Study On The Breeding Of High-Pyruvate-Producing Mutant And Fermentation Conditions

Pyruvic acid is an important organic acid extensively used in industry. Compared with con- ventional methods, biological fermentation process is found to be less energy intensive and more environmental friendly, and nowadays more and more attentions are being paid on its fermentative way. According to the theory of metabolic control fermentation, the dissertation mainly focuses on the breeding of high-pyruvate-producing mutant and its fermentation conditions.One high-pyruvate-producing strain Torulopsis glabrata TK418 was selected from original strain T. glabrata TK101 by means of mutagenic treatment with U.V. and metabolic control. Its genetic labels are the auxotroph of nicotinic acid, thiamim-HCl, biotin, and pyridoxine-HCl andβ-FP and DG-resistant mutant. It was also a low ADH enzyme activity.The batch fermentation mediums and conditions of T. glabrata TK418 in shake flask were studied. The factors, such as seed medium, fermentation medium and fermentation conditions were determined. The initial glucose concentration was 106g/L,(NH4)2SO4, 8g/L, the carbon and nitrogen ratio was 25:1, Pyruvate production 58.20g/L andconversion rate 55.43% were achieved after 40hr batch fermentation in shake flask on the super conditions and productivity of 1.46g/(L·h).By using Box-Behnken center-united design and the method of response surface analysis together with SAS system, the optimum concentration conditions of thiamine, nicotinic acid, bio- tin, pyridoxine which were selected are 0.0125mg/L, 9.7mg/L, 0.022mg/L and 0.54mg/L. And thiamine was confirmed to be the most important factor affecting the production of pyruvic acid. It designed a way that can easily screen high-pyruvate-producing strain. It is a intelligently selected method which can highly improve the efficiency of strain screening. The principle can be described as the following: on the CaCO3 medium, a transparent ring can be exhibited based on the reaction of PYR produced by the strain and CaCO3 in the medium for pyruvate-calcium is a kind of soluble substance, it is obviously that the high-pyruvate-producing strain has a bigger dimension of the transparent ring. On the other hand, color reaction between TTC and ADH indi- cate the enzyme activities which have a proportional relation with color, our object strain is a weak-ADH-enzyme- activities type with a weak metabolic flux from PYR to alcohol. So the white color strain may be the right choice.