Environ. Sci. Technol. 2005, 39, 9286-9294
Reductive Biotransformation of Tetrachloroethene to Ethene during Anaerobic Degradation of Toluene: Experimental Evidence and Kinetics H A I S H E N † A N D G U Y W . S E W E L L * ,‡ National Research Council/Ground Water and Ecosystems Restoration Division, U.S. EPA Robert S. Kerr Environmental Research Center, Ada, Oklahoma 74820, and Department of Environmental Health Sciences, East Central University, Ada, Oklahoma 74820
Reductive biotransformation of tetrachloroethene (PCE) to ethene occurred during anaerobic degradation of toluene in an enrichment culture. Ethene was detected as a dominant daughter product of PCE dechlorination with negligible accumulation of other partially chlorinated ethenes. PCE dechlorination was linked to toluene degradation, as evidenced by the findings that PCE dechlorination was limited in the absence of toluene but was restored with a spike of toluene again in the cultures. PCE was effectively dechlorinated in cultures amended with a wide range of concentrations of PCE and toluene. PCE dechlorination can be described by a Monod-like equation but followed a zero-order kinetic at high levels of PCE. In addition to toluene, benzoate and lactate were also able to be used as sole electron donors for reductive dechlorination of PCE in the cultures. In terms of dechlorination rates, lactate was the best electron donor followed by benzoate and then toluene. The kinetic characteristics of PCE dechlorination were retained in the cultures regardless of electron donors used, but the kinetic constant values were unique to each electron donor. The dechlorination rate was found to be closely correlated with the level of H2 produced during fermentation of the three organic compounds. Nitrate and sulfate were observed to be favorable electron acceptors in this culture, and their presence completely blocked electron flow to PCE. However, the presence of nitrate and sulfate did not destroy the capability of PCE dechlorination by the culture. PCE dechlorination was immediately reestablished after depletion of nitrate and sulfate in the culture. This anaerobic process provides an opportunity for concurrent remediation of chlorinated solvents and certain fuel hydrocarbons, and recognition of this process is also important in understanding the subsurface fate and transport of these contaminants under natural conditions.
Introduction Contamination of the subsurface by chlorinated aliphatic solvents and petroleum hydrocarbons is a major public health * Corresponding author phone: (580)310-5547; fax: (580)310-5606; e-mail:
[email protected]. † U.S. EPA Robert S. Kerr Environmental Research Center. ‡ East Central University. 9286
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concern. Hundreds of sites where groundwater is contaminated with chlorinated solvents have been identified, and biotic and abiotic techniques have been widely examined and explored to cleanup these pollutants (1). Bioremediation, due to its low cost and effectiveness, has emerged as one of the active approaches for in situ treatment of the subsurface contamination (1, 2). Biologically mediated oxidation and cometabolism of trichloroethene (TCE), dichloroethenes (DCEs), and vinyl chloride (VC) under aerobic conditions have been well studied (2). It has been documented that anaerobic oxidation of VC can occur during Fe(III)-reducing conditions (3). However, tetrachloroethene (PCE) is resistant to microbial oxidation, and anaerobic processes are required for PCE dechlorination through reductive mechanisms (4). Anaerobic reductive transformation of PCE to less chlorinated ethenes has been intensively studied using soil microcosms, enrichment cultures, and pure culture (5-19). However, some of the observed reductive biotransformations of PCE stopped at cis-DCE and VC with accumulation of these partially halogenated daughter products. The incomplete dechlorination is not considered beneficial in the environment because cis-DCE and VC are still regulated compounds and VC is a proven human carcinogen, and thus further degradation of cis-DCE and VC is desirable. Complete reductive dechlorination of PCE to an environmentally acceptable compound such as ethene or ethane has been achieved using alcohols, fatty acids, and hydrogen as electron donors (1, 6, 11, 15, 16, 18, 19). Although complete sequential dechlorination of PCE to ethene was generally associated with the Dehalococcoides groups (15), mixed cultures that contained at least two species also demonstrated the capability of complete dechlorination of PCE to ethene (19). Contamination by mixtures of chlorinated solvents and petroleum hydrocarbons has been repeatedly detected at contaminated sites, and increasing efforts have been devoted to development of remedial processes to clean up these contaminants concurrently (4, 7, 8, 12). Because of limitations of delivering molecular oxygen in the subsurface, anaerobic microbial processes have been considered as an efficient remedy for degradation of fuel contamination in the subsurface environment (2). The recognition that various subsurface microbial populations may use fuel hydrocarbons as electron donors and chlorinated solvents as electron acceptors suggests the possibility of linking microbial degradation of fuel compounds to reductive dechlorination of PCE, so that concurrent bioremediation of the contaminant mixtures can be achieved through the same anaerobic process. Microcosm studies already showed that anaerobic consortia are capable of oxidizing toluene with transport of electrons to PCE for reductive dechlorination (7). However, only incomplete dechlorination occurred in the previous study, and significant accumulations of TCE and DCEs were observed as the transformation daughter products in the microcosms. In this study, we report that after adaptation by continuously exposing the dechlorination cultures to both toluene and PCE, an enrichment culture capable of completely dechlorinating PCE to ethene with accumulation of negligible amounts of other daughter products was obtained. This article presents the experimental results and kinetic analysis to demonstrate the complete dechlorination of PCE by the enrichment culture.
