Modern geochemical and molecular tools for monitoring in-situ biodegradation of MTBE and TBA

Methyl tert-butyl ether (MTBE) is a major gasoline oxygenate worldwide and a widespread groundwater contaminant. Natural attenuation of MTBE is of practical interest as a cost effective and non-invasive approach to remediation of contaminated sites. The effectiveness of MTBE attenuation can be difficult to demonstrate without verification of the occurrence of in-situ biodegradation. The aim of this paper is to discuss the recent progress in assessing in-situ biodegradation. In particular, compound-specific isotope analysis (CSIA), molecular techniques based on nucleic acids analysis and in-situ application of stable isotope labels will be discussed. Additionally, attenuation of tert-butyl alcohol (TBA) is of particular interest, as this compound tends to occur alongside MTBE introduced from the gasoline or produced by (mainly anaerobic) biodegradation of MTBE.     

Challenges in Monitoring the Natural Attenuation of Spatially Variable Plumes

Monitored natural attenuation may be applied as a risk-based remediation strategy if it can be established that contaminants are or will be reduced to some acceptable level at or before a compliance point. Contaminant attenuation is often attributed to intrinsic biodegradation, which in some circumstances may occur only at the plume fringes where electron acceptors from the surrounding uncontaminated zones mix by dispersion and diffusion with the plume. However, due to the common spatial and temporal variability exhibited by many plumes, the centreline monitoring approaches advocated in many natural attenuation protocols may be unable to detect natural attenuation occurring primarily by fringe processes. Snapshot data from a multilevel sampling well transect across an MTBE plume at Vandenberg Air Force Base, CA, USA, illustrate the difficulty of centreline monitoring and the challenge of providing sufficient detail to detect attenuation processes that may be occurring primarily at plume fringes. In a study of a phenols plume in Wolverhampton, UK, high-resolution multilevel wells demonstrated that the key biodegradation processes were restricted spatially to the upper fringe of the plume and were rate-limited by transverse dispersion and diffusion of electron acceptors into the plume. Thus the overall extent of biodegradation was considerably less than suggested by a plume-scale analysis of total electron acceptor and contaminant budgets. These examples indicate that more robust and cost-effective MNA assessments can be obtained using monitoring strategies that focus on the location of key biodegradation processes.     

Naturally occurring bacteria similar to the methyl tert-butyl ether (MTBE)-degrading strain PM1 are present in MTBE-contaminated groundwater

Methyl tert-butyl ether (MTBE) is a widespread groundwater contaminant that does not respond well to conventional treatment technologies. Growing evidence indicates that microbial communities indigenous to groundwater can degrade MTBE under aerobic and anaerobic conditions. Although pure cultures of microorganisms able to degrade or cometabolize MTBE have been reported, to date the specific organisms responsible for MTBE degradation in various field studies have not be identified. We report that DNA sequences almost identical (99% homology) to those of strain PM1, originally isolated from a biofilter in southern California, are naturally occurring in an MTBE-polluted aquifer in Vandenberg Air Force Base (VAFB), Lompoc, California. Cell densities of native PM1 (measured by TaqMan quantitative PCR) in VAFB groundwater samples ranged from below the detection limit (in anaerobic sites) to 10(3) to 10(4) cells/ml (in oxygen-amended sites). In groundwater from anaerobic or aerobic sites incubated in microcosms spiked with 10 microg of MTBE/liter, densities of native PM1 increased to approximately 10(5) cells/ml. Native PM1 densities also increased during incubation of VAFB sediments during MTBE degradation. In controlled field plots amended with oxygen, artificially increasing the MTBE concentration was followed by an increase in the in situ native PM1 cell density. This is the first reported relationship between in situ MTBE biodegradation and densities of MTBE-degrading bacteria by quantitative molecular methods.     

