Accepted Manuscript

Title: Effect of compression combined with steam treatment

on the porosity, chemical compositon and cellulose crystalline
structure of wood cell walls

Author: Jiangping Yin Tongqi Yuan Yun Lu Kunlin Song

Hanyin Li Guangjie Zhao Yafang Yin

PII: S0144-8617(16)30948-1
DOI: http://dx.doi.org/doi:10.1016/j.carbpol.2016.08.013
Reference: CARP 11438

To appear in:

Received date: 11-6-2016

Revised date: 3-8-2016
Accepted date: 4-8-2016

Please cite this article as: Yin, Jiangping., Yuan, Tongqi., Lu, Yun., Song,
Kunlin., Li, Hanyin., Zhao, Guangjie., & Yin, Yafang., Effect of compression
combined with steam treatment on the porosity, chemical compositon and
cellulose crystalline structure of wood cell walls.Carbohydrate Polymers
http://dx.doi.org/10.1016/j.carbpol.2016.08.013

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Effect of compression combined with steam treatment on the porosity,

chemical compositon and cellulose crystalline structure of wood cell

walls
Jiangping Yina,†, Tongqi Yuanb,†, Yun Lua, Kunlin Songc, Hanyin Lid, Guangjie Zhaod,
Yafang Yina,*
a
Department of Wood Anatomy and Utilization, Research Institute of Wood Industry, Chinese
Academy of Forestry, Beijing 100091, China
b
Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing 100083,
China
c
School of Renewable Natural Resources, Louisiana State University, Baton Rouge, LA 70803, USA
d
College of Materials Science and Technology, Beijing Forestry University, Beijing 100083, China
*
Corresponding authors: Yafang Yin, E-mail: yafang@caf.ac.cn; yinyafangcaf@gmail.com
Tel: +86 10 62889468; Fax: +86 10 62881937
†
These authors contributed equally.
Highlights

 CS-treatment of Spruce wood cell walls was studied on a macromolecular level.

