Biosynthesis Mechanism Of Polysaccharide-based Microbial Flocculants In Biology And Heavy Metal Removal

As the eco-friendly water treatment agent,the research,development and application of microbial flocculants have been paid a high attention,which has been the research hotspot in the field of environmental biotechnology for many years.However,the bottleneck problem of low yield of microbial flocculants cannot be fundamentally solved due to the unclear biosynthesis mechanism of microbial flocculants,which limited the application of microbial flocculants in the water treatment.At the same time,traditional flocculants are not ideal for the removal of dissolved pollutants in water.Polysaccharide-based microbial flocculants contained a large amount of negative charge,which cannot only flocculate suspended pollutants in water,but also adsorb dissolved pollutants.Therefore,in view of the the low-yield problem of microbial flocculants and its poor removal efficiency for soluble pollutants,this work carried out the study on the biosynthesis pathway of polysaccharide-based microbial flocculants to guide the gene regulation for their biosynthesis process,aiming to reveal their biosynthesis mechanism and increase their production yield.Polysaccharide-based microbial flocculants were applied in the removal of heavy metal to lay the foundation for solving the problem of poor removal efficiency for dissolved pollutants and to broaden their application scope.For the unclear biosynthesis mechanism of microbial flocculants,the genes and metabolites involved in the biosynthesis of microbial flocculants were analyzed by transcriptome,metabolome,and gene knockout technology,thereby constructing the biosynthesis pathway of polysaccharide-based microbial flocculants.Transcriptome data showed that the expression level of 20 genes related to glucose,galactose,mannose,and rhamnose metabolism was up-regulated during the biosynthesis of polysaccharide-based microbial flocculants in Agrobacterium tumefaciens F2compared with no production of microbial flocculants.Quantitative PCR results demonstrated the accuracy of transcriptome data.Metabolome detection results showed that the content of 118 metabolites significantly increased,while the content of other 27 metabolites significantly decreased.Among them,the content of glucose-6-phosphate significantly increased about 13 times,and the content of UDP-glucose significantly increased about 187 times.They were the key metabolites involved in the biosynthesis of polysaccharide-based microbial flocculants.Based on p<0.05,KEGG enrichment analysis showed that carbohydrate metabolism,amino acid metabolism,energy metabolism,and membrane trasnsport were closely related to the biosynthesis process of microbial flocculants.The genes were disrupted by gene knockout technology in order to verify the genes involved in the biosynthesis of polysaccharide-based microbial flocculants.The results showed that exo A,exo C,exo F,exo P,exo Q,exo Y gene-disrupted strains cannot produce microbial flocculants,and the mutant strain recovered the flocculant-producing ability after the complementation of the corresponding gene,demonstrating that these genes were involved in the biosynthesis of polysaccharide-based microbial flocculants in strain F2.According to the genes and metabolites involved in the biosynthesis of polysaccharide-based microbial flocculants,the biosynthetic pathway was revealed.For the bottleneck problem of the low yield of microbial flocculant produced by wild flocculant-producing strain,the recombinant flocculant-producing strains F2-exo A-O,F2-exo C-O,F2-exo F-O,F2-exo Q-O,and F2-exo Y-O were constructed in order to increase the yield of polysaccharide-based microbial flocculants by the gene regulation for the biosynthetic process.The yield of microbial flocculants produced by strain F2 was 686 mg/L,in which the polysaccharide content was 304mg/L.Compared with wild strain F2,the flocculant-producing ability of F2-exo Y-O significantly increased,and the yield of microbial flocculants reached 1219 mg/L,in which the polysaccharide content was 720 mg/L.Compred with polysaccharide-based microbial flocculants produced by strain F2,the content of polysaccharide in polysaccharide-based microbial flocculants produced by recombinant strain F2-exo Y-O increased about 1.37 times,which were more negatively charged at p H 6.0?7.0 and thereby more beneficial to flocculate suspended pollutants and adsorb dissolved pollutants in water.In addition,the flocculation activity per unit volume of the fermentation broth of the recombinant strain F2-exo Y-O was enhanced.Polysaccharide-based microbial flocculants contained rich functional groups,such as hemiacetal groups and hydroxyl groups,which was composed of the main sugar monomer glucose,galactose,mannose,and rhamnose.Compared with structure of microbial flocculants produced by strain F2,the charge,functional group and sugar monomer types of microbial flocculants produed by strain F2-exo Y-O did not change,but the proportion of glucose monomer increased from 48.24%to 75.40%,and galactose monomer increased from 6.66%to13.70%,while the monomer proportion of mannose and rhamnose decreased.Among the imported exo genes,only exo Y gene imported into the bacterial strain resulted in its significantly increased flocculant-producing ability.Combined with the gene function and flocculant structure analysis,exo Y gene coded undecaprenyl-phosphate galactose phosphotransfer and participated in the key step in the biosynthesis of polysaccharide-based microbial flocculant in strain F2,which determined the biosynthesis level of polysaccharide-based microbial flocculant.Polysaccharide-based microbial flocculants produced by the recombinant strain F2-exo Y-O were applied in Ag⁺removal,and nano silver was prepared under ultraviolet light based on the reduction ability of polysaccharide-based microbial flocculants,which was favorable for the resource recovery in the wastewater treatment,guiding the study of dissolved pollutants removal and broadening the application of microbial flocculants.The binding ability of polysaccharide-based microbial flocculants for Ag⁺was 85.40 mg/g.The results of kinetics and thermodynamics showed that k₂was 0.279×10^-3 mg g^-1 min^-1 in the binding of Ag⁺on polysaccharide-based microbial flocculants,and K_Fwas 16.56 mg g^-1.Adsorption mechanism of polysaccharide-based microbial flocculants produced by recombinant strain F2-exo Y-O for binding Ag⁺showed that the hemiacetal group was oxidized to carboxyl group,and Ag⁺was reduced to Ag⁰,and the hydroxyl group was also involved in the binding process of polysaccharid-based microbial flocculants with Ag⁺.Compared with the absence of ultraviolet light,and k₂and K_Fwere higher in the presence of ultraviolet light,indicating that ultraviolet light can promote the binding process of polysaccharide-based microbial flocculants with Ag⁺and accelerate the reaction.Nano silver were prepared based on polysaccharide-based microbial flocculants in the presence of ultraviolet light.The diameter size of the formed silver nanoparticles was about 12 nm,and the nano silver were obtained by loading silver nanoparticles on the surface of polysaccharid-based microbial flocculants.UV-vis showed the solution color changed from milk-white to brown.XRD and XPS demonstrated the occurrence of Ag⁰ and the presence of minor Ag₃PO₄.FTIR verified that the hemiacetal group in the polysaccharide-based microbial flocculant was oxidized into carboxyl group and Ag⁺was reduced into Ag⁰.Ag⁰ underwent the nucleation growth,and ultraviolet light accelerated the formation of silver nanoparticles.