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Applied and Environmental Microbiology, August 1998, p. 2853-2858, Vol. 64, No. 8
Division of Industrial Microbiology,
Received 10 February 1998/Accepted 12 May 1998
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).
Organisms used.
The white rot fungus
Bjerkandera sp. strain BOS55 (ATCC 90940) was
maintained on peptone-yeast extract agar slants (17) at
10°C. Malt extract plates were inoculated 5 days prior to the experiments, and fungal cultures were inoculated with an agar plug as
described previously (16).
Culture conditions.
Fungal cultures were cultivated on the
standard high-nitrogen, manganese-free medium modified from the medium
described by Tien and Kirk (30). This medium contained 33 mM
N as mycological peptone (5 g liter Additions to fungal cultures.
Benzo[a]pyrene
was added on day 6 as a 100×-concentrated stock solution in acetone,
which resulted in a final benzo[a]pyrene concentration of
20 mg liter
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Successive Mineralization and Detoxification of
Benzo[a]pyrene by the White Rot Fungus
Bjerkandera sp. Strain BOS55 and Indigenous
Microflora
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
1) and was
used without further treatment. Samples of two soils were collected
from forests from the litter layer (forest 1) (pH 4.5) and from a
decomposed beech log (forest 2) (pH 4.8). The forest soils were diluted
1:1 (wt/wt) with water to prepare slurries and were filtered through
cheesecloth. A 50-year-old PAH-polluted sediment sludge dredged from
Rotterdam Harbor was diluted 1:10 in buffer (pH 7) containing 0.02%
yeast extract and incubated for 10 days at 21°C in shake flasks
before it was used as an inoculum. An enrichment culture on
2,2'-diphenic acid, an intermediate of phenanthrene oxidation by
P. chrysosporium (11), was prepared from
activated sludge (from a municipal wastewater treatment plant in Delft,
The Netherlands) containing four different bacteria (as determined on
the basis of morphology on yeast-glucose agar plates). This enrichment
culture was incubated for 10 days in a pH 7 buffer containing 1 g
of 2,2'-diphenic acid liter1 at 21°C before it was used
as an inoculum. In all cases, approximately 2 × 108 to 5 × 108 CFU was added to each
fungal culture on day 15 in a volume of 5 to 10 ml.
1) and 10 g of
glucose liter1 buffered at pH 6 with 40 mM phosphate.
Manganese-containing medium was obtained by adding 66 µM Mn (as
MnSO4). The media were autoclaved for 30 min at 115°C.
After sterilization, 10 ml of a filter-sterilized thiamine solution
(200 mg liter1) was added. Sterile 250-ml serum bottles
containing 5 ml of medium were loosely capped to facilitate aeration
during incubation for 6 days at 30°C. Preparations containing
bacteria were incubated in a shaking water bath (80 rpm) at 21°C.
1 and an acetone concentration of 1%. Glucose
(5 g liter1) and Tween 80 (2.5 g
liter1) were added as 100×-concentrated stock solutions
in water. The bottles were then tightly capped, and an oxygen
atmosphere was supplied by flushing the bottles for 5 min with pure
oxygen. This was repeated once every 2 or 3 days.
[14C]benzo[a]pyrene experiments.
In the 14C experiments, 80,000 to 100,000 dpm of
[7,10-14C]benzo[a]pyrene (specific activity,
2.24 MBq µmol1; Amersham, Amersham, United Kingdom) was
added to each culture together with 20 mg of
benzo[a]pyrene liter1. Production of
14CO2 was measured by flushing the headspace
once every 2 or 3 days for 8 min through a three-stage trap in which
each stage contained 6 ml of 2 M NaOH. The
14CO2 production values for triplicate cultures
were pooled. Control experiments revealed a CO2-trapping
efficiency of more than 99%. For detection of volatile metabolites, a
fourth trap containing acetonitrile was added. The total recovery of
label remaining in the culture medium was measured by adding 3 volumes
of acetone to each bottle; the bottles were then shaken for 1 h.
