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.     

Microbial community analyses of three distinct, liquid cultures that degrade methyl tert-butyl ether using anaerobic metabolism

Methyl tert-butyl ether (MTBE) is a prevalent groundwater contaminant. In this study, three distinct MTBE-degrading, anaerobic cultures were derived from MTBE-contaminated aquifer material: cultures NW1, NW2 and NW3. The electron acceptors used are anthraquinone-2,6-disulfonate (AQDS; NW1), sulfate (NW2) and fumarate (NW3), respectively. About 1–2 mM MTBE is consistently degraded within 20–30 days in each culture. The 16S rDNA-based amplified ribosomal DNA restriction analysis (ARDRA) was used to analyze the microbial community in each culture. Results indicate novel microorganisms (i.e. no closely related known genera or species) catalyze anaerobic MTBE biodegradation, and microbial diversity varied with different electron acceptors. Tert-butyl alcohol (TBA) accumulated to nearly stoichiometric levels, and these cultures will be critical to understanding the factors that influence TBA accumulation versus degradation. The cultures presented here are the first stable anaerobic MTBE-degrading cultures that have been characterized with respect to taxonomy.     

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.     

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.     

Enzymes and genes involved in the aerobic biodegradation of methyl tert-butyl ether (MTBE)

Fuel oxygenates, mainly methyl tert-butyl ether (MTBE) but also ethyl tert-butyl ether (ETBE), are added to gasoline in replacement of lead tetraethyl to enhance its octane index. Their addition also improves the combustion efficiency and therefore decreases the emission of pollutants (CO and hydrocarbons). On the other hand, MTBE, being highly soluble in water and recalcitrant to biodegradation, is a major pollutant of water in aquifers contaminated by MTBE-supplemented gasoline during accidental release. MTBE was shown to be degraded through cometabolic oxidation or to be used as a carbon and energy source by a few microorganisms. We have summarized the present state of knowledge about the microorganisms involved in MTBE degradation and the MTBE catabolic pathways. The role of the different enzymes is discussed as well as the rare and recent data concerning the genes encoding the enzymes involved in the MTBE pathway. The phylogeny of the microorganisms isolated for their capacity to grow on MTBE is also described.     

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.     

Chemoinformatics-assisted development of new anti-biofilm compounds

The fuel oxygenate, methyl tert-butyl ether (MTBE), although now widely banned or substituted, remains a persistent groundwater contaminant. Multidimensional compound-specific isotope analysis (CSIA) of carbon and hydrogen is being developed for determining the extent of MTBE loss due to biodegradation and can also potentially distinguish between different biodegradation pathways. Carbon and hydrogen isotopic fractionation factors were determined for MTBE degradation in aerobic and anaerobic laboratory cultures. The carbon isotopic enrichment factor (εC) for aerobic MTBE degradation by a bacterial consortium containing the aerobic MTBE-degrading bacterium, Variovorax paradoxus, was −1.1 ± 0.2‰ and the hydrogen isotope enrichment factor (εH) was −15 ± 2‰. This corresponds to an approximated lambda value (Λ = εH/εC) of 14. Carbon isotope enrichment factors for anaerobic MTBE-degrading enrichment cultures were −7.0 ± 0.2‰ and did not vary based on the original inoculum source, redox condition of the enrichment, or supplementation with syringic acid as a co-substrate. The hydrogen enrichment factors of cultures without syringic acid were insignificant, however a strong hydrogen enrichment factor of −41 ± 3‰ was observed for cultures which were fed syringic acid during MTBE degradation. The Λ = 6 obtained for NYsyr cultures might be diagnostic for the stimulation of anaerobic MTBE degradation by methoxylated compounds by an as yet unknown pathway and mechanism. The stable-isotope enrichment factors determined in this study will enhance the use of CSIA for monitoring anaerobic and aerobic MTBE biodegradation in situ.     

