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Degradation of polycyclic aromatic hydrocarbons bynewly isolated Curvularia sp. F18, Lentinus sp. S5, andPhanerochaete sp. T20Kanokpan Juckpecha, Onruthai Pinyakonga,b, Panan Rerngsamrana,b,∗
a Bioremediation Research Unit, Department of Microbiology, Faculty of Science, Chulalongkorn University,
Bangkok 10330 Thailandb National Centre of Excellence for Environmental and Hazardous Waste Management (NCE-EHWM),
Chulalongkorn University, Bangkok 10330 Thailand
∗Corresponding author, e-mail: [email protected] 9 Nov 2011
Accepted 19 Apr 2012
ABSTRACT: Three chromogenic substances with structures resembling those of polycyclic aromatic hydrocarbons (PAHs)
were incorporated in culture medium in order to screen for fungi capable of degrading PAHs. Curvularia sp. F18,
Lentinus sp. S5, and Phanerochaete sp. T20 were isolated and shown to have the ability to degrade both low- and high-
molecular weight PAHs, with the most prominent degradation being observed with Phanerochaete sp. T20. Preliminary
metabolite analysis of fluorene degradation by Phanerochaete sp. T20 using HPLC and GC-MS revealed that one of the
early metabolites was 9-fluorenol, which is a less toxic substance. This fungus survived in 500 mg/l of PAH for at least
30 days. The fungus could degrade a mixture of four PAHs (25 mg/l each), resulting in the reduction of 97, 59, 39, and
47% of fluorene, phenanthrene, fluoranthene, and pyrene, respectively. This work demonstrates that Phanerochaete sp. T20
could be used to bioremediate environments contaminated with high concentrations and/or mixtures of PAHs.
KEYWORDS: biodegradation, bioremediation, fungi, mixed PAHs
INTRODUCTION
Polycyclic aromatic hydrocarbons (PAHs) are a group
of environmental pollutants that are composed of
carbon and hydrogen with fused benzene rings in
linear, angular, and clustered arrangements. Based
on the molecular weight of these hydrocarbons, PAHs
can be classified into two broad groups: (i) the low-
molecular weight PAHs that contain 2–3 benzene
rings, such as naphthalene, fluorene, and phenan-
threne, and (ii) the high-molecular weight PAHs, such
as fluoranthene, pyrene, and chrysene1. PAHs are
generated as byproducts of incomplete combustion
of organic substances, which are found in burnt fos-
sil fuels, forest fires, volcano eruptions, and motor
vehicle emissions, as well as in grilled and smoked
foods2. PAHs can also be found as contaminants
at industrial sites, especially those associated with
petroleum or gas production and wood preserving
processes3, 4. PAHs and their metabolites are reported
to possess mutagenic and carcinogenic properties for
humans and other animals5, 6. Consequently, the
US Environmental Protection Agency has listed some
PAHs as priority pollutants1. Generally, the high-
molecular-weight PAHs are less water-soluble and
more recalcitrant to degradation than low-molecular-
weight PAHs7. Investigations of the content of PAHs
found in several contaminated areas reveal that con-
tamination is the result of a mixture of PAHs rather
than a single type of contaminant4, 8–10. Due to the
long half life of PAHs and the human activities that
cause the emissions of these contaminants into the
environment every day, PAHs continuously increase
and accumulate in the soil, water, and sediments and
thus appropriate treatment is required to reduce the
concentration and toxicity of these substances.
Chemical methods, such as chemical oxidation
and liquid solvent extraction, and physical meth-
ods, such as incineration and microwave energy
treatments, have been shown to have high levels
of efficiency in remediating sites contaminated with
PAHs11. However, these methods require complex
technologies, have high treatment cost, tend to use
excessive amounts of organic solvents, and may harm
living organisms12. Bioremediation, a safe, envi-
ronmentally friendly, and effective method, uses the
ability of organisms, such as bacteria, fungi, algae,
or plants, to reduce the concentrations of PAHs to
an acceptable level by transforming them into less
toxic forms or to completely mineralize them into
Reproduced from ScienceAsia 38: 147-156 (2012).
Panan Rerngsamran: Participant of the 22nd UM, 1994-1995.
394
CO213. Fungi have advantages over other organisms
in that they produce classes of enzymes, such as lignin
peroxidase, manganese peroxidase, and laccase, that
can interact with several types of PAHs with a fairly
high degree of non-specific activity14. They also have
other enzymatic systems, such as cytochrome P450
monooxygenase and epoxide hydrolase that oxidize
PAHs15, 16. Fungi are also tolerant to high concen-
trations of recalcitrant compounds and are able to
flourish in extreme conditions, such as at high temper-
atures and under low pH conditions. In addition, the
fact that fungi form large, branching mycelia makes
it possible for them to grow and distribute through
a solid matrix to degrade PAHs within contaminated
areas (in situ) by virtue of secreting extracellular
enzymes or by sequestration of PAHs17–19. Fungi can
also degrade PAHs under microaerobic and very-low-
oxygen conditions20. In addition to biodegradation
and mineralization of the PAHs, fungi adsorb PAHs
onto their hydrophobic cell wall21 and/or store them
in vacuoles or other organelles inside the cells22, 23.
