The Selection Of Klebsiella Oxytoca XCH-1 Strain And The Study On Its Exopolysaccharide

The bacterium Klebsiella oxytoca XCH-1 strain studied in this thesis was isolated and obtained from the water extract of Laminaria Japonica in Qingdao Algae Chemical Plant. It produces a large quantity of viscous exopolysaccharide while growing. For further study and exploitation of the exopolysaccharide, intensive analyses on it have been made and depicted in this paper.In this study, exopolysaccharide was prepared by fermentation of K .oxytoca XCH-1 strain.Many factors, such as, aeration, initial pH of cultivating substrate and nutritional composition of the substrate, can affect the yield of exopolysaccharide by fermentation of K. oxytoca XCH-1 strain. Experiment results show that as to fermentation of AT. oxytoca XCH-1 strain for producing exopolysaccharide, mannite is the best carbon source, and (NH4)SO4 is the best nitrogen source. Fermentation conditions have been studied by Uniform Design Test. The conclusion is that the yield of exopolysaccharide from K. oxytoca XCH-1 is 5.93 mg/g on the condition of initial mannite concentration as 1%, C : N as 50 : 1, temperature as 23 C, initial pH value as 8.18, inoculation quantum as 9%.After fermentation of K.oxytoca XCH-1 strain, crude exopolysaccharide was then obtained by precipitating it in ethanol. The crude product was purified in gradation by anion-exchange ( Q-Sepharose FF ), thus the main fraction of it was obtained. The purified product was thoroughly analyzed for its physical and chemical properties. The exopolysaccharide is a white powder, soluble in water, and can be dissolved into acid or alkaline solution, but shows no solubility in organic solvent. The exopolysaccharide water solution is viscous and translucent. Its average molecular weight is examined as 156000 or so by gel filtration chromatography with Sephaeryl S-300HR column. The exopolysaccharide is free from sulphate group and protein, and with 66.45% of total carbohydrates, 15.89% of uronic acids, so it presents acidic polyssacharide characteristic. With 0.1mol/l of NaCl solution as solvent, 0.5% of the exopolysaccharide solution was prepared, its intrinsic viscosity tested as 6.77 at 25C. The exopolysaccharide is of high viscosity and of significant toleraice to heat. It can stand the temperature of 100C for a short time. The relative viscosity is constant in the range of pH 4-8. Electrolytes have impact on the exopolysaccharide to some extent. The relative viscosity of the exopolysaccharide solution is declining as concentration of NaCl or CaCl2 or Na2B4O7 increases. When concentration of the above electrolytes respectively exceed 3%, 1%, 1.5%, the relative viscosity of the exopolysaccharide solution basically keep unchanged, hence shows some tolerance to electrolytes.In this thesis, removal effect of the exopolysaccharide for lead (PbII), cadmium (CdII), chromium (CrVI), iron (FeIII), as well as the effect of reaction time, concentrations ofheavy metal solutions, pH value of the solutions on its removal capability for these heavy metals have been studied.The results show excellent removal effect of the exopolysaccharide for those heavy metals. The sequence from high to low for capability of binding heavy metals of the exopolysaccharide is Fe(III)>Pb(II)> Cd(II)>Cr(VI). Weak acid or neutral condition is more favorable for heavy metal binding than strong acid condition.According to graded standards of acute oral toxicity ( LD50 ) regulated by "Toxicological evaluation procedure and method for food safety", the exopolysaccharide and its sulphatised products were tested of no toxicity by acute oral toxicity test in mice and they are most probably toxic free substance. The exopolysaccharide in this study was modified by sulphatizing and the sulphatised exopolysaccharide was then tested for the successful substitution of -OSO3- by infrared spectroscopy and also studied for its anticoagulant activity. The experiment reveals that the sulphatised exopolysaccharide have significant anticoagulant activity. The acute toxicological test for lymphocyte was carried out by MTT method, aad conte...