Methane-driven microbial fuel cell for simultaneous electrogenesis and biological nitrogen fixation: a syntrophic division of labor mechanism.

Anaerobic methane oxidation coupled to extracellular electron transfer (EET) has shown promising application in bioenergy recovery, yet whether this process can simultaneously drive biological nitrogen fixation (BNF) remains unexplored. Here, we demonstrated that methane-fed microbial fuel cells (M-MFCs) under nitrogen-limited conditions achieve concurrent electrogenesis and BNF via a syntrophic division of labor mechanism. The M-MFC system generated a peak current of 1.17 ± 0.18 mA with a Coulombic efficiency of 78.1%, and the ammonium in the medium accumulated to 0.22 ± 0.01 mg/L over a 30-day operation. 15N2 stable isotope probing (SIP) confirmed direct methane-driven BNF, with the 15N abundances in biomass and aqueous ammonium reaching 13.55% and 7.15%, respectively. Raman spectroscopy revealed 15N incorporation into cytochrome c, while electrochemical analyses and inhibitor assays identified cytochrome c-mediated electron transfer as the dominant pathway for electrogenesis. DNA-SIP combined with high-throughput sequencing further revealed the distinct metabolic specialization of the consortium: Methanobacterium mediated anaerobic methane oxidation, Geobacter and Desulfovibrio specialized in EET, and Sulfurivermis, Desulfobulbus and Azoarcus acted as the core diazotrophs. This work establishes a new paradigm for multifunctional bioelectrochemical systems, which enables energy recovery from methane while simultaneously co-producing bioavailable nitrogen, thereby achieving a trinity of benefits for sustainable environmental biotechnology.