米根霉发酵产L-乳酸的中和剂研究及代谢通量分析
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摘要
在米根霉发酵产L-乳酸过程中,一般采用CaCO3作为酸中和剂,不仅L-乳酸损失大,且环境污染严重。为降低L-乳酸生产成本,寻求替代CaCO3的新型中和剂,本文通过摇瓶发酵优化得出以CaCO3、NaOH溶液和氨水作为中和剂的最适培养基组成,通过罐发酵实验确定了CaCO3、NaOH溶液和氨水的最适中和剂浓度,并在最适浓度基础上考察了米根霉的菌体形态和发酵动力学,实现了NaOH溶液和氨水作为中和剂的半连续发酵,同时对其进行代谢通量分析。主要研究结论如下：
1.通过单因素和正交试验,优化得出采用CaCO3、NaOH溶液和氨水作为中和剂时的培养基组成：(1)以CaCO3作为中和剂的最优培养基组成：葡萄糖120g/L, (NH4)2SO4 4.0g/L, KH2PO4 0.15g/L, NaH2PO4 0.20g/L, ZnSO4·7H2O 0.22g/L, MgSO4·7H2O 0.35g/L。(2)以NaOH溶液作为中和剂的最优培养基组成：葡萄糖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)以氨水作为中和剂的最优培养基组成：葡萄糖120g/L, (NH4)2SO41.0g/L, MgSO4·7H2O 0.35g/L, ZnSO4·7H2O 0.22g/L, NaH2PO4 0.15g/L, KH2PO4 0.15g/L。
2. NaOH溶液和氨水作为中和剂时,添加Ca2+后L-乳酸产量平均提高7.3倍；通过罐发酵得出米根霉发酵产L-乳酸的最适pH值为5.5±0.2, NaOH溶液、氨水溶液作为中和剂的最佳浓度分别为10mol/L、25%,通过摇瓶发酵试验得出CaCO3最佳浓度为60g／L；在最适NaOH溶液、氨水、CaCO3浓度基础上进行罐发酵得菌丝体小球直径分别为0.2-1.2mm、1.2-2.2mm、0.8-1.8mm,残糖分别为2.58g/L、1.37g/L、22.78 g/L, L-乳酸产量分别为74.34g/L、80.61g/L、75.80g/L,发酵强度分别为1.03g/(L-h)、1.12g/(L·h)、1.40g/(L·h);而使用NaOH溶液和CaCO3复合中和剂、氨水和CaCO3复合中和剂时发酵强度皆为1.18 g/(L·h),高于NaOH溶液、氨水的发酵强度,低于CaCO3的发酵强度。
3.在米根霉半连续发酵过程中,使用NaOH溶液作为中和剂时,L-乳酸平均发酵强度为1.53g/(L·h),以氨水作为中和剂时,L-乳酸平均发酵强度为1.65g/(L·h),高于分批发酵的产酸强度。
4.简单构建了米根霉As3.819胞内代谢网络模型。应用代谢通量分析法,分别以10mol/LNaOH溶液、25%氨水、60g/LCaCO3作为中和剂时不同发酵阶段胞内代谢通量分布的变化。以NaOH溶液为中和剂,第12h、36h、60h产物L-乳酸的流量速率分别为0.93mmol/g/h、2.05mmol/g/h和5.83mmol/g/h;以氨水为中和剂时,第12h、36h、60h产物L-乳酸的流量速率分别为1.48mmol/g/h、2.02 mmol/g/h、5.94mmol/g/h;以CaCO3作为中和剂,第12、30h、48h产物L-乳酸的流量速率分别是10.54mmol/g/h、4.72mmol/g/h、7.30mmol/g/h。
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.
1.通过单因素和正交试验,优化得出采用CaCO3、NaOH溶液和氨水作为中和剂时的培养基组成：(1)以CaCO3作为中和剂的最优培养基组成：葡萄糖120g/L, (NH4)2SO4 4.0g/L, KH2PO4 0.15g/L, NaH2PO4 0.20g/L, ZnSO4·7H2O 0.22g/L, MgSO4·7H2O 0.35g/L。(2)以NaOH溶液作为中和剂的最优培养基组成：葡萄糖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)以氨水作为中和剂的最优培养基组成：葡萄糖120g/L, (NH4)2SO41.0g/L, MgSO4·7H2O 0.35g/L, ZnSO4·7H2O 0.22g/L, NaH2PO4 0.15g/L, KH2PO4 0.15g/L。
2. NaOH溶液和氨水作为中和剂时,添加Ca2+后L-乳酸产量平均提高7.3倍；通过罐发酵得出米根霉发酵产L-乳酸的最适pH值为5.5±0.2, NaOH溶液、氨水溶液作为中和剂的最佳浓度分别为10mol/L、25%,通过摇瓶发酵试验得出CaCO3最佳浓度为60g／L；在最适NaOH溶液、氨水、CaCO3浓度基础上进行罐发酵得菌丝体小球直径分别为0.2-1.2mm、1.2-2.2mm、0.8-1.8mm,残糖分别为2.58g/L、1.37g/L、22.78 g/L, L-乳酸产量分别为74.34g/L、80.61g/L、75.80g/L,发酵强度分别为1.03g/(L-h)、1.12g/(L·h)、1.40g/(L·h);而使用NaOH溶液和CaCO3复合中和剂、氨水和CaCO3复合中和剂时发酵强度皆为1.18 g/(L·h),高于NaOH溶液、氨水的发酵强度,低于CaCO3的发酵强度。
3.在米根霉半连续发酵过程中,使用NaOH溶液作为中和剂时,L-乳酸平均发酵强度为1.53g/(L·h),以氨水作为中和剂时,L-乳酸平均发酵强度为1.65g/(L·h),高于分批发酵的产酸强度。
4.简单构建了米根霉As3.819胞内代谢网络模型。应用代谢通量分析法,分别以10mol/LNaOH溶液、25%氨水、60g/LCaCO3作为中和剂时不同发酵阶段胞内代谢通量分布的变化。以NaOH溶液为中和剂,第12h、36h、60h产物L-乳酸的流量速率分别为0.93mmol/g/h、2.05mmol/g/h和5.83mmol/g/h;以氨水为中和剂时,第12h、36h、60h产物L-乳酸的流量速率分别为1.48mmol/g/h、2.02 mmol/g/h、5.94mmol/g/h;以CaCO3作为中和剂,第12、30h、48h产物L-乳酸的流量速率分别是10.54mmol/g/h、4.72mmol/g/h、7.30mmol/g/h。
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.
引文
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