Experimental kS estimation: A comparison of methods for Corynebacterium glutamicum from lab to microfluidic scale.

Knowledge about the specific affinity of whole cells toward a substrate, commonly referred to as kS , is a crucial parameter for characterizing growth within bioreactors. State-of-the-art methodologies measure either uptake or consumption rates at different initial substrate concentrations. Alternatively, cell dry weight or respiratory data like online oxygen and carbon dioxide transfer rates can be used to estimate kS . In this work, a recently developed substrate-limited microfluidic single-cell cultivation (sl-MSCC) method is applied for the estimation of kS values under defined environmental conditions. This method is benchmarked with two alternative microtiter plate methods, namely high-frequency biomass measurement (HFB) and substrate-limited respiratory activity monitoring (sl-RA). As a model system, the substrate affinity kS of Corynebacterium glutamicum ATCC 13032 regarding glucose was investigated assuming a Monod-type growth response. A kS of <70.7 mg/L (with 95% probability) with HFB, 8.55 ± 1.38 mg/L with sl-RA, and 2.66 ± 0.99 mg/L with sl-MSCC was obtained. Whereas HFB and sl-RA are suitable for a fast initial kS estimation, sl-MSCC allows an affinity estimation by determining tD at concentrations less or equal to the kS value. Thus, sl-MSCC lays the foundation for strain-specific kS estimations under defined environmental conditions with additional insights into cell-to-cell heterogeneity.

dMSCC: a microfluidic platform for microbial single-cell cultivation of Corynebacterium glutamicum under dynamic environmental medium conditions.

In nature and in technical systems, microbial cells are often exposed to rapidly fluctuating environmental conditions. These conditions can vary in quality, e.g., the existence of a starvation zone, and quantity, e.g., the average residence time in this zone. For strain development and process design, cellular response to such fluctuations needs to be systematically analysed. However, the existing methods for physically imitating rapidly changing environmental conditions are limited in spatio-temporal resolution. Hence, we present a novel microfluidic system for cultivation of single cells and small cell clusters under dynamic environmental conditions (dynamic microfluidic single-cell cultivation (dMSCC)). This system enables the control of nutrient availability and composition between two media with second to minute resolution. We validate our technology using the industrially relevant model organism Corynebacterium glutamicum. The organism was exposed to different oscillation frequencies between nutrient excess (feasts) and scarcity (famine). The resulting changes in cellular physiology, such as the colony growth rate and cell morphology, were analysed and revealed significant differences in the growth rate and cell length between the different conditions. dMSCC also allows the application of defined but randomly changing nutrient conditions, which is important for reproducing more complex conditions from natural habitats and large-scale bioreactors. The presented system lays the foundation for the cultivation of cells under complex changing environmental conditions.