Screening And Indentification Of Sphingomonos Sp.w4-3and The Study On Its Exopolysaccharide

Exopolysaccharides （EPS） are widely used in many fields such as food、medicine、oil anddaily light etc., and have attracted more and more concern. At present, the growth rate per yearof microbial polysaccharide production is about10%, while some new polysaccharide, of whichbacterial exopolysaccharide accounting for a large share, can be up to more than30%. In thispaper, a strain named W4-3screened from samples was introduced,which could produce highyiled exopolysaccharide. The main contents of the study include the screening and identificationof the strain、optimization of the fermentation conditions、extraction and purification ofexopolysaccharide as well as its some physical and chemical properties, the main results wereshowed as follows:Isolating, screening and identification of EPS-producing strain：15target strains wereselected from samples, among which, one strain named W4-3with most strong ability ofEPS-producing was harvested after rescreening, and based on the physiological and biochemicalcharacteristics as well as16S rDNA sequence, this strain was identified as Sphingomonos sp. itsphylogenetic dendrogram was built based on its16S rDNA gene sequence homology.Optimization of culture medium fermentation conditions: The impacts of main basiccomponents of culture medium including carbon, nitrogen, KH₂PO₄,and MgSO₄on theproduction of EPS were studied and analyzed by single factor experiment with shake flask in labconditions, then those basic components of culture medium were selected and optimized bySurface Response Method （RSM）, the results were obtained as follows: sucrose as carbonresource with concentration40g/L, beef extract as nitrogen resource with C/N mass ratio1:10,KH₂PO₄concentration1.0g/L, MgSO₄concentration0.80g/L. With this optimal conditions, theproduction of exopolysaccharide could reach up to7.158g/L, closed to the estimated value7.235g/L. Regression analysis and verification experiment showed that this RSM was reasonable andfeasible, then some of the fermentation conditions were optimized by single factor experimentwith results as: broth volume30mL/250mL, initial pH value7.0.Extraction and purification of exopolysaccharide and the study of some of its physical andchemical properties: The refined exopolysaccharide power was obtained by freeze-drying afterthe procedure of sterilization, precipitation with ethanol, deproteinization with Sewage reagent、 dialysis and concentration. By the methods of single factor and orthogonal tests, the optimalparameters of ethanol precipitation were obtained as follows: final ethanol concentration75%,temperature4℃, time duration20h..The ultraviolet spectrum showed that no protein andnucleic acid were contained in EPS, DEAE-52column chromatography. showed that it did notcontain neutral polysaccharide, but mainly composed of acidic polysaccharides which could beeluted by0.04M NaCl. The IR spectra showed that the exopolysaccharides had the typicalpolysaccharide absorption peak in5000-3000cm-1、3000-2800cm-1、1400-1200cm-1and1000-1100cm-1, and it was determined as β-D-glucopyranoside bond configuration, with nomannose included.The refined exopolysaccharide was milk-white, insoluble in acetone,95%ethanol andanhydrous ether, difficult to dissolve in distilled water at room temperature, easy to dissolve withboiling distilled water and1M HCl, easier to dissolve in1M of NaOH. The polysaccharide colorreaction experiment showed that the exopolysaccharide didn’t contain starch but contain uronicacid. Viscosity experiments showed that it had a good property of viscosifying.