The oxidation of pyrene and benzo[a]pyrene by nonbasidiomycete soil ...

The oxidation of pyrene and benzo[a]pyrene by nonbasidiomycete soil fungi Loren Launen, Linda Pinto, Christine Wiebe, Eberhard Kiehlmann, and Margo Moore

Abstract: The purpose of this study was to determine the ability of nonbasidiomycete soil fungi to oxidize pyrene (four rings) and benzo[a]pyrene (BaP) (five rings). Fungi were isolated from five different soils in which the polycyclic aromatic hydrocarbon content ranged from 0.8 to 80 pg/g dry soil. Approximately 50% of the isolates in all sites were able to oxidize pyrene. The pyrene-oxidizing species belonged to all fungal divisions except basidiomycetes. The most common were Penic-illiumspp. of the subgenus Furcaturn and I-ac~enzosum these dominated the more contaminated soils. Penicillium jc~nthinellumand Spin~ephalc161~trum exhibited the most rapid rates of pyrene oxidation. The major pyrene metabolites were identified by proton NMB and mass spectrometry as 1-pyrenol, I ,6- and 1,8-pyrenediol,and the 1,6- and 1.8-pyrenequinones. A high correlation was found between the ability to oxidize pyrene and B e . As with pyrene, approximately 50% of the fungal isolates tested oxidized BaP to 9-hydroxy-BaP. Eighty percent of the pyrene-oxidizing strains were also able to metabolize BaP. Key wet-d,~: fungi, polycyclic aromatic hydrocarbons, biotransformation. bioremediation, cytochrome P-450.

RCsumCI : Determiner l'aptitude des champignons du sol non-basidiomycktes B oxyder le pyrkne (quatre anneaux) et le benzo[a]pyrkne (BaQ) (cinq anneaux), tel a CtC le but de cette Ctude. Les champignons ont CtC isolCs de einq sols diffkrents dans lesquels les teneurs en hydrocarbures aromatiques polycycliques ont variC de 0,8 B 80 pg/g de sol sec. Approximativement 50% des isolats de tous les sols ont kt6 capables d'oxyder le pyrbne. Les espkces qui ont oxydC le pyrkne appartenaient B toutes les divisions fongiques, sauf les basidiomycktes. Les espbces de Penicillium du sous-genre Furcatunz ont kt6 les plus communes: elles ont domink les sols les plus contaminks. Le Perzicilll'z~rnjanthinellum et le Syncephcilastrum lucenzosurn ont prCsentC les taux les plus rapides d'oxydation du pyrkne. Les principaux metabolites du pyrkne ont CtC identifiCs par rCsonance magnktique nuclCaire et par spectrographic de masse comme suit : 1-pyrCnol. 1,6- et 1,8-pyrknediols et 1,6- et 1,8-pyritnequinones.Une corrClation ClevCe a CtC trouvCe entre l'aptitude B oxyder le pyrkne et le BaP. Comme pour le pyrkne, environ 50% des isolats fongiques testes ont oxydC le BaP en 9-hydroxy-BaP. Des diverses souches qui ont oxydC le pyrbne, 80% d'entre elles oilt Cgalement pu mCtaboliser le BaQ. mot,^ ~ 1 : champignons, 4 ~ ~ hydrocarbures aromatiques polycycliques, biotransformation, biodkgradation, cytochrome P-450. [Traduit par la RCdaction]

Introduction Polycyclic aromatic hydrocarbons (PAH) comprise a class of compounds consisting of three or more benzene rings fused in linear, angular, or cluster foimations, composed solely of car-

Received October 5, 1994. Revision received February 8, 1995. Accepted February 13, 1995.

