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.     

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

A bacterial strain, PM1, which is able to utilize methyl tert-butyl ether (MTBE) as its sole carbon and energy source, was isolated from a mixed microbial consortium in a compost biofilter capable of degrading MTBE. Initial linear rates of MTBE degradation by 2 x 10(6) cells ml(-1) were 0.07, 1.17, and 3.56 microg ml(-1) h(-1) for initial concentrations of 5, 50, and 500 microg MTBE ml(-1), respectively. When incubated with 20 microg of uniformly labeled [(14)C]MTBE ml(-1), strain PM1 converted 46% to (14)CO(2) and 19% to (14)C-labeled cells within 120 h. This yield is consistent with the measurement of protein accumulation at different MTBE concentrations from which was estimated a biomass yield of 0.18 mg of cells mg MTBE(-1). Strain PM1 was inoculated into sediment core material collected from a contaminated groundwater plume at Port Hueneme, California, in which there was no evidence of MTBE degradation. Strain PM1 readily degraded 20 microg of MTBE ml(-1) added to the core material. The rate of MTBE removal increased with additional inputs of 20 microg of MTBE ml(-1). These results suggest that PM1 has potential for use in the remediation of MTBE-contaminated environments.     

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.     

Biodegradation of the gasoline oxygenates methyl tert-butyl ether, ethyl tert-butyl ether, and tert-amyl methyl ether by propane-oxidizing bacteria.

Several propane-oxidizing bacteria were tested for their ability to degrade gasoline oxygenates, including methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME). Both a laboratory strain and natural isolates were able to degrade each compound after growth on propane. When propane-grown strain ENV425 was incubated with 20 mg of uniformly labeled [14C]MTBE per liter, the strain converted > 60% of the added MTBE to 14CO2 in < 30 h. The initial oxidation of MTBE and ETBE resulted in the production of nearly stoichiometric amounts of tert-butyl alcohol (TBA), while the initial oxidation of TAME resulted in the production of tert-amyl alcohol. The methoxy methyl group of MTBE was oxidized to formaldehyde and ultimately to CO2. TBA was further oxidized to 2-methyl-2-hydroxy-1-propanol and then 2-hydroxy isobutyric acid; however, neither of these degradation products was an effective growth substrate for the propane oxidizers. Analysis of cell extracts of ENV425 and experiments with enzyme inhibitors implicated a soluble P-450 enzyme in the oxidation of both MTBE and TBA. MTBE was oxidized to TBA by camphor-grown Pseudomonas putida CAM, which produces the well-characterized P-450cam, but not by Rhodococcus rhodochrous 116, which produces two P-450 enzymes. Rates of MTBE degradation by propane-oxidizing strains ranged from 3.9 to 9.2 nmol/min/mg of cell protein at 28 degrees C, whereas TBA was oxidized at a rate of only 1.8 to 2.4 nmol/min/mg of cell protein at the same temperature.     

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.     

Single-walled carbon nanotubes as an effective adsorbent in solid-phase microextraction of low level methyl tert-butyl ether,ethyl tert-butyl ether and methyl tert-amyl ether from human urine

Carbon nanotubes (CNTs) are a kind of new carbon-based nano-materials which have drawn great attention in many application fields. The potential single-walled carbon nanotubes (SWCNTs) as solid-phase microextraction (SPME) adsorbents for the preconcentration of environmental pollutants have been investigated in recent years. The goal of this work was to investigate the feasibility of SWCNTs used as adsorbents for solid-phase microextraction of methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME) in human urine. SWCNTs were attached onto a stainless steel wire through organic binder. Potential factors affecting the extraction efficiency were optimized, including extraction time, extraction temperature, desorption time, desorption temperature, and salinity. The developed method showed good performance according to the ICH performance criteria for bioanalytical methods. The calibration curves of the ethers were linear (r² ≥ 0.992) in the range from 10 to 5000 ng L^?1. The limits of detection at a signal-to-noise (S/N) ratio of 3 were 10 ng L^?1 for all the analytes. In addition, compared with the commercial carboxen/polydimethylsiloxane (CAR/PDMS) fiber, the SWCNT fiber showed better thermal stability (over 350 °C) and longer life span (over 150 times). The developed method was applied successfully to determine trace level of the ethers in urine of 10 healthy male volunteers.     

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.     

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.     

汽油添加剂甲基叔丁基醚(MTBE)污染的植物修复

甲基叔丁基醚(MTBE)是北美燃料市场最常用的汽油添加剂。由于其在土壤中的不吸附性和极高的水溶性,MTBE已成为一种蔓延性的地下水污染物。本实验用一自行设计的植物反应器来观察和测定在不同温度条件下柳树(Salix alba)对MTBE污染水溶液的修复潜力。长出新根须和嫩叶的柳树枝条在一容积500mL的锥型瓶中生长12d(其中MTBE溶液450mL)来观察MTBE对柳树生长的影响,同时测定柳树对MTBE的吸收和降解。MTBE及其主要降解产物叔丁基醇(TBA)用气相色谱来检测。本实验结果表明在为期12d的时间内,水溶液中24．84～53．27％MTBE可以通过柳树的蒸腾作用去除。同时在15℃～25℃的温度范围内,：MTBE的去除率(％)和柳树的蒸腾量(g)之间存在着明显的线性关系。由于没有发现TBA和其它可能的降解产物,植物挥发是MTBE植物修复技术中主要的作用机理。尽管植物在MTBE污染的修复过程中只是起着中间传输媒介的作用,大量的MTBE通过植物的蒸腾作用以气态的形式释放到大气中,但由于MTBE在气态下的光氧化非常快,MTBE不会造成空气污染。我们认为植物修复技术对MTBE污染的土壤和地下水仍不失为一个有效的修复手段。     

