Hierarchical structure and dynamics of plant cell wall studied

UNIVERSITY OF HELSINKI
REPORT SERIES IN PHYSICS
HU-P-D190
HIERARCHICAL STRUCTURE AND DYNAMICS OF
PLANT CELL WALL STUDIED USING X-RAYS
Kirsi Svedström
Division of Materials Physics
Department of Physics
Faculty of Science
University of Helsinki
Helsinki, Finland
ACADEMIC DISSERTATION
To be presented, with the permission of
the Faculty of Science of the University of Helsinki,
for public criticism in Auditorium A129
of the Department of Chemistry (Chemicum),
A.I. Virtasen aukio 1, on May 2nd 2012 at 12:15.
Helsinki 2012
Supervisor
Prof. Ritva Serimaa
Department of Physics
University of Helsinki
Helsinki, Finland
Pre-examiners
Prof. Kristofer Gamstedt
Department of Engineering Sciences
Ångström Laboratory
Uppsala University
Uppsala, Sweden
Dr. Tancrède Alméras
Laboratoire de Mécanique et Génie
Civil
Université Montpellier
Montpellier, France
Opponent
Custos
Prof. Martin Müller
Institute of Materials Research
Helmholtz-Zentrum Geesthacht
Geesthacht, Germany
Prof. Ritva Serimaa
Department of Physics
University of Helsinki
Helsinki, Finland
Report Series in Physics HU-P-D190
ISSN 0356-0961
ISBN 978-952-10-7078-5 (printed version)
ISBN 978-952-10-7079-2 (pdf version)
http://ethesis.helsinki.fi/
Helsinki University Print
Helsinki 2012
i
Preface
This thesis is based on the research carried out at the Dept. of Physics, University of Helsinki.
I wish to thank Prof. Juhani Keinonen for providing me the possibility to work there. This
study would not have been possible without collaboration with the Dept. of Biosciences
(University of Helsinki), Finnish Forest Research Institute, Technical Research Centre of
Finland, Dept. of Fiber and Polymer Technology (Royal Institute of Technology), Dept. of
Forest Genetics and Plant Physiology (Swedish University of Agricultural Sciences), MAXlab (Lund University), Ghent University Centre for X-ray Tomography, Chinese Academy of
Forestry, and Institute of Macromolecular Compounds (Russian Academy of Sciences); all
the contributing researchers are greatly acknowledged. I thank National Graduate School
in Materials Physics, Academy of Finland (project 1127759), University of Helsinki, COST
Action FP0802, and Finnish Cultural Foundation for the financial support.
I own definetely my sincerest gratitude to my supervisor, Prof. Ritva Serimaa, for her
great encouragement, help and support. She introduced me to the field of the thesis and
guided me wisely and heartily throughout all the stages.
I thank the former and present personnel at the Laboratory of Electronic Structure for
providing a stimulating, warm and friendly working atmosphere. Prof. Keijo Hämäläinen is
acknowledged for having a great influence on creating that atmosphere. I am very grateful
to Dr. Merja Blomberg, Phil.Lic. Pasi Lintunen and Dr. Mika Torkkeli for their help at
the laboratory and for answering always to my questions. I thank Dr. Seppo Andersson for
his kind and professional guidance into the field of wood science using 4-circle goniometer,
regarding especially the secrets of the microfibril angle. I am thankful to Dr. Ulla Vainio and
Dr. Kari Pirkkalainen for leading my first steps with experimental work and showing how
to be a proper scientist. I appreciate Dr. Marko Peura, M.Sc. Jussi-Petteri Suuronen and
M.Sc. Aki Kallonen for their valuable help with the tomography experiments. I feel joy and
gratitude for being a colleague with Dr. Arto Sakko and M.Sc. Paavo Penttilä, and I thank
them sincerely for all the discussions.
I have had the priviledge to work with many great researchers. Especially, I thank warmly
Dr. Ingela Bjurhager for her co-authorship and friendship. I own gratitude also to Dr. Jan
Van den Bulcke for both his excellent help considering x-ray microtomography experiments
and his kind hospitality during my visit in Ghent.
My warmest thanks are to my parents Osmo and Merja due to their support and help
considering so many aspects of my life. I thank from the bottom of my heart my sisters and
brother, Maarit, Marja and Matti, for all joyful moments, which have brightened the serene
physicist’s life. The same is valid with my friends; especially, I would like to thank Janika,
Minna and Eve for their friendship over decades, and Suvi, Toni and Antti for sharing the
delight of studying physics.
Most of all, I am truly, deeply grateful to my beloved husband Jan. Thanks due to his
kind encouragement, I was brave enough to start this exciting endeavour over four years ago.
During these years his love and patience were the strongest and most substantial support for
me. Kiitos rakas.
Helsinki, 21.02.2012
Kirsi
ii
K. Svedström: Hierarchical structure and dynamics of plant cell wall studied using x-rays,
University of Helsinki, 2012, 50 pages + appendices. University of Helsinki, Report Series in
Physics, HU-P-D190.
Abstract
The interest in wood and its main constituent, cellulose, is growing continuously. This can be
explained by the demands of sustainable development and the extending scope of applications
enabled by novel materials like nanocrystalline cellulose. Outstanding mechanical properties
are one of the main reasons for the usability of wood. However, details on the ultrastructure
of wood cell wall are still lacking, and thus also the origin for the mechanical properties
is partly unknown. The various properties of plant materials arise from their hierarchical
structure. Thus information at several size scales is needed for revealing the structure-function
relationships. In this study, the structure of plant cell wall and cellulose based materials
was characterized from the micro- to nanometer scale: using x-ray microtomography, the
three-dimensional cellular structure was revealed and using x-ray scattering methods, the
nanostructure was investigated.
In the cell wall, parallel cellulose chains form microfibrils, which are partially crystalline
and embedded in an amorphous hemicellulose-lignin matrix. The orientation, width and
length of the crystalline parts of microfibrils vary between the plant species and materials. In
this thesis, the cellulose structure was characterized in various plant species, and the effects
due to drying, chemical pulping and acid hydrolysis were determined. It was found that
the properties of the amorphous matrix played a role in all the effects studied. The wide
spectrum of the samples, including native cotton, flax, bamboo, various wood species, pulp,
microcrystalline and nanofibrillated cellulose (MCC, NFC), enabled interesting comparisons.
It was observed, that in tension wood, pulp, MCC, and NFC, the cellulose crystallite width
was significantly larger than in native normal wood. The high cellulose content and lack
of lignin were typical for all the samples with a larger crystallite width. It was also found
that the lateral and longitudinal order of the cellulose chains in crystallites was higher in
never-dried wood compared to the corresponding air-dried samples. This indicated that the
moisture had impact on the structures formed by crystalline cellulose although the water
molecules can not enter the crystallites. One of the objects of the thesis was the study of the
cellulose structure of oak from the Swedish warship Vasa. The results gave information on
the preservation of the historically important wood throughout the centuries at the bottom
of the sea.
Classification (INSPEC): A6110F, A6140K, A8170J
Keywords: x-ray scattering, x-ray microtomography, xylem, cellulose crystallite, microcrystalline cellulose, nanofibrillated cellulose
Cover photo: Jan Svedström
iii
List of papers
This thesis consists of an introductory part and six research articles, which are referred
to by the Roman numerals I–VI throughout the text.
I K. Leppänen, I. Bjurhager, M. Peura, A. Kallonen, J.-P. Suuronen, P. Penttilä, J. Love, K. Fagerstedt, R. Serimaa (2011) X-ray scattering and microtomography study on the structural changes of never-dried silver birch, European aspen and hybrid aspen during drying. Holzforschung 65:865-873. DOI
10.1515/HF.2011.108.
II K. Svedström, J. Lucenius, J. Van den Bulcke, D. Van Loo, P. Immerzeel, J.-P.
Suuronen, L. Brabant, J. Van Acker, P. Saranpää, K. Fagerstedt, E. Mellerowicz,
R. Serimaa (2012) Hierarchical structure of juvenile hybrid aspen xylem revealed
using x-ray scattering and microtomography. Submitted for publication.
III K. Svedström, I. Bjurhager, A. Kallonen, M. Peura, R. Serimaa (2012) Structure
of oak wood from the Swedish warship Vasa revealed by x-ray scattering and
microtomography. Holzforschung. DOI 10.1515/HF.2011.157.
IV Y. Wang, K. Leppänen, S. Andersson, R. Serimaa, H. Ren, B.H. Fei (2012)
Studies on the nanostructure of the cell wall of bamboo using x-ray scattering.
Wood Science and Technology 46:317-332.
V K. Leppänen, S. Andersson, M. Torkkeli, M. Knaapila, N. Kotelnikova, R. Serimaa (2009) Structure of cellulose and microcrystalline cellulose from various wood
species, cotton and flax studied by x-ray scattering. Cellulose 16:999-1015.
VI K. Leppänen, K. Pirkkalainen, P. Penttilä, J. Sievänen, N. Kotelnikova, R. Serimaa (2010) Small-angle x-ray scattering study on the structure of microcrystalline
and nanofibrillated cellulose. Journal of Physics: Conference Series 247:012030.
The papers I-VI are included as appendices in the printed version of this thesis. The
papers I and III are reprinted with permission by De Gruyter, the papers IV and V
are reprinted with kind permission from Springer Science+Business Media, and the
paper VI is reprinted with permission by IOP.
Author’s (K.S.) contribution
I: K.S. conducted the WAXS and SAXS measurements on the birch wood samples.
The µCT measurements were conducted by K.S., A. Kallonen and J.-P. Suuronen.
K.S. evaluated all the WAXS, SAXS and µCT results and wrote the paper.
iv
II: K.S. performed the WAXS experiments and data analysis together with J. Lucenius.
The µCT measurements and the µCT data analysis were done by K.S. with help from
J. Van den Bulcke and D. Van Loo. The samples were obtained from P. Immerzeel, E.
Mellerowicz, P. Saranpää, and K. Fagerstedt. K.S. wrote the paper.
III: K.S. planned and conducted the WAXS measurements together with I. Bjurhager,
carried out the SAXS experiments, evaluated all the data, and wrote most of the paper.