 The porosity in CS-treated and steam-treated wood cell walls was investigated.
 Changes of chemical composition were caused by CS- and steam treatment.
 Cellulose crystalline structure changes occurred after CS- and steam-treatment.
Abstract: The changes of porosity, chemical composition and cellulose crystalline structure of
Spruce (Picea abies Karst.) wood cell walls due to compression combined with steam treatment
(CS-treatment) were investigated by nitrogen adsorption, confocal Raman microscopy (CRM) and
X-ray diffraction (XRD), respectively. A number of slit-shaped mesopores with a diameter of 3.7
nm was formed for the CS-treated wood, and more mesopores were found in the steam-treated
wood. CRM results revealed cellulose structure was affected by treatment and β-aryl-ether links
associated to guaiacyl units of lignin was depolymerized followed by re-condensation reactions.
The crystallinity index (CrI) and crystallite thickness (D200) of cellulose for CS-treated wood were
largely increased due to crystallization in the semicrystalline region. Higher degree of increase in
both CrI and D200 was observed in both the earlywood and latewood of steam-treated wood,
ascribing to the greater amount of mesopores in steam-treated wood than CS-treated wood.
Keywords: Compression combined with steam treatment, Mesopores, Cellulose, Lignin,
Cellulose crystalline structure
1. Introduction
Wood is the most abundant natural and renewable materials on earth, and has a long history
to be widely used for construction, furniture and packaging industry. However, the utilization of
wood, especially the plantation wood, is restricted by its inherent limitations in its properties, such
as poor dimensional stability and durability, low density and strength (Wikberg & Maunu, 2004;
Drafz, Franz, Namyslo, & Kaufmann, 2015; Wei, Stark, & McDonald, 2015). Considerable wood
modification technologies were attempted in order to improve the performances of wood based
materials. Great efforts are devoted to develop more environmentally benign and efficient
modification methods (Fang, Mariotti, Cloutier, Koubaa, & Blanchet, 2012). Among them,
compression combined with steam treatment (CS-treatment, or thermo-hydro-mechanical
treatment) was able to improve the dimensional stability, durability as well as the mechanical
strength of wood without deteriorating its advantages (Kutnar & Kamke, 2012). During
CS-treatment, high temperature steam with more effective heat transfer than conventional heat
treatment was applied to obtain a permanent fixation of compressive deformations. This can
overcome the problem of typical compression treated wood that the compressive deformation is
unstable and the structure can be easily recovered (completely/partially) after reheating and
water/moisture exposure (Dwianto, Morooka, & Norimoto, 2000; Kekkonen, Ylisassi, & Telki,
2014). Hence, CS-treatment could transform wood into a new material with desired stability
(Popescu, Lisa, Froidevaux, Navi, & Popescu, 2014).
Significant strides have been made in elucidating the changes in the macroscopic
characteristics e.g., color and shape, dimensional stability and mechanical properties of wood
materials after CS-treatment (Inoue, Norimoto, Tanahashi, & Rowell, 1993; Navi & Girardet,
2000; Lesar, Humar, Kamke, & Kutnar, 2013; Cai, Yang, Cai, & Sheldon, 2013). However, it was
unclear about the mechanism caused by steam degradation and the rearrangement of
bio-molecules under compressive condition. Changes in physical and chemical structure on a
molecular level have not been fully characterized and understood. It is therefore desirable to
monitor the decomposition of the polymer structure in the cell wall and gain knowledge of
property changes in CS-treated wood.
In addition, wood is a cellular biomaterial with complex multi-component structure. The cell
wall is primarily comprised of cellulose, hemicellulose and lignin. Cellulose molecules form
microfibrils aggregates that are embedded in a soft matrix of hemicellulose and lignin (Salmén &
Burgert, 2009). The properties of wood are highly dependent on the chemical compositions, the
orientation of cellulose microfibrils, and the molecular interactions of the cell wall polymer
assembly (Burgert, 2006; Wei & McDonald, 2016).
In our previous studies (Yin, Berglund, & Salmén, 2011; Guo, Song, Salmén, & Yin, 2015), it
was shown that CS-treatment and steam treatment led to the changes of chemical components,
especially hemicelluloses, in wood cell walls. Specifically, a progressive degradation of the
carbonyl groups in the glucuronic acid unit of xylan and the mannose units in the glucomannan
would occur. This resulted in the decrease of moisture absorbing sites and the linkages among the
polymers in the wood cell wall, which are wholly accounted for changes of the hygroscopicity and
indentation modulus of the wood cell wall. However, the degradation pathways of cellulose and
lignin have not been clarified. It is known that the origins of cellulose molecular chains, such as
the crystalline, semicrystalline and amorphous regions, have various effects on the characteristics
of wood (Inagaki, Siesler, Mitsui, & Tsuchikawa, 2010). In addition, the crystalline regions of
cellulose exhibit higher density and lower chemical reactivity, and can determine the mechanical
properties of plant cell wall (Wei, McDonald, & Stark, 2015; Wei & McDonald, 2016). Moreover,
each glucopyranose ring of cellulose has three hydroxyl groups on average, implying cellulose is
highly hydrophilic material (Kulasinski, Guyer, Keten, Derome, & Carmeliet, 2015; Wei, Liang, &
McDonald, 2015). Lignin is biosynthesized from three basic monomeric units, syringyl, guaiacyl,
and p-hydroxyphenyl alcohol. Lignin is a highly amorphous macromolecule that plays an
important role of supporting material in wood cell wall (Song, Yin, Salmén, Xiao, & Jiang, 2014).
Therefore, gaining more knowledge about the structure of cellulose and lignin should provide
insight into the mechanism of cell wall changes responding to CS-treatment. Furthermore, wood
cell walls exhibit large mesopores because the spaces between the cellulose microfibrils could be
partially filled by lignin, hemicellulose and extractive (Yin, Song, Lu, Zhao, & Yin, 2015). The
structure of mesopores in wood cell wall could affect the sorption and transport of moisture. Any
changes in the mesopore structure due to CS-treated modification could determine the properties
of modified wood.
In this paper, the effects of CS-treatment on porosity, chemical composition and cellulose
crystalline structure on a molecular level were studied in details. Nitrogen adsorption was studied
for the mesopore structure upon treatments. Confocal Raman microscopy (CRM) was used to
investigate the chemical composition distribution in wood cell wall including the secondary cell
walls (S) and cell corners (CC). X-ray diffraction (XRD) was used to characterize the cellulose
crystalline structure. Differences between the earlywod and latewood were explored as well. The
results would promote the understanding of the effect and mechanism of degradation and
rearrangement of chemical composition, especially cellulose, during CS-treatment.

2. Experimental
2.1. Materials

Small specimens (dimensions were 20 × 20 × 25 mm in the tangential (T), radial (R), and
longitudinal (L) directions, respectively) cut from Spruce (Picea abies Karst.) sapwood section
were subjected to CS-treatment in a laboratory-scale autoclave as described in our previous
studies (Guo, Song, Salmén, & Yin, 2015). To be more specific, the CS-treatment was conducted
with a compression ratio of 50% in radial direction (the percentage of the decrease in thickness to
the initial thickness of the specimen) at 110°C for 6 min, followed by a steaming process at 160°C
for 30 min. For comparison, specimens were treated with steam only without using compression
treatment in the autoclave at 160°C for 30 min. All the treated samples were put into a preheated
autoclave, with pressurized steam being applied to regulate the corresponding prescribed
temperature. The treated samples were cooled down to room temperature inside the autoclave and
then conditioned to an equilibrium moisture content close to 12% by storing them in a
conditioning room maintained at 20°C and 65% relative humidity for > 20 days.
Small wood pieces containing whole growth rings with an age of 30 years were cut from the
surface of the treated samples and then seperated into five samples, see Fig. S1 (Supplementary).
Sample 1 (S1) was divided into earlywood and latewood with aid of a stereo-microscope (S6E,
Leica, Germany) and then cut into wood sticks (5 mm in longitudinal direction, 1 × 1 mm2 in
cross section) for nitrogen adsorption measurement. Sample 2 (S2) was sliced in radial directions
(200 μm thickness) for examination with a field emission scanning electron microscope (FE-SEM).
Sample 3 (S3) was sliced in radial directions (10 μm thickness) for atomic force microscope
(AFM). Sample 4 (S4) was sliced in cross sections (10 μm thickness) for CRM analysis.
Earlywood and latewood of Sample 5 (S5) were isolated in the same way as S1, and then were
milled into powder for X-ray diffraction (XRD) measurement.

