Remediation of Dense Non-Aqueous Phase Liquids Using Biostimulation and Bioaugmentation

Demonstration of Enhanced Bioremediationin a TCE Source Area at Launch Complex 34,

Cape Canaveral Air Force Stationby E.D. Hood, D.W. Major, J.W. Quinn, W.-S. Yoon, A. Gavaskar, and E.A. Edwards

AbstractThe ability of bioremediation to treat a source area containing trichloroethene (TCE) present as dense nonaqueous phase

liquid (DNAPL) was assessed through a laboratory study and a pilot test at Launch Complex 34, Cape Canaveral Air ForceCenter. The results of microcosm testing indicate that the indigenous microbial community was capable of dechlorinatingTCE to ethene if amended with electron donor; however, bioaugmentation with a dechlorinating culture (KB-1; SiREM,Guelph, Ontario, Canada) significantly increased the rate of ethene formation. In microcosms, the activity of the dechlorinat-ing organisms in KB-1 was not inhibited at initial TCE concentrations as high as 2 mM. The initially high TCE concentra-tion in ground water (1.2 mM or 155 mg/L) did not inhibit reductive dechlorination, and at the end of the study, the averageconcentration of ethene (2.4 mM or 67 mg/L) was in stoichiometric excess of this initial TCE concentration. The productionof ethene in stoichiometric excess in comparison to the initial TCE concentration indicates that the bioremediation treatmentenhanced the removal of TCE mass (either sorbed to soil or present as DNAPL). Detailed soil sampling indicated that thebioremediation treatment removed greater than 98.5% of the initial TCE mass. Confirmatory ground water samples collected22 months after the bioremediation treatment indicated that chloroethene concentrations had continued to decline in theabsence of further electron donor addition. The results of this study confirm that dechlorination to ethene can proceed at thehigh TCE concentrations often encountered in source areas and that bioremediation was capable of removing significantTCE mass from the test plot, suggesting that enhanced bioremediation is a potentially viable remediation technologyfor TCE source areas. Dehalococcoides abundance increased by 2 orders of magnitude following biostimulation andbioaugmentation.

Some figures in this paper are available in color in the online version of the paper.

IntroductionThe application of anaerobic bioremediation of chlori-

nated ethenes, such as tetrachloroethene (PCE) and tri-chloroethene (TCE), through reductive dechlorination toethene was historically limited to the treatment of dis-solved-phase plumes rather than the source areas, whichare associated with the release of dense nonaqueous phaseliquids (DNAPLs). Several studies have demonstrated thatchloroethenes can be completely degraded to ethene at thefield scale (e.g., Major et al. 2002; Lendvay et al. 2003;Song et al. 2002). However, the perceived limitation of bio-remediation for the treatment of source areas is the beliefthat microbial activity is inhibited by the high concen-trations of PCE and TCE within source areas and theexpectation that biodegradation rates were too low to

meaningfully impact the rate of contaminant mass removal(Pankow and Cherry 1996).

However, several studies have demonstrated that dech-lorinating microorganisms are active at chloroethene con-centrations that are much higher than those commonlyobserved in source areas. At least one laboratory study re-ports PCE and TCE dechlorination at the solubility limitsfor these compounds (Nielsen and Keasling 1999), withother studies demonstrating dechlorination at the highconcentrations often encountered in source areas (e.g.,DiStefano et al. 1991; Isalou et al. 1998; Yang andMcCarty 2000; Duhamel et al. 2002; Adamson et al. 2003).Complete dechlorination of PCE, TCE, cis-1,2-dichloroethene(cis-DCE), and vinyl chloride (VC) at initial concentrationsof 0.8, 1.5, 0.8, and 1.4 mM, respectively, occurred in mi-crocosms bioaugmented with KB-1, a dechlorinatingmicrobial culture (Duhamel et al. 2002, 2004). Dechlorina-tion of PCE (more than 0.9 mM) by a mixed culture wasnot inhibited by 1.0 mM ethene, while dechlorination of

Copyright ª 2008 The Author(s)Journal compilationª 2008National GroundWater Association.

98 Ground Water Monitoring & Remediation 28, no. 2/ Spring 2008/pages 98–107

cis-DCE was not inhibited by an initial cis-DCE concentra-tion of 0.7 mM (Yang and McCarty 2000). In fact, highchloroethene concentrations may improve the efficiencyof dechlorinating organisms by inhibiting competitiveelectron donor utilization by methanogens (DiStefano et al.1991; Yang and McCarty 2000).

