摘 要：解脂耶氏酵母（Yarrowia lipolytica）在氮饥饿条件下能够分泌大量柠檬酸，此过程受到发酵培养基溶解氧浓度的极大影响。本研究中，我们选择油酸作为氧载体来提高Y. lipolytica的柠檬酸发酵产率。当培养基油酸添加量为2%时，柠檬酸合成过程保持较高的溶氧量（溶氧> 42.1%），柠檬酸产量达到32.1 g l-1，菌体生物量为8.8 g l-1。我们还测定了发酵过程中底物吸收速率、产物合成速率及柠檬酸合成关键酶活性。结果表明，添加油酸后柠檬酸合成效率显著增强。同时发现油酸在发酵过程中能够被细胞吸收利用，这意味着油酸可同时作为氧载体及共基质参与Y. lipolytica柠檬酸合成。在发酵罐中扩大培养时，柠檬酸产量可达到36.4 g g l-1，发酵时间为192 h，证实了油酸可用于Y. lipolytica柠檬酸的大规模生产。71522

毕业论文关键词：柠檬酸，溶解氧，发酵，油酸，优化，氧载体

Abstract: Yarrowia lipolytica is able to secrete large mounts of citric acid (CA), which is greatly affected by the dissolved oxygen concentration (DOC) in the fermentation medium. In this study, oleic acid was selected as oxygen-vector to improve DOC during CA fermentation. When 2% (v/v) of oleic acid was added to the culture broth, higher DOC (>42.1%) was determined throughout the CA synthesis phase. The yield of CA reached a maximum of 32.1 g l-1 (25.4% higher than the control) and the biomass was 8.8 g l-1. The substrate uptake rate, products formation rate and key enzyme activities were also determined, and the results indicated that CA synthesis was strengthened with oleic acid added. Furthermore, it was detected that oleic acid could be assimilated by the cells, which meaned that oleic acid could be served both as oxygen-vector and co-substrate for CA synthesis by Y. lipolytica. In a bioreactor with working volume of 3 L, the highest concentration of CA reached to 36. 4 g l-1 in the presence of 2% (v/v) oleic acid after 192 h of fermentation. These results confirmed that oleic acid could be applied in the large-scale production of CA production by Y. lipolytica.

Keywords: Citric acid, Dissolved oxygen, Fermentation, Oleic acid, Optimization, Oxygen-vector 

目录

1前言4

2材料和方法5

2.1实验材料5

2.1.1菌种5

2.1.2培养基与试剂5

2.2实验方法6

2.3统计分析8

3结果与讨论8

3.1 CA发酵中氧载体的选择8

3.2油酸对CA发酵过程的影响8

3.3油酸浓度对柠檬酸发酵的影响9

3.4油酸对Y. lipolytcia SWJ-1b生理特征的影响10

3.5油酸添加时间的优化12

3.6油酸对生物反应器中生产CA的影响13

结论15

参考文献16

致谢19

1 前言

柠檬酸（Citric acid，CA）是三羧酸循环的中间产物，有许多工业用途，如作为食物、化妆品、饮料行业和制药行业中的的增味剂、酸化剂和防腐剂，动植物油的稳定剂，或洗涤剂中的起泡及漂白成分[1]。CA的工业生产主要是黑曲霉（Aspergillus niger）以蔗糖、糖蜜或葡萄糖浆为碳源通过深层发酵获得。然而这一过程通常伴随着一些环境问题，如固体有机及金属废弃物的产生。此外，众所周知，A. niger菌株在是毒理学上属于危险菌株，在过去的几年中，操作人员因接触黑曲霉孢子而感染得曲霉病的案例被不断被报道[2]。因此，探索其它更为经济安全的CA生产过程来取代A. niger发酵迫在眉睫。最近人们发现，解脂耶氏酵母（Yarrowia lipolytcia）在CA发酵中具有很大优势。Y. lipolytica最初从食物中分离，经过美国食品药品管理局（FDA）认证，被认为对人体健康完全无害。除此之外，Y. lipolytica较A. niger具有更为广泛的原料范围（这使得低价碳源的选择成为可能）[3]，其对重金属的敏感性较小，因此非精制碳源（如粗甘油）可用于Y. lipolytica CA的发酵生产，而酵母菌易于基因改造的遗传特性使得其在近年的CA发酵研究中备受关注[4-6]。论文网