Final Report: Aerobic Cometabolism of Ether-bonded Compounds

EPA Grant Number: R823426
Title: Aerobic Cometabolism of Ether-bonded Compounds
Investigators: Hyman, Michael R.
Institution: Oregon State University , North Carolina State University
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1995 through September 1, 1998
Project Amount: $358,953
RFA: Exploratory Research - Environmental Biology (1995) RFA Text |  Recipients Lists
Research Category: Biology/Life Sciences , Health , Ecosystems


Ether-bonded compounds are an important but diverse group of chemicals. For example, simple ethers such as diethyl ether are used extensively as industrial solvents while branched alkyl ethers such as methyl tertiary butyl ether are consumed in enormous quantities as gasoline oxygenates. In addition to the widespread use of ether-bonded compounds, many of these compounds are persistent in the environment. One reason for this persistence is that relatively few microorganisms appear to be able to utilize ether-containing compounds as growth substrates. However, the inability of microorganisms to mineralize ether-bonded compounds should not be taken to indicate that the ether bond is resistant to enzymatic degradation. The scientific literature provides many examples of ether-bonded compounds that are oxidized by microorganisms that cannot further metabolize the products of those reactions. The ability of microorganisms to degrade compounds that they cannot utilize as growth substrates is known as cometabolism. The overall aim of this research project was to conduct a systematic investigation of the aerobic cometabolic degradation of a variety of ether-bonded compounds. The research focused primarily on the oxidation of ether-bonded compounds by organisms that express non-specific oxygenase enzymes.

The research was comprised of four objectives. First, we aimed to determine the structural features that influence the reactivity of a single non-specific monooxygenase enzyme towards a variety of ethers including a range of alkyl, alicyclic, and aromatic compounds. The non-specific oxygenase we employed for these studies was ammonia monooxygenase (AMO), a highly active enzyme found in the soil nitrifying bacterium, Nitrosomonas europaea. The second objective was to investigate the reactivity of AMO towards a series of potentially toxic compounds, including several compounds that are recognized by the U.S. Environmental Protection Agency (EPA) as Priority Pollutants. These compounds included selected chlorinated alkyl ethers, haloaryl ethers, and cyclic and branched alkyl ethers. The third objective was to examine the rates of ether cometabolism and the products obtained from the oxidation of a series of six representative ethers by a range of physiologically diverse bacteria and fungi. These organisms have included species grown on alkanes, alkenes, and aromatics. The fourth objective was to examine the physiological consequences of ether cometabolism on bacteria. These studies investigate the toxic effects and potentially beneficial aspects of ether cometabolism in a variety of bacterial strains.

Summary/Accomplishments (Outputs/Outcomes):

Objective 1: Cometabolism of Unsubstituted Alkyl, Alicyclic and Aromatic Ethers by Nitrosomonas europaea

The specific aims of this objective were to determine the rate of oxidation and to identify the oxidation products generated by N. europaea from several series of structurally similar ether-bonded compounds. These groups of compounds included symmetrical alkyl ethers, non-symmetrical and branched ethers, and alicyclic and aromatic ethers. The oxidation of the simplest linear alkyl ethers (dimethyl and diethyl ether) involves an O-dealkylation reaction that gives rise to alcohol and aldehyde products in close to equimolar ratios (1:1.4). The deviation from the expected ratio of 1:1 was shown to be due to the unexpected ability of N. europaea to reduce acetaldehyde to ethanol. Unlike diethyl ether, longer chain ethers such as n-propyl and n-butyl ethers were consistently hydroxylated, leaving the ether bond uncleaved. Using diethyl ether as a model substrate, we determined that the maximal rate of ammonia-dependent ether oxidation occurs when approximately 0.65 additional electrons are diverted to AMO and away from the respiratory chain. Our studies of the oxidation of non-symmetrical ethers was limited to ethyl methyl ether, ethyl propyl ether, and methyl propyl ether. All of these compounds were oxidized by N. europaea and generated products that were compatible with O-dealkylation reactions. Our studies with branched ethers provided interesting insights into the potential factors that control the microbial cometabolism of the gasoline oxygenate, methyl tertiary butyl ether (MTBE). Our initial studies indicated that AMO in N. europaea is unreactive towards MTBE. However, at very high concentrations we observed a slow oxidation reaction that could be attributed to AMO activity. In contrast to this, N. europaea very rapidly oxidizes and O-dealkylates simple alkyl ethers. These results suggest that one factor that probably influences the biodegradability of MTBE is not an intrinsic inability of non-specific oxygenases to oxidize ether-bonded compounds, but rather a steric hindrance that prevents binding of MTBE to these enzymes. We have investigated this effect in a separate project and have now demonstrated that there is a close association between the ability of an organism to metabolize branched alkanes and the ability to cometabolically degrade MTBE.