L. Launen, L. Pinto, and Christine Wiebe. Department of Biological Sciences, Simon Fraser University, Bumaby, BC V5A 1 S6, Canada. E. Kiehlmann. Department of Chemistry, Simon Fraser University, Bumaby, BC V5A 1 S6, Canada. M. Moore.' Department of Biological Sciences, Simon Fraser University, Burnaby, BC V5A 1S6, Canada. Author to whom all comspondence should be addressed. Can. 3. Microbial. 41: 477-488 (1995). Printed in Canada / ImprimC au Canada

bon and hydrogen atoms. They form as a result of the incomplete coinbustion of fossil fuels and are therefore present in a variety of products such as tar, coal, soot, petroleum, cutting oils, and tobacco smoke (International Agency for Research on Cancer 1983). A number of PAH are known mutagens possessing significant genotoxici ty (Bos et al. 1988; MerschSundermann et al. l992), and several have been shown to be powerful chemical carcinogens (Levin et al. 1976; Buening et al. 1978). PAH released into the air may be dispersed into groundwater by rain or gravity and, owing to their hydrophobicity, accumulate in sediments. As abiotic factors do not contribute significantly to the loss of PAH with more than three rings froin soil (Park et al. 1990), much effort has been directed toward examining the contribution of microorganisms to PAH degradation. However, when compared with one- to three-ring compounds

Can. J. Microbial. Voi. 41, 1995

such as benzene, naphthalene, biphenyl, phenanthrene, and fluorene (Boldrin et al. 1993; Cerniglia 1981; MacGillivray and Shiaris 1993; Pothuluii et al. 1993; Shiaris 1989), compounds that contain more than three fused aromatic rings are relatively resistant to microbial degradation. Half-lives of the five- and six-membered ring PAH have been estimated to be on the order of years (Park et al. 1990; Herbes and Schwa11 19'78; Shiaris 1989). Bacterial species that can oxidize PAH containing more than three rings have been isolated. For example, a Beijeriltckia sp. has been found that metabolized benzo[a]pyrene and benz[a]anthracene to cis-dihydrodiols (Cerniglia 1981), and two different Mycohacteriunz strains have been isolated that can mineralize the four-ring PAH, pyrene (Boldiin et al. 1993; Heitkamp et al. 1988). Soil fungi have been shown to effectively degrade longchain, aliphatic hydrocarbons (reviewed by Leahy and Colwell 1990) and aromatic hydrocarbons with two or three rings; however, the range of fungi capable of oxidizing PAW containing four or more rings is less well characterized. For example, recent work on PAH biotransfomation by yeasts isolated from coastal sediments indicated that approximately 50% of the isolates were capable of phenanthrene biotransformation (three rings). In contrast, only two of the yeast strains were able to oxidize benz[a]anthracene (four rings) and this oxidation was very limited (MacGillivray and Shiaris 1993). Exteilsive studies of PAH oxidation by the zygomycete filamentous fungus Cunninghamella elegans have been done by Cerniglia and his colleagues. They demonstrated that pure cultures of this fungus were capable of regio- and stereoselective oxidation of naphthalene (Cerniglia et al. 19'78), fluorene (Pothuluri et al. 1993), fluoranthene (Pothuluri et al. 1990), benzo[a]pyrene (Cerniglia and Gibson 1980), pyrene (Cerniglia et al. 1986), as well as methylated anthracenes (Cerniglia et al. 1990). Other studies of PAH metabolism by yeast reported that Sacclaaromyces cerevisiue and Candidu lipolytica metabolized benzo[a]pyrene (Cerniglia and Gibson 19'79; Cerniglia and Crow 1981; King et al. 1984). The pattern of the fungal PAH metabolites observed in the above studies was similar to that of compounds which are known to arise from mammalian cytochrome P-450 monooxygenase oxidation of PAH, suggesting that fungal oxidation is mediated by a similar monooxygenase enzyme. Another oxidation pathway for PAH in fungi has also been demonstrated: extracellular lignin peroxidase enzymes produced by basidiomycete white-rot fungi (e.g. Bha~zemchrrete,Tranzetes, Bjerkandel-a) have been shown to oxidize a variety of PAH including phenanthrene, anthracene, and benzo[a]pyrene (Bumpus 1989; Field et al. 1992; Haemmerli et al. 1986; Hammel et al. 1992, 1986). The oxidation has been shown to result in ring fission, suggesting that these fungi may be able to contribute to PAW mineralization of these compounds under certain conditions (Bumpus et al. 1985; Hammel et al. 1991, 1992). The purpose of this study was to screen fungi isolated from a range of petroleum-contaminated soils and determine their ability to oxidize pyrene and benzo[a]pyrene. Because we wished to determine the ecological importance of nonbasidiomycete soil fungi in the oxidation of sediment-borne PAH, we focussed on these genera only. In addition to the determination of the identity and distribution of PAH-metabolizing

strains in the soil sites, the major metabolites of pyrene and benzo[a]pyrene were isolated and identified for selected species.

