Benz[a]anthracene Biotransformation and Production of Ring Fission Products by Sphingobium sp. Strain KK22

Abstract

A soil bacterium, designated strain KK22, was isolated from a phenanthrene enrichment culture of a bacterial consortium that grew on diesel fuel, and it was found to biotransform the persistent environmental pollutant and high-molecular-weight polycyclic aromatic hydrocarbon (PAH) benz[a]anthracene. Nearly complete sequencing of the 16S rRNA gene of strain KK22 and phylogenetic analysis revealed that this organism is a new member of the genus Sphingobium. An 8-day time course study that consisted of whole-culture extractions followed by high-performance liquid chromatography (HPLC) analyses with fluorescence detection showed that 80 to 90% biodegradation of 2.5 mg liter⁻¹ benz[a]anthracene had occurred. Biodegradation assays where benz[a]anthracene was supplied in crystalline form (100 mg liter⁻¹) confirmed biodegradation and showed that strain KK22 cells precultured on glucose were equally capable of benz[a]anthracene biotransformation when precultured on glucose plus phenanthrene. Analyses of organic extracts from benz[a]anthracene biodegradation by liquid chromatography negative electrospray ionization tandem mass spectrometry [LC/ESI(−)-MS/MS] revealed 10 products, including two o-hydroxypolyaromatic acids and two hydroxy-naphthoic acids. 1-Hydroxy-2- and 2-hydroxy-3-naphthoic acids were unambiguously identified, and this indicated that oxidation of the benz[a]anthracene molecule occurred via both the linear kata and angular kata ends of the molecule. Other two- and single-aromatic-ring metabolites were also documented, including 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid and salicylic acid, and the proposed pathways for benz[a]anthracene biotransformation by a bacterium were extended.

INTRODUCTION

High-molecular-weight polycyclic aromatic hydrocarbons (HMW PAHs) are commonly occurring environmental pollutants that are generally considered to be more resistant to biodegradation than their lower-molecular-weight aromatic counterparts (1–4). Many are suspected carcinogens and display genotoxic and immunotoxic properties in addition to causing oxidative cell damage (5, 6). The HMW PAH benz[a]anthracene is considered to be environmentally recalcitrant, is classified as a group 2A carcinogen by the International Agency for Research on Cancer, and is included in the U.S. Environmental Protection Agency's Priority Pollutant List. As such, there is much interest in understanding the environmental fate of benz[a]anthracene and the mechanisms by which it may be transformed.

Few studies have documented the bacterial biotransformation of benz[a]anthracene even though many studies have documented the biotransformation of the structurally similar three-ring angular kata-annelated PAH phenanthrene (7–16) and, although less so, also the structurally similar three-ring linear kata-annelated PAH anthracene (7, 13, 17–19). The benz[a]anthracene molecule itself is comprised of four aromatic rings that are bonded via both linear and angular kata annelation, and it may be thought of as a benzannelated derivative of either phenanthrene or anthracene. Initial enzymatic oxidation of the aromatic ring system of benz[a]anthracene may occur at various locations on the molecule, including via the 1,2- or 3,4-carbon positions, an angular kata-type initial dioxygenation, via the 8,9- or 10,11-carbon positions, a linear kata-type initial dioxygenation, or via the K-region at the 5,6-carbon positions. If metabolites that represent the initial oxidation steps are not directly recovered in metabolism studies, identification of downstream metabolites may allow for predicting whether an angular kata-, linear kata-, or K region-type initial dioxygenation originally occurred. For example, 2-hydroxy-3-naphthoic acid may occur as a downstream metabolite of benz[a]anthracene biotransformation through an angular kata-type initial dioxygenation event.

To date, metabolites from the biotransformation of benz[a]anthracene by bacteria have been identified from only four organisms. In chronological order, they are (i) Sphingobium yanoikuyae mutant strain B8/36 (20–22), (ii) S. yanoikuyae strain B1 (23), (iii) Mycobacterium sp. strain RJGII-135 (24), and (iv) Mycobacterium vanbaalenii strain PYR-1 (25). Additionally, biotransformation of benz[a]anthracene through cloned/expressed proteins from Sphingomonas sp. strain CHY-1 (26–28) were also documented. Biodegradation of benz[a]anthracene without documentation of metabolites has been reported to have occurred by members of the genera Alcaligenes (29), Stenotrophomonas (30–32), Sphingomonas (33), and Pseudomonas (34, 35).

Among the identified metabolites of benz[a]anthracene, cis-1,2-, -5,6-, -8,9-, and -10,11-dihydrodiols were identified from strain B8/36 (20–22), cis-5,6-, -8,9-, and -10,11-dihydrodiols were identified from strain RJGII-135 (24), and 1-hydroxy-anthranoic acid, 2-hydroxy-3-phenanthroic acid, and 3-hydroxy-2-phenanthroic acid were identified from strain B1 (23). These studies were instrumental in documenting the initial steps in benz[a]anthracene biostransformation by sphingomonads and by a mycobacterium; however, only from Mycobacterium vanbaalenii strain PYR-1 has downstream biotransformation after the production of o-hydroxy-triaromatic acids been shown (25). In that case, four biotransformation pathways were proposed where initial benz[a]anthracene oxidation occurred at the 1,2-, 5,6-, 10,11-, and 7,12-positions, and it was via the cis-1,2-dihydrodiol that biotransformation through 1,2-dihydroxyanthracene to 6-hydrofuran[3,4-g]chromene-2,8-dione was documented. In all reports of benz[a]anthracene metabolism by bacteria, however, the recovered metabolites consisted of four-ring and three-ring products, and downstream metabolites with less than three rings were not documented. In the case of strain PYR-1, three metabolites, benzo[g]chromen-2-one, 3-hydrobenzo[f]isobenzofuran-1-one, and 6-hydrofuran[3,4-g]chromene-2,8-dione were identified as three-ring closure products that appeared to have been derived from two-ring and single-ring metabolites (25).

