Grantee Research Project Results
2004 Progress Report: Microbial Transformation of Fluorinated Environmental Pollutants
EPA Grant Number: R830249Title: Microbial Transformation of Fluorinated Environmental Pollutants
Investigators: Loeffler, Frank E. , Sohn, Rosa , Song, Ryoung
Institution: Georgia Institute of Technology
EPA Project Officer: Aja, Hayley
Project Period: September 1, 2002 through August 31, 2004 (Extended to August 31, 2005)
Project Period Covered by this Report: September 1, 2003 through August 31, 2004
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:
The objectives of this research project are to: (1) explore whether fluorinated organic compounds (FOCs) can serve as metabolic terminal electron acceptors; (2) use model compounds to investigate the microbial strategies to degrade (poly)fluorinated hydrocarbons; and (3) demonstrate that environmentally relevant polyfluorinated and perfluorinated hydrocarbons can undergo microbially mediated defluorination/transformation reactions.
Progress Summary:
Anaerobic Degradation of FOCs in Microcosms and Dechlorinating Consortia
Defluorination of Monofluoroacetate (MFA). Anaerobic microcosms established with aquifer material collected from the Lucent Kearney site in Milledgeville, GA, 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 (see Figure 1).
Figure 1. Microbial Defluorination of MFA in Anaerobic 30-ml Cultures Following Transfers From Active Microcosms Established With Sediment Material From the Lucent Kearney Site
Fluoride release also was observed in the pentachloronitrobenzene (PCNB) dechlorinating PCNB consortium amended with butyrate as an electron donor, but the reaction occurred slowly and long incubation periods (>150 days) were required (see Figure 2). No degradation of MFA was observed in all other cultures tested.
Figure 2. Microbial Defluorination of MFA by the PCNB Center Consortium
Degradation of 4-Nitro-3-Trifluoromethyl Phenol (TFM). TFM is the active ingredient in a restricted-use pesticide for controlling sea lamprey (Petromyzon marinus) in waters of the Great Lakes Basin. TFM has chemical characteristics that impart stability to organic compounds, and the fate of this compound and possible intermediates is poorly understood. TFM does not readily undergo chemical hydrolysis.
Despite its intensive use in the Great Lakes area, relatively few studies assessed the degradation of TFM. Under anaerobic conditions, TFM can be reduced to 4-amino-3-trifluoromethylphenol (RTFM) over periods of 5 to 30 days by microbial activity. RTFM is reported to be stable, although there is some evidence suggesting that further breakdown might occur under anoxic conditions. The enzymatic breakdown of TFM also was investigated under aerobic conditions in natural water sediments. No fluoride release was observed over a 10-week incubation period, and the trifluoromethyl-substituted aromatic ring system remained intact.
TFM transformation was observed in microcosms established with seven different sediments and aquifer materials. 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 (see Figure 3). Fluoride release from RTFM has been observed in microcosms derived from four sampling sites. Defluorination activity was maintained upon transfers to fresh medium amended with TFM and lactate.
Five consortia (BioDechlor Inoculum [BDI], PCNB, MB, CH, and GSI) reduced TFM to RTFM (see Table 1). Fluoride was released from RTFM only in the BDI and PCNB consortia.
Figure 3. Microbial Defluorination of TFM by the Microcosms Established With Pristine Samples From Patagonia
Table 1. Pure Cultures and Dechlorinating Consortia Tested for Defluorinating Activity
The BDI consortium has been sequentially transferred to new medium without loss of defluorinating activity (see Figure 4). Fluoride release in the PCNB culture occurred slowly, and long incubation periods (>150 days) were required.
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 (see Table 2). No further
Figure 4. Microbial Defluorination of TFM by the BDI Consortium After Five Consecutive Transfers With TFM
transformation was observed, and no fluoride release occurred. Reductive dehalogenation also occurred in cultures amended with cis- and trans-1,2-dichloro-1,2-difluoroethene. All three consortia dechlorinated 2-chloro-1,1-difluoroethene and chlorotrifluoroethene (CTFE), and produced 1,1-difluoroethene and trifluoroethene, respectively. Trichlorofluoroethene (TCFE) was dehalogenated by all cultures and a number of as yet unidentified products were formed.
Table 2. Products of Dehalogenation of Chlorofluorohydrocarbons by Chloroethene Dechlorinating Consortia
Dehalogenation of Chlorofluorohydrocarbons by Pure Cultures That Use Chlorinated Compounds as a Growth-Supporting Electron Acceptor
Pure cultures capable of using chlorinated ethenes as metabolic electron acceptors (i.e., chlororespiring cultures) were challenged with chlorofluorohydrocarbons. Growth of three PCE- to-cis-DCE dechlorinating pure cultures (i.e., Desulfuronmonas michiganensis strain BB1, Sulfurospirillum multivorans, and Geobacter sp. strain SZ) was tested with 1,1-dichloro-2,2-difluoroethene (see Table 3).
