The Cell Wall Ultrastructure of Wood Fibres – Effects of the Chemical Pulp Fibre Line

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The cell wall ultrastructure of wood fibres – effects of the chemical pulp fibre line

Jesper Fahlén

Akademisk avhandling som med tillstånd av Kungliga Tekniska Högskolan framlägges till offentlig granskning för avläggande av teknologie doktorsexamen fredagen den 18 februari 2005 klockan 10:00 i STFI-salen, STFI-Packforsk, Drottning Kristinas väg 61, Stockholm. Avhandlingen försvaras på engelska.

STFI-Packforsk is one of the world’s leading R&D companies in the fields of pulp, paper, graphic media, packaging and logistics. The activities range from basic research to direct assignments, where the competence is utilised to find solutions applicable at the customers.

The Wood Ultrastructure Research Centre (WURC) is a competence centre which was initiated in 1996 by NUTEK (Swedish National Board of Technical and Industrial Development) and today established in co-operation with VINNOVA, SLU (Swedish University of Agricultural Science), eight companies from the Swedish pulp and paper industry (StoraEnso, SCA, Korsnäs, M-Real, Kappa Kraftliner, Sveaskog, Södra Cell, Holmen) and one chemical company (EKA Chemicals AB). NUTEK was replaced by VINNOVA (The Swedish Agency for Innovation Systems) January 2001. The centre is based at SLU, but within WURC’s structure, close co-operation occurs with STFI- Packforsk, KTH (Royal Institute of Technology), UU (Uppsala University), and CTH (Chalmers University of Technology). WURC is financed jointly by VINNOVA, the industrial companies, and by the various research organisations.

To my late grandparents

The cell wall ultrastructure of wood fibres – effects of the chemical pulp fibre line

Jesper Fahlén, Royal Institute of Technology (KTH), Department of Fibre and Polymer Technology, Stockholm, Sweden Abstract Knowledge of the ultrastructural arrangement within wood fibres is important for understanding the mechanical properties of the fibres themselves, as well as for understanding and controlling the ultrastructural changes that occur during pulp processing. The object of this work was to explore the use of atomic force microscopy (AFM) in studies of the cell wall ultrastructure and to see how this structure is affected in the kraft pulp fibre line. This is done in order to eventually improve fibre properties for use in paper and other applications, such as composites. On the ultrastructural level of native spruce fibres (tracheids), it was found that cellulose fibril aggregates exist as agglomerates of individual cellulose microfibrils (with a width of 4 nm). Using AFM in combination with image processing, the average side length (assuming a square cross-section) for a cellulose fibril aggregate was found to be 15–16 nm although with a broad distribution. A concentric lamella structure (following the fibre curvature) within the secondary cell wall layer of native spruce fibres was confirmed. These concentric lamellae were formed of aligned cellulose fibril aggregates with a width of about 15 nm, i.e. of the order of a single cellulose fibril aggregate. It was further found that the cellulose fibril aggregates had a uniform size distribution across the fibre wall in the transverse direction. During the chemical processing of wood chips into kraft pulp fibres, a 25 % increase in cellulose fibril aggregate dimension was found, but no such cellulose fibril aggregate enlargement occurred during the low temperature delignification of wood into holocellulose fibres. The high temperature in the pulping process, over 100 ºC, was the most important factor for the cellulose fibril aggregate enlargement. Neither refining nor drying of kraft or holocellulose pulp changed the cellulose fibril aggregate dimensions. During kraft pulping, when lignin is removed, pores are formed in the fibre cell wall. These pores were uniformly distributed throughout the transverse direction of the wood cell wall. The lamellae consisting of both pores and matrix material (“pore and matrix lamella”) became wider and their numeral decreased after chemical pulping. In holocellulose pulp, no such changes were seen. Refining of kraft pulp increased the width of the pore and matrix lamellae in the outer parts of the fibre wall, but this was not seen in holocellulose. Upon drying of holocellulose, a small decrease in the width of the pore and matrix lamellae was seen, reflecting a probable hornification of the pulp. Refining of holocellulose pulp led to pore closure probably due to the enhanced mobility within the fibre wall. Enzymatic treatment using hemicellulases on xylan and glucomannan revealed that, during the hydrolysis of one type of hemicellulose, some of the other type was also dissolved, indicating that the two hemicelluloses were to some extent linked to each other in the structure. The enzymatic treatment also decreased the pore volume throughout the fibre wall in the transverse direction, indicating enzymatic accessibility to the entire fibre wall. The results presented in this thesis show that several changes in the fibre cell wall ultrastructure occur in the kraft pulp fibre line, although the effects of these ultrastructural changes on the fibre properties are not completely understood.

Keywords: atomic force microscopy, cellulose, cell wall, drying, fibre, fibril, fracture, hemicellulose, holocellulose, kraft pulping, lamellar structure, Norway spruce, Picea abies, pores, refining, secondary wall, ultrastructure, wood Svensk sammanfattning För att förstå vedfiberns mekaniska egenskaper och för att kunna kontrollera de fiberförändringar som sker vid pappersmassaframställning är kunskap om fiberns ultrastruktur grundläggande. Syftet med detta arbete var att utnyttja atomkraftsmikroskopi (AFM) för undersökningar av cellväggens ultrastruktur och ultrastrukturella förändringar under kemisk massaframställning. Detta görs i syfte att kunna förbättra fiberegenskaperna för användning i papper och andra tillämpningar som till exempel kompositmaterial. Vid ultrastrukturella underökningar med AFM på nativa vedfibrer (trakeider) observerades cellulosafibrillaggregat uppbyggda av agglomerat av individuella cellulosamikrofibriller, (cirka 4 nm breda). Med AFM i kombination med bildbehandling bestämdes medelsidlängden (med antagandet om kvadratiska tvärsnitt) för cellulosafibrillaggregaten till mellan 15–16 nm dock med en relativt vid storleksfördelning. En koncentrisk lamellstruktur (som följer fiberns kurvatur) i det sekundära cellväggsskiktet bekräftades. Dessa koncentriska lameller är uppbyggda av uppradade cellulosafibrillaggregat med en bredd på ungefär 15 nm det vill säga av samma storleksordning som ett individuellt cellulosafibrillaggregat. Vidare konstaterades det att cellulosafibrillaggregatens storleksfördelning var homogen tvärs fiberväggen i transversell led. Vid kemisk massaframställning ökade cellulosafibrillaggregatens storlek med 25 % vilket däremot inte skedde vid lågtemperatur-delignifiering av ved till holocellulosa. Den höga temperaturen, över 100ºC, som används vid kemisk massaframställning var den enskilt viktigaste faktorn för cellulosafibrillaggregatens storleksökning. Varken vid malning eller torkning av kemisk massa eller av holocellulosa påvisades någon förändring av cellulosafibrillaggregatens storlek. Vid framställning av kemisk massa löses lignin ut och porer bildas i fiberväggen. En jämn fördelning av dessa porer tvärs fiberväggen i transversell riktning påvisades. Lamellerna i fiberväggen efter kemisk massaframställning som består av både porer och matrismaterial (”por- och matrislamellerna”) utvidgades och deras antal minskade under framställningen av kemiska massa. För holocellulosa upptäcktes däremot inga sådana förändringar. Vid malning av kemisk massa ökade bredden hos por- och matrislamellerna i fiberns ytterskikt, vilket inte skedde för holocellulosa. Vid torkning av holocellulosa minskade bredden av por- och matrislamellerna, sannolikt beroende på förhorning. Malning av holocellulosa gav upphov till porförslutning förmodligen på grund av en högre mobilitet i fiberväggen. Efter enzymatisk hydrolys av xylan och glukomannan med hemicellulaser observerades att nedbrytningen av en hemicellulosa även gav upphov till utlösning av den andra. Detta indikerar att xylan och glukomannan på något sätt är kopplade till varandra i fiberväggen. Fibrernas porvolym minskade tvärs hela fiberväggen i transversell led efter den enzymatiska behandlingen, vilket indikerar tillträde till hela fiberväggen. I detta arbete har flera ultrastrukturella fiberförändringar påvisats under kemisk massaframställning, emellertid är kunskapen om de ultrastrukturella förändringarnas inverkan på fiberegenskaperna ännu inte helt klarlagda.

List of publications

This thesis is based on the following papers, which in the text are referred to by their Roman numerals:

Paper I Fahlén J, Salmén L (2002) “On the Lamellar Structure of the Tracheid Cell Wall”. Plant Biology 4(3): p. 339–345 Reprinted with kind permission of Georg Thieme Verlag

Paper II Fahlén J, Salmén L (2003) “Cross-sectional structure of the secondary wall of wood fibers as affected by processing”. Journal of Material Science 38(1): p. 119–126 Reprinted with kind permission of Springer Science and Business Media

Paper III Fahlén J, Salmén L “Pore and Matrix Distribution in the Fiber Wall Revealed by Atomic Force Microscopy and Image Analysis”. Biomacromolecules, in press. (Published on the internet 2004-12-04)

Paper IV Fahlén J, Salmén L “Ultrastructural changes in the transverse direction of a holocellulose pulp revealed by enzymes, thermoporosimetry and Atomic Force Microscopy”. Submitted to Holzforschung

Other relevant publications

Fahlén J, Salmén L (2001) “Cross-sectional structure of the secondary wall of wood fibers as affected by processing” From the 11th International Symposium on Wood and Pulping Chemistry (ISWPC): Nice, France: p. 585–588

Fahlén J, Salmén L (2004) “Ultrastructural differences in the transverse direction of the wood fibre wall” From the 8th European Workshop on Lignocellulosics and Pulp (EWLP): Riga, Latvia: p. 21–24

Table of content 1. Introduction...... 1 1.1 Background ...... 1 1.2 Objectives ...... 1 2. The structure of wood...... 3 2.1 From the tree to the cell level ...... 3 2.2 The molecular level ...... 4 2.2.1 Cellulose ...... 4 2.2.2 Hemicelluloses...... 5 2.2.3 Lignin...... 6 2.3 The ultrastructural level ...... 8 2.3.1 Primary cell wall layer ...... 8 2.3.2 Secondary cell wall layer ...... 9 3. Microscopy techniques for investigation of wood and fibre structures...... 13 3.1 Light microscopy ...... 13 3.2 Electron microscopy...... 13 3.2.1 Transmission electron microscopy...... 13 3.2.2 Scanning electron microscopy ...... 14 3.3 Scanning probe microscopy...... 14 3.3.1 Atomic force microscopy...... 14 3.3.2 The use of atomic force microscopy in wood and fibre ultrastructural research ...... 17 4. The chemical pulping process...... 19 4.1 Chemical pulping...... 19 4.1.1 Kraft pulping...... 19 4.1.2 Low temperature delignification ...... 20 4.2 Bleaching...... 21 4.3 Refining ...... 21 4.4 Pores and water present within fibres ...... 22 4.5 Techniques for determining pore water...... 23 4.5.1 Water retention value...... 23 4.5.2 Solute exclusion...... 24 4.5.3 Inverse size exclusion chromatography ...... 24 4.5.4 Thermoporosimetry...... 24 5. Atomic force microscope investigations of the wood and fibre ultrastructure ...... 27 5.1 Sample preparation for atomic force microscopy investigations ...... 27 5.2 Size determination of ultrastructural features investigated with atomic force microscopy...... 28 5.2.1 Cellulose fibril aggregates...... 28 5.2.2 Matrix lamella width...... 32 5.3 Lamella structure of the secondary cell wall layer...... 33 6. Ultrastructural changes in the kraft pulp fibre line ...... 39 6.1 Changes in cellulose fibril aggregate dimensions...... 39 6.2 Visualisation of pores and the transverse pore structure with atomic force microscopy ...... 45 6.3 Changes in pore and matrix lamella dimensions...... 47 6.4 Ultrastructural changes during drying...... 50 7. Conclusions ...... 53 8. Future aspects...... 55 9. Acknowledgements...... 57 10. References ...... 59

1. Introduction

1.1 Background The composition and structure of wood are a masterpiece of evolutionary design, which enable trees to grow tall and live for many years. The wood structure also enables the trees to withstand strong natural forces such as wind and gravity and the structure even provides for a remarkably efficient transport of water from the roots to the crown. Wood is a complex biocomposite built up of cells whose own building blocks, the wood polymers and their ideal composition, give rise to a superior weight-to-strength ratio for the wood material. Separated into individual fibres during pulping wood is the raw material for paper production.

