International Biodeterioration & Biodegradation 52 (2003) 1 – 5

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Degradation of organic pollutants by the white rot basidiomycete

Trametes trogii
L. Levina; ∗ , A. Vialeb , A. Forchiassina
a MicologaExperimental, Departamento de Cs. Biologicas y, Facultad de Ciencias Exactas Naturales, Universidad de Buenos Aires,
1428 Capital Federal, Argentina
b Microbiologa, Departamento de Qumica Biol
ogica, Facultad de Ciencias Exactas Naturales, Universidad de Buenos Aires,
1428 Capital Federal, Argentina

Abstract

The ability of the white rot basidiomycete Trametes trogii (strain BAFC 463) to degrade in vitro concentrations of 250 –500 ppm of
nitrobenzene and anthracene was analyzed. Within 12 – 24 days, more than 90% of the organic pollutants added to the fungal cultures were
removed, as demonstrated by gas–liquid chromatography. Enzyme estimations indicated a high and relatively stable activity of laccase, as
well as a lower production of manganese peroxidase. Laccase activity could be implicated in the degradation of these xenobiotic compounds
by T. trogii. Earlier work showed that this fungus almost completely removed anthraquinone dyes and polychlorinated biphenyls. In view
of the results obtained, this strain seems promising for detoxi5cation. ? 2002 Elsevier Science Ltd. All rights reserved.

1. Introduction (Beaudette et al., 1998), dioxins (Joshi and Gold, 2000),

Polycyclic aromatic hydrocarbons (PAHs) are pollu-
tants, often carcinogenic, mutagenic, or toxic, found in
most terrestrial ecosystems, that arise from industrial oper-
ations and from natural events such as forest 5res. Major
components of petroleum, they are continuously released
into natural environments, posing a serious risk to human
health (Sack et al., 1997). Bioremediation of PAH-polluted
soil is severely hampered by the low rate of degradation
of higher PAHs, particularly the four- and 5ve-ring PAHs.
These higher PAHs 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 PAHs rapidly with their ex-
tracellular ligninolytic enzyme systems has therefore raised
interest in the use of these organisms for bioremediation
of PAH-polluted soils (Kotterman et al., 1998). Oxidation
of PAHs by white rot fungi to more water-soluble prod-
ucts with greater bioavailability resulted in higher rates of
mineralization by bacteria than those of the parent com-
pounds (Meulenberg et al., 1997). The nonspeci5c oxidases
involved in lignin degradation (mainly laccases, lignin-
and manganese-peroxidases) are able to transform PAHs
(Johannes and Majcherczyk, 2000), chlorinated phenols
(Reddy and Gold, 2000), polychlorinated biphenyls (PCBs)
∗ Corresponding author.
E-mail address: aaviale@qb.fcen.uba.ar (L. Levin).
pesticides (Kullman and Matsumura, 1996), explosives
(Hawari et al., 1999) and dyes (Abadulla et al., 2000).
Intracellular systems that are generally present in most
fungi, such as cytochrome P-450 monooxygenase, may
also be involved in organopollutant degradation (Bezalel
et al., 1996). However, as ligninolytic enzymes are active
extracellularly, white rot fungi are better candidates for the
bioremediation of highly apolar pollutants than nonligni-
nolytic microorganisms (Field et al., 1993).
BTEX compounds (benzene, toluene, ethylbenzene, and
o-, m- and p-xylenes) are an important family of organic
pollutants that are components of gasoline and aviation
fuels; they and their nitro derivatives are widely used in
industrial syntheses. They are carcinogenic and neurotoxic
and are classi5ed as priority pollutants (Spain, 1995). Both
aerobic and anaerobic bacteria have been shown to degrade
BTEX compounds, but most of these studies on bacterial
degradation of BTEX have used microbial consortia and
no native strain of bacterium is known to degrade all the
components of BTEX eFciently. O-xylene particularly has
been shown to be recalcitrant to bacterial degradation (Tsao
et al., 1998). On the other hand, the white rot fungus Phane-
rochaete chrysosporium simultaneously degraded all the
BTEX components (Yadav and Reddy, 1993; Song, 1997).
Most of the research on bioremediation by white rot fungi
has focused on this single species which is known to metab-
olize a wide range of xenobiotic compounds (Paszczynski
and Crawford, 1995). Recently, however, other fungi have

