Wood Sci Technol (2007) 41:443–460

DOI 10.1007/s00226-006-0121-6

ORIGINAL

Cellulose microfibril aggregates and their size variation

with cell wall type

Lloyd Donaldson

Received: 3 August 2006 / Published online: 19 January 2007

Springer-Verlag 2007

Abstract The organization of wood cell wall components involves aggregates

of cellulose microfibrils and matrix known as macrofibrils. A combination of
field emission electron microscopy and environmental scanning electron
microscopy was used to visualise the organization of macrofibrils in different
cell wall types comparing normal and reaction wood of radiata pine and
poplar as examples of a typical softwood and hardwood. The size of macro-
fibrils is shown to vary among cell wall types with the smallest structures
occurring in the gelatinous layer of tension wood (14 nm) and the largest
structures in the S2L layer of compression wood (23 nm). A positive corre-
lation between macrofibril size and degree of lignification is observed, with
macrofibrils apparently increasing in size in more highly lignified cell wall
types. The fibrillar structure of the secondary wall varies from microfibril-
sized structures of 3–4 nm up to large aggregates of 60 nm diameter. The size
of macrofibrils also varies slightly among adjacent cells of the same cell wall
type. Macrofibrils occur predominantly in a random arrangement, although
radial and tangential lamellae may sometimes be seen in individual cells.

Introduction

The organization of the components comprising the wood cell wall has long
been a subject of investigation because of its importance in understanding the
mechanical properties of wood. The first detailed investigation of cell wall

L. Donaldson (&)
Cellwall Biotechnology Centre, Scion, Private Bag 3020, Rotorua, New Zealand
e-mail: lloyd.donaldson@scionresearch.com

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ultrastructure to investigate the arrangement of cellulose and matrix used

potassium permanganate staining of the lignified matrix combined with
transmission electron microscopy (Kerr and Goring 1975). This led to the
proposal of an ‘‘interrupted lamellae’’ model, which describes cellulose mi-
crofibrils in discontinuous tangential lamellae that are embedded in a lignin–
hemicellulose matrix. This model was supported by a number of subsequent
studies and was not challenged until 1993 when Sell and Zimmermann pub-
lished a description of radially arranged fibrils on the transverse fracture
surfaces of spruce and white fir wood using field emission scanning electron
microscopy (Sell and Zimmermann 1993a, b; Zimmermann and Sell 1997). In
contrast to radial and tangential structures, random arrangements have also
been described by Donaldson and Frankland (2004) and Zimmermann et al.
(2006).
In addition to the debate on the arrangement of cell wall components, there
has been a parallel discussion of exactly what constitutes a cellulose micro-
fibril. This discussion has been clouded by method-specific differences, and by
the tendency of microfibrils to form aggregates, which was not recognised
until relatively recently (Kataoka et al. 1992; Hult et al. 2001a). At the present
time, the 3–4 nm fibrils observed by electron microscopy are thought to cor-
respond to the 2 nm fibrils observed by NMR and X-ray diffraction techniques
(Jakob et al. 1995; Newman 1994, 1999). These are the smallest fibril struc-
tures that have been observed in cell walls and are thus considered to be
microfibrils (Preston 1974), also known as elementary fibrils (Frey-Wyssling
1968). At least in the case of electron microscopy, these microfibrils are
usually visualised by virtue of the associated matrix since cellulose itself
probably is not stained by conventional heavy metal salts (Heyn 1966). Thus,
these microfibrils likely have both cellulose and hemicellulose components
(Preston 1974; Salmén and Olsson 1998; Awano et al. 2000).
Microfibril aggregates (macrofibrils) have been described using a range of
techniques often applied to developing cells (Kataoka et al. 1992; Awano
et al. 2000), delignified fibres (Kraft pulp) (Daniel and Duchesne 1998; Bar-
dage et al. 2003), or decayed cell walls (Daniel et al. 2004a, b). Techniques
used include NMR (Hult et al. 2001a, b), scanning probe microscopy (Hanley
and Gray 1994, 1999; Fahlén and Salmén 2002, 2003), field emission scanning
electron microscopy (Kataoka et al. 1992; Daniel and Duchesne 1998;
Duchesne and Daniel 1999; Awano et al. 2000; Duchesne et al. 2001; Daniel
2003; Daniel et al. 2004a, b) and transmission electron microscopy (Fengel
1970; Bardage et al. 2003; Daniel et al. 2004b). These studies have shown that
microfibril aggregates are common in chemically or physically altered cell
walls, but little information is available on cellulose aggregates in native cell
walls or for different types of cell wall such as reaction wood, for example.
The properties of wood-based materials, including solid wood and fibre-
based products, depend on the properties of the chemical constituents and
their arrangement and interaction within the cell wall. Information on these
interactions at the macroscopic level is well documented, but at the micro-
scopic level, more work is needed to understand the interaction of wood

