Stabilization of microbial cell population for long-term plasmid production in continuous biomanufacturing.

Bacterial cells such as Escherichia coli are generally considered insensitive to shear stress in stirred bioreactors, primarily because their small size is assumed to place them below the length scales at which mechanical forces become damaging. This assumption underlies the widespread use of high agitation rates in microbial bioprocesses. However, cellular physiology can strongly modulate this apparent robustness. Metabolic burden associated with high-copy plasmid maintenance and replication is known to alter cell morphology, including increases in cell size and changes in envelope integrity, potentially modifying the mechanical vulnerability of cells. In this study, we investigated the impact of agitation intensity on plasmid-bearing E. coli cultivated in continuous mode. Using automated flow cytometry coupled to fluorescent reporters, we observed a rapid and reproducible population collapse in continuous bioreactors operated at standard stirring rates in 1 L bioreactors i.e., 1000 rpm. This collapse occurred despite growth conditions that are classically considered safe for bacterial systems, highlighting that physiological state rather than nominal cell size alone determines sensitivity to mechanical stress. Our results demonstrate that metabolic burden can indirectly expose bacterial populations to shear-related instabilities, challenging standard assumptions in continuous cultivation. Importantly, by accounting for this effect, we developed a robust operating protocol that enables stable continuous cultivation of plasmid DNA-producing cells for more than 100 h. These findings have direct implications for the design and operation of future continuous biomanufacturing processes.