In the last 10 years, liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) has become a valuable analytical tool in the field of proteomics and in the pharmaceutical industry for metabolite identification, partly because ESI, one of the softest ionization techniques, is highly amenable for the analyses of polar and ionic compounds. LC/ESI-MS/MS has been utilized much less so in the field of environmental microbiology for the determination of microbial biotransformation products; however, its application in this field is expected to increase as more research groups begin to develop analytical methods. Some of the main advantages of LC/ESI-MS/MS in the study of bacterial biotransformations are that coeluting peaks may be isolated through mass selectivity and are not always constrained by chromatographic resolution, that molecular mass and structural information may be obtained through controlled fragmentation, and that both quantitative and qualitative data may be obtained with relatively limited sample preparation.

In this investigation, various LC/ESI-MS(/MS) techniques were applied to investigate a newly characterized bacterial isolate, Sphingobium sp. strain KK22, in the context of its ability to biodegrade the recalcitrant environmental pollutant benz[a]anthracene. Benz[a]anthracene biotransformation was demonstrated through both quantitative and qualitative analytical approaches.

MATERIALS AND METHODS

Chemicals and growth media.

Benz[a]anthracene (1,2-benzanthracene; 99% purity) and phenanthrene (97% purity) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Benzo[a]pyrene (≥97% purity), pyrene (≥98% purity), salicylic acid (99.5% purity), and the organic solvents chloroform, ethyl acetate, acetonitrile, and methanol (high-performance liquid chromatography [HPLC]-grade or higher) were purchased from Wako Chemical (Osaka, Japan). Diesel fuel was purchased from Jomo Oil Company in Kyoto, Japan (March 2005), and separated by heat distillation into a heavy fraction and light fraction; the heavy fraction consisted of 67.5% (vol/vol) of the original diesel fuel and was stored at 4°C in the dark. Noble agar was purchased from Difco Bacto (Sparks, MD, USA).

Isolation and maintenance of strain KK22.

Strain KK22 was isolated from a bacterial consortium that was originally recovered from cattle pasture soil (36–38) and was maintained by growth on the heavy fraction of diesel fuel. From this consortium, an enrichment culture was created that consisted of phenanthrene as the sole source of carbon and energy (300 mg liter⁻¹) in Stanier's Basal Medium (SBM) (39). Strain KK22 was isolated from this culture by streaking of culture fluids on SBM Noble agar plates, followed by incubation at 28°C with phenanthrene crystals contained in the plate lid as the sole carbon source. Following isolation, strain KK22 was maintained in 300 mg liter⁻¹ phenanthrene by continuous rotary shaking at 150 rpm at 30°C in the dark and transferred approximately every 10 days or was revived from −80°C storage. For biodegradation assays, phenanthrene prepared in chloroform was applied to the bottoms of sterilized flasks under aseptic conditions, followed by solvent evaporation via filter-sterilized nitrogen gas. SBM and inocula were added last.

Benz[a]anthracene biodegradation assays with and without N,N-dimethylformamide.

Strain KK22 was grown on 300 mg liter⁻¹ phenanthrene for 6 days as described above and harvested by centrifugation (8,700 × g for 10 min at 4°C), resuspended in phosphate buffer (50 mM, pH 7), and washed and centrifuged in three steps at 5,700 × g at 4°C for 10 min, 8 min, and 8 min each. Cells were resuspended in SBM and incubated by rotary shaking for approximately 12 h at 30°C and 150 rpm, and cultures were prepared in 100-ml Erlenmeyer flasks that contained 20 ml of SBM each plus 2.5 mg liter⁻¹ benz[a]anthracene in N,N-dimethylformamide (<0.05%, vol/vol; Wako, Osaka, Japan). Ten cultures were prepared with strain KK22, and the optical density at 620 nm (OD₆₂₀) of harvested and washed cells was adjusted to 0.10 by analysis of cell suspensions using a V-530 model UV-visible light (UV-Vis) spectrophotometer (Jasco, Tokyo, Japan). Ten cultures that consisted of benz[a]anthracene without cells served as abiotic controls. All cultures were incubated by rotary shaking at 30°C at 150 rpm in the dark for 8 days, and whole-flask extractions were conducted in duplicate for 8 days. Benzo[a]pyrene was added as an extraction standard via microsyringe (Hamilton, Reno, NV, USA), followed by addition of an equal volume of ethyl acetate to each culture and overnight shaking at 23°C and 150 rpm in the dark. Organic and aqueous phases were separated in glass separating funnels, passed through anhydrous sodium sulfate that was prepared by overnight drying at 50°C, and extracted a second time. Ethyl acetate extracts were pooled and concentrated en vacuo via rotary evaporation (Eyela, Tokyo, Japan). Residues were resuspended in ethyl acetate and passed through 0.45-μm-pore-size polytetrafluoroethylene (PTFE) syringe filters (Whatman, NJ, USA) into brown glass vials.

Quantitative analyses were performed by high-performance liquid chromatography (HPLC) with fluorescence detection using a Jasco HPLC system (Tokyo, Japan) that consisted of a PU-2089 quaternary pump in-line with an FP-2020 Plus fluorescence detector. Extracts were eluted isocratically in 85% methanol-water and separated on a Crestpak C₁₈S 150-mm by 4.6-mm column (Jasco). The flow rate was 0.8 ml/min, and sample injection was conducted by a Jasco AS-2057 Plus autoinjector. Detection was conducted at an excitation wavelength of 246 nm and an emission wavelength of 412 nm. The retention times of benz[a]anthracene and benzo[a]pyrene were 7.5 min and 11.0 min, respectively, under these conditions.