Naturally Occurring Bacteria Similar to the Methyl tert-Butyl Ether (MTBE)-Degrading Strain PM1 Are Present in MTBE-Contaminated Groundwater

Methyl tert-butyl ether (MTBE) is a widespread groundwater contaminant that does not respond well to conventional treatment technologies. Growing evidence indicates that microbial communities indigenous to groundwater can degrade MTBE under aerobic and anaerobic conditions. Although pure cultures of microorganisms able to degrade or cometabolize MTBE have been reported, to date the specific organisms responsible for MTBE degradation in various field studies have not be identified. We report that DNA sequences almost identical (99% homology) to those of strain PM1, originally isolated from a biofilter in southern California, are naturally occurring in an MTBE-polluted aquifer in Vandenberg Air Force Base (VAFB), Lompoc, California. Cell densities of native PM1 (measured by TaqMan quantitative PCR) in VAFB groundwater samples ranged from below the detection limit (in anaerobic sites) to 10³ to 10⁴ cells/ml (in oxygen-amended sites). In groundwater from anaerobic or aerobic sites incubated in microcosms spiked with 10 μg of MTBE/liter, densities of native PM1 increased to approximately 10⁵ cells/ml. Native PM1 densities also increased during incubation of VAFB sediments during MTBE degradation. In controlled field plots amended with oxygen, artificially increasing the MTBE concentration was followed by an increase in the in situ native PM1 cell density. This is the first reported relationship between in situ MTBE biodegradation and densities of MTBE-degrading bacteria by quantitative molecular methods.     

Treatment of volatile organic contaminants in a vertical flow filter: Relevance of different removal processes

Vertical flow filters and vertical flow constructed wetlands are established wastewater treatment systems and have also been proposed for the treatment of contaminated groundwater. This study investigates the removal processes of volatile organic compounds in a pilot-scale vertical flow filter. The filter is intermittently irrigated with contaminated groundwater containing benzene, MTBE and ammonium as the main contaminants. The system is characterized by unsaturated conditions and high contaminant removal efficiency. The aim of the present study is to evaluate the contribution of biodegradation and volatilization to the overall removal of benzene and MTBE. Tracer tests and flow rate measurements showed a highly transient flow and heterogeneous transport regime. Radon-222, naturally occurring in the treated groundwater, was used as a gas tracer and indicated a high volatilization potential. Radon-222 behavior was reproduced by numerical simulations and extrapolated for benzene and MTBE, and indicated these compounds also have a high volatilization potential. In contrast, passive sampler measurements on top of the filter detected only low benzene and MTBE concentrations. Biodegradation potential was evaluated by the analysis of catabolic genes involved in organic compound degradation and a quantitative estimation of biodegradation was derived from stable isotope fractionation analysis. Results suggest that despite the high volatilization potential, biodegradation is the predominant mass removal process in the filter system, which indicates that the volatilized fraction of the contaminants is still subject to subsequent biodegradation. In particular, the upper filter layer located between the injection tubes and the surface of the system might also contribute to biodegradation, and might play a crucial role in avoiding the emission of volatilized contaminants into the atmosphere.     

Natural Attenuation Rate Clarifications: The True Picture is in the Details

The term “Natural Attenuation” (NA) has been defined as naturally occurring processes in soil and groundwater environments that act without human intervention to reduce the mass, toxicity, mobility, volume, or concentration of contaminants in those media. Monitored natural attenuation (MNA) protocols generally involve the collection of biogeochemical data from groundwater monitoring wells at sites. The data are correlated in time and space with the various chemicals of concern (COC's) to establish predominant biodegradation mechanisms. Modelers using the first-order decay expression typically use the rate coefficient as a calibration parameter and adjust it until the transport model results match field data. With this approach, uncertainties with a number of parameters (e.g., dispersion, sorption, biodegradation, etc.) are lumped together in a single calibration parameter. The problems associated with the lumped parameter approach are illustrated using two commonly used models, BIOSCREEN and Buscheck/Alcantar Analytical Solution, in a variety of practical examples. The natural attenuation decay rate estimated using the lumped parameter approach is distinguished from a biodegradation rate established by isolating processes and examining biodegradation lines of evidence. The half-life determined from empirical data using the lumped parameter approach is often mistakenly interchanged with a biodegradation half-life when it is an all-encompassing half-life based on the interaction of numerous processes. Isolation of the processes, as they are represented in the governing transport equation, and a rationale approach at parameter estimation to avoid the potential pitfalls of the all-inclusive “attenuation rate,” are provided. In closing, it is imperative to implement the following steps to dissern lumped process degradation rates from biodegradation half-lives: (a) be sure the rate/half-life processes are clarified as to what they encompass, (b) establish exactly how the rate/half-life was determined, (c) make certain other processes, such as dispersion, were estimated correctly, and (d) if the half-life is presented as a first-order biodegradation rate, examine the available lines of evidence to substantiate it.     

