Final Report: Microbial Transformation of Fluorinated Environmental Pollutants

EPA Grant Number: R830249
Title: Microbial Transformation of Fluorinated Environmental Pollutants
Investigators: Loeffler, Frank E. , Sohn, Rosa , Song, Ryoung
Institution: Georgia Institute of Technology
EPA Project Officer: Lasat, Mitch
Project Period: September 1, 2002 through August 31, 2004 (Extended to August 31, 2005)
Project Amount: $198,936
RFA: Futures Research in Natural Sciences (2001) RFA Text |  Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , Land and Waste Management , Hazardous Waste/Remediation

Objective:

Fluorinated organic compounds (FOCs) have been extensively manufactured and used for the past several decades as surfactants, pesticide formulations, lubricants, refrigerants, fire retardants, drugs, cosmetics, paints, adhesives, and other compounds. Recent studies indicated that FOCs are not biologically inert and exhibit toxic effects on humans and animals and impact overall ecosystem health. The strength of the carbon-fluorine bond and the anthropogenic origin of FOCs are often used to explain the inability of biological systems to degrade FOCs. There is convincing evidence, however, that microbes are capable of degrading and detoxifying monofluorinated and polyfluorinated hydrocarbons and that enzyme systems that break carbon-fluorine bonds exist.

The objective of this research project was to explore the microbial degradation of alipahtic and aromatic hydrocarbons carrying one or more fluorine substituents. Among the FOCs investigated in this research project, monofluoroacetate (MFA) is a restricted use pesticide and has been placed in Toxicity Category I by the U.S. Environmental Protection Agency because of its high acute toxicity. MFA has great physicochemical stability because of its fluorine-carbon bond, withstanding boiling and treatment with concentrated sulfuric acid (Saunders and Stacey, 1948). The fate of MFA under anaerobic conditions has not been adequately explored.

4-nitro-3-trifluoromethylphenol (TFM) is the active ingredient in a restricted-use pesticide for controlling sea lamprey ( Petromyzon marinus) in waters of the Great Lakes basin (Hubert, 2003). TFM has chemical characteristics that impart stability to organic compounds, and the fate of this compound and possible intermediates is poorly understood. In this research project, microbial defluorination of TFM and several other trifluoromethylphenol compounds, which share structural similarity with TFM (i.e., 2-, 3-, 4­trifluoromethylphenol and 2-nitro-4-trifluoromethylphenol; 2-TFMP, 3-TFMP, 4-TFMP, and 2-N-4-TFMP, respectively), were explored.

Another focus was on the microbial degradation of medium chain length fluorinated alkanes. Poly- and perfluorinated medium chain length fluorinated hydrocarbons are of particular concern because of their widespread distribution in the environment. To elucidate the microbial strategies that transform such aliphatic FOCs under aerobic conditions, 1-fluorodecane (1-FD) was chosen as a model compound, and the degradation of this compound was studied with Pseudomonas sp. strain 273.

This research effort explored the microbial degradation of fluorinated model compounds and environmentally relevant FOCs to improve our understanding of the fate of these chemicals in the environment. Specifically, the following objectives were addressed:

  • Objective 1. Explore if FOCs are reductively defluorinated and serve as metabolic terminal electron acceptors in anaerobic respiration.
  • Objective 2. Use model compounds to investigate the microbial strategies to degrade (poly)fluorinated hydrocarbons.
  • Objective 3. Demonstrate that environmentally relevant polyfluorinated and perfluorinated hydrocarbons can undergo microbially mediated defluorination/transformation reactions.

Summary/Accomplishments (Outputs/Outcomes):

Anaerobic Degradation of FOCs

Reductive Defluorination of Trifluoroacetate (TFA), Difluoroacetate (DFA) and Ethyl-4,4,4-Trifluoroacetoacetate. Reductive defluorination of TFA, DFA, and ethyl-4,4,4-trifluoroacetoacetate was not observed in any of the microcosms established with sediment, soil, and aquifer materials listed in Table 1. Also, no fluoride release from these compounds occurred in cultures that were inoculated with the dechlorinating pure and mixed cultures.

