Biodegradation of methyl tert-butyl ether and other fuel oxygenates by a new strain,Mycobacterium austroafricanum IFP 2012

A strain that efficiently degraded methyl tert-butyl ether (MTBE) was obtained by initial selection on the recalcitrant compound tert-butyl alcohol (TBA). This strain, a gram-positive methylotrophic bacterium identified as Mycobacterium austroafricanum IFP 2012, was also able to degrade tert-amyl methyl ether and tert-amyl alcohol. Ethyl tert-butyl ether was weakly degraded. tert-Butyl formate and 2-hydroxy isobutyrate (HIBA), two intermediates in the MTBE catabolism pathway, were detected during growth on MTBE. A positive effect of Co2+ during growth of M. austroafricanum IFP 2012 on HIBA was demonstrated. The specific rate of MTBE degradation was 0.6 mmol/h/g (dry weight) of cells, and the biomass yield on MTBE was 0.44 g (dry weight) per g of MTBE. MTBE, TBA, and HIBA degradation activities were induced by MTBE and TBA, and TBA was a good inducer. Involvement of at least one monooxygenase during degradation of MTBE and TBA was shown by (i) the requirement for oxygen, (ii) the production of propylene epoxide from propylene by MTBE- or TBA- grown cells, and (iii) the inhibition of MTBE or TBA degradation and of propylene epoxide production by acetylene. No cytochrome P-450 was detected in MTBE- or TBA-grown cells. Similar protein profiles were obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude extracts from MTBE- and TBA-grown cells. Among the polypeptides induced by these substrates, two polypeptides (66 and 27 kDa) exhibited strong similarities with known oxidoreductases.     

Oxidation of methyl tert-butyl ether by alkane hydroxylase in dicyclopropylketone-induced and n-octane-grown Pseudomonas putida GPo1

The alkane hydroxylase enzyme system in Pseudomonas putida GPo1 has previously been reported to be unreactive toward the gasoline oxygenate methyl tert-butyl ether (MTBE). We have reexamined this finding by using cells of strain GPo1 grown in rich medium containing dicyclopropylketone (DCPK), a potent gratuitous inducer of alkane hydroxylase activity. Cells grown with DCPK oxidized MTBE and generated stoichiometric quantities of tert-butyl alcohol (TBA). Cells grown in the presence of DCPK also oxidized tert-amyl methyl ether but did not appear to oxidize either TBA, ethyl tert-butyl ether, or tert-amyl alcohol. Evidence linking MTBE oxidation to alkane hydroxylase activity was obtained through several approaches. First, no TBA production from MTBE was observed with cells of strain GPo1 grown on rich medium without DCPK. Second, no TBA production from MTBE was observed in DCPK-treated cells of P. putida GPo12, a strain that lacks the alkane-hydroxylase-encoding OCT plasmid. Third, all n-alkanes that support the growth of strain GPo1 inhibited MTBE oxidation by DCPK-treated cells. Fourth, two non-growth-supporting n-alkanes (propane and n-butane) inhibited MTBE oxidation in a saturable, concentration-dependent process. Fifth, 1,7-octadiyne, a putative mechanism-based inactivator of alkane hydroxylase, fully inhibited TBA production from MTBE. Sixth, MTBE-oxidizing activity was also observed in n-octane-grown cells. Kinetic studies with strain GPo1 grown on n-octane or rich medium with DCPK suggest that MTBE-oxidizing activity may have previously gone undetected in n-octane-grown cells because of the unusually high K(s) value (20 to 40 mM) for MTBE.     

Biodegradation of ethyl t-butyl ether (ETBE), methyl t-butyl ether (MTBE) and t-amyl methyl ether (TAME) by Gordonia terrae

Gordonia terrae strain IFP 2001 was selected from activated sludge for its capacity to grow on ethyl t-butyl ether (ETBE) as sole carbon and energy source. ETBE was stoichiometrically degraded to t-butyl alcohol (TBA) and the activity was inducible. A constitutive strain, G. terrae IFP 2007, derived from strain IFP 2001, was also selected. Methyl t-butyl ether (MTBE) and t-amyl methyl ether (TAME) were not used as carbon and energy sources by the two strains, but cometabolic degradation of MTBE and TAME was demonstrated, to TBA and t-amyl alcohol (TAA) respectively, in the presence of a carbon source such as ethanol. No two-carbon compound was detected during growth on ETBE, but formate was produced during cometabolic degradation of MTBE or TAME. A monooxygenase was involved in the degradation of ethers, because no degradation of ETBE was observed under anaerobic conditions and the presence of a cytochrome P-450 was demonstrated in G. terrae IFP 2001 after induction by cultivation on ETBE.     

