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Enzymatic modification of Douglas-fir pulp Mansfield, Shawn Denton 1997

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ENZYMATIC MODIFICATION OF DOUGLAS-FIR PULP by SHAWN DENTON MANSFIELD B.Sc. (Hons.), Mount Allison University, 1992 M.Sc, Dalhousie University, 1994 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department ofWood Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November, 1997 © Shawn Denton Mansfield In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of WfloD -Sf \^H/ t> The University of British Columbia Vancouver, Canada DE-6 (2/88) 11 Abstract A cellulase and a xylanase enzyme preparation were assessed for their potential to enhance the fiber characteristics of both mechanical and kraft pulps derived from Douglas-fir wood chips. The effects of cellulase treatments on the pulp properties were dependent on enzyme dosage and resulted in improvements in handsheet density and smoothness, pulp freeness and fiber coarseness. However, this was achieved at the expense of both strength and yield loss. Enzymatic treatments of individual fiber length fractions indicated that, in general, all fiber length fractions demonstrated similar trends to those observed with the cellulase treated unfractionated pulp. In contrast, the individual fiber length fractions responded differently to xylanase treatments, as indicated by the solubilization of high-molecular-mass, UV-absorbing material released during enzyme treatments. Xylanase treatments enhanced the handsheet density and smoothness as well as some strength properties. However, the response of the different fiber length fractions to xylanase treatments was not uniform, indicating that fiber composition plays a role in determining the efficacy of the treatments. The application of hydrolytic enzymes to the pulp fibers resulted in changes in both fiber and paper properties. Monitoring pore volume, degree of polymerization, crystallinity, FT-IR spectra, and scanning electron microscopy helped elucidate changes in fiber composition and morphology. There was strong evidence that the reduction in paper strength resulted from the collective effects of decreased intrinsic fiber strength and the reduction in the degree of polymerization of a large portion of the hemicellulose component Ill of the fibers. Other contributing factors included fiber defibrillation and fines hydrolysis. Since the traditional hydrolases (cellulases/xylanases) appeared to modify the fibers by changes to the fiber surface, cellobiose dehydrogenase. (CDH), purified from Phanerochaete chrysosporium was assayed to see if its oxidoreductase activity could further enhance access of the enzymes to a Douglas-fir kraft pulp. Although the addition of cellobiose dehydrogenase alone had little effect, supplementation with cellobiose and iron resulted in a substantial reduction in the degree of polymerization of the pulp cellulose. This indicated that cellobiose dehydrogenase generated hydroxyl radicals via Fenton's chemistry, which subsequently resulted in the depolymerization of the cellulose. In this way a substantial reduction in the degree of polymerization of the cellulose could be achieved without a significant release of sugar or yield loss. Having established that cellulase enzymes could cause the greatest alterations in fiber morphology and that their attack was most noticeable on smaller, thinner fibers, further studies were carried out on the selective treatment of the larger coarser fibers. These cellulase treated fibers were then recombined with the untreated fibers and refined or, alternatively, first refined and then recombined with the untreated fibers. Laboratory scale fractionation treatments resulted in significant improvements in both the tensile (17 %) and burst indexes (24 %) (at 100 PFI revolutions) with minimal enzyme addition and little yield loss. A subsequent study was carried out to determine if this combined approach, using industrial scale fractionation, could also provide such positive effects. Improvements in both tensile and burst indexes were observed, however, not to the same extent as was observed when the fibers were separated by laboratory fractionation. IV Table of Contents Page Abstract ii Table of contents i v List of tables x List of figures xi List of abbreviations xiv Acknowledgments xvii Chapter 1 1.1 General introduction 1 1.2 Cellulases .