The overall goals of this project are (i) to explore whether major nitrogen-containing polyfluoroalkyl substances (N-precursors) found at sites impacted by aqueous film-forming foam (AFFF) serve as N sources supporting microbial growth while being transformed, and (ii) to test if available molecular biological tools (MBTs) for N cycling processes can inform N-precursor transformation potential. The project team will test the central hypothesis that a link exists between the biotransformation of N-precursors, production of perfluoroalkyl acids (PFAA), and microbes that utilize N-precursors to fulfill their nutritional requirement for N. The project team will explore a novel proof-of-concept that the N-precursor biotransformation potential can be assessed by measuring already available gene targets related to N cycling in the environment. The initial focus will be on four AFFF-derived N-precursors that have the potential to transform into perfluoroalkyl sulfonates (PFSA), including two precursors to perfluorooctane sulfonate (PFOS) and two precursors to perfluorohexane sulfonate (PFHxS). The specific objectives are:

• to demonstrate that N-precursors can serve as N sources for microbes,
• to measure the extent of transformation of the N-precursors in microcosms maintained under controlled geochemical conditions,
• to establish enrichment cultures and to monitor the changes in microbial community composition in cultures that utilize N-precursors through sequencing approaches, and
• to apply available quantitative polymerase chain reaction (qPCR) assays to measure 10 N cycle gene targets and decipher their links to N-precursor biotransformation and potential PFSA formation.

The project will be structured through three tasks. Task 1 will explore N-precursor transformation in microcosms established with N-deplete soil, sediment, or aquifer materials collected from geographically distinct locations (including at least one AFFF-impacted DoD site). The project team will determine N-precursor transformation rates and the formation of PFOS/PFHxS and other transformation products and derive enrichment cultures of microorganisms capable of utilizing N-precursors to fulfill their nutritional requirement for N. Task 2 will apply 16S ribosomal ribonucleic acid gene amplicon sequencing approaches to assess microbial community composition in microcosms and enrichment cultures that utilize N-precursor as the sole source of N to identify taxa involved in N-precursor transformation. In Task 3, the project team will survey 10 N cycle gene targets using existing qPCR assays in an attempt to link target gene abundances to N-precursor biotransformation and PFSA formation potential.

This research will support assessment of PFAS impacts caused by the historical use of legacy electrochemical fluorination based-AFFFs. The project team will advance fundamental scientific understanding of microbial N-precursor transformation and demonstrate the value of MBTs to assess N-precursor transformation and PFSA formation potentials. Knowledge of relevant in situ processes involved in precursor transformation is critical to site management at many impacted sites. (Anticipated Project Completion - 2024)