解纤维梭菌培养条件的优化

Optimization of culture conditions for Clostridium cellulolyticum

摘要:

解纤维梭菌Clostridium cellulolyticum是产纤维小体的专性厌氧菌，由于其培养困难，目前仍难以实现高效培养。文中采用响应面法对产纤维小体的解纤维梭菌C. cellulolyticum高细胞密度培养的条件进行了优化。首先用Plackett-Burman实验设计对影响因素效应进行评价，筛选出的显著影响因素分别为：酵母提取物浓度、纤维二糖浓度及培养温度。之后用最陡爬坡实验设计逼近菌体最佳生长条件的区域范围。最后通过中心组合实验设计和响应面分析方法确定显著影响因素的水平和C. cellulolyticum的最优培养条件。优化后的显著影响因素酵母提取物浓度、纤维二糖浓度和培养温度分别为3 g/L、7 g/L和34 ℃。在最优条件下，摇瓶培养的菌体浓度OD600值由0.303提高到了0.586，增加了93.4％。在发酵罐批次培养条件下，菌体OD600值达到了3.432，比文献报道值高出了2.8倍。研究结果为C. cellulolyticum培养及应用研究提供了基础。

Abstract:

Clostridium cellulolyticum, as one of obligate anaerobic bacteria capable of secreting cellulosome, has not been efficiently cultured due to its strict requirement of growing conditions. In this study, culture conditions of C. cellulolyticum were optimized using response surface methodology. Plackett-Burman design was first used to screen the dominant impact factors for the growth of C. cellulolyticum, which were determined as yeast extract concentration, cellobiose concentration and culture temperature. The steepest ascent path design was then applied to gain the suitable range close to the optimal culture conditions for obtaining high cell density. The central composite design and the response surface analysis were finally used to determine the optimal levels of the influential factors, which were 3 g/L for yeast extract concentration, 7?g/L cellobiose concentration and 34 °C for culture temperature. The optimized medium was used for flask culture, and OD600 of C. cellulolyticum was increased from 0.303 to 0.586. With a pH-controlled fermentor at batch mode, OD600 reached 3.432, which was 2.8 times higher than elsewhere reported. These results support further study on the high-density culture of C. cellulolyticum and its application.