Fil: Sack, Ute. Institute of Microbiology. Friedrich Schiller University; GermanyFil: Heinze, Thomas M.. National Center for Toxicological Research, Food and Drug Administration. Arkansas; ArgentinaFil: Deck, Joanna. National Center for Toxicological Research, Food and Drug Administration. Arkansas; ArgentinaFil: Cerniglia, Carl E.. National Center for Toxicological Research, Food and Drug Administration. Arkansas; ArgentinaFil: Cazau, María Cecilia. Institute of Botany. University of La Plata; ArgentinaFil: Fritsche, Wolfgang. Institute of Microbiology. Friedrich Schiller University; German

Cazau, María Cecilia

Cerniglia, Carl E.

Deck, Joanna

Fritsche, Wolfgang

Heinze, Thomas M.

Sack, Ute

PubMed

Naturalis

APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/97/$04.0010July 1997, p. 2906–2909 Vol. 63, No. 7Copyright © 1997, American Society for MicrobiologyNovel Metabolites in Phenanthrene and PyreneTransformation by Aspergillus nigerUTE SACK,1* THOMAS M. HEINZE,2 JOANNA DECK,2 CARL E. CERNIGLIA,2MARIA C. CAZAU,3 AND WOLFGANG FRITSCHE1Institute of Microbiology, Friedrich Schiller University, 07743 Jena, Germany1;National Center for Toxicological Research, Food and Drug Administration,Jefferson, Arkansas 720792; and Institute of Botany, Universityof La Plata, La Plata 1900, Argentina3Received 11 December 1996/Accepted 10 April 1997Aspergillus niger, isolated from hydrocarbon-contaminated soil, was examined for its potential to degradephenanthrene and pyrene. Two novel metabolites, 1-methoxyphenanthrene and 1-methoxypyrene, were iden-tified by conventional chemical techniques. Minor metabolites identified were 1- and 2-phenanthrol and1-pyrenol. No 14CO2 evolution was observed in either [14C]phenanthrene or [14C]pyrene cultures.Polycyclic aromatic hydrocarbons (PAHs) are hydrophobicenvironmentally persistent compounds and are genotoxic at upto four or five fused aromatic rings (6). Several wood-decayingfungi are capable of detoxifying and mineralizing PAHs (3, 4,19, 21, 22). Little is known about the ability of soil fungi tometabolize PAHs. Most attention has been focused on deu-teromycetes, such as Penicillium spp. (14, 18) and Aspergillusspp. (5, 20, 23). The zygomycete Cunninghamella elegans hasbeen studied extensively to investigate possible pathways forthe detoxification of PAHs. In the case of phenanthrene andpyrene, C. elegans is able to transform these PAHs to trans-di-hydrodiols, phenols, and quinones and to form glycoside con-jugates (7–9). Wunder et al. (23) reported that an Aspergillusniger strain transformed pyrene to 1-pyrenol, 1,6- and 1,8-py-renequinone, 1,6- and 1,8-dihydroxypyrene, 1-pyrenyl sulfate,and 1-hydroxy-8-pyrenyl sulfate. In soil, such ring oxidationproducts are able to form bound residues through enzymatic orspontaneous reactions with soil organic matter (17). Ring ox-idation products of PAHs are also substrates for bacteria orother indigenous soil microorganisms to mineralize and thusenhance mineralization of PAHs (10). This paper describesnovel metabolites of phenanthrene and pyrene degradation byan A. niger strain isolated from mineral oil-contaminated soil inLa Plata, Argentina.Metabolism of [14C]phenanthrene and [14C]pyrene. Kinet-ics of [ring U-14C]phenanthrene (4.33 GBq/mmol; radiochem-ical purity after thin-layer chromatography in benzene, 96.5%;Amersham Buchler, Braunschweig, Germany) and [4,5,9,10-14C]pyrene (2.07 GBq/mmol; radiochemical purity, 98%; Am-ersham Buchler) metabolism were determined with 300-mlErlenmeyer flasks containing 100 ml of Czapek-Dox medium(12) that were inoculated with 15-mycelium 1-cm2 plugs ofmalt agar cultures of A. niger van Tieghem (strain b52, DSM11167) isolated