Study On Bioflocculant-producing Origin Of Ruditapes Philippinarum Conglutination Mud Based On Microbiome

Ruditapes philippinarum conglutination mud?RPM?used to be a typical byproduct waste from manila clam R.philippinarum aquaculture.However,RPM from an aquaculture farm at Zhoushan in China was newly reported as a promising natural bioflocculant resource.With an intent to figure out whether RPM flocculation activity is ubiquitous to the manila clam across a wide geographical range or just Zhoushan location defined,explore the flocculation production basis and ultimately widen their exploitation scope,an extensive survey of bioflocculant-producing origin of RPM and bioflocculant-producing strains isolated from RPM were systematically studied in this study.The RPMs from Dalian,Weihai,Zhoushan and Zhanjiang had flocculation activities.The flocculation rates of freezing-treated RPM samples reached the highest at 8 g�L^-1?61.9�2.4%to 73.2�0.9%?,and the highest flocculation rates of fresh RPM samples reached to 91.34.�1.18%,indicating that the flocculation activity of RPM is widespread.The crude polysaccharides and diversity of RPM samples from different locations were analyzed.The results showed that regional differences led to differences in bacterial community structure and polysaccharide composition of RPMs,and the polysaccharides produced by bacterial communities may be the basis of flocculation activity of RPMs.Overlapping OTUs in all samples accounted for 44.6-62.22%of overall sequences.TOP 25belonged to overlapping OTUs and 82%of TOP 50 belonged to overlapping OTUs.These OTUs may be the"core microbiome"responsible for producing flocculant and undertaking flocculating activities in RPMs.Inhibitory growth of bacteria significantly reduced the decolorization rate of RPM,while fungal inhibition had no significant change,while the polysaccharides produced by bacteria in RPMs may be the main contributor on flocculation activity,suggesting that bacterial community in RPM directly affected polysaccharide compositions and decolorization rates.The members of family Flavobacteriaceae may be the main contributors of flocculation activity,such as Lutibacter?3.99-16.13%?and Maribacter?4.77-15.13%?.Some low-abundant communities may play an important role in determining the RPM community structures,while some high abundance communities had no high discriminative power in all groups.The ecological environment function between these communities still need to be further studied in the future.Two strains JP and GHS18 with high flocculation activity were isolated from RPM,and the flocculation rates were 80.0%and 75.1%respectively.Based on 16S rDNA sequence alignment and biochemical results,strains JP and GHS18 were identified as Pseudoalteromonas undina and P.paragorgicola,respectively.Genome frame scanning and gene prediction were carried out for strains JP and GHS18,which predicted 3746 and4342 genes,14 rRNA/87 tRNA and 11 rRNA/94 tRNA,respectively.The predicted genes were annotated into the databases of COG,GO,KEGG,NR and Swiss-Prot.The precursor genes?e.g.UDP-Glucose,GDP-mannose and UDP-glucuronate?and oligosaccharide repeating unit genes?e.g.UDP-Gal,dNTP-D-Glu,dNTP-L-Rham and UDP-ManNAcA?in the biosynthesis of extracellular polymeric substances were found in the genomes of both strains.However,the eps gene cluster which polymerized repeating units was not found,it is speculated that they had similar gene cluster instead of functioning.This study revealed that symbiotic and epiphytic bacteria of RPM played a major role in flocculating activity and were the origin of RPM flocculant production.It provides a theoretical basis for the future development of RPM and its symbiotic flocculating active strains for fermentation products,and lays a foundation for further research on gene regulation of flocculant biosynthesis pathway of flocculating active strains.