Effects of hot water extraction and fungal decayon wood crystalline cellulose structure
Caitlin Howell • Anne Christine Steenkjær Hastrup •
Rory Jara • Flemming Hofmann Larsen •
Barry Goodell • Jody Jellison
Received: 22 March 2011 / Accepted: 15 June 2011 / Published online: 5 July 2011
� Springer Science+Business Media B.V. 2011
Abstract The effect of hot-water extraction and
two types of fungal decay, brown rot and white rot,
on wood crystalline cellulose structure was examined
using a combination of X-ray diffraction (XRD) and13C solid-state nuclear magnetic resonance (NMR)
spectroscopy. Although having opposite effects on
the overall crystallinity of the wood, the XRD results
revealed that both extraction and brown-rot decay
caused a significant decrease in the 200 crystal plane
spacing (d-spacing) not seen for the white-rotted
samples. This effect was found to be additive, as
samples that were first extracted, then decayed
showed a double decrease in d-spacing compared to
that caused by extraction alone. This suggested that,
despite having a similarly directed effect on the
spacing of the crystalline planes, the two treatment
methods facilitate a decrease in d-spacing in different
ways. NMR results support the conclusion of differ-
ing structural effects, suggesting that the hot-water
extraction procedure was causing a co-crystallization
of existing crystalline domains, while the brown rot
decay was depolymerizing the cellulose chains of the
crystals, possibly allowing the remaining crystalline
material the freedom to relax into a more energeti-
cally favorable, tightly packed state. These findings
could have important implications for those seeking
to understand the effects of modification treatments
or biodegradation of crystalline cellulose nanostruc-
tures in their native states.
Keywords Wood cellulose � Hot-water extraction �Fungal decay � Crystallinity � X-ray diffraction �13C CP/MAS NMR � Brown-rot
Introduction
Understanding the nature of cellulose nanostructures in
the fibers of higher plants and how these structures are
modified and degraded is one of the key challenges in
the use of woody biomass. This includes breakdown
and conversion of biomass into biofuels and other
derivative products as well as protection of building
C. Howell (&) � J. Jellison
School of Biology and Ecology, University of Maine,
311 Hitchner Hall, Orono, ME 04469, USA
e-mail: [email protected]
A. C. S. Hastrup
Department of Biology, University of Copenhagen,
Sølvgade 83H, 1307 Copenhagen K, Denmark
R. Jara
Department of Chemical and Biological Engineering,
University of Maine, 5737 Jenness Hall, Orono,
ME 04469, USA
F. H. Larsen
Department of Food Science, University of Copenhagen,
Rolighedsvej 30, 1958 Frederiksberg C, Denmark
B. Goodell
Department of Wood Science and Forest Products,
Virginia Polytechnic Institute and State University,
230 Cheatham Hall, Blacksburg, VA 24061, USA
123
Cellulose (2011) 18:1179–1190
DOI 10.1007/s10570-011-9569-0
materials from biological, chemical, or physical deg-
radation. To address these issues in detail requires a
thorough understanding of the arrangement of the
cellulose chains relative to each other and to the
surrounding hemicellulose and lignin matrix. Of
particular interest is the structural impact on cellulose
when exposed to different processes related to biolog-
ical, chemical, and mechanical degradation.
In wood and other higher plants cellulose is
organized mainly into long, thin fibers of the cellulose
I allomorph, surrounded by a sheet of hemicellulose
and lignin (Zabel and Morrell 1992; Daniel 2003).
Cellulose I consists of a mixture of two distinct
crystalline forms: Ia (triclinic) and Ib (monoclinic)
(Atalla and Vanderhart 1984). There is little consensus
regarding the ratio of cellulose Ia to Ib in wood.
However, it is generally agreed upon that the Ib form is
the dominant polymorph in higher plant cellulose
(O’Sullivan 1997), with even higher levels possible in
processed wood as the metastable Ia form can be
converted to the more stable Ib form (Yamamoto and
Horii 1994; Hult et al. 2003). The crystalline regions in
wood are accompanied by regions of less order,
although the cellulose/hemicelluloses/lignin composi-
tion of the non-crystalline or amorphous regions is not
well documented. Furthermore, the existence of less-
ordered paracrystalline structures surrounding the
interior crystalline regions of cellulose microfibrils
has been proposed (Newman 1999; Ding and Himmel
2006). Such a transitional region between crystalline
and paracrystalline structures may partly explain the
difficulty in determining the exact arrangement of
cellulose structures in native wood.
Both degradation by fungi and hot-water extrac-
tion are known to affect the crystalline cellulose in
wood. Brown rot fungi are the only organisms known
to be able to circumvent the lignin barrier in order to
access and degrade hemicelluloses and celluloses, a
fact which is currently being investigated with a view
to improve cellulose-based bioprocessing and bio-
technology (Schilling et al. 2009). Hot-water extrac-
tion has been assessed as a method for removing
hemicelluloses or partly degraded hemicelluloses for
industrial fermentation while preserving the remain-
ing wood for use as construction material and other
purposes. This process has been shown to increase the
overall crystallinity of the wood material, mainly due
to the removal of amorphous material, i.e. hemicel-
luloses (Paredes et al. 2009).
