Bioremediation of groundwater contaminated with gasoline hydrocarbons and oxygenates using a membrane-based reactor

The objective of this study was to operate a novel, field-scale, aerobic bioreactor and assess its performance in the ex situ treatment of groundwater contaminated with gasoline from a leaking underground storage tank in Pascoag, RI. The groundwater contained elevated concentrations of MTBE (methyl tert-butyl ether), TBA (tert-butyl alcohol), TBF (tert-butyl formate), BTEX (benzene, toluene, ethyl benzene, and xylene isomers), and other gasoline additives (tert-amyl methyl ether, di-isopropyl ether, tert-amyl alcohol, methanol, and acetone). The bioreactor was a gravity-flow membrane-based system called a Biomass Concentrator Reactor (BCR) designed to retain all biomass within the reactor. It was operated for six months at an influent flow rate that ultimately reached 5 gpm. The goal was to achieve a removal of all contaminants to <5 microg/L, which is the California Drinking Water advisory for MTBE. The concentration of TBA, an MTBE biodegradation byproduct, was consistently lower than that of MTBE. The other daughter compound detected in the influent, TBF, was degraded to concentrations below the detection limit of 0.02 microg/L. BTEX were consistently degraded to significantly lower levels in the effluent throughout the duration of the study (<1 microg/L). A similar high removal efficiency of the other gasoline oxygenates present in the groundwater (TAME, DIPE, and TAA) was also achieved. Dissolved organic carbon analysis demonstrated the ability of the bioreactor to produce high quality effluents with nonpurgeable organic carbon (NPOC) averaging approximately 50% lowerthan the NPOC concentrations in the influent contaminated groundwater.     

Evaluation of the impact of fuel hydrocarbons and oxygenates on groundwater resources

The environmental behavior of fuel oxygenates (other than methyl tert-butyl ether [MTBE]) is poorly understood because few data have been systematically collected and analyzed. This study evaluated the potential for groundwater resource contamination by fuel hydrocarbons (FHCs) and oxygenates (e.g., tert-butyl alcohol [TBA], tertamyl methyl ether [TAME], diisopropyl ether [DIPE], ethyl tert-butyl ether [ETBE], and MTBE) by examining their occurrence, distribution, and spatial extent in groundwater beneath leaking underground fuel tank (LUFT) facilities, focusing on data collected from over 7200 monitoring wells in 868 LUFT sites from the greater Los Angeles, CA, region. Excluding the composite measure total petroleum hydrocarbons as gasoline (TPHG), TBA has the greatestsite maximum (geometric mean) groundwater concentration among the study analytes; therefore, its presence needs to be confirmed at LUFT sites so that specific cleanup strategies can be developed. The alternative ether oxygenates (DIPE, TAME, and ETBE) are less likely to be detected in groundwater beneath LUFT facilities in the area of California studied and when detected are present at lower dissolved concentrations than MTBE, benzene, or TBA. Groundwater plume length was used as an initial indicator of the threat of contamination to drinking water resources. Approximately 500 LUFT sites were randomly selected and analyzed. The results demonstrate MTBE to pose the greatest problem, followed by TBA and benzene. The alternative ether oxygenates were relatively localized and indicated lesser potential for groundwater resource contamination. However, all indications suggest the alternative ether oxygenates would pose groundwater contamination threats similar to MTBE if their scale of usage is expanded. Plume length data suggest that in the absence of a completely new design and construction of the underground storage tank (UST) system, an effective management strategy may involve placing greater emphasis on UST program for ensuring adequate enforcement and compliance with existing UST regulations.     

Enrichment of stable carbon and hydrogen isotopes during anaerobic biodegradation of MTBE: microcosm and field evidence