Further, there is an increasing evidence that biodegra-dation can enhance mass removal from source areas,including a theoretical foundation for enhanced nonaque-ous phase liquid dissolution (Seagren et al. 1993), whichhas been complemented by multiple bench-scale studies. Incolumn studies, pentanol amendment resulted in at leasta fivefold increase in the rate of PCE DNAPL removal(Yang and McCarty 2000). In mesocosm studies performedat the University of Toronto, a threefold increase in therate of removal of PCE DNAPL from an electron donor–amended, bioaugmented mesocosm was observed (Sleepet al. 2006), while in Cope and Hughes (2001) the removalof PCE from bioaugmented columns was enhanced 6.5-fold by pyruvate addition in comparison to abiotic col-umns. In conjunction with studies demonstrating PCE andTCE dechlorination at typical source zone concentrations,these studies suggest that the use of biodegradation pro-cesses to enhance DNAPL dissolution may be a feasibleapproach for source area treatment.

Coupled with the recognition that bioremediation maybe applied in source areas is an improved understanding ofthe key dechlorinating microorganisms. Bacteria of thegenus Dehalococcoides are the only known organisms capa-ble of metabolic reduction of cis-DCE and VC to ethene(Cupples et al. 2003; He et al. 2003; Duhamel et al. 2004;Sung et al. 2006). Some subspecies of Dehalococcoidesappear to possess distinct dehalogenase genes that code forenzymes that dechlorinate VC to ethene (Muller et al. 2004;Waller et al. 2005; Krajmalnik-Brown et al. 2004). To sup-plement indigenous dechlorinating communities, the use ofbioaugmentation cultures containing the requisite Dehalo-coccoides organisms has been a highly successful strategyfor remediation of contaminant plumes (e.g., Ellis et al.2000; Major et al. 2002; Lendvay et al. 2003).

Test Site DescriptionIn this study, the ability of bioremediation to treat

a TCE source area was assessed through a laboratory studyand in a field-scale pilot test at Launch Complex 34(LC34), Cape Canaveral Air Force Station, Florida. LC34was a launch site for Saturn rockets from 1960 to 1968.Approximately 40,000 kg of TCE DNAPL is present in thesubsurface at the site as a result of historical operations atLC34; TCE concentrations within this source zone rangefrom 100 mg/L to the solubility (1100 mg/L) limit for thiscompound (Eddy-Dilek et al. 1998). Lower concentrationsof cis-DCE and VC are present in ground water as a resultof intrinsic biodegradation processes (Battelle 1999).

HydrogeologyAn unconfined aquifer and a semiconfined aquifer

underlying a clay unit comprise the major water bearingunits at LC34. The pilot test was conducted in the

unconfined aquifer, which extends from the water table(depth to water was 10 feet below ground surface [ft bgs])to a depth of approximately 45 ft bgs. The surficial aquiferis further divided into the upper sand unit (USU), the mid-dle fine-grained unit, and the lower sand unit. The studywas performed in the USU, which is composed of mediumto coarse-grained sand and crushed shells and extendsfrom ground surface to 26 ft bgs. The ambient hydraulicgradient at the site is flat, with reported gradients rangingfrom 0.0001 to 0.0007 ft/ft (CRA Services 1999).

GeochemistryBackground geochemical conditions (Battelle 1999)

indicate that (1) concentrations of total dissolved solids areas high as 1220 mg/L (predominantly Na+, K+, Mg2+, Ca2+,Cl�, and SO4

2�), including 300 mg/L SO42�; (2) the

ground water pH is near neutral with an alkalinity of up to360 mg/L (as CaCO3); (3) the dominant electron acceptorsin the ground water are sulfate and TCE; (4) the oxidation-reduction potential is generally reducing (i.e., �100 mV);and (5) there is little dissolved organic carbon (less than3 mg/L). Under intrinsic conditions, TCE is partially bio-degraded to cis-DCE, which represents only 5% (molefraction) of the total chloroethene concentration. Theground water temperature is typically 29 �C.

