Organic Geochemistry 39 (2008) 945–951

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Organic Geochemistry
journal homepage: www.elsevier.com/locate/orggeochem

A comparison of lignin oxidation, enzymatic activity and fungal

growth during white-rot decay of wheat straw
Steven A. Robertson a, Sharon L. Mason b, Ethan Hack b, Geoffrey D. Abbott a,*
a
School of Civil Engineering and Geosciences, Newcastle University, Drummond Building, Newcastle Upon Tyne, NE1 7RU, United Kingdom
b
School of Biology and Psychology, Division of Biology, Newcastle University, Ridley Building, NE1 7RU, United Kingdom

a r t i c l e i n f o a b s t r a c t

Article history: This paper reports a laboratory study where multiple variables have been monitored in
Received 30 September 2007 parallel as a function of white-rot incubation time. A model system with Pleurotus ostreatus
Received in revised form 3 February 2008 growing on unamended wheat (Triticum aestivum) straw under solid-state cultivation con-
Accepted 14 March 2008
ditions was used to investigate changes in lignin oxidation, amounts of the fungal bio-
Available online 4 April 2008
marker ergosterol and activity of manganese peroxidase (MnP) at regular intervals over
84 days. Lignin oxidation was monitored with thermally assisted hydrolysis and methyla-
tion (THM) using tetramethylammonium hydroxide (TMAH) where 3,4-dimethoxybenzoic
acid, methyl ester (G6) to 3,4-dimethoxybenzaldehyde (G4) [Ac/Al]G and 3,4,5-trimethoxy-
benzoic acid, methyl ester (S6) to 3,4,5-trimethoxybenzaldehyde (S4) [Ac/Al]S were used as
relative lignin decomposition state proxies. [Ac/Al]G, [Ac/Al]S and ergosterol production
show little change during the first 21 days of incubation. MnP activity, however, rises rap-
idly and peak activity is reached during the same time interval. This is followed by an
increase in both [Ac/Al]G, [Ac/Al]S and ergosterol formation where most of the increase
in these variables takes place after 21 days. Therefore a rapid early rise in manganese-
dependent peroxidase activity precedes significant changes both in lignin oxidation and
fungal growth. We have demonstrated that it is possible to analyse lignin oxidation using
THM in the presence of TMAH and, in the same system, measure enzyme activity and
amounts of fungal biomarkers as a function of incubation time. This approach will be useful
when investigating compositional changes in litter and soil layers.
Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Martínez et al., 1994; Hakala et al., 2004) and to degrade

Lignin is a complex three-dimensional biopolymer
formed by dehydrogenation of up to three types of precur-
sor alcohol with cinnamyl backbones (Adler, 1977). Its
structure is resistant to effective, rapid degradation by
most biotic processes and its location in the cell walls of
many land plants lends resistance to these structures.
White-rot basidiomycete fungi have the ability to degrade
lignin extensively (Kirk and Farrell, 1987). As such, white-
rot fungi have received considerable attention for their po-
tential both to remove lignin in industrial applications (e.g.
* Corresponding author. Tel.: +44 (0) 191 222 6608; fax: +44 (0) 191
222 5431.
E-mail address: geoff.abbott@ncl.ac.uk (G.D. Abbott).
pollutants having structures related to that of lignin (e.g.
Reddy, 1995; Pointing, 2001). The activity of white-rot fun-
gi also represents a key step in the global carbon cycle and
this influences the forms in which carbon is released from
lignocellulosic macrostructures such as tree trunks and
grass stems (Hammel, 1997). This release of carbon is a
biotic process and has been shown in the laboratory to
vary with time as the fungi grow, decomposing the lignin
component (e.g. Hedges et al., 1988), and producing en-
zyme suites (e.g. Valmaseda et al., 1991). An investigative
approach that links together these different processes al-
lows interactions between the variables to be observed.
Understanding how the products of lignin degradation
change during the stages of the fungal life cycle is a neces-
sary step in improving our knowledge of the dynamics of

