Research

Aim: The aim of this study is to generate ultrastructural models for tracheid cell walls of Norway spruce (Picea abies L. Karst.) including data on chemical and physical properties.

Background: Earlier generated models are out of date and do not consider new research findings. Therefore they are partly inaccurate and at high resolution the structure of the cell is shown as a small undefined area in most models.

Project description: Literature studies and results generated within WURC by this and other projects should make it possible to generate integrated fibre models of spruce tracheids.

lnitially there will be a literature survey of wood anatomy with special focus on the tracheids in spruce xylem. After that data for macro-, micro- and ultrastructure will be collected, for example on tracheid dimensions, cell wall layers, microfibrillar orientation etc. In cases where information is lacking in the literature, TEM and SEM studies will be carried out. Data for ultrastructure may come from studies on conifer species, other than Norway spruce. Further on, there will be a survey to find software dealing with structure modelling. The first model generated will show the whole tracheid at low resolution. The first study will focus on the distribution of pits in tracheid walls. The data will be used for creating models for pit distribution. Thereafter more models should be generated at higher resolution and over smaller, well defined, areas. Since the other projects within WURC will contribute their results it should be possible to generate a model at very high resolution.

Project group:
Thomas Nilsson, SLU (project leader)
Jonas Brändström, SLU (Ph. D student)
Stig Bardage, SLU
Geoffrey Daniel, SLU
Lennart Salmén, STFI.

Industrial mentors:
Inger Eriksson, SCA
Gunilla Söderstam, StoraEnso

IFP Pulp 2000

Aim: The project's overall aim is to characterise the surface ultrastructure of "wood fibres" and investigate how this changes after various enzymatic, mechanical and chemical treatments.

Background: The surface structure of wood fibres has been insufficiently characterised previously, partly because of the unavailability of good analytical techniques and partly because of a lack of information on the native ultrastructure of wood fibres.

Project description: In the present project the surface characteristics of wood fibres will be studied using a variety of complementary microscopical methods including: high resolution scanning electron microscopy (FE-SEM), high resolution SEM in conjunction with cryo-techniques, transmission electron microscopy (TEM), environmental scanning electron microscopy (ESEM), and atomic force microscopy (AFM). Emphasis will be placed on spruce wood fibres which will be used to develop a model representing a "reference surface structure of softwood fibres". This reference wood fibre structure will then be compared with standard fibres subjected to prior enzymatic, mechanical or chemical treatments.

1. Frozen hydrated fibre surface (FE-SEM)
2. Freeze dried fibre surface (FE-SEM)
3. Air dried fibre surfaces (FE-SEM)

Project group:
Geoffrey Daniel, SLU
lsabelle Duchesne, SLU
Thomas Nilsson, SLU
Bert Pettersson, STFI

Industrial mentors:
Inger Eriksson, SCA
Lars Wågberg, SCA

Aim: The aim of the project is to elucidate important relations between structural characteristics of wood cellulose and hemicelluioses and the reactivates and properties of fibre substrates.

Background: Although cellulose is a simple homopolysaccharide. natural celluloses have a solid state architecture with a high degree of individuality, depending on their biological origin and the isolation procedure used. The solid state structure is expected to greatly influence the reactivity and physical properties of cellulosic materials.

Project description: The work is focused on the influence of supramolecular and surface structure of cellulose fibrils and the association to hemicelluloses on pulp and paper properties and accessibility/ reactivity in enzyme-assisted fibre modification strategies.

1 3 C - CP/MAS - NMR - spectroscopy (high resolution solid state NMR) and quantification of the individual cellulose forms by line fitting is a method well suited for investigation of different solid state structures in cellulose substrates. A cluster with a distribution between 86 and 92 ppm contains fairly sharp signals corresponding to C-4 carbons situated in crystalline cellulose l (X and l 0 domains together with paracrystalline cellulose. The C-4 carbons of more disordered regions are distributed in a broad band ranging from 80 to 86 ppm. In the disordered region a pair of signals resolved at 84.0 ppm and 84.9 ppm assigned to surfaces of cellulose fibrils are also visible. This methology allows analyses of the bulk composition of different celluloses and also supplies information about fibril dimensions, e.g. core versus surface structures.

Project group:
Tommy Iversen, STFI
Eva-Lena Hult, STFI
Ants Teder, KTH
Göran Gellerstedt, KTH
Karl Hult, KTH (activity 2)
Mikael Lindström, STFI (activity 3).
Thomas Nilsson, SLU
Geoffrey Daniel, SLU

Industrial mentors:
Ulla Johansson, Södra
Monika Ek, StoraEnso
Torsten Nilsson, Korsnäs

Aim: The ultimate aim of this project is to deliver background knowledge to the redesign of chemical pulping processes in such a way that pulp fibres of higher strength can be obtained. The direct aim is to increase the knowledge on how conditions in the chemical pulping process affect the polymeric structure in the fibre wall and how these changes in turn affect the fibre strength.

Background: A vast knowledge has been collected on this subject over decades by pulp and paper scientists world-wide, but still large gaps remain. According to known essential factors such as: wood species, mechanical fibre damage, pulp yield and cellulose chain length (measured as pulp viscosity), pulps should be of equal strength, but in reality can differ greatly in strength.

