Investigation of growth responses in saprophytic fungi to charred biomass

Abstract

We present the results of a study testing the response of two saprophytic white-rot fungi species, Pleurotus pulmonarius and Coriolus versicolor, to charred biomass (charcoal) as a growth substrate. We used a combination of optical microscopy, scanning electron microscopy, elemental abundance measurements, and isotope ratio mass spectrometry ((13)C and (15)N) to investigate fungal colonisation of control and incubated samples of Scots Pine (Pinus sylvestris) wood, and charcoal from the same species produced at 300 degrees C and 400 degrees C. Both species of fungi colonise the surface and interior of wood and charcoals over time periods of less than 70 days; however, distinctly different growth forms are evident between the exterior and interior of the charcoal substrate, with hyphal penetration concentrated along lines of structural weakness. Although the fungi were able to degrade and metabolise the pine wood, charcoal does not form a readily available source of fungal nutrients at least for these species under the conditions used in this study.

Full text

Ascough, P.L., Sturrock, C.J. and Bird, M.I. (2010) Investigation of
growth responses in saprophytic fungi to charred biomass. Isotopes in
Environmental and Health Studies, 46 (1). pp. 64-77. ISSN 1025-6016
http://eprints.gla.ac.uk/25716/
Deposited on: 12 August 2010
Enlighten – Research publications by members of the University of Glasgow
http://eprints.gla.ac.uk

1
Investigation of growth responses in saprophytic fungi to charred biomass
P. L. Ascough1*, C. J. Sturrock2, M. I. Bird3.
1SUERC, Scottish Enterprise Technology Park, Rankine Avenue, East Kilbride, G75
0QF, UK
2South Eastern Applied Materials Research Centre, Waterford Institute of
Technology, Cork Road, Waterford, Ireland.
3School of Earth and Environmental Sciences, James Cook University, Cairns,
Queensland, 4870, Australia
Abstract
We present results of a study testing the response of two saprophytic white-rot fungi
species, Pleurotus pulmonarius and Coriolus versicolor, to charred biomass
(charcoal) as a growth substrate. We used a combination of optical microscopy,
Scanning Electron Microscopy (SEM), elemental abundance measurements, and
Isotopic Ratio Mass Spectrometry (IRMS) to investigate fungal colonization of
control and incubated samples of Scots Pine (Pinus sylvestris) wood, and charcoal
from the same species produced at 300°C and 400°C. Both species of fungi colonize
the surface and interior of wood and charcoals over time periods of less than 70 days,
however distinctly different growth forms are evident between the exterior and
interior of the charcoal substrate, with hyphal penetration concentrated along lines of
structural weakness. Although the fungi were able to degrade and metabolize the pine
wood, charcoal does not form a readily available source of fungal nutrients at least for
these species under the conditions used in this study.
Keywords
Charcoal, Fungi, Degradation, Biochar, Stable Isotope
*Corresponding author. Tel.: +44 1355 270179; fax: +44 1355 229898. E-mail:
p.ascough@suerc.gla.ac.uk (P. L. Ascough).

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Introduction
Fire plays a central role in the dynamics of many ecosystems, with over 8.6 x 1012 kg of
biomass burnt globally each year [1]. Consequently, fire strongly influences vegetation
development and soil microbial function, driving patterns of succession and species
composition [2]. A major product of biomass burning is charcoal, formed as a result of
biomass pyrolysis when material is exposed to elevated temperatures (over c.300°C),
where flaming combustion is inhibited by low oxygen availability. The importance of
charcoal to global biogeochemical cycles is highlighted by the fact that at present, total
global production of pyrogenically altered carbon (including charcoal) from biomass
burning has been estimated at 50-270 Gt yr-1 [3]. Therefore, the products of biomass
pyrolysis, including charcoal, make up an important proportion of many soils,
comprising up to 35% of total organic content [4].
The molecular changes during production of charcoal, particularly reorganization of the
carbon structure into condensed aromatic ring configurations [5] both raise the carbon
content and the resistance to environmental degradation of charcoal relative to un-
pyrolysed biomass. As a result, the mineralization rates of some forms of charcoal are
extremely slow, apparently resisting degradation over geological timescales [1]. On the
other hand, it is also apparent that some charcoal components undergo environmental
degradation on decadal timescales (e.g. [6]). The potential environmental resistance to
mineralization has resulted in a recent interest in the potential for net sequestration of C
over extended time periods, via the large scale amendment of soils with charcoal, also
termed ‘biochar’ [7]. A central question for both specific initiatives such as biochar,
and for understanding of wider general ecosystem dynamics, is the interaction between
charcoal and soil microbial communities.
Fungi are an important part of dynamic soil systems, with the majority of the over
80,000 presently known species spending at least part of their life-cycle in the soil [8],
and fungal hyphae contents estimated in some environments as high as 67 km per 1g of
dry soil [9]. Fungi therefore play an essential role in soil ecosystems. For example, the
ramification of hyphae through soil has the beneficial effect of stabilizing soil structure,
both through the adhesive properties of exo-polysacharides and the physical entrapment
of soil particles [10]. These actions have significant influence on soil water infiltration

