Successive mineralization and detoxification of benzo[a]pyrene by the white rot fungus Bjerkandera sp. strain BOS55 and indigenous microflora

Abstract

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.

FIG. 1

Oxidation of [¹⁴C]benzo[a]pyrene by Mn-sufficient fungal cultures. Zero time was the time when benzo[a]pyrene (20 mg liter⁻¹) was added to 6-day-old fungal cultures. Symbols: • and □, distribution of ¹⁴C label from benzo[a]pyrene in the dibutyl ether and water phases, respectively; ▴, total recovery of label in the culture medium after addition of acetone; ■, mineralization to ¹⁴CO₂.

FIG. 2

TLC profiles of benzo[a]pyrene metabolites during incubation in fungal cultures, as visualized by UV illumination. (A) Preparation developed twice with chloroform-methanol (97:3). (B) Metabolites which were retained near the origin in panel A after an additional run with chloroform-methanol (75:25). Lane 1, zero time; lanes 2 and 3, 1 and 15 days after addition of benzo[a]pyrene, respectively; lane 4, control fungal culture that did not receive benzo[a]pyrene.

FIG. 3

Mineralization of [¹⁴C]benzo[a]pyrene by fungal cultures, by activated sludge, and by both. Zero time was the time when benzo[a]pyrene was added to the 6-day-old fungal cultures; at day 15 activated sludge was added. Symbols: ■, fungus; ▴, fungus plus activated sludge; •, dead fungus plus activated sludge plus intact [¹⁴C]benzo[a]pyrene.

FIG. 4

Mineralization of [¹⁴C]benzo[a]pyrene by fungal cultures, by PAH adapted sludge, and by both. Zero time was the time when benzo[a]pyrene was added to the 6-day-old fungal cultures; at day 15 activated sludge was added. Symbols: ■, fungus; ▴, fungus plus PAH-adapted sludge; •, dead fungus plus PAH-adapted sludge plus intact [¹⁴C]benzo[a]pyrene.

FIG. 5

Mutagenic activity of benzo[a]pyrene and benzo[a]pyrene metabolites towards S. typhimurium TA100 and TA98. At zero time benzo[a]pyrene (20 mg liter⁻¹) was added to 6-day-old Mn-sufficient fungal cultures. At different times samples were taken and tested. The number of spontaneous revertants in the controls was subtracted from the values shown. The arrow indicates when activated sludge was added. Symbols: •, fungal cultures plus benzo[a]pyrene plus S-9 mix; ▴, fungal cultures plus benzo[a]pyrene; ○, effect of activated sludge addition.