PUB - Publikationen an der Universität Bielefeld

In large-scale bioprocesses, the production organism is subject to fluctuating environmental conditions, e.g., dissolved oxygen, substrate concentration, and pH value, which are negligible at the laboratory scale. The gradients resulting from the fluctuating environmental conditions are generated by insufficient mixing of the volume. This can not only affect individual cells as they travel through the bioreactor in time periods ranging from a few seconds to minutes, depending on the size of the bioreactor, but also the metabolism, production yield, and growth of the production organism. The present work contributes to the study of these gradient zones and cellular movement through bioreactors. A new microfluidic system, dynamic microfluidic single-cell cultivation (dMSCC), was developed and established, which allows fluctuating medium oscillations up to 5 seconds. Other advantages of the new dMSCC device are the high degree of parallelization and control measurements of the media parameters simultaneously with the oscillation. For this purpose, the widely used, biotechnologically relevant bacterium *Corynebacterium glutamicum* was used to investigate the influence of different fluctuating environmental conditions on its growth and morphology. An initial study of nutrient oscillations with intervals between 10 seconds and 1 hour showed significant drops in the growth rate of *C. glutamicum*, especially in the oscillation range between 5 and 15 minutes. This indicated that regulatory and metabolic processes of the cell are severely affected by oscillation. Moreover, a systematic investigation of a bioprocessrelevant and often neglected parameter, pH value, was performed by analysing the cell response after single stress pulses. As a result, cell viability was found to decrease with increasing stress duration from 2 to 9 hours, with ~9% viability after 9 hours of stress at pH 5. In this context, a scale comparison between the traditional scale-down reactor (STR-STR) and the dMSCC device was performed for a specific pH 6/7 oscillation. Here, both devices showed a similar decrease in growth rate under oscillating conditions compared to a reference measurement under constant perfusion conditions. Following the comparability of the dMSCC device with traditional devices, cellular lifelines representing cells travelling through the different substrate zones of a large-scale bioreactor, created using computational fluid dynamics simulations, were analysed in the dMSCC system for the first time. It was found that the fluctuations of the different concentrations had a significant impact on the decrease in the growth rate of *C. glutamicum*. In this work, a system for dynamic microfluidic cultivation was developed and established as a future single-cell scaledown reactor, which provides a high potential for studying gradients to determine more robust strains to fluctuating conditions, further optimizing the bioprocess. Furthermore, the presented device can be optimized to enable additional applications such as long-term cultivations and investigation of other important bioprocess-relevant organisms, e.g., *Escherichia coli* and *Bacillus subtilis*. To achieve improved comparability with traditional devices, such as scale-down systems, the device needs to be optimized and characterized for multiparameter studies. Limitations such as new oscillation limitations of the device need to be considered and evaluated.