INTRODUCTION

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The quality of petroleum is progressively deteriorating as the highest-quality petroleum deposits are preferentially produced (10, 17). Consequently, concern about the concentration of compounds and contaminants such as sulfur, nitrogen, and metals in petroleum will intensify. These contaminants not only contribute to environmental pollution resulting from the combustion of petroleum but also interfere with the processing of petroleum by poisoning catalysts and contributing to corrosion (8, 17). The selective removal of contaminants from petroleum while retaining the fuel value is a difficult technical challenge. New processes are needed (10). The selective removal of sulfur from dibenzothiophene and from petroleum by various microbial cultures has been demonstrated, and this technology is now being commercialized for the biorefining of petroleum (13). Biorefining can also potentially be used to remove nitrogen and metals from petroleum, but so far this area of research has received very little attention (12).

Quinoline is perhaps the most widely studied organonitrogen compound as regards biodegradation, and quinoline is considered to be representative of many organonitrogen compounds typically found in petroleum (6). Many aerobic and anaerobic microbial cultures that can degrade quinoline have been found (9). The majority, if not the entirety, of microbial cultures described in the literature that metabolize quinoline do so by fully degrading it and can therefore utilize quinoline as a sole source of carbon, energy, and nitrogen (2, 5, 7, 9, 14, 18-22). However, for use in a petroleum biorefining application, it would be preferable if nitrogen was selectively removed from quinoline, leaving the carbon and the calorific value of the molecule intact. The metabolic pathways utilized by various aerobic quinoline-degrading microorganisms have been investigated, and even though the cultures were capable of fully degrading or mineralizing quinoline, some metabolic pathways were shown to initiate the degradation of quinoline by selectively oxidizing and removing nitrogen from quinoline (9, 19). While the biodegradation of quinoline has been reasonably well studied, there is very little information concerning the use of quinoline-degrading microorganisms to remove nitrogen from petroleum. Several quinoline-degrading Pseudomonas cultures were found to have no ability to remove significant levels of nitrogen from crude oil or asphaltene fractions of petroleum (2). The removal of 20 to 45% of nitrogen from crude oil by unspecified mixed cultures has been claimed, but the ability of these cultures to metabolize quinoline is unknown (12).

The purpose of this investigation was to isolate aerobic bacterial cultures capable of utilizing quinoline as a nitrogen source, but incapable of utilizing quinoline as a carbon source, and subsequently to examine the metabolic pathway of quinoline degradation and the ability of such cultures to selectively remove nitrogen from petroleum.

	MATERIALS AND METHODS

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Bacterial cultures and growth conditions. Environmental samples of soil and/or water were obtained from petroleum and coal processing sites, compost, and other sites where contamination with petroleum hydrocarbons exist.

Nitrogen bioavailability assay. The nitrogen bioavailability assay used defined mineral salts medium in growth tests in which organonitrogen model compounds such as quinoline, pyridine, carbazole, and porphyrin served as sources of carbon and/or nitrogen. Growth tests were performed using six conditions:

Spectrophotometric scans to detect metabolites. UV/visible spectrophotometric scans were performed at 240 to 900 nm on a Beckman DU-65 spectrophotometer. Supernatants from cultures grown using ammonia or quinoline as sole nitrogen sources were obtained by centrifugation at 10,000 × g for 15 min and then compared spectrophotometrically to identify new peaks formed due to accumulation of metabolites specific to the metabolism of quinoline.

