INTRODUCTION

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Widely used petroleum products, such as gasoline, kerosene, and diesel oil, are common pollutants of the environment which have been shown to be biodegradable to some extent. It has been shown that the overall biodegradation of gasoline by microflorae from soil or groundwater of contaminated sites is efficient, and the level of biodegradation is as high as 90% (13, 23, 30, 32, 33). However, detailed information concerning the fate of components of gasoline is rather scarce; the only exceptions are some individual hydrocarbons. It has been demonstrated that benzene, toluene, ethylbenzene, m- and p-xylenes, and n-alkanes are readily biodegradable (7, 11, 14, 17, 20, 22, 26), whereas -branched and quaternary branched alkanes, o-xylene, and cyclohexane have been found to be less susceptible to biodegradation (3, 4, 8, 15, 16, 20, 22).

Gasoline is a complex hydrocarbon mixture that can contain more than 200 individual compounds, as shown by gas chromatography (GC). Detailed analysis is tedious because only minor amounts of some components are present. In order to facilitate studies of biodegradation of individual hydrocarbons by microfloræ in the environment, we have developed specific methods in which hydrocarbon mixtures are used as substrates (20). The use of these gasoline model mixtures, which are simpler to handle than gasoline, has been validated by the finding that they yield results which are quite similar to the results obtained with gasoline (21). Degradation tests performed with gasoline model mixtures revealed that the degradation capacities of microfloræ obtained from various environments were usually high (total extent of degradation, at least 85%). However, the limitations of the microfloræ were apparent when degradation of compounds such as cyclohexane and trimethylpentanes, which appeared to be the most recalcitrant compounds in gasoline, was examined. For this reason, microfloræ that specifically degrade cyclohexane and 2,2,4-trimethylpentane were selected from samples obtained from the environment. When cyclohexane was the carbon source, no pure strain could be isolated (22), which is consistent with the accepted view that cometabolism and mutualism phenomena are involved in cyclohexane biodegradation (4, 15). The capacity to degrade isooctane (2,2,4-trimethylpentane) was found to be rare in natural samples, and only one microflora and one pure strain with this characteristic were obtained by Solano-Serena et al. (22).

In the present work, we investigated the degradation capacities of the new isolate obtained by Solano-Serena et al., which utilized isooctane, a component of gasoline that is usually considered recalcitrant, as a carbon source. Culture tests were performed under nonlimiting conditions. The carbon sources used were the main gasoline hydrocarbons, which were supplied individually or in mixtures. In order to simulate the possible bacterial strategies for attenuation of pollutants in the environment, the cometabolism capacities of the strain were also characterized by using cyclohexane as a cosubstrate.

	MATERIALS AND METHODS

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Culture media. Vitamin-supplemented mineral salt medium MSM (6) was used in this study. The mineral solution was autoclaved for 20 min at 120°C, and vitamins were added by sterile filtration (pore size, 0.22 µm). The only carbon sources used were individual hydrocarbons and two hydrocarbon mixtures that were representative of gasoline. For analytical purposes, two synthetic mixtures were prepared by using equal quantities of several commercial products. The first mixture was designated light fraction model 9 (LFM9), which contained the following nine hydrocarbons having 4 to 6 carbon atoms: butane, 2-methylpropane (isobutane), pentane, isopentane (2-methylbutane), 2- and 3-methylpentanes, 2,2- and 2,3-dimethylbutanes, and methylcyclopentane. LFM9 could be adequately analyzed by headspace sampling. The second mixture was designated gasoline model mixture 23 (GM23), which contained 23 hydrocarbons having 6 to 9 carbon atoms (20). This mixture could be appropriately analyzed after solvent extraction.

Isolation of 2,2,4-trimethylpentane- and cyclohexanone-degrading bacteria. 2,2,4-Trimethylpentane-degrading strain IFP 2173 was isolated from gasoline-contaminated groundwater (22). Cyclohexanone-degrading strain IFP 2149 was isolated from an activated sludge sample obtained from an urban wastewater treatment plant. Enrichment cultures were grown in 250-ml flasks containing 50 ml of medium MSM supplemented with 500 mg of cyclohexanone per liter and 300 mg (dry weight) of activated sludge per liter. After five subcultures at 30°C with shaking at 250 rpm, single loopfuls of the cell suspension were streaked onto agar plates containing the medium MSM supplemented with cyclohexanone and incubated at 30°C. Isolated colonies were used to inoculate medium MSM containing cyclohexanone in order to confirm that this carbon source was utilized.

