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.     

Biostimulation and Phytoremediation Treatment Strategies of Gasoline-Nickel Co-Contaminated Soil

This study investigated the potential effect of poultry dung (biostimulation) and stubborn grass (Sporobolus pyramidalis) (phytoremediation) on microbial biodegradation of gasoline and nickel uptake in gasoline-nickel-impacted soil. In addition, the potential stimulatory effects of nickel on hydrocarbon utilization were investigated over a small range of nickel concentrations (2.5–12.5 mg/kg). The results showed that an increase in nickel concentration increased hydrocarbon degraders in soil by a range of 8.4–17.2% and resulted in a relative increase in gasoline biodegradation (57.5–62.4%). Also, under aerobic conditions, total petroleum hydrocarbons’ (TPH) removal was 62.4% in the natural gasoline-nickel microcosm (natural attenuation), and a maximum of 78.5%, 85.7%, and 95.8% TPH removal was obtained in phytoremediation, biostimulation, and a combination of biostimulation- and phytoremediation-treated microcosms, respectively. First-order kinetics described the biodegradation of gasoline and nickel uptake very well. Half-life times obtained were 28.88, 18.24, 14.44, and 8.56 days for gasoline degradation under natural attenuation, phytoremediation, biostimulation, and combined biostimulation and phytoremediation treatment methods, respectively. The results indicate that these remediation methods have promising potential for effective remediation of soils co-contaminated with petroleum hydrocarbons and heavy metals.     

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.     

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.     

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.     

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-(14)C]MTBE was mineralized to (14)CO(2). 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.     

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.     

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.     

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.     

Biodegradation of gasoline by gellan gum-encapsulated bacterial cells

Encapsulated cell bioaugmentation is a novel alternative solution to in situ bioremediation of contaminated aquifers. This study was conducted to evaluate the feasibility of such a remediation strategy based on the performance of encapsulated cells in the biodegradation of gasoline, a major groundwater contaminant. An enriched bacterial consortium, isolated from a gasoline-polluted site, was encapsulated in gellan gum microbeads (16-53 microm diameter). The capacity of the encapsulated cells to degrade gasoline under aerobic conditions was evaluated in comparison with free (non-encapsulated) cells. Encapsulated cells (2.6 mg(cells) x g(-1) bead) degraded over 90% gasoline hydrocarbons (initial concentration 50-600 mg x L(-1)) within 5-10 days at 10 degrees C. Equivalent levels of free cells removed comparable amounts of gasoline (initial concentration 50-400 mg x L(-1)) within the same period but required up to 30 days to degrade the highest level of gasoline tested (600 mg x L(-1)). Free cells exhibited a lag phase in biodegradation, which increased from 1 to 5 days with an increase in gasoline concentration (200-600 x mg L(-1)). Encapsulation provided cells with a protective barrier against toxic hydrocarbons, eliminating the adaptation period required by free cells. The reduction of encapsulated cell mass loading from 2.6 to 1.0 mg(cells) x g(-1) bead caused a substantial decrease in the extent of biodegradation within a 30-day incubation period. Encapsulated cells dispersed within the porous soil matrix of saturated soil microcosms demonstrated a reduced performance in the removal of gasoline (initial concentrations of 400 and 600 mg x L(-1)), removing 30-50% gasoline hydrocarbons compared to 40-60% by free cells within 21 days of incubation. The results of this study suggest that gellan gum-encapsulated bacterial cells have the potential to be used for biodegradation of gasoline hydrocarbons in aqueous systems.     

