Unraveling the influence of environmental and operational conditions on the methanogenic pathways in full-scale anaerobic digesters

Anaerobic digestion (AD) technology integrates waste treatment, nutrient recycling, and renewable energy generation, aligning with circular economy principles. During AD, methane is predominantly produced via acetoclastic (AM) and hydrogenotrophic methanogenesis (HM). Understanding and managing these methanogenic pathways can prevent instability, improve process performance, and ensure gas quality. This study assessed the methanogenic pathways across 29 full-scale anaerobic digesters with diverse feedstock composition and operational conditions. The fraction of methane produced from HM (f HM ) was estimated using stable isotopes in parallel incubations with 2- 13 C-acetate – an approach rarely applied at this scale. The results revealed a significant influence of feedstock composition and temperature on methanogenic pathway dominance. Thermophilic digesters showed predominantly HM (f HM > 0.88), whereas mesophilic systems displayed mixed AM and HM contributions. Regarding feedstock composition, sewage sludge and agricultural biomass favored AM, food waste- and manure-fed digesters leaned toward HM. Notably, pH, volatile fatty acids (VFA), dissolved organic carbon, and elements such as sodium and calcium, were revealed as factors that correlate with the methanogenic pathway. While total ammonia nitrogen showed a weaker correlation with HM (ρ = 0.49) than previously reported, free ammonia correlation was stronger (ρ = 0.71). These findings were supported by hierarchical clustering, which identified four distinct digester groups with similar methanogenic pathways, feedstocks, and characteristics (e.g., temperature, pH, VFA, sodium). The results provide a comprehensive overview of methanogenic pathway distribution under full-scale conditions and highlight the multifactorial nature of pathway selection in AD systems. • Methanogenic pathways were assessed in 29 full-scale anaerobic digesters. • Acetoclastic pathway was dominant in sewage sludge and agricultural biomass digesters. • Methane production in manure and food waste digesters was mostly hydrogenotrophic. • Temperature and ammonia were strongly correlated to hydrogenotrophic methanogenesis. • Na, Ca and volatile fatty acids also correlated with the hydrogenotrophic pathway.