MicrobiologyAlthough there is some evidence that microbial growth

at LC34 is inhibited by the presence of DNAPL (Eddy-Dilek et al. 1998), a more recent study reports that themicrobial community is diverse (Azadpour-Keeley et al. 2004).The presence of a member of the genus Dehalococcoidescapable of mediating complete dechlorination of TCE toethene has been reported at Facility 1381, an adjacent site(Fennell et al. 2001). Baseline sampling and 16S ribosomalRNA (rRNA) gene analysis confirmed that Dehalococ-coides organisms are present at LC34; however, the resultsof microcosm studies completed using samples from fivelocations demonstrated that organisms from only three offive locations were capable of complete dechlorination toethene, indicating that dechlorinating activity was not uni-formly distributed (data not presented). A similarly hetero-geneous distribution of dechlorinating activity was reportedby Fennell et al. (2001).

Methods

Microcosm StudyA microcosm study was carried out to evaluate the ef-

fects of high concentrations of TCE on the rate and extentof TCE dechlorination. A summary of the treatments andcontrols included in the microcosm study is presented inTable 1. A description of the microcosm study is providedin the supplementary material.

Ground Water Sample CollectionField parameters (dissolved oxygen concentration, pH,

ORP, temperature, and specific conductance) were moni-tored using a YSI 556 multiparameter meter. Prior to

E.D. Hood et al./ Ground Water Monitoring & Remediation 28, no. 2: 98–107 99

collecting ground water samples for chemical analysis,stagnant water in the well casing (greater than three wellvolumes) was purged until field parameters stabilized.Ground water samples were collected directly into thesample bottles and were stored at 4 �C until analysis.

Soil Sample CollectionPaired pre- and posttreatment cores were collected

from four randomly selected locations in each quadrantof the test plot. The subsampling procedure (includingmethanol extraction) is described by Quinn et al. (2005).Methanol extracts were shipped on ice to CAS Laboratory(Jacksonville, Florida) for TCE analysis (U.S. EPA method8260).

Analytical MethodsChloroethenes, dissolved hydrocarbon gases (methane,

ethane, and ethene), Dehalococcoides-specific polymerasechain reaction (PCR) assays, and anion analyses were per-formed by SiREM (Guelph, Ontario, Canada). Volatilefatty acid (i.e., acetate, lactate, pyruvate, propionate, andbutyrate) concentrations were determined by MicroseepsLaboratory (Pittsburgh, Pennsylvania). Descriptions of theanalytical methods are provided in the supplementarymaterial.

Pilot Test Construction and OperationThe test plot was constructed entirely within the much

larger source area at LC34. Well installation was com-pleted using a direct push rig. Injection and extractionwells (2-inch inside diameter) were constructed with10 feet screens completed at the bottom of the USU (26 ftbgs); monitoring wells were constructed of polyvinyl chlo-ride with 5-feet screens completed at the bottom of theUSU. Native material was permitted to collapse around thewell screen and casing as the drive casing was removed.Wells were developed by purging 15 casing volumes fromeach well. Each well was equipped with a dedicatedWaterra� (Bellingham, Washington) pump consisting ofa foot valve attached to 5/8-inch high-density polyethylene

tubing. Multilevel wells were constructed of 1.5-inch out-side diameter continuous multichannel tubing with five 6-inch screened sample ports spaced vertically at 2.5 feet in-tervals such that monitoring points were uniformly distrib-uted across the fence of multilevel wells.

The system includes injection and extraction wells andaboveground infrastructure, including piping, process instru-mentation, and process controls. The locations of monitor-ing and recirculation wells are presented in Figure 1. Thesystem was operated in three phases, including baseline(recirculation of unamended ground water from August 7,2002, to October 22, 2002, 76 d), biostimulation (recir-culation of ground water amended with electron donor fromOctober 22, 2002, to February 7, 2003, 108 d), and bio-augmentation (bioaugmented on February 7, 2003, with re-circulation of ground water amended with electron donoruntil October 14, 2003, 249 d). In accordance with regula-tory requirements, ground water was treated using activatedcarbon to remove volatile organic compounds (VOCs) dur-ing the baseline phase only.

A tracer test was performed during the baseline phaseof the study. Reinjected ground water was amended for5 d with 50 mg/L bromide (KBr). Programmable autosam-plers were employed to collected ground water samplesfrom MW-6 and FL-2 (two samples per day) for the pur-pose of generating breakthrough curves at these monitor-ing locations.

Electron Donor Amendment and BioaugmentationElectron donor dosing was based on providing a four-

fold excess of the electron donor concentration required toreduce all electron acceptors in the recirculated ground water(predominantly TCE, 261 mg/L, and sulfate, 285 mg/L).Starting on day 76, weekly electron donor amendment con-sisted of a 5-min pulse of 10% denatured ethanol (SDA-3;Ashland Chemical, Dublin, Ohio) into each injection wellcorresponding to a time-weighted average ethanol concen-tration of 520 mg/L (9.3 mM). Electron donor additionwas performed throughout both the biostimulation and thebioaugmentation phases of the study.

