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...

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Abstract-Anaerobic biotransformation of several aromatic hydrocarbons found in gasoline including benzene, toluene, ethylbenzene, m-xylene, p-xylene, and o-xylene (BTEX) was studied in batch anaerobic laboratory microcosms. Aquifer sediment and ground water were obtained from the site of a historic gasoline spill at Seal Beach, California. Sulfate is present in the site ground water at 80 mg/ L, and sulfate-reducing activity appears to be the dominant intrinsic BTEX bioremediation process where nitrate is absent. In the laboratory, the microcosms were set up with different electron acceptors (sulfate and nitrate) in site ground water and various defined anaerobic media to estimate intrinsic biodegradation rates and to suggest conditions under which anaerobic bioremediation could be enhanced. In unamended microcosms, anaerobic biotransformation of toluene and mϩp-xylene (m-xylene and p-xylene were measured as a summed parameter) occurred at a rate of 7.2 and 4.1 g L Ϫ1 h Ϫ1 , respectively, with sulfate as the apparent electron acceptor. Addition of nitrate stimulated nitrate-reducing conditions and increased rates of toluene and mϩp-xylene biotransformation to 30.1 and 5.4 g L Ϫ1 h Ϫ1 , respectively. The catabolic substrate range was altered to include ethylbenzene in the nitrate-amended microcosms, suggesting an apparent preferential use of different BTEX compounds depending on the electron acceptor available. Under all the conditions studied, more than twice the amount of nitrate or sulfate was used than could be accounted for by the observed BTEX degradation. Benzene transformation was not observed under the conditions studied. Although methane was detected in microcosms prepared with anaerobic media lacking nitrate and sulfate, methanogenic biotransformation of BTEX compounds was not observed. The results of these experiments indicate that indigenous microorganisms from the Seal Beach aquifer have significant capability to degrade BTEX hydrocarbons and that intrinsic processes in the Seal Beach aquifer may remediate a portion of the hydrocarbon contamination in situ without intervention. However, the data also suggest that intervention by nitrate addition would enhance the rate and extent of anaerobic BTEX biotransformation.

Residues of octylphenol polyethoxylates (OPEO) are frequently found in wastewater effluents. OPEO are commonly used surfactants. The polyethoxy chain of OPEO may be shortened or carboxylated during biological wastewater treatment and the aromatic ring chlorinated or brominated during wastewater disinfection with chlorine. Mass spectral data obtained using electron ionization (EI) and chemical ionization (CI) are presented which positively characterize these residues. For CI, methane was used as the reactant gas and protonated molecular ions were observed. The most prominent ions formed under EI resulted from benzylic cleavage while the prominent ions formed under CI resulted from alkyl ion displacement and olefin displacement. EI and CI mass spectra are summarized and fragmentation mechanisms are proposed. Response factors are presented for quantitative analysis by single ion monitoring.CH,C(CHJ3, R,=CH,.In NPEO compounds, R, and R, are variable as a consequence of commercial synthesis procedure^.'^

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