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Most methods for investigating bacterial diversity of soil microorganisms was lack of the ability to establish a causal relationship between function and community compositions.Recently,using stable isotopes to label phylogenetically informative biomolecules(phospholipid fatty acids,DNA,or RNA),has the advantage of providing more definitive evidence that a detected population is active in a specific process,if that process leads to assimilation of C or N into cellular constituents.In practice,Nucleic acid SIP is considerably more sensitive and more informative for taxonomic purposes,particularly among of those related organisms,and thus is more generally useful. Shawan cold spring provides a typical non-sulfide cold spring environment in the arid and semi-arid areas of Xinjiang.In the previous research,the first overview of microbial diversity in the Shawan cold spring had provided,and the results showed that bacterial communities in the Shawan cold spring were diverse which comprised of 11 phyla and maybe exist a diversity of functional groups.In this paper,we surveyed the carbon sources utilization functional bacteria and their diversity in the sediment of the cold spring by stable isotope probing(SIP) and 16S rDNA clone library.The functional bacteria were enriched using 13C-labeled glucose as substrate,and then 13C-labeled genomic DNA was separated directly from the sediment samples and then it used as template for the PCR amplification of bacterial 16S rRNA gene and construction of the gene clone library.Based on restriction fragment length polymorphism(RFLP),417 positive clones were picked up and grouped into 27 operational taxonomic units(OTUs) by Hae III digestion.Subsequently,unique clones were sequenced.The blast search in Genbank and phyogenetic sequence analysis showed that 27 OTUs were divided into 9 phyla of bacterial domain i.e.,Firmicutes,Chloroflexi,Nitrospirae,Actinobacteria,Proteobacteria,Bacteroidetes,Planctomycetes,Cyanobacteria and Acidobacteria.Of those,Firmicutes and Proteobacteria were the dominant groups,and accounted for 28.3% and 38.6% of the total clone library respectively. Compared with 86 OTUs in the previous publication,Gemmatimonadetes,Chlorobi and Verrucomicrobia were not captured in glucose-utilizing bacteria,and the number of OTUs in other phyla decreased evidently,especially in Acidobacteria,only 6.45% of OTUs was observed in this research(48% in the previous research).In sum,that only 31% OTUs utilized glucose as carbon source in the system,suggesting there maybe exist other carbon sources utilization bacteria in the environment.In addition,we found that the incubating time and substrate concentration are key factors for the detection of some species that in low lever in the environment.As the result of enriching in this paper,a lots of OTUs were detected for the first time,such as the phylum Chloroflexi and genus Desulfuromonas.Particularly,genus Desulfuromonas was an important supplement to previous research,because strains belonged to the genus could reduce the sulfur to H2S,and the reason why the genus exited in a non-sulfide cold spring need further study.Additionally,compared with previous research,we found that the proportion of OTUs in Firmicutes was increased obviously from 1 to 7,which were divided into order Clostridiales and order Lactobacillales,and replaced Acidobacteria as the second dominant groups.Hence,the results indicated that an appropriate and reasonable pre-culture could be a useful supplement to study microbial composition and diversity in environmental samples by using culture-independent methods. |