from mineral oil-contaminated soil of an oilrefinery in La Plata, Argentina, as described previously (1).After 24 h, 100 ml of phenanthrene or pyrene solution (37 kBqof radiolabelled and unlabelled PAH with a final concentrationof 5 mg/100 ml, dissolved in dimethylformamide) was added toeach sample. The flasks were aerated continually with CO2-free, humidified air. The air was passed through dimethylfor-mamide to trap volatile organic substances. The liberated14CO2 was trapped in 2 N NaOH and quantified as describedpreviously (19). Sterile controls to detect abiotic PAH decreasewere prepared. The experiments were conducted in triplicatein the dark at 24°C. For mass balance analysis, culture fluid andmycelia were extracted with ethyl acetate after an incubationtime of 63 days by the procedure of Sack et al. (19). Thecombined extracts were dried and analyzed by high-pressureliquid chromatography (HPLC) as described previously (19).One-milliliter HPLC fractions were collected every 1 min inscintillation vials and mixed with scintillation cocktail, and theradioactivity of each fraction was determined by liquid scintil-lation counting (19). After extraction, mycelia were air dried,homogenized, and combusted in an oxidizer (Biological oxi-dizer OX 500; Zinsser Analytic) (19).Table 1 illustrates the mass balance analysis of [14C]phenan-threne and [14C]pyrene after 63 days of incubation. No 14CO2evolution in either phenanthrene or pyrene cultures was ob-served; since A. niger does not contain extracellular radicalgenerating enzymes, such as ligninases, the lack of CO2 liber-ation is not surprising. In phenanthrene metabolism, 3.1%(abiotic control, 19.4%) of radioactivity was from volatile or-ganic substances, and in pyrene metabolism, 0.1% (abioticcontrol, 1.1%) was from volatile organic substances. Less than10% of initial radioactivity was detected in the aqueous phases.A total of 33.9% (phenanthrene) and 10.1% (pyrene) of ra-dioactivity was cell associated in mycelia (final dry weight, 3.5g/liter) and was considered to be unreacted phenanthrene andpyrene. When A. niger was grown in the presence of [14C]phenanthrene, 51.4% of the radioactivity initially added wasfound in the ethyl acetate extract; 20% of this extract was frompolar metabolites, and 10% was from metabolites with a re-tention time higher than that of phenanthrene. A total of88.2% of the radioactivity initially added was ethyl acetateextractable after cultivation of A. niger in the presence of [14C]pyrene; 2.5% of this extract was polar metabolites, and 3% wasless polar than pyrene.Isolation, detection, and identification of metabolites. Toidentify phenanthrene and pyrene metabolites, the fungus wascultivated in 1,000-ml Erlenmeyer flasks containing 400 ml ofCzapek-Dox medium and inoculated with 50-mycelium plugs.Phenanthrene or pyrene (20 mg) was dissolved in 500 ml ofdimethylformamide and added to one flask after 24 h of incu-* Corresponding author. Mailing address: Institute of Microbiology,Friedrich Schiller University, Philosophenweg 12, 07743 Jena, Ger-many. Phone: 49-3641-630950. Fax: 49-3641-631237. E-mail: usack@merlin.biologie.uni-jena.de.2906 on April 18, 2016 by guesthttp://aem.asm.org/Downloaded from bation in darkness. The combined extracts were dried overanhydrous sodium sulfate, and the solvent was evaporatedunder reduced pressure at 40°C. The residue was dissolved inmethanol and analyzed by HPLC (Merck, Darmstadt, Ger-many) with a model L 4500 diode array detector operating at254 nm as described previously (19). Gas chromatography-mass spectrometry analyses were performed with a VarianSaturn II (Varian Instrument Group, Walnut Creek, Calif.) iontrap mass spectrometer (electron ionization mode with an elec-tron energy of 70 eV; manifold temperature, 210°C). The in-strument was scanned from 50 to 450 Da in 1 s. A DB 5 mscapillary column (0.25 by 30 m, 0.25 mm thick) (Durabond) wasused for gas chromatography separation. 