0146-6380/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.orggeochem.2008.03.017
946 S.A. Robertson et al. / Organic Geochemistry 39 (2008) 945–951
lignin-derived carbon in soils. This is important because re-
cent studies have suggested that lignin from plant residues
is not as recalcitrant in soil as conventionally perceived
(Rasse et al., 2006). Numerous authors have described
changes over time in one or more parts of the fungal deg-
radation process and with various white-rot fungi or com-
binations of fungi (e.g. Hedges et al., 1988; Vane et al.,
2001b; Chi et al., 2007), although in some cases the studies
made use of substrates plus additional nutrients, including
nitrogen sources (e.g. Valmaseda et al., 1991). In this study
we have made use of a model system where the white-rot
fungus Pleurotus ostreatus is grown on wheat (Triticum aes-
tivum) straw under solid-state conditions, without addi-
tional nutrient sources. This has allowed us to track the
progress of lignin oxidation using thermally assisted
hydrolysis and methylation (THM) with tetramethylam-
monium hydroxide (TMAH), which is also known as TMAH
thermochemolysis (Hatcher et al., 1995), along with
changes in the amounts of ergosterol ([22E]-ergosta-
5,7,22-trien-3b-ol, a fungal biomass proxy) and manga-
nese-dependent enzyme activity over time, in a system
that represents the conditions likely to be found in envi-
ronmental material such as lignocellulosic macrostruc-
tures in soil litter layers.
2. Materials and methods
2.1. Fungal incubation
Wheat (T. aestivum L.) straw was supplied by Goodwills
Ltd. (Ponteland, Northumberland, UK). Any remaining ears
were removed from the straw and a mixture of stem and
leaf material was divided into 2 cm lengths. Straw (4 g
dry weight) was then placed into 250 ml amber glass jars
(separate jars were used for each time point in the series)
and soaked in 100 ml 15 MX cm distilled water for 2 h;
84 ml of the excess water was then removed, leaving be-
hind a solid residue. The jars were capped with aluminium
foil and pasteurised at 62.5 ± 2.5 °C for 4 h followed by
52.5 ± 2.5 °C for 3 days. Jars were inoculated with 0.2 g of
P. ostreatus grain spawn (Ann Miller’s Speciality Mush-
rooms Ltd., Inverurie, Aberdeenshire, UK) and incubated
at 25 °C and ambient humidity in the dark. Jars were re-
moved for analysis after 7, 14, 21, 28, 42, 56 and 84 days.
2.2. Separation of extracellular fluid and incubation solids
Extracellular fluid was separated from the incubation
solids (comprising degraded straw and fungal mycelium)
by centrifugation through muslin in a Centaur 2 swing-
ing-bucket centrifuge (MSE, UK) at 1600g for 10 min. Fol-
lowing extraction of the fluid the remaining solids were
freeze-dried and homogenized with a Cyclotec 1092 sam-
ple mill (Tecator, Sweden) to pass through a 63 lm mesh.
2.3. Thermally assisted hydrolysis and methylation (THM) in
the presence of tetramethylammonium hydroxide (TMAH)
THM was performed on line using a pulsed mode open
pyrolysis system, specifically a CDS 1000 pyroprobe unit
(Chemical Data Systems, USA) fitted with a platinum coil
probe and a CDS1500 valved interface. Homogenized,
freeze-dried cultivation solids (0.2 mg) were weighed into
quartz pyrolysis tubes plugged with glass wool. A volume
of TMAH solution (25% w/w in methanol) was added
immediately before thermochemolysis to give a TMAH/
cultivation solids ratio of 10/1 (w/w). Prepared tubes were
then loaded into the pyroprobe and thermochemolysis
performed at 600 °C for 10 s (20 °C/ms probe heating pro-
gramme) with the products passing into an HP 5890 gas
chromatograph (Agilent, USA) with an open split and a
30 m HP5 column (0.25 mm internal diameter, 0.25 lm
film thickness). The carrier gas was helium at a flow rate
of 1 ml/min. The oven temperature was held at 40 °C for
10 min and then increased at 5 °C/min to 300 °C where it
was maintained for 3 min. Product detection was carried
out using an HP 5000 series mass selective detector (Agi-
lent, USA) in full scan mode (m/z 50–700). The naming of
THM products follows the conventions established by
Hatcher et al. (1995), Clifford et al. (1995) and del Rio
et al. (1998). Two replicates were analysed for each point
in the time series. A table showing the THM product
assignments with their characteristic mass spectral frag-
ment ions (Vane et al., 2001a) and a figure of their structures
(Vane et al., 2001b) have been published by our group.
2.4. Ergosterol extraction
Ergosterol extraction was performed using a method
based on a combination of techniques described by Stahl
and Parkin (1996) and Eash et al. (1996). Homogenized,
freeze-dried cultivation solids (250 mg) were weighed into
30 ml screw topped vials and hydrated with 2 ml
15 MX cm distilled water. An aliquot (100 ll) of a 5a-
androstan-3b-ol recovery standard (0.52 mg/ml in dichlo-
romethane) was added to each 30 ml vial. Methanol
(10 ml) and 4 ml potassium hydroxide in ethanol (4 g solid
potassium hydroxide in 100 ml 95% v/v ethanol/water)
were added to each sample, and the samples ultrasonically
agitated for 5 min in a Hilsonic ultrasonic bath (Hilbre
Ultrasonics Ltd., UK). The vials were heated for 2 h at
85 °C and immediately cooled by immersion in cold water.
n-Hexane (2 ml) was added to each vial and the vials were
rotated end over end for 10 min at 48 r.p.m. The phases
were separated by centrifugation at 1000 g for 3 min. The
hexane layer was transferred to autosampler vials, and re-
duced in volume under a stream of dry nitrogen. The
hydrocarbon 5a-androstane internal standard (100 ll of a
0.53 mg/ml solution in dichloromethane) was added and
the mixture was derivatised using N,O-bis(trimethyl-
silyl)trifluoroacetamide (BSTFA, purum P 98.0%). GC anal-
ysis was performed using an HP 6890 autosampler
connected to an HP 5890 gas chromatograph with an open
split, 30 m DB5 column (0.25 mm internal diameter,
0.1 lm film thickness), hydrogen carrier gas (2 ml/min)
and a flame ionisation detector. The oven temperature
was held at 50 °C for 2 min and then increased at 4 °C/
min to 300 °C where it was maintained for 20 min. Mix-
tures of pure standards and pure ergosterol were analysed
alongside the samples for calculation of relative response
factors and for peak identification. Three replicates were
 S.A. Robertson et al. / Organic Geochemistry 39 (2008) 945–951 947