Biotransformation assays were conducted with 100 mg liter⁻¹ benz[a]anthracene without N,N-dimethylformamide. To examine induction effects on biotransformation, strain KK22 was cultivated on 20 mM glucose in SBM with and without 500 mg liter⁻¹ phenanthrene for 3 days and harvested, washed, and incubated overnight as described above. Flasks were prepared in duplicate and consisted of 20 ml of SBM each plus benz[a]anthracene, which was applied to the bottoms of each flask in diethyl ether by microsyringe and evaporated as described above. Abiotic controls that consisted of benz[a]anthracene were prepared similarly but without cells. Cells were incubated by rotary shaking at 30°C and 150 rpm in the dark, whole-flask extractions were conducted after 3 and 8 days, and extracts were treated as described above. Total protein was monitored by using the bicinchoninic method according to the manufacturer's instructions (Sigma-Aldrich). Organic extracts were resuspended in methanol and analyzed by HPLC with UV detection at 254 nm using a Waters 2690 Separations Module delivery system in-line with a Shimadzu SPD-10A UV-Vis detector (Kyoto, Japan) with a flow rate of 0.3 ml min⁻¹. Analytes were separated on a Shimadzu Shim-pack XR-ODS column (3.0-mm internal diameter [ID] by 75 mm) that was in-line with a Security Guard Cartridge Precolumn (Phenomenex, CA, USA). A gradient program was used as follows: 60:40 methanol-water that was changed to 95:5 methanol-water over 35 min, held for 20 min, and then returned to the original starting conditions for a total run time of 60 min.

Detection of metabolites of benz[a]anthracene biotransformation by LC/ESI(−)-MS(/MS).

Metabolites of benz[a]anthracene biotransformation were investigated after exposure of strain KK22 to 30 mg liter⁻¹ benz[a]anthracene as described above except that cells were grown in 70 ml of SBM in 500-ml conical flasks. Cultures were sampled 10 times over a period of 30 days such that 3 ml of culture supernatant liquid was aseptically removed at each sampling time and extracted at neutral pH with ethyl acetate similarly as described previously. To recover polar metabolites, culture fluids were reextracted in an identical manner at pH 2 following acidification of the extracted culture medium with concentrated hydrochloric acid. Neutral and acidified sample extracts were analyzed separately by liquid chromatography negative electrospray ionization mass spectrometry LC/ESI(−)-MS in full-scan mode using a Waters 2690 Separations Module delivery system in-line with a Shimadzu SPD-10A UV-Vis detector that was interfaced with a Quattro Ultima triple-stage quadrupole mass spectrometer (Micromass, Manchester, United Kingdom). Sample extracts were eluted isocratically in 77% methanol and 23% water at a flow rate of 0.3 ml min⁻¹ through a Shimadzu Shim-pack XR-ODS column (3.0-mm ID by 75 mm) that was in-line with a Security Guard Cartridge Precolumn. Total run times varied by sample but were generally 30 to 45 min. Full-scan analyses were conducted over a range of 50 to 500 m/z in electrospray negative ionization mode. Nitrogen was used as the nebulizing gas, the ion source temperature was 130°C, the desolvation temperature was 350°C, and the cone voltage was operated at a constant 40 V. Nitrogen gas was also used as the desolvation gas (600 liters/h) and the cone gas (60 liters/h).

Results of the full-scan analyses were examined to determine putative mass ions of interest by comparing the results from analyses of extracts from benz[a]anthracene biotransformation cultures with the results from analyses of extracts from biotic (strain KK22 only) and abiotic (benz[a]anthracene only) controls. After selection of putative mass ions of interest, sample extracts were analyzed again by LC/ESI-tandem mass spectrometry in negative ionization mode [LC/ESI(−)-MS/MS] by using collision-induced dissociation (CID) product ion scanning and precursor ion scanning modes under mass conditions similar those described above. Argon gas was used as the collision cell gas, and various collision cell energies were employed generally over a range of 1 to 20 eV, depending upon the sample. The mass spectral fragmentation patterns resulting from product ion and precursor ion scanning at various collision energies were analyzed to aid in the determination of the molecular structures of unknown benz[a]anthracene biotransformation products.

Identification of hydroxy-naphthoic acid biotransformation products by LC/ESI(−)-MS/MS in selected reaction monitoring (SRM) mode.

Authentic standards of 1-hydroxy-2-naphthoic acid (Kanto Chemical Co., Tokyo, Japan), 2-hydroxy-1-naphthoic acid (Tokyo Chemical Industries, Tokyo, Japan), and 2-hydroxy-3-naphthoic acid (Wako) were prepared in methanol and separated on a 150-mm by 4.6-mm Crestpak C₁₈S column (Jasco) by gradient program elution using a mobile phase consisting of a mixture of acetonitrile and water that contained 0.1% (vol/vol) formic acid. The initial conditions of the gradient were 20% acetonitrile and 80% water-formic acid, and this was changed to 95% acetonitrile over 35 min, returned to the initial starting conditions in 5 min, and held for 25 min at a flow rate of 0.3 ml min⁻¹ for a total run time of 45 min. Based upon the results of LC/ESI(−)-MS/MS product ion scanning analyses of the three hydroxy-naphthoic acid authentic standards, a tandem MS method that utilized the SRM mode and mass transition of 187.0 → 142.9 m/z was developed to identify hydroxy-naphthoic acids in sample extracts. Mass conditions were similar to those described above, and the collision energy was 8 eV.

