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

Marie Kunihiro,a Yasuhiro Ozeki,a Yuichi Nogi,b Natsuko Hamamura,c Robert A. Kanalya

Department of Life and Environmental System Science, Graduate School of Nanobiosciences, Yokohama City University, Kanazawa, Kanagawa, Yokohama, Japana;Extremobiosphere Research Program, Japan Agency for Marine-Earth Science and Technology, Yokosuka, Japanb; Center for Marine Environmental Studies, EhimeUniversity, Matsuyama, Japanc

A soil bacterium, designated strain KK22, was isolated from a phenanthrene enrichment culture of a bacterial consortium thatgrew on diesel fuel, and it was found to biotransform the persistent environmental pollutant and high-molecular-weight polycy-clic aromatic hydrocarbon (PAH) benz[a]anthracene. Nearly complete sequencing of the 16S rRNA gene of strain KK22 and phy-logenetic analysis revealed that this organism is a new member of the genus Sphingobium. An 8-day time course study that con-sisted of whole-culture extractions followed by high-performance liquid chromatography (HPLC) analyses with fluorescencedetection showed that 80 to 90% biodegradation of 2.5 mg liter�1 benz[a]anthracene had occurred. Biodegradation assays wherebenz[a]anthracene was supplied in crystalline form (100 mg liter�1) confirmed biodegradation and showed that strain KK22cells precultured on glucose were equally capable of benz[a]anthracene biotransformation when precultured on glucose plusphenanthrene. Analyses of organic extracts from benz[a]anthracene biodegradation by liquid chromatography negative electro-spray ionization tandem mass spectrometry [LC/ESI(�)-MS/MS] revealed 10 products, including two o-hydroxypolyaromaticacids and two hydroxy-naphthoic acids. 1-Hydroxy-2- and 2-hydroxy-3-naphthoic acids were unambiguously identified, andthis indicated that oxidation of the benz[a]anthracene molecule occurred via both the linear kata and angular kata ends of themolecule. 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 wereextended.

High-molecular-weight polycyclic aromatic hydrocarbons(HMW PAHs) are commonly occurring environmental pol-

lutants that are generally considered to be more resistant to bio-degradation than their lower-molecular-weight aromatic coun-terparts (1–4). Many are suspected carcinogens and displaygenotoxic and immunotoxic properties in addition to causing ox-idative cell damage (5, 6). The HMW PAH benz[a]anthracene isconsidered to be environmentally recalcitrant, is classified as agroup 2A carcinogen by the International Agency for Research onCancer, and is included in the U.S. Environmental ProtectionAgency’s Priority Pollutant List. As such, there is much interest inunderstanding the environmental fate of benz[a]anthracene andthe mechanisms by which it may be transformed.

Few studies have documented the bacterial biotransformationof benz[a]anthracene even though many studies have docu-mented 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 linearkata-annelated PAH anthracene (7, 13, 17–19). The benz[a]an-thracene molecule itself is comprised of four aromatic rings thatare bonded via both linear and angular kata annelation, and it maybe thought of as a benzannelated derivative of either phenan-threne or anthracene. Initial enzymatic oxidation of the aromaticring system of benz[a]anthracene may occur at various locationson the molecule, including via the 1,2- or 3,4-carbon positions, anangular kata-type initial dioxygenation, via the 8,9- or 10,11-car-bon positions, a linear kata-type initial dioxygenation, or via theK-region at the 5,6-carbon positions. If metabolites that representthe initial oxidation steps are not directly recovered in metabolismstudies, identification of downstream metabolites may allow forpredicting whether an angular kata-, linear kata-, or K region-type

initial dioxygenation originally occurred. For example, 2-hy-droxy-3-naphthoic acid may occur as a downstream metabolite ofbenz[a]anthracene biotransformation through an angular kata-type initial dioxygenation event.

To date, metabolites from the biotransformation of ben-z[a]anthracene by bacteria have been identified from only fourorganisms. In chronological order, they are (i) Sphingobiumyanoikuyae mutant strain B8/36 (20–22), (ii) S. yanoikuyae strainB1 (23), (iii) Mycobacterium sp. strain RJGII-135 (24), and (iv)Mycobacterium vanbaalenii strain PYR-1 (25). Additionally, bio-transformation of benz[a]anthracene through cloned/expressedproteins from Sphingomonas sp. strain CHY-1 (26–28) were alsodocumented. Biodegradation of benz[a]anthracene without doc-umentation of metabolites has been reported to have occurred bymembers 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 fromstrain B8/36 (20–22), cis-5,6-, -8,9-, and -10,11-dihydrodiols wereidentified from strain RJGII-135 (24), and 1-hydroxy-anthranoicacid, 2-hydroxy-3-phenanthroic acid, and 3-hydroxy-2-phenan-

Received 8 April 2013 Accepted 8 May 2013

Published ahead of print 17 May 2013

Address correspondence to Robert A. Kanaly, kanaly@yokohama-cu.ac.jp.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.01129-13.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.01129-13