The recovery of benzo[a]pyrene in autoclaved fungal
controls, as determined by a high-performance liquid chromatography
(HPLC) analysis of unlabeled benzo[a]pyrene, was more than
98% when this method was used. The distribution of label remaining in
the culture medium between the water-soluble and organic
compound-soluble phases was measured by adding 10 ml of dibutyl ether.
After dibutyl ether was added, the cultures were vigorously shaken for
1 h. After phase separation, the amount of label in both the water
phase and the solvent phase was measured. The amount of water-soluble label was sometimes also measured quickly by filtering the culture fluids through hydrophilic Schleicher & Schuell type FP 030/3 0.2-µm-pore-size filters, which resulted in levels of recovery that
were 5 to 10% higher than those obtained after dibutyl ether extraction. The amount of label associated with fungal biomass was
measured by incubating acetone-extracted fungal biomass with Soluene
(Packard). One milliliter of the partially homogenized biomass was then
transferred to a microvial with 5 ml of scintillation cocktail.
Quenching by the biomass was corrected by adding a known amount of
label to control vials with and without equal amounts of
Soluene-treated biomass. All samples (volume, up to 1 ml) were added to
5 ml of scintillation cocktail (Ultima-gold; Packard) in microvials,
and the radioactivity was measured with a liquid scintillation analyzer
(model Tri-Carb 1600 TR; Packard) with appropriate controls.
Salmonella typhimurium revertant test (Ames
test).
To monitor the mutagenic potential of
benzo[a]pyrene metabolites, the Salmonella
typhimurium revertant plate test, first described by Ames et al.
(1), was used. Experiments were performed basically by using
the plate incorporation test described by Maron and Ames (21). Both strain TA98 for point mutations and strain TA100 for frameshift mutations were used. Overnight cultures were inoculated directly from a 
80°C stock, and the genotypes were tested after preparation of the 80°C stock. To mimic liver biotransformation, a
rat liver homogenate (S9-mix) (Boehringer, Mannheim, Germany) from
Araclor-induced rats was used. Benzo[a]pyrene
dissolved in acetone was used as a positive control in experiments
performed with S-9 mix, and 4-nitroquinoline-N-oxide was
used as a positive control in experiments performed without S-9 mix. A
preincubation step (10 min, 37°C) included in initial tests resulted
in no significant increase in the mutagenic response and was therefore
omitted in the subsequent experiments.
Analytical methods.
For the analysis of residual unlabeled
benzo[a]pyrene, triplicate cultures were sacrificed by
adding 3 volumes of acetone to each bottle. The bottles were then
sealed with Teflon liners, and shaken for 1 h, and samples were
centrifuged in an Eppendorf centrifuge (10 min, 13,000 × g). The analysis was conducted with a HPLC equipped with a
diode array detector as described previously (8). The range
of benzo[a]pyrene concentrations was 0 to 5 mg
liter1, the detection limit was around 0.05 mg
liter1, and the reproducibility of the HPLC was more than
99.5%. Fungal cultures were used to monitor abiotic losses of
benzo[a]pyrene. The abiotic losses never exceeded 2% of
the benzo[a]pyrene added.
Chemicals. All of the chemicals used were commercially available and were analytical grade or higher. The solvents used for TLC were chromatography grade. All chemicals were used without further treatment.
Statistical procedures. Unless indicated otherwise, the data shown are the means and standard deviations of values obtained from triplicate cultures.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Degradation of benzo[a]pyrene by the white rot fungus
Bjerkandera sp. strain BOS55.
In the first
experiments, degradation of benzo[a]pyrene was monitored
in both Mn-sufficient and Mn-deficient cultures in the presence of the
surfactant Tween 80, since the presence of Mn has been shown to affect
PAH oxidation by Bjerkandera sp. strain BOS55
(20). For Mn-sufficient cultures (Fig.
1), the level of recovery of
14CO2 after 15 days was 8.4% of the initial
amount of label added; in manganese-deficient cultures a slightly lower
level of recovery of 14CO2 (7.9%) was observed
(results not shown). The distribution of the 14C label in
the culture fluid in the hydrophobic solvent and water phases was
monitored in Mn-sufficient cultures (Fig. 1). The amount of
14C label extractable with dibutyl ether decreased very
rapidly, while water-soluble polar metabolites quickly accumulated.