Review of MTBE Biodegradation and Bioremediation

Conclusive evidence of methyl tert-butyl ether (MTBE) biotransformation and complete mineralization under aerobic conditions in environmental samples and enrichment cultures is reviewed, in addition to increasing evidence of MTBE biotransformation under anaerobic conditions. The metabolic pathway of MTBE appears to have two key intermediates, tert-butyl alcohol (TBA) and 2-hydroxy isobutyric acid (HIBA). The first enzyme in MTBE biodegradation has been identified as either a cytochrome P450 or a nonhemic monooxygenase in different isolates. Mixed and pure cultures of microorganisms have utilized MTBE as a sole carbon and energy source. Cometabolism of MTBE with n-alkanes at rates of 3.9 to 52 nmol/min/mg protein has been documented. The presence of co-contaminants such as BTEX has either not affected or seemed to limit MTBE biodegradation. Some studies of MTBE natural attenuation have attributed mass loss to biodegradation, while others have attributed mass loss to dilution and dispersion. Recent advances in the assessment of MTBE biodegradation have indicated the potential for natural anaerobic transformation of MTBE. In situ bioremediation of MTBE has been enhanced by adding air or oxygen, or by adding microorganisms and air or oxygen. Bioreactors have attained significant removal of MTBE from MTBE-contaminated influent. Despite historical concerns about the biodegradability of MTBE, several biological methods can now be used for MTBE remediation.     

Review of MTBE Biodegradation and Bioremediation

Conclusive evidence of methyl tert-butyl ether (MTBE) biotransformation and complete mineralization under aerobic conditions in environmental samples and enrichment cultures is reviewed, in addition to increasing evidence of MTBE biotransformation under anaerobic conditions. The metabolic pathway of MTBE appears to have two key intermediates, tert-butyl alcohol (TBA) and 2-hydroxy isobutyric acid (HIBA). The first enzyme in MTBE biodegradation has been identified as either a cytochrome P450 or a nonhemic monooxygenase in different isolates. Mixed and pure cultures of microorganisms have utilized MTBE as a sole carbon and energy source. Cometabolism of MTBE with n-alkanes at rates of 3.9 to 52 nmol/min/mg protein has been documented. The presence of co-contaminants such as BTEX has either not affected or seemed to limit MTBE biodegradation. Some studies of MTBE natural attenuation have attributed mass loss to biodegradation, while others have attributed mass loss to dilution and dispersion. Recent advances in the assessment of MTBE biodegradation have indicated the potential for natural anaerobic transformation of MTBE. In situ bioremediation of MTBE has been enhanced by adding air or oxygen, or by adding microorganisms and air or oxygen. Bioreactors have attained significant removal of MTBE from MTBE-contaminated influent. Despite historical concerns about the biodegradability of MTBE, several biological methods can now be used for MTBE remediation.     

