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

According to the theory of metabolic control fermentation, the dissertation mainly focuses on the breeding of high-pyruvate-producing mutant and its fermentation conditions.1. The method of HPLC was defined to determine pyruvate concentration qualitatively and quantitatively in the fermentation broth. The optimal chromatographic conditions was SCR-101H chromatographic column, room temperature, flow phage , flow rate 0.8mL/min, pH3.8. It proved that HPLC was fit for the determination of pyruvate in broth because of its accuracy and speediness by the tests of reproductivity and recovery rate on this condition,. Furthermore, it can determine other organic acids simultaneously. Consequently, it is a reliable analytic method for the study on breeding and fermentation conditions2. One high-pyruvate-producing strain Torulopsis glabrata TGP19 was derived by mutagenic treatment with EMS. A strain T.glabrata TG-06 which produce most pyruvate was selected from seven original strains by means of fermentation. Then, T.glabrata TG-06 was treated by mutagenic EMS, and one high-pyruvate-producing strain T.glabrata TGP19 was selected, which doesn't grow on the medium consisted of pyruvate as sole carbon source. The production of pyruvate was 30.5g/L, which enhanced 28.7%. Its genetic labels are the auxotroph of nicotinic acid, thiamin-HCl, biotin, and pyridoxine-HCl.3. The batch fermentation mediums and conditions of T.glabrata TGP19 in shake flask were studied. The factors, such as seed medium, fermentation medium and fermentation conditions were determined. Pyruvate production 39.6g/L and conversion rate 49.5% were achieved after 48h batch fermentation in shake flask on the super conditions.4. The fermentation of T.glabrata TGP19 in 5L bioreactor was studied preliminary. On the basis of single factor experiment in shake flask and batch fermentation 5L bioreactor, the following experiment was carried out. The initial glucose concentration was decreased to 60g/L, and feed glucose twice in the course of fermentation. Meanwhile, the carbon and nitrogen ratio was maintained constantly, namely 25: 1. Consequently, pyruvate of 61.9g/L, conversion rate of 53.8% and productivity of 1.29g/(L-h) were achieved after 48h fermentation in 5L bioreactor. The maximum levels were yield 67.8g/L, conversion rate 49.4%, productivity 1.08g/(L-h) after 63h feed-batch fermentation on conditions that total glucose concentration was 140g/L. One can see that both conversion rate and productivity exceed overseas' excellent yield. Determination by HPLC of productsin bioreactor shows that the product only consists of pyruvate, which is very beneficial to extraction downstream, and no other acids.