Evolution of predictive tools for in situ bioremediation and natural attenuation evaluations

REMEDIATION Autumn 2010

Evolution of Predictive Tools for In SituBioremediation and Natural AttenuationEvaluations

Matthew Burns

Colleen Myers

Proving the viability of in situ bioremediation technologies and gathering data for its full-scale

implementation typically involves collecting multiple rounds of data and often completing micro-

cosm studies. Collecting these data is cumbersome, time-consuming, costly, and typically difficult

to scale. A new method of completing microcosm studies in situ using an amendable sampling

device deployed and incubated in groundwater monitoring wells provides actionable data to ex-

pedite site cleanup. The device, referred to as a Bio-Trap® sampler, is designed to collect actively

colonizing microbes and dissolved organic compounds from groundwater for analysis using con-

ventional analytical techniques and advanced diagnostic tools that can answer very specific design

and viability questions relating to bioremediation.

Key data that can be provided by in situ microcosm studies using Bio-Trap® samplers include

definitively demonstrating contaminant destruction by using compound-specific isotope analysis

and providing data on the mechanism of the degradation by identifying the responsible microbes.

Three case studies are presented that demonstrate the combined flexibility of Bio-Trap® samplers

and advanced site diagnostics. The applications include demonstrating natural attenuation of dis-

solved chlorinated solvents, demonstrating natural attenuation of dissolved petroleum compounds,

and using multiple Bio-Trap® samplers to comparatively assess the viability of bioaugmentation at

a chlorinated solvent release site. At each of these sites, the in situ microcosm studies quickly and

cost-effectively answered key design and viability questions, allowing for regulatory approval and

successful full-scale implementation. Oc 2010 Wiley Periodicals, Inc.

INTRODUCTION

The interests of remediation professionals, property owners, regulatory authorities, andother contaminated site stakeholders align when remediation, by any technology,progresses in a deliberate, cost-effective, and successful manner. Outside of theseconditions, conflicting interests often lead to project delays and dissatisfaction for allinvolved. For in situ remediation technologies, conflicting interests are often encounteredwhile justifying or proving that an alternative is viable. Specifically, proving actualdegradation and the mechanism of degradation using the weight-of-evidence approachadvocated by the U.S. Environmental Protection Agency (US EPA Office of Solid Wasteand Emergency Response, 1999), the American Society for Testing and Materials (ASTM;1998), the Interstate Technology Regulatory Cooperation Workgroup (1998), and others

c© 2010 Wiley Periodicals, Inc.View this article online at wileyonlinelibrary.com. DOI: 10.1002/rem.20259 5

Evolution of Predictive Tools for In Situ Bioremediation and Natural Attenuation Evaluations

has, until recently, relied on numerous rounds of costly and time-consuming datacollection. This level of data is understandable from some stakeholder perspectives whenmechanisms of destruction are unseen and not as well understood, and when thenonreactive effects of amendment application (e.g., dilution and displacement) affectconcentration data in an identical manner as the desired reactive mechanism.

Protocols to demonstrate applicability of microbial-mediated in situ technologieshistorically require collection of multiple layers of data and the completion of ex situmicrocosm studies, which are typically difficult to scale to the field. The results of theseactivities are often tangential to the information necessary to demonstrate success in thefield. While techniques, such as the Air Force Center for Engineering and theEnvironment (AFCEE) protocol (Wiedemeier et al., 1998), remain pertinent as apreliminary screening tool of concentration data, new analytical tools, which are based ongenetic and stable isotope fingerprinting and are broadly classified as advanced sitediagnostics (ASDs), can more directly and cost-effectively demonstrate contaminantdegradation and the mechanism of the degradation. Combining ASDs with microcosmscompleted in situ creates a powerful technique for predictive analysis, thereby providingan aide in aligning stakeholder interests and expediting site cleanup.

Protocols to demonstrateapplicability of microbial-mediated in situ tech-nologies historically requirecollection of multiple layersof data and the completionof ex situ microcosm stud-ies, which are typically diffi-cult to scale to the field.

MICROCOSM TESTING

In situ microcosm testing was initially conducted by injecting groundwater spiked with acompound of interest into an aquifer, collecting samples for ex situ analysis from the slugof spiked groundwater, and observing changes in concentration with time. In thissingle-well method, the studies were limited by local heterogeneity, the reaction raterelative to advection velocity, and the large volumes of injected amendment fluid andmonitoring points required for sampling.