Cometabolism of methyl tert-butyl ether (MTBE) with alkanes

The release of methyl tert-butyl ether (MTBE) to the environment, mainly from damaged gasoline underground storage tanks or distribution systems spills, has provoked extended groundwater pollution. Biological treatments are, in general, a good alternative for bioremediation of polluted sites; however, MTBE elimination from environment has constituted a challenge because of its chemical structure and physicochemical properties. The combination of a stable ether link and the branched moiety hinder biodegradation. Initial studies found MTBE to be highly recalcitrant but, in the last decade, reports of its biodegradation have been published first under aerobic conditions and just recently under anaerobic conditions. Microbial MTBE degradation is characterized by bacteria having low growth rates (0.35 day⁻¹) and biomass yields (average value 0.24 g biomass/g MTBE). Alternatively, cometabolism (defined as the transformation of a non-growth substrate in the obligate presence of a growth substrate), has been considered since it uncouples biodegradation of the contaminant from growth, reducing the long adaptation and propagation period. This period has been reported to be of several months in systems where it is degraded as sole carbon source. Cometabolic degradation rates are between 0.3 and 61 nmol/min/mg protein (in the same range of direct aerobic metabolism). However, a major concern in MTBE cometabolism is that the accumulation of tert-butyl alcohol (TBA) may, under certain cases, result in an incomplete site cleanup. This paper reviews in detail the implicated enzymes and field treatments for the cometabolism of MTBE degradation with alkanes as growth substrates.     

Natural Biological Attenuation of Phenoxy Herbicides in Groundwater: Dow AgroSciences Paritutu Site, New Zealand

Groundwater beneath a manufacturing site previously used for herbicide production has been shown to contain low levels of chlorinated phenols and phenoxy herbicides. The importance of biological processes in the natural attenuation of the groundwater contaminants was examined as part of an ongoing investigation. Analysis of the groundwater chemistry indicated that the aquifer is essentially aerobic in the area of interest. Laboratory microcosm experiments demonstrated that the naturally occurring microorganisms rapidly degraded a mixture of the predominant organic contaminants under conditions that simulate those in the aquifer. The time required for 50% degradation ranged from 7 to 27 days for 2,4-dichlorophenoxyacetic acid (2,4-D) and 9 to 49 days for 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). The rapid biodegradation rates were consistent with the results of microbiological analyses, which demonstrated that a substantial proportion of the culturable bacteria were capable of growth on 2,4-D as a sole carbon source. Results of gene probe assays suggested the numbers of bacteria with the potential to degrade 2,4-D were one to two orders of magnitude higher than were detected using plate counts. Computer model simulations illustrated that biodegradation would be expected to significantly contribute to the attenuation of 2,4-D and 2,4,5-T in the aquifer. On the basis of the various lines of evidence and the distances the groundwater must travel, the groundwater contaminants would be expected to naturally biodegrade to below levels of concern before the plume reaches potential environmental receptors.     

Comparison of Source Zone Natural Attenuation Rates At Crude Oil and Ethanol–Blended Fuel Release Sites

Understanding differences in source zone natural attenuation (SZNA) rates occurring among field sites impacted by the same contaminant and across field sites impacted by different contaminants is critical for developing management strategies. For example, unique site conditions can favor or inhibit biodegradation, whereas differences between contaminants can lead to variations in biodegradation potential. However, the implications of these effects for real-world release scenarios remain ambiguous. To better understand the implications of these effects, the authors investigated differences in SZNA rates at two crude oil and two denatured fuel-grade ethanol (DFE) spill sites using on-site measurements of surficial gas effluxes. At the crude oil sites, CH₄ effluxes were below detection, whereas CH₄ effluxes occurred at both DFE sites. Similarly, SZNA rates among sites impacted by the same contaminant were comparable, whereas order of magnitude differences existed between sites impacted by crude oil or DFE. At DFE sites, results also revealed source zone expansion in relation to the initial mass in place, suggesting that extended spatial monitoring may be required to characterize risk potential. Overall, key differences between crude oil and DFE release sites demonstrated the importance of site-specific interactions between hydrogeology and contaminant composition for mediating gas emissions and SZNA rates, and modulating gas transport regimes under field conditions.     

