Biochar‐Enhanced Methane‐Dependent Denitrification: Insights Into Pyrolysis Temperature‐Driven Microbial Electron Transfer Pathways and Regulatory Mechanisms

Denitrifying anaerobic methane oxidation (DAMO) is a promising carbon-neutral technology for synchronous nitrogen pollution control and methane mitigation, yet its engineering application is severely limited by inefficient microbial interspecies electron transfer. Although biochar can promote microbial extracellular electron transfer (EET), the mechanisms by which pyrolysis temperature-driven changes in biochar properties modulate EET pathways in DAMO systems remain unclear. Herein, sludge-derived biochars prepared at 300°C (BC300) and 800°C (BC800) were used to explore their effects on DAMO performance and EET pathways. Both biochars significantly enhanced nitrate removal, with BC300 and BC800 achieving 1.7-fold and 2.2-fold higher nitrate removal rates than the unamended control, respectively. BC300 with abundant redox-active functional groups acted as an electron shuttle to mediate indirect electron transfer via upregulating Rnf complex genes. BC800 with high graphitization and conductivity functioned as an electron conduit to establish efficient direct interspecies electron transfer (DIET) via enriching Methanospirillum and upregulating pilA gene, exhibiting a superior promotional effect on electron transfer compared to BC300. This work elucidates the differentiated EET regulatory mechanisms of pyrolysis-tailored biochars in DAMO systems, providing critical theoretical support for carbon-neutral wastewater nitrogen removal.