The Materials section and parts of the Introduction were written by I. Bjurhager. The
µCT experiments were conducted by A. Kallonen and M. Peura.
IV: The WAXS experiments, the data analysis, and writing of the paper were performed together by K.S., Y. Wang and S. Andersson. Y. Wang took and analyzed the
light microscopy and SEM images.
V: K.S. conducted the SAXS measurements and the corresponding data analysis. N.
Kotelnikova prepared the samples, conducted the SEM and FTIR measurements, analyzed the corresponding data, and wrote the corresponding sections of the paper. S.
Andersson conducted the WAXS measurements and the corresponding data analysis.
K.S. wrote most of the paper.
VI: K.S. conducted the SAXS experiments together with K. Pirkkalainen and P. Penttilä. K.S. evaluated all the data and wrote the paper. The MCC samples were obtained
from N. Kotelnikova and the NFC samples were obtained from J. Sievänen.
v
Other related publications
List of publications which are relevant to this thesis but not included in it:
• K. Pirkkalainen, M. Peura, K. Leppänen, A. Salmi, A. Meriläinen, P. Saranpää,
R. Serimaa (2012) Simultaneous x-ray diffraction and x-ray fluorescence microanalysis on secondary xylem of Norway spruce. Accepted for publication in Wood
Science and Technology.
• U. Vainio, R.A. Lauten, S. Haas, K. Svedström, L.S.I. Veiga, A. Hoell, R. Serimaa (2012) Distribution of counterions around lignosulfonate macromolecules in
different polar solvent mixtures. Langmuir 28:2465-2475.
• T. Virtanen, K. Svedström, S. Andersson, L. Tervala, M. Torkkeli, M. Knaapila,
N. Kotelnikova, S.L. Maunu, R. Serimaa (2012) A physico-chemical characterisation of new raw materials for microcrystalline cellulose manufacturing. Cellulose
19:219-235.
• T. Hänninen, P. Tukiainen, K. Svedström, R. Serimaa, P. Saranpää, E. Kontturi, M. Hughes, T. Vuorinen (2012) Ultrastructural evaluation of compression
wood-like properties of common juniper (Juniperus communis). Holzforschung
DOI 10.1515/HF.2011.166.
• T. Hänninen, E. Kontturi, K. Leppänen, R. Serimaa, T. Vuorinen (2011) Kraft
pulping of Juniperus communis results in paper with unusually high elasticity.
BioResources 6:3824-3825.
• P.A. Penttilä, A. Várnai, K. Leppänen, M. Peura, A. Kallonen, P. Jääskeläinen,
J. Lucenius, J. Ruokolainen, M. Siika-aho, L. Viikari, R. Serimaa (2010) Changes
in submicrometer structure of enzymatically hydrolyzed microcrystalline cellulose. Biomacromolecules 11:1111-1117.
• K. Pirkkalainen, K. Leppänen, U. Vainio, M.A. Webb, T. Elbra, T. Kohout,
A. Nykänen, J. Ruokolainen, N. Kotelnikova, R. Serimaa (2008) Nanocomposites of magnetic cobalt nanoparticles and cellulose. European Physical Journal D
49:333-342.
Contents
1 Introduction
1.1 Hierarchical structure of wood: from macro- to nanoscale . . . . . . . .
1
1
2 Materials and aims of the study
2.1 Native plant materials . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2 Archaeological oak wood from Vasa . . . . . . . . . . . . . . . . . . . .
2.3 Micro- and nanocrystalline cellulose . . . . . . . . . . . . . . . . . . . .
6
6
7
8
3 Theory and methods
3.1 X-ray microtomography . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2 Small-angle x-ray scattering . . . . . . . . . . . . . . . . . . . . . . . .
3.3 Wide-angle x-ray scattering . . . . . . . . . . . . . . . . . . . . . . . .
10
10
16
19
4 Results and discussion
4.1 Cellulose crystallite dimensions in native samples . . . . . . . . . .
4.2 Changes due to drying (papers I and II) . . . . . . . . . . . . . . .
4.3 Changes due to degradation of Vasa oak (paper III) . . . . . . . . .
4.4 Changes as a function of the radial position in bamboo (paper IV)
4.5 Changes due to pulping and hydrolysis (papers V and VI) . . . . .
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30
5 Conclusions and future aspects
References
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34
1 INTRODUCTION
1
1.1
1
Introduction
Hierarchical structure of wood: from macro- to nanoscale
The main component of plants is cellulose. The arrangement of semicrystalline cellulose
with amorphous hemicellulose, lignin and pectin into the hierarchical structure of cell
wall enables that the vegetation on the Earth is found in diversified appearances, as
algae, grass, flowers, and trees, instead of being just a formless pile of protoplasm [1].
The complex biological systems give rise to many difficulties for the research and so,
despite all the scientific efforts, many details of the ultrastructure of the plant cell wall
remain still unknown.
Trees are an impressive example of the biological structures: the largest stems can
grow over 100 meters of height and the oldest stems have been able to stand in wind,
rain and storms for centuries, even for millenaries. This is possible due to the intriguing
hierarchical structure being made up from the nanoscale cellulose crystallites to the
macroscopic organization of different types of cells across the stem. The transverse
cross-section of the stem consists of the pith, the xylem, the vascular cambium, the
inner (phloem) and outer bark (epidermis) (Fig. 1 A). The wood tissue produced during
the early season of the growth period is called earlywood, and the tissue produced after
that is called latewood. These two tissue types can be observed in the stem cross-section
as macroscopically different concentric rings, which together form the annual ring.
The inner part of tree trunk, heartwood, consists only of dead cells. The living
wood cells are in the vascular cambium, where the cell division and the other steps
of cell development take place. All trees have a bifacial cambium, which produces
secondary xylem towards the stem axis and phloem to the opposite direction. So the
cambial cells are programmed to differentiate either into vascular tissue of xylem or
phloem [2]. The vascular cells accomplish the development steps of the cell expansion
and elongation, the cellulose deposition in the secondary cell wall, the lignification, and
finally the programmed cell death. Thus the xylem consists of the walls of the dead
cells taking out the parenchyma cells, which die later.
A bamboo, being a monocot and belonging to grass plants, has another type of
cambium. The monocot cambium produces lignified fibers in well-ordered, discrete
vascular bundles containing both xylem and phloem (Fig. 1 B), and the monocots
have no secondary xylem.
All the embryotic land plants are believed to have developed from green algae [3].
The differentiation of unspecified plant cells into tracheary elements is studied by using
model species such as Arabidopsis and P opulus, whose genomes are already fully
sequenced [4, 5]. Trees are members of two phylogenetic groups, gymnosperms and
angiosperms. Gymnosperms, i.e. conifers, are considered as softwood, whereas birch,
aspen, poplar, and oak belong to angiosperms and are considered as hardwood. The
cellular structure of hardwood is more complicated than that of softwood: hardwood
2
1 INTRODUCTION
Figure 1: A: The cross-section of a young tree stem from the inner to outer part:
the pith (p), the xylem (x), the vascular cambium (c), and the outer bark (b). The
length of the scale bar is 1 mm. The image is a cross-section of a volume obtained
by x-ray microtomography at the Centre for X-ray Tomography of the University of
Ghent. B: The cellular structure of bamboo: the parenchyma cells and the vascular
bundles consisting of the lignified fibers (f), the metaxylem (mx), the protoxylem (px),
and the phloem (ph). The length of the scale bar is 0.2 mm. The scanning electron
microscopy image is taken by Yurong Wang.
consists of several cell types, which have own specific functions, whereas softwood
consists mainly of tracheids, which take care of both water transport and support
of the stem simultaneously. In most hardwoods, the vessels, multicellular conduits,
transport water while the fiber cells support the stem. The vessel diameter can range
up to 0.5 mm and the length up to 2 m [2]. The different cell types of hardwood are
presented in Fig. 2. In conifers, tracheids are 0.5-4 mm in length and 8-80 µm in
diameter [6] depending on their location and function. For instance, the root tracheids
are longer and wider than the stem tracheids [7].
The wood cell wall consists of multiple layers. Middle lamella consists mainly of
pectins and it is the layer, which glues the separate cells together. The first cell wall
layer formed is the primary wall. During the primary wall stage, the cells grow to
their final dimensions by a combination of the process of neighboring cells expanding
together and elongating past another [8]. The next layer formed is the secondary
cell wall, which consists of three inner layers called S1, S2 and S3. The S2 layer is
the thickest part of the cell wall, which must be borne in mind, when x-ray scattering
measurements are conducted on wood samples: the main contribution in the scattering
patterns arises thus from the S2 secondary cell wall layer.
1 INTRODUCTION
3
Figure 2: The different cell types in
hardwood: vessels (V), fibers (F) and
ray cells (R).
Cellulose microfibrils
The cell walls consist of semicrystalline cellulose microfibrils formed by parallel cellulose chains and embedded in an amorphous matrix of hemicelluloses and lignin. The
microfibrils are winding helically around the cell axis, and the angle between the axis
and the microfibrils (microfibril angle, MFA) is different in each cell wall layer. In the
primary wall of the wood cell, the microfibrils are oriented almost randomly, whereas
in the S1 layer, the mean MFA is around 90◦ [9]. In mature normal wood (with few
exceptions such as juniper [10]) the mean MFA in the S2 layer is low, 0-30◦. The
MFA in the S2 layer is considered to be one of the main factors contributing to the
mechanical properties of wood [11]. It is also well known that the mean MFA varies as
a function of the cambial age [9, 12], and that the mean MFA in reaction wood differs
from that of normal wood of the same species [13]. Thus the MFA is inevitably linked
to the structural function of wood.
Cellulose is a non-branching polymer of (1,4)-linked β-D-glucan rings, which forms
the microfibrils by crystallization. The shape and size of the microfibrils vary in nature,
ranging from thin membranes to square structures consisting of more than 1200 glucan
chains in V alonia ventricosa [14]. In vascular plants, cellulose is generated in the
plasma-membrane of the cell by the cellulose-synthesizing complexes called rosettes.
