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International Biodeterioraton & Biodegradation 43 (1999) 125133

Decontamination of aged creosote polluted soil: the inuence of


temperature, white rot fungus Pleurotus ostreatus, and pretreatment
Trine Eggen a,*, Per Sveum b
Jordforsk, Centre for Soil and Environmental Research, N-1432 As, Norway
b
Deconterra, Gamle Snary vei 53, N-1330 Fornebu, Norway

Received 28 September 1998; received in revised form 13 November 1998; accepted 27 January 1999

Abstract
This study investigated the eect of inoculation of white rot fungus, Pleurotus ostreatus, temperature and two pre-treatment
methods on PAH degradation in aged creosote contaminated soil. It is shown that Pleurotus ostreatus has an overall positive
eect on PAH degradation, and that temperature and soil pre-treatment aect this degradation. In general, adding bark and
incubating at 228C before inoculation with white rot fungi has a better eect on PAH degradation than no pre-treatment, or
pre-treatment with fertilizer. At low temperature (88C) fungal inoculation had best eect when fertilizer was not added, and
signicant eect on degradation on dierent groups of PAH compounds, except for the more easily degradable compounds, 3ring PAHs and heterocyclic compounds was obtained. Pre-treatment with fertilizer stimulated microbial activity at low
temperature and enhanced PAH degradation even without addition of fungi. # 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Bioremdiation; White rot fungi; Composting; Microbial activity; PAH; Creosote

1. Introduction
Polycyclic aromatic hydrocarbons (PAH) are pollutants typically found at wood preservation plants and
gas work sites. Low molecular weight PAH are usually
readily degraded by indigenous microbes, while high
molecular weight PAHs are more recalcitrant (Lamar
and Glaser, 1994; Mueller et al., 1991).
Contaminants in articially or newly polluted soil
are degraded faster than in aged contaminated soils
(Erickson et al., 1993). Field et al. (1995) found that
the refractory fraction of Benzo(a)pyrene doubled
when the pollutant age increased from zero to three
months. This reduced biodegradation over time can be
explained by reduced bioavailability (immobilization in
* Corresponding author.

micropores or changes in binding forms e.g. oxidative


coupling reactions) (Bollag et al., 1992; Bollag and
Myers, 1992; McFarland et al., 1992).
White rot fungi secrete non-specic extracellular
enzymes, which are involved in the degradation of lignin (Barr and Aust, 1994). The same mechanisms that
give these fungi the ability to degrade lignin are also
used to degrade a wide range of pollutants (Higson,
1991), such as DDT (Barr and Aust, 1994; Fernando
et al., 1989), TNT (Majcherczyk et al., 1994), PCBs
(Sasek et al., 1993; Zeddel et al., 1993) and PAHs
(Bumpus, 1989; Morgan et al., 1993).
Degradation of high molecular weight compounds
by white rot fungi requires a suitable carbon co-substrate to be successful. Examples of such co-substrates
are potato pulp, wheat straw, peat, bark and wood
chips (Lamar and Glaser, 1994; Zeddel et al., 1993).
Adding compost reduces the amount of extractable

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T. Eggen, P. Sveum / International Biodeterioraton & Biodegradation 43 (1999) 125133

Table 1
Characterization of the soil. Concentration of PAHs are given as mean ppm2std (n = 3) (n.d. = not detected)
Soil after addition of amendments (after pre-treatment)
Parameters
Total Organic Carbon (g/kg)
Kjeldahl Nitrogen (g/kg)
Phosphorus (g/kg)
C:N:P
2-ring PAHs (ppm)a
3-ring PAHs (ppm)b
4-ring PAHs (ppm)c
5- and 6-ring PAHs (ppm)d
Sum PAH (ppm)e
Sum heterocyclic (ppm)f
Sum main compounds (ppm)g
Sum identied (ppm)h

