Multilayer graphene reshapes syntrophic interactions and enhances methane production during anaerobic digestion of food waste

The efficiency of anaerobic digestion (AD) is often constrained by the kinetic bottleneck of interspecies hydrogen transfer. This study investigates multilayer graphene (MG), a conductive carbon material, as a strategy to promote direct interspecies electron transfer (DIET). Batch AD experiments were conducted with MG dosages of 0, 1, 2.5, 5, and 10 g/L. All MG-amended reactors demonstrated accelerated volatile fatty acid (VFA) consumption and higher methane yields compared to the control. However, a saturation effect was observed above 2.5 g/L, with no statistically significant increase in methane production (p ' 0.05), establishing this concentration as the most effective under the tested conditions. Microbial community analysis revealed a mechanistic shift: MG addition suppressed the hydrogenotrophic methanogen Methanoculleus while enriching the DIET-associated syntroph Syntrophomonas and mixotrophic Methanosarcina . These findings suggest that MG redirects metabolism from a sensitive hydrogen-based syntrophy to a more direct and resilient DIET pathway, providing a strong proof-of-concept for enhancing AD performance.