One rosette generates 36 cellulose chains [15]. The number of the chains corresponds
well to the width of cellulose crystallites in wood (about 3 nm) determined using x-ray
diffraction (XRD) [16]. In bacteria and some algae cellulose is synthesized by linearly
arranged terminal complexes [15].
Cellulose has multiple crystalline allomorphs, e.g. cellulose Iα , Iβ , II, and IIII [17].
The higher plants consist mainly of cellulose Iβ , whereas algae consist mainly of cellulose
Iα [18]. Cellulose II is the rarest form of cellulose found in nature: it can be found only
in few species of algae and bacteria [14]. The glucan chains are oriented parallel in
both allomorphs of cellulose I, while they are antiparallel in cellulose II. The unit cell
4
1 INTRODUCTION
Figure 3: The cellulose molecule (below) and the unit cell for the cellulose Iβ crystal
structure from two different views (above). In the unit cell, black dots denote carbon
atoms, red dots oxygen atoms and gray dots hydrogen atoms. The lattice dimensions
for the unit cell are: a = 7.78 Å, b = 8.20 Å, c = 10.38 Å, and γ = 96.5◦ (the monoclinic
angle between ā and b̄) [19].
of cellulose Iβ is monoclinic [19] (Fig. 3), whereas that of cellulose Iα is triclinic [20].
In the analysis of the XRD data of this study, only the crystal structure of cellulose Iβ
was taken into account.
Approximately half of the cellulose in the wood cell wall exists in amorphous
form [16, 21]. The exact presence of amorphous cellulose and its relation to the crystalline regions are not known [22]. According to the latest views, the microfibrils are
partially amorphous: the surface layers consist of amorphous chains, and the amorphous and crystalline regions alter also in the longitudinal direction of the microfibril.
The amorphous parts may be lattice defects and/or larger regions of non-ordered chains.
The lattice defects and other anomalies in the chains could be explained by the fact,
that the cellulose chains fail periodically to coalesce into the crystalline structure [23].
It has been stated that these regions of the cellulose microfibrils may be the ones, which
are connected to the hemicellulose chains [1].
Matrix polymers
Lignin and hemicelluloses are synthesized in the Golgi-apparatus. The backbones of
the hemicellulose molecules are very similar to that of cellulose, but hemicelluloses
have branches. The main hemicellulose in the secondary walls of woody angiosperms
(including hardwoods) is xylan, and in the secondary walls of conifers the main hemicelluloses are mannans [24, 25].
1 INTRODUCTION
5
Lignin is the most branching cell wall polymer. It consists of phenylpropane
units with no, one or two methoxyl-groups. Three different monolignols are called pcoumaryl, coniferyl and sinapyl alcohols [26]. Lignin is different in hard- and softwoods.
Softwood lignin consists mainly of coniferyl alcohols (with little p-coumaryl alcohols),
whereas in hardwoods lignin consists of equal amounts of coniferyl and sinapyl alcohols.
Thus hardwood has higher concentration of methoxy groups. The higher content of
3-substituted sinapyl alcohol residues in hardwoods leads to less condensed structure
and less networking for polymerization. Thus hardwood lignin is less branched than
softwood lignin [27].
Microfibril-matrix relations
Awano et al. studied the secondary cell wall formation in F agus crenata (Japanese
beech) [28]. Based on their results, the cellulose microfibrils are released into the inner
surface of the secondary cell wall after cellulose has been synthesized on the plasma
membrane. Xylan is transported there from the Golgi-apparatus across the plasma
membrane. Xylan deposition into the cell wall continues after cellulose microfibril
deposition; xylan penetrates the cell wall and accumulates on the microfibrils simultaneously with the lignin deposition [28]. Lignin precursors are polymerized into the
gaps between microfibrils stiffening the xylan-cellulose structures and preserving the
initial geometry [29].
Reis et al. stated that glucuronoxylans form a tight charged layer around microfibrils, and by doing that, glucuronoxylan, which is an acidic surfactant, causes the
spacing pattern of the carbohydrate components by preventing the aggregation of microfibrils [29, 30]. Vian et al. hypothesized that xylan acts also as a twisting agent for
cellulose microfibrils [31], and according to Reis et al. the crystallization of cellulose
and glucuronoxylan direct the cellulose microfibrils in a helicoidal array [29]. However,
it has also been proposed that the MFA is controlled by microtubules, which determine
the trajectories of the rosettes, and/or by the geometry of cell wall [8, 32, 33].
Dammström et al. presented based on their studies on hardwood cell wall [34],
that one fraction of xylan is associated with cellulose, while the other fraction (the
more branched type [35]) is associated with lignin. Based on the dynamic Fourier
transform infrared spectroscopy studies on Norway spruce wood, glucomannan had a
close contact with cellulose in the softwood cell wall while no mechanical interaction
was observed between xylan and cellulose [36].
6
2
2.1
2 MATERIALS AND AIMS OF THE STUDY
Materials and aims of the study
Native plant materials
Never-dried wood
At macroscopic scale, wood swells and shrinks as a function of the moisture content
anisotropically: the dimensional change is largest tangentially, second largest radially
and smallest longitudinally. The smaller radial deformation compared to the tangential one can be explained by the restraining strength of the rays [27]. In industrial
applications usually dry wood is used, so most of the structural studies have been
conducted using dry wood samples. Recently, irreversible deformations of cellulose
crystallites have been detected upon drying of never-dried wood samples [37–39]. The
results have been diverse, depending on wood species, and no explicit explanation for
the detected phenomena exists yet. In papers I and II, never-dried samples of silver
birch (Betula pendula Roth.), European aspen (P opulus tremula) and hybrid aspen
(P opulus tremula x tremuloides) were studied using x-ray scattering in situ during
their drying in ambient conditions. Never-dried birch wood was measured also using
x-ray microtomography. The aim was to study the drying deformations of hardwood
xylem both at the micrometer level and at the nanometer level.
Reaction wood
Trees respond to mechanical stress caused by strong wind, uneven soil or other similar
factor by generating reaction wood. In the upper part of a leaning hardwood stem,
reaction wood called tension wood (TW) is formed in order to tense the stem or branch
upwards. It has also been found out, that in saplings grown upright in a greenhouse,
TW formation is typical [40–42]. Also, TW has been detected in young fast growing
Eucalyptus globulus trees grown straight upright in plantation conditions [43]. In
many wood species, TW cells have different cell wall structure from normal wood cells:
TW cells have a thick layer called gelatinous (G-) layer [44]. Another reaction wood
type is compression wood (CW), which is formed in the lower part of a leaning softwood
stem to generate compressive stress.
TW formation can be studied by XRD, because the cellulose crystallites are larger
in width, the MFA is lower, and the crystallinities are higher in TW with G-layers than
in normal wood [45,46]. The macroscopic drying deformations are different in TW from
those in normal wood. In TW, with the low MFA, the longitudinal swelling/shrinkage
is relatively large, whereas in normal wood the higher MFA is linked to the larger
longitudinal deformation [47]. This observation has lead to many ultrastructural studies by XRD, which have aimed to map the origin for the peculiar drying behavior
2 MATERIALS AND AIMS OF THE STUDY
7
of TW [47–49]. In paper I, the drying deformations of never-dried hybrid aspen and
European aspen TW were determined and compared to normal wood samples of the
same species.
Juvenile and mature wood
Juvenile wood is produced in a tree during its first years of growth. Young trees consist
only of juvenile wood, while in older stems juvenile wood is found in the central core
(in approximately the first 5-20 annual rings) [50]. Based on the studies conducted
on mature stems studied as a function of the annual ring from the pith to the bark,
the properties and structure of wood change as a function of the cambial age [51–53].
However, the ultrastructure of young saplings had not been systematically studied
using x-ray methods. Thus, in paper II the nano- and microstructure of 3-month-old
hybrid aspen saplings was characterized using x-ray scattering and microtomography.
The hybrid aspen saplings were grown in a greenhouse, so it was also interesting to
check, if the saplings had formed TW.
2.2
Archaeological oak wood from Vasa
The mechanical and physical properties of wood can stay nearly unaltered for even
thousands of years depending strongly on the storage conditions. The severe enemy
for the wood cells is the wood-degrading fungi. The fungi can not stand anaerobic
conditions, thus storage in water is advantageous for wood.
The historical Swedish warship Vasa is one of the great examples of the extreme
durability of wood. The ship was built in 1626-27 to be one of the prides of the Swedish
navy. In 1628 the ship was set to sail her maiden voyage. However, after only a short
distance at open water, she capsized and sank into the Stockholm harbor. The ship
was salvaged in 1961 after spending 333 years at the bottom of the sea, and a 26year-long pioneering preservation process started. To prevent the cracking and drying
deformations of wet wood, the ship was spray impregnated with aqueous solutions
of polyethylene glycol (PEG) with different molecular weights (Mw): PEG 1500 and
PEG 600 (the number indicates the mean Mw) were used, and finally PEG 4000 was
infused to the surface of the hull [54]. It has been stated that the severely devastated
water of the Stockholm harbor of the 18th and 19th centuries was one of the factors,
which enabled the relatively good macroscopic condition of Vasa oak [55]. Also the
absence of shipworm due to the brackish sea water, the anaerobic conditions and the
low temperature helped in the maintenance of the Vasa [56].
The chemical, anatomical and mechanical properties of Vasa oak have been extensively studied since the recovering [57–61]. However, the structure of cellulose of Vasa
had not been characterized previously. In paper III, the samples of Vasa oak (Fig. 4)
8
2 MATERIALS AND AIMS OF THE STUDY
Figure 4: The piece of Vasa oak
studied was from the orlop deck.
The piece had originally two inner (indicated by dashed line)
and four outer surfaces. The positions of the samples are indicated by the spots and the numbers correspond to the distances
in horizontal and vertical directions (in mm) from the outer surfaces. The image of the block is
taken by Ingela Bjurhager.
were studied by determining the cellulose crystallite structure using x-ray scattering
and by observing the cellular level structure using x-ray microtomography. The results
on the Vasa samples were compared to those of recent oak (Quercus robur), which
gave information on the degradation state of the historically important oak wood cell
walls.