In combination, all of these mechanisms lead to the
reduction of PAHs in the environment. Several reports
have demonstrated that fungi, such as Phanerochaetechrysosporium, Cunninghamella elegans, Trametesversicolor, Bjerkandera adusta, and Pleurotus ostrea-tus, play an important role in the degradation of a wide
variety of xenobiotic compounds, including PAHs18.
Most of the current research in the field has studied
the ability of a specific fungus to degrade a particular
PAH compound24–27. However, contaminated sites
are commonly contaminated with a mixture of PAHs.
Therefore, the objective of this study was to screen
for fungi that could degrade a mixture of four PAHs,
including fluorene and phenanthrene, and fluoran-
thene and pyrene, as representatives of low- and high-
molecular weight PAHs, respectively. These fungi
have the potential to be used for in situ bioremediation
at environmental sites where contamination is caused
by several types of PAHs, a more common scenario.
MATERIALS AND METHODS
Polycyclic aromatic hydrocarbons (PAHs)
Fluorene was obtained from the Wako Pure Chemical
Industries Co. (Japan). Fluoranthene was obtained
from the Kanto Chemical (Japan). Phenanthrene,
pyrene, benomyl, and the three chromogenic PAH-
like substances (guaiacol, azureB, and phenol red)
were obtained from the Sigma-Aldrich Co. (USA),
9-fluorenone was obtained from Nacalai Tesque Co.
(Japan), and 9-fluorenol was obtained from the TCI
Co. (Japan). All chemicals were of analytical grade.
Media
Two percent malt extract agar (MEA), used for the
preliminary isolation of fungi, contained (per litre):
20 g malt extract, 5 g peptone, 20 g glucose, 15 g agar,
3 mg benomyl, and 50 mg chloramphenicol28.
Minimal medium (MM), used for peroxidase en-
zyme screening, contained (per litre): 0.5 g KH2PO4,
0.5 g MnSO4, 0.1 g NH4NO3, 18 g agar, and 200 ml
of trace elements solution containing the following
reagents (per litre): 5 g Na2EDTA, 0.5 g FeCl3, 0.05 g
ZnCl2, 0.01 g CuCl2, 0.01 g CoCl2 · 6 H2O, 0.01 g
H3BO3, and 1.6 g MnCl225.
Modified GPY (mGPY), used for the preparation
of fungal inocula, contained (per litre): 10 g glucose,
3 g peptone, 2 g yeast extract, 1 g KH2PO4, 1 g
MgSO4 · 7 H2O, and 0.4 g Na-tartrate25.
N-limited medium, used for the biodegradation
experiments, contained (per litre): 10 g glucose,
0.1 g NH4NO3, 1 g KH2PO4, 1 g MgSO4 · 7 H2O,
0.01 g FeSO4 · 7 H2O, 0.01 g ZnSO4 · 7 H2O, 0.001 g
MnSO4, and 0.001 g CuSO4 · 5 H2O29.
Fungal isolation
Wood-rot fungi or mushrooms growing on rotten
wood and soil contaminated with petroleum oil were
collected from five provinces in Thailand (Bangkok,
Chonburi, Nakhon Pathom, Phatthalung, and Ra-
yong). Pieces of the inner tissue of the mushroom
or rotten wood fungi were placed on MEA media
containing 3 mg/l benomyl and 50 mg/l chloram-
phenicol to inhibit fast-growing fungi and bacteria,
respectively. Fungi from soil samples were isolated
using the soil dilution plate technique on MEA media
bearing benomyl and chloramphenicol. All plates
were incubated at room temperature for 5–7 days.
The fungi were isolated as pure cultures using the
same media. The purified isolates were kept as stock
cultures at 4 °C on MEA slants until used.
Screening for potential fungi using chromogenicsubstances
0.1% Guaiacol, 0.1% azureB, and 0.0025% phenol
red were used for the screening of potential fungi that
could produce peroxidase and laccase enzymes30–33.
Three 7-mm agar plugs containing fungal mycelia
from 7-day-old cultures on MEA plates were placed
on an MM plate that contained each chromogenic
substance. All MM plates were incubated in the
dark at room temperature for 3 days. The fungi that
were able to change these chromogenic substances,
as determined by visual appearance of a different
coloured halo around the fungal colony, were selected
395
for further study.
Fungal identification was performed by ITS1 - 5.8
RNA - ITS2 DNA sequence identity using standard
conditions and primers ITS1 and ITS4 or ITS1-F
and ITS434. Direct sequencing of both strands of
each purified amplicon was commercially performed
by 1st BASE DNA Sequencing Service (Malaysia).
The consensus nucleotide sequences were compared
to those available in the GenBank database using the
BLASTn algorithm.
Degradation of PAHs in liquid medium andmetabolite analysis
Fungi that exhibited positive results from the screen-
ing step were prepared for inocula in 100 ml of mGPY
liquid medium at 30 °C with shaking at 120 rpm for
5 days. Mycelia were harvested by centrifugation at
1120g for 15 min and washed twice with 0.85% (w/v)
NaCl. Three grams of fresh mycelia were added into
30 ml of N-limited medium in 125-ml Erlenmeyer
flasks containing three glass marbles. Each PAH
(100 mg/l) was added to these cultures and shaken
at 120 rpm in the dark at 30 °C. For the control
experiment, the flasks containing fungal mycelia were
autoclaved at 121 °C for 15 min prior to adding the
PAH. Samples were collected 15 days after incuba-
tion.
