Light-Independent Fe₃O₄–<i>Methanosarcina acetivorans</i> Biohybrid Enhances Nitrogen Fixation and Methanogenesis

Biohybrid systems that integrate microorganisms with nanomaterials have emerged as promising solutions for sustainable nitrogen fixation. However, key challenges, such as the dependence on light and the vulnerability of nitrogenase to oxidative damage, have limited their application. Here, we report a novel, light-independent biohybrid system integrating Fe₃O₄ nanoparticles with <i>Methanosarcina acetivorans</i> C2A, resulting in a significant enhancement of both nitrogen fixation activity and methane production. Fe₃O₄ nanoparticles facilitate directional electron transfer, thereby enhancing methanogenesis and nitrogenase function. Furthermore, the biohybrid system enhances adenosine triphosphate (ATP) synthesis through improved electron flow along membrane-bound electron chains, further supporting nitrogenase activity. Our findings provide a new strategy for advancing nitrogen fixation in archaea, offering an efficient and sustainable approach for biological nitrogen fixation without reliance on solar energy.