Table 1Summary of Microcosm Study Results

Amendments Initial TCE (mg/L)

Half-Life2 (d)

Methanogenic Activity % Ethene1 (d)TCE VC

Sterile control 23 ND ND Not detected 0 (274)Unamended control 21 92 ± 77 199 ± 129 Not detected 6 (274)MEAL 23 17 ± 13 9 ± 4 Present 100 (154)MEAL + KB-1 26 5 ± 7 2 ± 0 Not detected 100 (31)MEAL + KB-1 74 5 ± 1 5 ± 1 Not detected 97 (52)MEAL + KB-1 132 1 ± 0 25 ± 3 Not detected 82 (653)MEAL + KB-1 267 20 ± 14 69 ± 37 Not detected 73 (653)MEAL + KB-1 637 ND ND Not detected 0 (653)

Notes: Half-lives presented for TCE and VC biodegradation assume first-order reaction kinetics. MEAL is an acronym for methanol, ethanol, acetate, and lactate (details ofelectron donor addition are provided in the supplementary material). The absence of measurable compound degradation is denoted by ‘‘ND.’’ Results shown are based on trip-licate microcosms. ‘‘% ethene’’ represents the average mole fraction of the total chloroethenes present as ethene following the incubation period specified in parentheses.

E.D. Hood et al./ Ground Water Monitoring & Remediation 28, no. 2: 98–107100

Culture enrichment and production are described in thesupplementary material. The test plot was bioaugmentedon day 184 of the study after 108 d of electron donor addi-tion. To minimize oxygen exposure, the culture was dis-pensed by pressurizing the vessel with argon, pushing theculture through a submerged delivery line into the injec-tion well. Each of the three injection wells were bioaug-mented with 13 L of KB-1.

Results and Discussion

Microcosm StudyHalf-lives for TCE biodegradation and ethene forma-

tion (equivalent to the half-life for VC dechlorination) foreach set of control and treatment microcosms are summa-rized in Table 1. No losses of volatile compounds occurredin sterile controls. In the unamended microcosms, slowTCE biodegradation resulted in the accumulation of cis-DCE more than 274 d. The limited extent of dechlorinationto cis-DCE in the unamended controls is consistent withthe extent of reductive dechlorination under intrinsic con-ditions at LC34 (CRA Services 1999).

In nonbioaugmented, electron donor–amended micro-cosms, complete dechlorination of all added TCE (0.2 mM)to ethene occurred after 154 d (Table 1), confirming thatdechlorination in the intrinsic microcosms was electrondonor limited and that indigenous Dehalococcoides organ-isms present in this specific soil sample were capable ofcomplete TCE dechlorination to ethene.

Addition of KB-1 to the microcosms significantlyincreased dechlorination rates, with complete dechlorina-tion of all added TCE to ethene occurring after only 31 dof incubation (Table 1). Half-lives for TCE and VC dechlor-ination were comparable in the bioaugmented treatmentmicrocosms with initial TCE concentrations of 0.2, 0.6,and 1.0 mM; however, TCE dechlorination was markedlyslower in microcosms with an initial TCE concentration of2.0 mM and completely inhibited in microcosms with thehighest initial TCE concentration of 4.8 mM (637 mg/L).Similarly, VC dechlorination was slower at TCE concen-trations of 1.0 mM and was completely inhibited at 4.8mM. Methanogenesis occurred in the nonbioaugmentedtreatment microcosms only at the lowest initial TCE con-centration (0.2 mM) and appeared to be inhibited in allother treatments.

Characterization of Indigenous Dehalococcoides OrganismsA 1390 bp PCR product was obtained from a soil

sample using Dehalococcoides-specific primers (see supple-mentary material). This sequence aligns with Dehalococ-coides in the Pinellas group (Hendrickson et al. 2002) butwas distinct with a 20-bp difference (98% identity) fromthe most similar group of sequences that include Dehalo-coccoides KB-1/PCE (AY146780), BAV-1 (CP000688),and CBDB1 (AJ965256). The National Aeronautics andSpace Administration (NASA) indigenous sequence wasonly 97% similar with strain VS (AY323233) and strain195 (CP000027) with 36- to 38-bp differences, respectively.Since 16S rRNA gene–based phylogenetic designations are

