Environ. Sci. Technol. 1996, 30, 136-142
Degradation of Polynuclear Aromatic Hydrocarbons by Sphingomonas paucimobilis †
DINGYI YE, M. AKMAL SIDDIQI, ALEXANDER E. MACCUBBIN,‡ SUBODH KUMAR, AND HARISH C. SIKKA* Laboratory of Environmental Toxicology and Chemistry, Center for Environmental Research and Education, State University of New York College at Buffalo, 1300 Elmwood Avenue, Buffalo, New York 14222
The ability of Sphingomonas paucimobilis, strain EPA 505 (a soil bacterium capable of utilizing fluoranthene as the sole source of carbon and energy for growth) to metabolize a variety of high molecular weight polynuclear aromatic hydrocarbons (PAHs) was investigated. After 16 h of incubation with 10 ppm of a PAH, a resting cell suspension (1 mg wet cells/ mL) of S. paucimobilis grown on fluoranthene degraded 80.0, 72.9, 31.5, 33.3, 12.5, and 7.8% of pyrene, benz[a]anthracene (B[a]A), chrysene, benzo[a]pyrene (B[a]P), benzo[b]fluoranthene (B[b]F), and dibenz[a,h]anthracene (DB[a,h]A), respectively. No degradation of dibenzo[a,l]pyrene was detected under these conditions. 1-Nitropyrene was degraded at a lower rate than pyrene. The extent of degradation of the PAHs increased with an increase in cell density. Studies with [7-14C]B[a]P and [5,6,11,12-14C]chrysene showed that, after 48 h of incubation, the cells degraded nearly 28 and 42% of [14C]B[a]P and [14C]chrysene to 14CO2, respectively, suggesting that the bacterium is able to metabolize B[a]P and chrysene via ring cleavage. No evolution of 14CO2 was detected from cultures incubated with [4,5,9,10-14C]pyrene or [1,2,3,4,4a,4b-U-14C]dibenz[a,l]pyrene. Analysis of the ethyl acetate extracts of the culture medium by reverse-phase HPLC showed that B[a]P, B[b]F, and B[a]A were each degraded to the major, high polar metabolite(s). The degradation of B[a]P with S. paucimobilis significantly reduced the mutagenic activity associated with the hydrocarbon. The addition of solubilizing agents such as Tween 80 or cyclodextrin to the incubation medium did not enhance the biodegradation of B[a]P by EPA 505. The addition of 5 ppm of B[a]A, chrysene, fluoranthene, or DB[a,h]A to the incubation medium containing 5 ppm of B[a]P had no effect on the degradation of B[a]P by EPA 505. However, the biodegradation of B[a]P was reduced by nearly 30% in the presence of 5 ppm of B[b]F. The results demonstrate that S. paucimobilis
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EPA 505 has the ability to degrade several four- and fivering PAHs ranging in molecular size, shape, and chemical structure.
Introduction Polynuclear aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants found in air, in soil, and in freshwater and marine environments (1). These chemicals are found in particularly high levels at coal gasification sites (2) and creosote-contaminated sites (3). Environmental contamination by PAHs presents a serious risk to both human health and the environment because a number of these chemicals are potent carcinogens (1). It is well recognized that microorganisms have the ability to degrade a wide variety of organic compounds (4). In recent years, the use of biodegradation technologies for the treatment of hazardous wastes has received considerable attention. Thus, the discovery of microorganisms having the ability to degrade PAHs to less toxic products will greatly help in the development of processes for remediation of PAH-contaminated sites. Information on the metabolism of PAHs by microorganisms is also useful for assessing the fate of these chemicals in the environment. During the past several years, numerous reports on the metabolism of PAHs by microorganisms have been published (5-8). Most of these studies using pure cultures or mixed cultures of microorganisms pertain to lower molecular weight PAHs (two or three aromatic rings) that are relatively less persistent and are not genotoxic. In contrast, only a limited amount of information is available on the bacterial degradation of higher molecular weight PAHs (having four or more aromatic rings) (9-16). It is particularly the higher molecular weight PAHs that are of great environmental concern because of their persistence and mutagenic and carcinogenic properties. Mueller et al. (17) have recently isolated Sphingomonas paucimobilis strain EPA 505 from soil highly contaminated with coal tar creosote. This bacterium can use fluoranthene as a sole source of carbon and energy and is able to degrade a variety of PAHs. However, most of the PAHs examined by these investigators contained less than four rings. The objective of our studies was to evaluate the ability of S. paucimobilis EPA 505 to degrade high molecular weight PAHs, including those containing four, five, or six aromatic rings. We selected chrysene, benz[a]anthracene (B[a]A), pyrene, 1-nitropyrene (1-NO2-Pyr), benzo[a]pyrene (B[a]P), dibenz[a,h]anthracene (DB[a,h]A), benzo[b]fluoranthene (B[b]F), and dibenzo[a,l]pyrene (DB[a,l]P as model compounds for our studies. These compounds were selected because they vary in their molecular size, shape, and physicochemical properties, which can influence the overall metabolism of a PAH (18).
