APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1994, p. 4567-45720099-2240/94/$04.00+0Copyright © 1994, American Society for Microbiology

Dechlorination of Trichlorofluoromethane (CFC-11) by Sulfate-Reducing Bacteria from an Aquifer Contaminated with

Halogenated Aliphatic CompoundsDYANE N. SONIER,t NORMA L. DURAN, AND GEOFFREY B. SMITH*

Biology Department, New Mexico State University, Las Cruces, New Mexico 88003

Received 6 June 1994/Accepted 29 September 1994

Groundwater samples were obtained from a deep aquifer contaminated with halogenated aliphaticcompounds. One-milliliter samples contained 9.2 x 105 total bacteria (by acridine orange microscopic counts)and 2.5 x 103 sulfate-reducing bacteria (by most probable number analysis). Samples were incubatedanaerobically in a basal salts medium with acetate as the electron donor and nitrate and sulfate as the electronacceptors. Residual levels of trichlorofluoromethane (CFC-11) in samples were biotically degraded, whiletrichloroethylene was not. When successively higher levels of CFC-11 were added, increasingly rapiddegradation rates were observed. Concomitant with CFC-11 degradation was the near stoichiometricproduction of fluorodichloromethane (HCFC-21); the production of HCFC-21 was verified by mass spectrom-etry. CFC-11 degradation was dependent on the presence of acetate (or butyrate) and sulfate but was

independent of nitrate. Other carbon sources such as lactate and isopropanol did not support the degradation.The addition of 1 mM sodium sulfide completely inhibited CFC-11 degradation; however, degradation occurredin the presence of 2 mM 2-bromoethanesulfonic acid. These results indicate that the anaerobic dechlorinationof CFC-11 is carried out by sulfate-reducing bacteria and not by denitrifying or methanogenic bacteria.

Halogenated aliphatic hydrocarbons such as trichloroflu-oromethane (CFC-11 or Freon-11) and trichloroethylene(TCE) have been widely used as solvents, refrigerants, andaerosol and polystyrene propellants. The extensive use of thesecompounds has resulted in contamination of the environmentin the form of volatilized gases, industrial wastewater contam-ination, or leakage from underground storage tanks. TCE is a

known carcinogen (20) and is frequently found as a majorcontaminant of groundwater supplies (16). The effects ofCFC-11 on biological systems have not been determined (20).There is evidence that when airborne, CFC-11 and CFC-12(dichlorodifluoromethane) are the major chlorofluorocarbons(CFCs) responsible for the depletion of stratospheric ozone (4,13).These compounds were once considered inert and without a

biological or chemical sink. Biological degradation of TCE hasbeen demonstrated under aerobic and anaerobic conditions (1,14), and a pathway via sequential reductive dechlorination hasbeen observed (2, 3). There are fewer data available describingbiodegradation of CFC-11. The reductive dehalogenation ofCFC-11 by coenzyme F430 and by corrinoids in the presence oftitanium citrate has been demonstrated, and a potential reac-tion mechanism has been proposed by Krone et al. (6-8). It hasalso been suggested that a reduced hematin is involved in a

nonenzymatic mechanism of CFC-11 degradation (11). Re-cently, anaerobic groundwater contaminated by landfillleachate containing CFC-11 and other halogenated com-

pounds exhibited the ability to degrade CFC-11, as haveanaerobic soils and sediments (9-11). However, no particular

* Corresponding author. Mailing address: Biology Department,New Mexico State University, Box 3AF, Las Cruces, NM 88003.Phone: (505) 646-6080. Fax: (505) 646-5665. Electronic mail address:GSMITH@NMSU.EDU.

t Present address: Microbiology Department, Washington StateUniversity, Pullman, WA 99164.

microbial group has been identified as responsible for theCFC-11 degradation reaction.

Traditionally, nonbiological treatment of sites contaminatedwith volatile hydrocarbons has involved airstripping the con-

taminated media. While this is a somewhat reasonable processfor TCE, it would result only in the increase of CFC-11'scontribution to ozone depletion if it were applied to CFC-contaminated sites. Therefore, in situ bioremediation ofCFC-11 resulting in complete mineralization or production ofless harmful intermediates would be more environmentallyappropriate than traditional methods in eliminating the CFCcontaminants.

In this study, we examined whether the microorganismspresent in anaerobic samples obtained from an aquifer con-taminated with CFC-11 and TCE were capable of degradingthese halogenated aliphatic compounds. CFC-11 but not TCEbiodegradation was observed, and we determined that theanaerobic process was dependent on the presence of acetateand sulfate but independent of nitrate and bromoethanesulfonic acid, indicating that sulfate-reducing bacteria are theresponsible agents.