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throic acid were identified from strain B1 (23). These studies wereinstrumental in documenting the initial steps in benz[a]anthra-cene biostransformation by sphingomonads and by a mycobacte-rium; however, only from Mycobacterium vanbaalenii strainPYR-1 has downstream biotransformation after the production ofo-hydroxy-triaromatic acids been shown (25). In that case, fourbiotransformation pathways were proposed where initial ben-z[a]anthracene oxidation occurred at the 1,2-, 5,6-, 10,11-, and7,12-positions, and it was via the cis-1,2-dihydrodiol that bio-transformation through 1,2-dihydroxyanthracene to 6-hydro-furan[3,4-g]chromene-2,8-dione was documented. In all reportsof benz[a]anthracene metabolism by bacteria, however, the re-covered metabolites consisted of four-ring and three-ring prod-ucts, and downstream metabolites with less than three rings werenot 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 asthree-ring closure products that appeared to have been derivedfrom two-ring and single-ring metabolites (25).

In the last 10 years, liquid chromatography coupled with elec-trospray ionization tandem mass spectrometry (LC/ESI-MS/MS)has become a valuable analytical tool in the field of proteomicsand in the pharmaceutical industry for metabolite identification,partly because ESI, one of the softest ionization techniques, ishighly amenable for the analyses of polar and ionic compounds.LC/ESI-MS/MS has been utilized much less so in the field of en-vironmental microbiology for the determination of microbial bio-transformation products; however, its application in this field isexpected to increase as more research groups begin to developanalytical methods. Some of the main advantages of LC/ESI-MS/MS in the study of bacterial biotransformations are that coe-luting peaks may be isolated through mass selectivity and are notalways constrained by chromatographic resolution, that molecu-lar mass and structural information may be obtained throughcontrolled fragmentation, and that both quantitative and qualita-tive data may be obtained with relatively limited sample prepara-tion.

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

MATERIALS AND METHODSChemicals 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 solventschloroform, ethyl acetate, acetonitrile, and methanol (high-performanceliquid chromatography [HPLC]-grade or higher) were purchased fromWako Chemical (Osaka, Japan). Diesel fuel was purchased from Jomo OilCompany in Kyoto, Japan (March 2005), and separated by heat distilla-tion into a heavy fraction and light fraction; the heavy fraction consisted of67.5% (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 isolatedfrom a bacterial consortium that was originally recovered from cattlepasture soil (36–38) and was maintained by growth on the heavy fractionof diesel fuel. From this consortium, an enrichment culture was createdthat consisted of phenanthrene as the sole source of carbon and energy

(300 mg liter�1) in Stanier’s Basal Medium (SBM) (39). Strain KK22 wasisolated from this culture by streaking of culture fluids on SBM Noble agarplates, followed by incubation at 28°C with phenanthrene crystals con-tained in the plate lid as the sole carbon source. Following isolation, strainKK22 was maintained in 300 mg liter�1 phenanthrene by continuousrotary shaking at 150 rpm at 30°C in the dark and transferred approxi-mately every 10 days or was revived from �80°C storage. For biodegra-dation assays, phenanthrene prepared in chloroform was applied to thebottoms of sterilized flasks under aseptic conditions, followed by solventevaporation via filter-sterilized nitrogen gas. SBM and inocula were addedlast.

Benz[a]anthracene biodegradation assays with and without N,N-dimethylformamide. Strain KK22 was grown on 300 mg liter�1 phenan-threne 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 10min, 8 min, and 8 min each. Cells were resuspended in SBM and incu-bated by rotary shaking for approximately 12 h at 30°C and 150 rpm, andcultures were prepared in 100-ml Erlenmeyer flasks that contained 20 mlof SBM each plus 2.5 mg liter�1 benz[a]anthracene in N,N-dimethylfor-mamide (�0.05%, vol/vol; Wako, Osaka, Japan). Ten cultures were pre-pared with strain KK22, and the optical density at 620 nm (OD620) ofharvested and washed cells was adjusted to 0.10 by analysis of cell suspen-sions using a V-530 model UV-visible light (UV-Vis) spectrophotometer(Jasco, Tokyo, Japan). Ten cultures that consisted of benz[a]anthracenewithout cells served as abiotic controls. All cultures were incubated byrotary shaking at 30°C at 150 rpm in the dark for 8 days, and whole-flaskextractions were conducted in duplicate for 8 days. Benzo[a]pyrene wasadded as an extraction standard via microsyringe (Hamilton, Reno, NV,USA), followed by addition of an equal volume of ethyl acetate to eachculture and overnight shaking at 23°C and 150 rpm in the dark. Organicand aqueous phases were separated in glass separating funnels, passedthrough anhydrous sodium sulfate that was prepared by overnight dryingat 50°C, and extracted a second time. Ethyl acetate extracts were pooledand concentrated en vacuo via rotary evaporation (Eyela, Tokyo, Japan).Residues were resuspended in ethyl acetate and passed through 0.45-�m-pore-size polytetrafluoroethylene (PTFE) syringe filters (Whatman, NJ,USA) into brown glass vials.