After 15 days of incubation, the major products of fungal
benzo[a]pyrene oxidation were water-soluble products,
which accounted for 68% of the initial amount of label, as shown in
Fig. 1. In separate experiments, the water-soluble products at day 15 accounted for 67 to 73% of the initial amount of label. HPLC analysis
of simultaneously grown cultures containing unlabeled
benzo[a]pyrene showed that benzo[a]pyrene (20 mg liter1) was eliminated for 91, 96, and 100% after 1, 5, and 15 days, respectively. The total amount of 14C label
recovered in the gas and water phases decreased slightly with time,
whereas the amount of label associated with the fungal biomass
increased from zero at the start of the experiment to around 8% at day
15. Volatile compounds other than CO2 were not observed. In
the Mn-deficient cultures, polar water-soluble products were also
found to be the major products, accounting for 70% of the label
(results not shown). After 15 days, no
14CO2 production was detected in the
autoclaved fungal controls; the level of recovery of label with acetone
was 100%, and only 0.1% of the label was recovered from the water
phase.
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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.
Characterization benzo[a]pyrene metabolites. The spectrum of oxidized benzo[a]pyrene products produced by Mn-sufficient cultures of Bjerkandera sp. strain BOS55 was investigated further. When the ethyl acetate-extracted metabolites were examined by using TLC with nonpolar eluents, such as petroleum ether 40-60-ethyl acetate (3:1) (results not shown) and chloroform-methanol (97:3), most of the metabolites remained in the origin, while benzo[a]pyrene exhibited significant migration (Fig. 2A). A more polar eluent, such as chloroform-methanol (75:25), was required for any significant migration of the polar metabolites (Fig. 2B). Use of the eluents chloroform-methanol (65:35) and ethyl acetate-methanol (65:35) resulted in migration of all of the metabolites, but the resolution was very poor (results not shown). Clearly, extensive oxidation of benzo[a]pyrene to polar metabolites took place during the first day of incubation. After 15 days no intact benzo[a]pyrene was detected, and some metabolites present on day 1 were apparently oxidized further. Ethyl acetate extracts of 15-day-old control fungal cultures (without added benzo[a]pyrene) produced only two faint blue spots under UV light (not visible in Fig. 2), which were tentatively identified as the secondary metabolites veratryl alcohol and veratraldehyde. Identical benzo[a]pyrene metabolite profiles were observed after TLC with an autoradiogram of 14C-labeled benzo[a]pyrene metabolites (results not shown). Acidification of the culture fluids with HCl was necessary for a high level of recovery of the metabolites by ethyl acetate extraction. Still, the extraction efficiency decreased during the incubation period; around 15% of the water-soluble label was not extractable after 15 days. Extensive degradation of benzo[a]pyrene and accumulation of polar metabolites were also observed when benzo[a]pyrene was incubated for 1 day in extracellular culture fluids from 6-day-old fungal cultures (results not shown).
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Mineralization of benzo[a]pyrene by Bjerkandera sp. strain BOS55 and indigenous microflora. Mineralization of benzo[a]pyrene metabolites by natural mixed cultures of microorganisms not previously adapted to PAH from four different inoculum sources was examined. The inocula included activated sludge, two acidic forest soils, and an enrichment culture grown on 2,2'-diphenic acid. The results of this kind of experiment are shown in Fig. 3; in this experiment [14C]benzo[a]pyrene was oxidized for 15 days by Mn-sufficient cultures of Bjerkandera sp. strain BOS55 before activated sludge was added to the fungal cultures. Just before the activated sludge was added, the fungal cultures had already mineralized 8% of the benzo[a]pyrene. Just after the activated sludge was added, the level of mineralization rapidly increased to 20% in a few days, and thereafter it increased slowly to 27% by day 56 of the experiment. Adding 5 ml of fresh activated sludge and 0.02% yeast extract at this point had no effect on 14CO2 production. In the control fungal cultures that did not receive activated sludge, the level of mineralization was only 12% on day 56. Adding autoclaved sludge to the fungal cultures had no effect on 14CO2 production (data not shown).