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polymers and the effects of processing (Salmén 2004). For example, several
studies have shown that microfibril aggregates vary in size with different types
of treatment, especially chemical pulping. Bardage et al. (2003) found that
macrofibril diameter among various Kraft pulps showed a distinct peak of
fibril size at 18–20 nm, similar to the size found by Fengel (1970). Several
studies have shown that cellulose aggregation is related to hemicelluloses
content in pulp where cellulose aggregate size increases with decreasing
hemicelluloses content (Hult 2001a, b; Duchesne et al. 2001). Fahlén and
Salmén (2002) measured cellulose aggregate diameters of 15–25 nm using
scanning probe microscopy. For unprocessed spruce wood, the average
diameter was 18 nm, while for Kraft pulp fibres, the diameter was 23 nm, with
both samples varying from less than 10 nm to more than 40 nm (Fahlén and
Salmén 2002).
This study attempts to determine the relationship between macrofibrils,
microfibrils and matrix and how these components vary among cell wall types,
including normal and reaction wood of radiata pine and poplar as examples of
a typical softwood and hardwood, respectively.

Materials and methods

Samples of normal and reaction wood of Pinus radiata D. Don and Populus
deltoides Bartr. ex Marsh. were collected and stored in formalin aceto-alcohol
(FAA)1 for a minimum of 6 months. Sample blocks were washed in water and
sectioned in the transverse plane using a sledge microtome at a thickness of
100 lm. Sections were washed in ethanol and clamped between two micro-
scope slides to prevent curling before vacuum drying at room temperature for
1 h. Sections were mounted on aluminium stubs using colloidal silver and
coated with approximately 12 nm of chromium in a sputter coater. Sections
were examined using a JEOL 6700F field emission scanning electron micro-
scope (FESEM) at an accelerating voltage of 3 kV.
An additional sample of never-dried radiata pine sapwood was prepared as
above, but without exposure to fixative or to ethanol. Some sections were
examined, fully hydrated, using a FEI Quanta environmental scanning elec-
tron microscope (ESEM) at 7 kV in a water vapour atmosphere at 4.5 Torr
and a temperature of 2C. The same sections were then air dried and exam-
ined by FESEM, either with or without chromium coating. In the case of
uncoated samples the accelerating voltage was 0.5–1 kV.
Samples of radiata pine holocellulose were prepared using peracetic acid at
90C for 4 h and embedded in Spurr resin, followed by sectioning at a
thickness of 2 lm using a glass knife. Sections were mounted on coverslip
glass and briefly etched for 20 s in sodium ethoxide to expose microfibrils at
the surface of the section while retaining the supporting resin within the
section to avoid collapse and loss of structure. Sections were coated with 5 nm

1
A mixture of 90% ethanol, 5% formaldehyde, and 5% glacial acetic acid.

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of chromium rather than 12 nm to give improved resolution, and examined by