Nowadays there is an increasing demand for more paper and also for more specialised paper grades that call for a better understanding of the variations in pulp strength that arise during fibre processing. Explanations for the differences in pulp strength have mostly been searched for on the molecular level or on the fibre level. On the intermediate nanoscale level or ultrastructural level, our knowledge is however still limited, especially with regard to important structural features within the fibre wall. An understanding of the arrangement of the wood polymers within the fibre wall is nevertheless important for a full understanding of the mechanical properties of the fibres. Due to their high load-bearing ability, the arrangement of the cellulose fibril aggregates within the cell wall is of special interest. After chemical pulp processing, structural defects in the fibres reduce their load- bearing capacity and the reasons for this are probably to be found on the ultrastructural level.

1.2 Objectives The objectives of this work have been to study the ultrastructure of spruce wood fibres in order to understand how the fibre ultrastructure is affected upon chemical processing and to improve the use of wood fibres in paper and other applications such as composite materials.

2. The structure of wood

2.1 From the tree to the cell level Fully grown trees can vary from a few centimetres in height, as for the very slow growing Tiny Dwarf willow found on frozen tundra in the Arctic, up to 110 meters for a rapidly growing Coast redwood found in California. Trees also have the ability to grow for more than a thousand years. Indeed, the world’s oldest tree, a Bristlecone pine, has lived for over 4700 years. In Sweden, the tallest tree is a Norwegian spruce which is 47 meters high and the oldest tree is an English oak which is over 1000 years old. All tree species are seed-bearing plants, which are further divided into gymnosperms and angiosperms. Softwood belongs to the gymnosperms and hardwood to the angiosperms. From an evolutionary point of view, softwoods are much older, around 300 million years, than hardwoods which are about 150 million years old (Raven et al. 1999[111]). Common to the two groups is that the main cell type (tracheid/fibre) in the wood has at least some supporting function that gives mechanical strength to the tree. Hardwoods, that developed much later than softwoods, have a different arrangement of the cells building up the trees and there are differences in both their general function and their chemical composition. However, since this thesis has focused on softwoods, the concentration will be on the structure of these species.

A cross-section of a tree trunk reveals the different parts of the stem (Figure 1). The outer layer is of course the bark which can be divided into the outer dead bark and the inner living bark. Adjacent to the inner bark is the cambium layer which is the growth zone in wood. The inner parts of the steam consist of dead cells in the sapwood and in the heartwood. Softwoods consist mainly of longitudinal tracheids (90–95 %) and a small number of ray cells (5–10 %) (Fengel and Grosser 1976[40]). The longitudinal tracheids are herein for simplicity referred to as fibres. As a consequence of seasonal changes in growth, fibres produced during the rapid cell development in the spring (earlywood fibres) are thin-walled and the fibres created later in the growth period i.e. in the summer (latewood fibres) are thick-walled. The earlywood and latewood fibres produced within the same year form an annual ring (Figure 1).

All fibres are formed as tubes and the hollow void inside them is called the lumen, which provides for the water transport within the fibre. The distribution of fluids between adjacent cells is achieved through a system of pits. The average length of a Scandinavian softwood (Norwegian spruce and Scots pine) fibre is 2–4 mm and the width is 0.02–0.04 mm. Earlywood fibres have an average cell wall thickness of 2–4 µm and latewood fibres have an average cell wall thickness of 4–8 µm (Sjöström 1993[126]).

Figure 1. Schematic illustration of the morphological structure of softwood and the three principal directions of wood: longitudinal (L), radial (R), and tangential (T).

2.2 The molecular level The wood fibre is basically built up of the polymers: cellulose, hemicelluloses, and lignin. Pectin, inorganic compounds, and extractives are also present in wood, although only as minor components.

2.2.1 Cellulose Cellulose is the most abundant biopolymer on earth. It is synthesised in plants (trees, grass etc.), algae (Valonia, Cladophora etc.) and even in some animals (Tunicates), and it can also be synthesised by some bacteria (Acetobacter xylinum). Around 40 % of the dry weight of wood consists of cellulose. Cellulose is a linear polymer built up of D-glucose units linked together by β-(1-4)-glycosidic bonds (Figure 2). The degree of polymerisation ( DP ) is normally 9000–10000 glucose units, but DP values as high as 15000 glucose units have been reported (Goring and Timell 1962[44]). Most of the cellulose found in wood fibres has approximately the same molecular size, i.e. a very low polydispersity (Goring and Timell 1962[44]). In living plants, the crystalline cellulose structure is denoted cellulose I, which has two different unit cells: Iα and Iβ (Atalla and VanderHart 1984[10]; Sugiyama et al. 1991[135]). In spruce, the two structures co-exist and the [85] composition is species specific (Maunu et al. 2000 ). During pulping, cellulose Iα is to some [101] extent converted to Iβ in an alkaline but not in a sulphite environment (Page and Abbot 1983 ; Hult et al. 2002[61]; Åkerholm et al. 2004[156]), which indicates that both a high temperature and a high alkalinity are required for a transformation from the Iα to the thermodynamically more stable Iβ form.

Figure 2. Chemical structure of cellulose. The cellulose molecule is linear and it is therefore capable of forming strong intra- and intermolecular hydrogen bonds and aggregated bundles of molecules. In the literature, these bundles of cellulose molecules have been given many different names such as, elementary fibrils, microfibrils, protofibrils etc. (Barber and Meylan 1964[15]; Kerr and Goring 1975[64]; Heyn 1977[56]). Here the term cellulose microfibrils will be used. These cellulose microfibrils have crystalline and para-crystalline regions (Figure 3). The crystalline cellulose is located in the interior of the cellulose microfibrils whereas the para-crystalline parts are located on their surfaces (Larsson et al. 1997[72]; Wickholm et al. 1998[143]). The lateral dimension for a cellulose microfibril varies significantly between different species. In wood, the lateral dimensions are around 4×4 nm (Wickholm 2001[142]).

2.2.2 Hemicelluloses Hemicelluloses are a group of heterogeneous polymers that play a supporting role in the fibre wall. 20–30 % of the dry weight of wood consists of hemicelluloses. The hemicellulose polymers are built up of several different monomers, such as mannose, arabinose, xylose, galactose, and glucose. Some acidic sugars like galacturonic acid and glucuronic acid are also constituents of hemicelluloses. One, two or several types of monomer usually build up the backbone of the hemicellulose polymers. Most of the hemicelluloses also have short branches containing types of sugars other than those of the backbone. The degree of polymerisation for the hemicelluloses is between 100 and 200 (Fengel and Wegener 1984[41]).

In softwood, the hemicellulose galactoglucomannan, makes up about 16 % of the dry weight of the wood. Galactoglucomannans have a backbone of (1-4)-linked β-D-glucose and β-D-mannose units, with α-D-galactose linked to the chain through (1-6)-bonds. An important structural feature is that the hydroxyl groups at C(2) and C(3) positions in the chain units are partially substituted by O-acetyl groups, on the average one group per 3–4 hexose units (Figure 4). The hemicellulose arabinoglucuronoxylan constitutes about 8–9 % of the dry weight of softwood. It has a backbone of (1-4)-β-D-xylose, where most of the xylose residues have an acetyl group at C(2) or C(3). About every tenth xylose unit also has a 4-O-methyl-α-D-glucuronic acid residue linked by a (1-2)-bond (Figure 5). The backbone substitution and degree of branching can vary considerably between hemicelluloses of the same category (Sjöström 1993[126]).

Figure 4. Chemical structure of galactoglucomannan, R = CH3CO or H.

Figure 5. Chemical structure of arabinoglucuronoxylan.

2.2.3 Lignin Lignins are heterogeneous three-dimensional polymers that constitute approximately 30 % of the dry weight of wood. Lignin limits the penetration of water into the wood cells and makes wood very compact. Lignins are complex polymers based on the three monolignols: p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol (Figure 6). The monolignols in various proportions are the building blocks for the 3-D structure of native lignin in higher plants (Sjöström 1993[126]). The monolignols units are considered to polymerise mainly according to a radical polymerisation process.

Figure 6. The three monolignols: left-hand side p-coumaryl alcohol, middle coniferyl alcohol, and right-hand side sinapyl alcohol. The complex and irregular structure and the difficulty of isolating native lignin have led to problems in determining its chemical composition. Many hypothetical lignin structures have been proposed over the years, where Figure 7 shows a recent model of a lignin segment in softwood proposed by Brunow et al. (1998[28]). The most frequent intermonomeric linkage in lignin is the β- O-4 aryl ether bond.

2.3 The ultrastructural level The tracheid fibres in wood are attached to each other through a region rich in lignin called the middle lamella (M.L.). The middle lamella is 0.2–1.0 µm thick and its main function is to bind fibres together (Sjöström 1993[126]).

The fibre ultrastructure is the hierarchic level ranging from the molecular level up to the fibre cell wall layers. The structure of the actual wood cell wall is very complex and it consists of several layers. For this reason it has been difficult to illustrate the whole cell wall with one model, although Brändström (2002[29]) recently presented a good approach as displayed in Figure 8.

Figure 8. Schematic model of the cell wall layers present in a softwood latewood fibre. The lines in the different layers represent the organisation of the cellulose microfibrils in the different layers. [29] Note the dominance of the S2 layer. Reprinted with permission from Brändström (2002 ). Copyright © 2002 Jonas Brändström.

2.3.1 Primary cell wall layer The primary (P) cell wall layer exists in all plants because it is the basic structural unit of the living cell. Most detailed chemical and structural studies of the primary cell walls have been carried out on non-woody plants. McCann and co-workers presented a detailed model of the primary wall in onions were they suggested that the pectin network is independent of the hemicellulose/cellulose structure (McCann et al. 1990[87]; McCann and Roberts 1991[86]). In wood, the primary wall in the dead fibre, with a developed secondary cell wall layer, consists of cellulose, hemicellulose, pectin, and protein embedded in lignin (Sjöström 1993[126]). Albersheim (1975[5]) proposed a model for the primary cell wall in wood where all the wood polymers were included. In this model, it was suggested that all the wood polymers (except cellulose) are covalently linked together to form one big macromolecule (Figure 9).

Figure 9. A schematic drawing of the primary cell wall layer in wood slightly modified from Albersheim (1975[5])

2.3.2 Secondary cell wall layer

The thick secondary cell wall consists of three different layers, S1, S2, and S3, from the middle lamella towards the lumen. The cellulose microfibrils are arranged in different directions in these layers and it is therefore possible to distinguish between them visually (Figure 10).

Figure 10. An image of a kraft pulp fibre surface where cellulose microfibrils in the S1 layer is evident although some cellulose microfibrils running in the perpendicular direction belonging to the S2 layer are also detectable. The arrow indicates the fibre direction.

[24] The S1 layer is between 0.1 and 0.2 µm thick (Booker and Sell 1998 ) and this layer is the one [30] adjacent to the primary cell wall layer. Recently Brändström et al. (2003 ) proposed that the S1 layer consists mainly of a single lamella where the cellulose microfibril orientation is more or less perpendicular to the cell axis (70–90º). They further emphasised that the S1 layer is rather homogeneous and rigid. The S1 layer is important for the transverse elastic modulus of fibres (Bergander and Salmén 2002[17]).

The central S2 layer is about 1 µm thick in earlywood and 5 µm thick in latewood, and forms the main portion of the cell wall. The S2 is therefore the layer contributing most to the mechanical strength of the fibre in the longitudinal direction. The angle of the cellulose microfibrils to the longitudinal direction within this layer is between 0° and 30°. This is dependent on where the fibre is situated in the tree. Latewood fibres tend to have a slightly lower mean microfibril angle than earlywood fibres (Marton and McGovern 1970[84]; Paakkari and Serimaa 1984[99]; Kyrkjeeide 1990[68]; Sahlberg et al. 1996[115]).

It has been proposed that the cellulose microfibrils within the S2 layer are surrounded by some of the hemicelluloses and are aggregated into a larger structural unit that has been termed a macrofibril, cellulose fibril, or cellulose fibril aggregate (Fengel 1970[39]; Duchesne and Daniel 2000[36]; Hult 2001[58]). In this thesis the term “cellulose fibril aggregate” is used. Mechanical studies of the wood polymers have shown that there are strong interactions between the hemicelluloses, xylan and glucomannan, and the other wood polymers, cellulose and lignin. After studies of the softening behaviour of glucomannan and xylan, it was suggested that xylan is more associated with lignin and that glucomannan is more associated with cellulose (Salmén and Olsson 1998[116]). This hypothesis was supported by the results of spectroscopic studies using so-called dynamic FT–IR (Åkerholm and Salmén 2001[157]). It has therefore been proposed that the cellulose microfibrils surrounded by some of the glucomannan are indeed aggregated into a cellulose fibril aggregate. The matrix material consisting of the hemicellulose xylan and lignin together with some of the glucomannan surrounds these cellulose fibril aggregates. A recent study has also indicated that the phenyl-propane units in lignin have a preferred orientation along the fibre axis (Åkerholm and Salmén 2003[158]). In Paper IV, enzymes acting on glucomannan and xylan were used for investigations on the fibre wall. It was found that when only one of the hemicelluloses was chemically attacked by the enzyme, the other one was also partly dissolved. Thus, even though the glucomannan is assumed to be more associated with cellulose and the xylan more associated with lignin, they may not be fully independently distributed. Figure 11 illustrates the ultrastructure of the wood fibre wall including the organisation of the wood polymers.