2.3
Micro- and nanocrystalline cellulose
Microcrystalline cellulose (MCC) has been produced and used already from the 50s
on [62], and nowadays it is used widely in pharmaceutical applications, food industry,
cosmetics, and as a reinforcement material in composites [63,64]. Acid hydrolysis is one
of the methods to produce MCC. Using acid hydrolysis, hemicellulose and lignin are
degraded from the fibers and the amorphous and crystalline regions of cellulose microfibrils are altered. In hydrolysis, a hydronium ion (H3 O+ ) breaks the bond between
oxygen and carbon atom 1 (presented in Fig. 3) by attaching to the oxygen atom,
which is connected to the next cellulose subunit. The extent of these effects depends
on the used acid, hydrolysis temperature and time. The fibers of MCC are in the scale
of micrometer in diameter, and they are formed by elementary cellulose microfibrils,
whose crystalline parts have a width of about 5-9 nm and a length of about 20-30 nm
depending on the source of cellulose (paper V, [65]).
Novel cellulose based materials, cellulose nanowhiskers, nanocrystalline and nanofibrillated cellulose (NCC, NFC), have been developed since the 80s, which have gained
much attention recently. They can be considered as nanomaterials, because they consist
of fibrils, whose diameters are down to a few nanometers. The length of whiskers, which
are rigid cellulose rods, is usually of the order of 100 nm, while in NFC the exact length
is difficult to determine, because it exceeds usually 1 µm and the NFC fibrils entangle
2 MATERIALS AND AIMS OF THE STUDY
9
strongly.
Nanocelluloses are produced from native cellulose using various top-down methods
including enzymatic, chemical and/or physical treatments and bottom-up methods
including bacterial producing of nanofibrils from glucose [62, 66–68]. Nanowhiskers
are produced using acid or enzymatic hydrolysis of MCC or pulp, often followed by
ultrasonic treatment, while NFC is obtained usually simply by mechanical grinding of
pulp.
Due to the dimensions in the nanoscale, whiskers, NCC and NFC have different optical, magnetic and mechanical properties from conventional cellulose based materials.
It has been found out that in aqueous colloidal suspensions cellulose whiskers form chiral nematic phases, and that the whiskers can be aligned by a magnetic field [62]. NFC
has been used to make optically transparent paper [69] and to enhance the mechanical
strength of paper [67]. Especially the mechanical properties of nanocellulose, for example the elastic modulus being close to the high theoretical value of crystalline cellulose
(167.5 GPa [70]), have gained much attention. The superior properties of nanocellulose
enable numerous application possibilities in security papers, electronic devices, bioartificial implants, bio-NMR, biodegradable composites, films, barriers, and packaging
materials [68].
Nanocellulose can be hydrophilic due to the hydroxy groups of cellulose or if hydrolysis has been conducted using sulfuric acid, which creates sulfate ester units. Cellulose
nanocrystals have shown limited dispersibility in water, some dispersibility in aqueous
mixtures and in organic solvents with high dielectric constants (e.g. ethylene glycol)
and tendency to aggregate in highly hydrophobic solutions [68]. In paper VI, MCC
and NFC samples were immersed in different solvents (water, ethanol and acetone)
and characterized using small-angle x-ray scattering. The crystallite dimensions were
determined for the dry NFC powder samples using XRD (paper VI), and the results
obtained were used in the atomistic molecular dynamics simulations to study the structure of NFC [71]. As a result in those simulations, twisting of the nanofibrils traced
down to the hydrogen bonding between the cellulose chains was detected [71].
10
3
3.1
3 THEORY AND METHODS
Theory and methods
X-ray microtomography
X-ray microtomography (µCT) has turned out to be a suitable method to study the
anatomy and morphology of plants at the cellular level. Among the first µCT studies
on wood, Steppe et al. characterized the structure of vessels in oak and beech with
the resolution of 10 µm [72]. After that several studies with a resolution of the order
of 1 µm have been conducted on numerous wood species using both synchrotron and
laboratory set-ups: for instance, Scots pine, beech, movingui, afzelia [73], oak, Norway
spruce, common yew [74], loblolly pine, Douglas fir, eucalyptus, and teak [75] have been
characterized. In these studies, the anatomical features of wood, the vessel and fiber
size distributions, the cell wall thicknesses and the porosities, have been determined
with high statistical accuracy enabled by the 3D data. The interest is now aiming
towards the possibilities of this technique to study the dynamics of wood as a function
of moisture or mechanical changes. Derome et al. studied spruce wood as a function
of moisture changes using phase-contrast synchrotron µCT, and they detected that
swelling and shrinkage were larger and more hysteretic for latewood than for earlywood
[76]. They explained these observations by the larger amount of cell wall materials in
the latewood compared to earlywood.
The µCT technique is based on the x-ray images taken at different angles of the
sample, so either the sample is or the detector and the source are rotated during the
measurements. The intensities of the x-rays are measured by the detector at various
angles of the sample, and from the stack of the two-dimensional images obtained, the
three-dimensional structure of the sample can be reconstructed using the backprojection algorithm. The contrast in the images arises from the differences in the linear
attenuation coefficients of the materials. Attenuation can be assumed to be a sum of
two effects, photoelectric absorption and scattering, because pair production does not
occur below 1 MeV photon energy [77]. For the samples including heavy elements, the
scattering cross-section is small compared to that of photoelectric absorption, and thus
the images taken of these samples can be considered as pure absorption images. For
the samples consisting of only light elements, the phase shift due to the scattering of
the rays has to be considered, and it can be used to enhance the contrast of the images.
On the other hand, if the phase shift is not taken into account, it causes artifacts in
the images.
When a slice of the density matrix of the sample, f (x, y), is considered, the measured
projection is its Radon transformation, pθ (t) (θ denotes here the projection angle and
t the distance of the ray from the rotation axis, so that t = x cos θ + y sin θ) [78]. The
computation of the image from the projection is based on the Fourier slice theorem:
the Fourier transform of the projection, Gθ (w), is equal to a radial line of the twodimensional Fourier transform of the object, F (u, v) [78]. The object slice f (x, y) can
3 THEORY AND METHODS
11
Figure 5: The coordinates, the projection angle θ and the detector plane
for the cone beam geometry. The
relation between a and a0 is a =
a0 S/(S + D) and that between b and
b0 is b = b0 S/(S + D), where S is the
distance between the source and the
rotation center and D is the distance
between the detector and the rotation center.
thus be solved from F (u, v) by using the inverse Fourier transform:
Z ∞Z ∞
f (x, y) =
F (u, v) exp (i2π(ux + vy))dudv.
−∞
(1)
−∞
In practice, a finite number of projections is taken, which means that F (u, v) consists of
points along a finite number of radial lines, and interpolation must be used to get these
points on a square grid. The practical algorithm used mostly for the reconstructions
is the filtered back projection. In the case of the parallel beam geometry, the filtering
is applied by weighting the N projections in the frequency domain by 2π|w|/N, where
w is a given frequency. The factor |w| represents the Jacobian for the variable change
between polar and rectangular coordinates needed for the inverse Fourier transform [78].
The resulting reconstruction is obtained by summing up the inverse Fourier transforms
of the weighted projections [78]:
Z πZ ∞
f (x, y) =
Gθ (w)|w| exp (i2πwt)dwdθ.
(2)
0
−∞
A volume (f (x, y, z)) could then be reconstructed from the stack of the projections of
the slices f (x, y) obtained by small movements in the z direction.
The projections in this study were obtained using a cone beam geometry. The
geometry has to be taken into account by coordinate changes, which transform the
projections to correspond to the parallel geometry. This leads to the weighted filtered
back projection algorithm, where the weights depend on the positions of the source,
the sample and the detector. Feldkamp et al. presented the first practical formulation
for the backprojection for the cone beam geometry [79]. According to this algorithm,
12
3 THEORY AND METHODS
the reconstruction is computed by [78]:
1
f (x, y, z) =
2
Z
2π
0
S2
(S + x cos θ + y sin θ)2
S
√
p(θ, a, b) ∗ h(a)dθ,
S 2 + a2 + b2
(3)
where θ is the angle of the projection, S is the distance between the source and the
rotation center, p(θ, a, b) is the projection data obtained by the (virtual) plane detector
(see Fig. 5), ∗ denotes convolution, h(a) is defined as:
Z
h(a) = |w| exp (i2πwa)dw.
(4)
and
a=
S(−xsinθ + ycosθ)
;
S + x cos θ + y sin θ
b=
zS
.
S + x cos θ + y sin θ
(5)
The coordinates are presented in Fig. 5.
Phase-contrast technique
The refractive index describing the interactions of x-rays with a medium is n(λ) =
1 − δ(λ) + iβ(λ), where the imaginary part takes into account the absorption and the
real part corresponds to the change in the phase velocity, and λ is the wavelength of
radiation. The δ and β are related to the following factors:
δ(λ) =
re λ 2 N
(Z + f 0 (λ)),
2π V
and
(6)
re λ2 N 00
f (λ),
(7)
2π V
where re is the classical radius of electron, N is the number of the atoms, V is the
volume of the sample, Z is the atomic number, f 0 is the real part of the anomalous
scattering factor, and f 00 is the imaginary part of the anomalous scattering factor [80].
Methods, which are able to detect effects of refraction and scattering, are referred
as phase contrast techniques, because refraction and scattering depend on the phase
differences, which occur in the sample [81]. These methods are sensitive to changes
in refractive index at the boundaries of object, and thus the visibility of e.g. thin cell
walls is enhanced [75].
In this study, the measured data consisted of images, whose gray values were computed based only on absorption. For the dry wood samples, the contrast between the
cell wall materials and air was relatively high compared to that of the wet wood samples,
whose low contrast arose from the small differences in the absorption between water
and cell wall elements (paper I). The information on the phase would have enhanced
the contrast in the images. Especially for the organic samples the phase information
β(λ) = −
3 THEORY AND METHODS
13
Figure 6: The x-ray microtomography set-up at the University of Helsinki. A: nanofocus x-ray tube, B: computer controlled sample stage, C: CMOS flat panel detector.
Photo: Seppo Andersson.