Materials and Methods Enrichment Culture. The enrichment culture for PCE dechlorination was developed by Sewell and Gibson from aquifer solids collected from an area that was exposed to 10.1021/es050390v CCC: $30.25
2005 American Chemical Society Published on Web 10/21/2005
both alkylbenzenes and chlorinated ethenes at the U.S. Coast Guard Air Station in Traverse City, MI (7). Toluene was used as the sole carbon source to sustain PCE dechlorination. The enrichment contained no solids and was maintained in 2-L bottles with routine feeding of toluene (100 µM) and PCE (40 µM). The biomass was estimated at 7 g protein per liter. The bottles were fully filled with freshly prepared sterile liquid medium using sterile pipets and were incubated in an anaerobic chamber at 23 °C in the dark. The medium was prepared with Byrd’s Mill spring water (Ada, OK) diluted with distilled water (1:1) as described before (7). The medium was supplemented with 10 mM (NH4)2HPO4. Resazurin was added at a final concentration of 0.0001% as a redox indicator, and then the pH was adjusted to 7 with 1 M solutions of NaOH or HCl. The medium was autoclaved at 121 °C for 40 min and then placed in the anaerobic glovebox to cool after purging with a gas mixture of the chamber atmosphere (98% N2/2% H2). The fully filled bottles and warm liquid minimizes the transfer of hydrogen from the chamber gases in the container. Sodium sulfide was added at a final concentration of 1 mM to maintain reducing conditions prior to use of the medium. Batch Experiments. The batch experiments were conducted in duplicate to evaluate reductive dechlorination of PCE by the enrichment cultures with toluene, benzoate, and lactate as sole electron donors. The enrichment was withdrawn from the source bottles and mixed with the fresh medium at the ratio of 1:4 in the anaerobic chamber. The diluted mixture culture (120 mL) was delivered to 160 mL serum bottles and sealed with Teflon-coated butyl rubber septa and aluminum crimp caps to maintain anaerobic conditions. Then the serum bottles were moved outside of the anaerobic chamber, and each bottle was purged for 20 min to remove residual volatile compounds in the bottles using nitrogen gas (99.999%) that had been passed through a column of hot reduced copper filings to remove trace oxygen. After sitting overnight to ensure no residue of trace molecular oxygen in the bottles, the cultures were spiked with PCE and toluene using syringes to demonstrate PCE dechlorination and toluene degradation. The syringes were flushed with the sterilized nitrogen gas three times before dosing the compounds. To evaluate other organic compounds as sole electron donors for PCE dechlorination, benzoate and lactate, along with PCE, were added to the cultures using the same procedures as described before. To determine the influences of other electron acceptors on PCE dechlorination, nitrate and sulfate were added together with PCE and toluene. Benzene, due to its resistance to microbial attack by this culture, was added as an inert reference to make sure no leaking of volatile compounds from the serum bottles occurred during the incubation period. The neat PCE, toluene and benzene, and stock solutions (1.0 M each) of benzoate, lactate, nitrate, and sulfate were used for the spikes according to the various amounts indicated in the figures to be discussed later. All bottles were also protected from light and were incubated outside the anaerobic chamber at 23 °C with liquid phase in contact with the septum to minimize losses of volatile compounds. Abiotic controls were prepared following the same procedure but were not inoculated with the active cultures. Analytical Methods. A gas chromatography system (Shimadzu GC-14a, Dallas, TX) equipped with a flame ionization detector was used to analyze volatile organic compounds by injecting 100 µL of headspace gas to each column. A 30-m capillary column with 0.32 mm i.d. and 1.5 µm film thickness (Restek, Bellefonte, TX) was employed to separate benzene, toluene, TCE, cis-DCE, trans-DCE, and PCE. The oven temperature was programmed to start at 40 °C with a linear increase at a rate of 20 °C per minute to 140 °C at which it was held for 4 min. To quantify methane, ETH, VC, and