Soil before addition of amendments


95
2.4
0.465
100:2.5:5

Reference soil

BTF-treatment

BT-treatment

104
4.7
0.99
100:4.5:1
16.123.1
370.0293.6
1606.5268.5
332.1241.4
2308.5285.1
114.3215.4
1813.0280.0
2439.32103.6

116
11.2
4.38
100:10:4
12.621.3
422.7246.8
1753.42138.3
273.3235.3
2449.52216.0
102.929.6
1991.32167.5
2564.92226.3

110
4.9
1.08
100:4.5:1
20.723.3
363.52128.3
1280.2285.1
512.6227.6
2156.5218.9
84.523.0
1616.9223.8
2261.6219.2

2-ring PAHs: Naphthalene, 2-Methylnaphthalene, 1-Methylnaphthalene, Biphenyl.


3-ring PAHs: Acenaphthylene, Acenaphthene, Fluorene, Phenanthrene, Anthracene, 3-Methylphenanthrene, 2-Methylphenanthrene, 2Methylanthracene, 4,5-Methylenephenanthrene, 4-9-Methylphenanthrene, 1-Methylphenanthrene.
c
4-ring PAHs: Fluoranthene, Benz)e)acenaphthylene, Pyrene, Ethylmethylphenanthrene, Benzo(a)uorene, Benzo(b)uorene, 2Methylpyrene/methyluoranthene, 1-Methylpyrene, Benzo(ghi)uoranthene, Benzo(c)phenanthrene, Benz(a)anthracene, Chrysene/Triphenylene.
d
5- and 6-ring PAHs: Benzo(j)uoranthene, Benzo(e)pyrene, Benzo(a)pyrene, Perylene, Indeno(1,2,3cd)pyrene, Dibenz(ac/ah)anthracene,
Benzo(ghi)perylene, Antanthrene.
e
Sum PAHs: sum (2-, 3-, 4-, 5-, 6-ring PAHs).
f
Sum hetrocyclic compounds (HC): Dibenzofuran, Dibenzothiophene, Carbazol, Benzothionaphthene, Benzophenanthridine.
g
Sum main compounds (startconc.>100ppm): Anthracene, 4-,5-Methylenephenanthrene, Fluoranthene, Pyrene, Benzo(a)anthracene, Chrysene,
Benzo(b,j,k)uoranthene.
h
Sum identied compounds: sum (PAHs, HC).
b

PAHs and other xenobiotics, either by stimulating biodegradation or by stimulating binding of intermediate
degradation product to organic matter in the soil
matrix (Mahro et al., 1994).
Assuming that the total degradation of PAH in contaminated soil is determined by the ratio between
extracellular enzyme concentration and the concentration of PAH compounds, it may be desirable to
remove the most readily degradable compounds by stimulating the bacterial degradation of PAH before inoculation with white rot fungi. This pre-treatment
might increase the subsequent degradation of the most
recalcitrant PAHs by white rot fungi, as the ratio
between extracellular enzymes and PAHs increases.
Earlier studies focused mainly on the degradation of
xenobiotics by Phanerochaete chrysosporium in liquid
medium or articially contaminated soil. For
Phanerochaete, the degradation process of lignin and
xenobiotics is inhibited by nitrogen concentrations
above a certain value (Bumpus et al., 1985; Kirk et al.,
1978). Recently, it has been shown that other species,
e.g. Pleurotus ostreatus (Majcherczyk and Huttermann,
1993; Zeddel et al., 1993; Morgan et al., 1993) and
Bjerkandera adusta (Kotterman et al., 1994; Kaal et
al., 1993) also degrade xenobiotics in the presence of
nitrogen. Thus, these species may be more suitable to
remediate contaminated soil where nitrogen concen-