The inner S3 layer (thickness 0.1–0.2 µm) lies adjacent to the cell lumen and consists of a thin layer of cellulose microfibrils. The cellulose microfibril angle varies from about 30° to 90° and the cellulose microfibrils in the S3 layer are thus essentially perpendicular to the cell axis (Abe et al. 1992[1]; Booker and Sell 1998[24]).

Figure 11. Schematic illustration of the organisation of the wood polymers in the secondary cell wall layer modified from Åkerholm and Salmén (2003[158]).

3. Microscopy techniques for investigation of wood and fibre structures

3.1 Light microscopy From the seventeenth century until the middle of the twentieth century, the light microscope (LM) was the only technique available for the magnification of objects for scientific studies, particularly in medicine, physics, chemistry, and biology. In wood anatomy studies, the light microscope was also adopted in the seventeenth century and in 1665 Robert Hooke[57] (as in Hooke’s law) was able to distinguish between vessles and fibres in wood cross-sections. The biological term “cell” is attributed to Hooke, which he coined because his observations of plant cells reminded him of monks’ cells. Today the light microscope is used for fibre investigations on the micrometer scale including fibre dimensions and some of the main elements of the wood cell wall (Booker and Sell 1998[24]; Brändström 2002[29]). In a light microscope, visible light is focused through a specimen by a condenser lens, and is then passed through two more lenses placed at each end of a light-tight tube. These two lenses magnify the image. Limitations to what can be seen in light microscopy are related not so much to magnification as to resolution, illumination, and contrast. “Resolution” is the ability to distinguish points lying close together as separate objects. “Contrast” is the difference in visual properties that makes an object (or its representation in an image) distinguishable from other objects and the background. However, even with perfect lenses and perfect illumination, no light microscope can be used to distinguish objects that are smaller than half the wavelength of light. White light has an average wavelength of 550 nm, half this wavelength is 275 nm and any object with a diameter smaller than 275 nm will be invisible or, at best, show up as a blur. This restricts the use of light microscopy in ultrastructural investigations of wood fibres.

3.2 Electron microscopy The introduction of the electron microscope (EM) in the middle of the twentieth century, where the wavelength of electrons instead of white light is used, gave high resolution and gave wood researchers a new and effective tool for structural investigation of wood fibres even down to the ultrastructural level. Now it became possible to study almost all aspects of wood anatomy with an accurate sample preparation. However, the drawback of electron microscope is that it is more or less restricted to observations under vacuum and the use of a highly energetic electron beam. This requires dry samples and samples insensitive to electron bombardment. A number of methods are available for the preparation of dry wood and fibre samples prior to electron microscopy investigations in vacuum. Among these methods are air-, freeze-, and critical-point drying and replica techniques (Daniel and Duchesne 1998[33]).

3.2.1 Transmission electron microscopy In a conventional transmission electron microscope (TEM), the primary electrons are generated by a metal filament under high voltage, typically between 20 and 200 kV. The primary electron beam generated is then focused on the sample by a series of electromagnetic lenses placed in a vacuum chamber. Only electrons that are able to pass through the sample are used for image creation. In order for the electrons to pass through the sample, ultra-thin (50–100 nm) sample sections are required. The TEM images are two-dimensional and the micrographs are acquired on

13 monochrome paper or nowadays also in digital form. A TEM has an approximate resolution of 0.2 nm.

3.2.2 Scanning electron microscopy Scanning electron microscopy (SEM) provides information about the topography of a specimen. The electrons used in the microscope are emitted from an electron source, typically of tungsten, under the influence of a current. The electron beam is focused on the sample in a vacuum chamber using rotational electromagnets. Non-conductive samples are coated with a conducting metal layer prior to investigation in order to prevent charge accumulation and specimen damage during electron irradiation. These metal coatings may obscure rough surfaces. The electrons that strike the surface of the specimen generate secondary electrons from the specimen surface. These secondary electrons have a rather low energy and cannot escape from a depth greater than 10 nm. Secondary electrons are detected by secondary electron detectors, which amplify and change the signal to an electric one. A three-dimensional image is built up from the number of electrons emitted from different spots on the sample giving a resolution around 1–5 nm.

A special more modern type of scanning electron microscope, the so-called environmental scanning electron microscope (ESEM), is also worth mentioning. Whereas the conventional scanning electron microscope requires high vacuum, an ESEM may be operated under low vacuum. In such “wet mode” imaging, the specimen chamber is isolated from the rest of the vacuum system and this permits the direct examination of specimens in the presence of high vapour pressure components such as water or gases for example CO2 or N2. Polymers, biological cells, plants, soil bacteria, concrete, wood, asphalt, and liquid suspensions have been observed in the ESEM without prior specimen preparation or metal coating. The ESEM is usually also equipped with heating and cooling stages that can extend the temperature control over a much wider range. Although the ESEM has a lot of advantages, the resolution is limited for samples in their hydrated state due to the presence of a watery film on the sample surface that more or less obstructs the passage of secondary electrons. The resolution, about 5–10 nm, in the ESEM mode is therefore lower than that in conventional SEM instruments and not sufficient for ultrastructural investigations on wood and fibres (Duchesne and Daniel 1999[35]).

3.3 Scanning probe microscopy In 1982 the scanning tunneling microscope (STM) was invented, and this provided unique opportunities for obtaining three-dimensional images of surfaces with atomic resolution (Binnig and Rohrer 1984[22]). For the invention of the STM, Gerd Binnig and Heinrich Rohrer were awarded the Nobel Prize in Physics in 1986. The STM works only with conducting surfaces since a voltage must be applied to both surfaces, creating a current between the tip and the surface. The limited application for the STM to conducting materials inspired Binning to develop the atomic force microscope (AFM) with a novel force sensing technique operating regardless of material composition (Binnig et al. 1986[21]; Binnig 1987[20]). The commercial scanning probe microscope (SPM) family nowadays includes instruments specialising in image topographical, chemical, magnetic, viscoelastic, frictional, electrical, and thermal features of a surface on the atomic to microscopic scale (Poggi et al. 2004[105]).

3.3.1 Atomic force microscopy The basic principle of the atomic force microscope (AFM) is the measurement of forces between the sample surface and a sharp tip. The sample is mounted on a piezoelectric scanner that provides

14 sub-Angstrom motion of the sample. The tip is secured to the end of a cantilever and, as the sample passes beneath the tip, changes in topography cause the tip and cantilever to deflect. This deflection is measured by reflecting a laser beam from the back of the cantilever to a position- sensitive photodiode (Figure 12) (Meyer and Amer 1988[88]; Alexander et al. 1989[6]). Advantages of the AFM technique include the non-destruction of samples and the ability to study the surfaces in vacuum, air, and liquids, and this in combination with a resolution of about 0.1 nm has contributed to its widespread use for imaging (Engel 1991[38]; Radmacher et al. 1992[110]). In addition to the imaging capabilities of AFM, progress in the ability of the technique to measure forces – of the same order as molecular interactions has emerged. In AFM, the choice of mode of operation determines what data is collected and analysed. There is a vast range of modes resulting in: images based on the topography; images based on viscoelastic properties; images based on hardness; force-distance curves; friction loops etc.

Figure 12. A schematic drawing of the principle involved in the atomic force microscope (AFM).

In the Contact Mode, the repulsive force (≈10-7 N) between the tip and sample surface is kept constant by the feedback loop, and the piezoelectric scanner adjusts the separating distance. In the Contact Mode, the signal of primary interest is the cantilever deflection signal and the choice of cantilever determines the range and resolution of applicable forces between tip and substrate. Since the tip is in contact with the substrate while they move laterally relative to each other, the shear force can rupture a soft surface. When imaging, the computer-controlled feedback from the detector to the scanner is adjusted to minimise damage of the surface by the tip and maximise the image quality. For instance, when the topography is the information of interest, the sample is scanned back and forth in the x-y-plane, while the feedback determines to what extent the sample is moved in the z-direction. When the 15 image is generated from the x-y-z movement of the scanner the resulting image is called a height image. However, in practice, the feedback loop cannot follow the surface topography perfectly and any height change along the scan path will result in an error. Because of errors and delays in the scanner movement, the images appear slightly indistinct compared to the so-called deflection image. A deflection image, very often acquired simultaneously with the height image, shows the error induced by the imperfection of the feedback loop. The height and the deflection images are complementary and should therefore always be viewed at the same time.

The Tapping Mode™ (Zhong et al. 1993[152]) is a development of the contact mode AFM. In the Tapping Mode, the cantilever on which the tip is mounted is oscillated at a frequency near its resonance (typically a few hundred kHz) while separated from the sample surface. The oscillation is driven by a constant driving force. Amplitude oscillation of the cantilever is monitored and, in contrast to the contact mode, it is the primary signal of the Tapping Mode. The tip touches the surface at the bottom of each oscillation, and this reduces the oscillation amplitude of the cantilever (Puttman et al. 1994[109]). The feedback control loop of the system then maintains this new amplitude constant as the oscillating, or tapping, tip scans the surface. The information in a Tapping Mode height image corresponds in principle to the information in the Contact Mode. Since the tapping frequency is so high, there is however virtually no lateral force between the tip and the substrate, and this is a huge advantage over the shearing contact in the Contact Mode. Compliant and soft substrates are therefore preferably imaged in the Tapping Mode. The Tapping Mode equivalent to the deflection image is the amplitude image, in which the amplitude change information from the detector is used directly (not using the feedback loop) to create an image. Amplitude images have a much better contrast than height images.

An extension in the Tapping Mode is the so-called phase imaging mode, where the phase shift between the driven oscillation of the cantilever and the detected oscillation signal is used to create an image. This requires the oscillation and amplitude feedback loop of the tapping mode and phase imaging is therefore not possible in the Contact Mode. The acquisition of a phase image is also based on information directly from the detector, which makes the image distinct. Combined with the fact that the phase image includes the information from the topography, this makes it a convenient way of obtaining clear images. The phase differences between the driven and the resulting oscillation can also be interpreted to yield information about the viscoelastic and adhesive properties of the substrate. Recent development in Tapping Mode AFM allows the detections of shifts in phase angles of vibration when the oscillating cantilever interacts with the sample surface. The detection of phase angle shifts provides enhanced image contrasts, especially for heterogeneous samples. However, care must be taken to assign the features of height and phase images to different chemical components, since the images are sensitive to changes in the set-point amplitude ratio (Bar et al. 1997[14]).

3.3.2 The use of atomic force microscopy in wood and fibre ultrastructural research The application of atomic force microscopy for surface characterisation in the field of wood, fibres, pulp and paper is still at an early stage; although the technique has been tested in all most every part of the value chain ranging from wood to printed paper and the number of papers in this field is steadily increasing (Hanley and Gray 1995[50]; Béland 1997[16]; Niemi and Paulapuro 2002[94]). The features observed for fibres range from the detection of fibre dimensions to imaging molecules at the atomic level. Measurements on fibrillation and the size of algae cellulose microfibrils have been reported and compared with transmission electron microscopy (Hanley et al. 1992[48]; 1997[52]). Individual glucose molecules have also been identified with AFM (Kuutti et al. 1995[66]). AFM has been used to illustrate the existence of lignin on pulp fibre surfaces, and images of kraft pulp fibres surfaces have been taken both in air and in water (Pereira and Claudio- da-Silva 1995[103]; Kuys 1996[67]; Furuta and Gray 1998[42]; Hanley and Gray 1998[51]; Börås and Gatenholm 1999[31]; Okamoto and Meshitsuka 1999[97]; Snell et al. 2001[127]; Gray 2003[45]; Sasaki et al. 2003[118]; Koljonen et al. 2004[65]). Nevertheless only a limited number of studies have yet been made on cross-sections of wood and fibres (Hanley and Gray 1994[49]; Niemi and Mahlberg 1999[95]; Yan et al. 2004[149]).

4. The chemical pulping process In order to make paper from wood, the individual fibres must be separated into a wood pulp and then refined to some extent. In the paper machine, the pulp is evenly distributed on a drainage wire where the fibres are oriented and consolidated into a paper sheet. The wood pulp can basically be produced in two different ways; either mechanically or chemically. In mechanical pulp production, the fibres are softened and separated from each other using temperature and mechanical forces (shear). Since there is virtually no loss of material in mechanical pulping, the pulp produced has a high yield usually over 90 %. Chemical pulping on the other hand gives a pulp of lower yield but with better strength properties.