To mitigate these limitations, the in situ microcosm (ISM) method was developed bythe University of Waterloo (Nielsen et al., 1996). The ISM consists of a pipe with screensthat allow water to be pumped into or out of the interior, which contains a test chamber,an equipment chamber, and two screens. Installation involves advancing a boring to thedesired interval, driving the ISM unit beyond the base of the boring, and plumbing it tothe surface. Upon installation, groundwater from the ISM chamber is extracted, spiked,and reinjected through the main screen. Temporal groundwater samples are thencollected from the ISM for ex situ analysis to quantify the effects of the amendment. At theend of the study, the amended saturated soils can also be retrieved with the ISM unit todetermine its physical and chemical characteristics. Additionally, as the majority of themicrobial biomass in aquifers is associated with the sediment, and only a small fraction ispresent in the groundwater, microbial observations have been made on sedimentcollected during retrieval of the ISM.

The ISM method was a significant improvement on the previous in situ testingmethods and required much less time and equipment; however, ISM testing applicationwas limited because the test zone is isolated from the natural advective and dispersiveprocesses in the aquifer and can only be conducted in reasonably permeable geologicmaterials; soil biomass sampling cannot be conducted during, only after, the groundwatersampling is complete; and developing an appropriate sampling plan requires advancedknowledge of reaction rates.

6 Remediation DOI: 10.1002/rem c© 2010 Wiley Periodicals, Inc.


An ex situ design for microcosm tests for the purpose of estimating the in situ rateconstant for the biodegradation of chemicals of concern (COCs) was presented by the USEPA in Wiedemeier et al. (1998). This design involves collecting samples consisting ofsoil and adding groundwater to closely simulate subsurface conditions. These tests arecumbersome and expensive, require sacrifice of entire microcosms for extraction andanalysis at each time point, and can require 12 months to complete. Simplified microcosmtest procedures were also developed by the Air Force (Morse et al., 1998) to determine ina more cost-effective manner whether natural attenuation is occurring at a site, and toevaluate the potential for enhancing the natural attenuation by testing amendments thatwould speed up the natural process. The testing methods are similar to the US EPA’s;however, analyses can be conducted during several time intervals, and the results fromspiked samples can usually be obtained in three to six months.

An ex situ design for mi-crocosm tests for the pur-pose of estimating the insitu rate constant for thebiodegradation of chemi-cals of concern (COCs) waspresented by the US EPA inWiedemeier et al. (1998).

As described in the aforementioned microcosm studies, flaws are inherent whenattempting to replicate macro-scale processes in micro-scale experiments. Controllingscaling variables using these and other “traditional” microcosm approaches is either toocostly, time-consuming, or difficult to accomplish to be practical at many sites. MicrobialInsights of Rockford, Tennessee, has developed a novel approach to in situ microcosmsthat unambiguously identifies contaminant destruction and the mechanism of destructionin a timely and cost-effective manner (Sublette et al., 2006). The approach replacescollecting groundwater samples using a conventional approach, and combines the benefitsof ex situ protocols with many of the benefits of the in situ protocols. The results providemicrobial, chemical, and geochemical evidence to screen remedial alternatives andevaluate biodegradation as a treatment mechanism.

Microbial Insights’ approach is based on a unique sampling tool called a Bio-Trap®

sampler. Bio-Trap® samplers are polyvinyl chloride (PVC) screen enclosures that houseBio-Sep® beads. The beads are 2 to 4 millimeters in diameter and are an engineeredcomposite of Nomex® and powdered activated carbon (Sublette et al., 2006). TheBio-Trap® samplers are amendable with biostimulants, microbial cultures, contaminantanalogs, and many other materials that may or may not have an effect on site microbialecology. When a Bio-Trap® sampler is deployed in a monitoring well, the Bio-Sep® beadspassively adsorb contaminants present in the aquifer and provide a large surface area forbiofilm growth, similar to the sediment particles comprising aquifer materials. After 30 to90 days of incubation, the Bio-Trap® samplers and any other passive samplers deployedconcurrently with the Bio-Traps® (e.g., grab and passive diffusion samplers) are retrievedand are subjected to any number of conventional or ASD analyses.

