Environ. Sci. Technol. 2010, 44, 1085–1092
Monitoring Bioaugmenation with Single-Well Push-Pull Tests in Sediment Systems Contaminated with Trichloroethene JAE-HYUK LEE,† MARK DOLAN,‡ JENNIFER FIELD,§ AND J O N A T H A N I S T O K * ,| California Department of Transportation, Marysville, California, School of Chemical, Biological, and Environmental Engineering, Department of Environmental and Molecular Toxicology, and School of Civil and Construction Engineering, Oregon State University, Corvallis, Oregon 97331
Received July 2, 2009. Revised manuscript received October 18, 2009. Accepted October 20, 2009.
Bioaugmentation to enhance the rate and extent of reductive dechlorination of chlorinated ethenes was investigated in intermediate (∼1 m) scale physical aquifer models (PAMs) designed to simulate the groundwater flow field near an injection well. Push-pull tests were used to quantify the reductive dechlorination of injected trichloroethene (TCE) and trichlorofluoroethene (TCFE) in prepared sediment packs with and without an added dechlorinating culture containing Dehalococcoides spp. Distribution of the added culture throughout the sediment pack was confirmed by microscopic observation. Repeated additions of TCE (100-350 µM) were completely transformed to ethene in 14 days and a subsequent TCFE addition (114 µM) was completely transformed to fluoroethene (FE) in 24 days. Similar transformation rates, product distributions, and time courses for TCE and TCFE transformation were observed when these compounds were added together at similar initial concentrations. In the control PAM (nonbioaugmented), TCE and TCFE were transformed to only cis-DCE and cisDCFE, respectively, and transformation rates were 6-12 times slower than those in the bioaugmented PAM. The use of TCFE for push-pull tests is shown to be an effective tool for detecting and quantifying the effects of bioaugmentation on TCE transformation.
Introduction Chlorinated aliphatic hydrocarbons (CAHs) cause environmental problems due to their recalcitrance and toxicity. As a result of spills and disposal of these compounds into the environment, trichloroethene (TCE) is one of the most common organic groundwater contaminants in the United States (1). Previous laboratory and field studies (2, 3) have shown that the destruction of chlorinated compounds can be achieved via reductive dechlorination by stimulating the activity of indigenous microorganisms. However, complete reductive dechlorination of TCE to ethene is not always * Corresponding author e-mail:
[email protected]; phone: 541-737-8547. † California Department of Transportation. ‡ School of Chemical, Biological, and Environmental Engineering. § Department of Environmental and Molecular Toxicology. | School of Civil and Construction Engineering. 10.1021/es9019645
2010 American Chemical Society
Published on Web 12/23/2009
observed and this has been attributed to the absence of dehalorespiring microorganisms and/or the absence of a suitable electron donor (4, 5). In particular, accumulation of vinyl chloride (VC) due to incomplete reductive dechlorination is a concern. While reductive dechlorination supports growth, the transformation of VC to ethene is cometabolic and half-velocity coefficient (Ks) values for VC are larger than for the higher chlorinated compounds (6-8), thus the transformation of VC to ethene is considered one of the more difficult steps in TCE mineralization. Bioaugmentation is defined as the introduction of exogenous microorganisms to an environment to promote the degradation of targeted contaminants (9-14). Laboratory and field studies have shown that bioaugmentation can increase the rate and extent of reductive dechlorination of chlorinated ethenes (10-14). Despite these findings, the effectiveness of bioaugmentation remains uncertain because of uncertainties about the feasibility of distributing exogenous microorganisms in the subsurface and about the ability of exogenous microorganisms to survive and compete with indigenous microorganisms for nutrients and substrates and to maintain the targeted metabolic activity (14). Furthermore, robust methods for assessing rate and extent of reductive dechlorination after bioaugmentation are needed in the presence of mixed waste streams containing TCE, DCE, and VC. Vancheeswaran et al. reported that TCFE could be a useful surrogate