Isolation,Genomic Analysis,and Activity Optimization Of Complex Seaweed Polysaccharide Degrading Bacteria

The seaweed resources are abundant in China.However,the current products of the seaweed industry are mainly seaweed with a narrow application range and low economic added value.Seaweed polysaccharides can be degraded to algal oligosaccharides with different degree of polymerization(DP).Seaweed oligosaccharides have the activities of anti-inflammatory,anti-cancer,food preservation,and whitening functions,and can be widely used in medicine,cosmetics,health products,crops,fertilizers and preservation,and increase the economic added value of products.Compared with the traditional acid hydrolysis method for the degradation of seaweed polysaccharides,the microbial degradation method has several advantages,such as stable product polymerization,mild conditions,less equipment loss,and no acid-base wastewater pollution.Therefore,microbial hydrolysis is used in the degradation of seaweed polysaccharides and the preparation of oligosaccharides.However,many existing polysaccharide-degrading strains only have limited activity for a single kind of polysaccharide;therefore,multiple strains need to be fermented to degrade complex seaweed polysaccharides,thereby increasing the production cost of seaweed oligosaccharide.Thus,the field of seaweed oligosaccharide preparation urgently needs to develop a single-strain-based method for the degradation of complex seaweed polysaccharide to reduce the production cost.This thesis aims to isolate strains with the degradation ability for complex seaweed polysaccharides in marine environments,and performs the genome analysis and biochemical activity optimization to reduce the degradation cost of complex seaweed polysaccharides,which lays the foundation for complex seaweed polysaccharide degradation at low cost.The results of this thesis are summarized as follows:(1)Isolation,cultivation and identification of complex polysaccharide degrading strains.YPW1?YPW6 and WPAGA9 were isolated by using sample 2216E medium.Furthermore,YPW1 was assigned into genus Microbulbifer based on the 99.44%maximum similarity of16S r RNA gene with Microbulbifer mangrovi DD-13.The 16S r RNA gene of WPAGA9 has a maximum similarity of 99.87%with Shewanella pacifica KMM3597;therefore,WPAGA9was assigned into genus Shewanella.YWP6 was assigned into genus Streptomyces because of the maximum similarity of 99.65%of 16S r RNA gene with that Streptomyces sundarbansensis MS1/7.Among them,YPW1 and WPAGA9 have polysaccharide degradation activity,YPW6 has the ability to degrade a single polysaccharide.(2)The genomic analysis of degrading strains for complex seaweed polysaccharides.The genome size of YPW1 was 4,578,595 bp,and the average GC content was 57.64 mol%.A total of 3,680 coding genes,12 r RNA genes and 52 t RNA genes have been annotated in the genome.The genome size of WPAGA9 is 4,942,986 bp and the average GC content is 40.82mol%.A total of 4,127 coding genes,25 r RNA and 97 t RNA are annotated in the genome.The genome of YPW1 strain has a variety of seaweed polysaccharide degradation genes.A total of 7 agarase genes,3 algin lyase genes,3 xylanase genes,1 chitosanase gene,3 amylase genes,3 cellulase genes,and 3 genes were found in YPW1 genome.Similarly,3 agarase genes,1 alginate lyase gene,1 carrageenase gene,2 xylanase genes,4 amylase genes and 1pullulanase gene were found in the genome of WPAGA9.(3)The genome size of YPW6 was 7,695,589 bp,and the GC content was 72.02%.The total length of the repeat sequence was 179,759 bp,the content was 2.34%,the annotation was 6,760 coding protein genes,and the average length was 988 bp.In addition,there were18 r RNA genes,66 t RNA genes,29 nc RNA genes,1 pseudogene with length of 87 bp,167CRISPR genes,19 gene island genes,4 prophage genes,and 8 chitinase genes.(4)Optimization of the degradation activity of WPAGA9.The optimal conditions for the reaction and production of agarase,carrageenase and alginate lyase in the WPAGA9 strain are very similar,including the optimal temperature(50°C),influence metal ion(Na~+),carbon source(maltose)and carbon source concentration(1.5%;w/v),indicating that the conditions for WPAGA9 to degrade different seaweed polysaccharides show a convergent trend.Under the optimal conditions,the maximum enzymatic activities of WPAGA9 for the degradations of agar,alginate and carrageenan were 71.63,76.4 and 735.13 U/m L.Moreover,WPAGA9has advantages in the degradation activity of three kinds of seaweed polysaccharides compared to other strains.Therefore,the fermentation broth of WPAGA9 can simultaneously degrade complex seaweed polysaccharides and prepare corresponding seaweed oligosaccharides,thus greatly reducing the production cost of related products.In general,this study performed genomic analysis on the three isolated strains,and optimized the enzyme production and enzyme reaction conditions of Shewanella sp.WPAGA9 which has the ability to degrade complex seaweed polysaccharides.The results proved that The strain has a great application prospect in the production of active seaweed oligosaccharides.This work is also the first time to study the complex polysaccharide degradation ability of the members in genus Shewanella from the perspectives of genomics and biochemistry,which deepens our understanding of the ecological functions of genus Shewanella and reveals the industrial value of WPAGA9 in complex polysaccharide degradation and seaweed oligosaccharide production.