3
and water holding capacity. The fungi of interest in this study are the large group of
fungi that function as primary degraders of organic material to forms more bioavailable
for plants and other soil biota (saprophytic fungi).
There is a growing body of evidence that some fungal communities respond positively
to the presence of charcoal in soils, for example, increased activity of arbuscular
mycorrhizal fungi due to changes in soil chemistry has been demonstrated following
charcoal additions [11]. However, direct empirical evidence for the mechanisms by
which charcoal presence may enhance fungal growth is often lacking [12, 13]. Different
hypotheses include the availability of C and nutrients from the charcoal surface [14], or
the provision of micro-habitats for fungal communities within the char structure itself
[15]. In the case of saprophytic fungi, many species are able to utilize aromatic
macromolecules, such as lignin, as an energy source, by catabolic degradation with a
variety of exoenzymes [16]. In addition to compounds in uncharred biomass,
saprophytic species have also been observed to co-metabolise recalcitrant materials
such as black shales and charcoal over relatively short timescales of <4 months [17].
However, questions remain surrounding the role of pyrolysed biomass as a growth
substrate, and specifically whether fungi are able to utilize carbon from charred
material as an energy source.
In this study, we investigate the response of two saprophytic fungal species to charcoal,
produced under different pyrolysis conditions. These results feed directly into enhanced
understanding and quantification of the role charcoal plays in global biogeochemical
cycles via soil microorganism- charcoal interactions. In order to address these research
goals, we have used a combination of stable isotopic analysis via Isotope Ratio Mass
Spectrometry (IRMS), fungal growth analysis, and observation by Scanning Electron
Microscopy. The combination of IRMS with visual techniques represents a new
approach to studies of this type, and appears a promising methodological approach to
deriving direct empirical evidence for fungi- charcoal interaction mechanisms.
1. Materials and methods
The approach used in this study involved quantitative examination of (i.) the effect on
fungal growth of charcoal additions to a minimal nutrient medium, and (ii.) the

4
utilization of charcoal particles as a growth substrate by soil fungi. The charcoal was
prepared from samples of Scots Pine (Pinus sylvestris), obtained from Tentsmuir
Forest, Fife, in 2005. The bark was removed, and a sub-section of the first 15 rings was
taken, and processed to 1.5cm cubes. Charcoal was prepared from the cubes using a
Carbolite™ rotary furnace in an inert (N2) atmosphere for a period of 60 minutes at
either 300°C or 400°C, as described in [18]. Molecular structural differences between
charcoals produced at these temperatures results from differential thermal degradation
of wood structural components. Cellulosic material degrades very rapidly over 250-
400°C, while lignin whereas slower degradation of lignin proceeds over the interval
200°C to 720°C [19, 20].
Two species of white-rot, lignin-degrading fungi were used in the study; Pleurotus
pulmonarius and Coriolus versicolor which have a demonstrated ability to break
down complex macromolecular lignin structures that contain a relatively large number
of aromatic rings. Furthermore, the ability of these fungi to co-metabolise a range of
macromolecules from polycyclic aromatic hydrocarbons (PAHs) and
pentachlorophenols (PCPs) to tars and oils has also been reported [21, 22, 23, 24].
Therefore, it is hypothesised that the exocelluar enzymes produced by these fungi
may also be capable of the biodegradation charcoal.
To determine fungal growth rate response, a study of the two fungal species was
conducted on minimal essential media (MEM) [25] with differing carbon sources. The
carbon sources were 0 and 0.5 % D-glucose, 0.5% 300°C powdered charcoal and
0.5% 400°C powdered charcoal. The base media components were selected in an
attempt reflect the low levels of available nutrient for growth found in the charcoals.
Fungi were incubated at 20 °C on the respective carbon sources for a period of 8 days,
during which hyphal extension and morphology of filaments were assessed daily.
In the second experiment, charcoal blocks and uncharred blocks of pine wood were
incubated with both fungal species for 70 days. At least 12 replicates were used for
fungal incubations on charcoals, and 5 replicates for incubations on wood. The blocks
were placed upon MEM agar, which contained a starting carbon (0.5 % D-glucose)
and nitrogen (0.02% L-aspargenine) source to initiate fungal colonization. Prior to,

