Evaluation of porous polyethylene-supported zeolite carriers to support feammox-based perfluorooctanoic acid biotransformation

Traditional treatment methods for removal of per- and polyfluoroalkyl substances (PFASs) from water are costly and often generate secondary waste, prompting interest in biological alternatives. Acidimicrobium sp. Strain A6 (A6) conducts feammox, or anaerobic ferric iron reduction paired with ammonium oxidation. These bacteria have been shown to defluorinate perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) under acidic, anoxic conditions. Its practical application, however, is limited by slow growth, sensitivity to environmental conditions, and specific nutrient requirements. This study explored the potential of porous polyethylene-supported zeolite (PEZ) carriers as a nutrient-integrated support system intended to promote A6 growth and PFOA biotransformation. It was hypothesized that the carrier would provide both ammonium and ferric iron to localize A6 bacteria growth and PFOA degradation. Iron-enhanced PEZ carriers were successfully synthesized and exhibited ammonium sorption capacities greater than normal PEZ, confirming their suitability for feammox nutrient delivery. Four unique culture environments were tested in batch incubation experiments spiked with 10 mg/L (24 μM) PFOA, and degradation was monitored over 100 days. Incubations revealed no significant differences in ammonium removal or A6 growth between cultures with carriers and controls, and no PFOA removal was observed, even in the positive control. A6 was detectable on PEZ carriers (10 3 copies/carrier) at the end of the incubations, suggesting that these carriers provided localized microenvironments supportive of A6 retention, though insufficient for measurable feammox activity or PFOA defluorination, which limits assessment of the carrier design. The minimal activity observed may be attributed to low initial A6 concentrations, a suboptimal iron phase/limited availability of iron, or the presence of other bacteria outcompeting A6. This study indicates that PEZ carriers have potential to support the maintenance of A6 in engineered systems even though no clear support of feammox metabolism or PFOA degradation was observed and outlines parameters that require further optimization to improve anaerobic PFAS biotransformation.

Per- and poly-fluoroalkyl substances (PFASs) are highly fluorinated compounds with widespread uses as a result of their thermal and chemical stability (Lenka et al., 2021). PFASs are used as lubricants, in aqueous film-forming foams (AFFF), and in cookware, personal care products, and clothing (Kwiatkowski et al., 2020). PFAS are widely detected in groundwater, drinking water, and soils, prompting regulatory agencies to set limits for their presence in drinking water (U.S. EPA, 2025). Moreover, their chemical stability renders them persistent and difficult to degrade or remove from water. Long-chain PFAS compounds like perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) have toxic effects on biota and humans (Sunderland et al., 2019; Li et al., 2022; Hossain et al., 2025). Effectively removing and destroying PFAS, however, remains a pressing challenge in environmental engineering.

Recent work has explored biological degradation as a potential complementary strategy for PFAS treatment. A meta-analysis reviewed 97 studies from 1989 to 2023 on fluorinated compound biotransformation, with 80% of those including PFAS compounds (Skinner et al., 2025). Across the aerobic experiments included, 82% documented biotransformation, but only 41% observed fluoride production (Skinner et al., 2025). Incubation periods ranged from 3 h to 730 days (average 36 ± 80 days; Skinner et al., 2025). For the limited anaerobic experiments (incubated 1.25–341 days; average 90 ± 93 days), 72% documented PFAS biotransformation, but only 46% showed evidence of fluoride release (Skinner et al., 2025). Studies of pure and enrichment cultures documented PFAS biotransformation using Pseudomonas oleovorans and Pseudomonas butanovora, Acidimicrobium sp. Strain A6, and the dechlorinating enrichment culture KB-1 (Tiedt et al., 2017; Huang and Jaffé, 2019; Yan et al., 2022; Yu et al., 2022). In anaerobic experiments using KB-1, ɑ,β-unsaturation was critical for reductive defluorination and hydrogenation of fluorinated carboxylic acids (Yu et al., 2022).

These findings reinforce the difficulty in biologically transforming fully fluorine-saturated PFASs and highlight the long incubation time needed, representing a barrier using biological methods for PFAS treatment in water. Indeed, the effectiveness of treatment varies depending on the PFAS, with shorter-chain and unsaturated compounds broken down more successfully (Hamid et al., 2020). These limitations apply to PFOA and PFOS, two of the five individual regulated PFAS compounds (U.S. EPA, 2025). Despite these barriers, recent studies indicate that bacteria capable of the feammox process, or anaerobic ammonia oxidation under iron-reducing conditions, may facilitate successful (partial) defluorination of PFOA and PFOS (Huang and Jaffé, 2019).