PAHs and their metabolites were extracted from
5 ml of samples using ethyl acetate, as previously de-
scribed24, and analysed by HPLC. HPLC analysis was
performed with a liquid chromatograph system (Shi-
madzu) equipped with an LC-3A pump, an SPD-2A
UV-Vis detector and a C-RIA recorder. The separation
column was 4.6× 150 mm (Inersil ODS-3) and the
mobile phase was methanol:water (80:30 (v/v)) at a
flow rate of 1 ml/min. The reduction of each PAH
was calculated as (1−AT/AC), where AC is the area
under the peak of the substrate from the control set and
AT is the area under the peak of the substrate from the
test set. Prior to extraction step of some experiments,
100 mg/l of pyrene was added as an internal standard
to monitor the extraction efficiency.
For metabolite analysis, the metabolites were
collected at the optimum production time. These
metabolites were acidified with hydrochloric acid to
pH 2–3 and extracted by ethyl acetate, evaporated,
resuspended in ethanol, and evaporated a second
time24. The precipitate was resuspended in methanol
and these were analysed for the presence of PAHs and
metabolites by HPLC and GC-MS. HPLC analyses
were performed as described above. Samples for
GC-MS analyses were analysed using gas chromatog-
raphy with time of flight mass spectrometry (Pegasus
III, LECO). The GC used a 50 m long HP-5 column
of 320 μm in diameter and was coated to a 0.25-μm
film thickness with 5% phenyl-methyl-syloxane.
Survival of selected fungi in differentconcentration of PAHs
Three grams of fresh mycelia of each selected fungus
were inoculated into N-limited medium supplemented
with each single PAH at 25, 50, 100, 300, and
500 mg/l and was incubated in the dark at 30 °C for
30 days. To test for the survival of the fungus, 100 μl
of fungal culture was dropped on to MEA medium.
The plate was incubated for 7 days and fungal growth
was observed.
Ability of fungi to grow on solid mediumcontaining mixed PAHs
The four PAHs, including fluorene, phenanthrene,
fluoranthene, and pyrene, were incorporated into MM
agar plates at a concentration of 25 mg/l each. Three
7-mm agar plugs containing fungal mycelia from 7-
day-old cultures grown on MEA plates were placed
on each of the MM plates. All plates were incubated
in the dark at room temperature for 10 days.
Degradation of mixed PAHs in liquid medium
Each fungal inoculum was prepared and 3 mg of fresh
mycelia were added into 30 ml of N-limited medium
in 125 ml Erlenmeyer flasks containing three glass
marbles. A mixture of the four PAHs (25 mg/l each)
was added to each flask. All flasks were shaken
at 120 rpm in the dark at 30 °C. Flasks containing
fungal mycelia that were autoclaved at 121 °C for
15 min prior to the addition of the PAHs were used
as controls. Samples were collected at 15 days after
incubation, whereupon PAHs were extracted and anal-
ysed as previously described24. All data are presented
as the mean value derived from duplicate samples.
RESULTS
Fungal isolation and screening
From the initial 55 fungal isolates, 47 isolates (85.5%)
were unable to change the colour of any of the
three indicator compounds. However, eight isolates
(14.5%) were able to change at least one of the three
chromogenic substances. Among the latter, three
isolates consisting of F18, S5, and T20 gave the
widest zone of colour changes and, therefore, were se-
lected for further characterization because the colour
changes of these substances have been reported to be
related to the production of peroxidase and laccase
enzymes, which are responsible for the degradation of
PAHs30–33.
396
Nucleotide sequencing of the ITS regions of the
rRNA genes (ITS1 - 5.8S RNA - ITS2) was per-
formed for the F18, S5, and T20 isolates followed
by BLASTn searches to identify close relatives (high
sequence identity) in the GenBank database. The F18
isolate had 100% nucleotide sequence identity to the
sequence designated as Curvularia sp. F SMR-2011
(HQ909079). Isolates S5 and T20 showed 98% and
93% sequence identity to those designated as Lentinussquarrosulus strain 7-4-2 (GU001951) and Phane-rochaete sp. ATT215 (HQ607891), respectively. In
this study, we refer to these isolates as Curvulariasp. F18, Lentinus sp. S5 and Phanerochaete sp. T20.
The consensus ITS nucleotide sequences for isolates
F18, S5, and T20 have been submitted to the Gen-
Bank database under accession numbers JN253597,
JN253598, and JN253599, respectively.
Degradation of single PAH in liquid medium
These three fungi were tested for their ability to
degrade the four representative PAHs of fluorene,
phenanthrene, fluoranthene, and pyrene in nitrogen-
limited liquid media (100 mg/l). After analysis of the
metabolites by HPLC, the areas under the peaks were
compared to that of the control set using the killed
fungus. The reduction for each PAH was calculated
using the aforementioned formula. Curvularia sp. F18
was found to be unable to degrade fluoranthene,
phenanthrene, and pyrene, as the substrate peaks were
still present (data not shown). However, Curvulariasp. F18 was able to degrade fluorene, as it showed a
90% reduction in the initial level added after 15 days
of incubation. In addition, the analysis of Curvulariasp. F18 showed the appearance of four major new
peaks (Fig. 1) that are assumed to be intermediate
metabolites. Lentinus sp. S5 was unable to degrade
fluoranthene under these conditions (data not shown)
but degraded fluorene, phenanthrene, and pyrene at
60, 86, and 85% relative to the control cultures,
respectively, (Fig. 2). Finally, Phanerochaete sp. T20
was able to degrade 83% of fluorene, 87% of phenan-
threne, and 31% of fluoranthene (Fig. 3) but was
unable to degrade pyrene (data not shown).