Project description: As a first step eight different pulps will be produced from the same, carefully selected, wood sample according to processes that in previous studies have shown the strength anomalies mentioned above. The pulping will be carried out at two of our industrial partners. The pulps will be ODEDD bleached by a third industrial partner and tested by two additional partners regarding more "conventional" properties.

Project group:
Ants Teder, KTH
Göran Gellerstedt, KTH,
Ulrika Molin, KTH
Tommy Iversen, STFI
Lars Ödberg, STFI,
Thomas Nilsson, SLU
Geoffrey Daniel, SLU.

Industrial mentors:
Stefan Högman, Korsnäs
Ann Marklund, Modo
Sture Backlund, SCA
Frank Peng, StoraEnso
Martin Waubert de Puiseau, Södra

Aim: The aim is to determine occurrence, localization and extractability of different metal ions in wood which has been treated in different ways with chemicals in aqueous solution.

Background: The content of inorganic materials in wood is relatively low
(1%), but it is important for processes such as the production and bleaching of mechanical pulp, ECF- and TCF- bleaching of chemical pulp, and in preservative treatment of wood.

Project description: This project will involve analyses of the metal ion distribution in wood samples subjected to different metal-removing treatments to gain information on extractability and mode of chemical attachment for different metal ions.

Project group:
Rune Simonson, CTH
Harald Brelid, CTH
Annica Sundén, CTH
Ants Teder, KTH
Thomas Nilsson, SLU
Geoffrey Daniel, SLU

Industrial mentors:
Ann Marklund, MoDo
Jiri Basta, EKA
Per Larsson, Södra

Aim: To clarify the chemical structure of lignin and hemicellulose in spruce wood, Picea abies, using advanced mass spectrometry (MS).

Background: The traditionel way to analyse biopolymers like lignin has been different kinds of wet chemical analyses. Our present knowledge about lignin structure is mainly based on identification of monomeric and dimeric fragments formed by controlled chemical degradation of isolated lignin preparations e.g. Björkman lignin. Most of our knowledge on lignin chemical structure is based on work from the -60s and the -70s. In the polysaccharide area the picture is similar. Wet chemical degradation followed by separation and identification of single sugars was the important analytical principle.

Nowadays it is possible to selectively degrade the wood polymers and through the development of MS it has been possible to analyse even more high molecular weight compounds. The advantage here is that extremely small samples are necessary in comparison with for example NMR.

Project group:
Göran Gellerstedt, KTH
Hans Önnerud, KTH
Tord Eriksson, KTH

Aim: The aim is to clarify to what extent the wood polymers hemicellulose, lignin and cellulose cooperate mechanically in the wood structure and how the polymers are affected by the pulp processes. This will increase the knowledge of the mechanical properties and open up possibilities for a better utilisation of the fibre.

Background: In order to better control the physical properties of wood fibres in different processes it is important to understand how the different wood polymers contribute to fibre properties. Through combination of optical spectroscopy with dynamic mechanical analysis called dynamic FTIR spectroscopy, it is possible to correlate the movement of the separate funtional groups with an applied stretching. The meaning is to apply this technique in studies of wood fibres in order to gather more knowledge about the structural organisation of wood polymers and the extent of cooperation between them.

Project group:
Lennart Salmén, STFI
Margaretha Åkerholm, STFI
Göran Gellerstedt, KTH

Aim: To clarify mechanisms which govern changes in the super-molecular structure of cell wall polysaccharides during kraft pulping, and to give a knowledge background which expands the possibilities of the industry to optimise pulp production.

Background: We are studying the super-molecular structure of cellulose and hemicellulose using high resolution solid phase NMR. The project is based on earlier results in WURC project 4. These structural studies means that NMR signals from two non-equivalent, chemically accessible cellulose surfaces and one signal from a non-available cellulose surface, can be used to study surface reactivity and interchange between cellulose and other molecules. In the project, restructuring of cell wall polysaccharides and factors which governs this change will be studied during different pulping conditions.

Project group:
Tomas Larsson, STFI
Tommy Iversen, STFI
Kristina Wickholm, STFI
Ants Teder, KTH
Göran Gellerstedt, KTH
Geoffrey Daniel, SLU
Thomas Nilsson, SLU

IFP  Effects of refining on wood fibre structure

Aim: The aim of the project is to study formation, properties and ultrastructure of dislocations in spruce wood fibres and to relate this to pulp and paper properties.

Background: A dislocation is a structure easily seen in polarised light microscopy and is the result of a localised change or distorsion of the microfibrils in either the S1 or both S1 and S2 walls. They occur regularly along the spruce fibre. Fibre deformations in the form of dislocations are less common in fresh spruce wood mildly treated and delignified. Dislocations, earlier called nodes, can appear during chipping, pulping and bleaching and if large enough they can decrease paper strength. Small dislocations, earlier called slip planes, affect fibre flexibility and are not considered dangerous. Larger dislocations also give fibre flexibility, but due to the less ordered or more open structure in the dislocations, they can be the target for chemical, mechanical or enzymatic attack giving fibre shortening and inferior paper properties.