Decay patterns of Basidiomycete fungi can be
grouped into white-rot, in which lignin, cellulose, and
hemicelluloses are degraded, and brown-rot, in which
only cellulose and hemicelluloses are degraded, while
the lignin is modified and left behind (Zabel and
Morrell 1992). Although the effects of white-rot on the
structure of crystalline cellulose have not yet been
thoroughly investigated, degradation by brown-rot
fungi has been shown initially to increase the overall
percent crystallinity of wood, presumably due to
removal of hemicelluloses and non-crystalline cellu-
lose (Howell et al. 2009a). In the early stages of decay,
brown-rot fungi use hydroxyl radicals generated by
Fenton chemistry. These reactive oxygen species
randomly attack compounds within close proximity
causing a rapid depolymerization (Kim et al. 2002,
Hastrup et al. 2011). Degradation of the wood struc-
tures in this way creates openings in the cell wall large
enough for enzymes. The non-enzymatic processes
used by brown rot fungi are further enhanced by the
production of low molecular weight chelators such as
oxalic acid and phenolate-catechol siderophores.
These facilitate the availability, and in the case of the
latter, also the reduction of iron compounds from the
surrounding environment which are essential for the
radical-generation process (Goodell et al. 1997; Aran-
tes et al. 2009, 2010). The initial decrease in the
hemicelluloses and non-crystalline cellulose by hydro-
xyl radicals (Kleman-Leyer et al. 1992), with a near-
complete removal of the hemicelluloses occurring at
about 20% weight loss (Curling et al. 2001) cause an
increase in percent crystallinity early in the decay
process which is followed by a gradual decrease
(Highley and Dashek 1998; Howell et al. 2009a).
However, the molecular-level changes that occur
during these processes are still not well understood.
The purpose of this study was to examine struc-
tural modifications of crystalline cellulose in wood on
the molecular scale when exposed to two different
types of treatments: biological decay and hot-water
extraction.
Materials and methods
Sample preparation
Extracted wood material similar to what was used in
these experiments has been thoroughly described in
1180 Cellulose (2011) 18:1179–1190
123
terms of treatment procedures, composition, and
microstructure. These results have been published
elsewhere (Paredes et al. 2008, 2009). Briefly, Red
Maple (Acer rubrum) strands (10 9 0.9 ± 0.05 cm)
were extracted in tap water in a high-pressure reactor.
Extractions were performed in an M/K Digester
consisting of two high-pressure cookers. A liquid to
wood ratio of 4 was used for each extraction. The
vessel was heated to 160� from room temperature in
50 min, then held at a constant temperature for 0, 25,
45, or 90 min. These conditions of varying severity of
treatment were labeled a, b, c, and d, corresponding
to strand weight losses determined to be 3.4 (±0.5),
6.2 (±2.0), 9.9 (±1.2), and 17.2 (±0.8)%, respec-
tively. Weight loss was determined according to the
procedures of Paredes et al. 2008. The extracted
wood material was dried in air and ground to less
than 70 mesh. Analysis of total carbohydrate mono-
meric content of the hydrolizate was performed by
High Performance Anion Exchange Chromatography
with Pulse Amperometric Detection (HPAEC-PAD,
Dionex) as described by Davis (1998) (Table 2).
Approximately 100 mg of wood was subjected to a
standard sequential double acid hydrolysis at 72 and
4% sulfuric acid concentration. Wood samples were
tested in duplicate and are presented as an average
value.
Decayed material included in this work has also
been thoroughly characterized (Howell et al. 2009b).
The primary organism used was Meruliporia incrass-
ata (MFStoner-1). Gloeophyllum trabum (ATCC
11539) and Irpex lacteus (ATCC 60993) were also
used. Decay tests were performed using a modified
AWPA soil block jar method (AWPA 2003) accord-
ing to previously described procedures (Howell et al.
2007, 2009b), using oriented strand board blocks
without adhesive. For decay tests performed on
extracted material, strands with severity level d were
used. There were five replicates per treatment per
time point, as well as five uninoculated controls.
Control values for decay test were averaged across
the entire experiment.
XRD
Wood wafers were prepared and scanned using a
panalytical X-ray diffraction machine (Panalytical,
Netherlands) with symmetric h-2h Bragg–Brentano
scattering geometry as previously described (Howell
et al. 2009b). Due to the variability of published
procedures on the analysis of XRD spectra from wood
(Park et al. 2010), the spectra from these experiments
were processed and analyzed using two different
methods: a standard least-squares peak fitting method
with an amorphous standard (Andersson et al. 2003;
Thygesen et al. 2005), and a Rietveld analysis (Riet-
veld 1969) using the cellulose Ib crystal structure
published by Nishiyama et al. (2002). No significant
differences were found for percent crystallinity calcu-
lated by the two methods. Average distance between
the crystal planes was calculated using Bragg’s law:
2d sin h ¼ nk ð1Þ
where d represents the distance between the crystal
planes, h the angle between the planes and the
incoming X-rays, k the wavelength of the X-rays and
n an integer. The obtained value for the d-spacing
was then multiplied by two to take into account the
body-centered crystal arrangement and give a value
reflecting the distance between b-D-glucan molecules
in a single unit cell.
13C CP/MAS NMR
The 13C cross-polarization (CP) magic-angle-spin-
ning (MAS) NMR spectra of the dried, ground wood
powder (420 lm) were recorded on a Bruker Avance
400 (9.4 T) spectrometer (Bruker Biospin Gmbh,
Rheinstetten, Germany), operating at Larmor fre-
quencies of 400.13 and 100.62 MHz for 1H and 13C,
respectively. The experiments were carried out using
a double-tuned (CP/MAS) probe equipped with a
4 mm (o.d.) rotor. 1H and 13C rf-field strengths of
80 kHz were used during both TPPM-1H-decoupling
(Bennett et al. 1995) and cross-polarization. The
variable amplitude CP scheme (Peersen et al. 1993)
was employed to enhance the CP performance during
fast spinning. All spectra were acquired at room
temperature using a spin-rate of 8 kHz, a contact time
of 1.0 ms, an acquisition time of 37.3 ms, a recycle
delay of 3 s and 1,000 scans. Prior to Fourier
transformation the free induction decays (FID) were
apodized by a Lorentzian line broadening of 10 Hz.