Materials and Methods Chemicals. [G-3H]benz[a]anthracene (188 mCi/mmol), [G-3H]benzo[b]fluoranthene (570 mCi/mmol), [G-3H]chry* Corresponding author telephone: (716) 878-5422; fax: (716) 8785400. † Present address: Environmental Research Laboratory, U.S. Environmental Protection Agency, Athens, GA 30605. ‡ Roswell Park Cancer Institute, Buffalo, NY 14263.
0013-936X/96/0930-0136$12.00/0
1995 American Chemical Society
sene (1275.35 mCi/mmol), [G-3H]dibenz[a,h]anthracene (933.9 mCi/mmol), [7-14C]benzo[a]pyrene (57.16 mCi/ mmol), [5,6,11,12-14C]chrysene (46.8 mCi/mmol), [1,2,3,4,4a,4b-U-14C]dibenzo[a,l]pyrene (40.8 mCi/mmol), 1-nitro[4,5,9,10-14C]pyrene (58.8 mCi/mmol, and [4,5,9,10-14C]pyrene (32.3 mCi/mmol) were purchased from NCI Radiochemical Carcinogen Reference Standard Repository at Chemsyn Science Laboratories, Lenexa, KA. [G-3H]BaP was obtained from Amersham Corporation, Arlington Heights, Il. All labeled PAHs were purified to >98% purity by HPLC. Unlabeled PAHs were obtained from the following sources: B[a]A from Fluka AG, Buchs, Switzerland; B[a]P from Sigma Chemicals, St. Louis, MO, and was recrystallized from glacial acetic acid; BbF, chrysene, DB[a,l]P, 1-NO2Pyr, and DB[a,h]A from NCI Chemical Carcinogen Reference Standard Repository at Midwest Research Institute, Kansas City, MO. The purity of all the PAHs was >98%. Tween 80 (polyoxyethylene sorbitan monooleate) was purchased from Sigma Chemicals, St Louis, MO. Cyclodextrin (hydroxypropyl-β-cyclodextrin) was obtained from Aldrich Chemical Co., Milwaukee, WI. Degradation of PAHs by Resting Cells. Sphingomonas paucimobilis strain EPA 505 originally isolated from soil from a creosote waste site was obtained from the Environmental Research Laboratory, U.S. Environmental Protection Agency, Gulf Breeze, FL. The organism was grown in a mineral salt medium containing 100 mg/L fluoranthene as the sole source of carbon and energy and 200 mg/L Tween 80 to solubilize fluoranthene (MSF medium) at 30 °C in the dark on a rotary shaker operating at 160 rpm (17). After 72 h, the culture (used as the source of inoculum) was serially diluted and inoculated on nutrient agar (Difco Laboratories, Detroit, MI) plates containing 0.4 g/L glucose, and the plates were incubated at 30 °C. The cells in the growth phase were harvested and washed three times with 30 mM phosphate buffer (pH 7.0) and then resuspended in the buffer to a final concentration of 1 mg (wet weight)/mL (108 cfu/mL). Biodegradation experiments were performed with 2 mL of the cell suspension in 20-mL autoclaved glass scintillation vials. The cell suspension was incubated at 30 °C in the dark with 10 ppm of a PAH (supplemented with 0.05 µCi of the labeled PAH) dissolved in acetone. Triplicate vials were periodically removed at random from a series of vials, and the entire contents of the vial was analyzed for the parent hydrocarbon and total metabolites using a procedure based on the method of Van Cantfort et al. (19). The entire contents of a vial was mixed with 1 vol of a solution of 0.15 M KOH in 85% DMSO. The alkaline mixture was then extracted twice with 2 vol of hexane to remove selectively the parent hydrocarbon. Aliquots of both the organic layer containing the parent compound and the aqueous layer containing PAH metabolites were assayed for radioactivity. Nonbiological degradation of the PAH was assessed using the same incubation medium, but containing autoclaved cells. The extraction and analysis of PAHs were done under yellow light to minimize photodegradation of the hydrocarbons. Degradation of B[a]P by Growing Cells. The organism was grown in a mineral salts medium supplemented with 100 mg/L fluoranthene as described above in 125-mL Erlenmeyer flasks. After 72 h of incubation, 3H-B[a]P dissolved in acetone was added to the flasks at a concentration of 10 ppm, and the cell suspension was incubated at 30 °C in the dark. The cell density, as determined by
