Culture Of The Bacteria By Culturomics In Baiyangdian

Bacterial are an important part of the lake ecosystem,and they play an irreplaceable role in the process of cycling of C,N,P and S material and energy metabolism in lake ecosystems.It's necessary to understanding the community structure and species diversity of culturable bacterial in lakes.Culturomics is the basis for constructing a library of lake bacterial and studying how bacterial participate in ecological functions.In this paper,the species diversity and community structure of bacterial in Baiyang Lake were analyzed by combining culture-dependent and culture-independent methods,and the taxonomic classification of new species isolated from Baiyang Lake was identified by polyphasic approach.The main results are as follows:(1)The bacterial diversity and community structure of 40 samples from Baiyang Lake water were studied using Illumina Hiseq2500 high-throughput sequencing.A total of 1907830 valid Tags sequences were obtained by sequencing all samples,and the mass fraction of each base in the sequence was higher than 30,indicating that the sequencing results can be used for further analysis.The sequences after quality filtering and rarefaction(including 32,240 sequences in each sample)were clustered into 3200 OTUs according to 97%sequence similarity using usearch V8.0.1517software,and all OTU sequences were aligned using Greengenes database.And the classification of species affiliated with 34 phyla,79 classes,218 orders,395 families and 632 genera(including unknown groups),and of which Actinobacteria,Bacteroidetes,Verrucomicrobia and Proteobacteria occupy important group,which is consistent with the main group of reported freshwater lake community structure.Among the 3200 OTUs,except for cyanobacteria,the OTU with a relative abundance of more than 0.5%is defined as the dominant OTU in the community structure,and its relative abundance accounts for 78%–96%(including 106 OTUs in total).Among the dominant OTUs,nine groups are included Actinobacteria,Armatimonadetes,Bacteroidetes,Chloroflexi,Firmicutes,Patescibacteria,Planctomycetes,Proteobacteria,and Verrucomicrobia,the Actinobacteria,Bacteroidetes,and Proteobacteria are the major groups.(2)The strains were isolated by using various oligotrophic culture media.Isolation 1002 strains from the Baiyang Lake water samples,belongs to 5 phyla(Actinomycetes,Bacteroides,Firmicutes,Deinococcus-Thermus and Proteobacteria),10 classes,28 orders,48 families,101 genera,305 species and 19 potential new species(the highest similarity of species is less than 98.65%).Through the blastn software(threshold value is 97%),the 16S rRNA sequences and the OTU sequence were blast found the culture-independent methods can not only cultivate the dominant and rare of the Baiyang lake water community structure,but also isolate the group that was not detected by culture-dependent methods.Moreover,comparing the dominant OTU species and pure culture isolates at the taxonomic level,the culture-independent methods was able to isolate 52.8%of the dominant communities in the Baiyang Lake.It can be seen that through the pure culture method,most of the dominant group in the community structure can be isolated.(3)Two new species were identified by polyphasic taxonomic studies,and two new species were named Rufibacter latericius R-22-1c-1~T and Azospirillum griseum L-25-5w-1~T.The research process found that the strains of the genus Azospirillum have the function of nitrate reduction.In addition,by studying the physiological and biochemical characteristics of these new species,it is helpful to explore the ecological functions of the bacterial microorganisms in the lake as well as to obtain the bacterial resources with potential application value.