5
and following, the incubation period, the cubes were lyophilized and weighed to
estimate the rate of decay of the blocks. In the case of P. pulmonarius this was
accompanied by visual qualitative analysis of the degree of surface colonization
following 56 days incubation. The extent and nature of fungal colonization of the
blocks was investigated using scanning electron microscopy (SEM). Lyophilized
blocks were split open, and the freshly split surfaces sputter-coated with Au. The
outer and inner surfaces of the blocks were then examined at the St. Andrews
University Facility for Earth and Environmental Analysis (FEEA), using a JEOL
JSM-5410 scanning electron microscope at 10 kV accelerating voltage, with a
variable working distance of between 15 and 28 mm.
Isotopic and elemental analyses were performed on samples of untreated wood and
charcoal and of the material following fungal incubation. To obtain samples for
analysis, dried blocks of both control and incubated material were split open across
the centre and material for analysis was extracted to a depth of 2mm at a minimum of
four points across the interior cross-section of each block, extending to 2mm from the
outer edges of the block surface. Additionally, samples were obtained from the
surface 0.5 mm of blocks that had been subject to P. pulmonarius fungal incubation
(see Figure 2C). This ensured extraction of material with a homogeneous isotopic
signal prior to incubation, excluding the effect of inter-ring isotopic variation within
the original plant sample. Isotopic and elemental analyses were also made of both the
D-glucose and L-Asparagine carbon and nitrogen sources and the two species of fungi
separately grown on MEM without the addition of any charcoal. This allowed an
assessment of the degree of fractionation between these substrates and the fungi, and
of the isotopic signature of the fungi when these nutrient sources were accessed.
Stable isotopic ratios of carbon (13C/12C) and nitrogen (15N/14N) were measured using
continuous flow via a Costech elemental analyser (EA) fitted with a zero-blank
autosampler, interfaced by a ConFlo II to a ThermoFinnegan Deltaplus XL at the
University of St. Andrews FEEA. The carbon (%C) and nitrogen (%N) content of the
samples was determined following combustion in the EA. Each sample was measured
in duplicate, and individual machine runs included a mix of sample material, blanks
and laboratory standards. Three internal standards were measured, where the primary
standard was acetanilide (IAEA/Sigma Aldrich, %C: 71.09%, δ13C: -30.11‰; %N:

6
10.36, δ15N: -0.03), and secondary standards were a commercially available protein
(B2155, %C: 47.02, δ13C: -26.98‰, %N: 13.63, δ15N: 5.94) and a C4 cane sugar/uric
acid mix (Tesco, %C: 40.20, δ13C: -12.02‰, %N: 9.1, δ15N:15.07). Results are
expressed in standard notation (δ13C and δ15N) as parts per thousand (‰) relative to
Vienna PeeDee Belemnite (VPDB) for δ13C and atmospheric N2 for δ15N.
Measurement precisions were better than 0.1‰ for δ13C, 0.3‰ for δ15N, 1% for %C
and 0.05% for %N.
As it was not possible to both analyse a single block for elemental/isotopic
composition, and also incubate the same block with the fungal species, it is likely that
slight differences existed in the composition of the control and incubated blocks prior
to fungal incubation. SEM analysis indicates that most colonization on the exterior of
the blocks with the number of hyphae decreasing progressively into the interior of the
blocks. Therefore, individual measurements in a single block are normalized to the
composition obtained from the centre of each block to make the results comparable
between blocks. This allows an assessment of the extent of variability within
incubated and control blocks, and whether isotopic variations were randomly
distributed throughout the blocks, or followed a spatial pattern.