tration is naturally high; or if the addition of nitrogen


is desirable within the total bioremediation strategy
selected.
The eect of inoculation with the white rot fungus
P. ostreatus, temperature and pre-treatment strategies
on PAH degradation in aged creosote contaminated
soil were investigated in the present study. Selected
temperatures are chosen to reect spring/autumn and
summer periods in a Nordic climate.
2. Material and methods
Creosote contaminated soil was collected from an
abandoned wood preservation site in southern
Norway. The content of 2-ring compounds was low
(<1%) and reects the removal of readily degradable
compounds due to ageing of the pollution in the
soil. P. ostreatus was isolated from rotting wood
(Mycoteam #91033) and maintained on malt agar
(Difco) at 308C. Fungal substrate was produced in 20 l
bags. To each bag 750 g chopped straw was added, humidied to 75% water content and steamed at 1008C
for 2 h. Then 200 g P. ostreatus inoculum was added
to each bag. The P. ostreatus inoculum was grown on
autoclaved barley grain mixture (2000 g grain, 20 g
gypsum, 40 g bone meal and 4 l water) for two weeks

T. Eggen, P. Sveum / International Biodeterioraton & Biodegradation 43 (1999) 125133


Table 2
Characterization of soil based on distribution of groups of PAH
(mean2std, n = 3)
Content of PAHs

Reference soil BTF-treatment BT-treatment

2-ring PAHs (%)


3-ring PAHs (%)
4-ring PAHs (%)
5- and 6-ring PAHs (%)

0.720.1
15.223.6
65.923.4
13.620.6

0.520.1
16.520.7
68.420.8
10.520.3

0.820
11.621.1
62.826.3
19.728.0

at 258C before inoculated in the substrate. The inoculate straw was incubated at room temperature until
mycelium completely colonized the substrate (three
weeks).
The experiment was conducted in two steps: pretreatment and fungal treatment.
2.1. Pre-treatment
Soil was screened (25 mm) and 3 pile batches were
treated as followed:
. Reference soil: Incubated at 588C, no amendments.
. BTF(Bark Temperature Fertilizer)-treatment soil:
25 kg commercial fertilizer (N:P:K = 12:4:16) was
added to 0.3 m3 soil mixed with 0.2 m3 pine bark
(Commercial cover-bark:compost-bark in ratio 2:1).
Water was added to adjust the humidity in the soil
to approx. 55% of the water holding capacity before
the BTF soil was incubated at 228C for 2 months.
. BT(Bark Temperature)-treatment soil: Same treatment as for BTF but without fertilizer addition.
Concentration and distribution of PAHs in the three
dierent soil piles (reference, BTF and BT) after pretreatment are shown in Tables 1 and 2.
2.2. Fungal treatment
After pre-treatment, each pile was sieved (15 mm
aperture) and mixed with 20% dry weight sieved
(15 mm aperture) compost (municipal biowaste compost >6 months) and added to the soil/compost mixture (1:4 ratio by weight) in a four layered sandwichlike manner in 55 l polyethylene boxes with a bottom
screen for aeration (each fungal layer 1.4 kg; each soil
layer 5.5 kg). Boxes without fungal inoculation had
autoclaved straw added and were organized in the
same manner as described above. Liquid potato pulp
(2000 ml) was spread on the top of each batch. Air
was supplied 30 min each day from a fan. The experiment included 32 batches and evaluated the parameters temperature (8 and 228C) and fungal
inoculation for the three soils types (reference soil, pretreatment BTF and BT) (Fig. 1).

