Monitoring Gene Expression To Evaluate Oxygen Infusion at a

Aug 3, 2010 - Increasingly, molecular biological tools, most notably quantitative polymerase ... genes can aid in site management decisions, evaluatio...
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Environ. Sci. Technol. 2010, 44, 6829–6834

Monitoring Gene Expression To Evaluate Oxygen Infusion at a Gasoline-Contaminated Site BRETT R. BALDWIN,† ANITA BIERNACKI,† JOEL BLAIR,‡ MICHAEL P. PURCHASE,‡ JEFFREY M. BAKER,§ KERRY SUBLETTE,| G R E G D A V I S , † A N D D O R A O G L E S * ,† Microbial Insights, Inc., Rockford, Tennessee 37853-3044, Arctos Environmental, Berkeley, California 94702-1128, Tesoro Companies, Inc., Auburn, Washington 98001-9540, and Center for Applied Geosciences, University of Tulsa, Tulsa, Oklahoma 74104

Received April 26, 2010. Revised manuscript received July 8, 2010. Accepted July 20, 2010.

Increasingly, molecular biological tools, most notably quantitative polymerase chain reaction (qPCR), are being employed to provide a more comprehensive assessment of bioremediation of petroleum hydrocarbons and fuel oxygenates. While qPCR enumeration of key organisms or catabolic genes can aid in site management decisions, evaluation of site activities conducted to stimulate biodegradation would ideally include a direct measure of gene expression to infer activity. In the current study, reverse-transcriptase (RT) qPCR was used to monitor gene expression to evaluate the effectiveness of an oxygen infusion system to promote biodegradation of BTEX and MTBE. During system operation, dissolved oxygen (DO) levels at the infusion points were greater than 30 mg/L, contaminant concentrations decreased, and transcription of two aromatic oxygenase genes and Methylibium petroleiphilum PM1-like 16S rRNA copies increased by as many as 5 orders of magnitude. Moreover, aromatic oxygenase gene transcription and PM1 16s rRNA increased at downgradient locations despite low DO levels even during system operation. Conversely, target gene expression substantially decreased when the system was deactivated. RT-qPCR results also corresponded to increases in benzene and MTBE attenuation rates. Overall, monitoring gene expression complemented traditional groundwater analyses and conclusively demonstrated that the oxygen infusion system promoted BTEX and MTBE biodegradation.

Introduction Bioremediation, whether under monitored natural attenuation (MNA) conditions or through engineered corrective actions (e.g., oxygen addition), is a common and often effective remedy at sites impacted by petroleum hydrocarbons and fuel oxygenates. Typically, site managers have focused on chemical and geochemical lines of evidence for site assessment and evaluation of corrective measures designed to promote biodegradation. Temporal monitoring * Corresponding author phone: (865)573-8188 x107; fax: (865)5738133, e-mail: [email protected]. † Microbial Insights, Inc. ‡ Arctos Environmental. § Tesoro Companies, Inc. | University of Tulsa. 10.1021/es101356t

