| As a C4-dicarboxylic acid, succinic acid is valuable due to its widely use in food, chemical and pharmaceutical industries. It is currently produced primarily from fossil fuels by chemical synthetic process. This process causes environmental pollution easily and the development is limited by the shortage of fossil fuels. Therefore, the biological synthetic process for succinic acid is more economical and acceptable. The commercial production of succinic acid by biological process will become one sustainable model of development due to the consumption of greenhouse gas-CO2, meanwhile, the microbial transformation of renewable resources to produce succinic acid can get rid of the dependence on petrochemical raw materials. Scholars have done a lot of effective research on the microbial production of succinic acid, however, there are still some problems to solve, such as the unclear metabolic network and the formation of by-product, particularly the low succinic acid productivity.Corynebacterium crenatum is one of the main glutamic acid production microorganisms used in the practical production for many years. It is known that the metabolic flux will be changed during the production of glutamic acid when the medium dissolved oxygen is insufficient. When the culture condition is changed to anaerobic, the carbon flow was directed into the succinic acid biosynthesis pathway. In this report, in order to enhance the succinic acid yield and productivity, the features of succinic acid production by Corynebacterium crenatum CICC202019under anaerobic conditions were investigated, and the intracellular key enzyme activity was analyzed. On the basis of above results and combined with metabolic inhibition analysis, the metabolic net of C. crenatum under anaerobic condition was constructed. Meanwhile, the metabolic net was modified by genetically engineering for decreasing the production of by-product and enhancing succinic acid productivity. In order to improve succinic acid productivity, the pH control process was also optimized. The main conclusions and results are as follows.(1) The characters of lactic acid and succinic acid production by C. crenatum were studied by comparing the products and enzyme activities under different aerobic conditions. When C. crenatum cells were cultured under anaerobic conditions, the cells could produce succinic acid and lactic acid from glucose even through their proliferation was arrested. About19.8g/L succinic acid and46g/L lactic acid were produced in22h, with the yield of succinic acid and lactic acid as0.27g/g and0.62g/g, respectively. In the dual-phase fermentation of C. crenatum, the cells proliferated quickly in the aerobic phase without the production of succinic acid and lactic acid. When aerobic conditions were changed to anaerobic phase by aerating with CO2gas, the concentration of succinic acid and lactic acid increased significantly, which were15g/L and51g/L, respectively. The results of high-cell-density fermentation indicated that a proximate linear relationship existed between cell concentration and the production rates of lactic acid and succinic acid. When dry cell concentration was60g/L,59g/L of lactic acid and23g/L of succinic acid were produced within2h. The productivity of lactic acid and succinic acid was29.5g/(L-h) and11.5g/(L-h), respectively. During cell-recycling repeated fermentation, about310g lactic acid and423g succinic acid were produced from780g glucose in100h. The volumetric productivities of succinic acid and lactic acid could maintain above4.2g/(L-h) and3.1g/(L-h) for at least100h. The characters of acid production by C. crenatum demonstrated that C. crenatum cells had sustained succinic acid production ability and high acid volumetric productivity under anaerobic conditions, those conclusions in this part are beneficial to produce succinic acid subsequently with C. crenatum.(2) Through analyzing the key enzyme activity in metabolic pathway, it was found that the phosphoenolpyruvate carboxylase and malate dehydrogenase enzyme activity in anaerobic conditions were higher than the value detected in aerobic conditions, meantime, isocitrate lyase enzyme activity was low. However, the isocitric dehydrogenase enzyme activity could not be detected. The effect of inhibitor on succinic acid production indicated that succinic acid concentration decreased43%with propane diacid concentration increasing from0to2g/L, while fluoroacetic acid had no obvious effect on succinic acid production. From the effect of ORP on succinic acid production, it was found that when reducing the ORP level from-40mv to-300mv, succinic acid concentration increased from14.4g/L to21.5g/L, meanwhile, the yield of succinic acid increased from0.18g/g to0.31g/g. The conclusions stated above in this part indicated that succinic acid was produced through C4pathway and glyoxylate cycle. Combined with Corynebacterium glutamicum genome information, the metabolic network of C. crenatum under anaerobic conditions was constructed. Metabolic flux calculation results indicated that the C4pathway was the main metabolic pathway for succinic acid production, the succinic acid produced by C4pathway accounted for93%of the whole succinic acid produced.(3) Lactic acid is the primary by-product when C. crenatum is used to produce succinic acid. The effect of lactate dehydrogenase(1dhA) knockout on the succinic acid production was studied. The IdhA gene was amplified and the knockout plasmid pMKA was constructed and electrotransformed. Compared with the wild strain, the lactate dehydrogenase (LDH) enzyme activity in mutant could not be detected, and glucose consumed by mutant decreased50%. The yield of succinic acid produced by mutant is0.62g/g, which is higher than the value (0.22g/g) obtained from the wild strain. The biosynthetic pathway was removed to enhance the yield of succinic acid by knocking out the1dhA gene.(4) The effect of ppc and pyc overexpression on the succinic acid production in1dhA mutant was investigated. Although the phosphoenolpyruvate carboxylase activity increased by1.5fold when ppc gene was overexpressed through pJA expression vector, but.the glucose consumption rate and the yield of succinic acid had no obvious change, implying that the phosphoenolpyruvate carboxylase was not the rate-limiting enzymes in the production of succinic acid by C. crenatum AldhA. The pyruvate carboxylase activity increased by8fold when pyc gene was overexpressed through pJC expression vector and the glucose consumption rate increased46%compared with C. crenatum AldhA, meanwhile, the yield of succinic acid increased from0.64g/g to0.68g/g.(5) The succinic acid biological production cost can be reduced by improving succinic acid production efficiency through optimizing appropriate pH and neutralizing agent. The results indicated, when the pH was6.8, the glucose consumed and succinic acid productivity was the maximum compared with the other pH, which were43.6g/L and1.88g/(L-h), respectively. The succinic acid concentration in broth was also the maximum, which achieved to30g/L. The fermentation efficiency obtained at pH6.8was better than that of the other pH. For controlling the pH during C. crenatum AldhA(pJC) fermentation, Mg(OH)2was superior to Ca (OH)2、KOH and NaOH. When Mg(OH)2as neutralizing agent, the yield of succinic acid reached the maximum, the glucose consumption rate and succinic acid productivity were obviously higher than that of Ca (OH)2, KOH and NaOH. When pH was6.8and with Mg (OH)2as neutralizing agent, there was78.5g/L glucose was consumed within22h, and the final succinic acid concentration reached53.8g/L, with the average succinic acid productivity as of2.45g/(L-h). Through optimizing the pH control process during succinic acid anaerobic fermentation, the suitable pH and neutralizing agent for C. crenatum△IdhA (pJC) anaerobic fermentation were obtained, which can provide technical support to large scale fermentation.A new succinic acid production bioprocess by C. crenatum was investigated in mineral medium under anaerobic conditions. The results indicated that C. crenatum cells can produce succinic acid with sustained acid production ability and high acid volumetric productivity. The biological process was more efficient than some of the conventional fermentations in terms of volumetric productivity and dispensability of complex nutrients. Through this project, the characteristics of succinic acid production by C. crenatum under anaerobic conditions were known, the obtained results in this paper have theoretical and practical significance for succinic acid industrial production. |
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