FESEM.
FESEM images showing macrofibrils were examined to determine the
arrangement and size of the macrofibrils among all of the samples, except for
the wet samples examined by ESEM where the structure was partially ob-
scured by the water film. Size was determined using Digital Optics V++
software. Briefly, the image was marked close to each side of a macrofibril to
be measured. The tool uses an edge-finding algorithm, and determines the full
width at half-maximum height of each macrofibril along a brightness profile,
as shown in Fig. 1. This method was found to be more accurate than
attempting to either segment the SEM image directly, due to local variations
in image contrast that prevent accurate segmentation, or to manual marking
of the edges of each macrofibril, which was too subjective. Fifty macrofibrils
were measured for each of five different tracheids or fibres for normal and
compression wood of radiata pine, as well as normal and tension wood of
poplar, for a total of 1,000 measurements. Additional measurements were

Fig. 1 A method for detection and measurement of macrofibrils that estimates the full width at
half-maximum height (FWHM) of selected objects. Two markers are placed on either side of the
object to be measured. A brightness profile is generated and the FWHM is measured giving an
unbiased estimate of macrofibril diameter. Multiple objects can be measured on a single profile

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made in the cell corner middle lamella region of normal wood samples of both
species for comparison. Results were analysed using nested analysis of vari-
ance (Sokal and Rohlf 1981).

Results and discussion

Macrofibrils

Our simple method of preparation was found to reliably reveal macrofibrils on

the transverse cut surfaces of all of the tracheids in our sections for both

Fig. 2 Field emission scanning electron micrographs of macrofibrils in normal wood coated with
12 nm of chromium: a S2 region of Pinus radiata tracheid. Macrofibrils extend deep into the cell
wall (arrow). Scale bar 1 lm; b S2 region of Populus deltoides fibre showing randomly arranged
macrofibrils. Scale bar 0.5 lm

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radiata pine and poplar (Fig. 2). Macrofibrils were readily observed in the S2
region because they are visible in cross-section (except for cells with high
microfibril angle). However, these structures could also be seen in the S1 and
S3 layers in some views where these layers were exposed longitudinally. The
primary wall also contained macrofibrils that were easily seen due to stretching
and fracture between the secondary wall and middle lamella (Fig. 2). Some
views indicated that macrofibrils were not only surface features, but extended
deep into the cell wall (Fig. 2).
Macrofibrils were remarkably similar in appearance between radiata pine
and poplar. The radiata pine cell wall appeared less porous than poplar
(Fig. 2), which may reflect the greater lignification of the cell wall in radiata
pine tracheids (Donaldson 1985; Donaldson et al. 2001). It is assumed that
because lignification occurs by infilling of pores (Donaldson 1994), that a more
highly lignified cell wall region will be less porous giving a denser more
compact appearance by FESEM imaging as it does by TEM imaging (Don-
aldson and Frankland 2004). Macrofibrils in tension wood were slightly
smaller than in normal wood of poplar (Fig. 3), while compression wood in
radiata pine showed larger macrofibrils compared to normal wood with some
variation in the organisation of macrofibrils (Fig. 3). The inner S2 region of
radiata pine compression wood contained macrofibrils with a tendency to-
wards radial alignment, while in some cells there was a distinct inner region
adjacent to the lumen where macrofibrils were randomly arranged and
noticeably smaller (not shown). In the outer S2L region, macrofibrils were
distinctly larger and more randomly arranged and the cell wall was less porous
(Fig. 3), probably due to the greater lignification in this region of the sec-
ondary wall (Donaldson et al. 1999).
The G-layer of tension wood fibres was very sensitive, showing a 10–30%
radial shrinkage within a few seconds of exposure to the electron beam. Most
of this shrinkage appeared to be in the outer part of the G-layer, suggesting
that this region is very porous and delicate due to the minimal amount of
supporting matrix material (Clair et al. 2005). The microfibril orientation in
the G-layer could often be determined by observation of the macrofibril
structure and appeared to be more or less vertical, in contrast to the main part
of the secondary wall, which had a larger microfibril angle. Microfibril ori-
entation did not seem to affect the appearance of macrofibrils or their
arrangement but this requires more detailed investigation. The G-layer of
poplar may sometimes be very weakly lignified (Joseleau et al. 2004; Pilate
et al. 2004), especially at the lumen surface (Gierlinger and Schwanninger
2006). In Fig. 3, the lumen surface of the G-layer shows an amorphous, more
solid structure without any noticeable fibrillar texture, suggesting that it may
be lignified (Joseleau et al. 2004; Pilate et al. 2004; Gierlinger and Schwann-
inger 2006).
The arrangement of either cellulose microfibrils or their aggregates has
been a controversial topic. Several reports have indicated the presence of a
radial pattern of macrofibrils or of matrix particles on transverse fracture faces
of wood (Sell and Zimmermann 1993a, b), or in thin sections of compression