2.2. Nitrogen adsorption

The samples were supercritically dried using a critical point drier (EM CPD300, Leica,
Germany). More specifically, wood sticks were firstly dehydrated using a series of ethanol with
different concentration, 80%, 90%, 95%, and 100%. The dehydrated samples were introduced into
the drier apparatus, which was filled with liquid carbon dioxide. During supercritical drying, the
liquid carbon dioxide was transformed into a supercritical fluid with a null surface tension. When
the temperature was increased beyond its critical point (304.25 K at 7.39 MPa), the valve of the
drier was gradually opened to allow gas to escape. In this way, shrinkage due to capillary pressure
was avoided.
Nitrogen adsorption test was carried out using a surface area and pore-size analyzer
(Autosorb iQ, Quantachrome, USA) at 77 K. Before the adsorption measurements, samples (1.0 g
~ 1.5 g) were degassed at 80°C for 10 h under a high vacuum (<10-5 Pa) to remove any moisture
or adsorbed contaminants from sample surfaces. By means of the nitrogen adsorption isotherms,
pore volume was derived from the amount of adsorbed nitrogen at a relative pressure close to
unity, assuming the pores were filled with liquid adsorbate. Shapes of pores were calculated using
the hysteresis loops. The total pore volume (Vtotal) was calculated from the volume of the liquid
nitrogen, maintained at the relative pressure of 0.99. The Brunauer-Emmett-Teller (BET) surface
area (SBET) of the samples was determined using an accelerated surface area (Brunauer, Emmett, &
Teller, 1938). The pore-size distribution was calculated using the Barrett-Joyner-Halenda (BJH)
method (Barrett, Joyner, & Halenda, 1951).

2.3. Field emission scanning electron microscope (FE-SEM)

The morphology of samples was examined by FE-SEM (JSM-6301F, JEOL, Japan) with a
secondary electron detector operating at 5 kV ~ 10 kV. Radial section of wood samples were cut
and supercritically dried. Surfaces were coated with a thin layer of platinum using a sputter coater
(JSM-5500LV).

2.4. Atomic force microscope (AFM)

AFM (Dimention Icon, Bruker, Germany) was used to characterize the surfaces of treated
samples with a NanoScope V controller. Radial sections of wood samples were cut and
supercritically dried. A standard 5 μm scanner was used with tapping mode etched silicon probes
(TESP, Veeco NanoProve, USA). In addition, a customized Nikon optical microscope with a deep
focus was used to aid the positioning of the AFM tip to the desired location on the cell wall. An
autotuning resonance frequency was in the range of 250 kHz ~ 300 kHz and a scan rate of 0.5 Hz
~ 2.0 Hz was used.

2.5. Confocal Raman microscopy (CRM)

For sample preparation, 10 μm thickness cross sections were cut from the samples using a
rotary microtome (RM 2255, Leica, Germany). The cross sections were placed on glass slides and
wetted with deuterium oxide. To avoid evaporation and drying out during measurements, the
sections were covered with glass coverslips (0.17 mm thickness) and sealed with nail polish, as
described in previous studies (Wang, Keplinger, Gierlinger, & Burgert, 2014; Li, Sun, Zhou, Peng,
& Sun, 2015).
The Raman spectra were acquired with a LabRam Xplora confocal Raman microscope
(Horiba JobinYvon, Longjumeau, France) equipped with a confocal microscope (BX51, Olympus,
Japan) and a motorized scan stage. To achieve high spatial resolution, an MPlan 100×oil
immersion microscope objective (numerical aperture (NA) = 1.4, Olympus) and a laser in the
visible wavelength range (λ = 532 nm) were used. The linearly polarized laser light was focused
with diffraction limited spot size of 0.61λ/NA onto samples and the Raman light was detected with
an air cooled back-illuminated CCD behind the spectrograph. The laser power on the samples was
~8 mW. Labspec 5 software was used for image processing and spectra analyses. The
wavenumber ranged from 3000 cm−1 to 1000 cm−1 with a confocal aperture of 100 μm and a slit
width of 100 μm. The reported depth resolution based on the silicon (standard) phonon band at
520 cm−1 was 2 μm. Chemical images were generated using a sumfilter by integrating over defined
wavenumber regions in the spectra. The filter calculated the intensities within the chosen borders
and the background was subtracted by taking the baseline from the first to second border. Samples
in Spruce earlywood and latewood regions were selected for Raman mapping at 0.7 μm steps and
an integration time of 2 s. The overview chemical images allowed to separate cell wall layers (S
and CC) with different chemical compositions, and to mark distinct cell wall regions for
constructing average spectra.
Lignin and carbohydrates can be imaged simultaneously by selecting the characteristic
Raman bands (Supplementary Table S1). Cellulose and hemicellulose are carbohydrate polymers
that possess similar chemical bonds, resulting in the signals of hemicelluloses would be obscured
by those of cellulose (Agarwal & Ralph, 1997). Therefore, only Raman images and spectra of
cellulose and lignin of earlywood and latewood in untreated, CS-treated and steam-treated wood
cell walls were analyzed in this study. Three vibration bands of cellulose, the C-O-C linkages
stretching at 1096 cm-1, the C-C and C-O stretching plus the HCC and HCO bending at 1149 cm-1,
the C-H and C-H2 stretching at 2897 cm-1, were monitored (Agarwal, 1999; Agarwal, Reiner, &
Ralph, 2010; Wang, Keplinger, Gierlinger, & Burgert, 2014; Li, Sun, Zhou, Peng, & Sun, 2015).
The characteristic band of lignin was the aromatic skeletal vibrations close to 1600 cm-1, which
was accompanied by a shoulder at 1650 cm-1 assigned to the conjugation to C=C stretch of conifer
alcohol and a C=O stretch of coniferaldehyde. Furthermore, the lignin signals of guaiacyl ring
showed at 1271 cm-1 and aliphatic O-H bending was at 1333 cm-1 (Agarwal & Atalla, 1986;
Agarwal, 1999; Agarwal, McSweeny, & Ralph, 2011; Gierlinger, Keplinger, & Harrington, 2012).

