Long-term responses to caproic acid in anaerobic digestion systems: Methanogenic enhancement, extracellular polymer dynamics and microbial metabolic remodeling.

Caproic acid (CA)-rich waste streams generated during chain elongation severely inhibit methanogenesis, posing a major challenge for anaerobic digestion (AD) systems. However, the long-term physicochemical and microbial adaptation processes that sustain methanogenesis under prolonged CA stress remain poorly understood. Here, the physicochemical restructuring, microbial adaptation, and energy conservation remodeling underlying long-term adaptation to CA stress were investigated. Long-term CA exposure increased VS-based methane yields by 11.1-12.8% besides degrading CA, accompanied by restructuring of sludge physicochemical properties. Floc disintegration was accompanied by a shift from particulate COD toward more bioavailable colloidal fractions, potentially increasing substrate accessibility for methanogenesis. Protein-rich soluble microbial products (SMP) increased by 54.0-64.7%, together with reduced relative abundances of phosphatidylcholine biosynthesis genes (pssA, psd, and CHK), suggesting membrane homeostasis perturbation. SMP-proteins may mitigate CA toxicity through hydrophobic sequestration. Metagenome-inferred functional analysis suggested remodeling of microbial energy conservation toward F420H2-dependent electron transfer in methanogenic archaea. This interpretation was supported by coordinated enrichment of the fpoA-O operon, consistent with the 258-294% increase in hydrogenotrophic specific methanogenic activity. Nevertheless, enhanced methanogenic performance was accompanied by aggravated membrane fouling, revealing an engineering trade-off in the anaerobic membrane bioreactor, with the average TMPmax growth rate increasing by 75.5% and effective membrane productivity decreasing by 35.1%. These findings establish a mechanistic framework for understanding microbial adaptation to prolonged CA stress, providing new insights into improving methane recovery from medium chain fatty acid-rich waste streams.