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Fig. 3 Field emission scanning electron micrographs of macrofibrils in reaction wood: a S2 and
S2L regions of P. radiata compression wood tracheid. Samples are coated with 12 nm of chromium.
Scale bar = 0.5 lm; b G-layer of P. deltoides tension wood fibre. Scale bar 0.25 lm

wood (Singh et al. 1998), and decayed wood (Larsen et al. 1995). Fahlén and
Salmén (2002) suggested that the radial aggregates observed on fracture faces
are the result of energy release during fracturing, and presumably a tangential
pattern might arise in the same way. In spite of reports of radial patterns in
decayed wood and in reaction wood, there is little evidence of radial patterns
in normal or untreated wood from either softwoods or hardwoods other than
for fracture surfaces (Sell and Zimmermann 1993a, b; Fahlén and Salmén
2002). In comparison, there are numerous reports of tangential arrangements
in normal wood (Kerr and Goring 1975; Ruel et al. 1978; Fahlén and Salmén
2002). While some results could be due to patterns formed by overlying mi-
crofibrils in thin sections observed by TEM (Donaldson 2001a), these patterns

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have also been observed by FESEM (Terashima et al. 2004) and by scanning
probe microscopy (Fahlén and Salmén 2002, 2003) where such artefacts
cannot occur. In the current study, macrofibrils in both radiata pine and poplar
were mostly randomly arranged. In some cells, tangential patterns could be
seen (Fig. 2a), but this was uncommon. In compression wood, there was some
indication of radial patterns (Fig. 3), which was more pronounced in some
cells than in others. It is likely that the process of cutting and/or fracturing
during the preparation of samples for microscopy may result in slight distor-
tions that can change a random arrangement of small objects into an apparent
tangential or radial arrangement (Zimmermann et al. 2006). Until some
alternative method is developed for preparation of cross-sections of cell walls
without localised chemical or physical disruption, the evidence from the
present study suggests that macrofibrils are usually randomly arranged, but
that radial or tangential patterns can occur in some individual cells or wood
types (Donaldson and Frankland 2004; Zimmermann et al. 2006). It should be
emphasised that there is no reason why one cell cannot differ from its
neighbours with respect to cell wall organization. Certainly this is the case for
microfibril angle (Donaldson 1998).
In general, descriptions of cell wall arrangement seem to emphasise a
model that must apply to all cells of a particular type (Kerr and Goring 1975;
Larsen et al. 1995; Fahlén and Salmén 2002). A quantitative approach, which
measures the number of cells with random, tangential or radial patterns, might
prove worthwhile in determining cell to cell variation in the arrangement of
cell wall components provided that these features are not related to prepa-
ration method. The observation that different patterns seem to vary among
adjacent cells suggests that these are real variations rather than artefacts of
specimen preparation.