Metabolite analysis
Although the results indicate that Lentinus sp. S5 and
Phanerochaete sp. T20 are potentially good candi-
dates for PAHs degradation, isolate S5 exhibited a
slow growth rate. Thus we selected Phanerochaetesp. T20 for further characterization. This fungus
was also closely related to P. chrysosporium, which
is the most reported PAH-degrading fungus35–38. In
order to determine the preliminary PAH degradation
Area
Time: 13.0114 Minutes - Amplitude: -- Volts
Detector A (275nm)
Retention Time
0.30
0.25
0.20
0.15
0.10
0.05
0.00
1.7
67
1
71
65
6
2.3
62
4
04
81
3
2.6
96
1
37
01
9
3.1
16
3
47
76
3.3
37
3
61
83
4
4.5
72
6
46
89
5.3
48
9
02
5.6
78
9
39
0
6.1
74
2
16
59
7.4
09
4
33
23
04
Minutes
9.5
91
8
92
3
10
.54
0
81
78
0
0 1 2 3 4 5 6 7 8 9 10 11 12
(a)
1.8
87
2
62
18
5
2.3
67
8
98
46
2.6
13
5
47
55
52
.98
1
24
48
82
4.0
81
7
21
4.5
78
2
56
33
5.6
66
5
67
1
7.3
95
1
01
10
1
10
.51
7
57
56
4
12
.40
9
76
27
7
Minutes0 1 2 3 4 5 6 7 8 9 10 11 12
0.06
0.05
0.04
Retention Time
Area
Time: 13.0114 Minutes - Amplitude: -- Volts
Detector A (275 nm)
0.03
0.02
0.01
0.00
-0.01
(b)
Fig. 1 HPLC chromatograms of fluorene degradation by
Curvularia sp. F18. Representative HPLC chromatograms
from a 15-day-old culture of (a) autoclaved and (b) live
mycelia of Curvularia sp. F18 grown in N-limited media
containing fluorene at 100 mg/l. Arrows indicate the peaks
of fluorene. A circle specifies the intermediate peaks.
pathway used by Phanerochaete sp. T20, we de-
termined the early metabolites that arose from the
degradation of fluorene. The Phanerochaete sp. T20
fungus was grown in N-limited medium supplemented
with 100 mg/l of fluorene. The first intermediate
peak was observed on the third day of incubation and
was extracted, purified, and analysed by HPLC and
GC-MS. In each case, 9-fluorenol and 9-fluorenone
were used as external standards because these prod-
ucts have been reported to be the first metabolites
of fluorene degradation in most white rot fungi39.
The HPLC analysis also revealed that the purified
intermediate had the same retention time (RT =0.71) as 9-fluorenol (Fig. 4), while GC-MS analysis
revealed that the purified intermediate gave m/z ratio
values of 63, 76, 91, 126, 152, and 181, which were
the same as those for 9-fluorenol. Taken together,
the results from HPLC and GC-MS methods strongly
suggested that the first observed intermediate from
fluorene degradation by Phanerochaete sp. T20 was
likely to be 9-fluorenol.
397
4.2
65 2
84666
5.2
45 1
09383
5.5
74 1
16582
6.8
10 5
598579
7.5
47 4
63225
8.7
59 9
7132
9.5
84 9
563630
10.6
85 1
023032
13.1
90 6
333
14.4
69 7
810
15.3
81 5
40
6.1
92 2
5706
Minutes
1.4
68 5
1520
2.2
69 6
77277
2.4
92 9
99375
2.2
926 3
29021
3.1
37 5
56040
3.7
42 1
81691
Time: 16.0138 Minutes - Amplitude: -- Volts
Detector A (275 nm)
Retention Time
Area
0.5
0.4
0.3
0.2
0.1
0.0
0 2 4 6 8 10 12 14 16
(a)
2.7
97
524163
3.0
29
752727
3.4
17
446100
3.9
43
87089
4.1
09
247823
4.4
83
151927
4.9
82
373342
6.5
18
9070795
7.3
86
209171
8.3
84
25513
9.1
66
9912338
10.4
20
2282909
13.6
43
7411
14.6
43
924
0.4
20
1225
1.5
14
15805
Time: 16.0138 Minutes - Amplitude: -- Volts
