Enriching Fluorotelomer Carboxylic Acids-Degrading Consortia from Sludges and Soils

Enriching Fluorotelomer Carboxylic Acids-Degrading Consortia from Sludges and Soils | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enriching Fluorotelomer Carboxylic Acids-Degrading Consortia from Sludges and Soils Chen Wu, Mengyan Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4824417/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Fluorotelomer carboxylic acids (FTCAs) has drawn increasing attention due to their prevalent occurrence, high toxicity, and bioaccumulating effects. In this study, we enriched and characterized microbial consortia with sustainable FTCA removal abilities from two activated sludges and five soils when no external carbon sources were supplemented. After four generations of enrichment, stable 6:2 FTCA and 5:3 FTCA biodegradation were achieved, reaching 0.72~0.98 and 0.53~1.05 µM/day, respectively. Coupling with 6:2 FTCA biotransformation, fluoride release co-occurred, conducive to approximate 0.19 fluoride per 6:2 FTCA molecule that was biodegraded. In contrast, minimal free fluoride was detected in 5:3 FTCA-amended consortia, indicating the dominance of “non-fluoride releasing pathways”. Microbial community analysis revealed the dominance of 13 genera across all consortia. Among them, 3 genera, including Hyphomicrobium, Methylorubrum, and Achromobacter , were found more enriched in consortia amended with 6:2 FTCA than those with 5:3 FTCA from an identical inoculation source, suggesting their involvement in biodefluorination. This study uncovered that microbial consortia can degrade FTCAs without the supplement of external carbon sources, though with low biotransformation and biodefluorination rates. Further research is underscored to investigate the involved biotransformation pathways and biodefluorination mechanisms, as well as effects of external carbon sources. PFAS precursor biotransformation biodefluorination fluorotelomer carboxylate acids Hyphomicrobium Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Fluorotelomer carboxylic acids (FTCAs) represent a class of per- and polyfluoroalkyl substances (PFASs) that have garnered significant attention in recent years due to their prevalent occurrence and potential environmental impacts (Fenton et al. 2021 ). Structure of FTCAs is featured by a perfluorinated tail, a carboxylic acid head, and a few non-fluorinated alkyl carbons in between (Figure S1 ). 6:2 FTCA and 5:3 FTCA are two most concerned FTCAs considering their widespread detection in landfill leachates, wastewater, and other environmental matrixes (Allred et al. 2014 , Fuertes et al. 2017 , Lang et al. 2017 , Wu et al. 2022 ). Furthermore, FTCAs have also been frequently detected at aqueous film-forming foam (AFFF)-impacted sites, as they are key intermediates and end products from the biotransformation of PFAS precursors, such as fluorotelomer sulfonates (FTSs) (Shaw et al. 2019 , Zhang et al. 2016 ) and fluoroalkyl phosphates (PAPs) (Lewis et al. 2016 ). Though not regulated, FTCAs are found more toxic than their corresponding perfluorocarboxylic acids (PFCAs), particularly those that have received primary public and regulatory attentions (e.g., PFOA) (Phillips et al. 2007 , Shi et al. 2017 ). Therefore, it is of significant value to investigate the fate and behavior of FTCAs and develop feasible treatment approaches to mitigate their impacts to the environment. As compared to PFCAs, FTCAs contain non-fluorinated carbons, which may facilitate microbial enzymatic reactions that activate subsequent biodefluorination (Ross et al. 2018 , Wackett 2022 ). Recent studies on pure cultures and environmental microbiomes (e.g., activated sludges and soils) revealed that biotransformation of FTCAs and their precursors can occur and generate shorter chain PFCAs, such as perfluorohexanoic acid (PFHxA), perfluoropentanoic acid (PFPeA), and perfluorobutanoic acid (PFBA) (Che et al. 2021 , D'Agostino and Mabury 2017 , Evich et al. 2022 , Lee et al. 2010 , Li et al. 2018 , Qiao et al. 2021 , Wang et al. 2011 , Wang et al. 2012 , Wu et al. 2024 , Zhang et al. 2013 , Zhao et al. 2013 ). Several biotransformation pathways have been proposed. For instance, Wang et al. postulated "one-carbon removal pathways", through which 5:3 FTCA is transformed to 4:3 FTCA or shorter chain PFCAs via the formation of α-OH 5:3 acid and 5:2 FTCA (Wang et al. 2012 ). In contrast to these degradative pathways, an alternative conjugative pathway that is analogous to fatty acid β oxidation has recently been reported based on the formation of CoA adducts for FTCAs with non-fluorinated β carbons (Mothersole et al. 2023 ). A medium-chain acyl-CoA synthetase in Gordonia sp. strain NB4-1Y was responsible for the catalysis of such reactions with 2:3 FTCA, 2:3 fluorotelomer unsaturated carboxylic acid (FTUCA), and 1:4 FTCA (Mothersole et al. 2023 ). These findings indicated that FTCAs can be biodegradable. However, knowledge remain elusive regarding microorganisms that can degrade FTCAs without the supplement of external carbon sources. In this study, we seek to acclimate microbial consortia from diverse sources (two activated sludges and six soils) that were repetitively fed with high concentrations of 6:2 and/or 5:3 FTCAs (~ 80 µM) without the supplement of external carbon sources. The microbial community structures of the enriched consortia were analyzed and compared when constant FTCA removal (and fluoride release, if any) were observed in a 1-year long enrichment. Dominant bacteria that may contribute to FTCA biotransformation and biodefluorination were identified for future investigation. 2. Methods AND MATERIALS 2.1. Chemicals and Reagents 6:2 FTCA standard, 5:3 FTCA standard, and mass-labeled M2-6:2 FTCA standard for calibration were all purchased from Wellington Laboratories Inc. (Guelph, Ontario, Canada) with the purity of > 98%. Bulk 6:2 FTCA (3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanoic acid, > 97%) and 5:3 FTCA (2H,2H,3H,3H-perfluorooctanoic acid, > 97%) for enrichment experiments were purchased from Synquest Laboratories (Alachua, FL, USA). LCMS grade methanol (> 99%) was purchased from Fisher Chemicals (Hampton, NH, USA). Ultrapure water obtained from the Milli-Q RC Synthesis water purification system (Millipore, Bedford, MA, USA) was used in the experiments. 2.2. Enrichment of FTCA-degrading Consortia Biosolids were collected from 8 different sources, including two activated sludges from wastewater treatment plants (WWTPs) and 6 soils in New Jersey (Table S1 ). Biosolids were washed three times with phosphate buffer saline solution (PBS, 20 mM sodium phosphate, pH 7.0) to remove organic matter before enrichment. First, 120 µL of 20 mM FTCA(s) stock solution was spiked to 160-mL sterile amber serum bottles, and the methanol solvent was fully evaporated using nitrogen flow. Then, 1 g of each washed biosolid (wet weight) was inoculated into individual FTCA-spiked serum bottle, followed by adding 30 mL ammonium mineral salts (AMS) media. All seeded bottles were incubated under 30°C while being shaken at 120 rpm. At time 0 and select intervals (as indicated Table S1 ), supernatants (~ 3 mL) were collected, centrifuged at 12,000 rpm for 3 min, and then filtrated via a PES membrane filter for the monitoring of the removal of FTCAs and release of free fluoride as described below. In parallel, analytical controls were prepared without biosolid inoculation to discern the baseline of the initial FTCA dosage. After 60 ~ 130 days of incubation, biosolids in the serum bottles was collected and washed three times with PBS before being transferred to sterile bottles with fresh AMS media spiked with FTCA(s) for the 2nd, 3rd, and 4th generations of enrichment as shown in Fig. 1 . Four generations of enrichment were prepared and monitored in the same fashion. At the end of the 4th generation, biosolids were collected by centrifugation at 12,000 rpm for 15 min for DNA extraction and microbial community analysis to reveal the dominant species that might contribute to FTCA biotransformation and biodefluorination. Before further analysis, all samples were sealed and stored in the − 20°C freezer. 2.2. FTCA Quantification by LC/MS/MS To monitor FTCA removal, 1 mL of the filtered supernatant was diluted by 100 times with pure methanol and then spiked with 20 µg/L M2-6:2 FTCA as the internal standard before FTCA quantification analysis based on a protocol adapted from EPA Method 537 (Liu et al. 2021 , Wu et al. 2022 ). 