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Mutagenicity studies.
Ames tests were performed to monitor the
changes in the mutagenic potential of
benzo[a]pyrene during its oxidation and
mineralization. In the experiments performed with S. typhimurium TA100, no increase in the number of revertants was
observed in the fungal culture broth itself in the absence of the rat
liver activation mixture (S-9 mix) compared to the spontaneous number
of revertants (65 revertants per plate). However, the fungal culture
broth did cause a small increase (10 to 20 revertants) in the presence
of S-9 mix throughout the experiment compared to the number of
spontaneous revertants in the presence of S-9 mix (72 revertants per
plate). Addition of benzo[a]pyrene (20 mg
liter1) to the fungal cultures resulted in high mutagenic
activity only when S-9 mix was added, and this mutagenic activity was
reduced to background levels within 15 days when cultures were
incubated with Bjerkandera sp. strain BOS55 (Fig.
5). No decrease in the mutagenic
potential of benzo[a]pyrene was caused by
autoclaved fungal cultures (results not shown).
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, effect of activated
sludge addition.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In this study, mineralization of the recalcitrant pollutant
benzo[a]pyrene was investigated by successively
incubating [14C]benzo[a]pyrene
with the white rot fungus Bjerkandera sp. strain BOS55 and natural mixed cultures of microorganisms from soil, sediment,
and sludge. Bjerkandera sp. strain BOS55 can oxidize PAH very rapidly in high-nitrogen cultures supplied with adequate H2O2 for maximal peroxidase activity and the
surfactant Tween 80 to improve PAH bioavailability. Under such
conditions, benzo[a]pyrene supplied at a
concentration of 50 mg liter1 was oxidized at rates of up
to 450 mg liter1 day1 (19). In
the experiments performed here with 14C-labeled
benzo[a]pyrene, we found that with a level of
recovery of 8% 14CO2 at an initial
mineralization rate of 0.3 mg liter1 day1,
water-soluble metabolites accounting for up to 73% of the label accumulated after 15 days. These results correlate well with the results of other studies in which oxidation of PAH was examined, although the results depend strongly on the white rot species and
strain used. For
[14C]benzo[a]pyrene, levels of
14CO2 recovery between 0.17 and 19% have been
reported in studies with different species (4, 5, 26). The
initial level of accumulation of water-soluble metabolites observed in
this study is high compared to the levels found in other studies. This
difference can be attributed to both the species used and the
definition of the term water soluble.
The metabolites of peroxidase-mediated benzo[a]pyrene oxidation have not been identified yet. The initial oxidation products have been identified as benzo[a]pyrenequinones (10), but these quinones are rapidly further oxidized to more water-soluble products with unknown structures by cultures of P. laevis and P. chrysosporium (4, 5, 26). Peroxidase-mediated oxidation of the low-molecular-weight PAH, such as anthracene and phenanthrene, also results in quinones. Depending on the strain used, anthraquinones are either accumulated as metabolites (2, 8) or are further oxidized to unidentified products (2, 8) or phthalate (12). Oxidation of phenanthrenequinone to 2,2'-diphenic acid by ligninolytic cultures of P. chrysosporium has been reported (11). The observation in this study that all benzo[a]pyrene metabolites detected by autoradiography also were highly fluorescent under UV light indicates that the polyaromatic structure of benzo[a]pyrene was not completely destroyed. The high water solubility of these metabolites can presumably be attributed to the presence of carboxyl and/or hydroxyl groups. The presence of carboxyl groups is suggested because the acidification of the culture media greatly increased the efficiency of extraction of these metabolites with ethyl acetate.