Microbial degradation and fate in the environment of methyl tert-butyl ether and related fuel oxygenates

Oxygenates, mainly methyl tert-butyl ether (MTBE), are commonly added to gasoline to enhance octane index and improve combustion efficiency. Other oxygenates used as gasoline additives are ethers such as ethyl tert-butyl ether (ETBE), tert-amyl methyl ether (TAME), and alcohols such as tert-butyl alcohol (TBA). As a result of its wide use, MTBE has been detected, mainly in the USA, in groundwater and surface waters, and is a cause of concern because of its possible health effects and other undesirable consequences. MTBE is a water-soluble and mobile compound that generates long pollution plumes in aquifers impacted by gasoline releases from leaking tanks. Field observations concur in estimating that, because of recalcitrance to biodegradation, natural attenuation is slow (half-life of at least 2 years). However, quite significant advances have been made in recent years concerning the microbiology of the degradation of MTBE and other oxygenated gasoline additives. The recalcitrance of these compounds results from the presence in their structure of an ether bond and of a tertiary carbon structure. For the most part, only aerobic microbial degradation systems have been reported so far. Consortia capable of mineralizing MTBE have been selected. Multiple instances of the cometabolism of MTBE with pure strains or with microflorae, growing on n-alkanes, isoalkanes, cyclohexane or ethers (diethyl ether, ETBE), have been described. MTBE was converted into TBA in all cases and was sometimes further degraded, but it was not used as a carbon source by the pure strains. However, mineralization of MTBE and TBA by several pure bacterial strains using these compounds as sole carbon and energy source has recently been reported. The pathways of metabolism of MTBE involve the initial attack by a monooxygenase. In several cases, the enzyme was characterized as a cytochrome P-450. After oxygenation, the release of a C -unit as formaldehyde or formate leads to the production of TBA, which can be converted to 2-hydroxyisobutyric acid and further metabolized. Developments in microbiology make biological treatment of water contaminated with MTBE and other oxygenates an attractive possibility. Work concerning ex situ treatment in biofilters by consortia and by pure strains, and involving or not cometabolism, is under way. Furthermore, the development of in situ treatment processes is a promisinggoal.     

Mineralization of methyl tert-butyl ether and other gasoline oxygenates by Pseudomonads using short n-alkanes as growth source

Biodegradation of methyl tert-butyl ether (MTBE) by cometabolism has shown to produce recalcitrant metabolic intermediates that often accumulate. In this work, a consortium containing Pseudomonads was studied for its ability to fully degrade oxygenates by cometabolism. This consortium mineralized MTBE and TBA with C3-C7 n-alkanes. The highest degradation rates for MTBE (75 +/- 5 mg g(protein) (-1) h(-1)) and TBA (86.9 +/- 7.3 mg g(protein) (-1) h(-1)) were obtained with n-pentane and n-propane, respectively. When incubated with radiolabeled MTBE and n-pentane, it converted more than 96% of the added MTBE to (14)C-CO(2). Furthermore, the consortium degraded tert-amyl methyl ether, tert-butyl alcohol (TBA), tert-amyl alcohol, ethyl tert-butyl ether (ETBE) when n-pentane was used as growth source. Three Pseudomonads were isolated but only two showed independent MTBE degradation activity. The maximum degradation rates were 101 and 182 mg g(protein) (-1) h(-1) for Pseudomonas aeruginosa and Pseudomonas citronellolis, respectively. The highest specific affinity (a degrees (MTBE)) value of 4.39 l g(protein) (-1) h(-1) was obtained for Pseudomonas aeruginosa and complete mineralization was attained with a MTBE: n-pentane ratio (w/w) of 0.7. This is the first time that Pseudomonads have been reported to fully mineralize MTBE by cometabolic degradation.     

Biodegradation of methyl tert-butyl ether by a pure bacterial culture.