6:2 and 5:3 FTCAs were analyzed using a 1290 Infinity II HPLC system in tandem with 6470A triple quadrupole mass spectrometer (Agilent, Santa Clara, CA). Aliquots (10 µL) were injected into this LC/MS/MS system equipped with a Symmetry C18 column (ID 2.1 mm, length 100 mm, particle size 3.5 µm) (Waters, Milford, MI) at a flow rate of 0.3 mL/min. The mobile phase initially consisted of 80% solvent A (5 mM ammonia acetate in 10% (v/v) methanol), decreased to 40% A with 60% solvent B (pure methanol) in 2.0 min, and further reduced to 20% A in 1 min and kept for 5 min, and then changed back to 80% A in 1 min and held for 4.5 min. Triple quadrupoles mass spectrometer was set in the negative-ion electrospray mode, and multiple reaction mode (MRM) was set for ion collection. The pressure in the nebulizer was set at 25 psi with a capillary voltage of -3.5 kV. The desolvation gas temperature was 350°C, with a flow rate of 8 L/min. MS data were processed using the MassHunter QQQ quantitative analysis software (Agilent Technologies, USA). Laboratory reagent blanks (LRBs) were prepared with 250 mL reagent water in HDPE bottles in the laboratory. LRBs were preserved, stored, and processed at the same conditions as all samples. PFAS calibration and lower limits of detection were determined following the procedures described in our previous publication (Wu et al. 2022 ). All calibration solutions were prepared in triplicate, and their average mass intensities were used for constructing the calibration curves when the regression linearity met R 2 > 0.99. Calibration verification checks were performed every 15–20 samples and at the end of analysis. 2.3. Fluoride Analysis To measure free fluoride, 2.0 mL of the filtered supernatant was collected and filtered through a 0.22-µm PES membrane. After mixing with 2.0 mL of TISAB II buffer solution (Thermo-Fisher Scientific), fluoride concentration was measured using the Orion Star Meter equipped with the fluoride electrodes (Thermo-Fisher Scientific). 2.4. Microbial Community Analysis Phylogenetic structures were analyzed using the 16S rRNA amplicon-based sequencing and associated bioinformatics tools in order to investigate the microbial community profiles of the consortia after the 4th generation of enrichment. Genomic DNA was extracted from the obtained biosolids using the Qiagen DNeasy PowerSoil Kit (Qiagen, Germany) following the manufacturer's protocol. Primers 338F (5'- ACTCCTACGGGAGGCAGCA-3') and 806R (5'- GGACTACHVGGGTWTCTAAT-3') were used to amplify the 16S rRNA variable regions V3 and V4 (Kumar et al. 2011 ). Paired-end high-throughput sequencing was performed by the HiSeq Sequencing System (Illumina, San Diego, CA) (Nelson et al. 2014 ). 16S rRNA gene reads were processed using the QIIME2 pipeline (v2021.4) (Bolyen et al. 2019 ) with the Divisive Amplicon Denoising Algorithm 2 (DADA2) (Callahan et al. 2016 ) for sequence pairing, denoising, and chimera elimination. 3. Results And Discussion 3.1. Consistent FTCA Biotransformation in Enriched Consortia Over four generations of enrichment, consistent removal of 6:2 FTCA was observed in consortia from two activated sludges (BH and V) and five soils (B to F) while little removal was observed in analytical controls (Figure S2). As normalized to the enrichment durations of each generation, 6:2 FTCA biotransformation rates (Fig. 2 ) were in range between 0.28 ~ 0.53, 0.05 ~ 0.40, 0.50 ~ 1.34, 0.72 ~ 0.98 µM/day in the 1st, 2nd, 3rd, and 4th generations, respectively. For enrichments prepared with Soil A, 6:2 FTCA biotransformation rates were consistently lower than 0.04 µM/day. Therefore, this consortium was discontinued after the 3rd generation of enrichment. Similarly, consistent 5:3 FTCA removal was also evident in those consortia that exhibited 6:2 FTCA removal (Figure S3). In general, 5:3 FTCA biotransformation was slower than 6:2 FTCA, particularly in the first two generations of enrichment. 5:3 biotransformation rates (Fig. 2 ) were in range between 0.07 ~ 0.42, 0.02 ~ 0.54, 0.78 ~ 1.38, 0.53 ~ 1.05 µM/day in the 1st, 2nd, 3rd, and 4th generations, respectively. With BH, B, and E as the inocula, their consortia exhibited low 5:3 biotransformation with rates below 0.2 µM/day in the 1st and 2nd generations. However, 5:3 biotransformation removal in these enrichment consortia escalated in the 3rd generation. In contrast, the consortium obtained from Soil A continued to show a low 5:3 FTCA removal, which was thus discontinued at the end of the third generation. For both 6:2 and 5:3 FTCAs, biotransformation was mostly slowest in the 2nd generation and then increased in the 3rd and 4th generations (Figs. 2 and 3 ). In the 1st generation, even though biosolids were thoroughly washed and amended only with FTCAs, endogenous metabolism may occur due to the decay of the biomass from different inoculation sources. Some microorganisms will break down and release metabolites and cellular compositions that become available to other microorganisms, particularly those can participate in FTCA biotransformation. Such mechanism can fuel the cells to degrade FTCAs over the 1st generation. However, after the transfer to fresh AMS media in the 2nd generation without the supplement of external carbon sources other than FTCAs, the effects of endogenous metabolism became minimal. This explained the dip in FTCA biotransformation observed in the 2nd generation of enrichment. After the 2nd generation, the microbiomes of the consortia continued to adapt to FTCAs as the sole carbon supplement and thus exhibit greater FTCA biotransformation in the 3rd and 4th generations of enrichment. These results indicated the acclimation of microbial communities with stable FTCA biotransformation abilities in the enriched consortia. Coupling with FTCA biotransformation, metabolites based on “one-carbon removal pathways” were detected by a new PFAS analytical approach developed in our lab, nano-electrospray ionization and high-resolution mass spectrometry (Nano-ESI-HRMS) (Wu et al. 2022 , Wu et al. 2024 ), since their concentrations were mostly close to or even below the limits of detection by LC/MS/MS. Such metabolites included PFCAs (e.g., PFHxA, PFPeA, and PFBA) and FTCAs (e.g., 5:2 FTCA and 4:3 FTCA). We also detected some novel metabolites that were more dominant, which will be reported in a separate work by the group. 3.2. Biodefluorination Only for 6:2 FTCA, But Not 5:3 FTCA Biodefluorination was evident as the liberation of free fluoride in the media. As shown in Fig. 2 and S5, minimal fluoride was detected in consortia that were fed with 5:3 FTCA only regardless of inoculation sources and enrichment generations. This is in good agreement with our recent report on 5:3 FTCA biotransformation by four activated sludges collected from four other WWTPs (not BH or V as reported in this study) in the New York and New Jersey area. Minimal fluoride release was observed within 7 days of incubation, though with the low detection of less fluorinated products, such as 5:3 FTUCA and PFBA (Wu et al. 2024 ). In a separate study by Che et al ., free fluoride was not detected during the aerobic biodegradation of 1:3 FTCA, 1:3 FTUCA, and 2:3 FTCA by activated sludge (Che et al. 2021 ). These results corroborated that aerobic biotransformation of 5:3 FTCA and other n:3 FT(U)CAs may be dominated by “non-fluoride releasing pathways” (Wu et al. 2024 ), rather than “one-carbon removal pathways”. The availability of non-fluorinated α and β carbons in n:3 FTCAs may enable the entrance to β oxidation via the conjugation with CoA without the liberation of free fluoride (Che et al. 2021 , Mothersole et al. 2023 ). Furthermore, assimilation of less-fluorinated molecules and their adducts may also enter cell metabolism and lead to the buildup of fluorinated macromolecules, such as fluorinated anabolites and phospholipids (Xie et al. 2022 ). Given the fact that little fluoride was released over 5:3 FTCA biotransformation in all samples in this and previous studies, we accounted all the detected fluoride for 6:2 FTCA biotransformation in the first three generations of enrichment for soil samples (A to F) when 6:2 and 5:3 FTCAs were concurrently amended. For two sludge samples BH and V, 6:2 and 5:3 FTCAs were amended separately in all four generations of enrichment (Table S1 ). As shown in Figure S4 and 2, constant free fluoride accumulation, varying from 37.2 ~ 46.8 µM for 1st generation to 6.8 ~ 12.8 µM for 2nd to 4th generation, was observed in the consortia from two activated sludges. Fluoride release was also observed in the soil enrichments C to F through the four generations of enrichment, accumulating to 6.0 ~ 75.1 µM for the 1st generation and 3.6 ~ 18.0 µM for 2nd to 4th generations (Figure S4). At the same time, little fluoride release was detected in the soil enrichment A from the 1st to 3rd generations and the soil enrichment B in the 1st and 2nd generations as the defluorination rates were below 0.02 µM/day (Fig. 2 ). However, the defluorination rate in the soil enrichment B started to accelerate in the 3rd generation (0.09 µM/day). Considering the consensus of 6:2 FTCA removal and fluoride release in the first three generations of enrichment, consortia B was continued for the 4th generation of enrichment. No fluoride accumulation was observed in the analytical controls. The observed fluoride accumulation corresponded to the removal of 6:2 FTCA in all consortia (Fig. 2 ), supporting that 6:2 FTCA biotransformation was accompanied by free fluoride release. A significant correlation (Fig. 3 ) was established between the 6:2 FTCA biotransformation rates and biodefluorination rates based on all consortia across four generations of enrichment ( p < 0.01). Notably, a correlation factor of 0.1943 was estimated as the slope of the linear regression forced through the origin. This suggested an average of ~ 0.19 fluoride was released per 6:2 FTCA molecule biotransformed by the consortia. This is much lower than our recent work on 6:2 FTCA biotransformation by four other activated sludges that demonstrated 0.61 ~ 1.92 fluoride release per molecule of 6:2 FTCA that was biodegraded (Wu et al. 2024 ). This was probably due to the initial supplement of external carbons (i.e., glucose and acetate) in the previous study, while no external carbons were amended in this study. Biodefluorination can be energy consuming (Wackett 2022 ). Thus, without the supplement of external carbon sources, 6:2 FTCA biodefluorination extent and efficiency can be greatly dampened. 