Effects of environmental settings on MTBE removal for a mixed culture and its monoculture isolation

A mixed culture was utilized to evaluate methyl tert-butyl ether (MTBE) removal under various conditions and to isolate a MTBE-degrading pure culture. The results showed that high MTBE removal efficiencies can be reached even in the presence of other substrates. The biodegradation sequence of the target compounds by the mixed culture, in order of removal rate, was toluene, ethyl benzene, p-xylene, benzene, MTBE, ethyl ether, tert-amyl methyl ether, and ethyl tert-butyl ether. In addition, preincubation of the mixed cultures with benzene and toluene showed no negative effect on MTBE removal; on the contrary, it could even increase the degradation rate of MTBE. The kinetic behavior showed that the maximum specific growth rate and the saturation constant of the mixed culture degrading MTBE are 0.000778 h⁻¹ and 0.029 mg l⁻¹, respectively. However, a high MTBE concentration (60 mg l⁻¹) was slightly inhibiting to the growth of the mixed culture. The pure culture isolated from the enrichments in the bubble-air bioreactor showed better efficiency in MTBE removal than the mixed culture; whereas, tert-butyl alcohol was formed as a metabolic intermediate during the breakdown of MTBE.     

Aerobic Biotransformation of Gasoline Aromatics in MultiComponent Mixtures

The primary objective of this study was to evaluate the impact of substrate interactions on the biotransformation rates and mineralization potentials of gasoline monoaromatics and methyl tert-butyl ether (MTBE), compounds that commonly co-exist in groundwater contaminant plumes. A mixed culture was derived from gasoline-contaminated aquifer material using toluene as the enrichment substrate. Two pure cultures, Rhodococcus sp. RR1 and RR2, were isolated from the mixed culture. The three toluene-grown cultures were shown to biotransform all of the six BTEX compounds (benzene, toluene, ethylbenzene, o-xylene, m-xylene, and p-xylene), both individually and in mixtures, over a broad range of concentrations. The mixed culture was shown to degrade all of the BTEX compounds to ¹⁴CO₂, while the two isolates mineralized BTE(m-/p-)X, but biotransformed o-xylene without production of carbon dioxide. Studies to evaluate substrate interactions caused by the concurrent presence of multiple BTEX compounds during their biodegradation revealed a number of patterns,including competitive inhibition and cometabolism. Ethylbenzene was shown to significantly inhibit BTX degradation in mixtures. MTBE was not biodegraded by any of the three toluene-grown cultures over a range of MTBE concentrations. Furthermore, the presence of MTBE at concentrations of 2 to 100?mg/L had no effect on BTEX biotransformation rates.     

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.     

Carbon Isotope Fractionation during Anaerobic Degradation of Methyl tert-Butyl Ether under Sulfate-Reducing and Methanogenic Conditions

Methyl tert-butyl ether (MTBE), an octane enhancer and a fuel oxygenate in reformulated gasoline, has received increasing public attention after it was detected as a major contaminant of water resources. Although several techniques have been developed to remediate MTBE-contaminated sites, the fate of MTBE is mainly dependent upon natural degradation processes. Compound-specific stable isotope analysis has been proposed as a tool to distinguish the loss of MTBE due to biodegradation from other physical processes. Although MTBE is highly recalcitrant, anaerobic degradation has been demonstrated under different anoxic conditions and may be an important process. To accurately assess in situ MTBE degradation through carbon isotope analysis, carbon isotope fractionation during MTBE degradation by different cultures under different electron-accepting conditions needs to be investigated. In this study, carbon isotope fractionation during MTBE degradation under sulfate-reducing and methanogenic conditions was studied in anaerobic cultures enriched from two different sediments. Significant enrichment of ¹³C in residual MTBE during anaerobic biotransformation was observed under both sulfate-reducing and methanogenic conditions. The isotopic enrichment factors () estimated for each enrichment were almost identical (−13.4 to −14.6; r² = 0.89 to 0.99). A value of −14.4 ± 0.7 was obtained from regression analysis (r² = 0.97, n = 55, 95% confidence interval), when all data from our MTBE-transforming anaerobic cultures were combined. The similar magnitude of carbon isotope fractionation in all enrichments regardless of culture or electron-accepting condition suggests that the terminal electron-accepting process may not significantly affect carbon isotope fractionation during anaerobic MTBE degradation.     