Materials and methods Soil sampling Petroleum-contaminated soil was collected from an oil disposal site at a refinery in Burnaby, B.C. The disposal site was an area of land of approximately 2500 m3 designated for the disposal of postdistillation water and had been in continuous use from 1976 until sampling in 1992. Topsoil analysis performed by the refinery had shown that the soil contained all components of petroleum hydrocarbons: asphaltenes, paraffins, resins, and aromatics. Soil pH ranged from 5.9 to 6.6. Soil was collected from five different sites using gloves and sterile scoops and stored in sterile plastic bags at 4OC. The five samples were collected according to their proximity to the presumed most heavily contaminated area (see Fig. 1). Sample 1 was taken at the base of the site (approximately 1.0 In below the surface of the topsoil that had been removed by a tractor on the same day). Sample 2 was taken from the immediate periphery of the site. Sample 3 contained the most oily topsoil that resembled tarballs. Sample 4 was less oily soil from approximately 0.5 m depth, and sample 5 was collected 1.5 m away from the disposal site on a grassy embankment. Chemical analyses of the soils are listed in Table 1. Isolation of fungi from soil samples For each site, approximately 0.1 g of soil was sprinkled per MYPD agar plate (0.3%) malt extract, 0.3% yeast extract, 0.5% peptone, and 1% dextrose, pH 6.0) supplemented with filter-sterilized penicillin G (60 kg/mL) and streptomycin sulphate (100 pg/mL). All plates were incubated at room temperature. After various periods of growth (up to several weeks), fungal colonies were selected and serial replated on MYPD agar (without penicillin or streptomycin) until pure cultures were obtained. As no fungi were isolated from site 3 using the above method, site 3 soil was also sprinkled onto minimal salts plus 1% glucose agar supplemented with pyrenebenzo[a]pyrene (0.05 mg1mL of each) and incubated as above. This technique yielded four isolates that were replated on MYPD without added PAH until pure cultures were obtained. Screening of fungal strains for PAH oxidation MYPD (10 mL, pH 6.0) in 50-mL Erlenmeyer flasks was inoculated with an agar plug from fresh fungal cultures, and the cell suspension was allowed to grow for 48 h at 24°C at 240 rpm on a rotary shaker. Pyrene was then added to a final concentration of 0.1 mglmb, from a freshly prepared 100x concentrated stock solution made as follows: pyrene was dissolved in dimethylformamide (114 volume) and a sterile 25% solution of Tween 80 in water was added (314 volume). Two separate control experiments were run for each fungal species tested. One contained only pyrene in MYPD without cells (autooxidation control) and the other contained cells but no pyrene (cell material control). Metabolism studies; screening fungi for pyrene and benzo[a]pyrene metabolites Thin-layer cl~romatograpl~y The cultures were grown for '7 days and then acidified to approximately pH 4.0 with HC1. The suspensions were

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Fig. 1. The proximity of sites 1-5 (which were the source of the soils used in this study) to the area that received the petroleum-contaminatedrefinery waste.