4.1 Chemical pulping The chemical pulping process has two main purposes: to cause a chemical attack on the lignin in the middle lamella in order to separate the individual fibres and to remove lignin from the cell wall to make the fibres sufficiently flexible to give high strength to the paper produced.

4.1.1 Kraft pulping The kraft pulping process was invented in the late nineteenth century and is the general name for an alkaline process were wood chips are heated between 140 and 170 °C in a liquor composed of sodium hydroxide and sodium sulphide or, more correctly, hydroxide ions and hydrosulfide ions. The aim of the kraft cooking process is to degrade and dissolve lignin in the wood to such an extent that fibres can be librated with a minimum of mechanical force. The chemistry of the process is not completely selective towards lignin and as a result carbohydrates, especially the hemicelluloses, are also degraded to some extent. The removal of lignin and carbohydrates results in a pulp with quite a low yield, usually around 50 %. The kraft pulping process can be divided into three phases with respect to lignin dissolution (Figure 13). In the initial phase, the lignin dissolution is slow whereas the carbohydrate loss (especially of glucomannan) is rapid. In the bulk phase, most of the delignification occurs and the carbohydrates are relatively stable against dissolution. In the residual phase, lignin dissolution is slow even though the polysaccharide degradation occurs to the same extent. Although some cellulose may be dissolved during the process, the crystallinity is not changed during the process if hemicelluloses still remain (Hattula 1986[55]). The glucomannan left in the structure after the pulping process still contains small amounts of galactose. A lot of the xylan present in the wood also dissolves into the pulping liquor. The remaining pulp xylan also contain some arabinose residues (Sjöström 1993[126]). Some of the dissolved xylan is resorbed onto the fibre surfaces at the end of the kraft pulping process (Yllner and Enström 1956[150]; Yllner and Enström 1957[151]; Mitikka et al. 1995[89]).

Figure 13. Example of processing conditions used for a kraft pulping sequence (solid line) and lignin content (dotted line).

4.1.2 Low temperature delignification Low temperature delignification is here defined as a laboratory process with the aim of delignifying wood in order to prepare so-called holocellulose. Holocellulose is the carbohydrate part of a plant tissue. Some low temperature methods, i.e. below 100 ºC, of preparing holocellulose have been developed of which two, the chlorite and peracetic acid methods, will be briefly discussed here.

Chlorite delignification using sodium chlorite was introduced in the beginning of the 1940’s as a modification of the previous method based on chlorine dioxide (Wise et al. 1946[145]). Chlorite delignification was also used by Ahlgren (1970[4]) in a thorough study of holocellulose preparation from wood. Of course, the intention when preparing a holocellulose pulp is to remove all the lignin without degrading any of the carbohydrates. No holocellulose preparation is however ideal, and it was discovered early that some carbohydrates were lost during the chlorite treatment of wood (Wise et al. 1946[145]; Browning and Bublitz 1953[27]; Timell 1959[138]). Ahlgren (1970[4]) showed that a chlorite procedure is selective to lignin only during the first 60 % of delignification and that some carbohydrates were dissolved during later stages of the delignification.

Peracetic acid delignification has been recommended as a method for holocellulose preparation by both Leopold (1961[75]) and Haas et al. (1955[46]) based on pioneering research by Poljak (1951[106]). Leopold (1961[75]) compared holocelluloses delignified with peracetic acid and chlorite. It was found that the peracetic acid method had a superior recovery of carbohydrates compared with the chlorite method. This result was confirmed by Thompson and Kaustinen (1964[137]) who compared the yield and composition of pulps prepared using peracetic acid and chlorite.

4.2 Bleaching Unbleached kraft pulp is brown in colour. Thus in order to produce a paper product that can be used for printing the pulp has to be bleached. The residual lignin in the pulp, although it accounts for only some 2–5 % of the dry pulp weight, is intensely coloured (Hartler and Norrström 1969[53]) and accounts for more than 90 % of the colour in the unbleached pulp. The residual lignin is difficult to remove in the kraft cook without severe carbohydrate degradation. Therefore the residual lignin is modified or removed in several bleaching stages. Chlorine gas was used extensively as a bleaching agent until the early 1990’s when its use was dramatically reduced due to environmental regulations and customer pressure. Pulp mills today use a one- or a two-stage oxygen (O) delignification prior to bleaching. The actual bleaching is thereafter done in several stages predominantly using chlorine dioxide (D) and peroxide (P) as bleaching agents, although ozone (Z) and peracetic acid (T) are also used in small amounts. A typical bleaching sequence free of elemental chlorine, so-called ECF-bleaching, could be D(EOP)DD, where E is alkali extraction. A typical sequence totally free of chlorine (TCF- bleaching) could be Q(OP)Q(PO), where Q is a complex binder stage.

4.3 Refining A paper sheet produced from fibres of unbeaten pulp has a low strength and irregular surfaces, is bulky and contains many opening gaps (Hartman and Higgins 1983[54]). These properties are not desirable for commercial paper. Therefore the mechanical action of refining or beating is considered to be one of the most important treatments of a chemical pulp to improve its papermaking properties. Until the 1960’s, the main fibre treatment was beating carried out in a batch process using so-called Hollander beaters. The beating of virgin pulps was carried out in most cases by experienced beater men and it was considered an art with little quantification. Nowadays the beater is replaced with continuous refiners of the conical or disc type and theories behind refining have been developed (Baker 2000[13]).

During the mechanical action in a refiner, the pulp fibres’ potential and conformability for good bonding in a paper sheet is increased. Refining is done to a certain level where the bonding capacity of the fibres is as high as possible without any decrease in the single fibre strength. Some of the major effects of the refining process on the fibre level are listed by Page (1989[100]):

• cutting or fibre shortening • fines production and the complete removal of parts of the fibre wall • external fibrillation, the partial removal of the fibre wall (Figure 14) • internal fibrillation – internal changes in the cell wall ultrastructure also described as delamination or swelling • straightening of the fibre • dissolution of colloidal material • redistribution of hemicelluloses from the interior of the fibre to the exterior • scraping of the fibre surface at the molecular level producing a gelatinous surface

Figure 14. Photomicrographs showing fibres before (a) and after refining (b) where severe external fibrillation are obvious.

4.4 Pores and water present within fibres The papermaking properties of chemical pulps are determined by the composition and structure of the fibres after the pulping process, when most of the lignin has been removed from the cell wall. During the removal of lignin and to some extent of hemicelluloses, large pores are produced in the fibre wall and these facilitate a more rapid dissolution of lignin from the secondary cell wall layer (Stone and Scallan 1967[129]; Kerr and Goring 1975[63]). The effect of this gradual delignification on the hemicellulose-lignin matrix as interpreted by Goring (1977[43]) is illustrated in Figure 15. The size and distribution of these pores affect many fibre properties; including their swelling in water (Stone et al. 1968[132]), their accessibility to macromolecules (Stone et al. 1969[134]), the diffusion of chemicals into and out of the fibres, colloidal interactions (Alince and van de Ven 1997[8]), fibre shrinkage (Weise and Paulapuro 1995[140]), and water removal (Maloney et al. 1997[80]).

The total amount of water present in the fibre wall, determined as the so-called fibre saturation point (FSP), can be divided into three different fractions: non-freezing water, freezing bound water, and bound water (Yamauchi and Murakami 1991[148]). The fibre wall contains small amounts of water which do not freeze at all, the non-freezing water. This non-freezing water is present in the hemicellulose-lignin matrix gel bound to the polymers. The non-freezing water [91] [117] typically represents 0.2–0.3 ml H2O/g dry wood (Nakamura et al. 1981 ; Salmén et al. 1985 ). The freezing bound water is water present in pores having volumes with a diameter greater than 1 nm but smaller than 1000 nm, usually referred to as micropores. This freezing bound water is under pressure and therefore has a depressed freezing temperature. The bulk water is water with ordinary thermodynamic properties found in pores with a diameter greater than 1000 nm, the so-called macropores, which is a term coined in the 1960’s to describe all the large cell wall pores which do not collapse during solvent-exchange drying (Stone and Scallan 1967[129]). Water in the macropores can also be referred to as free water.

Pore sizes are measured as an attempt to describe the geometry of a solid filled with voids. Basic pore analysis includes the measurement of the pore size distribution (PSD) and related information such as the average pore size. Nevertheless, such measurements are not easy since the pores are often irregular in shape and difficult to define by a single number. So, for simplicity, pores are often assumed to be represented as cylinders or spheres, with a certain pore diameter. Several techniques have been developed for determining the pore volume and pore size distribution in pulp fibres, e.g. solute exclusion (Stone et al. 1969[134]), electron microscopy (Dolmetsch and Dolmetsch 1968[34]), gas adsorption (Porter and Rollins 1972[108]), X-ray diffraction (Lenz et al. 1988[74]), nuclear magnetic resonance (NMR) (Li et al. 1993[76]), inverse size exclusion chromatography (Berthold and Salmén 1997[19]), and thermoporosimetry (Maloney 2000[79]). The disadvantage of all these techniques is that they are indirect methods. However, four of the more important of these methods are briefly described below.

4.5 Techniques for determining pore water

4.5.1 Water retention value A technique that is often used to measure swelling is the water retention value (WRV), which gives a value of the fibre saturation point or the total amount of water (ml/g) held by a pulp (total pore volume). In the WRV test, a pulp pad is centrifuged under conditions that are assumed to remove only water present between the fibres. The moisture content of the pad after centrifuging is a measure of the fibre swelling. The WRV is an empirical test method, the result of which depends on the specific test conditions (Lebel et al. 1979[73]; Abson and Gilbert 1980[2]). The WRV technique is fast, easy, and accurate and also gives reproducible results. A recently found drawback with the WRV method is that, although water is supposed to be removed from between fibres, some water is always retained and, depending on the pulp, the measured water is sometimes even pressed out from the cell wall (Maloney et al. 1999[81]). For these reasons, the WRV value can overestimate or underestimate the amount of cell water or the fibre saturation point although, for some pulps and test conditions, the WRV value may correlate with the fibre saturation point. However the WRV technique should preferably be used only when values of the total amount of water in the fibres are required or to measure relative swelling changes in pulps (Maloney et al. 1999[81]).

4.5.2 Solute exclusion Solute exclusion is one of the oldest methods available to measure the fibre pore size distribution (Stone et al. 1968[132]). Solute exclusion is based on the principle of size exclusion where the accessibility of pores to non-interacting probe molecules can be measured (Porath and Flodin 1959[107]). For wood and pulp fibres soluble mono- and polysaccharides ranging in size from glucose to Dextran with a molar mass of 2×106 constitute a suitable family of probes. The idea of solute exclusion is that, when a solution of the probe is mixed with a quantity of wet pulp fibres, the change in solution concentration can be used to calculate the inaccessible water. However it is not easy to determine the true size of pores by solute exclusion, since water near the pore walls is not available for diluting the probe molecule and some pores also have limited accessibility from the outside (Lindström 1986[77]; Alince 1991[7]; Alince and van de Ven 1997[8]). Osmotic pressure can also exclude the probe from the cell wall. This limits the reproducibility of the solute exclusion method and a large number of measurements have to be made in order to get statistically significant data. Despite these limitations, the technique can give information about the total pore volume (ml/g), the amount of inaccessible water (ml/g), the maximum pore size (nm) as well as the apparent pore size distribution (nm).

4.5.3 Inverse size exclusion chromatography Inverse size exclusion chromatography (ISEC) was first developed by Aggerbrandt and Samuelsson (1964[3]) and it is also based on size exclusion (Porath and Flodin 1959[107]), although the mobile phase is used to characterise the stationary phase and not the opposite, which is usual. The ISEC method can provide the same data as the solute exclusion technique i.e. the total pore volume (ml/g), the amount of inaccessible water (ml/g), the maximum pore size (nm) as well as the apparent pore size distribution (nm), although with the huge advantage that this information can be obtained with just one experiment. The ISEC-method has previously been used to determine differences in pore structure for cotton fibres (Martin et al. 1969[83]; Rowland et al. 1984[112]). The factor limiting the overall quality of the results in ISEC is the preparation of a column suitable for chromatographic analysis although Berthold presented a somewhat automatic packing procedure which made it possible to measure never-dried wood and pulp fibres (Berthold and Salmén 1997[19]).