viable plate counts before adding the hydrocarbon, ranged from 105 to 106 cfu/mL. At appropriate intervals, aliquots of the culture medium were removed from triplicate flasks and assayed for the parent hydrocarbon and total metabolites as described above. Mineralization of 14C-Labeled PAHs. These studies were conducted using the resting cell suspension. The amount of 14CO2 resulting from the degradation of 14Clabeled PAHs was determined using a biometer flask (20). Twenty milliliters of resting cell suspension (see above) containing 5 mg of wet cells/mL were incubated with 10 ppm of a PAH supplemented with 1 µCi of 14C-labeled PAH, and the 14CO2 evolved from the incubation mixture was trapped in 2.5 mL of 0.1 N KOH in the side arm. The CO2 trapping solution was removed at appropriate intervals and assayed for 14C by liquid scintillation counting. The radioactivity collected in the KOH trap was verified as 14CO2 by acidifying the CO2 trapping solution with HCl. An autoclaved cell suspension incubated with the PAHs served as a control. Analysis of PAH Metabolites. These experiments were carried out by incubating a resting cell suspension at 30 °C on a rotary shaker with 3H-labeled PAHs. The entire contents of the flask was extracted twice with equal volumes of ethyl acetate. The aqueous phase was then adjusted to pH 2.0 and extracted with 2 equal vol of ethyl acetate. The extracts were pooled, dried over anhydrous sodium sulfate, concentrated under vacuum at 27 °C, and evaporated to dryness under nitrogen. The residue was dissolved in a small volume of methanol and analyzed by HPLC using a DuPont Zorbax ODS column (4.6 × 250 mm), which was eluted with various linear gradients of methanol in water according to the published procedures for B[a]P (21), B[a]A (22), and B[b]F (23). One-minute fractions were collected using a fraction collector and assayed for radioactivity by liquid scintillation counting. Mutagenicity of B[a]P Biodegradation Products. A resting cell suspension (100 mL) of S. paucimobilis EPA 505 (1 mg of wet cells/mL) was incubated with 10 ppm of B[a]P in an Erlenmeyer flask for 24 h as described above. Autoclaved cell suspension incubated with 10 ppm of B[a]P served as a control. The entire contents of the flask was extracted with ethyl acetate as described above. The combined organic extract was dried over anhydrous sodium sulfate, concentrated under vacuum at 30 °C, and evaporated to dryness under nitrogen. The residue was dissolved in 4 mL of DMSO. The DMSO extracts from both control and experimental incubations were diluted with DMSO to five identical dilutions. Each dilution of the DMSO stock solution (100 µL) was evaluated for its ability to induce mutations in the histidine-dependent strain of Salmonella typhimurium TA98 both with and without a metabolic activation system as described by Maron and Ames (24). The controls contained 100 µL of DMSO. Each dilution of the extract was assayed in triplicate.
Results Degradation of PAHs by Growing Cells of S. paucimobilis. Figure 1 shows the degradation of B[a]P by the growing cells of S. paucimobilis. The degradation of B[a]P started apparently without any lag. After 7 days, the hydrocarbon was degraded to a limited extent by the cells growing on 100 mg of fluoranthene/L. The addition of yeast extract (100 mg/L) to the culture medium significantly enhanced the degradation of B[a]P by the bacterium.