7
2. Results
Figure 1 shows a comparison of the mean hyphal extension rates for each fungal
species on each growth medium. Hyphal extension rates for both species were
significantly higher for media with 0 and 0.5 % glucose than was the case for any
media that included charcoal. In media without charcoal, C. versicolor appeared to
show faster growth rates than P. pulmonarius and hyphal extension was greatest with
0% glucose for both fungi. However it is important to note that the growth forms on 0
and 0.5% glucose were very different. On 0% glucose, growth was very sparse, with
few branched hyphae, typical of an exploratory growth form [26]. In contrast, much
denser growth, typical of a more exploitative growth form, occurred on 0.5 %
glucose.
Growth of fungal hyphae on the charcoal-containing media was generally very sparse,
with a lag period of 3 days, and it was difficult to visualise the hyphae against the
black coloured agar. The slow hyphal extension rate meant it was difficult to measure
hyphal branching angles, however small fan-like flushes of hyphal growth were
observed, suggestive of an exploratory growth form. There were differences between
the two fungal species in growth response to the two different charcoal-containing
media. In the case of P. pulmonarius there was a clearly faster growth rates on the
media containing 300°C powdered charcoal compared to the 400°C powdered
charcoal (Figure 1). In contrast, C. versicolor showed no significant difference in
hyphal extension rate between the two charcoal treatments.
The weight gain recorded for some charcoal blocks following fungal incubation
(Table 1) demonstrates colonization of the surface of the charcoal by a significant
mass of fungi. However there is no evidence for removal of any significant proportion
of the charcoal structure itself via fungal degradation. Weight losses in the wood
blocks on the other hand, indicate that >9.50% by weight of the lignocellulosic
structure has been metabolized over the fungal incubation period. Differences in
colonization were also evident in visual analysis. After 8 weeks incubation, fungal
growth of P. pulmonarius was visible on the outer surfaces of both the 300°C and

8
400°C charcoals, however, a greater degree of colonization had occurred on the
300°C charcoal blocks (Figure 2A and 2B).
Both species of fungi appear to be able to colonize the outer surface of both the wood
and charcoal blocks (Figure 3, A-D). A relatively dense mass of branched,
anastomosed fungal hyphae was observed on the outer surface of all samples, with
some morphological features suggestive of an exploitative growth form. In the pine
wood, there is evidence for substantial fungal degradation of interior structures within
the blocks (Figure 4 A-B), with numerous cracks, small cavities, and holes in the cell
walls that are not present in the non-fungal controls. In the charcoal blocks, similar
evidence for structural degradation is absent, and penetration of hyphae in significant
quantities into the interior of the blocks appears to occur mainly along pre-existing
physical cracks and fissures that extend from the surface to varying depths. Fungal
colonization of these involves a sparse, exploratory growth form (Figure 4 C-D). SEM
comparison of the fungal colonization of the interior of the charcoal blocks again
shows differences in colonization degree between 300°C and 400°C charcoal for C.
versicolor, but a similar degree of colonization for the two charcoals by P.
pulmonarius.
Internal %C variability through the control blocks is low, with a range of <0.45% in
the wood and 300°C charcoal, and <0.85% in the 400°C charcoal (Table 2). Isotopic
variation across the control blocks is also relatively small, with a variation of 0.2‰ in
the 300°C charcoal, and 0.3‰ in the wood and 400°C charcoal. The nitrogen content
of all three sample types is low; in wood and 400°C charcoal, this was lower than the
measurement precision, however in the 300°C charcoal %N content averaged 0.3%
(giving an average C:N ratio of 197), with a range of 0.04%. The δ 15N values of these
samples had a range of 0.66‰ with an average value of -4.27‰ (Table 2). The
internal isotopic and elemental variability in the control blocks is slightly greater than
the instrument precision, and therefore the maximum range in values across control
blocks is taken as the significance level for further interpretation. These values are
±0.85% for %C, ±0.31‰ for δ13C, 0.04% for %N and 0.66‰ for δ 15N.
The δ13C of the D-glucose is -11.25‰, and of the L-Asparagine is -24.23‰, while the
δ13C of C. versicolor and P. pulmonarius grown on MEM is -8.61‰ and -10.44‰

9
respectively, showing no significant incorporation of L-Asparagine carbon by the
fungi (Table 2). The elemental composition of the two fungi species for C was
37.05% in C. versicolor and 41.54% in P. pulmonarius, and for N was 9.02 % in C.
versicolor and 3.22% in P. pulmonarius. The D-glucose is obtained from C4 sugars,
that do not contain nitrogen, so the L-Asparagine (δ15N = +4.23‰) comprises the
entirety of the fungal ON source, as reflected in the δ15N of fungi grown on MEM;
+2.93‰ for C. versicolor and +3.08‰ for P. pulmonarius. This gives fungi δ15N
~1‰ lower than that of the L-Asparagine, indicating that both species discriminate
against uptake of 15N when grown on this substrate, as previously observed [27].
Variation in %C increased following incubation with P. pulmonarius and C.
versicolor in the wood and 300°C charcoal, for example variations of up to 2.57%
were observed in the 300°C charcoal incubated with C. versicolor. However, in the
400°C charcoal %C variation was only slightly larger than the control samples (Table
2). Fungal incubation raised the %N of all samples to measurable levels, with the
highest %N concentrations in the majority of samples were recorded on the outside of
the blocks, where fungal hyphae concentration was greatest. Overall though, %N
values across the tested samples remained relatively low.
After incubation with both P. pulmonarius and C. versicolor the range in δ13C from
the exterior to interior of the blocks showed an overall increase relative to the control
samples, up to a maximum of 1.40‰ (Figure 5). Variability of δ13C in charcoal
samples incubated with P. pulmonarius increased relative to the control in both the
300°C and 400°C charcoal, whereas following incubation with C. versicolor,
increased δ13C variability was only observed in the 300°C charcoal. Increases in δ13C
variability appear to relate to a pattern of isotopic offset between samples from the
exterior and interior of the blocks, however these are inconsistent in direction between
sample types. For δ15N, there was considerable variation in values across the
incubated blocks, with differences of up to 3.6‰ in δ15N (Figure 6). Again, these
variations appear to follow a pattern, with separation in values between the exterior
and interior of the blocks, where values on the exterior tend to be higher. Average
values of δ15N in all samples decrease in the order wood > 300°C charcoal > 400°C
charcoal (Table 2); for example, following incubation with P. pulmonarius the
average sample δ15N values are -3.04‰, -4.7‰ and -6.6‰, respectively. In all