ethane, a 2.1-m glass column (2.6 mm i.d. and 5 mm o.d.) packed with 80/100 mesh Porapac Q (Shimadzu, Dallas, TX) was used. The oven temperature started at 55 °C and was linearly increased to 140 °C at a rate of 20 °C per minute. After holding for 3 min at 140 °C, the temperature was further increased to 200 °C at a rate of 40 °C per minute. The injector and detector temperatures were constantly maintained at 200 and 230 °C, respectively. Helium was supplied as the carrier gas for both columns, while hydrogen and air were used for FID operation with flow rates at 29 and 250 mL/ min, respectively. The method detection limit for each compound was 0.01 µM in the liquid phase. Hydrogen was determined using a reduction gas analyzer equipped with a reduction gas detector (RGA3, Trace Analytical, Menlo Park, CA). A 2.0 mL sample of headspace gas from each bottle was introduced into a 3 m × 32 mm i.d. column, and hydrogen was separated from other reducing gases at 100 °C. The nitrogen carrier gas (99.999%), purified through a heated metallic oxide to remove any trace of reducing gas before entering the column, passed through the column at a rate of 20 mL per minute. The detection limit for hydrogen was 0.4 nM. Benzoate was determined using a thermo separation capillary electrophoresis (CE) unit equipped with a UV detector and a 70 cm × 75 µm i.d. capillary cassette (Spectra Phoresis 500, San Jose, CA). The supernatant of liquid samples was analyzed following centrifugation at 5000g for 15 min. Separation of benzoate from other compounds was performed at 30 °C by applying a negative voltage of 25 kV between platinum and gold electrode contacts according to the procedure developed previously (20). A sodium sulfate (5.0 mM) and tetradecyl trimethylammonium bromide (0.5 mM) solution was employed as the carrier electrolyte. After separation, benzoate was determined at the wavelength of 220 nm; the quantification limit was 4 µM. Sulfate, nitrate, and nitrite were analyzed using the same CE unit with a similar capillary cassette. The separation was conducted at 45 °C and -25 kV using a carrier electrolyte containing 3 mM pyromellitic acid adjusted to pH 7.0 with tetramethylethylenediamine. The quantification of nitrate, nitrite, and sulfate was performed at the wavelength of 254 nm. Separation and quantification of lactic, acetic, propionic, formic, and butyric acids were carried out using a Dionex DX 500 ion chromatography (IC) system equipped with a suppressed conductivity detector and an anion micromembrane suppressor for ion chromatography exclusion. A HPICE AS-1 column (Dionex) was employed for separation with 1.0 mM heptafluorobutyric acid as mobile phase and 5.0 mM tetrabutylammonium hydroxide as regenerant for the suppressor. The temperature was maintained at 30 °C, and the flow rate was set at 0.8 and 5.0 mL per minute for the mobile phase and the regenerant, respectively. The lowest detectable concentration for lactic acid and other carboxylic acids was approximately 0.5 µM. Protein analysis was conducted with a Protein Assay Reagent Kit which uses bicinchoninic acid as a reagent and bovine serum albumin as a standard (Pierce, Rockford, IL). The detection limit of this method was about 6 mg/L. Kinetics of PCE Dechlorination. The rate of PCE dechlorination in a reductive process may be described by a Monodlike equation as follows:
kmP dP ) X dt Kp + P
-
(1)
where P (µmol) is the amount of PCE in the bottle at the incubation day of t (d), km [µmol PCE (g protein)-1 d-1] and Kp (µmol) are the maximum specific dechlorination rate and the half-saturation constants, respectively. The variable X (g protein) is the biomass amount in the bottle and may be VOL. 39, NO. 23, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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described by a linear equation in a diluted culture,
X ) Y(P0 - P) + X0
(2)
where P0 and X0 are the initial amounts of PCE and biomass in the bottle, respectively, and Y (g protein/µmol PCE) is the yield coefficient which indicates the ratio of the increased amount of dechlorinating microorganisms to the amount of PCE consumed. After replacing X with eq 2, eq 1 becomes:
-
kmP dP ) [Y(P0 - P) + X0] dt Kp + P
(3)
Equation 3 can be integrated to give the following analytical solution:
t)
[
(YP0 - YP + X0)P0 Kp 1 ln + km YP0 + X0 PX0 1 YP0 - YP + X0 (4) ln Y X0
]
Equation 4 illustrates the time course of reductive dechlorination of PCE in which the rate-limiting factor is the PCE amount. To determine the values of kinetic parameters in eq 4, the Marquardt algorithm of nonlinear regression analysis was used. Trial values of the parameter values (km and Kp) were initially estimated, and the equation was solved by minimizing the residual sum of squares (RSS) between the dependent variable (t) in eq 4 and the experimental data in Figure 3. Weight variable, as the reciprocal of the dependent variable (1/t), was employed to homogenize error variance. Constraints were also enforced to set positive parameter values so that nonsensical or invalid parameter data were omitted.