Anaerobic ferric iron (Fe(III)) reduction coupled with anaerobic ammonium oxidation is mediated by Acidimicrobium sp. Strain A6 (A6) (Huang and Jaffé, 2019). If goethite is the ferric iron source, the overall reaction is Equation 1

Beyond nitrogen cycling, A6 has the ability to cometabolize PFASs, including PFOA, under iron-reducing conditions (Huang and Jaffé, 2019; Sima et al., 2023). This transformation likely arises from expression of reductive dehalogenase genes (rdhA, rdhB, and rdhTKZECD) found through a metagenomic analysis of the feammox cultures that defluorinated PFASs (Jaffé, Peter et al., 2021). The rdhA gene, which translates a catalytically active enzyme, was twice as high in PFAS incubations than in control incubations without PFASs (Jaffé et al., 2021). The use of A6 for PFOA remediation, however, has been limited by the slow growth of these bacteria, strict redox requirements, and the need to supply both ammonium and Fe(III) to sustain activity (Huang and Jaffé, 2019).

Engineered carriers may provide a means of overcoming barriers to using A6 for PFOA treatment. Polyethylene-supported zeolite (PEZ) carriers can concentrate ammonium and host biofilms, potentially creating favorable microenvironments for A6 (Huff Chester et al., 2023). Recent work suggests that A6 can survive in the presence of PEZ carriers, potentially benefiting from their ammonium-retentive properties. Nevertheless, PEZ carriers have not integrated iron (III) delivery nor have they been evaluated for their ability to actively enhance A6 growth (Huff Chester et al., 2023).

This study evaluated the feasibility of incorporating ferric minerals (goethite; α-FeOOH) into the PEZ structure, enabling them to serve as localized iron and ammonium sources, co-delivering key nutrients for feammox and thus PFAS transformation. It was hypothesized that these PEZ carriers would aid A6 growth and PFOA biotransformation. Iron-enhanced PEZ carriers were synthesized and their ability to sorb ammonium, support A6 growth, and promote PFOA transformation in enrichment cultures was tested. The cultures were monitored over a 103-day incubation period and sampled for A6 numbers, total bacteria numbers, ammonium, PFOA, and fluoride. The goal was to assess whether iron-enhanced PEZ carriers could provide a suitable foundation for cultivating A6 and advancing PFAS bioremediation strategies.

Chemicals and reagents

A list of chemicals and reagents, the composition of the growth media, and the synthesis method of goethite particles are provided in the Supplementary Material. While 6-line ferrihydrite has shown to lead to greater activity of A6 than goethite (Huang and Jaffé, 2018), this mineral may not be amenable to incorporation into a polymer matrix due to limited stability. X-ray diffraction was used to verify goethite was successfully synthesized (Supplementary Figure S1).

Carrier synthesis and characterization

PEZ carriers were prepared using a large batch manufacture method (Feinberg et al., 2021). Polyethylene (PE), polyethylene oxide (PEO), and zeolite were loaded into a HAAKe Rheomix lab mixer set to 150 °C and 100 rpm. For normal carriers (prepared without goethite inclusion), raw materials were added in equal masses (15 g each of PE, PEO, and zeolite), and for iron-enhanced carriers, 15 g PE, 15 g PEO, 14 g zeolite, and 1 g of dried goethite particles were added. These materials were melt-blended for 10 min, then the product was hot pressed at 150 °C and 2,000 lb-ft (2711 N-m) for 1 min in a Wabash hot press to a thickness of 1.5 mm. The product was removed, allowed to cool to room temperature, and manually punched into discs using a 10-mm hole punch. Carriers were added to deionized water to etch the soluble PEO for 48 h, replacing water as needed to encourage rapid removal. After etching, water was replaced with methanol for another 24 h, and PEZ carriers were then removed and air-dried for at least 24 h. The availability of iron in the carriers was assessed by dissolution experiments (ultrapure water, 0.01 M pH 4.5 acetic acid, 0.1 M pH 4.6 oxalic acid, or 1 M HCl) (Voelz et al., 2019). One iron-enhanced carrier was placed in each of triplicate vials containing 5 mL of the chosen solution, and the vials were agitated on an orbital shaker (60 rpm) at room temperature overnight. The iron released was measured using the ferrozine method (Viollier et al., 2000) after reducing the iron using hydroxylamine, and absorbance of the resulting colored complex for the samples and ferrous iron standards was measured on a BioTek Synergy LX microplate reader.

