Two-stage process and strain engineering for continuous bioconversion of CO₂ to butanol

The efficient valorization of gaseous C1 feedstocks, such as CO₂, into liquid fuels presents a significant process engineering challenge. Here, we design and demonstrate an integrated chemostat system for the continuous synthesis of butanol from CO₂ and H₂ in a tandem process. Our system architecture sequentially couples two bioreactors housing complementary microorganisms. The first stage utilizes Sporomusa ovata to produce acetate from CO₂ and H₂ autotrophically, which then serves as the sole carbon source for metabolically engineered Escherichia coli in the second stage. Multi-level metabolic engineering of the biocatalyst resulted in a butanol titer of 422 ± 4 mg L^-1 in batch cultures. The two-stage continuous system serves as a proof-of-concept, producing 4.8 mg L^-1 h^-1 of butanol from CO₂ via acetate as an intermediate. While not yet meeting established economic benchmarks, this study reveals critical optimization targets and establishes a foundational and scalable framework for converting CO₂ to butanol.