Transformation of 6:2 fluorotelomer sulfonic acid in laccase-mediator systems

6:2 Fluorotelomer sulfonic acid (FTS) has been used as an alternative to perfluorooctane sulfonate (PFOS) as chrome mist suppressant in electroplating, emulsion aid in fluoropolymer processing, etc. Its biotic degradation is slow and incomplete, generating short chain perfluoroalkyl acids (PFAAs) and it is frequently detected in the environment. This proof-of-concept study reports possible transformation of 6:2 FTS by enzyme-catalyzed oxidative humification reactions (ECOHRs) in laccase-mediator systems (LMS) using crude or purified laccases, either mediated by a synthetic chemical (1-hydroxybenzotriazole) or a natural organic material (soybean meal extract; SBMe). The LMS with crude laccase, SBMe and 10 mM CuSO 4 exhibited a 23.8% reduction in concentration (mean of three technical replicates) relative to its control. A 34.8% reduction in adsorbable organic fluorine provided a supporting observation for transformation and potentially degradation. High-resolution mass spectrometry (HRMS) analysis unveiled a suite of tentative transformation products ranging from C8 to C3, including C 3 F 7 O 3 S, a previously unreported candidate formula for 6:2 FTS transformation products and potentially consistent with perfluoropropanesulfonate (PFPrS). Additionally, the calculated bond dissociation energies (BDEs) of the C-C bonds and the C-S bond in the 6:2 FTS were higher than those of PFOS, which could potentially explain the variable degradation observed in different studies. These findings underscore the potential of ECOHRs to transform per-and polyfluoroalkyl substances (PFAS) precursors. Future investigations into the transformation of PFAS mixtures in complex soil systems could provide actionable insights into ECOHRs as a potentially cost-effective remediation technique for vast crop lands, and pastures.

PFAS have been defined by the Organization for Economic Cooperation and Development (OECD) as fluorinated substances that contain at least one fully fluorinated methyl or methylene carbon atom (Wang et al., 2021; OECD, 2021). The U.S. Environmental Protection Agency (EPA) CompTox Chemicals Dashboard lists over 21,000 known PFAS species (USEPA, 2026). PFAS were widely used in numerous consumer products and industrial processes, ranging from non-stick pans to fire-extinguishing foams (Kissa, 2001), for their stability at high temperatures, chemical inertness, and surfactant properties (Quiñones and Snyder, 2009; Arora and Verma, 2026). The production and use of these compounds for decades, combined with their recalcitrance, have led to their pervasive presence in the ecosystem (Worley et al., 2017; Abunada et al., 2020; Wang et al., 2023b). PFAS were found to accumulate in various organs, leading to liver and kidney diseases, adverse effects on fertility, neurological dysfunctions, and thyroid cancer (Manojkumar et al., 2023; Espartero et al., 2022; Fenton et al., 2021; van Gerwen et al., 2023).

Increasing scientific evidence on the adverse health effects of PFAS led to their close scrutiny and eventual ban of many commonly detected PFAS, such as perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) (Brennan et al., 2021). The industry shifted to polyfluorinated alternatives, which were perceived as less toxic, less bioaccumulative, and less persistent (Poulsen et al., 2011). They often have shorter fluorinated carbon chains (six carbons or fewer), for instance, 6:2 FTS (six perfluorinated carbons) has been used as a substitute for PFOS in the aqueous film forming foams (AFFFs) as co-formulants (Place and Field, 2012), chrome mist suppressants in electroplating (Yang et al., 2014; Poulsen et al., 2011), emulsion aid in fluoropolymer processing (Yang et al., 2022), and food packaging (Trier et al., 2011). However, the degradation of 6:2 FTS is slow under aerobic conditions and negligible under anaerobic conditions (Wang et al., 2011). The slow degradation of 6:2 FTS, combined with its extensive use, has led to its frequent detection in environmental matrices, including groundwater, surface water, and soil (Oakes et al., 2010; Schultz et al., 2004; Houtz et al., 2013). Furthermore, polyfluorinated compounds such as 6:2 FTS are perfluoroalkyl acid (PFAA) precursors, known to yield persistent, terminal perfluorocarboxylic acids (PFCAs) following their environmental transformation (Grimberg et al., 2024).