Quantitative analyses were performed by high-performance liquidchromatography (HPLC) with fluorescence detection using a Jasco HPLCsystem (Tokyo, Japan) that consisted of a PU-2089 quaternary pumpin-line with an FP-2020 Plus fluorescence detector. Extracts were elutedisocratically in 85% methanol-water and separated on a Crestpak C18S150-mm by 4.6-mm column (Jasco). The flow rate was 0.8 ml/min, andsample injection was conducted by a Jasco AS-2057 Plus autoinjector.Detection was conducted at an excitation wavelength of 246 nm and anemission wavelength of 412 nm. The retention times of benz[a]anthra-cene and benzo[a]pyrene were 7.5 min and 11.0 min, respectively, underthese conditions.

Biotransformation assays were conducted with 100 mg liter�1 ben-z[a]anthracene without N,N-dimethylformamide. To examine inductioneffects on biotransformation, strain KK22 was cultivated on 20 mM glu-cose in SBM with and without 500 mg liter�1 phenanthrene for 3 days andharvested, washed, and incubated overnight as described above. Flaskswere prepared in duplicate and consisted of 20 ml of SBM each plusbenz[a]anthracene, which was applied to the bottoms of each flask indiethyl ether by microsyringe and evaporated as described above. Abioticcontrols that consisted of benz[a]anthracene were prepared similarly butwithout cells. Cells were incubated by rotary shaking at 30°C and 150 rpmin the dark, whole-flask extractions were conducted after 3 and 8 days, andextracts were treated as described above. Total protein was monitored byusing the bicinchoninic method according to the manufacturer’s instruc-tions (Sigma-Aldrich). Organic extracts were resuspended in methanoland analyzed by HPLC with UV detection at 254 nm using a Waters 2690Separations Module delivery system in-line with a Shimadzu SPD-10A

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UV-Vis detector (Kyoto, Japan) with a flow rate of 0.3 ml min�1. Analyteswere separated on a Shimadzu Shim-pack XR-ODS column (3.0-mm in-ternal diameter [ID] by 75 mm) that was in-line with a Security GuardCartridge Precolumn (Phenomenex, CA, USA). A gradient program wasused 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 originalstarting conditions for a total run time of 60 min.

Detection of metabolites of benz[a]anthracene biotransformationby LC/ESI(�)-MS(/MS). Metabolites of benz[a]anthracene biotransfor-mation were investigated after exposure of strain KK22 to 30 mg liter�1

benz[a]anthracene as described above except that cells were grown in 70ml of SBM in 500-ml conical flasks. Cultures were sampled 10 times overa period of 30 days such that 3 ml of culture supernatant liquid wasaseptically removed at each sampling time and extracted at neutral pHwith ethyl acetate similarly as described previously. To recover polar me-tabolites, culture fluids were reextracted in an identical manner at pH 2following acidification of the extracted culture medium with concen-trated hydrochloric acid. Neutral and acidified sample extracts were ana-lyzed separately by liquid chromatography negative electrospray ioniza-tion mass spectrometry LC/ESI(�)-MS in full-scan mode using a Waters2690 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, UnitedKingdom). Sample extracts were eluted isocratically in 77% methanol and23% water at a flow rate of 0.3 ml min�1 through a Shimadzu Shim-packXR-ODS column (3.0-mm ID by 75 mm) that was in-line with a SecurityGuard Cartridge Precolumn. Total run times varied by sample but weregenerally 30 to 45 min. Full-scan analyses were conducted over a range of50 to 500 m/z in electrospray negative ionization mode. Nitrogen was usedas the nebulizing gas, the ion source temperature was 130°C, the desolva-tion temperature was 350°C, and the cone voltage was operated at a con-stant 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 putativemass ions of interest by comparing the results from analyses of extractsfrom benz[a]anthracene biotransformation cultures with the results fromanalyses of extracts from biotic (strain KK22 only) and abiotic (ben-z[a]anthracene only) controls. After selection of putative mass ions ofinterest, sample extracts were analyzed again by LC/ESI-tandem massspectrometry in negative ionization mode [LC/ESI(�)-MS/MS] by usingcollision-induced dissociation (CID) product ion scanning and precursorion scanning modes under mass conditions similar those described above.Argon gas was used as the collision cell gas, and various collision cellenergies were employed generally over a range of 1 to 20 eV, dependingupon the sample. The mass spectral fragmentation patterns resulting fromproduct ion and precursor ion scanning at various collision energies wereanalyzed to aid in the determination of the molecular structures of un-known benz[a]anthracene biotransformation products.