Ammonium sorption and desorption experiments and PFOA sorption experiments

Aqueous solutions (10 mL, n ≥ 7) containing ammonium chloride (range of 5–100 mg/L as NH 4 +) were used in sorption experiments. The dry mass of each PEZ carrier was recorded, and one carrier was added to each solution. After 24 h of rotating, the carriers were removed from the solution, and the concentration of ammonium measured using an ion-selective electrode (ThermoFisher Scientific Orion High Performance ammonia Combination Selective Electrode). Taking care to keep carriers separate, they were each then placed into a new 10 mL solution of experimental feammox medium (pH ∼ 4.5) to assess desorption. For normal carriers, the medium included iron (III) in solution, and for iron-enhanced carriers, the medium did not contain iron (III), to simulate culture conditions. After 24 h on the rotator, the concentration of ammonium in solution was measured to evaluate desorption.

For PFOA sorption tests, PEZ carriers were preloaded with ammonium by mixing carriers in 1 L of 100 mg/L as NH 4 + solution for 24 h, followed by air-drying for 48 h. Experimental solutions (10 mL, n ≥ 7) of feammox medium (pH ∼4.5) were spiked with PFOA ranging in concentration from 1 to 10 mg/L (2.4–24 μM) PFOA. Initial PFOA concentrations were measured prior to carrier addition. The dry mass of each carrier was recorded, and a single carrier was added to each solution. After 24 h, carriers were removed and aqueous samples analyzed for PFOA using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Isotherm parameters were determined by nonlinear fitting to the Langmuir isotherm.

The mass sorbed/desorbed on the carriers was calculated and normalized to PEZ carrier mass, with q e the amount of sorbate sorbed per unit mass of carrier (mg/g), q m the maximum sorption capacity of the carrier (mg/g), C e the equilibrium concentration of sorbate in the aqueous phase (mg/L) and K L the Langmuir constant (L/mg).

Batch culture experiments with Acidimicrobium sp. strain A6

A6 cultures were obtained directly from Princeton University (Huang and Jaffé, 2019). These “parent cultures,” were confirmed to have feammox activity by measuring ammonium removal, then shipped to the University of Minnesota where they were inoculated into fresh media. Feammox medium for A6 cultures was prepared following the procedure in Huang and Jaffé (2019), but goethite was used as the iron (III) source in place of ferrihydrite. Concentrated nutrient solutions (10× and 10× No NH 4 +) and a trace element solution were prepared (see Supplementary Material) and stored 4 °C in autoclaved containers:

Feammox medium was freshly prepared on the benchtop the day of each culture transfer. To prepare 1 L of culture medium, 100 mL of 10× nutrient solution, 2 mL of 25 mM anthroquinone disulfonate, and 1 mL of 1× trace elements solution were added to a 1-L volumetric flask. Ultrapure water was added to the fill line. This solution was transferred to a 1-L bottle and stirred on low heat until any solids dissolved and then filtered through a 0.22 µm filter (Sigma-Aldrich Steritop Quick Release-Gv Sterile Vacuum Filtration System) into a clean bottle. Vitamin supplement (1 mL, ATCC) was added, and goethite solution was added to achieve a final concentration of ∼6 mM Fe(III). The pH was adjusted to ∼4.5 using 1 M HCl.

This media preparation was adjusted to test the four unique media types in experimental triplicates shown in Figure 1 (photographs in Supplementary Figure S2). The “Positive Control” culture medium was prepared as stated above, containing standard concentrations of goethite and ammonium (Huang and Jaffé, 2019) and no carriers. The “Positive Control” cultures used these conditions, for which successful PFOA defluorination has been previously observed, to compare against the activity of cultures with PEZ carriers. The “Negative Control” medium was prepared using 10× “No NH 4 + ” nutrient solution and removing the goethite addition step. These conditions removed the two key nutrients (Fe(III) and NH 4 +) that A6 needs for the feammox process. The “No NH 4 + ” medium was prepared with 10× No NH 4 + nutrient solution and goethite. Nine normal PEZ carriers preloaded with ammonium were added to these cultures. This culture condition tested the use of PEZ carriers as the sole source of ammonium to A6. Finally, the “Experimental” culture medium was prepared in the same manner as the “Negative Control” cultures, containing no ammonium or goethite in the liquid medium. Instead, nine iron-enhanced PEZ carriers preloaded with ammonium were added to the cultures as the sole source of ammonium and iron (III).