Both biotic and abiotic degradation routes of 6:2 FTS have been studied for treatment purposes. Biotic transformation is regarded as more sustainable and cost-effective to implement. Transformation of 6:2 FTS with microbial community mixtures from different sources has been studied, such as activated sludge, landfill leachate, wastewater treatment plant (WWTP) sludge, and wetland rhizosphere, with variable success (Yin et al., 2019; D'Agostino and Mabury, 2017; Hamid et al., 2020; Wang et al., 2011). However, the effect of fungal bioremediation, specifically fungal extracellular enzymes, on 6:2 FTS is limited to very few studies. For instance, Wu et al. (2026) depicted 99.97% ± 0.21% degradation within 7 days by Phanaerochaete chrysosporium and Grimberg et al. (2024) depicted 50 mol% of the applied 6:2 FTS removed after 30 days by Trametopsis cervina. Both studies used live white-rot fungal strains that secrete extracellular enzymes, including laccase, lignin peroxidase, manganese peroxidase, and versatile peroxidase. These enzymes are secreted under nutrient starvation and are commonly involved in lignin oxidative transformation during the natural humification process (Datta et al., 2017). The process is governed by enzymatic reactions that involve the interaction of extracellular enzymes with small-molecular-weight substrates, which serve as mediators, generating highly reactive, unstable intermediate compounds. These intermediates are non-specific and react with lignin as well as various xenobiotics, thereby involving them in humification processes. These enzymatic reactions are coined as Enzyme-Catalyzed Oxidative Humification Reactions (ECOHRs) (Munir et al., 2025). Laccase-mediator systems (LMS) are most frequently employed to induce ECOHRs for remediation purposes, because laccase utilizes molecular oxygen as the terminal electron acceptor, whereas lignin and manganese peroxidases require hydrogen peroxide (Munir et al., 2025; Sellami et al., 2022).

Previous studies have shown that PFOS and PFOA were successfully transformed by ECOHRs induced by LMS (Luo et al., 2015; Luo et al., 2018b). In these studies, a purified laccase from Pleurotus ostreatus (EC 420-150-4) in combination with a synthetic mediator 1-hydroxybenzotriazole (HBT) transformed approximately 50% PFOA and 59% PFOS. Additionally, different metal cations were found to enhance the transformation of PFAS by acting as a bridge between negatively charged PFAS compounds and laccase enzyme (Luo et al., 2015; Luo et al., 2017).

Although the potential of ECOHRs for the remediation of PFAAs has been indicated by the studies with PFOA and PFOS, how PFAA precursors like 6:2 FTS may behave during ECOHRs has not been investigated. This study systematically examined the transformation of 6:2 FTS during ECOHRs induced by LMS. In this study, the LMS was established with two different laccase sources, a purified laccase from Aspergillus sp. and a crude laccase from Pycnoporus sp. SYBC-L3, with two different mediator sources, the synthetic mediator HBT and a natural product, soybean meal extract (SBMe). It was found in our previous study that SBMe can effectively mediate laccase reactions because it contains a suite of natural mediator compounds, including vanillin, apocynin (acetovanillone), and daidzein (Liang et al., 2017). Additionally, the synergistic effect of divalent metal cations (Cu +2) was also examined for both enzymatic systems. This study will address a critical research gap regarding the efficacy of ECOHRs in transforming a ubiquitous PFAS precursor, i.e., 6:2 FTS. Using crude laccase and the natural mediator SBMe could potentially be cost-effective, but the influence of complex soil matrices on transformation efficiency remains unexplored. Evaluating these dynamics is essential for guiding field-scale application of PFAS remediation across croplands, and pastures.