Effect of metal coating

Conventional sample preparation for scanning electron microscopy requires

coating the sample with a thin layer of metal such as gold or platinum. For
FESEM, chromium is recommended because a very thin, featureless layer can
be applied by sputtering. However, with modern electron microscopes, it is
also possible to examine samples at low accelerating voltage (0.1–1 kV)
without metal coating. Although resolution may be reduced by a few na-
nometers under these conditions, there is also the possibility of observing
more fine structure without the interfering layer of metal.
Because macrofibrils are relatively small objects, typically 10–60 nm in
diameter, a layer of chromium 12 nm thick would be expected to have some
slight effect on the appearance of these objects. Comparison of chromium
coated samples with similar samples prepared without metal coating indicated
some difference in the appearance of macrofibril structures (Fig. 4b). Mac-
rofibrils in uncoated samples appeared sharper and less rounded, suggesting
that in coated samples, chromium was accumulating around the exposed fibril
tips making them appear rounder and slightly larger. Uncoated samples

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(Fig. 4b) had significantly less resolution and contrast making a detailed
comparison difficult. In addition, uncoated samples show charging artefacts at
higher magnifications and may also show greater damage from the electron
beam so while it is possible to image uncoated wood surfaces this is not an
ideal method.

Fig. 4 a Environmental scanning electron micrograph of P. radiata tracheids in the hydrated state
showing the presence of macrofibrils (arrows) visible through the film of water on the surface of
the sample. Imaging conditions are shown on the data bar. The sample had equilibrated to these
conditions for approximately 1.5 h. Scale bar 5 lm. b Field emission scanning electron micrograph
of the same sample as above after drying. The sample has no chromium coating so resolution is
reduced. Macrofibrils can still be detected, although they have a more feather-like appearance.
Scale bar 1 lm

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Wet cell walls

Little information is currently available on the ultrastructure of wet cell walls.

However, reports have recently begun to appear in the literature describing
the use of ESEM (Turkulin et al. 2005a, b). Additionally, a study using
scanning probe microscopy reported ultrastructural details of wet cell wall
surfaces, including the presence of macrofibrils, but this method has not been
able to resolve individual cellulose microfibrils to date (Hanley and Gray
1999). Daniel et al. (2004b) have used cryo-FESEM to study macrofibrils in
decayed wood, showing that such fibrils are present after freeze etching.
Bardage et al. (2003) have observed macrofibrils on the surface of pulp fibres
after quick-freeze deep etching and transmission electron microscopy, also
suggesting that macrofibrils occur in wet cell walls. ESEM has the advantage
of being able to image the sample during drying and thus determine if the
surface structure changes with moisture content, but has the disadvantage that
detail may be obscured by the water film on surfaces.
Fully hydrated sections of radiata pine were examined in an ESEM
(Fig. 4a). Under conditions which promote slow drying, water is first lost from
the lumens. At the point where all lumen water has gone, the cell wall
transverse surface appears completely smooth due to a film of water bound to
its surface. This film seemed to remain for several hours in the water vapour
atmosphere inside the microscope. After some time, details of the underlying
cell wall were gradually revealed. At this stage it was possible to identify some
larger macrofibrils protruding through the water film (Fig. 4a).
After removing the sample from the microscope, it was completely air dried
without any subsequent fixation and examined by FESEM, first uncoated
(Fig. 4b) and then coated. This sample of sapwood also revealed the same
macrofibril texture that we observed on fixed cell walls (Fig. 4b). This suggests
that macrofibrils also occur in wet cell walls and are not artefacts induced by
drying of the exposed section surface. Exposure to fixative and ethanol were
ruled out as contributing to the presence of macrofibrils, since macrofibrils
were observed even without these treatments (Fig. 4b). However, washing the
section in ethanol prior to drying was found to reduce the incidence of
extractive deposits on the cut surface, which sometimes obscured the mac-
rofibrils in unwashed samples.

