Study On Characteristics Of Flocculant-Producing Bacteria With Plasma Mutation And Its Flocculation

Microbial flocculant(MBF)with no secondary pollution and high flocculating activity is a safe and non-toxic new flocculant,which has become a research hotspot and attracted more and more attention to scholars in recent years.However,the bioflocculant is greatly restricted in the practical application,due to its high production cost and low yield.At present,the study on microbial flocculant is limited to the laboratory stage.Because of this background,the mutagenesis and breeding of the bioflocculant-producing bacteria A9 was implemented by plasma mutation to increase the yield of MBF A9.Besides,the extraction,separation and purification conditions of MBF A9 were optimized and its physicochemical characteristics were discussed preliminarily,providing the research foundation for the commercial application of MBF.(1)The A9 strain of the plasma mutation breeding is carried out by plasma mutation,then the optimal mutation time was 108s and the optimum bacterial concentration is 10-4 times of the starting strain.With the method of the initial screen and shake flask fermentation,combined with the colony morphology,the flocculation and polysaccharide production of the colony,2 positive mutant strains and 2 negative mutant strains were selected.Compared with the starting strain A9,the flocculation of fermentation liquid for the strain A9-108-3(1)and A9-108-3(2)increased by 4.6%and 4.8%respectively,and the yield of polysaccharide increased by 10.8%and 15.9%respectively,indicating the yield of the strain was positive mutation.The flocculation and polysaccharide production of the strain A9-109-3(1)and A9-109-3(3)weredeclined obviously,and the mutation was negative.(2)The separated conditions of MBF A9 were optimized using single factorexperiments,then the anhydrous ethanol with relatively low price was selected as organic solvent and the volume ratio of the fermentation liquid and ethanol was 1:3,and the optimal yield of 1.98 g/L was obtained after 20 mins' centrifugation at the 10000 r/min speed and precipitation for 6 h.(3)The optimum conditions for removing free proteins,which were that Vsample:Vsevag was 2:3,Vchloroform:Vbutanol was 4:1,the time was 30min,were determined by orthogonal experiment.(4)The chemical properties of MBFA9 werepreliminarily determined.Ninhydrin was blue in coloring experiment;the distinct purple ring was found on the interface between the concentrated sulfuric acid and samples reagent mixture in the sugar Molish reaction;anthrone reaction also appeared bright yellow.The results showed that the contents of polysaccharide and protein in MBFA9 were significantly higher than that of protein,and the polysaccharide played a main role in flocculation function.(5)The images of Scanning electron microscopy showed that the porous structure of MBF A9 was porous and cross-linked.Furthermore,the samples surfaces became more smooth and clearer,and the pores were connected more closely with larger porous density,and the crosslinking among the chains also became more obvious,as the purification was advancing from the crude extracts to dialysates,which indicated that the structure of MBF A9 was linear long-chain molecular with some branched chains and the network was formed among the long chains.Meanwhile,the dry MBF A9 has strong toughness and was not easy to grind into powders,which also confirmed its linear long-chain molecular structure.(6)The dialysates of MBF A9 with molecular weight of 9.66×106 Da was tested to be non homogeneous polysaccharide by high performance liquid chromatography(HPLC).In addition,the further purified product of MBF A9 was proved to be a single peak and the molecular weight of the corresponding component was 6.56×104 Da.