All spectra were referenced (externally) to the
carbonyl resonance in a-glycine at 176.5 ppm. Con-
trol samples, extracted samples (severity d) and
samples decayed by fungi for 9 weeks (both extracted
and non-extracted) were analyzed in triplicate.
Cellulose (2011) 18:1179–1190 1181
123
Selected regions of the 13C CP/MAS spectra were
examined by Principal Component Analysis (PCA)
(Wold et al. 1987) using the built-in PCA procedure
in PLStoolbox 5.5 in Matlab 7.9.0.529. The data were
mean centered prior to the PCA.
Statistical analysis
Statistical analyses of percent crystallinity and d-
spacing values were performed employing either one-
way ANOVAs and protected Fisher LSD post-hoc
tests using SySTAT v.12 (Systat Software Inc., San
Jose, California, USA).
Results and discussion
Hot-water hemicellulose extraction
Table 1 shows the wood sugar analysis values
obtained for the control and extracted material. The
amounts of the main hemicellulose sugars (xylan,
mannan, arabinan, and galactan) decrease as the
extraction intensity increases, while the amount of
lignin stays largely the same, with only a 12%
removal at the highest severity (d). Over 50% of
xylan, which is the major hemicellulose in Red
Maple, is removed at severity d. The amount of
glucan, the basic unit of cellulose, remains nearly
constant at all extractions conditions.
X-ray diffraction (XRD) provides information on
crystal structure based on the creation of an interfer-
ence pattern by X-rays when they encounter the
regularly-spaced crystal matrix. Cellulose crystallin-
ity (%) and d-spacing values calculated from the
major peak located at approximately 22� 2h, corre-
sponding to the (200) crystal plane oriented perpen-
dicular to the fiber axis, are shown in Fig. 1.
The percent crystallinity of the extracted samples
was found to increase with increasing severity of
extraction, leveling out before the most severe
treatment (Fig. 1b). This pattern has previously been
observed in other studies using similar samples
(Howell et al. 2009a; Paredes et al. 2008, 2009),
and was attributed mainly to the removal of hemi-
celluloses and other non-crystalline matter during the
extraction process, as shown in Table 1. However, it
is also known that adjacent crystalline domains can
join together (co-crystallize) when certain conditions,
such as the removal of the non-crystalline material
that separates them, are met. This phenomenon has
been documented during Kraft pulping (Newman
2004), during hemicellulose removal using NaOH
(Wan et al. 2010), and during steaming (Inagaki et al.
2010). Other studies have suggested that heating can
increase absolute crystallinity via a crystallization of
initially semi-crystalline cellulose, especially under
moist conditions (Bhuiyan et al. 2000). Both of these
processes may also play a role in the observed
increase in crystallinity in the extracted samples
examined in this work.
Upon extraction, the d-spacing between the (200)
planes decreased from 0.799 (±0.002) to 0.792
(±0.001) nm at severity d (Fig. 1e), becoming
statistically significant at severity c (P = 0.046).
As was observed for the percent crystallinity in
these samples, the decrease in d-spacing appeared to
reach a maximum before the highest severity of
extraction. A change in d-spacing, as detected by
XRD, can be caused either by: (1) an actual
reduction of the spacing between the crystal planes
due to compression of the crystal, (2) a relaxation of
the crystal into a more energetically favorable
compact state, or (3) removal of non-crystalline or
paracrystalline material, which distorts or strains the
crystal structure. In the extracted samples, it may be
that the decrease is due to some combination of the
three. Extraction removes the hemicelluloses, which
are known to be in close association with cellulose
(Salmen 2004; Neagu et al. 2006) and potentially
cause strain on the crystals by disrupting their
ability to form a regular structure (O’Sullivan 1997).
Furthermore, the heat and water involved in the
extraction process would likely introduce both
energy and flexibility into the cellulose chains,
which could permit a rearrangement into a more
energetically favorable Ib dominant state (Hult et al.
2003). It should also be noted that while a
significant shift was only clear for the 200-plane,
it is also possible that changes were occurring in the
(110)- and (1�10)- planes located at around 16� 2h as
well. However, in XRD spectra from wood using a
non-synchrotron X-ray source, these peaks overlap
to a large degree with the contribution from non-
crystalline material, making it impossible to distin-
guish them clearly (Hill et al. 2010).
1182 Cellulose (2011) 18:1179–1190
123
13C CP/MAS NMR experiments were used to
examine the differences in the wood composition of
these samples. This experimental approach has
previously proven invaluable in elucidating different
crystalline forms of cellulose present in native
cellulose (Atalla and Vanderhart 1984; Sugiyama
Table 1 Amount of cellulose, lignin, and hemicellulose components remaining in the solid wood material after the hot water
extraction procedure
Cellulose Lignin Hemicelluloses
Glucan Phenols Mannan Xylan Arabinan Galactan
Control 455 242.9 30.4 183 6.3 6.1
Extracted
a 466 236.2 30.2 192 4.2 5.0
b 474 241.1 28.3 191 3.2 4.7
c 505 250.0 19.4 166 2.7 4.6
d 552 258.5 20.9 107 1.4 2.8
All values are given as mg/g extracted material
Fig. 1 Plots of cellulose crystal parameters as determined
from XRD data for wood decayed by the brown-rot fungus
M. incrassata (triangles in a, d), hot-water extracted wood
(squares in b, e), wood that had been first extracted, then
decayed (circles in c, f), and untreated (non-extracted) controls
(diamonds in all). Percent crystallinity for decayed wood (a),
extracted wood (b) and extracted-decayed wood (c) versus
controls. Average transverse (200) d-spacing of the crystalline
cellulose planes for decayed (d), extracted (e), and extracted-
decayed wood (f) versus controls. The horizontal grey line with
squares in (c) and (f) represent the average values for the
extracted controls (severity d) used in decay experiments
conducted on extracted blocks. It should be noted that the a, b,
c, and d labels for the extracted materials do not indicate a
linear relationship between these points and that the trend line
between them is only to guide the eye as to the general pattern,
not to indicate a linear relationship
Cellulose (2011) 18:1179–1190 1183
123
et al. 1991; Hult et al. 2003), as well as assessing the
structural changes due to treatments such as Kraft
pulping and thermal modification (Newman 2004;
Wikberg and Maunu 2004).
