Schulte Andreas, Brockmann Janik, Müller Nina, Anderlei Tibor, Büchs Jochen
AVT - Biochemical Engineering, RWTH Aachen University, Forckenbeckstr. 51, D-52074, Aachen, Germany.
Kuhner Shaker GmbH, Kaiserstr. 100, 52134, Herzogenrath, Germany.
J Biol Eng. 2025 Jan 28;19(1):11. doi: 10.1186/s13036-025-00480-5.
Shake flasks are essential tools in biotechnological development due to their cost efficiency and ease of use. However, a significant challenge is the miniaturization of process analytical tools to maximize information output from each cultivation. This study aimed to develop a respiration activity online measurement system via off-gas analysis, named "Transfer rate Online Measurement" (TOM), for determining the oxygen transfer rate (OTR), carbon dioxide transfer rate (CTR), and the respiration quotient (RQ) in surface-aerated bioreactors, primarily targeting shake flasks.
Sensors for off-gas analysis were placed in a bypass system that avoids the shaking of the electronics and sensors. An electrochemical oxygen sensor and an infrared CO sensor were used. The bypass system was combined with the established method of recurrent dynamic measurement phases, evaluating the decrease in oxygen and the increase in CO during stopped aeration. The newly developed measurement system showed high accuracy, precision and reproducibility among individual flasks, especially regarding CTR measurement. The system was compared with state-of-the-art RAMOS technology (Respiration Activity Monitoring System, see explanation below) and calibrated with a non-biological model system. The accuracy of RQ measurement was +-4% for the tested range (8% filling volume, OTR and CTR: 0-56 mmol/L/h), allowing for the determination of metabolic switches and quantitative analysis of metabolites. At ambient CO levels, a CTR resolution of less than 0.01 mmol/L/h was possible. The system was applied to the microbial model systems S. cerevisiae, G. oxydans, and E. coli. Physiological states, such as growth vs. protein production, could be revealed, and quantitative analysis of metabolites was performed, putting focus on RQ measurements.
The developed TOM system showcases a novel approach to measuring OTR, CTR, and RQ in shaken bioreactors. It offers a robust and accurate solution for respiration activity analysis. Due to its flexible design and tunable accuracy, it enables measurement in various applications and different shake flasks.
由于成本效益高且易于使用，摇瓶是生物技术开发中的重要工具。然而，一个重大挑战是将过程分析工具小型化，以最大限度地提高每次培养的信息输出。本研究旨在开发一种通过尾气分析进行呼吸活性在线测量的系统，称为“转移速率在线测量”（TOM），用于测定表面曝气生物反应器中的氧转移速率（OTR）、二氧化碳转移速率（CTR）和呼吸商（RQ），主要针对摇瓶。
用于尾气分析的传感器放置在一个旁路系统中，该系统可避免电子设备和传感器的晃动。使用了一个电化学氧传感器和一个红外CO传感器。旁路系统与已建立的循环动态测量阶段方法相结合，评估停止曝气期间氧气的减少和CO的增加。新开发的测量系统在各个摇瓶之间显示出高精度、精密度和可重复性，尤其是在CTR测量方面。该系统与最先进的RAMOS技术（呼吸活性监测系统，见下文解释）进行了比较，并用非生物模型系统进行了校准。在测试范围内（8%填充体积，OTR和CTR：0-56 mmol/L/h），RQ测量的准确度为±4%，可用于确定代谢转换和代谢物的定量分析。在环境CO水平下，CTR分辨率可低于0.01 mmol/L/h。该系统应用于微生物模型系统酿酒酵母、氧化葡萄糖酸杆菌和大肠杆菌。可以揭示生理状态，如生长与蛋白质生产，并进行代谢物的定量分析，重点是RQ测量。
所开发的TOM系统展示了一种在振荡生物反应器中测量OTR、CTR和RQ的新方法。它为呼吸活性分析提供了一种强大而准确的解决方案。由于其灵活的设计和可调的精度，它能够在各种应用和不同的摇瓶中进行测量。
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