Fe stabilizes syntrophic volatile fatty acid oxidizing consortia under high organic load in chicken manure anaerobic digestion.

Volatile fatty acid (VFA) accumulation driven by thermodynamic constraints on syntrophic oxidation is a critical bottleneck in high-load anaerobic digestion (AD) of nitrogen-rich feedstocks. Using metagenomic and metatranscriptomic analyses of 172 bacterial genomes, thermodynamic calculations, and granule morphology, we investigated how Fe reshaped syntrophic VFA-oxidizing consortia during 310-day AD of chicken manure under escalating organic loading rates (OLR 1-6  g VS/L/d). Fe attenuated VFA accumulation by 65.0-46.5 % (OLR 4-6), and acted as a stabilizer of core syntrophic consortia: Pelotomaculum persisted as the dominant propionate oxidizer (67.7 % of methylmalonyl-CoA pathway transcription at OLR 6), whereas the control shifted to Fermentimonas; Gallicola and unclassified Clostridiaceae maintained acetate oxidation redundancy; and Syntrophomonas and unclassified Bacteroidales sustained butyrate oxidation capacity. This guild-level stabilization was corroborated at the system level, where methylmalonyl-CoA pathway transcription remained 20.9 % higher (OLR 6), while β-oxidation twofold higher (OLR 4) in the Fe reactor. From this root, a cascade followed: preserved consortia kept VFA concentrations substantially lower, producing more favorable thermodynamic conditions with wider allowable H2 partial pressure windows for all four VFAs, and limiting bicarbonate alkalinity consumption, thereby delaying acidification. Granule analysis further revealed Fe-dependent structural changes, with more uniform granules at moderate load and larger-irregular aggregates at extreme load. These findings suggest Fe sustains syntrophic VFA oxidation by stabilizing the syntrophic consortia network that integrates VFA oxidation, thermodynamics, alkalinity buffering, and granule architecture into a coherent stress-response system, offering a rational basis for microbiome-targeted strategies to improve AD resilience.