for estimating rates of reductive dechlorination of PCE and TCE (15). Anaerobic transformation of TCFE proceeded by sequential reductive dechlorination to form DCFEs, CFEs, and FE, analogous to the products formed from the reductive dechlorination of TCE (i.e., DCEs, VC, and ethene) (15-19). In two field studies, Hageman et al. (16) demonstrated that TCFE reductive dechlorination rates were similar to those of TCE and also reported the successful enhancement of TCFE reductive dechlorination to FE in TCE contaminated groundwater with additions of fumarate as a stimulant (18). Lee et al. (19) reported that fumarate and succinate were effective stimulants for reductive dechlorination of trichloroethene (TCE) and trichlorofluoroethene (TCFE) in the presence of high (2.5 mM) initial sulfate concentrations in laboratory microcosm studies using sediments and groundwater from the same TCE-contaminated aquifer used by Hageman et al. (16-18). Field et al. also used TCFE to detect, quantify, and compare TCFE reductive dechlorination activity at the same site in large-diameter permeable columns treated with Fe(0), H2, and lactate (20). Ennis et al. demonstrated that trans-CFE was a good surrogate for monitoring VC reductive dechlorination (21). Push-pull tests have been used to obtain in situ, quantitative information on microbial activity and contaminant transformations in the subsurface (24, 25) including methane oxidation (26), aerobic cometabolism (27), anaerobic transformation of petroleum hydrocarbons (28), reductive precipitation of radionuclides (29), and anaerobic transformations of chlorinated solvents (16-21). In this study, we used laboratory push-pull tests to evaluate the effectiveness of bioaugmentation for quantifying the rate and the extent of reductive dechlorination of TCE and TCFE using identical bioaugmented and nonbioaugmented (control) physical aquifer models (PAMs). To assess reductive dechlorination rates, the forced mass balance (FMB) technique (17, 18, 20, 21) was applied. Through a series of tests, we developed procedures that should be useful for evaluating the effectiveness of bioaugmentation for assessing complete reductive dechorination of chlorinated ethenes. VOL. 44, NO. 3, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Physical aquifer models used in bioaugmentation experiments: (A) rationale and (B) plan view.
Materials and Methods Chemicals. TCE was purchased from Fisher (99.9%, Fair Lawn, NJ). PCE (99.9%, HPLC grade, Sigma-Aldrich Inc., St. Louis, MO), cis-1,2-DCE (97%, Acros Organics, Pittsburgh, PA), trans-DCE (98%, Aldrich Chemical, Milwaukee, WI), 1,1DCE (99%, Aldrich Chemical), VC (99.5%, Aldrich Chemical), and ethene (99.5%, Aldrich Chemical) were purchased for use as analytical standards. TCFE (97%), DCFE (98% mixture consisting of 50% of cis and 50% of trans isomers), and CFE (97% mixture consisting of 69% of cis and 31% of trans isomers) were purchased from SynQuest Laboratories, Inc. (Alachua, FL) and FE (98%) was purchased from Lancaster synthesis (Pelham, NH). Culture and Growth Medium. The dehalogenating culture used in this study was harvested in a closed semibatch reactor (Kimax, NJ) with total volume 1.2 L containing a liquid volume of 1 L. The culture was enriched from groundwater and sediment from a TCE-contaminated site at the Evanite site in Corvallis, OR (8). The Evanite culture is known to contain Dehalococcoides spp. and has been extensively tested for reductive dechlorination of chlorinated ethenes (8, 22). A detailed description of the Evanite culture was provided elsewhere (8, 22). Physical Aquifer Models. Laboratory experiments were conducted in two identical physical aquifer models (PAMs) constructed in a wedge shape to approximate the radial flow field near an injection/extraction well during field push-pull tests (Figure 1A). The PAMs were constructed of polypropylene with interior dimensions of 5 cm (width at narrow end), 50 cm (width at wide end), 125 cm (length), and 20 cm (height) (Figure 1B). The PAMs were packed with sediment from the Hanford Formation, an alluvial deposit of sands and gravels of mixed basaltic and granitic origin (19) after removing particles >0.5 cm in diameter by sieving. The sediment contained less than 0.001 wt % organic matter and had a particle density of 2.9 g/cm3. The porosity and bulk density of the packed sediment were 0.39 and 1.77 g/cm3, respectively. Total internal volume was 69 L and the pore volume was 27 L. Tap water was used in all laboratory experiments. The narrow ends of the PAMs contained injection/extraction ports covered with a screen to allow water to be pumped in or out during push-pull tests. After 1086
