Magnetite Nanoparticles Enhancing H₂-Driven Biomethanation in a Mixed Microbial Community

Biological methanation is increasingly considered for biogas upgrading. Here, the supplementation of conductive magnetite (Fe₃O₄) nanoparticles is investigated as a strategy to enhance H₂-driven biomethanation in a mixed hydrogenotrophic methanogenic community. An enrichment culture, maintained for over 180 days in a fill-and-draw anaerobic bioreactor under H₂/CO₂ feeding, is used to inoculate batch microcosms containing 0, 1.25, and 2.5 gFe L^-1 of magnetite. Magnetite addition resulted in a dose-dependent increase in maximum methane production rates-up to 13-fold compared to controls-and sustained high hydrogen-to-methane conversion yields (78-107%). 16S rRNA gene sequencing reveals that archaeal community composition remained dominated by hydrogenotrophic Methanobrevibacter and Methanobacterium spp., whereas bacterial populations shifted from acetogenic Sporomusa and Acetobacterium spp. toward H₂-oxidizing Paracoccus and Thauera spp. at higher magnetite concentrations. Electron microscopy and energy-dispersive X‑ray spectroscopy show that magnetite nanoparticles formed conductive networks bridging microbial cells, and fluorescence in situ hybridization confirmed co-localization of methanogens and Paracoccus within these aggregates. The findings support a direct interspecies electron transfer (DIET) mechanism facilitated by magnetite, whereby Paracoccus spp. oxidize H₂ and shuttle electrons to methanogens, accelerating biomethanation. These results highlight the potential of magnetite-mediated DIET to improve power-to-methane processes and advance biogas upgrading technologies.