Degradation of methyl tert-butyl ether by gel immobilized Methylibium petroleiphilum PM1

Cells of Methylibium petroleiphilum PM1 were immobilized in gel beads to degrade methyl tert-butyl ether (MTBE). Calcium alginate, agar, polyacrylamide and polyvinvyl alcohol were screened as suitable immobilization matrices, with calcium alginate demonstrating the fastest MTBE-degradation rate. The rate was accelerated by 1.8-fold when the beads had been treated in physiological saline for 24h at 28 degrees C. MTBE degradation in mineral salts medium (MSM) was accompanied by the increase of biomass. The half-life of MTBE-degradation activity for the encapsulated cells stored at 28 degrees C was about 120 h, which was obviously longer than that of free cells (approximately 36 h). Efficient reusability of the beads up to 30 batches was achieved in poor nutrition solution as compared to only 6 batches in MSM. The immobilized cells could be operated in a packed-bed reactor for degradation of 10 mg L(-1) MTBE in groundwater with more than 99% removal efficiency at hydraulic retention time of 20 min. These results suggested that immobilized cells of PM1 in bioreactor might be applicable to a groundwater treatment system for the removal of MTBE.     

Biomarkers of exposure to aromatic hydrocarbons and methyl tert-butyl ether in petrol station workers

This cross-sectional study was aimed at reconstructing the exposure to gasoline in 102 petrol station attendants by environmental and biological monitoring of benzene, toluene, ethylbenzene and xylene (BTEX) and biomonitoring of methyl tert-butyl ether (MTBE). Airborne BTEX were higher for manual refuelers than self-service assistants and were highly correlated with each other. Significant relationships were found between airborne BTX and the corresponding urinary solvents (U-BTX) and beween airborne B and urinary MTBE (U-MTBE). Smokers eliminated higher values of U-B, trans,trans-muconic (t,t-MA) and S-phenylmercapturic (S-PMA) acids but not U-MTBE. All these biomarkers were, however, significantly raised during the shift, independently from smoking. Linear regression confirmed that occupational exposure was a main predictor of U-MTBE, U-B and S-PMA values, both the latter confounded by smoking habits. The study supports the usefulness of biomonitoring even at low exposure levels.     

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.     

汽油添加剂甲基叔丁基醚(MTBE)对环境的危害性

甲基叔丁基醚(MTBE)是欧美燃料市场最常用的汽油添加剂。由于其在土壤和地下水中的特殊理化行为,MTBE对人体健康和自然环境的负面影响正受到研究人员的关注。本文根据现有资料就MTBE的环境行为及其对生物的危害性影响进行综合评估。我们认为尽管大部分的MTBE是以气态的形式释放到大气中,但由于其光氧化速度非常快,所以MTBE不会造成空气污染。MTBE在土壤中的不吸附性和极高的水溶性,使其正在成为一种蔓延性的地下水污染物,MTBE在地下水中的半衰期至少需要二年。在适宜的环境条件下,MTBE的有氧微生物降解是可以发生的,但其厌氧降解的机率几乎为零。MTBE对动物是一种致癌物质,目前还没有足够的证据证明其对人类的致癌作用,但是MTBE对人体健康的负面影响是非常明显的。当MTBE浓度大于7.4 mg·L-1,其对水生生物能产生急性毒性作用,而在低浓度条件下(<0.1 mg·L-1),MTBE它们的急性毒性作用是非常有限的。MTBE对陆生植物的毒理学研究目前还十分有限。我们认为全面综合地评估MTBE对生物的毒性作用还需要大量的亚急性和慢性试验数据作为依据。     

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.     

Biodegradation of methyl t-butyl ether by pure bacterial cultures

Three pure bacterial cultures degrading methyl t-butyl ether (MTBE) were isolated from activated sludge and fruit of the Gingko tree. They have been classified as belonging to the genuses Methylobacterium, Rhodococcus, and Arthrobacter. These cultures degraded 60 ppm MTBE in 1–2 weeks of incubation at 23–25 °C. The growth of the isolates on MTBE as sole carbon source is very slow compared with growth on nutrient-rich medium. Uniformly-labeled [¹⁴C]MTBE was used to determine ¹⁴CO₂ evolution. Within 7 days of incubation, about 8% of the initial radioactivity was evolved as ¹⁴CO₂. These strains also grow on t-butanol, butyl formate, isopropanol, acetone and pyruvate as carbon sources. The presence of these compounds in combination with MTBE decreased the degradation of MTBE. The cultures pregrown on pyruvate resulted in a reduction in ¹⁴CO₂ evolution from [¹⁴C]MTBE. The availability of pure cultures will allow the determination of the pathway intermediates and the rate-limiting steps in the degradation of MTBE. Received: 8 December 1995 / Received last revision: 5 August 1996 / Accepted: 12 August 1996     

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.     

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.     

Fungal degradation of benzoic acid and related compounds

Most knowledge of the degradation of aromatic compounds has been gained through investigation of the pathways in bacteria. In recent years, however, significant developments have been made in the understanding of the degradation of these compounds in yeasts and moulds. Many similarities have been identified between the bacteria and the yeasts and moulds but some significant differences occur. This review highlights these differences and discusses the current understanding of the fungal degradation of benzoate and some substituted benzoates. The pathways for the further conversion of the ring-fission substrates, which are common to all fungi capable of degrading these aromatic compounds, are also presented.J.D. Wright was with the Department of Applied Biology, University of Hull, Hull HU6 7RX, UK and is now with Castrol International, Castrol Technology Centre, Whitchurch Hill, Pangbourne, Reading RG8 7QR, UK.     

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.