Biodegradation of methyl tert-butyl ether (MTBE) by the hydrogen-oxidizing bacterium Hydrogenophaga flava ENV735 was evaluated. ENV735 grew slowly on MTBE or tert-butyl alcohol (TBA) as sole sources of carbon and energy, but growth on these substrates was greatly enhanced by the addition of a small amount of yeast extract. The addition of H(2) did not enhance or diminish MTBE degradation by the strain, and MTBE was only poorly degraded or not degraded by type strains of Hydrogenophaga or hydrogen-oxidizing enrichment cultures, respectively. MTBE degradation activity was constitutively expressed in ENV735 and was not greatly affected by formaldehyde, carbon monoxide, allyl thiourea, or acetylene. MTBE degradation was inhibited by 1-amino benzotriazole and butadiene monoepoxide. TBA degradation was inducible by TBA and was inhibited by formaldehyde at concentrations of >0.24 mM and by acetylene but not by the other inhibitors tested. These results demonstrate that separate, independently regulated genes encode MTBE and TBA metabolism in ENV735.     

Characterization of the initial reactions during the cometabolic oxidation of methyl tert-butyl ether by propane-grown Mycobacterium vaccae JOB5

The initial reactions in the cometabolic oxidation of the gasoline oxygenate, methyl tert-butyl ether (MTBE), by Mycobacterium vaccae JOB5 have been characterized. Two products, tert-butyl formate (TBF) and tert-butyl alcohol (TBA), rapidly accumulated extracellularly when propane-grown cells were incubated with MTBE. Lower rates of TBF and TBA production from MTBE were also observed with cells grown on 1- or 2-propanol, while neither product was generated from MTBE by cells grown on casein-yeast extract-dextrose broth. Kinetic studies with propane-grown cells demonstrated that TBF is the dominant (> or = 80%) initial product of MTBE oxidation and that TBA accumulates from further biotic and abiotic hydrolysis of TBF. Our results suggest that the biotic hydrolysis of TBF is catalyzed by a heat-stable esterase with activity toward several other tert-butyl esters. Propane-grown cells also oxidized TBA, but no further oxidation products were detected. Like the oxidation of MTBE, TBA oxidation was fully inhibited by acetylene, an inactivator of short-chain alkane monooxygenase in M. vaccae JOB5. Oxidation of both MTBE and TBA was also inhibited by propane (K(i) = 3.3 to 4.4 microM). Values for K(s) of 1.36 and 1.18 mM and for V(max) of 24.4 and 10.4 nmol min(-1) mg of protein(-1) were derived for MTBE and TBA, respectively. We conclude that the initial steps in the pathway of MTBE oxidation by M. vaccae JOB5 involve two reactions catalyzed by the same monooxygenase (MTBE and TBA oxidation) that are temporally separated by an esterase-catalyzed hydrolysis of TBF to TBA. These results that suggest the initial reactions in MTBE oxidation by M. vaccae JOB5 are the same as those that we have previously characterized in gaseous alkane-utilizing fungi.     

Isolation and characterization of two aerobic bacterial strains that completely degrade ethyl tert-butyl ether (ETBE)

Two bacterial strains, E1 and E2, isolated from gasoline-polluted soil completely degraded ethyl tert-butyl ether (ETBE), as the sole source of carbon and energy, at specific rates of about 80 mg g(-1) and 58 mg g(-1) of cell protein day(-1), respectively. On the basis of morphological and phenotypic characteristics, strain E1 was tentatively identified as Comamonas testosteroni and strain E2 as belonging to Centre for Disease Control group A-5. The inhibitory effect of metyrapone on the degradative ability of both strains was the first evidence indicating the involvement of a soluble cytochrome P-450 in the cleavage of the ETBE ether bond. This observation was confirmed by spectrophotometric analysis of reduced cell extracts that gave, in the presence of carbon monoxide, a major absorbance peak at about 450 nm. Both strains were also able to degrade, as the sole source of carbon and energy, ETBE's major metabolic intermediates (tert-butyl alcohol and tert-butyl formate) and other gasoline oxygenates (methyl tert-butyl ether and tert-amyl methyl ether). The degradation rates varied considerably, with both strains exhibiting a preferential activity for ETBE's metabolic intermediates.     

Biodegradation of tert-butyl alcohol and related xenobiotics by a methylotrophic bacterial isolate

A new aerobic bacterial strain, CIP 1-2052, isolated from an activated sludge sample, was able to use tert-butyl alcohol (TBA), a product of methyl tert-butyl ether (MTBE) and ethyl tert-butyl ether (ETBE) degradation, as its sole carbon and energy source. Cobalt ions stimulated TBA mineralization. The maximum growth and TBA degradation rates were 0.032 +/- 0.004 h(-1) and 35.8 +/- 8.5 mg TBA x g(-1) (cell dry mass) per h, respectively. The growth yield on TBA was 0.54 +/- 0.02 g x g(-1). Strain CIP 1-2052 exhibited a particular substrate specificity towards alcohols. It degraded tertiary alcohols, TBA and tert-amyl alcohol (TAA), but neither their primary and secondary alcohol homologues, nor ethanol. However, one-carbon compounds, namely methanol and formate, were degraded by strain CIP 1-2052, showing the methylotrophic nature of this isolate. The properties of this new strain suggest that it could be used for bioremediation of contaminated aquifers.     