127

At the end of the experiment, eight soil samples


were withdrawn from each batch, combined and mixed
in a blender.
Chemical analysis: Internal standards (d8-naphthalene, d10-biphenyl, d10-phenanthrene, d10-pyrene, d12chrysene and d12-perylene) were added to the mixed
samples before extraction with dichloromethane
(DCM:water (1:1)) using sonication and shaking.
Extraction was repeated. Extracts were combined and
the water separated. Dichloromethane was replaced
with cyclohexane. The cyclohexane extract was cleaned
with liquid/liquid extraction with dimethyl formamide,
DMF:water (9:1), and PAH compounds were extracted
back to cyclohexane. The extract was rinsed with
water, dried with sodium sulphate, and analyzed by
GC/MSD (GC Hewlett Packard 5890 with auto injection 7673A, MS Hewlett Packard 5970) in a single ion
monitoring mode. The separation of PAHs were
achieved on GC-column J&W, DB 5 (30 m  0.25 mm,
0.1 mm). After 3 min at 408C, the temperature was
increased to 1008C at a rate of 88C/min, then to 2008C
(48C/min) and nally to 3008C (16 min).
When evaluating the data set, focus was given to the
groups of PAHs listed in Table 1.
Data were analyzed statistically using JMP (SAS
institute Inc. Cary, USA). A probability level of 0.10
was assumed to be the critical signicance level.
3. Results and discussion
The experiment was designed to investigate the eect
of a two-step biological treatment, fungal inoculation
and incubation temperature, and not degradation kinetics. The experiment was conducted under non-sterile
conditions. Thus, other microorganisms present in the
soil might have participated in the PAH degradation.
Although the established mycelia of inoculated white
rot fungi were able to withstand competition from the
native microora and penetrated the surrounding soil,
the amount of visible mycelial growth in soil varied in
the batches. The degradation data presented was calculated using the concentration immediately after the
pre-treatment period as initial concentration. The
degradation rate during the pre-treatment was not considered.
The degradation of the selected groups of compounds showed a high variability in the 32 batches
(Fig. 1). The factorial design was used in this experiment to reect a possible interaction of the factors
(pre-treatment, temperature and fungal inoculation),
measured as response (i.e. % degradation) for any
batch. The comparison of mean values based on univariate grouping for each factor did not give signicant dierences that could be ascribed to the inuence
of the individual factors or interaction between them.

128

T. Eggen, P. Sveum / International Biodeterioraton & Biodegradation 43 (1999) 125133

Fig. 1. Percentage degradation for the studied groups of compounds in each of the 32 batches. Each batch treatment is shown below (Fungal
inoculum: Yes or No; Soil treatment: Reference, BTF or BT; Temperature: 8 or 228C).

Thus factorial statistical analysis was used. The data


were tted to nominal factorial models with two (pretreatment and fungal inoculation) and three (pre-treatment, temperature and fungal inoculation) factors

included. When two-factor models were used, soils


incubated at 8 and 228C were treated separately.
All of the tested three-factor models were statistically signicant (Table 3). However, for some selected

MT

BT

BTF

4.523.3
38.123.3

18.723.3
31.523.3

15.524.0
45.724.0

88C
228C

88C
228C

12.922.0
38.422.0

88C
228C

88C
228C

29.322.0
22.122.0

Yes
No

Reference

21.322.3
25.122.3
30.622.8

Reference
BTF
BT

Least square mean2std. error (% degradation)

Eect tests

60.924.9
60.824.9

32.224.0
32.824.0

24.924.0
68.424.0

39.322.5
54.022.5

n.s.

46.722.8
32.522.8
60.923.4

0.845
<0.0001
<0.0001
(0.324)
(0.206)
0.0005
<0.0001
(0.399)
(0.278)

0.846
<0.0001
0.0598
0.0209
(0.275)
<0.0001
0.0110
(0.947)
0.0967

R2
Whole Model
M
F
MF
T
MT
FT
MFT

S heterocyclic

S identied

Factor

14.024.2
43.924.2

17.123.4
32.023.4

4.123.4
37.423.4

11.722.1
37.822.1

28.822.1
20.722.1

n.s.

0.829
<0.0001
(0.129)
0.0154
(0.319)
<0.0001
0.0353
(0.995)
(0.230)

S PAH

14.524.2
48.624.2

22.123.5
36.423.5

6.223.5
41.123.5

14.322.2
42.022.2

32.122.2
24.122.2

n.s.