4.5.4 Thermoporosimetry A differential scanning calorimetry (DSC) technique called thermoporosimetry has recently been adapted to measure the pore size distribution of pulp fibres (Maloney et al. 1998[82]). The thermoporosimetry technique is based on the fact that water retained within small pores shows a depressed melting temperature, due to the increased pressure of water in cavities with a curved interface. The pore diameter, D, is calculated from the Gibbs-Thomson equation:

−4Vmσ ls k D = = − Equation 1 Tm Tm ∆H m ln T0 where Vm is the molar volume of ice, σls is the surface energy at the ice-water interface, T0 is the melting point of water at normal pressure, Tm is the melting temperature and ∆Hm is the latent heat of melting. In this form of the Gibbs-Thomson equation, it is assumed that the pores are spherical. The Gibbs-Thomson equation assumes that the depression of melting temperature of water is caused solely by the pressure difference across a curved interface. It does not take into account

24 partial dissolution of cell wall polymers and the presence of ions in the fibre, which may also affect the melting temperature.

In conventional thermoporosimetry, the common procedure involves heating at a constant rate from below the melting temperature of the sample to a point well above it. A constant heating rate can however result in thermal lag, where there is a temperature difference between the sample and the measurement sensor just below the sample holder. For samples with large pores, including pulp fibres, the thermal lag means that a melting peak may be spread over several degrees although the melting transition is almost an isothermal event. In order to eliminate the thermal lag, an isothermal step melting procedure (Maloney et al. 1998[82]) can be used instead of a conventional constant heating rate program (Figure 16). In the isothermal step melting procedure, the sample is heated in discrete increments and, at the end of each heating step, the temperature is held constant until equilibrium is reached, i.e. until the water in the pores has finished melting.

Figure 16. Schematic drawing of a conventional DSC constant heating program and an isothermal step melting procedure. ∆t is the length of an isothermal segment, ∆T is size of a step and ∆T/∆t is the heating rate.

The peak areas in the isothermal step melting plot (Figure 17) consist of contributions from both sensible (required to increase the temperature in the sample) and latent (required to melt the water) heat. The latent heat alone is required to calculate the amount of melted water. In order to determine the sensible heat gain, the first peak in the temperature program must have a so low temperature that no melting occurs. The heat associated with the first step is then subtracted from each of the other steps taking into account the sizes of the respective steps. The result is the latent heat, given by: ∆T H = H − H n Ln Tn T1 Equation 2 ∆T1 where HLn is the latent heat of step n, HTn is the total heat of step n, HT1 is the total heat of the first step, ∆T1 is the size of step 1 and ∆Tn is the size of step n. This equation applies only when the heating rate between each step in the program is the same.

The resulting latent heat HL can be converted to pore volume according to: H V = Ln n Equation 3 H mW where Vn is the total volume of pores covered in step n, Hm is the heat of fusion, or latent heat, of the liquid and W is the dry mass of the sample. Since the volume calculated is for a complete step, it is the volume associated with an interval of pore sizes. Smaller steps in the program lead to smaller intervals and greater accuracy concerning specific pore sizes.

Figure 17. Example of an isothermal step melting plot used to calculate pore size distribution of water-saturated wood and pulp samples. The temperature, in ºC, (top label) and the corresponding pore diameter, in nm, at the end of each step is marked.

Besides eliminating the thermal lag the isothermal step melting procedure is also able to solve another problem in the measurement of the pore size distribution of pulp fibres or other materials with relatively large pores. For pulp fibres saturated with water, the water melting temperature is depressed only slightly below that of the bulk or free water, so the peaks from bulk and pore water overlap. In the isothermal step melting procedure, very small and precise changes to the temperature may be made and this permits excellent resolution of overlapping transitions.

5. Atomic force microscope investigations of the wood and fibre ultrastructure

5.1 Sample preparation for atomic force microscopy investigations In order for the tip to track the surface, investigations under the atomic force microscope require a very smooth sample surface. Smooth surfaces of flat transverse cross-sections of small wood pieces could sometimes be made with a razor-blade. For individual fibres, however, embedding in a supporting media was required, involving aligning the fibres along the fibre axis and preparing thin sections. A common preparation technique that has been used for studying wood morphology is ultramicrotomy. Specimens are embedded in a resin and thin sections are prepared with an ultramicrotome. For TEM observations, the ultramicrotome sections are about 50–100 nm thick and very fragile; but for easier handling of these sections they were 0.5 µm thick in the work described in this thesis. The embedding resins are used to prevent collapse of the specimen while cutting.

Since most of fibre interactions during processing occur in the swollen hydrated state, it is important to try to study the fibre ultrastructure in this swollen state. However, in order to ensure a satisfactory hardening of the embedding media (here an epoxy resin) the samples have to be dry. A number of different fibre drying techniques are available for the preparation of fibres with a more or less preserved ultrastructure such as air-drying, freeze-drying, and critical-point drying. It has been shown that freeze-drying and critical-point drying preserve the fibre wall very well and give similar results, while air-drying changes the ultrastructure considerably (Daniel and Duchesne 1998[33]). The fibre wall can also be dehydrated with a successive exchange of water using different organic solvents such as ethanol, methanol, and acetone; this method is called solvent- exchange drying. This procedure has been shown to preserve the microvoid structure of partly delignified wood although microvoids collapse in native wood (Papadopoulos et al. 2003[102]). In the present work, the solvent-exchange procedure was found to distort the fibre structure in delignified wood leading to delamination (Figure 18b) that was not seen in fibres after freeze- drying (Figure 18a). However when AFM investigations were made on a nanoscale on undistorted areas, indicated with white boxes in Figure 18, in solvent-exchanged holocellulose fibres and freeze-dried holocellulose fibres no significant ultrastructural differences were observed. Since solvent-exchange drying caused delamination which makes AFM measurements difficult, freeze- drying was the method chosen to dry wood and pulp fibres in the present work. In Paper IV, thermoporosimetry showed that freeze-drying of a holocellulose pulp had no significant effect on the pore volume for pores with a diameter between 1 and 500 nm.

Figure 18. AFM images of transverse holocellulose fibres after freeze-drying (a) and after solvent- exchange drying (b).

5.2 Size determination of ultrastructural features investigated with atomic force microscopy During chemical processing, fibre changes occur on the ultrastructural level. In order to understand and control the ultrastructural changes, features on the nanoscale level have to be identified and their dimensions determined. Ultrastructural images of transverse cross-sections of wood obtained using AFM are shown in Figure 19. In Figure 19a, two adjacent fibres with the intermediate middle lamella are clearly seen as well as the lumen of the fibre to the left-hand side filled with epoxy resin. A scan within the marked white box in Figure 19a generated the image displayed in Figure 19b where structures following the curvature of the fibre were noted. In a similar way a scan within the marked white box in Figure 19b generated Figure 19c where small bright features appear to generate a structure following the curvature of the fibre. This is confirmed by Figure 19d where it is clear that the structure is built up of these small bright features. In an AFM phase contrast image, lighter areas correspond to regions of greater stiffness. These bright areas were also found to be higher than the surrounding regions. The small bright features are therefore probably associated with cellulose fibril aggregates, which are known to be significantly stiffer than the hemicellulose-lignin matrix.

5.2.1 Cellulose fibril aggregates Image processing has been made with the IMP software based on watershed segmentation, developed at the Centre for Image Analysis in Uppsala, Sweden (Nordin 1997[96]; Wählby et al. 2002[147]) and adopted at STFI-Packforsk to evaluate the AFM images with regard to cellulose fibril aggregate dimensions. Although the purpose of this thesis is not to discuss image processing, some background and general comments on the image processing routine used must be given. More detailed information of the image processing steps used here was presented in the doctoral thesis by Carolina Wählby (2003[146]).

Figure 19. AFM phase images of wood in the transverse direction at different magnifications. The white box indicates the area where the following image was scanned. The digital images acquired by the atomic force microscope are not continuous but consist of discrete picture elements, so-called pixels. The first step in the image processing routine used is a pre-processing that reduces the effects of undesired imperfections induced by the imaging system. In this case the non-uniformities in the image acquired with the atomic force microscope have to be reduced, especially if intensity measures from different parts of the image are to be compared. This was done here with a data-driven approach that approximates the image background with a surface. The algorithm iteratively computes a better and better estimate of the intensity variations of the image background by fitting a cubic B-spline surface to the image. The distance between the spline surface and the image is minimised by least squares regression. To obtain a first estimate of the background, the spline surface is initially fitted to the whole image. This first approach will give a too bright estimate of the background, as even the bright objects are considered. All image pixels that deviate more than a constant number of standard deviations from the background estimate are considered to belong to the objects and are masked away. The iterative procedure continues until the average change in pixel value between two successively calculated backgrounds is less than half the original quantisation step of the image. This criterion is usually reached after 4–10 iterations.

Segmentation is a process by which an image is divided into its constituent objects and the background. This is often the most vital and most difficult step in an image analysis task. The success of the image analysis is usually depending on the segmentation result. For this reason, many segmentation techniques and segmentation algorithms have been developed over the years. The construction of a segmentation algorithm can be thought of as defining a model of the objects that are to be detected in the image. This model is then the basis for the segmentation algorithm (Wählby 2003[146]). In the easiest model, the objects are well separated and are brighter than the background. If there is a suitable intensity threshold that separates the background and objects, the segmentation is completed. However, if the objects are brighter than the background but clustered, like the cellulose fibril aggregates in Figure 19d, thresholding will only separate the objects from the background and not separate the individual objects from each other. In this case, a model is needed that says that the objects have a high intensity and are less intense at the borders towards other objects. If image intensity represents height, the cellulose fibril aggregates can be seen as mountains separated by valleys in an intensity landscape.

A segmentation algorithm that has proven to be very useful for many areas of image segmentation where landscape-like image models are used is watershed segmentation (Vincent and Soille 1991[144]), which has been used throughout this study for the identification of the assumed cellulose fibril aggregates. If the intensity of the image is thought of as the height in a landscape, watershed segmentation can be described as submerging the image landscape in water, and allowing water to rise from each minimum in the landscape. Each minimum will thus give rise to a so-called catchment basin and when the water rising from two different basins meet, a border, or watershed, is created in the image landscape. If it is necessary to separate brighter mountains, such as the cellulose fibril aggregates, from less bright valleys, the landscape is simply turned upside down by inverting the image.

The watershed segmentation used can give rise to both over- and under-segmentation. Over- segmentation means that an object in the image will be divided into several parts. Under- segmentation means that the watershed segmentation is not able to distinguish objects from a cluster. Both over- and under-segmentation can be reduced by including a priori information, in this case so-called seeds, in the model before running the actual watershed algorithm. Seeds are starting regions given as input to the watershed segmentation. Water is then allowed to rise only from these seeded regions, and all other image minima are flooded by the water rising from the seeds. The water will continue to rise until each catchment basin originating either from a foreground or a background seed meets another catchment basin. Due to the seeding, all catchment basins originating from the background have the same level (in this case 1) and can easily be separated from foreground objects.

Two kinds of seeds are needed; seeds that represent the different objects (image maxima) and a seed for the background (image minimum). Seeds can be set manually or in an automatic routine. In the watershed segmentation routine used in this thesis, a so-called h-maxima transform was used to automatically set the seeds. The h-maxima transform finds the regional maxima i.e. the objects. The input to the h-maxima transform defines the level of variation allowed within a local maximum and this value was held low and constant in order to have many seeds. It is better to have many seeds since neighbouring regions can be merged afterwards although, if too few seeds are used, some objects will not be detected at all. Since a large number of seeds is created, there will be more seeds than objects. However, if two seeds are in the same object the border between them will be weak. In this work the strength of a border between objects was measured as the

30 mean value of all border pixels of two neighbouring objects. After the merging procedure, the mean value is updated by adding together the mean values and the number of pixels of the merged objects to give a new and larger object and its neighbours. The merging procedure is continued until each remaining object border is stronger than a new given threshold. This threshold was tested for some images in order to have as good a merging of neighbouring object as possible. This threshold value was thereafter used and held constant in the subsequent image processing.

The resolution of an image determines how small details of the real world can be detected and the number of pixels in an image determines the limit of spatial resolution. In order to achieve a high resolution the AFM images were acquired with the largest number of pixels possible, i.e. 512×512. In addition the set-point amplitude during AFM imaging was optimised to achieve the best possible contrast and no changes were made in the AFM images before they were exported to tiff-format. Figure 20 shows an example of the way in which the image processing software detected and indicated the individual cellulose fibril aggregates by circling them. From the image processing program the areas, in pixels, of each object are determined. All objects found on the image border were removed. Since the size of each AFM image is known with regard to both the number of the pixels and the area, the pixel area obtained can be transformed into a relevant area in nm. The widths of the cellulose fibril aggregates can thereafter be calculated in different ways assuming circular, square, or other forms for the aggregates. The geometrical arrangement of a cellulose fibril aggregate cross-section is not known, although the cellulose fibril aggregates seems to be more or less circular, as seen in Figure 19c and Figure 19d. In this thesis, the cellulose fibril aggregates are however assumed to have a square cross-section for easy comparison with literature data.

