Article pubs.acs.org/est
Biotransformation of 6:2 Fluorotelomer Alcohol (6:2 FTOH) by a Wood-Rotting Fungus Nancy Tseng,† Ning Wang,‡ Bogdan Szostek,‡ and Shaily Mahendra*,† †
Department of Civil and Environmental Engineering, University of California, Los Angeles, California 90095, United States DuPont Haskell Global Centers for Health and Environmental Sciences, Newark, Delaware 19711, United States
‡
S Supporting Information *
ABSTRACT: Biotransformation of 6:2 FTOH [F(CF2)6CH2CH2OH] by the white-rot fungus, Phanerochaete chrysosporium, was investigated in laboratory studies. 6:2 FTOH is a raw material increasingly being used to replace products that can lead to long-chain perfluoroalkyl carboxylic acids (PFCAs, ≥ 8 carbons). During a product’s life cycle and after final disposal, 6:2 FTOH-derived compounds may be released into the environment and potentially biotransformed. In this study, P. chrysosporium transformed 6:2 FTOH to perfluorocarboxylic acids (PFCAs), polyfluorocarboxylic acids, and transient intermediates within 28 days. 5:3 Acid [F(CF2)5CH2CH2COOH] was the most abundant transformation product, accounting for 32−43 mol % of initially applied 6:2 FTOH in cultures supplemented with lignocellulosic powder, yeast extract, cellulose, and glucose. PFCAs, including perfluoropentanoic (PFPeA) and perfluorohexanoic (PFHxA) acids, accounted for 5.9 mol % after 28-day incubation. Furthermore, four new transformation products as 6:2 FTOH conjugates or 5:3 acid analogues were structurally confirmed. These results demonstrate that P. chrysosporium has the necessary biochemical mechanisms to drive 6:2 FTOH biotransformation pathways toward more degradable polyfluoroalkylcarboxylic acids, such as 5:3 acid, with lower PFCA yields compared to aerobic soil, sludge, and microbial consortia. Since bacteria and fungi appear to contribute differently toward the environmental loading of FTOH-derived PFCAs and polyfluorocarboxylic acids, wood-rotting fungi should be evaluated as potential candidates for the bioremediation of wastewater and groundwater contaminated with fluoroalkyl substances.
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INTRODUCTION Fungi are capable of degrading a wide range of contaminants, including halogenated compounds, pharmaceuticals, and munitions wastes.1 Wood-rotting fungi naturally degrade complex plant polymers resistant to degradation by other microorganisms.2 A well-studied wood-rotting fungus is Phanerochaete chrysosporium, which is found in the environment and can mineralize lignin to CO2 and H2O.1,2 This species has also been reported to degrade a wide range of xenobiotics, including chlorinated compounds,3 and endocrine disrupters.4 However, fungal transformation of fluorotelomer alcohols has not yet been reported. Fluorotelomer alcohol [FTOH, CnF2n+1CH2CH2OH]-based products have valuable properties, such as water, oil, and stain resistance, and are widely used in consumer and industrial applications.5 If FTOH-based products were released into the environment and subjected to microbial biodegradation, FTOHs are likely to be the initial transformation products, followed by further transformation to perfluoroalkyl carboxylic acids (PFCAs) and polyfluoroalkyl carboxylic acids in various environmental matrices.6−9 Owing to the environmental persistence and toxicological properties associated with longchain PFCAs including perfluorooctanoic acid (PFOA),10,11 © 2014 American Chemical Society
PFOA and its potential precursors, such as 8:2 FTOH, are being phased out in industrialized countries.12 As a result, 6:2 FTOH-based products are increasingly being used to replace 8:2 FTOH-based products,13 and it is necessary to understand the potential contribution of 6:2 FTOH toward PFCAs in the environment. In addition, understanding the various 6:2 FTOH biotransformation pathways and mechanisms can be used to devise microbial-based remediation strategies that enhance the degradation potential of polyfuoroalkyl substances. Aerobic biotransformation of 6:2 FTOH has been studied in soils, sediments, activated sludge, and microbial consortia. The major products of 6:2 FTOH biotransformation in these matrices were PFCAs [e.g., PFBA [F(CF2)3COOH], PFPeA, and PFHxA] and x:3 acids, such as 5:3 acid [F(CF2)5CH2CH2COOH] and 4:3 acid [F(CF2)4CH2CH2COOH]. The yields of PFCAs and of x:3 acids were quite similar, while other transient intermediates were also observed with considerably lower yields.8,14,15 6:2 Received: Revised: Accepted: Published: 4012
December 25, 2013 March 1, 2014 March 4, 2014 March 4, 2014 dx.doi.org/10.1021/es4057483 | Environ. Sci. Technol. 2014, 48, 4012−4020
Environmental Science & Technology
Article
FTOH biotransformation in Pseudomonas bacterial strains generated lower amounts of PFCAs and x:3 acids but higher levels of transient intermediates.16 These results show that a single bacterial strain may lack effective biochemical mechanisms (e.g., enzymes or cofactors) to fully degrade transient intermediates to PFCAs and x:3 acids. Instead, complete 6:2 FTOH transformation in the environment may rely on cooperation among multiple bacterial or fungal species. The objectives of this study were to determine 6:2 FTOH biotransformation potential of P. chrysosporium, to identify and quantify transformation products, and to establish 6:2 FTOH biotransformation pathways. Additionally, this work aimed to understand the differences between 6:2 FTOH biotransformation processes in various bacterial or fungal species and in environmental matrices containing mixed populations.
