Ability of a microfluidic droplets system to assess the dynamics of Green Fluorescence Protein (GFP) production by Escherichia coli for modelling and bioprocess purposes

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Understanding microbial growth and metabolism under controlled environments is critical for both fundamental research and bioprocess development. In this study, we present a cost-effective droplet-based microfluidic device enabling high-throughput screening of GFP expression in Escherichia coli ( E. coli ) under varying glucose concentrations. Fluorinated Ethylene Propylene (FEP) tubing was selected for its low cost and compatibility with stable water-in-oil emulsification, facilitating robust droplet generation. The system achieved a 33333-fold reduction in media consumption compared to traditional Erlenmeyer flask cultures. Growth kinetics and GFP expression were assessed in both Erlenmeyer flasks and microdroplets, showing high qualitative correlation between platforms. Low glucose levels (5–10 g/L) supported rapid initial growth and early GFP production, followed by a fluorescence decline due to nutrient depletion. In contrast, higher glucose concentrations (25–50 g/L) prolonged the exponential phase and enhanced GFP production per unit biomass, though growth was slowed by overflow metabolism. In microdroplets, delayed GFP expression at 25 and 50 g/L were observed, and parallel bioreactor experiments confirmed that this delay is caused by oxygen limitation at high glucose concentrations. Importantly, the microfluidic device enables controlled variation of oxygen availability simply by adjusting droplet size or generation frequency, providing a powerful means to probe oxygen-sensitive metabolic behaviors. These results validate the microfluidic platform’s ability to mimic Erlenmeyer flask-scale dynamics, while uniquely allowing precise modulation of oxygen transfer conditions at the microscale . The system offers a reliable, miniaturized alternative for optimizing microbial bioprocesses with drastically reduced reagent use and increased experimental throughput. • Growth and GFP expression profiled across 5–50 g/L glucose in flasks and droplets • Microdroplet GFP trends mirrored flask results, supporting bioprocess validation • Low-cost microfluidics enabled a 33k-fold volume saving vs flask in E. coli GFP screens • Low glucose led to fast early growth; high glucose supported longer exponential phase • Droplet size and frequency enable novel, precise tuning of oxygen availability.

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