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FIGURE 1. Degradation of B[a]P by S. paucimobilis growing in a mineral salts medium containing 100 mg/L of fluoranthene (MSF). The error bars represent the standard deviation of triplicates. If error bar is not evident, it is obscured by the symbol. The data are corrected for degradation observed with autoclaved cells. The extent of B[a]P degradation in MSF medium containing autoclaved cells was 3.5 ( 2.8, 1.2 ( 0.3, and 1.0 ( 0.3% at 48, 96, and 168 h of incubation, respectively.
FIGURE 2. Degradation of PAHs by a resting cell suspension of S. paucimobilis. The error bars represent the standard deviation of triplicates. The data are corrected for PAH degradation observed with autoclaved cells. The percentage of PAH degradation after 16 h of incubation with autoclaved cells was 1.3 ( 0.4 (pyrene), 2.7 ( 0.3 (1-NO2-pyrene), 1.8 ( 0.1 (B[a]A), 9.6 ( 1.0 (B[a]P), 0.9 ( 0.5 (chrysene), 5.0 ( 0.6 (B[b]F), 5.4 ( 1.1 (DB[a,h]A), and 2.4 ( 1.0 (BD[a,l]P).
Degradation of PAHs by Resting Cells of S. paucimobilis. Washed resting cells of S. paucimoblis that had been grown with fluoranthene as the sole carbon source and then plated on nutrient agar were able to degrade several higher molecular weight PAHs. Figure 2 presents the time course of degradation of pyrene, B[a]A, B[a]P, chrysene, B[b]F, DB[a,h]A, and DB[a,l]P by a resting cell suspension (1 mg/mL) of S. paucimobilis incubated with 10 ppm of a PAH. The bacterium was able to degrade the four- and five-ring PAHs apparently without any lag. However, the rate and extent of degradation varied depending upon the chemical. After 16 h of incubation, S. paucimobilis had degraded pyrene, B[a]A, chrysene, B[a]P, B[b]F, and DB[a,h]A by 80.0, 72.9, 33.3, 31.5, 12.5, and 7.8%, respectively. No degradation of DB[a,l]P, a six-ring PAH, was detected under these conditions. In order to verify the PAH degradation results that we obtained using the procedure of Van Cantfort et al. (19), we also determined the total metabolism of selected PAHs directly by HPLC. We noted that the data obtained using the two procedures were comparable.
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FIGURE 3. Effect of cell density on the degradation of B[a]A by S. paucimobilis. The error bars represent the standard deviation of triplicates.
Influence of Cell Density on Biodegradation of PAHs. In order to determine if cell biomass has an effect on the biodegradation of PAHs, we examined the degradation of B[a]A, B[a]P, and DB[a,l]P by the resting cells of S. paucimobilis at varying cell densities. The data presented in Figure 3 show that cell density had a significant influence on the biodegradation of B[a]A. Both the rate and extent of degradation of B[a]A increased with an increase in cell density from 0.4 to 20 mg of wet cells/mL. An increase in cell density also enhanced the extent of degradation of B[a]P and DB[a,l]P by the bacterium. For example, no degradation of DB[a,l]P was detected when S. paucimobilis was incubated with the chemical for 16 h at a cell density of 1 mg of wet cells/mL. When the cell density was increased to 5 mg of wet cells/mL, 8.9% of DB[a,l]P was degraded over a period of 16 h. Similarly, 66.9% of BaP was degraded after 16 h of incubation at a cell density of 5 mg of wet cells/mL compared to 33.3% at a cell density of 1 mg of cells/mL. HPLC Analysis of the Culture Medium. After 96 h of incubation of a resting cell suspension (5 mg/mL of medium) with 10 ppm of B[a]P, B[a]A, or B[b]F, the cell suspension was analyzed for the parent hydrocarbon and its possible metabolites. Analysis of the ethyl acetate extract of the cell suspension by reverse-phase HPLC showed that B[a]P, B[a]A, and B[b]F were each degraded to major, highly polar metabolite(s) by S. paucimobilis. The elution time of the metabolites ranged from 4 to 7 min. These peaks represented 85.2, 66.4, and 17.9% of the ethyl acetateextractable radioactivity from the culture media incubated with B[a]A, B[a]P, and B[b]F, respectively. Two additional peaks with retention times of 19.0 and 32.0 min were detected in the extracts of the culture medium incubated with B[b]F. These two peaks represented 23.9 and 9.9% of the extractable radioactivity. BaP, B[a]A, and B[b]F eluted at 48, 29, and 38 min, respectively. A representative HPLC elution profile of 3H-B[a]A metabolites formed by S. paucimobilis is shown in Figure 4. Mineralization of PAHs. In order to determine whether S. paucimobilis EPA 505 is able to cause ring fission in a PAH having more than four aromatic rings and to assess the regioselectivity involved in this metabolic reaction, we monitored the evolution of 14CO2 from a resting cell suspension (5 mg of cells/mL) incubated with 10 ppm of [7-14C]B[a]P, [5,6,11,12-14C]chrysene, [4,5,9,10-14C]pyrene, 1-nitro[4,5,9,10-14C]pyrene, and [1,2,3,4,4a,4b-U-14C]DB[a,l]P. These 14C-labeled PAHs were selected for mineralization studies because they are commercially available.