After the media was prepared, it was moved, uncapped, into an anaerobic glove bag (98% N 2 /2% H 2) and allowed to equilibrate with the glovebag atmosphere to remove oxygen. A6 was introduced to all treatments by adding the active, anoxic A6 parent enrichment culture at a 1:10 volumetric ratio. The dilution was necessary based on the culture available and the number of treatments needed, and it was expected there would be sufficient activity retained from the culture with verified activity. PFOA was then added into each culture to achieve an initial concentration of 10 mg/L (24 μM) PFOA, based on past studies that used this concentration with A6 (Huang and Jaffé, 2019; Jaffé et al., 2024). Bottles (120 mL) containing the different treatments were sealed with butyl rubber stoppers and crimped before being removed from the glove bag. The headspace was then simultaneously vacuumed and flushed with 20% CO 2 /80% N 2 gas to ensure anoxic conditions and provide CO 2 for A6 growth (Huang and Jaffé, 2019) by connecting a vacuum line to one 23g × 1″ regular bevel needle and inserting it through the rubber butyl stopper into the headspace of the bottle, then connecting the gas tank to another needle and inserting it in the same way. Each culture was flushed/vacuumed for at least 2 min. Serum bottles were placed on a bench top shaker at a speed of 140 rpm.

On Days 16 and 50, additional (4 mL and 6 mL, respectively) active, anoxic A6 enrichment parent culture spiked with ∼10 mg/L PFOA was added to all cultures to stimulate feammox activity. This was performed in the anaerobic glovebag, and the bottles were resealed following the addition. Samples were taken immediately before and 24 h after the addition of the culture to account for any dilution effects. On Day 16, resazurin, a redox indicator, was added (1 mg/L) to one culture of each media type to help assess whether anaerobic culture conditions were present, and sodium acetate was added at a concentration of ∼2 μg/L to help lower the redox potential further. From Day 16 onward, each culture was flushed/vacuumed for 2 min, or until the resazurin indicated reduced conditions, after each sampling event.

The Day 0 media was sampled for initial concentrations of PFOA prior to splitting it into the different treatments. The individual cultures were sampled for ammonium and DNA on Day 2 after ammonium equilibrium had been reached in the cultures with PEZ carriers. Samples were taken using 23g × 1″ regular bevel needles and syringes on Days 2, 10, 16, 17, 24, 31, 38, 50, 51, 60, 71, 80, and 103 and the headspace was flushed/vacuumed as described above. On Day 103, the cultures were opened and carriers removed for analysis of DNA and PFOA. Once the remaining liquid medium was removed, each bottle was rinsed with methanol to test for any PFOA sorption to the glass.

Four main parameters were tracked over the 103-day incubation period: A6 numbers, total bacteria numbers, and ammonium and PFOA concentrations. Measurements were taken of all three experimental triplicates and averaged for each culture type. For 12% of biological samples and 67% of PFOA samples, replicate PCR or analytical runs were conducted. These numbers were averaged before averaging the experimental triplicates. Fluoride was also measured on select dates. Analytical methods for A6 and total bacteria (quantitative polymerase chain reaction (qPCR)), ammonium (ion specific electrode), PFOA (LC-MS/MS), and fluoride (ion chromatography) are described in the Supplementary Material and Supplementary Tables S1-S3.

Data fitting and statistical analysis

Data fitting and statistical analyses were performed in R (R Core Team, n. d.). Isotherms were fit to Equation 2 and 95% confidence intervals obtained for the parameters. Analysis of variance (ANOVA) was used to evaluate differences in fitted Langmuir model parameters for sorption and desorption experiments. Averages, standard deviations, and interquartile ranges (IQR) for microbial abundance and PFOA data were determined. The statistical significance used for comparing experimental treatments or conditions used α = 0.05, and, unless otherwise stated, T-tests were performed as two-sample, two-sided tests. Outliers were identified by the IQR method. For iron dissolution from the carriers, averages and standard deviations of triplicates are reported.

