Preparation And Characterization Of The Bioflocculant Based On Ruditapes Philippinarum Conglutination Mud

The bioflocculant is a natural polymer compound which is non-toxic,harmless and easily degradable by fermentation,extraction and purification by microorganisms such as bacteria and fungi.The chemical composition analysis of the microbial flocculant showed that the main components were polysaccharides and a small amount of polypeptides,proteins,lipids and their complexes.Bioflocculants have good flocculation and decolorization functions,and have wide applications in the food industry and environmental protection fields.However,the preparation process of the general microbial flocculant is cumbersome,the production cost is high,and the development process is limited.The Ruditapes philippinarum conglutination mud has been reported to be a very promising natural bioflocculant resource with typical bioflocculant functions such as flocculation,decolorization and heavy metal removal,and it can be naturally precipitated and suitable for fresh water and seawater treatment.Studies have shown that the flocculation activity of the conglutination mud of the Ruditapes philippinarum can be related to the microbes.However,the Ruditapes philippinarum conglutination mud has a low natural yield and is difficult to collect and apply to actual production.Based on this,according to the strategy of ecological biomimetic compounding,the biomimetic preparation of the conglutination mud of Ruditapes philippinarum was carried out,in order to produce a microbial flocculant which has the characteristics of Ruditapes philippinarum conglutination mud and can be used for fermentation production.To this end,this article has done the following work:1.Shaking bottle preparation of compound bioflocculant（BBF）In this study,the compound bioflocculant（BBF）,which is similar to the Ruditapes philippinarum conglutination mud,was prepared by using the RPM as the inoculum.Single factor experiments were used to investigate the effects of inoculum volume,sandy soil addition,culture time and rotation rates on the flocculation rate of BBF.The BBF was cultured in sequential 11 batches under the optimal conditions(inoculation volume（0.5%,v/v）,sand soil dosage（7%,w/v）,culture time（36 h）,rotation rate(180 r min^-1)).During the sequential batch fermentation,the average yield of flocculant was 73.77±1.79 g L^-1,and the average flocculation rate was 80.28±2.10%.Under the same dosage(8 g L^-1)as the RPM,the flocculation rate of the BBF can reach 87.92±0.65%.2.Characterization of BBFThrough electron microscopy analysis,we found that the surface of the BBF is uneven,and various bacteria of different shapes are attached to the depression.At the same time,the decolorization rate of the BBF to methylene blue,crystal violet and malachite green dye liquor can reach 98.78±0.46%,89.37±0.35%and 99.11±0.17%,the harvest efficiency of microalage can reach 85.65±0.15%.We analyzed the polysaccharide content of 5 to 11batches with stable flocculation rate,and found that the polysaccharide content of the 8th batch was the highest,and the polysaccharide content of the 10th batch was the lowest,which was consistent with the change trend of the flocculation rate.3.Microbial diversity analysis of BBF based on high-throughput sequencingBased on the results of high-throughput analysis,it was found that at the genus level,the seven genus with the highest relative abundance in RPM were Pseudoalteromonas（6.28%-23.04%）and Neptuniibacter（8.14%-24.24%）.Maribacter（3.63%-4.72%）,Paraglaciecola（1.70%-6.28%）,Marine monomonas（1.17%-3.18%）,Winogradskyella（1.81%-2.62%）and Vibrio（1.49%-2.72%）.However,there are two mainly genera of Vibrio（58.73%-91.05%）and Bacillus（0.39%-30.61%）in BBF.Under the sequencial batch fermentation,therelative abundance of Vibrio and Bacillus in the 10th batch was 75.09%,and theflocculation rate in the 10th batch was 77.54±1.17%.It is inferred that Vibrio and Bacillus are polysaccharide-producing strains which mainly flocculate in BBF.4.Research on the production of compound bioflocculant（MBF-JPS）fermentation tankBased on the research of BBF,taking into account the actual production efficiency and production safety,two strains（JP and S5）and polyurethane biofillers were combine to produce the MBF-JPS.JP and S5 were selected from the RPM.A compound bioflocculant MBF-JPS which can be further applied to production is prepared by compounding.The response surface method was used to optimize the production conditions of the MBF-JPS.The results showed that the optimum production conditions were 26.33°C,pH 8.33,and dissolved oxygen concentration was 61.11%.Under the selected condition,the flocculation rate could reach 81.76±0.26%and the yield was 5.15 g L^-1.The efficiency of MBF-JPS for adsorbing heavy metal lead is 0.2686 mg g^-1.The sedimentation efficiency of MBF-JPS to Chlorella Vulgaris and Chlorella salina was 96.98±1.02%and 97.44±0.98%,respectively.The decolorization efficiency of methylene blue was extremely significant,reaching at99.26±0.34%,and the decolorization rate of crystal violet solution was 68.47±0.49%.5.Extraction,separation and purification of MBF-JPSThe method of polysaccharide extraction and hydrolysis of MBF-JPS was centrifugation,concentration,dialysis,alcohol precipitation series.The results of high performance liquid chromatography showed that the monosaccharide components of MBF-JPS were mannose,glucosamine,ribose,rhamnose,galacturonic acid,glucose,galactose,arabinose and fucoal molar ratio is 3:3.5:9.2:0.8:1.2:63.9:2.2:3.6:12.6.Comparing the results of chemical analysis and components of BBF to previous study of RPM,it was found that the quantitative analysis and the monosaccharide composition showed a high degree of similarity.In this paper,two kinds of bioflocculants were prepared by means of ecological biomimetic research through shake flask test and fermenter pilot test.These two bioflocculants have higher flocculation activity than the RPM and are easier to collect.BBF is self-settling and the compound bioflocculant（MBF-JPS）can be collected in suspension.Among them,Vibrio and Bacillus are the main producing strains of polysaccharides in BBF.In MBF-JPS,two strains of Pseudomonas can also be produced active polysaccharides for flocculation.It lays an experimental foundation for the industrial production of ecological biomimetic bioflocculants based on RPM.