Species Diversity In Hypersaline Environments And Polyphasic Taxonmic Identification Of A Haloarcheal New Species

Halophiles,including halophilic archaea and halophilic bacteria,are extremophiles that live in hypersaline environments.Due to their special physiological and metabolic characteristics,they are used to produce biodegradable plastic and to degrade organic pollutants in the wastewater with a high concentration of salt.The composition of halophiles is different in various hypersaline environments.In three typical high-salinity environments:salt lake,marine solar saltern,and subterranean salt mine,the fluidity of the salt mine is poor and the interspecific gene exchange is blocked.It provides favorable conditions for speciation.Therefore,a salt mine is a well preserved gene and species pool,full of halophiles.But recent biological research about the salt mine is lacking.Moreover,the comparative study on species diversity of the three high-salinity habitats is rarely reported.In this study,the prokaryotic biodiversity composition of one salt lake?Chaka Salt Lake?,two marine solar salterns?Hainan Saltern and Lianyungang Saltern?and three salt mines?Dingyuan Salt Mine,Xiangyan Salt Mine and Yuanyongjing Salt Mine?was revealed by culture-dependent and culture-independent approaches.The effects of fluidity of environment,geographical isolation and buried depth of the salt mine on the diversity of prokaryotic species in hypersaline environments were investigated.A total of 657 strains from 25 genera of culturable halophiles were isolated from these 8 samples collected from three hypersaline environments via culture-dependent approach.There were a total of 441 strains of haloarchaea?67.12%?from 14genera,Halorubrum,Natronoarchaeum,Natrinema,Haloterrigena,Halovivax,Natronomonas,Halopenitus,Halobacterium,Halococcus,Halolamina,Haloarcula,Halobaculum,Haloferax,and Natrinema.There were 216 strains of halophilic bacteria,accounting for 32.88%from 11 genera,Pseudomonas,Halomonas,Aliifodinibius,Halobacillus,Halospina,Aquisalimonas,Rhodovibrio,Oceanobacillus,Chromohalobacter,Halovibrio and Virgibacillus,respectively.Halophiles were widespread,and haloarchaea were the dominant species in the above hypersaline environment.Among the three types of samples,the number of strains isolated from the salt mine was the most abundant group,accounting for 85.24%of the total number of isolates from 22 genera.The effects of fluidity of environment,geographical isolation and buried depth of the salt mine on the diversity of prokaryotic species in hypersaline environments were analyzed by clone library and culture-independent high-throughput sequencing strategies.The results were shown as follows:?1?from the perspective of liquidity,salt lake,solar salterns and salt mine were decreased successively.Simpson index was0.6150,0.0206 and 0.0774,while Shannon index was 0.9757,4.3608 and 3.5724,suggesting that the diversity of prokaryotic species in the salt lake was the lowest,and the dominant species was obvious.But in salt mines and solar salterns,the phenomenon was opposite to that of salt lake.Among them,the diversity index of solar salterns was the highest one;?2?from the perspective of geographical isolation,the Yunnan Salt Mine samples XY and YYJ were both collected from Yunnan province,which are relatively close to each other.The Dingyuan Salt Mine samples were collected from Anhui province,which was far away from the other two samples.The number of OTUs obtained from XY and YYJ was 478 and 493,respectively.The dominant species were Proteobacteria,Epsilonbacteraeota and Lentisphaerae,and the number of the same OTUs between XY and YYJ samples reached nearly half of the total OTUs in the both of two habitats.There were 2356 OTUs in the sample of Z24,which was significantly higher than that of Yunnan Salt Mine,and the dominant species were Proteobacteria,Firmicutes and Bacteroidetes.Simpson index of XY,YYJ and Z24 samples was 0.0537,0.0589 and 0.0075,respectively.Shannon index was 4.0829,4.0753 and 6.6506,respectively.In addition,Dingyuan Salt Mine was rich in elements of Fe,Mg and Mn,poor in Ca and SO₄^2-.Furthermore there were some soli and stone in Yunnan Salt Mine.Yunnan Salt Mine was different from solar salterns or salt lake.The difference between XY and YYJ samples was not significant.It showed that the species in Dingyuan Salt Mine was more abundant.The difference between Dingyuan Salt Mine and Yunnan Salt Mine was significant.The prokaryotic species compositions of the two Yunnan samples were similar.Ion composition and concentration as well as geographical isolation played an important effect on species diversity composition in hypersaline environments;?3?from the perspective of buried depth,the samples of Z241,Z246 and Z248 were collected from 404-409 m,367-371m and 305-310 m subsurface.The number of OTUs was 1328,1303 and 1304,respectively.The dominant species of three samples was the same,Firmicutes,Proteobacteria and Bacteroidetes.Shannon index was all nearly 6.1.It showed that the effect of buried depth on the composition of prokaryotic species was not significant in Dingyuan Salt Mine.The heterogeneity of salt mine blocked the interspecific gene exchange which is beneficial to speciation.We isolated a possible new species from Yunnan Salt Mine.Polyphasic taxonomy was applied to identify this haloarchaeal strain.Phenotypic and chemotaxonomic characterization,phylogenetic analysis of 16S rRNA gene and rpoB'gene,polar lipids profiling,DNA G+C content and DNA–DNA relatedness clearly indicated that strain ZY8 represents a novel species in the genus Halorubrum.Since it can grow normally in the basic medium supplemented with glutamate as the sole carbon source and energy source,we named this new species as Halorubrum glutamatedens.The type strain is ZY8^T?=CGMCC 1.16026^T=NBRC 112866^T?In this study,a large number of halophilic strains were isolated by culture-dependent method.In addition,the influence on the distribution of prokaryotic species in high salinity habitats was analyzed from three perspectives:fluidity,geographical isolation and burial depth.And,a new species of haloarchaea which isolated from Yunnan Salt Mine was successfully identified.This research increased our understanding of species diversity and enabled better exploitation and utilization of microbial resources from hypersaline environments.