Substrate range tests. Microbiological tests were performed to determine the range of organonitrogen compounds that could serve as sole sources of nitrogen for growth for the various pure cultures obtained in the project. Tests were also performed to determine if various organonitrogen compounds are inhibitory to the growth of these microbial cultures. Growth tests were performed according to the nitrogen bioavailability assay procedures previously described. The organonitrogen compounds and control compounds used included 2-methyl--naphthothiazole, 2-methyl benzothiazole, 2(methylmercapto) benzimidazole, 1,1-methylene bis(3-methyl piperidine), thiazole, 1-butylpyrrolidine, 2-methylene-1,3,3-trimethyl indoline, 2-methyl-3-propylpyrazine, 2-phenylbenzothiazole, 2-methyl quinoxaline, 2-methyl indoline, carbazole, quinoline, 8-hydroxyquinoline, 5-hydroxyisoquinoline, 3,4-dihydro-2(1H)-quinoline, quinazoline, 2,4-quinolinediol, isoquinoline, 3-methyl isoquinoline, isocarbostyril, protoporphyrin, pyridine, phenyl benzothiazole, nicotinic acid, imidazole, indole, HEPES buffer, urea, guanine, lysine, tryptophan, 4-hydroxycoumarin, 7-hydroxycoumarin, and ammonium sulfate. These chemicals were chosen to determine the range of organonitrogen compounds that could be used as nitrogen sources by bacterial cultures. All chemicals were obtained from Sigma (St. Louis, Mo.) or Aldrich Chemical Company (Milwaukee, Wis.) and were of the highest purity obtainable.

TLC for identification of metabolites. Thin-layer chromatography (TLC) was performed on Whatman silica C₁₈ plates by the method described by Watson and Cain (22). The running-phase solvents used were hexane, acetic acid, and xylene in the ratio of 5:1:2. Supernatants from bacterial cultures grown with quinoline as the sole source of nitrogen were obtained after centrifugation at 10,000 × g for 15 min and were then acidified to pH 2 with HCl. Typically 100 ml of aqueous supernatant was extracted with 0.5 to 1.0 volume of ethyl acetate which was recovered from the aqueous phase using a separatory funnel. The ethyl acetate extract was then evaporated in a hood, resulting in concentration of the sample from 20- to 1,000-fold prior to the analysis of the extracts by TLC. Typically 10 to 50 µl of ethyl acetate sample that had been concentrated 100-fold relative to the volume of aqueous supernatant extracted was spotted onto TLC plates. The TLC plates typically were run for about 20 min and then observed under normal lighting and under short-wave (245 nm) and long-wave (366 nm) UV light. Some experiments also utilized resting cells which were prepared by centrifuging from 100 to 500 ml of log-phase cultures grown with either quinoline or ammonia as a nitrogen source. Then the washed cell pellets were resuspended in 5 to 50 ml of mineral salts medium to final cell densities of 10⁹ to 10¹⁰ cells/ml. These cell suspensions were incubated with 20 mM quinoline for periods ranging from 15 min to 16 h at 30°C with shaking. Extraction of the supernatants from resting cells as well as growing cells was carried out either by ethyl acetate solvent extraction or with C₁₈ solid-phase extraction cartridges (Waters Associates, Milford, Mass.).

GC-MS. GC-MS analysis was performed on extracts derived from growing and resting cell cultures exposed to quinoline and on compounds eluted from spots observed on TLC plates.

Tests with petroleum. Pseudomonas ayucida IGTN9m was grown in mineral salts medium using quinoline as the sole nitrogen source to produce 1 liter of culture at an optical density at 600 nm of 1.67, which was harvested by centrifugation at 10,000 × g for 15 min; the cell pellet was resuspended in 100 ml of mineral salts medium. The culture was divided into two 50-ml portions, and 3 ml of shale oil (1.7% nitrogen) was added to each. The cultures were incubated in 250-ml Erlenmeyer flasks shaking at room temperature overnight (16 h). Then the oil was recovered using a separatory funnel and the centrifugation of any emulsion phase at 2,000 × g for 10 min. The water-free oil phase was removed by syringe or pipette. Nitrogen analyses were performed by a modified ASTM D-3177 method. The allowed replicate tolerances and reproducibility of these analyses were 0.05%, and the detection limit was 0.002% N. Carbon-hydrogen analyses were performed by a modified ASTM D-3177 method. The allowed replicate analysis tolerances and reproducibility of these analyses were 0.5% C and 0.1% H, and the detection limits were 0.3% C and 0.1% H. Sulfur concentrations in oil samples were determined using a LECO SC-132 analyzer, which has a working range of 0.002 to 100%, with an accuracy of ±61% (relative). The detection limit for sulfur is 0.002%. The amount of quinoline present in oil samples was determined by GC using an HP 5890 series II GC equipped with a 5921A atomic emission detector and calculating the area under the peak corresponding to a retention time for quinoline.