Bioremediation of MTBE: a review from a practical perspective

The addition of methyl tert-butyl ether (MTBE) to gasoline has resulted in public uncertainty regarding the continued reliance on biological processes for gasoline remediation. Despite this concern, researchers have shown that MTBE can be effectively degraded in the laboratory under aerobic conditions using pure and mixed cultures with half-lives ranging from 0.04 to 29 days. Ex-situ aerobic fixed-film and aerobic suspended growth bioreactor studies have demonstrated decreases in MTBE concentrations of 83% and 96% with hydraulic residence times of 0.3 hrs and 3 days, respectively. In microcosm and field studies, aerobic biodegradation half-lives range from 2 to 693 days. These half-lives have been shown to decrease with increasing dissolved oxygen concentrations and, in some cases, with the addition of exogenous MTBE-degraders. MTBE concentrations have also been observed to decrease under anaerobic conditions; however, these rates are not as well defined. Several detailed field case studies describing the use of ex-situ reactors, natural attenuation, and bioaugmentation are presented in this paper and demonstrate the potential for successful remediation of MTBE-contaminated aquifers. In conclusion, a substantial amount of literature is available which demonstratesthat the in-situ biodegradation of MTBE is contingent on achieving aerobic conditions in the contaminated aquifer.     

The use of stable isotope probing techniques in bioreactor and field studies on bioremediation

Stable isotope probing (SIP) is a molecular technique that allows investigators to follow the flow of atoms in isotopically enriched molecules through complex microbial communities into metabolically active microorganisms. Thus, SIP has immense promise for discovering microorganisms responsible for ecologically important biogeochemical reactions in nature. Applications of SIP to biodegradation and bioremediation processes are still in their infancy. In the past few years, approximately a dozen biodegradation studies using SIP based on the analysis of labeled DNA, RNA or phospholipid fatty acids have been completed. Results have begun to link biomarkers (especially sequences of 16S ribosomal RNA and functional genes) to biodegradation reactions in naturally occurring microbial communities. As extensive compilations of ecologically important genotypes and phenotypes accrue, predictive abilities for contaminant metabolism in particular habitats may be achieved.     

Biodegradation of methyl <Emphasis Type="Italic">tert</Emphasis>-butyl ether by newly identified soil microorganisms in a simple mineral solution

Methyl tert-butyl ether (MTBE) is a widely used fuel ether, which has become a soil and water contaminant. In this study, 12 microbial strains were isolated from gasoline-contaminated soils and selected because of their capacity to grow in MTBE. The strains were identified by 16S/ITS rDNA gene sequencing and screened for their ability to consume MTBE aerobically in a simple mineral solution. Solid phase microoextraction and gas chromatography were used to detect MTBE degradation. High levels of MTBE biodegradation were obtained using resting cells of the bacteria Achromobacter xylosoxidans MCM1/1 (78%), Enterobacter cloacae MCM2/1 (50%), and Ochrobactrum anthropi MCM5/1 (52%) and the fungus Exophiala dermatitidis MCM3/4 (14%). Our phylogenetic analysis clearly shows that bacterial MTBE biodegraders belong to the clade of Proteobacteria. For further insight, MTBE-degrader strains were profiled by denaturing gel gradient electrophoresis (DGGE) of PCR-amplified 16S rRNA gene sequences. This approach could be used to analyse microbial community dynamics in bioremediation processes.     

Effect of biostimulants on 2,4,6-trinitrotoluene (TNT) degradation and bacterial community composition in contaminated aquifer sediment enrichments

2,4,6-Trinitrotoluene (TNT) is a toxic and persistent explosive compound occurring as a contaminant at numerous sites worldwide. Knowledge of the microbial dynamics driving TNT biodegradation is limited, particularly in native aquifer sediments where it poses a threat to water resources. The purpose of this study was to quantify the effect of organic amendments on anaerobic TNT biodegradation rate and pathway in an enrichment culture obtained from historically contaminated aquifer sediment and to compare the bacterial community dynamics. TNT readily biodegraded in all microcosms, with the highest biodegradation rate obtained under the lactate amended condition followed by ethanol amended and naturally occurring organic matter (extracted from site sediment) amended conditions. Although a reductive pathway of TNT degradation was observed across all conditions, denaturing gradient gel electrophoresis (DGGE) analysis revealed distinct bacterial community compositions. In all microcosms, Gram-negative γ- or β-Proteobacteria and Gram-positive Negativicutes or Clostridia were observed. A Pseudomonas sp. in particular was observed to be stimulated under all conditions. According to non-metric multidimensional scaling analysis of DGGE profiles, the microcosm communities were most similar to heavily TNT-contaminated field site sediment, relative to moderately and uncontaminated sediments, suggesting that TNT contamination itself is a major driver of microbial community structure. Overall these results provide a new line of evidence of the key bacteria driving TNT degradation in aquifer sediments and their dynamics in response to organic carbon amendment, supporting this approach as a promising technology for stimulating in situ TNT bioremediation in the subsurface.     