Relationship of macrofibrils to microfibrils

To determine if macrofibrils are in fact bundles of microfibrils, resin etching of

2 lm sections was used. Because microfibrils are less likely to be visible in
lignified cell walls due to encrustation with matrix substances, delignified
material was used. The macrofibrils revealed in gently delignified cell walls
appeared larger at 31 nm average diameter, than those observed in wood. The
resin etching technique provided finer resolution, allowing details less than
10 nm in size to be resolved. As shown in Fig. 5a, the macrofibrils appear to
be made up of finer structures, which are assumed to be the exposed ends of

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cellulose microfibrils, based on their size and abundance. The size of these
small fibrils is larger (6–7 nm) than the 3–4 nm microfibrils that can be ob-
served by transmission electron microscopy (Donaldson and Xu 2005),
probably because of the masking effect of the thin chromium coating (in this
sample reduced to 5 nm). In this example, macrofibrils can be seen to form
larger aggregates separated by relatively large pores (Fig. 5a). Other studies
have also provided evidence that macrofibrils are bundles of microfibrils and
matrix materials (Donaldson and Xu 2005; Xu et al. 2006).

Fig. 5 a High resolution field emission scanning electron micrograph of embedded P. radiata
tracheids showing the S2 region after delignification, with macrofibrils and their constituent
microfibrils (arrows) revealed after resin etching for 20 s. This sample has a reduced chromium
coating thickness of 5 nm. Scale bar = 0.1 lm. b High resolution image of the cell corner middle
lamella region of P. radiata tracheids showing the granular texture. Scale bar = 0.1 lm

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Measurements

Macrofibril diameter was found to vary significantly among the wood types
examined, as shown in Table 1, with the smallest macrofibrils 14 nm in
diameter found in the G-layer of tension wood fibres in poplar. Normal poplar
wood fibres had macrofibrils with an average diameter of 16 nm compared to
radiata pine tracheids with an average macrofibril diameter of 19 nm. The S2L
region of compression wood tracheids had the largest macrofibrils at 22 nm
diameter. The confidence intervals for these average values are ±1 nm
(p = 0.05).
The above trend suggests a relationship to the degree of lignification among
these cell wall types. This hypothesis was investigated using previously pub-
lished values for lignin concentration in comparable wood types. The G-layer
of poplar tension wood is thought to be unlignified (Bentum et al. 1969;
Blanchette et al. 1994; Donaldson and Bond 2005) or very slightly lignified
(Joseleau et al. 2004; Pilate et al. 2004; Gierlinger and Schwanninger 2006).
The lignin concentration of normal poplar fibre secondary walls has been
measured as 6–8% for the S2 region, and 63% for the middle lamella (Don-
aldson et al. 2001). In radiata pine normal wood, the S2 region has a lignin
concentration of 21% and the middle lamella has 75% (Donaldson 1985),
while in compression wood, the S2L region has 29-46% (Donaldson et al.
1999). Macrofibril diameter appears to show an approximately linear rela-
tionship with lignin concentration, ranging from 14 nm in the non-lignified G-
layer of poplar tension wood, to 22 nm in the highly lignified outer S2 layer of
compression wood tracheids in radiata pine as shown in Fig. 6. For compar-
ison, the values for the granular structure of the middle lamella (Table 1;
Fig. 5b) are shown to follow a similar relationship (Fig. 6), even though these
structures are not entirely analogous to macrofibrils. This suggests that the
tendency to form aggregate structures is more a property of the cell wall
matrix than of the cellulose microfibrils. The analysis of variance (Table 2)
shows a significant variance component among cells within each wood type
accounting for 3% of the total variation, compared to 33% of the variation
among cell wall types. Most of the variation, 64%, is among macrofibrils

Table 1 Macrofibril diameter variation among wood types and relationship with lignin
concentration

Cell number 1 2 3 4 5 Mean (nm) Lignin (%)