Results PCE Dechlorination and Toluene Degradation. When toluene was added as a sole carbon source in the batch cultures, PCE losses were observed with concurrent disappearance of toluene (Figure 1). PCE dechlorination became much slower during days 46-89 following depletion of toluene in the bottles. A respike of toluene at day 94 immediately restored the rate of PCE dechlorination in the cultures, and toluene was continuously degraded almost at the same rate throughout the incubation. Coupling to the PCE loss, ethene was detected along with partially chlorinated transformation daughter products including TCE, cis-DCE, and VC. However, TCE, cis-DCE, and VC vanished eventually after reaching a peak at day 40, and then ethene accumulated in the cultures as the sole product of PCE transformation. PCE dechlorination continued following respike of PCE, and ethene production leveled off until complete consumption of added PCE at day 140. Methane was detected in the cultures, but no ethane was observed. Methane production was negligible following an initial rise, and even the respike of toluene stimulated insignificant production of methane. During the same period of incubation, abiotic controls only showed limited decreases in the amounts of PCE and toluene, and no detection of PCE transformation daughter products or methane. The characteristics of PCE dechlorination linked to toluene degradation were further investigated using different levels of toluene (8, 15, and 37 µmol/bottle) and PCE. All three levels of toluene amendments were effective in sustaining PCE dechlorination (Figure 2), and PCE dechlorination proceeded nearly at the same initial rate at all three levels, and so did ethene production. However, when toluene was totally depleted at day 45 in the cultures receiving the lowest level of toluene (8 µmol/bottle), the rates of PCE loss and 9288
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FIGURE 1. PCE dechlorination (A), toluene degradation (B), and formation of daughter products (TCE, cis-DCE, VC, and ethene) (C) in the active cultures and abiotic controls. Toluene and PCE were readded at day 94. The data are means of duplicate batch tests, and the error bars indicate the relative errors. ethene production started to decrease. Methane production demonstrated a small initial increase with the increased toluene levels. In addition, the cultures receiving the highest level of toluene (37 µmol/bottle) showed a significant increase in methane production following completion of dechlorination after day 59 as evidenced by the termination of ethene production. The sharp increase in methane production occurred concurrently with rapid degradation of toluene, indicating the predominance of methanogenesis in the culture after PCE depletion. Despite little impact on PCE dechlorination by the high level of toluene, toluene degradation was obviously inhibited by a high level of PCE. Figure 3 shows that the rate of toluene degradation decreased with the increase in PCE concentrations from 17 to 25 and then 37 µmol/bottle, and the rate of toluene degradation increased significantly after PCE was reduced below approximately 20 µmol/bottle. PCE losses appeared independent of PCE concentrations, but the high level of PCE delayed the appearance of ethene although significant production of ethene was observed subsequently. Large amounts of VC were also detected in the culture receiving the highest level of PCE, but the accumulated VC was eventually transformed to ethene following depletion of
FIGURE 2. PCE dechlorination (A), toluene degradation (B), and formation of daughter products (C) in the active cultures amended with initial toluene (µmol/bottle) at 8 (O,b), 15 (3,1), and 37 (],[). The data are means of duplicate batch tests. PCE. Methane production virtually ceased during PCE dechlorination, and only an initial jump was repeatedly observed under all three conditions. The culture receiving the medial PCE concentration exclusively exhibited a further increase in methane production, but that increase occurred only following depletion of PCE and respike of toluene at day 125. PCE Dechlorination during Degradation of Other Electron Donors. PCE dechlorination in the cultures amended with lactate, benzoate, and toluene as the sole electron donor is compared in Figure 4. Figure 4a shows PCE dechlorination with formation of daughter products; Figure 4b shows degradation of lactate, benzoate, and toluene; and Figure 4c shows variations of H2 and methane concentrations during the incubation period. Despite amendments with different electron donors, all cultures demonstrated immediate PCE dechlorination coupling with formation of daughter products among which ethene was again the final compound with a brief appearance of VC (Figure 4a). As evaluated by PCE dechlorination rates, lactate was the best electron donor followed by benzoate and then toluene although the culture was only exposed to toluene previously. Concurrently with PCE dechlorination, the removal of lactate, benzoate, and
