Isolation,Identification And Denitrification Characterizing Of Novel Species From BaP Enrichment Culture

Mangrove wetland sediments deposited abundant microbial resources.However,99%of the microorganisms in the environment were unculturable[1],it greatly impeded scientists'understanding of the physiological and ecological functions of environmental microorganisms.The microbial community from mangroves sediment was simplified by enrichment process under the pressure of BaP(benzopyrene)and microbes were isolated.However,large number of these isolates had unclear classification status at the level of genus,and even the family level.In order to precisely classified these bacteria,we carried out high-throughput culturing of microorganisms fromm the enrichment culture,and selected strains with lower 16S rRNA similarity for microbial taxonomic identification,and then reclassificate their framework based on phylogenomic tree.Finally,through the prediction of genomic metabolic pathways,microbes with special metabolic functions are excavated and verified by experimental ways for further study.The main findings of this thesis were as follows:1.By high-throughput isolation of microorganisms from the enrichment culture system,102 strains were grasped.Of these strains,15 were classified as potential species and/or novel genera,and strains M11-4T,C9-11 T,X9-2-2T and M9-3-2T had maximum similarity of the 16S rRNA gene sequences which were lower than others.Phylogenetic analysis based on 16S rRNA gene,physiological and biochemical characterization,comparison of fatty acid components,phospholipid components indicated that both strain M11-4t and X9-2-2T represented novel genera,while strain C9-11T represented a novel species and have been published on IJSEM(International Journal of Systematic and Evolutionary Microbiology).2.Analysis of a maximum likelihood tree which were constructed from 16S rRNA sequences of almost all Gammaproteobactria type strains revealed that genus Marinobacter,Mangrovitalea and Tamilnaduibacter should be reclassified as a novel family and named as Marinobacteriaceae fam.nov.,Further,evolutional trees were constructed based on the concatenated core genome and ubiquitous 120 protein in bacteria,then AAI and POCP values were quantified.The results exhibited that Oceanospirillales,Cellvibrionales,Pseudomonadales and Marinobacteriaceae should be integrated into the same Pseudomonadales except the families Moraxellaceae and Kangiellaceae.In addition,Alishewanellaceae fam.nov.,Agarivoransaceae fam.nov.,Zooshikellaceae fam.nov.,Catanovulumaceae fam.nov.,Moraxellales ord.nov and Kangilillares ord.nov.,a total of four novel families and two new orders were also first proposed.3.Through analysis of genomic properties of the four novel strains,we found that strain M9-3-2 has a complete denitrifying metabolic pathway,and the strain M9-3-2 shared closest similarity of 16S rRNA with Azoarcus buckelii U120T only?94.5%.Moreover,16S rRNA gene phylogenetic and core-genome phylogenomic tree analysis,and the experimental verification of the genomic metabolic pathway indicated that the strain M9-3-2 represents a new genus belonged to Zoogloeaceae.Besides,M9-3-2 exhibited both aerobic denitrification and anaerobic denitrification activities,with maximum removal efficiency was 0.46 mmol/h and 0.06 mmol/h in the anaerobic environment and aerobic environment respectively,while the regulation mechanisms of switch from aerobic to anaerobic denitrification needed further research.