Project description: Dislocations are induced during industrial conditions and in this case often negatively affect paper strength. In the laboratory, normal stirring in water does not induce dislocations and during shearing they disappear and the fibres are more or less swelled and disintegrated. In HCl fibres are not cleaved at pH 1.8, while at pH 1 or at lower pHs, fibres are cleaved at dislocations. In addition to fibre surface erosion, cellulases also cleave fibres at dislocations giving shorter fibres, while xylanase give delamination. Balloon swelling is obtained in LiCl/DMAc and in phosphoric acid, and the balloon formation and stability reflect the properties of the used pulp fibre. After phosphoric acid swelling stable balloons which can be studied with EM, are obtained in fibres with 3-4% lignin. Cellulase treated, bleached or otherwise mechanically affected fibres give rapid ballooning and also further dissolution of the fibre. Spruce pulp fibres will be further investigated regarding influence of dislocations using cellulases and HCl. Appearance of dislocations in industrial pulps during manufacturing will be investigated and correlated with pulp and paper properties.

Project group:
Paul Ander, SLU
Geoffrey Daniel, SLU
Stig Bardage, SLU
Nasko Terziev, SLU
Bert Pettersson, STFI
Gunnar Henriksson, KTH

Industrial mentors:
Ann Marklund, M-real

IFP  Effects of refining on wood fibre structure

Aim: To clarify how milling and drying of fibres is affecting ultrastructural and fracture-mechanical properties of the fibres in the paper structure. The main point is on investigating the lamellar structure of the fibre wall.

Background: Milling, pressing and drying strongly influence the properties of the pulp fibres and of the resulting paper. Knowledge of fibre ultrastructure in native or processed pulp fibres is missing. Thus the lamellar structure, pore volume and distribution during swelling and drying are needed to study in more detail. This will be done using AFM, TEM and different SEM techniques.

Project group:
Lennart Salmén, STFI
Jesper Fahlén, STFI
Ulf Gedde, Polymer Technology, KTH

IFP  Effects of refining on wood fibre structure

Aim: To determine how structural and chemical factors of the hemicelluloses as well as its co-operation with lignin affects the elastic properties of the matrix cell wall material. With more reliable values of elastic properties of native hemicelluloses within the fiber cell wall matrix material of lignin and hemicelluloses, a better understanding of the role of the hemicelluloses can be obtained.

Background: The mechanical properties of the hemicelluloses in the wood fiber have been very sparsely studied. Even on dissolved hemicelluloses studies of mechanical properties are very limited, the only existing data being those of Cousins. In order to be able to model the fiber wall properties in the transverse direction the value of the hemicellulose stiffness must be better determined. The effect of water is also very important for the hemicellulose properties, therefore the moisture sorption behaviour for different hemicelluloses is needed to understand how the fiber wall properties are affected.

Project group:
The group consists of Lennart Salmén (project leader) and Anne-Mari Olsson,
STFI, Box 5604, 114 86 Stockholm, Sweden

IFP  Effects of refining on wood fibre structure

Ultrastructural studies of post harvest changes in wood

Aim: This project strives to elucidate post harvest pre process mechanisms that negatively affect the quality properties of the wood raw material intended for pulp and paper production. The project focuses mainly on mechanisms causing discoloration of wood.

Background: The properties of raw wood raw material (i.e. the native trees) are known to significantly affect the final properties of the pulp and paper produced, however product quality is also affected by the treatment of the raw material after felling. It is often necessary to store wood intended for industrial use in sawmills and pulp mills for shorter- or longer periods. During this storage period, the logs are often water sprinkled during the warm season in order to prevent quality losses due to insect infestations and by staining fungi etc. Sprinkling is an efficient method to prevent losses related to the drying-out of logs. One drawback with water sprinkling and wet storage of wood in general, is the discolouration of the outer part of the xylem caused by the inward movement of substances from the bark. Water sprinkling and wet storage is also known to facilitate bacterial degradation of pit membranes in the wood. Several substances (e.g. tannins) in the bark are thought to be involved in the discolouration of stored wood.

Project description: Wood stored under different conditions is studied using light- and scanning electron microscopy (LM and SEM) in order to study how microorganisms deteriorate the wood and facilitate the penetration of substances from the bark into the wood. Fluorescence microscopy is used to study how these discolouring substances from the bark are located within the wood structure and how different storage systems affect their localisation. The ultrastructural cause for differences in bark substance allocation is studied using fluorescence microscopy in combination with the use of specific enzymes and pre-treatment systems. Scanning electron microscopy is used to reveal how these enzymes have affected the wood structure. Possible enzymatic and abiotic systems for removing discoloured substances are also being tested.

References:
Brändström, J. & E. Persson. 2003. Spatial distribution of fluorescent substances in stored Norway spruce pulpwood. Submitted to Paperi ja Puu.

Project group:
Jonas Brändström, SLU
Erik Persson, Holmen Paper,
Lars Hildén, Uppsala Univ.
Jing Zhang, Uppsala Univ.
Gunnar Johansson, Uppsala Univ.

For more information contact:
Jonas Brändström, jonas.brandstrom@trv.slu.se
or Erik Persson, erik.persson@holmenpaper.com

IFP  Effects of refining on wood fibre structure

Aim: The project's overall aim is to characterise the surface ultrastructure of "wood fibres" and investigate how this changes after various enzymatic, mechanical and chemical treatments.