BIO-TRAP® In Situ MICROCOSM STUDIES

Bio-Trap® in situ microcosm studies have been used as tools to assess many contemplatedremedies at sites with widely varying contaminants, geochemical characteristics, andlithology. Below, case studies for natural attenuation of chlorinated ethenes, naturalattenuation of petroleum, and enhanced bioremediation of chlorinated ethenes arepresented to show some of the tool’s versatility in both application and the range of ASDsthat can be applied to provide definitive and actionable data to better assess and expediteimplementation of site-closure alternatives.

c© 2010 Wiley Periodicals, Inc. Remediation DOI: 10.1002/rem 7


Combining Bio-Traps® and ASDs in Support of Monitored NaturalAttenuation of Chlorinated Ethenes

In their simplest form, Bio-Trap® samplers can be deployed without amendments within asite monitoring well to assess the microbial ecology as it pertains to monitored naturalattenuation (MNA). For example, Bio-Trap® samplers were deployed in this manner toassess MNA at a central Georgia manufacturing facility. A historic release of coolantcontaining chlorinated solvents at the facility resulted in a 1,200-foot-long dissolvedvolatile organic compound (VOC) plume. The VOCs consist primarily of trichloroethene(TCE), 1,1,1-trichloroethane, and their respective daughter products formed byreductive dechlorination.

Temporal VOC concentration trends at the site vary based on the position within theplume; source-area concentrations increased (50 mg/L, maximum), indicating acontinuing vadose-zone source while downgradient concentrations decreased, showing astable to decreasing footprint. An MNA evaluation completed in general accordance withthe AFCEE protocol showed ambiguous evidence of traditional reductive dechlorinationdata. Specifically, the terminal electron accepting data were contradictory but generallyindicated slightly aerobic conditions that are not consistent with biologically mediatedreductive dechlorination.

Temporal VOC concen-tration trends at the sitevary based on the po-sition within the plume;source-area concentra-tions increased (50 mg/L,maximum), indicating acontinuing vadose-zonesource while downgradientconcentrations decreased,showing a stable todecreasing footprint. As part of the MNA evaluation, nonamended Bio-Trap® samplers were deployed and

incubated for seven weeks before being retrieved. The microbes that colonized on theBio-Sep® beads were assessed using phospholipid fatty acid (PLFA) and quantitativepolymerase chain reaction (qPCR) analysis techniques. PLFAs are components of allcellular membranes and can be readily extracted from environmental samples andanalyzed using conventional analytical methods. PLFA measurements provide informationon total microbial biomass and indicate the presence of specific microbial groups thatshare membrane biochemical signatures. PLFA data from the Georgia site identified amoderate population of microbes (2.18 × 104 cells/bead to 6.64 × 104 cells/bead) thatincluded hydrogen-producing firmicutes (average of 5.4 percent of the total population)that many dechlorinating microbes use as an energy source. The PFLA data also identifieda low fraction of metal- and sulfur-reducing microbes, which can compete withdechlorinating microbes.

The qPCR analysis is an ASD tool that quantifies signature DNA sequences specific fora certain microorganism or functions of interest. The qPCR analysis is an important toolin assessing bioattenuation because it enables the quantification of key microbesresponsible for degradation. For example, qPCR is used to identify the presence ofMethylibium Petroleiphilum (PM1), which is one of the only known microbes capable ofaerobic growth on methyl tertiary butyl ether (Hanson et al., 1999), and Dehalococcoidesspp. (DHC), which is the only known genera of microbes known to completely degradechlorinated ethenes (Lu et al., 2006). The qPCR data at the Georgia site showed thepresence of a significant population of DHC (range of 2.57 × 102 cells/bead to 6.31 ×104 cells/bead). The presence of native DHC is a significant finding because it identifiesthe mechanism by which the VOCs present in the downgradient portion of the plume arebeing reduced. Furthermore, because DHC are obligate anaerobes (i.e., they cannotsurvive in aerobic environments), at a minimum, anaerobic microenvironments thatsupport microbial attenuation of the chlorinated VOCs must be present. Thus, theelectron acceptor data indicating aerobic conditions were refuted.



Based on these data, regulatory approval was secured in 2007 for an integratedremediation approach consisting of vadose-zone source excavation, in situ chemicaloxidation (ISCO) for source-area groundwater containing total VOC concentrationsgreater than 0.5 mg/L, and MNA for downgradient groundwater. Currently,groundwater concentrations in the source area have been treated to total VOCconcentrations of less than 0.5 mg/L, and the downgradient portion of the plume remainsstable. Performance monitoring of select wells is performed annually to document theplume stability.