A PCA of the 13C CP/MAS spectra using the
spectral range 0–200 ppm is presented in Fig. 2. PC1
captures 83.7% of the variation and is mainly due to
cellulose as seen from loading one in Fig. 2b when
compared to spectra of pure cellulose (Kolodziejski
et al. 1982; Atalla and Vanderhart 1984). PC2 captures
9.2% of the variation and contains information about
lignin and hemicelluloses. Comparson of this spectrum
to spectra of pure lignin and pure hemicelluloses
(Kolodziejski et al. 1982; Bardet et al. 2009) shows that
the loading for PC2 is similar to a difference spectrum
between lignin and hemicelluloses. The most intense
characteristic resonances for lignin are located at 147.5
and 55 ppm. The former originate from aromatic
carbons in non-esterified syringyl (S3 and S5) and
guaiacyl (G1 and G4) whereas the latter originate
from the methoxy groups. For Red Maple, the hemi-
celluloses primarily consist of O-acetyl-4-O-methyl-
glucorono-xylan (Timell 1967). The characteristic
resonances from the hemicelluloses originate therefore
from the carboxylic acid group (*172 ppm) and the
acetyl group (*21.2 ppm). It is observed that the
resonances from the lignins have positive intensity in
the loading for PC2 whereas the intensity for the
hemicelluloses is negative. The dispersive peaks in
loading two in the area of 60–110 ppm are due to
overlapping resonances from lignin and hemicellu-
loses with positive and negative intensity, respectively.
From the score plot (Fig. 2a) it can be observed that the
extracted samples (labeled E) have a significantly
higher lignin content and lower hemicellulose content
compared to the non-extracted (labeled N) samples.
This is in agreement with Table 1, which shows that
the extraction process is removing mostly hemicellu-
loses and small amounts of lignin, leaving the majority
of the cellulose behind.
Generally, the most informative regions in terms of
the cellulose crystallinity in NMR spectra of wood are
those associated with C-4 and C-6 of cellulose (Fig. 3)
(Vanderhart and Atalla 1984). Both of these regions
can be divided into interior cellulose chains of the
microfibril (86–92 ppm and 64–68 ppm for C-4 and
C-6, respectively), which are expected to be primarily
crystalline, and surface chains (80–86 ppm and
Fig. 2 The PCA score
(upper) and scree (lower)
plots (a) and loadings
(b) for the 13C CP/MAS
spectra (0–200 ppm) for
extracted (labeled E) and
non-extracted (labeled N)
wood either undecayed
(controls, triangles) or
decayed by M. incrassata(stars), G. trabeum(circles), or I. lacteus(squares) after 9 weeks
1184 Cellulose (2011) 18:1179–1190
123
60–64 ppm for C-4 and C-6, respectively), which
contain more para- or non-crystalline structures
(Newman 2004). The loading for PC1 in the PCA
(Fig. 3b) captures 78% of the variation and is
attributed to the total amount of cellulose present in
the sample, with a positive score being equivalent to a
high content. There is no detectable separation of the
extracted and non-extracted samples along this axis,
indicating that no significant amounts of cellulose are
being removed during the hot-water extraction pro-
cess, in agreement with the wood sugar analysis
(Table 1) and previous studies (Paredes et al. 2008).
Furthermore, the lack of change between the extracted
and unextracted decayed samples along this axis
suggests that the effects caused by the extraction
method are not interfering significantly with the
degradation, also in agreement with previous findings
(Howell et al. 2009a). Decay by I. lacteus causes a
slightly greater reduction in crystallinity in the non-
extracted compared to the extracted samples, which
correlated with higher weight loss (21.4% ± 1.4% vs.
15.2% ± 8.1% for the non-extracted and extracted
samples, respectively). The loading for PC2 (captur-
ing 16.3% of the variation) is primarily due to the
change within the crystalline cellulose. A positive
score on PC2 is equivalent to a higher number of
interior crystalline chains than the mean (greater peak
intensity at 86–92 ppm and 64–68 ppm for C-4 and
C-6, respectively), whereas a negative score indicates
higher content of para- and non-crystalline material
(peaks at 80–86 ppm and 60–64 ppm for C-4 and C-6,
respectively). The extracted and non-extracted sam-
ples are separated due to the scores on PC2, indicating
that the amount of interior chain material is increasing
upon extraction. This result is consistent with the
concept of crystalline domains coming together in a
co-crystallization process upon removal of the hemi-
celluloses, in agreement with the XRD data.
The possibility of gathering information on the
presence of the two cellulose I allomorphs, Ia and Ib,
by examining the splitting of the peaks attributed to
the crystalline region of C-1, C-4 and C-6 has been
proposed (Sugiyama et al. 1991; VanderHart and
Atalla 1984). However, distinguishing these peaks in
wood is often difficult, if not impossible, as the non-
crystalline cellulose and hemicelluloses are present in
sufficient quantities to obscure the smaller Ia and Ibpeaks (Hult et al. 2003; Newman 2004).
