Environ. Sci. Technol. 1996, 30, 575-585
Carbazole Degradation by Pseudomonas sp. LD2: Metabolic Characteristics and the Identification of Some Metabolites LISA M. GIEG,† ALBIN OTTER,‡ AND P H I L L I P M . F E D O R A K * ,† Departments of Biological Sciences and Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2E9
A carbazole-degrading bacterium was isolated by enrichment from a creosote-contaminated soil. This organism, designated Pseudomonas sp. LD2, utilized carbazole as a sole source of carbon, nitrogen, and energy. When isolate LD2 was grown in nitrogenfree mineral medium with 14C-labeled carbazole, 43% was recovered as 14CO2 after 3 days of incubation. Numerous aromatic and heterocyclic compounds were tested as growth substrates for isolate LD2, but few supported the growth of this bacterium. Anthranilic acid and catechol served as growth substrates and were positively identified as intermediates of carbazole degradation by isolate LD2. In addition, 10 nitrogencontaining metabolites were observed in acidified extracts of LD2 culture supernatants, four of which were unequivocally identified. These included indole-3-acetic acid, 5-(2-aminophenyl)-5-oxopentanoic acid, and the cyclized products of 5-(2-aminophenyl)5-oxopent-3-enoic acid and 6-(2-aminophenyl)-2-hydroxy-6-oxohexa-2,4-dienoic acid.
Introduction Nitrogen, sulfur, and oxygen heterocycles have been found in soils and groundwaters at sites contaminated with petroleum and wood-preserving wastes (1-4). Although many of these heterocycles have been found to be biodegraded or biotransformed in laboratory cultures (47), little is known about their fate in the environment (1, 2). Some laboratory and field studies have examined the fate of N-heterocycles in a creosote-contaminated aquifer (1, 8-10). Of the N-heterocycles present in creosote and petroleum wastes, the biodegradation of quinoline and its methyl derivatives has been the most frequently studied (9-14). Carbazole has received less attention. Like quinoline, carbazole has been found to be a predominant N-hetero* Corresponding author telephone: (403)492-3670; fax: (403)4929234; e-mail address: Phil
[email protected]. † Department of Biological Sciences. ‡ Department of Chemistry.
0013-936X/96/0930-0575$12.00/0
1996 American Chemical Society
cycle in coal tar creosote (2) and found to be present in groundwater and soil resulting from creosote contamination (1, 15, 16). Finnerty et al. (17) used carbazole as a model compound to study the microbial denitrogenation of petroleum materials. Four bacterial strains were isolated that could use carbazole as a sole source of carbon and energy, one of which could also use this compound as a sole nitrogen source. Several water-soluble metabolites were detected by thin-layer chromatography (TLC), although they were not identified. Fedorak and Westlake (18) reported that carbazole enrichment cultures were capable of degrading a wide range of alkylcarbazoles present in Norman Wells crude oil. Grosser et al. (19) reported the mineralization of [14C]carbazole in soils that had been inoculated with a previously isolated carbazole-degrading Xanthomonas species, whereby 45% of the label was recovered as 14CO2. Recently, Ouchiyama et al. (20) proposed a pathway for the metabolism of carbazole by two Pseudomonas isolates, although only anthranilic acid and catechol were positively identified as metabolites. Resnick and Gibson (21) also reported anthranilic acid as a metabolite of carbazole degradation as well as 1-hydroxycarbazole formation by a mutant Pseudomonas strain. At the time this research project began, no intermediates of carbazole metabolism had been reported, and the goal was to isolate a bacterium that could degrade carbazole and to identify some of the metabolites. The ability of the isolate to grow on or transform a variety of aromatic compounds, carbazole analogs, and possible intermediates was tested to help elucidate the manner in which carbazole was metabolized. This paper describes some of the metabolic capabilities of a bacterium that uses carbazole as its sole source of carbon, nitrogen, and energy. In addition, several metabolites were identified.