Chemicals and reagents

Purified laccase from Aspergilus sp (CAS no. 80498-15-3), 1-hydroxybenzotriazole monohydrate (HBT), 2,6-dimethoxyphenol (DMP), citric acid (C 6 H 8 O 7), disodium phosphate (Na 2 HPO 4), and anhydrous copper sulphate (CuSO 4) were purchased from Sigma-Aldrich (St. Louis, MO). The crude laccase enzyme was concentrated from the fermentation broth of the fungus Pycnoporus sp. SYBC-L3 using a published procedure (Liu et al., 2013). All HPLC-grade organic solvents, including acetonitrile and methanol, were obtained from Fisher Scientific (Pittsburgh, PA). The native fluorotelomer 6:2 FTS and the surrogate standard sodium 1H, 1H, 2H, 2H-perfluoro-1-[1,2- 13 C 2 ] octanesulfonate (M2- 6:2 FTS) were purchased from Wellington Laboratories (Ontario, Canada). Organic material, soybean meal, was purchased from Walt’s Organic Fertilizer Co.

Experiment setup

The experiment was carried out in 250 mL HDPE (high-density polyethylene) bottles with a working solution of 200 mL, incubated at 22 °C with continuous agitation on a rotary shaker at 130 RPM (revolutions per minute). Two enzymes (purified laccase from Aspergilus sp. and crude laccase from Pycnoporus sp. SYBC-L3) were investigated with a synthetic mediator, HBT, and a natural product, soybean meal extract (SBMe), in matching treatment setups (Table 1). Four treatment reactors were prepared for each enzyme, and four control reactors were prepared for the experiment. Two of the four treatment reactors for each enzyme were designed to compare the efficacy of mediators. They contained 1 µM 6:2 FTS (0.428 mg/L), 1 U/mL enzyme from one of the two laccase enzymes (enzyme activity is defined in Supplementary Material Text S1), and either received 2 µM HBT (40 µL of 10 mM HBT dissolved in acetonitrile) as supplementation or used SBMe as reaction medium (CLSM, CLNM, PLSM, PLNM). The remaining two reactors were supplemented with 10 mM CuSO 4 (1.59 g/L), in addition to the aforementioned composition (CLSMC, CLNMC, PLSMC, PLNMC). Control reactors consisted of a synthetic mediator control (SMC; 1 µM 6:2 FTS, 2 μM HBT, 10 mM CuSO 4), a natural mediator control (NM; 1 µM 6:2 FTS, 200 mL SBMe), and two enzyme-only controls for both enzymes (EN; 1 µM 6:2 FTS, 1 U/mL purified or crude laccase) to quantify any mass loss due to surface binding. However, some matrix-matched controls were not included in the experimental design, for instance, Cu supplemented NM-control and a non-Cu supplemented SMC control. The experimental components were dissolved in DI water for the HBT-mediated treatments, whereas SBMe was used as the reaction medium in SBMe-mediated treatments.

Although the reactors were not sterilized, the NM control (SBMe and 6:2 FTS only) was included to evaluate any potential contribution of SBMe-associated microbial activity to 6:2 FTS transformation. The reactors were opened only during sampling, and triplicate samples (1 mL each) were collected every 7 th day from within a single reactor for each treatment group at preselected time intervals. The samples were split into two, 0.5 mL each, for 6:2 FTS and HBT quantification. HBT was not detected at any sampling time; therefore, no data is available for it. Concentration correction was performed to address any variation in solution volume or concentration from evaporation or dilution from supplementation over time (see the Supplementary Material Text S1 for more information on the sampling and supplementation procedures).

Soybean meal extract (SBMe) preparation

SBMe was prepared according to a published protocol (Luo et al., 2018a). Non-sterile organic soybean meal (20 g) was added to 600 mL DI (deionized) water and equilibrated on a rotary shaker for 5 days before filtration. The filtration was a multistep process, starting with separating the coarse soybean meal particles by passing the mixture through a Buchner funnel without filter paper and vacuum. In the next step, the extract was filtered through Whatman no. 42 filter paper, followed by a 0.45 µm cellulose acetate membrane under vacuum. The extract for the entire experiment was prepared at the beginning, homogenized, and stored at 4 °C.