10
samples following incubation, the highest δ15N values occur on the outer surface of
the blocks.
3. Discussion
Fungal colonization occurred after 70 days on the surface of all samples, with a
particular concentration of fungal hyphae on the outer surface of the blocks, and it is
only in this zone that an exploititative growth form is observed, indicating either a
concentration of available nutrients on these surfaces, or that the substrate surface
forms a favourable habitat for the fungi to exploit the D-glucose and L-asparagine C
and N sources. Penetration of the fungi to the interior of the charcoal blocks appeared
limited to physical cracks in the structure. The preferential exploitation of structures
such as cracks in the charcoal blocks indicates heterogeneity in the presence of fungal
hyphae within the blocks, rather than a progressive radiation of fungi from the
exterior to the interior of the blocks. This suggests that colonization of charred
organic material by saprophytic fungi may therefore occur more rapidly when the
material contains greater amounts of cracks that the fungi can easily penetrate. The
resulting spatial heterogeneity in fungal hyphae within the blocks observed by SEM
imaging appears to correspond to the heterogeneity observed in the elemental and
isotopic values within incubated samples. Overall, both image based, elemental and
isotopic analyses suggest that colonization of the samples by the fungi exterior and
interior colonization is distinctly different. During the three month period of this
study, the majority of fungal growth occurred within the top few mm of the charcoal
block surfaces.
Increased variability in measured isotopic and elemental values between the control
and incubated samples suggests that for both species, fungal growth on different
samples produced sufficient mycelium mass to be detectable by these techniques. In
the majority of incubated samples, decreased %C at the block exterior is consistent
with a greater contribution of fungal mass to samples taken from these locations.
Increases in %N following incubation are also suggestive of the presence of fungal
hyphae through the samples, however there is no consistent trend in values with
location in the blocks. This may be due to the fact that the C: N ratios of the
unincubated substrates are so high. The introduction of even a small proportion of

11
fungal mass, with a far lower C: N ratio would therefore produce a measurable
increase in %N. However, as the %N values of the samples remain uniformly low, it
is evident that hyphae comprise a relatively small proportion of the overall sample
mass. This is consistent with observations by SEM of only limited penetration by the
fungi to the block interiors.
Increased δ13C variation across the blocks following incubation appears due to
significant differences between the outer block, where fungal colonization is greatest,
and the interior (Figure 5). In wood and 400°C charcoal following P. pulmonarius
incubation, δ13C values are higher on the block exterior, relative to the interior,
consistent with a contribution from the isotopically heavier fungi. However, in 300°C
charcoal incubated with either species, block exterior values tend to be lower than in
the interior. In the case of δ15N, higher values on the block exteriors (Figure 6) are
consistent with a contribution from fungi δ15N, which is significantly enriched in 15N
relative to the charcoal substrate. The greater observed variability in δ15N relative to
δ13C in the incubated blocks (figures 5 and 6) may again result from the high C:N
ratios of the unincubated control samples. C:N values for the fungi are 4.1 for C.
versicolor and 12.9 for P. pulmonarius, compared to values of 181 to 215 in the
control 300°C charcoal. As the %C of the control substrate is so high, a higher
proportion of fungal mass would be required to significantly influence the δ13C of the
samples than would be required to influence δ15N.
Comparison of δ13C and δ15N values indicates apparent differences between the effect
of fungal incubation on wood, 300°C and 400°C charcoal samples, as highlighted in
figure 7, where several different sample groups are evident. This includes the control
300°C charcoal, and comparison of the scatter in values between this and the
incubated sample groups again emphasises the higher isotopic variability of incubated
versus control samples. In wood and 400°C charcoal following P. pulmonarius
incubation, increases in δ13C generally correspond to higher δ15N values. In contrast,
for 300°C charcoal incubated with both fungal species the trend appears to be in the
opposite direction. The negative correlation between δ13C and δ15N in 300°C
incubated charcoal samples, in contrast to the positive association in 400°C charcoal
(Figure 7) may suggest variation in the isotopic composition of the fungi within
samples, possibly as a function of resource partitioning within the hyphae.