Table 1. Sample Materials Tested for Degradation/Defluorination of FOCs

Site

Activity

Wastewater treatment facility, Atlanta GA

Defluorination of MFA and TFM

FMC Corp., CA (2 locations)

- (a)

Mangrove swamp, FL

-

Kalamazoo River , MI

-

Surgeon fish guts

TFM → RTFM

Young-Rainey Science, Technology, and Research (STAR) Center, FL (3 locations)

-

Stone Mountain State Park , GA

-

Milledgeville , GA (2 locations)

Defluorination of MFA and TFM

Visteon site, MN (2 locations)

-

Hydrite Chemical site, WI (2 locations)

-

Suzi Creek , South Korea

TFM → RTFM

Wastewater treatment facility, South Korea

TFM → RTFM

TRW Minerva, OH

Defluorination of TFM

Pinellas , FL (3 locations)

-

Patagonia , Chile (4 pristine locations)

Defluorination of TFM (b)

Buford Dam, GA (2 locations)

Defluorination of TFM

Occidental Chemical site, Montague, MI

-

Creek sediment, Urbana-Champaign, IL

Defluorination of MFA and TFM

Neckar River, Stuttgart, Germany

Defluorination of MFA and TFM

Savannah River , GA (2 locations)

Defluorination of TFM

Streambed sediment, Cancun, Mexico

Defluorination of MFA and TFM

Lake sediment, Fargo, ND

-

(a) - = No activity
(b) Defluorination was observed in one out of four replicate microcosms.

Defluorination of Monofluoroacetate (MFA). Anaerobic microcosms established with aquifer material collected from a chloroethene-contaminated aquifer in Milledgeville, Georgia, defluorinated MFA and stoichiometric amounts of fluoride were released. Subsequent transfers (3%, vol/vol) to fresh reduced basal salts medium amended with MFA and lactate yielded a sediment-free, MFA defluorinating culture. Defluorination of MFA occurred in four more anaerobic microcosms established with digestor sludge from a wastewater treatment facility in Atlanta, Georgia; creek sediment collected near the University of Illinois at Urbana-Champaign; creek sediment collected near Cancun, Mexico; and sediment from the Neckar River in Germany (Table 1).

MFA degradation and fluoride release also was observed in the pentachloronitrobenzene dechlorinating PCNB consortium amended with butyrate as an electron donor, but the degradation rates were slow, requiring long incubation periods (>150 days; see Figure 1). No degradation of MFA was observed in all other microcosms, consortia, and pure cultures tested.

Microbial Defluorination of MFA by the PCNB Consortium

Figure 1. Microbial Defluorination of MFA by the PCNB Consortium

Degradation of 4-nitro-3-trifluoromethyl phenol (TFM). TFM transformation was observed in microcosms established with 12 different sediments and aquifer materials (Table 1). These microcosms transformed TFM to RTFM over periods of 5 to 30 days. This reduction results in a loss of the characteristic yellow color of TFM as measured by the decrease in absorption at 392 nm. Fluoride release from RTFM has been observed in microcosms derived from nine sampling sites. Defluorination activity was maintained upon transfers to fresh medium amended with TFM and lactate, and sediment-free enrichment cultures were obtained.

Six consortia (BDI, PCNB, MB, CH, GSI, and SHAW) reduced TFM to RTFM (Table 2). Subsequent degradation of RTFM and the release of fluoride were observed only in the BDI and PCNB consortia. The BDI consortium has been sequentially transferred to fresh medium five times without loss of defluorinating activity (Figure 2). The concentration of RTFM is not shown in Figure 2 after 55 days of the incubation because of the overlap of the RTFM peak with an unknown peak in HPLC analysis. Fluoride release in the PCNB culture occurred slowly and required long incubation periods (>150 days).

Table 2 . Dechlorinating Consortia Tested for Defluorinating Activity

Culture

Dechlorinating activity

Defluorinating activity

BDI

PCE

→ ethene

Defluorination of TFM

PCNB

PCNB → 2,5-dichloroaniline

Defluorination of TFM and MFA

MB

PCE

→ ethene

TFM → RTFM

CH

PCE

→ ethene

TFM → RTFM

GSI

PCE

→ ethene

TFM → RTFM

SHAW

PCE

→ ethene

None detected

Microbial Defluorination of TFM by the BDI Consortium Following Five Consecutive Transfers in Basal Salts Medium Amended With TFM

Figure 2. Microbial Defluorination of TFM by the BDI Consortium Following Five Consecutive Transfers in Basal Salts Medium Amended With TFM

The BDI consortium also defluorinated 2-, 3-, 4-trifluoromethylphenol and 2-nitro-4-trifluoromethylphenol (2-TFMP, 3-TFMP, 4-TFMP, and 2-N-4-TFMP, respectively). In 85 days following inoculation, 2.30 mM of fluoride was released from 1 mM of 2-TFMP, 0.05 mM from 3-TFMP, 0.83 mM from 4-TFMP, and 2.34 mM from 2-N-4-TFMP. For each trifluoromethylphenol compound, several novel peaks were observed in HPLC results, but none of the intermediates/end products have been identified (Figure 3).