Fungal decay
XRD data revealed a constantly decreasing crystal-
linity in the wood decayed by the brown-rot fungus
M. incrassata (Fig 1a). This decrease is expected, as
Fig. 3 The PCA score
(upper) and scree (lower)
plots of the 13C CP/MAS
spectra for the two spectral
regions representing C-4
(80–92 ppm) and C-6
(60–68 ppm) (a) with
corresponding loadings for
the selected principal
components (b). The ‘N’
and ‘E’ in the score plotindicate non-extracted and
extracted samples,
respectively, while the
markers represent either
undecayed samples
(triangles), or samples
decayed for 9 weeks by the
two brown-rot species
M. incrassata (stars) and
G. trabeum (circles), and
the white-rot species,
I. lacteus (squares)
Cellulose (2011) 18:1179–1190 1185
123
the fungus is in the process of depolymerizing the
wood components in order to absorb and metabolize
the sugars. This decrease has been observed in
multiple studies using XRD and other techniques
(Goodell et al. 1997; Howell et al. 2009a; Jellison
et al. 1991; Kleman-Leyer et al. 1992). A previous
study conducted with the same organism grown on
soft wood reported a brief initial increase in crystal-
linity around 3% weight loss (Howell et al. 2007).
This was not observed in this study, possibly due to
the relatively advanced state of decay at the first
sampling point at week 3 (Table 2).
The wood decayed by M. incrassata showed the
same change in d-spacing observed in the extracted
wood, decreasing from 0.799 nm (±0.002) to
0.793 nm (±0.008) nm after six weeks of decay
(P = 0.026). This decrease then remained stable
through week 12 (Fig. 1d). Similar observations have
been made in softwood decayed by three different
species of brown-rot fungi (Howell et al. 2009a). In
that work, this phenomenon was hypothesized to be
caused by removal of hemicelluloses and non-crys-
talline cellulose more easily accessible by fungal
degradative compounds, leaving behind the more
tightly packed crystalline cellulose. This theory was
supported by the observation that the decrease
appeared to occur consistently around 20% weight
loss, the point in decay at which nearly all of the
hemicelluloses are removed (Curling et al. 2001).
The results presented in Fig. 1d support that theory,
as the decrease in d-spacing in the decayed samples
becomes significantly different from the controls in
the range between 12.8 and 33.8% weight loss
(Table 2). It is interesting to note that the patterns
of crystallinity change observed in these experiments
for M. incrassata decaying hardwood (Red Maple)
are similar to what was observed for the same and
other species of brown rot fungi (S. lacrymans,
C. puteana, and G. trabeum) decaying softwood
(Pine) (Howell et al. 2009a), while the pattern for
M. incrassata growing on softwood are significantly
faster (Howell et al. 2007). This may be due to the
aggressiveness of this organism.
The PCA score plot of the 13C CP/MAS spectra
yielded a clear separation of the samples decayed by
M. incrassata from their respective controls along
PC1, indicating that the total cellulose content in
these samples decreased after 9 weeks of decay
(Figs. 2, 3). This is consistent with the concept of the
breakdown and digestion of non-, para- and crystal-
line cellulose as decay progresses (Kleman-Leyer
et al. 1992; Curling et al. 2001; Howell et al. 2009a).
Combined hot-water extraction and decay
In order to more thoroughly investigate these changes
in the cellulose crystallinity, in particular the
d-spacing, we performed experiments in which wood
samples were first extracted, then decayed. For
extracted wood degraded by M. incrassata, the
crystallinity decreased rapidly, reaching a final level
similar to that of the untreated decayed wood
(Fig. 1c). This process may have been aided by the
hot-water extraction as this treatment is known to
increase the porosity of the wood (Paredes et al.
2009), thus potentially improving the accessibility of
the cellulose fibrils to the fungal enzymes.
The d-spacing in the extracted-decayed samples
showed a pattern similar to the non-extracted decayed
samples, decreasing to a minimum level by week 6
and remaining static thereafter (Fig. 1f). However,
this minimum (&0.789 nm) was significantly lower
than the value achieved as a result of either extraction
or fungal decay alone (P = 0.038). It should also be
noted that there is a slight difference between the
d-spacing of extraction severity d and the starting
Table 2 Percent weight loss for samples decayed by M. incrassata, extracted, and extracted then decayed by M. incrassata
Decayed Extracted Extracted and decayed
Weeks of decay Weight loss (%) Severity of extraction Weight loss (%) Weeks of decay Weight loss (%)
3 12.8 (4.4) a 3.4 (0.5) 3 8.2 (6.4)
6 33.8 (5.9) b 6.2 (2.0) 6 43.3 (1.8)
9 37.7 (3.9) c 9.9 (1.2) 9 45.5 (10.1)
12 37.5 (7.2) d 17.2 (0.8) 12 46.3 (12.4)
Standard deviations are given in parentheses
1186 Cellulose (2011) 18:1179–1190
123
point of the extracted controls in the extracted and
decayed samples. This discrepancy is most likely due
to the fact that the controls of the extracted and
decayed samples were treated exactly as the decayed
samples in this set, i.e. they were subjected to moist
soil block jars conditions for up to 12 weeks before
being re-dried at 95� for 48 h. Hill et al. (2010)
examined changes in d-spacing upon wetting and
drying of wood samples, and found that although the
changes were very small, there was a slight increase
in the spacing of the (200) plane as the sample was
wet and re-dried. This distance change for a complete
unit square (twice the value published in that article)
works out to be about 0.004 nm, about the amount of
the discrepancy that was observed in this work.