Monitoring of indicators of cell growth.

Absorbance monitoring of strain KK22 was conducted by UV-visible spectrophotometry using an optical density equal to 620 nm. Culture supernatant fluids in 600-μl aliquots were transferred to a quartz cuvette and analyzed on a V-530 UV-Vis Spectrophotometer (Jasco, Tokyo, Japan).

16S rRNA gene PCR and DNA sequence determination.

Colony PCR was conducted using the following primers: PrOR, 5′-AGAGTTTGATCCTGGCTCAG-3′ (Escherichia coli 16S rRNA gene positions 8 to 27); 9Rev, 5′-AAGGAGGTGATCCCAGCC-3′ (positions 1532 to 1551); 1070F, 5′-ATGGCTGTCGTCAGCT-3′ (positions 1055 to 1070); and 534Rev, 5′-ATTACCGCGGCTGCTGG-3′ (positions 518 to 534). A Dice TP600 Thermal Cycler (TaKaRa Bio, Inc., Shiga, Japan) was used with the following program: 98°C for 30 s, followed by 35 cycles of three steps each (denaturation at 98°C for 1 min, annealing at 60°C for 2 min, and extension at 72°C for 3 min), and a final elongation step at 72°C for 10 min. PCR products were purified and sequenced using a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, CA, USA), and the products were analyzed with a model 3130 Genetic Analyzer (Applied Biosystems). Database queries were conducted by using the BLAST program (40) with the GenBank database. 16S rRNA gene sequences of the strains nearest to strain KK22 were retrieved from the NCBI database, aligned by Clustal W (41), and refined by visual inspection. An unrooted neighbor-joining tree was constructed by using MEGA, version 5.0, software (42).

Nucleotide sequence accession number.

The nucleotide sequence of strain KK22 has been deposited in the GenBank database under accession number HQ830159.

RESULTS

Sphingobium sp. strain KK22.

Strain KK22 was isolated from an enrichment culture of a bacterial consortium by spreading dilutions of enrichment culture fluids on Noble agar plates where phenanthrene crystals were aseptically placed into the lid. Afterwards, this strain was grown and maintained on phenanthrene as the sole source of carbon and energy up to concentrations as high as 500 mg liter⁻¹. Phylogenetic analyses based upon the 16S rRNA gene sequence of strain KK22 indicated that it was most closely related to Sphingobium fuliginis strain TKP (99.8% identity) (43) and was less related to members of the other Sphingomonadaceae genera, Sphingomonas, Sphingopyxis, Sphingosinicella, and Novosphingobium.

Quantitation of benz[a]anthracene biodegradation.

Strain KK22 biodegradation of 2.5 mg liter⁻¹ benz[a]anthracene occurred rapidly with almost 50% biotransformation in the first 48 h, as indicated in Fig. 1. After 8 days, approximately 85% of benz[a]anthracene was removed from solution by strain KK22 compared to levels in the abiotic controls. Recoveries for the abiotic controls were greater than 90% throughout the incubation period. Phenanthrene induction effects on benz[a]anthracene biotransformation by strain KK22 were investigated in 100 mg liter⁻¹ benz[a]anthracene cultures, and after 8 days, the overall rates of benz[a]anthracene biotransformation by cells precultured on glucose or precultured on glucose plus phenanthrene differed by approximately only 10% or less. The average rates of benz[a]anthracene catabolism were 6.6 ± 1.3 and 7.2 ± 1.5 μg of benz[a]anthracene per mg of cell protein per day, respectively (summarized in Table S1 in the supplemental material). These results showed that induction on phenanthrene appeared to be unnecessary for effective benz[a]anthracene biotransformation and that strain KK22 cells were capable of benz[a]anthracene biotransformation at high concentrations.

Fig 1.

Recoveries of benz[a]anthracene after whole-flask extractions of 20-ml cultures that contained 2.5 mg liter⁻¹ benz[a]anthracene and were incubated with (●) or without (■) strain KK22 cells. Cultures were extracted with ethyl acetate, and extracts were analyzed by HPLC with fluorescence detection. Each point represents the average of duplicate cultures, and the error bars indicate ranges.

Detection of hydroxy-triaromatic and hydroxy-naphthoic acid by product ion scanning analyses.

Results of LC-UV/ESI(−)-MS analyses of 8-day acidified sample extracts from exposed KK22 cells are shown in Fig. 2, where two large peaks were revealed to occur in high abundance by UV detection (retention times [t_Rs] of 4.35 and 4.78 min) (Fig. 2A) compared to the controls (Fig. 2B and C). These two peaks were first detected after 30 min from the start of the experiment (data not shown) and were not detected in the controls at that time or at any time afterwards. Results of full-scan analyses revealed two peaks with retention times equal to 4.51 and 4.94 min that matched well with the two peaks detected by UV.

Fig 2.

Results of LC/ESI(−)-MS analysis of day 8 acidified sample extracts of strain KK22 plus 30 mg liter⁻¹ benz[a]anthracene (A), a biotic control, strain KK22 without benz[a]anthracene (B), and the abiotic control, 30 mg liter⁻¹ benz[a]anthracene without strain KK22 (C). (D) The extracted ion mass chromatogram (EIC) for [M − H]⁻ = 237 from the same sample extract as shown in panel A. UV chromatograms are normalized for peak size comparisons. The retention time of benz[a]anthracene was 25.5 to 26.0 min (data not shown).

These peaks were found to correspond to the deprotonated molecules [M − H]⁻ = 237, as shown in the extracted-ion chromatogram (EIC) in Fig. 2D.