analysed for each point in the time series. Data was ana-
lysed using Atlas 8.1 software (Thermo Electron Corpora-
tion, USA).
2.5. Enzyme assay
MnP activity was assayed using oxidation of phenol red
and a modification of the method of de Souza-Cruz et al.
(2004). The analyses were performed in duplicate.
1.25 ml 20 mM sodium succinate buffer (pH 4.5), 1.5 ml
50 mM sodium lactate, 0.5 ml 1.0 M manganese sulfate,
0.5 ml 1 g/l phenol red and 0.25 ml 2.0 mM hydrogen per-
oxide were placed in a glass beaker. At time zero, 0.5 ml of
sample extract (50 ll of extract + 450 ll of deionised
water) was added to the beaker. After 0, 5, 10 and
15 min, 1 ml aliquots were removed and the reaction ter-
minated by adding it to a 1.5 ml microcentrifuge tube con-
taining 30 ll of 6.5 M sodium hydroxide. After terminating
the reaction, the absorbance of each 1 ml solution was
measured at 610 nm using a Unicam 8625 UV/Visible spec-
trophotometer (Unicam Ltd., UK) and the absorbance was
plotted as a function of time (0–15 min). Replicates were
carried out in the absence of manganese sulphate to check
for manganese-independent activity and blanks were run
with 15 MX cm distilled water in place of the sample
extract.
3. Results and discussion
3.1. Analysis of lignin
3.1.1. Lignin oxidation during growth of P. ostreatus
In common with other studies using THM with TMAH to
investigate lignin degradation by fungi (e.g. Filley et al.,

100 G18
Relative intensity (%)

Day 7
P18

G14 S14
S15
G6 G15
G3 S4
G4 S6

100 G18
Relative intensity (%)

Day 42
P18

G14
G15 S14
G6 S6
G3 S4 S15
G4

100 P18 G18

Relative intensity (%)

