Experimental k_S 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 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.