3.3. Potential FTCA Degraders Enriched in the Consortia A total of 14 consortia from 7 different sources were incubated over 1 year under the exposure of 6:2 or/and 5:3 FTCAs without adding external carbon sources. Given the continuous FTCA removal (and fluoride accumulation for 6:2 FTCA-spiked consortia), it’s plausible to hypothesize that some dominant taxa in the 4th generation enrichments were FTCA degraders via metabolism or co-metabolism and through fluoride releasing pathways for 6:2 FTCA and non-fluoride releasing pathways for 5:3 FTCA. Hence, 16S rRNA sequencing was conducted for consortia obtained after the 4th generation of enrichment to identify the dominant OTUs at the genus level. As shown in Table S2, 36 OTUs were found prevalent across 14 consortia with maximum relative abundances (RA) of 0.1% or higher. Though 5 out of these 36 OTUs were unclassified, the rest 31 OTUs were classified into 13 genera and 10 families according to nucleotide-based BLAST from NCBI. These 13 genera were all gram-negative, similar to our previous work on FTCA-degrading activated sludge communities (Wu et al. 2024 ). The bias from the varied DNA extraction efficacies for gram-positive and gram-negative strains cannot be fully excluded even though sonication was applied to enhance the cell lysis before DNA extraction. To compare the microbial structure of 6:2 FTCA and 5:3 FTCA degrading consortia, an index (Ω) was introduced to evaluate the impact of different FTCAs, calculated by the relative abundance (RA) ratio of the OTUs in the consortia amended with 6:2 FTCA to that with 5:3 FTCA of the same source. A greater Ω value (green) indicates that the taxa were possibly more specific to 6:2 FTCA biotransformation, while a smaller Ω value (brown) indicates the microbe is more affinitive to 5:3 FTCA biotransformation (Fig. 4 ). Three genera were more enriched in 6:2 FTCA-enriched consortia, including Hyphomicrobium, Methylorubrum, and Achromobacter (Fig. 3 ). Notably, Hyphomicrobium was strongly correlated with 6:2 FTCA biodegradation after a short-term incubation (5 days) in our previous study on 6:2 FTCA-exposed activated sludge (Wu et al. 2024 ). Members of Hyphomicrobium have been reported with dechlorination activities via the expression of oxidative dichloromethane dehalogenase (Kohler-Staub et al. 1995 ). However, no prior report has associated Hyphomicrobium species with defluorination, warranting further investigation. Similarly, Methylorubrum extorquens DM4 can grow on dichloromethane as the sole carbon source and perform dechlorination (Maucourt et al. 2022 ). Achromobacter can degrade the polychlorinated biphenyls (PCB) (Ahmed and Focht 1973 ) and Lindane (formerly known as gamma benzene hexachloride) (Egorova et al. 2021 ) and utilize 2-chlorobenzoate and 2,5-dichlorobenzoate as the sole source of energy (Jencova et al. 2004 , Strnad et al. 2011 ). Five genera were more dominant when the consortia were amended with 5:3 FTCA, including Phenylobacterium, Xanthobacter, Methylotenera, Pseudomonas , and Rhodanobacter (Fig. 4 ). The genus Phenylobacterium comprises a species named P. immobile , which is remarkable for its extremely limited nutritional spectrum. So far, all strains isolated and described under this species can grow optimally only on artificial compounds like chloridazon (Lingens et al. 1985 ), antipyrin, and pyramidon (Eberspächer and Lingens 2006 ). Xanthobacter has been reported as an abundant genus in PFOA-spiked activated sludge (Huang et al. 2022 ), and members of this genus consist of degraders of many resistant contaminants, such as halogenated aliphatic by Xanthobacter autotrophicus GJ10 (Janssen et al. 1985 ) and 1,4-dichlorobenzene by Xanthobacter flavus 14p1 (Spiess et al. 1995 ). Specifically, Xanthobacter autotrophicus GJ10 contains a dehalogenase specific to halogenated carboxylic acid, which is relatively heat-stable and shows a broad substrate specificity (Janssen et al. 1985 , Keuning et al. 1985 , Van Der Ploeg et al. 1991 ). Pseudomonas demonstrates great metabolic diversity and has been widely reported for its dehalogenation capability. For instance, genera Pseudomonas were recorded as being able to degrade FTOHs (Kim et al. 2012 ), 2,2,2-trifluoroethane sulfonate (TES) (Key et al. 1998 ), and 6:2 FTS (Key et al. 1998 ). Pseudomonas putida was also found to contain dehalogenase activity against chlorinated aliphatic acids, including monochloroacetate, dichloroacetate, 2-monochloropropionate, 2,2’-dichloro-propionate (Slater et al. 1979 ), 4-chlorobenzoic acid (Banta and Kahlon 2007 ), and 2,2-fluoro-1,3-benzodioxole (DFBD) (Bygd et al. 2021 ). In addition, defluorination of 4-deoxy-4-fluoro-d-glucose (4-FDG) was reported in the cytoplasmic membrane of Pseudomonas putida , which was fully inhibited with the presence of glucose. Rhodanobacter is reported as one of the dominant bacteria in PFAS-contaminated soil (Senevirathna et al. 2022 ), and its family Rhodanobacteraceae was also found enriched in AFFF-impacted soil (Cao et al. 2022 ). 3.4. Environmental Implications and Limitations After almost one year of enrichment, we obtained 14 consortia exhibiting stable FTCA biodegradation. Though these consortia were enriched from diverse inoculation sources, they exhibited distinctive biodefluorination patterns when amended with 6:2 versus 5:3 FTCAs. Little fluoride release was observed for 5:3 FTCA, while 6:2 FTCA biotransformation couples with consistent fluoride release. Approximately 0.19 fluorine was liberated as fluoride per molecule of 6:2 FTCA that was degraded. This is the first to establish microbial consortia that can degrade FTCAs without the supplement of external carbon sources in long term. It is very possible that these consortia can subsist on FTCAs considering the consistency across long enrichment cycles. However, autotrophic mechanisms may take place and support the consortia by sequestering CO 2 in the headspace. Even though the consortia were washed thoroughly at the beginning of each enrichment cycle, our experimental setup cannot fully preclude endogenous metabolism by utilizing organic matters leaching from dead biomass. Thus, future studies are needed to track the carbon assimilation and mineralization when FTCAs were fed as the sole carbon and energy source. Furthermore, the consistently low defluorination efficiencies in our consortia also underscore the importance of investigating external carbon sources that can accelerate FTCA biotransformation and enhance defluorination extent (particularly for 6:2 FTCA). Dominant genera were also uncovered given their possible roles in FTCA biotransformation and biodefluorination. To gain a better understanding of the FTCA biotransformation mechanisms and exploit feasible remedial approaches, it is of significant value to initiate attempts to assess FTCA biotransformation in isolates or available pure cultures and characterize genes and enzymes involved in biodefluorination. To address the fluorine mass discrepancies over FTCA biotransformation, identifying novel transformation products via non-target analysis is also needed to advance our knowledge of FTCA biotransformation pathways and their fate once released to the environment. Declarations ACKNOWLEDGEMENTS This work was funded by National Science Foundation (NSF, CBET-1903597), US Geological Survey (USGS, G24AP00026), Strategic Environmental Research and Development Program (SERDP, ER21-3556), and New Jersey Water Resources Research Institute (NJWRRI, 2019NJ183B). Chen Wu was sponsored by the Mark B. Bain Graduate Fellowship from the Hudson River Foundation. Summary of the results about the FTCA biotransformation metabolites was provided by Boyuan Su from NJIT. Ethnical Approval The submission of this manuscripts was approved by all authors. We declare no competing financial interest. Funding Funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication. References Ahmed, M. and Focht, D. (1973) Degradation of polychlorinated biphenyls by two species of Achromobacter. Canadian Journal of Microbiology 19(1), 47-52. Allred, B.M., Lang, J.R., Barlaz, M.A. and Field, J.A. (2014) Orthogonal zirconium diol/C18 liquid chromatography-tandem mass spectrometry analysis of poly and perfluoroalkyl substances in landfill leachate. J Chromatogr A 1359, 202-211. Banta, G. and Kahlon, R.S. (2007) Dehalogenation of 4 — Chlorobenzoic Acid by Pseudomonas isolates. Indian Journal of Microbiology 47(2), 139-143. 