Long-term humic substances exposure favoring chain elongation rather than methanogenesis during the anaerobic digestion.

Humic substances, including fulvic acid (FA) and humic acid (HA), are major refractory organic pollutants in landfill leachate. Although their individual effects on anaerobic digestion (AD) are well studied, FA and HA coexist in leachate with concentrations varying by landfill age. Herein, this study systematically investigated the successive changes of FA/HA concentrations across different landfill ages on AD performance and mechanisms. Results showed FA addition alone decreased methane yield by 67.04% with caproic acid reaching 2.43 ± 0.05 g/L. Co-addition of FA and HA further suppressed methane yield to 12.71 ± 3.91 mL/(gCOD·d), while caproic acid peaked at 2.59 ± 0.08 g/L. Microbial analysis revealed that FA enriched chain elongating bacteria of Caproiciproducens (44.41%) and Ethanoligenens (26.12%) with hydrogenotrophic methanogens Methanobacterium (>95%) dominating, while co-addition further enriched lactic acid producing bacteria (Olsenella) with Caproiciproducens and Methanobacterium remained dominant. FA channeled acetyl-CoA toward the fatty acid biosynthesis pathway to promote caproic acid production, while coenzyme M/B synthesis genes for methane production declined. Co-addition inhibited methanogenesis by blocking methane precursor (5-methyl-THMPT) production, as indicated by reduced key gene abundances (e.g., frhA, frhB, frhG). Thermodynamic calculations validated that FA/HA addition triggered H2 accumulation, making chain elongation thermodynamically preferable to butyric acid oxidation and consequently repressing acetic acid formation and methanogenesis. These findings provide mechanistic insights into the metabolism change that caused by microbial metabolites of FA/HA in AD process.