Background: The surface structure of wood fibres has been insufficiently characterised previously, partly because of the unavailability of good analytical techniques and partly because of a lack of information on the native ultrastructure of wood fibres.

Project group:
Geoffrey Daniel, SLU

IFP  Effects of refining on wood fibre structure

Aim: The aim of the project is to increase the knowledge about the ultrastructure and composition of wood cells and wood fibres by using specific enzymes.

Background: The information about the ultrastructure of wood fibres is constantly increasing. The picture has drifted from simple models with only a few layers to models with a lamellar structure with more than 20 sublayers in one fibre. Several methods to reveal the composition of the individual layers exist. Usually lignin or hemicellulose is marked in some way. Cellulose is considered to be more difficult to mark. We think that well characterized wood degrading enzymes can be a useful tool for studying different layers and especially their cellulose parts. We will use both structurally and mechanically well defined cellulases from wood degrading fungi e.g. Phanerochaete chrysosporium and Trichoderma reesei. By first degrading a very thin piece of intact or delignified wood with one or more specific enzymes and then analyze both the wood slice and the solution surrounding it after incubation we hope to get information about what has been removed from where in the slice. The degradation patterns of the enzyme treated samples will be analyzed with electron microscopy, primarily SEM. The saccharide content of the surrounding solution will be analyzed using an HPLC-method. The project started 000103 and is planned to continue for four years.

Project description:
1. Determining the practical parameters of for studying fibres with enzymes (incubation times, necessary enzymes, photographic techniques etc.)

2. Investigate the cellulose content and orientation in wood fibres or delignified wood.

3. Investigate the content and orientation of other substances, mainly lignin and hemicellulose, in wood fibres.

Project group:
Geoffrey Daniel, SLU
Lars Hildén, SLU/UU
Gunnar Johansson, UU
Thomas Nilsson, SLU

Competence linked to the group:
Christina Divne, UU
Gunnar Henriksson, KTH
Hongbin Henriksson, UU
Jiebing Li, KTH
Anu Nutt, UU
Sven Oscarsson, MDH
Göran Pettersson, UU
Jerry Ståhlberg, SLU/UU
Priit Veljamäe, UU
Jing Zhang, UU

Links:
Lars Hildén, Gunnar Johansson, Göran Pettersson, Anu Nutt, Priit Veljamäe, Hongbin Henriksson and Jing Zhang are located at the Department of Biochemistry, Uppsala University, Uppsala:
http://www.biokem.uu.se

Gunnar Henrikson and Jiebing Li are located at Pulp & Paper Chemistry &
Technology, division of Wood Chemistry, The Royal Institute ofTechnology,
Stockholm:
http://www.pmt.kth.se/wood/index.htm

Jerry Ståhlberg and Christina Divne are located at the Joint Structural
Biology Labs at the Biomedical Centre, Uppsala:
http://www.xray.bmc.uu.se

Sven Oscarsson is located at the Department of Biological and Chemical
Engineering, Mälardalen University, Eskilstuna:
http://www.mdh.se/organisation/ibk

IFP  Effects of refining on wood fibre structure

Biosynthesis of the Cell Wall

Enzymes Determining Wood Fibre Composition and Ultrastructure

Aim: This project strives to elucidate the mechanisms and enzymes responsible for the physico-chemical properties of wood fibres. It will focus on the carbon skeleton of the fiber, i.e. cellulose and hemicellulose.

Background: Wood fibers are composite structures consisting of a network of cellulose and hemicellulose encrusted with lignin and structural proteins. These components form predetermined patterns resulting in characteristic fiber ultrastructure. The fiber morphology, ultrastructure and chemical composition differ between types of fibers, e.g. compression wood fibers, juvenile fibers and early wood/late wood fibers, giving them their specific physico-chemical properties. These properties are dictated by strictly controlled expression of a number of different enzyme systems acting in developing xylem cells. Despite their importance, the enzymes involved in the wood fiber formation are not very well understood, partially since they were not easily isolated using traditional biochemical techniques. High through-put sequencing of genes expressed in the wood-forming tissues of a model tree species, the hybrid aspen, created an opportunity to identify enzymes involved in fiber formation and to elucidate their role. This in turn opens the prospects for the development of new biotechnical applications in fiber modification.

Methods: The project can be divided into three major tasks.

Task 1. Identification and characterization of enzymes potentially involved in fiber formation In this task we will take advantage of the EST-sequence database of the wood forming tissues of poplar (www.biochem.kth.se/PopulusDB/) in order to identify and characterize the enzymes involved in the different steps of cell wall synthesis and assembly. Based on sequence similarities with enzymes in other organisms, we have identified several different enzyme families, which may play an important role in the fiber biosynthesis, particularly in the formation of carbohydrate components of wood fibers. These include e.g. cellulose synthases, cellulases, xyloglucan endo transglycosylases (XET), expansins and a putative xylan endohydrolase. At KTH, a number of such clones are currently being full-length sequenced followed by detailed expression analysis using the microchip technology.

