Although biochar (BC)-assisted microbial electrolysis cells (MEC) has been shown to improve anaerobic digestion (AD) performance of waste activated sludge (WAS), the underlying mechanisms remain unclear. This study conducted an in-depth investigation into the mechanism based on analyses of extracellular polymeric substances (EPS) characteristics, enzyme activities and multi-omics. The results showed that compared with the control group, methane production improved by 16.73 %, 21.32 %, and 29.37 % in the BC, MEC, and BC-assisted MEC (BC-MEC)- MEC) groups, respectively. The reconfiguration of the protein secondary structure increased the hydrophobicity of the EPS, thereby promoting microbial aggregation. In addition, partial least-squares path modeling (PLS-PM) and mantel test based on the enzyme activity and multi-omics analyses revealed that the promotional effect of MEC on the hydrolysis of WAS was superior to that of BC, while BC was more advantageous in promoting electron transfer and biofilm formation regulated by quorum sensing. The synergistic effects of BC and MEC were exemplified in the BC-MEC group. g_norank_Aminicenantales responsible for the hydrolysis of WAS was enriched (29.6 %), and the activities of hydrolytic enzymes including alpha-glucosidases and proteases were increased by 29.1 % and 43.6 %, respectively. Further, the expressions of genes related to acyl homoserine lactones (AHLs) and diffusible signal factor (DSF) in quorum sensing systems, as well as the genes related to hydrogenase involved in electron transfer ( mbhJKL, hyfB-JR, hypA-F, , and hoxFHUY), ), were up-regulated in the BC-MEC group. This facilitated electron transfer and microbial communication, consequently enhancing methane production. This research significantly advances the understanding of the mechanism by which BC- assisted MEC enhances AD performance and provides valuable insights into strategies for improving energy recovery from WAS.