FIGURE 3. PCE dechlorination (A), toluene degradation (B), and formation of daughter products (C) in the active cultures amended with initial PCE (µmol/bottle) at 17 (O,b), 25 (3,1), and 37 (],[). VC (9) was detected in the cultures with initial PCE at 37 µmol/ bottle. The data are means of duplicate batch tests. toluene was also observed in the active cultures (Figure 4b). Benzoate was degraded almost at the same rate as toluene, while lactate degradation was coupled to formation of acetate and propionate as intermediates. The two intermediates appeared and then were further degraded in the cultures amended with lactate. During the same period of incubation, abiotic controls showed little or no reduction in the amounts of lactate, benzoate, and toluene. Although all of the three compounds showed the ability to drive reductive dechlorination of PCE, different patterns were observed in the production of methane and H2 (Figure 4c). No significant amounts of methane were produced in the cultures amended with toluene. However, the culture with addition of benzoate resulted in a continuous accumulation of methane, while the culture amended with lactate showed the highest production of methane. The level of H2 also demonstrated diverse patterns in the cultures receiving the three different electron donors. Toluene as the electron donor maintained the lowest level of H2 with most measured H2 values below 1 nmol/bottle in the cultures. A steady level of H2 was observed in the cultures amended with benzoate, and the value was maintained at about 2 nmol/bottle. The cultures amended with lactate, however, VOL. 39, NO. 23, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 5. The analysis of electron flows for PCE dechlorination and methane production under the assumptions of complete oxidation of toluene, benzoate, and lactate to carbon dioxide.
FIGURE 4. PCE dechlorination with formation of daughter products (A), substrate degradation with formation of intermediates (B), and production of H2 and methane (C), in the active cultures amended with toluene, benzoate, and lactate as the electron donors. The abiotic controls contain PCE, toluene, benzoate, and lactate. The data are means of duplicate batch tests. 9290
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accumulated a substantial amount of H2 with the value immediately over 20 nmol/bottle. Despite observation of decrease in the H2 level following elimination of lactate after day 35, the lactate-amended culture still retained a much higher H2 concentration compared with the cultures amended with the two aromatic compounds. Mass and Electron Balance Analyses. The mass balance between PCE and its transformation daughter products during degradation of toluene, benzoate, and lactate was analyzed using the data reported in Figures 1-4. The mass balance analysis observed that PCE losses, after deduction of abiotic decreases, were recovered as the transformation daughter products (ETH, VC, and cis-DCE) at average percentages of 86 ( 15, 92 ( 18, and 87 ( 22% (average ( standard deviations), respectively, in the active cultures amended with toluene, benzoate, and lactate. However, the electron balance analysis showed different patterns of electron flow with the degradation of different electron donors (Figure 5). To perform the electron flow analysis, a complete oxidation of electron donors to carbon dioxide was assumed in all of the cultures amended with toluene, benzoate, and lactate. Toluene, among the tested three electron donors, demonstrated the highest capability of delivering electrons to PCE, and 12 ( 6% of the consumed toluene was observed to be used for the role of PCE dechlorination. Methane production in this system accounted for another 14 ( 5% of the consumed toluene, and the unidentified number was 74%. In the benzoate and lactate amended systems, the percentages of consumed electron donors that were used for methane production jumped to 45 ( 7 and 44 ( 21%, respectively, while the numbers that were used for PCE dechlorination reduced to 7 ( 1 and 6 ( 3%, respectively. The unexplained electron flow during consumption of both benzoate and lactate was around 50% each. The residual H2, due to its very low levels, would only provide insignificant portions of the electron flow in all the systems amended with different electron donors. The biomass and other fermentative products appear to be the other major sinks of the unexplained transport electrons, but no experimental data were available for further quantitative calculations. However, GC/MS analysis reveals that there were detectable amounts of aliphatic acids and aromatic compounds including propanoic acid, trimethylacetic acid, pentanoic acid, butyric acid, hexanoic acid, 2-ethylhexanoic acid, benzoic acid, phenol, o-cresol, p-cresol, m-cresol, and decanoic acid in the active cultures amended with PCE and toluene. Kinetic Analysis. Equation 4, along with the parameter values in Table 1, was used to simulate PCE losses in the batch cultures, and the statistical analysis of simulation results was provided in Table 2. Figure 1a shows that the model is
TABLE 1. Output of Parameter Values by Fitting Eq 4 to Experimental Data Using Nonlinear Regression Analysisa parameter carbon source
km, µmol PCE (g protein)-1 d-1
Kp, µmol PCE
data source
toluene lactate benzoate
1.23 ( 0.06 4.04 ( 2.07 1.85 ( 0.13
0.86 ( 0.71 0.23 ( 1.45 0.00
Figure 3A Figure 4A Figure 4A
a
The yield coefficient (Y) was estimated at 2.7 Fg protein /µmol PCE.