Poplar
G-layer 14 15 13 16 14 14 0
Normal S2 18 16 15 16 17 16 7
Middle lamella 21 23 22 23 26 23 63
Radiata
Normal S2 18 20 20 17 21 19 21
S2L compression wood 23 21 22 24 21 22 29-46
Middle lamella 29 24 34 28 21 27 75

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Fig. 6 The relationship between average macrofibril diameter of different wood types and lignin
concentration (Table 1) shows an approximately linear relationship. Values for the granularity of
the middle lamella region are included for comparison. The regression has an r2 value of 95%,
which is highly significant (p = 0.001)

Table 2 Analysis of variance for macrofibril diameter in the S2 region of fibres and tracheids for
wood type (normal and reaction wood), and among cells within wood type

Degrees of freedom Mean square F-statistic Probability

Cell wall type 3 2,982.33 36.95 2.0138E-07

Among cells 16 80.70 3.52 3.3719E-06
Residual 980 22.93

This analysis excludes the measurement values for the granularity of the middle lamella

within a particular region of cell wall. The variation among cells and among
cell wall types is highly significant.
While it is possible to show a relationship between lignin content and
macrofibril size, other cell wall components such as hemicelluloses, are also
known to vary in content and type among cell wall regions. Unfortunately
such variations are rather poorly understood in comparison to the extensive
literature on lignin topochemistry (Donaldson 2001b) making it difficult to
demonstrate any significant relationships between hemicelluloses content and
macrofibril size in native wood on a microscopic scale. Bulk hemicelluloses
content is known to have an effect on macrofibril size in processed wood (pulp
fibres) (Hult 2001a, b; Duchesne et al. 2001) but this is complicated by ther-
momechanical and delignification effects. Some qualitative information is
available on the types of matrix polysaccharides in different cell wall types
(Awano et al. 1998; Maeda et al. 2000; Hosoo et al. 2002; McCartney et al.
2005). In conifers, mannans predominate in the secondary wall while in
hardwoods, xylans predominate (Hamilton and Thompson 1959; Meier and
Wilkie 1959). The difference in macrofibril diameter between radiata pine and
poplar normal wood could therefore also be related to a difference in the type
of matrix polysaccharide in addition to lignin concentration. Investigation of
macrofibril diameter in developing xylem prior to lignification could resolve

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this point (Kataoka et al. 1992; Awano et al. 2000). Likewise, differences in
matrix polysaccharide type in reaction wood might also explain some of the
differences in macrofibril size observed in radiata pine compression wood and
in poplar tension wood. For example, the occurrence of galactan in the sec-
ondary wall of compression wood tracheids (Côté et al. 1968) may contribute
to the larger macrofibrils observed in the S2L region of radiata pine com-
pression wood tracheids.
Several studies have examined macrofibril size. Fengel (1970) examined
various types of polysaccharide fractions and concluded that elementary fibrils
(microfibrils) group together in bundles of 12 nm, which in turn group to-
gether in bundles of 24 nm. In the present study, it was found that larger
macrofibrils can be made up of smaller fibrils that are in turn made up of
microfibrils (Fig. 5). Fengel (1970) concluded that these fibril bundles contain
matrix material, supported by more recent studies showing that the amount of
matrix material is related to the aggregate size (Hult et al. 2001a). Awano
et al. (2000), using FESEM of developing secondary walls, also found fibril
aggregates of 12 nm diameter. Bardage et al. (2003) found that macrofibril
diameter among various Kraft pulps showed a distinct peak of fibril size at 18–
20 nm, similar to the size found by Fengel (1970). Several studies have shown
that cellulose aggregation is related to hemicelluloses content, where cellulose
aggregate size increases with decreasing hemicelluloses content (Hult 2001a,
b; Duchesne et al. 2001). Fahlén and Salmén (2002) measured cellulose
aggregates with diameters of 15–25 nm using scanning probe microscopy. For
unprocessed spruce wood, the average diameter was 18 nm, while for Kraft
pulp fibres the diameter was 23 nm, with both samples varying from less than
10 nm to more than 40 nm (Fahlén and Salmén 2002). Macrofibril size
therefore varies with material and preparation method, and macrofibrils may
be composites of smaller fibril aggregates and matrix materials. Evidence from
these studies and from measurements of delignified cell walls in the present
study, suggests that lignin removal causes swelling of the macrofibrils com-
pared to native wood.
There has been some speculation that macrofibrils may contain matrix of
different composition to any matrix between the macrofibrils (Singh et al.
2002; Terashima et al. 2004). Jarvis (2005) found uniform deuteration of hy-
droxyl groups using FTIR microscopy, suggesting that all of the matrix has
access to water and hence probably has more or less uniform composition
within and between macrofibrils. In contrast, Donaldson and Frankland (2004)
found some evidence for non-uniform density of the cell wall using precipi-
tation of iodine crystals, which appeared to form preferentially in slightly less
lignified regions on a scale of tens of nm. Using transmission electron
microscopy, Singh et al. (2002) found evidence for fine-scale variations in
lignin distribution that may be related to cellulose microfibril aggregation.
The results in this study indicate that lignin also has some influence on the
aggregation of cellulose microfibrils into macrofibrils. Increasing concentra-
tions of lignin correlate with increasing aggregate size. Lignin is known to
form aggregates of certain sizes. In a study of in vitro lignin formation Radotić