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(2022) Effects of perfluorooctanoic acid (PFOA) on activated sludge microbial community under aerobic and anaerobic conditions. Environmental Science and Pollution Research. Janssen, D.B., Scheper, A., Dijkhuizen, L. and Witholt, B. (1985) Degradation of halogenated aliphatic compounds by Xanthobacter autotrophicus GJ10. Applied and Environmental Microbiology 49(3), 673-677. Jencova, V., Strnad, H., Chodora, Z., Ulbrich, P., Hickey, W. and Paces, V. (2004) Chlorocatechol catabolic enzymes from Achromobacter xylosoxidans A8. International Biodeterioration & Biodegradation 54(2-3), 175-181. Keuning, S., Janssen, D.B. and Witholt, B. (1985) Purification and characterization of hydrolytic haloalkane dehalogenase from Xanthobacter autotrophicus GJ10. Journal of bacteriology 163(2), 635-639. Key, B.D., Howell, R.D. and Criddle, C.S. (1998) Defluorination of organofluorine sulfur compounds by Pseudomonas sp. strain D2. Environmental Science & Technology 32(15), 2283-2287. 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Environmental Science & Technology 44(9), 3305-3310. Lewis, M., Kim, M.H., Liu, E.J., Wang, N. and Chu, K.H. (2016) Biotransformation of 6:2 polyfluoroalkyl phosphates (6:2 PAPs): Effects of degradative bacteria and co-substrates. J Hazard Mater 320, 479-486. Li, F., Su, Q., Zhou, Z., Liao, X., Zou, J., Yuan, B. and Sun, W. (2018) Anaerobic biodegradation of 8:2 fluorotelomer alcohol in anaerobic activated sludge: Metabolic products and pathways. Chemosphere 200, 124-132. Lingens, F., Blecher, R., Blecher, H., Blobel, F., Eberspächer, J., Fröhner, C., Görisch, H., Görisch, H. and Layh, G. (1985) Phenylobacterium immobile gen. nov., sp. nov., a Gram-Negative Bacterium That Degrades the Herbicide Chloridazon. International Journal of Systematic and Evolutionary Microbiology 35(1), 26-39. Liu, N., Wu, C., Lyu, G. and Li, M. (2021) Efficient adsorptive removal of short-chain perfluoroalkyl acids using reed straw-derived biochar (RESCA). Science of The Total Environment, 149191. Maucourt, B., Roche, D., Chaignaud, P., Vuilleumier, S. and Bringel, F. (2022) Genome-Wide Transcription Start Sites Mapping in Methylorubrum Grown with Dichloromethane and Methanol. Microorganisms 10(7), 1301. Mothersole, R.G., Wynne, F.T., Rota, G., Mothersole, M.K., Liu, J. and Van Hamme, J.D. (2023) Formation of CoA Adducts of Short-Chain Fluorinated Carboxylates Catalyzed by Acyl-CoA Synthetase from Gordonia sp. Strain NB4-1Y. ACS Omega 8(42), 39437-39446. Nelson, M.C., Morrison, H.G., Benjamino, J., Grim, S.L. and Graf, J. (2014) Analysis, optimization and verification of Illumina-generated 16S rRNA gene amplicon surveys. PloS one 9(4), e94249. Phillips, M.M., Dinglasan-Panlilio, M.J.A., Mabury, S.A., Solomon, K.R. and Sibley, P.K. (2007) Fluorotelomer acids are more toxic than perfluorinated acids. Environmental Science & Technology 41(20), 7159-7163. Qiao, W., Miao, J., Jiang, H. and Yang, Q. (2021) Degradation and effect of 6:2 fluorotelomer alcohol in aerobic composting of sludge. Biodegradation 32(1), 99-112. Ross, I., McDonough, J., Miles, J., Storch, P., Thelakkat Kochunarayanan, P., Kalve, E., Hurst, J., S. Dasgupta, S. and Burdick, J. (2018) A review of emerging technologies for remediation of PFASs. Remediation Journal 28(2), 101-126. Senevirathna, S.T.M.L.D., Krishna, K.C.B., Mahinroosta, R. and Sathasivan, A. (2022) Comparative characterization of microbial communities that inhabit PFAS-rich contaminated sites: A case-control study. Journal of Hazardous Materials 423, 126941. Shaw, D.M., Munoz, G., Bottos, E.M., Duy, S.V., Sauvé, S., Liu, J. and Van Hamme, J.D. (2019) Degradation and defluorination of 6: 2 fluorotelomer sulfonamidoalkyl betaine and 6: 2 fluorotelomer sulfonate by Gordonia sp. strain NB4-1Y under sulfur-limiting conditions. Science of The Total Environment 647, 690-698. Shi, G., Cui, Q., Pan, Y., Sheng, N., Guo, Y. and Dai, J. (2017) 6:2 fluorotelomer carboxylic acid (6:2 FTCA) exposure induces developmental toxicity and inhibits the formation of erythrocytes during zebrafish embryogenesis. Aquat Toxicol 190, 53-61. Slater, J.H., Lovatt, D., Weightman, A.J., Senior, E. and Bull, A.T. (1979) The Growth of Pseudomonas putida on Chlorinated Aliphatic Acids and its Dehalogenase Activity. Microbiology 114(1), 125-136. Spiess, E., Sommer, C. and Görisch, H. (1995) Degradation of 1, 4-dichlorobenzene by Xanthobacter flavus 14p1. Applied and Environmental Microbiology 61(11), 3884-3888. Strnad, H., Ridl, J., Paces, J., Kolar, M., Vlcek, C. and Paces, V. (2011) Complete Genome Sequence of the Haloaromatic Acid-Degrading Bacterium Achromobacter xylosoxidans A8. Journal of bacteriology 193(3), 791-792. Van Der Ploeg, J., van Hall, G. and Janssen, D.B. (1991) Characterization of the haloacid dehalogenase from Xanthobacter autotrophicus GJ10 and sequencing of the dhlB gene. Journal of bacteriology 173(24), 7925-7933. Wackett, L.P. (2022) Nothing lasts forever: understanding microbial biodegradation of polyfluorinated compounds and perfluorinated alkyl substances. Microbial biotechnology 15(3), 773-792. Wang, N., Liu, J., Buck, R.C., Korzeniowski, S.H., Wolstenholme, B.W., Folsom, P.W. and Sulecki, L.M. (2011) 6:2 Fluorotelomer sulfonate aerobic biotransformation in activated sludge of waste water treatment plants. Chemosphere 82(6), 853-858. Wang, N., Buck, R.C., Szostek, B., Sulecki, L.M. and Wolstenholme, B.W. (2012) 5:3 Polyfluorinated acid aerobic biotransformation in activated sludge via novel "one-carbon removal pathways". Chemosphere 87(5), 527-534. Wu, C., Wang, Q., Chen, H. and Li, M. (2022) Rapid quantitative analysis and suspect screening of per-and polyfluorinated alkyl substances (PFASs) in aqueous film-forming foams (AFFFs) and municipal wastewater samples by Nano-ESI-HRMS. Water Research 219, 118542. Wu, C., Goodrow, S., Chen, H. and Li, M. (2024) Distinctive biotransformation and biodefluorination of 6:2 versus 5:3 fluorotelomer carboxylic acids by municipal activated sludge. Water Research 254, 121431. Xie, Y., May, A.L., Chen, G., Brown, L.P., Powers, J.B., Tague, E.D., Campagna, S.R. and Löffler, F.E. (2022) Pseudomonas sp. Strain 273 Incorporates Organofluorine into the Lipid Bilayer during Growth with Fluorinated Alkanes. Environmental Science & Technology 56(12), 8155-8166. Zhang, S., Szostek, B., McCausland, P.K., Wolstenholme, B.W., Lu, X., Wang, N. and Buck, R.C. (2013) 6:2 and 8:2 Fluorotelomer Alcohol Anaerobic Biotransformation in Digester Sludge from a WWTP under Methanogenic Conditions. Environmental Science & Technology 47(9), 4227-4235. Zhang, S., Lu, X., Wang, N. and Buck, R.C. (2016) Biotransformation potential of 6: 2 fluorotelomer sulfonate (6: 2 FTSA) in aerobic and anaerobic sediment. Chemosphere 154, 224-230. Zhao, L., Folsom, P.W., Wolstenholme, B.W., Sun, H., Wang, N. and Buck, R.C. (2013) 6:2 fluorotelomer alcohol biotransformation in an aerobic river sediment system. Chemosphere 90(2), 203-209. Additional Declarations No competing interests reported. Supplementary Files FTCAenrichmentSD2.docx GraphicalAbstract.png Graphic Abstract Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4824417","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":338653994,"identity":"ca1fa937-686d-4161-92ea-782711c7893b","order_by":0,"name":"Chen Wu","email":"","orcid":"","institution":"New Jersey Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Wu","suffix":""},{"id":338653995,"identity":"8bb35766-4f1a-443a-b968-b201bad1378f","order_by":1,"name":"Mengyan Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYDADPmbmAwwSBiBmApFa2JjZEoBaDEjRwsADUk6EFt323sOveWruMLCx83x7YFHwh4GfPccArxazM+fSrHmOPQM6jHe7Achhkj1vCGi5kWNmzMN2GKRlmwRIi8ENQrbcfwPU8g+khecZWIs9QS03eIwf87aBtbBBbJEg6JccM8a5fYd5gIFsBtRizCNx5lkBfi3Hzxh/ePPtsBw//+Fn0hJ/5OT425M34NUCBGxSPAwMPCAWswSUQQgwf/wBZTF+IEb9KBgFo2AUjDgAAF+5OlKSY2gVAAAAAElFTkSuQmCC","orcid":"","institution":"New Jersey Institute of Technology","correspondingAuthor":true,"prefix":"","firstName":"Mengyan","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-07-29 21:14:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4824417/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4824417/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63618583,"identity":"4abc1741-5a7a-483b-98b4-f140924b20b1","added_by":"auto","created_at":"2024-08-30 08:29:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":87124,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic of the experimental design.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4824417/v1/7061c40f4ea532174c6486e9.png"},{"id":63618581,"identity":"b0daba89-84cd-4fe5-8be3-6f66d488824d","added_by":"auto","created_at":"2024-08-30 08:29:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":152689,"visible":true,"origin":"","legend":"\u003cp\u003eFTCA biotransformation rates and biodefluorination rates in 1\u003csup\u003est\u003c/sup\u003e to 4\u003csup\u003eth\u003c/sup\u003e generation of enrichment from two activated sludges (BH and V) and six soils (A to F) that were amended with 6:2 or/and 5:3 FTCA (~80 µM). AC indicates the analytical controls without biosolids. NA indicates data are not available in corresponding experiments.