TABLE 2. Residual Sum of Squares (RSS) for Model Simulation of Experimental Data by Equation 4 figure
electron donor
1 (O) 1 (O) 2 (O) 2 (4) 2 (]) 3 (O) 3 (4) 3 (]) 4 (O) 4 (3) 4 (])
toluene toluene toluene toluene toluene toluene toluene toluene toluene lactate benzoate
a
X0, g protein
RSSa
0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.08 0.08 0.08
39.4 (days 0-89) 6.50 (days 94-160) 125 15.8 9.06 10.7 35.5 59.4 11.2 3.45 1.14
RSS ) ∑{(weight) (Yobserved - Ypredicted)2.
capable of describing PCE dechlorination when toluene was present in the cultures. Once toluene was entirely consumed during the days from 46 to 89, the model simulations yielded PCE values lower than the experimental data. A respike of toluene in the cultures at day 94 realigned the model simulations with the experimental data, as evidenced by the very small value of residual sum of squares (RSS) for the period from 94 to 160 (Table 2). To examine the applicability of the model to a wide range of PCE and toluene levels, eq 4 was used to simulate PCE dechlorination in Figures 2a and 3a. As shown by the curve with an initial toluene concentration at 8 µmol/bottle (Figure 2a), PCE dechlorination, after depletion of toluene in the cultures, deflected from the model simulation and proceeded at a lower rate. Although the absence of toluene restricted PCE dechlorination, a large increase in the amounts of toluene from 15 to 37 µmol/bottle could not further enhance the rate of PCE dechlorination, as demonstrated by consistent results of the experimental data with the model simulations (Figure 2a). Additionally, Figure 3a revealed that PCE dechlorination occurred almost at the same rate regardless of initial PCE amounts in the cultures. The three parallel lines predicted by the model simulations were also in agreement with the experimental data. The applicability of the model was subsequently displayed in Figure 4a by simulating PCE dechlorination, while toluene, lactate, and benzoate were individually used as the sole electron donors. A comparison of the parameter values in Table 1 reveals that the highest value of km was obtained when lactate was used as the electron donor, and this value was 2 and 3 times greater than that when benzoate and toluene were, respectively, used as the electron donors. The simulation results generated from eq 4 also explicitly predicted the continuous increases in the rate of PCE dechlorination when the electron donor was shifted from toluene to benzoate and then lactate. PCE Dechlorination Influenced by Other Electron Acceptors. The presence of nitrate and sulfate delayed the occurrence of PCE dechlorination (Figure 6). When PCE,
FIGURE 6. PCE dechlorination, toluene degradation, and formation of daughter products (A) as well as reduction of nitrate and sulfate (B) in the active cultures and abiotic controls. The data are means of duplicate batch tests, and the error bars indicate the relative errors. nitrate, and sulfate were added together to the same culture, nitrate reduction occurred first followed by sulfate reduction and then PCE dechlorination. During the time of nitrate and sulfate reduction (days 0-95), toluene was rapidly degraded, but no PCE dechlorination or methane production were observed. However, PCE dechlorination, along with the appearance of the transformation daughter products including ETH, VC, and cis-DCE, occurred immediately following the complete removal of sulfate. Methane production was also detected after depletion of sulfate in the culture. In the abiotic control, no transformation products were detected throughout the incubation period, and only limited losses of PCE, toluene, sulfate, and nitrate were observed.