MATERMILS AND METHODS

Groundwater sample incubation conditions. Groundwatersamples were obtained from a monitoring well with an auto-claved, weighted bottle sampler from a depth of 118 feet belowthe surface of a basin and range province dominated bylimestone in the western United States. The water sample was

kept at 4°C until the incubations were begun within 24 h.Nineteen milliliters of basal salt medium (BSM) was added to40-ml amber vials which were sealed with Mininert valves. Thevials were then flushed and evacuated four times with nitrogengas. BSM was based on medium 337 as described by Poindex-ter (15) and included the following (in grams per liter):KH2PO4, 0.136; Na2HPO4 7H20, 0.268; (NH4)2SO4, 0.502;FeSO4 7H20, 2.0 x 10-3; MnSO4 H20, 3.0 x 10-4; Na2MOO4

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2H20, 5.0 X 10-4; CaSO4 2H20, 0.069; MgSO4, 0.096; KN03,0.051; and yeast extract, 0.01. Carbon sources such as acetate,butyrate, lactate, and isopropanol were present at concentra-tions of 10 mM. Five of the 40-milliliter vials were eachinoculated with 1.0 ml of sample. Two of the inoculated vialswere autoclaved on 3 successive days and were used asheat-killed abiotic controls. This procedure was repeated witha duplicate groundwater sample.

Halogenated compound amendments. Because of the differ-ent Henry's constants of CFC-11 and TCE, different masses ofthe two compounds were added to give similar concentrationsin solution. A 1,000-,ug/ml aqueous stock of CFC-11 was madefrom pure CFC-11 in a cold room (4°C). For vials to whichCFC-11 was added as a contaminant, 50 [lI of this stock wasinjected by a gas-tight syringe through the Mininert valves ofthe 40-ml vials. By using the Henry's constant for CFC-11 of11.05 kPa m3/mol (12), this was calculated to give 450 ng/ml forthe 20-ml solution and 2.05 ,ug/ml for the 20-ml headspace. An800-,ug/ml stock of TCE was prepared at room temperature,and 25 [lI of this was added to the vials. By using the Henry'sconstant of 0.939 kPa m3/mol for TCE (12), this was calculatedto give 720 ng/ml in solution and 280 ng/ml in the headspace.All vials were then inverted and incubated at 25°C.

Analytical procedures. A 5-,ul headspace sample was takenwith a gas-tight syringe and was manually injected into aHewlett-Packard 5890 series II gas chromatograph equippedwith an electron capture detector and an HP-5 capillarycolumn (25 m by 0.32 mm by 0.52 ,um; Hewlett-Packardcatalog no. 19091J-112). At 35°C, the retention times ofCFC-11 and TCE were 1.2 and 2.7 min, respectively, withhelium carrier gas at a flow rate of 8 ml/min and nitrogenmakeup gas at 24 ml/min. Standards for both CFC-11 and TCEwere made, and headspace analysis detection limits were 0.1and 1.0 ng/ml, respectively. With these standard curves, theconcentrations reported below are in terms of the total con-centration present in each vial. To separate related CFCcompounds, an SP-1000 packed column (8 ft by 1/8 in. [ca. 244cm by ca. 0.32 cm]; Supelco catalog no. 1-2545) was used, andthe effluent was analyzed on the electron capture detector. Formass spectrometric verification of degradation products, EPAmethod 8240A was used (21). The presence of methane wasassayed by sampling 0.1 ml of headspace and injection onto aNukol capillary column (15 m by 0.53 mm by 0.5 ,um; Supelcocatalog no. 2-5326) linked to a flame ionization detector.

Preparation of hematin. A 1-mg/ml stock of hematin (Sigmano. H-3505) was prepared in ethanol, and from this a 10-,ug/mlstock was made in sterile distilled water. After 3 days ofincubation at room temperature, a brown precipitate formedwhich was removed by centrifugation (5 min at 2,000 x g). Theresultant brown supernatant was deemed water saturated, and1 ml of this was added to 19 ml of BSM for the oxidizedhematin treatment. For reduced hematin, the saturated solu-tion was boiled under nitrogen gas, and Na2S was added to givea final concentration of 0.5 mM sulfide. One milliliter of thissolution was added to 19 ml of BSM for the reduced-hematintreatment.