The conventional approach to evaluate biodegradation of organic contaminants in groundwater is to demonstrate an increase in the concentration of transformation products. This approach is problematic for MTBE from gasoline spills because the primary transformation product (TBA) can also be a component of gasoline. Compound-specific stable isotope analysis may provide a useful alternative to conventional practice. Changes in the delta13C and deltaD of MTBE during biodegradation of MTBE in an anaerobic enrichment culture were compared to the delta13C and deltaD of MTBE in groundwater at nine gasoline spill-sites. The stable isotopes of hydrogen and carbon were extensively fractionated during anaerobic biodegradation of MTBE. The stable isotope enrichment factor for carbon (epsilonC) in the enrichment cultures was -13 (-14.1 to -11.9 at 95% confidence level), and the hydrogen enrichment factor (epsilonH) was -16 (-21 to -11 at 95% confidence level). The isotope enrichment factors for carbon and hydrogen during anaerobic biodegradation indicate that the first reaction is enzymatic hydrolysis of the O-Cmethyl bond. The ratio of epsilonH to epsilonC was consistent between the enrichment culture and the field site that provided the inoculum, and with the other eight sites, suggesting a common degradation pathway. Compound-specific isotope evidence is discussed in terms of its utility for monitoring in situ biodegradation, in particular, for measuring how much MTBE was degraded. For the studied field sites, significant biodegradation of the original mass of MTBE is suggested, in some cases exceeding 90%.     

Impact of ethanol on the natural attenuation of benzene, toluene, and o-xylene in a normally sulfate-reducing aquifer

Side-by-side experiments were conducted in a sulfate-reducing aquifer at a former fuel station to evaluate the effect of ethanol on biodegradation of other gasoline constituents. On one side, for approximately 9 months we injected groundwater amended with 1-3 mg/L benzene, toluene, and o-xylene (BToX). On the other side, we injected the same, adding approximately 500 mg/L ethanol. Initially the BToX plumes on both sides ("lanes") extended approximately the same distance. Thereafter, the plumes in the "No Ethanol Lane" retracted significantly, which we hypothesize to be due to an initial acclimation period followed by improvement in efficiency of biodegradation under sulfate-reducing conditions. In the "With Ethanol Lane", the BToX plumes also retracted, but more slowly and not as far. The preferential biodegradation of ethanol depleted dissolved sulfate, leading to methanogenic/acetogenic conditions. We hypothesize that BToX in the ethanol-impacted lane were biodegraded in part within the methanogenic/acetogenic zone and, in part, within sulfate-reducing zones developing along the plume fringes due to mixing with sulfate-containing groundwater surrounding the plumes due to dispersion and/or shifts in flow direction. Overall, this research confirms that ethanol may reduce rates of biodegradation of aromatic fuel components in the subsurface, in both transient and near steady-state conditions.     

Environmental implications on the oxygenation of gasoline with ethanol in the metropolitan area of Mexico City

Motor vehicle emission tests were performed on 12 in-use light duty vehicles, made up of the most representative emission control technologies in Mexico City: no catalyst, oxidative catalyst, and three way catalyst. Exhaust regulated (CO, NOx, and hydrocarbons) and toxic (benzene, formaldehyde, acetaldehyde, and 1,3-butadiene) emissions were evaluated for MTBE (5 vol %)- and ethanol (3, 6, and 10 vol %)-gasoline blends. The most significant overall emissions variations derived from the use of 6 vol % ethanol (relative to a 5% MTBE base gasoline) were 16% decrease in CO, 28% reduction in formaldehyde, and 80% increase in acetaldehyde emissions. A 26% reduction in CO emissions from the oldest fleet (< MY 1991, without catalytic converter), which represents about 44% of the in-use light duty vehicles in Mexico city, can be attained when using 6 vol% ethanol-gasoline, without significant variation in hydrocarbons and NOx emissions, when compared with a 5% vol MTBE-gasoline. On the basis of the emissions results, an estimation of the change in the motor vehicle emissions of the metropolitan area of Mexico city was calculated for the year 2010 if ethanol were to be used instead of MTBE, and the outcome was a considerable decrease in all regulated and toxic emissions, despite the growing motor vehicle population.     