	RESULTS AND DISCUSSION

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Environmental samples obtained from petroleum-contaminated locations were used to inoculate nutristats and shake flask enrichment culture experiments in which 3 to 20 mM quinoline was supplied as the sole source of nitrogen. Quinoline was initially toxic to the nutristat cultures, making it difficult to establish and maintain bacterial growth. However, after several weeks bacterial growth was obtained. Initially the flow rates of the nutristats were adjusted to achieve hydraulic retention times of about 30 days. As the ability of the microbial culture to tolerate and utilize quinoline improved, the flow rates of the nutristats were increased to be as fast as possible without causing washout of all cells. Eventually, hydraulic retention times of 48 h were achieved. Mixed and pure cultures obtained from the quinoline nutristats were routinely tested using the nitrogen bioavailability assay to detect cultures capable of using quinoline as a nitrogen source but not as a carbon source. All of the quinoline-utilizing cultures initially obtained from the nutristats were found to fully degrade quinoline, utilizing it as a carbon as well as a nitrogen source. Subsequently, the flow rates of the nutristats were increased so that hydraulic retention times decreased from 96 to 4 h or less over a period of 4 months. During this time cells were routinely obtained from the nutristat effluent, mutagenized, and returned to the nutristat. Mutagenesis was employed because repeated enrichment culture experiments performed without mutagenesis failed to isolate any cultures that utilized quinoline as a nitrogen source, without also using it as a carbon source. Eventually, we obtained a pure culture that yielded nitrogen bioavailability assay results indicating that quinoline was used as a nitrogen but not a carbon source. A partial (500-bp) sequence of the 16S rRNA gene of this gram-negative, rod-shaped bacteria was determined, identifying it as P. ayucida. This culture was designated P. ayucida IGTN9m. P. monteilii, which shows 99% homology, and P. nitroreducens and P. pseudoalcaligenes, which both show 98.3% homology, are closely related but not identical to P. ayucida IGTN9m.

P. ayucida IGTN9m was determined to have a cell doubling time of 4.25 h when grown in defined salts medium at 30°C with quinoline as the sole nitrogen source. Substrate range and specificity tests were also carried out for P. ayucida IGTN9m, using prolonged incubations of 2 to 3 weeks. These substrate range tests were repeated several times using inocula derived from all of those organonitrogen compounds that yielded growth in a previous nitrogen bioavailability assay. This was done to address the possible need for adaptation to a given compound before growth could be obtained and to accurately determine the widest range of organonitrogen structures that could be utilized by P. ayucida IGTN9m. Agar plates were streaked after growth was observed on any of the test compounds to check culture purity. Repeated substrate range tests confirmed that the results obtained were representative of the abilities of P. ayucida IGTN9m and not a mutant derivative of the culture. The full range of compounds used in these tests is listed in Materials and Methods. Growth was obtained on urea, tryptophan, lysine, guanine, nicotinic acid, quinoline, 3,4-dihydro-2(1H)-quinolinone, 2,4-quinolinediol, 8-hydroxyquinoline, and quinoxaline. Growth with urea, tryptophan, lysine, guanine, and nicotinic acid as nitrogen sources is an ability possessed by many if not most aerobic bacteria and most likely has no relationship to metabolic pathways relevant to the utilization of quinoline. The culture grew in the presence of all test compounds except 1-butylpyrrolidine and 2-methylene-1,3,3-trimethyl indoline when quinoline was simultaneously present as an alternate nitrogen source. These results indicate that with the exception of the two compounds mentioned above, the organonitrogen compounds do not have a toxic effect on the culture, and so failure to grow on the other test compounds provides a true reflection of the substrate range of P. ayucida IGTN9m for the metabolism of organonitrogen compounds. Further tests are needed to determine if 1-butylpyrrolidine and/or 2-methylene-1,3,3-trimethyl indoline are intrinsically toxic or if they are metabolized by IGTN9m into inhibitory compounds.