Based upon these observations, product ion scanning of acidified 8-day extracts of [M − H]⁻ = 237 were conducted, and the results showed nearly identical fragmentation patterns for both [M − H]⁻ = 237 target peaks. Figure 3A shows the results of product ion scanning analysis of the earlier eluted peak, [M − H]⁻ = 237 with a t_R of 4.35 min; however, fragmentation data and relative intensity values for both [M − H]⁻ = 237 peaks are given as part of a detailed summary of all results of product ion scanning analyses in Table 1. Losses of 44 Da (m/z 193) and 72 Da (m/z 165) occurred and were indicative of losses of CO₂ and of CO₂ plus CO, respectively. Early detection of metabolites from benz[a]anthracene biotransformation with masses equal to 238 Da are due to the production of o-hydroxy-triaromatic acids, as was first shown by Mahaffey et al. (23). Our results reported herein, however, showed a strong ion fragment corresponding to losses of 44 plus 28 Da, m/z 165, and the lack of an ion fragment corresponding to a loss of H₂O, such as 18 Da. Because o-hydroxy-triaromatic acid standards are not commercially available, three authentic standards of the two-ring o-hydroxy-naphthoic acids, 2-hydroxy-1-naphthoic acid, and its 1,2- and 2,3-isomers were obtained and analyzed under identical LC/ESI(−)-MS/MS conditions. The results of product ion scanning analysis of 2-hydroxy-3-naphthoic acid are shown in Fig. 3B, where identical losses of 44 Da (m/z 143) and 72 Da (m/z 115) from the deprotonated molecule [M − H]⁻ = 187 were observed. These data confirmed that under these conditions, using ESI negative ionization, the loss of 28 Da occurred due to a loss of CO. By applying ESI negative ionization and CID MS/MS to fragment hydroxylated PAHs, losses of 28 Da each were also documented by Xu et al. (44), and these results were attributed to losses of CO. Using electron impact mass spectrometry, Mahaffey et al. (23) observed similar losses from the molecular ion m/z 238, in addition to H₂O.

Fig 3.

ESI(−)-MS/MS spectra acquired by product ion scanning analyses of benz[a]anthracene metabolites. (A) Fragmentation pattern acquired from [M − H]⁻ = 237 corresponding to the two largest metabolite peaks produced during the initial biotransformation of benz[a]anthracene by strain KK22 from acidified extracts with a t_R of 4.4 min; collision energy was 20 eV. (B) Fragmentation pattern acquired from the analysis of a 2-hydroxy-3-naphthoic acid authentic standard solution, [M − H]⁻ = 187; collision energy was 8 eV. (C) Fragmentation pattern acquired from [M − H]⁻ = 187 analysis from neutral extracts with a t_R of 2.7 min; collision energy was 8 eV. (D) Fragmentation pattern acquired from [M − H]⁻ = 291 analysis from acidified extracts with a t_R of 4.1 min; collision energy was 20 eV. (E) Fragmentation pattern acquired from [M − H]⁻ = 241 analysis from acidified extracts with a t_R of 3.4 min; collision energy was 8 eV. Proposed molecular formulae for each metabolite are also shown.

Table 1.

Fragmentation ions revealed by LC/ESI(−)-MS/MS product ion scanning analyses of metabolites produced from the biotransformation of benz[a]anthracene by strain KK22

Parent ion [M − H]− (m/z)	tR(s) (min)a	CID (eV)b	Diagnostic fragments from product ion scanning analyses (m/z [ion, % relative intensity(ies)])	Identity assignment(s)
237	4.4, 4.8c	20	237 (M−, 2, 4), 193 (M− − CO2, 100, 100), 165 (M− − CO2 − CO, 3, 1)c	1-(2-)Hydroxy-2-(1-)anthranoic acid and 2-(3-)hydroxy-3-(2-)phenanthroic acid
187	2.7	8	187 (M−, 100), 169 (M− − CO, 2), 143 (M− − CO2, 17), 125 (M− − CO2 − H2O, 3), 115 (M− − CO2 − CO, 1)	2-Hydroxy-3-naphthoic acide, 1-hydroxy-2-naphthoic acide
291	4.1	20	291 (M−, 6), 273 (M− − H2O, 2), 247 (M− − CO2, 11), 221 (M− − CO2 − C2H2, 7), 203 (M− − 2CO2, 100), 177 (M− − 2CO2 − C2H2, 19)	1-(2-)[3-Hydroxy-3-oxo-prop-1-enyl]anthracene-2-(1-)-carboxylic acid and/or 2-(3-)[3-hydroxy-3-oxo-prop-1-enyl]phenanthrene-3-(2-)-carboxylic acid
247	3.9	20	247 (M−, 4), 229 (M− − H2O, 100), 203 (M− − CO2, 1), 201 M− − CO − H2O, 4), 185 (M− − CO2 − H2O, 71), 173 (M− − 2CO − H2O, 3), 159 (M− − 2CO2, 11), 157 (M− − CO2 − CO − H2O, 25), 141 (M− − 2CO2 − H2O, 67), 129 (M− − CO2 − 2CO − H2O, 42)	1,6-Dihydroxynaphthalene-2,7-dicarboxylic acidf
241	3.4	8, 20	241 (M−, 72, 10), 197 (M− − CO2, 100, 7), 169 (<0.1, 5), 153 (M− − 2CO2, 64, 100), 127 (M− − 2CO2 − C2H2, <0.1, 15d)	3-(2-Carboxyvinyl)naphthalene-2-carboxylic acid
181	2.3	20	181 (M−, 10), 163 (M− − H2O, 11), 137 (M− − CO2, 3), 135 (M− − CO2, 26),g 119 (M− − CO2 − H2O, 100), 107 (M− − CO2 − CHOH, 85), 93 (M− − CO2 − CHOHCH2 − H2O, 4), 73 (M− − CO2 − CHOHCH2 − H2O, 11)	2-Hydroxy-3-(2-hydroxyphenyl)propanoic acidf
165	2.7	8	165 (M−, 69), 137 (M− − CO, 1), 121 (M− − CO2, 100), 93 (M− − CO2 − C2H4, 4)	3-(2-Hydroxyphenyl)propanoic acidf
137	2.6	17	137 (M−, 100), 109 (M− − CO, 1) 93 (M− − CO2, 29)	Salicylic acide

^at_R, retention time, corresponding to UV detection at 254 nm.