Biodegradation of ether pollutants by Pseudonocardia sp. strain ENV478

A bacterium designated Pseudonocardia sp. strain ENV478 was isolated by enrichment culturing on tetrahydrofuran (THF) and was screened to determine its ability to degrade a range of ether pollutants. After growth on THF, strain ENV478 degraded THF (63 mg/h/g total suspended solids [TSS]), 1,4-dioxane (21 mg/h/g TSS), 1,3-dioxolane (19 mg/h/g TSS), bis-2-chloroethylether (BCEE) (12 mg/h/g TSS), and methyl tert-butyl ether (MTBE) (9.1 mg/h/g TSS). Although the highest rates of 1,4-dioxane degradation occurred after growth on THF, strain ENV478 also degraded 1,4-dioxane after growth on sucrose, lactate, yeast extract, 2-propanol, and propane, indicating that there was some level of constitutive degradative activity. The BCEE degradation rates were about threefold higher after growth on propane (32 mg/h/g TSS) than after growth on THF, and MTBE degradation resulted in accumulation of tert-butyl alcohol. Degradation of 1,4-dioxane resulted in accumulation of 2-hydroxyethoxyacetic acid (2HEAA). Despite its inability to grow on 1,4-dioxane, strain ENV478 degraded this compound for > 80 days in aquifer microcosms. Our results suggest that the inability of strain ENV478 and possibly other THF-degrading bacteria to grow on 1,4-dioxane is related to their inability to efficiently metabolize the 1,4-dioxane degradation product 2HEAA but that strain ENV478 may nonetheless be useful as a biocatalyst for remediating 1,4-dioxane-contaminated aquifers.     

Roles of tert-butyl formate,tert-butyl alcohol and acetone in the regulation of methyl tert-butyl ether degradation by Mycobacterium austroafricanum IFP 2012

Mycobacterium austroafricanum IFP 2012 is a Gram-positive strain able to grow on methyl tert-butyl ether (MTBE) as a sole carbon and energy source. The effect of two downstream metabolites of MTBE, tert-butyl formate (TBF) and tert-butyl alcohol (TBA) on MTBE degradation was investigated using resting cells. The addition of low concentrations of TBF decreased the MTBE degradation rate by about 30%. In contrast, the addition of TBA did not have a significant effect on MTBE degradation rate, even at high concentrations; and it was also shown that TBA degradation occurred only once MTBE was exhausted. At neutral pH, TBF hydrolysis involved mainly an esterase-type activity regulated by the presence of TBA. The TBF degradation rate was about four times lower than the MTBE degradation rate. Furthermore, acetone was identified as an intermediate during TBA degradation. An acetone mono-oxygenase activity, inhibited by methimazole but not by acetylene, was suggested. It was different from the MTBE/TBA mono-oxygenase and, thus, acetone did not appear to compete with MTBE and TBA for the same enzyme. These new results show that the metabolic regulation of the early steps of MTBE degradation by M. austroafricanum IFP 2012 is complex, involving inhibition and competition phenomena.     

Methyl tert-butyl ether (MTBE) degradation by a microbial consortium

The widespread use of methyl tert-butyl ether (MTBE) as a gasoline additive has resulted in a large number of cases of groundwater contamination. Bioremediation is often proposed as the most promising alternative after treatment. However, MTBE biodegradation appears to be quite different from the biodegradation of usual gasoline contaminants such as benzene, toluene, ethyl benzene and xylene (BTEX). In the present paper, the characteristics of a consortium degrading MTBE in liquid cultures are presented and discussed. MTBE degradation rate was fast and followed zero order kinetics when added at 100 mg l(-1). The residual MTBE concentration in batch degradation experiments ranged from below the detection limit (1 microg l(-1)) to 50 microg l(-1). The specific activity of the consortium ranged from 7 to 52 mgMTBE g(dw)(-1) h(-1) (i.e. 19-141 mgCOD g(dw) (-1) h(-1)). Radioisotope experiments showed that 79% of the carbon-MTBE was converted to carbon-carbon dioxide. The consortium was also capable of degrading a variety of hydrocarbons, including tert-butyl alcohol (TBA), tert-amyl methyl ether (TAME) and gasoline constituents such as benzene, toluene, ethylbenzene and xylene (BTEX). The consortium was also characterized by a very slow growth rate (0.1 d(-1)), a low overall biomass yield (0.11 gdw g(-1)MTBE; i.e. 0.040 gdw gCOD(-1)), a high affinity for MTBE and a low affinity for oxygen, which may be a reason for the slow or absence of MTBE biodegradation in situ. Still, the results presented here show promising perspectives for engineering the in situ bioremediation of MTBE.     