Day 84
G3
S14
G4 S6
G6
S4
G14
G15
S15

25 30 35 40 45 50
Retention time (min)
Fig. 1. Partial chromatograms of the total ion current (TIC) of the THM products from day 7, day 42 and day 84 during the degradation of wheat straw by
Pleurotus ostreatus under solid-state cultivation conditions. The naming of the THM products follows the conventions established by Clifford et al. (1995)
and del Rio et al. (1998).
948 S.A. Robertson et al. / Organic Geochemistry 39 (2008) 945–951
2000; Vane et al., 2001a), a suite of THM products was
identified and their distributions changed with increasing
incubation time (Fig. 1). From the suite of products, we
have chosen four that have been used to indicate lignin
oxidation in previous studies through the measurement
of ratios highlighting lignin side chain oxidation. The ratio
of 3,4-dimethoxybenzoic acid, methyl ester to 3,4-dime-
thoxybenzaldehyde [Ac/Al]G (G6/G4, Fig. 2) has been used
to indicate lignin oxidation in guaiacyl lignin sub-units
(e.g. Hatcher et al., 1995) and the ratio of 3,4,5-trimethoxy-
benzoic acid, methyl ester to 3,4,5-trimethoxybenzalde-
hyde [Ac/Al]S (S6/S4, Fig. 2) to indicate oxidation in
syringyl moieties (e.g. Hedges et al., 1985). Fig. 2 shows
that [Ac/Al]G and [Ac/Al]S increase with increasing incuba-
tion time. There is, however, no significant increase in
either proxy during the initial 21 days of incubation time.
[Ac/Al]G takes a value of between  0.3 and  0.4 whereas
[Ac/Al]S is between  1 and  1.1 in this same time inter-
val. [Ac/Al]G then rises from 0.383 ± 0.003 at 21 days to
0.966 ± 0.035 after an incubation time of 84 days. [Ac/Al]S
increases from 1.12 ± 0.07 at 21 days to 2.50 ± 0.08 at
84 days. Fig. 2, therefore, clearly shows that most lignin
oxidation occurs after 21 days, with the two proxies more
than doubling between 21 and 84 days incubation.
The ratio of 3,4-dimethoxybenzoic acid, methyl ester
(G6) to the sum of threo and erythro 1-(3,4-dimethoxy-
phenyl)-1,2,3-trimethoxypropane (G14 and G15) has been
used to indicate oxidative cleavage of the side chain of gua-
iacyl lignin sub-units during fungal degradation (e.g. Filley
et al., 2006) and the ratio of 3,4,5-trimethoxybenzoic acid,
methyl ester (S6) to the sum of threo and erythro 1-(3,4,5-
trimethoxyphenyl)-1,2,3-trimethoxypropane (S14 and
S15) used to indicate cleavage of the side chain of syringyl
sub-units. On comparison of the 84 day chromatogram
with the 7 day and 42 day TIC traces (Fig. 1), the peak areas
of both G6 and S6 have increased relative to those of
(G14 + G15) and (S14 + S15) respectively after 84 days
incubation. This is therefore good qualitative evidence for
side chain oxidation in the straw lignin.
Fig. 2. Acid/aldehyde ratios ([Ac/Al]) in THM products from wheat straw
degraded by Pleurotus ostreatus under solid-state conditions. (N) ratio of
3,4-dimethoxybenzoic acid, methyl ester (G6) to 3,4-dimethoxybenzal-

