Mitigating Long-Chain Fatty Acid Inhibition in Anaerobic Digestion of Model LCFA Substrates with Ruminococcus flavefaciens Bioaugmentation

Anaerobic digestion of high-lipid waste possesses substantial biomethanation potential. However, its stability is readily inhibited by the accumulation of long-chain fatty acids (LCFAs). In this study, pure stearic acid and oleic acid were employed as model LCFA substrates to simulate LCFA inhibition conditions, and the mitigation effects of Ruminococcus flavefaciens (R. flavefaciens) bioaugmentation on LCFA inhibition were systematically evaluated; metagenomic analysis was conducted to analyse metabolic processes. The results demonstrated that a 1% (v/v) R. flavefaciens addition increased cumulative methane yield by up to 270.9% compared to the LCFAs-only reaction. R. flavefaciens addition was associated with enhanced LCFA conversion and promoted acetate formation. Metagenomic analysis suggested shifts in microbial community abundance, with modest increases in β-oxidation-associated Syntrophomonas and the acetotrophic methanogenic archaeon Methanosarcina. Analysis of metabolic pathway-related enzyme genes revealed that R. flavefaciens addition was associated with higher abundance of key genes involved in LCFA activation, β-oxidation, and acetate-utilising methanogenesis, potentially contributing to putative LCFA conversion, alleviating LCFA inhibition. Concurrently, carbohydrate-active enzyme analysis revealed that R. flavefaciens addition elevated glycosyltransferase and carbohydrate esterase potentials associated with extracellular polysaccharide synthesis and substrate deacetylation, which may enhance substrate accessibility and biofilm formation, thereby sustaining metabolic continuity.