2.6. X-ray diffraction (XRD)

The XRD patterns of samples were measured using the XRD (D/max 2200, Rigaku, Japan),
with Ni-filtered CuKα radiation (λ = 0.154 nm) at 40 kV and 30 mA. Scattered radiation was
detected in the range of 2θ = 5 ~ 40° at a scan rate of 2°/min. The background was subtracted and
peaks were resolved using PeakFit○ R (Sea-Solve Software Inc., Richmond, CA). The crystallinity

index (CrI) was determined using the peak area method as follows (Hult, Iversen, & Sugiyama,
2003):
 A1  10  A110  A012  A200  A004
CrI  100% (1)
A110  A110  A012  A200  A004  Aam
where Ahkl represents the peak areas of crystallographic reflections in wood denoted by the Miller
incides (hkl). CrI was calculated from areas Ahkl of the reflections (1-10), (110), (012), (200) and
(004) to the total area of both the crystalline and amorphous contributions (Aam).
The crystal size dimension Dhkl was evaluated by Scherrer’s equation (Wei, McDonald, &
Stark, 2015):

K
Dhkl  (2)
1/2 cos 
where K is the Scherrer constant (K = 0.9), λ is the wavelength of the X-ray, β1/2 is the peak full
width at half of maxima intensity (fwhm = 2 deg.) obtained by IGOR Pro, when peak fitting was
conducted with Gaussian function, and θ is the diffraction angle.

2.7. Data statistical analysis

The Analysis of Variance (ANOVA) was done on the data using SAS program (SAS Institute
9.0, USA). Least-significant-difference tests were used to test the significant difference at 95%
confidence interval of probability. Average was calculated from three replicates.

3. Results and discussion

3.1. Nitrogen adsorption

The nitrogen adsorption test allows the measurement of pore size of materials smaller than 50
nm without changing the original features of the materials (Clair, Gril, Di Renzo, Yamamoto, &
Quignard, 2008; Chang, et al., 2009). The nitrogen adsorption-desorption isotherms of both
earlywood and latewood in the untreated, CS-treated and steam-treated wood cell walls are shown
in Fig. 1a and Fig. 1b. The isotherm of earlywood in the untreated wood was intermediate between
typeⅡ and type Ⅳ, according to the international union of pure and applied chemistry (IUPAC)
classification (Sing et al., 1985). It presented the hysteresis loop that was typical of mesopore
adsorbents. The shape of the hysteresis loop indicated that the mesopores belonged to type H3
with slit-shaped pores, which is consistent with previous findings (Clair, Gril, Di Renzo,
Yamamoto, & Quignard, 2008; Chang et al., 2009). Earlywood after treatment adsorbed a larger
amount of nitrogen, especially for the steam-treated wood. A similar trend was observed in the
latewood.
Examples of pore volume distributions against pore diameter for untreated, CS-treated and
steam-treated wood cell walls are plotted in Fig. 1c and Fig. 1d. All the samples presented broad
pore size distributions, and the peak between 3 nm and 4 nm was sharp. This indicated that many
slit-shaped mesopores with a diameter of 3.7 nm existed in the samples. However, the intensities
of peak in the curves were in the order of steam-treated > CS-treated > untreated wood. This
suggested that there was a greater amount of mesopores in the treated samples than the untreated
sample, as indicated by the SEM (Fig. 1e) and AFM (Fig. 1f) micrographs of the radial sections of
cell walls in the latewood of CS-treated wood. Moreover, the amount of mesopores in the
CS-treated samples was less than those in the steam-treated samples.
Fig. 1. Nitrogen adsorption-desorption isotherms of earlywood (a) and latewood (b), and the mesopore-size
distribution of the earlywood (c) and latewood (d) in untreated, CS-treated and steam-treated wood cell walls.
SEM (e) and AFM (f) images of mesopores in the latewood of CS-treated wood.