Other analyses. For acridine orange direct counts, 0.5 mlwas withdrawn from each original groundwater sample, and10-1 and 10-2 dilutions were stained with 0.01% acridineorange and examined by epifluorescence microscopy (5). Sam-ple dilutions were also inoculated into Starkey's medium (19)to determine most probable numbers of sulfate-reducing bac-teria. After 3 weeks of incubation, the presence of sulfatereducers was obvious by the production of a black FeSprecipitate.

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FIG. 1. In a groundwater sample amended with 1,000 ppb of TCE,residual levels (10 ppb) of Freon-11 were degraded after 50 days. Ondays 70 and 80, 3.5 and 500 ppb of Freon-11, respectively, were added.Values are the means of six replicates.

RESULTS

CFC-11 biodegradation, kinetics, and conditions. In theTCE-amended samples containing acetate, no TCE degrada-tion was observed after 150 days of incubation (data notshown); however, the residual levels of CFC-11 present in thesample were degraded after 50 days (Fig. 1). When CFC-11was spiked in at increasing concentrations, the degradationrate increased from 0.2 ppb/day to 37 ppb/day, where ppb =ng/ml. In parallel incubations in which 2,500 ppb of CFC-11instead of TCE was amended, CFC-11 degradation proceededat a rate of 18 ppb/day without a lag period. No CFC-11degradation was observed in heat-killed controls (data notshown).

After the 4-month incubation shown in Fig. 1, 0.5 ml of thecultures was transferred to fresh BSM containing acetate, andCFC-11 degradation was observed again (data not shown).These cultures were transferred again into BSM without yeastextract and were tested for CFC-11 degradation in the pres-ence and absence of acetate or nitrate (Fig. 2). CFC-11degradation was dependent on acetate and independent ofnitrate. When acetate was added to one of the acetate-lacking

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FIG. 2. Freon-11-degrading cultures were transferred to BSM withand without 5 mM nitrate or 10 mM acetate. O, sterile control; 0, withacetate and nitrate; *, with acetate and without nitrate; *, withoutacetate and with nitrate. On day 48, 10 mM acetate was amended intoreplicate A of the treatment sample without acetate and with nitrate.Values are the means of two replicates.

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FIG. 3. Biodegradation of Freon-11 by duplicate cultures whichhad been pre-exposed to 0.01 mM sulfate for 60 days before beinginoculated into BSM with various concentrations of sulfate.

replicates on day 48, CFC-11 degradation was observed. TheseCFC-11-degrading cultures have since been repeatedly trans-ferred on acetate-amended BSM without nitrate or yeastextract, each time degrading CFC-11 at an average rate of 15to 30 ppb/day. Sulfate (5 mM) was present in all of theexperiments discussed above.One milliliter of the CFC 11-degrading cultures was inocu-

lated into medium containing 0.01 or 5 mM sulfate, and therate of CFC-11 degradation decreased to 7 ppb/day with 0.01mM sulfate, compared with 27 ppb/day with 5 mM sulfate. The0.01 mM sulfate cultures were used to inoculate tubes in anexperiment to test the effect of the sulfate concentration (Fig.3). The CFC-11 degradation rate was dependent on theconcentration of sulfate; in the absence of sulfate, no degra-dation was observed relative to sterilized controls (Fig. 3).

In separate experiments testing the effects of bromoethane-sulfonic acid (BES) or other carbon sources such as lactate,butyrate, or isopropanol, 1 ml of CFC-11-degrading cultureswas used to begin incubations. Positive controls having 10 mMacetate, 5 mM sulfate, and no BES degraded CFC-11 at 25ppb/day. When 2 mM BES was present, CFC-11 was degradedbelow detection limits (0.1 ppb) after 40 days. No methane hasbeen detected by gas chromatographic analysis in any of thetreatments (data not shown). Other than acetate, the onlycarbon source which supported CFC-11 degradation was bu-tyrate, although at a diminished rate of 10 ppb/day. The

1200'

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-- Expt 1. Hematin Effect --

butyrate-amended cultures have since been transferred repeat-edly into butyrate medium, and only after the fifth passage didthe CFC-11 degradation rate increase to that of the acetate-supported rate.

In correlation with the degradation of CFC-11, we observedthe production of a gas chromatographic peak with a retentiontime that was 0.15 min shorter than that of CFC-11. To betterseparate these related compounds, we used a packed columnto analyze the headspaces of these cultures. With this column,the unknown peak had the same gas chromatographic reten-tion time as fluorodichloromethane (HCFC-21 [retention time,8.7 min]), which was well separated from that of CFC-11(retention time, 11.3 min). In one experiment using acetate asthe primary carbon source, 3,780 ppb of CFC-1 1 was convertedby these enrichment cultures to 3,400 ppb of HCFC-21 asmeasured by headspace analysis. The identification of theHCFC-21 degradation product was verified by mass spectrom-etry.