dehyde (G4); ( ) 3,4,5-trimethoxybenzoic acid, methyl ester (S6) to 3,-
4,5-trimethoxybenzaldehyde (S4). Error bars are plus or minus one
standard error of the mean for two replicates.
At long incubation times, however, the relative mea-
surement errors were large because the absolute amounts
of the respective TMH products were very small. This
meant that there were large measurement errors espe-
cially after 84 days incubation. This made it impossible to
extract quantitative information of statistical significance
from plotting G(S)6/(G(S)14 + G(S)15) as a function of
incubation time.
3.1.2. The [Ac/Al]G and [Ac/Al]S variables
[Ac/Al]G and [Ac/Al]S have been used as relative
decomposition state proxies for guaiacyl and syringyl lig-
nin monomers respectively (Filley et al., 2006). Filley et al.
(2006) also advise that caution needs to be exercised
when acid/aldehyde ratios are used to infer fungal alter-
ation of lignin because of the potential contribution of
3,4,5-trimethoxybenzoic acid from hydrolysable tannins
to S6. However, wheat straw contains neither condensed
nor hydrolysable tannins (Mueller-Harvey, 2001 and per-
sonal communication) so interference with the [Ac/Al]S
proxy is not an issue here. In addition, changing the
THM conditions (e.g. time or temperature) has been
shown to affect the absolute yields of G4 and G6 (Kling-
berg et al., 2005), but with the use of a constant set of
conditions, changes in [Ac/Al]G can be reliably observed
over time. We are confident, therefore, that in this study
the time dependence of [Ac/Al]G and [Ac/Al]S represents
increasing extent of lignin oxidation during the growth
of P. ostreatus.
3.2. Ergosterol
Ergosterol is a prominent membrane sterol in all eumy-
cotan groups except chytrids, rusts and some yeasts and,
since it is not a vascular plant sterol, it is used to indicate
fungal colonisation as well as to measure fungal mass in
decaying litter (Newell, 1992).
In this study, ergosterol amounts (measured as milli-
grams ergosterol per gram freeze-dried cultivation solids)
do not change significantly (within experimental error) be-
fore 21 days have elapsed and then increase threefold
throughout the remaining incubation period (Fig. 3), which
indicates that the fungus is actively accumulating biomass
between about 21 and 84 days.
Stahl and Parkin (1996) note that a degree of error is in-
volved when living fungal biomass is estimated from
ergosterol content alone and recommend that ergosterol
measurements be combined with staining methods to give
an indication of living and non-living fungal biomass in
soils. In addition, Zhao et al. (2005) report that fungal bio-
mass is decomposed more rapidly than ergosterol in soils
following fungal death. They suggest that ergosterol mea-
surements are not, therefore, a good indicator of living fun-
gal biomass, although this suggestion is not universally
accepted (Young et al., 2006; Zhao et al., 2006). However,
the present study involves actively-growing mycelium col-
onising a large mass of substrate. Problems associated with
the presence of non-living fungal biomass will be reduced,
along with the degree of error noted by Stahl and Parkin
(1996). Thus, ergosterol amounts should provide a suitable
proxy for fungal biomass in this instance.
 S.A. Robertson et al. / Organic Geochemistry 39 (2008) 945–951 949

0.7

0.6
Ergosterol amounts (mg/g)

0.5

0.4

0.3

0.2

0.1

0
0 10 20 30 40 50 60 70 80 90
Time (days)

Fig. 3. Ergosterol ([22E]-ergosta-5,7,22-trien-3b-ol) amounts (mg ergosterol/g freeze-dried solids) measured during cultivation of Pleurotus ostreatus on
wheat straw. Error bars are plus or minus one standard error of the mean for three replicates.

3.3. Manganese-dependent peroxidase activity
Under appropriate conditions, P. ostreatus can carry out
Mn-independent oxidation of phenol red (Shin et al.,
1997). The activity measured in the present study is man-
ganese-dependent, since repeating the enzyme assay in the
absence of Mn2+ revealed little or no phenol red oxidation.
In contrast to lignin oxidation and ergosterol concentra-
tion, manganese-dependent peroxidase activity begins to
rise immediately from zero at time = 0 days to a maximum
at about 14 days and then decreases to about half its max-
imum value after 84 days incubation (Fig. 4). A maximum
in the time course of manganese peroxidise production
has also been observed in solid-state cultures of the
white-rot fungus Lentinula edodes on corn cob powder
(Gandolfi Boer et al., 2004).
Fungi use several different enzyme systems to degrade
lignin. Pleurotus species were reported to belong to a group
of organisms that produces manganese peroxidase and lac-
case but not lignin peroxidase, the classic lignin-degrading
enzyme extensively studied in Phanerochaete chrysosporium
(Hatakka, 1994). Camarero et al. (1999) showed that in

12.0

11.0

10.0
MnP activity/g of dry straw

9.0

8.0

7.0

6.0

5.0

4.0

3.0

2.0

1.0

0.0
0 10 20 30 40 50 60 70 80 90
Time (days)