Task 2. Localization of the enzymes active during fiber formation Localization of enzymes in the wood forming tissues can be done using antibodies specific of each different enzyme. Using a robust expression system for several of the target enzymes in E. coli, we have generated antibodies in egg yolk. The antibodies will specifically recognize each enzyme in the tissue extract or a section allowing us to follow its expression during the process of xylem cell development. We are currently performing immuno localization experiments for XET - an enzyme involved in the assembly of hemicellulose-cellulose network in developing fibers. An example of immunolocalisation of XET in the cambial region of hybrid aspen using confocal laser microscopy is shown below.

A

B

A. Immunlocalisation of XET in the cambial region of poplar using egg yolk anti- XET antibodies. Label (green) is evident in the peripheral cytoplasm of sieve tubes cells, cambium and developing xylem cells.
B shows the same region labeled with control pre-immune egg yolk proteins.

Task 3. Investigation of the role of enzyme function for cell wall ultrastructure
Transgenic techniques allow us to up- and down regulate the activity of any enzyme in planta. Careful phenotypic analysis of the clones allows us to determine the role of each enzyme and elucidate mechanisms behind assembly of the cell wall components into a functional structure. We are currently producing hybrid aspen trees with altered expression of selected cell wall enzymes. The ultrastructure and the chemical composition of the fiber in these trees will be characterized by light and electron microscopy as well as by chemical analysis.

Participants: The project is a part of a research network of Swedish groups in the fields of molecular biology and enzymology (KTH, Stockholm), wood biology (SLU, Umeå) as well as wood structural analysis and utilization in the pulp and paper industries (SLU, Uppsala).

Project group:
Björn Sundberg, SLU Umeå
Ewa Mellerowicz, SLU, Umeå
Geoffrey Daniel, SLU
Tuula Teeri, KTH
Kristina Blomqvist, KTH
Harry Brumer, KTH

IFP  Effects of refining on wood fibre structure

Background: Mechanical pulp fibres have a very heterogeneous structure, and the surface structure and composition depends on both the wood species and the processing method adopted. The quality of mechanical pulps is determined by the inherent properties of the pulp fibres, such as surface ultrastructure and chemical composition, as well as physical characteristics like shape. Project 15 aims at studying the fibre-surface structure and chemistry, fibre collapsibility, and their subsequent effects on final paper quality.

1) Collapsibility-structure
Previous studies have shown that compression wood fibres may contribute to surface roughness of printing papers based on thermomechanical pulp.1,2 A pilot plant study was conducted to evaluate the effects of a compression wood rich assortment (i.e. knots) on pulp and paper properties. Preliminary results indicate that the compression wood rich assortment has inferior strength properties compared to the reference assortment. Work in progress strives to elucidate the qualitative and quantitative effects of compression wood on paper surface properties. Results from these studies are expected during autumn 2004.

Fig. 1. Part of a compression wood fibre selected from a Norway spruce mechanical pulp and observed using scanning electron microscopy. The white arrow indicates the abrupt transition from the outer (i.e. S1) to the middle (i.e. S2) secondary cell wall layers. The cell corners (CC) are smooth due to the presence of intercellular spaces in native compression wood (from Brändström 2004).

2) Fibre surface
After mechanical pulping, residues of compound middle lamellae containing pectin which resists degradation – remains on the pulp-fibre surface. Pectin has a high negative charge density affecting the fibre-surface chemistry and pulp properties. Both TMP and CTMP pulps have localised patches of high concentrations of pectin (Fig. 2) on the fibre surface.3 A bioassay, developed for detecting pectin on pulp fibre surfaces has shown TMP to contain more methyl esterified galacturoanan (the main pectic substance in wood) than CTMP, 4 which may be attributed to the chemical treatment affecting the fibre surface in the case of CTMP processing. The presence and degree of esterification of fibre-surface pectin has recently been correlated to fibre charge in CTMP,5 and the colorimetric bioassay for pectin has been further developed, and used to show the de-esterification of surface-localised pectin during alkaline hydrogen peroxide bleaching of CTMP.6

Fig. 2. Immuno-fluorescence labelled pectin on mechanical pulp surface.

3) Ph.D project
Studies conducted on characterizing the cell wall damages of spruce TMP fibre fractions showed that fibre splitting and fibrillation do not occur in a random process, but rather are related to the original native structure/ultrastructure of the fibre cell wall. As a result, S1 and S2 secondary wall layers generate two different types of fibrillation namely “flake-like” and “sheet and/or ribbon-like respectively, which contribute to the particle size and shape distribution of the pulp. In both cases, fibrillation developed from the initial cracking at sites of weakness present on the cell wall and subsequent splitting of individual fibre wall layers along the orientation of the native cellulose microfibrils (i.e. along MFA) for both S1 and S2 layers.7

Fig. 3. Fibre fibrillation by the initiation of splits near sites of weakness (arrows) within the cell wall of pulp fibres such as cross-field (a, b.) and bordered pits (c.)

Fig. 4. Parts of the cell wall of the TMP fibres are easily recognized due to their typical fibrillation in S1 (flake-like particles) and S2 (ribbon-like materials)

Fig. 5. Native wood fibre cell wall architecture/ultrastructure and microfibrillar organization (S2) govern the type of fibre fibrillation (ribbon-like).

Industrial Practice
As part of the Ph.D. work, industrial practice was commenced in collaboration with Kappa Kraftliner, Piteå where there is a considerable problem with printed top birch kraftliner paper, which is thought to be due to wood extractives responsible for pitch problems in the pulp and paper.