Total pore volumes (Vtotal) and BET specific surface areas (SBET) of wood cell walls were
changed significantly after both CS-treatment and steam treatment. The Vtotal of the earlywood and
latewood of treated wood were greater than those of untreated wood (Table 1). The Vtotal values of
the native earlywood and latewood were 0.0046 cm3/g and 0.0026 cm3/g, respectively. Vtotal values
of the earlywood were increased by 17% for the CS-treated wood and 41% for the steam-treated
wood. For the latewood, larger increments were obtained due to treatment, for example, after
steam treatment Vtotal of the latewood was increased by 85%. The influences of treatment on SBET
had the similar trend to Vtotal. For earlywood, it displayed an increase of 35% and 72% for
CS-treated wood and steam-treated wood, respectively, while the SBET of latewood showed an
increase of 53% for the CS-treated wood and 122% for the steam-treated wood, respectively. More
mesopores are formed after CS-treatment and steam treatment, which could result in the
permeability and capillary condensed water uptake in wood cell walls.
Table 1

Pore characteristics of the wood cell wall subjected to CS-treatment and steam treatment.

Untreated CS-treated Steam- treated F probability

Vtotala EW 4.61×10-3Bd 5.40×10-3B 6.54×10-3C 0.299

(5.07×10-4)e (1.00×10-3) (2.63×10-3)

(cm3/g)
LW 2.64×10-3B 3.64×10-3BC 4.77×10-3C 0.012

(7.67×10-4) (1.00×10-3) (4.61×10-4)

SBETb EW 1.58B 2.13BC 2.71C 0.036

(cm2/g) (0.38) (0.49) (0.62)

LW 1.05B 1.61C 2.33C 0.005

(0.38) (0.39) (0.43)

DBJHc EW 3.89 B 3.72C 3.73C 0.016

(nm) (0.13) (0.02) (0.02)

LW 3.72B 3.72B 3.72B 0.994

(0.02) (0.02) (0.01)

a b c
The total pore volume. The BET specific surface area. The average mesopore diameter using the BJH method.
d
B-C: The same capital letter in the same row of the table indicates that there is no significant difference among
the sampling positions at P< 0.05. e The standard deviation is indicated in brackets. EW = earlywood; LW =
latewood.

3.2. Confocal Raman microscopy

Changes in cellulose distribution

Raman imaging was conducted to reveal the cell wall chemistry and structural details with a
resolution at the micro-level. False color images were generated by integrating over the intensity
of defined Raman spectra. The spatial distribution of cellulose was visualized, based on the peak
at 2897 cm-1, due to the stretching of the C-H and C-H2 (Fig. 2). For the untreated wood, the
highest concentration of cellulose was observed in the S regions and the lowest level in the CC
regions (Fig. 2a and Fig. 2d). This is consistent with the wood cell wall structure that had been
previously reported (Whiting & Goring, 1983; Schmidt et al., 2009; Wang, Keplinger, Gierlinger,
& Burgert, 2014). The distribution of cellulose in the CS-treated (Fig. 2b and Fig. 2e) and the
steam-treated wood (Fig. 2c and Fig. 2f) showed a similar pattern to that in the untreated wood.
However, the intensity of the cellulose signal in earlywood and latewood for treated wood
decreased slightly in the S and CC regions, which was further confirmed by Raman spectra (Fig.
3).
Fig. 2. Raman images showing the distribution of cellulose in earlywood (a-c) and latewood (d-f) in untreated (a
and d), CS-treated (b and e), and steam-treated (c and f) wood, based on 2897 cm-1 (2920-2830 cm-1). S = the
secondary cell wall; CC = the cell corner.

In order to determine the subtle dynamic changes in the cellulose from different morphological
regions related to different treatments, a set of average Raman spectra that were extracted from
different wood sections were analyzed shown in Fig. 3. For untreated wood, the peak at 2897 cm-1
was more prominent, when compared to that at 1149 cm-1 and 1096 cm-1 in the spectra of S
regions. In the spectra of the CC regions, the peak at 1149 cm-1 was more pronounced than that in
the spectra of the S regions. The relative intensities of the peak at 2897 cm-1 and 1149 cm-1
decreased for both S and CC regions of earlywood and latewood after CS-treatment and steam
treatment as compared to untreated wood. This indicated cellulose structure was affected during
treatment. This might be attributed to two main factors: (a) partly degradation of cellulose in the S
and CC regions during CS-treatment and steam treatment. The steaming caused a high level of
degradation in the hemicellulose in the CS-treated wood and steam-treated wood, releasing the
acetic acid and formic acid (Zhang, Ma, Ji, & Xu, 2012). Low pH and high temperature would
facilitate the acidic hydrolysis of polysaccharides (Fengel & Wegener, 1984; Weiland & Guyonnet,
2003). The depolymerization reaction occurred in the amorphous region through β-(1,4)-bonds
breaking between the saccharide units of cellulose and hemicellulose. (b) Cellulose fibril
reorienting. The microfibril helix could be deformed perpendicularly to its axis during treatments.
It is well known that the 2897 cm-1 band is sensitive to the orientation of the fibrils, and the
intensity is highest when the laser electric vector is perpendicular to the cellulose chain direction,
so that any changes in this geometry would cause peak intensity changes. In addition, it is worth
noting that the swelling of cell wall could occur during CRM examination. This could be one of
the factors causing the decline of peaks at 2897 cm-1 and 1149 cm-1. However, both untreated and
treated samples were wetted with deuterium oxide, and thus cell wall swelling is assumed to be
not important for the purpose to compare the changes due to treatment.
Moreover, a greater reduction in the relative intensities of the peak at 2897 cm-1 and 1149 cm-1
were observed in the earlywood and latewood after steam-treated wood when compared to the
CS-treated wood, probably due to a larger mesopores leading to better permeability of steam.
Hence, cellulose structure was degraded or deformed more seriously in steam-treated wood.
Fig. 3. Average spectra extracted from the S and CC of the earlywood (a) and latewood (b) in the untreated,
CS-treated and steam-treated wood, respectively. S = the secondary cell wall; CC = the cell corner.

