Acidimicrobium sp. Strain A6 (referred to as A6) is a slow growing autotroph, that requires acidic conditions, and uses either ammonium or hydrogen as its electron donor and ferric iron as its electron acceptor. It has recently been shown that the novel A6 is capable of defluorinating per- and polyfluoroalkyl substances (PFAS), including the more recalcitrant perfluoroalkyl acids (PFAA), such as perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS). The process of ammonium oxidation under these conditions is called Feammox, and A6 defluorinates PFAS cometabolically while conducting the Feammox reaction.

This effort is being conducted in two phases. The objectives of Phase I of this project were to confirm that A6 is capable of defluorinating PFAA, gain insights into the defluorination mechanism, and finally to identify Department of Defense (DoD) sites impacted with PFAS where A6 might be naturally present at low numbers and might be biostimulated for PFAS bioremediation. The results of the Phase I effort are available in the Phase I Final Report and are summarized in the Phase I Results section below.

The objectives for Phase II are based on the results of the incubations with the pure A6 culture, A6 enrichment culture, and the two constructed strains. Based on this interim data, the project team will gain new insights into which enzymes are key in the defluorination process.

During Phase I, incubations were conducted in vials containing either PFOA, PFOS, or perfluorohexanesulphonic acid (PFHxS), with pure cultures of A6 and A6 enrichment cultures. Incubations were conducted with these cultures while oxidizing NH4+ and reducing Fe(III). The parent compound, potential degradation intermediates, as well as end products including F-, SO42- for PFOS and PFHxS, and acetate were tracked over time. A6 numbers in both cultures and change in the microbial community of the enrichment culture were tracked, as well as the expression of genes for key oxygenases and dehalogenases, that are thought to play a role in the degradation of these PFAS. These incubations allowed for a careful monitoring of the expression of the rdhA (reductive dehalogenase homolog) and FceA (fluoroacetate dehalogenase homolog) genes. It was decided to use initial concentrations of ~ 10 mg/L, which is substantially lower than the 100 mg/L used previously, but still allowed for tracking of fluoride produced.

During Phase II, incubations will be conducted with PFOA, PFOS, and PFHxS and the pure A6 culture; production of the respective H-PFAA will be tracked. Incubations will then be conducted with H-PFBA, which is the only PFAA commercially available with the F missing from the alpha carbon. Triplicate treatments will be prepared with different inocula, including the pure A6 strain, the A6 enrichment culture, and the two A6 mutants that have either the rdhA or the FceA gene knocked out. Production of intermediates and gene expressions will be tracked. Results will give novel insights into the defluorination mechanism of PFAA by A6. Next, A6 enrichment culture incubations will be conducted with PFOA, PFOS, and PFHxS starting with concentrations that are typical for source areas and then decreasing the concentrations and track their concentrations with time as well as the rdhA gene expression. In parallel, the project team will screen and identify relatively clean sediments from DoD sites impacted with PFAS. DNA extraction and qPCR will be done to determine if A6 is present and can be stimulated. Incubations will then be conducted with slurries from sites identified as ideal for the growth of A6.

Phase I Results

During Phase I, results have confirmed that A6 is capable of degrading PFOA, PFOS, and PFHxS, with the buildup of shorter carbon chain PFAA and fluoride. Fluorine mass balances show that a large fraction of these compounds was completely defluorinated. The gene for a novel reductive dehalogenase (rdhA) is always expressed when A6 defluorinates PFAS and a strong correlation between that gene expression and the amount of fluoride produced was shown. The kinetics of PFAS defluorination by A6 were shown to be proportional to the rate of ammonium oxidation (the electron donor for the Feammox reaction). Results also showed a strong non-linearity between A6 biomass and both the ammonium oxidation and PFAS defluorination rate.  A kinetic model for PFAS defluorination by A6 based on these results was then proposed and tested.

This project will result in a better understanding of the factors that drive the biodegradation of PFAA by A6, including tracking the defluorination activity via the expression of genes for key reductive dehalogenases that are linked to PFAS defluorination. By identifying PFAS-impacted sites where A6 is present, even at very low numbers, and testing whether it can be biostimulated in laboratory incubations (i.e., via the addition of ammonium and/or ferric iron, and possibly pH and redox adjustment), this project will assess the feasibility of a field-scale biostimulation experiment for the biodegradation of PFAS, including PFAA, laying the foundation for PFAS bioremediation schemes at sites with the geochemical characteristics suitable for A6 biostimulation. (Anticipated Phase II Completion - 2023)

Sima, M.W., S. Huang, and P.R. Jaffé. 2023. Modeling the Kinetics of Perfluorooctanoic and Perfluorooctane Sulfonic Acid Biodegradation by Acidimicrobium sp. Strain A6 during the Feammox Process. Journal of Hazardous Materials, Vol. 448. doi.org/10.1016/j.jhazmat.2023.130903.