Fig. 4. Manganese-dependent peroxidase activity per gram dry straw (change in absorbance at 610 nm per minute due to oxidation of phenol red) in the
extracellular fluid extracted during solid-state cultivation of Pleurotus ostreatus on wheat straw. Error bars are the squared upper and lower limits of the 95%
confidence intervals for square root data.
950 S.A. Robertson et al. / Organic Geochemistry 39 (2008) 945–951
Pleurotus eryngii, a major contributor to manganese-depen-
dent peroxidase activity is an enzyme that they named
versatile peroxidase. Unlike the more specific manganese
peroxidase first described in P. chrysosporium, this enzyme
can oxidise a wide range of substrates including the char-
acteristic lignin peroxidase substrate veratryl alcohol,
although it is most active in the presence of Mn2+ (Camare-
ro et al., 1999; Ruiz-Dueñas et al., 2001). In P. ostreatus,
three distinct ‘‘manganese peroxidase” isozymes (MnP1,
MnP2, and MnP3) have been identified (summarised by
Kamitsuji et al., 2004) which are all of the versatile perox-
idase type (Martínez, 2002). MnP2 has much greater man-
ganese-independent activity than MnP3 (Kamitsuji et al.,
2004; Kamitsuji et al., 2005). The manganese-dependent
assay used in the present study detects the combined
activity of all forms. Valmaseda et al. (1991) did not detect
MnP activity during growth of P. ostreatus on wheat straw,
possibly due to the use of different growth conditions, in
particular the inclusion of a nitrogen source.
3.4. Lignin oxidation during growth of P. ostreatus
A great deal of effort has been spent studying the fungal
oxidation of lignin with a view to optimising activity for
industrial, agricultural or pollution control processes (e.g.
Martínez et al., 1994; Li et al., 2001; Gandolfi Boer et al.,
2004; Chi et al., 2007). Several of these studies have linked
together lignin alteration, enzyme activity, and other as-
pects of the oxidation process over time. In most instances
where several parts of the oxidation process (e.g. enzymes,
biomass and lignin content) have been studied at the same
time (e.g. Chi et al., 2007), lignin has only been character-
ised at a bulk, not a molecular, level. From a geochemical
perspective, the aim of studying lignin oxidation is not to
optimise it through strain selection or nutrient addition
but, ultimately, to investigate this decay process in the
field. In this study, three basic aspects of the process,
including analysis of lignin at the molecular level, have
been investigated using the same model system. The re-
sults indicate that P. ostreatus initially concentrates on pro-
ducing degradative enzymes, as MnP activity is seen to rise
rapidly and reaches a maximum in its activity-time func-
tion at about day 14 (Fig. 4). In contrast there is no signif-
icant change in both [Ac/Al]G and [Ac/Al]S (Fig. 2) before
21 days. This is also the case for the extent of ergosterol
formation (Fig. 3). Significant lignin oxidation and fungal
growth occurs only after peak enzyme activity has been
reached. The rapid early rise in manganese-dependent per-
oxidase activity, preceding measurable changes in lignin
oxidation, is consistent with the view that peroxidases
with manganese-dependent activity are important for the
initiation of lignin decomposition. The early period of en-
zyme production corresponds to the ‘‘colonisation phase”
described by Valmaseda et al. (1991) and the subsequent
increased lignin oxidation to the ‘‘degradation phase” pro-
posed by these authors for P. ostreatus solid-state cultiva-
tion on nitrogen-amended wheat straw. The colonisation
phase may be linked to a slow initial opening of the cell
wall and lignin degradation by smaller agents like reactive
oxygen species (Hammel et al., 2002), before a more exten-
sive, directly enzyme-catalysed degradation including cell
wall polysaccharides can occur. Ferraz et al. (2003) report
such a mechanism during the degradation of Eucalyptus
grandis wood by the white-rot fungus Ceriporiopsis
subvermispora.
4. Conclusions
Lignin degradation does not occur in isolation but pro-
ceeds via an array of processes including fungal metabo-
lism, growth, resource capture and dispersal. We have
demonstrated that it is possible to analyse lignin oxidation
using THM in the presence of TMAH and, in the same sys-
tem, measure enzyme activity and fungal growth as a func-
tion of incubation time. When P. ostreatus is grown on
unamended wheat straw, a rapid early rise in manga-
nese-dependent peroxidase activity precedes significant
changes in lignin oxidation and fungal growth. This ap-
proach will be useful when investigating compositional
changes in litter and soil layers and experiments are now
underway in our laboratory to include additional fungi
and lignocellulosic substrates.
Acknowledgements
This work was partly funded by the Natural Environ-
ment Research Council (NERC) through a studentship for
SAR. The authors would like to thank P. Donohoe, B. Bowler
and I. Harrison for assistance with GCMS and GC. The paper
benefitted from useful and constructive comments made
by an anonymous reviewer and P. Greenwood.
Guest Associate Editor—H. Knicker
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