Figure 1. Site layout, showing the locations of injection and extraction wells, monitoring wells, and multilevel monitoring wells(arrows on particle trajectories indicate 5 d travel time).


subject to significant phenotypic ambiguity (i.e., they donot predict the degradative abilities of the species [Duha-mel et al. 2004]), further testing of the baseline DNA wasperformed using a commercially available PCR test(SiREM, Gene-Trac VC) specific to a VC reductase (vcrA)gene found in dechlorinating cultures VS and KB-1(Muller et al. 2004). We did not detect DNA correspondingto vcrA in these LC34 samples based on detection of a PCRproduct by conventional gel electrophoresis and staining(see supplementary material).

Pilot Test HydraulicsAnalysis of the tracer breakthrough curves using the

first-moment method (Freyberg 1986) resulted in an esti-mated average linear ground water velocity along thecenterline of the test plot of 0.75 ft/d (274 ft/year) corre-sponding to a residence time of 24 d. Visual MODFLOWwas used to simulate steady-state ground water flow. Theaquifer was modeled using a hydraulic conductivity for theUSU of 5 3 10�5 m/s, a porosity of 0.33, and a regionalgradient of 0.0001 ft/ft. Particle trajectories indicated thatflow within the test plot was primarily horizontal at re-circulation rates of 1.5 gallons per minute (Figure 1).Based on this analysis, residence times in the test plotranged from 24 d through the centerline (which agreesclosely to that estimated by the tracer test) to 32 d alongthe edge.

An active ground water recirculation system was em-ployed to simplify geochemical data interpretation by min-imizing flow variability and to decrease the duration of thestudy by increasing the ground water velocity. However, asa consequence of the stagnant flow in the surroundingaquifer, much of the ground water captured by the extrac-tion wells originated from outside the test plot and con-tained significant concentrations of TCE and sulfate.These constituents, along with TCE biodegradation prod-ucts, were continuously reinjected into the test plot.

Geochemical ParametersA summary of test plot geochemistry representing

conditions at the end of each phase of the study is pro-vided in the supplementary material (Table A). Measur-able concentrations of ethanol were not observed in anyground water samples; however, increasing concentra-tions of acetate, propionate, and butyrate (Table A insupplementary material) suggest that ethanol was rapidlyfermented by indigenous microorganisms, presumably re-sulting in the production of hydrogen, the only knownelectron donor for Dehalococcoides (He et al. 2003).Acetate appeared to accumulate in the test plot and break-through at the extraction wells occurred 108 d followingthe start of electron donor addition. The concentration ofdissolved iron and manganese did not appear to increasewithin the test plot, suggesting that there may not havebeen a significant mineral-phase reservoir of these reduc-tants (data not shown).

The average baseline sulfate concentration in the testplot was 315 mg/L. Following electron donor addition, theconcentration of sulfate decreased concurrently with TCEdechlorination to 2 mg/L, indicating an increase in the

activity of sulfate-reducing microorganisms. During theperiod of electron donor addition, sulfate reduction wassufficiently rapid that sulfate was typically detected onlyin the two monitoring wells closest to the injection wells(MW-3 and PA-26).

The activity of methanogens was of particular inter-est, especially given the potential problems of inefficientelectron donor utilization and aquifer fouling (Yang andMcCarty 2002). The average methane concentration obser-ved during the baseline monitoring period was 0.4 mg/L.During most of the period of electron donor addition, meth-ane concentrations did not increase; however, during thebioaugmentation phase of the study, an increase in meth-anogenesis occurred subsequent to the onset of ethene pro-duction (average methane concentration, 14 mg/L). Theinhibition of methanogenesis prior to the onset of etheneproduction is consistent with the results of prior studiesindicating that methanogenesis is inhibited by high chlor-oethene concentrations (e.g., Nielsen and Keasling 1999;DiStefano et al. 1991; Yang and McCarty 2002; Lee et al.1997; Kennes et al. 1998).