TABLE 1
Mutagenicity of Benzo[a]pyrene Degradation Products Produced by S. paucimobilis EPA 505 mutants/plate culture extract in DMSO (µL) 100 60 10 6 controla a
FIGURE 4. HPLC elution profile by 3H-B[a] metabolites formed by S. paucimobilis. (A) B[a]A incubated with live cells; (B) B[a]A incubated wih autoclaved cells.
FIGURE 5. 14CO2 evolution from S. paucimobilis culture incubated with 14C-labeled PAHs. The error bars represent the standard deviation of triplicates. No 14CO2 evolution was detected in autoclaved controls.
Degradation of [14C]PAH to 14CO2 began within a few hours after addition of the chemicals (Figure 5). After 48 h of incubation with [14C]B[a]P and [14C]chrysene, nearly 28 and 42% of the initial 14C radioactivity was evolved as 14CO2, respectively. No evolution of 14CO2 was detected from cultures incubated with [14C]pyrene, [14C]1-nitropyrene, or [14C]DB[a,l]P. The PAHs used in this study were labeled with 14C at specific positions in the molecule, and the position of 14Clabeling in the ring varied with the PAH. Since the different sites in a PAH molecule may vary in their susceptibility to microbial attack, we cannot compare the extent of mineralization of the PAHs examined, since they were labeled with 14C at dissimilar positions. Mutagenicity of B[a]P Biodegradation Products. The microbial metabolism of PAHs may result in the formation of nontoxic products as well as mutagenic/carcinogenic products (7). In order to determine if the biodegradation of PAHs by S. paucimobilis EPA 505 reduces the mutagenicity associated with the parent PAH, we assessed the mutagenic activity of the culture medium following incu-
autoclaved cells + B[a]P +S9 -S9 240.0 ( 23.8 191.7 ( 14.1 49.0 ( 4.6 43.0 ( 1.0 30.0 ( 8.4
36.7 ( 4.9 30.3 ( 11.0 30.3 ( 2.5 23.7 ( 4.6 35.0 ( 5.6
live cells + B[a]P +S9 -S9 50.3 ( 0.6 36.3 ( 3.7 32.7 ( 1.2 41.3 ( 6.7 30.3 ( 8.4
37.3 ( 7.5 30.7 ( 9.0 35.1 ( 3.6 20.7 ( 4.7 35.0 ( 5.6
Contained 100 µL of DMSO only.