The final class of ether compounds we examined was the alicyclic and aromatic ethers. We observed that AMO is largely unreactive towards simple cyclic ethers such as tetrahydropyran and dioxane isomers, even though we had previously established this enzyme will oxidize several sulfur analogs of these compounds. In view of this, our main studies on these compounds concentrated on their oxidation by propane-oxidizing bacteria (see Objective 3). In contrast to the cyclic ethers, N. europaea rapidly oxidized anisole and several other methoxylated aromatics. The oxidation of anisole initially leads to the phenol and 4-methoxyphenol accumulation. Phenol then undergoes a further oxidation to hydroquinone or catechol, whereas 4-methoxyphenol is exclusively O-dealkylated to produce hydroquinone. We also detected the accumulation of formaldehyde and obtained a >95 percent mass balance for all of the reactants and sequential products. The various dimethoxy benzene isomers also were all oxidized by AMO whereas the trimethoxybenzene isomers were not substrates for this enzyme. All of the dimethoxybenzene isomers were subject to O-dealkylation reactions rather than the mixture of O-dealkylation and hydroxylation reactions observed with anisole. We also established that phenetole and butyl phenyl ether are substrates for AMO. In the case of phenetole, we observed similar sequence of intermediates to those described above for anisole. One important development to come from of our studies with ansiole oxidation is that we were able to establish the first real-time spectrophotometric assay for AMO activity. This assay relies on the fact that AMO appears to selectively O-dealkylate aromatic ethers that have substituents in the para position. Using 4-nitroanisole as a substrate, we demonstrated that AMO O-dealkylates this compound to generate formaldehyde and p-nitrophenol. The intense absorption of p-nitrophenol at 400 nm, therefore, allowed us to monitor AMO activity spectrophotometrically.

Objective 2: Cometabolism of Ethers of Environmental Concern by Nitrosomonas europaea. The main aim of this objective was to determine whether N. europaea has the ability to initiate the oxidation of selected ether-bonded compounds that are recognized as important pollutants. Several of these compounds are recognized by the EPA as Priority Pollutants. This work concentrated on three classes of compounds, chloroalkyl ethers, haloaryl ethers, and both dibenzofuran and dibenzo-p-dioxin. Several of the chloroalkyl ether compounds tested, including bis(2-chloroisopropyl) ether [BCIE] and bis(2-chloroethoxy) methane [BCEM], were not transformed by N. europaea, whereas their non-chlorinated analog ethers were both rapidly degraded by this organism. This suggested that chlorination significantly affected the biodegradability of the underlying ether structure. A similar influence of chlorine substituents on chloroethyl ethyl ether [CEEE] and bis chloroethyl ether [BCEE] also was observed. For example, the oxidation of the non-chlorinated analog diethyl ether produced roughly equivalent concentrations of alcohol and aldehyde products through a normal O-dealkylation reaction. In contrast, BCEE oxidation resulted only in the production of a monochlorinated aldehyde product. This product is compatible with a complete lack of O-dealkylation and a hydroxylation reaction directed at the chlorinated alkyl chain.

Several of the chlorinated ethers, as well as their non-chlorinated analogs, also produced a toxic effect on cells. These effects are described in more detail under Objective 4. Both 4-bromophenyl ether and 4-chlorophenyl ether were oxidized by AMO, although the products of these reactions were hydroxylated compounds rather than either dehalogenated compounds or products in which the ether bond had been broken. We also established that several other aromatic ethers including dibenzo-p-dioxin, dibenzofuran and xanthene are oxidized by N. europaea. Although our initial studies again demonstrated these compounds are typically hydroxylated by AMO, we also observed several other unique transformation products. For example, time course experiments revealed that all of these compounds are rapidly hydroxylated by AMO, but that further transformation of several of the hydroxylated products continues, even when AMO activity is inactivated or inhibited by compounds such as acetylene or thiourea. We subsequently established the cause of these transformations and demonstrated that the AMO-independent transformations involve a pH-dependent, abiotic nitration reaction. During ammonia oxidation, the pH of the reaction medium drops to a sufficiently low value that significant concentrations of undissociated nitrous acid accumulate in the medium. Nitrous acid is an effective nitrating agent for hydroxylated aromatic compounds and the reaction between these compounds leads to the production of nitro-hydroxylated products. Since our discovery of this reaction we subsequently have observed a similar effect with other aromatic AMO substrates, including naphthalene and other PAHs.