Use of compound-specific stable carbon isotope analyses to demonstrate anaerobic biodegradation of MTBE in groundwater at a gasoline release site

Currently it is unclear if natural attenuation is an appropriate remedial approach for groundwater impacted by methyl tertiary butyl ether (MTBE). Site-characterization data at most gasoline release sites are adequate to evaluate attenuation in MTBE concentrations over time or distance. But, demonstrating natural biodegradation of MTBE requires laboratory microcosm studies, which could be expensive and time-consuming. Recently, compound-specific carbon isotope ratio analyses (13C/12C expressed in delta13C notation) have been used to demonstrate aerobic biodegradation of MTBE in laboratory incubations. This study explored the potential of this approach to distinguish MTBE biodegradation from other abiotic processes in an anaerobic groundwater plume that showed extensive decrease in MTBE concentrations. To our knowledge, this is the first study to use delta13C of MTBE data in groundwater and laboratory microcosms to demonstrate anaerobic biodegradation of MTBE. The delta13C of MTBE in monitoring wells increased by up to 31 per thousand (-25.5 per thousand to +5.5 per thousand) along with a 40-fold decrease in MTBE concentrations. Anaerobic incubations in laboratory microcosms indicated up to 20-fold reduction in MTBE concentrations with a corresponding increase in delta13C of MTBE of up to 33.4 per thousand (-28.7 per thousand to +4.7 per thousand) in live microcosms. Little enrichment was observed in autoclaved controls. These results demonstrate that anaerobic biodegradation was the dominant natural attenuation mechanism for MTBE at this site. The estimated isotopic enrichment factors (epsilon(field) = -8.10 per thousand and epsilon(lab) = -9.16 per thousand) were considerably larger than the range (-1.4 per thousand to -2.4 per thousand) previously reported for aerobic biodegradation of MTBE in laboratory incubations. These observations strongly suggest that delta13C of MTBE could be potentially useful as an "indicator" of in-situ MTBE biodegradation.     

Role of volatilization in changing TBA and MTBE concentrations at MTBE-contaminated sites

Tertiary butyl alcohol (TBA) is commonly found as an impurity in methyl tertiary butyl ether (MTBE) added to gasoline. Frequent observations of high TBA, and especially rising TBA/MTBE concentration ratios, in groundwater at gasoline spill sites are generally attributed to microbial conversion of MTBE to TBA. Typically overlooked is the role of volatilization in the attenuation of these chemicals especially in the vadose zone, which is a source of contamination to groundwater. Here we show that volatilization, particularly through remediation by vapor extraction, can substantially affect the trends in TBA and MTBE concentrations and the respective mass available to impact groundwater aquifers, through the preferential removal of more volatile compounds, including MTBE, and the apparent enrichment of less volatile compounds like TBA. We demonstrate this phenomenon through numerical simulations of remedial-enhanced volatilization. Results show increases in TBA/MTBE concentration ratios consistent with ratios observed in groundwater at gasoline spill sites. Volatilization is an important, and potentially dominant, process that can result in concentration trends similar to those typically attributed to biodegradation.     

Impact of ethanol on the natural attenuation of MTBE in a normally sulfate-reducing aquifer

Side-by-side experiments were conducted in an aquifer contaminated with methyl-tert-butyl ether (MTBE) at a former fuel station to evaluate the effect of ethanol release on the fate of pre-existing MTBE contamination. On one side, for approximately 9 months we injected groundwater amended with 1-3 mg/L benzene, toluene, and o-xylene (BToX). On the other side, we injected the same, adding approximately 500 mg/L ethanol. The fates of BToX in both sides ("lanes") were addressed in a prior publication. No MTBE transformation was observed in the "No Ethanol Lane." In the "With Ethanol Lane", MTBE was transformed to tert-butyl alcohol (TBA) underthe methanogenic and/or acetogenic conditions induced by the in situ biodegradation of the ethanol downgradient of the injection wells. The lag time before onset of this transformation was less than 2 months and the pseudo-first-order reaction rate estimated after 7-8 months was 0.046 d(-1). Our results imply that rapid subsurface transformation of MTBE to TBA may be expected in situations where strongly anaerobic conditions are sustained and fluxes of requisite nutrients and electron donors allow development of an active acetogenic/methanogenic zone beyond the reach of inhibitory effects such as those caused by high concentrations of ethanol.     