bation of B[a]P with the bacterium. The ethyl acetate extract of the culture medium was tested for mutagenic activity in S. typhimurium strain TA98 with and without metabolite activation. The degradation of B[a]P by S. paucimobilis significantly reduced the mutagenic activity associated with B[a]P. After 24 h of incubation of 10 ppm of B[a]P with the bacterium, the mutagenic activity of the culture medium containing 10 ppm of B[a]P and live cells was approximately 20% of the mutagenic activity of the culture medium containing 10 ppm of B[a]P and autoclaved cells (Table 1). Biodegradation of B[a]P in the Presence of Solubilizing Agents. The biodegradation of hydrophobic compounds like PAHs is often limited by low water solubility and dissolution rates of these chemicals (25). The addition of surfactants increases the concentration of hydrophobic chemicals in the aqueous phase by solubilization or emulsification. Since the water solubility of B[a]P is only 2.5-5 ppb (18), it is likely that the low water solubility of B[a]P may limit its bioavailability and consequently its biodegradation. To determine whether the addition of a solubilizing agent to the incubation medium enhances the biodegradation of a PAH by increasing its concentration in the water phase, we examined the degradation of B[a]P by S. paucimobilis EPA 505 in the presence of Tween 80 or cyclodextrin. Both Tween 80 and cyclodextrin have been used previously to solubilize hydrophobic compounds in similar studies (17, 26, 27). The resting cells of this bacterium (1 mg of wet cells/mL) were incubated in a medium containing 3H-B[a]P (10 ppm) and either Tween 80 (0.2 g/L) or cyclodextrin (10 g/L). We noted that after 16 h of incubation, the extent of degradation of B[a]P in the presence and absence of Tween 80 or cyclodextrin was essentially similar. Effect of Co-occurring PAHs on the Biodegradation of B[a]P. The sites contaminated with PAHs contain a complex mixture of these hydrocarbons having different chemical structures. Our studies have shown that S. paucimoblis has a broad substrate specificity for PAHs. When S. paucimobilis is exposed simultaneously to more than one PAH, it is likely that interactions between the chemicals may occur, possibly as a result of competition of the substrates for the active site of the enzyme(s) involved in the metabolism of PAHs. In order to determine if the presence of a PAH can affect the biodegradation of another PAH by S. paucimobilis, we investigated the microbial degradation of B[a]P in the presence of another high molecular weight PAH. A resting cell suspension of S. paucimobilis (1 mg of cells/mL) was incubated with 5 ppm of 3H-B[a]P alone and in the presence of 5 ppm of chrysene, B[a]A, fluoranthene, B[b]F, or DB[a,h]A. The data presented
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TABLE 2 3
Degradation of H-B[a]P by S. paucimobilis EPA 505 in the Presence of Other PAHs
b
PAHa
% B[a]P degradationb
B[a]P B[a]P + B[a]A B[a]P + B[b]F B[a]P + chrysene B[a]P + DB[a,h]A B[a]P + fluoranthene
59.6 ( 6.9 53.4 ( 6.8 37.8 ( 4.9 59.3 ( 2.8 51.7 ( 3.6 53.4 ( 3.3
a Each PAH was added to the medium at a concentration of 5 ppm. Mean ( SD of three replications.
in Table 2 show that none of the PAHs tested, except B[b]F, had an effect on the biodegradation of B[a]P. The addition of 5 ppm of B[b]F to the incubation medium reduced the degradation of B[a]P by nearly 30%. Biodegradation of 1-Nitropyrene. Nitro-PAHs, like their unsubstituted analogs, are products of numerous incomplete combustion processes and have been found to be widely distributed in the environment (28, 29). As a group, nitro-PAHs generally are more mutagenic and carcinogenic than their parent hydrocarbons (30, 31). During the past several years, a number of studies on the metabolism of unsubstituted PAHs by bacteria have been published (7). However, information on the metabolism of nitro-PAHs by bacteria is extremely limited (11). For the microbial oxygenation of aromatic hydrocarbons, an electrophilic type of reaction has been proposed (32). This implies that electron-withdrawing substituents such as the nitro group, which tend to deactivate the aromatic ring in certain positions for the attack by oxygenases, would make the ring less susceptible to microbial attack. We have compared the metabolism of pyrene and 1-nitropyrene (1-NO2-Pyr) by S. paucimobilis to assess the effect of the nitro substituent on the degradation of the parent hydrocarbon. As expected, our studies showed that 1-nitropyrene was degraded at a lower rate than pyrene. After 6 h of incubation, the bacterium had degraded pyrene and 1-nitropyrene by 80 and 48.6%, respectively (Figure 2). However, despite the difference in the rates at which the two chemicals were degraded, 1-nitropyrene was degraded to the same extent as pyrene after 30 h of incubation (data not shown).