Detector A (275 nm)
Retention Time
Area
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0 2 4 6 8 10 12 14 16
Minutes
2.2
49
8619903
(b)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0 2 4 6 8 10 12 14 16Minutes
1.1
67
6
91
09
1.6
45
5
61
10
2.2
82
2
18
20
72
.56
2
42
40
75
2.9
78
4
35
43
3
3.8
32
7
36
75
4.2
45
1
90
98
4.6
31
1
66
07
4.9
00
5
39
5
5.3
10
1
91
51
5.8
04
5
80
96
.08
3
15
61
6.4
88
4
28
18
7.2
54
1
73
39
93
3
8.1
79
1
25
79
21
9.0
08
2
10
6
9.8
87
7
47
24
30
11
.16
8
64
78
43
13
.64
6
11
63
9
15
.16
2
86
63
Detector A (275 nm)
Retention Time
Area
Time: 16.0138 Minutes - Amplitude: -- Volts
(c)
0.4
0.3
0.2
0.1
0.0
0 2 4 6 8 10 12 14 16
Minutes
0.2
31 3
628
1.6
06 1
3234
2.3
59 6
339637
3.7
62 8
7040
3.9
80 2
77210
4.5
36 1
5890
5.0
85 3
348
5.7
68 1
001
6.4
61 7
760
7.2
25 3
886659
8.1
81 2
98067
8.9
92 2
9644
9.8
39 9
174522
11.1
51 1
972371
Time: 16.9914 Minutes - Amplitude: 0.001136 Volts
Detector A (275 nm)
Retention Time
Area
(d)
Time: 18.0156 Minutes - Amplitude: -- Volts
Detector A (275 nm)
Retention Time
Area
0.8
0.6
0.4
0.2
0.0
0 2 4 6 8 10 12 14Minutes
0.5
50 1
3148
1.4
87 6
5351
1.6
69 1
12518
2.2
48 3
48066
2.5
03 2
93992
2.6
13 5
42912
3.0
54 5
87542
3.4
32 6
84206
4.7
56 7
8652
5.2
30 1
29138
5.5
88 7
5325
6.1
95 4
6681
6.5
25 1
5213
6.7
46 2
1400
6.9
81 2
0084
7.3
61 1
394452
8.1
92 2
60524
9.3
18 8
6788
9.5
43 1
01070
10.5
09 2
0092068
12.1
25 1
947842
14.6
68 1
5406
(e)Time: 18.0156 Minutes - Amplitude: -- Volts
Detector A (275 nm)
Retention Time
Area
0.20
0.15
0.10
0.05
0.00
0 2 4 6 8 10 12 14Minutes
1.4
56
8
47
64
2.3
53
3
20
05
12
.65
4
14
92
42
72
.91
5
52
73
76
3.0
53
8
60
81
0
3.5
76
8
58
26
4
4.6
87
1
84
98
6
5.0
89
4
50
21
2
5.5
62
8
91
81
6.0
85
2
82
58
3
6.6
25
1
13
94
4
7.3
19
3
47
82
29
8.3
41
1
75
06
8.6
70
5
88
15
9.7
84
6
90
79
10
.46
3
25
84
07
2
12
.05
7
16
76
09
13
.17
5
10
43
8
14
.53
3
43
39
(f)
Fig. 2 HPLC chromatograms of fluorene, phenanthrene, and pyrene degradation by Lentinus sp. S5. Representative HPLC
chromatograms from a 15-day-old culture of (a, c, e) autoclaved and (b, d, f) live mycelia of Lentinus sp. S5 grown in
N-limited media containing (a, b) fluorene, (c, d) phenanthrene, and (e, f) pyrene at 100 mg/l. Arrows indicate the peaks of
fluorene. Circles specify the intermediate peaks. The internal loading standard of pyrene is found to the right of the substrate
peaks in the fluorene and phenanthrene degradations.
Survival of Phanerochaete sp. T20 at variousconcentrations of PAHs
To investigate the ability of this fungus to survive
under different concentrations of PAHs, fresh mycelia
from mGPY medium were cultured in media con-
taining different concentrations (25, 50, 100, 300,
or 500 mg/l) of fluorene, phenanthrene, fluoranthene,
or pyrene. After 30 days, survival of the fungus
was investigated by dropping the fungal culture onto
an MEA plate. Growth of Phanerochaete sp. T20
was observed on MEA plates from all five tested
concentrations of all four evaluated PAHs (data not
shown). The fungus was able to survive, at least to
some extent, in all four tested PAHs at concentration
of up to 500 mg/l for at least 30 days.
