Methanogenic community and pathway responses to iron addition in high-load chicken manure anaerobic digestion under ammonia stress.

Anaerobic digestion of chicken manure is often inhibited by high ammonia concentrations, particularly under increasing organic loading rates (OLR). We characterized the transcriptional responses of methanogenic communities and methanogenesis pathways to Fe addition in long-term (310 days) reactors operated at OLRs from 1 to 6 gVS/L/d using integrated metagenomic and metatranscriptomic analyses. Fe supplementation increased methane yield by 22.2% at OLR 4 gVS/L/d and 60.2% at OLR 5, raising the maximum sustainable OLR from 3 to 5 gVS/L/d, a 66.7% improvement in treatment capacity. Metagenomic assembly yielded three high-quality methanogenic rMAGs (Methanosarcina, Unclassified Methanomethylophilaceae, and Methanoculleus). Fe enhanced their transcriptional activity across all OLR. For Methanosarcina, Fe alleviated acetoclastic pathway transcriptional inhibition and diversified methylotrophic substrate transcription. For Unclassified Methanomethylophilaceae, Fe preserved monomethylamine as the primary transcriptional substrate, preventing stress-induced substrate transcription shift. System-level analysis confirmed these findings, with overall methanogenic pathway transcriptional activity in the Fe treatment reaching 2.22, 1.54, and 3.35 times that of the control at OLR 1, 4, and 6. At OLR 6, Fe maintained a balanced transcriptional distribution among methylotrophic (40.5%), acetoclastic (30.3%), and CO2 reduction (27.5%) pathways, while the control shifted to single-pathway transcription dominance (CO2 reduction, 79.9%) with complete acetoclastic transcription loss. Fe also upregulated Fe, Co, and Ni transporter genes in all three methanogens. These results provide transcriptional evidence that Fe addition is associated with higher methanogenic activity and a more balanced methanogenesis pathway distribution under high‑OLR, high‑ammonia stress.