HPLC Chromatogram of Transformation Products Formed During 4-TFMP Defluorination by the Consortium BDI (λ = 255 nm)

(a) 4-TFMP                         (b) Products of 4-TFMP

Figure 3. HPLC Chromatogram of Transformation Products Formed During 4-TFMP Defluorination by the Consortium BDI (λ = 255 nm)

Dehalogenation of Chlorofluorohydrocarbons by Chloroethene-Dechlorinating Consortia

The chloroethene-dechlorinating consortia CH, MB, and GSI dechlorinated 1,1-dichloro-2,2-difluoroethene to 2-chloro-1,1-difluoroethene and 1,1-difluoroethene. No further transformation was observed, and no fluoride release occurred. The other two chloroethene-dechlorinating consortia (BDI and SHAW) dechlorinated 1,1-dichloro-2,2-difluoroethene to 2-chloro-1,1-difluoroethene, and no further dehalogenation was observed over a 90-day incubation period.

Reductive dehalogenation also occurred in cultures amended with cis-/trans-1,2-dichloro-1,2-difluoroethene (Figure 4). Consortia CH, MB, and GSI dechlorinated cis-/trans-1,2-dichloro-1,2-difluoroethene to cis-/trans-2-chloro-1,2-difluoroethene and cis-/trans-1,2-difluoroethene. Interestingly, vinyl fluoride was detected indicating that a reductive defluorination reaction occurred in these cultures. The formation of dehalogenation products was confirmed by gas chromatography/mass spectrometry(GC/MS). Defluorination of vinyl fluoride to ethene or ethane was not observed. Although the GC system used for the analysis was able to separate cis-/trans-1,2-chloro-1,2-difluoroethene (Figure 4), quantification was not possible because standards were not available.

The effect of PCE on 1,2-dichloro-1,2-difluoroethene dehalogenation was explored with consortia CH, MB, and GSI. All three consortia were grown with methanol as a source of reducing equivalents and PCE as an electron acceptor. After all PCE had been dechlorinated to ethene, transfers (3%, vol/vol) occurred to fresh medium amended with lactate (5 mM) and 1,2-dichloro-1,2-difluoroethene and/or PCE. In the presence of PCE, the lag time before the defluorination of 1,2-dichloro-1,2-difluoroethene occurred was reduced to 11 days in cultures of consortium CH compared with a 30-day lag in cultures that did not receive PCE. Consortium MB, however, responded differently to the presence of PCE, and 1,2-dichloro-1,2-difluoroethene defluorination was observed in 15 days in the absence of PCE but required 65 days in vessels that received PCE. In both cases, all PCE was consumed within 7 days and ethene was produced. The addition of PCE to consortium GSI had no effect on 1,2-dichloro-1,2-difluoroethene defluorination, which started after an 8-day lag period.

Both the BDI and SHAW consortia dechlorinated cis-/trans-1,2-dichloro-1,2-difluoroethene only to cis-/trans-2-chloro-1,2-difluoroethene, and no formation of vinyl fluoride occurred during a 100-day incubation period. Consistent with the these observations was that the CH, MB, and GSI consortia dechlorinated 2-chloro-1,1-difluoroethene to 1,1-difluoroethene, but the BDI and SHAW consortia failed to dehalogenate 2-chloro-1,1-difluoroethene. All five consortia (i.e., CH, MB, GSI, BDI, and SHAW) dechlorinated CTFE to trifluoroethene. TCFE was dehalogenated by all cultures and a number of unidentified products were formed (Figure 5). Results of GC/MS analysis showed the formation of TCFE dechlorination products with one and two chlorines removed. Because each of these compounds have three isomers each, it was not possible to identify the exact structure of these intermediates/products.

Chromatogram of Products Formed During 1,2-Dichloro-1,2-difluoroethene Dehalogenation by the Methanogenic Consortium GSI

* 1,2-dichloro-1,2-difluoroethene (90 %) and 1,1-dichloro-2,2-difluoroethene (10 %).

