Large-Scale Recovery Of Genomes From Cultivable Bacteria In Coastal Sediments

Numerous microbial species live in the ocean and the diversity metabolic of marine microorganisms allows them to play crucial roles in the marine biogeochemical cycles.Two main strategies are used in the acquisition of marine microbial resources:(1)the conventional cultivation strategy,which is used to obtain pure culture of culturable microorganisms,and(2)the culture-independent strategy,represented by metagenomics,which can be used to recover the genomes of uncultured microorganisms.Culturable microorganisms play an important role in the biogeochemical cycles.However,due to technical limitations,the current understanding of the diversity and metabolic capacity of culturable microorganisms is still limited.Recently,our group developed a new method,referred to as ’Cultivation and Mixed Bacteria Sequencing(CMBS)’,which can be used to recover a large number of genomes of culturable microorganisms at a low cost.This thesis would use this new method to recover microbial genomes from coastal surface sediments.Firstly,eight surface sediment samples were collected from four sites along the coast of Shandong Province and a total of 144,036 colonies were obtained by large-scale cultivation with six media.A total of 3,314 Gb data were obtained by high-throughput sequencing.A total of 14,925 medium-and high-quality genomes(completeness ≥50%,contamination<10%)were recovered after assembling and binning,representing 5,529 strains from 2,714 species,including 2,238 potential new species.These results indicated that this method has great potentials in microbial genome recovery.Combined with data from a previous study,a microbial genome catalog of coastal surface sediments were constructed,containing genomes of 7,011 strains from 3,432 species,including 2,839 potential new species.Then,metagenomes were sequenced for two coastal surface sediment samples X200911 and X220413,resulting in 495 Gb(X200911)and 488 Gb(X220413)data,respectively.A total of 362(X200911)and 662(X220413)medium-and high-quality genomes(completeness≥50%,contamination<10%)were recovered after assembling and binning,representing 362 and 662 strains,respectively,and corresponding to 362 and 662 species,respectively.For these two samples,the genomes obtained by metagenomics and CMBS were compared.It was shown that only a small number of recovered genomes were shared by the two methods,indicating that there was an essential difference between CMBS and metagenomics methods.Based on the number of genomes obtained per unit of sequencing data,the efficiency of genome recovery by CMBS was higher than that of metagenomics.The combination of these two methods allowed the recovery of genomes from thousands of species in a sediment sample,which included both culturable and uncultured bacteria in the community,indicating that CMBS is a promising method in the study of microbial community structure.Based on the genome sequences,the abilities to participate in carbon,nitrogen,and sulfur cycles were predicted for the 2,839 potential new species,and it was found that these new species were involved in most steps of the biogeochemical cycles.The comparison revealed that the functions of coastal culturable bacteria in the biogeochemical cycles were substantially different among species and groups,and the engagement of different species and groups in the biogeochemical cycles was different.This thesis revealed the broad application potential of CMBS in large-scale genome recovery studies,provided a large number of reference genomes of new species for marine microbial research,and shed new light on the species diversity of the microbial community and the complexity of the biogeochemical cycles in coastal surface sediments.