| Fumaric acid is a kind of important C4 chemical materials. It is widely used in food, pharmaceutical, chemical, resin and plasticizer industries. Currently, fumaric acid is produced mainly by chemical methods in industry. This situation has caused great interest in the fumaric acid production by fermentation, which mainly includes the optimization of fermentation conditions, the control of mycelia morphology, genetic alterations of fixed-point, etc. However, it is relatively rare to regulate the production of fumaric acid by R.oryzae from the angle of microorganism metabolism. In this paper, Rhizopus oryzae CICC40351 were selected as the strain, obtaining the optimal culture medium of synthesis of fumaric acid by using response surface optimization. On this basis, the influencing mechanism of different exogenous additives on R.oryzae fermentation and key enzymes in the fermentation process were studied. The main content and results are as follows:(1) By single-factor experiments and response surface methods, the conditions(culture conditions and medium factors) for the production of fumaric acid fermented by Rhizopus oryzae CICC40351 was optimized. The optimum fermentation conditions of seed development stage were as follows: initial pH of seed medium was 2.6~2.8, spore concentrations 2x108 spores per liter seed culture medium. The optimum fermentation medium were(g/L): glucose 80, KH2PO4 0.6, MgSO4·7H2O 0.5,(NH4)2 SO4 0.71, ZnSO4·7H2O 0.01, FeSO4·7H2O 0.0004, CaCO3 60. The fermentation temperature and agitation speed of rotary shaker were 37℃ and 220 r/min respectively. Under this conditions, fumaric acid production reached 38.1 g/L after 60 h fermentation. The fumaric acid yield was 0.39 g/g and the sugar consumption reached 100%.(2) Adding different intermediate metabolites have a greater impact on the metabolism of R.oryzae. When the addition of malic acid at a concentration of 4.0 g/L, fumaric acid productionincrease by 24.5% than that without addition of malic acid, the fumaric acid yield increasedby 26%. Ethanol was only 5.4 g/L, showed a downward trend with the increase of malic acid concentration.When exogenously adding 1.0 g/L oxaloacetic acid, fumaric acid production increased by 19.0%, while the concentration of ethanol increased by 23.3%. Adding 0.5 g/L oxaloacetic acid, fumarase activity reached 2.08 U/mg, increased 44.4%. When 1.5 g/L pyruvate was exogenously added, fumaric acid production increased 41.8% when compared with the initial condition.The glucose conversion rate reached 0.35 g/g, the production of ethanol was almost free from the addition of pyruvate.(3) Fermentation inhibitors significantly inhibited the process of R.oryzae fermentation. When exogenously adding 0.5 g/L formic acid, fumaric acid concentration was only 4.8 g/L. Ethanol production showed a large scatter with the increase of formic acid concentration. The highest concentration of ethanol was up to 20.6 g/L. The inhibition effect of acetic acid on R.oryzae was weaker than that of formic acid. When acetic acid concentration reached 1.0 g/L, fumaric acid concentration decreased 54.9%, while ethanol concentration improved 115.1%.(4) It was discovered that Ca2+, Zn2+, Ba2+ and Co2+ have active effect on fumarase when investigating the influence of metal ions on R.oryzae fermentation process. Mg2+, Zn2+, Ba2+ and Co2+ have activation on alcohol dehydrogenase, while Cu2+, Ca2+, Fe2+, Ni2+ and Fe3+ have inhibiting effect on alcohol dehydrogenase. When CaCl2 concentration was 4 mmol/L, fumaric acid concentrations reached up to 37.7 g/L, ethanol concentration was only 5.2 g/L.(5) 3L fermentor experiment was conducted on the basis of shake flask experiments. The influence of fermentor agitation speed, ventilation rate and fermentation pH on fumaric acid production by R.oryzae fermentation. The results indicate that the fermenter agitation speed of 400 r/min, ventilation rate of 1.0 vvm and fermentation pH of 4.5 was the optimal condition for R.oryzae fermentation. Fumaric acid concentration reached 32.6 g/L, the glucose conversion rate was 0.44 g/g, compared with shake flask experiments, increased 12.8%, ethanol concentration was 5.9 g/L.(6) Rhizopus oryzae major cellular metabolic network was established, according biochemical information, previous studies and cell composition of the physiology of Rhizopus oryzae. The metabolic flux analysis of Rhizopus oryzae was made in adding pyruvic acid, acetic acid and calcium chloride conditions, results were: ?adding pyruvate can effectively promote the production of malic acid, but metabolism of fumaric acid was inhibited; ?the addition of acetic acid can inhibit the overall metabolism of the cell, but can promote the accumulation of biomass and ethanol; ?calcium chloride can regulate carbon flow distribution, help promote products accumulation. |
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