Objective 3: Ether Cometabolism by Other Aerobic Microorganisms

The aim of this objective was to determine how widely the ability to cometabolically degrade ether compounds is distributed among aerobic bacteria and fungi. We evaluated selected alkane-, alkene-, alkyne-, and aromatic-utilizing bacteria and fungi for their ability to oxidize a series of ether-bonded compounds. These compounds included, among others, a symmetrical alkyl ether (diethyl ether), a branched alkyl ether (MTBE), and an alicyclic ether (1,4-dioxane). A brief description of our key findings with each of these compounds is given below:

? Diethyl ether. During this study we demonstrated that a wide variety of bacteria grown on alkanes, alkenes, and aromatics can all oxidize DEE. A more unexpected discovery was that a filamentous fungus, a Graphium strain, can utilize diethyl ether as a sole source of carbon and energy for growth. In addition, this organism also can be grown on simple alkanes such as propane and n-butane. This Graphium species is able to cometabolically oxidize MTBE after growth on either alkanes or diethyl ether.

? MTBE. Our studies of MTBE oxidation initially concentrated on the activity observed in the filamentous fungus, Graphium. We observed that MTBE oxidation by this organism involves an initial cytochrome P-450-catalyzed monooxygenation that converts MTBE to tertiary butyl formate (TBF). This ester then undergoes both abiotic and biotic hydrolysis to yield tertiary butyl alcohol (TBA). We subsequently have demonstrated that the same pathway occurs in the propane-oxidizing bacterium Mycobacterium vaccae JOB5. Propane-grown cells of M. vaccae JOB5 also oxidize TBA and our evidence indicates that the oxidation of both MTBE and TBA is catalyzed by the same alkane monooxygenase. Our data also indicate this organism can express at least two different oxygenase enzymes in response to alkane growth substrates. One enzyme is expressed in the presence of C2?C8 alkanes and has an extremely wide cosubstrate range. A second enzyme, with a more restricted cosubstrate range, is expressed in response to C10 and higher alkanes. Only the enzyme expressed in response to C3?C8 alkanes oxidizes MTBE and TBA.

? 1,4-Dioxane. Mycobacterium vaccae JOB-5 also cometabolically degrades cyclic ethers after growth on either straight chain (C3?C8) and branched alkanes. The cyclic ethers degraded by this organism include tetrahydrofuran (THF), tetrahydropyran (THP), and hexamethylene oxide (HMO), as well as both 1,3-dioxane and 1,4-dioxane. The oxidation of all of these compounds apparently is catalyzed by alkane monooxygenase because all of the transformation reactions are inhibited by acetylene and only occur in cells grown on alkane substrates. We characterized the cometabolism of THF by propane-grown cells in some detail. The oxidation of THF by propane-grown cells led to the production of g-butyrolactone. This product is further degraded by M. vaccae to gamma-hydroxybutyric acid (GHB). This compound itself is rapidly consumed by cells and is mineralized by the activities of the central metabolism in this organism. Growth studies with M vaccae show that this organism cannot utilize cyclic ethers (THF, THP) as growth substrates. However, their lactone (butyrolactone, valerolactone) and hydroxy acid derivatives all support rapid growth of the bacterium. This presents an interesting situation in which the cometabolism of a group of compounds can produce resultant products that are able to sustain growth of the organism. The results of our studies with lactone metabolism also led us to investigate whether M. vaccae can produce lactones from their corresponding cyclic ketones by introducing oxygen into the alicyclic ring. This approach would stand in contrast to the approach described above where a hydroxylation reaction was imposed on a cyclic ether. We were able to demonstrate that M. vaccae can oxidize cyclopentanone. However, the product of the reaction was 1,3-cyclopentadione rather than valerolactone. This finding is significant because it has been previously reported that M. vaccae does not degrade cyclic ketones and it has been proposed that the lack of cyclopentanone degrading activity underlies the role of organisms like M. vaccae in the cometabolic "priming" of cyclic alkanes in the environment.