^bCollision-induced dissociation energy.

^cTwo peaks were detected with similar fragmentation patterns; relative intensities for both metabolites are given in order of elution.

^dIncreasing the collision energy revealed the presence of m/z 127 ([M − H]⁻ = 241).

^eStructural confirmation was conducted via analyses of authentic standards using the SRM mode.

^fThe positions of hydroxyl- and carboxy-groups on the aromatic ring(s) were not determined.

^gLoss of 46 Da from the parent molecule is representative of a loss of a carboxyl group during ESI(−) CID analysis of alpha-hydroxy carboxylic acids (see text for details).

From 8-day neutral sample extracts from exposed cells, product ion scanning analyses of [M − H]⁻ = 187 with a t_R of 2.7 min were conducted, and the results as shown in Fig. 3C revealed fragmentation patterns nearly identical to o-hydroxy-naphthoic acid authentic standards (Fig. 3B), incurring major losses of 44 Da (loss of CO₂, m/z 143) and 72 Da (losses of CO₂ and CO, m/z 115), as in the case of the biotransformation product corresponding to [M − H]⁻ = 237. Additionally, fragments m/z 169 and m/z 125 were also detected and indicated losses of H₂O (18 Da) and of H₂O plus CO₂ (62 Da), respectively, from [M − H]⁻ = 187 (Fig. 3C).

Based upon these results, it was concluded that metabolites with molecular masses of 238 Da and 188 Da with molecular formulae of C₁₅H₁₀O₃ and C₁₁H₈O₃, respectively, represented at least two o-hydroxy-triaromatic acid biotransformation products of benz[a]anthracene and at least one of three possible o-hydroxy-naphthoic acid biotransformation products of benz[a]anthracene.

Detection of other three- and two-ring metabolites by product ion scanning analyses.

In addition to [M − H]⁻ = 187 and [M − H]⁻ = 237, full-scan mass screening analyses resulted in six other putative target ions of interest with deprotonated molecule values ranging from [M − H]⁻ = 291 to [M − H]⁻ = 137. Shown in Fig. 3D are the results of product ion scanning of the metabolite corresponding to [M − H]⁻ = 291. The proposed molecular formula for this compound was C₁₈H₁₂O₄, and losses of CO₂ (m/z 247), 2CO₂ (m/z 203), CO₂ plus C₂H₂ (m/z 221), and 2CO₂ plus C₂H₂ (m/z 177) were revealed. This fragmentation pattern, which included double losses of CO₂ combined with losses of C₂H₂, provided evidence for ortho-type cleavage products derived from an upstream dihydroxy-benz[a]anthracene metabolite that would have originated from a 1,2-, 3,4-, 8,9-, or 10,11-carbon position initial enzymatic attack on the benz[a]anthracene molecule.

Figure 3E shows the fragmentation pattern from CID MS/MS analysis of the deprotonated molecule [M − H]⁻ = 241, obtained at 8 eV, where sequential losses of 44 Da each (CO₂), m/z 197 and m/z 153, were revealed and provided evidence that this may be a dicarboxylated biotransformation product of benz[a]anthracene. Further fragmentation at 20 eV showed that the fragment m/z 127 occurred at a relative intensity of 15% (Table 1), and this result was indicative of losses of 2CO₂ plus C₂H₂ (114 Da) and a molecular formula of C₁₄H₁₀O₄. It is understood that when meta cleavage products of PAH biotransformation are analyzed under such conditions, a loss of 72 Da from the parent ion, corresponding to losses of the carboxyl moiety and the alpha-keto moiety, are typically observed. Taken together, an ortho cleavage event of a dihydroxylated anthracene biotransformation product of benz[a]anthracene was proposed, and the metabolite corresponding to [M − H]⁻ = 241 was concluded to be the ortho cleavage product of 1,2-dihydroxyanthracene, 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid. Figure 4 illustrates the pathways proposed for the biotransformation of benz[a]anthracene including metabolites that are discussed in the following sections.

Fig 4.

Pathways proposed for the biotransformation of benz[a]anthracene by strain KK22. Metabolites in brackets were not identified in the culture medium.

Figure 5A and Table 1 present the results of [M − H]⁻ = 247 product ion scanning analyses where nine diagnostic fragments were observed at 20 eV. From the parent molecule, [M − H]⁻ = 247, losses of CO₂, m/z 203, 2CO₂, m/z 157, and 2CO₂ plus H₂O, m/z 141, occurred. At the same time, losses of 2CO plus H₂O, m/z 173, and 2CO and CO₂ plus H₂O, m/z 129, were also observed. Taken together, and by consideration of a compound with a molecular formula of C₁₂H₈O₆, these losses indicate that this molecule most likely possessed two carboxyl and two hydroxyl groups attached at various positions to a double aromatic ring structure. This type of metabolite may occur if the benz[a]anthracene molecule was oxidized via both the 1,2- or 3,4-carbon position and the 8,9- or 10,11-carbon position. This indicated that strain KK22 had initiated benz[a]anthracene molecule oxidation via both the kata-linear and kata-angular ends of the molecule. A representative structure is shown in Fig. 4.