Combining Bio-Traps® and ASDs in Support of MNAof Petroleum Compounds

As mentioned earlier, amended Bio-Trap® samplers are employed to evaluate remedialalternatives and evaluate biodegradation as a treatment mechanism. At a petroleumrelease site in Tennessee, amended Bio-Trap® samplers were used to assess naturalattenuation. At the site, multiple historic releases of petroleum products from varioussources resulted in a dissolved petroleum plume across a 6.5-acre area; free product wasnot observed. Concentration data showed a temporally decreasing dissolved total organiccarbon concentration trend, anaerobic conditions, and naturally high sulfateconcentrations present in karstic geology.

Based on these data,regulatory approval wassecured in 2007 for anintegrated remediationapproach consisting ofvadose-zone source ex-cavation, in situ chemicaloxidation (ISCO) forsource-area groundwatercontaining total VOCconcentrations greaterthan 0.5 mg/L, and MNAfor downgradient ground-water.

Bio-Trap® samplers were deployed and allowed to incubate for 55 days. TheBio-Sep® beads were then subjected to PLFA, stable isotope probing (SIP), and densitygradient gel electrophoresis (DGGE) procedures. The PLFA data identified a moderatepopulation of microbes (2.7 × 105 cells/bead average) and a community structureprimarily composed of proteobacteria (approximately 70 percent of the population),which include many hydrocarbon-utilizing bacteria.

The SIP data also identified the presence of native hydrocarbon-utilizing microbes.The SIP process involved amending Bio-Trap® samplers with carbon-13 (13C)-labeledbenzene and tracking the disposition of the 13C label to identify its ultimate fate. The basisfor using 13C as a label is that carbon is naturally present in two stable (i.e., notradioactive) isotopes, 13C and carbon-12 (12C). The natural abundance of 13C in benzeneand most organic compounds is approximately 1 percent, while the abundance of13C-labeled benzene used for SIP is typically between 10 and 15 percent. The differencein 13C abundance between naturally occurring organic compounds and the labeledbenzene is sufficient analytically to track 13C incorporation into cellular mass andinorganic carbon (by-product of respiration). SIP data for the Tennessee site are shown inExhibit 1. The data show a decrease in 13C-labeled benzene concentrations sorbed to theBio-Sep® beads from pre-deployment concentrations and enrichment of 13C within thebiomass. These data definitively prove contaminant degradation by native microbes.

DGGE is a genetic fingerprinting technique that provides a profile representing thegenetic diversity of a microbial community. It is an electrophoretic method that usesdifferences in each nucleotide’s affinity for a medium to separate different nucleotidesequences. After separation, bands of identical nucleotide sequences are amplified,sequenced, and compared to a database for identification. Exhibit 2 shows the DGGEband image for the Tennessee site Bio-Trap® samplers; major bands include Pseudomonasspp (bands 1.1, 3.1, 3.2, and 4.1), Azoarcus spp. (bands 3.1 and 2.2), and Geobacter spp.(bands 1.5, 1.6, 2.1, 3.3, and 4.3). These microbes use a wide variety of organic



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Exhibit 1. Tennessee site SIP results

compounds, including petroleum compounds, as electron donors (Atlas, 1981; Lovleyet al., 1989, 1990; Ridgway et al., 1990; Widdel & Rabus, 2001).

Temporal COC trends suggested that biodegradation was occurring at the site. TheSIP data provided definitive evidence that the decreasing temporal trends wereattributable to biodegradation as opposed to other nondestructive mechanisms, such asdilution, dispersion, and sorption. The PLFA and DGGE data provided sequentially moredetailed information about the microbial population and the presence of knownpetroleum-degrading microbes, further supporting the temporal COC data observationsand SIP results. These data were crucial in making a successful case for regulatoryapproval of MNA at the site.