 2010 American Chemical Society

Published on Web 08/03/2010

of groundwater contaminant concentrations is used in transport models, risk assessment, and trend analysis to evaluate dissolved plume stability. Evaluation of subsurface geochemistry at petroleum-contaminated sites emphasizes measurement of potential electron acceptors (dissolved oxygen, nitrate, sulfate, etc.) to establish a geochemical footprint of microbial activity. While providing a wealth of information critical for effective site management, chemical and geochemical results alone do not demonstrate biodegradation. Decreases in contaminant concentrations can result from physical processes such as dilution and consumption of electron acceptors, although an indirect indicator of microbial activity, does not necessarily indicate biodegradation of contaminants of concern. To more thoroughly evaluate biodegradation as a treatment mechanism, traditional chemical and geochemical monitoring have increasingly been coupled to characterization of the microbial community under field conditions in terms of abundance and activity of organisms responsible for biodegradation of contaminants of concern. During the past 15 to 20 years, the use of nucleic acidbased methods, most notably quantitative polymerase chain reaction (qPCR), has grown substantially in the environmental restoration industry. The reasons for increased application of qPCR for site assessment are 2-fold: nucleic acids can be extracted directly from site soil, sediment, and groundwater eliminating the biases associated with cultivation, and qPCR can quantify genes encoding the enzymes specifically responsible for biodegradation of contaminants of concern. PCR detection or qPCR quantification of functional genes involved in aerobic (1-7) and anaerobic (8-10) catabolism have been successfully employed to evaluate biodegradation of BTEX. Similarly, qPCR enumeration of Methylibium petroleiphilum PM1 16S rRNA genes has been used as an index to evaluate the feasibility and effectiveness of methyl tert-butyl ether (MTBE) biodegradation (11-13). Although enumeration of aromatic oxygenase genes in the aforementioned studies allowed quantitative assessment of the impact of corrective actions on the potential for aerobic BTEX biodegradation, qPCR results do come with the important caveat that the presence of a catabolic gene, even in high copy numbers, does not necessarily indicate expression and activity of a catabolic pathway. Organisms harboring genes for aerobic biodegradation of BTEX can and often are present in high abundance at petroleum-contaminated sites but due to subsurface conditions such as low oxygen availability may not be active. Increased activity of BTEX-utilizing bacteria in response to site activities at times can be inferred from increases in gene copy numbers (i.e., growth) as demonstrated by Nebe et al. (7). However, background populations, particularly those harboring aromatic oxygenase genes, may be elevated under MNA conditions, and enhanced biodegradation following an engineered corrective action may result from increased activity rather than increases in degrader populations. Ideally, evaluation of site activities conducted to stimulate contaminant biodegradation would include a direct measure of gene expression to demonstrate the desired microbial activity (14). Quantification of targeted in situ microbial activities depends on detection of a specific yet transient biomarker indicative of the desired function (15). RNA transcripts are short-lived and due to conserved but unique nucleic acid sequences can be readily detected with a high degree of specificity by reverse-transcriptase qPCR (RT-qPCR). Indeed, rapid RNA turnover while necessary to assess activity was one of the primary obstacles that once limited routine use VOL. 44, NO. 17, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. The site is an operating gasoline station with dissolved BTEX concentrations ranging from 22,780 µg/L to less than 5 µg/L. Monitoring wells impacted by BTEX or MTBE are displayed as solid circles (•). BTEX and MTBE have not been detected in groundwater samples from MW-7. System injection points (2) and corresponding monitoring points (∆) are shown as triangles. The oxygen infusion system monitored during the study consisted of infusion points IP-1 through IP-3 and monitoring points MP-1 and MP-2. of RT-qPCR analysis. However, improvements in RNA extraction (16) and field preservation protocols have largely circumvented these issues potentially permitting regular examination of gene expression in environmental samples. In the current study, RT-qPCR analysis of two aromatic oxygenase gene transcripts and PM1-like 16S rRNA was employed to evaluate the effectiveness of an oxygen infusion system at a gasoline-impacted site. Groundwater samples were obtained quarterly over a 2-year period from system infusion points and downgradient monitoring points for chemical and geochemical analyses. RT-qPCR results from passive microbiological samples (Bio-Traps) were then compared to system operational history, geochemical conditions, and trends in BTEX/MTBE concentrations within and outside the system zone of influence as a comprehensive assessment of impact of oxygen infusion as a corrective action.

Materials and Methods Study Site. The study site is an operating gasoline station located in northern California (Figure 1). During replacement of three single-walled gasoline underground storage tanks (USTs) in October 1987, benzene and gasoline range total petroleum hydrocarbons (TPHg) were detected in soil samples obtained from the tank excavation. In 1998, the dispenser islands and associated product piping were replaced. A phase II Environmental Investigation which included the installation of monitoring wells MW-1 through MW-3 revealed that groundwater was impacted by petroleum hydrocarbons and methyl tert-buyl ether (MTBE). Monitoring wells MW-4 through MW-9 were installed in February 2006 to delineate the dissolved plume. In March 2007, site managers submitted a work plan for installation of an oxygen 6830