Prob>F
0.843
<0.0001
(0.115)
0.0166
(0.458)
<0.0001
0.0136
(0.944)
(0.165)

S main compounds

19.626.7
25.526.7

12.825.4
25.525.4

13.025.4
46.625.4

15.123.4
30.523.4

27.323.4
18.323.4

29.823.8
16.123.8
22.524.7

0.639
0.0110
0.0639
0.0735
(0.469)
0.0043
0.0356
(0.311)
(0.201)

S 3-ring PAHs

20.524.6
59.224.6

27.123.7
44.123.7

11.223.7
48.323.7

19.622.3
50.522.3

38.622.3
31.522.3

29.722.6
35.622.6
39.823.2

0.849
<0.0001
0.0679
0.0452
(0.500)
<0.0001
0.0198
(0.932)
(0.110)

S 4-ring PAHs

Table 3
Factorial model identication for three factors. 8 and 228C treated together, ( p < 0.1) (Pre-treatment method = M, Temperature = T and Fungal inoculation = F). Not signicant p-values are
given in cursive and parenthesis (n.s. = not signicant)

T. Eggen, P. Sveum / International Biodeterioraton & Biodegradation 43 (1999) 125133


129

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T. Eggen, P. Sveum / International Biodeterioraton & Biodegradation 43 (1999) 125133

groups of compounds, some of the individual factors


and their interactions were not statistically signicant.
Statistically signicant two-factor models were
obtained for all selected groups of soils incubated at
88C, except for 3-ring PAHs (Table 4). At 228C, a statistically signicant model could only be tted for 3ring PAHs.
3.1. Three-factor model
3.1.1. Single factors
Inoculation of P. ostreatus had a signicant eect
on the degradation of all groups except heterocyclic
compounds (Table 3). For S identied compounds, S
PAHs and S main compounds, fungal inoculation
increased degradation by 3040%. For S 3-ring PAHs
and S 4-ring PAHs, the increases were 49 and 23%,
respectively.
It was shown that pre-treatment had a positive eect
on the degradation of S identied compounds, S heterocyclic compounds and S 4-ring PAHs (Table 3).
For S 3-ring PAHs, reference soil without pre-treatment gave the highest degradation. A suggested explanation for this result can be that a major part of the
available 3-ring compounds were degraded during the
pre-treatment period. Reference soil (incubated at 88C
during pre-treatment period) had a higher potential to
increase the degradation rate when temperature
increased and/or compost was added. In the cases,
pre-treatment enhanced the degradation rate, pre-treatment without addition of fertilizer (BT) gave better
results than with fertilizer (BTF). For S identied
compounds, degradation increased by 25 and 50% for
BTF and BT pre-treatment, respectively. For heterocyclic compounds, the degradation was 32% (BTF)
and 61% (BT). Also, for S 4-ring PAHs, BT treatment
resulted in a slightly higher degradation (40%) than
BTF treated soil (35%) and reference soil (30%). No
signicant eect of pre-treatment alone was found for
S PAH or S main compounds.
All soils received compost and potato pulp.
Compost addition has been shown to be advantageous
for biological degradation of oil-contaminated soil
(Stegmann et al., 1991) and PAH-contaminated soil
(Martens, 1982); potato pulp has been shown to be a
good substrate for white rot fungi (Majcherczyk and
Huttermann, 1993; Zeddel et al., 1993). Thus, in this
experiment, even reference soil and control soil without fungal inoculation were added stimulating factors.
Bark may act either as structural material (enhancing
soil gas exchange) or as a co-substrate. Both BTF and
BT pre-treatments included the addition of bark. Since
all boxes were aerated and added straw, this eect
would be most pronounced during the pre-treatment
period.
Increased temperature had a positive eect on the