cellulose, 34% hemicellulose, 7% protein, 12% water-soluble chemicals, and 14% other materials on a dry-weight basis.20 Since only dry stems were used in the experiments, the lignocellulosic powder likely had relatively higher lignin content and lower amounts of protein, water-soluble chemicals, and other materials. In general, lignin, cellulose, and hemicellulose contribute to the cell wall of a plant and thus are more abundant in the stems than blades.21,22 The stems were rehydrated in sterile water overnight and pulverized inside a coffee grinder with dry ice. The wet powder was air-dried inside a biological safety cabinet and then autoclaved at 121 °C for 30 min before use. Prior to initiating the experiments, the starter culture was blended as described previously, and centrifuged at ∼640 gforce for 15 min before decanting the supernatant. The remaining cell pellet was resuspended in equal volume of fresh Kirk medium without glucose and centrifuged again to decant the supernatant. The pellet was concentrated by resuspending in half the volume of fresh Kirk medium without glucose. The biodegradation experiments were conducted in 160-mL serum bottles containing 10-mL total volume per bottle crimp-sealed with gray butyl rubber stoppers. The 10-mL total volume consisted of resuspended fungi in Kirk medium without glucose as base medium and additional organic nutrient components, including 100 mg lignocellulosic powder (L), 0.25 mg yeast extract (Y), and 2 mg cellulose (C). Two different glucose conditions were tested (per 10-mL total volume as described previously): (1) addition of 10 mg of glucose (LYCG) and (2) addition of 0 mg glucose (LYC). These two conditions were tested to determine whether glucose starvation would affect 6:2 FTOH biotransformation. The sterile control group was prepared by substituting the live culture with autoclaved fungal culture supplemented with LYCG plus 0.05 M NaOH. Each experimental group consisted of triplicate samples prepared for sacrificing at each time point. To all bottles, 25 mg of activated C18 powder from MaxiClean C18 cartridges (Grace Davison Discovery Sciences) was added to reduce the vaporization of 6:2 FTOH into the headspace, since 6:2 FTOH is very volatile with a measured vapor pressure of 108 Pa at 35 °C.23 C18 cartridges were preconditioned by eluting with 5 mL of acetonitrile, followed by 5 mL of air, and let dry overnight as described previously.8,14−18 The experiments were initiated by adding 20 μL of 6:2 FTOH stock solution made in 50% ethanol (water:pure ethanol = 1:1, v/v) to the live and sterile culture medium to result in a final 6:2 FTOH concentration of 1.7 mg L−1. A live matrix control group was also prepared to account for background levels of PFCAs and polyfluoroalkyl substances in the test bottles. The live matrix control group contained live culture medium supplemented with LYCG and 20 μL of 50% ethanol instead of 20 μL of 6:2 FTOH stock solution. The bottles were stoppered with gray, rubber butyl stoppers and crimp-sealed after the addition of 6:2 FTOH or 50% ethanol. After the bottles were sealed, two C18 cartridges were inserted into the headspace of each bottle via two sterile 18Gauge needles by puncturing through the stoppers. The cartridges allowed for simultaneous aeration of the culture medium and capture of 6:2 FTOH, 5:2 ketone [F(CF2)5C(O)CH3], and 5:2 sFTOH [F(CF2)5CH(OH)CH3] that may partition into the headspace. Headspace oxygen concentration in the live matrix control bottles was measured by an oxygen analyzer (model 905, Quantek Instruments, Grafton, MA) equipped with a needle probe to approximate oxygen