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infusion system to promote aerobic biodegradation of dissolved petroleum hydrocarbons in the suspected source area (dispenser islands). Three infusion points (IP-1 through IP-3) and two monitoring points (MP-1 and MP-2) for the original oxygen infusion system were installed in the vicinity of the dispenser islands in May 2007 (Figure 1). An additional three infusion points (IP-4 through IP-6) and one additional monitoring point (MP-3) were installed in June 2008 to expand the oxygen infusion system. Groundwater flow is consistently toward the southeast with a typical hydraulic gradient of approximately 0.010 m/m. Oxygen Infusion Systems. Soil borings for system infusion and monitoring points were installed using a 25.4 cm (10in.) hollow stem auger to a total depth of 6.1 m below grade. The infusion points were constructed using 10.16 cm (4-in.) PVC casing and were screened (0.020-in. slots) from 1.2 to 5.8 m below grade. A Monterey No. 2/12 sand pack was installed from approximately 0.3 m below the screened interval to 0.3 m above the screened interval (0.9 to 6.1 m below grade). A 0.3 m-thick hydrated bentonite seal was installed on top of the sand pack. The remaining annular space was filled with Portland cement slurry (0.3 to 0.6 m below grade). All injection points were completed at the surface with a 45.7 cm-diameter traffic-rated vault set in concrete. Overall, the site is underlain primarily by silty and sandy clays with water-bearing layers of silty sands or gravel. In the vicinity of the infusion points, depth to groundwater typically ranges from approximately 1.6 to 2.8 m below grade. Sandy or silty clays were encountered to depths of 2.1 m below grade. Silty sands (fine to medium grained) were generally encountered from 2.1 to approximately 3.7 m below grade. The silty sand unit was typically underlain by sandy clay to a depth of approximately 5.2 m below grade. Silt with varying amounts of sand was encountered from 5.2 m to the bottom of the soil borings (6.1 m). Well screens for the infusion and associated monitoring points (0.9 to 6.1 m below grade) span the entire thickness of the silty sand and sandy clay units. The oxygen infusion system essentially consisted of oxygen cylinders, 2-stage regulators, manifolds, in-well emitters, and associated tubing. Two types of oxygen emitters were used over the course of the project: iSOC (inVenture Technologies, ON, Canada) and Waterloo (Solinst, ON, Canada) emitters. The systems were designed to deliver a total of 0.085 standard m3/day to each group of three infusion points. Oxygen was supplied to the manifolds at 50 psig, and each emitter delivered oxygen to the subsurface at estimated rates of 16 mL/min (iSOC) and 2.5 mL/min (Waterloo). The oxygen infusion system, consisting of infusion points IP-1 through IP-3 and monitoring points MP-1 and MP-2 (approximately 3 and 6 m downgradient), was activated on June 1, 2007. The expanded infusion system (IP-4 through IP-6 and MP-3) was activated on February 1, 2009 south of the original system. Groundwater Sampling for Chemical and Geochemical Parameters. Prior to sampling, low-flow purging with a submersible pump was performed until a minimum of three well volumes had been purged and pH, conductivity, turbidity, and temperature of the purge water had stabilized. Groundwater samples were obtained with disposable polyethylene bailers and transferred to three 40-mL glass vials containing a preservative. Groundwater samples were immediately placed on ice and delivered to a state-certified analytical laboratory within 24 to 48 h for BTEX and MTBE analysis by EPA Method 8260B. Additional groundwater samples were obtained for analysis of nitrate (EPA 300.0), ferrous iron (SM3500-FeD), sulfate (EPA 300.0), and dissolved methane (RSK-175M). Dissolved oxygen (DO) was measured in the field using a YSI DO probe. Mann-Kendall analysis of dissolved benzene and MTBE concentrations was conducted as described previously (5, 17). First order attenuation

