Abstract
The successful scale-up of biotechnological processes from laboratory to industrial scale is crucial for translating innovation to practice. Scale-down simulators have emerged as indispensable tools in this endeavor, enabling the evaluation of potential hosts' adaptability to the dynamic conditions encountered in large-scale fermenters. By simulating these real-world scenarios, scale-down simulators facilitate more accurate estimations of host productivity, thereby improving the process of selecting optimal strains for industrial production. Conventional scale-down systems for detailed intracellular analysis necessitate an elaborate setup comprising interconnected lab-scale reactors such as stirred tank reactors (STRs) and plug-flow reactors (PFRs), often proving time-consuming and resource-intensive. This work introduces a miniaturized bubble column reactor setup (60 mL working volume), enabling individual and parallel carbon-limited chemostat fermentations, offering a more efficient and streamlined approach. The industrially relevant organism Escherichia coli, chosen as a model organism, is continuously grown and subjected to carbon starvation for 150 s, followed by a return to carbon excess for another 150 s. The cellular response is characterized by the accumulation of the alarmone guanosine pentaphosphate (ppGpp) accompanied by a significant reduction in energy charge, from 0.8 to 0.7, which is rapidly replenished upon reintroduction of carbon availability. Transcriptomic analysis reveals a two-phase response pattern, with over 200 genes upregulated and downregulated. The initial phase is dominated by the CRP-cAMP- and ppGpp-mediated response to carbon limitation, followed by a shift to stationary phase-inducing gene expression under the control of stress sigma factors. The system's validity is confirmed through a thorough comparison with a conventional STR/PFR setup. The analysis reveals the potential of the system to effectively reproduce data gathered from conventional STR/PFR setups, showcasing its potential use as a scale-down simulator integrated in the process of strain development.