Graphitic biochar-anammox achieved by multi-heme-based extracellular electron transfer

The anammox process decarbonizes nitrogen removal by avoiding greenhouse gas emissions and organic carbon demand, yet its reliance on nitrification and denitrification undermines these advantages. To address this, we developed a biochar-assisted anammox system that leverages graphitic defects as redox-active sites to enable interspecies, multi-heme extracellular electron transfer (EET). Biochar produced at 800 °C for 4 h (BC800-4 h) exhibited the greatest graphitic defect density and the highest electron accepting capacity, uniquely exceeding the daily stoichiometric electron demand for complete ammonium oxidation in the present study. Metagenomic and in vitro assays revealed that BC800-4 h promoted hydroxylamine-dependent ammonium oxidation by anaerobic ammonia-oxidizing bacteria (AnAOB) via EET. A cooperative microbial network was identified: AnAOB in suspension supplied heme precursors, while ammonia-oxidizing and denitrifying bacteria colonizing the biochar facilitated heme assembly and transport. This partitioning enabled direct electron transfer to biochar, achieving 62 % nitrogen removal without exogenous nitrite and reducing N₂O emissions by 28 %. The pore-size-dependent reduction in graphitic defects suggests that large molecular-weight biological channels (>10 kDa) are essential for electron transfer between anammox consortia and biochar. Our findings indicate an opportunity to develop a biochar-anammox reactor-with suspended AnAOB and a fixed-bed biochar biofilm-to exploit this synergy for efficient and low-emission nitrogen removal.