constants for benzene and MTBE were estimated as described by Wilson et al. (18). Bio-Traps - Sampling for Microbiological Analyses. Samples for RT-qPCR analysis were obtained using Bio-Traps (Microbial Insights, Rockford, TN) containing Bio-Sep beads (University of Tulsa). Briefly, Bio-Sep beads are an engineered composite of 25% aramid (Nomex) polymer and powdered activated carbon (PAC) that provide a large surface area (∼600 m2/g) for microbial colonization and biofilm formation. During in-well deployment, biofilms characteristic of in situ aquifer conditions (19-21) are formed on and within the Bio-Sep beads. More detailed descriptions of the Bio-Traps are available elsewhere (19). Bio-Traps were suspended below the water table in monitoring wells and infusion points for approximately 30 days prior to retrieval. Recovered Bio-Traps were placed in sterile bags and shipped overnight on ice for nucleic acid extractions. RNA Extraction and RT-qPCR. RNA extractions from BioSep beads were performed using RNA PowerSoil Total RNA Isolation kits (MO BIO Laboratories, Inc., Solana Beach, California) according to the manufacturer’s recommendations except as noted. Based on ref 22, firefly (Coleoptra) luciferase mRNA (Promega, Madison, WI) was added to all samples (3.7 × 105 gene copies/µL) as an exogenous internal reference mRNA. RNA extracts were DNase treated using Turbo DNA-free (Ambion, Austin, TX) according to the manufacturer’s instructions. Reverse transcription was performed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster, CA) according to the manufacturer’s protocol. Positive controls, thermocycling conditions, and qPCR primer sets targeting phenol hydroxylase (PHE) and toluene dioxygenase (TOD) have been described previously (23). Quantification of Methylibium petroleiphilum PM1 was used as an index of aerobic MTBEutilizing bacteria as detailed elsewhere (11). Thermocycling conditions and the primer-probe set employed to quantify the luciferase reference standard (REF) were described by Johnson et al. (22). RT-qPCR for SYBR Green assays (PHE and TOD) was performed on an ABI 7000 Sequence Detection System (ABI Sequence Detector Software, v1.1) and included melt curve analysis as described by Baldwin et al. (23) for all samples. TaqMan based assays (PM1 and REF) were performed on an ABI 7300 Real-Time PCR System (ABI Sequence Detector software v1.3). Standard curves and associated quality control parameters for all RT-qPCR assays are provided in Table S1 in the Supporting Information. Quantification of the luciferase RNA reference standard (REF) demonstrated RNA isolation, reverse transcription, and amplification (mean percent recovery 94.8%, standard deviation 12%). All RT-qPCR experiments included appropriate negative (no reverse transcriptase and no DNA) and positive control reactions. No amplification was detected in negative controls (Ct > total cycles).

Results and Discussion Site Conditions Prior to System Activation. Dissolved BTEX and MTBE concentrations prior to activation of the oxygen infusion system generally reflected proximity to the dispenser islands (Figure 1; Table S1). Within this source zone, average total BTEX concentrations at the infusion points and associated monitoring points were on the order of 2300 to 23,000 µg/L. Similarly, MTBE concentrations ranged from approximately 280 µg/L at the northernmost infusion point to 10,000 µg/L within the dispenser island. BTEX and MTBE were also routinely detected at low concentrations (5 to 30 µg/L) in monitoring wells associated with the USTs and upgradient monitoring well MW-5. At nonimpacted well MW7, DO levels (1.4 to 3.6 mg/L), an average oxidation/reduction potential (ORP) of 85.5 mV, and the consistent presence of alternative electron acceptors (nitrate and sulfate) all indi-

cated mildly aerobic background conditions outside the dissolved BTEX/MTBE plume (Table S1). Within the dissolved plume, DO, nitrate, and sulfate concentrations were significantly lower, while methane production was significantly greater (t test; R ) 0.05). Overall, geochemical monitoring revealed that available electron acceptors other than CO2 were largely depleted within the dissolved plume prior to system activation. Geochemical Response to System Operation. After system startup, DO levels at the infusion points rapidly increased to over 30 mg/L and remained elevated through November 2007 (Figure 2A). During the period spanning December 2007 through March 2008, the system was periodically deactivated for general maintenance, the addition of a second oxygen cylinder, and the replacement of an oxygen emitter. DO levels while the system was deactivated rapidly decreased to less than 5 mg/L. From April through October 2008, the system operated continuously, and DO concentrations were consistently in the 30 to 50 mg/L range. In December 2008 and early January 2009, DO levels decreased due to a temporary deactivation of the system. By mid-January, the system was reactivated, and DO levels at the infusion points remained elevated. Despite high levels at the infusion points, however, DO concentrations at the downgradient monitoring points MP-1 and MP-2 did not increase (Figure 2B,C) and were typically less than 2 mg/L even during consistent system operation. Gene Expression in Response to System Operation. RTqPCR analysis of phenol hydroxylase (PHE) and toluene dioxygenase (TOD) transcripts and Methylibium petroleiphilum PM1 16S rRNA was used to evaluate the response of aerobic BTEX-utilizing bacteria and assess activity of a known MTBE-utilizing organism. Prior to system startup, PHE and TOD transcripts as well as PM1 16S rRNA transcripts were below laboratory detection limits at infusion point IP-3 (Figure 2A). Following system activation however, PHE transcripts and PM1 16S rRNA were detected between 103 and 105 copies/bead at IP-3 (Figure 2A). Bio-Trap samplers were not deployed in IP-3 during the first system shutdown (December 2007 through March 2008). Following system upgrades and reactivation, PHE transcripts were again detected on the order of 103 copies/bead, while PM1 16S rRNA copies/bead increased to nearly 106. During a second, unscheduled system shutdown, samplers which had been deployed in IP-3 were recovered for analysis. Consistent with system shutdown and the subsequent decrease in DO concentrations, PHE expression was no longer detected, and PM1 16S rRNA copies decreased by nearly 3 orders of magnitude at IP-3 (Figure 2A). PHE, TOD, and PM1 16S rRNA were again detected in elevated copy numbers following the reactivation of the system further indicating that gene expression was linked to system operation and suggesting that oxygen infusion would stimulate aerobic biodegradation in the source area. Ideally, the impact of the infusion system would extend beyond the infusion points and enhance biodegradation at downgradient locations. As mentioned previously however, DO concentrations at downgradient monitoring points MP-1 (Figure 2B) and MP-2 (Figure 2C) remained below 2 mg/L even during periods of continuous operation initially suggesting a rather limited system zone of influence. Likewise, neither PM1 16S rRNA nor aromatic oxygenase transcripts were detected at MP-1 and MP-2 during the initial five months of system operation. However, PM1 16S rRNA and PHE and TOD transcripts were detected at MP-1 after another 5 months of continuous system operation (August 2008) indicating enhanced activity of an MTBE utilizing strain and expression of two aerobic pathways for BTEX catabolism. While in relatively low copy numbers, PM1 16S rRNA was also detected at MP-2 during this sampling event indicating VOL. 44, NO. 17, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. First Order Attenuation Constantsa benzene -1