In order to be able to assess statistical significance of the objects characterised in this work, at least five different fibres from each sample were always analysed. The fibres that were to be analysed were randomly chosen although it was sometimes not possible to scan these chosen fibres under the AFM due to surface roughness, and a substitute had to be chosen, which somewhat limited the objectiveness. From each fibre, two 1×1 µm images were usually taken from the cross- section. Each image included about one thousand objects or assumed cellulose fibril aggregates.

Figure 20. a) An original AFM phase contrast image captured from the S2 layer of a wood sample. b) The same image after image processing using watershed segmentation. 31

The shape of the scanning AFM tip influences the apparent width and height of the cell wall features, with low areas appearing smaller and high features appearing larger. It is difficult to calculate the distortion of the structure map caused by the dimensions of the AFM tip with great accuracy because several factors are involved, such as the tip radius, the radius of the features imaged, and the depth of the valleys between individual objects. A rough estimate of the enlargement effect was however possible, using ordinary triangle trigonometry (Figure 21). The half-angle (α) of the AFM tip used was according to the manufacture 10º. The average height (h) of the individual cellulose fibril aggregate was determined from the roughness function included in the AFM software and the values were found to be between 3 and 11 nm for all samples investigated. From the simple trigonometry equation given in Figure 21, the enlargement effect was calculated to be between 0.5 and 2.0 nm depending on the sample investigated.

Figure 21. An illustration of how the enlargement effect (b) is calculated.

5.2.2 Matrix lamella width In order to determine the width of the matrix lamellae present between the cellulose fibril aggregates, a second image processing routine was adopted. In this case, each AFM image was transformed to a greyscale image and the image was thereafter inverted (Figure 22). A threshold value was tested in order to distinguish in a binary image between the white matrix lamellae and the black clustered cellulose fibril aggregates as clear as possible. This threshold value was then held constant for all images to maximise the objectiveness of the image processing routine. For these black and white images, a program was constructed capable of calculating the widths and the number of lamellae along one hundred lines equally distributed across the image as indicated by the red lines in Figure 22b.

The fibre cross-sections in the samples were oriented prior to AFM investigation in order to align the lamellae vertically within the images. However all lamellae are not perfectly aligned, as seen in Figure 22, where the lamellae in the middle of the image are rotated approximately 10º from the central line in the image, whereas the lamellae in the outer part are parallel to the central line in the image. The angular dependence on the lamellae width was investigated by rotating captured images and calculating the lamella width at different angles. This investigation indicated that angles below 10º had only a minor effect on the lamella width within the margin of statistical error. Images where the majority of lamellae had angles exceeding 10º were not considered and a new image was captured. As stated earlier, five different fibres from each sample were analysed 32 and 1×1 µm AFM images were captured. Each image contained between 500 and 3000 matrix lamellae, giving a total of 2500 to 15000 matrix lamellae investigated for each sample.

Figure 22. a) an original AFM phase contrast image captured from the S2 layer of a pulp sample. b) the same image inverted and as a binary image, where the matrix lamella structures are shown in white. The red lines indicate the one hundred lines equally distributed across the image from top to bottom for calculating the widths and the number of matrix lamellae.

5.3 Lamella structure of the secondary cell wall layer The debate as to whether the wood cell wall has a concentric or a radial lamellar arrangement has been lively during recent decades. With a concentric structure, the lamellae follow the curvature of the fibre, and with a radial structure, the lamellae follow the fibre radius as seen in Figure 23. Some of the early reports on this topic proposed radial structures for the wood fibre wall (Bailey 1938[12]; Wardrop and Dadswell 1950[139]). However, these early studies were carried out on compression wood, which is different both chemically and structurally from normal wood (Blanchette et al. 1994[23]). In the early 1970’s Stone et al. (1971[133]) suggested, based upon delignification and drying experiments on wood fibres, that the cross-sectional structure of a fibre had a concentric lamellar structure.

Figure 23. Schematic representation of a fibre with a concentric (a) and radial (b) lamella structure modified from Stone et al. (1971[133]).

A few years later, Scallan (1974[119]) proposed a modification of the lamellar structure of the fibre wall where radial cleavages in the wood cell wall where proposed and said to be sufficient for the transport of macromolecules through the cell wall and to provide the cell wall with cohesive strength in the radial direction. Scallan (1974[119]) also suggested that a perfect concentric lamella structure exists only after pulping and extensive swelling. One of the best known models of the wood cell wall with a concentric lamella structure is the one seen in Figure 24, which was proposed by Kerr and Goring (1975[64]). Their results were to a large extent in agreement with an earlier model proposed by Scallan (1974[119]). In a study using TEM to investigate cross-sections of black spruce and silver fir, Ruel et al. (1978[113]; 1979[114]) confirmed this concentric lamella structure.

During the 1990s, the idea of a radial lamella structure for the wood cell wall had a renaissance after several papers by Sell and Zimmermann (1993[123]; 1993[122]; Zimmermann et al. 1994[155]; Zimmermann and Sell 1997[153]). Sell and Zimmermann fractured wood in longitudinal bending and saw, using SEM, radial structures on transverse wood cross-sections. Their explanation for these radial structures was that the bonds between cellulose fibril aggregates are stronger in the radial than in the tangential direction. In an ultrastructural investigation using TEM in addition to SEM, radial striations of both cellulose fibril aggregate agglomerates and lignin in compression wood of pine were reported (Singh et al. 1998[125]). These radial structures were of the order of 30–300 nm thick. Sell and Zimmermann also noticed such radial agglomerations in the S2 layer of compression wood (Zimmermann and Sell 2000[154]). A similar kind of radial structure was found in the gelatinous G-layer of tension wood, but in this case there was no strictly perpendicular orientation relative to the middle lamella (Zimmermann and Sell 2000[154]). On the basis of literature data and specific studies of the tracheid cell wall of Yellow pine using different methodologies, including bacterial decay and chemical treatment, Larsen et al. (1995[71]) refined the model proposed by Kerr and Goring (1975[64]) into a model with a concentric laminar structure for the cellulose microfibrils but with a radial plane of symmetry. According to these authors, these radial planes could easily be exploited during degradation by fungal and thermochemical agents. They also suggested that the radial structure may be a revealed, rather than a modified, 34 structure of the underlying wood chemical composition and ultrastructure, which is not evident in undecayed wood (Larsen et al. 1995[71]). A radial lamella structure has thus never been observed unless the cell walls have been modified biologically, chemically, or distorted mechanically in some way.

The AFM images produced in this thesis, e.g. Figure 19c and Figure 19d, indicated a concentric lamella orientation for the fibre wall built up of aligned cellulose fibril aggregates. It is however clear that some radial structures are present after the chemical or mechanical treatment of wood. The question was therefore whether these two structures could co-exist? This was further investigated in Paper I where it was found that the fracturing of wood in both longitudinal bending and tensile testing gave severe damage of many individual wood fibres (Figure 25). Individual fibres showed mostly a preferred radial lamellar structure, although many fibres also had a dense and compact structure where it was impossible to see any preferred structure at all. The radial structures found were between 30 and 100 nm thick. The fibre damage found was in agreement with the results presented by Sell and Zimmermann (1993[122]; Zimmermann and Sell 1997[153]).

Figure 25. SEM images showing a) Overview of the fracture zone from wood fractured in tensile testing. b) Wood fibres with a brittle fracture surface with only part of the cellulose fibril aggregate/matrix loosened up. c) Structures of the order of 30–100 nm in size oriented in both a radial and a concentric manner.

In Paper I, AFM investigations of the wood fracture surfaces from 5–10 fibres showed both radial and concentric structures within the fibre wall (Figure 26a). However, some hundred microns below the actual fracture surface, only a concentric lamella structure within the fibre wall was evident in these fibres (Figure 26b). These concentric lamellar structures originated from aligned cellulose fibril aggregates interrupted by a more or less tangential layer of matrix material (Figure 26c).

Figure 26. a) AFM image from the fracture zone of a wood fibre fractured in tensile testing. The middle lamella (M) and lumen (L) as well as the S2 layer of the wood fibre wall are indicated in the figure. Radial striations due to fracture are marked with black arrows, and concentric lamellae are marked with white arrows. b) AFM image from the same fibre 300 µm below the fracture zone. The middle lamella (M) and lumen (L) as well as the S2 layer of the wood fibre wall are indicated in the figure. The concentric lamella structure is marked with white arrows. c) Close up of a part of the S2 layer in b) where it is evident that the concentric lamella structure consists of aligned cellulose fibril aggregates with a more or less straight region of matrix material in between. In the figure, some cellulose fibril aggregates are marked to clarify their appearance and arrangement inside the S2 layer of the wood fibre wall. The tangential direction of the fibre wall is indicated with the white arrow.

Paper I showed that radial structures are formed during wood fracturing. However these radial structures did not penetrate far into the cell wall structure in the longitudinal direction. In fact, in the samples with cracks examined by AFM, no underlying radial structure pointing to the location of these cracks was found. It is therefore suggested that these radial fracture structures are created during the fracture process itself. A large amount of energy has to be stored in the fibre wall before wood fracture; an energy which, when released, is able to distort the cell wall structure. It seems logical that the lamellar structure could appear to be both concentric and radial in this distorted wood, depending on the way in which the wood cell has been fractured. This is essentially in agreement with Sell (1994[121]) who viewed the cell wall structure as being sandwich-like.

If wood fibres are considered to be part of a fibre-reinforced composite material with the cellulose fibril aggregates as reinforcement in a matrix consisting of hemicelluloses and lignin, the fracture process can be compared to that of other types of composite materials. When composites with glass or carbon fibres in a polyester matrix are fractured in tensile testing, radial structures can appear and these can be seen as agglomerates of glass and carbon fibres in a more or less destroyed matrix (Piggott 1980[104]). This structural appearance has also been reported for graphite fibres in a matrix of epoxy resin (Whitcomb 1979[141]). A similar but more random structure was found in carbon-fibre-reinforced carbon matrix in which there is a very strong fibre-matrix bond (Cooper 1974[32]). Paper I showed that the cracks found in wood had no particular starting point but were more or less randomly situated (Figure 25 and Figure 26). The regular distance between the radial cracks (Figure 26a) was in agreement with a hypothesis regarding statistical fracture in composites presented by Argon (1974[9]). Radial structures are undoubtedly dependent on wood distortion and are thus evident only at positions adjacent to the fracture surface. The concentric lamellar structure, on the other hand, is present throughout the native wood structure. This is in accordance with most of the previous microscopic studies (Kerr and Goring 1975[64]; Scallan 1977[120]; Ruel et al. 1978[113]) and also with the results of other methods such as solvent-exchange (Stone and Scallan 1968[131]), fibre swelling (Stamm and Smith 1969[128]; Scallan 1974[119]), and the drying of wood (Stone et al. 1971[133]). Although the model presented by Kerr and Goring (1975[64]) and shown in Figure 24 basically represents this structure, some modifications based on the results presented in this thesis may be made. It is evident that the concentric lamella structure should be considered to be built up of individual cellulose fibril aggregates 15×15 nm in cross- section rather than of 2–4 cellulose microfibrils bonded at their radial faces.

6. Ultrastructural changes in the kraft pulp fibre line

6.1 Changes in cellulose fibril aggregate dimensions It is well known that changes occur in the ultrastructure during chemical processing of wood due to the dissolution of lignin and hemicelluloses. One of the ultrastructural features of particular interest is the cellulose fibril aggregate dimensions because they are known to correlate with pulp strength (Molin 2002[90]). The changes in the cellulose fibril aggregate dimensions were therefore followed through the kraft pulping process. The cellulose fibril aggregates investigated in Paper II–Paper IV within the S2 layer of native wood showed a distribution of aggregate sizes (Figure 27). Based on the size distribution seen in Figure 27 and considering that the cross-section of a single cellulose microfibril is 4 nm in width (section 2.2.1) it seems probable that one cellulose fibril aggregate could consist of from one up to about 7×7 cellulose microfibrils. Nevertheless, most of the cellulose fibril aggregates are in the size range from 8–16 nm and hence composed of 2×2 up to 4×4 cellulose microfibrils. There are no sharp size increments for the cellulose fibril aggregates, but a fairly smooth distribution of cellulose fibril aggregate dimensions as displayed in Figure 27. The less well- defined sizes are probably due to the incorporation of up to 30 % of the total amount of hemicelluloses inside a cellulose fibril aggregate, as indicated by Fengel (1970[39]). The varying size of the cellulose fibril aggregates is in agreement with the three-dimensional structure of cellulose microfibrils proposed by Boyd (1982[25]). A schematic illustration of the build up of individual cellulose fibril aggregates incorporating different numbers of cellulose microfibrils is seen in Figure 28.