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MATERIALS AND METHODS Chemicals. All medium components had a purity ≥98.0% and were purchased from either Sigma-Aldrich (St. Louis, MO) or Thermo Fisher Scientific (Waltham, MA). Cellulose was purchased from Sigma-Aldrich. The chemical names, acronyms, and formula of the poly- and perfluoroalkyl chemicals described in this article are listed in Supporting Information, Table S1. The parent compound, 6:2 FTOH (99% purity) was purchased from Fluka Analytical (St. Louis, MO). Standards for the biotransformation products are described elsewhere.8,18 Internal standards [1,1,2,2-D; 3-13C] 6:2 FTOH (C5F1113CF2CD2CD2OH, +97%; DuPont, Wilmington, DE) and [1,2-13C] PFHxA (C4F913CF213COOH, +98%; Wellington Laboratories, Ontario, Canada) were used for LC/MS/MS quantitative analysis. All solvents, including acetonitrile, water, and methanol, were HPLC grade or higher. Biotransformation Studies. Phanerochaete chrysosporium (ATCC 24725) was tested for its ability to degrade 6:2 FTOH. Fungal spores were obtained from axenic P. chrysosporium growing on potato dextrose agar plates (Fluka BioUltra, St. Louis, MO) for 1 week at 22 °C. The spores were collected by resuspension in 8-mL sterile deionized water and filtration through sterile glass wool. The filtered spore solution was counted on a hemacytometer, and about 104 spores were used to inoculate a starter culture. The starter culture was grown in ́ and co-workers,19 at 30 Kirk medium, as described by Ramirez °C with 150 rpm agitation for 6−8 days. During this time, the culture was aseptically blended about once every two days for ∼30 s duration (∼5 s intervals) to disperse the aggregated hyphae. Briefly, Kirk medium19 contained (per liter) 55.5 mM glucose, 1.19 mM diammonium tartrate, 20 mM acetate buffer, 0.38 mM Tween 80, 6.73 mM MgSO4·7H2O, 1.2 mM NaCl, 0.03 mM FeSO4·7H2O, 0.1 mM CoCl2·6H2O, 0.43 mM ZnSO4·7H2O, 0.04 mM CuSO4, 0.08 mM MnSO4, 0.001 mM AlK(SO4)2·12H2O, 0.01 mM H3BO3, 0.003 mM Na2MoO4· 2H2O, 0.36 mM EDTA−sodium salt, 0.003 mM thiamine·HCl, 0.4 mM veratryl alcohol, 14.7 mM KH2PO4, and 0.90 mM CaCl2·2H2O. The medium was prepared as described by ́ and co-workers.19 Ramirez Kirk medium without glucose was prepared as described by ́ and co-workers,19 except that glucose was eliminated. Ramirez This glucose-free medium was used to remove residual glucose in the established culture to assess the effect of glucose on 6:2 FTOH biotransformation. Lignocellulosic powder was prepared by selecting dry stems from Timothy hay (Phlem pratense) purchased from a local pet store in Newark, DE. Timothy hay, including leaf blades and stems, contains 7% lignin, 27% 4013
dx.doi.org/10.1021/es4057483 | Environ. Sci. Technol. 2014, 48, 4012−4020
Environmental Science & Technology
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described previously,18,24 including HPLC column, software to process acquired data, and application of mass-defect filter to identify novel transformation products bearing the signature of polyfluoroalkyl substances. Detailed instrumental conditions are listed in Supporting Information, Table S3. For the live and live matrix control extracts, full-scan (100−1000 m/z) mass spectra with a resolution of 30000 were acquired to identify potential biotransformation products. Mass-defect filtration (100−500 m/z, −50 to +10 mDa) was applied to the data in order to eliminate signals that are not pertinent to polyfluorinated transformation products. Molecular structural elucidation was based on accurate mass measurement of the deprotonated molecular ions and corresponding product ion mass spectra.