For the non-extracted decayed samples, it was
previously hypothesized that the change in d-spacing
was due to the removal of hemicelluloses by the fungi
(Howell et al. 2009a). In the extracted-decayed
samples, however, the majority of the hemicelluloses
were removed prior to decay (weight loss = 17.2
(±0.8) %). Nevertheless, the same relative decrease
in d-spacing occurs in these samples as during the
initial stages of the decay, indicating that removal of
the hemicelluloses by brown-rot decay may not, in
fact, be the only factor contributing to this change in
d-spacing.
In order to determine whether or not the observed
changes in crystallinity and d-spacing in the
extracted-decayed samples were due to decay by
M. incrassata or were a property of fungal decay in
general, we performed similar decay tests using a
second brown-rot species, Gloeophyllum trabeum,
and a white-rot species, Irpex lacteus (Fig. 4). Non-
extracted, decayed samples were also tested with
results similar to those obtained for extracted decayed
samples; however, for clarity of presentation only the
results from the extracted samples are shown here. As
shown in Fig. 4, both G. trabeum and I. lacteus
showed little change in the crystallinity, with only
I. lacteus becoming significant after 12 weeks of
decay (P = 0.020). This lack of change in percent
crystallinity may be in part due to the low weight
losses obtained in the wood blocks inoculated with
these fungi: 27.4% (±1.2) and 24.7% (±1.4) for
G. trabeum and I. lacteus at 12 weeks, respectively
(Table 3), compared to 46.3% for M. incrassata
(Table 2). However, decay mechanisms characteristic
for each fungal species may also be playing a role.
From the PCA score plot (Fig. 3) it can be
observed that both species of brown-rot fungi cause
a reduction in the total amount of cellulose compared
to the control samples in both extracted and non-
extracted wood. The amount of cellulose in the
extracted samples decayed by the white-rot fungus
I. lacteus, however, remains statistically unaltered.
This may be a result of the low weight loss at week 9
(Table 3), but is more likely due to the nature of
decay employed by I. lacteus, in which all of the
major wood components are simultaneously broken
down and digested. Non-extracted Red Maple wood
has been shown to be more susceptible to white-rot
fungal growth, resulting in higher weight losses
(Howell et al. 2009b), which is consistent with the
greater decrease in cellulose content observed in this
work (Fig. 3).
In the samples decayed by G. trabeum a decrease
in d-spacing (Fig. 4c), similar to what was observed
for M. incrassata, is evident (Fig. 1d). Samples
decayed by the white-rot I. lacteus, however, showed
no change (Fig. 4d), suggesting that among the
limited species tested here this phenomenon is
characteristic of brown-rot decay. It is likely that
the reduction in d-spacing is partly facilitated by
hydroxyl radicals generated by the Fenton reaction,
which are known to cause a drastic decrease in the
degree of polymerization (Hastrup et al. 2011). This
may well cause a release of some of the strain that
was originally in place in the cellulose chains,
resulting in imperfections in the crystalline structure.
This new freedom may allow the chains to rearrange
into a more energetically favorable, tightly packed
crystalline structure before being degraded by the
organism. Nevertheless, it is interesting to note that
the observed decrease in d-spacing appears to be
largely independent of what is going on with the
overall degree of crystallinity, as this phenomenon
can occur whether the crystallinity is increasing,
decreasing, or remaining static.
It is also possible that the changes observed in
these samples, as well as in the extracted samples, are
a result of changes in moisture content. It has been
previously shown that brown-rot decay can increase
the moisture content of the wood more than white rot
decay (Williams and Hale 2003). It is possible that
altered moisture contents are also contributing to
these results; however, no correlation was observed
between moisture content and changes in either
Cellulose (2011) 18:1179–1190 1187
123
percent crystallinity or d-spacing in these samples. In
a study using XRD to examine changes in cellulose
crystalline lattice structure at different moisture
contents, Abe and Yamamoto (2005) demonstrated
that the (200) peak from wood powder could be
shifted to a higher 2h value (and thus a lower
d-spacing) with an increase in moisture content. It
was hypothesized that this was due to a compression
of the cellulose structure by the swelling of the
surrounding hemicelluloses, as the crystalline cellu-
lose structure itself was too tightly packed to be
penetrated by water molecules. However, if this were
the main contributing factor to these results, then it
would be expected that the decrease in d-spacing
should be less for the samples which are first
extracted, then decayed, as these samples have
significantly fewer hemicelluloses to swell (Table 1).
Conclusions
Changes in crystalline cellulose structures in wood
undergoing hot-water extraction and brown-rot decay
were examined using a combination of XRD and 13C
CP/MAS NMR. Hot-water extraction increased the
crystallinity of the samples, primarily as a conse-
quence of hemicellulose removal but also because of
a co-crystallization of adjacent crystalline domains.
Fig. 4 Percent crystallinity
for extracted wood
undergoing decay by the
brown-rot fungus
G. trabeum (a) and the
white-rot fungus I. lacteus(b) versus extracted
controls (dark horizontalline with squares). Average
(200) plane d-spacing
values for the same samples
(c, d)
Table 3 Percent weight loss values for extracted blocks
decayed by the brown-rot fungus G. trabeum and the white-rot
fungus I. lacteus
Extracted and decayed:
G. TrabeumExtracted and decayed:
I. lacteus
Weeks of
decay
Weight loss
(%)
Weeks of
decay
Weight loss
(%)
3 9.7 (0.9) 3 7.0 (2.9)
6 17.1 (1.3) 6 11.3 (1.4)
9 22.5 (1.6) 9 15.2 (8.1)
12 27.4 (1.2) 12 24.7 (1.4)
Standard deviation values are given in parentheses
1188 Cellulose (2011) 18:1179–1190
123
Brown-rot decay by M. incrassata caused decreased
crystallinity associated with the breakdown of the
crystalline cellulose over time.