Degradation tests. The degradation capacities of strain IFP 2173 were determined in flasks inoculated (10%, vol/vol) with a preculture of the isolate grown on isooctane. The flasks were incubated for 28 days at 30°C with shaking at 250 rpm.

Cometabolism and commensalism experiments. Cultures were grown in 155-ml flasks that were hermetically closed and contained 10 ml of inoculated culture medium. In cometabolism experiments, the flasks were inoculated (10%, vol/vol) with a preculture of strain IFP 2173 that had been grown on isooctane, whereas in commensalism experiments, the flasks were inoculated with both a preculture of strain IFP 2173 that had been grown on isooctane and a preculture of strain IFP 2149 that had been grown on cyclohexanone (5% [vol/vol] each).

Chromatographic analyses. CO₂ contents were determined with a chromatograph equipped with a thermal conductivity detector and a Porapak Q column (80/100 mesh; length, 2 m) by using an external standard method. The carrier gas was helium, and the column temperature was 50°C (20).

Degradation and mineralization yields. Mineralization yields were calculated by determining the molar ratios of the difference between the amount of carbon in the total CO₂ produced in a test flask and the amount of carbon in the total CO₂ produced in a substrate-free flask to the amount of carbon in the substrate consumed.

GC-MS analyses. The metabolites were analyzed by performing electron impact mass spectrometry (MS) with an Autospec Ultimas apparatus (Micromass, Manchester, United Kingdom). The mass spectrometer was operated at 70 eV, and the temperature of the ion source was 250°C. For GC-MS analyses, we used a CP-Sil Pona CB column obtained from Chrompack (0.25 mm by 100 m). The temperature program was the same as the temperature program described above.

16S rDNA PCR and DNA sequencing. Strain IFP 2173 was grown on medium MSM plates containing isooctane as a carbon source. A colony was resuspended in 20 µl of sterile demineralized water. Five microliters of the cell suspension were directly used for 16S ribosomal DNA (rDNA) amplification. The sequence of forward primer fD1 was 5'-AGAGTTTGATCCTGGCTCAG-3', and the sequence of reverse primer rD1 was 5'-AAGGAGGTGATCCAGCC-3', as described by Weisburg et al. (27). The thermal profile was as follows: 30 cycles consisting of 94°C for 30 s, primer annealing at 53°C for 30 s, and extension at 72°C for 1.30 min. All experiments were performed in triplicate. The amplification products were purified after gel electrophoresis with a Gene Clean Kit II (Bio 101, Vista, Calif.), and both strands were sequenced (ESGS, Evry, France). The sequences were compared to sequences in the EMBL/GenBank database by using the BLAST alignment tool (1).

Phylogenetic analysis. 16S rDNA sequences (length, about 1,380 bp) of isolate IFP 2173 and 14 other Mycobacterium strains were aligned by using the CLUSTALV software (12). An unrooted tree was constructed by using the neighbor-joining method (18). A bootstrap analysis of the tree was performed with 1,000 resamplings (10).

Chemicals. Cyclohexane, benzene, toluene, xylenes, ethylbenzene, and trimethylbenzenes were purchased from Prolabo (Fontenay-sous-Bois, France). Other hydrocarbons, ethers, and vitamins were obtained from Sigma-Aldrich Chimie (Saint-Quentin-Fallavier, France).

Nucleotide sequence accession numbers. The nucleotide sequence of strain IFP 2173 has been deposited in the GenBank database under accession no. AF190800. The GenBank accession numbers of the other sequences used in the analyses are as follows: Mycobacterium aichiense, X55598; M. asiaticum, X55604; M. aurum, X55595; M. austroafricanum, X93182; M. fallax, M29562; M. flavescens, X52932; M. fortuitum, X52933; M. gadium, X55594; M. komossense, X55591; M. nonchromogenicum, X52929; M. obuense, X55597; M. vaccae, X55601; M. xenopi, X52928; and Mycobacterium sp. strain PYR-I, X84977.

