Screening Of Pigment-free Pullulan High Producing Microorganism And Research Of Optimal Culture Media

Pullulan is a kind of secondary metabolite producing by Aureobasidium pullulans. Pullulan is an unbranched homopolysaccharide which consists of maltotriose units with a-(1,6)-glucosidic bonds. Pullulan have extensive applying prospect in many fields. In this experiment,a UV mutant with no pigment and high yield of pullulan was obtained from wild strain A.pullulans NG.The yeast cells of NG which were cultivated in liquid YND medium with a certain concentration were irradiated by ultraviolet radiation. According to the survival rate,2.0 min was the optimum UV irradiation time. After 3 rounds of UV mutagenesis, we finally got a free pigment mutant UVMU3-1 with higher polysaccharide yield. The result of comparative analysis of morphologic between mutant and wild type strains showed that there was no cell polymorphism mutation of UVMU3-1. The difference between mutant strains and wild strains was not obvious in swollen differentiation rate at pH4.5,.The 96h grow curves in liquid PDA medium of mutant UVMU3-1 and wild had no significant difference. Dry biomass, polysaccharide yield, and polysaccharide-producing capacity per cell were little difference between two strains in PDA,YAD and YPD flask fermentation.Fermentation in 10 liters fermenter with YAD medium under controlling pH at 4.5 showed that dry biomass was nearly equal between two strains, but polysaccharide yield, polysaccharide-producing capacity per cell, and transformation rate of glucose of UVMU3-1 was 1.6,1.65, and 1.59 times above wild strains respectively. Polysaccharides of UVMU3-1 were identified as pullulan by infrared spectrum.Optimization of fermentation medium by response surface method for UVMU3-1 was carried out. PB experiment showed that glucose and potassium dihydrogen phosphate were chiefly postive effecting factors; sodium nitrate, yeast extract and Tween-80 were positive influencing factors; ammonium sulfate and magnesium sulfate were negative effecting factors.The maximum pullulan yield of UVMU3-1 was gained at 18.01g/L when glucose and potassium dihydrogen phosphate were 65g/L and 5.01g/L respectively in steepest ascent experiment. On the basis of steepest ascent experiment results, we design the central composite experimental with 2 factors and 5 levels for optimization of culture medium. The largest response value would be achieved at 19.765g/L when glucose and potassium dihydrogen phosphate were67g/L and 5.18g/L respectinely.Actual pullulan yield of UVMU3-1 was 19.975g/L under the optimum medium condition, it was similar with the value of forecast. A higher polysaccharide yield (30.43g/L) was gained through tank fermentation by controlling the pH with the conversion rate of sugar at 45.42%. Compareing with flask fermentation, tank fermentation showed higher crude polysaccharide production, sugar yield of per unit cell and sugar conversion rate significantly. It indicated that UVMU3-1 could be a potential industrial production strains.