A study has been conducted on birch aimed at localizing the extractives involved in pitch deposition in the native wood raw materials and at different stages of the kraft pulping process to understand the nature and behavior of its redistribution using variety of microscopical methods (LM, FM, SEM and TEM) in combination with chemical analysis.

Preliminary results have shown that the polyene antibiotic Filipin can be successfully applied for localizing sterols in both wood raw materials and the pulps at different stages during the pulping process as well to understand the redistribution of wood extractives. Other major birch wood extractive constituents, i.e. fats (neutral fats and fatty acids) that are also thought to be substantially responsible for the above problem, were successfully localized and their redistribution studied during different stages of the pulping process using both osmium tetroxide and Nile blue histochemical staining techniques.

Project group: Dinesh Fernando (Ph.D student), Jonas Brändström (project leader), Jonas Hafrén (project leader), Geoffrey Daniel (project leader), Hans Höglund (scientific advisor), Peter Sandström (industrial representative), Lennart salmén (STFI), Lars Ödberg (Sveaskog), Erik Persson (Holmen) and Magnus Paulsson (Stora-Enso).

Publications and manuscripts:

1. Brändström, J. 2004. Ultrastructure of compression wood fibres in fractions of a thermomechanical pulp. Nord. Pulp Paper Res. J., 19, 13-18

2. Brändström, J. 2004. Microfibril angle of the S1 cell wall layer of Norway spruce compression wood tracheids. IAWA J. In press.

3. Hafrén, J. and G. Daniel. 2003. Distribution of methyl-esterified galacturonan in chemical and mechanical pulp fibers. Journal of Wood Science 49, 361-365.

4. Hafrén, J. and G. Daniel. 2003. A bioassay for methylated galacturonan on pulp-fiber surfaces. Biotechnology Letters 25, 859-862.

5. Hafrén, J. and G. Daniel. 2004. Chemoenzymatic modifications of charge in chemithermomechanical wood pulp (submitted to Journal of Biotechnology).

6. Hafrén, J. 2004. Antibody-based assay for galacturonan de-esterification on wood-pulp fibers during bleaching (manuscript).

7. Fernando, D. and G. Daniel. 2004. Micro-morphological observations on spruce TMP fibre fractions with emphasis on fibre cell wall fibrillation and splitting. Nord. Pulp Paper Res. J. (In press).

IFP  Effects of refining on wood fibre structure

Aim: The aim of the project is to develop novel analytical tools based on carbohydrate binding modules (CBMs) for investigate the carbohydrate composition and spatial distribution on pulp fibre surfaces.

Background: Enzymes involved in carbohydrate metabolism and turnover often have modular structures with a catalytic domain linked to one or more substrate-binding modules. The first CBMs isolated showed affinity towards cellulose. Later on other CBMs have been found, with affinity towards other cell wall carbohydrates such as xylan, chitin and mannan. Since these are recognition elements evolved in nature, their specificity is better than for example that of antibodies generated against carbohydrates, which are often poorly antigenic.

Project description: Different CBMs are expressed in E. coli, fused to the engineered double ZZ-domain of the staphylococcal protein A. The ZZ-domain provides a specific binding site of antibodies and thus allows direct immunogold labeling of the fusion proteins (see Fig 1). Since no secondary antibodies are needed, better resolution can be achieved when transmisson electron microscopy (TEMI is used for imaging). When CBMs with different specificities are fused to the ZZ-module, the different carbohydrates located on pulp fibre surfaces can be mapped with very good precision. The project involves cloning and expression of CBMs with known substrate specificity as well as the characterization of CBMs with so far unknown binding specificity.

Figure 1. The principle of carbohydrate mapping of fibre surfaces using the CBM-fusion proteins

Project group:
Professor Tuula Teeri, Ph.D. student Åsa Kallas, Department of Biotechnology, Royal Institute of Technology, AlbaNova University Centre, 106 91 Stockholm, Sweden.

Professor Geoffrey Daniel, Lada Filonova, Wood Ultrastructure Research Centre (WURC), Dept. Wood Science, Swedish University of Agricultural Sciences, Box 7008, 750 07 Uppsala

IFP  Effects of refining on wood fibre structure

Aim: The object of this study is to increase the knowledge of metal ions in wood and their behavior in the fibre line of the kraft process.

Background: Wood is a complex cellular composite material. It has been shown that the metal ion content is high in certain morphological regions, such as, cell corners, the middle lamella, ray cells and epithelial cells around resin canals. The modes of attachment vary among different types of metal ions. Divalent metal ions such as Ca, Mg and Mn are to a large extent present as counter ions to carboxylic acids in the wood polymers whereas Fe (probably as Fe3+) has been found to be present as precipitates in the wood structure.

The wood used as raw material in kraft pulping is the main source of several non-process elements, NPEs, (e.g. Ca, Mn, Fe and Cu). NPEs in the kraft process are elements that have a negative impact on the chemical recovery cycle and the fiber line. As the degree of system closure in pulp mills is increased, the negative effects of NPEs are amplified, and thus the problems connected with NPEs have come in focus in the pulping industry. The behavior of the inorganic compounds in the wood chips during pulping is therefore of great importance to the kraft process. In the literature, several studies dealing with the mass balances of different metal ions in the kraft process can be found. The distribution and behavior of different metal ions in the wood material on a morphological level during pulping are, however, for the most part unknown. In the current project the removal and redistribution of metal ions in the wood material during kraft pulping is studied on a morphological level.