A water extraction, static headspace sampling, gas chromatographic method to determine MTBE in heating oil and diesel fuel

A method was developed to determine the fuel/water partition coefficient (KMTBE) of methyl tert-butyl ether (MTBE) and then used to determine low parts per million concentrations of MTBE in samples of heating oil and diesel fuel. A special capillary column designed for the separation of MTBE and to prevent coelution and a gas chromatograph equipped with a photoionization detector (PID) were used. MTBE was partitioned from fuel samples into water during an equilibration step. The water samples were then analyzed for MTBE using static headspace sampling followed by GC/PID. A mathematical relationship was derived that allowed a KMTBE value to be calculated by utilizing the fuel/water volume ratios and the corresponding PID signal. KMTBE values were found to range linearly from 3.8 to 10.9 over a temperature range of 5-40 degrees C. This analysis method gave a MDL of 0.7 ppm MTBE in the fuel and a relative average accuracy of +/-15% by comparison with an independent laboratory using purge and trap GC/ MS analysis. MTBE was found in home heating oil in residential tanks and in diesel fuel at service stations throughout the state of Connecticut. The levels of MTBE were found to vary significantly with time. Heating oil and diesel fuel from terminals were also found to contain MTBE. This research suggests thatthe reported widespread contamination of groundwater with MTBE may also be due to heating oil and diesel fuel releases to the environment. used extensively for the past 20 years as a gasoline additive (up to 15 wt %) to reduce automobile carbon monoxide and hydrocarbon emissions. The fact that MTBE is highly soluble in water (approximately 5 wt %) (3) and chemically inert when compared to other fuel constituents causes it to be often detected at high concentrations in groundwater in the vicinity of gasoline spills. The EPA has reported that low levels of MTBE in drinking water (above 40 microg/L) may cause unpleasant taste and odors and has designated MTBE as a possible human carcinogen (4). Past studies have concentrated on the reporting of MTBE levels in groundwater near gasoline spills. Happel et al. reported an MTBE occurrence rate of approximately 78% at locations where hydrocarbons have impacted groundwater (5). Johnson et al. estimate that 9,000 leaking underground fuel tanks have caused MTBE contamination at community water supplies in the 31 states surveyed (excluding California and Texas) (6). Robbins et al. reported finding a significant number of MTBE detections in groundwater samples taken at sites in Connecticut known to be contaminated by heating oil spills (7). Later, this same research group reported finding MTBE contamination to range from 9.7 to 906 mg/L in heating oil and from 74 to 120 mg/L in diesel fuel in samples collected from storage tanks in Connecticut (8). The method used to analyze these samples was based on fuel-water partitioning and GC analysis. This present study provides the detailed basis for that analytical method. MTBE fuel-water partition coefficients as a function of temperature, which are critical to the method, are also presented. This study also reports on variations in MTBE levels as a function of time observed at several residences and a service station. Analytical results are reported for samples taken from terminals as part of an effort to assess the sources of MTBE in heating oil and diesel fuel.     

Substrate interactions in BTEX and MTBE mixtures by an MTBE-degrading isolate

Groundwater contaminant plumes from recent accidental gasoline releases often contain the fuel oxygenate MTBE (methyl tert-butyl ether) together with BTEX (benzene, toluene, ethylbenzene, o-xylene, m-xylene and p-xylene) compounds. This study evaluates substrate interactions during the aerobic biotransformation of MTBE and BTEX mixtures by a pure culture, PM1, capable of utilizing MTBE for growth. PM1 was unable to degrade ethylbenzene and two of the xylene isomers at concentrations of 20 mg/L following culture growth on MTBE. In addition, the presence of 20 mg/L of ethylbenzene or the xylenes in mixtures with MTBE completely inhibited MTBE degradation. When MTBE-grown cells of PM1 were exposed to MTBE/benzene and MTBE/toluene mixtures, MTBE degradation proceeded, while the degradation of benzene and toluene was delayed for several hours. Following this initial lag, benzene and toluene were degraded rapidly, while the rate of MTBE degradation slowed significantly. MTBE degradation did not increase to previous rates until benzene and toluene were almost entirely degraded. The lag in benzene and toluene degradation was presumably due to the induction of the enzymes necessary for BTEX degradation. Once these enzymes were induced, sequential additions of benzene or toluene were degraded rapidly, and growth on benzene and toluene was observed. The results of this study suggest that BTEX and MTBE degradation occurs primarily via two independent and inducible pathways. If subsurface microbial communities behave similarly to the culture used in this study, the observed severe inhibition of MTBE degradation by ethylbenzene and the xylenes and the partial inhibition by benzene and toluene suggest thatthe biodegradation of MTBE in subsurface environments would most likely be delayed until MTBE has migrated beyond the BTEX plume.     