Changes in lignin distribution

The spatial distribution of lignin was visualized, based on the peak at 1600 cm-1 ascribed to the
symmetric stretching of the C=C bonds in the aromatic ring (Fig. 4). In comparison with the
distribution of the cellulose, the lignin distribution in the untreated wood showed an opposite
pattern with the highest level of lignin concentration occurred in the CC regions and the lowest
level was found in the S regions (Fig. 4a and Fig. 4d). Similar findings were reported by previous
studies (Ji, Ma, & Xu, 2014; Wang, Keplinger, Gierlinger, & Burgert, 2014). After treatment, the
intensity of the lignin signal in the CC and S regions displayed distinct changes. Compared to the
untreated sample, the intensities of the lignin signal clearly were increased in the CC regions,
indicating the relative concentration of lignin were much higher in the CC regions of both the
CS-treated (Fig. 4b and Fig. 4e) and the steam-treated wood (Fig. 4c and Fig. 4f). On the contrary,
an obvious reduction in intensities of lignin signal occurred in the S regions of treated wood,
especially in the CS-treated wood.

Fig. 4. Raman images showing the distribution of lignin in earlywood (a-c) and latewood (d-f) in untreated (a and
d), CS-treated (b and e), and steam-treated (c and f) wood around the peak at 1600 cm-1 (1690 - 1560 cm-1). S = the
secondary cell wall; CC = the cell corner.

As indicated in the Raman spectra (Fig. 3), the relative intensities of the absorption peaks at
1650 cm-1 and 1271 cm-1 for both S and CC regions decreased under CS-treatment and steam
treatment as compared to untreated wood. This indicated that losses of C=O and C=C groups
linked to the aromatic skeleton of guaiacyl units probably occurred. On the contrary, the relative
intensities of the absorption peak at 1333 cm-1 ascribed to aliphatic O-H bending was significantly
increased, suggesting that the thermal depolymerisation of lignin was associated with the
re-condensation reactions during CS-treatment and steam treatment. The β-aryl-ether linkages of
lignin were partially depolymerized in response to steaming, resulting in the formation of
carbonium ions (Weiland & Guyonnet, 2003; Windeisen, Stroble, & Wegener, 2007; Rousset,
Lapierre, Pollet, Quirino, & Perre, 2009). The presence of carbonium ions induced further
condensation reactions under the catalysis of the high temperature and the presence of acetic acid.
This confirmed our previous assumptions that cross-linking had been formed among the aromatic
units in the lignin during steam-treated wood (Yin, Berglund, & Salmén, 2011) and CS-treated
wood (Guo, Song, Salmén, & Yin, 2015).
The intensities of strong aryl ring stretching at 1600 cm-1 increased in the CC regions but
decreased in the S regions of wood cell wall after CS-treatment, which was in accordance with the
description of the lignin distribution images (Fig. 4). This might be derived from the different
lignin units from different morphological regions. As previously reported, lignin in the S regions
was more of a linear type with less branching which could give a looser structure (Donaldson,
2001; Wang et al., 2012). The matrix substance were softened by the high temperature steam and
became mobile (Kiemle et al., 2014). Therefore, it was easier for lignin units to be transited in the
S regions under compression combined with steam treatment.

3.3. Cellulose crystalline structure

Crystalline structure of cellulose after treatments was studied by XRD. XRD diffractograms
of both earlywood and latewood in the untreated, CS-treated and steam-treated wood are plotted in
Fig. 5. The untreated wood displayed a typical native cellulose Iβ pattern which had a parallel up
arrangement of cellulose chains. The typical peaks were observed at 14.8°, 16.5°, and 22.14°,
corresponding to (1-10), (110) and (200) planes (Guo, Guo, Wang, & Yin, 2016; Wei, McDonald,
& Stark, 2015). The diffraction peaks of the (1-10) and (110) planes merged together. Consistent
to this, the unit cell of native cellulose was found to be monoclinic, that the cellulose fiber axis
was parallel to the c-axis, while the cellulose chains were hydrogen bonded into planar sheets
along the b-axis. These sheets then assembled into stacks along the a-axis with van der Waals
interactions between the layers (Peura et al., 2006). After treatment, there was a significant
increase in the intensity of the (200) reflection, especially in the steam-treated wood. Interestingly,
the (004) reflection experienced no change in the earlywood and latewood of the CS-treated wood
and steam-treated wood, suggesting that the cellulose molecular chains remained largely
unaffected by the high temperature steam and compression.

