The Purification And Characterization Of Extracellular Polysaccharides From Flocculating Active Bacteria In Ruditapes Philippinarum Conglutination Mud

Microbial flocculant is a biological macromolecule with special flocculation effect obtained by fermentation of microorganisms such as bacteria,and its components mainly include polysaccharides,proteins and nucleic acids.The flocculating strain was derived from the microbial community in the sticky mud of the Philippine clam.Due to the purification of the surrounding waters by the Philippine clam,it was suspected that the phenomenon was caused by the microbial community in which the clam was located.In this paper,the extracellular polysaccharide polymers of three flocculating active strains were studied in depth.Due to its good flocculation effect,its structure was characterized by various means,and its structural information may be helpful to promote the flocculation effect.The following work was done from the screened strains:(1)Extraction and purification of microbial exopolysaccharideThree strains of Halomonas sp.GHF11,Pseudoalteromonas sp.GHS19 and Pseudoalteromonas sp.JP were selected from the flocculation active strains identified in the laboratory for initial screening.The crude polysaccharides were obtained by ethanol precipitation after the inoculation.The crude polysaccharide was deproteinized by Sevag method and further purified by anion exchange column DEAE and Sephadex gel permeation chromatography column to obtain pure polysaccharide.The obtained polysaccharide was subjected to flocculation activity test and characterization of structural information.(3)Activity test of pure polysaccharidesThe flocculation activity of Kaolin was investigated by microorganism polysaccharide pure product,and the flocculation activity of different stages of polysaccharide extraction and purification was tracked.The results showed that at the F11 polysaccharide dose of 0.5 mg/L,the optimal flocculation effect reached 71.2 %,while the flocculation effect was poor when the reaction dose was less than 0.1 mg/L or greater than 1.0 mg/L.In addition,the decolorization effect of F11 polysaccharide pure products on three dye-ing agents including methyl blue,crystalline purple and malachite green was investigated.The results showed that the polysaccharide had the best decolorization effect on malachite green and achieved the best decolorization effect of 92.4 % at the reaction dose of 1.8 mg / L.Similarly,if the reaction dose is too high or less,the decolorization effect will gradually weaken.Similarly,the flocculation activity of S19 polysaccharide has a similar relationship with its dose.At a dose of 0.9 mg/L,the optimal flocculation rate was 78.8 %,and at a reaction dose of 2.0 mg/L,the optimal decolorization activity was 87.9 %.(4)Analysis of polysaccharide structureThe separated and purified polysaccharides are characterized by a series of methods.Infrared spectroscopy shows that the main functional groups of this polysaccharide include hydroxyl groups,carbonyl groups,methylene groups,and carbon skeleton rings.Gel osmotic chromatography measured its molecular weight of 31 KDa and 1010 KDa.The monosaccharide group of F11 samples was measured by high performance liquid chromatography as mannose,Glucosamine,glucose,galactose,and ribose.Methylation of polysaccharides was performed using a gas phase mass spectrometer.The total ion flow and mass spectrometry showed that the glycosidic bonds were connected in Glc 1?2?3?4connection,and Man 1?2?3?4?6 connection.The monosaccharide group of the S19 sample became glucose and mannose,and the methylation analysis showed that the glycosidic bond connections were Glc1?3,Glc1?4,Glc1?3?4 connections,and Man 1? 2?3?4?6 connections.