Experimental Section Growth Media and Conditions. Experiments examining the biodegradation of carbazole were generally carried out in 500-mL Erlenmeyer flasks containing 200 mL of a nitrogen-free mineral medium (denoted B-N). This medium had the following composition (per liter of distilled water): 0.5 g of K2HPO4, 2.0 g of Na2SO4, 0.2 g of MgSO4‚7H2O, 2.0 g of KCl, and 1.0 mL of a trace metals solution (22). In some experiments, the inorganic nitrogen sources KNO3 (0.2 g) and NH4Cl (0.1 g) were added in place of the KCl, and this medium was denoted B+N. Carbazole (BDH, Poole, England) and other substrates were added directly to culture media in their crystalline forms. All cultures were incubated at 28 °C in the dark on a rotary shaker at 200 rpm, unless otherwise noted. Plate count agar (PCA; Difco, Detroit, MI) was used to isolate carbazole-degrading bacteria and to enumerate bacteria by the plate count method. Agar plates were incubated at 28 °C for at least 3 days before counting. Isolation of a Carbazole-Degrading Bacterium. A carbazole-degrading bacterium was isolated by enrichment from creosote-contaminated soil from a site in Edmonton, AB, Canada. Soil (10 g) was inoculated into 200 mL of mineral medium that contained 1 g of NH4Cl/L (8) and 20 mg of carbazole. Every 4 days for 16 days this shake-flask culture was transferred (inoculum 10% by volume) into
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fresh medium and substrate and then the culture was transferred into 200 mL of B-N medium containing 0.1 g of carbazole. Growth was evident by turbidity in this medium within 3 days. This mixed culture was streaked on PCA, and morphologically different colonies were selected and tested to see if they were capable of using carbazole as a sole growth substrate in B-N medium. One isolate, designated LD2 [previously named D2 (23)], was found that used carbazole as a sole growth substrate and was subsequently characterized by various taxonomic tests. Substrates Tested for the Growth of Isolate LD2. The following compounds were tested as growth substrates for isolate LD2, both in the presence and absence of carbazole: 9-methylcarbazole, 2-hydroxycarbazole, 1,2,3,4-tetrahydrocarbazole, 2-aminobiphenyl, 2-nitrobiphenyl, phenothiazine, iminostilbene, fluorene, dibenzofuran (all from Aldrich, Milwaukee, WI), dibenzothiophene (Fluka, Ronkonkoma, NY), anthranilic acid (BDH), and indole-3acetic acid (Sigma, St. Louis, MO). Other substrates tested as carbon sources included the following: catechol, benzoic acid, benzene (all from Fisher Scientific Co., Fair Lawn, NJ), biphenyl (Eastman Kodak Co., Rochester, NY), 2,2′dihydroxybiphenyl, indole-3-pyruvic acid, naphthalene, tryptophan (Sigma), 2,3-dihydroxybiphenyl (Wako Pure Chemical Industries Ltd., Osaka, Japan), indole (Matheson, Coleman, and Bell, Norwood, OH), indole-3-propanoic acid, indole-3-butanoic acid, indole-3-carboxaldehyde, indole3-carboxylic acid, trans-indole-3-acrylic acid, and phenanthrene (Aldrich). In the growth experiments without carbazole in the medium, isolate LD2 was inoculated into B+N and/or B-N medium containing 20-50 mg of the desired compound. If carbazole was present, B-N medium was used. The cultures were incubated from 2 to 4 weeks, then acidifed, and extracted with ethyl acetate or dichloromethane for gas chromatographic analysis to determine whether the compound had been consumed or transformed. Whenever cultures of isolate LD2 were incubated in the presence of an aromatic compound to test for growth or biotransformation, appropriate sterile controls were incubated and analyzed in the same manner as the test cultures to account for any abiotic transformations. None of the transformation products discussed in this paper was found in the sterile controls. The ability of isolate LD2 to fix N2 was evaluated using the acetylene reduction assay (24). Used as a positive control, Azotobacter vinelandii yielded ethylene under the same test conditions. Mineralization of Radiolabeled Carbazole. Mineralization of [U-14C]carbazole (Amersham Corp., Arlington Heights, IL) by isolate LD2 was studied in 500-mL Erlenmeyer flasks equipped with side-arms for sampling (25). Controls and cultures contained a mixture of both nonradiolabeled and ∼400 000 dpm [14C]carbazole per culture. Flasks were incubated and at various times the liquid and gas phases were sampled to recover 14CO2 that had been produced (25). Extraction of Metabolites and Chemical Synthesis. Metabolites from carbazole or other substrates were extracted from the culture medium with ethyl acetate either at neutral pH or after acidification to pH
