Abstract

Per- and polyfluoroalkyl substances (PFAS) such as GenX (HFPO-DA) are aerobically recalcitrant contaminants for which biological treatment options remain scarce; the best-characterized microbial degraders require strictly anaerobic conditions and external cofactors. We isolated Cladosporium halotolerans strain CsHGX-1 from activated sludge at a municipal wastewater treatment plant using GenX as the sole carbon source. Whole-genome sequencing (32.4 Mb; 11,201 genes) revealed a PFAS-degradation gene repertoire substantially expanded relative to congeneric Cladosporium species, including 26 dehalogenases (four type-II haloacid dehalogenases, HADs), 141 cytochrome P450s, and 558 esterases/hydrolases. Under aerobic conditions with GenX (50 mg L-1) as the sole carbon source, strain CsHGX-1 removed 47.6 {+/-} 1.7% of GenX within 48 h, accompanied by fluoride release (0.079 {+/-} 0.018 mM) that was absent in abiotic controls, confirming genuine C-F bond cleavage. Time-resolved RNA sequencing (0, 6, 24, 48 h; n = 6 biological replicates) revealed a phase-structured transcriptional program: oxidative genes, including cytochrome P450s, peaked first (6 h; up to 25.4-fold), hydroxylation and reactive-oxygen-species-management genes peaked next (24 h; up to 33.5-fold), and the three type-II HAD genes peaked last (48 h; up to 50.9-fold), coincident with fluoride accumulation. A parallel resazurin metabolic assay over 5 days confirmed sustained catabolic activity in GenX-exposed cultures relative to controls (1.37-1.52-fold; p [≤] 0.003). These findings identify strain CsHGX-1 as, to our knowledge, the first Ascomycete fungus for which genomic and transcriptomic evidence links oxidative activation to haloacid-dehalogenase-mediated defluorination of an aerobically recalcitrant PFAS, extending the known diversity of fungal PFAS degraders beyond Basidiomycota white-rot taxa.