** 2-chloro-1,1-difluoroethene and 1,1-difluoroethene are 1,1-dichloro-2,2-difluoroethene transformation products.

Figure 4. Chromatogram of Products Formed During 1,2-Dichloro-1,2-difluoroethene Dehalogenation by the Methanogenic Consortium GSI

TCFE Dehalogenation Products Formed by the Methanogenic Consortium GSI

Figure 5. TCFE Dehalogenation Products Formed by the Methanogenic Consortium GSI

Dehalogenation of Chlorofluorohydrocarbons by Pure Cultures That Use Chlorinated Compounds as Metabolic Electron Acceptors

Pure cultures capable of using chlorinated ethenes as metabolic electron acceptors (i.e., chlororespiring cultures) were challenged with chlorofluorohydrocarbons. Dehalogenation of 1,1-dichloro-2,2-difluoroethene was tested with three PCE-to-cis-DCE dechlorinating pure cultures (i.e., Desulfuromonas michiganensis strain BB1, Sulfurospirillum multivorans, and Geobacter sp. strain SZ). The experiments were performed in 160 mL serum bottles containing 100 mL of basal salts medium amended with 5 mM lactate as electron donor (Sung, et al., 2003). Cultures of Desulfuromonas michiganensis strain BB1 and Geobacter sp. strain SZ dechlorinated 1,1-dichloro-2,2-difluoroethene (30 µmol/bottle) to 2-chloro-1,1-difluoroethene (up to 10 µmol/bottle) in 4 days of incubation, and 1,1-difluoroethene was produced. All 1,1-dichloro-2,2-difluoroethene was converted to 2-chloro-1,1-difluoroethene, but not all 2-chloro-1,1-difluoroethene was converted to 1,1-difluoroethene for over 100-day incubation period. The dechlorination of 1,1-dichloro-2,2-difluoroethene by Sulfurospirillum multivorans is presented in Figure 6. No further transformation was observed, and no fluoride release occurred.

Dechlorination of 1,1-Dichloro-2,2-difluoroethene by Sulfurospirillum multivorans

Figure 6. Dechlorination of 1,1-Dichloro-2,2-difluoroethene by Sulfurospirillum multivorans

Reductive dechlorination also occurred in cultures of all three isolates amended with cis-and trans-1,2-dichloro-1,2-difluoroethene, but no fluoride release was observed even after extended incubation periods of 6 months. CTFE was dechlorinated by cultures of Desulfuromonas michiganensis strain BB1, Sulfurospirillum multivorans and Geobacter sp. strain SZ, and trifluoroethene accumulated. The formation of trifluoroethene was confirmed by GC-MS analysis. The highest CTFE dechlorination rates were measured in cultures of Sulfurospirillum multivorans whereas the transformation of CTFE in cultures of strain BB1 and strain SZ occurred at lower rates and was incomplete, even after prolonged incubation periods. TCFE was rapidly dechlorinated to several unidentified products by all three cultures, but no vinyl fluoride was formed. No dehalogenation was observed in cultures of Dehalococcoides sp. strain FL2, Dehalococcoides sp. strain BAV1, and Dehalococcoides sp. strain GT suggesting that these populations cannot transform these chlorofluorohydrocarbons.

Clostridium bifermentans DPH-1 dechlorinated 1,1-dichloro-2,2-difluoroethene only to 2-chloro-1,1-difluoroethene and cis-/trans-1,2-dichloro-1,2-difluoroethene to cis-/trans-2-chloro-1,2-difluoroethene. Clostridium bifermentans DPH-1 did not dehalogenate 2-chloro-1,1-difluoroethene and CTFE. TCFE was dechlorinated but no vinyl fluoride was formed and two unidentified products were observed by GC analysis.

Aerobic Degradation of Fluorinated Alkanes

Strain 273 grew with 1-FD as the sole source of carbon and energy, and stoichiometric amounts of fluoride were released into the growth medium (Figure 7; Song and Loeffler, 2004a). No intermediates were detected during growth with 1-FD. Strain 273 did not grow with MFA as the sole source of carbon and energy. When the organism was grown with a mixture of 1-FD and MFA, however, growth occurred, and both 1-FD and MFA were degraded (Figure 8).

Degradation and Defluorination of 1-FD by Pseudomonas sp. Strain 273

Figure 7. Degradation and Defluorination of 1-FD by Pseudomonas sp. Strain 273

Degradation of 1-FD and MFA by Pseudomonas sp. Strain 273

Figure 8. Degradation of 1-FD and MFA by Pseudomonas sp. Strain 273

Strain 273 also grew readily with decane, sebacic acid, glucose, and acetate as sole sources of carbon and energy. When MFA was provided together with each of these substrates, growth occurred at the expense of decane, sebacic acid, and glucose, and MFA was degraded concomitantly and fluoride was released. Similar results were obtained in acetate-fed cultures, although repeated additions of acetate (5 mM) were required to achieve stoichiometric fluoride release from MFA. The headspace of each culture vessel was purged with sterile air for 2-3 minutes following each acetate addition to ensure aerobic conditions. No MFA was detectable after 10 days, and approximately 0.65 mM fluoride was released from 1 mM of MFA after the 4-week incubation period. No other (fluorinated) intermediates were detected by ion chromatography (IC) and liquid chromatography/mass spectrometry (LC/MS) analyses. When the MFA concentration was increased to 2 mM, strain 273 grew on the expense of 1-FD and decane, but no MFA consumption occurred and no fluoride was released.