Objective 4: The Potential Toxic and Beneficial Effects of Aerobic Ether Cometabolism

The majority of the studies we conducted as part of Objective 4 focused on toxic effects associated with diethyl ether and chloroalkyl ether oxidation. During the time course of diethyl ether oxidation, we observed a slow, time-dependent inactivation of oxygenase activity in N. europaea. We were unable to reproduce this toxic effect by incubating cells with representative concentrations of the major products of diethyl ether oxidation (ethanol and acetaldehyde). We also failed to observe a general toxic effect when cells were incubated with diethyl ether under conditions where AMO activity was absent. Despite these observations, we were unable to clearly establish the mechanism of toxicity although our results strongly support the idea that a toxic and transient intermediate is generated during the oxidation of the ether by AMO. A clearer picture emerged from our studies of toxic effects associated with chloroalkyl ether oxidation. Both 2-chloroethyl vinyl ether [2CEVE] and its non-chlorinated analog, ethyl vinyl ether, were shown to act as mechanism-based inactivators of AMO activity. Evidence to support this conclusion included demonstrating that the loss of enzyme activity in the presence of these compounds is a pseudo-first order process and that the natural enzyme substrate, ammonia, protects the enzyme from inactivation. We also have demonstrated that recovery from the effects of enzyme inactivation required de novo protein synthesis. In contrast to the toxic effects associated with the cometabolism of ethers, beneficial features of this process were less obvious. The one instance where a potentially beneficial effect was observed was during the oxidation of diethyl ether by Xanthobacter Py2. Previously, we had noted that this reaction results in the accumulation of ethanol, while the second product, acetaldehyde, is rapidly consumed by the cells and does not accumulate. We subsequently showed that acetaldehyde represents a useful reductant source suitable for sustaining the cometabolic activity of this organism. This effect may account for the longevity of the ether oxidation reaction with this organism.

Significance of Findings. The findings of this research project have helped identify many of the structural features of ether-bonded compounds that affect their microbial biodegradability. Our studies with simple alkyl ethers, chloroalkyl ethers and methoxylated aromatics have demonstrated that scission of the ether bond can be achieved by many organisms when the target compound contains either C1 or C2 alkoxy groups. In contrast, larger alkoxy groups or additional substituents such as chlorine can dramatically alter the reactivity of oxygenase enzymes towards ether oxidation reactions. Our research into the factors that influence the oxidation of methyl tertiary butyl ether have been particularly rewarding and enabled us to characterize the initial steps in the oxidation process in some detail. One important finding is that MTBE oxidation to TBA does not appear to involve the production of formaldehyde. Formaldehyde is a recognized human carcinogen and the production of this compound during MTBE biodegradation in eukaryotes is one of many sources of concern with this compound. These studies also have allowed us to identify particular substrates that can promote the cometabolism of MTBE. It is notable that many of these compounds are components of gasoline. Because the majority of gasoline is introduced into the environment as part of gasoline formulations, our research conducted during this project will help identify environments where natural attenuation of this important pollutant compound might be occurring. Likewise, our research also has enabled us to identify compounds that could potentially be added to MTBE-contaminated environments to promote the cometabolic degradation of this compound. Our research also has demonstrated that the ability of microorganisms to degrade several ether-containing compounds is a widely-distributed trait. Our research has not only demonstrated that many of these compounds can be biodegraded but also has identified several substrates that could potentially be added to contaminated environments to promote cometabolic biodegradation processes. Our studies with M. vaccae and compounds such as 1,4- and 1,3-dioxane are a case in point. Finally, our research also has demonstrated some of the potential limitations of cometabolic processes for the degradation of ether-bonded compounds. We have shown that several ether-bonded compounds exert toxic effects on bacteria that can degrade these compounds. These findings suggest that there may be more than one reason why ether-bonded compounds often are recalcitrant in the environment. The latent toxic effects of these ether bonded compounds may limit their rate of biodegradation, even when appropriate organisms, cosubstrates, and environmental conditions are provided.

Journal Articles on this Report : 1 Displayed | Download in RIS Format

Other project views: All 12 publications 1 publications in selected types All 1 journal articles
Type Citation Project Document Sources
Journal Article Hardison LK, Curry SS, Ciuffetti LM, Hyman MR. Metabolism of diethyl ether and cometabolism of methyl tert-butyl ether by a filamentous fungus, a Graphium sp. Applied and Environmental Microbiology 1997;63(8):3059-3067. R823426 (Final)
R825689C020 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Full-text: AEM-Full Text PDF
  • Abstract: AEM-Abstract
  • Supplemental Keywords:

    groundwater, enzymes, petroleum, metabolism, dioxin, organics, bioremediation, microbiology., Scientific Discipline, Toxics, Waste, Ecosystem Protection/Environmental Exposure & Risk, pesticides, Fate & Transport, Environmental Microbiology, Biochemistry, Bioremediation, Biology, fate and transport, microbiology, dioxin, soil nitrifying bacterium, microbial degradation, reaction pathways, enzymes, aromatic pollutants, ether bonded compounds, aerobic cometabolism, contaminants in soil, contaminant release, aromatic ethers

    Progress and Final Reports:

    Original Abstract
  • 1996
  • 1997 Progress Report