anaerobic oxidation toluene, phenol, disssimilatory iron ... however, metabolism oftoluene, phenol,

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  • Vol. 56, No. 6APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1990, p. 1858-1864 0099-2240/90/061858-07$02.00/0 Copyright © 1990, American Society for Microbiology

    Anaerobic Oxidation of Toluene, Phenol, and p-Cresol by the Disssimilatory Iron-Reducing Organism, GS-15

    DEREK R. LOVLEY* AND DEBRA J. LONERGAN Water Resources Division, U.S. Geological Survey, 430 National Center, Reston, Virginia 22092

    Received 7 December 1989/Accepted 4 April 1990

    The dissimilatory Fe(III) reducer, GS-15, is the first microorganism known to couple the oxidation of aromatic compounds to the reduction of Fe(III) and the first example of a pure culture of any kind known to anaerobically oxidize an aromatic hydrocarbon, toluene. In this study, the metabolism of toluene, phenol, and p-cresol by GS-15 was investigated in more detail. GS-15 grew in an anaerobic medium with toluene as the sole electron donor and Fe(IH) oxide as the electron acceptor. Growth coincided with Fe(III) reduction. [ring-.4C]toluene was oxidized to '4C02, and the stoichiometry of '4C02 production and Fe(III) reduction indicated that GS-15 completely oxidized toluene to carbon dioxide with Fe(III) as the electron acceptor. Magnetite was the primary iron end product during toluene oxidation. Phenol and p-cresol were also completely oxidized to carbon dioxide with Fe(III) as the sole electron acceptor, and GS-15 could obtain energy to support growth by oxidizing either of these compounds as the sole electron donor. p-Hydroxybenzoate was a transitory extracellular intermediate of phenol and p-cresol metabolism but not of toluene metabolism. GS-15 oxidized potential aromatic intermediates in the oxidation of toluene (benzylalcohol and benzaldehyde) and p-cresol (p-hydroxybenzylalcohol and p-hydroxybenzaldehyde). The metabolism described here provides a model for how aromatic hydrocarbons and phenols may be oxidized with the reduction of Fe(III) in contaminated aquifers and petroleum-containing sediments.

    Aromatic hydrocarbons are among the most common groundwater contaminants (28, 34, 36, 45). Under aerobic conditions, microorganisms can readily degrade monoaro- matic hydrocarbons such as benzene, xylenes, and toluene, and the pathways for this metabolism have been studied intensively (10, 15). However, most groundwaters polluted with organic compounds are anaerobic. Geochemical evi- dence has indicated that aromatic hydrocarbons can be oxidized in anaerobic groundwater in which nitrate reduc- tion, methane production, or Fe(III) reduction is the termi- nal electron accepting process (21, 23, 28, 34; I. M. Cozza- relli, R. P. Eganhouse, and M. J. Baedecker, Environ. Geol. Water Sci., in press). Laboratory studies have indicated that the oxidation of aromatic hydrocarbons under these anaer- obic conditions is the result of microbial metabolism (17, 21-23, 28, 44-47). Although anaerobic microorganisms capable of metaboliz-

    ing aromatic acids and phenols under denitrifying, photosyn- thetic, sulfate-reducing, or methanogenic conditions have been described previously (for reviews, see references 5 and 13), until recently there were no microorganisms in pure culture that were known to anaerobically oxidize aromatic hydrocarbons. Two microbial isolates which can anaerobi- cally oxidize toluene have now been described. The dissim- ilatory Fe(III)-reducing microorganism, GS-15, was found to grow in an anaerobic medium with toluene as the electron donor and a poorly crystalline Fe(III) oxide as the electron acceptor (23). The extent of carbon dioxide production in the cultures as well as the stoichiometry of carbon dioxide production and Fe(III) reduction indicated that GS-15 could completely oxidize toluene to carbon dioxide with Fe(III) as the sole electron acceptor. More recently, a Pseudomonas sp. was found to oxidize [ring-'4C]toluene to '4Co2 with nitrate or N20 as the potential electron acceptor (46). It was not demonstrated whether the Pseudomonas sp. could ob-

    * Corresponding author.

    tain energy for growth from toluene oxidation, and the end product(s) of nitrate or N20 reduction was not determined.

    In this report, we detail the growth and metabolism of GS-15 on toluene as well as on two' other important aromatic contaminants, p-cresol and phenol. These results indicate that GS-15 may be a useful organism with which to study potential mechanisms for the anaerobic oxidation of aro- matic hydrocarbons and to model the metabolism of aro- matic contaminants in the Fe(III)-reducing zone of sedimen- tary environments.