Effects of co-substrates and inhibitors on the anaerobic O-demethylation of methyl <Emphasis Type="Italic">tert</Emphasis>-butyl ether (MTBE)

Methyl tert-butyl ether (MTBE) contamination is widespread in aquifers near urban areas around the world. Since this synthetic fuel oxygenate is resistant to most physical methods of treating fuel-contaminated water, biodegradation may be a useful means of remediation. Currently, information on anaerobic MTBE degradation is scarce. Depletion has been observed in soil and sediment microcosms from a variety of locations and under several redox conditions, but the responsible organisms are unknown. We are studying anaerobic consortia, enriched from contaminated sediments for MTBE-utilizing microorganisms for over a decade. MTBE degradation occurred in the presence of other fuel components and was not affected by toluene, benzene, ethanol, methanol, or gasoline. Many aryl O-methyl ethers, such as syringic acid, that are O-demethylated by acetogenic bacteria, were also O-demethylated by the MTBE-utilizing enrichment cultures. The addition of these compounds as co-substrates increased the rate of MTBE degradation, offering a potentially useful method of stimulating the MTBE degradation rate in situ. Propyl iodide caused light-reversible inhibition of MTBE degradation, suggesting that the MTBE degradation process is corrinoid dependent. The anaerobic MTBE degradation process was not directly coupled to methanogenesis or sulfidogenesis and was inhibited by the bactericidal antibiotic, rifampicin. These results suggest that MTBE degradation is mediated by acetogenic bacteria.     

Aerobic biodegradation of methyl tert-butyl ether (MTBE) by pure bacterial cultures isolated from contaminated soil

Summary Methyl tert-Butyl Ether (MTBE) has been used in gasoline as a substitute for lead-based additives, which have been demonstrated to be toxic. MTBE however, is persistent in soil and water, showing high affinity for water and low affinity for soil, and has become an important contaminant. Therefore, the aim of this work was to isolate and identify soil microorganisms capable of degrading MTBE. Two samples were taken from a gasoline-contaminated soil at a service station and 59 different bacterial strains were isolated by enrichment culture with three consecutive selective transfers. Biochemical and morphological characterization of the bacterial isolates classified them into the following groups: Bacillus, Rhodococcus, Micrococcus, Aureobacterium and Proteus. Twelve strains were selected for evaluation of MTBE biodegradation depending on visual growth and biomass production of the isolates in minimal salt broth. Six strains significantly reduced MTBE concentration (22–37%) compared to an abiotic control after 5 days of incubation. Although it has been considered that MTBE is degraded mainly by cometabolism, our results demonstrate that these microorganisms are able to reduce MTBE concentration when MTBE is the sole source of carbon.     

Biodegradation of methyl <Emphasis Type="Italic">t</Emphasis>-butyl ether by aerobic granules under a cosubstrate condition

Aerobic granules efficient at degrading methyl tert-butyl ether (MTBE) with ethanol as a cosubstrate were successfully developed in a well-mixed sequencing batch reactor (SBR). Aerobic granules were first observed about 100 days after reactor startup. Treatment efficiency of MTBE in the reactor during stable operation exceeded 99.9%, and effluent MTBE was in the range of 15–50 μg/L. The specific MTBE degradation rate was observed to increase with increasing MTBE initial concentration from 25 to 500 mg/L, which peaked at 22.7 mg MTBE/g (volatile suspended solids)·h and declined with further increases in MTBE concentration as substrate inhibition effects became significant. Microbial-community deoxyribonucleic acid profiling was carried out using denaturing gradient gel electrophoresis of polymerase chain reaction-amplified 16S ribosomal ribonucleic acid. The reactor was found to be inhabited by several diverse bacterial species, most notably microorganisms related to the genera Sphingomonas, Methylobacterium, and Hyphomicrobium vulgare. These organisms were previously reported to be associated with MTBE biodegradation. A majority of the bands in the reactor represented a group of organisms belonging to the Flavobacteria–Proteobacteria–Actinobacteridae class of bacteria. This study demonstrates that MTBE can be effectively degraded by aerobic granules under a cosubstrate condition and gives insight into the microorganisms potentially involved in the process.     

