Study On Succinic Acid Production By Corynebacterium Acetoacidophilum-Δldh

Succinic acid is regarded as one of the most important platform chemical, which isproduced during cell metabolism, as a metabolic intermedicate of the Krebs cycle in themitochondrion. In this work, we focused on the succinic acid production by C.acetoacidophilum-Δldh. The metabolic shift of C. acetoacidophilum-Δldh under oxygendeprivation conditions, the optimization of succinic acid production conditions were studied.Comparing with the wild strain ATCC13870, ldhA-deficent mutant C.acetoacidophilum-Δldh produced no lactate with glucose consumption rate decreased by29.3%, while succinate and acetate concentrations were increased by45.6%and182%,respectively. Moreover, the NADH/NAD+rate was less than1(about0.7), and the activitiesof phosphoenolpyruvate carboxylase and acetate kinase of the ldhA-deficent mutant wereenhanced by84%and12times, respectively. These results showed that succinicate andacetate production pathways are strengthened by blocking lactate synthesis.The conversion conditions of C. acetoacidophilum-Δldh in shake flast were studied inthis dissertation. The cell during the mid-logarithmic and stable growth stage, was appropriatefor producing succinic acid; the optimal cell concentration, initial glucose concentration,supplied bicarbonate salt, initial glucose and sodium bicarbonate ratio, temperature, pH were30g(DCW)·L-1,100g·L-1,1:1,33℃,8.0, respectively. Under the optimal conditions C.acetoacidophilum-Δldh can produce succinic acid110g·L-1after convertion for130hours bycontinuous feeding convertion.The succinic acid convertion process of C. acetoacidophilum-Δldh in3-liter fermentorwas studied. The cell culture medium was optimized. The medium contained: glucose25g·L-1, K2HPO41.5g·L-1, MgSO4·7H2O0.6g·L-1, FeSO4·7H2O5mg·L-1, thiamine0.2mg·L-1,carbamide2.5g·L-1, corn steep liquor10g·L-1, biotin0.2mg·L-1. In this work, we discoveredthat a trace amount of oxygen is needed during producing succinic acid. Then we optimizedthe micro-aerobic conditions of C. acetoacidophilum-Δldh of producing succinic acid in3-liter fermentor. The ventilation condition was0.42vvm continuously. Under this conditionC. acetoacidophilum-Δldh can produce succinic acid67g·L-1after convertion for48hours,the productivity and yield was enhanced to1.5g·L-1·h-1and0.63g·g-1, respectively.In this work, the operation conditions of hollow fiber membrane module applied to thesuccinic acid production were studied. We achieved repeated conversion batch for three times,and the average concentration of succinic acid of the three batches reached56.28g·L-1, theproductivity and yield were1.13g·L-1·h-1,0.7g·g-1, respectively. After80hours continuouscell-recycle conversion process under the dilution rate of0.02h-1, succinic acid concentrationwas61g·L-1, residual glucose was5g·L-1, productivity and yield was1.22g·L-1·h-1and0.75g·g-1, respectively.