endospore-forming,facultatively anaerobic,non-motile,Gram-positive bacterium.Growth occured at 10–45°C?optimum 30–35°C?,pH 6.0–11.0?optimum pH 8.0?with 0–20%?w/v?NaCl?optimum 5–8%?.The diagnostic amino acid was meso-Diaminopimelic acid in the cell wall peptidoglycan,and the major polar lipids were phosphatidylinositol,phosphatidylglycerol and diphosphatidylglycerol.With menaquinone 7 as the predominant respiratory quinone,and anteiso-C_15:0 and anteiso-C_17:0 as the major fatty acids?>10%of the total?.The phylogenetic analysis using 16S rRNA gene sequence comparison showed that,strain JSM 1685098 was member of the genus Bacillus,and that it was most closely related to Bacillus hwajinpoensis SW-72^T?98.99%16S rRNA gene sequence similarity?,Bacillus hemicentroti JSM076093^T?98.24%?and Bacillus algicola KMM 3737^T?97.44%?.These strains were clustered together on the phylogenetic tree,but strain JSM 1685098 formed a robust lineage supported by extremely high bootstrap value?100%?.The combination of results from the phylogenetic analysis,DNA–DNA hybridization and chemotaxonomic and phenotypic characterization supported the propose that,JSM 1685098 represents a novel Bacillus species,for which the name Bacillus tianmoensis is proposed,with JSM 1685098as the type strain.To sum up,the results above revealed that there are abundant phylogenetic diversity and some potential new taxa in the halophilic and halotolerant bacteria isolated from sandy soil samples collected from the Tianmo Desert in Zhangjiakou City,Hebei Province,China,and that most of the isolates show diverse exoenzyme-activity?amylase,gelatinase,esterase and/or urease?.