Enhancement Of Carboxylation Pathway And Process Optimization Of Fumaric Acid Production By Rhizopus Delemar

This thesis focuses on fumaric acid production by Rhizopus delemar NRRL1526. Effects of different medium compositions on fumaric acid production were studied. Components of pre-culture medium and culture temperature has significant effects on the pellet formation of the fungi, by which affected the final fumaric acid yield. Besides, by regulation of the pyruvate carboxylase (PC) activity, the fumaric acid yield was significantly improved. The main results in this thesis include:(1) Effects of medium compositions on R. delemar growth and fumaric acid production were studied. The optimum fumaric acid production medium were obtained (g/L): glucose, 140; peptone, 1.17; KH₂PO₄, 0.3; MgSO₄·7H₂0, 0.4; ZnSO₄·7H₂O, 0.044; FeSO₄·7H₂O, 0.01; CaCO₃, 60. By using the optimum medium, the fumaric acid yield was 46.19 g/L, which was 189.05% higher than the control. However, the final glucose concentration was 45 g/L when the initial glucose concentration was 140 g/L. This caused difficulties in the fumaric acid purification process and the waste of glucose. Therefore, the initial glucose concentration was changed into 100 g/L, with final glucose concentration and fumaric acid yield was 4.25 g/L and 27.10 g/L, respectively. Besides, addition of vitamin B1, vitamin B6 and vitamin B12 had negative effects on fumaric acid production.(2) The effects of temperature and medium composition, including glucose, soybean peptone, and inorganic ions, on pellet formation and pellet diameter of R. delemar NRRL1526 during pre-culture were studied. The initial glucose and soybean peptone concentrations significantly affected the pellet diameter. Within a certain range, pellet diameter decreased with increased initial substrate concentrations. However, above this range there was an opposite trend. Thus, optimal concentrations of substrate during pre-culture were beneficial for generation of small pellets of R. delemar. Furthermore, dry cell weight and yield of fumaric acid tended to increase with decreased pellet diameter. Based on the pellet morphology optimization, the final fumaric acid concentration was improved by 46.13% in the flask and 31.82% in the stirred bioreactor tank fermentation. Besides, the theoretical basis of different morphology formed also have been demonstrated based on fungal physiology, such as the spore germination, hyphal branching and mycelial intertwining. The relationship between morphology and broth reology was also studied. The viscosity of the broth was lower when the cell was formed in pellets than others, which was benefit for mass transfer. Besides, the model of glucoamylase secreted by A. niger was studied. The synthesis and secretion of glucoamylase located in the zone of leading hyphae in microscropic view and colony periphery in macroscopic view. Therefore, the pellet fungal, with the large specific surface area, would be benefit for fumaric acid production.(3) Deregulated glycolytic flux was caused by posttranslational modification of pyruvate carboxylase (PC) that resulted in formation of a highly active CO₂-fixed enzyme. In order to avoid complex posttranslational modification, the native pdcA promoter and terminator were used to start and end encode the S. cerevisiae PYC1 gene. The modified PYC1 ligated with pRS303H, which was inserted into the PacI/SalI site, was electroporated into germinated R. delemar spores. In R. delemar-pRS303H-PC transformants obtained, the specific pyruvate carboxylase activity was 5.1 times (0.228 U/(mg protein)) than the parent one. Furthermore, fumaric acid concentration achieved to 46.67 g/L, which was 1.19 times than the parents.