The alkyl tert-butyl ether intermediate 2-hydroxyisobutyrate is degraded via a novel cobalamin-dependent mutase pathway

Fuel oxygenates such as methyl and ethyl tert-butyl ether (MTBE and ETBE, respectively) are degraded only by a limited number of bacterial strains. The aerobic pathway is generally thought to run via tert-butyl alcohol (TBA) and 2-hydroxyisobutyrate (2-HIBA), whereas further steps are unclear. We have now demonstrated for the newly isolated beta-proteobacterial strains L108 and L10, as well as for the closely related strain CIP I-2052, that 2-HIBA was degraded by a cobalamin-dependent enzymatic step. In these strains, growth on substrates containing the tert-butyl moiety, such as MTBE, TBA, and 2-HIBA, was strictly dependent on cobalt, which could be replaced by cobalamin. Tandem mass spectrometry identified a 2-HIBA-induced protein with high similarity to a peptide whose gene sequence was found in the finished genome of the MTBE-degrading strain Methylibium petroleiphilum PM1. Alignment analysis identified it as the small subunit of isobutyryl-coenzyme A (CoA) mutase (ICM; EC 5.4.99.13), which is a cobalamin-containing carbon skeleton-rearranging enzyme, originally described only in Streptomyces spp. Sequencing of the genes of both ICM subunits from strain L108 revealed nearly 100% identity with the corresponding peptide sequences from M. petroleiphilum PM1, suggesting a horizontal gene transfer event to have occurred between these strains. Enzyme activity was demonstrated in crude extracts of induced cells of strains L108 and L10, transforming 2-HIBA into 3-hydroxybutyrate in the presence of CoA and ATP. The physiological and evolutionary aspects of this novel pathway involved in MTBE and ETBE metabolism are discussed.     

Metabolism of Diethyl Ether and Cometabolism of Methyl tert-Butyl Ether by a Filamentous Fungus, a Graphium sp

In this study, evidence for two novel metabolic processes catalyzed by a filamentous fungus, Graphium sp. strain ATCC 58400, is presented. First, our results indicate that this Graphium sp. can utilize the widely used solvent diethyl ether (DEE) as the sole source of carbon and energy for growth. The kinetics of biomass accumulation and DEE consumption closely followed each other, and the molar growth yield on DEE was indistinguishable from that with n-butane. n-Butane-grown mycelia also immediately oxidized DEE without the extracellular accumulation of organic oxidation products. This suggests a common pathway for the oxidation of both compounds. Acetylene, ethylene, and other unsaturated gaseous hydrocarbons completely inhibited the growth of this Graphium sp. on DEE and DEE oxidation by n-butane-grown mycelia. Second, our results indicate that gaseous n-alkane-grown Graphium mycelia can cometabolically degrade the gasoline oxygenate methyl tert-butyl ether (MTBE). The degradation of MTBE was also completely inhibited by acetylene, ethylene, and other unsaturated hydrocarbons and was strongly influenced by n-butane. Two products of MTBE degradation, tert-butyl formate (TBF) and tert-butyl alcohol (TBA), were detected. The kinetics of product formation suggest that TBF production temporally precedes TBA accumulation and that TBF is hydrolyzed both biotically and abiotically to yield TBA. Extracellular accumulation of TBA accounted for only a maximum of 25% of the total MTBE consumed. Our results suggest that both DEE oxidation and MTBE oxidation are initiated by cytochrome P-450-catalyzed reactions which lead to scission of the ether bonds in these compounds. Our findings also suggest a potential role for gaseous n-alkane-oxidizing fungi in the remediation of MTBE contamination.     

Temperature effect on tert-butyl alcohol (TBA) biodegradation kinetics in hyporheic zone soils

Background  

Remediation of tert-butyl alcohol (TBA) in subsurface waters should be taken into consideration at reformulated gasoline contaminated sites since it is a biodegradation intermediate of methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-butyl formate (TBF). The effect of temperature on TBA biodegradation has not been not been published in the literature.     