conditions in live culture systems containing 6:2 FTOH. The oxygen probe was calibrated against atmospheric O2 concentration at 20.9%. The headspace of the live culture bottles for samples collected on days 14−28 was purged once for 2−3 min with 0.5−0.75 L air through the cartridges to replenish O2. Bottles were sacrificed on days 0, 7, 14, and 28 for sampling, processing, and analysis for 6:2 FTOH and transformation products. Collection and Processing of Samples. At each sampling time point, triplicate bottles were analyzed. Sample processing was carried out as previously described.15,18 Briefly, sequential extractions were conducted to ensure quantitative recovery of 6:2 FTOH and transformation products from the aqueous media in each bottle. Prior to sample extraction, 2−3 L of ambient air was purged (250 mL min−1) through the two C18 cartridges connected to the headspace. 6:2 FTOH and potential volatile transformation products captured in the headspace by each C18 cartridge were eluted with 5 mL of acetonitrile. After pushing the septum into the culture medium, 10 mL of acetonitrile was added to each bottle, which was crimp sealed with a fresh stopper. The sample solution was then extracted overnight at 50 °C with 200 rpm horizontal shaking in an orbital shaker. After incubation, the first extract was collected by centrifuging the bottle at 1500 rpm for 30 min and filtering the supernatant (nylon filters with 0.45 μm pore size) for LC/ MS/MS and high-resolution mass spectrometry analysis. A second extraction was conducted by adding 5 mL of acetonitrile and 200 μL of 1 M NaOH to the cell pellet and processed in the same manner as the first extraction. All samples were stored at −20 °C before analysis. LC/MS/MS Quantitative Analysis. Before analyzing samples via LC/MS/MS, 1 mL of filtered sample was spiked with 50 μL of internal standards containing 10 ng [1,2-13C] PFHxA and 250 ng [1,1,2,2-D; 3-13C] 6:2 FTOH. Detailed methods on LC/MS/MS analysis of 6:2 FTOH and the transformation products were described previously.18,24 Quantitative analysis of 6:2 FTOH and its transformation products were carried out with a model 2795 HPLC/Micromass Quattro Micro tandem mass spectrometry system (Waters, Milford, MA) equipped with a C8 column (Zorbax RX-C8, 5-μm particle size, 2.1 mm × 150 mm) by Agilent (Santa Clara, CA). The mass spectrometer was operated in negative electrospray ionization mode using scheduled multiple reaction monitoring (MRM). Ion transitions are listed in Supporting Information, Table S2. Samples (20 μL) were injected via an autosampler and eluted with 0.15% acetic acid in HPLC grade water (solvent A) and 0.15% acetic acid in HPLC grade acetonitrile (solvent B) at a flow rate of 400 μL min−1. The gradient started with 90% A and 10% B (1.00 min), followed by 45% A and 55% B (1.10 to 2.00 min), 20% A and 80% B (2.00 to 7.50 min), 90% A and 10% B (7.50 to 7.60 min), and ending with 90% A and 10% B (7.60 to 8.00 min). A seven-point calibration curve ranging from 1 × LOD (limit of detection, Supporting Information, Table S2) to 250 × LOD was used for quantitation of 6:2 FTOH and its transformation products. PTFE and other fluoropolymer materials were avoided as much as possible to minimize background contamination. Identification of Novel Transformation Products. An LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA) connected to an Agilent 1200 HPLC was used for identification of novel transformation products from 6:2 FTOH biotransformation. The system was operated under electrospray negative ionization mode. Detailed methods were
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RESULTS AND DISCUSSION Fungal Transformation of 6:2 FTOH. Active cultures of P. chrysosporium transformed 6:2 FTOH to perfluoroalkyl carboxylic acids (PFCAs), polyfluoroalkyl carboxylic acids, and transient intermediates within 28 days. The total recovery (mass balance) of 6:2 FTOH and quantifiable transformation products from the culture medium and headspace over 28 days are summarized in Supporting Information, Figure S1. The total recovery ranged from 84−98 mol % for the live culture supplemented with LYC and from 98−109 mol % for the culture supplemented with LYCG. For the sterile control, 95− 124 mol % of initially applied 6:2 FTOH was recovered. The headspace oxygen concentrations in the live matrix control bottles were near 15% on day 7 and then increased to above 19% thereafter (Supporting Information, Figure S2). The satisfactory mass balance and aerobic condition in live culture systems demonstrate the integrity of the fungal culture incubation systems. 