Figure 7: The fiber lumina in the 99-dayold hybrid aspen sapling. The image was
prepared using Avizo 6.2 Fire software
(http://www.vsg3d.com/avizo/fire).
14
3 THEORY AND METHODS
Figure 8: The parenchyma ray cells segmented in air-dried silver birch xylem.
The selected parts corresponded together 3.27 mm in total length (in radial direction of the stem). The image was prepared using Avizo 6.2 Fire software
(http://www.vsg3d.com/avizo/fire).
3 THEORY AND METHODS
15
is important, because for the light elements the cross-section of scattering is significantly larger than the absorption cross-section considering the typical energies used in
µCT [75, 82].
The phase information can be measured indirectly by determining changes in the
phase differences caused by the medium. For instance, an x-ray interferometer can
be used, which creates two coherent beams. One of the beams is conducted to the
sample, and it causes the phase to advance with respect to the phase of the beam
in vacuum. An interferometer, which superimposes the waves, generates interference
pattern corresponding to the phase shift distribution. A phase change caused by the
structure of the sample alters the position of the pattern, which can be recorded by
a detector [77, 82]. For low-absorbing samples, the phase shift of the waves is always
present: it is visible as edge enhancements in the projection images. The phase information can be retrieved from the intensity data using e.g. the algorithm created by
Bronnikov [83]. He filtered the projections to separate the phase signal, and converted
the signal obtained so that the reconstruction of the phase data could be computed by
using the regular filtered backprojection algorithm.
Analysis of the µCT data on wood samples
The µCT data of this study was measured at the University of Helsinki using the set-up
presented in Fig. 6 and at the Centre for X-ray Tomography of the University of Ghent
(UGCT). The reconstructions of the data measured at the University of Helsinki were
computed using datos|x program (Phoenix|x-ray Systems and Services). The reconstructions were computed using the filtered backprojection algorithm for the cone beam
geometry, and the flat field, offset image and tilt compensation corrections were conducted. The volumes were rendered with VGStudio MAX (www.volumegraphics.com)
and Avizo 6.2 Fire (www.vsg3d.com/avizo/fire) softwares. The data measured at the
UGCT were reconstructed by Octopus program, which has been developed at the
UGCT [84–86].
The first steps in the data analysis were filtering and thresholding for the binarization of the reconstructed data. The data obtained at the UGCT was phase-filtered
using modified Bronnikov algorithm [85,86]. A suitable filter for the further processing
of the data of the wood samples was a bilateral filter, because it preserved the edges
in the image. The thresholding methods used had to be beneficial for the connected
structures like cell walls. Thus for example dual thresholding could be used.
From the µCT data on the hybrid aspen saplings, the fiber lumina were separated
using the tools of Morpho+ program [84] (Fig. 7). The ray and vessel cells were
excluded manually from the data. From the reconstructed fiber lumen volumes, the
width and length of lumina could be solved. The cell wall thicknesses were determined
using Matlab and the LocalThickness plug-in of ImageJ [87]. The latter determines the
16
3 THEORY AND METHODS
thickness distribution of the objects by computing the diameters of the largest spheres
that can be fitted inside the object. In Matlab the thickness distribution was computed
by the multiplication of the distance map and the skeleton of the image following the
formula given in [73].
The cross-sectional widths of the parenchyma ray cells for never-dried and air-dried
silver birch wood were determined using Avizo 6.2 Fire (www.vsg3d.com/avizo/fire).
The rays were segmented manually in every 5th -10th slice of the 3D data and interpolated in the intervening slices (Fig. 8). The mean of the thickness maxima of the
cross-sections was computed from the thickness distribution determined using the Euclidean distance transformation.
3.2
Small-angle x-ray scattering
Small-angle x-ray scattering (SAXS) is elastic scattering of the x-rays in the near
vicinity of the primary beam i.e. the scattering angle 0◦ . The scattering intensity of
the SAXS patterns arises from the electron density fluctuations of the sample. By
assuming the incoming and scattered waves as plane waves and by taking into account
only the single scattering, the amplitude of the elastic scattering can be computed as a
Fourier transform of the electron density. In x-ray scattering experiments, the intensity
measured by the detector is proportional to the square of the scattering amplitude.
The scaled (absolute) SAXS intensity (in units of cm−1 ) is defined as the differential
scattering cross-section per unit volume of the sample and counted as:
Iabs =
C
,
C0 ∆ΩT d
(8)
where C is the number of the scattered photons per second in the solid angle ∆Ω, C0
is the incoming flux of the photons, T is the transmission and d the thickness of the
sample [88].
The atomic scale inhomogeneities do not contribute to SAXS, so the resolution
of the technique is above the atomic scale. In most SAXS studies, the sample can be
regarded to consist of a matrix (phase I) with intrinsic inhomogeneities (phase II), which
can be either pores or particles. For example, in the case of wet wood, two phases are: I)
water in the lumina and cavities of the cell walls and the wet matrix and II) crystalline
cellulose. For dry wood, two different phases can be assumed to consist of I) cell wall
polymers and II) air. According to Babinet principle, the addition of a constant to the
scattering density does not alter the scattering curve, and thus the SAXS patterns of
the samples, which have the same contrast difference, are indistinguishable.
The scattered intensity of the system can be given as [89]:
< N >2
I(q) =< N >< F (q) > − < F (q) >
V
2
2
Z
0
∞
[1 − P (r, v)]
sin (qr)
4πr 2dr,
qr
(9)
3 THEORY AND METHODS
17
where N is the number of the scattering objects, F (q) is the scattering amplitude of
the object, V is the volume of the object, v is the mean sample volume per object,
and P (r, v) is the statistical distribution of the objects. The first term corresponds to
independent scattering from < N > objects, and the second term takes into account
the interparticle interference. For the isotropic, mono-disperse systems, the intensity
can be divided to the form factor, R(q), which depends on the particle shape, and to
the structure factor, Z(q), which arises from the interparticle interference effect, in a
simple way [90]:
I(q) ∝ R(q)Z(q).
(10)
In this case, the both factors depend only on the magnitude of the scattering vector q,
which is determined as q = 4π sin θ/λ, where θ is the half of the scattering angle and
λ is the wavelength of the radiation. If the interparticle interference does not play a
role (which is the case in dilute enough systems), Z(q) = 1, and the scattered intensity
depends only on the form factor.
The form factors for a sphere, ellipsoid and cylinder are presented in e.g. [90]. The
form factor for a cylinder with radius r and length l is [90]:
2
Z π/2 2J1 (qr sin α) sin (ql cos α/2)
sin αdα,
(11)
R(q) =
qr sin α
ql cos α/2
0
where J1 (x) is the first order Bessel function. For the analysis of the wood and cellulose
based samples, the crystalline parts of cellulose microfibrils were assumed as infinitely
long solid cylinders [91]. In this case, the form factor of infinitely long cylinder with a
circular cross-section was used and the intensity could be presented as [91, 92]:
2
1 J1 (qr)
I(q) ∝
Z(q).
(12)
q
qr
For only a few special cases, the structure factor can be computed analytically:
this is possible in the case of the particles interacting through hard-sphere potential or
screened Coulomb potential [90]. For the SAXS data analysis of hydrated pine wood
samples, Andersson et al. [92] used as a structure factor the function Z(q) = 1+J0 (qd),
which is the interference function for pairs of particles spaced with the mean distance
d, restricted to a plane and having a random orientation [93]. In this study, for wet
wood samples, a model based for a two-dimensional paracrystal [94, 95] was found out
to fit better the SAXS data (papers I and V) and using this model, it was possible to
study the dehydration of wood (paper I).
For most of the systems, it might not be possible to determine either the form or
structure factor. Still, several details of the sample structure can be found out using
SAXS. The SAXS intensities plotted in a logarithmic scale reveal the possible power
law behavior. If the power law is obeyed, the intensities follow the equation
I(q) ∝ q −α ,
(13)
18
3 THEORY AND METHODS
at enough wide q range (going beyond several order of magnitudes), and the exponent
(α) gives information on the morphology of the scattering objects. The value of α = 2
arises from the lamellar structures, the values α < 3 indicate that the objects are mass
fractals, and the values 3 < α < 4 correspond to surface fractals [96]. The value α = 4
indicates Porod law behavior implicating that the sample consists of two phases, which
have smooth and well-defined interfaces [88]. Porod law is usually obeyed at high q,
because it arises from the asymptotic behavior when q approaches infinity [89].
If the Porod region is found, then the specific surface of the phase I (or correspondingly, that of the phase II), Sp , can be computed in the case of the isotropic sample
using the equation:
A
,
(14)
Sp =
2π(∆p)2 φ1 ρ
where A is the proportional constant of the Porod law, ∆p is the scattering length
contrast between two phases, φ1 is the volume fraction of the phase I, and ρ is the
density of the phase I [88, 97]. The intensities in the equation above must be given in
absolute units. The scattering length contrast depends on the sample content. The
scattering length density for a molecule is computed as
P
re Z i
p=
,
(15)
Vm
where the sum is over the atoms of the molecule and Vm is the molecular volume.
For example, the value of 1.44 · 1011 1/cm2 was used for the ∆p for the dry sample
consisting of cellulose and air, and 5.0 · 1010 1/cm2 for the wet sample of cellulose and
water. The specific surface of cellulose could be solved using Eq. 14 for the dry and
wet MCC and NFC samples in paper VI.
For low q values close to zero, the Guinier approximation can be used, if the sample
can be assumed to be a dilute system of particles. In that case, the scattering function
is independent of particle shape and depends only on the size:
−rg2 q 2
I(q) = I0 exp (
),
3
(16)
where rg is the radius of gyration of the particle [98]. In paper VI, the Guinier approximation could be applied for the MCC powder immersed in water, ethanol and acetone
revealing differences in the values of rg of cellulose in different solvents. If both the
Guinier approximation (at low q) and Porod approximation (at high q) are applicable,
it is possible to compute the invariant Q
Z ∞
Q=
I(q)q 2dq,
(17)
0
which enables e.g. the computation of the average chord length (lc =
typical length scale in the structure of the object [98, 99].