0964-8305/03/$ - see front matter ? 2002 Elsevier Science Ltd. All rights reserved.
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2 L. Levin et al. / International Biodeterioration & Biodegradation 52 (2003) 1 – 5
begun to be evaluated for their pollutant-degrading abil-
ities, and notable diJerences with regard to the extent of
their pollutant transformation ability have been demon-
strated. The utilization of fungi for bioremediation requires
an understanding of the factors that enhance their ability to
detoxify, and clari5cation of the enzyme mechanisms used
(Cerniglia et al., 1992).
Trametes trogii is a white rot basidiomycete, distributed
worldwide. T. trogii strain BAFC 463, besides eFciently
degrading lignin in wood (Levin and Castro, 1998), has been
tested successfully in biomechanical pulping experiments
(Planes et al., 1986) and also demonstrated to be a good pro-
ducer of ligninases (Levin and Forchiassin, 2001). The si-
multaneous presence in this fungus of high ligninolytic and
hydrogen-peroxide-producing activities, essential for per-
oxidase activity and rate limiting for pollutant degradation
(Gramss et al., 1999), make it an attractive microorgan-
ism on which to base future biotechnological applications.
Previously it was shown to almost completely remove an-
thraquinone dyes and PCBs (Haglund, 1999; Levin et al.,
2001). In this study its ability to transform nitrobenzene and
anthracene in liquid cultures was analyzed in relation to its
complement of extracellular ligninolytic enzymes.
2. Materials and methods
Microorganism. Strain 463 (BAFC: Mycological Culture
Collection of the Department of Biological Sciences, Faculty
of Exact and Natural Sciences, University of Buenos Aires)
of T. trogii Berk. (Aphyllophorales, Basidiomycetes) was
used. Stock cultures were maintained on malt extract agar
slants at 4◦ C.
Basal culture medium. Was GA (Levin and Forchiassin,
2001) with glucose 10 g l−1 and asparagine 3 g l−1 . The 5nal
pH of the medium was adjusted to 4.5 with 0.5 M citric acid
and 0.5 M dibasic sodium phosphate buJer, diluted in the
medium to a 5nal concentration of 0.25 M. The kinetics of
growth and enzyme production was studied in this medium
and also in a medium containing glucose (20 g l−1 ) and malt
extract (20 g l−1 ).
Culture conditions. Erlenmeyer Masks of 250 ml contain-
ing 25 ml of medium were inoculated with 2 agar plugs (each
of 0:25 cm2 ) cut out from the margin of a colony grown on
Bacto-agar 2%. Incubation was carried out at 28 ± 1◦ C un-
der stationary conditions. Cultures were harvested after dif-
ferent incubation periods, 5ltered through 5lter paper using
a BNuchner funnel and dried overnight at 70◦ C. Dry weight
of mycelia was then determined. The culture supernatants
were used as enzyme sources.
Enzyme assays. Laccase activity was determined by mea-
suring the increase in A420 due to the oxidation of 0.5 mM
ABTS (2; 2 -azinobis(3-ethylbenzthiazoline-6-sulphonic
acid) in 0.1 M sodium acetate buJer (pH 5.0) (E420 =
36 mM−1 cm−1 (Bourbonnais et al., 1995). Manganese
peroxidase activity (MnP) was assayed with 0.1 mM
MnSO4 and H2 O2 and 0:01% phenol red as the substrate.
Reactions were halted by adding NaOH 5 N, and increase
in A610 measured (E610 = 22 mM−1 cm−1 ). Lignin peroxi-
dase activity (LiP) was assayed by the method of Tien and
Kirk (1988) monitoring the increase in absorbance at 310
nm due to oxidation of veratryl alcohol to veratryl aldehyde
(E310 = 9:333 mM−1 cm−1 ). One unit of enzyme activity
was de5ned as the amount of enzyme required to oxidize
1  mol of substrate min−1 . Enzyme activity is expressed as
U ml−1 of culture 5ltrate. The extracellular proteins were
measured by the Bradford method (Bradford, 1976) with
bovine serum albumin as the standard. The results are the
average of three triplicate experiments with a standard error
of ¡ 5%.
Degradation of xenobiotics. The degradation of an-
thracene (3-ring PAH) and nitrobenzene by the fungus was
measured by adding 250 –500 ppm to the 25 ml Erlenmeyer
cultures in their 4th day of growth. After a total incuba-
tion period of either 12 or 24 days, adding HClO4 to pH
1 stopped the growth. The remaining compounds were ex-
tracted either with methylene chloride (for nitrobenzene) or
benzene (for anthracene) and analyzed by gas chromatog-
raphy (GC) coupled to a mass spectrometer TRIO 2, in
a capillar column (50  m, 0.32 ID) with helium as car-
rier gas at 0.7 psi, temperature program: 150◦ C for 1 min,
temperature increase 5◦ C min−1 and 240◦ C, for 10 min.
Uninoculated medium controls and heat-killed culture con-
trols were also included. The identi5cation of the diJerent
chemicals was carried out by analyzing the fragmentation
pro5les with the aid of Mass Lynx v 2.1. This software al-
lows the quantitative analysis of the kinetics of degradation
by integrating the area under each peak, which is a direct
measure of the concentration of the chemical.
3. Results and discussion
The kinetics of in vitro production of extracellular ligni-
nolytic activities by T. trogii were studied in a synthetic
medium (glucose 10 g l−1 =asparagine 3 g l−1 ) and in a com-
plex medium (malt extract 20 g l−1 =glucose 20 g l−1 ). High
activities of laccase and MnP were detected in both media
(but especially in the synthetic medium), laccase activity
being predominant. Both activities appeared before mycelial
biomass peaked (primary phase of growth: trophophasic
cultures), but their highest levels were detected during the
phase of secondary metabolism (idiophasic growth). Maxi-
mal levels detected were: 0:55 U ml−1 laccase, 0:07 U ml−1
MnP in malt extract=glucose medium; 4:1 U ml−1 laccase,
0:22 U ml−1 MnP in glucose=asparagine (Fig. 1). Attempts
to detect LiP in the culture media were unsuccessful. The
negative LiP tests suggests that the fungus produces no
signi5cant levels of this enzyme or its production requires
diJerent growth conditions. LiP activity was detected pre-
viously, when this strain was grown in a wood-containing
medium (Levin and Forchiassin, 2001).
 L. Levin et al. / International Biodeterioration & Biodegradation 52 (2003) 1 – 5 3