Ammonium sorption capacity of normal and iron-enhanced PEZ carriers

Sorption isotherms (Figure 2A) revealed that the ammonium sorption capacity and Langmuir constant of the iron-enhanced PEZ was significantly different than those of the normal PEZ (ANOVA, p < 1 × 10 −8). Iron-enhanced PEZ had a >3-fold higher sorption capacity (q m = 7.24 mg/g vs. 2.24 mg/g), whereas normal PEZ had a higher Langmuir constant (K L = 2.04 L/mg vs. 0.1 L/mg), indicating ammonium had a stronger affinity for the normal PEZ carriers than the iron-enhanced carriers. Both carriers sorbed environmentally relevant ammonium concentrations (20–30 mg/L) with reasonable carrier volumes (5–7 carriers/50 mL culture). The lower affinity and higher capacity of the iron-enhanced PEZ carriers could be favorable for supplying available ammonium to bacteria.

Desorption studies (Figures 2B,C) showed hysteresis for both carrier types, with less ammonium released than sorbed (ANOVA, p < 1 × 10 −8 for normal, p < 0.005 for iron-enhanced), but similar Langmuir constants. This difference in the extent of desorption is likely due to differences in aqueous conditions: sorption was conducted in pure ammonium chloride at neutral pH, while desorption occurred in A6 medium at pH ∼4.5. Even so, carriers released levels of ammonium sufficient to support A6 growth (Huang and Jaffé, 2019).

PFOA sorption onto normal and iron-enhanced PEZ carriers

Minimal sorption of PFOA by PEZ carriers was detected (Supplementary Figure S3). Langmuir q m values were near zero, likely reflecting variability rather than sorption. This indicates PFOA remained in solution and available to A6.

Effect of carriers on A6 growth

It was hypothesized that over the course of ∼100-day incubations, A6 would see the most growth in the positive control, and the least amount in the negative control. If the iron (III) and ammonium were bioavailable to A6 from the iron-enhanced PEZ carrier, A6 would effectively facilitate feammox in the Experimental cultures as well. If, however, the iron was not bioavailable, and the Experimental cultures did not conduct feammox, the No NH 4 + cultures tested with normal PEZ carriers providing ammonium would enhance A6 growth.

Five outliers were removed from A6 qPCR data using the IQR method, and no outliers were detected for total bacteria qPCR data (Supplementary Figures S4a,b). Generally, all culture types followed similar trends in bacterial numbers and replicate analyses are consistent (Figure 3). A few time points have larger errors because one replicate was substantially different from the others, potentially due to heterogeneity/microenvironments in the reactors. The abundance of A6 (10 2 –10 4 A6 16S gene copies mL -1) is lower than previous cultures showing defluorination activity (10 6 gene copies mL -1; Huang and Jaffé, 2019). Over the first 16 days, A6 declined while total bacteria remained steady (Figures 3A,B). This is likely a result of the presence of some oxygen. Upon adding the resazurin redox indicator, cultures turned a bright pink/purple color, indicating oxygen was present, even after the vacuuming/flushing procedure had been followed (Supplementary Figure S2b). Anoxic conditions are required for A6 growth, but other organisms in the enrichment culture may have been able to grow in the presence of oxygen. After Day 16, low oxygen levels were maintained for the remainder of the experiment, with resazurin serving as a visual redox guide.

After the addition of A6 enrichment culture on Day 16, A6 levels initially increased or were maintained for 7–14 days, declining again by Day 31 (Day 38 for the No NH 4 + cultures). After the Day 50 amendment of additional A6, all cultures saw A6 growth between Days 60 and 80 before numbers declined again to Day 103 (Figure 3A). A6 numbers were not statistically different between culture types on most days as determined by T-tests. After the Day 16 amendment, the A6 numbers in the negative control were similar to those in the positive control, with few statistical differences (Supplementary Figure S4c).

Availability of iron in the carriers to the A6 bacteria is one possible limitation on growth. In the ultrapure water and acetic acid extraction tests, 8% ± 3% of the iron in the carriers was found in solution. For the complexing agent (oxalic acid) and stronger acid (HCl), 15% ± 1% and 22% ± 1% of iron dissolved. While dissolution by acid will not fully reflect the ability of the goethite in the iron-enhanced carriers to accept and conduct electrons transferred by A6 bacteria, these results reveal that all of iron is not readily accessible to the surrounding media.

