A Small-Scale Multibioreactor System for Fast and Scalable Bioprocess Optimization of Microbial Cell Factories for Small Molecule Production

The fast advance of recombinant DNA technologies allows for rapid development of novel microbial cell factories with native and artificial pathways for small molecule production. Due to the complexity of biological systems, strain design often ends in a variety of potential production strains that need to be evaluated and characterized. Additionally, the physiology of the production strain often has a tremendous impact on strain performance, which calls for cultivation conditions comparable to the final production process. To address these issues, various small-scale multibioreactor systems have emerged in the last decade. The main requirements for such systems are (i) flexibility with regard to substrates and forced nutrient limitations, control of growth rates, cultivation conditions like pH or temperature and (ii) transferability of these results to larger fermentation scales. Physiological characteristics like yields and rates are independent of the cultivation scale and thus well transferable into production scale bioreactors. Continuous cultivation is the method of choice for thorough and precise physiological characterization of microbial cell factories. Here we present a system of 24 parallel bioreactors with 7 mL working volume for continuous cultivation with online monitoring of dissolved oxygen. Stirring allows for high oxygen transfer rates with kLa values up to 120 h^-1. We show two case studies where this system was applied for (i) finding optimal cultivation conditions (feeding rate, pH and temperature) for production of the human molecule melatonin with engineered E. coli and (ii) investigating the impact of different nutrient limitations (C, N, P) on the physiology and productivity of 3-HP producing S. cerevisiae. In both cases, results from the small-scale system runs were confirmed in 1L fed-batch fermentations to demonstrate their scalability and show the applicability of the small-scale cultivation system for fast characterization and optimization of microbial bioprocesses.