degradation of all groups (Table 3). Degradation


increased 1.4 to 3.6 times as the incubation temperature increased from 8 to 228C (Table 3). Degradation
at 88C was generally low (1220%) except for heterocyclic compounds (40%). At both temperatures, 4-ring
PAHs were more degraded than 3-ring PAHs; 20 vs.
10% at 88C and 50 vs. 30% at 228C. Again, this probably reects a high degradation of available 3-ring
PAHs during pre-treatment.
3.1.2. Interacting factors
Statistically signicant interactions were found
between pre-treatment and temperature for all groups
(Table 3), while no statistically signicant interactions
were detectable between temperature and fungi, and
fungi and pre-treatment. The interaction eect of temperature was most pronounced for the reference soil,
where an increase in temperature from 8 to 228C
resulted in an increase in degradation ranging from 2.7
to 42 times for the dierent studied groups. Reference
soil inoculated at 88C showed very low degradation
for all groups of compounds (413%) except for S
heterocyclic compounds (25%). Pre-treated soil inoculated at 88C had, in general, a higher degradation;
BFT soil32% for S heterocyclic compounds and 13
28% for the other groups of compounds, and BT
soil60% for S heterocyclic compounds and 1420%
for the other groups. For the pre-treated soil, temperature increase gave no enhanced degradation of heterocyclic compounds. The degradation rate of heterocyclic
compounds at low temperature and, especially in soil
pre-treated without fertilizer, was unexpected high.
3.2. Two-factor model
When temperature was not considered as a factor,
i.e. when results obtained at 8 and 228C were treated
separately, both pre-treatment and fungal inoculation
were important and signicant factors, as well as the
interaction between the two factors (Table 4).
Statistically signicant models could be tted for all
groups of compounds at 88C, except 3-ring PAHs and
S heterocyclic compounds, and only for 3-ring PAHs
at 228C. This indicates that at the higher temperature,
factors not considered specically in this experiment,
e.g. indigenous microbes, compost, bark and potato
pulp, can be of greater importance. It should be noted
that models tted for the other groups of compounds
at 228C had the same general trend as for 88C,
although they were not statistically signicant.
The eect of pre-treatment at 88C showed the same
trend as for the interaction between pre-treatment and
temperature in the three-factor model as described
above; lower degradation in reference soil than in pretreated soil and, for pre-treated soil with fertilizer,
slightly higher degradation than pre-treated soil with-

BT

BTF

MF
Reference

4.522.3
18.722.3
15.522.8

16.622.0
9.222.0

6.023.2
2.923.2

16.523.2
20.923.2

27.224.0
3.724.0

Yes
No

Yes
No

Yes
No

Yes
No

66.825.5
55.025.5

25.324.5
39.124.5

26.724.5
3.124.5

n.s.

24.923.2
32.223.2
60.923.9

Least square mean2std. error (% degradation)

Eect tests

Reference
BTF
BT

0.8600
0.0050
0.0001
(0.898)
0.0661

Prob>F
0.797
0.0031
0.0038
0.0275
0.0099

r2
Whole model
M
F
MF

S heterocyclic

88C
S identied

Factor

25.324.3
2.724.4

16.323.5
17.923.5

5.623.5
2.623.5

15.722.2
7.722.2

4.122.5
17.122.5
13.923.1

0.741
0.095
0.0113
0.0276
0.0311

S PAH

25.325.4
3.825.4

20.324.4
23.924.4

7.924.4
1.424.4

17.822.8
9.722.8

4.723.1
22.123.1
14.523.8

0.715
0.0147
0.0088
0.0632
0.0818

S main compounds

32.425.3
8.725.3

24.924.3
29.224.3

12.424.3
9.924.3

23.522.7
15.922.7

11.223.1
27.123.1
20.523.7

0.709
0.0161
0.0133
0.0829
0.0438

S 4-ring compounds

38.827.8
12.227.8

29.726.3
9.226.3

44.126.3
49.226.3

37.523.9
23.523.9

46.624.5
19.524.5
25.525.5

0.758
0.0069
0.0042
0.0306
0.0862

228C
S 3-ring compounds

Table 4
Factorial model identication for two factors. Modeled separately for 8 and 228C. (Method = M and Fungal inoculation = F). Not signicant values are excluded from the table (n.s. = not signicant)

