The efficiency of microbial interspecies electron transfer between syntrophic bacteria and methanogens is considered a rate-limiting factor for the overall efficiency of anaerobic digestion (AD). Stimulating interspecies electron transfer by biochars has been demonstrated to be efficient to enhance AD. However, the enhancing effects vary significantly depending on biochar properties. The correlations between them are not fully under-stood. Herein, biochars with different physicochemical properties were produced from a whiskey by-product "draff" and subsequently applied in the digestion of draff. The biochar produced at 700 degrees C statistically (p less than 0.05) enhanced biomethane yield by 5%. In contrast, biochars produced at 500 and 900 degrees C did not increase biomethane yield. The addition of 700 degrees C-derived biochar in AD increased the relative abundance of the methanogen Methanosarcina, which may be the electron-accepting partner in direct interspecies electron transfer (DIET). The enrichment of Methanosarcina suggested the potential shift of the interspecies electron transfer pathway towards the DIET mode. The characterization of biochar properties suggested that moderate graphiti-zation degree and abundant active surface functional groups (such as -C = O, pyridinic-N, and graphitic-N) were correlated with a more stimulating interspecies electron transfer through both the carbon matrices and the charging - discharging cycles of surface functional groups.