LAUSR

Gradients negatively affect performance in large‐scale bioreactors; however, they are difficult to predict at laboratory scale. Dynamic microfluidics single‐cell cultivation (dMSCC) has emerged as an important tool for investigating cell behavior in rapidly changing environments. In the present study, dMSCC, biosensors of intracellular parameters, and robustness quantification were employed to investigate the physiological response of three Saccharomyces cerevisiae strains to substrate and pH changes every 0.75–48 min. All strains showed higher sensitivity to substrate than pH oscillations. Strain‐specific intracellular responses included higher relative glycolytic flux and oxidative stress response for strains PE2 and CEN.PK113‐7D, respectively. Instead, the Ethanol Red strain displayed the least heterogeneous populations and the highest robustness for multiple functions when exposed to substrate oscillations. This result could arise from a positive trade‐off between ATP levels and ATP stability over time. The present study demonstrates the importance of coupling physiological responses to dynamic environments with simultaneous characterization of strains, conditions, individual regimes, and robustness analysis. All these tools are a suitable add‐on to traditional evaluation and screening workflows at both laboratory and industrial scale, and can help bridge the gap between these two.