Potential abiotic mechanisms of CFC-11 degradation. Lov-ley and Woodward (11) have observed CFC-11 to be degradedabiotically by hematin, even after autoclaving, and Lasage et al.(9) have reported that chlorotrifluoroethene is hydrolyzedabiotically by sulfide. When we incubated 930 ppb of CFC-11in the presence of oxidized or reduced hematin for 90 days,little CFC-11 degradation was observed compared with thebiotic rates discussed above (Fig. 4 [experiment 1]). Similarly,various concentrations of sodium sulfide did not affect CFC-1 1concentrations after 90 days (Fig. 4 [experiment 2]). In aseparate experiment, 97% of the initial 1,235 ppb of CFC-11was biodegraded in the presence of the aquifer enrichmentcultures after only 35 days (Fig. 4 [experiment 3]). When thesecultures were incubated with 1 mM sodium sulfide, a completeinhibition of CFC-11 biodegradation was observed (Fig. 4[experiment 3]). In other experiments, the inhibition ofCFC-11 biodegradation observed after approximately 25 dayswas correlated with the accumulation of hydrogen sulfide (overtime, hydrogen sulfide at a concentration greater than 0.5 mMwas detected in the headspace).

Groundwater sample microbial populations. The originalaquifer sample contained 9.2 x 105 total bacteria (by acridineorange direct counting) and 2.5 x 103 sulfate-reducing bacteria(by most probable number analysis) per ml. Microscopically,there were numerous morphotypes present in the aquifersample, including spirilla, vibrios, rods, and cocci (Fig. 5A).After 4 months of incubation and two amendments of CFC-11,the community was still quite diverse (Fig. 5B). As describedabove, the cultures were transferred into medium without

--Expt 2. Sulfide Effect _Expt 3. Inoculation / Sulfide Effect

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none Oxid Red 0 0.01 0.1 1mM -/OmM +/OmM +/1 mM

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FIG. 4. Experiments showing the effects of abiotic or biotic incubation conditions on Freon-11 concentrations. The effects of oxidized (Oxid)or reduced (Red) hematin (experiment 1 [Expt 1]), various sodium sulfide concentrations (experiment 2 [Expt 2]), and inoculation of 1 ml of theenrichment culture with and without (+/-) 1 mM sodium sulfide (experiment 3 [Expt 3]) were examined. Experiments 1 and 2 began with 930ppb of Freon-11, and the levels of Freon-11 shown in the figure are after 90 days of incubation. Experiment 3 began with 1,235 ppb of Freon-11,and the levels of Freon-11 shown are after 35 days of incubation.

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FIG. 5. Epifluorescence photomicrographs of aquifer microorganisms stained with 0.01% acridine orange. (A) The microorganisms in theaquifer sample within 24 h of collection; (B) the microbial community after 4 months of anaerobic incubation in the presence of Freon-11 and TCE(this sample corresponds to the last time point in Fig. 1); (C) the community after removal of nitrate and yeast extract from the medium (thissample corresponds to the treatment sample with acetate and without nitrate shown in Fig. 2); (D) after an additional passage in medium withoutnitrate or yeast extract, the community has become dominated by spirilla and large rod morphotypes.

nitrate or yeast extract but with acetate and sulfate. Thischange in the medium resulted in a population shift whichfavored the presence of spirillum- and rod-shaped bacteria(Fig. 5C). An additional transfer on this medium furtherenriched for the large rods and spirillum-shaped bacteria (Fig.5D), both of which were found to be gram negative. Spirillaand large rods can be seen in both the original and 4-month-incubated samples (Fig. 5A and B).

DISCUSSION

Water samples from a deep aquifer contaminated withCFC-11 and TCE contained many morphologically distinctbacteria (by acridine orange direct counting), some of whichwere sulfate reducers (by sulfate-reducing most probable num-ber analysis). CFC-11 degradation was observed within 50 daysof anaerobic incubation of this aquifer sample in the presenceof acetate and sulfate. A separate sample taken 8 weeks laterfrom the same monitoring well again showed acetate-depen-dent CFC-11 degradation. These results indicate that there aremicroorganisms present in this aquifer which are capable ofdechlorinating CFC-11.During incubation of this sample in the absence of an

