Do seconds make a difference? Investigating strain-specific behavior of yeast in dynamic glucose environments

Background When applied for industrial-scale bioproduction, cells are subjected to ever-changing cultivation environments due to bioreactor heterogeneities, which can significantly influence their growth and production behavior. Cultivating and comparing cells and strains under conditions representative of an actual bioprocess instead of laboratory environments is therefore necessary for developing more reliable production strains. Scale-down bioreactors provide an experimental approach for this endeavor, yet they are limited in their temporal resolution and flexibility when it comes to environmental dynamics. Thus, the impact of second-scale differences in dynamic environments on microbial producers remains unexplored. This study uses the advantages of microfluidic single-cell cultivation to compare the growth behavior and intracellular parameters of three yeast strains under dynamic cultivation conditions with alternating phases of glucose excess and limitation at the timescale of seconds aiming to uncover strain-specific performance under as well as adaptation to fluctuating, bioprocess-relevant environments. Results Across all three strains, a consistent trend was observed: decreasing glucose availability resulted in reduced growth rates, lower ATP levels, diminished glycolytic flux, and smaller cell sizes. Notably, cells already reached approximately 50% of their maximal growth rate when exposed to growth-promoting glucose conditions for only 10% of the time. Importantly, both growth rate and cell size exhibited an adaptation phase rather than an immediate response to oscillatory glucose supply. Longer exposure to favorable conditions shortened the adaptation phase and resulted in higher adapted growth rates and larger cell sizes. Conclusion By leveraging microfluidic single-cell cultivation, this study provides unprecedented temporal resolution of dynamic cellular behavior, enabling direct, strain-specific comparison of cellular responses and adaptation to rapidly changing cultivation environments. Seconds do make a difference. Our findings demonstrate that growth rate is a highly conserved trait under environmental perturbations, which points to an intrinsic robustness of the investigated strains and highlights adaptation as a key determinant of performance in dynamic environments. Together, these insights have important implications for the design of future single-cell experiments and for the development of robust bioprocesses operating under dynamic conditions.