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4824417/v1/f1ead9ad0f52955d346243d0.png"},{"id":63618580,"identity":"79142f18-220c-4c09-b5b5-b8e23f05a006","added_by":"auto","created_at":"2024-08-30 08:29:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":67376,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between 6:2 FTCA biotransformation rates and biodefluorination rates in 1\u003csup\u003est\u003c/sup\u003e to 4\u003csup\u003eth\u003c/sup\u003e generation of enrichment from two activated sludges and six soils.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4824417/v1/82f4f5b118814ea5f3e28e78.png"},{"id":63618584,"identity":"f0b311c3-0a57-4141-8fc7-37f25040118c","added_by":"auto","created_at":"2024-08-30 08:29:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":114396,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4824417/v1/53e5c3e00378a6c6c437933d.png"},{"id":66670786,"identity":"8f5da95e-3d28-4d16-8081-3842af024d84","added_by":"auto","created_at":"2024-10-15 10:23:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":791385,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4824417/v1/06450c70-2bb4-4c08-a9d2-f2d651016450.pdf"},{"id":63618586,"identity":"f7ba0a87-0ef4-4d60-8003-748541d2990e","added_by":"auto","created_at":"2024-08-30 08:29:56","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1451094,"visible":true,"origin":"","legend":"","description":"","filename":"FTCAenrichmentSD2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4824417/v1/bffb271301c18ff764f85b9e.docx"},{"id":63618585,"identity":"62098202-6558-4115-8fc7-3da66e7e3bcf","added_by":"auto","created_at":"2024-08-30 08:29:55","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":86331,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphic Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-4824417/v1/4cbf4e2fcbd4536a89403256.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enriching Fluorotelomer Carboxylic Acids-Degrading Consortia from Sludges and Soils","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eFluorotelomer carboxylic acids (FTCAs) represent a class of per- and polyfluoroalkyl substances (PFASs) that have garnered significant attention in recent years due to their prevalent occurrence and potential environmental impacts (Fenton et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Structure of FTCAs is featured by a perfluorinated tail, a carboxylic acid head, and a few non-fluorinated alkyl carbons in between (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). 6:2 FTCA and 5:3 FTCA are two most concerned FTCAs considering their widespread detection in landfill leachates, wastewater, and other environmental matrixes (Allred et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Fuertes et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Lang et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Wu et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, FTCAs have also been frequently detected at aqueous film-forming foam (AFFF)-impacted sites, as they are key intermediates and end products from the biotransformation of PFAS precursors, such as fluorotelomer sulfonates (FTSs) (Shaw et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Zhang et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and fluoroalkyl phosphates (PAPs) (Lewis et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Though not regulated, FTCAs are found more toxic than their corresponding perfluorocarboxylic acids (PFCAs), particularly those that have received primary public and regulatory attentions (e.g., PFOA) (Phillips et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, Shi et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Therefore, it is of significant value to investigate the fate and behavior of FTCAs and develop feasible treatment approaches to mitigate their impacts to the environment.\u003c/p\u003e \u003cp\u003eAs compared to PFCAs, FTCAs contain non-fluorinated carbons, which may facilitate microbial enzymatic reactions that activate subsequent biodefluorination (Ross et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Wackett \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Recent studies on pure cultures and environmental microbiomes (e.g., activated sludges and soils) revealed that biotransformation of FTCAs and their precursors can occur and generate shorter chain PFCAs, such as perfluorohexanoic acid (PFHxA), perfluoropentanoic acid (PFPeA), and perfluorobutanoic acid (PFBA) (Che et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, D'Agostino and Mabury \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Evich et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Lee et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Li et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Qiao et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Wang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Wang et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Wu et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Zhang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Zhao et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Several biotransformation pathways have been proposed. For instance, Wang \u003cem\u003eet al.\u003c/em\u003e postulated \"one-carbon removal pathways\", through which 5:3 FTCA is transformed to 4:3 FTCA or shorter chain PFCAs via the formation of α-OH 5:3 acid and 5:2 FTCA (Wang et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In contrast to these degradative pathways, an alternative conjugative pathway that is analogous to fatty acid β oxidation has recently been reported based on the formation of CoA adducts for FTCAs with non-fluorinated β carbons (Mothersole et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). A medium-chain acyl-CoA synthetase in \u003cem\u003eGordonia\u003c/em\u003e sp. strain NB4-1Y was responsible for the catalysis of such reactions with 2:3 FTCA, 2:3 fluorotelomer unsaturated carboxylic acid (FTUCA), and 1:4 FTCA (Mothersole et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These findings indicated that FTCAs can be biodegradable. However, knowledge remain elusive regarding microorganisms that can degrade FTCAs without the supplement of external carbon sources.\u003c/p\u003e \u003cp\u003eIn this study, we seek to acclimate microbial consortia from diverse sources (two activated sludges and six soils) that were repetitively fed with high concentrations of 6:2 and/or 5:3 FTCAs (~\u0026thinsp;80 \u0026micro;M) without the supplement of external carbon sources. The microbial community structures of the enriched consortia were analyzed and compared when constant FTCA removal (and fluoride release, if any) were observed in a 1-year long enrichment. Dominant bacteria that may contribute to FTCA biotransformation and biodefluorination were identified for future investigation.\u003c/p\u003e"},{"header":"2. Methods AND MATERIALS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals and Reagents\u003c/h2\u003e \u003cp\u003e6:2 FTCA standard, 5:3 FTCA standard, and mass-labeled M2-6:2 FTCA standard for calibration were all purchased from Wellington Laboratories Inc. (Guelph, Ontario, Canada) with the purity of \u0026gt;\u0026thinsp;98%. Bulk 6:2 FTCA (3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanoic acid, \u0026gt;\u0026thinsp;97%) and 5:3 FTCA (2H,2H,3H,3H-perfluorooctanoic acid, \u0026gt;\u0026thinsp;97%) for enrichment experiments were purchased from Synquest Laboratories (Alachua, FL, USA). LCMS grade methanol (\u0026gt;\u0026thinsp;99%) was purchased from Fisher Chemicals (Hampton, NH, USA). Ultrapure water obtained from the Milli-Q RC Synthesis water purification system (Millipore, Bedford, MA, USA) was used in the experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Enrichment of FTCA-degrading Consortia\u003c/h2\u003e \u003cp\u003eBiosolids were collected from 8 different sources, including two activated sludges from wastewater treatment plants (WWTPs) and 6 soils in New Jersey (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Biosolids were washed three times with phosphate buffer saline solution (PBS, 20 mM sodium phosphate, pH 7.0) to remove organic matter before enrichment. First, 120 \u0026micro;L of 20 mM FTCA(s) stock solution was spiked to 160-mL sterile amber serum bottles, and the methanol solvent was fully evaporated using nitrogen flow. Then, 1 g of each washed biosolid (wet weight) was inoculated into individual FTCA-spiked serum bottle, followed by adding 30 mL ammonium mineral salts (AMS) media. All seeded bottles were incubated under 30\u0026deg;C while being shaken at 120 rpm. At time 0 and select intervals (as indicated Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), supernatants (~\u0026thinsp;3 mL) were collected, centrifuged at 12,000 rpm for 3 min, and then filtrated via a PES membrane filter for the monitoring of the removal of FTCAs and release of free fluoride as described below. In parallel, analytical controls were prepared without biosolid inoculation to discern the baseline of the initial FTCA dosage.