Initial reactions in anaerobic ethylbenzene oxidation by a denitrifying bacterium, strain EB1

Abstract

Initial reactions in anaerobic oxidation of ethylbenzene were investigated in a denitrifying bacterium, strain EB1. Cells of strain EB1 mineralized ethylbenzene to CO 2 under denitrifying conditions, as demonstrated by conversion of 69% of [ 14 C]ethylbenzene to 14 CO 2 . In anaerobic suspensions of strain EB1 cells metabolizing ethylbenzene, the transient formation and consumption of 1-phenylethanol, acetophenone, and an as yet unidentified compound were observed. On the basis of growth experiments and spectroscopic data, the unknown compound is proposed to be benzoyl acetate. Cell suspension experiments using H 2 18 O demonstrated that the hydroxyl group of the first product of anoxic ethylbenzene oxidation, 1-phenylethanol, is derived from water. A tentative pathway for anaerobic ethylbenzene mineralization by strain EB1 is proposed.Anaerobic degradation of aromatic hydrocarbons receives significant attention for the unusual biochemical reactions involved and for its importance in intrinsic bioremediation. Benzene, toluene, ethylbenzene, and xylenes are relevant pollutants resulting from surface spills of gasoline or leaking underground storage tanks (32). Because of their relatively high solubility in water (8) and their toxicity (10, 30), these compounds often come in contact with groundwater and can create a potential health hazard. As oxygen concentrations in groundwater decrease at these sites as a result of degradation of gasoline components by aerobic microorganisms (16, 28), contaminated aquifers become anoxic. Subsequent biological removal of aliphatic and aromatic hydrocarbons at these sites depends on the activity of bacteria capable of metabolizing hydrocarbons under anoxic conditions.In all presently known pathways for mineralization of aromatic hydrocarbons under aerobic conditions, the first oxidation reactions are catalyzed by mono-or dioxygenases (16,31). In oxygenases, molecular oxygen acts as a strong oxidizing agent and is incorporated into the aromatic hydrocarbon (16, 31). The oxygenated aromatic metabolites are then further oxidized via conventional metabolic pathways. Until first postulated in 1984 (27), degradation of aromatic hydrocarbons was not thought to occur in the absence of molecular oxygen. However, in the last 6 years, several pure cultures that are capable of degrading alkylbenzenes under denitrifying (11,12,26,29,35), sulfate-reducing (5, 25), and iron-reducing (20) conditions have been isolated. Evidently, these bacteria are capable of catalyzing the oxidation of aromatic hydrocarbons in the absence of molecular oxygen, yet the biochemical mechanisms underlying the initial enzymatic oxidation reactions are unresolved.We have been investigating the anaerobic degradation of ethylbenzene by a denitrifying bacterium, strain EB1. Strain EB1 was isolated from treatment pond sediment of an oil refinery (3). We report the characterization of strain EB1 and experiments elucidating the first metabolic intermediates formed in anoxic ethylbenzene oxidation. Isolation conditions. Anae...

Ethylbenzene Dehydrogenase, a Novel Hydrocarbon-oxidizing Molybdenum/Iron-Sulfur/Heme Enzyme

“…For one of these strains, Azoarcus sp. EB-1, ethylbenzene is the only known hydrocarbon utilized as growth substrate (1). The other two strains utilize either ethylbenzene or an alternative hydrocarbon compound, namely toluene (strain EbN1) or n-propylbenzene (strain PbN1) (2).…”

“…It is initiated by a novel biochemical reaction, namely an oxygenindependent oxidation of ethylbenzene to (S)-1-phenylethanol. This intermediate is then oxidized further to acetophenone by an alcohol dehydrogenase (1)(2)(3)(4). Activities of an ethylbenzeneoxidizing enzyme and an enantio-specific (S)-1-phenylethanol dehydrogenase have been reported in cell extracts of strain EB-1 (4), and a substrate-specific (S)-1-phenylethanol dehydrogenase has been purified and characterized from strain EbN1.…”

In situ Groundwater Remediation Using Enricher Reactor – Permeable Reactive Biobarrier

“…The hydroxyl group of this first intermediate is derived from water (Ball et al, 1996). 1-Phenylethanol is oxidized further to form acetophenone.…”

“…1-Phenylethanol is oxidized further to form acetophenone. Ball et al (1996) proposed that acetophenone may be carboxylated to form benzoyl acetate, which can be activated to benzoyl acetyl coenzyme A (benzoyl acetyl-CoA) and subsequently, a thiolytic cleavage of benzoyl acetyl-CoA would generate acetyl-CoA plus benzoyl-CoA.…”

Intrinsic Aromatic Hydrocarbon Biodegradation for Groundwater Remediation

“…With nitrate as TEA, biodegradation has been reported for ethylbenzene, toluene, and xylenes (Altenschmitt and Fuchs, 1991;Hutchins, 1991;Schocher et al, 1991;Evans et al, 1992;Fries et al, 1994;Seyfried et al, 1994;Biegert and Fuchs, 1995;Rabus and Widdel, 1995;Ball et al, 1996;Beller and Spormann, 1997). Benzene degradation under denitrifying conditions is still controversially discussed (Burland and Edwards, 1999;Lovley, 2000).…”