A Continuous Culture Millifluidic Device for the Study of Escherichia coli under Low and Fluctuating Nutrient...

Microbial habitats in nature are often characterized by low concentrations of mixed nutrients, spatial heterogeneity, and temporal fluctuations. However, traditional laboratory culturing methods fail to replicate these conditions. Batch cultures cannot sustain growth in low-nutrient environments, while chemostats maintain steady-state growth with a single limiting nutrient but are challenging to implement when the goal is to maintain defined low concentrations of nutrient mixtures or to introduce rapid fluctuations. Microfluidic systems generate dynamic environments but yield insufficient biomass for population-level omic analyses. To address these limitations, we introduce the millifluidic continuous culture device (MCCD), a versatile platform for studying microbial responses to stable and fluctuating nutrient conditions. The MCCD houses bacterial populations inside a Sterivex filter (0.45 µm polyvinylidene fluoride [PVDF] porous filtering membrane), where a continuous flow of media sustains stable culture conditions while preventing nutrient depletion. A three-way solenoid valve system, controlled via custom Matlab software, enables precise, minute-scale nutrient fluctuations. This protocol provides a step-by-step guide to operating the MCCD in two modes: (1) constant low-nutrient conditions and (2) fluctuating-nutrient conditions. Using this system, Escherichia coli grew exponentially in a mixture of amino acids and nucleobases present at tens to hundreds of nanomolar concentrations, reaching cell concentrations on the order of 10⁹ cells/mL. By recreating key features of natural microbial habitats, the MCCD enables the study of bacterial growth and physiology under controlled yet ecologically relevant conditions in E. coli and other microbial species.