12
Translocation of resources, including nutrients and water, within a continuous hyphal
network enables fungi to colonize spatially heterogeneous substrates [28, 29]. This
strategy may therefore be useful in colonization of substrates that are poor in certain
resources, such as charcoal with a high carbon to nitrogen ratio.
The quality of a fungal substrate is determined by the concentration and physical
availability of nutrients, as well their chemical form [29]. The results presented here
suggest that among the tested materials, charcoal produced at 300-400°C is a low-
quality growth substrate for both P. pulmonarius and C. versicolor fungal species.
The results of the growth rate study, specifically differences in hyphal growth forms,
demonstrate that that with 0% glucose the fungi are actively searching for energy
sources by devoting resources to rapid development of few long hyphae, whereas with
0.5% glucose the fungi are devoting resources to development of a more branched
hyphal network in order maximise resource capture from the nutrient agar plates. In
addition, the observed growth forms within the charcoal blocks suggest that an
exploratory form developed, and the hyphal penetration within the block is more
actively engaged in seeking readily available nutrient sources, rather than in any
utilization of the charcoal structure itself. This is supported by the absence of
significant weight loss following fungal incubation, with overall increases in mass
observed in some charcoal blocks. In contrast, some degradation of the wood structure
appears to have occurred, with significant mass loss in the measured samples, and a
clear difference in physical structural degradation revealed by SEM imaging of the
incubated samples. The destruction of anatomical structures in the wood sample by
fungal action contrasts with the apparent lack of evidence of structural degradation of
the charcoal samples.
Pyrolysis results in alterations to the wood macro- and micro-structure, with
progressive homogenization of the wood cell walls and disappearance of the middle
lamella [30]. This occurs in conjunction with increases in overall average carbon
abundances with increasing pyrolysis temperature, in this study from 46.0% in wood
to 78.8% for 400°C charcoal. The result is decreasing structural heterogeneity as
pyrolysis temperature increases, and it appears that this feature plays a major role in
inhibiting growth of some fungal species when charcoal is used as a substrate. These
results support the interpretation that changes in molecular structure induced by

13
pyrolysis of wood at temperatures of 300°C or more greatly inhibit the ability of these
species of saprophytic fungi to enzymatically degrade charcoal, at least over the
timeframe of this study. This is likely to be the result of lower bioavailability of the
carbon structure of the charcoal itself, however it is also possible that pyrolysis
produces certain substances that actively inhibit the activity of the fungi. Both
theories have previously been suggested to explain reduction of fungal attack in wood
following heat treatment [31].

14
4. Conclusions
It is clear from visual and SEM analysis that both species of fungi colonize the
surface, and to a lesser extent, the interior, of both 300°C and 400°C charcoals over
time periods of less than three months. Overall, it appears that a combined visual and
elemental/isotopic approach is a promising methodology for examining fungal
response to charcoal as a growth substrate. It is important to note that these results
represent a relatively short term experiment, and future work should concentrate on
determining whether over more extended time periods a more extensive degradation
of charcoal by fungi occurs. However, the results presented here suggest that for these
two common species of fungi, breakdown of the structure of charcoal itself does not
supply a readily available source of nutrients. This is in contrast to previous work,
which suggests that some saprophytic fungi species do degrade highly recalcitrant
condensed aromatic structures (e.g. [17]), and highlights the fact that the diversity of
microbial species and strategies in soils will likely result in a range of responses to
charcoal. Rather than using the charcoal structure itself as a source of nutrients, it is
also possible that colonization of the charcoal samples by the fungi could be the result
of different nutrients (e.g. P, K, Ca) that are present on the charcoal surface as a result
of pyrolytic degradation of the original biomass. Positive effects for soil saprophytic
fungi communities consisting of species similar to the ones tested here are therefore
more likely to result from pyrolysed material as a favourable habitat or source of
nutrients, than direct utilization of the charcoal structure itself, even in material
produced at relatively low temperatures (i.e. <300°C).
Further research within this field is clearly required in order to enhance understanding
of interactions between fungi and charcoal in soils, not least because of the truly
global importance of these within such systems. Future directions include a focus
upon variation in the range of conditions for fungal incubation, such as temperature-
dependent initiation of more rapid phases of fungal growth.