Use of Mycobacterium austroafricanum IFP 2012 in a MTBE-degrading bioreactor

Mycobacterium austroafricanum IFP 2012 is able to slowly grow on methyl tert-butyl ether (MTBE), a fuel oxygenate widely used as a gasoline additive. The potential of M. austroafricanum IFP 2012 for aerobic MTBE degradation was investigated in the presence of a secondary carbon source, isopropanol. The strain was then tested for MTBE biodegradation at the laboratory-scale in a fixed-bed reactor using perlite as the matrix, and isopropanol was injected once a week to maintain M. austroafricanum IFP 2012 biomass inside the perlite bed. The biofilter was operated for 85 days at an influent flow rate of 20 ml/h by varying the MTBE concentration from 10 to 20 mg/l. The hydraulic retention time was fixed at 5 days. The removal of MTBE depended on the inlet MTBE concentration and a MTBE removal efficiency higher than 99% was obtained for MTBE concentrations up to 15 mg/l. A set of 16S rRNA gene primers specific for M. austroafricanum species was used to analyze the DNA extracted from the biofilter effluent in order to detect the presence of M. austroafricanum IFP 2012 and to estimate the effect of periodic injections of isopropanol on the release of the strain from the perlite bed. The results demonstrated that the injection of isopropanol served to maintain an active MTBE degrading biomass in the biofilter and that this system could be used to effectively treat MTBE contaminated groundwater.     

Effect of Hydrologic and Geochemical Conditions on Oxygen-Enhanced Bioremediation in a Gasoline-Contaminated Aquifer

Oxygen addition to enhance bioremediation of gasoline-contaminated ground water was performed in two locations of a shallow aquifer in South Carolina characterized by benzene, toluene, and methyl tert-butyl ether (MTBE) at concentrations greater than 1 mg/L, respectively. Oxygen addition with an oxygen-release compound (a proprietary form of magnesium peroxide [MgO₂]) produced markedly different results with respect to dissolved oxygen (DO) generation and contaminant decrease in the two locations. Oxygen-release compound injected at the former underground storage tank (UST) source area did not significantly change measured concentrations of DO, benzene, toluene, or MTBE. Conversely, oxygen-release compound injected 200 m downgradient of the former UST source area rapidly increased DO levels, and benzene, toluene, and MTBE concentrations decreased substantially. The different results can be related to differences in hydrologic and geochemical conditions that characterized the two locations prior to oxygen addition. For example, the contaminated aquifer downgradient of the former UST source area was anoxic, but frequently received oxygen-saturated recharge during rainfall events. As such, the aquifer was characterized by low concentrations of reduced species (such as ferrous iron (Fe²⁺), as well as relatively high numbers of aerobic heterotrophic bacteria (as most probable number [MPN] per milliliter). In contrast, recharge does not occur in the paved, former UST source area. The anoxic aquifer was characterized by higher concentrations of Fe²⁺ that exerted a significant chemical oxygen demand on the oxygen injected, and much lower numbers of aerobic heterotrophic bacteria. The results of this investigation indicate the important role that pre-existing hydrologic, geochemical, and microbiologic conditions have on the outcome of oxygen-based remediation strategies, and suggest that these properties should be evaluated prior to the implementation of oxygen-based remedial strategies.     

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.     

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.     

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.     

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.     

Cometabolic biodegradation of methyl tert-butyl ether by a soil consortium: Effect of components present in gasoline

A soil consortium was tested for its ability to degrade reformulated gasoline, containing methyl tert-butyl ether (MTBE). Reformulated gasoline was rapidly degraded to completion. However, MTBE tested alone was not degraded. A screening was carried out to identify compounds in gasoline that participate in cometabolism with MTBE. Aromatic compounds (benzene, toluene, xylenes) and compounds structurally similar to MTBE (tert-butanol, 2,2-dimethylbutane, 2,2,4-trimethylpentane) were unable to cometabolize MTBE. Cyclohexane was resistant to degradation. However, all n-alkanes tested for cometabolic activity (pentane, hexane, heptane) did enable the biodegradation of MTBE. Among the alkanes tested, pentane was the most efficient (200 &mgr;g/day). Upon the depletion of pentane, the consortium stopped degrading MTBE. When the consortium was spiked with pentane, MTBE degradation continued. When the ratio of MTBE to pentane was increased, the amount of MTBE degraded by the consortium was higher. Finally, diethylether was tested for cometabolic degradation with MTBE. Both compounds were degraded, but the process differed from that observed with pentane.