TCE Dechlorination TrendsThe results of chloroethene and ethene monitoring at

the centerline monitoring wells are presented in Figure 2.In general, similar time trends were observed in each ofthese wells. During the baseline phase, TCE was the domi-nant chloroethene (average concentration of 1.2 mM or155 mg/L), comprising 96% of the total ethenes concentra-tion. The primary daughter product was cis-DCE; ethenewas not detected. Following the addition of electron donor,TCE concentrations rapidly decreased, while concen-trations of cis-DCE and VC increased, indicating that thehigh initial TCE concentration did not inhibit reductivedechlorination. During the bioaugmentation phase, theTCE concentration in each monitoring well continued todecline to concentrations approaching the detection limitfor this analyte (20 lg/L) while VC accumulated. Aftera 5-month lag, including approximately 3 months of re-circulation system downtime, VC dechlorination rates rap-idly increased, as shown by substantial increases in etheneconcentrations throughout the test plot. At the end of thedemonstration, the average ethene concentration in the testplot was 2.4 mM (67 mg/L), a stoichiometric excess incomparison to the initial TCE concentration. This result in-dicates that the bioremediation treatment enhanced theremoval of TCE mass (either sorbed to soil or present asDNAPL). This ethene concentration is approximately anorder of magnitude greater than the ethene concentrationreported to inhibit methanogenesis (Yang and McCarty2000); however, the rapid increase in ethene concentrationwas sequentially followed by an apparent increase in meth-anogenesis (described in the previous section), suggestingthat ethene did not exert an inhibitory effect.

Quantitative Analysis of the Extent of DechlorinationThe extent of reductive dechlorination was also moni-

tored at the downgradient fence of multilevel samplers.For each of the seven sampling events (25 sampleseach), the extent of dechlorination in each sample was


characterized by calculating the fraction of chlorineremoved from the initial concentration of the TCE (calcu-lated based on the sum of the concentration of all chloro-ethenes and ethene) using:

Dechlorination ð%Þ

=

1� 3½TCE� þ 2½cis-DEC� þ ½VC�

3ð½TCE� þ ½cis-DEC� þ ½VC� þ ½ethene�Þ

!100

where the parentheses indicate molar concentrations andscores of 33%, 66%, and 100% represent complete conver-sion to either cis-DCE, VC, or ethene, respectively. Sum-mary statistics of the dechlorination scores for eachsampling event (Figure 3) demonstrate that (1) TCE dechlo-rination occurring during the baseline phase was negligible;(2) dechlorination during the biostimulation phase princi-pally resulted in the accumulation of cis-DCE; (3) followingbioaugmentation, there was a gradual increase in the extentof dechlorination past cis-DCE; and (4) ethene was the prin-cipal dechlorination product in the final sampling event.These results suggest that during the bioaugmentation phase,there was a shift in the dechlorinating microbial communityfavoring organisms capable of efficient dechlorination ofboth cis-DCE and VC to ethene. However, it is not possibleto distinguish between the effects of bioaugmentation vs.other factors (e.g., electron donor addition) on dechlorinat-ing activity.

Confirmatory ground water samples were collectedfrom the centerline monitoring wells 22 months followingthe cessation of ground water recirculation. The results ofVOC/dissolved hydrocarbon gas analysis of these sam-ples are provided in the supplementary material (Table A).There were no detections of TCE in these samples, con-firming that VOC concentrations did not rebound after theperiod of active ground water treatment, and ethene re-mained the dominant degradation product in the test plot.However, there was a sevenfold decrease in the total chlor-oethene concentration, suggesting that there may havebeen some loss of ethene from the test plot. Under highlymethanogenic conditions, biodegradation of cis-DCE, VC,and ethene via anaerobic oxidation can occur (Dolfing1999; Bradley and Chapelle 1998, 1999), and this processmay be the cause of the apparent ethene sink.

Quantification of Dehalococcoides in Ground WaterSamples

Dehalococcoides 16S rRNA genes copies were quanti-fied in DNA extracted from ground water samples through-out the study, including baseline ground water samples.The initial Dehalococcoides concentration was approximately108 copies/L. Following the start of electron donor addi-tion, the Dehalococcoides concentration increased toapproximately 1011 copies/L, a significant increase of 2 to3 orders of magnitude that was sustained throughout theremainder of the study (Figure A, supplementary material).

Figure 2. Concentration trends in the centerline monitoring wells during the baseline, biostimulation, and bioaugmentation phasesof the study for TCE (s), cis-DCE (n), VC (n), ethene (d), methane (:), and total ethenes (unmarked line).


TCE Mass RemovalThe results of pre- and postdemonstration soil sam-

pling are summarized in Figure 4. In the predemonstrationdata set, TCE concentrations span 6 orders of magnitude(maximum 8300 mg/kg), with several samples exceedingthe bulk TCE concentration indicative of the presence ofDNAPL (Battelle 2004). Additional evidence of initialDNAPL presence in the test plot included TCE concen-trations in ground water samples exceeding the solubilitylimit (ML4-5, samples collected on August 19, 2002;February 7, 2003; April 3, 2003; and January 7, 2003) withTCE concentrations in samples collected from four othersampling locations (ML1-5, ML2-5, ML3-5, and ML5-1)exceeding 50% of the solubility limit.