	RESULTS

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Identification of strain IFP 2173. Using isooctane (2,2,4-trimethylpentane) as the sole carbon and energy source, Solano-Serena et al. isolated strain IFP 2173 from gasoline-contaminated groundwater by preparing successive enrichment cultures (22). This organism was tentatively identified as a Corynebacterium urealyticum strain by using classical methods, and it was deposited in the Collection Nationale de Cultures de Microorganismes (Institut Pasteur, Paris, France) as strain CIP-I-2126. A comparison of its complete PCR-amplified 16S rDNA sequence to previously published 16S rDNA sequences revealed high levels of similarity to the rapidly growing species of the genus Mycobacterium (Fig. 1). The level of 16S rDNA sequence identity between strain IFP 2173 and a reference strain of M. austroafricanum (ATCC 33464) was 100%.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Unrooted evolutionary distance tree based on the 16S rDNA sequence studied and sequences of bacteria belonging to the genus Mycobacterium. Bootstrap values greater than 50% are indicated at the nodes. Bar = 0.01 nucleotide difference per sequence position.

Hydrocarbon utilization. The degradative capacities of Mycobacterium sp. strain IFP 2173 were assessed by using a bacterial test in which growth on individual hydrocarbons was measured by measuring hydrocarbon consumption and production of CO₂ (mineralization). Most of the hydrocarbons tested were compounds detected in commercial gasoline (Table 1). We found that Mycobacterium sp. strain IFP 2173 degraded and mineralized n-alkanes with 5 to 16 carbon atoms. Propane was not degraded, and butane was only partially consumed. An analogous effect of carbon chain length on biodegradation was also observed with 2-methylalkanes; hydrocarbons with 5 to 8 carbon atoms in the main carbon chain were completely degraded, whereas isobutane (2-methylpropane) was only partially degraded (41%). Monomethylalkane isomers (2-methyl and 3-methyl alkanes) were degraded similarly.

Degradation capacities with hydrocarbon mixtures. The degradative capacities of Mycobacterium sp. strain IFP 2173 were also evaluated by using hydrocarbon mixtures in order to allow substrate interactions (competition and cometabolism) in cultures. As described above, two model gasoline mixtures were used.

View larger version (23K): [in this window] [in a new window]   FIG. 2.   Chromatographic patterns of residual hydrocarbons of GM23 after 28 days of incubation. (a) Abiotic flask. (b) Test flask.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Mass spectrum of the metabolic compound produced during incubation with GM23 (retention time, 41.3 min).

Cometabolism capacities. We confirmed that cometabolic degradation of ethylbenzene occurred. We found that during incubation with isooctane as the growth substrate, ethylbenzene was converted into acetophenone, as shown by GC-MS analysis, but not into phenylacetic acid, implying that there was preferential attack on the CH₂ group.

Syntrophic biodegradation of cycloalkanes. A cyclohexanone-degrading strain, designated IFP 2149, was isolated from activated sludge by using cyclohexanone as the sole carbon and energy source. This bacterium was identified by classical methods as an Acinetobacter lwoffii strain. Interactions between strains IFP 2173 and IFP 2149 during degradation of cyclohexane were investigated by using isooctane as an energy source (Table 5). A. lwoffii was not able to degrade isooctane or cyclohexane. Degradation of cyclohexane with Mycobacterium sp. strain IFP 2173 alone was similar to degradation of cyclohexane with strain IFP 2173 in association with A. lwoffii IFP 2149, although cyclohexanone accumulated in the former preparation but not in the latter preparation, in which it appeared to be used by strain IFP 2149. However, the final amount of cyclohexene was not different, confirming that cyclohexene was an end product of a side reaction. The amounts of CO₂ produced in the different cultures are shown in Fig. 4. The amount of CO₂ produced with both strains was significantly larger than the amount of CO₂ produced with Mycobacterium sp. strain IFP 2173 alone. Mineralization of cyclohexanone by A. lwoffii probably accounted for the increase in CO₂ production.

View larger version (30K): [in this window] [in a new window]   FIG. 4.   Production of CO2 during cyclohexane biodegradation. Open bars, hydrocarbon-free flasks; vertically striped bars, flasks containing cyclohexane; cross-hatched bars, flasks containing cyclohexane and isooctane. The average values (usually based on three replicates) are shown above the bars, and the error bars indicate standard deviations.

	DISCUSSION

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Mycobacterium sp. strain IFP 2173 was isolated from a gasoline-polluted groundwater sample because of its ability to degrade isooctane. In several ways, this strain biodegraded an unusual spectrum of hydrocarbon compounds.