Discussion Our studies have demonstrated that S. paucimobilis strain EPA 505 can degrade a number of tetracyclic and pentacyclic PAHs, including chrysene, B[a]A, pyrene, B[a]P, B[b]F, and DB[a,h]A. Since earlier studies have shown that this bacterium can not use B[a]P and chrysene as a sole source of carbon (17), our results suggest that EPA 505 degrades these PAHs cometabolically. Our data in conjunction with those reported by Mueller et al. (17) show that fluoranthene is capable of inducing the enzyme(s) responsible for the microbial degradation of a variety of PAHs. Previous studies with a Mycobacterium sp. have shown that pyrene also induces enzyme(s) that can metabolize PAHs having two to five aromatic rings (11). However, the enzyme(s) induced in S. paucimobilis EPA 505 by fluoranthene differ(s) from the one(s) induced in the Mycobacterium sp. in response to pyrene as evidenced by the differences in the relative degradation of the substrate tested. For example, S. paucimobilis is able to mineralize (degrade to CO2) [7-14C]B-
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[a]P, but mineralization of this chemical by the Mycobacterium sp. was not detected (11). On the other hand, [4,5,9 10-14C]pyrene and 1-nitro-[4,5,9,10-14C]pyrene were mineralized by the Mycobacterium sp. (11) but not by S. paucimobilis (this study). The data show that the rate and extent of degradation of individual PAHs by S. paucimobilis EPA 505 varied significantly. The extent of degradation of the PAHs after 16 h of incubation followed the order: pyrene > B[a]A > B[a]P ) chrysene > B[b]F ) DB[a,h]A > DB[a,l]P (p < 0.05 by ANOVA, followed by Duncans’ multiple range test). The molecular structure and size of a PAH are among the factors that can influence the biotransformation of a given chemical (18). Our data on the microbial degradation of the PAHs show that the extent of degradation of the PAHs generally decreased with an increase in the number of fused benzene rings (molecular size) as noted with (i) pyrene, B[a]P, and DB[a,l]P; (ii) BaA and DB[a,h]A; and (iii) fluoranthene and B[b]F. Previous studies have also reported an inverse relationship between the biodegradation of PAHs and the number of fused aromatic rings (33). However, our data on the microbial degradation of B[a]P and chrysene do not indicate such a relationship, since B[a]P (five rings) and chrysene (four rings) are degraded to a similar extent, suggesting that other molecular factors may also influence the microbial degradation of PAHs. In addition to the size of the PAH molecule, the shape of the molecule also appears to have an influence on the microbial degradation of PAHs. Our data show that the PAHs with the same number of fused rings differ in the extent to which they are degraded by S. paucimobilis. For example, the four-ringed PAHs were degraded in the following order: pyrene > B[a]A > chrysene. A comparison of the biodegradation of the two five-ring PAHs showed that B[a]P is degraded to a greater extent than DB[a,h]A. These data appear to suggest that among the PAHs with the same number of fused rings, those which are more condensed or clustered (e.g., pyrene and BaP) are more susceptible to biodegradation than those which are less clustered (e.g., B[a]A, chrysene, and DB[a,h]A). There appeared to be some correlation between the aqueous solubility of the PAHs examined and the extent of their degradation by S. paucimobilis. Pyrene and B[a]A are more water-soluble (18) and are metabolized to a greater extent than BaP and chrysene. Both DB[a,h]A and DB[a,l]P have extremely low water solubility and are biodegraded only to a minor extent. However, a correlation between the water solubility of PAHs and the extent of their biodegradation has not been noted with certain PAHs. For example, B[a]P has a higher water solubility than chrysene, but the two chemicals were degraded to a similar extent by S. paucimobilis. Boldrin et al. (14) noted that fluoranthene was degraded to a lesser extent than pyrene, although fluoranthene has a higher solubility than pyrene. It is generally believed that the biodegradation of PAHs is limited by their aqueous solubility and the rate of their dissolution (25). Mueller et al. (17) observed an increase in the rate of degradation of fluoranthene, phenanthrene, and 2,3dimethylnaphthalene by S. paucimobilis when Tween 80 (an emulsifying agent) was added to the incubation medium to enhance the aqueous solubility of the PAHs. However, in our studies, the addition of Tween 80 (0.2 g/L) or cyclodextran (10 g/L), an emulsifier used to solubilize hydrophobic compounds, failed to enhance the degradation of B[a]P by strain EPA 505. Our data indicate that the