Fig. 5. The XRD patterns of the earlywood (a) and latewood (b) in the untreated, CS-treated and steam-treated
wood.

The CrI was calculated as the ratio of the area arising from the crystalline phase to the total
area. The CrI of both earlywood and latewood after CS-treatment and steam treatment were larger
than those of the untreated wood. In addition, the steam-treated wood had the largest CrI (Table 2).
The CrI of the earlywood for untreated wood was calculated to be 25.8%. The values of the CrI
increased to 30.3% for the CS-treated wood and 49.2% for the steam-treated wood, indicating an
increase of 18% and 91%, respectively. A similar phenomenon was obtained with the latewood,
showing a growth of 9% for the CS-treated wood and 75% for the steam-treated wood, when
compared to the untreated latewood. ANOVA analysis was further used to investigate the
statistical significance for the CrI derived from CS-treatment and steam treatment. It was indicated
that both steam treatment and CS-treatment had a significant influence on the CrI at a significance
level of 5%, as showed by the different symbol letter towards CrI among woods in Table 2.
 The average crystallite thickness (D200) of the untreated wood was 2.73 nm for the earlywood
and 2.51 nm for the latewood. However, the values of the CS-treated wood increased to 2.89 nm
for the earlywood and 2.87 nm for the latewood, indicating an increase of 6% and 14%,
respectively. The values of the steam-treated wood increased to 3.03 nm for the earlywood and
2.96 nm for the latewood, indicating an increase of 11% and 18%, respectively. In accordance
with this, ANOVA analysis also showed that both CS-treatment and steam treatment resulted in a
significant increase of the cellulose crystallite dimensions.
It was clear seen that both CrI and D200 were increased after CS-treatment and steam treatment.
This agreed with earlier results for CS-treated wood (Ito, Tanahashi, Shigematsu, & Shinoda,
1998; Guo, Song, Salmén, & Yin, 2015) and hydrothermal treated wood (Bhuiyan, Hirai, & Sobue,
2000). The increase of CrI was probably due to the fact that cellulose became more crystalline
when wood was subjected to CS-treatment and steam treatment. This was further supported by the
indication that the native state of wood-cellulose was not-crystalline (Agarwal, Ralph, Reiner, &
Baez, 2016) and the increase of crystallinity index after CS-treatment was mainly due to the
increase or reordering in the crystalline region of cellulose using Wide-angle X-ray scattering
(WAXS) method, but not from the degradation of amorphous domains (Guo, Rennhofer, Yin, &
Lichtenegger, 2016). Cellulose macromolecules stored vivo stress due to their semicrystalline
nature (Kataoka & Kondo, 1998). The matrix substance was softened by the high temperature
steam and became mobile during the CS-treatment, which loosened the inner stress in the
crystalline region of the cellulose (Tanahashi, Goto, Horii, Hirai, & Higichi, 1989; Kiemle et al.,
2014). Under such conditions, the cellulose crystallite extended in the crystalline region and
crystallization occurred in the semicrystalline region. Moreover, a greater rise in both CrI and D200
were observed in the earlywood and latewood after steam-treated wood, when compared to the
CS-treated wood, probably due to a larger mesopores leading to the greater inner stress relaxation
during the steam treatement.

Table 2
The evolution of the crystallinity index and average crystallite thickness in the wood subjected to the CS-treatment
and steam treatment.

Untreated CS-treated Steam-treated F probability

Crystallinity EW 25.8Ba 30.4C 49.2D <0.0001

index (0.9)b (1.3) (0.9)

(%) LW 33.5B 36.7C 58.6D <0.0001

(1.6) (1.2) (1.1)

Crystallite EW 2.73B 2.89C 3.03D 0.0002

thickness (0.05) (0.03) (0.02)

(nm) LW 2.51 B 2.87C 2.96C <0.0001

(0.07) (0.03) (0.02)

a
B-D: The same capital letter in the same row of the table indicates that there is no significant difference among the sampling
positions at P< 0.05. b The standard deviation is indicated in brackets. EW = earlywood; LW = latewood.
3.4. Cell wall structure in response to CS-treatment and steam treatment

