The Diversity Of Bacteria In Karst Caves In Libo And The Identification And A Preliminary Study Of Two New Bacteria Species

Karst cave as darkness Highly oligotrophic typical habitat environment,with special adaptation of bacteria groups exist in the cave,to understand the extreme environmental conditions of bacterial community structure and distribution characteristics,and further excavation and research has the ecological adaptability of microbial groups,understand the cave microorganisms in the material and energy cycle In this study,we isolated and identified culturable bacteria from 6 samples from 3 karst caves in Libo,and explored the diversity of culturable bacteria community under extreme environmental conditions by analyzing the gradient of multi-culture base.By high-throughput sequencing of 16S r RNA gene in samples,the composition and function prediction of alpha diversity of Libo bacterial community were analyzed,and the culture-free diversity of the bacteria in the caevis was explored.At the same time,two potential new species(PO-11 and B4-66)found in the culturable isolation process were identified by polyphase taxonomy.The results are as follows.1.Isolation,identification and diversity analysis of culturable bacteria from karst cave samplesIn this study,9 kinds of media were used to isolate bacteria from karst cave samples,and a total of 202 strains of bacteria were obtained,according to 16S r RNA gene sequencing and sequence alignment,they belonged to 4 phyla,48classes,16 orders,28 families,50 genera and 91 species.Proteobacteria,Actinobacteria,Bacteroidetes and Firmicutes,respectively.Among them,Proteobacteria was the dominant phyla,and the dominant genera were Brevundimonas,Microbacterium,Flavobacterium,Bacillus and Staphylococcus.By analyzing the diversity,richness and evenness of bacterial community,it was found that the Shannon-Wiener diversity index and Margalef richness index of the sediment samples in cave A and cave C were the highest,which were 1.03,1.07 and6.22,6.03 respectively.The Pielou evenness index of bacteria in cave A and cave C was 0.29 and 0.30,respectively.The analysis of diversity,richness and evenness of bacteria isolated from different media showed that the Shannon-Wiener diversity index,Margalef richness index and Pielou evenness index of bacteria isolated from B4 medium were the highest,which were 1.05,6.59 and 0.29,respectively.2.Diversity analysis of no-culture bacteria in karst cave samplesA total of 64019 effective sequences with an average length of 444.04 bp were obtained by using Illumina Miseq high-throughput sequencing technology of 16S r RNA gene to analyze the bacterial diversity of karst cave sediment samples.The sequences were divided into 1405 OTUs,belonging to 376 genera of 29 phalla.The dominant bacteria belong to Actinobacteria,Proteobacteria,Chloroflexi,Firmicutes and Bacteroidetes,while the dominant bacteria belong to Pseudarthrobacter,Pseudomonas,Nitrospira,Acinetobacter and Flavobacteria.3.Polyphasic taxonomic identification of strains PO-11 and B4-66According to the morphological observation of strain PO-11 and B4-66,strain PO-11 was a round colony with white,translucent,neat edges and smooth surface,while strain B4-66 was a round colony with milky white,opaque,neat edges and smooth surface.Strain PO-11 and B4-66 were in 4??30?could grow,optimum temperature is 25?,p H tolerance range are 7?10,strain PO-11 and the optimal growth Na Cl B4-66 range 0%?1%and 0%?2.5%respectively,strain PO-11catalase,urease,such as experiment result is positive,but the oxidase,nitrate reduction,twain(40,80),citrate utilization,H₂S,gelatin liquefaction,indole test results were negative.The results of catalase,urease,twain(40,80)and nitrate reduction of strain B4-66 were positive,while the results of oxidase,citrate utilization,H₂S,gelatin liquefication and indole were negative.The major respiratory quinones of strains PO-11 and B4-66 were MK-9(H₂)and MK-7,respectively,and the major fatty acids were 15:0 anteiso,17:0 anteiso and iso-C_15:0 and anteiso-C_15:0,respectively,The polar lipid components all contain diphosphatidylglycerol(DPG),phospholipidglycerol(PG),phosphatidylethanolamine(PE),phosphatidylinositol(PI),unknown polar lipid(UL),3 glycolipids(GL)and doublephosphatidylglycerol(DPG),glycolipid(GL),sphingomyelin(SL),glycophosph olipid(GPL).The phylogenetic tree based on 16S r RNA sequences showed that strain PO-11was clustered into a branch with Arthrobacter methylotrophus TGA^T,and the similarity was 98.3%.The similarity between strain B4-66 and bacteriobacter Pedobacter frigoris RP-3-15^T was the highest with 97.8%.The DNA hybridization values of PO-11 and B4-66 and their closest relatives were 23.0%and 19.70%(DNA hybridization threshold of new species was 20%-60%),and ANI values were79.58%and 75.22%(interspecific threshold ANI was 95%-96%),respectively.The DNA G+C%content of PO-11 and B4-66 was 66.15%and 38.09%by the analysis of the genome rough.In conclusion,strains PO-11 and B4-66 should belong to a new species of Arthrobacter and Pedobacter,named Arthrobacter sedumentius sp.nov,and Pedobacter kstdxsis sp.nov.respectively.4.With a RAST(rapid annotation using subsystem technology)was carried out on the genome annotation,further through the Model SEED strains B4-66 and PO-11 genes scale metabolic network,for gene protein-reaction of column anti SMASH(https://antismash.secondarymetabolites.org/#!/start)to analyze and predict the gene clusters encoding secondary metabolites.5.The sediments of the scanning electron microscope(SEM),element analysis(EDS)and the formation of the sediment types(XRD)analysis,found that strains the PO-11 induced sediment of morphology under the electron microscope scanning is lumpy,EDS analysis of the sediment is mainly composed of C,O,Ca,P and other elements,XRD analysis of the sediments formed by crystals of apatite;The morphology of the sediments induced by strain B4-66 under electron microscope scanning is massive and mesh.EDS analysis shows that the sediments are mainly composed of C,O,Ca,P and other elements,while XRD analysis shows that the crystals formed by the sediments are calcite.