In contrast, bulk soil concentrations in the postde-monstration data set were much lower with numerousnondetects (Figure 4). Interestingly, decreases in TCEconcentration in the middle fine-grained unit underlyingthe test plot suggest that electron donor addition and bio-augmentation provided benefit outside the intended treat-ment zone.

The total mass of TCE (i.e., including aqueous, non-aqueous phase liquid, and adsorbed TCE) contained in thetest plot was determined using the linear interpolation andkriging methods described by Quinn et al. (2005). Usingthe linear interpolation method, pre- and postdemonstrationTCE mass estimates of 25.5 and 0.4 kg (corresponding to98.5% removal) were determined. Using the krigingmethod, pre- and postdemonstration TCE mass estimates of32.1 and 0.2 kg (corresponding to 99% removal) weredetermined. Although the reliability of total mass estimatesmade using either method is difficult to assess, the largereduction in total TCE mass in the USU and the occurrenceof multiple nondetects for TCE indicates that enhancedbioremediation effectively removed TCE mass from thetest plot.

Figure 4. Bulk concentrations of TCE in (a) pretreatment and(b) posttreatment test plot soil samples (open symbols representsamples below the method detection limit). Note that the re-circulation system targeted electron donor delivery only withinthe USU. The water table is located approximately 4 ft bgs.

Figure 3. The extent of dechlorination at the downgradient multilevel sampling fence. Box and whiskers represent the minimum,first quartile, median, third quartile, and maximum values (n ¼ 25; dashed lines at 33% and 66% represent complete conversion ofthe parent TCE to either cis-DCE or VC, respectively).


Mass removal was also assessed by integrating theeffluent ethene concentrations over the duration of thestudy to determine the cumulative mass of ethene con-tained in the extracted ground water. The extracted massof ethene, which was primarily removed in the final 2months of the study, was 36 kg (as an equivalent mass ofTCE), which is comparable to the mass of TCE initiallycontained with the test plot.

Further interpretation of the effluent monitoring data iscomplicated by the continuous reinjection of TCE capturedfrom outside the test plot and the recirculation of degrada-tion products. However, assuming that the final round ofsamples collected during the study correspond to a steady-state condition (a conservative assumption since dechlori-nating activity appeared to be increasing, as shown inFigure 2) permits a simple half-life estimate. Using theTCE concentration in the recirculated ground water (0.2mM) and the disappearance of TCE (detection limit 0.01mg/L) by the first monitoring well (MW-3) after a traveltime of 7 d (based on data from the tracer test) and assum-ing that first-order kinetics apply, the estimated TCE bio-degradation half-life is 1 d, which is comparable to theTCE half-lives observed in the microcosm studies. Notethat actual TCE biodegradation rates in the test plot werelikely higher since this estimate does not account for sour-ces of TCE and relies on nondetect concentrations at thenearest monitoring well.

Implications for Source Area RemediationThe emerging success of enhanced bioremediation ap-

proaches such as biostimulation and bioaugmentation (e.g.,Major et al. 2002; Lendvay et al. 2003) demonstrates thatthis technology can effectively enhance chloroethene bio-degradation at concentrations typical of those encounteredin ground water plumes. This study demonstrates that theapplication of this technology in DNAPL source areas,formerly considered infeasible due to microbial toxicitycaused by high VOC concentrations, may in fact be arelevant remediation technology capable of containmentVOC-impacted ground water within the source area and/orenhancing the removal of nonaqueous and sorbed contami-nant mass. In spite of very high initial TCE concentrations,the addition of electron donor and bioaugmentation re-sulted in the production of very high ethene concentrations.As previously suggested (Yang and McCarty 2002), highVOC concentrations appear to have had a beneficial effectby inhibiting the activity of methanogenic bacteria, therebyincreasing the efficiency of electron donor utilization bydechlorinating microorganisms.