Chemical analysis

The quantification of 6:2 FTS was performed on Waters ACQUITY UPLC (ultra-performance liquid chromatography) I-Class coupled with a triple-stage quadrupole mass spectrometer (UPLC-MS/MS, Xevo TQD, Waters Corp., United States). The detailed isocratic program parameters are described in Supplementary Material Text S2. Identification of the reaction products was performed on a Bruker solariX® Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer (Billerica, MA, United States), equipped with a 12 T magnet. Adsorbable organic fluorine analysis was performed on select treatments by combustion ion chromatography (CIC) by Eurofins Lancaster Laboratories (see the Supplementary Material Text S2 for more information).

Bond dissociation energy (BDE) computation

BDEs of C-C or C-O/C-S bonds in the main-chain of PFAS were calculated via the DMol3 module of Material Studio software. Spin-unrestricted are added in the radicals during the calculation processes. The dissociation energies are calculated using the following expression:

Where E radical_A and E radical_B represent the energies of the A and B radicals in the A-B bond, E PFAS is the energy of the specific PFAS. Further computational details related to BDE calculations are provided in Supplementary Material Text S3.

Change of 6:2 FTS concentration during ECOHRs

In this single-reactor setup, 6:2 FTS concentrations across various laccase-mediator systems (LMS) provide exploratory observations (Figure 1), utilizing technical replicate statistics (mean ± standard deviation) to depict measurement precision. It compares the efficacy of the LMS systems employing either the synthetic mediator; SM (HBT), or the natural mediator; NM (SBMe), with the crude laccase (Figure 1, Panel 2B: CLSM, CLNM) to those with the purified laccase (Figure 1, Panel 2A: PLSM, PLNM). The systems with the addition of CuSO 4 have also been studied and illustrated in Figure 1 as CLSMC and CLNMC (Panel 1B) for crude laccase, and as PLSMC and PLNMC (Panel 1A) for purified laccase. The 6:2 FTS concentration was compared with time zero and with the best available control reactors (see Table 1 for the compositions of the control reactors).

The concentration of 6:2 FTS was relatively stable in the control reactors over time but showed varying degrees of reduction in the treatment reactors compared with their respective controls. On day 119, the concentration of 6:2 FTS in treatments CLNMC with SBMe mediator and CLSMC with HBT mediator decreased by 23.8% and 13.0%, respectively, relative to SMC-Control (Figure 1, Panel 1B). A similar trend was observed for purified laccase, albeit to a lesser extent, an 8.0% drop in the treatment PLNMC with SBMe mediator and 4.6% increase in the PLSMC with HBT mediator relative to SMC-Control (Figure 1, panel 1A). The bridging role of metal ions (Cu 2+) observed in our previous study (Luo et al., 2017) was also observed in this study, as evidenced by better separation and a notable drop in the concentration of CuSO 4 supplemented treatments (specifically CLNMC, CLSMC, PLNMC) relative to the control (SMC-Control). However, CuSO 4 did not appear to assist the concentration reduction in PLSMC, as observed with its crude laccase analog in CLSMC. A possible reason could be the formation of benzotriazole, which is inactive as a mediator, from the reaction of the HBT radical with the structural aromatic amino acids of laccase (Hilgers et al., 2018). Additionally, treatments without CuSO 4 supplementation (CLSM, CLNM, PLNM, PLSM) exhibited a 7.6% decrease in CLSM with HBT and a 10% increase in the CLNM with SBMe for crude laccase, while a 20.4% decrease in PLSM with HBT and a 2.6% decrease in PLNM with SBMe for purified laccase relative to NM-Control (Figure 1, Panel 2B and 2A). The observed reduction in concentration is attributed to transformation rather than adsorption between the laccase enzyme and 6:2 FTS, as proposed by Steffens et al. (2023). This interpretation is supported by evidence from the enzyme-only controls (EN-control) for both enzymes, which contained only laccase (purified or crude, individually) and 6:2 FTS yet showed 99.86% recovery for crude laccase and 102% for purified laccase. Similarly, the stability of the NM-Control, which contained only 6:2 FTS and SBMe, suggests absence of a notable matrix effect of SBMe. Additionally, HRMS analysis identified tentative intermediates of commonly reported transformation products in both crude (CLNMC) and purified (PLNMC) laccase systems with SBMe (Supplementary Tables S3 and S4). The adsorbable organic fluorine analysis provided additional supporting observation with a 34.8% decrease in CLNMC relative to the NM control (Supplementary Table S4).

