Enhancing Anaplerosis and Glutamate Conversion Yield by Adding a Co2Source and Regulating pH in Glutamate Fermentation

论文摘要

在谷氨酸发酵过程中，回补反应通过固定CO2，将丙酮酸转化为草酰乙酸为谷氨酸合成提供中间代谢产物，此途径的强弱直接影响谷氨酸的转化率，因此，强化回补反应是提高谷氨酸转化率的必需条件。在回补反应中，CO2并不直接参与反应，而是以HCO3-形式参与反应，由于CO2容易从液体中溢出，理论上可以通过添加碳酸氢钠增加HCO3-强化回补反应。而碳酸氢钠溶液pH在8.0左右，CO2在偏碱性溶液中更容易形成HCO3-，且谷氨酸棒杆菌的最适pH生长范围在6.5-8.5，因此，本研究分别对在谷氨酸产酸期单独添加碳酸氢钠及添加碳酸氢钠和调节pH耦联条件下的发酵性能进行了研究，并分析和比较了不同操作条件下关键酶活性和代谢流分配，找出了谷氨酸转化率提高的原因。论文主要内容和结论如下：1)通过摇瓶实验确定碳酸氢钠最适添加量为25mmol/L，最终谷氨酸浓度比对照提高23%，转化率比对照提高36%。碳酸氢钠在进入产酸期后开始添加，每隔2h添加一次，共添加4次。2)在5L发酵罐条件下，碳酸氢钠采用与摇瓶同样的添加方式，在谷氨酸产酸期单独添加碳酸氢钠和升高pH条件下，最终谷氨酸浓度可以达到对照正常水平，但是转化率没有明显提高。而只有在产酸期同时添加碳酸氢钠、升高pH和先升高pH后添加碳酸氢钠条件下，谷氨酸转化率显著提高（分别提高41%和35%），此时谷氨酸最终产量也可以达到对照水平。3)不同操作条件下代谢主途径中关键酶活性分析表明，仅仅提高丙酮酸羧化酶（PC）活性并不能有效提高谷氨酸转化率，只有在PC和异柠檬酸脱氢酶（ICDH）活性同时增强、且α-酮戊二酸脱氢酶（ODHC）和丙酮酸脱氢酶（PDH）活性弱化条件下，谷氨酸转化率才能显著提高。而不同操作条件下的异柠檬酸裂解酶（ICL）活性没有明显变化。4)不同操作条件下代谢计算分析表明，在单独添加碳酸氢钠条件下，进入回补反应的代谢流增加，异柠檬酸→α-酮戊二酸的代谢流没有明显变化，α-酮戊二酸进入继续氧化途径的代谢流降低，进入谷氨酸合成的代谢流增加。而在同时添加碳酸氢钠、升高产酸期pH条件下，进入回补反应的代谢流增加，虽然异柠檬酸→α-酮戊二酸的代谢流降低，α-酮戊二酸进入继续氧化途径的代谢流降低，进入谷氨酸合成的代谢流增加。

论文目录

• ABSTRACT
• 摘要
• CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW
• 1.1. Brief background
• 1.2. Applications of glutamate
• 1.2.1. Food industry
• 1.2.2. Medical/Pharmaceutical industries
• 1.2.3. Research Products, Analytical Standards and Chemical Intermediate
• 1.2.4. Other applications
• 1.3. Industrial fermentation of glutamate
• 1.4. Literature review
• 1.4.1. The microorganism: Corynebacterium glutamicum
• 1.4.2. Central metabolism of Corynebacterium glutamicum in brief
• 1.4.3. The problem
• CHAPTER 2 MATERIALS AND METHODS
• 2.1. Strain
• 2.2. Shake flasks seed and fermentation media composition and conditions
• 2.3. Fermentation media composition and conditions in 5 L bioreactor
• 2.3.1. Fermentation conditions
• 2.3.2. Fermentation medium
• 2.4. Analytical methods
• 2.4.1. Concentrations of cell, glucose and glutamate
• 2release amount and yields of glutamate/CO₂on glucose'>2.4.2. Determination of CO₂release amount and yields of glutamate/CO₂on glucose
• 2.4.3. Enzyme activity assays
• CHAPTER 3. RESULTS AND DISCUSSION
• 3.1. Preliminary experiments in shake flasks
• 3 addition were singly regulated'>3.2. Fermentation performance and enzymatic activities changes when pH and NaHCO₃ addition were singly regulated
• 3addition only'>3.2.1. NaHCO₃addition only
• 3.2.2. pH increase only
• 3and HEPE'>3.2.3. Addition of both NaHCO₃and HEPE
• 3addition were jointly regulated'>3.3. Fermentation performance and enzymatic activities changes when pH and NaHCO₃addition were jointly regulated
• 3addition'>3.3.1. Simultaneous increase of pH and commencement of NaHCO₃addition
• 3addition later'>3.3.2. Increase of pH first and commencement of NaHCO₃addition later
• 3addition first and increase of pH later'>3.3.3. Commencement of NaHCO₃addition first and increase of pH later
• 3regulation modes'>3.4. Metabolic flux analysis of glutamate fermentations under different pH/NaHCO₃regulation modes
• 3.4.1 Calculation principle or methods of metabolic flux analysis
• 3.4.2 The results of metabolic flux analysis
• GENERAL CONCLUSION
• RECOMMENDATIONS FOR FURTHER STUDY
• ACKNOWLEDGEMENTS
• REFERENCES
• List of publications