Identification of hydroxy-naphthoic acid biotransformation prod-ucts by LC/ESI(�)-MS/MS in selected reaction monitoring (SRM)mode. Authentic standards of 1-hydroxy-2-naphthoic acid (KantoChemical Co., Tokyo, Japan), 2-hydroxy-1-naphthoic acid (TokyoChemical Industries, Tokyo, Japan), and 2-hydroxy-3-naphthoic acid(Wako) were prepared in methanol and separated on a 150-mm by4.6-mm Crestpak C18S column (Jasco) by gradient program elution usinga mobile phase consisting of a mixture of acetonitrile and water that con-tained 0.1% (vol/vol) formic acid. The initial conditions of the gradientwere 20% acetonitrile and 80% water-formic acid, and this was changed to95% acetonitrile over 35 min, returned to the initial starting conditions in5 min, and held for 25 min at a flow rate of 0.3 ml min�1 for a total runtime of 45 min. Based upon the results of LC/ESI(�)-MS/MS product ionscanning analyses of the three hydroxy-naphthoic acid authentic stan-dards, a tandem MS method that utilized the SRM mode and mass tran-sition of 187.0 ¡ 142.9 m/z was developed to identify hydroxy-naphthoic

acids in sample extracts. Mass conditions were similar to those describedabove, and the collision energy was 8 eV.

Monitoring of indicators of cell growth. Absorbance monitoring ofstrain KK22 was conducted by UV-visible spectrophotometry using anoptical density equal to 620 nm. Culture supernatant fluids in 600-�laliquots were transferred to a quartz cuvette and analyzed on a V-530UV-Vis Spectrophotometer (Jasco, Tokyo, Japan).

16S rRNA gene PCR and DNA sequence determination. Colony PCRwas 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); and534Rev, 5=-ATTACCGCGGCTGCTGG-3= (positions 518 to 534). A DiceTP600 Thermal Cycler (TaKaRa Bio, Inc., Shiga, Japan) was used with thefollowing 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 exten-sion at 72°C for 3 min), and a final elongation step at 72°C for 10 min. PCRproducts were purified and sequenced using a BigDye Terminator CycleSequencing Kit (Applied Biosystems, CA, USA), and the products wereanalyzed with a model 3130 Genetic Analyzer (Applied Biosystems). Da-tabase queries were conducted by using the BLAST program (40) with theGenBank database. 16S rRNA gene sequences of the strains nearest tostrain KK22 were retrieved from the NCBI database, aligned by Clustal W(41), and refined by visual inspection. An unrooted neighbor-joining treewas constructed by using MEGA, version 5.0, software (42).

Nucleotide sequence accession number. The nucleotide sequence ofstrain KK22 has been deposited in the GenBank database under accessionnumber HQ830159.

RESULTSSphingobium sp. strain KK22. Strain KK22 was isolated from anenrichment culture of a bacterial consortium by spreading dilu-tions of enrichment culture fluids on Noble agar plates wherephenanthrene crystals were aseptically placed into the lid. After-wards, this strain was grown and maintained on phenanthrene asthe sole source of carbon and energy up to concentrations as highas 500 mg liter�1. Phylogenetic analyses based upon the 16S rRNAgene sequence of strain KK22 indicated that it was most closelyrelated to Sphingobium fuliginis strain TKP (99.8% identity) (43)and was less related to members of the other Sphingomonadaceaegenera, Sphingomonas, Sphingopyxis, Sphingosinicella, and No-vosphingobium.

Quantitation of benz[a]anthracene biodegradation. StrainKK22 biodegradation of 2.5 mg liter�1 benz[a]anthracene oc-curred rapidly with almost 50% biotransformation in the first 48h, as indicated in Fig. 1. After 8 days, approximately 85% of ben-z[a]anthracene was removed from solution by strain KK22 com-pared to levels in the abiotic controls. Recoveries for the abioticcontrols were greater than 90% throughout the incubation period.Phenanthrene induction effects on benz[a]anthracene biotrans-formation by strain KK22 were investigated in 100 mg liter�1 ben-z[a]anthracene cultures, and after 8 days, the overall rates of ben-z[a]anthracene biotransformation by cells precultured on glucoseor precultured on glucose plus phenanthrene differed by approx-imately only 10% or less. The average rates of benz[a]anthracenecatabolism were 6.6 � 1.3 and 7.2 � 1.5 �g of benz[a]anthraceneper mg of cell protein per day, respectively (summarized in TableS1 in the supplemental material). These results showed that in-duction on phenanthrene appeared to be unnecessary for effectivebenz[a]anthracene biotransformation and that strain KK22 cellswere capable of benz[a]anthracene biotransformation at highconcentrations.