5:3 Acid was the Major Transformation Product of 6:2 FTOH by Fungal Cultures. 6:2 FTOH biotransformation by P. chrysosporium produced 5:3 acid as the most abundant transformation product along with 11 other short-chain PFCAs (PFBA, PFPeA, and PFHxA) and transient transformation products (Figures 1−3, Supporting Information, Table S4). Molar yields of individual transformation products at each time point are reported in Supporting Information, Table S4. In the culture medium supplemented with LYCG, the concentration of 5:3 acid reached a plateau by day 7, and it subsequently ranged from 32−43 mol % (average of 38 mol %). The sum of the PFCAs (PFBA, PFPeA, and PFHxA) was only 5.8 mol % by day 28 (Figure 2 and Table S4). 5:3 Acid levels accounted for 16−17 mol % by day 14−28 in the culture medium supplemented with LYC, whereas the total PFCAs (PFBA, PFPeA, and PFHxA) accounted for 2.8 mol % and 1.9 mol % by day 14 and 28, respectively (Figure 3 and Supporting Information, Table S4). By day 28, 4:3 acid was observed in the culture medium supplemented with LYCG and LYC at 0.9 mol % and 0.51 mol %, respectively (Table S4). 5:2 sFTOH, the direct precursor of PFPeA and PFHxA,8 was observed as the major transient intermediate, accounting for 9− 11 mol % by day 28 in the culture medium supplemented with LYC or LYCG (Figures 2 and 3 and Supporting Information, Table S4). Most of the 5:2 sFTOH observed by day 28 was found in the headspace and may not be readily available for further biotransformation. Low levels of 6:2 FTCA [F(CF 2 ) 6 CH 2 COOH, < 0.5 mol %], 6:2 FTUCA [F(CF 2 ) 5 CFCHCOOH, < 2 mol %], 5:3 Uacid [F(CF2)5CHCHCOOH, < 1.5 mol %], α-OH 5:3 acid 4014
dx.doi.org/10.1021/es4057483 | Environ. Sci. Technol. 2014, 48, 4012−4020
Environmental Science & Technology
Article
Figure 1. Molar yields of 6:2 FTOH in P. chrysosporium grown in 10 mL of Kirk medium under two organic nutrient conditions: LYC and LYCG. LYC contains 100 mg of lignocellulosic powder (L), 0.25 mg of yeast extract (Y), 2 mg of cellulose (C), while LYCG also contained 10 mg of glucose (G). For each bottle, 17 μg of 6:2 FTOH in 20 μL of 50% ethanol (water:pure ethanol = 1:1, v/v) was added to the 10 mL of live or sterile culture of P. chrysosporium. For live matrix control, 20 μL of 50% ethanol was used to replace 6:2 FTOH in the culture supplemented with LYCG. Error bars represent standard deviation of triplicate samples.
Figure 3. Molar yields and mass balance of 6:2 FTOH and quantifiable transformation products in P. chrysosporium under LYC condition as described in Figure 1. Graph B is a magnified view of graph A to show the time trend of transformation products with lower molar yields. Error bars represent standard deviation of triplicate samples.
Information, Table S4). This indicates that these transient intermediates have been transformed to downstream stable short-chain PFCAs (PFBA, PFPeA, and PFHxA) and 5:3 and 4:3 acids. In general, the addition of glucose to the culture medium generated 2−3 times more 5:3 acid and short-chain PFCAs in 28 days as compared to the culture without additional glucose (Table S4). PFHpA [F(CF2)6COOH] was not observed in the culture medium supplemented with LYCG or LYC over 28 days. After 28 days of incubation, 16 mol % of 6:2 FTOH remained in the culture medium supplemented with LYC and 36 mol % was observed in the headspace (Supporting Information, Table S3). In comparison, 2 mol % of 6:2 FTOH still remained in the culture medium supplemented with LYCG and 46 mol % was found in the headspace (Figure S3), indicating that most of 6:2 FTOH in the culture medium was transformed to downstream products. 6:2 FTOH in the headspace may be able to repartition into the culture medium when its aqueous concentration was reduced after transformation. The differences observed in the metabolite yields between the two tested conditions suggest that glucose starvation had an effect on 6:2 FTOH biotransformation. Glucose is essential for fungal growth.19 However, in our preliminary work, P. chrysosporium growing on a continuous supply of 1% glucose barely biotransformed 6:2 FTOH. As the present work has shown, P. chrysosporium transformed 6:2 FTOH much faster with 0.1% glucose as a cosubstrate than without glucose. This
Figure 2. Molar yields and mass balance of 6:2 FTOH and quantifiable transformation products in P. chrysosporium under LYCG condition as described in Figure 1. Graph B is a magnified view of graph A to show the time trend of transformation products with lower molar yields. Error bars represent standard deviation of triplicate samples.
[F(CF2)5CH2CH(OH)COOH, < 0.2 mol %], and 5:2 ketone (
