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: Caitlin.Howell@kit.edu

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