The cellulose molecules form microfibrils aggregation that are embedded in matrix of
hemicellulose and lignin (Salmén & Burgert, 2009), constituting the solid fraction of wood cell
wall (Fig. 6A). Although a high intermixing of the matrix wood polymer among the cellulose, the
wood cell wall exhibits a porosity of molecular scale dimensions, due to the partial filling of
spaces among the cellulose microfibrils with lignin, hemicellulose and extractive (Yin, Song, Lu,
Zhao & Yin, 2015). It has been speculated that the physical, chemical and mechanical properties
of wood are governed by the complex arrangement of the solid fraction and porosity of wood cell
walls. Recently, the changes in hygroscopicity and micromechanical properties after CS-treatment
and steam treatment were investigated in our previous studies (Yin, Berglund, &Salmén, 2011;
Guo, Song, Salmén, & Yin, 2015). It was revealed that hygroscopicity significantly decreased with
both CS-treatment and steam treatment, while the micromechanical properties decreased for the
steam treatment and increased for the CS-treatment. In this study, direct experimental evidence
was replenished when changes were observed in the porosity, chemical composition and cellulose
crystalline structure of CS-treated and steam-treated wood cell walls.
The hygroscopicity in wood cell walls could be correlated with absorbed water and capillary
condensed water. However, it should be noted that both CS-treatment and steam treatment showed
a greater proportion of mesopores in wood cell walls. The presence of these mesopores promoted
capillary condensed water uptake, which would result in increased hygroscopicity. However, the
hygroscopicity of both CS-treatment and steam treatment decreased as our previous studies.
Therefore, the effect of capillary condensed water on the hygroscopicity was ignored. As
illustrated diagrammatically in Fig. 6C, steam can depolymerize lignin by hydrolyzing the
β-aryl-ether links associated to the guaiacyl units, and then carbonium ions are generated. The
presence of carbonium ions induced further re-condensation reactions, resulting in a significant
decrease in the C=O and the C=C linked to the lignin aromatic skeleton. Moreover, the
depolymerization reaction occurred in the amorphous regions through β-(1,4)-bonds breakage
between the saccharide units of cellulose and hemicellulose, resulting in a reduction in the
availability of the overall carbohydrate -OH groups. Together with our previous findings by FT-IR
analysis (Yin, Berglund, & Salmén, 2011; Guo, Song, Salmén, & Yin, 2015), the clear progression
of C=O, assigned to the O=C-OH in the glucuronic acid unit of xylan, and a loss of mannose units
in the glucomannan backbone indicated that C=O were lost in the hemicellulose. Therefore, it was
assumed that the reduction in hygroscopicity after the CS-treatment and steam treatment was
highly related to the decrease of accessible sorption groups, e.g., hydroxyl and carbonyl groups,
and thus provided the fixation of the compression combined with steam treatment with limited
structure deformation recovery.
The strength and rigidity of the cell wall are probably related to the crystallinity index of
cellulose, the cross-linking of lignin and the cell wall density (Guo, Song, Salmén, & Yin, 2015).
The increase in the relative crystalline cellulose content (Fig. 6B) and the cross-linking of lignin
after CS-treatment and steam treatment would lead to an improvement in mechanical properties.
However, a larger number of mesopores was formed due of the degradation of amorphous
polysaccharides, leading a lower cell wall density during steam treatment. Consequently, a
decrease in the micromechanical properties was observed with steam-treated wood when
compared to the untreated wood. In contrast, the applied compression forces resulted in a stiffer
and stronger structure probably as a result of the higher cell wall density.
 Fig. 6. A diagram to illustrate the microfibril structure of wood cell walls (A), the cross section of cellulose
crystallite structure after CS-treatment and steam treatment (B) and the main structure and alignment of cellulose
and lignin (C). EW = earlywood; LW = latewood.

4. Conclusions
Compression combined with steam treatment (CS-treatment) caused obvious changes on the
porosity, chemical composition and cellulose crystalline structure of wood cell walls. A large
number of slit-shaped mesopores with a diameter of 3.7 nm was formed in the CS-treated wood
cell walls, and more mesopores were found in the cell walls of the steam-treated wood. The
cellulose structure was affected and β-aryl-ether links associated to guaiacyl units of lignin was
depolymerized followed by re-condensation reactions. The chemical reaction led to the decreases
of hydroxyl and carbonyl groups in the treated wood cell walls, and hence less moisture sorbing
sites were available. This provided better fixation of the compression combined steam treatment.
The CrI and D200 of cellulose in CS-treated wood cell wall were largely increased due to
crystallization that occurred in the semicrystalline region, but cellulose molecules chains in the
crystalline region remained unaffected by CS-treatment. Furthermore, the larger increase in the
CrI and the D200 were observed in the earlywood and latewood for steam-treated wood, which was
probably contributed to the greater amount of mesopores as compared to CS-treated wood. This
study can provide practical guidance to the modification of low quality plantation wood
potentially utilized for environment-friendly functional biomaterials.

Acknowledgments
This work was supported financially by the Chinese National Natural Science Foundation
(No.31370559) and the Wallenberg Wood Science Center (WWSC) of the Royal Institute of
Technology (KTH) of Sweden. The technical help from Dr. Toshiro Morooka of Research Institute
for Sustainable Humanosphere, Kyoto University, Japan for the compression combined with steam
treatment and the steam treatment, Prof. Lennart Salmén, Prof. Lars Berglund of KTH and Dr.
Juan Guo of Research Institute of Wood Industry, Chinese Academy of Forestry for the discussion
of experimental design are gratefully acknowledged. The assistance with language editing done by
Kevin Austin of BizTech English AB is also acknowledged.
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