Biodefluorination of Unsaturated Perfluorinated Carboxylic Acid by Anaerobic Digestion Sludge: Who and How?

Per- and polyfluoroalkyl substances (PFAS) are widespread environmental pollutants that are notoriously recalcitrant to biodegradation. We explored the biotransformation and defluorination of <i>(E)</i>-perfluoro-4-methylpent-2-enoic acid (PFMeUPA), a perfluorinated compound with diverse C-F bonds, using municipal anaerobic digestion sludge. Defluorination was stimulated with various substrate amendments and characterized by the release of fluoride (F^-) and the formation of corresponding byproducts. Methanol, formate, acetate, and lactate enhanced reductive defluorination as electron donors. However, the addition of vitamin B₁₂ (a cobalt-corrin complex), which commonly enhances reductive dehalogenation, had no effect. The methanogenesis inhibitor 2-bromoethanesulfonate had no significant effect, ruling out direct participation by methanogens. After 80 days of incubation, <i>Sulfurospirillum</i> and <i>Asaccharospora</i> exhibited significantly higher relative abundance in all substrate-amended treatment groups compared to those in both the original inoculum and control groups (no substrate or no PFAS added), indicating selective enrichment under defluorinating conditions. Comparative analysis of genomes that were enriched relative to the inoculum and that harbored the fluoride exporter <i>crcB</i> gene (<i>n</i> = 23) versus genomes lacking <i>crcB</i> (<i>n</i> = 233) revealed two novel defluorinating candidates belonging to the <i>Sulfurospirillum</i> genus (A_bin.69 and M_bin.68). Overall, these findings advance the understanding of anaerobic PFAS biodegradation and suggest <i>crcB</i> as an auxiliary biomarker to discover putative defluorinating species.