The interest in white rot fungi for PAH degradation is mainly fueled by the slow breakdown of PAH by bacteria. It has been reported many times that low bioavailability of PAH is the main factor limiting bacterial PAH degradation (27, 31), and until now, the attempts to improve PAH bioavailability and degradability by using surfactants have not been very successful (25). Oxidation of PAH by white rot fungi to more water-soluble products with greater bioavailability could therefore result in rates of mineralization of these metabolites by bacteria higher than the rates of mineralization of the parent PAH compounds. A study performed with the three-ring PAH anthracene has confirmed that all known oxidation products of this compound are mineralized by activated sludge more rapidly than anthracene itself is mineralized (24).
Addition of undefined microbial inocula, irrespective of the source, to
oxidized benzo[a]pyrene metabolites clearly
resulted in initially rapid mineralization rates, comparable to 1.0 mg of benzo[a]pyrene liter1
day1. Benzo[a]pyrene not previously
subjected to fungal oxidation was not mineralized at all by the inocula
not adapted to PAH, whereas PAH-adapted sludge mineralized intact
benzo[a]pyrene at a rate of only 0.1 mg of
benzo[a]pyrene liter1
day1. This confirms that fungal preoxidation of PAH
increases the rate of mineralization by bacteria. A similar synergistic
effect of a combination of white rot fungi and soil microorganisms has also been observed for pyrene mineralization by Pleurotus
sp. and Dichomitus squalens (14). Andersson and
Henrysson (2) showed that the dead-end metabolites of both
anthracene oxidation and benzo[a]anthracene oxidation
by P. chrysosporium were slowly further degraded in the
presence of nonadapted soil microorganisms.
By the end of the successive mineralization experiments, the maximum yields of 14CO2 were between 39 and 47%, and at least 16% of the initial label remained in water-soluble metabolites. The lack of mineralization of this fraction could not be attributed to toxicity of the metabolites or a lack of trace elements or vitamins. This suggests that not all of the fungus-oxidized metabolites were easily mineralized by indigenous microflora. If these metabolites are cometabolically degraded by bacteria, as has been described for higher PAH, such as benzo[a]pyrene and dibenzo[a,h]anthracene (13, 15), the absence of a suitable cosubstrate could explain the lack of complete mineralization. However, addition of glucose, autoclaved spent fungal medium, or activated sludge as a cosubstrate did not have any significant effect. This suggests that the remaining metabolites have structures that are poorly degradable.
Degradation of PAH like benzo[a]pyrene is desired since oxidation by eukaryotic monooxygenases can result in metabolites with high carcinogenic activity (28). The lack of complete mineralization of benzo[a]pyrene after sequential treatment by fungi and bacteria made it necessary to monitor the mutagenic activity during this treatment. The high mutagenic activity of S-9 mix-activated benzo[a]pyrene towards S. typhimurium TA100 and TA98 rapidly decreased during fungal incubation without any significant accumulation of direct or indirect mutagens. This suggests that either the intracellular monooxygenases were not involved in the oxidation of benzo[a]pyrene or their oxidation products did not accumulate. In any case, involvement of the extracellular peroxidases, which previously have been shown to rapidly oxidize anthracene and benzo[a]pyrene (19), was in this study demonstrated by the extensive oxidation of benzo[a]pyrene in the extracellular culture fluids of 6-day-old cultures of Bjerkandera sp. strain BOS55.
This research showed that benzo[a]pyrene is quickly oxidized into polar, water-soluble compounds by the white rot fungus Bjerkandera sp. strain BOS55. Most of these metabolites could be mineralized by non-PAH-adapted microbial indigenous communities under aerobic conditions. The lack of complete mineralization is not a serious setback for the use of white rot fungal techniques in PAH bioremediation, since the highly mutagenic potential of the parent compound was eliminated.
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ACKNOWLEDGMENT |
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This work was funded by an IOP project from Senter, an agency of the Dutch Ministry of Economics.
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Industrial Microbiology, Department of Food Science, Bomenweg 2, P.O. Box 8129, 6700 EV Wageningen, The Netherlands. Phone: 31 (0)317-484993. Fax: 31 (0)317-484978. E-mail: Michiel.Kotterman{at}algemeen.im.wau.nl.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Applied and Environmental Microbiology, August 1998, p. 2853-2858, Vol. 64, No. 8
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