Study On Biodiversity And Degradation Potential Of Cycloalkane Degrading Microbes In Marine Cold Water Habitats

In recent years,marine oil spills have occurred frequently,and oil pollution has become one of the most serious problems of global environmental pollution.The oil contains between 20-45% cycloalkanes,of which methylcyclohexane is one of the most abundant cycloalkane species.Cycloalkanes have a high concentration and danger,and cycloalkanes are clearly hazardous to both humans and aquatic life.A lot of research has been conducted on alkanes and aromatic hydrocarbons in China and abroad,but microbial degradation studies related to cycloalkanes are insufficient.In previous research work,we identified psychropoilic,uncultured microbial taxa in bottom cold water habitats as the primary drivers of cycloalkanes degradation.In addition,it has been proposed that key microorganisms responsible for cycloalkane-degrading bacteria and the oligotrophic marine gammaproteobacteria(OMG)may have a tight phylogenetic relationship.Therefore,understanding the composition distribution,physiological and biochemical characteristics and functional degradation potential of cyclic alkane degrading bacteria in cold water habitats such as the bottom cold water off China and the Antarctic surface water environment with cold climate is important for marine environmental protection and exploitation of microbial resources.This thesis addresses the microbial diversity and degradation potential of cycloalkane degrading bacteria in cold water environments.The research contents and results are as follows:(1)Structure and biodiversity of cycloalkane-degrading microbial communities in cold water habitats.a.Structure and biodiversity of cycloalkane degrading microbial communities in bottom waters of BCMS: three different cycloalkanes cyclohexane,methylcyclohexane and dodecylcyclohexane were used as the sole carbon source and energy source for enrichment of BCMS samples.BCMS samples were analyzed for microbial community structure and diversity using 16 Sr RNA highthroughput sequencing technology.The composition of bacterial species at the genus level,Colwellia,Pseualteromonas,and Cobtia were the main cycloalkane degrading bacteria.The bloomed culture also demonstrates that the presence of bacteria that can efficiently degrade MCH in BCMS can be proliferated by using MCH as a carbon and energy source.b.AASW microbial community structure and biodiversity: We enriched MCHdegrading bacteria from AASW samples by using MCH as the sole carbon source and energy source.We analyzed the microbial diversity and community composition by enrichment culture of AASW samples and Full-length 16 S r RNA gene amplicon sequencing after the cultures had bloomed.In EC and SC treatment,Pseudophaeobacter,Gilvimarinus,Pseudomonas,Cycloclasticus,and Roseovarius were higher than the relative abundance in BLK that represent natural microbial communities,indicating that these bacteria are capable of effective degradation and utilization of MCH.In comparison with the work on BCMS cycloalkane degradation bacteria,the present study found that the BCMS MCH-specific degrading psychropoilic bacteria C1-B045 did not occur in the enrichment culture in AASW,which also revealed that cycloalkanes degradation in AASW was mainly driven by its unique local microbial community in Antarctica.(2)Metagenome sequencing analysis and functional degradation potential MCHdegrading bacteria in cold water habitats.a.Metagenome sequencing analysis and functional degradation potential of BCMS MCH-degrading bacteria C1-B045: In this study,two rounds of dilution-toextinction culture experiments were conducted on the respectative sample from BCMS to find the key species involved in MCH-degradation and to perform metagenome sequencing treatment.After two rounds of dilution-to-extinction,the relative abundance of the new genus C1-B045 increased significantly from 15.9% to 97.5%.C1-B045 was significantly more dominant among MCH-degrading bacteria.The absolute dominance of C1-B045 among the MCHdegrading bacteria indicates that it is likely to be the key active microorganism involved in MCH degradation.In this study,50 high-quality metagenomic assembled genomes(MAGs)were constructed by metagenetic sequencing of eight samples from the first round of dilution-to-extinction,and C1-B045 was annotated with key genes involved in MCH degradation,and a complete MCH degradation pathway was found to exist in C1-B045.b.Metagenome sequencing analysis and functional degradation potential of AASW MCH-degrading microorganisms: In this study,seven cultures of Roseovarius,Nocardioides,Salinicola,and Colwellia,with MCH as the sole carbon source,were obtained by dilution-to-extinction culture.15 high-quality MAGs were constructed by metagenome sequencing.The 15 MAGs were annotated with species and functional genes through the GTDB-Tk database and RASTtk database,respectively.This study proposed two MCH degradation pathways,including lactone formation and aromatization degradation pathways.In addition,this study also revealed a possible metabolic division of labor and synergistic interactions among the MCH degrading microorganisms of AASW.