4.2 240

MnP [mU/ml]/ Dry weight [mg/25ml]

3.6 210

180
3.0
Laccase [U/m l]

150
2.4
120
1.8
90
1.2
60
0.6 30

0.0 0
0 3 6 9 12 15 18 21 24 27
Culture age [days]

Fig. 1. Kinetics of growth and enzyme production by Trametes trogii

in a synthetic medium (glucose 10 g l−1 =asparagine 3 g l−1 ) (A) and in
a complex medium (malt extract 20 g l−1 =glucose 20 g l−1 ) (B). (
)
Growth (mycelial dry weight (mg=25 ml)) in (A), () growth in (B),
( ) laccase activity (U=ml) in (A), () laccase activity (U=ml) in (B),
•
(
) MnP activity (mU=ml) in (A), ( ) MnP activity in (B).

Fig. 3. Consume of 250 and 500 ppm of anthracene by trophophasic

cultures of T. trogii grown in glucose=asparagine medium: (A) abiotic
control, (B) 500 ppm, (C) 250 ppm. The remaining hydrocarbon was
extracted with benzene.

cultures of T. trogii grown in glucose=asparagine medium

in the presence of 500 ppm of nitrobenzene. Only 3% of
the nitrobenzene was recovered from trophophasic cultures
(Fig. 2B; abiotic control Fig. 2A: 100%) and none from id-
iophasic cultures (Fig. 2C).
On Fig. 3 the consumption of 250 and 500 ppm of an-
thracene by trophophasic cultures of T. trogii grown in
glucose=asparagine medium is shown. Either 10% or 5%,
of respectively, 500 and 250 ppm of anthracene, added to
viable cultures, were recovered at trophophasic phase (Fig.
3B and C). Similar results were obtained in the complex
medium.
The resolved GC peaks were identi5ed and matched with
the fragmentation pro5les in the MassLynx spectral library
(Fig. 4). Chromatograms after 12 days of incubation in malt
extract=glucose medium with anthracene showed the pres-
Fig. 2. Chromatograms of methylene chloride extracts obtained from cul- ence of a peak (retention time 10.07 min) (Fig. 4B), absent
tures of T. trogii grown in glucose=asparagine medium in the presence of in the control incubated without anthracene (Fig. 4A). This
500 ppm of nitrobenzene: (A) abiotic control (12 days), (B) trophophasic peak was identi5ed as 4-methoxybenzaldehyde.
culture (12 days), (C) idiophasic culture (24 days). Most of the white rot fungi tested to date (Bjerkandera,
Phanerochaete and others) produce quinone intermediates
T. trogii showed considerable tolerance to the xenobi- during the degradation of PAHs, but it was demonstrated
otic compounds assayed; concentrations of up to 500 ppm previously that strains of the genus Trametes degraded an-
anthracene and 250 ppm nitrobenzene did not signi5cantly thracene without any accumulation of anthraquinone (Field
aJect its growth (data not shown). With 500 ppm of ni- et al., 1992). Identical results were obtained in the present
trobenzene, mycelial dry weight decreased 50%. study.
Fig. 2 shows the chromatograms obtained from the ex- A number of nonligninolytic fungi have intracellular
traction of trophophasic (12 days) and idiophasic (24 days) mechanisms for PAH degradation that usually lead to
4 L. Levin et al. / International Biodeterioration & Biodegradation 52 (2003) 1 – 5
Fig. 4. Mass spectra of the metabolite detected after incubation of T. trogii
in the presence of anthracene. In the corner: chromatograms of benzene
extracts obtained from T. trogii grown in malt extract=glucose medium
(A) for 12 days without anthracene, to which 250 ppm of anthracene were
added immediately before the extraction, (B) 250 ppm of anthracene were
added to the cultures at 4th day of growth, and extracted 8 days later.
dihydrodiol and hydroxy PAH metabolites. These prod-
ucts are suspected to originate from the activity of cy-
tochrome P-450 monooxygenases in conjunction with