Methyl tert-butyl ether biodegradation by indigenous aquifer microorganisms under natural and artificial oxic conditions

Microbial communities indigenous to a shallow groundwater system near Beaufort, SC, degraded milligram per liter concentrations of methyl tert-butyl ether (MTBE) under natural and artificial oxic conditions. Significant MTBE biodegradation was observed where anoxic, MTBE-contaminated groundwater discharged to a concrete-lined ditch. In the anoxic groundwater adjacent to the ditch, concentrations of MTBE were > 1 mg/L. Where groundwater discharge occurs, dissolved oxygen (DO) concentrations beneath the ditch exceeded 1.0 mg/Lto a depth of 1.5 m, and MTBE concentrations decreased to <1 microg/L prior to discharge. MTBE mass flux calculations indicate that 96% of MTBE mass loss occurs in the relatively small oxic zone prior to discharge. Samples of a natural microbial biofilm present in the oxic zone beneath the ditch completely degraded [U-14C]MTBE to [14C]CO2 in laboratory liquid culture studies, with no accumulation of intermediate compounds. Upgradient of the ditch in the anoxic, MTBE- and BTEX-contaminated aquifer, addition of a soluble oxygen release compound resulted in oxic conditions and rapid MTBE biodegradation by indigenous microorganisms. In an observation well located closest to the oxygen addition area, DO concentrations increased from 0.4 to 12 mg/L in <60 days and MTBE concentrations decreased from 20 to 3 mg/L. In the same time period at a downgradient observation well, DO increased from <0.2 to 2 mg/L and MTBE concentrations decreased from 30 to <5 mg/L. These results indicate that microorganisms indigenous to the groundwater system at this site can degrade milligram per liter concentrations of MTBE under natural and artificial oxic conditions.     

Simultaneous determination of fuel oxygenates and BTEX using direct aqueous injection gas chromatography mass spectrometry (DAI-GC/MS)

A direct aqueous injection-gas chromatography/mass spectrometry (DAI-GC/MS) method for trace analysis of gasoline components in water is presented. The method allows for the simultaneous quantification of the following solutes: methyl tert-butyl ether (MTBE), its major degradation products (tert-butyl formate, tert-butyl alcohol (TBA), methyl acetate, and acetone), and possible substitutes of MTBE as an octane enhancer in gasoline (tert-amyl methyl ether, ethyl tert-butyl ether) as well as benzene, toluene, ethylbenzene, p-xylene, m-xylene, and o-xylene (BTEX). No enrichment or pretreatment steps are required, and sample volumes of only 50 microL are needed for analysis. The detection limits in two different matrixes (spiked lake water and contaminated groundwater) are < or = 2 microg/L for most analytes and < 0.2 microg/L for MTBE, benzene, and toluene. The accuracy of the DAI-GC/MS method was excellent as determined from comparison with headspace-GC/MS and purge-and-trap-GC/MS. The DAI-GC/MS method has been applied to various environmental studies, which demonstrated its versatility. The applications comprised both laboratory (MTBE degradation in water treatment, quantification of polar gasoline components) and field (MTBE degradation ata gasoline spill site) investigations.     

Monitoring biodegradation of methyl tert-butyl ether (MTBE) using compound-specific carbon isotope analysis

Methyl tert-butyl ether (MTBE), the most common gasoline oxygenate, is frequently detected in surface water and groundwater. The aim of this study was to evaluate the potential of compound-specific isotope analysis to assess in situ biodegradation of MTBE in groundwater. For that purpose, the effect of relevant physical and biological processes on carbon isotope ratios of MTBE was evaluated in laboratory studies. Carbon isotope fractionation during organic phase/gas-phase partitioning (0.50 +/- 0.15@1000), aqueous phase/gas-phase partitioning (0.17 +/- 0.05@1000), and organic phase/aqueous-phase partitioning (0.18 +/- 0.24@1000) was small in comparison to carbon isotope fractionation measured during biodegradation of MTBE in microcosms based on aquifer sediments of the Borden site. In experiments with MTBE as the only substrate and a cometabolic experiment with 3-methypentane as primary substrate, MTBE became enriched in 13C by 5.1 to 6.9@1000 after 95 to 97% degradation. For both experiments, similar isotopic enrichment factors were obtained (-1.52 +/- 0.06 to -1.97 +/- 0.05@1000). Biodegradation of TBA, which accumulated transiently in the cometabolic microcosms, was also accompanied by carbon isotope fractionation, with an isotopic enrichment factor of -4.21 +/- 0.07@1000. This study suggests that carbon isotope analysis is a potential tool to trace in situ biodegradation of MTBE and TBA and thus to better understand the fate of these contaminants in the environment.     