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the sediment pack was water saturated, the PAMs were sealed with a lid containing sampling ports (Figure 1B) connected to “well” screens that fully penetrated the sediment pack’s saturated thickness. Experiments were performed under confined conditions. A schematic diagram of the experimental setup is in Figure 2. For sampling, aqueous samples were collected during the experiments using plastic syringes connected to the sampling ports. Three mL aliquots of sample were dispensed into 7-mL headspace vials with butyl rubber septas (Supelco, PA) for gas chromatograph (GC) headspace analysis. Samples were stored at 20 °C and analyzed within 2 days. One mL was dispensed into an eppendorf tube (1.5 mL, Brinkmann Instruments Inc.) and centrifuged for 10 min at 14,000 rpm (Eppendorf microcentrifuge model 5415C): 0.5 mL of the supernatant was then transferred into an autosampler polyvial for ion chromatography (IC; Dionex Series DX-320, Sunnyvale, CA) analysis. IC samples were stored at 4 °C and analyzed within 3 days. Bioaugmentation of the PAM. Six weeks prior to bioaugmentation, ∼1.3 µM of titanium(III) citrate solution (30) was pumped into the PAM followed by 6 pore volumes of oxygen-free water containing sodium lactate (∼1 mM) to stimulate microbial activity and to create initial anaerobic and reducing conditions. Test solutions were prepared in a 48-L glass carboy (Figure 2). Dissolved oxygen was removed by vigorous bubbling (flow rate ∼100-200 mL/min) with N2 gas that had passed through a gas purifier (Supelco, PA) to remove trace O2. The carboy was sealed with a black rubber stopper containing holes for sampling, sparging, and ventilation lines. Polyetheretherketone (PEEK) tubing was used for gas and water delivery lines. A piston pump and 5-port manifold (Upchurch Scientific, WA) were used to inject test solutions into the sediment pack; injection rates varied between 40 and 100 mL/min depending on the test. The concentrated aqueous TCE and/or TCFE solutions in the bag were prepared by adding 1.5 mL of neat TCE or 0.5 mL of neat TCFE to 1.1 L of oxygensfree tap water in 1.2-L glass bottles (Kimax, NJ). Approximately 1 L of this solution was transferred from a bottle to a bag containing 7 L of oxygen free water using positive nitrogen pressure applied thorough a rubber septa. The bioaugmentation injectate was prepared by adding sodium carbonate (37.6 mM), sodium sulfide (22 µM), and 1-butanol (10 mM) to the 48-L glass carboy and purging with carbon dioxide. The final pH was 7-8. Evanite culture (9 L) was mixed with the injectate by bubbling with N2; the initial concentration of the bioaugmented cells was ∼1 × 107 cells/ mL. During injection (∼6 h), the bioaugmentation injectate was combined with TCE and/or TCFE by metering concentrated aqueous solutions contained in an 8-L collapsible, metalized bag (Chromatography Research Supplies, KY) using a piston pump connected to a PEEK Y connector. Samples for background cell counts were collected from ports 5 and 8; samples to monitor the migration of bioaugmented cells were collected from ports 2, 5, 6, and 8. An identical control PAM was prepared and incubated with the same condition including TCE, TCFE, butanol, and the injectate without the bioaugmented culture and sampling was performed to check TCE and TCFE transformation and fermentation products. Analytical. TCE, TCFE, DCE, DCFE, CFE, VC, FE, and ethene headspace concentrations were determined on 100µL headspace samples using a Hewlett-Packard (Palo Alto, CA) 5890II GC connected to a flame ionization detector. Separation was performed using a 30 m × 0.32 mm × 4.0 µm Supelco SPB-1 capillary column (Supelco, PA). The column was operated under a thermal gradient with helium as the carrier gas. The GC oven was initially set at 40 °C for 4.5 min, heated at 20 °C/min to 160 °C, and kept at 160 °C for 0.5 min. A five-point (5-120 µM) external standard calibration curve
FIGURE 2. Laboratory setup used for physical aquifer model experiments.