Spectrophotometric scans (UV/visible) were carried out on the supernatants of P. ayucida IGTN9m cultures that were grown with quinoline as the sole nitrogen source. This was done to determine if we could detect spectrophotometric peaks other than quinoline that might correspond to the accumulation of metabolites. Control supernatants from the cultures grown with ammonium sulfate were included to rule out artifacts arising from normal metabolism of the culture. Quinoline has an absorption maximum peak at 310 nm. This peak can be observed to decrease with time during the growth of P. ayucida IGTN9m at the expense of quinoline; however, we observed no new peak that might correspond to a metabolite of quinoline (data not shown).

TLC was performed on extracts derived from the culture supernatants of P. ayucida IGTN9m grown with quinoline as the sole nitrogen source, as well as with resting cells incubated for various times in the presence of quinoline. Controls consisting of P. ayucida IGTN9m grown using ammonium sulfate rather than quinoline as a nitrogen source were included in all experiments. Additionally, pure chemicals that are possible metabolites of quinoline such as protocatechuate, catechol, pyruvic acid, p-hydroxybenzoic acid, formamide, 8-hydroxyquinoline, and succinic acid dimethyl ester were included on the TLC plates to determine if any of these compounds were formed during the microbial degradation of quinoline. Two spots which have R_f values of 0.73 and 0.88 were identified as possible metabolites of quinoline by P. ayucida IGTN9m, as these compounds were found only in samples derived from the incubation of P. ayucida IGTN9m cells with quinoline. P. ayucida IGTN9m cells incubated with ammonia did not produce these compounds (data not shown). These two compounds could not be accurately identified by TLC analysis alone, and so these spots were scraped from TLC plates, eluted with ethyl acetate, and subjected to GC-MS analysis.

GC-MS analysis was performed not only on compounds obtained from TLC spots but also on extracts of culture supernatants from growing cell as well as resting cells of P. ayucida IGTN9m. The GC-MS analysis of extracts of culture supernatants allowed for the possible detection of metabolites that did not yield detectable spots in TLC. The results from the GC-MS analyses provided probable structures for two compounds as metabolites of quinoline produced by P. ayucida IGTN9m: 2-quinolinone and 8-hydroxycoumarin. MS data comparing these metabolites with authentic 2-quinolinone and 7-hydroxy-2H-1-benzopyran-2-one are shown in Fig. 1 and 2, respectively. 8-Hydroxycoumarin is not commercially available, but 7-hydroxycoumarin (7-hydroxy-2H-1-benzopyran-2-one) is, so 7-hydroxycoumarin was used as a standard in these experiments. The metabolite of quinoline produced by P. ayucida IGTN9m had a somewhat shorter retention time in GC analysis than 7-hydroxycoumarin, but it yielded a mass spectrum apparently identical to that of 7-hydroxycoumarin and to published spectra of 8-hydroxycoumarin (18).

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Comparison of MS data of a metabolite of quinoline produced by P. ayucida IGTN9m with 2-quinolinone.

View larger version (30K): [in this window] [in a new window]   FIG. 2.   Comparison of MS data of a metabolite of quinoline produced by P. ayucida IGTN9m with 7-hydroxy-2H-1-benzopyran-2-one (7-hydroxycoumarin).

