Lüchtrath Clara, Forsten Eva, Polis Romeos, Hoffmann Maximilian, Genis Aylin Sara, Kuhn Anna-Lena, Hövels Marcel, Deppenmeier Uwe, Magnus Jørgen, Büchs Jochen
AVT- Biochemical Engineering, RWTH Aachen University, Aachen, Germany.
Institute for Microbiology and Biotechnology, University of Bonn, Bonn, Germany.
Bioprocess Biosyst Eng. 2025 Jun;48(6):1007-1024. doi: 10.1007/s00449-025-03159-9. Epub 2025 Apr 18.
Vibrio natriegens is a fast-growing microbial workhorse with high potential for biotechnological applications. However, handling the bacterium in batch processes is challenging due to its high overflow metabolism and mixed acid formation under microaerobic conditions. For early process development, technologies enabling small-scale fed-batch cultivation of V. natriegens Vmax are needed. In this study, fed-batch cultivations in 96-well microtiter plates were successfully online-monitored for the first time with a µTOM device. Using the online-monitored oxygen transfer rate, a scale up to membrane-based fed-batch shake flasks was performed. The overflow metabolism was efficiently minimized by choosing suitable feed rates, and mixed acid formation was prevented. A glucose soft sensor using the oxygen transfer rate provided accurate estimates of glucose consumption throughout the fermentation, eliminating the need for offline sampling. Analyzing the impact of the inducer IPTG on the recombinant production of the enzyme inulosucrase revealed concentration-dependent effects in batch processes. In contrast, fed-batch operating mode resulted in high inulosucrase activity even without induction. Overall, an inulosucrase titer of 80 U/mL was achieved. In conclusion, the advantages of small-scale fed-batch technologies supported by a glucose soft sensor have been demonstrated for early process development for V. natriegens Vmax.
嗜盐弧菌是一种生长迅速的微生物，在生物技术应用方面具有很高的潜力。然而，由于其在微需氧条件下具有高溢流代谢和混合酸形成，在分批培养过程中处理这种细菌具有挑战性。对于早期工艺开发，需要能够对嗜盐弧菌Vmax进行小规模补料分批培养的技术。在本研究中，首次使用µTOM设备对96孔微量滴定板中的补料分批培养进行了在线监测。利用在线监测的氧传递速率，扩大规模至基于膜的补料分批摇瓶培养。通过选择合适的进料速率有效地最小化了溢流代谢，并防止了混合酸的形成。使用氧传递速率的葡萄糖软传感器能够在整个发酵过程中准确估计葡萄糖消耗，无需进行离线取样。分析诱导剂IPTG对酶菊粉蔗糖酶重组生产的影响，揭示了分批培养过程中的浓度依赖性效应。相比之下，补料分批操作模式即使在没有诱导的情况下也能产生高菊粉蔗糖酶活性。总体而言，菊粉蔗糖酶滴度达到了80 U/mL。总之，葡萄糖软传感器支持的小规模补料分批技术的优势已在嗜盐弧菌Vmax的早期工艺开发中得到证明。