\u003c/p\u003e \u003cp\u003eAfter 60\u0026thinsp;~\u0026thinsp;130 days of incubation, biosolids in the serum bottles was collected and washed three times with PBS before being transferred to sterile bottles with fresh AMS media spiked with FTCA(s) for the 2nd, 3rd, and 4th generations of enrichment as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Four generations of enrichment were prepared and monitored in the same fashion. At the end of the 4th generation, biosolids were collected by centrifugation at 12,000 rpm for 15 min for DNA extraction and microbial community analysis to reveal the dominant species that might contribute to FTCA biotransformation and biodefluorination. Before further analysis, all samples were sealed and stored in the \u0026minus;\u0026thinsp;20\u0026deg;C freezer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.2. FTCA Quantification by LC/MS/MS\u003c/h2\u003e \u003cp\u003eTo monitor FTCA removal, 1 mL of the filtered supernatant was diluted by 100 times with pure methanol and then spiked with 20 \u0026micro;g/L M2-6:2 FTCA as the internal standard before FTCA quantification analysis based on a protocol adapted from EPA Method 537 (Liu et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Wu et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). 6:2 and 5:3 FTCAs were analyzed using a 1290 Infinity II HPLC system in tandem with 6470A triple quadrupole mass spectrometer (Agilent, Santa Clara, CA). Aliquots (10 \u0026micro;L) were injected into this LC/MS/MS system equipped with a Symmetry C18 column (ID 2.1 mm, length 100 mm, particle size 3.5 \u0026micro;m) (Waters, Milford, MI) at a flow rate of 0.3 mL/min. The mobile phase initially consisted of 80% solvent A (5 mM ammonia acetate in 10% (v/v) methanol), decreased to 40% A with 60% solvent B (pure methanol) in 2.0 min, and further reduced to 20% A in 1 min and kept for 5 min, and then changed back to 80% A in 1 min and held for 4.5 min. Triple quadrupoles mass spectrometer was set in the negative-ion electrospray mode, and multiple reaction mode (MRM) was set for ion collection. The pressure in the nebulizer was set at 25 psi with a capillary voltage of -3.5 kV. The desolvation gas temperature was 350\u0026deg;C, with a flow rate of 8 L/min. MS data were processed using the MassHunter QQQ quantitative analysis software (Agilent Technologies, USA). Laboratory reagent blanks (LRBs) were prepared with 250 mL reagent water in HDPE bottles in the laboratory. LRBs were preserved, stored, and processed at the same conditions as all samples. PFAS calibration and lower limits of detection were determined following the procedures described in our previous publication (Wu et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). All calibration solutions were prepared in triplicate, and their average mass intensities were used for constructing the calibration curves when the regression linearity met R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.99. Calibration verification checks were performed every 15\u0026ndash;20 samples and at the end of analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Fluoride Analysis\u003c/h2\u003e \u003cp\u003eTo measure free fluoride, 2.0 mL of the filtered supernatant was collected and filtered through a 0.22-\u0026micro;m PES membrane. After mixing with 2.0 mL of TISAB II buffer solution (Thermo-Fisher Scientific), fluoride concentration was measured using the Orion Star Meter equipped with the fluoride electrodes (Thermo-Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Microbial Community Analysis\u003c/h2\u003e \u003cp\u003ePhylogenetic structures were analyzed using the 16S rRNA amplicon-based sequencing and associated bioinformatics tools in order to investigate the microbial community profiles of the consortia after the 4th generation of enrichment. Genomic DNA was extracted from the obtained biosolids using the Qiagen DNeasy PowerSoil Kit (Qiagen, Germany) following the manufacturer's protocol. Primers 338F (5'- ACTCCTACGGGAGGCAGCA-3') and 806R (5'- GGACTACHVGGGTWTCTAAT-3') were used to amplify the 16S rRNA variable regions V3 and V4 (Kumar et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Paired-end high-throughput sequencing was performed by the HiSeq Sequencing System (Illumina, San Diego, CA) (Nelson et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). 16S rRNA gene reads were processed using the QIIME2 pipeline (v2021.4) (Bolyen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) with the Divisive Amplicon Denoising Algorithm 2 (DADA2) (Callahan et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) for sequence pairing, denoising, and chimera elimination.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results And Discussion","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Consistent FTCA Biotransformation in Enriched Consortia\u003c/h2\u003e \u003cp\u003eOver four generations of enrichment, consistent removal of 6:2 FTCA was observed in consortia from two activated sludges (BH and V) and five soils (B to F) while little removal was observed in analytical controls (Figure S2). As normalized to the enrichment durations of each generation, 6:2 FTCA biotransformation rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) were in range between 0.28\u0026thinsp;~\u0026thinsp;0.53, 0.05\u0026thinsp;~\u0026thinsp;0.40, 0.50\u0026thinsp;~\u0026thinsp;1.34, 0.72\u0026thinsp;~\u0026thinsp;0.98 \u0026micro;M/day in the 1st, 2nd, 3rd, and 4th generations, respectively. For enrichments prepared with Soil A, 6:2 FTCA biotransformation rates were consistently lower than 0.04 \u0026micro;M/day. Therefore, this consortium was discontinued after the 3rd generation of enrichment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilarly, consistent 5:3 FTCA removal was also evident in those consortia that exhibited 6:2 FTCA removal (Figure S3). In general, 5:3 FTCA biotransformation was slower than 6:2 FTCA, particularly in the first two generations of enrichment. 5:3 biotransformation rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) were in range between 0.07\u0026thinsp;~\u0026thinsp;0.42, 0.02\u0026thinsp;~\u0026thinsp;0.54, 0.78\u0026thinsp;~\u0026thinsp;1.38, 0.53\u0026thinsp;~\u0026thinsp;1.05 \u0026micro;M/day in the 1st, 2nd, 3rd, and 4th generations, respectively. With BH, B, and E as the inocula, their consortia exhibited low 5:3 biotransformation with rates below 0.2 \u0026micro;M/day in the 1st and 2nd generations. However, 5:3 biotransformation removal in these enrichment consortia escalated in the 3rd generation. In contrast, the consortium obtained from Soil A continued to show a low 5:3 FTCA removal, which was thus discontinued at the end of the third generation.\u003c/p\u003e \u003cp\u003eFor both 6:2 and 5:3 FTCAs, biotransformation was mostly slowest in the 2nd generation and then increased in the 3rd and 4th generations (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In the 1st generation, even though biosolids were thoroughly washed and amended only with FTCAs, endogenous metabolism may occur due to the decay of the biomass from different inoculation sources. Some microorganisms will break down and release metabolites and cellular compositions that become available to other microorganisms, particularly those can participate in FTCA biotransformation. Such mechanism can fuel the cells to degrade FTCAs over the 1st generation. However, after the transfer to fresh AMS media in the 2nd generation without the supplement of external carbon sources other than FTCAs, the effects of endogenous metabolism became minimal. This explained the dip in FTCA biotransformation observed in the 2nd generation of enrichment. After the 2nd generation, the microbiomes of the consortia continued to adapt to FTCAs as the sole carbon supplement and thus exhibit greater FTCA biotransformation in the 3rd and 4th generations of enrichment. These results indicated the acclimation of microbial communities with stable FTCA biotransformation abilities in the enriched consortia.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCoupling with FTCA biotransformation, metabolites based on \u0026ldquo;one-carbon removal pathways\u0026rdquo; were detected by a new PFAS analytical approach developed in our lab, nano-electrospray ionization and high-resolution mass spectrometry (Nano-ESI-HRMS) (Wu et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Wu et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), since their concentrations were mostly close to or even below the limits of detection by LC/MS/MS. Such metabolites included PFCAs (e.g., PFHxA, PFPeA, and PFBA) and FTCAs (e.g., 5:2 FTCA and 4:3 FTCA). We also detected some novel metabolites that were more dominant, which will be reported in a separate work by the group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Biodefluorination Only for 6:2 FTCA, But Not 5:3 FTCA\u003c/h2\u003e \u003cp\u003eBiodefluorination was evident as the liberation of free fluoride in the media. