AVT-Biochemical Engineering, RWTH Aachen University, Forckenbeckstraße 51, 52074, Aachen, Germany.
Microb Cell Fact. 2018 May 9;17(1):69. doi: 10.1186/s12934-018-0917-8.
Screening in the fed-batch operation mode is essential for biological cultivations facing challenges as oxygen limitation, osmotic inhibition, catabolite repression, substrate inhibition or overflow metabolism. As a screening tool on shake flask level, the membrane-based fed-batch shake flask was developed. While a controlled supply of a substrate was realized with the in-built membrane tip, the possibilities for replenishing nutrients and stabilizing pH values was not yet exploited. High buffer concentrations were initially used, shifting the medium osmolality out of the biological optimum. As the growth rate is predefined by the glucose release kinetics from the reservoir, the resulting medium acidification can be compensated with a controlled continuous supply of an alkaline compound. The focus of this research is to establish a simultaneous multi-component release of glucose and an alkaline compound from the reservoir to enable cultivations within the optimal physiological range of Escherichia coli.
In combination with the Respiratory Activity MOnitoring System, the membrane-based fed-batch shake flask enabled the detection of an ammonium limitation. The multi-component release of ammonium carbonate along with glucose from the reservoir resulted not only in the replenishment of the nitrogen source but also in the stabilization of the pH value in the culture medium. A biomass concentration up to 25 g/L was achieved, which is one of the highest values obtained so far to the best of the author's knowledge with the utilization of a shake flask and a defined synthetic medium. Going a step further, the pH stabilization allowed the decrease of the required buffer amount to one-fourth establishing an optimal osmolality range for cultivation. As optimal physiological conditions were implemented with the multi-component release fed-batch cultivation, the supply of 0.2 g glucose in a 10 mL initial culture medium volume with 50 mM MOPS buffer resulted in a twofold higher biomass concentration than in a comparable batch cultivation.
The newly introduced multi-component release with the membrane-based fed-batch shake flask serves a threefold purpose of replenishing depleted substrates in the culture medium, stabilizing the pH throughout the entire cultivation time and minimizing the necessary amount of buffer to maintain an optimal osmolality range. In comparison to a batch cultivation, these settings enable to achieve higher biomass and product concentrations.
在面临氧气限制、渗透压抑制、分解代谢物抑制、基质抑制或代谢物溢出等挑战的生物培养中，分批补料操作模式的筛选至关重要。作为摇瓶水平上的筛选工具，开发了基于膜的分批补料摇瓶。虽然内置的膜尖端实现了基质的受控供应，但尚未利用补充营养物质和稳定 pH 值的可能性。最初使用高缓冲浓度，使培养基渗透压超出生物最佳范围。由于生长速率由储液器中葡萄糖释放动力学预先定义，因此可以通过控制连续供应碱性化合物来补偿由此产生的培养基酸化。本研究的重点是建立从储液器中同时释放葡萄糖和碱性化合物的多组分，以实现大肠杆菌在最佳生理范围内的培养。
结合呼吸活性监测系统，基于膜的分批补料摇瓶能够检测到铵限制。从储液器中同时释放碳酸铵和葡萄糖不仅补充了氮源，还稳定了培养基中的 pH 值。生物量浓度达到 25 g/L，这是作者所知的迄今为止在使用摇瓶和定义合成培养基时获得的最高值之一。更进一步，pH 值的稳定允许减少所需缓冲液的量至四分之一，为培养建立了最佳渗透压范围。由于多组分释放分批补料培养实现了最佳生理条件，与可比的分批培养相比，在 10 mL 初始培养基体积中用 50 mM MOPS 缓冲液供应 0.2 g 葡萄糖可使生物量浓度提高一倍。
新引入的基于膜的分批补料多组分释放有三重目的：补充培养基中耗尽的底物、在整个培养过程中稳定 pH 值以及最小化维持最佳渗透压范围所需的缓冲液量。与分批培养相比，这些设置可以实现更高的生物量和产物浓度。