    MATERIALS AND METHODS Culture conditions. As previously described (26), strict

    anaerobic culturing and sampling techniques were used throughout. The basic growth medium for GS-15 was the same as that previously described for growth of GS-15 on acetate (26). The medium contained (in grams per liter of deionized water): NaHCO3 (2.5), CaCI2 2H20 (0.1), KCI (0.1), NH4Cl (1.5), and NaH2PO4 H20 (0.6), as well as a mixture of vitamins and trace minerals. The medium con- tained ca. 100 mmol of Fe(III) in the form of a poorly crystalline Fe(III) oxide. This was synthesized, as previ- ously described (24), by neutralizing a solution of FeCl3 and collecting the Fe(III) oxide precipitate. The gas phase was N2-C02 (80:20). The pH was 6.7. In order to examine the formation of magnetite during toluene metabolism, without potential interference of siderite formation (26), GS-15 was grown in a modified medium in which the NaHCO3 was omitted and the gas phase was N2. The medium was dispensed in 10-ml volumes in anaerobic

    pressure tubes or 80- or 100-ml volumes in 160-ml serum bottles. The medium was bubbled for at least 6 (pressure tubes) or 15 (serum bottles) min with the appropriate gas phase (N2-C02 or N2) to remove dissolved oxygen. The culture vessels were then sealed with thick butyl rubber stoppers (Bellco Glass, Inc., Vineland, N.J.) and an alumi- num crimp. The medium was sterilized by autoclaving

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  • ANAEROBIC TOLUENE OXIDATION 1859

    (121°C, 20 min). Unless noted, no reducing agent was added, because the Fe(II) that was transferred with the inoculum was more than sufficient to remove any traces of oxygen that might not have been removed by sparging the medium (27). However, metabolism of toluene, phenol, and p-cresol was also studied in the presence of added sulfide to further ensure anaerobic conditions. In these instances, sodium sulfide was added to sterilized medium from an anaerobic stock solution to provide a final concentration of 0.5 g of sodium sulfide per liter.

    Phenol, p-cresol, benzoate, benzylalcohol, benzaldehyde, p-hydroxybenzoate, and p-hydroxybenzaldehyde were add- ed to sterilized medium from anaerobic stock solutions (5 or 50 mM) to provide an initial concentration of ca. 0.5 mM. Toluene was added to the medium with a microsyringe to provide either 1 or 10 mmol of toluene per liter of medium. Because of the low solubility of p-hydroxybenzylalcohol, it was added directly to the medium prior to bubbling and sterilization in order to provide an initial concentration of 0.5 mM.

    In order to test the ability of GS-15 to metabolize toluene, phenol, and p-cresol, an inoculum of GS-15 that had been grown with benzoate as the electron donor and Fe(III) oxide as the electron acceptor was added to media containing the various electron donors. GS-15 was grown for nine transfers (10% inoculum) on toluene or p-cresol and for four transfers on phenol prior to conducting the cell growth, Fe(III) reduction, and radiotracer studies reported here. All incuba- tions were at 30°C in the dark. To determine whether the reduction of Fe(III) with tolu-

    ene resulted from the oxidation of the aromatic ring, GS-15 was grown in the presence of [ring-'4C]toluene. Medium (80 ml) was inoculated with 8 ml of a culture of GS-15 which had been grown on toluene, and 8 p.l of [ring-`4C]toluene (10 mCi/ml; Du Pont, Boston, Mass.) was added to initially provide 1 mmol of toluene per liter of medium. As outlined below, '4C02production was measured after 7 weeks of incubation.

    In order to measure '4C02 production from [U-'4C]phe- nol-, [U-4C]phenol (213 mCi/mmol; Sigma Chemical Co., St. Louis, Mo.) was dissolved in a solution of 50 mM phenol. The resultant solution contained 85 ,uCi per mmol of phenol. Dissolved oxygen was removed from the solution by bub- bling it with N2. The phenol solution (0.1 ml) was then added to medium (10 ml) to provide an initial phenol concentration of 0.5 mM.

    Analytical techniques. Cell numbers were monitored with epifluorescence microscopy (18). Samples (1 ml) were anaer- obically removed over time with a syringe and needle and were fixed with glutaraldehyde (final concentration, 2.5%). The iron forms were dissolved with an acidic oxalate solu- tion as previously described (26), and the cells were treated with an acridine orange solution to give a final acridine orange concentration of 0.01%. After 2 min, the sample was filtered onto a black Nuclepore filter (0.2-p.m pore diameter), and the filters were observed under oil immersion (x 1,000) with a Zeiss epifluorescence microscope. All fluorescent cells were counted.

    Fe(III) reduction was monitored by measuring the accu- mulation of Fe(II) over time. As previously described (25), subsamples were extracted in 0.5 N HCl for 15 min to dissolve Fe(II) minerals, and Fe(II) was determined with ferrozine. To measure aromatic compounds, a subsample of the

    culture was filtered (Gelman filter; 0.45-p.m pore diameter) and the aromatics were separated on a SupelCosil LC-18

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