Further Development of a Spectrophotometric Assay for Detection of In vitro Defluorination of PFAS by a Recombinant Reductive Dehalogenase (A6 RdhA)

Abstract

Per- and polyfluoroalkyl substances (PFAS) are a class of highly stable fluorinated compounds widely used in industrial and consumer applications, resulting in persistent global contamination of water systems and biological tissues. Their exceptional resistance to degradation arises from strong carbon–fluorine bonds, which have historically limited the effectiveness of conventional remediation strategies. While microbes are known to dehalogenate many chemicals, the capacity for biological defluorination has only recently begun to emerge as a viable pathway for PFAS degradation. In this thesis, we investigate the potential for enzymatic defluorination of PFAS using a recombinant reductive dehalogenase (RdhA) derived from Acidimicrobiaceae sp. A6. A xylenol orange fluoride detection assay adapted in the Avalos lab (Seth- Pasricha et al., in prep for publication) was further applied and tested for additional PFAS degradation. Recombinant fast-growing lab hosts expressing RdhA were used to create cell extracts, which were incubated anaerobically with representative PFAS, including GenX, PFNA, PFPrA, and PFPeA. Our results demonstrate that RdhA catalyzes measurable carbon–fluorine bond cleavage across multiple PFAS substrates within 24 hours, achieving defluorination efficiencies of up to 13.5% for GenX and over 12% for PFPeA, exceeding the activity observed for PFOA controls. These findings expand the known substrate scope of RdhA beyond previously identified compounds and establish in vitro defluorination as a rapid and scalable process compared to prior in vivo studies. Overall, this work highlights the potential of engineered enzymatic systems for PFAS bioremediation and provides foundational insight into the substrate specificity of microbial defluorination in order to mitigate the environmental and health impacts of persistent fluorinated pollutants.