Table 3. Products of Dehalogenation of Chlorofluorohydrocarbons by Pure Cultures
All cultures rapidly produced 2-chloro-1,1-difluoroethene, and 1,1-difluoroethene accumulated in cultures of S. multivorans (see Figure 5). No further transformation was observed and no fluoride release occurred. Reductive dechlorination also occurred in cultures 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 S. multivorans and Geobacter sp. strain SZ, and trifluoroethene was accumulated. The formation of the transformation product, trifluoroethene, was confirmed by gas chromatography-mass spectrometry analysis. Interestingly, D. michiganensis failed to dechlorinate CTFE. TCFE was rapidly dechlorinated by all cultures. No dehalogenation was observed in cultures of Dehalococcoides sp. strain FL2 and Dehalococcoides sp. strain BAV1, suggesting that these populations cannot transform these chlorofluorohydrocarbons.
Figure 5. Dechlorination of 1,1-Dichloro-2,2-Difluoroethene by Sulfurospirillum multivorans
Aerobic Degradation of Fluorinated Alkanes
Little is known about the fate 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 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. 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 (see Figure 6). No intermediates such as MFA were detected during growth with 1-FD.
Figure 6. Defluorination of 1-FD by Pseudomonas sp. Strain 273
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. Strain 273 also grew readily with decane, sebacic acid, glucose, and acetate as a sole source of carbon and energy. When MFA was provided with each of these substrates, growth occurred at the expense of decane (see Figure 7), sebacic acid, glucose, and acetate (see Figure 8), and MFA was degraded releasing stoichiometric amounts of fluoride.
Figure 7. Degradation of Decane and MFA by Pseudomonas sp. Strain 273
Figure 8. Degradation of Acetate and MFA by Pseudomonas sp. Strain 273
Strain 273 grew with 1,10-dichlorodecane (1,10-DCD) as a sole source of carbon and energy and released stoichiometric amounts of chloride. 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 (see Figure 9). 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 alkanes via β-oxidation, suggesting that MFA or MFA-CoA are intermediates in the degradation of 1-FD. Interestingly, MFA was degraded in cultures that were amended with 1-FD, and stoichiometric amounts of fluoride were released. 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 a sole source of carbon and energy. When the organism was grown with a mixture of 1-FD and DFA or TFA, growth occurred at the expense of 1-FD, but DFA or TFA was not transformed. Similarly, no DFA or TFA degradation occurred in cultures growing with decane, 1,10-DCD, sebacic acid, glucose, or acetate.
Figure 9. Pseudomonas sp. Strain 273 Failed to Degrade a Mixture of 1,10-DCD and MFA
No growth occurred in strain 273 cultures amended with perfluoro-n-octanoate, 4,4,4-trifluorobutyric acid, and ethyl-4,4,4-trifluoroacetoacetate as the sole source of carbon and energy. In the presence of 1-FD, growth occurred on the expense of 1-FD oxidation, 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 a mixture of decane and each of these compounds, decane was consumed but perfluoro-n-octanoate, 4,4,4-trifluorobutyric acid, and ethyl-4,4,4-trifluoroacetoacetate were not degraded and no fluoride release occurred.
Future Activities:
We will completedehalogenation studies with the pure and mixed cultures. Attempts made to identify the products formed from chlorofluorohydrocarbon dehalogenation will be identified by GC/MS analysis.
Several enrichment cultures that reductively defluorinate environmentally relevant FOCs were developed and now are available for in-depth studies of the microbiology involved in the dehalogenation process. This will be a focus in the next several months.
Efforts to elucidate fluoroalkane degradation with strain 273 will continue. We have begun to prepare a manuscript detailing our findings on the degradation of fluorinated aliphatic hydrocarbons by strain 273, and we anticipate submitting this paper in spring 2005.
Journal Articles:
No journal articles submitted with this report: View all 6 publications for this projectSupplemental Keywords:
fluorinated hydrocarbons, sediments, human and ecosystem health, biodegradation, detoxification, restoration, halorespiration, defluorination, halogenated hydrocarbons, ecosystem protection/environmental exposure and risk, water, aquatic ecosystem restoration, ecology, ecology and ecosystems, environmental chemistry, environmental monitoring, research/future, futures, biodiversity, conservation, contaminant uptake, ecological pollutants, ecotoxicology, environmental rehabilitation, environmental stress, exploratory research,, 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, ecotoxicologyProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.