Chloroethene Biodegradation in Sediments at 4{degrees}C

Microbial reductive dechlorination of [1,2-¹⁴C]trichloroetheneto [¹⁴C]cis-dichloroethene and [¹⁴C]vinyl chloride was observedat 4°C in anoxic microcosms prepared with cold temperature-adaptedaquifer and river sediments from Alaska. Microbial anaerobicoxidation of [1,2-¹⁴C]cis-dichloroethene and [1,2-¹⁴C]vinylchloride to ¹⁴CO₂ also was observed under these conditions.

INTRODUCTION

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Introduction

Understanding the effects of low temperature on contaminantbiodegradation is fundamental to the application of bioremediationtechnologies in low-temperature (<5°C), surface water,and groundwater systems. Water temperatures of 5°C or lessare, based largely on the characteristic inhibition of microbialcultures exposed to suboptimal growth temperatures, widely expectedto yield low to insignificant rates of contaminant biodegradation(11). Nevertheless, recent investigations have reported activebiodegradation of petroleum hydrocarbons (4, 8-10) and chlorophenol(1, 2) at temperatures of circa 5°C. The potential biodegradationof the widespread chloroethene contaminants at temperaturesbelow 5°C has not been directly assessed. In this study,the potential for reductive and oxidative microbial degradationof trichloroethene (TCE), cis-dichloroethene (DCE), and vinylchloride (VC) was examined at 4°C in cold-adapted sedimentsfrom Alaska.

Chloroethene biodegradation in sediments from chloroethene contaminatedsites in Soldotna, Alaska, and Fairbanks, Alaska, was investigated.Soldotna aquifer sediments (SO-AQ) were collected from an areaof prior electron donor injection and bioaugmentation and consistedof alluvial sands, gravels, and cobbles. The presence of significantconcentrations of dissolved CH₄ (1.7 mM), Mn(II) (255 µM),and Fe(II) (1.4 mM), detectable concentrations of SO₄ (<5µM) and sulfide (<2 µM), dissolved H₂ concentrations(5.6 nM) above the reported threshold for methanogenesis, andno detectable O₂ (minimum detection limit [MDL], <2 µM)or NO₃ (MDL, <2 µM) indicated that mixed anaerobicconditions predominated in these sediments. Surface water sediments(SO-Kenai) were collected from the Kenai River at the down-gradientend of the chloroethene plume and consisted of alluvial silts,sands, gravels, and cobbles. The sediment interstitial waterand the overlying water column contained greater than 94 µM(3 mg/liter) and 313 µM (10 mg/liter) dissolved O₂, respectively.The annual mean groundwater temperature at the Soldotna sitewas 5 to 7°C. Fairbanks aquifer sediments (FB-AQ1 and FB-AQ2),composed of silts, sands, and gravels, were collected from up-gradientand down-gradient portions, respectively, of a TCE plume. Atthe time of sample collection, FB-AQ1 contained 96 M (3.0 mg/liter)dissolved O₂, while FB-AQ2 appeared to be anoxic/metal-reducing,as indicated by significant concentrations of dissolved Mn(II)(7 µM) and Fe(II) (100 µM), H₂ concentrations below0.8 nM and no evidence of dissolved O₂, NO₃^– reduction,SO₄^2– reduction, or methanogenesis. The annual mean groundwatertemperature at the Fairbanks site was 3 to 5°C.