Both hot-water extraction and brown-rot decay
were found to decrease the distance between the
crystalline planes in the transverse (200) direction.
However, when samples were first treated with the hot-
water extraction procedure and then decayed by fungi,
the d-spacing was found to decrease even further.
A decrease in d-spacing was also found in wood
degraded by a second brown-rot species, G. trabeum,
but not by the white-rot fungus I. lacteus, despite
similar weight losses. This suggested that the decrease
d-spacing is unique to brown-rot decay.
This observation could be due to the production of
reactive oxygen species early in the decay process,
which are known to significantly depolymerize
cellulose, and may allow the remaining cellulose
chain fragments additional freedom of movement to
rearrange into a more energetically favorable state.
Acknowledgments The authors thank J. J. Paredes for
providing extracted material, J. Perkins for technical support,
Annelise Kjøller, PhD, for technical editing, and Dr. D. Frankel
of LASST at the University of Maine for XRD assistance. CH
acknowledges support from a US NSF Graduate Research
Fellowship. ACSH acknowledges support from the University
of Copenhagen PhD Scholarship.
References
Abe K, Yamamoto H (2005) Mechanical interaction between
cellulose microfibril and matrix substance in wood cell wall
determined by X-ray diffraction. J Wood Sci 51:334–338
American Wood Preserver’s Association (2003) Standard
method of testing wood preservatives by laboratory soil-
block cultures. In: Book of standards, American Wood
Preserver’s Association, Granbury, TX, pp. 206–212
Andersson S, Serimaa R, Paakkari T, Saranpaa P, Pesonen E
(2003) Crystallinity of wood and the size of cellulose
crystallites in Norway spruce (Picea abies). J Wood Sci
49:531–537
Arantes V, Qian Y, Milagres A, Jellison J, Goodell B (2009)
Effect of pH and oxalic acid on the reduction of Fe3? by a
biomimetic chelator on Fe3? desorption/adsorption onto
wood: implications for brown-rot decay. Int Bioterior
Biodegrad 63:478–483
Arantes V, Milagres A, Filey T, Goodell B (2010) Lignocel-
lulosic polysaccharides and lignin degradation by wood
decay fungi: the relevance of nonenyzmatic Fenton-based
reactions. J Ind Microbiol Biotechnol 38:541–555
Atalla RH, VanderHart DL (1984) Native cellulose: a com-
posite of two distinct crystalline forms. Science
223:283–285
Bardet M, Gerbaud G, Giffard M, Doan C, Hediger S, Le Pape
L (2009) 13C high-resolution solid-state NMR for struc-
tural elucidation of archaeological woods. Prog Nucl
Magn Reson 55:199–214
Bennett AE, Rienstra CM, Auger M, Lakshmi KV, Griffin RG
(1995) Heteronuclear decoupling in rotating solids.
J Chem Phys 103:6951–6958
Bhuiyan TR, Hirai N, Sobue N (2000) Changes of crystallinity
in wood cellulose by heat treatment under dried and moist
conditions. J Wood Sci 46:431–436
Curling S, Clausen C, Winandy J (2001) The effect of hemi-
cellulose degradation on the mechanical properties of
wood during brown rot decay. Int Res Group Wood Pres
IRG/WP 01-20219
Daniel G (2003) Microview of wood under degradation by
bacteria and fungi. In: Goodell B, Nicholas D, Schultz T
(eds) Wood deterioration and preservation: advances in
our changing world. American Chemical Society Pub-
lishing, Washington, DC, pp 35–72
Davis M (1998) A rapid modified method compositional car-
bohydrate analysis of lignocellulosics by high pH anion
exchange chromatography with pulsed amperometric
detection (HPAEC/PAD). J Wood Chem Technol 18(2):
235–252
Ding S, Himmel M (2006) The maize primary cell wall
microfibril: a new model derived from direct visualiza-
tion. J Ag Food Chem 54:597–606
Goodell B, Jellison J, Liu J, Daniel G, Paszczynski A, Fekete F,
Krishnamurthy S, Jun L, Xu G (1997) Low molecular
weight chelators and phenolic compounds isolated from
wood decay fungi and their role in the fungal biodegra-
dation of wood. Invited paper for special issue on pulp and
paper biotechnology. J Biotechnol 53:133–162
Hastrup ACS, Howell C, Jensen B, Green F III (2011) Non-
enzymatic depolymerization of cotton cellulose by fungal
mimicking metabolites. Int Biodeterior Biodegrad 65:
553–559
Highley T, Dashek W (1998) Biotechnology in the study of
brown- and white-rot decay. In: Bruce A, Palfreyman J
(eds) Forest products biotechnology. Taylor and Francis
Publishing, London, pp 15–36
Hill SJ, Kirby NM, Mudie ST, Hawley AM, Ingham B, Franich
RA, Newman RH (2010) Effect of drying and rewetting of
wood cellulose on molecular packing. Holzforshung
64:421–427
Howell C, Hastrup ACS, Jellison J (2007) The use of X-ray
diffraction for analyzing biomodification of crystalline
cellulose by wood decay fungi. Int Res Group Wood Pres
IRG/WP 07-10622
Howell C, Hastrup ACS, Goodell B, Jellison J (2009a) Tem-
poral changes in wood crystalline cellulose during deg-
radation by brown rot fungi. Int Biodeterior Biodegrad
63:414–419
Howell C, Paredes J, Jellison J (2009b) Decay resistance
properties of hot water extracted oriented strandboard.
