活细胞在线监控L-羟脯氨酸补料发酵工艺的研究
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摘要
为了研究活细胞在线监控补料技术对L-羟脯氨酸发酵的影响,在30 L发酵罐上安装活细胞在线检测仪,实时监测发酵液中活细胞数量,根据活细胞数量调整补料策略,分析活细胞在线监控补料对L-羟脯氨酸发酵补糖速率、产量、糖酸转化率和代谢流量分布的影响。结果表明,采用活细胞在线监控补料发酵工艺生产L-羟脯氨酸的最佳补糖策略是在发酵中后期及时降低补糖速率,最终降到9 g/(L·h),L-羟脯氨酸产量提高了11.39%,乙酸积累量减少了48.69%,糖酸转化率提高了14.92%,糖酵解途径和乙醛酸循环的代谢流分别减少了8.50%和16.89%,磷酸戊糖途径的代谢流增加了14.73%,乙酸合成的代谢流减少了4.97%,L-羟脯氨酸合成的代谢流提高了16.21%。上述结果说明采用活细胞在线监控补料技术生产L-羟脯氨酸,可以有效降低副产物的积累,提高L-羟脯氨酸产量及糖酸转化率。
In order to study the effect of online monitoring fed-batch fermentation of living cells on L-hydroxyproline fermentation,the live cell online monitor was used in the 30 L fermentor to monitor the number of living cells to adjust the feeding strategy,and the effect of online monitoring fed-batch fermentation of living cells on sugar supplement rate,glucose conversion rate and the metabolic flux channeled were analyzed. The results showed that the best sugar supplementation strategy for the production of L-hydroxyproline by the online monitoring of the living cells was to reduce the rate of sugar supplementation timely in the mid and later phase of fermentation, and finally to 9 g/( L·h). The production of L-hydroxyproline increased by 11. 39%,the accumulation of acetic acid decreased by 48. 69%,and glucose conversion rate increased by 14. 92%. At the same time,the flux channeled to EMP and glyoxyl circle decreased by 8. 50% and 16. 89%,respectively. However,the flux channeled to HMP increased by 14. 73%. The flux channeled to acetic acid decreased by 4. 97%,and the metabolic flux channeled to L-hydroxyproline increased by 16. 21%. These results indicated that online monitoring fed-batch fermentation of living cells could significantly reduce the accumulation of by-product,increase the production of L-hydroxyproline,and improve the glucose conversion rate.
In order to study the effect of online monitoring fed-batch fermentation of living cells on L-hydroxyproline fermentation,the live cell online monitor was used in the 30 L fermentor to monitor the number of living cells to adjust the feeding strategy,and the effect of online monitoring fed-batch fermentation of living cells on sugar supplement rate,glucose conversion rate and the metabolic flux channeled were analyzed. The results showed that the best sugar supplementation strategy for the production of L-hydroxyproline by the online monitoring of the living cells was to reduce the rate of sugar supplementation timely in the mid and later phase of fermentation, and finally to 9 g/( L·h). The production of L-hydroxyproline increased by 11. 39%,the accumulation of acetic acid decreased by 48. 69%,and glucose conversion rate increased by 14. 92%. At the same time,the flux channeled to EMP and glyoxyl circle decreased by 8. 50% and 16. 89%,respectively. However,the flux channeled to HMP increased by 14. 73%. The flux channeled to acetic acid decreased by 4. 97%,and the metabolic flux channeled to L-hydroxyproline increased by 16. 21%. These results indicated that online monitoring fed-batch fermentation of living cells could significantly reduce the accumulation of by-product,increase the production of L-hydroxyproline,and improve the glucose conversion rate.
引文
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[3]李兰,王泽建,金勇,等.电容法在线测定发酵过程中毕赤酵母浓度的研究[J].中国生物工程杂志,2014,34(3):91-95.
[4]R?NNEST N P,STOCKS S M,LANTZ A E,et al.Introducing process analytical technology(PAT)in filamentous cultivation process development:comparison of advancedonline sensors for biomass measurement[J].Journal of Industrial Microbiology&Biotechnology,2011,38(10):1 679-1 690.
[5]DUCOMMUN P,BOLZONELLA I,RHIEL M,et al.On-line determination of animal cell concentration[J].Biotechnology&Bioengineering,2001,72(5):515-522.
[6]KIVIHARJU K,SALONEN K,MOILANEN U,et al.Biomass measurement online:the performance of in situ measurements and software sensors[J].Journal of Industrial Microbiology and Biotechnology,2008,35(7):657-665.
[7]杨梦晨,户红通,蔡萌萌,等.L-色氨酸清液发酵工艺研究[J].食品与发酵科技,2017,53(2):29-33;39.
[8]陈宁,刘辉.柠檬酸钠对L-亮氨酸发酵代谢流分布的影响[J].高校化学工程学报,2008(3):478-483.
[9]宋翔,谢希贤,徐庆阳,等.苹果酸对L-谷氨酸发酵代谢流迁移的影响[J].生物加工过程,2009,7(5):58-62.
[10]马雷,程立坤,徐庆阳,等.柠檬酸钠对L-异亮氨酸发酵及代谢流量分布的影响[J].天津科技大学学报,2010,25(3):14-18.
[11]朱文泽,谢希贤,徐庆阳,等.柠檬酸钠对L-组氨酸发酵代谢流分布的影响[J].生物技术通讯,2009,20(2):202-204+212.
[12]刘力,桑跃,武永超,等.活细胞在线检测器在长双歧杆菌BBMN68培养中的应用研究[J].中国奶牛,2015(15):36-39.