biodegradation of high molecular weight PAHs is not limited by their aqueous solubility. The available information on the biodegradation of a variety of PAHs seems to suggest that, in addition to water solubility, steric and electronic factors also contribute to the overall rate at which various PAHs are degraded by microorganisms. On the basis of their studies regarding the rates of dissolution and biodegradation of water-insoluble organic compounds, Thomas et al. (34) also suggested that spontaneous dissolution rates are only one of the factors that deermine the rate of biodegradation. A possible explanation for the differences bewteen the results of our studies and those of Mueller et al. (17) regarding the effect of Tween 80 on PAH biodegradation by EPA 505 is the type of medium used for culturing cells in the two studies. In our studies, the cells were initially grown on fluoranthene and then plated on nutrient agar containing 0.4 g/L glucose to generate biomass whereas Meuller et al. (17) used cells grown strictly on fluoranthene. Fluoranthene, but not B[a]P, is a growth substrate for EPA 505. It appears that increasing the aqueous soluability of a PAH that is a growth substrate results in an increased biodegradation of the hydrocarbon. However, this may not be true in the case of a PAH that is not a growth substrate. A significant evolution of 14CO2 from the cultures incubated with [7-14C]B[a]P or [5,6,11,12-14C]chrysene indicates that S. paucimobilis degrades B[a]P via cleavage of the 7,8,9,10-benzo ring and chrysene via cleavage of the K-region. Since [14C]B[a]P was labeled only at the C-7 position and [14C]chrysene was labeled at the C-5, C-6, C-11, and C-12 positions, the possibility that other aromatic rings in the two molecules are also cleaved by this bacterium cannot be excluded. Although pyrene was extensively degraded by S. paucimobilis, no evolution of 14CO2 from the cultures incubated with [4,5,9,10-14C]pyrene was detected, suggesting that the K-region of this hydrocarbon in contrast to the K-region of chrysene is not susceptible to metabolic attack by EPA 505. We did not characterize the metabolites resulting from the degradation of the PAHs, but other investigations have reported the formation of dihydrodiols as initial ring oxidation products and of hydroxypolyaromatic acids as ring fission products resulting from the bacterial degradation of PAHs such as pyrene, B[a]P, and B[a]A (9, 10, 12). The data from the experiments that examined the degradation of B[a]P in the presence of another PAH indicate that the presence of a co-occurring PAH, except B[b]F, did not affect the biodegradation of B[a]P. These results suggest that the PAHs investigated in this study do not compete with B[a]P for the active site(s) of the enzyme involved in the degradation of B[a]P by S. paucimobilis. An alternate explanation for this observation is that the enzyme responsible for the degradation of B[a]P is different from the one(s) involved in the metabolism of other PAHs used in this study. Depending on the metabolic pathway, micobial degradation of a PAH may result in the formation of products that are more toxic or less toxic than the parent compound. Although we did not identify the products resulting from the microbial degradation of B[a]P, our data show that S. paucimobilis degrades the hydrocarbon to non-genotoxic products. The mutagenicity response in the culture medium incubated with B[a]P was most likely due to the presence of undegraded B[a]P, because a reduction in
mutagenicity corresponded to the chemical’s disappearance from this medium. In conclusion, our studies have demonstrated that S. paucimobilis strain EPA 505 has the ability to degrade several four- and five-ring PAHs varying in molecular size, shape, and chemical structure. Therefore, this organism may be useful for enhancing the microbial degradation of PAHs at sites contaminated with these chemicals as pointed out by Mueller et al. (17). Further studies are needed to examine whether S. paucimobilis strain EPA 505 can degrade PAHs in soil from a coal gasification site.
Acknowledgments This research was supported by Project C002418 from the New York Center for Hazardous Waste Management. We thank Dr. P. Hap Pritchard, U.S. Environmental Protection Agency, Gulf Breeze, FL, for providing a culture of S. paucimobilis and Ron Steppian for his technical assistance.
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Received for review March 22, 1995. Revised manuscript received July 24, 1995. Accepted August 7, 1995.X ES9501878 X
Abstract published in Advance ACS Abstracts, November 1, 1995.