Fig 5.

ESI(−)-MS/MS spectra acquired by product ion scanning analysis of benz[a]anthracene biotransformation product with a t_R of 3.9 min, corresponding to [M − H]⁻ = 247 (A) and benz[a]anthracene biotransformation product with a t_R of 2.3 min, corresponding to [M − H]⁻ = 181 (B). Both products were from organic extracts derived from acidified culture medium; collision energies were 20 eV in each case. Proposed molecular structures and fragmentation map are shown in panel B.

Detection of single-aromatic-ring metabolites by product ion scanning analyses.

Product ion scanning analyses of metabolites corresponding to [M − H]⁻ = 181, 165, and 137 were also conducted, and based upon the fragmentation pattern results given in Table 1, the molecular formulae C₈H₆O₄, C₈H₆O₅, and C₇H₆O₃, respectively, were assigned. The common fragment ion, m/z 93, was detected from all three of these metabolites, providing evidence for a phenolic-type fragment, and indicated that these metabolites appeared to represent single-aromatic-ring metabolites of benz[a]anthracene biotransformation.

In the case of [M − H]⁻ = 181, a major product ion scan diagnostic fragment, m/z 135 (26%), indicating a loss of 46 Da was revealed (Fig. 5B). When the MS is operated in negative ionization mode, a loss of 46 Da from the deprotonated molecular ion is indicative of an alpha-hydroxy carboxylate ion. Alpha-hydroxy carboxylic acids have been shown to dissociate to produce product ions that represent a neutral loss of 46 Da (CH₂O₂) and the production of an enolate ion when hydrogen(s) are present beta to the carboxyl moiety (45, 46). That the metabolite corresponding to [M − H]⁻ = 181 may be an alpha-hydroxy carboxylic acid was further confirmed by the observation of a major fragment corresponding to m/z 107 (85%), which indicated a net loss of 74 Da (M⁻ − CO₂ − CHOH) and represented a bond cleavage event between the alpha carbonyl and the beta carbon on the propanoic acid side chain of the proposed metabolite. Other CID fragmentation products were also revealed, and after all fragments were considered, this metabolite was proposed to be 2-hydroxy-3-(2-hydroxyphenyl)propanoic acid. The fragmentation pattern, molecular structure, and proposed fragmentation map for this biotransformation product are summarized in Fig. 5B.

Diagnostic fragmentation pattern analyses of [M − H]⁻ = 165 showed that major fragments were detected at m/z 121, m/z 137, and m/z 93 corresponding to losses of CO₂, CO, and CO₂ plus CO, respectively (Table 1). Based upon the identity assignment for the single-aromatic-ring metabolite corresponding to [M − H]⁻ = 181, combined with the diagnostic fragmentation pattern observed from analyses of [M − H]⁻ = 165, the identity of [M − H]⁻ = 165 was proposed to be 3-(2-hydroxyphenyl)propanoic acid. Finally, in the case of [M − H]⁻ = 137, major fragments which indicated losses of 44 Da, m/z 93, and 28 Da, m/z 109, were observed, and the identity of [M − H]⁻ = 137 was confirmed to be salicylic acid after comparison to the results of a product ion scanning analysis of a salicylic acid authentic standard solution in which the retention times and mass spectra were identical. The detailed results of all product ion scanning analyses for metabolites discussed are assembled in Table 1.

Identification of 1,2- and 2,3-hydroxy-naphthoic acids by LC/ESI(−)-MS/MS SRM mode.

At least one peak corresponding to the presence of [M − H]⁻ = 187 was revealed by LC/ESI(−)-MS to be a major ion in sample extracts. As described above, product ion scanning of [M − H]⁻ = 187 revealed that m/z 143 occurred as a major fragmentation ion, and this was confirmed by analyses of authentic hydroxy-naphthoic acid standards. Based upon the matching fragmentation patterns and the lack of major precursor ions, it was concluded that the biotransformation product that corresponded to [M − H]⁻ = 187 was a hydroxy-naphthoic acid, yet it was not clear if the peak represented multiple coeluting isomers of hydroxy-naphthoic acids or was a single hydroxy-naphthoic acid metabolite. At the same time, unambiguous identification of this key metabolite would also allow for a better understanding of the upstream biotransformation pathway of benz[a]anthracene by strain KK22. To investigate this question further, new HPLC separation conditions were devised, and a mass method that employed the SRM mode to allow for selective identification of metabolites using the transition 187 to 143 m/z was developed. Results of SRM mode analyses of sample extracts revealed two peaks and indicated that coelution had originally occurred. As shown in Fig. 6, after comparisons of the elution times of three authentic hydroxy-naphthoic acid standards to benz[a]anthracene biodegradation sample extracts, 1-hydroxy-2-naphthoic acid and 2-hydroxy-3-naphthoic acid were identified as metabolites of benz[a]anthracene.

Fig 6.

LC/ESI(−)-MS/MS SRM mode of transition 187 → 143 m/z of acidified sample extracts after exposure of strain KK22 to benz[a]anthracene (A), 1-hydroxy-2-naphthoic acid standard solution (B), 2-hydroxy-3-naphthoic acid solution (C), and 2-hydroxy-1-naphthoic acid solution (D). The identities of two biotransformation products, [M − H]⁻ = 187, of benz[a]anthracene by strain KK22 were established by this retention time comparison.