4Q
)
πA
revealing the
3 THEORY AND METHODS
19
Figure 9: The SAXS set-up at the University of Helsinki. A: a sealed x-ray tube, B:
a monochromator, C: slits, D: a sample holder (in this case: a stretching device which
enables in situ mechanical testing during the scattering experiments), E: a vacuum
tube, F: HI-Star area detector. The exactly same set-up, except without the vacuum
tube and with a larger area detector, can be used for wide-angle x-ray scattering studies
using the perpendicular transmission geometry. Photo: Seppo Andersson.
The SAXS experiments of the paper V were performed at the beamline ID117 at the
synchrotron facility MAX-lab [100]. The other SAXS measurements were conducted
using the set up of the University of Helsinki, which is presented in Fig. 9.
3.3
Wide-angle x-ray scattering
XRD (or wide-angle x-ray scattering, WAXS) pattern is related to the Fourier transform of the electron density of the material similarly as in the case of SAXS, but in
WAXS large scattering angles are taken into consideration. The larger scattering angles
correspond to smaller structures observed in the sample: so, using WAXS, the structure
of the sample is revealed down to the atomic scale. When the material is crystalline,
the diffraction pattern can be predicted based on the crystal lattice. In reverse, the
diffraction maxima reveal the crystal structure. The position of the diffraction peak
hkl in the q axis arising from the monoclinic lattice (e.g. cellulose Iβ ) can be computed
from:
s 2
2
2
k
h
2hk
l
2π
+
−
cos γ +
sin2 γ,
(18)
|qhkl | =
sin γ
a
b
ab
c
where a, b and c are the lattice dimensions, and γ is the monoclinic angle.
The measurement geometry has to be chosen carefully for the anisotropic samples,
such as wood. In this study, WAXS measurements were conducted using three different
geometries: perpendicular transmission, symmetrical transmission and symmetrical
reflection. In the first mentioned case, the sample surface is positioned perpendicular
to the incoming beam and the scattering pattern of the transmitted x-rays is measured.
20
3 THEORY AND METHODS
Figure 10: Diffracting planes in
symmetrical transmission geometry.
In the case of the symmetrical transmission mode, the angle between the incoming beam
and the normal of the sample surface and the angle between the (point) detector and
the normal of the sample surface are kept the same (Fig. 10). The situation in the case
of the symmetrical reflection geometry is exactly the same, but the sample surface is
turned 90 degrees compared to the symmetrical transmission case.
WAXS is a widely used and established method to determine the crystallinity index
of partially crystalline materials, e.g. wood, pulp and microcrystalline cellulose [21,101,
102]. The crystallinity index is determined as a ratio of the integrated intensities of
the reflections of the crystalline material and the total experimental intensity curve.
The separation of the contributions of the amorphous and crystalline components to
estimate the crystallinity index was established by Ruland [103] and Vonk [104]. In
the case of wood, the ratio is computed between the scattering curve of the crystalline
cellulose and the total scattering intensity of the sample including both crystalline and
amorphous cellulose, hemicelluloses, lignin, and extractives. The crystallinity index
corresponds to the weight fraction of crystalline cellulose among the whole sample.
The texture of the sample, e.g. the fiber orientation of wood, has to be taken into
account in the determination of crystallinity. To solve the absolute crystallinity value
for solid wood samples, they should be powdered, and the powder samples should be
measured using two different geometries, symmetrical transmission and reflection. The
crystallinity is then computed as a weighted mean of the crystallinities determined
using these two geometries, so that the weights of one-third and two-third are used
for the reflection and transmission data, respectively [101]. Relative crystallinities, i.e.
the values determined using only one geometry, can be compared, if the samples have
about the same texture: if solid wood samples were measured using only transmission
geometry, the samples should have about the same MFA so that their crystallinity
values could be compared reliably.
For dry wood and cellulose samples, the measured intensity curve was presented
as a linear combination of the theoretical diffraction pattern of crystalline cellulose Iβ
(based on the lattice parameters by Nishiyama et al. [19]) and the intensity curve of
3 THEORY AND METHODS
21
the amorphous components of cell wall. An experimental intensity curve of a sulfate
lignin sample was used as a model intensity for the amorphous background including
amorphous cellulose, lignin and hemicellulose [21]. The cellulose reflections were presented as Gaussians. For the most significant cellulose reflections, 200, 11̄0, 110, and
004, the full widths at half maximum (FWHM) and the positions were fitted, while
for all the other reflections only the intensities (scaling constants) were used as fitting
parameters. Also, the scaling constant for the amorphous background was one of the
fitting parameters.
For wet wood and the PEG impregnated Vasa samples, one extra component, the
scattering curve of water or PEG, respectively, had to be taken into account. Thus
water and pure PEG 1500 samples were measured and their WAXS patterns were used
as components in the fitting (Fig. 11). Water and PEG are organic molecules, so it was
possible to solve also their weight fractions among the samples using the corresponding
relations of the integrated curves. The free water content of the wet wood samples was
computed in papers I and II, and the PEG 1500 content was solved for the Vasa oak
samples in paper III.
For the wet wood samples, it was also noticed that by shifting the scattering curve
of the amorphous background related to q axis, the goodness of fit could be refined. So
it was possible to gain information also on the changes of the amorphous background
during drying. It was found that upon drying (from the never-dried to air-dried sample), the curve shifted about 0.1 Å−1 towards lower q values. This shift implicated that
changes occurred in the short range order of the matrix polymers.
The average dimensions of cellulose crystallites were computed based on the Scherrer equation [93], assuming that the diffraction peak and the instrumental broadening
were Gaussians:
Kλ
Bhkl = p
,
(19)
2
(∆2θ) − (∆2θinst )2 cos θ
where Bhkl is the average size of crystallite based on the reflection hkl, K is a constant
(between 0.9-1.0), λ is the wavelength of the radiation, ∆2θ is the FWHM of the
reflection hkl, 2θ is the position of the reflection in the scattering angle axis, and
∆2θinst is the instrumental broadening of the reflection determined using a calibration
sample (e.g. silicon or hexamethylenetetramine). The dimensions of the crystallites
were determined along the cellulose chain direction by using the reflection 004 and
perpendicular to the chain direction by using equatorial reflections 110, 110 and 200.
The MFA was computed using the azimuthal intensity profile of cellulose reflection
004 and/or 200 by fitting a model to the profile. It was assumed that the angle of cell
rotation respect to incident beam and the tilt were zero. When the whole azimuthal
intensity profile (360◦ ) of the 200 reflection arising from one wall layer of rectangular
cell with the MFA of Z is observed, at least four peaks are detected. The peaks at
the angles of 180◦ +Z and at +Z arise from the microfibrils of the front cell wall, and
22
3 THEORY AND METHODS
Figure 11: A: For the wet wood samples, the experimental intensity curve was presented
as a linear combination of the computed diffraction pattern of crystalline cellulose
Iβ (the stars denote the positions of the Gaussians), the measured intensity curve
of a sulfate lignin sample (amorphous background), and water. B: For the PEGimpregnated Vasa samples, the experimental intensity curve was presented as a linear
combination of the intensities of crystalline cellulose Iβ , amorphous background and
pure PEG 1500.
the peaks at -Z and 180◦ -Z arise from the back cell wall. Diffraction from (200) lattice
planes from two side cell walls causes narrow peaks at 0◦ and 180◦ [51, 105]. The MFA
distribution was modeled as a sum of Gaussians. The azimuthal intensity profiles were
modeled using pairs of Gaussians, so that the fitting parameters were the positions,
the widths and the intensities of the Gaussians. For a macroscopic wood sample, the
shape of the MFA distribution can be complex due to the fact, that many cell wall
layers contribute to the pattern and the cell shape can be irregular. Thus the use of
both reflections, 004 and 200, is advantageous, because they give information on the
cell shapes [105].
4 RESULTS AND DISCUSSION
4
4.1
23
Results and discussion
Cellulose crystallite dimensions in native samples
Table 1 presents the average lengths and widths of cellulose crystallites both in native
plant materials of various species and in chemically treated cellulose samples (pulp,
MCC, NFC, and whiskers). In all normal wood samples, the crystallite width was
3.0 ± 0.1 nm, while for TW larger crystallite widths (3.1-4.7 nm) were obtained. The
largest crystallite width among the native plant materials was obtained for cotton
linter (7.1 nm). It can be noted, that the larger crystallite widths were obtained for
the samples with higher cellulose content and lower lignin content. The crystallite
widths determined using the cellulose reflection 200 are presented as a function of the
lignin content of the samples in Fig. 12.
A similar kind of trend could not be found when considering the crystallite lengths.
The longest crystallites were found in Norway spruce and hybrid aspen wood (up to
40 nm), while in cotton linter and flax fibers with high cellulose content compared to
matrix materials, the crystallites were shorter (17-18 nm), but also in CW and juniper
with low cellulose content, the average crystallite length was found to be small (20 ± 2
nm) [10]. It was also found, that the crystallite length values varied strongly between
young saplings of hybrid aspen (between 26 and 40 nm). As high variation between
the samples of the same species as here has not been detected for mature trees in the
previous studies [16, 106].
Previous studies on the effects of the cell wall polysaccharides, especially hemicellulose, on cellulose microfibrils have been conducted by Tokoh et al. [107,108]. According
to their results on cellulose synthesized by Acetobacter xylinum, xylan [108] and mannan [107] affected the cellulose crystallite size, caused discontinuous disordered regions
along the microfibrils and changed cellulose Iα /Iβ ratio. Based on their results, mannan
caused more changes than xylan [108]. From this point of view, it would be interesting to compare the results of softwood species to those of hardwoods. No significant
difference in the crystallite dimensions between hard- and softwood species was found
in this study. However, definitely more results on hardwood species would be needed
to cover this topic more reliably.