Enzyme activity during ECOHRs

Figure 2 illustrates enzyme activity at each sampling time across all treatment reactors for both enzymes, i.e., crude (Panel A) and purified laccase (Panel B). Enzyme supplementation was performed only if the enzyme activity was detected at a level below 1 U/mL. The cumulative amount of enzyme and mediator added throughout the experiment is provided in Supplementary Table S2. The treatments that showed greater concentration reduction (CLSMC, CLNMC, PLSM, PLNMC) exhibited low residual enzyme levels (below the experimental level of 1 U/mL) at all sampling time points. Specifically, crude laccase treatments CLNMC with the SBMe mediator and CLSMC with the HBT mediator showed enzyme levels ≤0.5 U/mL and 6:2 FTS concentration reduction at 23.8% and 13%, respectively. Similarly, purified laccase treatment PLSM with HBT mediator (20.4% concentration reduction) and treatment PLNMC with SBMe mediator (8% concentration reduction) depicted enzyme levels at ≤ 1 U/mL and <0.5 U/mL, respectively. The enzyme activity data also revealed that the reactors supplemented with copper sulphate (CLSMC, CLNMC, PLSMC, PLNMC) exhibited lower residual enzyme activity than their unsupplemented analogs (Figure 2). As reported earlier, this trend is likely due to the bridging effect induced by Cu 2+ ions between PFAS and the enzyme (Luo et al., 2017). The only anomaly was PLSMC with HBT mediator of purified laccase, which exhibited low residual enzyme activity but did not show a corresponding 6:2 FTS concentration reduction. This limitation likely stemmed from the transformation of HBT radicals into benzotriazole, a known inactive mediator that suppresses the overall efficiency of the LMS (Hilgers et al., 2018; Sealey and Ragauskas, 1998).

Tentative transformation products of 6:2 FTS by ECOHRs

The treatments containing purified and crude laccase mediated by the natural mediator SBMe (Figure 1, PLNMC and CLNMC) were selected to investigate the reaction products. Although MS/MS fragmentation data was unavailable to confirm specific structural assignments, the tentative transformation products and possible structures are highlighted with CAS numbers in the Supplementary Tables S3 and S4. The mMass (version 5.5.0) was used to process the FTICR mass spectrometry. The m/z peaks were extracted from the HRMS full-scan spectrum by setting the signal-to-noise ratio (S/N) threshold to 5 and the relative intensity to 0.1%. Subsequently, all peaks were compared with a PFAS mass library with a mass error tolerance of 1.0 ppm. The mass library comprised of all the entries retrieved from the USEPA Chemistry Dashboard, over 10,000 PFAS. Molecular formulas were assigned to the matched peaks with Confidence Level 4 (CL-4) according to Schymanski et al. (2014) (Wang et al., 2023a). The tentative reaction products were identified in the laccase mediator treatment samples by excluding the m/z detected in the control samples, with molecular formulas assigned within 5 ppm of the m/z (see the Supplementary Material Text S2 for more information).