Dump Site

extracted overnight with 1 volume of ethyl acetate followed by two further extractions the next day. Extracts were dried over sodium sulphate and evaporated in vacuo. Samples were redissolved in ethyl acetate and spotted onto alumina-backed silica gel 60 F254 thin-layer chromatography plates using hexane : ethyl acetate (1 :1) as the mobile phase. Polar metabolites were detected with ultraviolet (UV) light and ammonium molybdate spray. The minimum detectable levels sf pyrene (I) and the major metabolite, 1-pyreno1 (2), were 50-1 00 ng. For the benzo[a]pyrene (BaP) metabolism studies. the samples were prepared in the same manner as for pyrene, with the exception that all manipulations were done in dim light and all incubations and extractions were carried out in covered flasks. The final BaP concentration was also 0.1 mg/mL in MYPD. An autooxidation control flask was included for each strain tested to ensure that abiotic factors did not cause BaP oxidation. Radio high-pelformance liquid chromatography Erlenmeyer flasks (50 mL) containing 5 mL of MYPD were inoculated with 2 x 106 spores/mL of Perzicillium janthinellurn (No. 403) taken from MYPD agar plates. After 48 h at 24OC, 240 rpm, unlabelled pyrene (0.1 mg/mL) was added to the suspension as described above. Radiolabelled [14C]pyrene (32.2 mCi/mmol; 1 Ci = 37 SBq) (Chemsyn Laboratories, Lenexa, Kans.) was added such that each flask contained 5 pCi of [14C]pyrene and the cultures were incubated at 24OC for a further 36 h. Cell suspensions were extracted exhaustively with ethyl acetate and the pyrene metabolites were separated by high-performance liquid chromatography (HPLC) using a Novapak C18 column and a Waters model 600 controller. Metabolites were detected at 254 nm using a Waters model 486 tunable absorbance detector. The gradient was as follows: 50:50 methanol:H20 to 100% methanol as a linear gradient over 35 min and followed by 100% methanol for a further 15 min (flow rate = 1.0 mL/min at ambient temperature). Column

eluate was collected every 26) s, BCS scintillation cocktail was added (5 mL) to the fractionated samples, and 14Cwas counted using a Beckman LS 3801 liquid scintillation counter with quench correction. Residual particulates were dried and combusted for 4 min, using a Biological Harvey Ox 300 oxidizer and counted as above. Aqueous material (postextraction) was sparged with N2 and 14C counts were done.

Isolation and identification of PAH metabolites Pyrene metubnlites Two litres of MYPD was inoculated with 200 mL of 48-h-old liquid cultures of Syncephalastrum mcemosum (site 2) or Penicillium janthinellunz (No. 119) and cultures were allowed to grow a further 48 h. Cells were then washed with minimal salts medium plus glucose (per litre: 0.50 g KC1, 0.14 g KH2P04, 1.20 g K2HP04, 2.00 g NaN03, 0.25 g MgS04, 0.01 g FeS04.7H20, 10.00 g glucose, pH 7.0) and resuspended in 2 L of fresh medium. Pyrene (0.1 mg/mL) was added at this time and growth was continued at 24OC for 6 days (Syncephalustrum I-acemosum) or 4 days (Penicilli~ana janthinellurn) until metabolites were detected by thin-layer chromatography (TLC) monitoring of the broth. Cells and media were exhaustively extracted with ethyl acetate (the pattern of metabolites was identical in the broth and in the washed cells; therefore, the extracts were later combined), and the extracts were pooled, dried over sodium sulphate, and evaporated in vacuo. Individual metabolites were fractionated by flash chromatography (silica gel 230-400 mesh) using the following solvents: hexane : ethyl acetate (2: 1, 1:1, and B:2), 100% ethyl acetate, and ethyl acetate : acetone (9: 1). The fractions obtained were further purified by flash chromatography and analyzed by proton NNIR and mass spectroscopy. lH-NMR spectra of pure compounds were run in acetone-d6 on a Bruker AMX 400 instrument at 400 MHz using a 5-mm inverse proton probe. Mass spectra (electron impact, E.I.) were obtained using a Hewlett-Packard 5985 gas chromatography - mass

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Table 1. Chemical characteristics of the five soil sites.

Characteristic

Site 2 (Topsoil, immediate periphery)

Site 5 (Topsoil, 1.5 m periphery)

Site 1 (1 m depth, dump site)

Site 3 (Topsoil, tarball, dump site)

Site 4 (0.5 m depth, dump site)

No. of rings in PAH

Mineral oil and grease (pg/g) Total oil and grease (pg/g) Sodium adsorption ratio Moisture (96, w/w) Total PAH (pg/g) Total high MW PAH Total low MW PAH 3-Methylcholanthrene 7,12-Dimethylbenz[n]anthracene Dibenzo[u,h]pyrene Dibenzo[a,i]pyrene Dibenzo[a,b]pyrene Acenaphthylene Anthracene Beilzo[jlfluoranthene Fluorene Acenaphthene Naphthalene Individual PAH present in levels > 1 pg/g Fluoranthene Indeno[1,2,3-c.dIpyrene Phenanthrene Dibenz[a,h]anthracene Benzo[c]pheilanthrene Benz[u]anthracene Dibenzo[h,k]fluoranthene Benzo[alpyrene Pyreile Benzo[g,b~,i]perylene Chrysene NOTE:nd, not determined.

spectrometry (GC-MS) instrument at 70 eV equipped with a direct insertion probe.

