Abstract: Methane fermentation is the core process for wastewater treatment units in food waste treatment plants. However, the rapid hydrolysis and acidogenesis rate of the wastewater can cause volatile fatty acids (VFA) accumulation, which largely limits the methane production rate under high organic load conditions. To cope with this issue, the organic solid waste (OSW) derived from the three-phase separation unit of a food waste treatment plant was used to prepare biochar (OSW-BC). The benefits of OSW-BC promoting methane fermentation of the wastewater were investigated by batch experiments. The results showed that a rich pore structure and aromatic organic functional groups were formed on the surface of the OSW-BC prepared by pyrolysis at 500 ℃. When the substrate/inoculum ratio (SIR) of the methane fermentation system was 2, 3, 5, respectively, OSW-BC addition could shorten the methanogenic lag time by 53.7%-68.1% and meanwhile elevated the maximum methane production rate by 6.1%-62.9%. Microbial metabolic activity and community succession analysis revealed that the addition of OSW-BC dramatically elevated the electron transfer system activity of microbes by enhancing microbial metabolism. Under high SIR conditions, OSW-BC enabled the rapid enrichment of Syntrophomonas and Methanoculleus of the CO₂ reduction metabolism. Meanwhile, the expression abundance of related functional genes, such as formylmethanofuran dehydrogenase and formylmethanofuran-tetrahydromethanopterin N-formyltransferase, increased by 74.2%-118.0%. It was indicated that the redox-active groups and graphite-like structure of OSW-BC might accelerate direct interspecies electron transfer (DIET) between VFA oxidizer and Methanoculleus under high SIR conditions, which facilitated the conversion of VFA to methane and thus achieved an increase in methane production capacity of the kitchen wastewater fermentation system under high SIR conditions.