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).     

Aerobic MTBE biodegradation: an examination of past studies, current challenges and future research directions

With the current practice of amending gasoline with up to 15% by volume MTBE, the contamination of groundwater by MTBE has become widespread. As a result, the bioremediation of MTBE-impacted aquifers has become an active area of research. A review of the current literature on the aerobic biodegradation of MTBE reveals that a number of cultures from diverse environments can either partially degrade or completely mineralize MTBE. MTBE is either utilized as a sole carbon and energy source or is degraded cometabolically by cultures grown on alkanes. Reported degradation rates range from 0.3 to 50 mg MTBE/g cells/h while growth rates (0.01–0.05 g MTBE/g cells/d) and cellular yields (0.1–0.2 g cells/g MTBE) are generally low. Studies on the mechanisms of MTBE degradation indicate that a monooxygenase enzyme cleaves the ether bond yielding tert-butyl alcohol (TBA) and formaldehyde as the dominant detectable intermediates. TBA is further degraded to 2-methyl-2-hydroxy-1-propanol, 2-hydroxyisobutyric acid, 2-propanol, acetone, hydroxyacteone and eventually, carbon dioxide. The majority of these intermediates are also common to mammalian MTBE metabolism. Laboratory studies on the degradation of MTBE in the presence of gasoline aromatics reveal that while degradation rates of other gasoline components are generally not inhibited by MTBE, MTBE degradation could be inhibited in the presence of more easily biodegradable compounds. Controlled field studies are clearly needed to elucidate MTBE degradation potential in co-contaminant plumes. Based on the reviewed studies, it is likely that a bioremediation strategy involving direct metabolism, cometabolism, bioaugmentation, or some combination thereof, could be applied as a feasible and cost-effective treatment method for MTBE contamination.     

Aerobic Biodegradation of Methyl tert-Butyl Ether by Aquifer Bacteria from Leaking Underground Storage Tank Sites

The potential for aerobic methyl tert-butyl ether (MTBE) degradation was investigated with microcosms containing aquifer sediment and groundwater from four MTBE-contaminated sites characterized by oxygen-limited in situ conditions. MTBE depletion was observed for sediments from two sites (e.g., 4.5 mg/liter degraded in 15 days after a 4-day lag period), whereas no consumption of MTBE was observed for sediments from the other sites after 75 days. For sediments in which MTBE was consumed, 43 to 54% of added [U-¹⁴C]MTBE was mineralized to ¹⁴CO₂. Molecular phylogenetic analyses of these sediments indicated the enrichment of species closely related to a known MTBE-degrading bacterium, strain PM1. At only one site, the presence of water-soluble gasoline components significantly inhibited MTBE degradation and led to a more pronounced accumulation of the metabolite tert-butyl alcohol. Overall, these results suggest that the effects of oxygen and water-soluble gasoline components on in situ MTBE degradation will vary from site to site and that phylogenetic analysis may be a promising predictor of MTBE biodegradation potential.     

The Comparative and Combined Inhibitory Effects of MTBE and TBA on the Activities of Soil Exoenzymes

Methyl tert-butyl ether (MTBE) and tert-butyl alcohol (TBA) are major soil contaminants, and they have been actively investigated for their toxic effects on living organisms in soil ecosystems. Although previous studies have been used as tools to evaluate the health of soil, they have been limited in scope and ability to analyze the overall microbial activity. In the present study, the effects of MTBE and TBA on the activity of soil exoenzymes including urease, acid phosphatase, arylsulfatase, β-glucosidase, dehydrogenase, and fluorescein diacetate hydrolase, which are involved in nutrient cycles and overall microbial activities, were investigated. Soil samples were treated with 0–2% of MTBE and TBA solutions, and the comparative effects and combined effects on quantity of active soil exoenzymes were determined. The activity of six exoenzymes exposed solely to MTBE and TBA did not significantly change with dose concentration or exposure time, but did show significant changes when exposed to high concentrations of MTBE and TBA combined, with dehydrogenase being the most affected. Therefore, we proposed dehydrogenase as a potential biomarker to assess the risk of co-contamination of MTBE and TBA.     

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.