Chloroethene biodegradation was investigated by using [1,2-¹⁴C]TCE (5.4 µCi/µmol; Sigma Chemical Co., St. Louis,Missouri), [1,2-¹⁴C]cis-DCE (4.0 µCi/µmol; MoravekBiochemicals, Brea, California) and [1,2-¹⁴C]VC (1.6 µCi/µmol;Perkin-Elmer Life Sciences, Boston, Massachusetts). Radiochemicalpurities were greater than 98%, 98%, and 99%, respectively.Microcosms were prepared under a nitrogen atmosphere and werecomposed of 10-ml serum vials with 10 ± 0.5 g of saturatedsediment. Quadruplicate experimental microcosms, triplicateautoclaved control microcosms, and a single sediment-free controlmicrocosm were prepared for each treatment. Microcosms wereamended with [1,2-¹⁴C]TCE, [1,2-¹⁴C]DCE, or [1,2-¹⁴C]VC to yieldinitial dissolved substrate concentrations of 2, 2, and 0.6ppm TCE, cis-DCE, and VC, respectively, and were incubated inthe dark at 4°C for 200 days. Headspace concentrations of¹⁴C-substrates and ¹⁴C-products, including [¹⁴C]ethene and [¹⁴C]ethane,were assessed at the outset and at approximately 50, 100, and200 days by radiometric detection gas chromatography (5-7).Anoxic conditions were confirmed throughout the study by headspacethermal conductivity detection gas chromatography. Dissolved-phaseconcentrations of ¹⁴C-analytes were estimated based on experimentallydetermined Henry's partition coefficients as described in detailpreviously (5-7). Sorbed-phase concentrations of ¹⁴C-analyteswere estimated based on experimentally determined sorption coefficientsas described in detail previously (6) and were found to be lessthan 1% of the total microcosm ¹⁴C recovery. Time series recoverydata for SO-AQ and FB-AQ1 microcosms amended with [¹⁴C]TCE or[¹⁴C]VC are presented (Fig. 1 and 2). For brevity, summary resultsfor all treatments are presented as final percentages of ¹⁴Crecoveries (Tables 1 and 2). Dissolved concentrations of NO₃^–and SO₄^2– were determined by ion chromatography, and dissolvedconcentrations of Mn(II) and Fe(II) were determined colorimetrically(6, 7).

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FIG. 1. Percentages of recovery of ¹⁴C-radiolabeled products [¹⁴C]TCE ( ${blacksquare}$ ), [¹⁴C]DCE ( ${blacktriangleup}$ ), [¹⁴C]VC ( ${blacktriangledown}$ ), and ¹⁴CO₂ (•) in SO-AQ microcosms amended with [1,2-¹⁴C]TCE (A) or [1,2-¹⁴C]VC (B). Data are means ± standard deviations for quadruplicate experimental microcosms. Corresponding open symbols represent means ± standard deviations for triplicate autoclaved control microcosms. Only the initial ¹⁴C-substrate was observed in control microcosms.

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FIG. 2. Percentages of recovery of ¹⁴C-radiolabeled products [¹⁴C]TCE ( ${blacksquare}$ ), [¹⁴C]DCE ( ${blacktriangleup}$ ), [¹⁴C]VC ( ${blacktriangledown}$ ), and ¹⁴CO₂ (•) in FB-AQ1 microcosms amended with [1,2-¹⁴C]TCE (A) or [1,2-¹⁴C]VC (B). Data are means ± standard deviations for quadruplicate experimental microcosms. Corresponding open symbols represent means ± standard deviations for triplicate autoclaved control microcosms. Only the initial ¹⁴C-substrate was observed in control microcosms.

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TABLE 1. Final percentages of distribution of ¹⁴C-radiolabeled products in Soldotna microcosms^a

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TABLE 2. Final percentages of distribution of ¹⁴C-radiolabeled products in Fairbanks microcosms^a

Reductive dechlorination at 4°C.

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Reductive dechlorination at...