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and S5, minimal fluoride was detected in consortia that were fed with 5:3 FTCA only regardless of inoculation sources and enrichment generations. This is in good agreement with our recent report on 5:3 FTCA biotransformation by four activated sludges collected from four other WWTPs (not BH or V as reported in this study) in the New York and New Jersey area. Minimal fluoride release was observed within 7 days of incubation, though with the low detection of less fluorinated products, such as 5:3 FTUCA and PFBA (Wu et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In a separate study by Che \u003cem\u003eet al\u003c/em\u003e., free fluoride was not detected during the aerobic biodegradation of 1:3 FTCA, 1:3 FTUCA, and 2:3 FTCA by activated sludge (Che et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These results corroborated that aerobic biotransformation of 5:3 FTCA and other n:3 FT(U)CAs may be dominated by \u0026ldquo;non-fluoride releasing pathways\u0026rdquo; (Wu et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), rather than \u0026ldquo;one-carbon removal pathways\u0026rdquo;. The availability of non-fluorinated α and β carbons in n:3 FTCAs may enable the entrance to β oxidation via the conjugation with CoA without the liberation of free fluoride (Che et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Mothersole et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Furthermore, assimilation of less-fluorinated molecules and their adducts may also enter cell metabolism and lead to the buildup of fluorinated macromolecules, such as fluorinated anabolites and phospholipids (Xie et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven the fact that little fluoride was released over 5:3 FTCA biotransformation in all samples in this and previous studies, we accounted all the detected fluoride for 6:2 FTCA biotransformation in the first three generations of enrichment for soil samples (A to F) when 6:2 and 5:3 FTCAs were concurrently amended. For two sludge samples BH and V, 6:2 and 5:3 FTCAs were amended separately in all four generations of enrichment (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). As shown in Figure S4 and 2, constant free fluoride accumulation, varying from 37.2\u0026thinsp;~\u0026thinsp;46.8 \u0026micro;M for 1st generation to 6.8\u0026thinsp;~\u0026thinsp;12.8 \u0026micro;M for 2nd to 4th generation, was observed in the consortia from two activated sludges. Fluoride release was also observed in the soil enrichments C to F through the four generations of enrichment, accumulating to 6.0\u0026thinsp;~\u0026thinsp;75.1 \u0026micro;M for the 1st generation and 3.6\u0026thinsp;~\u0026thinsp;18.0 \u0026micro;M for 2nd to 4th generations (Figure S4). At the same time, little fluoride release was detected in the soil enrichment A from the 1st to 3rd generations and the soil enrichment B in the 1st and 2nd generations as the defluorination rates were below 0.02 \u0026micro;M/day (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, the defluorination rate in the soil enrichment B started to accelerate in the 3rd generation (0.09 \u0026micro;M/day). Considering the consensus of 6:2 FTCA removal and fluoride release in the first three generations of enrichment, consortia B was continued for the 4th generation of enrichment. No fluoride accumulation was observed in the analytical controls.\u003c/p\u003e \u003cp\u003eThe observed fluoride accumulation corresponded to the removal of 6:2 FTCA in all consortia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), supporting that 6:2 FTCA biotransformation was accompanied by free fluoride release. A significant correlation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) was established between the 6:2 FTCA biotransformation rates and biodefluorination rates based on all consortia across four generations of enrichment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Notably, a correlation factor of 0.1943 was estimated as the slope of the linear regression forced through the origin. This suggested an average of ~\u0026thinsp;0.19 fluoride was released per 6:2 FTCA molecule biotransformed by the consortia. This is much lower than our recent work on 6:2 FTCA biotransformation by four other activated sludges that demonstrated 0.61\u0026thinsp;~\u0026thinsp;1.92 fluoride release per molecule of 6:2 FTCA that was biodegraded (Wu et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This was probably due to the initial supplement of external carbons (i.e., glucose and acetate) in the previous study, while no external carbons were amended in this study. Biodefluorination can be energy consuming (Wackett \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Thus, without the supplement of external carbon sources, 6:2 FTCA biodefluorination extent and efficiency can be greatly dampened.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Potential FTCA Degraders Enriched in the Consortia\u003c/h2\u003e \u003cp\u003eA total of 14 consortia from 7 different sources were incubated over 1 year under the exposure of 6:2 or/and 5:3 FTCAs without adding external carbon sources. Given the continuous FTCA removal (and fluoride accumulation for 6:2 FTCA-spiked consortia), it\u0026rsquo;s plausible to hypothesize that some dominant taxa in the 4th generation enrichments were FTCA degraders via metabolism or co-metabolism and through fluoride releasing pathways for 6:2 FTCA and non-fluoride releasing pathways for 5:3 FTCA. Hence, 16S rRNA sequencing was conducted for consortia obtained after the 4th generation of enrichment to identify the dominant OTUs at the genus level.\u003c/p\u003e \u003cp\u003eAs shown in Table S2, 36 OTUs were found prevalent across 14 consortia with maximum relative abundances (RA) of 0.1% or higher. Though 5 out of these 36 OTUs were unclassified, the rest 31 OTUs were classified into 13 genera and 10 families according to nucleotide-based BLAST from NCBI. These 13 genera were all gram-negative, similar to our previous work on FTCA-degrading activated sludge communities (Wu et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The bias from the varied DNA extraction efficacies for gram-positive and gram-negative strains cannot be fully excluded even though sonication was applied to enhance the cell lysis before DNA extraction.\u003c/p\u003e \u003cp\u003eTo compare the microbial structure of 6:2 FTCA and 5:3 FTCA degrading consortia, an index (Ω) was introduced to evaluate the impact of different FTCAs, calculated by the relative abundance (RA) ratio of the OTUs in the consortia amended with 6:2 FTCA to that with 5:3 FTCA of the same source. A greater Ω value (green) indicates that the taxa were possibly more specific to 6:2 FTCA biotransformation, while a smaller Ω value (brown) indicates the microbe is more affinitive to 5:3 FTCA biotransformation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThree genera were more enriched in 6:2 FTCA-enriched consortia, including \u003cem\u003eHyphomicrobium, Methylorubrum, and Achromobacter\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Notably, \u003cem\u003eHyphomicrobium\u003c/em\u003e was strongly correlated with 6:2 FTCA biodegradation after a short-term incubation (5 days) in our previous study on 6:2 FTCA-exposed activated sludge (Wu et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Members of \u003cem\u003eHyphomicrobium\u003c/em\u003e have been reported with dechlorination activities via the expression of oxidative dichloromethane dehalogenase (Kohler-Staub et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). However, no prior report has associated \u003cem\u003eHyphomicrobium\u003c/em\u003e species with defluorination, warranting further investigation. Similarly, \u003cem\u003eMethylorubrum extorquens\u003c/em\u003e DM4 can grow on dichloromethane as the sole carbon source and perform dechlorination (Maucourt et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eAchromobacter\u003c/em\u003e can degrade the polychlorinated biphenyls (PCB) (Ahmed and Focht \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1973\u003c/span\u003e) and Lindane (formerly known as gamma benzene hexachloride) (Egorova et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and utilize 2-chlorobenzoate and 2,5-dichlorobenzoate as the sole source of energy (Jencova et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Strnad et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFive genera were more dominant when the consortia were amended with 5:3 FTCA, including \u003cem\u003ePhenylobacterium, Xanthobacter, Methylotenera, Pseudomonas\u003c/em\u003e, and \u003cem\u003eRhodanobacter\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The genus \u003cem\u003ePhenylobacterium\u003c/em\u003e comprises a species named \u003cem\u003eP. immobile\u003c/em\u003e, which is remarkable for its extremely limited nutritional spectrum. So far, all strains isolated and described under this species can grow optimally only on artificial