TABLE 1. Summary of the Test Sequence injection bioaugmentation + 1st addition of TCE
date
purpose
materials
July 15, 2004
bioaugmentation of dechlorinating culture into PAM
TCE, butanol, diluted media including diluted Evanite culture
2nd addition of TCE
August 3, 2004
3rd addition of TCE
August 18, 2004
mass balance of ethene production and TCE reduction mass balance of ethene production and TCE reduction
TCE, butanol, and diluted media TCE, butanol, and diluted media
4th addition of TCE
September 4, 2004
mass balance of ethene production and TCE reduction
TCE, butanol, and diluted media
1st addition of TCFE
September 22, 2004
co-injection of TCE and TCFE
October 19, 2004
quantifying TCFE reductive dechlorination TCFE feasibility to track TCE reductive dechlorination as a surrogate
TCFE, butanol, and diluted media TCE, TCFE, butanol and diluted media
was used for quantification (R2 > 0.99); the detection limit was about 0.5 µM. A one-point standard check was performed for every sampling activity. Methane in the headspace was determined using a Hewlett-Packard (HP) 5890 Series II (GC) with thermal conductivity detector (TCD) and a Supelco 60/ 80 Carboxen 1000 packed column. Grade 4.5 helium was used as a carrier gas. Concentrations of inorganic and organic anions including bromide, chloride, acetate, butyrate, propionate, and sulfate were determined using a Dionex Series DX-320 IC (Sunnyvale, CA) equipped with a Dionex IonPac AS11-HC analytical column (4 × 250 mm), an IonPac AG11-HC guard column (4 × 50 mm), and an ATC-1 trap column in series. The gradient elution consisted of a 1-30 mM sodium hydroxide solution (50% w/w; Fisher Scientific, Fair Lawn, NJ) flowed at 1.5 mL/ min. A six-point (1-100 mg/L) external standard calibration curve was used for quantification (R2 > 0.99). A one-point standard check was performed for every sampling activity. 1-butanol was determined by extracting a 0.5-mL aqueous sample with 0.5 mL of DCM and vigorously mixing for 2 min on a vortex mixer. The DCM extract (10 µL) was analyzed using a HP-5890II GC connected to an FID. Test Sequence and Data analysis. A series of activity tests was conducted to monitor TCE and TCFE transformation
under no flow conditions (i.e., after TCE or TCFE was injected and distributed throughout the PAM flow was stopped and CAH concentrations were monitored by sampling). The bioaugmentation culture was coinjected with TCE (∼50 µM). Five subsequent injections of TCE, TCFE, and/or TCE/TCFE were conducted to quantify the activity of the bioaugmented PAM over a 4 month period (Table 1). The bioaugmented PAM was incubated under no-flow condition after each TCE and/or TCFE injection. Sampling of TCE, TCFE, TCE/TCFE byproducts, and anion analysis was performed 2-3 times a week by the sampling ports until complete transformation of TCE to ethene and/or TCFE to FE was observed (Figure 2); more than 1.5 pore volumes of new injectate flushed out the bioaugmented PAM for each TCE and/or TCFE injection. After the activity tests described above, a series of push-pull tests was conducted. Each push-pull test consisted of (1) a controlled injection (“push phase”) of a prepared test solution including a nonreactive tracer and TCFE, (2) no pumping (“rest phase”) for a certain time to allow TCFE reductive dechlorination of the test solution in the PAM, and (3) extraction (“pull phase”) of the test solution/ pore water mixture from the same location. In situ rates of TCE and TCFE transformation were computed from push-pull test data by removing the effects VOL. 44, NO. 3, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. Reductive dechlorination of TCE and TCFE in bioaugmented PAM (A) and fermentation products of butanol (B) detected at port 8 after the second addition of TCE. Arrows indicate additions of TCE (1st-4th), TCFE (5th), or both (6th): s[s TCE, s0s cis-DCE, s∆s VC, s3s ethene, sbs TCFE, s9s cis-DCFE, s-s trans-DCFE, s · s 1,1-CFE, s+s trans-CFE, sOs FE.
