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

Abstract Knowledge about the specific affinity of whole cells toward a substrate, commonly referred to as k S , 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 k S . In this work, a recently developed substrate‐limited microfluidic single‐cell cultivation (sl‐MSCC) method is applied for the estimation of k S 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 k S of Corynebacterium glutamicum ATCC 13032 regarding glucose was investigated assuming a Monod‐type growth response. A k S 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 k S estimation, sl‐MSCC allows an affinity estimation by determining t D at concentrations less or equal to the k S value. Thus, sl‐MSCC lays the foundation for strain‐specific k S estimations under defined environmental conditions with additional insights into cell‐to‐cell heterogeneity.