Figure 27. The size distribution of cellulose fibril aggregates within the S2 layer of spruce wood.

Figure 28. A schematic drawing of the variation in cellulose fibril aggregate dimensions.

The average side length for a cellulose fibril aggregate present within the S2 layer in native spruce wood, assuming square cross-sections, was found to be 16.0 nm (Paper II–Paper IV). This value is somewhat higher than the value of about 15 nm obtained by NMR (Newman 1992[93]; Hult et al. 2000[59]). The discrepancy between the AFM and NMR results is probably due to the enlargement effect of the AFM tip. If the side lengths for the cellulose fibril aggregates in wood are corrected for the tip enlargement effect the average side length for a cellulose fibril aggregate is 15.4 nm (Paper II–Paper IV), which is close to the data obtained by NMR (Newman 1992[93]; Hult et al. 2000[59]). With other microscopy techniques such as SEM and TEM it is difficult to study cellulose microfibrils within the S2 layer without delignification of wood or staining of wood polymers. In a study using SEM and rapid-freezing deep-etching techniques, cellulose microfibrils were however observed within the S2 layer, and bundles of these cellulose microfibrils were found to aggregate into cellulose fibril aggregates between 9 and 20 nm in width (Hafrén et al. 1999[47]). Since no suitable method for staining of cellulose exists, conclusions regarding the width of aggregated cellulose microfibrils using TEM are based on chemically stained lignin and literature values of cellulose microfibrils. Using this procedure, it has been suggested that aggregates of 5– 10 cellulose microfibrils are formed in spruce wood with a diameter of between 20 and 40 nm (Singh and Daniel 2001[124]).

AFM enables ultrastructural studies at different locations in the transverse direction of the fibre wall (Paper III and Paper IV). Figure 29 shows AFM images covering whole fibre wall structure from the fibre surface side to the lumen side. In Paper III and Paper IV, it was evident that the fibre wall ultrastructure in the outermost parts of the fibre wall was different from that in the centre of the fibre wall. These parts appeared to have a slightly different cellulose fibril aggregate size, which was taken as an indication that the outer part closest to the middle lamella corresponded to the S1 layer and that the outer part closest to the lumen corresponded to the S3 layer of the fibre wall. No significant differences in cellulose fibril aggregate sizes across the native wood fibre wall within the S2 layer were observed (Table 1).

Figure 29. a) Part of a fibre revealed with AFM. b–d) Three AFM images cover the complete cross-section of the same fibre wall in the transverse direction; from the fibre surface side (b) to the lumen side (d).

Table 1. Average cellulose fibril aggregate size and standard deviation for different locations in the transverse direction of spruce wood before and after correction for the tip enlargement effect. Cellulose fibril Cellulose Total number of Tip aggregate size fibril cellulose fibril Location of aggregates enlargement corrected for the aggregate size aggregates (nm) tip enlargement (nm) investigated (nm) Near M.L. 16.0 ± 1.4 11605 0.7 ± 0.3 15.4 ± 1.4

Interior of the S2 layer 16.2 ± 1.6 24612 0.4 ± 0.1 15.8 ± 1.6 Lumen side 15.7 ± 1.7 12437 0.6 ± 0.2 15.2 ± 1.7

Paper II and Paper III showed that there was a large increase, over 25 %, in cellulose fibril aggregate dimensions during the kraft pulping of spruce wood. However, during the low temperature delignification of wood to produce holocellulose pulp, there was no change in cellulose fibril aggregate dimensions (Paper IV, Figure 30, and Table 2). In Table 2, it is also evident that all the chemical pulping processes investigated increased the cellulose fibril

41 aggregates to the same extent i.e. about 25 %. It was further found that bleaching had no effect on the cellulose fibril aggregate dimensions (Table 2). The cellulose fibril aggregate dimensions increased to the same extent throughout the fibre wall in the transverse direction (Figure 30). An increase in cellulose fibril aggregate dimensions during kraft pulping has been reported, with other methods such as NMR and SEM supporting the findings presented in this work (Duchesne and Daniel 2000[36]; Hult 2001[58]). During kraft pulping, more than 50 % of the hemicelluloses are dissolved and it has been suggested that the hemicellulose dissolution induces cellulose fibril aggregate enlargement (Duchesne et al. 2001[37]; Hult et al. 2001[60]). In Paper IV, it was found that during holocellulose pulp preparation 40 % of the hemicelluloses were lost, although no cellulose fibril aggregate enlargement was found. It is therefore probable that the difference in processing temperature between the holocellulose and kraft pulp process is the key to cellulose fibril aggregate enlargement. This was further investigated in Paper II where small wood chips were heated to different temperatures with or without chemicals. Cellulose fibril aggregate enlargement occurred in wood treated in pure water at 150 ºC with minimum loss of hemicelluloses. Alkali treatment at temperatures below 100 ºC had no effect on the cellulose fibril aggregate dimensions, although substantial amounts of hemicelluloses were dissolved.

Table 2. Average cellulose fibril aggregate size within the S2 layer of spruce wood, a holocellulose pulp, and in different pulps. The pulps were an unbleached isothermal continuous cooking (ITC) kraft pulp and three chemical pulps (ITC, rapid displacement heating (RDH), and polysulphide (PS)) bleached in a D(E)D sequence. Cellulose fibril aggregate size Total number of corrected for the cellulose fibril Tip enlargement Type of sample tip enlargement aggregates (nm) effect investigated (nm) Spruce wood 15.5 ± 1.5 48654 0.5 ± 0.2 Holocellulose pulp 15.7 ± 0.5 3409 1.5 ± 0.5 ITC - unbleached pulp 20.0 ± 1.3 5799 1.3 ± 0.4 ITC - unbleached refined pulp 19.2 ± 1.2 4470 1.5 ± 0.5 ITC - bleached pulp 20.9 ± 2.1 2485 1.1 ± 0.1 RDH - bleached pulp 19.6 ± 1.4 3535 1.2 ± 0.5 PS - bleached pulp 19.4 ± 1.3 3329 1.1 ± 0.1

Figure 30. Cellulose fibril aggregate dimensions at different positions across the fibre wall in spruce wood, holocellulose pulp, and kraft pulp. The average increase in cellulose fibril aggregate dimensions of 25 % observed in kraft pulps corresponds to an actual increase in side length of 4 to 5 nm. This rather small enlargement can to some extent be explained by the three-dimensional model proposed by Boyd and Foster (1975[26]), featuring cellulose microfibril bridges between adjacent cellulose fibril aggregates. These cellulose microfibril bridges probably associate with one of the existing cellulose fibril aggregates and form a 4–5 nm larger structure. However this cannot explain the broad distribution of cellulose fibril aggregate dimensions found after kraft pulping (Figure 31). Therefore, it is also probable that small fibril cellulose aggregates merge together or are incorporated into existing cellulose fibril aggregates, see below.

The softening temperature for the amorphous wood polymers is very high in the dry state, between 180 and 250 ºC (Back and Salmén 1982[11]). A pronounced lowering of the softening temperature is observed in the water-saturated state. Spruce wood in the water-saturated state softens at about 90 ºC (Olsson and Salmén 1997[98]), a behaviour assigned mainly to the lignin (Back and Salmén 1982[11]; Irvine 1984[62]; Olsson and Salmén 1997[98]). Hemicelluloses are already well above their softening temperature which in the water-saturated state occurs below 0 ºC (Back and Salmén 1982[11]). Thus, under kraft pulping conditions, the native lignin exhibit a substantially increased mobility; and this increases the probability for adjacent cellulose microfibril or small cellulose fibril aggregate surfaces to come into close contact and form a larger structural unit i.e. cellulose fibril aggregate enlargement. Although refining has a major impact on both the fibre level and the ultrastructural level, no dimensional changes in the cellulose fibril aggregates have been observed (Laine et al. 2004[69]). This is in agreement with the results of AFM measurements (Figure 32) where refining had no effect on the cellulose fibril aggregates at any location in the transverse direction of the fibre wall of either kraft or holocellulose pulps (Paper III and Paper IV). This indicates, as has been suggested by Laine et al. (2004[69]), that refining is not able to split the cellulose fibril aggregates. In addition the energy or temperature in the refiner is not sufficient to cause further cellulose fibril aggregate enlargement. 43

Figure 31. The size distribution of cellulose fibril aggregates investigated within the S2 layer of spruce wood, holocellulose pulp, and kraft pulp.

Figure 32. Cellulose fibril aggregates dimensions at different positions across the fibre wall in a holocellulose and a kraft pulp before and after refining.

6.2 Visualisation of pores and the transverse pore structure with atomic force microscopy As was stated in section 4.5, several techniques have been developed for determining the pore volume and the pore size distribution in pulp fibres. None of these methods is however able to show the actual pores, to give a measure of the pore sizes across the fibre wall or even to determine where in the fibre wall the pores are located. In Paper III, pulp fibres were impregnated with poly(ethylene glycol), with a molar mass of 5000–7000, in order to reveal the pore structure present in pulp fibres. A temperature increase during the AFM measurements changed the phase contrast images, indicating a softening of the poly(ethylene glycol) present in the cell wall pores (Figure 33). The change in phase contrast seen as an increase in dark areas occurred at positions all across the sample surfaces indicating that pores filled with poly(ethylene glycol) were randomly distributed between the cellulose fibril aggregates throughout the fibre wall. In wood impregnated with poly(ethylene glycol), where virtually no pores exist, no changes were observed when the temperature was increased.

Figure 33. AFM phase contrast images of a part of the S2 layer from two different pulp fibres one without (a and b) and one impregnated with poly(ethylene glycol) (c and d) at two different temperatures: 25 ºC (a and c) and 50 ºC (b and d). Images a) and b) show no visible differences. In the sample impregnated with poly(ethylene glycol), there was an obvious increase in the amount of dark areas between the image captured at 25 ºC (c) and the image captured at 50 ºC (d). In all the samples, the individual cellulose fibril aggregates were clearly seen as bright features.

Figure 34. A schematic drawing illustrating the enlargement of cellulose fibril aggregates due to the incorporation of individual cellulose microfibrils or small cellulose fibril aggregates and the formation of pores within the hemicellulose-lignin matrix during kraft pulping.

Unfortunately, it was not possible to make a distinction between the hemicellulose-lignin matrix and the pores impregnated with poly(ethylene glycol) and the dark areas consisting of both matrix material and pores are hereafter referred to as “pore and matrix lamellae” (Figure 34). Kraft pulp fibres impregnated with poly(ethylene glycol) showed a small and uniform increase in pore and matrix lamella width at all positions across the fibre wall, also indicating that pores are present throughout the fibre wall in the transverse direction (Table 3).

Table 3. Average pore and matrix lamella widths and standard deviations in five kraft pulp fibres both impregnated with poly(ethylene glycol) and without impregnation measured with AFM at two scanning temperatures. Average lamella width Change from (nm) 25 to 50 ºC (nm) 25 ºC 50 ºC Kraft pulp fibres Near M.L. 20.0 ± 0.3 20.0 ± 0.1 0.0

without Interior of S2 layer 20.0 ± 0.2 20.0 ± 0.4 0.0 poly(ethylene glycol) Lumen side 19.5 ± 0.1 19.6 ± 0.1 0.1

Kraft pulp fibres Near M.L. 21.0 ± 0.5 22.7 ± 0.6 1.6

impregnated with Interior of S2 layer 19.5 ± 0.6 21.1 ± 0.5 1.7 poly(ethylene glycol) Lumen side 19.0 ± 0.4 20.6 ± 0.2 1.5

6.3 Changes in pore and matrix lamella dimensions During the chemical treatment of wood, the dissolution of lignin and to some extent hemicelluloses creates pores in the structure and also induces ultrastructural changes. In Paper III, it was found that the widths of the pore and matrix lamellae were significantly enlarged during kraft pulping and that the numbers of these pore and matrix lamellae were considerably reduced during pulping. However, in the low temperature delignification of wood to yield a holocellulose pulp, only a very small increase in lamella width was found (Figure 35 and Paper IV).

Figure 35 Widths (top) and number (bottom) of pore and matrix lamellae at different positions across the fibre wall for wood, a holocellulose pulp, and a kraft pulp.