Controlled Experiments to Optimize Bioremediation UsingMultiple Bio-Traps®

By deploying amended and nonamended Bio-Trap® samplers simultaneously, controlledin situ microcosm experiments can be completed to compare natural conditions withspecific engineered conditions. These experiments can be expanded by using multipleamended Bio-Trap® samplers to evaluate effects of biostimulation and biostimulation incombination with bioaugmentation, screen a variety of bioremediation alternatives (e.g.,aerobic and anaerobic biostimulants), or serve as a platform for efficiency testing of



Exhibit 2. Tennessee site DGGE band image

products that perform similar functions, such as electron donors (e.g., HRC®, EOS®,lactate, etc.) and electron acceptors (e.g., ORC®, PermeOx Plus®).

Microbial Insights refers to these multi-Bio-Trap® configurations as assemblies. Anassembly typically consists of two to three Bio-Trap® samplers strung together fordeployment within a single well. Each “unit” of the assembly is isolated by baffles and canincorporate continuous-feed amendment devices, passive diffusion samplers, and simplescreen-capped vials to collect aqueous samples for contaminant or geochemical analysis.

Exhibit 3 is a schematic of a typical Bio-Trap® sampler assembly. Above theBio-Trap® sampler (labeled MICRO) are examples of aqueous sample-collection devices.The septa on these vials have been replaced with a passive diffusion membrane (labeledCOC) and a screen (labeled GEO). Below the Bio-Trap® is an example of a continuousamendment delivery device. Each of the samplers shown and the amendment suppliershown in the right side of Exhibit 3 comprise a stand-alone experiment that is housed in a1.25-inch-by-15-inch slotted PVC housing shown in the center of Exhibit 3. The left ofExhibit 3 shows the units strung together into an assembly, each of which is labeled with apotential experimental construct.



Exhibit 3. Bio-Trap® assembly configuration

A multiple-Bio-Trap® sampler microcosm study was completed at a site in Californiawhere a prior application of an electron donor to treat TCE-affected groundwater wasineffective due to dichloroethene (DCE) stall. Rather than blindly apply an augment to theelectron donor–applied area, a Bio-Trap® sampler assembly was deployed to assess thepresence of conditions that inhibit DHC. The assembly consisted of two units, one thatwas not amended and one that was amended with an augment that included DHC.Because an electron donor had been previously applied, application of a biostimulant withthe bioaugment was not necessary. The assembly was incubated for 62 days, retrieved,and subjected to qPCR to assess the natural and augmented microbial population andcompound-specific stable isotope analysis (CSIA) to track the isotopic signature ofnaturally occurring carbon isotopes 12C and 13C comprising the TCE and its daughterproducts for evidence of contaminant degradation. The results are presented in Exhibit 4.

The qPCR data show a high population of microbes as represented by total eubacteriaand a high population of DHC. The data also show that the DHC includes DNA sequencesfor key functional genes that code for enzymes responsible for specific degradation stepsalong the reductive dechlorination pathway. In addition, the data show a low populationof methanogens that can compete with DHC for hydrogen, the common electron donorof the two groups of microbes. The qPCR data demonstrated that conditions that inhibitDHC are not present and that a bioaugment applied at the site would likely be a successfulremedy.



Exhibit 4. California site in situ microcosm data

Control Bioaugment

Microbial Population (Cells/Bead) by qPCRTotal Eubacteria 3.87E+07 3.97E+07Dehalococcoides spp. 5.07E+01 1.25E+07

tceA Reductase ND 5.68E+05bvcA Reductase 9.00E+00 (J) 7.44E+04vcrA Reductase 3.86E+01 1.05E+07

Dehalobacter spp. 9.60E+03 2.43E+05Desulfuromonas spp. 2.86E+02 1.85E+03Methanogen 1.00E+03 2.38E+03

Isotopic Signature (13C[‰]) by CSIATrichloroethene ND −22.16Cis-1,2-dichloroethene −22.08 −6.16Trans-1,2-dichloroethene −22.06 −34.96Vinyl chloride ND −6.08

ND = analytical target not detected.

J = result between the method detection limit and the reporting limit.

