Successive Mineralization and Detoxification of Benzo[a]pyrene by the White Rot Fungus Bjerkandera sp. Strain BOS55 and Indigenous Microflora

White rot fungi can oxidize high-molecular-weight polycyclic aromatic hydrocarbons (PAH) rapidly to polar metabolites, but only limited mineralization takes place. The objectives of this study were to determine if the polar metabolites can be readily mineralized by indigenous microflora from several inoculum sources, such as activated sludge, forest soils, and PAH-adapted sediment sludge, and to determine if such metabolites have decreased mutagenicity compared to the mutagenicity of the parent PAH. ¹⁴C-radiolabeled benzo[a]pyrene was subjected to oxidation by the white rot fungus Bjerkandera sp. strain BOS55. After 15 days, up to 8.5% of the [¹⁴C]benzo[a]pyrene was recovered as ¹⁴CO₂ in fungal cultures, up to 73% was recovered as water-soluble metabolites, and only 4% remained soluble in dibutyl ether. Thin-layer chromatography analysis revealed that many polar fluorescent metabolites accumulated. Addition of indigenous microflora to fungal cultures with oxidized benzo[a]pyrene on day 15 resulted in an initially rapid increase in the level of ¹⁴CO₂ recovery to a maximal value of 34% by the end of the experiments (>150 days), and the level of water-soluble label decreased to 16% of the initial level. In fungal cultures not inoculated with microflora, the level of ¹⁴CO₂ recovery increased to 13.5%, while the level of recovery of water-soluble metabolites remained as high as 61%. No large differences in ¹⁴CO₂ production were observed with several inocula, showing that some polar metabolites of fungal benzo[a]pyrene oxidation were readily degraded by indigenous microorganisms, while other metabolites were not. Of the inocula tested, only PAH-adapted sediment sludge was capable of directly mineralizing intact benzo[a]pyrene, albeit at a lower rate and to a lesser extent than the mineralization observed after combined treatment with white rot fungi and indigenous microflora. Fungal oxidation of benzo[a]pyrene resulted in rapid and almost complete elimination of its high mutagenic potential, as observed in the Salmonella typhimurium revertant test performed with strains TA100 and TA98. Moreover, no direct mutagenic metabolite could be detected during fungal oxidation. The remaining weak mutagenic activity of fungal cultures containing benzo[a]pyrene metabolites towards strain TA98 was further decreased by subsequent incubations with indigenous microflora.Bioremediation of polycyclic aromatic hydrocarbon (PAH)-polluted soil is severely hampered by the low rate of degradation of the higher PAH, particularly the four- and five-ring PAH (6, 32). These higher PAH have very low water solubility and are often tightly bound to soil particles. This results in very low bioavailability for bacterial degradation. The observation that white rot fungi can oxidize PAH rapidly with their extracellular ligninolytic enzyme systems has therefore raised interest in the use of these organisms for bioremediation of PAH-polluted soils (3, 9). Although PAHs are extensively oxidized by white rot fungi, the degree of mineralization to CO₂ is always limited. In various studies evaluating the degradation of the potent carcinogen benzo[a]pyrene by several white rot fungal species, from 0.17 to 19% of the radiolabeled PAH was recovered as ¹⁴CO₂ (4, 5, 26). The major products of the oxidation were both nonpolar and polar metabolites. The accumulation of such metabolites could be a reason for concern, since mammalian and fungal monooxygenases can oxidize benzo[a]pyrene to epoxides and dihydrodiols, which are very potent carcinogens (28, 29). However, peroxidase-mediated extracellular oxidation of benzo[a]pyrene in cultures of white rot fungi results initially in benzo[a]pyrenediones, which show weak mutagenic activity (29). These primary metabolites are rapidly oxidized further to unidentified metabolites by Phanerochaete laevis and Phanerochaete chrysosporium (5, 26). Furthermore, the oxidized benzo[a]pyrene metabolites have a higher aqueous solubility. Since the low bioavailability of PAH is a major rate-limiting factor in the degradation of these compounds by bacteria (27, 31), the increased bioavailability of oxidized PAH metabolites suggests that these compounds can be more easily mineralized by bacteria.The aim of this study was to investigate the degradation and mineralization of the five-ring PAH benzo[a]pyrene by the white rot fungus Bjerkandera sp. strain BOS55 and the subsequent mineralization of the metabolites by natural mixed cultures of microorganisms. During the oxidation and mineralization of benzo[a]pyrene, the decrease in the mutagenicity of the metabolites was monitored. The white rot fungal strain Bjerkandera sp. strain BOS55 was used because of its outstanding ability to rapidly oxidize PAH (8, 19) and because extensive information concerning its physiology is available (7, 18, 20, 22, 23).