Substantial reductive dechlorination of [¹⁴C]TCE was observedat 4°C under favorable redox conditions. Microbial reductivedechlorination of [1,2-¹⁴C]TCE to [¹⁴C]cis-DCE was apparentby day 50 in SO-AQ microcosms and evidence of subsequent reductivedechlorination of [¹⁴C]cis-DCE to [¹⁴C]VC was detected by day108 (Fig. 1A). In total, approximately 80% reductive dechlorinationof [1,2-¹⁴C]TCE to [¹⁴C]DCE and [¹⁴C]VC was observed in SO-AQmicrocosms after 200 days incubation at 4°C (Fig. 1A; Table1). The lack of dissolved O₂ ([O₂], <2 µM), NO₃ ([NO₃^–],<1.0 µM), and SO₄ ([SO₄^2–], <20 µM),the lack of production of dissolved sulfide (not detected; [HS^–],<0.2 µM), and the substantial accumulations of dissolvedMn(II) (160 ± 13 nmol), dissolved Fe(II) (930 ±100 nmol), and dissolved CH₄ (780 ± 510 nmol) indicatedthat mixed metal-reducing and methanogenic conditions predominatedin this sediment, regardless of the ¹⁴C-substrate. None of thepotential nonchlorinated degradation products ([¹⁴C]ethene,[¹⁴C]ethane, ¹⁴CO₂, and ¹⁴CH₄) were observed. [1,2-¹⁴C]TCE lossand ¹⁴C-product accumulation were not detected in autoclavedcontrol or sediment-free control microcosms. Limited reductivebiodegradation of [1,2-¹⁴C]TCE also was observed in SO-Kenaimicrocosms, in spite of the relatively oxidized character ofthis sediment. The lack of dissolved O₂ ([O₂], <2 µM),NO₃ ([NO₃^–], <1.0 µM), and SO₄ ([SO₄ ^2–],<20 µM), the lack of production of dissolved sulfide(not detected; [HS^–], <0.2 µM) and CH₄ (not detected;[CH₄], <1-µmol/liter headspace) and the substantialaccumulations of dissolved Mn(II) (15 ± 3 nmol) and dissolvedFe(II) (120 ± 11 nmol) indicated that metal-reducingconditions predominated in SO-Kenai microcosms. Transient productionof trace amounts of [¹⁴C]cis-DCE was detected in SO-Kenai microcosmsat day 108 (data not shown). The final loss of [1,2-¹⁴C]TCEin SO-Kenai microcosms was less than 10% and the sole productof [¹⁴C]TCE degradation that was detected at 200 days was [¹⁴C]VC.No [1,2-¹⁴C]TCE loss or ¹⁴C-product accumulation was observedin autoclaved control or sediment-free control microcosms. Nobiodegradation of [1,2-¹⁴C]TCE was observed in FB-AQ microcosms(Fig. 2A; Table 2). The lack of dissolved O₂ ([O₂], <2 µM)and NO₃ ([NO₃^–], <1.0 µM), the lack of changein dissolved SO₄^2– concentrations, the lack of dissolvedsulfide (not detected, [HS^–], <0.2 –M) or CH₄(not detected; [CH₄], <1-µmol/liter headspace), andthe substantial accumulations of dissolved Mn(II) (56 ±22 and 11 ± 7 nmol, respectively) and dissolved Fe(II)(87 ± 33 and 11 ± 3 nmol, respectively) indicatedthat Mn/Fe-reducing conditions predominated in FB-AQ treatments,regardless of the ¹⁴C-substrate. The mean loss of [1,2-¹⁴C]TCEwas less than 3% for both sediments. Inefficient reductive dechlorinationof TCE under oxidized conditions is consistent with numerousreports (for a review, see reference 3). The results of the[1,2-¹⁴C]TCE biodegradation study demonstrated that microbialreductive dechlorination of chloroethenes can be important attemperatures less than 5°C.

Anaerobic oxidation at 4°C.

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Anaerobic oxidation at...