Testing the ability of Phanerochaete sp. T20 togrow on solid medium containing mixed PAHsand to degrade mixed PAHs in liquid medium
To further test for the potential of Phanerochaetesp. T20 to degrade a mixture of PAHs, this isolate was
evaluated for its ability to grow on MM agar medium
containing an equal concentration of fluorene, phenan-
threne, fluoranthene, and pyrene as the sole carbon
398
0.8
0.6
0.4
0.2
0.0
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Minutes
1.5
67 7
2251 1
1.9
26 8
5120 2
2.0
74 6
5174 3
2.3
08 8
2940 4
2.6
58 1
36437 5
3.2
54 2
57776 6
3.8
78 3
4508 7
4.2
51 4
08461 8
4.8
18 1
44984 9
5.7
83 1
4461 10
6.6
83 5
07 11
7.9
20 8
37 12
9.6
75 8
713597 13
11.5
77 1
74780 14
Retention Time
Area
Time: 13.0112 Minutes - Amplitude: -- Volts
Detector A (275 nm) (a)
0.5
04 7
37 1
1.6
43 1
656857 2
1.9
41 9
454 3
2.2
27 9
202 4
2.4
77 2
0987 5
2.6
56 2
6508 6
3.8
86 8
7021 8
4.2
53 5
31512 9
4.8
20 1
98965 10
5.7
15 1
4356 11
6.1
45 1
2350 12
6.5
16 5
473 13
8.6
42 1
428 14
9.1
67 7
22 15
9.6
78 2
49442 16
11.5
69 4
322 17
3.2
57 3
939075 7
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Minutes
Retention Time
Area
Time: 13.0112 Minutes - Amplitude: -- Volts
Detector A (275 nm) (b)
2.0
24
2
55
40
4
2.5
17
1
05
73
65
2.9
33
8
22
46
1
4.0
63
1
82
13
7
4.4
59
1
57
20
84
.67
7
28
81
47
5.3
09
3
02
89
6
6.1
59
3
83
85
7
6.7
16
1
95
93
3
7.5
41
1
06
14
81
3
9.3
91
2
08
73
10
.26
2
19
63
74
87
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Minutes
Time: 13.0114 Minutes - Amplitude: -- Volts
Detector A (275 nm)
Retention Time
Area
(c)
1.9
19 2
37774
2.4
51 3
330303
2.9
44 5
84459
3.2
96 1
436026
3.8
91 1
18337
4.0
58 2
14915
4.5
12 4
037412
5.3
42 2
50871
5.7
24 1
18748
6.4
18 5
81072
7.5
54 1
410230
9.4
05 2
0094
10.2
79 1
9520198
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0 1 2 3 4 5 6 7 8 9 10 11 12 13Minutes
Retention Time
Area
Time: 12.9929 Minutes - Amplitude: -0.001647 Volts
Detector A (275 nm) (d)
Retention Time
Area
Detector A (275 nm)
1.4
92
1
6
1.9
30
8
90
55
2.3
58
4
79
30
6
2.6
88
6
28
01
2.9
39
1
82
27
4
3.3
05
2
84
07
1
4.5
18
3
79
12
2
5.3
21
1
47
58
3
6.6
08
1
83
45
7.2
75
8
56
13
8.3
27
1
05
4
8.7
29
1
83
9
9.4
04
1
76
99
00
4
10
.29
5
20
58
56
63
12
.11
1
34
89
5
0.8
0.6
0.4
0.2
0.0
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Minutes
Time: 13.0114 Minutes - Amplitude: -- Volts
(e)
2.0
10
1
09
33
3
2.5
72
1
01
69
89
2.7
67
2
25
54
42
.96
2
17
64
21
4
3.3
04
3
77
56
51
3.8
65
3
78
79
74
.05
2
36
07
28
4.5
12
1
04
95
97
5
5.3
47
1
20
59
63
5.7
51
7
69
68
3
6.3
81
1
56
86
05
7.2
44
7
85
45
5
8.0
83
2
58
99
8
8.4
22
5
49
39
7
9.4
05
1
36
71
56
9
10
.29
5
21
11
69
23
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0 1 2 3 4 5 6 7 8 9 10 11 12 13Minutes
Retention Time
Area
Detector A (275 nm)
Time: 13.0114 Minutes - Amplitude: -- Volts
(f)
Fig. 3 HPLC chromatograms of fluorene, phenanthrene, and fluoranthene degradations by Phanerochaete sp. T20.
Representative HPLC chromatograms from a 15-day-old culture of (a, c, e) autoclaved and (b, d, f) live mycelia of
Phanerochaete sp. T20 grown in N-limited media containing (a, b) fluorene, (c, d) phenanthrene, and (e, f) fluoranthene
at 100 mg/l. Arrows indicate the peaks of fluorene. Circles specify the intermediate peaks. The internal loading standard of
pyrene is located to the right of the substrate peak.