The isotopic signature of TCE and its reductive daughter products was obtained byextracting VOCs that originated from the aquifer and sorbed to the Bio-Sep® beads duringincubation and subjecting the extract to CSIA. Similar to the SIP process described earlier,the foundation of CSIA relies on the fact that the majority of all carbon is present as the12C isotope, but a small percentage of carbon is naturally present as the stable (i.e., notradioactive) 13C isotope. Whereas SIP testing consists of introducing anthropogenicallyaltered COCs enriched with the 13C isotope into the aquifer system and measuringthe increase in 13C abundance in the end products of biodegradation (biomass anddissolved inorganic carbon), CSIA involves measuring the naturally occurring ratio of the13C isotope to the 12C isotope for selected COCs. Chemical bonds involving the 13Cisotope are slightly stronger than those of the 12C isotope and, as a result, react slower inbond-breaking reactions, including biodegradation reactions. The slower reaction rateleads to an accumulation of the 13C isotope in the residual contaminant. The accumulationof the 13C isotope is referred to as fractionation. During CSIA testing, the 13C isotope ismeasured and reported as a fractionation or part per thousand ratio of 13C to 12C relativeto an international standard ratio, or δ13C (‰). Therefore, because reduction of a parentcompound requires a chemical reaction, the 13C isotope within the residual pool of parentcompound would be expected to be enriched (a less negative ratio) and the daughterproducts would be expected to be depleted (a more negative ratio).

At the California site, the isotopic signature of TCE in the bioaugment microcosmunit (−22.16 ‰) is more enriched or less negative than the range of nondegradedmanufactured TCE (−27.37 ‰ to −31.57 ‰; Shouakar-Stash et. al., 2003). These datadefinitively prove that the TCE has been biodegraded. However, complete degradation of



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Initial Electron Donor Application: May-08

Exhibit 5. California site representative full-scale data

TCE to end-products requires multiple reductions (simultaneous enrichment anddepletion of the 13C isotope within the carbon pool comprising each of the reductionpathway intermediates). Therefore, when assessing the completeness of the degradationpathway, 13C enrichment of a specific daughter product to an extent greater than that ofthe parent compound provides evidence that the pathway is complete through thatenriched daughter product. When degradation stalls (e.g., DCE stall) or completedegradation occurs (e.g., to ethene), the isotopic signature of the most reduced daughterproduct approaches the initial isotopic signature of the parent compound. At the site, 13Cenrichment of daughter-product carbon was also observed in the bioaugment microcosmunit; the 13C comprising the vinyl chloride (−6.08 ‰) is enriched as compared to theisotopic signature of the fractionated TCE parent compound (−22.16 ‰) and the likelyinitial isotopic signature of nondegraded manufactured TCE (−27.37 ‰ to −31.57 ‰),thereby demonstrating a complete degradation pathway through vinyl chloride (i.e., noDCE stall). The isotopic signature of ethene was not measured at the site, but it isexpected that it has approached the initial isotopic signature of the parent TCE.

With the evidence of complete biodegradation to innocuous end products(fractionation of vinyl chloride to a greater extent than parent compounds) and theindication that a bioaugment would not be inhibited by site conditions provided by thein situ microcosm tests, bioaugmentation proceeded to full-scale application (additionalelectron donor was also applied). Three months and continuing to six months followingfull-scale bioaugment application, VOC concentrations decreased throughout the site.Exhibit 5 is a plot of historic groundwater VOC trends in a representative well. The plot



shows minimal concentration effects following the initial application of an electron donoralone and greater than 90 percent VOC concentration reduction after bioaugmentapplication.

CONCLUSIONS

Knowledge of in situ processes is improving, but to a large extent bioremediation iscompleted blindly as compared to many conventional technologies, such as groundwaterextraction and treatment, where design variables are well understood and easilyquantified. Accordingly, many protocols to assess microbial-mediated technologies relyon a weight-of-evidence approach that in most cases is time-consuming and costly. Onecomponent to the weight-of-evidence approach is the completion of microcosm studies.Traditional microcosm studies, however, have proven to be cumbersome, costly, anddifficult to scale. In situ microcosms using Bio-Traps® provide a framework to use ASD toprovide definitive data to assess contaminant destruction via isotopic analysis and identifythe mechanism of the destruction via a variety of molecular biological tools. As shownin the three case studies presented herein, Bio-Trap® -based in situ microcosm studieshave the potential to cost-effectively assess the viability of bioremediation and therebyalign interests of contaminated-site stakeholders and expedite site cleanup.

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Hanson, J. R., Ackerman, C. E., & Scow, K. M. (1999). Biodegradation of methyl tert-butyl ether by a bacterial

pure culture. Applied and Environmental Microbiology, 65, 4788–4792.