A diverse range of C 8 –C 3 fluorinated compounds were detected, with C 8 species being the most abundant for both enzymes. Notably, PLNMC yielded substantially more unique products (38) than CLNMC (12), as detailed in Supplementary Tables S3 and S4. This divergence could be driven by the co-factors present in the crude laccase solution, including additional proteins (aldolase, glucanase, and exo-cellobiohydrolase), organic acids, and trace-level metal ions from the nutrient growth culture (Liu et al., 2013; Yu et al., 2025). These co-factors can potentially act synergistically and drive an exhaustive, deep transformation as well as potential breakage of the fluorinated backbone, resulting in the detection of fewer (12) end-products (Supplementary Table S4). Degradation may also be inferred from the structural profile of entries 1, 5, and 6 (C 8 H 4 F 8 N 2, C 8 H 2 F 6 N 2, and C 8 H 4 Cl 2 F 4 O 2) in CLNMC, and PLNMC products such as entries 1, 2, and 5 (C 8 H 8 F 7 NO 3, C 8 H 4 F 8 N 2 O 2, and C 8 H 8 F 7 NO 2) that retained C8 carbon count of 6:2 FTS but display headgroup modification and diminished fluorine counts; however, fluoride release data is not available to confirm defluorination. The transformation is also supported by targeted measurements within the CLNMC, which showed a 34.8% drop in total organic fluorine (Supplementary Table S5) and a 23.8% reduction in 6:2 FTS concentration. In contrast, no substantial drop in total organic fluorine was measured within PLNMC (Supplementary Table S5), consistent with the 8% reduction in concentration.

CLNMC entry 2 (C 8 F 17 N) and PLNMC entry 3 (C 8 H 2 F 17 NO 2 S) exhibited higher fluorine count than 6:2 FTS, indicating a potential radical recombination event driven by upstream C-C cleavage fragments. Headgroup substitution with halogens, phosphonates, and thiocyanates observed in the products might be derived from the SBMe matrix (Barbosa et al., 2013). Additionally, a suite of tentative transformation products was identified in PLNMC, C 8 H 3 F 14 I (entry 4, m/z 490.8981) ─> C 6 H 2 F 11 I (entry 29, m/z 408.8949) ─> C 5 F 9 IO (entry 33, m/z 372.8777) ─> C 4 F 9 I (entry 35, m/z 344.8828), whose decreasing carbon chain length is consistent with a putative sequential degradation pathway. However, single-time-point analysis is insufficient to establish a sequential degradation pathway, and confirmation requires time-resolved intermediate profiling or isotope tracing. Additionally, the entry 37, i.e., C 3 F 7 O 3 S represents, to the best of our knowledge, a previously unreported candidate formula which is potentially consistent with perfluoropropanesulfonate (PFPrS) as a tentative 6:2 FTS transformation product. The retention of the sulfur atom on the candidate formula (C 3 F 7 O 3 S) is an indication of the non-specific action of ECOHRs on the 6:2 FTS structure; however, the absence of structural confirmation limits the certainty in this structural assignment and, by extension, this interpretation.

While conventional terminal PFCAs, including PFHpA (perfluoroheptanoic acid), PFHxA (perfluorohexanoic acid), PFPeA (perfluoropentanoic acid), 5:3 acids, PFBA (perfluorobutanoic acid) were not detected in either enzyme treatment (Zhao et al., 2021; Wang et al., 2011; Zhang et al., 2016), several tentative products were identified that are structurally similar to the terminal PFCA products of 6:2 FTS. These tentative products are presented here as hypothetical transient precursors. C 8 H 5 F 11 O and C 6 HF 11 O could possibly represent a 5:3 telomer aldehyde/alcohol and undecafluorohexanal, respectively, which are single-oxygen deficit relative to 5:3 FTCA (C 8 H 5 F 11 O 2) and PFHxA (C 6 HF 11 O 2). The entry 34 (C 5 HF 8 NaO 2) could be matrix-driven sodium salt stabilization alongside a single-fluorine atom deficit relative to PFPeA (C 5 HF 9 O 2). Likewise, entry 8 (C 7 BrF 13 O) relative to PFHpA (C 7 HF 13 O 2) could result from the transient perfluoroheptanoyl radical intermediate being capped by SBMe-driven bromide ions.

Overall, the tentative products detected across both PLNMC and CLNMC highlight potentially similar degradation mechanisms as observed for PFOA and PFOS during ECOHRs in previous studies, including C-C cleavage, headgroup modification, and recombination (Luo et al., 2018b; Luo et al., 2015).