Additionally, when taking samples after Day 60, a strong sulfur odor was present and a yellow tint was observed (Supplementary Figure S2c). On Day 100, it was observed that the positive control cultures contained a black substance coating the sides of the bottles where goethite had previously been observed (Supplementary Figure S2d). The black material was only found on the side of the bottles not in contact with the aqueous medium when the vials were incubated horizontally.

Total bacteria growth trends generally mirrored the A6 growth trends, though with copies several orders of magnitude higher than A6 numbers (Figure 3B). Similarly, total bacteria count was not statistically different between culture types on most days (Supplementary Figure S4d) as determined by T-tests. A6 was a low percentage of the total bacteria count, ranging from 0.00% to 0.24% in the Positive Control, 0.00%–0.17% in the Negative Control, 0.00%–3.00% in the No NH 4 + treatment, and 0.0003%–0.26% in the Experimental treatment over the 103-day incubation.

DNA extracted from both carrier types on Day 103 (Supplementary Figure S5) showed low but detectable A6 growth on the carrier surface and/or within the porous structure. Iron-enhanced PEZ supported a slightly higher relative abundance of A6 (mean: 0.049%) than normal PEZ (mean: 0.022%), but there was no statistical difference in A6 or total bacteria concentrations between the two carrier types (p > 0.05). Because the iron-enhanced carriers provided delivery for iron and ammonium, they were hypothesized to provide a niche favoring feammox compared to normal carriers, which provided ammonium that other bacteria in the enrichment culture could also utilize. This data, while limited, suggests that iron-enhanced carriers could provide a more effective site for A6 growth than normal PEZ carriers; nevertheless, this needs to be verified with a more active and concentrated culture of A6.

Effect of carriers on feammox activity

Across all cultures, there was very little ammonium removal (Figure 4A). The varying baseline concentrations of ammonium in different cultures was expected—the iron-enhanced PEZ had a higher ammonium sorption capacity than normal PEZ, leading to the release of higher concentrations of ammonium. The only source of ammonium in the negative control cultures came from the amendment of the parent culture on Days 16 and 50, leading to low ammonium concentrations (0.003 mg/L to 5.0 mg/L) over the course of the experiment.

Putting aside values for the negative control, there are several key trends in ammonium concentration across the other three treatments. The longest period of ammonium removal for all three culture types occurs between Day 17 and 31, which is consistent with the A6 growth that was observed during that same period (Figure 3). The amount of ammonium utilized, however, was less than 20% for all cultures, which is much smaller than previous studies have found in active A6 cultures (Huang and Jaffé, 2019). Minimal ammonium removal was observed from Day 60–80 during the second period of A6 activity. While the qPCR data suggests potential A6 retention, there is no clear evidence of active feammox metabolism.

There was no PFOA removal across all treatments (Figure 4B), with six outliers detected and three removed (Supplementary Figure S6a). PFOA concentrations were relatively constant during the period from Day 17–50 where some A6 growth was observed (Supplementary Figure S6b). Previous work has shown that PFOA defluorination rates by A6 are proportional to the ammonium oxidation rate (Sima et al., 2023), and thus paired with the relatively constant ammonium levels observed, it appears that there was little feammox activity, even in the positive control, and A6 was not utilizing PFOA as a secondary electron acceptor. This indicates there are limitations of the carrier system and/or experimental conditions that limit PFOA degradation.

To further assess if there was any defluorination of PFOA, fluoride was measured on Days 0, 31, and 103. Fluoride results showed initial fluoride contamination at Day 0 in all the media (<0.5 mg/L) except for the No NH 4 + cultures, and concentrations of ∼0.5 mg/L (25 μM) and 3.5 mg/L (180 μM) after amendment with the additional parent A6 culture on Days 16 and 50 (Supplementary Table S4). Given little evidence of PFOA transformation, we attributed the detected fluoride to contamination present in the parent A6 cultures. Because fluoride is often used as evidence of PFAS transformation, assessment of potential fluoride contamination is important. Neither the glass vials nor the PEZ carriers sorbed PFOA from the cultures (<0.1 mg (<0.24 µM) of PFOA removed per vial, Supplementary Figure S7).