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Detection of hydroxy-triaromatic and hydroxy-naphthoicacid by product ion scanning analyses. Results of LC-UV/ESI(�)-MS analyses of 8-day acidified sample extracts from ex-posed KK22 cells are shown in Fig. 2, where two large peaks wererevealed to occur in high abundance by UV detection (retentiontimes [tRs] of 4.35 and 4.78 min) (Fig. 2A) compared to the con-trols (Fig. 2B and C). These two peaks were first detected after 30min from the start of the experiment (data not shown) and werenot detected in the controls at that time or at any time afterwards.Results of full-scan analyses revealed two peaks with retentiontimes equal to 4.51 and 4.94 min that matched well with the twopeaks detected by UV.

These peaks were found to correspond to the deprotonatedmolecules [M � H]� � 237, as shown in the extracted-ion chro-matogram (EIC) in Fig. 2D.

Based upon these observations, product ion scanning of acid-ified 8-day extracts of [M � H]� � 237 were conducted, and theresults showed nearly identical fragmentation patterns for both[M � H]� � 237 target peaks. Figure 3A shows the results ofproduct ion scanning analysis of the earlier eluted peak, [M �H]� � 237 with a tR of 4.35 min; however, fragmentation data andrelative intensity values for both [M � H]� � 237 peaks are givenas part of a detailed summary of all results of product ion scanninganalyses 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]anthra-cene biotransformation with masses equal to 238 Da are due to theproduction of o-hydroxy-triaromatic acids, as was first shown byMahaffey et al. (23). Our results reported herein, however, showeda 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 ofH2O, such as 18 Da. Because o-hydroxy-triaromatic acid stan-dards are not commercially available, three authentic standards ofthe two-ring o-hydroxy-naphthoic acids, 2-hydroxy-1-naphthoicacid, and its 1,2- and 2,3-isomers were obtained and analyzedunder identical LC/ESI(�)-MS/MS conditions. The results ofproduct ion scanning analysis of 2-hydroxy-3-naphthoic acid areshown in Fig. 3B, where identical losses of 44 Da (m/z 143) and 72

Da (m/z 115) from the deprotonated molecule [M � H]� � 187were observed. These data confirmed that under these conditions,using ESI negative ionization, the loss of 28 Da occurred due to aloss of CO. By applying ESI negative ionization and CID MS/MSto fragment hydroxylated PAHs, losses of 28 Da each were alsodocumented by Xu et al. (44), and these results were attributed tolosses of CO. Using electron impact mass spectrometry, Mahaffeyet al. (23) observed similar losses from the molecular ion m/z 238,in addition to H2O.

From 8-day neutral sample extracts from exposed cells, prod-uct ion scanning analyses of [M � H]� � 187 with a tR of 2.7 minwere conducted, and the results as shown in Fig. 3C revealed frag-mentation patterns nearly identical to o-hydroxy-naphthoic acidauthentic standards (Fig. 3B), incurring major losses of 44 Da (lossof CO2, m/z 143) and 72 Da (losses of CO2 and CO, m/z 115), as inthe case of the biotransformation product corresponding to [M �H]� � 237. Additionally, fragments m/z 169 and m/z 125 werealso detected and indicated losses of H2O (18 Da) and of H2O plusCO2 (62 Da), respectively, from [M � H]� � 187 (Fig. 3C).

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

FIG 1 Recoveries of benz[a]anthracene after whole-flask extractions of 20-mlcultures that contained 2.5 mg liter�1 benz[a]anthracene and were incubatedwith (�) or without (�) strain KK22 cells. Cultures were extracted with ethylacetate, and extracts were analyzed by HPLC with fluorescence detection. Eachpoint represents the average of duplicate cultures, and the error bars indicateranges.

FIG 2 Results of LC/ESI(�)-MS analysis of day 8 acidified sample extracts ofstrain KK22 plus 30 mg liter�1 benz[a]anthracene (A), a biotic control, strainKK22 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 chro-matogram (EIC) for [M � H]� � 237 from the same sample extract as shownin panel A. UV chromatograms are normalized for peak size comparisons. Theretention time of benz[a]anthracene was 25.5 to 26.0 min (data not shown).

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Detection of other three- and two-ring metabolites by prod-uct ion scanning analyses. In addition to [M � H]� � 187 and[M � H]� � 237, full-scan mass screening analyses resulted in sixother putative target ions of interest with deprotonated moleculevalues ranging from [M � H]� � 291 to [M � H]� � 137. Shownin Fig. 3D are the results of product ion scanning of the metabolitecorresponding to [M � H]� � 291. The proposed molecular for-mula for this compound was C18H12O4, and losses of CO2 (m/z247), 2CO2 (m/z 203), CO2 plus C2H2 (m/z 221), and 2CO2 plusC2H2 (m/z 177) were revealed. This fragmentation pattern, whichincluded double losses of CO2 combined with losses of C2H2, pro-vided evidence for ortho-type cleavage products derived from anupstream dihydroxy-benz[a]anthracene metabolite that wouldhave originated from a 1,2-, 3,4-, 8,9-, or 10,11-carbon positioninitial 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, obtainedat 8 eV, where sequential losses of 44 Da each (CO2), m/z 197 andm/z 153, were revealed and provided evidence that this may be adicarboxylated biotransformation product of benz[a]anthracene.Further fragmentation at 20 eV showed that the fragment m/z 127occurred at a relative intensity of 15% (Table 1), and this resultwas indicative of losses of 2CO2 plus C2H2 (114 Da) and a molec-ular formula of C14H10O4. It is understood that when meta cleav-age products of PAH biotransformation are analyzed under suchconditions, a loss of 72 Da from the parent ion, corresponding tolosses of the carboxyl moiety and the alpha-keto moiety, are typ-ically observed. Taken together, an ortho cleavage event of a dihy-droxylated anthracene biotransformation product of benz[a]an-thracene was proposed, and the metabolite corresponding to[M � H]� � 241 was concluded to be the ortho cleavage productof 1,2-dihydroxyanthracene, 3-(2-carboxyvinyl)naphthalene-2-