15
Acknowledgements
This work was partly funded by NERC standard grant NE/C004531/1 ‘Charcoal
Degradation in Natural Environments’. This paper represents a contribution from the
Scottish Alliance for Geosciences, Society and Environment (SAGES). The authors
acknowledge Donald Herd at St. Andrews School of Geography and Geoscience for
assistance with SEM imaging and photography.

16
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20
Tables
Table 1: Weight changes within fungal decay study, showing changes in weight of
samples of wood and two charcoals following fungal incubation with Pleurotus
pulmonarius and Coriolus versicolor.
Species
P. pulmonarius
C. versicolor
Media
Before
Incubation
(g)
After
Incubation
(g)
% wt
loss
Before
Incubation
(g)
After
Incubation
(g)
% wt
loss
2.622
2.220
15.332
2.392
2.264
5.351
1.724
1.584
8.121
1.553
1.431
7.856
1.756
1.608
8.428
1.688
1.572
6.872
1.961
1.804
8.006
2.423
2.343
3.302
Wood
2.336
2.157
7.663
2.654
2.577
2.901
1.219
1.218
0.082
1.235
1.235
0.000
1.740
1.739
0.057
1.470
1.469
0.068
1.904
1.909
-0.263
0.968
0.969
-0.103
1.481
1.478
0.203
1.612
1.613
-0.062
0.958
0.965
-0.731
1.227
1.229
-0.163
1.496
1.503
-0.468
1.502
1.501
0.067
1.190
1.194
-0.336
1.714
1.717
-0.175
3.101
3.106
-0.161
1.339
1.344
-0.373
0.865
0.870
-0.578
1.428
1.432
-0.280
0.999
1.353
-35.435
1.361
1.363
-0.147
1.590
1.597
-0.440
0.896
0.897
-0.112
300°C
charcoal
3.678
3.696
-0.489
0.763
0.763
0.000
0.326
0.333
-2.147
0.563
0.562
0.178
0.522
0.527
-0.958
0.579
0.581
-0.345
0.426
0.439
-3.052
0.483
0.484
-0.207
0.381
0.383
-0.525
0.513
0.511
0.390
0.476
0.481
-1.050
0.364
0.361
0.824
0.391
0.399
-2.046
0.374
0.376
-0.535
0.367
0.378
-2.997
0.601
0.602
-0.166
0.430
0.439
-2.093
0.569
0.568
0.176
0.340
0.350
-2.941
0.396
0.397
-0.253
0.346
0.345
0.289
0.328
0.327
0.305
0.484
0.500
-3.306
0.469
0.470
-0.213
400°C
charcoal
0.488
0.493
-1.025
0.547
0.548
-0.183

