Mutagenesis Breeding Of Actinobacillus Succinogenes For High-producing Aciduric And Optimization Of Mutant Fermentation Conditions

Succinic acid, a common natural organic acid, is one of the products among the Krebs cycle of organism, it exists widely in the human body, animals, plants and microbes. Succinic acid is used in following fields: surface active agent, chemicals, additives and foaming agent; chelator that is used in electroplating industry; acidification agent in the food industry, pH improver, flavor substances and antiseptics; and it is also used in the production of Medicine, antibiotics, amino acids and vitamins; it is the main raw materials of biodegradable plastic PBS. In recent years, producting succinic acid by microorganism fermentation becomes the focus of research, most research are focused on Actinobacillus succinogenes at home and abroad, Research about Actinobacillus succinogenes in the domestic are mainly includes the screening of strains, Mutagenesis, kinetic model and batch fermentation. In this paper, Actinobacillus succinogenes ATCC55618was irradiated by Mutagenesis Ultraviolet radiation, and through that procedure we obtained a strain M1that can tolerance low pH, then M1was irradiated by both UV and nitrosoguanidine (NTG), strainsRl, R2that are of high producing are obtained, then the strain R2was analyzed among culture media optimization.This study includes:(1) Using plate counting method to determine the proper UV mutagenesis time is50s,0.1mL of culture is at logarithmic phase is coated at medium plates that with pH7.0, pH6.7, pH6.4, pH6.1, pH5.8, pH5.5, pH5.2, pH4.9, pH4.6and of pH4.3, pH4.0, the critical pH4.6of Actinobacillus succinogenes ATCC55618is determined, after UV mutagenesis, a strain M1is obtained, whose yield is17.01g/L, increased by10.53%than the original strain.(2) Using the plate counting method to determine the proper NTG concentration500?g/ml, then M1was irradiated by both UV and nitrosoguanidine (NTG). After two rounds of the compound mutation, high producing strains R1, R2were obtained, the yield of succinic acid of R1is20.87g/L, improved22.69%than M1, improved35.61%than original strain,the yield of succinic acid of R2is24.41g/L, improved16.96%than R1,43.50%than M1,58.61%than the original strain ATCC55618.(3) The influence of the culture media factors on the growth of R2were carbon source, nitrogen source, incipient pH, pH regulator, and metalion pH of fermentation broth. The consequence of the analysis showed that glucose as carbon source, yeast powder as nitrogen source, incipient pH7.0, ammonia water as pH regulator, Mn2+as metalion.(4) Design single factor experiment of the factors:glucose, yeast extract, Mn2+. Addition of glucose was20g/L,25g/L,30g/L,35g/L,40g/L, addition of yeast extract was4g/L,7g/L,10g/L,13g/L,16g/L, addition of Mn2+was0.2g/L,0.3g/L,0.4g/L,0.5g/L,0.6g/L. Succinic acid production among single factor experiments were analyzed. The optimal amount of each component to add, as follows:glucose30g/L, yeast extract10g/L, and Mn2+0.3g/L.(5) To further optimize the conditions of the medium, the amount of the three factors were optimized by orthogonal experiments. Addition of glucose was27g/L,30g/L,33g/L; addition of yeast extract was8g/L,10g/L,12g/L; addition of Mn2+was0.2g/L,0.3g/L,0.4g/L, respectively. Succinic acid production are analyzed. The optimal amount of glucose27g/L, yeast extract10g/L, Mn2+0.2g/L.