Many sites are impacted by per- and polyfluoroalkyl substances (PFAS), and their management represents a substantial burden to the broader community. Polyfluoroalkyl substances (aka precursors) are often the major PFAS within soil and groundwater at sites impacted with aqueous film-forming foam (AFFF), but their fate and transformation, including the formation of more toxic and persistent perfluoroalkyl acids (PFAA), cannot be consistently predicted. This project will address the following topics:

• explore the metabolic transformation and defluorination of sulfur-containing PFAS (SPFAS), including S-containing precursors, by the soil isolate Pseudomonas sp. strain 273;
• measure the products of these transformation reactions;
• demonstrate that the covalent incorporation of precursors into microbial membranes is a relevant attenuation process;
• determine how geochemical conditions (e.g., temperature, pH, salinity, oxygen, co-occurring chemicals, sulfate) impact these processes;
• develop new, process-specific molecular biological tools (MBTs); and
• apply these new tools at sites to assess their utility for monitoring in situ precursor transformation and fate.

To advance a fundamental understanding of microbial precursor transformation and attenuation, the project team will conduct laboratory experiments with fluoroalkane-degrading Pseudomonas isolates, including Pseudomonas sp. strain 273. A high-throughput microtiter plate approach will determine the range of SPFAS strain 273 can utilize as a source of sulfur. Follow-up batch culture experiments will focus on relevant SPFAS that support growth and determine transformation products. Based on the observation that strain 273 incorporates exogenous 8:3 fluorotelomer carboxylic acid (8:3 FTCA) into its membrane lipids, lipidomic measurements will be performed to determine the range of FTCAs and perfluoroalkyl carboxylic acids (PFCAs) that strain 273 and other bacteria incorporate into their lipids. Laboratory incubations will determine the impact of geochemical conditions on the rates and extent of these transformation processes. Comparative genome-wide expression studies will reveal genes involved in sulfur extraction from SPFAS, defluorination, and FTCA (and possibly PFCAs) incorporation into the lipid bilayer, enabling the design of targeted quantitative polymerase chain reaction (qPCR) assays. Bioinformatic analysis will explore the feasibility of designing process - rather than strain 273-specific - qPCR assays with broad utility. The optimized lipidomic workflows and qPCR assays will be applied to microbial biomass samples collected from AFFF-impacted sites to demonstrate their value as prognostic and diagnostic MBT.

The laboratory studies will generate new scientific understanding about microbial processes that contribute to SPFAS transformation, defluorination, and PFAA formation. Further, this work will demonstrate that covalent incorporation of FTCAs, and possibly PFCAs, into bacterial membranes is a heretofore unrecognized pathway for PFAS attenuation. Evaluation of process performances under different geochemical conditions will provide information about transformation rates and extents that can be expected in different field settings. Novel MBT will emerge, including qPCR assays targeting process-specific biomarker genes (e.g., genes implicated in SPFAS transformation and covalent FTCA/PFCA incorporation), and a novel lipidome measurement targeting fluorinated phospholipids in microbial biomass. Application of the new MBT to samples collected from AFFF-impacted sites will demonstrate their utility for prognostic and diagnostic site assessment. The research outcomes will lead to a better understanding of dynamic in situ precursor transformation processes, improve risk assessment, support site management decision-making, and potentially realize substantial cost savings for the DoD. (Anticipated Project Completion - 2026)