Extracellular Electron Transfer Enables Methanol Fermentation in Acetogen Eubacterium limosum

Global warming driven by carbon emissions makes transitioning to a circular carbon economy increasingly urgent. Methanol is a promising energy carrier and a favorable substrate for biotechnology applications due to its high energy conversion efficiency and easy integration with existing infrastructure. Eubacterium limosum has the native ability to convert methanol into valuable chemicals, including acetate, butyrate, and hexanoate. However, oxidized cosubstrates such as CO 2 or formate are generally required due to limitations in energy and redox balance. In this study, we demonstrate a novel approach using extracellular electron transfer (EET) with ferric iron (ferrihydrite) as an electron acceptor to enable methanol fermentation without carbon cosubstrates. This ferrihydrite-assisted methanol fermentation yielded a product spectrum like methanol-formate cofermentation, producing more reduced chemicals such as hexanoate. Physiological evidence indicated that cysteine (Cys-SH), an amino acid containing a thiol group (R-SH) and typically included in anaerobic media, was essential in enabling the indirect EET process between E. limosum and ferrihydrite. Thiol regeneration from cystine (Cys–S–S–Cys) confirmed that E. limosum catalyzed the cysteine/cystine recycling. Proteomic analysis revealed the metabolic pathways of this cysteine-mediated EET process. This EET-driven process advances our understanding of microbial carbon cycling and provides a sustainable biotechnology for chemical production.