external carbon source, CFC-11 degradation did not occur(18). It was determined that an external carbon source, specif-ically acetate or butyrate, was necessary for the dechlorinationof CFC-11 to HCFC-21. The butyrate-supported rates wereinitially one-half of the acetate-supported rates. Recently, afterfive passages of the enrichment culture on 10 mM butyrate, thedechlorination rates are now equal to the acetate-supportedrates. Interestingly, a carbon source utilized by many sulfate-reducing bacteria, lactate, did not support the CFC-11 dechlo-rination. The reaction also proceeded in the absence of nitrateand in the presence of 2 mM BES, respectively, ruling out theinvolvement of denitrifying and methanogenic bacteria. Thereaction's dependence on the sulfate concentration indicatesthat sulfate-reducing bacteria are responsible for the degrada-tion of CFC-11. The reaction is probably cometabolic, since itis dependent on both an electron donor (two or four carbonfatty acids) and an electron acceptor (sulfate).

Incubation conditions have decreased the diversity of themicrobial population. Specifically, we have observed the en-richment of two morphologically distinct bacteria: a spirillumand a large rod, both of which were evidently present in theoriginal sample. The spirillum-shaped organism may be (relat-ed to) Desulfovibrio baarsii, a spirillum-shaped sulfate reducerwhich utilizes acetate or butyrate as a carbon and energysource (22). The large rod may be Desulfotobacterpostgatei, anacetate-utilizing, sulfate-reducing large rod (22). Although it isnot likely that a consortium is required for the single dechlo-rination step of transforming CFC-11 to HCFC-21, repeatedattempts to isolate a single organism which degrades CFC-11have failed. We are presently using a combination of metabol-ic-enrichment and molecular techniques to isolate and identifythe causative agent.

Sulfide has been proposed to mediate the degradation of arelated fluorinated compound, chlorofluoroethene (9). Inthese experiments, sulfide is not the reductant responsible for

the CFC-11 dechlorination; in fact, sulfide actually proved tobe inhibitory to the process. When the medium was prere-duced with Na2S or when biologically produced H2S accumu-lated, it inhibited the CFC-11 degradation reaction. Lovley andWoodward (11) have reported that CFC-11 was consumedabiotically in the presence of reduced hematin. This abioticdechlorination mechanism is evidently not responsible for thereaction reported here, since neither autoclaved aquifer sam-ples nor reduced or oxidized hematin degraded CFC-11.However, our experiments are not directly comparable tothose described by Lovley and Woodward, because the latterused a higher concentration of a different reductant (0.1 Mcysteine, whereas we used 0.01 M Na2S).

Recently, groundwater-saturated sediment samples fromtwo different sites from the aquifer sampled in this study wereincubated anaerobically in the presence of CFC-11. After 6weeks, no degradation was observed, indicating that theseaquifer sediments were not abiotically consuming CFC-11.Finally, in a comparison of the activity reported here withthose of other abiotic mechanisms such as those mediated bycorinoid compounds, the near-stoichiometric product ob-served in this study was dichlorofluoromethane (HCFC-21),whereas in corrinoid-mediated CFC-11 degradation, the majorend product was carbon monoxide (8). We are presentlyinvestigating the mechanism of this potentially novel dechlori-nation step which is intimately associated with biologicalactivity (i.e., acetate oxidation and sulfate reduction).

This is the first report that aquifer sulfate-reducing bacteriaare able to dechlorinate CFC-11. The aquifer sample exhibitedthe ability to biotransform a CFC into HCFC-21, a hydrochlo-rofluorocarbon. The biodegradation of CFC-11 did not exhibita lag phase upon initial incubation, indicating that the micro-organisms indigenous to the contaminated aquifer possess theability to dechlorinate CFC-11 in the environment. The addi-tion of acetate has been shown to stimulate the degradation ofhalogenated aliphatic compounds, including CFC-11, in ashallow aquifer (17). These results indicate that stimulating thegrowth of the CFC-11 degraders in the aquifer through theaddition of an electron donor such as acetate could result inthe in situ dechlorination of a CFC to a hydrofluorocarbon,thus preventing potential atmospheric ozone destruction.

ACKNOWLEDGMENTS

We acknowledge the helpful discussions and provision of aquifersamples by Gregory Contaldo and Valda Terouds, Geosciences Con-sultants, Ltd., Albuquerque, N. Mex., the mass spectrometry analysesby Betty Hoffman, Allied Signal, Las Cruces, N. Mex., and thetechnical assistance of Angelica Duarte, particularly with the abioticdegradation work.

This work was supported in part by MBRS-NIH grant no.GM08136-20.

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