sources. Clear growth was observed on this medium,
although with a slower growth rate compared to its
growth on MEA medium alone (data not shown). This
observation serves as a preliminary indication that this
fungus should be able to use or degrade these mixed
PAHs, therefore, we evaluated its ability to degrade
mixed PAHs (25 mg/l each) in liquid medium. After
15 days in culture, PAHs and their metabolites were
extracted from the culture media, analysed by HPLC
and the results were compared with those obtained
from the control cultures (autoclaved mycelia). A
decrease in the area of the substrate peaks and the
appearance of new peaks (assumed metabolites) as-
sociated with the live mycelia cultures, but not in the
autoclaved controls, were indicators of the ability of
the fungus to degrade multiple PAHs (Fig. 5). These
results indicated that Phanerochaete sp. T20 could
degrade a mixture of four different PAHs with initial
concentrations of 25 mg/l each. Next, we calculated
the reduction in the level of each PAH relative to
the level in the corresponding control (autoclaved
mycelia), and the results revealed that Phanerochaete
399
Retention Time
Area0.20
0.15
0.10
0.05
0.00
0 1 2 3 4 5 6 7Minutes
0.1
25 7
20.2
50 4
20.3
23 1
60
0.4
94 1
67
0.8
28 1
30
1.0
07 1
31
1.1
29 8
0
1.3
52 2
706
1.7
66 8
071
1.9
35 1
8782
2.2
95 6
887
2.6
70 1
8706
3.2
57 1
245368
4.0
97 1
2538
4.5
92 6
34.6
92 2
7
4.8
59 1
768
5.1
42 3
2
5.3
18 2
80
5.5
08 3
3
5.7
12 2
92
5.8
75 8
56.0
17 3
98
6.2
00 5
67
6.3
08 3
78
6.4
82 2
20
6.6
12 7
14
6.7
75 2
61
6.9
07 1
89
Time: 7.00605 Minutes - Amplitude: -- Volts
Detector A (275 nm) (a)
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0 1 2 3 4 5 6 7Minutes
0.1
58
1
33
0.2
17
7
40
.30
3
73
0.5
55
1
66
0.8
33
3
22
1.0
26
2
58
1.0
94
3
67
1.2
81
1
31
8
1.7
65
4
06
2
2.0
00
1
11
2.1
30
9
2
2.6
18
3
50
3.2
58
2
89
33
46
4.1
04
4
04
78
4.7
31
2
55
4.8
77
3
50
4.9
49
1
30
5.1
41
2
88
5.3
30
1
39
5.5
92
8
35
.65
3
63
5.8
25
8
35
.95
8
99
6.1
70
3
46
.30
8
12
36
.45
8
44
6.5
37
5
76
.66
4
14
96
.79
9
25
6
Retention Time
Area
Time: 0.0801945 Minutes - Amplitude: 2.5e-005 Volts
Detector A (275 nm) (b)
Fig. 4 HPLC chromatograms of purified intermediate and 9-
fluorenol. Representative HPLC chromatograms illustrating
(a) the purity of the purified intermediate compound from
a three-day-old culture of Phanerochaete sp. T20 grown in
N-limited media containing fluorene at 100 mg/l and (b) 9-
fluorenol.
sp. T20 was able to remove fluorene, phenanthrene,
pyrene, and fluoranthene by 97, 59, 47, and 39%, re-
spectively, (Fig. 5). Thus this fungus has the potential
to degrade and remove a mixture of at least four PAHs
(fluorene, phenanthrene, fluoranthene, and pyrene).
DISCUSSION
The white-rot fungi are a group of organisms ca-
pable of degrading a wide variety of environmental
pollutants, including PAHs, due to the production
of extracellular and relatively nonspecific (for the
substrate) ligninolytic enzymes, including lignin per-
oxidase, manganese peroxidase, and laccase13. In this
study, we initially isolated fungi that had the potential
to degrade PAHs by screening for the ability of these
isolates to degrade the chromogenic substrates phenol
red, guaiacol, and azureB, which have structures that
resemble PAHs. We used these substrates as indicators
for the potential production of extracellular peroxidase
and/or laccase enzymes. Two white-rot fungi, which
were identified as Lentinus sp. S5 and Phanerochaetesp. T20, and one Ascomycetes fungus, Curvulariasp. F18, were isolated using this criterion. These fungi
1.6
46 1
32184
1.9
06 3
31692
2.2
58 4
32066
2.6
77 1
47849
3.2
76 3
41917
3.9
06 1
03042
4.2
85 4
72491
4.6
22 8
2766
4.9
58 5
5566
5.5
47 3
0921
5.8
36 2
5229
6.2
24 7
3710
7.2
89 7
174
8.0
10 2
282
8.8
98 8
078 9.7
83 1
559661
10.4
07 3
886008
11.3
18 1
2632
12.1
17 5
6799
14.0
01 5
065412
15.4
07 6
004147
0.4
0.3
0.2
0.1
0.0
0 2 4 6 8 10 12 14 16 18 20Minutes
Time: 20.0173 Minutes - Amplitude: -- Volts
Detector A (275 nm)
Retention Time
Area
(a)
0.25
0.20
0.15
0.10
0.05
0.00
0 2 4 6 8 10 12 14 16 18 20
Minutes
Retention Time
Area
Time: 20.0173 Minutes - Amplitude: -- Volts
Detector A (275 nm)
1.6
56 1
08294
1.9
89 1
24833
2.3
31 1
44938
2.7
21 3
20928 3.2
79 1
132226
3.8
21 1
5124
4.2
40 1
245042
4.8
64 4
15885
5.9
82 7
6727
6.1
92 3
5151
6.6
08 1
74331
8.5
88 9
68.8
82 8
40
9.2
75 7
9
9.7
88 4
6166
10.4
06 1
605813
12.1
57 8
5666
13.9
98 3
114917
15.4
07 3
203119
(b)
Fig. 5 HPLC chromatograms of mixed-PAHs degradation
by Phanerochaete sp. T20. Representative HPLC chro-
matograms from a 15-day-old culture of (a) autoclaved
and (b) live mycelia of Phanerochaete sp. T20 grown in
N-limited media containing fluorene, phenanthrene, fluoran-
thene, and pyrene at 25 mg/l each. The retention time of
fluorene, phenanthrene, fluoranthene, and pyrene were 9.6,
10.3, 13.9, and 15.3 min, respectively. Arrows indicate the
peaks of these four substrates.
were then tested for the ability to degrade two low-
molecular weight PAHs (phenanthrene and fluorene)
and two high-molecular weight PAHs (fluoranthene
and pyrene), both alone and together. All three fungal
isolates were found to be good candidates for PAH
bioremediation, showing good degradation of the low-
molecular weight PAHs. In addition, S5 and T20
also showed good and moderate degradation of the
high-molecular weight pyrene and fluoranthene, re-
spectively. Further characterization of Phanerochaetesp. T20 revealed that it can withstand and survive in
high concentrations of PAHs (at least up to 500 mg/l
for 30 days) and that the degradation of fluorene
produced a less toxic compound, 9-fluorenol, as the
major early intermediate. In addition, this strain
showed the potential ability to degrade a mixture of
all four of these PAHs.