Strain 273 grew with 1,10-dichlorodecane (1,10-DCD) as a sole source of carbon and energy and released stoichiometric amounts of chloride (Wischnak, et al., 1998). When the organism was grown with a mixture of 1,10-DCD and MFA, no growth was observed and neither 1,10-DCD nor MFA were degraded (Song and Loeffler, 2004b). Although MFA had no inhibitory effect on growth of strain 273 with 1-FD, decane, sebacic acid, glucose, and acetate, MFA completely inhibited growth with 1,10-DCD. Strain 273 metabolizes (halogenated) alkanes via β-oxidation, indicating that MFA or MFA-CoA are possible intermediates in the degradation of 1-FD. It is interesting to note MFA inhibited growth with 1,10-DCD but not with 1-FD or other substrates tested. This observation is relevant and emphasizes that the complex interaction between microbes and their substrates must be understood before meaningful and reliable predictions on the fate of FOCs in the natural environment are possible.

Strain 273 failed to grow with MFA, DFA, and TFA as sole sources of carbon and energy. When the organism was grown with a mixture of 1-FD plus DFA or TFA, growth occurred at the expense of 1-FD, but DFA or TFA were not transformed. Similarly, neither DFA nor TFA degradation occurred in cultures growing with decane, 1,10-DCD, sebacic acid, glucose, or acetate.

No growth occurred in cultures of strain 273 amended with perfluoro-n-octanoate, 4,4,4-trifluorobutyric acid, or ethyl-4,4,4-trifluoroacetoacetate as the sole sources of carbon and energy. In the presence of 1-FD, growth occurred at the expense of 1-FD, and perfluoro-n-octanoate (1 mM), 4,4,4-trifluorobutyric acid (1 mM), and ethyl-4,4,4-trifluoroacetoacetate (1 mM) were partially degraded and additional fluoride (0.3 mM) was released. Similar results were obtained with 1,10-DCD as the primary substrate. When the organism was grown with decane in the presence of perfluoro-n-octanoate (1 mM), 4,4,4-trifluorobutyric acid (1 mM), and ethyl-4,4,4-trifluoroacetoacetate (1 mM), decane was consumed, but the FOCs were not degraded and no fluoride release occurred.

References:

Wischnak C, Loffler FE, Li J, Urbance JW, Muller R. Pseudomonas sp. Strain 273, an aerobic α, ω-dichloroalkane degrading bacterium. Applied and Environmental Microbiology 1998;64(9):3507-3511.

Hubert TD. Environmental fate and effects of the lampricide TFM: a review. Journal of Great Lakes Research 2003;29(Suppl 1):456-474.

Saunders BC, Stacey GJ. Toxic fluorine compounds containing the C-F link. Part I. Methyl fluoroacetate and related compounds. Journal of the Chemical Society 1948:1773-1779.


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Other project views: All 6 publications 1 publications in selected types All 1 journal articles
Type Citation Project Document Sources
Journal Article Sung Y, Ritalahti KM, Sanford RA, Urbance JW, Flynn SJ, Tiedje JM, Loffler FE. Characterization of two tetrachloroethene-reducing, acetate-oxidizing anaerobic bacteria and their description as Desulfuromonas michiganensis sp. nov. Applied and Environmental Microbiology 2003;69(5):2964-2974. R830249 (Final)
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Supplemental Keywords:

fluorinated hydrocarbons, sediments, human and ecosystem health, biodegradation, detoxification, restoration, halorespiration, defluorination,, RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Environmental Chemistry, Restoration, Environmental Monitoring, Aquatic Ecosystem Restoration, Futures, Exp. Research/future, biodiversity, biodegradation, defluorination, conservation, contaminant uptake, ecological pollutants, exploratory research, environmental rehabilitation, environmental stress, ecotoxicology

Progress and Final Reports:

Original Abstract
  • 2003 Progress Report
  • 2004 Progress Report