Microbial cells are often exposed to rapidly fluctuating environmental conditions. A novel microfluidic system for the cultivation of single cells and small cell clusters is presented under dynamic environment conditions.

Microbial cells in industrial large-scale bioreactors are exposed to fluctuating conditions, e.g., nutrient concentration, dissolved oxygen, temperature, and pH. These inhomogeneities can influence the cell physiology and metabolism, e.g., decelerate cell growth and product formation. Microfluidic systems offer new opportunities to study such effects in great detail by examining responses to varying environmental conditions at single-cell level. However, the possibility to reproduce large-scale bioreactor conditions in microscale cultivation systems has not yet been systematically investigated. Hence, we apply computational fluid dynamics (CFD) simulations to analyze and compare three commonly used microfluidic single-cell trapping and cultivation devices that are based on (i) mother machines (MM), (ii) monolayer growth chambers (MGC), and (iii) negative dielectrophoresis (nDEP). Several representative time-variant nutrient concentration profiles are applied at the chip entry. Responses to these input signals within the studied microfluidic devices are comparatively evaluated at the positions of the cultivated cells. The results are comprehensively presented in a Bode diagram that illustrates the degree of signal damping depending on the frequency of change in the inlet concentration. As a key finding, the MM can accurately reproduce signal changes that occur within 1 s or slower, which are typical for the environmental conditions observed by single cells in large-scale bioreactors, while faster changes are levelled out. In contrast, the nDEP and MGC are found to level out signal changes occurring within 10 s or faster, which can be critical for the proposed application.

The biotechnological production of fine chemicals, proteins and pharmaceuticals is usually hampered by loss of microbial performance during scale-up. This challenge is mainly caused by discrepancies between homogeneous environmental conditions at laboratory scale, where bioprocesses are optimized, and inhomogeneous conditions in large-scale bioreactors, where production takes place. Therefore, to improve strain selection and process development, it is of great interest to characterize these fluctuating conditions at large-scale and to study their effects on microbial cells. In this paper, we demonstrate the potential of computational fluid dynamics (CFD) simulation of large-scale bioreactors combined with dynamic microfluidic single-cell cultivation (dMSCC). Environmental conditions in a 200 L bioreactor were characterized with CFD simulations. Computational lifelines were determined by combining simulated turbulent multiphase flow, mass transport and particle tracing. Glucose availability for Corynebacterium glutamicum cells was determined. The reactor was simulated with average glucose concentrations of 6 g m−3, 10 g m−3 and 16 g m−3. The resulting computational lifelines, discretized into starvation and abundance regimes, were used as feed profiles for the dMSCC to investigate how varying glucose concentration affects cell physiology and growth rate. In this study, each colony in the dMSCC device represents a single cell as it travels through the reactor. Under oscillating conditions reproduced in the dMSCC device, a decrease in growth rate of about 40% was observed compared to continuous supply with the same average glucose availability. The presented approach provides insights into environmental conditions observed by microorganisms in large-scale bioreactors. It also paves the way for an improved understanding of how inhomogeneous environmental conditions influence cellular physiology, growth and production.