Although fungi in the genus Curvularia have been
reported to be able to degrade tricyclic PAHs, such
400
as phenanthrene and anthracene40, 41, the degradation
of fluorene (which has a more complex structure) by
fungi in this genus had never been demonstrated prior
to this study. Our results indicate that, among the
four PAHs tested, Curvularia sp. F18 could only de-
grade fluorene, a tricyclic PAH with a five-membered
ring, at high efficiency. Although the intermediate
metabolites of fluorene were not investigated in this
study, several previous reports have shown that for
non-white-rot fungi fluorene and a number of other
PAHs are generally metabolized by cytochrome P450
monooxygenase and epoxide hydrolase to form trans-
dihydrodiols39. Due to its robust ability to degrade
fluorene, Curvularia sp. F18 should be further investi-
gated for its potential use in situ bioremediation where
high levels of fluorene contamination are present.
Lentinus sp. S5 is a member of the white-rot
Basidiomycetes fungi. The fungi in this genus have
been reported to degrade several types of PAHs using
both extracellular ligninolytic enzymes and intracellu-
lar P450 monooxygenase systems42, 43. In this study,
Lentinus sp. S5 was capable of degrading phenan-
threne, fluorene, and pyrene. However, it could not
degrade fluoranthene. The chromatograms from the
phenanthrene, fluorene, and pyrene degradations by
Lentinus sp. S5 (Fig. 2) revealed new peaks with the
same retention times for all three PAHs, indicating
that Lentinus sp. S5 used the same pathway or the
same mechanism to degrade these PAHs, giving rise
to the same intermediate metabolites. This possibility
remains to be confirmed. However, if this hypothesis
is correct, these metabolites would probably be the
PAH-quinone compounds, which, in general, are the
products of PAHs degradation by white rot fungi
including Lentinus via the ligninolytic and laccase
enzymatic reactions42. In the case of complete miner-
alization, this PAH-quinone will pass the ring fission
process and produce CO2 as the final product44.
Phanerochaete sp. T20 is a white-rot Basid-
iomycetes fungus that belongs to the same genus
as the well-known PAHs degrader, P. chrysospo-rium35, 45. In the degradation of each PAH alone,
Phanerochaete sp. T20 appeared to be capable of
degrading fluorene, phenanthrene, and fluoranthene;
and several intermediate peaks were seen in the chro-
matograms of the degradation. However, it could not
degrade pyrene, which is a fused tetracyclic aromatic
high-molecular weight hydrocarbon. In contrast,
when Phanerochaete sp. T20 was grown in N-limiting
medium containing all four PAHs, it was able to
degrade pyrene, as well as fluorene, phenanthrene, and
fluoranthene, and at a higher level than fluoranthene
alone. The apparent degradation of pyrene in the
presence of other easily degradable substrates would
probably result from the interactions between sub-
strates that subsequently enhance the degradation of
compounds that are more difficult to degrade, such as
pyrene46. Synergistic effect between PAHs and co-
metabolism were also proposed for this feature47. In
addition to its ability to degrade a mixture of low- and
high-molecular weight PAHs, Phanerochaete sp. T20
was able to grow and survive (at least to some extent)
at relatively high concentrations of PAHs (500 mg/l)
for at least 30 days. These results indicate a notable
PAH degradation efficiency of Phanerochaete sp. T20
and suggest that this fungus might be useful for in
situ bioremediation at sites where mixed and/or high
concentrations of PAHs are a problem.
The early metabolites of fluorene degradation
were further investigated to preliminarily determine
the degradation pathway of Phanerochaete sp. T20.
The results from HPLC and GC-MS indicated that
this metabolite was most probably 9-fluorenol, which
is a less mutagenic compound than fluorene39. This
observation is considered to be an indicator for the
first step of fluorene detoxification11. Thus Phane-rochaete sp. T20 likely degrades fluorene under non-
ligninolytic conditions such that it degrades fluorene
via cytochrome P450 monooxygenase, as is frequently
found with P. chrysosporium and other white-rot
fungi39, 48, 49.
The three PAH-degrading fungi that were isolated
in this study can potentially be used for bioremedi-
ation. Curvularia sp. F18 may be suitable for condi-
tions where the contamination is fluorene, while Lenti-nus sp. S5 and Phanerochaete sp. T20 may be suitable
for contamination sites where more than one type of
PAHs is the main concern. This study reports on the
preliminary isolation and investigation of three PAH-
degrading fungi. However, further detailed studies are
required to investigate the degradation pathways and
the degradation products of these useful fungi.
Acknowledgements: This study was financially sup-
ported by a grant for the development of new faculty at
Chulalongkorn University; the Thai Government Stimulus
Package 2 (TKK2555), under the Project for Establishment
of Comprehensive Centre for Innovative food, Health Prod-
ucts and Agriculture; and a CU graduate school thesis grant.
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