Biodiversity Of Halophilic And Halotolerant Bacteria Isolated From Forest Soil From Tongguling In Wenchang City,hainan Province

In order to investigate the biodiversity of halophilic and/or halotolerant bacteria from non-saline forest soil samples collected from Tongguling(19.40.32N?19.40.34N,111.00.40E?111.00.43E),halophilic and halotolerant bacteria were isolated.Based on the 16S rRNA gene sequences,phylogenetic analysis was performed to investigate the biodiversity of the strains isolated.By means of hydrolysis of polymers the extracellular enzymatic characteristics were observed to understand the physiological diversity.By using the polyphasic taxonomic method some novel bacteria representing potential new taxa was studied to make sure their taxonomic position.Baded on the phylogenetic data from this study and 2 former researches,the distribution pattern of halophilic and halotolerant from 3 representative non-saline environments in China,i.e.Tongguling,Tianmo and Dehang,was preliminarily analysised.A total of 213 halophilic and halotolerant bacteria were separated from the non-saline forest soil samples from Tongguling on 4 isolation media(MH,NA,JMC611and MA)added with 15 or 20%(w/v)Na Cl.On the basis of the results of some phenotype studies(colony,cell,salt tolerance test),98 representative strains were selected for further study.The results of salt tolerance test showed that,most of the 98strains tested were halophiles(94 strain,95.9%),and of which 26 stains(26.5%),52(53.1%)and 16(16.3%)tolerated up to 30,25 and 20%(w/v)Na Cl,respectively.The other 4 strains(4.1%)were halotolerant bacteria tolerating up to 15%(w/v)Na Cl.The phylogenetic analysis results showed that,the 98 halophilic/halotolerant strains representated 26 species,belonging to 14 genera in 6 families in 2 phyla.The most dominant isolates belongd Firmicutes(95 strains,96.9%),and the most dominant family was Bacillaceae(89,90.8%,and the most abundant genus was the genus Halobacillus(53,54.1%).Only 3 strains belonged to Actinobacteria.At least 5JSM strains(1781026,1781065,1781072,1781083,1781138)represented 5 potential novel species.The exoenzyme observation results showed that,out of the 98representative isolates tested,79(80.6%)could produced at least 1 of the 3 exoenzymes tested(amylase,esterase,gelatinase),and 35(35.7%),32(32.7%)and 12(12.2%)were positive respectively for 1,2 or 3 kinds of the exoenzymes tested.In which 42 strains(42.9%)were amylase-positive,and 34 strains(34.7%)showed esterase-positive,and 62strains(63.3%)were gelatinase-positive strains.The results of extracellular enzymatic tests also showed that there were at least 8 JSM strains having relatively strong amylase-activity,and there were 7 JSM strains having relatively high esterase-activity,and there were 8 JSM strains exhibiting relatively strong gelatinase-activity,and they all belonged to the family Bacillaceae.Strain JSM 1781072 was studied by using the polyphasic taxonomic approach,and the results showed that,strain JSM 1781072 was Gram-positive,endospore-forming,slightly,motile,facultatively anaerobic,halophilic rod.The growth condition were0–20%(w/v)Na Cl(optimum 5–8%),15–40?(optimum 30?)and pH 6.5–10.5(optimum pH 7.5–8.0).The G+C was 40.3 mol%.The major cellular fatty acids were were iso-C_15:0and anteiso-C_15:0.Possessed menaquinone-7(MK-7)as the major respiratory isoprenoid quinone.The polar lipid pattern consisted of phosphatidylglycerol,diphosphatidylglycerol,phosphatidylglycerol and 2 unidentified phospholipid.There were also some significant differences between the novel strain and the most relative Alkalihalobacillus species,and the DNA–DNA hybridization data also supported JSM 1781072 representing a novel Alkalihalobacillus species.So we proposed that strain JSM 1781072 represent a novel species of the genus Alkalihalobacillus,for which the name Alkalihalobacillus hainanensis was proposed.Strain JSM 1781072(=CGMCC 1.13823^T=DSM 107895^T)is the the type strain of Alkalihalobacillus hainanensis.Based on the data of phylogenetic analysis from this study and 2 former researches,the distribution pattern of halophilic/halotolerant bacteria from 3 representative non-saline environments in China,i.e.Tongguling,Tianmu and Dehang,was preliminarily investigated.The results showed that,a total of 308 halophilic/halotolerant bacteria isolates were individuals of 77 psecies,belonging to 25 genera of 9 families in3 phyla.In terms of distribution pattern of species,out of 77 species 18 were polytopic and 59 sampling-zone-specific.Among 18 polytopic species,only 3 were found in all 3sampling zones;the other 15 polytopic species were found in 2 out the 3 sampling zones,i.e.8 species were found in Dehang-Tianmo,4 in Tongguling-Dehang,and 3 in Tongguling-Tianmo;In terms of distribution pattern of genera,out of 25 genera 12 were polytopic and 13 sampling-zone-specific.Among 12 polytopic genera,7 were found in all 3 sampling zones;the other 5 polytopic genera were found in 2 out the 3sampling-zones,i.e.3 were found in Tianmo-Dehang,2 in Tongguling-Dehang.In terms of distribution pattern of families,out of 9 families 6 were polytopic and 3sampling-zone-specific.Among 6 polytopic families,4 were found in all 3 sampling zones,and the other 2 were found in Tongguling-Dehang.Among 3sampling-zone-specific families,the family Halomonadaceae was found in Dehang,and Dietziaceae and Streptomycetaceae in Tianmo.The research results above showed that there is an abundant biodiversity of halophilic and halotolerant bacteria isolated from the non-saline forest soil samples from Tongguling in Wenchang City,Hainan Province,and that most of the isolates exhibit diverse exoenzyme-activity.This study also partly revealed that there is diverse distribution pattern of halophilic and halotolerant bacteria from 3 representative non-saline environments in China.