Discussion PCE losses in the cultures appear to be predominantly attributed to microbial dechlorination since the mass balance analysis indicated that more than 86% of PCE losses were recovered as the microbial transformation daughter products. Ethene was the terminal product in the microbial processes, while other partially chlorinated ethenes came out only as transitional intermediates at low levels during PCE dechlorination (Figure 1). Despite significant accumulation of VC in the culture with the highest levels of added PCE, VC was subsequently transformed to ethene following removal of PCE (Figure 3). The observed inhibitory influences of the VOL. 39, NO. 23, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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high dose PCE on VC degradation, but not on PCE transformation, may suggest that different groups of microorganisms in the mixed cultures contributed to the complete sequential dechlorination of PCE to ethene. It should not be a surprise if there are different population groups in the mixed culture, which accounted for dechlorination of certain chlorinated ethenes in the transformation of PCE to ethene. Dechlorination of PCE to ethene in the mixed cultures is normally attributed to different microbial groups (18, 19). On the other hand, the accumulation of VC may only suggest that VC degradation rate became lower than VC production rate at a higher PCE concentration. Since one single isolate, Dehalococcoides ethenogenes, has demonstrated complete dechlorination of PCE to ethene (15), we cannot eliminate the possibility of this species in this mixed culture. The complete dechlorination of PCE to ethene in this culture was determined not to be due to a specific substrate category or a high substrate concentration, as identified by Figures 2 and 4. On the other hand, in a highly diluted culture (with 5% inoculation), the accumulation of ethene, along with cis-DCE and VC, was observed throughout the incubation time (160 days) even if toluene or lactate was added as the electron donors (data not shown). In addition, the stimulation of the denitrifying and sulfate-reducing activities by addition of nitrate and sulfate to the cultures not only postponed PCE dechlorination but also resulted in the accumulation of ethene as well as VC and cis-DCE (Figure 6). These findings suggest that the size of the dechlorinating populations and the structure of the microbial consortia determine the performance and products of the dechlorination processes. Previous analyses of the changes in community compositions of different enrichments by Flynn et al. (19) indicate that at least two populations are responsible for the sequential dechlorination of PCE to ethene in their cultures, and those populations can cooperate to form consortia for the complete dechlorination of PCE. The literature search also indicates that complete reductive dechlorination of PCE primarily occurs in enrichment cultures with preexposure to PCE and selected electron donors (11, 13, 16, 18, 19) but seldom takes place in microcosms prepared from fresh soils and sediments, in which the PCE dechlorination normally stopped at TCE, DCEs, or VC (7, 8, 10, 14, 18). It is commonly agreed that microbial dechlorination of PCE to TCE and cis-DCE can be carried out by a few ubiquitous subsurface bacteria, including a variety of dechlorinating microorganisms as well as methanogens and sulfate-reducers (18). Therefore, the adaptation of the microbial cultures by exposure to PCE and appropriate organic compounds, such as toluene, appears to provide the opportunity to form the proper microbial community compositions for complete dechlorination of PCE to ethene without accumulation of harmful daughter products. Toluene as the primary electron donor appears to sustain PCE dechlorination in this culture. This is because absence of toluene limited the reductive dechlorination of PCE and respike of toluene was able to restore the dechlorination activity (Figures 1-3). However, toluene is unlikely to be used as the direct electron donor to drive PCE dechlorination since PCE dechlorination rates were not directly linked to toluene degradation rates (Figures 2 and 3). It is known that H2 produced from fermentation of organic compounds can be utilized as a direct electron donor for reductive dechlorination of PCE under anaerobic conditions, and many studies have confirmed that H2 is able to serve as the sole electron donor for PCE dechlorination in microbial cultures (10, 15, 18). In this culture, H2 may also serve as an electron donor to directly drive microbial dechlorination of PCE. This is evidenced by the findings that a high rate of PCE dechlorination was observed, while a high level of H2 was maintained 9292
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in the cultures (Figure 4). In addition, the findings that virtually no methane was generated during PCE dechlorination and significant production of methane occurred immediately following completion of the dechlorination processes (Figures 2 and 3) also support that PCE dechlorinators compete with methanogens for the same electron donor which apparently is H2. The preference of microbial dechlorination to methanogenesis at low levels of H2 has been observed during fermentation of other organic compounds (10, 11, 16). On the other hand, once the available H2 increased to higher levels due to change of the electron donors from toluene to benzoate and lactate, methane production was detected concurrently with PCE dechlorination (Figure 4). These results explain that H2, an essential intermediate pool to sustain methanogenic activity, also played the same role as a direct electron donor in the microbial reductive dechlorination of PCE. Furthermore, the findings that PCE dechlorination was inhibited by sulfate (Figure 6) provide an additional line of evidence that the dechlorinating microorganism compete for the same electron donor (H2) with sulfate reducers which are a known competitor even at a very low level of H2 (21). Acetate is also a potential direct electron donor for microbial dechlorination of PCE to ethene (17). The continuous production of the daughter products coupled with degradation of acetate as observed in Figure 4 also suggests that acetate may be another electron donor that can be directly used by this culture for complete PCE dechlorination to ethene. The use of H2 and acetate as the direct electron donors for driving PCE dechlorination implies that the interactions of microbial consortia play a critical role