MTBE -1

well

kB (yr )

upper 80%

kM (yr )

upper 80%

MW-3 MW-4 IP-3 MP-1 MP-2

-0.20 -0.51 0.73 0.82 -0.04

-0.30 -0.61 0.55 0.71 -0.13

0.19 0.64 1.52 1.34 1.13

0.08 0.56 1.35 1.25 1.01

a First order attenuation constants based on historical groundwater monitoring results for benzene (kB) and MTBE (kM) were calculated as described previously (18). To be considered a decreasing trend (bold), the upper 80% confidence limit on the slope of the linear regression must be greater than zero.

FIGURE 2. (A) IP-3, (B) MP-1, (C) MP-2, and (D) MW-4. RT-qPCR quantification of phenol hydroxylase transcripts (black bars), toluene dioxygenase transcripts (gray bars), and PM1-like 16S rRNA (white bars) correspond to the left y-axis. Error bars represent the standard deviation of analytical duplicates. System runtime (solid arrows) and downtime (dashed lines) are indicated at the top of each figure. Dissolved oxygen (DO) concentrations (mg/L) displayed on right y-axis. Solid circles (•) represent the average DO concentrations for the system injection points, whereas DO concentrations at system monitoring points are displayed as open circles. 6832

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a microbial community response at least 6 m downgradient of the infusion points. Moreover, PHE and TOD expression along with PM1 16S rRNA copies decreased to below detection limits at both MP-1 and MP-2 after system shutdown, subsequently rebounded after system reactivation, and in essence mirrored observations at infusion point IP-3. The RT-qPCR results for monitoring well MW-4 were less definitive (Figure 2D) perhaps due to its location somewhat sidegradient of infusion point IP-1. PHE and TOD transcripts were not detected at MW-4 during any sampling event indicating that the system did not promote expression of these two aromatic oxygenase genes at least during the study period. PM1 16S rRNA was detected during the last sampling event, however, suggesting that the system zone of influence may eventually encompass this location and enhance biodegradation with continued operation. Recent studies have placed renewed emphasis on evaluating in situ diversity of aromatic oxygenases and have indicated that less well characterized groups are also selected at BTEX-impacted sites (24-27). Although continued investigation of functional diversity will lead to more comprehensive characterization of the microbial community, little is currently known about the environmental prevalence and especially the in situ expression of even the more well characterized aromatic oxygenases. Granted, monitoring expression of only two aromatic oxygenase genes may have underestimated the true microbial response to system operation by ignoring pathways initiated by other (e.g., TOL plasmid xylene monooxygenases) and novel oxygenases. However, the PHE genotype has been frequently detected at other petroleum-impacted sites (2, 5, 6), and increases in both PHE (7) and TOD (28) gene copies have been reported following injection of a slurry-type oxygen releasing material. In the current study, PHE gene abundance (Figure S1) and expression in particular corresponded to system operation and enhanced biodegradation confirming previous reports of the environmental relevance of the ring-hydroxylating toluene monooxygenase/phenol hydroxylase pathway. In our experience, detection of the TOD genotype in environmental samples from petroleum-impacted sites is common (unpublished data). However, reports in the literature include the following: not detected (1, 7, 29, 30), infrequently detected (2), detected in source areas (5), and detected in response to oxygen amendment (28). In the current study, TOD expression was frequently but not always noted confirming the environmental relevance of the pathway but also suggesting that host-strain-specific or environmental factors also play a role in TOD abundance and expression. Contaminant Attenuation Rate Constants. From a risk reduction perspective, the value of the oxygen system will ultimately be judged based on long-term trends in contaminant concentrations. Therefore, benzene and MTBE first order attenuation constants (18) were compared between