To further analyze the metabolites of quinoline produced by P. ayucida IGTN9m, the relative abundance of these two metabolites was quantified with resting cells exposed to quinoline for various times. The results (Table 1) strongly indicate that quinoline is converted first to 2-quinolinone and then to 8-hydroxycoumarin. There are undoubtedly other metabolites, as the direct conversion of 2-quinolinone to 8-hydroxycoumarin is unlikely. However, no other intermediates relevant to this conversion were observed in these experiments. The structures of 2-quinolinone and 8-hydroxycoumarin (Fig. 3) suggest a partial pathway for the biotransformation of quinoline by P. ayucida IGTN9m. The oxygenation of the carbon atom adjacent to the nitrogen atom in quinoline to form 2-quinolinone is consistent with the selective cleavage of C-N bonds in quinoline by P. ayucida IGTN9m. Moreover, the results of the substrate range tests which indicate that P. ayucida IGTN9m can utilize quinoline, 3,4-dihydro-2(1H)-quinolinone, 2,4-quinolinediol, 8-hydroxyquinoline, and quinoxaline are also consistent with the partial pathway depicted in Fig. 3. The identification of 2-quinolinone and 8-hydroxycoumarin as metabolites of quinoline produced by P. ayucida IGTN9m is also consistent with published results of other investigations of the bacterial metabolism of quinoline (9, 19). Nonetheless, the partial pathway for the metabolism of quinoline by P. ayucida IGTN9m pictured in Fig. 3 must be considered tentative, as the structures of the metabolites have not been identified with absolute certainty.

View larger version (8K): [in this window] [in a new window]   FIG. 3.   Suggested partial pathway for the degradation of quinoline by P. ayucida IGTN9m.

Since 8-hydroxycoumarin does not contain nitrogen, these data demonstrate that P. ayucida IGTN9m is capable of selective removal of nitrogen from quinoline. The subsequent decreased abundance of 8-hydroxycoumarin with longer incubation times (Table 1) indicates that this compound is in turn converted to other, as yet unidentified metabolites. However, it is important to keep in mind that this culture does not use quinoline as a carbon source and is unable to completely degrade it efficiently. Also, many quinoline-degrading microorganisms have been reported to produce pink (18, 19), green (14, 18), and brown (19) metabolites, but P. ayucida IGTN9m produces no colored metabolites derived from quinoline.

The metabolism of quinoline by microbial cultures is complicated by the use of different chemical names by various researchers. The initial aerobic metabolites of quinoline are sometimes called 2-quinolinone (14), 2(1H)-quinolinone (15), 2-hydroxyquinoline (7, 18, 19), or 2-oxo-1,2-dihydroquinoline (4, 16). However, these various names refer to the same metabolite, which tautomerizes between different structures as shown in Fig. 3. The oxygen involved in the conversion of quinoline to 2-quinolinone/2-hydroxyquinoline/2-oxo-1,2-dihydroquinoline has been shown to be derived from water (6, 11, 15). Quinoline 2-oxidoreductase has been shown to be responsible for this initial metabolism of quinoline (4, 16). The subsequent conversion of 2-quinolinone/2-hydroxyquinoline/2-oxo-1,2-dihydroquinoline to 2,6-dihydroxyquinoline or 2,8-dihydroxyquinoline has been reported for various microbial cultures (7, 18, 19). The further metabolism of 2,6-dihydroxyquinoline has been reported to result in the degradation of the benzene ring portion of the molecule (7), while the further metabolism of 2,8-dihydroxyquinoline has been reported to result in the formation of 8-hydroxycoumarin with concomitant release of nitrogen and the subsequent degradation of the molecule to 2,3-dihydroxyphenyl-propionic acid (19), cis,cis-muconic acid, and catechol (6). The production of 8-hydroxycoumarin by P. ayucida IGTN9m is similar to the metabolism of quinoline reported for an unidentified Pseudomonas culture (19); however, the inability to use quinoline as a carbon source and the lack of production of colored metabolites make the metabolism of quinoline by P. ayucida IGTN9m unique.

Results with petroleum. A test for the removal of organic nitrogen from petroleum was performed to determine the ability of P. ayucida IGTN9m to remove nitrogen from shale oil. Duplicate washed, concentrated P. ayucida IGTN9m cell suspensions were incubated with shale oil samples for 16 h at 30°C. The control sample consisted of shale oil added to sterile mineral salts medium which was incubated for 16 h at 30°C. After incubation, the petroleum samples were recovered and analyzed. The results (Table 2) indicate that resting cells of P. ayucida IGTN9m are capable of removing about 5% of the total organic nitrogen and about 68% of quinoline from shale oil during an overnight (16-h) incubation.