【作者】 李晓华；

【导师】 张克旭；

【作者基本信息】 天津科技大学 ， 发酵工程， 2004， 硕士

【摘要】 本论文根据代谢控制发酵和代谢流分析理论，研究了L-缬氨酸高产菌5L罐的发酵条件。主要研究内容和结果如下： (1)以已有的摇瓶分批发酵最优条件为基础，对菌株TV2564进行了5L发酵罐分批发酵试验。首先研究了不同碳氮比对缬氨酸发酵的影响，结果表明高碳氮比或高浓度初糖不利于缬氨酸发酵。 (2)根据5L发酵罐中溶氧变化情况及不同K_La对缬氨酸发酵的影响，结合缬氨酸发酵的生长与生产耦合性研究及发酵过程中代谢流分配的特点，得到了两阶段供氧的控制方式。以5L罐分批发酵试验数据为依据，利用MATLAB工具软件建立了菌体生长、产物形成和底物消耗的动力学模型，实验值与拟合值接近，拟合情况较好。 (3)构建L-缬氨酸生产菌TV2564的代谢网络，依据代谢流分析原理，应用MATLAB线性规划计算得到了L-缬氨酸生产菌不同发酵时期的代谢流分配。通过分析葡萄糖经EMP、HMP与TCA循环以及关键性节点GLC6P、PEP和PYR的代谢流分配，为发酵过程控制提供理论指导。 (4)以分批发酵最优条件为依据，对菌株TV2564进行了5L罐补料分批发酵研究。进一步研究了溶氧在补料发酵方式下的变化情况，寻找到适宜的供氧方式，即根据菌体生长的四个不同阶段：延滞期、对数生长期、稳定期和衰亡期分别调节不同转速，以满足菌体生长和产物合成的溶氧需求。 (5)确定了最佳发酵工艺，即初始低糖低铵，产酸期控制糖浓度在2.0g／L以下，发酵前期及后期补加少量玉米浆，自动流加氨水控制pH值，发酵过程根据菌体生长的四个阶段分别调节不同转速，以满足生长和产酸的溶氧需求，在此工艺下，菌体生长良好，发酵后劲足，产酸期延长，到后期流加80％的浓糖，发酵72h产L-缬氨酸52.68g／L。 (6)利用不锈钢膜过滤技术对缬氨酸发酵液进行前处理，去除菌体和蛋白质，效果良好，去除蛋白率可达79.89％。并初步研究了缬氨酸的提取工艺。更多还原

【Abstract】 According to the theory of metabolic control fermentation and metabolic flux analysis, the dissertation focuses on the fermentation conditions of L-valine high-yielding strain .The main research contents and results are as follows:(1) Based on the optimized conditions of flask, Fermentation in 5L fermenter of L-valine by TV2564 has proceeded. The effect of different C/N on fermentation was studied, and the results show high C/N or high concentration of initial glucose is adverse to fermentation of valine.(2) According to the change of DO in 5L fermenter, with dynamics analysis during fermentation process, two stages oxygen supply was provided .The L-valine batch fermentation kinetics was studied by MATLAB tool based on the experimental data from 5-liter fermenter’s batch fermentation. Three kinetic models were constructed which could reflect the regularity of growth, product formation and substrate consumption in the process of batch fermentation.(3) The metabolic network was proposed for L-valine producing strain. The metabolic flux distributions of Brevibacterium flavum TV2564 during the different fermentation period were calculated by linear program of MATLAB software according to metabolic flux analysis theory. The metabolic flux distributions of EMP, HMP, TCA cycle and the key principle nodes of GLC6P, PEP and PYR were analyzed. At the same time it can be indicated to provide theory guide for the fermentation process.(4) Fed batch fermentation was studied with TV2564 according to the optimum condition of batch fermentation. According to the change of DO in fed-batch fermentation, the optimized oxygen supply was provided. The optimum conditions was obtained, which were the technology of low glucose and amino, feeding corn syrup liquid in the former and later phase of fermentation, controling pH by feeding amino liquid, feeding 80% glucose in later phase, production of L-valine reached 52.68g/L after fermentation for 72 hours under the optimized conditions.(5) By membrane filtration, the cells and protein were leached. The results are perfect, and the removal rate of protein is 79.89%.更多还原