within the system zone of influence indicate that oxygen infusion enhanced benzene and MTBE biodegradation. Gene Expression as a Site Management Tool. Although decreases in contaminant concentrations ultimately validated system installation and operation in terms of risk reduction, monitoring gene expression provided site managers with more immediate feedback of system impact and the link between contaminant concentrations, system operation, and biodegradation. For example, during the first six months of operation, RT-qPCR evidence of PHE expression at IP-3 provided rapid evidence that the infusion system would achieve its primary goal of enhancing biodegradation in the source area before a clear trend in contaminant concentrations would have been evident (Figure 3A). Gene expression results for downgradient monitoring points were even more enlightening from a site management perspective. Benzene and MTBE concentrations at MP-1 have decreased overall but not steadily over the course of the study (Figure 3B). For the first ten months of system operation, benzene and MTBE concentrations fluctuated and expression of target genes was not detected. In August 2008 however, quantification of PHE and TOD transcripts and PM1 16S rRNA demonstrated that the relatively consistent decreases in contaminant concentrations were likely a result of enhanced biodegradation prompted by more continuous system operation since April reactivation. The observed lag but eventual expression of targeted aromatic oxygenase genes at MP-1 also improved stakeholder confidence that system operation would ultimately enhance benzene biodegradation at MP-2 when concentrations at the time (August 2008) were increasing (Figure 3C). In May 2009, PHE and TOD expression was noted at MP-2 likely leading to the observed decreases in benzene concentrations by the end of the study. Furthermore, detection of PM1 16S rRNA at MP-2 corresponds to a distinct increase in the MTBE attenuation rate. Over the first ten months after system startup when PM1 rRNA was not detected, the estimated attenuation constant (0.53 yr-1) was within the range estimated for wells outside the system zone of influence. Conversely, the MTBE attenuation constant for the remainder of the study (1.54 yr-1), when PM1 rRNA was detected, was substantially greater. From an overall site management point of view, RT-qPCR quantification of target gene expression more accurately delineated the system zone of influence, provided direct evidence of enhanced biodegradation at times not evident in chemical or geochemical results, and instilled greater stakeholder confidence in the remediation strategy. FIGURE 3. (A) IP-3, (B) MP-1, and (C) MP-2. RT-qPCR quantification of phenol hydroxylase transcripts (black bars), toluene dioxygenase transcripts (gray bars), and PM1-like 16S rRNA (white bars) correspond to the left y-axis. Dissolved benzene (9) and MTBE (∆) concentrations (µg/L) displayed on right y-axis. System runtime (solid arrows) and downtime (dashed lines) are indicated at the top of each figure. monitoring wells within (IP-3, MP-1, and MP-2) and outside (MW-3 and MW-4) the system zone of influence (Table 1). Outside the system zone of influence, benzene concentrations increased over the course of the project, while concentrations within the zone of influence (IP-3 and MP-1) decreased substantially. Perhaps due to a combination of physical processes and biodegradation under MNA conditions, MTBE concentrations at wells outside the system zone of influence decreased over the course of the project with attenuation constants ranging from 0.19 to 0.64 yr-1. By comparison, MTBE attenuation constants at points within the system zone of influence were substantially greater, ranging from 1.13 to 1.52 yr-1. When combined with gene expression monitoring during the project, greater contaminant attenuation rates

Acknowledgments The authors wish to acknowledge Ms. Charlotte Taylor for performing RT-qPCR analyses.

Supporting Information Available A summary of qPCR standard curve quality control parameters (Table S1), a summary of BTEX, MTBE, and electron acceptor concentrations (Table S2), and comparison of qPCR and RT-qPCR results for the last three monitoring events (Figure S1). This material is available free of charge via the Internet at http://pubs.acs.org.

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