4.2
Changes due to drying (papers I and II)
Changes in silver birch wood structure due to drying were studied at the cellular level
using µCT in paper I. As a result it was found that the cross-sectional width of the
parenchyma ray cells decreased due to drying. In paper II, dry samples of 3-month-old
hybrid aspen saplings were studied using µCT. In a few samples, regions of collapsed
cells were observed; these deformed cells may indicate TW cells, which have collapsed
24
4 RESULTS AND DISCUSSION
Table 1: The average width (W ) and length (L) of cellulose crystallites in different plant
species, in pulp and MCC made of them, and in NFC and whiskers. The accuracy is 0.1
nm for the widths and 2.0 nm for the lengths. CW=compression wood, TW=tension
wood, *=pulp made of spruce and pine wood and MCC made of that, + =never-dried
samples. References: a =V, b = [10], c = [110], d =I, e =II, f =III, g =IV, h = [109], i =VI,
j
= [111]
Species
Sprucea,b
Spruce CWb
Juniperb,c
Bircha,d
Hybrid aspene
Hybrid aspen TWd,e
European oakf
Bamboog
Cotton lintera
.
Flax fibersa
Hemp stalkh
Rice huskh
Nanocellulose
samples
NFCi
Whisker,
freezedried (f)j
Whisker,
airdriedj
Whisker,
f+neutralizedj
Native
Pulp
MCC
W [nm] L [nm] W [nm] L [nm] W [nm] L [nm]
3.1
35-40 4.4-5.1* 23-26* 5.3-5.7*
27*
3.1
20
2.8-2.9
21
4.0
16
+
+
3.0 -3.2
4.3
23
4.6
22
3.0
26-40
+
3.2-4.7
28-39
3.0
22-28
3.0-3.2
21-33
7.1
18
8.8
30
3.9
18
4.7
21
4.7
24
4.8
19
5.2
23
5.2
21
4.1
12
4.6
8.3
4.7
6.0
W [nm] L [nm] Aspect
ratio
5.3-5.4
14
2.6
5.4
19
3.5
5.3
20
3.8
5.3
22
4.2
4 RESULTS AND DISCUSSION
25
Figure 12: Crystallite widths as a function of lignin contents of the samples. Stars
denote native plant samples: A) cotton linter [V] B) flax fibers [V] C) hemp stalks [109]
D) hybrid aspen (lignin content from [42], width from [II]) E) Norway spruce [10] F)
juniper [10]. Open spheres denote the pulp and MCC samples: a) MCC from cotton
linter [V] b) MCC from flax [V] c) MCC from conifer kraft pulp [V] d) conifer kraft
pulp [V] e) flax pulp [V]. The accuracy for the crystallite widths is about 0.1 nm.
due to drying. According to Bariska, many biological and physical factors can cause
collapse of wood, and one of these factors is the TW formation [112]. The collapse of
the TW cells upon drying may be caused due to the lack of lignin in the G-layer [112].
The drying of TW with G-layer has been studied by Fang et al. [113]. They observed
that in G-fiber, the lumen size increased upon drying, so the G-layer shrank outwards
due to drying. These drying behaviors of TW fibers might explain, why significantly
thicker cell walls corresponding to G-layers were not detected in the µCT images of
the dry hybrid aspen saplings, which should have included TW based on the WAXS
results (paper II).
The drying deformations of cellulose crystallites were studied in normal wood of approximately 12-year-old silver birch, 7-year-old European aspen and in young saplings
of hybrid aspen (3-month-old), silver birch, Norway spruce, and Scots pine (2-yearold). TW samples of European aspen and hybrid aspen were included in the study and
so it was possible to compare effects of different cell wall compositions on the drying
changes. The normal wood samples represented various ages and also various MFA
values.
Young’s modulus is known to decrease as a function of the increasing moisture
content in wood [114]. Also other mechanical properties are strongly dependent on
moisture content. These properties arise from the wood structure. For instance, it is
known that these effects depend on the MFA. The high MFA increases the dependence
of the structure on hydrogen and van der Waals bonding and makes the mechanical
26
4 RESULTS AND DISCUSSION
weakening effect of the water molecules larger [27]. By comparing the drying results
obtained for the samples with different MFAs, it was possible to get information on
that, if the differences in the MFAs could be linked also to the drying deformations at
the level of the elementary cellulose crystallites.
In the different wood samples studied, the cellulose unit cell shrank 0-0.3% longitudinally and expanded 0-0.5% in transverse direction upon drying based on the changes
in the positions of the cellulose reflections 004 and 200, respectively. The deformations
were explained by the behavior of the water-filled hemicellulose-lignin matrix: the wet
matrix induced stresses, which released upon drying and caused the cellulose crystal
lattice expansion and/or shrinkage. Also the widths of the cellulose reflections 200 and
004 changed during drying: in the both reflections the FWHM increased, which was
explained by the increased strain and disorder of cellulose chains. The change was
larger parallel to the chains (5-20%) than perpendicular to the chains (0-6%), which
was explained by the larger strain along the chains caused by the wet matrix supporting
the native ordering of the microfibril network.
The increase in the transverse disorder of the chains upon drying was larger in
TW than in corresponding normal wood, but the axial unit cell shrinkage was not as
significant in TW as in normal wood. The smaller amount of lignin in TW cell wall
may explain these differences. Lignin consolidates the matrix, thus in the TW cell wall
with less lignin the matrix might not be as rigid for the microfibrils as in normal wood.
Also, changes in the MFA distributions were not detected upon drying in the normal
wood samples, but for TW samples a small increase was observed in the mean MFA
during drying. This could also be connected to the lower lignin content of TW matrix,
leading to reduced rigidity and allowing the change in the orientation of microfibrils.
To conclude, the drying deformations in the cellulose chain direction were similar
in the normal wood samples of all the species and ages studied. Perpendicular to
the cellulose chains differences were observed between the species but not between the
ages: small or no deformations were detected in aspen wood in this direction, whereas
in other species (birch, spruce and pine) significant changes both in the d200 spacing
(dhkl = 2π/qhkl ) and in the degree of the order of the chains in the corresponding
direction were observed. Very small or no transverse deformations of the aspen samples
might be connected to the low MFA values of that species. As a conclusion it can be
stated, that the matrix properties took part in the drying deformations of cellulose
crystallites, whereas the age did not play a role for the samples studied here. When
regarding normal wood, the MFA might play a role in the drying deformations of
crystallites.
4 RESULTS AND DISCUSSION
27
Figure 13: The three-dimensional structure of the cells in Vasa oak visualized using
x-ray microtomography. The length of the scale bar is 1 mm.
28
4.3
4 RESULTS AND DISCUSSION
Changes due to degradation of Vasa oak (paper III)
The structures of the cells in Vasa oak at the micrometer level (Fig. 13) corresponded to
those in recent oak [115] based on the µCT images. Even the tyloses (thin membranes in
the earlywood vessels) were still detectable in the Vasa oak cells. In the nanostructure
of the cell wall, differences between Vasa and recent oak were observed. The SAXS
patterns indicated a declined short range order between the cellulose microfibrils and a
higher porosity of Vasa oak compared to recent oak. These observations may be linked
to the changes that have occurred in the hemicellulose-lignin matrix. Degradation of
hemicellulose has been observed previously in Vasa oak using chemical methods [60].
The relative crystallinity values computed from the WAXS patterns revealed, that
the fraction of crystalline cellulose was lower in the Vasa samples compared to that of
recent oak. However, the average cellulose crystallite dimensions (the length of about
25 ± 2 nm and the width of 3.0 ± 0.1 nm) and the mean MFAs (4-9◦) showed no differences indicating that the crystalline parts of cellulose microfibrils were well preserved in
Vasa oak. One of the possible severe threats for Vasa is supposed to be the hydrolysis
of cellulose due to sulfuric, oxalic or other acids in Vasa [57]. Sulfuric acid was formed
in Vasa by oxidation of hydrogen sulfide (which was generated during the anaerobic
conditions under the sea) due to the atmospheric oxygen and humidity and catalyzed
by iron [57]. Based on the results of paper V, in which a clear increase in the crystallite width due to the acid hydrolysis of cellulose was detected, the cellulose crystallite
dimensions in Vasa oak showed no signs for the hydrolysis reactions. However, the
chemistry of Vasa is supposed to be complicated and thus this kind of comparison may
be too simple.
According to the size exclusion chromatography (SEC) results by Bjurhager [61],
the average molecular weight of holocellulose was lower in Vasa oak than in recent
oak. The lower average molecular weight of holocellulose correlated with the reduced
tensile strength of Vasa oak: down to 60% lower longitudinal tensile strength values
were obtained for the Vasa oak samples compared to recent oak [61]. Taking into
account both the WAXS results on the cellulose crystallite dimensions and the SEC
results on the decreased molecular weight of holocellulose, these results could indicate
that the changes have occurred in the surface chains and in the amorphous regions of
microfibrils. The WAXS results on Vasa oak may be taken as a sign of the durability of
crystalline structures of cellulose: the cellulose crystallites can survive and stay nearly
intact regardless of 333 years at the bottom of the sea.
4.4
Changes as a function of the radial position in bamboo
(paper IV)
The microscopic structure of bamboo is dependent on the radial position in the culm:
it is well known that the density of the fibers increases as a function of the distance
4 RESULTS AND DISCUSSION
29
Figure 14: Left axis: The mean MFA (circles) in bamboo as a function of the radial
position in the stem. Right axis: The length of crystallites (squares) in bamboo as
a function of the radial position in the stem. The samples are from the stems of five
different ages (0.5-10.5 years old). The values are from the paper IV.
from the inner part of the culm [116, 117]. In this study, interesting relations between
the nanostructure of bamboo and the radial position in a culm were detected. It was
observed, that both the average width and length of cellulose crystallites increased as
a function of the position from the inner part to the outer part in the bamboo stem.
This same phenomenon was detected in all the stems representing three different ages
(10.5, 4.5 and 0.5 years old). Also the crystallinities increased from the inner part to
the outer part, which is in harmony with the increase in the crystallite dimensions.