The primary aerobic biodegradation of gasoline hydrocarbons

We describe the primary aerobic biodegradation of an unleaded, unoxygenated, regular gasoline by inocula from unacclimated fresh and sea water, and from a domestic sewage treatment plant. Biodegradation was rapid and complete in all inocula, with an overall median "half-life", at approximately 70 ppm gasoline and low levels of inorganic nutrients, of 5 days. The biodegradation of 131 individual hydrocarbons in the gasoline followed a relatively consistent pattern. The larger n-alkanes and iso-alkanes, and simple and alkylated aromatic compounds were the most readily degraded compounds, followed by the smaller n-alkanes and isoalkanes and the naphthenes. The last compounds to be degraded were butane, iso-butane, and 2,2-dimethylbutane, but even these disappeared with an apparent half-life of <30 days. The fact that the aqueous concentration of many of the individual components was in the sub ppb level is a remarkable demonstration of the ability of unadapted indigenous aerobic microorganisms to respond to and effectively biodegrade gasoline range hydrocarbons.     

Carbon isotopic fractionation during anaerobic biotransformation of methyl tert-butyl ether and tert-amyl methyl ether

The fuel oxygenate methyl tert-butyl ether (MTBE) has been frequently detected in groundwater and surface water. Since contaminated sites are often subsurface, anaerobic degradation of MTBE will likely be significant for remediation. As traditional approaches to evaluate biodegradation generally involve laboratory microcosm studies which require time and resources, innovative approaches are needed to demonstrate active in situ biodegradation of MTBE. This study was conducted to gather information at the laboratory level to evaluate the potential of applying carbon isotope fractionation as an indicator for in situ biodegradation of the fuel oxygenates MTBE and tert-amyl methyl ether (TAME). In this study, MTBE utilization was observed in a methanogenic sediment microcosm after a lengthy lag period of about 400 days. MTBE utilization was sustained upon refeeding and subculturing. tert-Butyl alcohol (TBA) was found to accumulate after propagation of cultures. The MTBE-grown cultures also utilized TAME and produced tert-amyl alcohol (TAA). The detection of TBA and TAA indicated that ether bond cleavage was the initial step in degradation for both compounds. Carbon isotope fractionation during anaerobic MTBE and TAME degradation was studied, and isotopic enrichment factors (epsilon) with 95% confidence intervals of -15.6 +/-4.1% and -13.7+/-4.5% were estimated for anaerobic MTBE and TAME degradation, respectively. Addition of 2-bromoethanesulfonic acid, an inhibitor of methanogenesis, substantially prolonged the lag period before transformation, but did not influence carbon isotope fractionation. Our experiment provided strong evidence of significant carbon isotope fractionation during anaerobic MTBE and TAME degradation, demonstrating that this technique can be used as an indicator for in situ MTBE and TAME degradation.     

New evaluation scheme for two-dimensional isotope analysis to decipher biodegradation processes: application to groundwater contamination by MTBE