21
Table 2: Results of elemental and isotopic analysis of control samples and material
following fungal incubation with Pleurotus pulmonarius and Coriolus versicolor.
PW: pine wood, PC-300: 300°C charcoal, PC-400: 400°C charcoal. 1: Exterior block
surface, 2: 1-2mm depth from top, 3: 7-8 mm depth from top, 4: 9-10 mm depth from
top, 5: 7-8 mm depth from base, 6: 1-2mm depth from base.
Sample
%C
δ13C
%N
δ15N
CN
D-glucose
39.40
-10.98
-
-
-
L-aspargenine
35.89
-23.95
16.00
4.23
2.24
C. versicolor
37.05
-8.61
9.02
2.93
4.11
P. pulmonarius
41.54
-10.44
3.22
3.08
12.90
PW_2
45.75
-25.07
-
-
-
PW_3
46.09
-25.05
-
-
-
PW_5
46.19
-24.76
-
-
-
PW_6
46.06
-24.82
-
-
-
PW_ P. pulmonarius _1
44.26
-23.93
0.31
-1.42
143.01
PW_ P. pulmonarius _2
45.16
-24.71
0.10
-4.73
465.35
PW_ P. pulmonarius _3
46.44
-25.19
0.04
-2.98
319.62
PW_ P. pulmonarius _5
46.29
-25.28
-
-
-
PW_ P. pulmonarius _6
46.83
-25.33
-
-
-
PC-300_2
58.17
-25.37
0.28
-3.93
215.33
PC-300_3
58.34
-25.57
0.31
-4.56
189.59
PC-300_5
58.62
-25.52
0.29
-4.00
201.72
PC-300_6
58.54
-25.39
0.32
-4.59
181.64
PC-300- C. versicolor _2
56.75
-26.65
0.26
-0.36
217.19
PC-300- C. versicolor _3
56.05
-26.15
0.24
-1.43
238.54
PC-300- C. versicolor _4
58.25
-25.85
0.31
-3.81
187.10
PC-300- C. versicolor _5
57.77
-25.98
0.34
-1.92
174.46
PC-300- C. versicolor _6
56.51
-26.14
0.24
-3.28
241.77
PC-300- P. pulmonarius _1
60.84
-26.41
0.27
-2.27
223.12
PC-300- P. pulmonarius _2
60.49
-26.60
0.19
-4.98
324.66
PC-300- P. pulmonarius _3
61.48
-26.01
0.21
-4.56
293.13
PC-300- P. pulmonarius _5
61.42
-26.30
0.20
-5.88
311.94
PC-300- P. pulmonarius _6
61.63
-26.55
0.19
-5.67
321.19
PC-400_2
78.77
-27.70
-
-
-
PC-400_3
79.19
-27.93
-
-
-
PC-400_5
78.34
-27.63
-
-
-
PC-400_6
78.74
-27.94
-
-
-
PC-400- C. versicolor _2
70.51
-27.71
0.25
-4.74
289.89
PC-400- C. versicolor_3
70.33
-27.91
0.19
-6.35
376.27
PC-400- C. versicolor _5
70.50
-27.81
0.19
-6.49
368.28
PC-400- C. versicolor _6
70.93
-27.97
0.19
-7.25
368.02
PC-400- P. pulmonarius _1
72.36
-26.02
0.34
-4.61
214.37
PC-400- P. pulmonarius _2
72.50
-26.91
0.24
-7.90
303.24
PC-400- P. pulmonarius _3
73.49
-27.03
0.24
-7.59
306.32
PC-400- P. pulmonarius _4
73.14
-26.86
0.24
-6.75
310.06
PC-400- P. pulmonarius _5
73.36
-26.20
0.33
-6.68
223.17
PC-400- P. pulmonarius _6
72.58
-25.79
0.28
-6.03
257.21

22
Figure captions
Figure 1. Charts showing comparison of mycelial extension rates of Pleurotus
pulmonarius(Pp) and Coriolus versicolor (Cv) on minimal essential media (MEM)
with differing carbon sources.

23
Figure 2: A. Charcoal blocks produced using furnace at 300°C inoculated with P.
pulmonarius after 8 weeks incubation; B. Charcoal blocks produced using furnace at
400°C inoculated with P. pulmonarius after 8 weeks incubation; C. Schematic
representation of sampling scheme for incubated and control blocks; homogenized
sampled were obtained from the surface 0.5 mm (1), and at 1-2 mm and 7-8 mm depth
from the top and bottom of each block (2, 3, 5, and 6, respectively). In two instances,
a further sample was obtained at a depth of 9-10 mm within the block (4, not shown).

24
Figure 3: A: Surface colonization of pine wood by P. pulmonarius; B: Surface
colonization of 400°C charcoal by P. pulmonarius; C: Surface colonization of 400°C
charcoal by C. versicolor; D: Surface colonization of 300°C charcoal by C. versicolor

25
Figure 4: A: Interior structures of pine wood prior to fungal incubation, B: Interior
structures of pine wood showing degradation by P. pulmonarius fungi following 3
months fungal incubation, C: growth structures of C. versicolor fungi on surfaces of
fissures extending to the block interior in 300°C charcoal, D: growth structures of C.
versicolor fungi on surfaces of fissures extending to the block interior in 400°C
charcoal.

26
Figure 5: Variation in δ13C with sampling location; 1: Exterior block surface, 2: 1-
2mm depth from top, 3: 7-8 mm depth from top, 4: 9-10 mm depth from top, 5: 7-8
mm depth from base, 6: 1-2mm depth from base. PW = pine wood, PC-300 = 300°C
charcoal, PC-400 = 400°C charcoal, Pp= P. pulmonarius, Cv=C.versicolor.

27
Figure 6: variation in δ15N with sampling location 1: Exterior block surface, 2: 1-2mm
depth from top, 3: 7-8 mm depth from top, 4: 9-10 mm depth from top, 5: 7-8 mm
depth from base, 6: 1-2mm depth from base. PW = pine wood, PC-300 = 300°C
charcoal, PC-400 = 400°C charcoal, Pp= P. pulmonarius, Cv=C.versicolor.

28
Figure 7: Isotopic variation in both δ13C and δ15N in samples. PW = pine wood, PC-
300 = 300°C charcoal, PC-400 = 400°C charcoal. Circles show groupings of values
for different substrates and fungi species, Pp= P. pulmonarius, Cv=C.versicolor.