Defined Electrosynthetic Microbial Consortia Reveal Electron Transfer Modes Governing Acetate Production

Precise manipulation of interspecies electron transfer (IET) is critical for advancing microbial electrosynthesis (MES) toward efficient CO 2 bioconversion. Here, defined synthetic consortia are constructed by pairing Shewanella oneidensis MR‐1, a model bidirectional electroactive bacterium, with Clostridium aceticum (electroactive acetogen) or Acetobacterium woodii (nonelectroactive acetogen), mimicking functional guilds commonly observed in natural MES communities. Co‐cultivation markedly boosts acetate production by up to 88%, achieving 1.16 ± 0.01 and 1.05 ± 0.01 g L −1 , with carbon conversion efficiencies exceeding 84%. Comprehensive electrochemical, spectroscopic, and biomass analyses reveal distinct spatial electron transfer modes: DIET via cytochrome c and riboflavin dominates at the biofilm‐electrode interface in S. oneidensis–C. aceticum consortia, whereas S. oneidensis–A. woodii consortia prefer H 2 /formate‐mediated IET in the planktonic phase. This metabolic stratification enables S. oneidensis to function as an “ecosystem engineer,” orchestrating electron flow to optimize CO 2 ‐to‐acetate conversion across biofilm and suspension niches. The proposed synthetic ecology strategy provides a blueprint for designing high‐efficiency MES consortia, paving new avenues for sustainable carbon capture and bio‐based chemical production.