Benzo[a]pyrene metabolite Benic.illiua;~zjanthincllunz (No. 403) was incubated with 0.1 mg/mL BaP for 7 days as outlined above and the extracts were separated by preparative TLC. The major metabolite (Rf = 0.59) was scraped and eluted from the silica gel using ethyl acetate. After drying and evaporation under vacuum, the metabolite was redissolved in 95% ethanol and the UV-VIS spectrum recorded on a Milton-Roy Spectronic 3000 diode array spectrophotometer and compared with the spectrum of monohydroxy-BaP standards. Soil analyses Soil analyses were performed Laboratories, Bumab~, B.C. PAH concentrations were determined by GC-MS using U.S. EPA protocol 8270B (Environmental Protection Agency 1992). Briefly, to determine the total oil and grease, mineral oil and grease, and sodium adsorption ratio, sediments were acidified and extracted with dichloromethane in a Soxhlet apparatus overnight. The extract was dried with sodium sulphate and one

portion mixed with excess silica gel. After both filtrates were reduced to dryness, the residue from the silica-treated extract was weighed and expressed as mineral oil and grease. The other residue was weighed and expressed as total oil and grease. The sodium adsorption ratio (SAR) was determined by air drying the soil and mixing it with deionized water in a ratio of 1:2 (w/v) soi1:water. The concentrations (meq) of calcium, magnesium, and sodium ions were measured with an ICAB instrument and used to calculate the SAR: SAR = 1.414 - [Na9] . ([Ca2+] + [hIg2+])-0.5

G hemicals Pyrene and benzo[a]pyrene were purchased from Sigma. Unlabelled benzo[a]pyrene metabolite standards were obtained from Midwest Research Institute (Kansas City, Kans.) and [4,6,9,10-14C]pyrene (32.2 mCi/mmol) was from Chemsyn Science Laboratories (Lenexa, Kans.).

Results Soil characteristics The chemical characteristics of the five soil sites are listed in Table 1. There is a 100-fold range in PAH levels between the

Launen et al.

Table 2. The distribution of fungi within the soil sampling sites and the proportion of pyreneoxidizing strains. No. of pyrene oxidizers/total no. of isolates Fungal strains

Site 2

Site 5

Site 1

Site 3

Site 4

All sites

Mucor sp. Synceplaalastrum racemosl~m Cuiadida parapsilopsis Yeast (red pigment) Chaetorniumglc~hosunt Trichoderma harziaitum Ti-ichoderma sp. Fusarildm oxysporum Fldsarium sp. Penicillium janczewskii Penic.illium hrevicompact~dnl Penicillium jcrazthinellum Peizicilli~kmsp. 1 Peizicillium sp. 2 Penicillium sp 3 Unidentified sp. 1 Unidentified sp. 2 Unidentified sp. 3 Unidentified sp. 4 Unidentified sp. 5 Proportion of isolates that oxidized pyrene No. of different genera isolated in each site No. of different species isolated in each site pg/g high MW PAH in soil

