Isolation, Identification, Mutation Breeding And Fermentation Condition Optimization Of A Strain For Producing Succinic Acid

There are some advantages in succinic acid fermentation by bacteria, such as low processing cost and little pollution for environment. Because of using renewable agriculture resources and green house gas CO2, the research for succinic acid fermentation has become the hotspot of bioconversion research at home and abroad in last years. In high selectivity enrichment culturing process and rapid screening procedure, the succinic acid producers with starchness and saccharide were isolated from bovine rumen. After the related physiological and biochemical reaction identification, the parent strain was irradiated by UV. During some screening plates the mutant strains were obtained with high yield of succinic acid. And then, the culture medium ingredient and fermentation condition were optimized. The main results were as following:1. The monensin and fumarate sodium were added in enrichment culture for selective culturing most of bacteria, which would produce succinic acid from bovine ruminal contents. Five hundred strains producing organic acid were isolated with yellow hoop as selective mark presented in the bromocresol green medium. After simple fermentation six stains with high yield were selected by TLC preliminary screening and the improved HPCE secondary screening. Among them B-15 had the highest succinic acid product with 11.5 g·L^-1, meanwhile the by-product yield such as formic acid(4.65 g·L^-1) and acetic acid(6.25 g·L^-1) was fewer. The strain B-15 was identified as Actinobacillus genus by physiological and biochemical characteristics identification.2. The parent strain B-15 was irradiated by UV. The mutant FB-4 was obtained through the bromomcresol green plate, the fluoroacetate plate and the allyl alcohol plate in turn. The fermentation product succinic acid yield (19.90 g·L^-1) of FB-4 increased 70%, and the by-product ethanol, formate and acetate were 7.55 g·L^-1, 2.05 g·L^-1 and 4.85 g·L^-1, respectively.3. The optimal culture composing were glucose 50 g·L^-1, yeast extract 10 g·L^-1,corn starch liquid 5 g·L^-1, K₂HPO₄ 3 g·L^-1, MgCl₂·6H₂O 0.4 g·L^-1, CaCl₂ 0.3 g·L^-1, NaCl 0.5 g·L^-1, MgCO₃ 15 g·L^-1 for mutant FB-4 fermentation. Complex N sources affected the fermentation greatly. The optimal shake flask fermentation conditions were 37℃, inoculation 5%, rotational rate 100 r·min^-1 and 40% CO₂-5% H₂ gas condition. In initial pH 7.0 and fermentation time 48 hours, the final succinic acid concentration in broth was 24 g·L^-1.