Wood Fiber Sci 41:201–208
Hult E, Iverson T, Sugiyama J (2003) Characterization of the
supermolecular structure of cellulose in wood pulp fibres.
Cellulose 10:103–110
Inagaki T, Siesler HW, Mitsui K, Tsuchikawa S (2010) Dif-
ference of the crystal structure of cellulose in wood after
Cellulose (2011) 18:1179–1190 1189
123
hydrothermal and aging degradation: a NIR spectroscopy
and XRD study. Biomacromol 11:2300–2305
Jellison J, Chandhoke V, Goodell B, Fekete F (1991) The
action of siderophores isolate from Gloeophyllum trabe-
um on the structure and crystallinity of cellulose com-
pounds. Int Res Group Wood Pres IRG/WP 1479
Kim YS, Wi SG, Lee KH, Singh AP (2002) Cytochemical
localization of hydrogen peroxide production during wood
decay by brown-rot fungi Tyromyces palustris and Con-iophora puteana. Holzforschung 56:7–12
Kleman-Leyer K, Agosin E, Conner AH, Kirk TK (1992)
Changes in molecular size distribution of cellulose during
attack by white rot and brown rot fungi. Appl Environ
Microbiol 58:1266–1270
Kolodziejski W, Frye JS, Maciel GE (1982) Carbon-13 nuclear
magnetic resonance spectrometry with cross polarization
and magic-angle spinning for analysis of lodgepole pine
wood. Anal Chem 54:1419–1424
Neagu C, Gamstedt E, Kristofer B, Stig L, Lindstrom M (2006)
Ultrastructural features affecting mechanical properties of
wood fibres. Wood Mat Sci Eng 1:146–170
Newman RH (1999) Estimation of the lateral dimensions of
cellulose crystallites using 13C NMR signal strengths. Sol
State Nuc Magn Res 15:21–29
Newman RH (2004) Carbon-13 NMR evidence for cocrystal-
lization of cellulose as a mechanism for hornification of
bleached kraft pulp. Cellulose 11:45–52
Nishiyama Y, Langan P, Chanzy H (2002) Crystal structure
and hydrogen-bonding system in cellulose Ib from syn-
chrotron X-ray and neutron fiber diffraction. J Am Chem
Soc 124:9074–9082
O’Sullivan AC (1997) Cellulose: the structure slowly unravels.
Cellulose 4:173–207
Paredes J, Jara R, van Heiningen A, Shaler S (2008) Influence
of hot water extraction on the physical and mechanical
behavior of OSB. Forest Prod J 58(12):56–62
Paredes J, Mills R, Howell C, Shaler S, Gardner D, van
Heiningen A (2009) Surface characterization of Red
Maple strands after hot water extraction. Wood Fiber Sci
41:38–50
Park S, Baker JO, Himmel ME, Parilla PA, Johnson DK (2010)
Cellulose crystallinity index: measurement techniques and
their impact on interpreting cellulase performance. Bio-
technol Biofuels 3:1–10
Peersen OB, Wu XL, Kustanovich I, Smith SO (1993) Vari-
able-amplitude cross-polarization MAS NMR. J. Magn
Reson A 104:334–339
Rietveld HM (1969) A profile refinement method for nuclear
and magnetic structures. J Appl Crystallogr 2:65–71
Salmen L (2004) Micromechanical understanding of the cell-
wall structure. CR Biologies 327:873–880
Schilling JS, Tewalt JP, Duncan SM (2009) Synergy between
pretreatment lignocellulose modifications and saccharifi-
cation efficiency in two brown rot fungal systems. Appl
Microbiol Biotechnol 84:465–475
Sugiyama J, Persson J, Chanzy H (1991) Combined infrared
and electron diffraction study of the polymorphism of
native celluloses. Macromol 24:2461–2466
Thygesen A, Oddershede J, Lilholt H, Thomsen A, Stahl K
(2005) On the determination of crystallinity and cellulose
content in plant fibers. Cellulose 12:563–576
Timell TE (1967) Recent progress in the chemistry of wood
hemicelluloses. Wood Sci Technol 1:45–70
VanderHart D, Atalla R (1984) Studies of microstructure in
native celluloses using solid-state carbon-13 NMR. Mac-
romol 17:1465–1472
Wan J, Wang Y, Xiao Q (2010) Effects of hemicelluloses
removal on cellulose fiber structure and recycling char-
acteristics of eucalyptus pulp. Bioresource Technol
101:4577–4583
Wikberg H, Maunu SL (2004) Characterisation of thermally
modified hard- and softwoods by 13C CPMAS NMR.
Carbohydr Polym 58:461–466
Williams FC, Hale MD (2003) The resistance of wood chem-
ically modified with isocyanates: the role of moisture
content in decay suppression. Int Biodeterior Biodegrad
52:215–221
Wold S, Esbensen K, Geladi P (1987) Principal component
analysis. Chemometric Intell Lab Syst 2:37–52
Yamamoto H, Horii F (1994) In situ crystallization of bacterial
cellulose I. Influences of polymeric additives, stirring and
temperature on the formation celluloses Ia and Ib as
revealed by cross polarization/magic angle spinning (CP/
MAS) 13C NMR spectroscopy. Cellulose 1:57–66
Zabel RA, Morrell JJ (1992) Wood microbiology: decay and its
prevention. Academic Press Inc., San Diego, pp 21–194
1190 Cellulose (2011) 18:1179–1190
123
Top Related