磷酸根对海洋克雷伯氏菌合成1,3-丙二醇代谢的影响

摘要

Klebsiella pneumoniae HSL4 is an excellent strain for the fermentation of 1,3-propanediol(1,3-PDO), which was isolated from coastal mangrove sediment environment. Many nutrient factors are involved in the culture medium ofK. pneumoniae HSL4. Among them, phosphate is one of the main factors, which have a significant impact on the fermentation. The effects of the low concentration of phosphate (LP: 1.6×10?4mol·L?1 PO43?) and the high concentration phosphate (HP: 5.0×10?3mol·L?1 PO43?) on cell growth, 1,3-PDO and by-product (acetic succinic acid, lactic acid, acid, 2,3-butanediol, and ethanol) production were studied. The impacts of phosphate on the metabolism ofK. pneumoniae HSL4 were characterized by metabolic flux analysis. The results showed that the final concentration of 1,3-PDO was 75g·L?1 under both LP and HP in the culture medium. Significantly, the 1,3-PDO conversion was higher, but the substrate glycerol consumption and by-products (except for 2,3-butanediol) accumulation were less under LP condition compared to HP condition. The productions of acetic acid and ethanol were higher under HP condition. The accumulations of acetic acid and ethanol will change the environment of fermentation and increase the difficulty of product separation in the downstream. Metabolic flux distributions in four fermentation periods (adjustmental phase, log phase, stationary phase, and decline phase) were analyzed. This study will help researchers to choose reasonable phosphate concentration in the culture broth and provide information for process control and metabolic regulation in 1,3-PDO fermentation byK. pneumoniae HSL4.%海洋克雷伯氏菌Klebsiella pneumoniaeHSL4是一株分离于海洋沉积物环境的用于发酵生产1,3-丙二醇(1,3-propanediol, 1,3-PDO)的优良菌株.K. pneumoniaeHSL4发酵生产1,3-PDO的培养基包含多种营养因子,其中磷酸根对发酵过程影响较大.利用5L发酵罐进行补料发酵实验,分别考察培养基中低磷(1.6×10–4mol·L–1 PO43–)和高磷(5.0×10–3mol·L–1 PO43–)浓度条件下K. pneumoniaeHSL4发酵生产1,3-PDO过程中细胞生长、1,3-PDO及多种副产物生成的规律,分析初始磷酸根浓度差异对K. pneumoniaeHSL4不同发酵阶段代谢流分布的影响.结果表明,培养基中高、低初始磷酸根浓度条件下, 1,3-PDO的终浓度均在75g·L–1左右.在低磷条件下,底物甘油消耗量较少,具有较高的1,3-PDO转化率; 除2,3-丁二醇外, 其他副产物在发酵液中的积累均小于高磷条件下副产物的积累.高磷条件促进乙酸、乙醇、乳酸等副产物生成, 改变中、后期发酵环境, 增加下游分离提取1,3-PDO的压力.根据高、低初始磷酸根浓度条件下菌体生物量、产物、副产物合成的差异, 对细菌4个发酵时期(延滞期、对数生长期、平台期和衰亡期)的代谢流分布及其变化进行了分析.研究结果可以为合理配比培养基中磷酸盐浓度、进行发酵生产1,3-PDO过程控制与代谢调控提供理论依据.