epoxide hydrolase. Epoxides and dihydrodiols are very po-
tent carcinogens. Even P. chrysosporium has been shown
to metabolize phenanthrene under nonligninolytic condi-
tions to dihydrodiol and hydroxylated PAH metabolites
(Cerniglia et al., 1992). Such compounds were not detected
in this study. As suggested by Bezalel et al. (1996), it is
probable that both ligninolytic and cytochrome P-450 en-
zymes are involved in the degradation of PAHs in vivo.
Cytochrome P-450 enzymes perform the initial modi5ca-
tion of the aromatic groups followed by degradation of
the PAH derivative by the ligninolytic system. The pres-
ence of both a PAH-degrading cytochrome P-450 and a
ligninolytic system in P. chrysosporium supports this hy-
pothesis (Masaphy et al., 1996). Aryl metabolites, such as
4-methoxybenzaldehyde, which are produced simultane-
ously with the extracellular ligninolytic enzymes, have an
important role in the ligninolytic system of white rot fungi.
The most commonly occuring aryl compounds are veratryl
(3,4 -dimethoxybenzyl) and p-anisyl (4-methoxybenzyl)
alcohol–aldehyde. Veratryl alcohol is known to be a co-
factor involved in the degradation of lignin, lignin model
compounds and xenobiotic pollutants by LiP. P-anisyl al-
cohol is thought to be involved in H2 O2 generation as a
substrate for the extracellular aryl alcohol oxidase. The
addition of known precursors and aromatic acids represent-
ing lignin degradation products stimulated the production
of aryl metabolites (Mester et al., 1997). Similarly the pres-
ence of anthracene or its degradation products could induce
the synthesis of the aryl compound detected in our study.
T. trogii in contrast to the well studied model organism
P. chrysosporium, is N-unregulated. SuFcient or excess
N-nutrients stimulate high MnP and laccase titres in parallel
with the high biomass production. This characteristic of the
fungus makes it an outstanding candidate for large-scale
fermentation to produce ligninolytic enzymes in bulk for
bioremediation. It produces high amounts of MnP, and
higher laccase levels than those reported for most other
white rot fungi under favorable conditions, accompanied
by high levels of the hydrogen-peroxide-producing enzyme
glyoxal oxidase (Levin and Forchiassin, 2001). The pres-
ence of extracellular peroxidases, but also of laccase, may
be crucial to an eJective oxidation of PAH adsorbed to soil
substrates. An eJective conversion of PAH in biotechno-
logical soil remediations may nevertheless greatly depend
on the presence of H2 O2 in the substrate or its generation
by the fungus itself (Gramss et al., 1999). Although the
role of individual ligninolytic enzymes in PAH-degradation
is still not completely understood, it was shown recently
that both laccase and MnP are capable or PAH degra-
dation in the presence of appropriate mediators secreted
by ligninolytic fungi (Gramss et al., 1999; Johannes and
Majcherczyk, 2000). The BTEX-degradation system is ex-
pressed during primary metabolism in P. chrysosporium
(Yadav and Reddy, 1993; Song, 1997). Extracellular lignin-
and manganese-peroxidase are not produced during this
stage; probably laccase or other hitherto uncharacterized
enzyme(s) may be involved in their oxidation. In view of
the results currently being obtained in our laboratory on the
biodegradation of diJerent organopollutants by T. trogii,
this strain seems promising for detoxi5cation.
Acknowledgements
To CONICET and University of Buenos Aires for 5nan-
cial support.
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