This study investigated whether PEZ carriers could be engineered to incorporate ferric iron and support A6 growth for potential application in PFAS bioremediation. Iron-enhanced PEZ carriers were successfully synthesized, exhibiting a nearly three-fold increase in ammonium sorption capacity compared to normal PEZ carriers. The availability of ammonium via desorption suggested that carriers could potentially act as a localized nutrient “hot spot” for A6, and the carriers retained low levels of A6.

Despite this potential, A6 enrichment cultures showed limited growth, even in the positive control, and carrier addition did not significantly change population dynamics/A6 abundance or PFOA removal relative to controls under the tested conditions. The most likely constraint was the low initial A6 abundance, and the culture may not have been sufficiently active (as shown by lack of activity in the positive control) to fully test the carrier concept. Prior studies used A6 enrichment cultures containing 10 6 initial 16S gene copies (Huang and Jaffé, 2019), whereas the quantity of A6 in the experiment described herein began with only 10 2 –10 4 A6 16S gene copies and never reached 10 6 copies, indicating the initial dilution of the cultures may have limited growth. Acetate was added to lower redox potential due to low observed initial activity but may have shifted the microbial community from A6 early on, and even subsequent dosing of A6 may have not allowed recovery. Even so, A6 was consistently detected on both normal and iron-enhanced carriers after 103 days (∼10 3 copies/carrier), suggesting that the carriers may provide a more favorable habitat for these organisms than the bulk aqueous medium, but further optimization is needed.

The sulfur odor and the black material, which may be an iron sulfide resulting from the chemical reaction between hydrogen sulfide gas and goethite (Alguacil et al., 2022), suggest that sulfur- or sulfate-reducing bacteria (SRB) were present. These reactions have been observed in cultures that contain sulfate-reducing bacteria, transforming goethite into ferrous iron sulfide minerals such as mackinawite, which is black (Li et al., 2006). SRB could have outcompeted the A6 bacteria, particularly with the addition of acetate to decrease the redox potential. The transformation of available ferric iron in goethite into ferrous iron by sulfide and subsequent precipitation of an iron sulfide mineral could have further reduced the availability of the ferric iron needed by the A6 bacteria. Additional community analysis of the biomass and characterization of the black material would be necessary to confirm the hypothesis that SRB were present and iron sulfide minerals formed, but this was beyond the scope of the efforts herein. Consistent with the low A6 abundance, feammox activity was also minimal, and PFOA transformation was not significant. The acid dissolution studies showed limited iron availability, and there may be limitations on access due to location in the PEZ matrix, particle accessibility, and surface contact with cells that require elucidation. Besides limited iron availability, additional barriers to the growth of A6 in this experiment may include the crystallinity of goethite compared to ferrihydrite and microbial competition. Past successful defluorination has been observed with ferrihydrite (Huang and Jaffé, 2019), and while both iron oxides can be the iron source for A6, previous work has shown greater ammonium oxidation and iron reduction with the less crystalline ferrihydrite (Huang and Jaffé, 2018). Surface modification of the goethite may be needed to better facilitate the feammox process (Jaffé et al., 2024). A6, however, has recently been shown to transform crystalline goethite into less crystalline forms (Park et al., 2026). More work is needed to assess the viability of goethite and other iron oxide phases in the PEZ carriers.

Based on past work using this initial concentration (Huang and Jaffé, 2019; Jaffé et al., 2024), PFOA availability was likely not an issue, but evaluation at more broadly relevant environmental concentrations is also needed. Microbial competition could have occurred and been exacerbated by the addition of acetate to decrease the redox potential. While no measurable PFAS degradation was achieved, this study identified key considerations for future work.

This study demonstrated that carrier-based strategies can successfully integrate capture (ammonium) or incorporation (ferric iron) of nutrients and retain A6. While ammonium removal and A6 growth were briefly observed upon addition of A6 enrichment culture, a localized environment for A6 growth and PFOA transformation, however, was not established. There was no evidence of sustained A6 growth, ammonium removal, or PFOA biotransformation. The findings indicate that more concentrated A6 enrichment cultures, optimized iron delivery/availability, and improved culture conditions will be required to realize enhanced feammox activity and PFAS transformation. These findings underscore both the promise and limitations of applying A6 in engineered systems, and they provide a foundation for refining carrier design and cultivation strategies intended to support PFAS biotransformation. Future research should emphasize methods to increase A6 colonization and explore multifunctional sorbents that co-localize ammonium, iron, and PFAS.