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 acidifiedextracts with a tR of 4.4 min; collision energy was 20 eV. (B) Fragmentation pattern acquired from the analysis of a 2-hydroxy-3-naphthoic acid authenticstandard solution, [M � H]� � 187; collision energy was 8 eV. (C) Fragmentation pattern acquired from [M � H]� � 187 analysis from neutral extracts witha tR of 2.7 min; collision energy was 8 eV. (D) Fragmentation pattern acquired from [M � H]� � 291 analysis from acidified extracts with a tR of 4.1 min; collisionenergy was 20 eV. (E) Fragmentation pattern acquired from [M � H]� � 241 analysis from acidified extracts with a tR of 3.4 min; collision energy was 8 eV.Proposed molecular formulae for each metabolite are also shown.

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carboxylic acid. Figure 4 illustrates the pathways proposed for thebiotransformation of benz[a]anthracene including metabolitesthat are discussed in the following sections.

Figure 5A and Table 1 present the results of [M � H]� � 247product ion scanning analyses where nine diagnostic fragmentswere 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/z173, and 2CO and CO2 plus H2O, m/z 129, were also observed.Taken together, and by consideration of a compound with a mo-lecular formula of C12H8O6, these losses indicate that this mole-cule most likely possessed two carboxyl and two hydroxyl groupsattached at various positions to a double aromatic ring structure.This type of metabolite may occur if the benz[a]anthracene mol-ecule was oxidized via both the 1,2- or 3,4-carbon position and the8,9- or 10,11-carbon position. This indicated that strain KK22 hadinitiated benz[a]anthracene molecule oxidation via both the kata-linear and kata-angular ends of the molecule. A representativestructure is shown in Fig. 4.

Detection of single-aromatic-ring metabolites by production scanning analyses. Product ion scanning analyses of metab-

olites corresponding to [M � H]� � 181, 165, and 137 were alsoconducted, and based upon the fragmentation pattern resultsgiven in Table 1, the molecular formulae C8H6O4, C8H6O5, andC7H6O3, respectively, were assigned. The common fragment ion,m/z 93, was detected from all three of these metabolites, providingevidence for a phenolic-type fragment, and indicated that thesemetabolites appeared to represent single-aromatic-ring metabo-lites of benz[a]anthracene biotransformation.

In the case of [M � H]� � 181, a major product ion scandiagnostic fragment, m/z 135 (26%), indicating a loss of 46 Da wasrevealed (Fig. 5B). When the MS is operated in negative ionizationmode, a loss of 46 Da from the deprotonated molecular ion isindicative of an alpha-hydroxy carboxylate ion. Alpha-hydroxycarboxylic acids have been shown to dissociate to produce productions that represent a neutral loss of 46 Da (CH2O2) and the pro-duction of an enolate ion when hydrogen(s) are present beta to thecarboxyl moiety (45, 46). That the metabolite corresponding to[M � H]� � 181 may be an alpha-hydroxy carboxylic acid wasfurther confirmed by the observation of a major fragment corre-sponding to m/z 107 (85%), which indicated a net loss of 74 Da(M� � CO2 � CHOH) and represented a bond cleavage event

TABLE 1 Fragmentation ions revealed by LC/ESI(�)-MS/MS product ion scanning analyses of metabolites produced from the biotransformationof benz[a]anthracene by strain KK22

Parent ion[M � H]� (m/z)

tR(s)(min)a

CID(eV)b

Diagnostic fragments from product ionscanning analyses (m/z [ion, % relativeintensity(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

a tR, retention time, corresponding to UV detection at 254 nm.b Collision-induced dissociation energy.c Two peaks were detected with similar fragmentation patterns; relative intensities for both metabolites are given in order of elution.d Increasing the collision energy revealed the presence of m/z 127 ([M � H]� � 241).e Structural confirmation was conducted via analyses of authentic standards using the SRM mode.f The positions of hydroxyl- and carboxy-groups on the aromatic ring(s) were not determined.g Loss 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).