Project description: During kraft pulping, organic wood components are fragmented and dissolved in the alkaline pulping liquor. Kraft pulping also leads to a partial redistribution of metal ions originally present in the wood material into the black liquor. It can also be expected that metal ions, to a certain extent, are redistributed in the wood material. Multivalent metal ions, such as Ca and Mn, have a strong tendency to form inorganic precipitates under kraft cooking conditions. Computer simulations have shown that after a completed kraft cook, almost all Ca and Mn should be present as inorganic precipitates. A substantial part of the metal ions is, however, distributed from the wood material into the black liquor during cooking. In the black liquor, these metal ions are probably, at least partly, present in the form of colloidal precipitates. In order to study the redistribution of metal ions, and by that gain a more in depth knowledge of the behavior of different metal ions in kraft cooking, we use analysis methods such as, m-XRF, SEM-EDXA and X-ray diffraction.

Project group:
Harald Brelid, CTH
Anders Rindby CTH
Geoffrey Daniel, SLU

Industrial mentors:
Jiri Basta, EKA
Ann Marklund, M-real

IFP  Effects of refining on wood fibre structure

IFP  Effects of refining on wood fibre structure

Aim: To supply a knowledge base which facilitates the industrial optimisation of pulp fibre production with regard to the influence of cell wall supramolecular structures on fibre properties.

Background: In preceding projects methods based on CP/MAS 13C-NMR spectroscopy were developed for quantifying the states of order, cellulose crystallinity, and aggregation pattern, hemicellulose and cellulose fibril assembly, found in fibre cell walls. Studies were performed to characterize the supramolecular assemblies present in native wood and in kraft and sulphite pulp fibres prepared from Norway spruce (Picea abies). It was shown that during the kraft pulping of spruce wood, in contrast to sulphite pulping, the aggregation pattern of the polysaccharides changes. Three different structural transformations are consecutively observed during kraft cooking; an increase in lateral cellulose fibril aggregate dimension, an increase in cellulose crystallinity and a change in order or aggregation pattern of hemicelluloses. These structural transformations seem to be closely associated with changes in both the kinetics of the hemicellulose dissolution and the delignification. The changes in the aggregation pattern during drying of pulp, i.e. formation of larger cellulose fibril aggregates (hornification), were also revealed.

If supramolecular cell wall structures can be intentionally altered this will allow a controlled organisation of the polysaccharides into superstructures with channels and pores suitable for ion and molecule transport, or with controlled shapes and symmetries. A design of the structure of the cell wall should thus give rise to new challenging possibilities to optimise industrial pulp fibre processing and to exploit molecular self-assembly for the production of pulp fibres with a wide range of designed properties.

Project description:

1. Investigate the relationship between fibril aggregation (by CP/MAS 13C-NMR) and pore (ultra)structure (by mineralization/molecular imprint, see attached pre-project).
   
   
2. 2. Study the influence of water activity/dynamics/structure on fibril aggregation (cell wall water ultrastructure)
3. Study the influence of the molecular structure, ultrastructure and localisation of hemicelluloses (and lignin) on fibril aggregation.

Project group:
Tommy Iversen (project leader)
Tomas Larsson
Kristina Wickholm

Industrial relevance:
This project aims at supplying an in-depth understanding of the chemical and physical phenomena governing the aggregation, and de-aggregation, of the cell wall components and how fibre processing may influence the aggregation pattern. An understanding which will enable the Swedish forest products industries to develop radically new approaches to the manufacture of both commodity products, for example based on pulp fibres optimised with respect to fibre properties such as stiffness, fracture resistance, hygrostability, and speciality products such as cellulose whiskers and other structural or functional materials.

IFP  Effects of refining on wood fibre structure

Aim: Isolate and characterize lignin from outer cell wall layers

Background: The projects goal is to obtain information regarding the differences in the lignin structure within the individual cell walls, i.e., the cell wall layers in spruce and aspen. It has been suggested that such differences occur, but the experimental evidence for this have so far been limited, to a large extent due to the difficulties in obtaining sufficient amounts of material for lignin analysis of separate cell wall layers. In this project we prepare plant materials enriched in primary cell wall/middle lamella/S1 layers and analyze the lignin structures with GC-MS, GPC and NMR. To verify that the material has an increased proportion of outer cell wall material AFM and confocal microscopy are employed. Carbohydrate monomer distribution is also determined since it is diagnostic for different cell wall layers.

Project description: So far three different sources for plant materials with an increased amount of outer cell layers as compared to woody tissue, which contain mostly secondary cell, wall have been analyzed.

Firstly poplar cell suspension cultures, have been investigated.

Secondly, three approximately 10 year old aspen trees were collected at the end of June 2003, frozen and de-barked while semi frozen. Thereafter the phloem rich region inside the bark and the xylem rich side of the log were collected separately by scraping the semi frozen tissue with scalpels. This area, the vascular region, produces new cells for the tree and even though some of these cells contain secondary cell walls the hypothesis was that in early summer most of the cells that produce secondary cell walls would not have had time to lignify the inner layers yet.