Aerobic MTBE biodegradation in the presence of BTEX by two consortia under batch and semi-batch conditions

This study explores the effect of microbial consortium composition and reactor configuration on methyl tert-butyl ether (MTBE) biodegradation in the presence of benzene, toluene, ethylbenzene and p-xylenes(BTEX). MTBE biodegradation was monitored in the presence and absence of BTEX in duplicate batch reactors inoculated with distinct enrichment cultures: MTBE only (MO—originally enriched on MTBE) and/or MTBE BTEX (MB—originally enriched on MTBE and BTEX). The MO culture was also applied in a semi-batch reactor which received both MTBE and BTEX periodically in fresh medium after allowing cells to settle. The composition of the microbial consortia was explored using a combination of 16S rRNA gene cloning and quantitative polymerase chain reaction targeting the known MTBE-degrading strain PM1^T. MTBE biodegradation was completely inhibited by BTEX in the batch reactors inoculated with the MB culture, and severely retarded in those inoculated with the MO culture (0.18 ± 0.04 mg/L-day). In the semi-batch reactor, however, the MTBE biodegradation rate in the presence of BTEX was almost three times as high as in the batch reactors (0.48 ± 0.2 mg/L-day), but still slower than MTBE biodegradation in the absence of BTEX in the MO-inoculated batch reactors (1.47 ± 0.47 mg/L-day). A long lag phase in MTBE biodegradation was observed in batch reactors inoculated with the MB culture (20 days), but the ultimate rate was comparable to the MO culture (0.95 ± 0.44 mg/L-day). Analysis of the cultures revealed that strain PM1^T concentrations were lower in cultures that successfully biodegraded MTBE in the presence of BTEX. Also, other MTBE degraders, such as Leptothrix sp. and Hydrogenophaga sp. were found in these cultures. These results demonstrate that MTBE bioremediation in the presence of BTEX is feasible, and that culture composition and reactor configuration are key factors.     

A Field Application of Hydrogen-Releasing Compound (HRCTM) for the Enhanced Bioremediation of Methyl Tertiary Butyl Ether (MTBE)

