铜绿假单胞杆菌O-2-2产鼠李糖脂的发酵培养基优化及LC-MS／MS分析

Medium Optimization for Rhamnolipids Production by Pseudomonas aeruginosa O-2-2 and LC-MS/MS Analysis

鼠李糖脂是一种性能优良的生物表面活性剂,在生物医药、环境保护、二次采油等方面具有很高的应用潜力。采用响应面分析法,对铜绿假单胞杆菌O-2-2的培养基进行了优化。Plackett-Burman(PB)实验设计表明,磷酸盐、硝酸盐和微量元素对鼠李糖脂的产量具有显著影响。Box-Behnke(BB)优化确定最佳培养基组成为磷酸盐、硝酸盐和微量元素用量分别为3.2g/L、13.76g/L和5.17ml,理论的最大产量为8.48g/L,与实测糖脂产量8.85g/L接近。摇瓶优化后的鼠李糖脂产量较优化前的6.24g/L提高了30.8%。最优化条件下采用10%的接种量逐级放大,并通过补料发酵,最终200L罐的鼠李糖脂产量达到70g/L,发酵时间仅为110h。采用新发明的二次蒸馏工艺,鼠李糖脂纯度达86.6%。液质联用(LC-MS)分析表明所生产的鼠李糖脂成分及含量为:双糖单脂32.9%、双糖双脂17.02%、单糖单脂3.16%、单糖双脂33.54%。

Rhamnolipids are excellent biosurfactants which are widely used in biomedicine, oil recovery and environmental protection. Response surface methodology （RSM） was employed to optimize the medium for rhamnolipids production by Pseudomonas aeruginosa 0-2-2. The result of the Plackett-Burman design showed that phosphate, nitrate and trace elements had significant effects on rhamnolipids production. The Box-Behnke design suggested that the optimum values of phosphate, nitrate and trace elements were 3. 2g/L, 13. 76g/L and 5.17ml, respectively. Rhamnolipids production reached 8.85g/L, which was in agreement with the predicted production of 8.48 g/L. Compared with the production of original level （6.24g/L）, 30.8% increment had been obtained. By scaling up the fermentation to 200L fermentor with the optimal medium, the concentration of rhamonlipids reached to 70g/L, and the fermentation period was kept in ll0h. When the secondary-distillation was used as a new separation process, the rhamnolipid purity was 86.6%. In general, relative content of each ingredient in this product was： mono-rhamno-mono-lipid 1.79%, mono-rhamno-di-lipid 36.83%, di-rhamno- mono-lipid 39.64% , di-rhamno-di-lipid 20.70%.