The activity to metabolize the polycyclic aromatic hydrocarbons (PAH) phenanthrene, anthracene, pyrene, fluorene and fluoranthene by Trametes versicolor, Pleurotus ostreatus (white rot fungi), Laetiporus sulphureus, Daedaela quercina, Flamulina velutipes (brown rot fungi), Marasmiellus sp. (litter decaying fungus) and Penicillium sp. M 1 (isolated from a PAH contaminated soil sample) were compared. Screening methods for the presence of exoenzymes (peroxidases, polyphenoloxidases, "radical generating" enzymes) were evaluated for their use in screenings for fungi degrading PAH. Laetiporus sulphureus and Penicillium sp. M 1 cometabolize several PAH with rates comparable to white rot fungi. In most of the cases the patterns of extracellular peroxidases indicate the potential of fungi to degrade PAH.

The degradation of phenanthrene and pyrene was investigated by using five different wood-decaying fungi. After 63 days of incubation in liquid culture, 13.8 and 4.3% of the [ring U-14 C]phenanthrene and 2.4 and 1.4% of the [4,5,9,10-14 C]pyrene were mineralized by Trametes versicolor and Kuehneromyces mutabilis, respectively. No 14 CO 2 evolution was detected in either [ 14 C]phenanthrene or [ 14 C]pyrene liquid cultures of Flammulina velutipes, Laetiporus sulphureus, and Agrocybe aegerita. Cultivation in straw cultures demonstrated that, in addition to T. versicolor (15.5%) and K. mutabilis (5.0%), L. sulphureus (10.7%) and A. aegerita (3.7%) were also capable of mineralizing phenanthrene in a period of 63 days. Additionally, K. mutabilis (6.7%), L. sulphureus (4.3%), and A. aegerita (3.3%) mineralized [ 14 C]pyrene in straw cultures. The highest mineralization of [ 14 C] pyrene was detected in straw cultures of T. versicolor (34.1%), which suggested that mineralization of both compounds by fungi may be independent of the number of aromatic rings. Phenanthrene and pyrene metabolites were purified by high-performance liquid chromatography and identified by UV absorption, mass, and 1 H nuclear magnetic resonance spectrometry. Fungi capable of mineralizing phenanthrene and pyrene in liquid culture produced enriched metabolites substituted in the K region (C-9,10 position of phenanthrene and C-4,5 position of pyrene), whereas all other fungi investigated produced metabolites substituted in the C-1,2, C-3,4, and C-9,10 positions of phenanthrene and the C-1 position of pyrene.

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Aspergillus niger, isolated from hydrocarbon-contaminated soil, was examined for its potential to degrade phenanthrene and pyrene. Two novel metabolites, 1-methoxyphenanthrene and 1-methoxypyrene, were identified by conventional chemical techniques. Minor metabolites identified were 1-and 2-phenanthrol and 1-pyrenol. No 14 CO 2 evolution was observed in either [ 14 C]phenanthrene or [ 14 C]pyrene cultures.

The white-rot fungus Nematoloma frowardii was examined for the ability to degrade [ring U-i4C]-phenanthrene and [4,5,9,10-i4C]pyrene in liquid and solid (straw) cultures in a period of 63 days. 3.2% phenanthrene and 8.6% pyrene were mineralized to 14C0, in liquid cultures, respectively. A considerable higher mineralization of 1 1.2% (phenanthrene) and 46.5% (pyrene) was detected in straw cultures. Metabolites were identified by their UV absorption spectra. N. frowardii transformed phenanthrene to phenanthrene 9,lO-dihydrodiol. Pyrene 4,5-dihydrodiol was identified as major metabolite in pyrene degradation.Polycyclic aromatic hydrocarbons (PAHs), like phenanthrene (three ring PAH) and pyrene (four ring PAH) are widely distributed pollutants in terrestrial and aquatic ecosystems (BLUMER 1976). The ability of wood-decaying fungi to mineralize PAHs seems to be connected with the activity of ligninolytic enzymes, since mineralization in liquid or soil cultures has mostly been described for wood-decaying fungi like the white-rot fungi Phanerochaete chrysosporium (SANGLARD et al. 1986, BUMPUS et al. 1985, BUMPUS 1989, MORGAN et al. 1991, Trametes versicolor (SACK et al. 1995), and Kuehneromyces mutabilis (SACK and FRITSCHE 1997).A screening of wood-decaying fungi for producing radical generating extracellular enzymes has selected the South American white-rot fungus Nematoloma frowardii with a high manganese peroxidase (MnP) activity (HOFRICHTER and FRITSCHE 1997). We used phenanthrene and pyrene as model PAHs for investigations on the mineralization of these substances in liquid and solid (straw) substrates by N. frowardii. Materials and methods

The degradation of polycyclic aromatic hydrocarbons by a manganese peroxidase crude preparation of Nematoloma forwardii was demonstrated for a mixture of eight different polycyclic aromatic hydrocarbons, and the five individual polycyclic aromatic hydrocarbons phenanthrene, anthracene, pyrene, fluoranthene, and benzo[alpha]pyrene. Oxidation of polycyclic aromatic hydrocarbons was enhanced by the addition of glutathione, a mediator substance, able to form reactive thiyl radicals. Glutathione-mediated manganese peroxidase (1.96 U ml(-1)) was capable of mineralizing [14C]pyrene (7.3%),[14C]anthracene (4.7%), [14C]benzo[alpha]pyrene (4.0%), [14C]benz(alpha)anthracene 2.9%), and [14C]phenanthrene (2.5%) in a period of 168 h. This is the first description of direct enzymatic mineralization of polycyclic aromatic hydrocarbons by manganese peroxidase, and indicates their important role in the oxidation of polycyclic aromatic hydrocarbons by wood-decaying fungi.

The mineralization of [14C]pyrene in sterilized and non‐sterile soil was investigated using the wood‐decaying fungi Kuehneromyces mutabilis and Agrocybe aegerita in a period of 63 days. In sterilized soil 5.1% and 1.5% of the pyrene was mineralized to 14CO2 by K. mutabilis and by A. aegerita, respectively. In non‐sterile soil, 27.3% of pyrene was mineralized by indigenous soil microflora including a Mycobacterium gilvum strain. During soil inoculation with fungi the mineralization was higher (47.7% for K. mutabilis and 38.5% for A. aegerita). For a mass balance analysis the soil was extracted with toluene and methanolic KOH (humic acid extraction). Considering the sum of mineralization and formation of bound residues (non‐extractable radioactivity), about 50% (sterilized soil) and 75% (non‐sterile soil) of pyrene were eliminated by K. mutabilis. In comparison with indigenous soil microflora, K. mutabilis enhanced pyrene elimination up to 42%.

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