Thirdly, white spruce that have been preserved under anaerobic conditions for 10 000 years has been investigated. The sample has an intact outer cell wall and middle lamella, but the secondary cell wall have been selectively removed by microorganisms.

Project group:
Gunnar Henriksson, KTH
Maria Christiernin, Ph.D student, KTH

Competence linked to the group:
Anna Ohlsson, KTH
Torkel Berglund, KTH
Liming Zhang, KTH
Shannon Notley, KTH

IFP  Effects of refining on wood fibre structure

Fibre models Part II

Aim: increase knowledge on the structure and ultrastructure odf wood and pulp fibres.

Project description: 3D modelling and reconstruction will be accelerated trough combined semi-automatic image analysis, CAD and visualisation. The project will concentrate in the following steps:

1: Morphological 3D models

• Cell wall structure: S1, S2, S3.
• Pits and cross-field regions.
• Pulp fibre surfaces.
• Structure of cellulosic macrofibrils and interfibrillar spaces.

2: FEA of fibre mechanics

3: Visualisation projects

• Compilation of results in CD-ROM presentations containing interactive 3D models.
• Development of Internet based presentation of the results and findings containing interactive 3D models (limited to WURC partners).

3D modelling by Dr. Stig L. Bardage
A methodology for computerised 3D reconstruction and visualisation of wood fibres has been developed within the Wood Ultrastructure Research Centre. 3D reconstructions are generated from stacks of micrographs obtained from serial sectionings of wood fibres. The micrographs are processed and integrated in a computerised CAD/NURBS-modelling system, which produces 3D models consisting of surfaces or volumetric bodies. The 3D models can be moved in the X, Y and Z directions allowing tilts and rotations. This freedom of manipulation allows the examination of a model from different angles. The models can also be digitally sectioned, dismembered or deformed, and provide spatial and volumetric measurement data. Norway spruce wood and pulp fibres are currently being modelled. 3D modelling will hopefully become a useful tool in the study of wood ultrastrucuture.

Project group:
Stig Bardage, SLU
Geoffrey Daniel, SLU
Lennart Salmén, STFI
Leif Eriksson , UU
Lloyd Donaldson, Forest Research, NZ

Industrial mentors:
Inger Eriksson, SCA

IFP  Effects of refining on wood fibre structure

Aim: To understand at a detailed molecular level the fundamental interactions and reactions leading to lignin formation in the cellulose/hemicellulose matrix, and to describe the key reactions for lignin removal in pulping processes. Alternative methods for removal of lignin (new bleaching chemicals, photochemistry, and enzymatic degradation) will also be investigated.

Background: Lignin is the second most abundant biopolymer on earth (after cellulose), and is generally found associated with hemicelluloses in the spaces between cellulose microfibrils in the primary and secondary cell walls of vascular plants. The randomly distributed lignin polymer is crucial for the plants in providing mechanical support and as a physiochemical barrier against pathogens.

Lignin is also of significant economic importance, from a number of perspectives. The key factor is the ability to easily and efficiently remove lignin in pulping processes, in order to generate high quality paper. At the same time, the high variability in the molecular structure of the lignin biopolymer makes lignin degradation in alkaline pulping and chemical bleaching a difficult task. In addition a number of questions still remain unresolved regarding the exact mechanisms for polymer formation, and the rate determining steps in pulping/bleaching processes.

Project description: Theoretical chemical methodologies have during the past decade evolved into a highly accurate ‘toolbox’, able to reproduce experimental data, with very high predictive power. In the present project, we will set up and investigate different types of models for lignin formation and, primarily, for lignin degradation. However, in order to understand how to best attack lignin at the molecular level, a fundamental understanding of the mechanisms for formation is also vital. We have thus initiated the project by exploring different aspects of monolignols and the first steps in the polymerization process. To this end we are investigating using classical and hybrid quantum/classical mechanics molecular dynamics simulations, lignin monomers (closed shell and radical forms) in water and the energy barriers required for lignin transport across a lipid bilayer (cell membrane); see Fig 1. These investigations will provide insight into the interactions between lignin and its surrounding, and the diffusion mechanism inside the cellulose/hemicellulose matrix. On the more detailed quantum level, we have investigated the mechanisms for dimerisation of lignin momomers, leading to the 7 different cross linkages observed. The relative stabilities of these were also determined (see Fig 2).

In addition, the reaction mechanisms of existing bleaching chemicals with lignin will be explored, in order to determine rate determining steps, and possibly provide suggestions for alternative chemicals or degradation routes. We are also investigating the mechanisms of photochemical bleaching through excited state calculations and comparison of computed and experimental data for radical fragments resulting from these processes (EPR spectroscopy). Finally, the reaction mechanisms of lignin degrading enzymes will be explored using a combination of quantum chemistry and molecular dynamics simulations.

Fig. 1. Model used in MD simulations of lignin monomer diffusion through lipid a membrane.

Fig. 2. Optimized structure of Guaiacyl-glycerol-b-coniferyl ether b-O4 linkage.

Project group:
Leif Eriksson , UU
Yanni Wang, postdoc, UU
Bo Durbeej, Ph.D. student, UU