1H-nuclear magneticresonance (1H-NMR) spectra were obtained at ambient tem-perature with a Bruker AM500 spectrometer (Bruker Instru-ments, Billerica, Mass.). Samples were dissolved in deuteratedacetone, with the residual proton signal assigned as 2.04 ppm.Data acquisition conditions were as follows: spectrometer fre-quency, 500 MHz; data size, 32K; sweep width, 7,042 Hz; filterwidth, 8,900 Hz. Data were zero filled to 64K for determina-tion of coupling constants. 1H-NMR resonance assignmentswere made by proton integration, homonuclear decouplingexperiments, and nuclear Overhauser effect experiments.Phenanthrene was metabolized to small amounts of 1- and2-phenanthrol by A. niger. The metabolites were identified bycomparing their relative HPLC retention times (23.1 and 21.7min), UV-visible absorption spectra, and mass spectra to pre-viously published spectra and electron-impact mass-spectralproperties (2, 5) after 63 days of incubation. The major me-tabolite (which made up 8.2% of the radioactivity among themetabolite fraction of the ethyl acetate extract) identified hada retention time of 36.7 min. This indicated a metabolite lesspolar than phenanthrene. It had a UV absorption spectrumwith maximum wavelength (lmax) values at 251, 292, 302, 331,and 346 nm, and a mass spectrum (Fig. 1) with a molecular ion(M1 z) at m/z 208 and fragment ions at m/z 193 (M1 2 zCH3)and m/z 165 (M1 2 zCH3 and 2CO), which is similar to thespectrum reported for 1-methoxyphenanthrene, formed by themarine cyanobacterium Agmenellum quadruplicatum PR-6(15). 1H-NMR spectroscopy analysis was conducted to deter-mine the configuration of this metabolite. Resonance assign-ments (parts per million) and coupling constants (hertz) forthe metabolite are as follows: 4.05 (s, 3, 2OCH3), 7.15 (d, 1,J2,3 5 8.0, J2,4 5 0.6, H2), 7.61 (t, 1, J3,4 5 8.2, H3), 7.62 (m, 1,J7,8 5 7.8, H7), 7.67 (m, 1, J6,8 5 1.4, H6), 7.80 (d, 1, J9,10 5 9.2,H9), 7.96 (d, 1, H8), 8.21 (d, 1, H10), 8.37 (d, 1, H4), 8.77 (d,1, J5,6 5 8.2, J5,7 5 1.4, H5). The metabolite was identified as1-methoxyphenanthrene.In pyrene metabolism by A. niger, two major metabolites andone minor metabolite were identified. The minor metabolitehad an HPLC retention time of 13.5 min and a UV absorptionspectrum with lmax values at 244, 267, 275, 333, 345, 372, and389 nm, which were comparable to those published for 1,6-dihydroxypyrene (13, 23). The metabolite was not formed inquantities sufficient for isolation or further structural charac-terization. Major metabolite I had an HPLC retention time(27.8 min), a UV absorption (with lmax at 241, 276, and 341nm), and a mass spectrum consistent with those of synthetic1-pyrenol. Major metabolite II (which made up 10.2% of ra-FIG. 1. Mass spectrum of 1-methoxyphenanthrene produced by A. niger.TABLE 1. Mass balance analysis of radioactivity initially added(37 kBq) as [14C]phenanthrene and [14C]pyrene to liquidcultures of A. niger after a period of 63 daysaSource ofradioactivityRadioactivity (%)[ring U-14C]phenanthrene[4,5,9,10-14C]pyreneA. niger Control A. niger Control14CO2 0.7 0.3 0.2 0.1Volatile organic substances 3.1 19.4 0.1 1.1Ethyl acetate extract 51.4 67.9 88.2 97.3Aqueous phase 9.6 7.8 1.2 0.1Mycelia 33.9 0 10.1 0Balance 98.7 95.4 99.8 98.6a All values are means (n 5 3) with a standard deviation of ,3%.VOL. 63, 1997 PHENANTHRENE AND PYRENE TRANSFORMATION BY A. NIGER 2907 on April 18, 2016 by