High cell density media for Escherichia coli are generally designed for aerobic cultivations -: consequences for large-scale bioprocesses and shake flask cultures
High cell density media for Escherichia coli are generally designed for aerobic cultivations -: consequences for large-scale bioprocesses and shake flask cultures
复制标题
DOI：
10.1186/1475-2859-7-26
复制
发表时间：
2008-08-07
影响因子：
6.4
通讯作者：
Neubauer, Peter
中科院分区：
文献类型：
--
作者：
Soini, Jaakko;Ukkonen, Kaisa;Neubauer, Peter
背景：对于在生物反应器中培养大肠杆菌，微量元素溶液通常设计用于在有氧条件下实现最佳生长。它们通常不含硒和镍。其中只有少数含有钼。这些元素是在厌氧条件下诱导的甲酸氢裂解酶 (FHL) 复合物的一部分。众所周知，氧限制出现在摇瓶培养物和局部大型生物反应器中，FHL 复合体的功能可能会影响过程行为。据描述，甲酸盐会在大规模培养物中积累，并可能对大肠杆菌产生毒性作用。虽然大肠杆菌的厌氧代谢已得到充分研究，但迄今为止尚未发表估计 FHL 复合物对限氧大肠杆菌生物过程影响的参考数据，但这对于更好地理解过程很重要。结果：进行了两组在触发限氧和甲酸盐积累的条件下的补料分批培养。摇瓶培养物中常见的永久性氧气限制是由于搅拌速率的降低而在生物反应器中引起的。瞬时氧限制被描述为最终发生在大型生物反应器的进料区，在由搅拌釜反应器和活塞流反应器 (PFR) 组成的双隔室规模缩小的生物反应器中进行模拟，并连续向 PFR 中加入葡萄糖。在两种模型中，在不添加硒、钼和镍的情况下，培养基中甲酸累积量高达约 20 mM。通过添加这些微量元素，甲酸积累量降低到低于在充分混合的实验室规模培养物中观察到的水平。有趣的是，在具有永久限氧的模拟器中，添加额外的微量元素会导致大量乳酸的积累，并降低生物量产量，但在缩小规模的两室生物反应器中却不会。结论：通过添加FHL复合体功能所必需的微量元素硒、镍和钼，可以完全防止大肠杆菌限氧培养物中甲酸的积累。对于大规模培养，如果可能存在葡萄糖梯度，则两室缩小生物反应器的结果表明，添加额外的微量元素是有益的。如果细胞反复暴露于瞬时氧限制，则在含有额外微量元素的培养物中不会观察到对生物量产量或任何其他生物过程参数的负面影响。
Background: For the cultivation of Escherichia coli in bioreactors trace element solutions are generally designed for optimal growth under aerobic conditions. They do normally not contain selenium and nickel. Molybdenum is only contained in few of them. These elements are part of the formate hydrogen lyase (FHL) complex which is induced under anaerobic conditions. As it is generally known that oxygen limitation appears in shake flask cultures and locally in large-scale bioreactors, function of the FHL complex may influence the process behaviour. Formate has been described to accumulate in large-scale cultures and may have toxic effects on E. coli.Although the anaerobic metabolism of E. coli is well studied, reference data which estimate the impact of the FHL complex on bioprocesses of E. coli with oxygen limitation have so far not been published, but are important for a better process understanding.Results: Two sets of fed-batch cultures with conditions triggering oxygen limitation and formate accumulation were performed. Permanent oxygen limitation which is typical for shake flask cultures was caused in a bioreactor by reduction of the agitation rate. Transient oxygen limitation, which has been described to eventually occur in the feed-zone of large-scale bioreactors, was mimicked in a two-compartment scale-down bioreactor consisting of a stirred tank reactor and a plug flow reactor (PFR) with continuous glucose feeding into the PFR.In both models formate accumulated up to about 20 mM in the culture medium without addition of selenium, molybdenum and nickel. By addition of these trace elements the formate accumulation decreased below the level observed in well-mixed laboratory-scale cultures. Interestingly, addition of the extra trace elements caused accumulation of large amounts of lactate and reduced biomass yield in the simulator with permanent oxygen limitation, but not in the scale-down two-compartment bioreactor.Conclusion: The accumulation of formate in oxygen limited cultivations of E. coli can be fully prevented by addition of the trace elements selenium, nickel and molybdenum, necessary for the function of FHL complex. For large-scale cultivations, if glucose gradients are likely, the results from the two-compartment scale-down bioreactor indicate that the addition of the extra trace elements is beneficial. No negative effects on the biomass yield or on any other bioprocess parameters could be observed in cultures with the extra trace elements if the cells were repeatedly exposed to transient oxygen limitation.