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1997 Progress Report: Aerobic Cometabolism of Ether-bonded Compounds
EPA Grant Number: R823426Title: Aerobic Cometabolism of Ether-bonded Compounds
Investigators: Hyman, Michael R.
Institution: Oregon State University
Current Institution: Oregon State University , North Carolina State University
EPA Project Officer: Manty, Dale
Project Period: October 1, 1995 through September 1, 1998
Project Period Covered by this Report: October 1, 1996 through September 1, 1997
Project Amount: $358,953
RFA: Exploratory Research - Environmental Biology (1995)
Research Category: Biology/Life Sciences
Description:
Objective:The aim of this research project is to characterize the microbial aerobic cometabolic degradation of a variety of ether-bonded compounds. The research is separated into four objectives. First, we aim to determine which structural features which either promote or decrease the reactivity of a non-specific monooxygenase towards a variety of ether-bonded compounds. These compounds include a range of alkyl, alicyclic and aromatic ethers and these studies emphasize the use of the soil nitrifying bacterium Nitrosomonas europaea . This organism initiates the oxidation of its sole growth-supporting substrate, ammonia, through the activity of the membrane-bound enzyme, ammonia monooxygenase (AMO). The second objective is to investigate the reactivity of AMO towards a series of ether-bonded compounds which are recognized as environmental pollutants, including chlorinated alkyl ethers, haloaryl ethers, cyclic and branched alkyl ethers. The third objective aims to examine the rates of ether cometabolism and the products obtained from the oxidation of a series of 6 representative ethers by a range of physiologically distinct bacteria and fungi including species grown on gaseous n-alkanes, gaseous n-alkenes and aromatics. The fourth objective aims to examine the consequences of ether cometabolism on active microorganism. These studies aim to investigate the mechanism of toxicity of ether oxidation and to examine potential for beneficial aspects of ether cometabolism on a variety of bacterial strains. Progress Summary:
We have initiated studies in all of the objectives and have now largely completed the studies outlined in Objective 1. The cometabolism of simplest linear alkyl ethers (dimethyl and diethyl ether) by N. europaea has been shown to involve O-dealkylation reactions which give rise to alcohol and aldehyde products. In contrast, n-propyl and n-butyl ethers are hydroxylated and the ether bond is not cleaved. We have also completed a kinetic analysis of diethyl ether oxidation that demonstrated this compound does not behave as a typically substrate for AMO and that oxidation of this compound is associated with a toxic effect on N. europaea resulting from substrate turnover. We have recently demonstrated that N. europaea will also oxidize a wide variety of monoaromatic ethers including anisole, 1,3-dimethoxybenzene and 1,4-dimethoxybenzene but not 1,3,5-trimethoxybenzene. The oxidation of anisole has been shown to general phenol, catachol, hydroquione and formaldehyde. Our proposed studies examining the oxidation of cyclic ether compounds have established that furan and tetrahydrofuran are also oxidized by N. europaea and our studies of these compounds is ongoing.
We have also completed the studies described in Objective #2 and our results have revealed several surprising features of the catalytic capabilities of AMO. Our earlier studies concentrated on the oxidation of chloroalkyl ethers. We demonstrated that many of these compounds are oxidized by N. europaea and that the reactivity of towards these compounds follows a substantially similar pattern to the reactivity of AMO towards their non-chlorinated counterparts. Our most recent studies have demonstrated that AMO will also oxidize several haloaryl ethers (4-bromo and 4-chlorophenyl ether) and a wide selection of other aromatic ethers including dibenzo-p-dioxin, dibenzofuran and xanthene. We have also established that all of these compounds are rapidly hydroxylated by AMO, followed by a pH-dependent abiotic nitration reaction which leads to the production of nitro-hydroxylated ethers. This unusual reaction also occurs during the oxidation of several PAHs and we suggest that these nitration reactions may represent mechanisms by which nitrogen can be introduced into soil organic matter by nitrifying organisms. Our studies have also demonstrated that the O-dealkylation of aromatic ethers such as p-nitroanisole and 1-methoxynaphtalene can be used as very sensitive assay for AMO activity.
Our most recent studies in the third Objective have focused on characterizing the ability of various alkane-utilizing organisms to degrade the gasoline oxygenate MTBE. We have published one study describing the oxidation of MTBE by a filamentous fungus, Graphium and we have made significant progress with several bacterial systems. Our research demonstrates that MTBE is consistently oxidized by alkane-utilizing bacteria but is not susceptible to degradation by many other microorganisms that express non-specific monooxygenase enzymes (e.g. ammonia, propylene, toluene) even though we have also established that all of these organisms can catalyze simple O-dealkylation reactions with diethyl ether. Our studies also indicate that MTBE oxidation consistently involves the initial production of tert-butyl formate which subsequently undergoes abiotic and biotic hydrolysis to yield tert-butyl alcohol (TBA) Our inhibitor studies have also demonstrated that TBA itself is further oxidized by the same monooxygenase that initiates MTBE oxidation and that several additional proposed products of MTBE degradation (a diol, acetone and hydroxyacetone) are also substrates for the same monooxygenase. These observations have important implications for the kinetics of MTBE oxidation and may contribute to our observation that MTBE is generally a kinetically poor substrate for many bacterial monooxygenases. We have also extended our studies of MTBE oxidation to the molecular level and have identified a component polypeptide of the alkane-oxidizing monooxygenase in Mycobacterium vaccae and two other strains of MTBE-degraders. Our most recent finding is that MTBE oxidation is observed in certain bacterial strains after growth on many major components of gasoline including isopentane, pentane, and hexane. These observations suggest that many of the cosubstrates required for the cometabolism of MTBE are actually present in gasoline itself.
The proposed studies outlined in Objective #4 have concentrated on the toxic effects associated with diethyl ether and chloroalkyl ether oxidation. Our results indicate that many of these compounds produce turnover-dependent inactivating effects that are specific for the ammonia-oxidizing system in N. europaea. Our kinetic studies to date indicate that these compounds appear to behave as mechanism-based inactivators and that the inactivating effects of these compounds are directed selectively towards AMO. We have also examined potential beneficial effects of ethers on bacterial systems and have identified two alkane-utilizing species that can grow on diethyl ether as a sole source of carbon and energy. Our studies with these organisms are ongoing.
Future Activities:Our future studies will continue to examine the oxidation of several ether-bonded compounds by N. europaea and will focus on the possibility of using these reactions as possible priming processes for increasing their biodegradability in conjunction with denitrifying organisms. Our future studies will also concentrate on establishing the kinetics, pathway and underlying physiological effects of MTBE cometabolism by alkane-utilizing microorganisms, including both bacteria and selected eukaryotes. Journal Articles:
No journal articles submitted with this report: View all 12 publications for this project
Supplemental Keywords:ground water, enzymes, petroleum, metabolism, dioxin, organics, bioremediation, microbiology. , Ecosystem Protection/Environmental Exposure & Risk, Toxics, Scientific Discipline, Waste, Biology, Biochemistry, Environmental Microbiology, Fate & Transport, pesticides, Bioremediation, dioxins, aromatic pollutants, fate and transport, microbial degradation, aerobic cometabolism, soil nitrifying bacterium, enzymes, ether bonded compounds, contaminant release, contaminants in soil, microbiology, dioxin, reaction pathways
Progress and Final Reports:
Original Abstract
Final Report