TABLE 2. Summary of Computed Maximum Zero-Order Transformation Rates and Conversion for Activity Tests with a Stop-Flow in Bioaugmented PAM (Mean ± 95% Confidence Interval)a TCE or TCFE transformation rate
ethene or FE production rate
-1
(µM · day-1)
(µM · day ) TCE or TCFE injection
reactant
port 1
port 3
port 5
port 8
product
port 1
port 3
port 5
port 8
TCE 2nd injection TCE 4th injection TCFE 1st injection TCE/TCFE injection
TCE TCE TCFE TCE TCFE
11 ( 37 32 ( 41 21 ( 50 31 ( 6 39 ( 9
ND 24 ( 64 9 ( 26 34 ( 10 37 ( 10
30 ( 15 85 ( 24 8(8 31 ( 8 36 ( 11
ND 56 ( 40 6(3 26 ( 5 29 ( 5
ethene ethene FE ethene FE
0.5 ( 0.6 0.4 ( 0.2 3.3 ( 0.4 4.9 ( 1.1 7.3 ( 0.7
0.5 ( 0.4 1.3 ( 0.6 6.1 ( 4.5 5.8 ( 3.6 7.7 ( 4.2
17.3 ( 11.9 8.1 ( 5.4 7.0 ( 4.3 6.5 ( 1.0 5.2 ( 0.9
20.8 ( 13.2 10.0 ( 3.2 6.5 ( 1.2 5.7 ( 0.4 7.1 ( 1.2
a ND: No calculations were obtained due to R2 < 0.6. The 1st and 3rd injections were not included because TCE aqueous concentration was not detected for the following 2nd or 3rd sampling period after the injections.
of transport processes from measured aqueous concentrations using the forced mass balance (FMB) technique (17). Details of the FMB technique were described and tested elsewhere (17-21). The zero-order production rates of FE were obtained using linear regression after transforming progress curves for FE using the FMB technique. Progress curves for FE will appear greater than the aqueous injectate concentrations as a result of the FMB data treatment, which takes into account FE in the aqueous and solid phases. Because FE is the only known end product of TCFE transformation under anaerobic conditions, the appearance rate of FE is proportional to the disappearance rate of TCFE. An error analysis conducted by Hageman et al. (17) indicated that the actual in situ rates obtained using the FMB technique are within 10% of the true rates. 1088
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Results Activity Tests. TCE. Rapid transformation of injected TCE in the bioaugmented PAM indicated that conditions were favorable for dechlorination of chlorinated ethenes after bioaugmentation. Ethene production was observed in all ports indicating survival and activity of the bioaugmented culture. Four additional injections of TCE alone and an injection of TCE/TCFE together resulted in all CAHs being completely transformed at all ports. Data for port 8 are shown as examples (Figure 3A) and TCE concentrations in the following parentheses were at ports 1 and 8, respectively. The first TCE (24 and 56 µM) injection was completely transformed to ethene in 7 days at all ports (Figure 3A). The second (33 and 165 µM) and third (18 and 64 µM) injections
FIGURE 4. Result of TCE and TCFE coinjection from day 95 to 124 (A) and the linear regression for TCE and TCFE reductive dechlorination (B) at port 8 during the same period. of TCE were transformed to cis-DCE, VC, and ethene within 8 days at all ports (Figure 3A). Transformation rates ranged from 10.5 to 31.8 µM · day-1 and ethene production rates ranged from 0.5 to 17.3 µM · day-1 (2nd injection, Table 2 and Figure 3A); conversion calculations of TCE to ethene were likely inaccurate due to partitioning of less-dechlorinated products, VC and ethene, into possible gas pockets within the PAM. Furthermore, possible losses by sorption from syringes during sampling could be a factor to cause varied TCE and TCFE concentration. The recovery of ethene was not stoichimetically observed during TCE transformation. Some rate calculations were not performed (ND) due to highly variable TCE concentrations during incubation at several ports (3 and 8, Table 2). The rate calculations for TCE transformation were not included for the first and third injections due to no data points obtained under fast transformation with low TCE concentration (