Compound-specific analysis of stable carbon and hydrogen isotopes was used to assess the fate of the gasoline additive methyl tert-butyl ether (MTBE) and its major degradation product tert-butyl alcohol (TBA) in a groundwater plume at an industrial disposal site. We present a novel approach to evaluate two-dimensional compound-specific isotope data with the potential to identify reaction mechanisms and to quantify the extent of biodegradation at complex field sites. Due to the widespread contaminant plume, multiple MTBE sources, the presence of numerous other organic pollutants, and the complex biogeochemical and hydrological regime atthe site, a traditional mass balance approach was not applicable. The isotopic composition of MTBE steadily changed from the source regions along the major contaminant plume (-26.4% to +40.0% (carbon); -73.1% to +60.3% (hydrogen)) indicating substantial biodegradation. Constant carbon isotopic signatures of TBA suggest the absence of TBA degradation at the site. Published carbon and hydrogen isotope fractionation data for biodegradation of MTBE under oxic and anoxic conditions, respectively, were examined and used to determine both the nature and the extent of in-situ biodegradation along the plume(s). The coupled evaluation of two-dimensional compound-specific isotope data explained both carbon and hydrogen fractionation data in a consistent way and indicate anaerobic biodegradation of MTBE along the entire plume. A novel scheme to reevaluate empiric isotopic enrichment factors (epsilon) in terms of theoretically based intrinsic carbon (12k/13k) and hydrogen (1k/2k) kinetic isotope effects (KIE) is presented. Carbon and hydrogen KIE values, calculated for different potential reaction mechanisms, imply that anaerobic biodegradation of MTBE follows a SN2-type reaction mechanism. Furthermore, our data suggest that additional removal process(es) such as evaporation contributed to the overall MTBE removal along the plume, a phenomenon that might be significant also for other field sites at tropic or subtropic climates with elevated groundwater temperatures (25 degrees C).     

Hydrogen isotopic enrichment: an indicator of biodegradation at a petroleum hydrocarbon contaminated field site

Compound-specific carbon and hydrogen isotope analysis was used to investigate biodegradation of benzene and ethylbenzene in contaminated groundwater at Dow Benelux BV industrial site. delta13C values for dissolved benzene and ethylbenzene in downgradient samples were enriched by up to 2+/-0.5 per thousand, in 13C, compared to the delta13C value of the source area samples. delta2H values for dissolved benzene and ethylbenzene in downgradient samples exhibited larger isotopic enrichments of up to 27+/-5 per thousand for benzene and up to 50+/-5 per thousand for ethylbenzene relative to the source area. The observed carbon and hydrogen isotopic fractionation in downgradient samples provides evidence of biodegradation of both benzene and ethylbenzene within the study area at Dow Benelux BV. The estimated extents of biodegradation of benzene derived from carbon and hydrogen isotopic compositions for each sample are in agreement, supporting the conclusion that biodegradation is the primary control on the observed differences in carbon and hydrogen isotope values. Combined carbon and hydrogen isotope analyses provides the ability to compare biodegradation in the field based on two different parameters, and hence provides a stronger basis for assessment of biodegradation of petroleum hydrocarbon contaminants.     

An evaluation of compound-specific isotope analyses for assessing the biodegradation of MTBE at Port Hueneme, CA

The use of compound-specific isotope analysis (CSIA) as a diagnostic tool for MTBE biodegradation in aquifers was tested at the Port Hueneme, CA site. There, a 1500-m long dissolved MTBE plume and associated engineered aerobic flow-through biobarrier have been well-studied, leading to delineation of regions of known significant and limited bioattenuation. This allowed comparison of field-scale CSIA results with a priori knowledge of aerobic MTBE biodegradation, leading to conclusions concerning the utility of CSIA as a diagnostic tool for other aerobic biodegradation sites. Groundwater samples were collected and analyzed for both 13C and 2H (D) in MTBE through the bioactive treatment zone and within the larger MTBE plume. For reference, the 13C enrichment factor for MTBE biodegradation in laboratory-scale microcosms using site groundwater and sediments was also quantified. Aerobic microcosms showed a 13C enrichment of 5.5 to 6.4 +/- 0.2 per thousand over a two-order of magnitude concentration decrease, with an average isotope enrichment factor (epsilon(c)) of -1.4 per thousand, in agreement with other aerobic microcosm studies. Less 13C enrichment (about 25%) was observed for similar MTBE concentration reductions in groundwater samples collected within the aerobic biotreatment zone, and this enrichment was comparable to the scatter in delta13C values within the source zone. Increasing enrichment with decreasing MTBE concentration seen in microcosm data was not evident in either the 13C or D field data. The discrepancy between field and laboratory data may reflect small-scale (<1 m) spatial heterogeneity in MTBE biodegradation activity and the mixing of water from adjacent strata during groundwater sampling; for example, relatively nonattenuated MTBE-impacted water from one stratum could be mixed with highly attenuated/low-MTBE concentration from another, and this could produce a sample with both reduced MTBE concentration and low enrichment. Overall, the results suggest that 13C data alone may produce inconclusive results at sites where MTBE undergoes aerobic biodegradation, and that even with two-dimensional CSIA (13C and D), an increase in the confidence of data interpretation may only be possible with data sets larger than those typically collected in practice.     