If the cellulose fibril aggregate enlargement is due to individual cellulose microfibrils or small cellulose fibril aggregates associating with one of the cellulose fibril aggregates nearby, a space will be left. The pore area adjacent to the matrix lamella is then viewed as broadened and the number of pore and matrix lamellae is also decreased, which would explain the changes occurring in the kraft pulp. The difference in pore and matrix lamella width between the holocellulose and the kraft fibre is not reflected in the pore volume of the samples, indicating that only a redistribution of matrix material has occurred in the kraft pulp (Figure 36).

Figure 36. Cumulative pore volume measured with thermoporosimetry for wood, a holocellulose pulp before and after refining, and a kraft pulp.

During the refining of a kraft pulp, it was concluded that an increase in pore and matrix lamella width occurred, especially near the outer surface (Figure 37). It is probable that, since refining loosens up the outer parts of the fibre wall, expanded corridors consisting of pore and matrix materials can form in the outer part of the fibre wall (Szwarcsztajn and Przybysz 1972[136]; Page 1989[100]) although the number of lamellae remains constant. This hypothesis is in agreement with the results obtained by Nanko (1998[92]) who reported a successive loosening of the cell wall in the regions surrounding a crack during the refining of never-dried pulp fibres. Another contributing factor could be the release of the matrix polymers to the external liquid phase accompanying the opening of pores in the cell wall during the mechanical treatment in a refiner (Lindström et al. 1978[78]). The leakage of these wood polymers probably occurs from the outer part of the cell wall. In a holocellulose pulp, there was no increase in pore and matrix lamella width at any position during refining, and even a small reduction in pore volume was observed (Figure 36 and Figure 37). This does not reflect the main objective of refining i.e. to increase fibre swelling, but it may be a consequence of the mild refining performed here (2500 revolutions) which has been shown to slightly reduce the total pore volume by forcing pores to close, as indicated by inverse size exclusion chromatography (Berthold and Salmén 1997[18]). The pore closure that occurred probably also explains why there was no increase in lamella width in the outer part of the fibre wall, in contrast to the behaviour of the kraft pulp.

Figure 37. Widths (top) and number (bottom) of pore and matrix lamellae at different positions across the fibre wall for a holocellulose pulp and a kraft pulp before and after refining.

6.4 Ultrastructural changes during drying It is known that water removal form the fibre wall during pulp fibre drying results in a more or less irreversible pore closure. This pore closure leads to a reduction in the swelling ability of the fibres, so-called hornification (Stone and Scallan 1968[131]; Lindström 1986[77]; Laivins and Scallan 1993[70]; Berthold and Salmén 1997[18]). This should naturally affect the ultrastructural behaviour of wood and holocellulose and particularly the pore and matrix lamella structure. After air-drying of wood and embedding in the dry-state, a small decrease of about 1 nm in the cellulose fibril aggregate dimensions was observed (Figure 38). The reason for this is probably that water present within the cellulose fibril aggregates bound to glucomannan and disordered cellulose swells the cellulose fibril aggregates to some extent. After drying at room temperature no significantly changes were found in the cellulose fibril aggregate dimensions for holocellulose pulp (after rewetting) (Figure 38). The probable explanation for this finding is that the mobility of the fibre wall polymers is very low at temperatures below the glass transition and that individual cellulose microfibrils or cellulose fibril aggregates cannot be incorporated into an existing cellulose fibril aggregate. It has also been found with NMR that virtually no cellulose fibril aggregate enlargement occurred during pulp drying, where the hemicellulose content was above 15 % (Duchesne et al. 2001[37]; Hult et al. 2001[60]).

Figure 38. Cellulose fibril aggregate dimensions at different positions across the fibre wall in untreated, dried, dried, and refined holocellulose pulp (tested in the water swollen state), wood (tested in the water swollen state), and air-dried wood (tested in the dried state).

The pore and matrix lamella width increased moderately during the preparation of holocellulose pulp from wood. Drying and rewetting of the holocellulose resulted in a reduction of the pore and matrix lamella width by about 2 nm to that of native wood, a width that was partly recovered upon refining. Native wood showed a small decrease in the pore and matrix lamella width during air- drying at room temperature when tested in the dried state (Figure 39). In Paper IV, it was found that the width of the pore and matrix lamella also became narrower after enzymatic treatment of the hemicelluloses. The pore closure occurred throughout the fibre wall, in the same order as for the dried and rewetted holocellulose, indicating an enzymatic attack of the entire fibre wall,

50 causing hornification. These results further support the previous results indicating that pores are present throughout the fibre wall in the transverse direction of the cell wall (section 6.2). No significant change in the number of pore and matrix lamellae was found between the holocellulose samples and the native wood sample, although the scatter was rather large (Figure 39).

Figure 39. Widths (top) and number (bottom) of the pore and matrix lamellae at different positions across the fibre wall for untreated, dried, dried and refined holocellulose pulp (tested in the water swollen state), wood (tested in the water swollen state), and air-dried wood (tested in the dried state).

The pore volume, measured with thermoporosimetry, indicated that the untreated holocellulose pulp had a larger pore volume than the other holocellulose samples (Figure 40). Drying the holocellulose pulp decreased the pore volume considerably, apart from native wood, this pulp had the lowest pore volume in the whole pore range. This is in agreement with earlier studies (Stone and Scallan 1968[130]; Lindström 1986[77]; Berthold and Salmén 1997[18]). The pore and matrix lamella width for the dried holocellulose also decreased down to the level of native wood. This reduction in pore and matrix lamella width accounts for a relative volume decrease of 7 %, considering changes in length and in width perpendicular to the lamellae to be negligible. If the cell wall polymers are assumed to have a density of 1.5 g/cm3 with the holocellulose containing 0.3 ml/g non-freezing water in addition to the 1.28 ml/g freezing bound water here measured (Figure 40), this reduced swelling upon drying will account for only 0.16 ml/g. This is less than half the decrease in pore volume detected, indicating that a lot of the pore volume water may be related to water in the surface gel. Another possible explanation could be a total collapse of some of the pore and matrix lamellae, indicated by a small decrease in the number of pore and matrix lamellae for the dried holocellulose pulp (Figure 39).

Figure 40. Cumulative pore volume measured with thermoporosimetry for untreated, dried, dried and refined holocellulose pulp, and wood.

7. Conclusions Atomic force microscopy (AFM) in combination with image processing was the main tool used for studies of the wood cell wall ultrastructure and the ultrastructural changes occurring during different chemical pulping processes. Other methods such as scanning electron microscopy, enzymatic degradation, and thermoporosimetry were also used to support the ultrastructural investigations made with AFM. The following information relating to the wood cell wall ultrastructure and changes during processing was obtained:

• In native spruce fibres (tracheids), cellulose fibril aggregates exists as agglomerates of individual cellulose microfibrils (with a width of 4 nm). The average side length for a cellulose fibril aggregate was 15–16 nm (assuming square cross-sections), although there was a broad distribution where the side lengths varied from 5 up to 25 nm. It was further found that the cellulose fibril aggregate dimensions were uniform in size in the transverse direction across the wood fibre wall.

• In native wood, the secondary cell wall layer was found to have a concentric lamella structure (following the fibre curvature) built up of individual cellulose fibril aggregates and not, as suggested earlier of agglomerates of 2–4 cellulose microfibrils. The width of these lamellae is of the order of the width of a single cellulose fibril aggregate, i.e. about 15 nm. The matrix material, costing of hemicelluloses and lignin, present between cellulose fibril aggregates also forms a more or less concentric lamella structure. The width of these matrix lamellae was also found to be uniform in the transverse direction of the wood fibre wall.

• During kraft pulping, a 25 % increase in cellulose fibril aggregate dimensions was found, but no cellulose fibril aggregate enlargement was found during the low-temperature delignification of wood into holocellulose pulp. Neither refining nor drying of kraft or holocellulose pulp changed the cellulose fibril aggregate dimensions. It was found that a high temperature, over 100 ºC, increasing the mobility of the fibre wall polymers, is the most important factor contributing to cellulose fibril aggregate enlargement.

• When lignin is dissolved during the delignification of wood, pores are formed in the fibre cell wall. These pores have a uniform distribution throughout the transverse direction of the wood cell wall. After kraft pulping, the lamellae consisting of both pores and matrix materials (“pore and matrix lamella”) became wider and their number decreased. In holocellulose pulp, no such changes were seen. Refining increased the pore and matrix lamella width in the outer parts of the fibre wall of kraft pulps, but this was not seen in holocellulose pulp.

• Drying of holocellulose pulp had no impact on the cellulose fibril aggregate dimensions. However, a small decrease in the pore and matrix lamella width was observed upon drying reflecting the “hornification” of the pulp. Refining of holocellulose pulp led to pore closure probably due to the enhanced mobility within the fibre wall. Enzymatic treatment using hemicellulases on holocellulose pulp revealed that both the hemicelluloses present in spruce were always dissolved to some extent by hydrolysis of one of the two, this could indicate that the two hemicelluloses are some extent linked to each other in the structure. The enzymatic treatment also decreased the pore volume to some extent throughout the fibre wall in the transverse direction indicating enzymatic accessibility to the entire fibre wall.

8. Future aspects The results presented in this thesis have shown that several changes in the cell wall ultrastructure occur in the kraft pulp fibre line. However, knowledge of the effects of these ultrastructural changes on the strength of the pulp and paper produced is still limited. So far it has only been shown that the tear index of a kraft pulp at a given density correlates with the cellulose fibril aggregate dimensions (Molin 2002[90]). Therefore investigations that are designed to relate ultrastructural changes occurring during chemical pulping to pulp and paper strength properties are necessary. This is particularly important in order to increase pulp strength and pulp yield and in order to tailor fibre properties.

Atomic force microscopy (AFM) proved to be a useful tool for the ultrastructural investigation of wood and pulp fibres. It should therefore be possible to use the AFM for ultrastructural investigations of other chemical pulps or mechanical pulps. The AFM could also be used to explore the ultrastructure of other fibre types such as hardwood fibres or recycled fibres. In the work embraced by this thesis, AFM investigations were made under ambient and dry conditions at room temperature or somewhat higher temperatures, but it would also be of interest to utilise the AFM for ultrastructural investigations on fibres under moist conditions and at even higher temperatures exceeding the softening temperature of the individual wood polymers. If this could be done, ultrastructural changes could be observed in situ. Utilising the AFM for studying fibres both under wet and dry conditions would make it possible to explore ultrastructural changes during hornification.

Measurements of the pore volume in wood and pulp fibres by thermoporosimetry have been used in this work in order to compare changes in pore and matrix lamella dimensions with changes in pore volume. Ultrastructural changes occurring especially to the pore and matrix lamellae could usually be related to changes in the pore volume although these relations were sometimes obscure. It seemed that the thermoporosimetry method was sensitive to sample weight and moisture content, and this needs to be further studied. In this thesis, the total pore volume measured with thermoporosimetry appeared to be relatively high, approaching the literature values for the total water content present within the fibre wall. It is therefore important to compare thermoporosimetry data with measurements of the total pore volume i.e. the fibre saturation point, using, for instance, solute exclusion techniques.

9. Acknowledgements I am indebted to my supervisor, Associate Professor Lennart Salmén, for ideas, inspiration, guidance, support, encouragement, and patience throughout this thesis work. Professor Ulf W. Gedde is acknowledged for accepting me as his student and for valuable comments on all the manuscripts.

I would like to thank Ann-Catrine Hagberg, Joanna Hornatowska (STFI-Packforsk) and Owe Lidbrant (formerly STFI-Packforsk) for help with sample preparation and assistance with various microscopy techniques. Dr. Carolina Wählby (Centre for Image analysis in Uppsala), Hans Christiansson and Dr. Catherine Östlund (STFI-Packforsk) are acknowledged for help with image processing and Matlab® issues. I am also thankful to Niklas Jacobson for help with holocellulose preparation and enzymatic treatments. Associate Professors Peter Axegård and Nils-Olof Nilvebrant are acknowledged for valuable comments on the manuscript. Associate Professor Anthony Bristow is acknowledged for linguistic review.

I thank all former and present members of the Wood, fibre and Mechanical pulping group for interesting discussions, assistance and for making these years pleasant. I would also like to thank every one else at STFI-Packforsk for support and for providing a pleasant working environment.

I wish to thank friends and colleagues at the Department of Fibre and Polymer Technology at the Royal Institute of Technology (KTH) for technical assistance and for fruitful co-operation during courses in the field of fibres and polymers.

This work was carried out at STFI-Packforsk AB, to which I am very thankful for providing me the opportunity to write this thesis. Financial support from WURC (Wood Ultrastructure Research Centre) is especially acknowledged.

I would also like to express my sincere thank to my mother and father for all their love and support.

Finally, I wish to express my love to Sofi; your warm heart, support and patience made this thesis finally come through!

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