T. Eggen, P. Sveum / International Biodeterioraton & Biodegradation 43 (1999) 125133


131

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T. Eggen, P. Sveum / International Biodeterioraton & Biodegradation 43 (1999) 125133

out fertilizer addition for S identied compounds, S


PAHs, S main compounds and S 4-ring PAHs.
Degradation in reference soil and BT soil varied
between 1 and 10%, while it was found to be 1830%
in BTF soil. This generally higher degradation of
PAHs in soil with fertilizer added during pre-treatment, when fungal inoculation are not considered as a
factor, indicate a stimulating eect on indigenous
microorganisms by nitrogen/phosphorus addition.
It is also notable that this is not the case for heterocyclic compounds; pre-treatment without fertilizer addition (BT) had almost twice the degradation as pretreatment with fertilizer (BTF).
When investigating fungal inoculation as a factor
alone, P. ostreatus had a signicant eect on S identied compounds, S PAHs, S main compounds and S
4-ring PAHs at 88C (not for S heterocyclic compounds
and S 3-ring PAHs), and for S 3-ring PAHs at 228C
(for the other groups of compounds, the p values were
in the range 0.150.28). The increase in degradation
was in the range 1.52 times when inoculated with P.
ostreatus. When treating the fungal inoculation and
pre-treatment factors together at 88C, soil with added
bark (BT) and reference soil were positively aected
by fungi inoculation (Table 4). Fungal inoculation at
low temperature had no or even a slightly negative
eect in pretreated soil with nitrogen (BTF). Since the
reference and BT pre-treated soil had less nitrogen and
phosphorus than the BTF soil (Table 1), it is likely
that this eect can be ascribed to the dierences in the
nitrogen and/or phosphorus concentration in this soil.
P. ostreatus is one of the species found to degrade
xenobiotics even in an excess of nitrogen (Majcherczyk
et al., 1994) because it produces extracellular enzymes
in the growth phase. Comparison of nitrogen concentration in this study with previous studies is dicult,
since most of the experiments evaluating nitrogen concentration are performed in liquid cultures. In contrast
to the reduced eect of fungal inoculation at low temperature in soil with added fertilizer, there was a
slightly positive, but not signicant, eect with fungal
inoculation in the same soil (BTF) at 228C (data not
shown) for all the groups of compounds. It is dicult
to explain why fertilizer addition has a negative impact
on the eect of fungal inoculation at low temperature
but not at higher temperature. It would be expected
that such an eect is independent of temperature.
Fungal inoculation had a much lower eect on the
degradation in reference soil than in BT soil when
incubated at low temperature. The eect of fungal inoculation on degradation of 3-ring PAHs at 228C was
only obtained in pre-treated soil. This indicates that
indigenous microorganisms have degraded most of the
available fraction of 3-ring PAHs during the pre-treatment period, and further degradation was stimulated
with fungal inoculation.

4. Conclusions
The study shows that P. ostreatus has an overall
positive eect on the degradation of aged creosote in
soil and that degradation is enhanced by increased
temperature and pre-treatment. In general, adding
bark and incubating at 228C during pre-treatment,
before inoculating with P. ostreatus increases PAH
degradation compared to no pre-treatment, or treatment with fertilizer. At low temperature (88C), fungal
inoculation had the best eect when fertilizer was not
added and a signicant eect was obtained on degradation of dierent groups of PAH compounds, except
for the more easily degradable compounds, 3-ring
PAHs and heterocyclic compounds. Pre-treatment with
fertilizer showed stimulated microbial activity at low
temperature and enhanced PAH degradation even
without the addition of fungi.
Acknowledgements
The Norwegian State Railway (NSB), The
Norwegian State Pollution Control Authority (SFT)
and Jordforsk supported this work. We also thank
Sverre Hols (Mycoteam), Edgardo Araneda
(Jordforsk) and istein Vethe (Jordforsk) for their
invaluable contribution, and Dr. Roger Prince (Exxon
Research and Engineering) for comments on our early
version of the paper.
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