compounds like chloridazon (Lingens et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1985\u003c/span\u003e), antipyrin, and pyramidon (Ebersp\u0026auml;cher and Lingens \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). \u003cem\u003eXanthobacter\u003c/em\u003e has been reported as an abundant genus in PFOA-spiked activated sludge (Huang et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and members of this genus consist of degraders of many resistant contaminants, such as halogenated aliphatic by \u003cem\u003eXanthobacter autotrophicus\u003c/em\u003e GJ10 (Janssen et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1985\u003c/span\u003e) and 1,4-dichlorobenzene by \u003cem\u003eXanthobacter flavus\u003c/em\u003e 14p1 (Spiess et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Specifically, \u003cem\u003eXanthobacter autotrophicus\u003c/em\u003e GJ10 contains a dehalogenase specific to halogenated carboxylic acid, which is relatively heat-stable and shows a broad substrate specificity (Janssen et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1985\u003c/span\u003e, Keuning et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1985\u003c/span\u003e, Van Der Ploeg et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). \u003cem\u003ePseudomonas\u003c/em\u003e demonstrates great metabolic diversity and has been widely reported for its dehalogenation capability. For instance, genera \u003cem\u003ePseudomonas\u003c/em\u003e were recorded as being able to degrade FTOHs (Kim et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), 2,2,2-trifluoroethane sulfonate (TES) (Key et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), and 6:2 FTS (Key et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). \u003cem\u003ePseudomonas putida\u003c/em\u003e was also found to contain dehalogenase activity against chlorinated aliphatic acids, including monochloroacetate, dichloroacetate, 2-monochloropropionate, 2,2\u0026rsquo;-dichloro-propionate (Slater et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1979\u003c/span\u003e), 4-chlorobenzoic acid (Banta and Kahlon \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), and 2,2-fluoro-1,3-benzodioxole (DFBD) (Bygd et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, defluorination of 4-deoxy-4-fluoro-d-glucose (4-FDG) was reported in the cytoplasmic membrane of \u003cem\u003ePseudomonas putida\u003c/em\u003e, which was fully inhibited with the presence of glucose. \u003cem\u003eRhodanobacter\u003c/em\u003e is reported as one of the dominant bacteria in PFAS-contaminated soil (Senevirathna et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and its family \u003cem\u003eRhodanobacteraceae\u003c/em\u003e was also found enriched in AFFF-impacted soil (Cao et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Environmental Implications and Limitations\u003c/h2\u003e \u003cp\u003eAfter almost one year of enrichment, we obtained 14 consortia exhibiting stable FTCA biodegradation. Though these consortia were enriched from diverse inoculation sources, they exhibited distinctive biodefluorination patterns when amended with 6:2 versus 5:3 FTCAs. Little fluoride release was observed for 5:3 FTCA, while 6:2 FTCA biotransformation couples with consistent fluoride release. Approximately 0.19 fluorine was liberated as fluoride per molecule of 6:2 FTCA that was degraded. This is the first to establish microbial consortia that can degrade FTCAs without the supplement of external carbon sources in long term. It is very possible that these consortia can subsist on FTCAs considering the consistency across long enrichment cycles. However, autotrophic mechanisms may take place and support the consortia by sequestering CO\u003csub\u003e2\u003c/sub\u003e in the headspace. Even though the consortia were washed thoroughly at the beginning of each enrichment cycle, our experimental setup cannot fully preclude endogenous metabolism by utilizing organic matters leaching from dead biomass. Thus, future studies are needed to track the carbon assimilation and mineralization when FTCAs were fed as the sole carbon and energy source. Furthermore, the consistently low defluorination efficiencies in our consortia also underscore the importance of investigating external carbon sources that can accelerate FTCA biotransformation and enhance defluorination extent (particularly for 6:2 FTCA).\u003c/p\u003e \u003cp\u003eDominant genera were also uncovered given their possible roles in FTCA biotransformation and biodefluorination. To gain a better understanding of the FTCA biotransformation mechanisms and exploit feasible remedial approaches, it is of significant value to initiate attempts to assess FTCA biotransformation in isolates or available pure cultures and characterize genes and enzymes involved in biodefluorination. To address the fluorine mass discrepancies over FTCA biotransformation, identifying novel transformation products via non-target analysis is also needed to advance our knowledge of FTCA biotransformation pathways and their fate once released to the environment.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eACKNOWLEDGEMENTS\u003c/p\u003e\n\u003cp\u003eThis work was funded by National Science Foundation (NSF, CBET-1903597), US Geological Survey (USGS, G24AP00026), Strategic Environmental Research and Development Program (SERDP, ER21-3556), and New Jersey Water Resources Research Institute (NJWRRI, 2019NJ183B). Chen Wu was sponsored by the Mark B. Bain Graduate Fellowship from the Hudson River Foundation. Summary of the results about the FTCA biotransformation metabolites was provided by Boyuan Su from NJIT.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEthnical Approval\u003c/p\u003e\n\u003cp\u003eThe submission of this manuscripts was approved by all authors. We declare no competing financial interest.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eFunders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAhmed, M. and Focht, D. (1973) Degradation of polychlorinated biphenyls by two species of Achromobacter. Canadian Journal of Microbiology 19(1), 47-52.\u003c/li\u003e\n \u003cli\u003eAllred, B.M., Lang, J.R., Barlaz, M.A. and Field, J.A. 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(2016) Biotransformation potential of 6: 2 fluorotelomer sulfonate (6: 2 FTSA) in aerobic and anaerobic sediment. Chemosphere 154, 224-230.\u003c/li\u003e\n \u003cli\u003eZhao, L., Folsom, P.W., Wolstenholme, B.W., Sun, H., Wang, N. and Buck, R.C. (2013) 6:2 fluorotelomer alcohol biotransformation in an aerobic river sediment system. Chemosphere 90(2), 203-209.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"PFAS precursor biotransformation, biodefluorination, fluorotelomer carboxylate acids, Hyphomicrobium","lastPublishedDoi":"10.21203/rs.3.rs-4824417/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4824417/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFluorotelomer carboxylic acids (FTCAs) has drawn increasing attention due to their prevalent occurrence, high toxicity, and bioaccumulating effects. In this study, we enriched and characterized microbial consortia with sustainable FTCA removal abilities from two activated sludges and five soils when no external carbon sources were supplemented. After four generations of enrichment, stable 6:2 FTCA and 5:3 FTCA biodegradation were achieved, reaching 0.72~0.98 and 0.53~1.05 µM/day, respectively. Coupling with 6:2 FTCA biotransformation, fluoride release co-occurred, conducive to approximate 0.19 fluoride per 6:2 FTCA molecule that was biodegraded. In contrast, minimal free fluoride was detected in 5:3 FTCA-amended consortia, indicating the dominance of “non-fluoride releasing pathways”. Microbial community analysis revealed the dominance of 13 genera across all consortia. Among them, 3 genera, including \u003cem\u003eHyphomicrobium, Methylorubrum, \u003c/em\u003eand\u003cem\u003e Achromobacter\u003c/em\u003e, were found more enriched in consortia amended with 6:2 FTCA than those with 5:3 FTCA from an identical inoculation source, suggesting their involvement in biodefluorination. This study uncovered that microbial consortia can degrade FTCAs without the supplement of external carbon sources, though with low biotransformation and biodefluorination rates. Further research is underscored to investigate the involved biotransformation pathways and biodefluorination mechanisms, as well as effects of external carbon sources.\u003c/p\u003e","manuscriptTitle":"Enriching Fluorotelomer Carboxylic Acids-Degrading Consortia from Sludges and Soils","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-30 08:29:50","doi":"10.21203/rs.3.rs-4824417/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fdef9a73-f892-4fb6-8e71-efcfb347eaf7","owner":[],"postedDate":"August 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-15T10:23:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-30 08:29:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4824417","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4824417","identity":"rs-4824417","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}