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.