Bond dissociation energy (BDE) of 6:2 FTS

The degradation pattern observed for 6:2 FTS varies strikingly across different studies. For instance, Zhang et al. (2016) observed that 6:2 FTS transformed in aerobic sediment with a half-life of less than 5 days, whereas Wang et al. (2011) observed only 36.3% transformation after 90 days in aerobic soil. Similarly, the highest transformation observed in the current study is 23.8% after 119 days. To elucidate the underlying mechanism governing this localized recalcitrance, BDEs of 6:2 FTS were calculated and compared with reference PFAS compounds (Figure 3). Notably, the BDE of the 6:2 FTS backbone exhibited a significant energy spike at the junction connecting the perfluorinated tail and the hydrogenated linker, even exceeding the uniform BDE profile of PFOS. This localized bond stabilization may be rationalized through the application of Bent’s rule and the asymmetry of the 6:2 FTS structure (Bent, 1961; Buck et al., 2011). Due to the extreme electronegativity of the fluorine substituents, the junction carbon atoms preferentially direct higher p-character into the more electronegative substituent, i.e., fluorine. Consequently, the remaining atomic orbital budget forces a significant inflation of s-character into the adjacent C-C bond (Lemal, 2004). Because atomic s-orbitals feature a shorter radial distribution closer to the carbon nucleus than p-orbitals, this localized increase in s-character causes an intrinsic shortening and dramatic strengthening of the backbone junction (Grabowski, 2011). Furthermore, this electronic polarization may have been mechanically compounded by the structural mismatch between the rigid, helical perfluorinated tail and the flexible, zigzag hydrocarbon linker (Buck et al., 2011), creating a dense steric and electronic boundary that further elevates the thermodynamic barrier to bond cleavage.

Role of soybean meal extract as a natural mediator

The dosage of the natural mediator (20 g soybean meal in 600 mL DI water) used in the current study was previously tested for a relatively shorter duration, i.e., 36 days (Luo et al., 2018a). However, the current study evaluated its performance over an extended 119-day period relative to the synthetic mediator HBT. Critically, soybean meal extract was amended only once at the onset of the experiment, while 2 µM HBT was supplemented every week. As illustrated in Figure 1, treatments containing the natural mediator (PLNM, PLNMC, CLNM, CLNMC) maintained sufficient phenolic compounds to sustain ECOHRs across the entire 119-day duration. These systems exhibited better transformation efficiencies than systems with HBT as the mediator, particularly when supplemented with copper ions (PLNMC, CLNMC), as evidenced by consistently lower 6:2 FTS concentrations (Figure 1). The 200 mL extract dosage did not induce laccase deactivation, a phenomenon previously linked to excessive mediator concentrations and subsequent radical overproduction (Luo et al., 2017), depicting the stability of the optimized dose under long-term conditions. Furthermore, the low cost of soybean meal ($0.15/lb) (USDA, 2026), and the elimination of 80% downstream enzyme purification costs in crude laccase (Antecka et al., 2019; Akinyemi et al., 2022), are all potential advantages of this system.

This proof-of-concept study reported 6:2 FTS transformation under different laccase-mediator systems, one with the synthetic mediator HBT and the other with the natural mediator soybean meal extract. The crude laccase and SBMe combination, supplemented with CuSO 4 (CLNMC), exhibited higher efficiency, transforming ∼24% of the 6:2 FTS concentration, compared with its control. The statistical significance could not be assessed because the single-reactor setup lacked independent replicates. The CLNMC also showed a ∼35% reduction in adsorbable organic fluorine, alongside the observed concentration reduction, which indicates transformation and potential degradation. The tentative transformation products observed after HRMS analysis ranged from C 8 –C 3 carbon chains for crude and purified laccase in the LMS with SBMe and CuSO 4 supplementation. A previously unreported candidate formula (C 3 F 7 O 3 S) was also identified, which is potentially consistent with perfluoropropanesulfonate (PFPrS). Additionally, the bond dissociation energy calculation showed that 6:2 FTS has much higher BDEs for the C-C bond at the α-carbon and the C-S bond than PFOS, which might potentially explain its variable transformation across different studies. Future work should focus on transformation in the environmentally relevant soil matrices, and assessment of ecotoxic effects of CuSO 4 if its use is to be recommended for practical application. Additional priorities could include quantifying degradation products and defining the degradation pathway in ECOHRs.