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.     

Biotransformation of methyl tert-butyl ether by human cytochrome P450 2A6

Methyl tert-butyl ether (MTBE) is widely used as gasoline oxygenate and octane number enhancer for more complete combustion in order to reduce the air pollution caused by motor vehicle exhaust. The possible adverse effects of MTBE on human health are of major public concern. However, information on the metabolism of MTBE in human tissues is scarce. The present study demonstrates that human cytochrome P450 2A6 is able to metabolize MTBE to tert-butyl alcohol (TBA), a major circulating metabolite and marker for exposure to MTBE. As CYP2A6 is known to be constitutively expressed in human livers, we infer that it may play a significant role in metabolism of gasoline ethers in liver tissue.     

Enhancing transport of hydrogenophaga flava ENV735 for bioaugmentation of aquifers contaminated with methyl tert-butyl ether

The gasoline oxygenate methyl tert-butyl ether (MTBE) has become a widespread contaminant in groundwater throughout the United States. Bioaugmentation of aquifers with MTBE-degrading cultures may be necessary to enhance degradation of the oxygenate in some locations. However, poor cell transport has sometimes limited bioaugmentation efforts in the past. The objective of this study was to evaluate the transport characteristics of Hydrogenophaga flava ENV735, a pure culture capable of growth on MTBE, and to improve movement of the strain through aquifer solids. The wild-type culture moved only a few centimeters in columns of aquifer sediment. An adhesion-deficient variant (H. flava ENV735:24) of the wild-type strain that moved more readily through sediments was obtained by sequential passage of cells through columns of sterile sediment. Hydrophobic and electrostatic interaction chromatography revealed that the wild-type strain is much more hydrophobic than the adhesion-deficient variant. Electrophoretic mobility assays and transmission electron microscopy showed that the wild-type bacterium contains two distinct subpopulations, whereas the adhesion-deficient strain has only a single, homogeneous population. Both the wild-type strain and adhesion-deficient variant degraded MTBE, and both were identified by 16S rRNA analysis as pure cultures of H. flava. The effectiveness of surfactants for enhancing transport of the wild-type strain was also evaluated. Many of the surfactants tested were toxic to ENV735; however, one nonionic surfactant, Tween 20, enhanced cell transport in sand columns. Improving microbial transport may lead to a more effective bioaugmentation strategy for MTBE-contaminated sites where indigenous oxygenate degraders are absent.     

Cometabolism of methyl tertiary butyl ether and gaseous n-alkanes by Pseudomonas mendocina KR-1 grown on C5 to C8 n-alkanes

Pseudomonas mendocina KR-1 grew well on toluene, n-alkanes (C5 to C8), and 1 degrees alcohols (C2 to C8) but not on other aromatics, gaseous n-alkanes (C1 to C4), isoalkanes (C4 to C6), 2 degrees alcohols (C3 to C8), methyl tertiary butyl ether (MTBE), or tertiary butyl alcohol (TBA). Cells grown under carbon-limited conditions on n-alkanes in the presence of MTBE (42 micromoles) oxidized up to 94% of the added MTBE to TBA. Less than 3% of the added MTBE was oxidized to TBA when cells were grown on either 1 degrees alcohols, toluene, or dextrose in the presence of MTBE. Concentrated n-pentane-grown cells oxidized MTBE to TBA without a lag phase and without generating tertiary butyl formate (TBF) as an intermediate. Neither TBF nor TBA was consumed by n-pentane-grown cells, while formaldehyde, the expected C1 product of MTBE dealkylation, was rapidly consumed. Similar Ks values for MTBE were observed for cells grown on C5 to C8 n-alkanes (12.95 +/- 2.04 mM), suggesting that the same enzyme oxidizes MTBE in cells grown on each n-alkane. All growth-supporting n-alkanes (C5 to C8) inhibited MTBE oxidation by resting n-pentane-grown cells. Propane (Ki = 53 micromoles) and n-butane (Ki = 16 micromoles) also inhibited MTBE oxidation, and both gases were also consumed by cells during growth on n-pentane. Cultures grown on C5 to C8 n-alkanes also exhibited up to twofold-higher levels of growth in the presence of propane or n-butane, whereas no growth stimulation was observed with methane, ethane, MTBE, TBA, or formaldehyde. The results are discussed in terms of their impacts on our understanding of MTBE biodegradation and cometabolism.     

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.