为了提高1株光滑球拟酵母(Candida glabrata)高产突变菌株的丙酮酸产量和底物转化率,对发酵过程进行了系统优化。首先,对诱变筛选获得的7株菌株进行发酵验证,确定最佳菌株为C. glabrata 4H2。对种子培养基重要成分及浓度进行优化,确定最佳氮源为大豆蛋白胨,质量浓度为10 g/L。突变菌株在30 ℃条件下摇瓶发酵52 h,产量达到(48.56±0.46) g/L,生产强度为0.93 g/(L·h),糖酸转化率为0.46 g/g,比优化前分别提高了25.0%、52.5%和43.8%。基于上述结果,在15 L发酵罐中进行发酵条件优化,确定了以初始葡萄糖质量浓度为80 g/L,当葡萄糖质量浓度剩余55 g/L时,恒速流加70 g/L葡萄糖的补料发酵工艺,最终丙酮酸的产量达到最高,为(86.63±0.29) g/L,较摇瓶水平提高了78.4%,生产强度为1.07 g/(L·h),糖酸转化率为0.78 g/g。研究表明,发酵过程优化强化光滑球拟酵母生产丙酮酸是一种有效的方法,该研究为进一步提升工业水平丙酮酸发酵性能奠定了基础。
In order to improve the titer and yield of pyruvic acid, the fermentation conditions for pyruvic acid production by Candida glabrata mutants were systematically optimized. A strain C. glabrata 4H2 with better performance in accumulating pyruvic acid was firstly selected from seven screened strains. Then, various types and concentrations of nitrogen source were tested for seed cultivation in shake flasks. The optimum nitrogen source was determined to be soybean peptone and its optimal concentration was 10 g/L. As a result, the titer, yield and productivity of pyruvic acid of the optimized fermentation process reached (48.56±0.46) g/L, 0.46 g/g and 0.93 g/(L·h), respectively, after 52 h shake-flask cultivation at 30 ℃, which were increased by 25.0%, 43.8% and 52.5%, respectively. Further, the fermentation optimization was performed in a 15 L bioreactor. First, the optimum initial glucose concentration was determined to be 80 g/L. Then, when the concentration of residual glucose was lowered down to 55 g/L, 70 g/L glucose was supplemented into the bioreactor with a constant feeding approach. The final titer of pyruvic acid reached (86.63±0.29) g/L, which was 78.4% higher than that of shake-flask cultivation. The productivity and yield of pyruvic acid reached 1.07 g/(L·h) and 0.78 g/g, respectively. The results indicated that the fermentation optimization effectively improved pyruvic acid production in C. glabrata, which laid a foundation for the industrial production of pyruvic acid.
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