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FIG 4 Pathways proposed for the biotransformation of benz[a]anthracene by strain KK22. Metabolites in brackets were not identified in the culture me-dium.

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between the alpha carbonyl and the beta carbon on the propanoicacid side chain of the proposed metabolite. Other CID fragmen-tation products were also revealed, and after all fragments wereconsidered, this metabolite was proposed to be 2-hydroxy-3-(2-hydroxyphenyl)propanoic acid. The fragmentation pattern, mo-lecular structure, and proposed fragmentation map for this bio-transformation product are summarized in Fig. 5B.

Diagnostic fragmentation pattern analyses of [M � H]� � 165showed 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 thesingle-aromatic-ring metabolite corresponding to [M � H]� �181, combined with the diagnostic fragmentation pattern ob-served from analyses of [M � H]� � 165, the identity of [M �H]� � 165 was proposed to be 3-(2-hydroxyphenyl)propanoicacid. Finally, in the case of [M � H]� � 137, major fragmentswhich indicated losses of 44 Da, m/z 93, and 28 Da, m/z 109, wereobserved, and the identity of [M � H]� � 137 was confirmed tobe salicylic acid after comparison to the results of a product ionscanning analysis of a salicylic acid authentic standard solution inwhich the retention times and mass spectra were identical. Thedetailed results of all product ion scanning analyses for metabo-lites discussed are assembled in Table 1.

Identification of 1,2- and 2,3-hydroxy-naphthoic acids byLC/ESI(�)-MS/MS SRM mode. At least one peak correspondingto the presence of [M � H]� � 187 was revealed by LC/ESI(�)-MS to be a major ion in sample extracts. As describedabove, product ion scanning of [M � H]� � 187 revealed that m/z143 occurred as a major fragmentation ion, and this was con-

firmed by analyses of authentic hydroxy-naphthoic acid stan-dards. Based upon the matching fragmentation patterns and thelack of major precursor ions, it was concluded that the biotrans-formation product that corresponded to [M � H]� � 187 was ahydroxy-naphthoic acid, yet it was not clear if the peak repre-sented multiple coeluting isomers of hydroxy-naphthoic acids orwas a single hydroxy-naphthoic acid metabolite. At the same time,unambiguous identification of this key metabolite would also al-low for a better understanding of the upstream biotransformationpathway of benz[a]anthracene by strain KK22. To investigate thisquestion further, new HPLC separation conditions were devised,and a mass method that employed the SRM mode to allow forselective identification of metabolites using the transition 187 to143 m/z was developed. Results of SRM mode analyses of sampleextracts revealed two peaks and indicated that coelution had orig-inally occurred. As shown in Fig. 6, after comparisons of the elu-tion times of three authentic hydroxy-naphthoic acid standards tobenz[a]anthracene biodegradation sample extracts, 1-hydroxy-2-naphthoic acid and 2-hydroxy-3-naphthoic acid were identifiedas metabolites of benz[a]anthracene.

Detection of these two metabolites confirmed further that ben-z[a]anthracene biotransformation occurred via both linear kata-type and angular kata-type enzymatic attacks on the benz[a]an-thracene molecule at the 1,2- and/or 3,4-carbon position, whichresulted in 2-hydroxy-3-naphthoic acid, and at the 8,9- and/or10,11-carbon position which resulted in 1-hydroxy-2-naphthoicacid, respectively. Based upon these and the previously describedresults, a pathway for the biotransformation of benz[a]anthraceneby strain KK22 was proposed in Fig. 4. This pathway highlights

FIG 5 ESI(�)-MS/MS spectra acquired by product ion scanning analysis of benz[a]anthracene biotransformation product with a tR of 3.9 min, correspondingto [M � H]� � 247 (A) and benz[a]anthracene biotransformation product with a tR of 2.3 min, corresponding to [M � H]� � 181 (B). Both products were fromorganic extracts derived from acidified culture medium; collision energies were 20 eV in each case. Proposed molecular structures and fragmentation map areshown in panel B.

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that at least two o-hydroxy-triaromatic acids were formed leadingto 2-hydroxy-3- and 1-hydroxy-2-naphthoic acids, after whichone or both of these compounds were further biotransformed intosingle-aromatic-ring metabolites. At the same time, it also in-cludes metabolite(s) that occurred via both linear kata-type andangular kata-type oxidations on the same molecule, as repre-sented by the metabolite(s) corresponding to [M � H]� � 247.

