Study On The Separation And Cultivation Of Yet-to-be Cultured Marine Microorganism Based On Metabolomics

With the development of high-throughput sequencing technology and bioinformatics,more and more new groups of marine microorganisms have been discovered and recognized.However,in order to further study the physiological and biochemical functions of marine microorganisms and their role in marine ecosystems,there is still a need for Cultivate these microorganisms and analyze their morphological structure to phenotypic characteristicsIn the early stage,our laboratory established a relatively complete enrichment and separation culture process.We found that there are complex interactions between microorganisms,and it can efficiently separate the uncultivated microorganisms from the marine sediment by systematic study.But,about how to use the interaction between microorganisms to improve the separation and cultivation of uncultivated microorganisms after enrichment is still unknown,which hinders the in-depth research and application of enrichment culture methodsIn this study,we used 16S rRNA gene high-throughput sequencing and targeted metabonomics technology to focus on the key metabolites that are significantly related to the difficult-to-cultivate bacterial groups during the enrichment process,and carried out the separation and cultivation of marine hard-to-cultivate microorganisms.In this paper,metabonomics is used for the first time to study biological factors related to microbial community changes during enrichment culture,in order to find new biological factors that promote the isolation and culture of difficult-to-culture microorganisms.The main research results are as follows:(1)16S rRNA gene high-throughput sequencing analysis of bacterial community structure changes in different enrichment periods.The results showed that the abundance and diversity of microorganisms were the highest in XSDO period With the enrichment culture proceeded,the diversity of microorganisms decreased gradually.After enrichment and cultivation,the relative abundance of some microbial groups increased dramatically,such as Clostridiales,Marinilabiliales,Fusobacteriales,Spirochaetales,Desulfovibrionales,Synergistales and Desulfobacterales(2)Based on targeted metabolomics,the extracellular metabolites of the culture fluids were analyzed during different enrichment periods.A total of 43 metabolites were detected,and the contents of various metabolites changed significantly during the enrichment culture,such as 4-Pyridoxic acid,Adenine,5-Aminopentanoic acid,Pyridoxic acid,SCFA,etc.,which mainly involved cofactors and Vitamin metabolism,membrane transport,nucleotide metabolism,SCFA metabolism,amino acid metabolism,metabolism of terpenes and polyketides(Adenine and 5'-Methylthioadenosine),signal transduction pathways and other pathways.Further analysis found that the SCFA metabolites increased significantly as the enrichment culture progressed,and which was significantly correlated with a variety of difficult-to-cultivate bacterial groups.(3)Optimize the culture conditions and nutrient components of the medium,isolate the cultured bacteria in the enriched culture medium and compare and analyze the genomic metabolic pathways.Using anaerobic culture conditions,at the same time optimize the nutrient components of the culture medium of the experimental group(add SCFA compounds).A total of 444 strains were isolated in this study,including 206 strains isolated from the CK group,belonging to 6 families,31 families,69 species,14 potential novel strains,and 0 strains with 16S rDNA similarity less than 90%;The experimental group isolated 238 strains,belonging to 10 phyla,26 families,82 species,50 potential novel strains,8 strains with 16S rDNA similarity less than 90%.The analysis results showed that the experimental group significantly increased the diversity of cultivable bacteria and potential new bacteria,and the bacterial groups separated by the CK group and the experimental group were quite different.The analysis of the genomes of some potential novel bacteria showed that most of the potential novel bacteria isolated from the culture medium of the experimental group had different degrees of SCFA synthesis pathway defects,but had certain SCFA oxidative degradation ability(4)Complete the polyphase classification of a marine anaerobic bacteria.Through comparative analysis of phenotypic characteristics,genome comparison,phylogeny and other methods,the taxonomic status of strain S-5007T was determined.It represents a new class of the Kiritimatiellaeota phylum.It is proposed to be named Chinicoccuse classis.nov.,and a new species of the Chinese cocci class,named Chinicoccus aerotolerans gen.nov.,sp.nov.).The type strain is S-5007T(=MCCC 1H00402T=KCTC 15876T).