实现高密度发酵和高产孢率是当前丁酸梭菌发酵面临的重要挑战,为了解决这一问题,该研究通过单因素和响应面试验并结合分段pH调控策略优化了丁酸梭菌发酵条件及产孢条件。结果表明,丁酸梭菌三角瓶规模发酵培养基最佳组成为:10.34 g/L可溶性淀粉、30.96 g/L酵母浸粉、5.12 g/L磷酸氢二钾、4.00 g/L乙酸钠、0.50 g/L半胱氨酸盐酸盐、0.80 g/L氯化钙、0.60 g/L硫酸镁、0.20 g/L硫酸锰;最佳发酵条件为:温度37 ℃、接种量1%、初始pH 7.0。5 L发酵罐发酵实验发现,当pH值维持在5.5时,菌体量达到最大水平,而产孢最适pH值为6.0。因此采用分阶段pH调控策略,待培养基初始pH值由7.0降低至5.5,通过流加氨水保持该pH至菌体进入稳定期,随后将pH值调至6.0维持到发酵结束。该策略下,5 L发酵罐37 ℃培养20 h的丁酸梭菌菌体量达到最大,为3.3×109个/mL,比优化前提高了3.23倍,产孢率达到90%以上。
Achieving high-density fermentation and increased sporulation rates are critical challenges in the fermentation of Clostridium butyricum.To address this issue, the present study optimized the fermentation and sporulation conditions of C.butyricum through single factor and response surface experiments, combined with a segmented pH control strategy.Results indicated that the optimal composition of the C.butyricum triangular flask scale fermentation medium was as follows:10.34 g/L of soluble starch, 30.96 g/L of yeast extract, 5.12 g/L of dipotassium hydrogen phosphate, 4.00 g/L of sodium acetate, 0.50 g/L of cysteine hydrochloride, 0.80 g/L of calcium chloride, 0.60 g/L of magnesium sulfate, and 0.20 g/L of manganese sulfate.The optimal fermentation conditions included a temperature of 37 ℃, an initial pH of 7.0, and an inoculum of 1%.The experiment conducted on the 5 L fermentation tank showed that the maximum biomass level was achieved when the pH was maintained at 5.5.On the other hand, the optimal pH for sporulation was found to be 6.0.Therefore, a staged pH control strategy was employed.As the pH was decreased from an initial 7.0 to 5.5, ammonia solution was continuously added to maintain the pH level at 5.5 until the microbial culture entered the stable phase.The pH was then adjusted to 6.0 and maintained until the fermentation process was completed.Following this strategy, the biomass of C.butyricum cultured in a 5 L fermentation tank at 37 ℃ for 20 h, reached its maximum level of 3.3×109 cells/mL, which was 3.23 times higher than that before optimization, with a sporulation rate of over 90%.
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