Laboratory and field verification of a method to estimate the extent of petroleum biodegradation in soil

We describe a new and rapid quantitative approach to assess the extent of aerobic biodegradation of volatile and semivolatile hydrocarbons in crude oil, using Shushufindi oil from Ecuador as an example. Volatile hydrocarbon biodegradation was both rapid and complete-100% of the benzene, toluene, xylenes (BTEX) and 98% of the gasoline-range organics (GRO) were biodegraded in less than 2 days. Severe biodegradation of the semivolatile hydrocarbons occurred in the inoculated samples with 67% and 87% loss of the diesel-range hydrocarbons (DRO) in 3 and 20 weeks, respectively. One-hundred percent of the naphthalene, fluorene, and phenanthrene, and 46% of the chrysene in the oil were biodegraded within 3 weeks. Percent depletion estimates based on C(30) 17α,21β(H)-hopane (hopane) underestimated the diesel-range organics (DRO) and USEPA 16 priority pollutant PAH losses in the most severely biodegraded samples. The C(28) 20S-triaromatic steroid (TAS) was found to yield more accurate depletion estimates, and a new hopane stability ratio (HSR = hopane/(hopane + TAS)) was developed to monitor hopane degradation in field samples. Oil degradation within field soil samples impacted with Shushufindi crude oil was 83% and 98% for DRO and PAH, respectively. The gas chromatograms and percent depletion estimates indicated that similar levels of petroleum degradation occurred in both the field and laboratory samples, but hopane degradation was substantially less in the field samples. We conclude that cometabolism of hopane may be a factor during rapid biodegradation of petroleum in the laboratory and may not occur to a great extent during biodegradation in the field. We recommend that the hopane stability ratio be monitored in future field studies. If hopane degradation is observed, then the TAS percent depletion estimate should be computed to correct for any bias that may result in petroleum depletion estimates based on hopane.     

Anaerobic biodegradation of ethylene dibromide and 1,2-dichloroethane in the presence of fuel hydrocarbons

Field evidence from underground storage tank sites where leaded gasoline leaked indicates the lead scavengers 1,2-dibromoethane (ethylene dibromide, or EDB) and 1,2-dichloroethane (1,2-DCA) may be present in groundwater at levels that pose unacceptable risk. These compounds are seldom tested for at UST sites. Although dehalogenation of EDB and 1,2-DCA is well established, the effect of fuel hydrocarbons on their biodegradability under anaerobic conditions is poorly understood. Microcosms (2 L glass bottles) were prepared with soil and groundwater from a UST site in Clemson, South Carolina, using samples collected from the source (containing residual fuel) and less contaminated downgradient areas. Anaerobic biodegradation of EDB occurred in microcosms simulating natural attenuation, but was more extensive and predictable in treatments biostimulated with lactate. In the downgradient biostimulated microcosms, EDB decreased below its maximum contaminant level (MCL) (0.05 microg/L) at a first order rate of 9.4 +/- 0.2 yr(-1). The pathway for EDB dehalogenation proceeded mainly by dihaloelimination to ethene in the source microcosms, while sequential hydrogenolysis to bromoethane and ethane was predominant in the downgradient treatments. Biodegradation of EDB in the source microcosms was confirmed by carbon specific isotope analysis, with a delta13C enrichment factor of -5.6 per thousand. The highest levels of EDB removal occurred in microcosms that produced the highest amounts of methane. Extensive biodegradation of benzene, ethylbenzene, toluene and ortho-xylene was also observed in the source and downgradient area microcosms. In contrast, biodegradation of 1,2-DCA proceeded at a considerably slower rate than EDB, with no response to lactate additions. The slower biodegradation rates for 1,2-DCA agree with field observations and indicate that even if EDB is removed to below its MCL, 1,2-DCA may persist.