Terminal-Group-Associated Molecular Properties Shape Differential Methane Inhibition by Ultrashort-Chain PFAS in Anaerobic Codigestion

Abstract

Abstract Ultrashort-chain per- and polyfluoroalkyl substances (PFAS) are increasingly detected in aquatic environments and waste streams, yet their effects on complex anaerobic microbial consortia remain poorly understood. Here, two representative ultrashort-chain PFAS with distinct terminal groups, perfluoropropionic acid (PFPrA) and perfluoropropanesulfonic acid (PFPrS), were systematically compared during anaerobic codigestion of sludge and swine manure at 20 μg/g total solids. Results showed that PFPrA and PFPrS reduced cumulative methane yield by 11.5% and 23.7%, respectively, suggesting substantially stronger inhibition by PFPrS. Mechanistic analyses revealed that both compounds restricted substrate release and acetoclastic methanogenesis, whereas PFPrS caused stronger cellular injury and preferentially impaired hydrogenotrophic methanogenesis. PFPrS produced larger decreases in soluble organic carbon and hydrolytic enzyme activities, and its stronger interactions with proteinaceous extracellular polymeric substances coincided with greater reactive oxygen species accumulation, membrane leakage, and coenzyme F420 depletion. Microbial community and metagenomic analyses further indicated stronger inhibition of hydrogenotrophic methanogenesis by PFPrS, reflected by losses of hydrogenotrophic methanogens, reduced H2/CO2-derived methane potential, and decreased predicted functional profile for upstream CO2 reduction. This work clarifies previously unresolved detrimental pathways and microbial responses to ultrashort-chain PFAS, supporting risk assessment and management in complex anaerobic environments.