Multimetallic biochar as an ecosystem engineer: Orchestrating synergistic IHT-DIET pathways via spatial niche...

The efficacy of anaerobic digestion (AD) is limited by inefficient interspecies electron flow and metabolic pathways. While conductive materials promote direct interspecies electron transfer (DIET), their potential to engineer microbial ecosystems remains underexplored. This study engineered multimetallic biochar (Fe, Ni, Co, and Mn loaded) via mechanochemistry, integrating electron shuttle and microbial niche regulator functions. Physicochemical characterization confirmed redox-active metal-carbon interfaces and mesoporous structures, enhancing electron exchange and microbial colonization. In cow manure-corn straw co-digestion, optimized BC-Fe-Ni addition group achieved the highest cumulative methane yield (224.7 NmL/g.VS) and shortened the lag phase from 22.3 to 3.7 days. Microbial co-occurrence network analysis revealed phase-specific functional differentiation: the BC phase formed a DIET module centered on Methanosarcina as the core hub, while the slud phase established a syntrophic hydrogenotrophic (IHT) module with hydrolytic-acidogenic bacteria (Clostridium sensu stricto 1, Ruminofilibacter), syntrophic bacteria (Pelotomaculum, Synergistaceae), and hydrogenotrophic methanogens (Methanobacterium, Methanocorpusculum). Fisher's exact test confirmed significant phase-specific enrichment of these taxa (FDR-adjusted q < 0.05), validating non-random niche partitioning between electroactive archaea and hydrogenotrophic consortia. KEGG profiling showed biochar enriched DIET-related redox enzymes (EC 1.12.98.1) and downstream methanogenic enzymes (EC 2.8.4.1), while slud phase had more hydrolysis-acidification enzymes (EC 6.2.1.1) and IHT-related hydrogenases (EC 1.2.7.12). This niche partitioning establishes a division-of-labor strategy where DIET and IHT operate in parallel and synergistically, optimizing overall system efficiency. This work transcends biochar as a mere additive, presenting a new paradigm of tailored multimetallic biochar reprogramming microbial ecology to boost AD performance.