Abstract
Per- and polyfluoroalkyl substances (PFAS) represent a pervasive class of anthropogenic pollutants characterized by remarkable chemical stability, high environmental persistence, and significant biological toxicity. Conventional remediation strategies, including carbon adsorption and high-temperature incineration, remain energy-intensive and often generate hazardous secondary waste streams. In this study, we developed an engineered strain of Pseudomonas putida KT2440 designated strain Def-09, harboring a rationally engineered fluoroacetate dehalogenase variant (FAcD-M4) and a customized multi-component alkane monooxygenase system (AlkB/RubA/RubB) targeted to the periplasmic space. Strain Def-09 demonstrated robust tolerance to high concentrations of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) up to 250 mg/L. To translate this biocatalytic capability to realistic water treatment applications, we designed an aerobic, continuous-flow dynamic fluidized-bed bioreactor utilizing macroporous aminated carbon-silica composite carrier beads for stable bacterial immobilization. Under continuous operating conditions with an optimized hydraulic retention time of 18 hours, the system achieved a steady-state defluorination rate of 68.4 ± 3.1% for PFOA and 52.8 ± 2.6% for PFOS from synthetic and genuine groundwater matrices containing 100 μg/L baseline contaminants. High-resolution LC-MS/MS and 19F NMR analyses confirmed progressive defluorination through sequential chain-shortening and terminal decarboxylation/desulfonation intermediates without toxic byproduct accumulation. These findings establish a viable, scalable bioengineering framework for the sustainable in situ and ex situ destruction of recalcitrant fluorinated pollutants.