First, Mycobacterium sp. strain IFP 2173 could degrade various molecular structures, such as n-alkanes, isoalkanes, aromatic compounds, or oxygenates, which are commonly introduced into gasoline formulations. Second, it could also attack a wide range of compounds with chain lengths ranging from 4 to 16 carbon atoms. The degradation of short alkanes is noteworthy since these compounds are usually considered toxic to bacterial cells because of their solubility in culture media (7). Moreover, it is known that propane and butane are degraded by bacterial strains that specialize in short-chain alkane metabolism (2, 25, 29).

The most spectacular feature of strain IFP 2173 was its ability to degrade branched alkanes. Recently, workers have described quite notable advances in degradation of n-alkanes, and bacteria from specific habitats, such as marine (31) or cold (28) environments, have been characterized, but less effort seems to have been devoted to degradation of isoalkanes. We found that Mycobacterium sp. strain IFP 2173 was able to degrade monomethylated alkanes and almost all dimethylated alkanes. Even -methyl-branched (anteiso) structures, which usually prevent or hinder normal -oxidation (19), were found to be susceptible to biodegradation. In fact, 3-methylacyl coenzyme A intermediates which cannot undergo -oxidation might be degraded through a decarboxymethylation mechanism. In this pathway, which has been elucidated in some specialized microorganisms, CO₂ is first enzymatically fixed on the lateral methyl group, and the acetyl group formed is subsequently removed from the molecule. As a result, the initial methyl group on carbon 3 is eliminated from the molecule (8). The existence of such a pathway is strongly suggested by our results. Not surprisingly, -dimethyl-branched alkanes (3,3-dimethylpentane) could not be degraded through the decarboxymethylation process. The most remarkable property of strain IFP 2173, however, is its ability to degrade some isoalkanes that have a quaternary carbon structure (e.g., 2,2-dimethylpentane and 2,2,4-trimethylpentane). As the high mineralization yields determined for degradation of these isoalkanes indicated that the quaternary structure was attacked, the presence of a relevant unknown mechanism may be postulated.

We found that as a consequence of the bacterial activity, some methyl groups, such as the terminal CH₃ of the main hydrocarbon chains or substituents of branched alkanes or aromatic compounds, could be attacked. Furthermore, methylene groups, such as CH₂ included in aliphatic or alicyclic chains, were also attacked. In contrast, the CH groups of benzene and several quaternary carbon structures were found to be refractory, as pointed out previously by McKenna (16).

Mycobacterium sp. strain IFP 2173 also exhibited a substantial capacity for cometabolism. Cyclohexane was attacked by the initial oxidation system of this microorganism, which produced cycloalkanone. A similar cometabolism capacity has been found in M. vaccae JOB5, which produces cycloalkanones from cycloalkanes after growth on propane (5). With Mycobacterium sp. strain IFP 2173, cyclohexanone was produced when the energetic requirements were met by utilization of an appropriate substrate. We found that many hydrocarbon compounds, particularly isooctane, could satisfy the energetic needs of the strain. In addition to cyclohexanone, cyclohexene was identified as one of the cometabolic products. Hence, it seems likely that a common precursor of the cyclohexanone and cyclohexene that accumulated was cyclohexanol, although the latter compound was not identified. When we compared degradation of the gasoline mixtures by strain IFP 2173 and degradation of individual hydrocarbons, we observed a positive cometabolism effect for several compounds. In the standard test in which gasoline model mixtures were used, cyclohexane, methylcyclopentane, and 2,2- and 2,3-dimethylbutanes appeared to be cometabolized. Finally, up to 86% of GM23 was found to be biodegraded by strain IFP 2173. Therefore, the extent of degradation obtained with a pure strain was similar to the extent of degradation obtained with a complex microbial population obtained from a soil sample (i.e., 89%) (20).

The efficiency of using two selected strains together for cyclohexane degradation by cometabolism illustrates the extended potential of complex natural microfloræ in the environment for hydrocarbon attenuation. The synergistic relationship in the mixed culture containing strains IFP 2173 and IFP 2149 was clearly shown by the fact that strain IFP 2149 could utilize cyclohexanone produced by Mycobacterium sp. strain IFP 2173. Accordingly, the synergistic actions of the two bacterial populations resulted in mineralization of cyclohexane instead of cyclohexanone production.

In summary, Mycobacterium sp. strain IFP 2173, which was isolated from gasoline-polluted groundwater, is at present the only strain that has been described which can degrade isooctane, one of the major octane boosters in gasoline. This strain belongs to the rapidly growing Mycobacterium species group which has been shown to play an important role in the environment, particularly in xenobiotic compound biodegradation (24).