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Wood Sci Technol (2007) 41:443–460 457

et al. (1994) found that lignin forms ‘‘supermodules’’ containing ~500 mono-
mer units, which are 2–3 nm in size. Analogous studies of lignifying xylem in
Ginkgo using FESEM (Terashima et al. 2004) have shown bead-like modules
25 nm in size. These modules were found to be larger in the S1 layer and
middle lamella regions and are similar to the structures shown in Fig. 5b.
Similar modules were found in radiata pine using transmission electron
microscopy (Donaldson 1994). Lignin is assumed to infiltrate the cellulose
microfibril aggregates during lignification (Donaldson 1994) and this may
result in swelling of the aggregates, thus explaining the relationship in Fig. 6.
The larger structures in the middle lamella region may reflect the lack of
constraint on the growth of lignin modules by the pectic polysaccharide matrix
during lignification as described earlier by Donaldson (1994).

Conclusions

Macrofibrils occur in a range of cell wall types, including reaction wood of

both softwoods and hardwoods. Macrofibrils are shown to occur in both wet
and dry cell walls and are predominantly arranged in a random fashion, but
occasionally may show alignment in either the tangential or radial directions.
Matrix substances, particularly lignin appear to contribute to this organization
of structural components, as suggested by the significant correlation between
macrofibril diameter and degree of lignification among different cell wall
types. Macrofibrils are smallest in the unlignified gelatinous layer of poplar
tension wood fibres and largest in the highly lignified outer S2 region (S2L) of
compression wood tracheids in radiata pine. The middle lamella region, which
lacks cellulose, also forms granular aggregates of lignified matrix which appear
to show the same relationship between size and lignin concentration, sug-
gesting that the tendency to form aggregates is a property of the cell wall
matrix.

Acknowledgments I am grateful to Prof. Geoff Daniel and Assoc. Prof. Stig Bardage, Wood
Ultrastructure Research Centre, Swedish University of Agricultural Sciences, Uppsala, Sweden,
for hosting my visit to Sweden in 2001, during which time this work was initiated. This work was
partially funded by a grant from the ISAT Linkages fund of the Royal Society of New Zealand.
I gratefully acknowledge the assistance of Catherine Hobbis and Bryony James, University of
Auckland, for assistance with environmental scanning electron microscopy. I am also grateful
to Adya Singh and Tim Strabala, Scion, and two anonymous referees, for helpful comments on
the manuscript, and to Prof. Noritsugu Terashima, Nagoya University, for some discussion on
macrofibril size.

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