Phycosphere Microbial Diversity And Functional Potential Of Four Wild Macroalgae In The Coastal Of Weihai

The oceans play a significant role in absorbing CO2.Macroalgae are one of the primary producers in the ocean and are key members in coastal ecosystems,which play an important role in maintaining coastal biodiversity as shelters and food sources for many aquatic organisms.Macroalgae absorb CO2 through photosynthesis and release organic matter into the ocean.Macroalgal phycosphere microbes,microbes in surrounding seawater and sediments are important members of the recycling of organic matter in the ocean and an important contributor to slowing down global warming.Degradation and utilization of marine polysaccharides are an important part of the marine carbon cycle.Scientist have successively proposed that macroalgae have great potential in absorbing carbon dioxide and achieving carbon neutrality.Elucidating the ecological significance of microorganisms in macroalgal phycosphere environment,seawater,and sediments has positive implications for assessing the contribution of macroalgae in carbon neutralization.Currently,there is no comprehensive research on the composition and function of macroalgal phycosphere microbial communities in China.In this study,four common macroalgae including Ulva sp.(green algae),Saccharina sp.(brown algae),Grateloupia sp.,and Gelidium sp.(both red algae),surrounding seawater and sediments are the research objects.Through large-scale bacterial culture,16S rRNA(V3-V4)amplicon sequencing and next-generation metagenomic sequencing,the composition and function of microbial communities were analyzed.Aiming at the microbial composition and function of the macroalgal phycosphere environment,a systematic study of the macroalgal phycosphere environment was carried out through the combination of metagenomics and culturomics.The following results were obtained(1).By 16S amplicon sequencing,we obtained 92 amplicon datasets.Using 16S rRNA(V3-V4)amplicon sequencing,we identified the core bacterial community composition associated with the four macroalgae at the family and genus level.Our analysis showed that bacterial communities in different macroalgal intercalary microenvironments from the same site were similar between seasons.The amplicon data were also analyzed for alpha-and betadiversity.In addition,we defined the core microbial taxa of the four macroalgal groups and discussed the core phycosphere microbial taxa at the family and genus level.We found similar but diverse core algal microbial communities in the macroalgal phycosphere environment,with 10 families of Proteobacteria,Bacteroidetes,Verrucomicrobia,and Actinobacteriota consistently present in all macroalgae.And it has quite strong stability in different seasons.Although they represent only 5%of the number of taxa in the bacteria in family level,they accounted for more than 85%of the total relative abundance of bacteria.Based on 16S amplicon sequences analysis,we revealed the similarities and differences between the bacterial community composition of macroalgal phycosphere and seawater and sediments,which is helpful for targeted in-depth research.(2).Based on the highly eutrophic characteristics of the macroalgal phycosphere environment,we designed two culture media and carried out a large scale of isolation culture experiments to culture microorganisms in the macroalgal phycosphere environment.A large number of novel species resources,and a rich representative strains from macroalgal phycosphere were obtained,which built the foundation for future research.A total of 5,527 strains(4,473 were from the macroalgal phycosphere environment),representing 1,235 distinct species(689 were potential new species),were obtained by large-scale plating of the four kinds macroalgae samples and seawater and sediment samples..A large number of macroalgal phycosphere bacteria and new bacterial species have been isolated,which has reserved bacterial species resources for subsequent research on functional and metabolism,and enriched our bacterial species resource library.(3).956 genomes of strains isolated from different sample were sequenced.In order to investigate the diversity and potential of the polysaccharide utilization loci(PUL)and biosynthetic gene clusters for secondary metabolites,we sequenced the genomes of 965 strains isolated.And the 965 strains represented 832 different genomes as well as 820 different species.Through a comparative analysis of genome size,we found that the average genome size from the macroalgal phycosphere bacteria environment was the largest,followed by marine sediment samples,while the genomes from seawater samples were the smallest.A large number of genomes of macroalgal phycosphere bacteria and bacteria in other environments have been obtained,which has reserved a wealth of genetic resources for subsequent research on function and metabolism.(4).1,618 medium to high quality MAGs were extracted from sequences and analysis of 23 metagenomes(1.3 T of raw data in total).Although a large number of microbial taxa can be obtained by method of cultivation.However,the study of rare taxa and difficult-to-cultivate taxa needs to be supplemented by metagenome sequencing.Through binning of 23 metagenomes,a total of 1,618 medium to high quality metagenomic assembled genomes(MAGs)were obtained,representing 1,183 non-redundant genomes.The combined dataset includes genomes of 2,093 species,including 965 nearly complete genomes and 550 high-quality MAGs(completeness>90%,contamination≤5%),many of which represent core phycosphere taxa.These MAGs greatly improved the coverage of representative genomes of difficult-to-cultivate and rare taxa in the macroalgae phycosphere environment.It’s available to study and identify unculturable and rare taxa from a variety of environments by metagenome sequencing and binning.(5).Functional annotation analysis of 2568 genomes was carried out by using bioinformatics.The polysaccharide degradation of bacteria and the diversity of secondary metabolite gene clusters were mainly investigated.In total,we obtained more than 4,000 completed PUL in the phylum Actinobacteriota,which correspond to a wide variety of polysaccharide substrates.More than 8,000 biosynthetic gene clusters for secondary metabolites were predicted in 2,5 83 genomes.The diverse biosynthetic gene clusters for secondary metabolites provided abundant candidate materials for our future studies.Through bioinformatics,we analyzed the composition and culturability of these core bacteria and analyzed some of their potential functions,including the potential to degrade polysaccharides and the biosynthesis of secondary metabolites.We found that macroalgae phycosphere from four kinds of macroalgae had more CAZymes,while bacteria from seawater samples contained more sulfatase,especially bacteria in phylum Verrucomicrobiota,Planctomycetota and class Alphaproteobacteria.Bacteria of the phylum Bacteroidota have relatively high polysaccharide degradation potential.A large number of highly diverse biosynthetic gene clusters for secondary metabolites were found not only in phylum Firmicutes and Actinobacteria,but also in genus Aquimarina of Bacteroidetes,family Rhodobacteriaceae of class Alphaproteobacteria and the phylum Planctomycetota.This broadens our understanding of the function of macroalgae phycosphere bacteria and reserves valuable strains and genetic resources for mining potential resources of bacteria.