DISCUSSION

Biotransformation of benz[a]anthracene by strain KK22 pro-ceeded through initial oxidative steps via carbon positions 1,2and/or 3,4 and 8,9 and/or 10,11. Initial oxidations at some of thesecarbon positions of benz[a]anthracene have been reported previ-ously but only for three naturally occurring bacteria, a sphin-gomonad and two mycobacteria (23–25). The first ring fission bystrain KK22 occurred through ortho cleavage resulting in metab-olite(s) corresponding to [M � H]� � 291, which were proposedherein to be o-ethylenecarboxy-phenanthroic and -anthranoic ac-ids. In a pathway that is analogous to transformation of ben-z[a]anthracene by a 8,9- and/or 10,11-carbon position attack bystrain KK22, ortho cleavage of the anthracene biotransformationproduct 1,2-dihydroxyanthracene to 3-(2-carboxyvinyl)naphtha-

lene-2-carboxylic acid was proposed to occur by M. vanbaaleniistrain PYR-1 (13). At the same time, an initial ortho cleavage stepthat is analogous to a 1,2- and/or 3,4-carbon position attack onbenz[a]anthracene by strain KK22 was reported recently forphenanthrene biotransformation to 1-(2-)(2-carboxy-vinyl)-naphthalene-2-(1-)carboxylic acids by Arthrobacter sp. strainP-1-1 (15). Because the downstream metabolites of benz[a]an-thracene biotransformation by strain KK22, 1-hydroxy-2-naph-thoic acid and 2-hydroxy-3-naphthoic acid, were identified in theculture medium, the o-hydroxytriaromatic acids, phenanthroicand anthranoic acid, were also produced earlier. These o-hydroxy-triaromatic acids may have originated only from upstream metab-olites that were oxidized via both linear kata-type and angularkata-type attacks on the benz[a]anthracene molecule, and this isrepresented in the proposed pathway in Fig. 4. Indeed, two peakswhich were detected in the greatest abundance, corresponding to[M � H]� � 237, were proposed to be the products from thebiotransformation of the o-ethylenecarboxy-phenanthroic and-anthranoic acids: o-hydroxy-phenanthroic and o-hydroxy-an-thranoic acids. These acids have been identified only once beforeas products of S. yanoikuyae strain B1 (23).

At least in the case of the o-hydroxy-anthranoic acid(s) pro-

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 oftwo biotransformation products, [M � H]� � 187, of benz[a]anthracene by strain KK22 were established by this retention time comparison.

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duced by strain KK22 from benz[a]anthracene, a second orthocleavage event occurred to produce 2-hydroxy-3-naphthoic acidfrom 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 inthe biotransformation of anthracene by M. vanbaalenii strainPYR-1 in 2001 (13), and this is the first documentation of thismetabolite from benz[a]anthracene. An intermediate between theproduction of o-hydroxy-phenanthroic acid(s) and 1-hydroxy-2-naphthoic acid was not identified in this study. Additionally, therewas no evidence to support a 5,6-carbon position attack on ben-z[a]anthracene by strain KK22 as was demonstrated by Moody etal. (13), and this was not unexpected because strain KK22 ap-peared unable to biotransform the PAH pyrene (unpublisheddata). Pyrene does not possess any kata-annelated aromatic rings.It has been discussed that K-region PAH oxidation may be uniqueto pyrene-degrading nocardioform bacteria (among hetero-trophic organisms) (16).

The recovery of the metabolite corresponding to [M � H]� �247, whose mass analyses revealed that it was most likely the prod-uct of enzymatic attack via both ends of the benz[a]anthracenemolecule, provided another line of evidence that strain KK22metabolized 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-annelatedthree-ring PAH phenanthrene have been isolated (7–16), demon-stration of benz[a]anthracene biodegradation accompanied bymetabolite identification has been shown to occur in only threenaturally occurring isolates. From these studies, more than onering cleavage step in the biotransformation pathway of benz[a]an-thracene was shown to occur only by strain PYR-1 (25). Results ofexperiments with strain KK22 reported herein indicated that thefirst and second cleavage events appeared to have occurred byortho mechanisms ultimately leading to two- and one-ring metab-olites, which in addition to the identification of two o-hydroxy-naphthoic acids were representative of previously unreported me-tabolites of benz[a]anthracene biotransformation. These resultsallowed for the construction of a new pathway for the biodegra-dation of benz[a]anthracene by this bacterium.

Overall, this work extended our understanding of the pathwaysby which the HMW PAH benz[a]anthracene may be transformedby a sphingomonad bacterium and demonstrated the utility ofapplying LC/ESI-MS/MS to study bacterial biotransformations inthe field of environmental microbiology. There are few data avail-able in regard to the use of soft ionization techniques (ESI) toevaluate biotransformation products of bacterial pollutant bio-degradation, and this research required the development of meth-ods and data interpretation approaches that will be useful to applyto future studies in this field. Combined with nuclear magneticresonance or high-resolution mass investigations, for example,the utility of the approach will be strengthened.

ACKNOWLEDGMENTS

We thank Youko Utsuno for technical assistance. We are grateful to theanonymous reviewers for their detailed and helpful suggestions.

This work was supported in part by Yokohama City University Stra-

tegic Research Grant K2002 and the Japanese Society for the Promotion ofScience NEXT program (grant GS023).

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