Interstate Technology and Regulatory Cooperation Workgroup. (1998, December). Requirements for

enhanced in situ bioremediation of chlorinated solvents in groundwater: Final. Retrieved from

http://www.itrcweb.org/gd.asp

Lovley, D. R., Baedecker, M. J., Lonergan, D. J., Cozzarelli, I. M., Phillips, E. J. P., & Siegel, D. I. (1989).

Oxidation of aromatic contaminants coupled to microbial iron reduction. Nature, 339, 297–299.

Lovley, D. R., & Lonergan, D. J. (1990). Anaerobic oxidation of toluene, phenol, and p-cresol by the

dissimilatory iron-reducing organism, GS-15. Applied Environmental Microbiology, 56, 1858–1864.

Lu, X., Kampbell, D. H., & Wilson, J. T. (2006). Evaluation of the role of Dehalococcoides organisms in the

natural attenuation of chlorinated ethylenes in ground water. EPA/600/R-06/029. Ada, OK: U.S.

Environmental Protection Agency, Office of Research and Development, National Risk Management

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Morse, J., Alleman, B., Gossett, J., Zinder, S., Fennell, D., Sewell, G., & Vogel, C. (1998). Draft technical

protocol: A treatability test for evaluating the potential applicability of the reductive anaerobic

biological in situ treatment technology (RABITT) to remediate chloroethenes. Appendix C.3.4: Design,

implementation, and interpretation of microcosm studies.

Nielsen, P. H., Christensen, T. H., Albrechtsen, H., & Gillham, R. W. (1996). Performance of the in situ

microcosm technique for measuring the degradation of organic chemicals in aquifers [Electronic version].

Ground Water Monitoring and Remediation, 16(1), 130–140. doi: 10.1111/j.1745-6592.1996.tb00580.x

Ridgway, H. F., Safarik, J., Phipps, D., Carl, P., & Clark, D. (1990). Identification and catabolic activity of

well-derived gasoline degrading bacteria from a contaminated aquifer. Applied Environmental

Microbiology, 56, 3565–3575.

Shouakar-Stash, O., Frape, S. K., & Drimmie, R. J. (2003). Stable hydrogen, carbon and chlorine isotope

measurements of selected chlorinated organic solvents. Journal of Contaminant Hydrogeology, 60(3–4),

211–228.

Sublette, K., Peacock, A., White, D., Davis, G., Ogles, D., Cook, D., . . . Yang, X. (2006). Monitoring subsurface

microbial ecology in a sulfate-amended, gasoline-contaminated aquifer. Groundwater Monitoring and

Remediation, 26(2), 70–78.

U.S. Environmental Protection Agency, Office of Solid Waste and Emergency Response. (1999, April). Use of

monitored natural attenuation at Superfund, RCRA corrective action, and underground storage tank

sites. Directive Number: 9200.4-17P. Retrieved from http://www.cluin.org/download/reg/d9200417.pdf

Widdel, F., & Rabus, R. (2001). Anaerobic biodegradation of saturated and aromatic hydrocarbons. Current

Opinion in Biotechnology, 12, 259–276.

Wiedemeier, T., Swanson, M., Mouton, D., Gordon, E., Wilson, W., Wilson, B., . . . Chapelle, F. (1998).

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EPA/600/R-98/128. Washington, DC: U.S. Environmental Protection Agency, Office of Research and

Development. Retrieved from http://www.epa.gov/superfund/health/conmedia/gwdocs/protocol.htm

Matthew Burns is a senior project director with WSP Environment & Energy. He has more than 15 years of

professional chemistry and engineering experience. He holds a BS in environmental science from the University of

Massachusetts Amherst, and an MS in civil/environmental engineering from the University of Maryland College

Park. He is a member of the scientific advisory board for the International Conference on Soils, Sediments,

Water, and Energy and the advisory board for the Advanced Tools Web site (advancedtools.us).

Colleen Myers , P.G., is a technical manager with WSP Environment & Energy. She has more than 13 years of

experience as a hydrogeologist with expertise in geology, hydrogeology, and engineering geology. She earned BS

degrees in geosciences and earth science secondary education from the State University of New York, College

at Buffalo, and an MS in engineering geology from Kent State University in Kent, Ohio. She is a registered

professional geologist licensed in Alabama, Florida, Georgia, Texas, Pennsylvania, South Carolina, and Virginia,

and is an active member of the National Ground Water Association.


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Evolution of predictive tools for in situ bioremediation and natural attenuation evaluations