Detection of these two metabolites confirmed further that benz[a]anthracene biotransformation occurred via both linear kata-type and angular kata-type enzymatic attacks on the benz[a]anthracene molecule at the 1,2- and/or 3,4-carbon position, which resulted in 2-hydroxy-3-naphthoic acid, and at the 8,9- and/or 10,11-carbon position which resulted in 1-hydroxy-2-naphthoic acid, respectively. Based upon these and the previously described results, a pathway for the biotransformation of benz[a]anthracene by strain KK22 was proposed in Fig. 4. This pathway highlights that at least two o-hydroxy-triaromatic acids were formed leading to 2-hydroxy-3- and 1-hydroxy-2-naphthoic acids, after which one or both of these compounds were further biotransformed into single-aromatic-ring metabolites. At the same time, it also includes metabolite(s) that occurred via both linear kata-type and angular kata-type oxidations on the same molecule, as represented by the metabolite(s) corresponding to [M − H]⁻ = 247.

DISCUSSION

Biotransformation of benz[a]anthracene by strain KK22 proceeded through initial oxidative steps via carbon positions 1,2 and/or 3,4 and 8,9 and/or 10,11. Initial oxidations at some of these carbon positions of benz[a]anthracene have been reported previously but only for three naturally occurring bacteria, a sphingomonad and two mycobacteria (23–25). The first ring fission by strain KK22 occurred through ortho cleavage resulting in metabolite(s) corresponding to [M − H]⁻ = 291, which were proposed herein to be o-ethylenecarboxy-phenanthroic and -anthranoic acids. In a pathway that is analogous to transformation of benz[a]anthracene by a 8,9- and/or 10,11-carbon position attack by strain KK22, ortho cleavage of the anthracene biotransformation product 1,2-dihydroxyanthracene to 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid was proposed to occur by M. vanbaalenii strain PYR-1 (13). At the same time, an initial ortho cleavage step that is analogous to a 1,2- and/or 3,4-carbon position attack on benz[a]anthracene by strain KK22 was reported recently for phenanthrene biotransformation to 1-(2-)(2-carboxy-vinyl)-naphthalene-2-(1-)carboxylic acids by Arthrobacter sp. strain P-1-1 (15). Because the downstream metabolites of benz[a]anthracene biotransformation by strain KK22, 1-hydroxy-2-naphthoic acid and 2-hydroxy-3-naphthoic acid, were identified in the culture medium, the o-hydroxytriaromatic acids, phenanthroic and anthranoic acid, were also produced earlier. These o-hydroxytriaromatic acids may have originated only from upstream metabolites that were oxidized via both linear kata-type and angular kata-type attacks on the benz[a]anthracene molecule, and this is represented in the proposed pathway in Fig. 4. Indeed, two peaks which were detected in the greatest abundance, corresponding to [M − H]⁻ = 237, were proposed to be the products from the biotransformation of the o-ethylenecarboxy-phenanthroic and -anthranoic acids: o-hydroxy-phenanthroic and o-hydroxy-anthranoic acids. These acids have been identified only once before as products of S. yanoikuyae strain B1 (23).

At least in the case of the o-hydroxy-anthranoic acid(s) produced by strain KK22 from benz[a]anthracene, a second ortho cleavage event occurred to produce 2-hydroxy-3-naphthoic acid from an o-ethylenecarboxy-naphthoic acid intermediate, 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid, [M − H]⁻ = 241 (Fig. 4). As mentioned above, 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid was first identified as an ortho cleavage product in the biotransformation of anthracene by M. vanbaalenii strain PYR-1 in 2001 (13), and this is the first documentation of this metabolite from benz[a]anthracene. An intermediate between the production of o-hydroxy-phenanthroic acid(s) and 1-hydroxy-2-naphthoic acid was not identified in this study. Additionally, there was no evidence to support a 5,6-carbon position attack on benz[a]anthracene by strain KK22 as was demonstrated by Moody et al. (13), and this was not unexpected because strain KK22 appeared unable to biotransform the PAH pyrene (unpublished data). Pyrene does not possess any kata-annelated aromatic rings. It has been discussed that K-region PAH oxidation may be unique to pyrene-degrading nocardioform bacteria (among heterotrophic organisms) (16).

The recovery of the metabolite corresponding to [M − H]⁻ = 247, whose mass analyses revealed that it was most likely the product of enzymatic attack via both ends of the benz[a]anthracene molecule, provided another line of evidence that strain KK22 metabolized benz[a]anthracene via 1,2- and/or 3,4- and 8,9- and/or 10,11-carbon position initial dioxygenation. Finally, o-hydroxynaphthoic acids were converted to various single-ring metabolites.

Although many organisms that biodegrade the kata-annelated three-ring PAH phenanthrene have been isolated (7–16), demonstration of benz[a]anthracene biodegradation accompanied by metabolite identification has been shown to occur in only three naturally occurring isolates. From these studies, more than one ring cleavage step in the biotransformation pathway of benz[a]anthracene was shown to occur only by strain PYR-1 (25). Results of experiments with strain KK22 reported herein indicated that the first and second cleavage events appeared to have occurred by ortho mechanisms ultimately leading to two- and one-ring metabolites, which in addition to the identification of two o-hydroxy-naphthoic acids were representative of previously unreported metabolites of benz[a]anthracene biotransformation. These results allowed for the construction of a new pathway for the biodegradation of benz[a]anthracene by this bacterium.

Overall, this work extended our understanding of the pathways by which the HMW PAH benz[a]anthracene may be transformed by a sphingomonad bacterium and demonstrated the utility of applying LC/ESI-MS/MS to study bacterial biotransformations in the field of environmental microbiology. There are few data available in regard to the use of soft ionization techniques (ESI) to evaluate biotransformation products of bacterial pollutant biodegradation, and this research required the development of methods and data interpretation approaches that will be useful to apply to future studies in this field. Combined with nuclear magnetic resonance or high-resolution mass investigations, for example, the utility of the approach will be strengthened.