The mean MFA decreased from about 45◦ in the inner part to about 9◦ in the outer
part (Fig. 14). Similar decreasing trend in the mean MFA has been detected for
the wood stems studied from the pith to bark [21, 51–53]. In wood, the large MFA
in juvenile wood (in about ten first formed annual rings) has been connected to the
structural function of the fibers, which have to support the young thin sapling. The
smaller the cross-sectional area of the stem, the larger the stress e.g. due to wind
to an individual tracheid, and the higher MFA enables higher flexibility of the young
stem [12]. It must be noted, that the development of bamboo is different compared to
that of wood: bamboo produces one shoot, which grows to its ultimate height in a few
months. However, the dependence of the MFA on the radial position in the bamboo
stem may also be connected to the structural function of the supporting fibers similarly
as in the case of wood fibers.
30
4 RESULTS AND DISCUSSION
Figure 15: The cross-section
of the cellulose microfibril in
normal wood (left) and in tension wood (right) of hybrid aspen based on the WAXS results on the cellulose reflections 200, 11̄0 and 110.
4.5
Changes due to pulping and hydrolysis (papers V and VI)
In cotton, hemp and flax, the width and length of cellulose crystallites increased during the acid hydrolysis and pulping when compared to the crystal dimensions of the
corresponding native materials. In wood and rice husk, only the crystallite width enlarged in the case of pulping, whereas the crystallite length was largest in the native
samples (paper V, [109]). Interestingly, only in the wet samples of wood and rice husk,
the interference term arising from the short range order of cellulose microfibrils was
detected in the SAXS patterns (papers I and V, [109]). This might indicate that the
differences in the dimensional changes of crystallites due to pulping/acid hydrolysis
could be linked to the microfibril-matrix relationships.
The SAXS patterns indicated that in the wood and rice husk cell walls, microfibrils
are embedded in a hemicellulose-lignin matrix with a moderate short range order between them. Based on the WAXS results, when hemicelluloses and lignin are degraded
due to pulping and hydrolysis, cellulose crystallites are able to grow larger transversely,
but not longitudinally: the longitudinal order of the cellulose chains seemed to decrease
leading to shorter crystallite lengths in pulp and MCC than in the native materials.
In native cotton, hemp and flax the amorphous matrix did not have an impact on the
crystallites in the longitudinal direction, and no signs of the short range order between
the microfibrils was detected in the SAXS patterns. Thus in these plants, the crystalline parts of microfibrils were able to enlarge both transversely and longitudinally
upon the making of pulp and MCC (Table 1).
Also, an increase both in crystallite length and width was detected when MCC was
made of wood pulps (Table 1), which strengthens the interpretation, that here, in the
case of pulps, the matrix did not play a role anymore. It was also found out that the
crystallite widths in the NFC samples were about the same as the widths in the MCC
samples made of wood pulps (paper VI).
5 CONCLUSIONS AND FUTURE ASPECTS
5
31
Conclusions and future aspects
In the TW cell wall the average cellulose crystallite width was larger than in normal
wood. Fig. 15 presents the cross-sections of cellulose microfibrils in TW and normal
wood of hybrid aspen based on the WAXS results on the average crystallite dimensions
computed from the cellulose reflections 200, 11̄0 and 110. For TW with G-fibers,
cellulose content is higher and lignin content lower than those in normal wood [47].
The smaller amount of amorphous matrix materials with respect to crystalline cellulose
may be linked to the size of the cellulose crystallites in the TW cell wall: with smaller
amount of the bound matrix around the microfibrils, the crystalline regions may be able
to aggregate during or after the cellulose crystallization. Correspondingly, the increase
in crystallite widths upon pulping and hydrolysis was explained by the degradation of
the amorphous matrix materials, i.e. the decrease in hemicellulose and lignin content
(paper V).
It was found that the crystalline regions of microfibrils were in better order (corresponding to larger crystallite dimensions) in the never-dried samples compared to those
in the air-dried samples. This was explained by the hydrated hemicellulose-lignin matrix, which preserved the native ordering. Furthermore, in the never-dried normal
wood cell wall, the wet matrix maintained the microfibril network so, that this support
was more pronounced in the axial direction. Thus the drying deformations of cellulose crystallites and also the differences detected between the TW and normal wood
samples upon drying could be explained by the microfibril-matrix relations (paper I).
All the variations in the average crystallite widths observed in varying conditions
studied here (pulping, acid hydrolysis, drying, and reaction wood) were in accordance
with each others. The changes in the crystallite lengths detected due to the chemical
treatments (pulping and acid hydrolysis) and due to drying could be possibly linked to
each others, because the crystallite lengths were largest in the wet native wood samples,
in which the matrix was supposed to support the native ordering of the microfibrils
most. However, this kind of connection was not valid in the case of the reaction wood
samples studied: the results on the crystallite lengths in reaction wood showed that in
CW of Norway spruce, the average crystallite lengths were significantly shorter (around
20 nm) than in normal wood of spruce [10, 118], while in TW of hybrid aspen similar
values than in normal wood were obtained. Thus no connection between the different
matrix contents of reaction wood samples and the crystallite lengths could be detected
based on the results of this study.
The average crystallite width was the same in normal wood of all the species studied
(Norway spruce, Scots pine, silver birch, European aspen, hybrid aspen, European oak),
whereas the crystallite lengths varied significantly between the species (between 25 ± 2
nm obtained for oak and 40 ± 2 obtained for Norway spruce and hybrid aspen). It was
found that the average crystallite lengths varied strongly between 3-month-old saplings
32
5 CONCLUSIONS AND FUTURE ASPECTS
of hybrid aspen (between 26 ± 2 and 40 ± 2 nm), the average crystallite width being the
same in these young trees (3.0 ± 0.1 nm) (paper II). The number of the cellulose chains
generated in the cellulose synthesis has to be the same (36) in all wood species [15]. The
results of this study implicate, that in normal wood the crystallization of the chains
is stable and controlled transversely, whereas longitudinally more variation (lattice
defects and chain disorder) is generated both between the various species and in young
trees of the same species.
It was an interesting result, that the mean MFAs of the juvenile hybrid aspen
saplings grown in a greenhouse were significantly low, around 10◦ . For juvenile saplings
of other species (spruce, pine and birch) and grown at an outdoor field, the MFA
was determined to be significantly higher, around 30◦, compared to the corresponding
mature wood materials (paper II). The decreasing trend in the MFAs as a function of
the cambial age has been observed generally in softwoods and diffuse-porous hardwoods
[50] such as birch [52] and poplar [53]. However, in tropical wood species different kinds
of radial trends exist [50, 119].
It has been speculated that the high MFA in juvenile wood is connected to the mechanical response of young, thin stems [12,50], and thus the low MFA results obtained
for the hybrid aspen saplings were interesting. Wood with large MFA is more flexible,
thus the large MFA of juvenile trees has often been interpreted as an adaptation to
wind load on thin stems, which tend to bend along wind. Larger stems resist wind
instead of bending, and for this they need stiffness provided by the low MFA [50]. The
low MFA values of the greenhouse-grown saplings could be an indication of adjustment
to the growth conditions: in a greenhouse, saplings do not need to adjust their properties to wind load, but instead of that, they may adapt to fast growth rate and high
mechanical stability [120]. The increased stiffness is achieved by a low MFA. Other
possible explanation for the low MFA values detected in the hybrid aspen saplings
may be the TW formation, which is typical in young hardwood stems. Arcs of TW
cells were detected in the hybrid aspen saplings using microscopy with histochemical
staining and microtomography (paper II). However, other properties linked to the TW
formation, i.e. the larger crystallite widths and higher crystallinities, were detected
only in a few saplings among 21 saplings studied. Thus it could be speculated that the
G-layer was not fully developed in the young hybrid aspen saplings. More studies on
similar hybrid aspen trees grown in different conditions (at an outdoor field and in a
greenhouse) would be valuable for better understanding of the effects on the growing
conditions on the MFA and TW formation.
Studies trying to link the mechanical properties to the ultrastructure of the wood
cell wall are still needed. In situ x-ray scattering experiments as a function of mechanical tension have been conducted already on never-dried silver birch wood [121],
wet kraft cooked Norway spruce pulp [122], dry Norway spruce wood [123], CW of
Norway spruce [124], and single pine wood tracheid cell [125]. On-going in situ SAXS
5 CONCLUSIONS AND FUTURE ASPECTS
33
studies with mechanical tests on wet CW samples of Scots pine at varying temperatures will give further information on the nature of the structure-function-relationships
of the wood cell wall. Similar measurements using WAXS would also be extremely
interesting.
The research on Vasa continues by several research groups using wide spectrum of
experimental techniques. For instance, on-going studies on Vasa oak are trying to link
the chemical degradation of oak wood to the mechanical response [61]. Those results
reveal connections between the matrix materials and the mechanical properties of oak
wood, giving at the same time extremely important information on the state of the
historically valuable Vasa ship.
To understand the structure-function relationships of the hierarchical biomaterials, information on several different size scales is needed. Thus usually at least three
different experimental methods are needed to cover all the size scales relevant for the
samples studied. The drying of wood was studied here both at the cellular and the
nanometer level of the cell wall using µCT, SAXS and WAXS. However, it would be
interesting to cover also the scale between these two levels. For instance, results on the
drying behavior of the wood cell wall using phase-contrast tomography with resolution
down to 100 nm could be valuable. Results on tomography studies on the drying of
never-dried CW and TW can not be found yet, so it would be interesting to conduct
these experiments with the resolution down to 100 nm.
Phase-contrast tomography experiments with high resolution are needed also for
the structural studies of nanocellulose based materials. The three-dimensional information on the nanocellulose fibril network is needed for the modeling of the optical and
mechanical properties of the NFC applications, for instance papers and films made of
NFC. These models depend strongly on the physical structure of the fibril networks,
and three-dimensional data with enough high resolution could be obtained using phasecontrast x-ray tomography technique.
Plant cell walls form an abundant renewable resource on the Earth, and there exists nowadays a strong intention for the fuel generation from this biomass. However,
currently the yield of fermentable sugars is low and the whole process is inefficient.
Deeper understanding on the three-dimensional design and structure of the cell walls
with knowledge on the structure-function relationships would pave the way for redesigning of the plant cell walls: increasing the cell wall biomass and changing the cell
wall structure more susceptible to degradation are examples of the goals, which could
be achieved [3]. This requires combining physical, chemical, biological, and genetical
knowledge on the composition, function and structure of the plant cell wall.
34
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