Bioremediation of Per- and Poly-Fluoroalkyl Substances (PFAS) – development of a disparity mutator system in Pseudomonas and strain improvement towards enhanced PFAS biodegradation capabilities

PFAS are persistent organic compounds which consist of a hydrophilic head group and a hydrophobic alkyl chain of variable length (4-16) partially (poly-) or completely (per-) fluorinated. They are contaminants of soil and water, and can cause harm to the human health and the environment. PFAS have been widely used in industrial and commercial products such as fire-fighting foams, materials for cook-ware, high-temperature lubricants, ski wax, water repellant clothing and many more products since the 1940s. Various Pseudomonas spp. have been shown to degrade perfluoro-octane-sulfonate (PFOS) and it was observed that Pseudomonas strains have developed a strong tolerance to fluoride. In our project we will use a customized variant of the described method of Luan et al. (2013) to obtain Pseudomonas spp. mutants with improved PFAS degradation abilities. We will generate three plasmids encoding variants of DnaQ to provide strong, medium and weak mutator abilities. Pseudomonas. spp. obtained from strain collections or isolated from PFAS-contaminated environments (collaboration with AIT, Thomas Reichenauer) will be transformed with these plasmids and will be grown in inducing conditions, at the same time expressing inactive DnaQ protein which results in a mutator phenotype. Increasing concentrations of PFAS in the culture medium over time will lead to adaption by improved enzyme sets/degradation pathways in specific clones which can be isolated by high throughput screening methods and identified by whole genome sequencing. Once a feasible clone is identified, it can be grown in medium in absence of the inducer, so that expression of the inactive DnaQ protein is repressed or the obtained strain can be cured of the mutator plasmid, and genetic stability is restored. Cured strains will be tested for their PFAS reducing abilities in different environmental matrices like water or soil. Finally, degradation and transformation products emerging during PFAS-degradation will be analyzed by LC-HRMS/MS and LC-ion mobility-HRMS/MS.