guesthttp://aem.asm.org/Downloaded from dioactivity among the metabolite fraction of the ethyl acetateextract) had an HPLC retention time of 40.6 min and a UVabsorption spectrum with lmax values at 232, 266, 275, 341, and372 nm. The mass spectrum (Fig. 2) had a molecular ion (M1 z)at m/z 232 and fragment ions at m/z 217 (M1 2 zCH3) and m/z189 (M1 2 zCH3 and 2CO). The similarity of the UV spec-trum to that of 1-pyrenol and the similarity of the mass-spectralfragmentation to that of methoxyphenanthrene indicated thatthe metabolite was methoxypyrene. The structure was identi-fied by 1H-NMR spectroscopy as 1-methoxypyrene. Resonanceassignments (parts per million) and coupling constants (hertz)for the metabolite are as follows: 4.20 (s, 3, 2OCH3), 7.73 (d,1, J2,3 5 8.4, H2), 7.94 (d, 1, H5), 7.99 (t, 1, J7,8 5 7.5, H7), 8.03(d, 1, J4,5 5 9.0, H4), 8.10 (d, 1, J9,10 5 9.2, H9), 8.15 (d, 1,J6,7 5 7.7, J6,8 5 1.1, H6), 8.18 (d, 1, H8), 8.23 (d, 1, H3), 8.43(d, 1, H10).To our knowledge, 1-methoxyphenanthrene and 1-methoxy-pyrene are novel metabolites in the transformation of PAHs byfungi which have not previously been isolated. Narro et al. (15)described the formation of 1-methoxyphenanthrene by the ma-rine cyanobacterium A. quadruplicatum PR-6 and postulated amethylation of 1-phenanthrol. 1-Phenanthrol and 1-pyrenolproduction by A. niger were identified, and these PAHs couldbe precursors of the methoxylated PAH derivatives. To date,methylation reactions of hydroxyl groups of monoaromaticcompounds (chloro- and nitrocatechols) have been reportedonly for fungi such as Penicillium frequentans (11) and Phan-erochaete chrysosporium (16).A. niger metabolized phenanthrene and pyrene like othersoil fungi. Casillas et al. (5) reported 1-phenanthrol, 2-phenan-throl, and phenanthrene trans-9,10-dihydrodiol as major me-tabolites from the metabolism of phenanthrene by A. niger.Glycoside, glucuronide, sulfate, or xyloside conjugate forma-tion, as described by Wunder et al. (23), was not observed withA. niger after a cultivation period of 63 days. The formation ofthe conjugates may be growth medium dependent. Cerniglia etal. measured degradation of phenanthrene by C. elegans; they(7) reported that a maximum accumulation of the glycosideconjugate of 1-phenanthrol occurred 72 h after the addition ofphenanthrene and subsequently declined.The formation of methoxylated metabolites may be one wayfor fungi to detoxify 1-phenanthrol and 1-pyrenol, since Narroet al. (15) have demonstrated inhibition of A. quadruplicatumgrowth by 9-phenanthrol with an alga lawn bioassay. However,no effect on growth after addition of 9-methoxyphenanthrenehas been observed.This work was supported by Bundesministerium fu¨r Bildung, Wis-senschaft, Forschung und Technologie (grant 0310394A) and by agrant from DAAD.We thank A. Henderson (Jefferson, Ark.) for valuable suggestions.REFERENCES1. Alef, K. 1994. Biologische Bodensanierung, p. 96–99. VCH Press, Weinheim,Germany.2. Bao, Z., and S. K. Yang. 1991. Liquid chromatographic separation of iso-meric phenanthrols on monomeric and polymeric C18 columns. J. 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Sterner, and H. Anke. 1994. Metabolism of thepolycyclic aromatic hydrocarbon pyrene by Aspergillus niger SK 9317. Appl.Microbiol. Biotechnol. 42:636–641.VOL. 63, 1997 PHENANTHRENE AND PYRENE TRANSFORMATION BY A. NIGER 2909 on April 18, 2016 by guesthttp://aem.asm.org/Downloaded from 

Novel Metabolites in Phenanthrene and Pyrene Transformation by Aspergillus-Niger

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Novel Metabolites in Phenanthrene and Pyrene Transformation by Aspergillus-Niger

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