| CaCO3 was commonly used as an acid neutralizer in the process of producing L-lactic acid by Rhizopus oryzae. CaSO4 residue from the recovery procedure not only caused the loss of L-lactic acid during filtration but also brought enormous environmental pressures. Therefore, in order to reduce production cost, to seek a new neutralizer instead of CaCO3, CaCO3,NaOH solution and ammonia were researched respectively. The optimal medium composition of the three different neutralizers were optimized through fermenting by shake flasks; the optimal neutralizer concentration of CaCO3,NaOH solution and ammonia were derived by tank fermentation, morphology of Rhizopus oryzae and fermentation kinetics were studied based on the optimal neutralizer concentration; Achieved the semi-continuous fermentation with NaOH solution and ammonia as the neutralizer, and made research based on metabolic flux analysis. The main findings were as follows:1. The culture medium ingredient with CaCO3,NaOH solution and ammonia as the neutralizer were optimized by the combination.of single factor experiments and orthogonal experiments. (1) The optimal medium composition with CaCO3 as the neutralizer:glucose 120g/L, (NH4)2SO4 4.0g/L, KH2PO4 0.15g/L, NaH2PO4 0.15g/L, ZnSO4·7H2O 0.22g/L, MgSO4·7H2O 0.35g/L, CaCO3 60g/L. (2) The optimal medium composition with NaOH solution as the neutralizer:glucose 120g/L, (NH4)2SO4 4.0g/L, MgSO4·7H2O 0.35g/L, ZnSO4·7H2O 0.22g/L, NaH2PO4 0.10g/L, KH2PO4 0.15g/L. (3)The optimal medium composition with ammonia as the neutralizer:glucose 120g/L, (NH4)2SO4 1.0g/L, MgSO4·7H2O 0.35g/L, ZnSO4·7H2O 0.22g/L, NaH2PO4 0.15g/L, KH2PO4 0.15g/L.2. Effects of Ca2+ on L-lactic acid production were studied with NaOH solution and ammonia as the neutralizer. The results showed that after adding Ca2+, the average production of L-lactic acid was 7.3 times more than the control one; The optimum pH value of L-lactic acid production by Rhizopus oryzae was 5.5±0.2, the optimal concentration of NaOH solution ammonia as the neutralizer were 10mol/L,25% based on tank fermentation respectively, the optimum CaCO3 adding amount was 60g/L based on shake flask fermentaton; fermentation on the optimum NaOH,ammonia,CaCO3 concentration, the physical form of Rhizopus oryzae were uniform pellet with diameter of 0.2-1.2mm,1.2-2.2 mm,0.8-1.8mm, residual sugar were 2.58g/L,1.37g/L,22.78g/L, L-lactic acid yields were 74.34g/L,0.61g/L 75.80g/L, fermentation intensity were 1.03g/(L·h),1.12g/(L·h),1.40g/(L·h); both of the fermentation intensity of NaOH'solution and CaCO3 composite neutralizer,ammonia and CaCO3 composite neutralizer were 1.18g/(L·h), higher than the fermentation intensity of NaOH solution ammonia but lower than fermentation intensity CaCO3.3. The semi-continuous fermentation using NaOH solution,ammonia as the neutralizer were studied. The results were as following:when using NaOH solution as the neutralizer, the average L-lactic fermentation intensity was 1.53g/(L·h), when the ammonia as the neutralizer, the average L-lactic fermentation was 1.65g/(L·h), higher than fermentation intensity of batch fermentation.4. The intracellular metabolic network model of Rhizopus oryzae As3.819 was built through literature review and biochemical information of Rhizopus oryzae and experimental determination. Application of metabolic flux analysis, the changes of distribution in intracellular metabolic flux were researched with 10mol/LNaOH solution,25% ammonia,60g/L CaCO3 as the neutralizer. The results showed that: the flow rate of L-lactic acid at 12h,36h,60h with NaOH solution as the neutralizer were 0.93mmol/g/h,2.05mmol/g/h and 5.83mmol/g/h respectively; the flow rate of L-lactic acid at 12h,36h,60h with ammonia as the neutralizer were 1.48mmol/g/h,2.02mmol/g/h,5.94mmol/g/h respectively; the flow rate of L-lactic acid at 12h,30h,54h with CaCO3 as the neutralizer were 10.54mmol/g/h,4.72mmol/g/h,7.30mmol/g/h respectively. |
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