in PCE dechlorination and toluene degradation in the cultures. The adaptation of an anaerobic process is typically governed by several mechanisms including induction, mutation, growth of transformation populations, formation of microbial consortia, and formation of metabolic intermediate pools, among which the formation of metabolically competent consortia is determined to be a limiting step (22). In terms of dechlorinating processes, a substrate capable of releasing H2 at a slow rate during fermentation can selectively enhance formation of dechlorinating microbial consortia (6, 16). According to the findings observed in this study, toluene, possibly along with other fuel hydrocarbons, appears to be the preferred substrates to accomplish the adaptation process for complete dechlorination of PCE to ethene. Previous studies have reported that microorganisms are capable of degrading toluene under methanogenic conditions (23, 24). This study further shows that when PCE was available, dechlorinating organisms were capable of outcompeting methanogens to achieve PCE dechlorination. In terms of the efficiency of electron utilizations for PCE dechlorination, degradation of toluene was the most effective process compared with degradation of benzoate and lactate. A total of 12% of electrons released from toluene degradation can be used for dechlorinating activity, while degradations of benzoate and lactate only deliver 7 and 6% of electrons to PCE dechlorination (Figure 5). Yang and McCarty (11) have observed a similar result that dechlorinating microorganisms were able to obtain 9% of transport electrons for DCE dechlorination when benzoate was used as the primary electron donor. The highest utilization percentage of transport electrons for PCE dechlorination during degradation of toluene further suggests that toluene is one of the potential substrates that are suitable for selectively stimulating the formation of dechlorinating microbial consortia. The findings that PCE dechlorination in this culture can be described by a Monod-like equation provide a mathematical approach to evaluate the design of a remedial
process. The culture was able to retain the kinetic features of PCE dechlorination regardless of electron donors used (Table 1). At high concentrations of PCE, the reductive dechlorination of PCE proceeded following a zero-order kinetic as evidenced by both the virtually parallel lines of PCE losses (Figure 3) and the very low values of the halfvelocity constant (Table 1). As long as sufficient electron donors were available in the cultures, experimental data were adequately described by the model simulations (Table 2). That PCE dechlorination showing a Monod-like kinetic performance was not unique for this culture. It was broadly observed in other cultures that reductively dechlorinate PCE (14, 16, 26, 27). However, the reported maximum specific dechlorination rate constants varied over a wide range depending on the bacterial cultures, the electron donors, and the terminal transformation products (14). In this study, the kinetic parameter values also showed variations with electron donors used in the cultures (Table 1). The predictability of PCE dechlorination by a mathematical model will aid to optimize the design and operation of a remedial system. The findings that PCE dechlorination was temporarily inhibited by nitrate and sulfate but resumed immediately following removal of these electron acceptors (Figure 6) indicate that the dechlorination may be a less competitive process compared with denitrification and sulfate reduction. Regardless of the temporary change of respiratory systems, the findings that the bacterial culture was capable of restarting dechlorination activity after removal of nitrate and sulfate suggest that this process still holds the potential for application even in subsurface environments containing sulfate and nitrate. However, achievement of PCE remediation at such contaminated sites may require longer treatment times and larger amounts of electron donors. It is interesting to note that the multiple-year old toluene/ PCE enrichments used as the source inoculum for the batch culture maintained the capacity to rapidly utilize nitrate and sulfate as terminal electron acceptors. The long-term maintenance of nonselected metabolic capabilities in the mixed consortia, presumably via support of subpopulations, has implications to our view of how microorganisms compete, interact, and achieve commensal balance. The rapid onset of toluene degradation linked to nitrate and sulfate reduction suggests the maintenance of these nonselected capabilities and the microorganisms that they reside in, and/or the capacity for the same microorganisms to act in various consortia roles based metabolisms of opportunity. This microbial process showing PCE dechlorination with concurrent degradation of toluene has extraordinary significance for remediation of the subsurface environment contaminated with both chlorinated solvents and petroleum compounds. The frequent detection of these toxic mixtures at hazardous waste sites indicates the preference for applying joint treatment techniques for simultaneous remediation of these contaminants (8, 12). A recent study that assessed natural attenuation at a site contaminated with both chlorinated solvents and BTEX concluded that toluene, ethylbenzene, and xylenes not only have been degraded but also probably served as electron donors for microbial reductive dechlorination of TCE that coexisted in the groundwater (8). Since the contaminated subsurface environment is commonly oxygen-limited following bacterial consumption of available molecular oxygen and since PCE is resistant to microbial attack under aerobic conditions, the process combining degradation of fuel compounds with transport of electrons to PCE for reductive dechlorination holds the promise for cleaning up these contaminated sites, and the recognition of this process is also important in defining and predicting the subsurface fate and transport of these contaminants under both active treatment and natural conditions.
Acknowledgments We thank Dr. Dennis Fine for GC/MS analysis. The U.S. Environmental Protection Agency funded the research described here. It has not been subjected to Agency review and therefore does not necessarily reflect the views of the Agency, and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
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Received for review February 24, 2005. Revised manuscript received June 8, 2005. Accepted June 10, 2005. ES050390V