Approximately 40% and 70% removals of [1,2-¹⁴C]cis-DCE and [1,2-¹⁴C]VCwere observed in Kenai River sediment microcosms after 200 daysat 4°C (Table 1). For both substrates, ¹⁴CO₂ accumulationwas apparent in SO-Kenai microcosms by 61 days (data not shown).The sole product of [1,2-¹⁴C]cis-DCE and [1,2-¹⁴C]VC biodegradationthat was detected in these treatments was ¹⁴CO₂. No [¹⁴C]chloroetheneloss or ¹⁴C-product accumulation was observed in autoclavedcontrol or sediment-free control microcosms. Likewise, substantialbiodegradation of [1,2-¹⁴C]cis-DCE and [1,2-¹⁴C]VC at 4°Cin FB-AQ microcosms was observed (Fig. 2B; Table 2). Approximately25% and 70% removals of [1,2-¹⁴C]cis-DCE and [1,2-¹⁴C]VC wereobserved in FB-AQ-1 sediment microcosms, respectively. Approximately30% and 20% removals of [1,2-¹⁴C]cis-DCE and [1,2-¹⁴C]VC wasobserved in FB-AQ-2 sediment microcosms, respectively. In bothsediments, degradation of [1,2-¹⁴C]cis-DCE and [1,2-¹⁴C]VC wasapparent by day 61 (Fig. 2B) and was stoichiometric to ¹⁴CO₂(Fig. 2B; Table 2). The degradation of [1,2-¹⁴C]cis-DCE and[1,2-¹⁴C]VC observed in SO-Kenai and Fairbanks treatments wasattributable to biological activity because no detectable ¹⁴C-substrateloss or ¹⁴C-product accumulation was observed in autoclavedcontrol or sediment-free control microcosms. The fact that mineralizationof [1,2-¹⁴C]DCE and [1,2-¹⁴C]VC to ¹⁴CO₂ also was observed inSO-AQ microcosms indicated that a distinct potential for anaerobicmicrobial oxidation of cis-DCE and VC remained in these sedimentseven after in situ electron donor injection and the onset ofactive methanogenesis (Fig. 1B; Table 1). Interestingly, accumulationof ¹⁴CO₂ was not observed in [¹⁴C]TCE-amended Soldotna microcosmsdespite the accumulation of [¹⁴C]DCE and [¹⁴C]VC in these treatments.This result suggests that the presence of TCE may have suppressedthe anaerobic oxidation of DCE and VC under these conditions.The net anaerobic oxidation of DCE and VC observed in Soldotnaand Fairbanks microcosms is consistent with reports of microbialchloroethene oxidation in Mn(IV)/Fe(III)-reducing sedimentsunder temperate conditions (for a review, see reference 3) anddemonstrates that anaerobic oxidation of chloroethene contaminantscan be important at temperatures below 5°C.

Concluding remarks.

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Concluding remarks.

Although the inhibitory effect of suboptimal temperatures onmicrobial activity is well established, the a priori assumptionof low to insignificant microbial activity at water temperaturesless than 5°C does not consider the possible presence ofcold-adapted (psychrotolerant and psychrophilic) microorganisms.The potential for microbial degradation of chloroethene contaminantsis well known in temperate groundwater and surface water systems(3). The results of this study demonstrate that chloroethenebiodegradation via reductive or oxidative mechanisms also canbe significant at temperatures less than 5°C. These resultsextend the growing list of environmental contaminants demonstratedto be susceptible to microbial degradation at temperatures lessthan 5°C and suggest that bioremediation strategies forchloroethene-contaminated groundwater are feasible in cold-temperatureenvironments.

ACKNOWLEDGMENTS

We thank D. Bauer, J. Lindstrom, T. McDougall, J. Paris, andR. Sundet for sediment collection and critical evaluation ofthe research.

This research was supported by the Alaska Department of EnvironmentalConservation and the U.S. Geological Survey Toxic SubstancesHydrology Program.

FOOTNOTES

* Corresponding author. Mailing address: U.S. Geological Survey, 720 Gracern Rd., Suite 129, Columbia, SC 29210. Phone: (803) 750-6125. Fax: (803) 750-6181. E-mail: pbradley{at}usgs.gov.

REFERENCES

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