Breeding Of Succinic Acid Producer And Study Of Its Bioconversion Conditions

In this dissertation, a succinic acid producting strains was treated by mutagen, and asuccinic acid producer GL-03was obtained. the conditions of batch cultivation andbioconversion conditions were studied. Moreover, the fed-batch bioconversion and the cellreuse was researched under oxygen deprivation conditions. At last, the metabolic fluxdistribution was compared between the original strain and this obtained stain. The detailedresults are as follows:(1) The mutant strain GL-03was obtained after treating by UV and DES, and theproduction of succinic acid was44.9g·L-1in the initial fermentation condition,which wasincreased by25.1%.(2) The composition and conditions of seed media was conducted. the optimal mediumwere glucose30g·L-1, corn steep liquor45g·L-1, urea4g·L-1, K2HPO4·3H2O2g·L-1,MgSO4·7H2O0.6g·L-1by experimental results. The conditions have been found: initial pH7.0, loading volume50mL/500mL. These factors were optimised by the Box-Behnkendesign method according to single factor experiments. the optimum medium were got(g·L-1):glucose89.1, K2HPO4·3H2O2.2, KH2PO41, MgSO4·7H2O0.6, MnSO4·4H2O0.02,FeSO4·7H2O0.02, VB10.2mg·L-1, biotin0.38mg·L-1, and fermentation conditions:seed age10h, dry cell concentration40g·L-1, temperature33℃. The yeild of succinic acid reached48.9g·L-1under the above-mentioned fermentation conditions.(3) The reuse of cell to produce succinic acid by Corynebacterium glutamicum GL-03was carried out under oxygen deprivation conditions. The results showed that the productivityof the reuse cell showed little change,the effects of inorganic salts on the second and thirdanaerobic transformation were studied, indicating that there was high yield of succinic acidalthough they had little influence on the anaerobic conversion.(4) In order to improve the yield of succinic acid, the conditions of fed-batchfermentation were studied. The production of acid was the highest when the original glucosewas40g·L-1. In this condition,35g·L-1glucose was added every12hours during fermentationprocess, the succinic concentration reached69.2g·L-1during48h.(5) The metabolic network of Corynebacterium glutamicum was established and themetabolic flux distribution was compared between the original strain and the obtained stain.Itwas found that the CO2fixed equation was improved, the flux to succinic acid increased by26.3%,the flux to pyruvate reduced by29.4%.