Due to a greater understanding of the behavior of the fuel oxygenate Methyl Tertiary Butyl Ether (MTBE) in groundwater, the United States Environmental Protection Agency (EPA) and the American Petroleum Institute (API) recently have acknowledged the need for the development and application of additional remedial strategies to address the more extensive, longer lived, and faster moving dissolved MTBE plumes often associated with oxygenated fuel releases (API, 2000 and USEPA, 2000a). The need for alternative methods for managing dissolved MTBE plumes is particularly evident in the case of the Upper Glacial aquifer of Long Island, New York. Hydrogeologic conditions in the this water table aquifer (i. e., high hydraulic conductivity, high average pore velocities, low organic carbon, and high rates of recharge) have been found to contribute to the formation of extensive, long, narrow, and three-dimensional dissolved MTBE plumes that plunge into the aquifer in response to recharge (Weaver et. al. 1999). The characteristics of MTBE plumes in the Upper Glacial aquifer in combination with abundant sensitive receptors (mainly drinking water supply wells), often renders monitored natural attenuation (MNA) plume management strategies inappropriate, resulting in the need for plume control, frequently via pumping and treating (NYSDEC, 2000). In such cases, remedial costs can rise well beyond those associated with similar fuel releases that did not contain MTBE (USEPA, 1998a). Consequently, the application of remedial technologies for MTBE other than MNA, or pumping and treating, are of great interest to those responsible for the management of dissolved MTBE plumes on Long Island or in similar hydrogeologic settings. An alternative strategy for the remediation of dissolved MTBE plumes was recently field tested at an oxygenated fuel spill site on Long Island. The strategy was enhanced biodegradation via the application of Hydrogen Release Compound (HRC^TM). HRC^TM is a form of polylactate ester that slowly releases biodegradation stimulating constituents into the aquifer and has been shown in other studies to foster methanogenic conditions that advance the reductive dechlorina-tion of perchloroethene (PCE) and trichloroethene (TCE) (Koenigsberg, 1998). Numerous reports have been written that discuss the biodegradation of MTBE under aerobic conditions, as well as microcosm studies in which MTBE biodegradation was observed under anaerobic conditions. However, there are limited reports that document the natural anaerobic biodegradation of dissolved MTBE (McLoughlin, 2000). Despite the lack of documented natural anaerobic biodegradation of MTBE, it has been observed that MTBE transport often occurs under anoxic conditions at oxygenated fuel releases as the result of the biodegradation of other fuel constituents, such as benzene, toluene, ethylbenzene and xylene (BTEX), which deplete the available dissolved oxygen as well as other electron acceptors (nitrate, ferric iron, manganese, etc.) (USEPA, 2000c and API, 1996). Therefore, an anaerobic biodegradation strategy is attractive due to its synergy with the existing geochemical conditions. Consequently, the study was conceived and designed to test the ability of HRC(tm) to foster the anaerobic bio-degradation of MTBE under methano-genic conditions (McLoughlin, 2000). The application of HRC(tm) did result in the formation of a large area of enhanced reducing conditions in the vicinity and down gradient of the application zone. However, under these site conditions, the HRC(tm) application did not induce measurable methanogenic conditions with the associated elevated dissolved hydrogen concentrations required for significant MTBE anaerobic biodegradation. The high hydraulic conductivity and high average pore velocity at the site were likely responsible. Despite this, the study can be viewed as a success since much was learned that can be used in future studies of anaerobic biodegradation of MTBE and the application of HRC(tm).     

Modeling natural attenuation of benzene with analytical and numerical models

Natural attenuation of benzene is a much-accepted technology for remediating low risk sites. To date, numerous protocols have been developed for assessing natural attenuation and measuring indicator parameters. Many models have additionally been developed to describe the advection, dispersion, sorption and biodegradation processes involved. It is evident that while there is extensive guidance in natural attenuation protocols for field sampling methodologies, less emphasis is placed on analyzing natural attenuation data for supporting appropriate model development. This paper presents methodologies for data analysis and interpretation that may be undertaken to achieve data reduction for modeling purposes. A case study is presented to illustrate the use of an analytical and a numerical natural attenuation model at the same site for predicting the time required to achieve the remedial goal at the site.This paper was originally intended for the Special Issue on Natural Attenuation Volume I published in Biodegradation 15(6), 2004.     

Characterization of Methyl tert-Butyl Ether-Degrading Bacteria from a Gasoline-Contaminated Aquifer

Molecular microbial community analysis was combined with traditional cultivation strategies to investigate the presence of methyl tert-butyl ether (MTBE)-degrading bacteria in a gasoline-contaminated aquifer (Ronan, MT). A bacterial consortium, RS24, which is capable of complete mineralization of MTBE as a sole carbon and energy source was enriched from soil and aquifer materials taken from the contaminated site. The consortium was capable of degrading MTBE at rates up to 0.66 mg d^-1, with corresponding gross biomass yields of 0.25±0.02 mg dry biomass (mg MTBE)^-1. Two MTBE-degrading isolates identified as Pseudomonas Ant9 and Rhodococcus koreensis were obtained from the consortium. However, both isolates required the presence of 2-propanol as a cosubstrate for MTBE degradation. Denaturing gradient gel electrophoresis (DGGE) of Poly-merase Chain Reaction (PCR)-amplified 16S rDNA confirmed the presence of both isolates in the initial consortium and indicated their disappearance with transfer and subculturing. MTBE degradation and cell growth by the consortium was stimulated by the presence of spent culture medium, suggesting the production of a growth factor during MTBE degradation. These results indicate the presence of naturally occurring MTBE-degrading bacteria in a contaminated aquifer and suggest the potential for natural attenuation or enhanced aerobic oxidation.