Although the presence of a native Dehalococcoidesorganism capable of dechlorinating both cis-DCE and VCin ground water at LC34 implies that bioaugmentation wasnot strictly required, the slow intrinsic rates of dechlorina-tion observed at both the field scale and in laboratorymicrocosms and the below detection limit levels of the vcrAgene perhaps indicate that the indigenous Dehalococcoideswere not efficient VC degraders. While it is true that thevcrA gene is not necessary for VC degradation (e.g., in strainBAV-1 [He et al. 2003]), it certainly is found in many

VC-degrading cultures (Muller et al. 2004; Lee et al. 2006).Although the strict necessity for test plot bioaugmentationwas not assessed as part of this study, the apparently hetero-geneous distribution of these dechlorinating organisms inground water (Fennell et al. 2001), the absence of dechlori-nating activity under intrinsic conditions at LC34, the slowrate of ethene formation in nonbioaugmented microcosms,and the relatively minimal cost of bioaugmentation indicatethat bioaugmentation was a prudent measure to ensure effec-tive treatment. Accordingly, bioaugmentation should be con-sidered at sites where the indigenous Dehalococcoidespopulation has no demonstrated capability to efficiently useVC as a metabolic electron acceptor or where competentDehalococcoides strains are heterogeneously distributed orat low concentration, in addition to those sites where Deha-lococcoides are absent, to increase the likelihood of success-ful bioremediation.

The rapidly expanding regulatory interest in the appli-cation of source area bioremediation is the focus of theInterstate Technical Regulatory Council’s Bioremediationof DNAPL working group. Although, currently, there arefew well-documented studies evaluating this technology atthe field scale, it is being increasingly applied at industrialsites and it is likely that more case studies will emerge inthe near future. Significant issues associated with this tech-nology include its long-term cost and performance and thepotential benefits of partial dechlorination with respect tothe aquifer’s attenuation capacity for cis-DCE and VC viadegradation through alternative degradation pathways (e.g.,aerobic oxidation).

Supplementary MaterialThe following supplementary material is available for

this article:Demonstration of Enhanced Bioremediation in

a TCE Source Area at Launch Complex 34, Cape Can-averal Air Force Station

This material is available as part of the online articlefrom: http://www.blackwell-synergy.com/doi/abs/10.1111/j.1745-6592.2008.00197.x

(This link will take you to the article abstract).Please note: Blackwell Publishing is not responsible

for the content or functionality of any supplementary mate-rials supplied by the authors. Any queries (other than miss-ing material) should be directed to the correspondingauthor for the article.

AcknowledgmentsThe authors would like to acknowledge the contribu-

tions of the Geosyntec staff (L. MacKinnon, D. Bertrand,J. Coughlin, R. Sosa, and M. Wissler) and Phil Dennis(SiREM). Some chloroethene monitoring data were pro-vided by M. Annable (University of Florida). Microcosmswere conducted by Christina Heidorn (University of Toronto).Quantitative PCR analyses were conducted with the helpof Alison Waller (University of Toronto). The project wasfunded by the NASA Small Business Innovation Researchprogram (contract no. NAS10-00069) with supplementary


funding provided by the Environmental Security Technol-ogy Certification Program under project CU-0016.

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Editor’s Note: The use of brand names in peer-reviewedpapers is for identification purposes only and does not con-stitute endorsement by the authors, their employers, or theNational Ground Water Association.

Biographical SketchesEric D. Hood, Ph.D., P.Eng., corresponding author, is a

Senior Scientist at Geosyntec Consultants Inc., 130 Research Lane,Suite 2, Guelph, Ontario, Canada N1G 5G3; (519) 822-2230;fax (519) 822-3151; [email protected].

David W. Major, Ph.D., is a Principal at Geosyntec Con-sultants Inc., 130 Research Lane, Suite 2, Guelph, Ontario,Canada N1G 5G3.

Jacqueline W. Quinn, Ph.D., is an Environmental Engineerat the National Aeronautics and Space Administration, Mail StopYA-C3-C, Kennedy Space Center, FL 32899.

W.-S. Yoon, is an Environmental Engineer in the Environmen-tal Restoration Section at the Battelle Memorial Institute, 505King Ave., Columbus, OH 43201.

Arun Gavaskar, M.Sc., is an Associate Manager in the Envi-ronmental Restoration Section at the Battelle Memorial Institute,505 King Ave., Columbus, OH 43201.

Elizabeth A. Edwards, is a Professor in the Department ofChemical Engineering and Applied Chemistry at the University ofToronto, 200 College Street, Toronto, Ontario, Canada, M5S 3E5.


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Remediation of Dense Non-Aqueous Phase Liquids Using Biostimulation and Bioaugmentation