five sites (0.8 (site 2) to 80 (site 4) kg/g soil) and the trends between the sites in the low molecular weight (MW) (two and three rings), high MW (more than three rings), and total PAH values were similar for all of the sites and generally correlated with the levels of the individual compounds. The site with the highest PAH content was 0.5 m below the topsoil surface (site 4). Individual PAH compounds are listed in order of increasing concentration from the top to the bottom of the table and the substrates used in these studies, pyrene and benzo[n]pyrene, were found to be present in relatively high concentrations. (Note that site 3 contained more BaP (7.8 pg/g) but less pyrene (8.8 pg/g) than site 4 (4.7 and 22 kg/g, respectively).) Structures with four or more rings predominated in the contaminated soils, confirmiilg that the soil is relatively weathered, i.e., that volatilization and microbial degradation of the smaller compounds had already occurred. This was confirmed by the ratio of the masses of high MW PAH/low MW PAH (approximately 10-25). The moisture content of the soil was approximately 5% in sites 2,4. and 5,9% in site 1, and 19% in site 3. It is doubtful that the lower yield of fungi from site 3 was related to the higher water content, since this level of moisture would be expected to stimulate the growth of fungi. The total oil and grease concentrations of sites 3 and 4 were 100 000 and 74 000 pg/g, respectively (not measured in the other three sites). The mineral oil and grease content was also higher in site 3 (55 000 pg/g)

than in site 4 (44 000 pglg). The sodium adsorption ratios of the two sites were similar.

The proportion of isolated fungal strains that oxidized pyrene and their distribution according to soil PAH level Table 2 shows the genus and species distribution of the fungal isolates as well as the proportion of isolates found to oxidize pyrene after 7 days of incubation in liquid culture. Identification of the isolated fungi revealed the following genera and species: Zygomyc.etes (three isolates) (Mucor cf. hiernalis van Tiegham, Syncephalastrum racemosum Cohn ex Schrot), Ascomycetes (three isolates) (Chaetornium globosum Kunze (subdivision Ascomycotiraa), Candidn parapsilopsis), Beutclumycetes (51 isolates) (Fusnrium oxysporurn Schlecht, plus six other Fusnriunz spp., Trichodernza hnrzinnum Rifai aggr., plus eight other Trichoderma spp., Penicillium janczewskii Zaleski, Pe17ic-illium hrcvicc~mpnctum Dierckx, Pe~zicillium janthinellum Biourge (this strain could also be identified as Pe~zicilliumsimplic.issimum (Oud.) Thom. (K. Seifert, personal communication), plus three other species of Pet7icillium). In addition, there were five unidentified species including one red yeast. The fungi that were isolated reflect not only the actual distribution of species present in the soils but also the isolation method used (selection of nonbasidiomycetes only). Approximately 50% of the isolates in sites 5, 1, 3, and 4 with levels of 3,28,71, and 79 pg/g total high MW PAH, respectively, were

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Table 3. Proton NMR and mass spectral data of pyrene (1) and its metabolites (2-6) isolated from Syncephalastrum racernosum (2,s) and Penicillium janthinellurn (3,4,6). Proton or m/z

Pyrene (1) 8.274 (d, 7.6) 8.055 (t, 7.6) 8.165 (s)

I -Pyrenola (2)

1,6-Pyrenediol (3)

1,8-Pyrenediol

7.622 (d, 8.4) 8.099(d,8.2) 7.903 (d, 9.0) 8.005 (d, 8.9) 8.128 (d, 7.6) 7.964 (t, 7.6) 8.137 (d, 7.5) 8.054 (d, 9.2) 8.423 (d, 9.2) 9.488 (s)

7.553 (d, 8.2) 7.938(d,8.2) 7.871 (d, 9.3) 8.135 (d, 9.2)

7.546 (d, 8.2) 7.923(d,8.2) 7.717 (s)

(4)

8.290 (s)

1,6-Quinone (5)

1,8-Quinone (6)

6.675 (d, 9.3) 7.962(d,9.5) 8.106 (d, 7.5) 8.454 (d, 7.3)

6.638 (d, 9.9) 7.942(d,10.2) 7.959 (s)

8.589 (s)

-

NOTE:Proton NMR spectra were recorded in acetone-d, (2.040 ppm) at 400 MHz. Chemical shift values (6) are reported in ppm downfield from TMS. Signal multiplicities and coupling constants (Hz) are shown in parentheses. For mass spectral data: molecular ion (EI, 70 eV). a The H-4/H-5 and H-6B-8 chemical shift assignments, respectively, may be reversed.

capable of oxidizing pyrene to polar metabolites. A smaller proportion (25%) of the fungi isolated from site 2 (