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BACKGROUND Co-culture, fermentation with more than two microbial strains, is a potential flexible method for optimizing the metabolic conversion process in bio-production. However, maintaining an ideal population throughout the fermentation process remains a challenge. METHODS AND RESULTS In this study, we developed a proportional control system for controlling the population ratio of Escherichia coli strains to a set value during continuous co-culture. Two E. coli strains were distinguished by expressing different fluorescent proteins, and their population ratio was determined by culture fluorescence. Furthermore, different types of amino acid auxotrophs were provided to each strain, and among these, growth was controlled by the amino acid concentrations in the feed medium. An Arduino-based device was developed using light-emitting diodes and cadmium sulfide light sensors for the in-line monitoring of culture fluorescence. Two E. coli strains of methionine auxotroph green fluorescent protein (GFP) expressing (met-GFP) strain and arginine auxotroph red fluorescent protein (RFP) expressing (arg-RFP) strain were co-cultured using a jar-fermenter. The amounts of methionine and arginine in the feed medium were altered to guide the population ratio to a set value. During the continuous culture, the population ratio between the met-GFP and arg-RFP strains was successfully maintained at approximately the setpoint values. CONCLUSION This study demonstrated the development of a home-made device for controlling the reactor of E. coli based on fluorescent proteins using inexpensive parts. This article is protected by copyright. All rights reserved.