• 4 1.2.1 Cellulolysis 4 1.2.2 Quantification of cellulase activity 9 1.2.3 Cellulase activities and its natural substrate 10 1.3 Substrate 14 1.3.1 Cellulose 15 1.3.2 Hemicellulose 15 1.3.3 Lignin 18 1.3.4 Component organization and structure 19 1.4 Fiber networks in paper formation 21 1.5 Hydrolases in the pulp and paper industry 23 1.5.1 Xylanases 24 1.5.2 Cellulases 26 V Page 1.5.2.1 Dewatering 27 1.5.2.2 Refining energy 30 1.5.2.3 Fibrillation 34 1.5.2.4 Deinking 36 1.5.2.5 Reduction in vessel picking 38 1.5.2.6 Fiber modification 39 1.6 Research approach and objectives 43 Chapter 2 Methods and Materials 46 2.1 Pulps 4 6 2.2 Pulp composition 46 2.3 Laboratory fractionation 47 2.4 Industrial scale fractionation 47 2.5 Fiber analysis 48 2.6 Physical pulp testing 48 2.7 Optical pulp testing 48 2.8 Pulp kappa 49 2.9 Pulp bleaching 49 2.10 Pulp chelation 5 0 2.11 Fungal growth conditions 5 0 2.12 Cellobiose dehydrogenase protein purification 51 v i Page 2.13 Ce l lob iose dehydrogenase enzyme assays 51 2.14 Commerc i a l enzyme characterization 52 2.15 Ce l lu lase treatment o f pulp 52 2.16 En z ymat i c treatment o f fractionated pulp 53 2.17 En z ymat i c treatment o f industr ia l ly fractionated pulp 53 2.18 C D H pulp treatment conditions 54 2.19 Carbohydrated determination 55 2.20 F i l t rate analysis 55 2.21 S i ze exclus ion chromatography 56 2.22 Pore vo lume determination 56 2.23 F T - I R spectroscopy 57 2.24 Degree o f po lymer izat ion determination 58 2.25 Crys ta l l in i ty 59 2.26 M i c r o s c o p y 59 2.27 Statist ical analysis 60 Chapter 3 The mod i f i ca t i on o f Doug las - f i r pulps by cel lulase enzymes 61 3.1 Backg round 61 3.2 Results and discussion 62 3.2.1 K r a f t pulp 62 3.2.2 Mechan i ca l pu lp 65 Vll Page 3.2.3 Enzymatic treatment of fractionated pulps. 67 3.2.4 Fractionated mechanical pulp 70 3.2.5 Fractionated kraft pulp 71 3.3 Conclusion 74 Chapter 4 The potential of xylanases to modify Douglas-fir kraft pulp 76 4.1 Background 76 4.2 Results and discussion 78 4.2.1 Optical properties 78 4.2.2 Chemical composition of the fiber length fractions 81 4.2.3 Solubilized material 83 4.2.4 Papermaking properties 87 4.3 Conclusion 90 Chapter 5 Physical characterization of enzymatically modified kraft pulp fibers 93 5.1 Background 93 5.2 Results and discussion 95 5.2.1 Carbohydrate solubilization 95 5.2.2 Pore volume determination 97 5.2.3 Scanning electron mircroscopy 99 vm Page 5.2.4 FT-IR spectroscopy 99 5.2.5 Degree of polymerization 102 5.2.6 Crystallinity 106 5.3 Conclusion 106 Chapter 6 Changes in cellulose ultrastructure using cellobiose dehydrogenase 109 6.1 Background 109 6.2 Results and discussion 111 6.3 Conclusion 122 Chapter 7 Improvements in the paper properties of Douglas-fir kraft pulp by cellulase treatments of different fiber length fractions...124 7.1 Background 124 7.2 Results and discussion 126 7.2.1 Selective treatments and reconstitution 126 7.2.2 Selective treatments, reconstitution and refining 131 7.2.3 Selective treatments, refining and reconstitution 136 7.3 Conclusion 140 Chapter 8 Enhancement of pulp properties by industrial fractionation of kraft pulp followed by cellulases treatments 142 8.1 Background 142 ix Page 8.2 Results and discussion 143 8.2.1 Fiber separation 144 8.2.2 Cellulase treatment of the rejects 144 8.2.3 Cellulase treatment of the rejects followed by refining.... 150 8.3 Conclusion 156 General conclusions and future work 157 Conclusions 157 Future work 159 Applied studies 159 Fundamental studies ....161 References 162 X List of tables Page Table 1 Percent composition (by weight) of different fiber length fractions of Douglas-fir kraft and mechanical pulps 65 Table 2 The composition (percent by weight) of different fiber length fractions of Douglas-fir kraft pulp 82 Table 3 Composition of the Douglas-fir pulp and filtrates obtained after a 5mg/g pulp cellulase treatment 96 Table 4 Average DP N and DPW values of cellobiose dehydrogenase treated Douglas-fir kraft pulp 117 Table 5 Polysaccharides liberated (mg/mL) by cellobiose dehydrogenase after 18 hours incubation ...119 Table 6 Polysaccharides liberated (mg/mL) by cellobiose dehydrogenase (CDH, 0.1 IU/mL or 0.2 IU/mL) supplemented with 20 mM cellobiose and 0.2 mM FeCl3 over time 120 Table 7 Hydrolysis and yield loss resulting from different cellulase treatments of the specific fiber length fractions of Douglas-fir kraft pulp separated by Bauer-Mcnett fractionation 130 Table 8 Hydrolysis and yield losses of cellulase treated reject fibers collected by the industrial fractionation of a high-fir market kraft pulp 152 xi List of figures Page Figure 1 Putative mechanism for enzymatic enhancement of fiber collapsibility 5 Figure 2 The molecular architecture of the cellulose molecule showing its relationship to the microfibrils and to the total cell wall 17 Figure 3 A simplified structure of a softwood tracheid cell wall, showing the middle lamella (ML), primary wall (P), secondary wall layers (S b S2 and S3) and the warty layer (W) 20 Figure 4 Average effect of cellulase treatments on (A) hydrolysis yield, (B) pulp freeness and (C) fiber coarseness of kraft and mechanical pulps 63 Figure 5 Average effect of cellulase treatments on (A) tensile index, (B) burst index, (C) tear index and (D) zero-span breaking length of handsheets produced from kraft and mechanical pulps 64 Figure 6 Average effect of cellulase treatments on (A) density, (B) porosity and (C) roughness of handsheets produced from kraft and mechanical pulps 66 Figure 7 Effects of cellulase treatment on hydrolysis yield for fiber length fractions of mechanical and kraft pulps 69 Figure 8 Average effect of cellulase treatments on (A) pulp freeness and (B) fiber coarseness of the different fiber length fractions of kraft and mechanical pulps 72 Figure 9 Average effect of cellulase treatments on (A) density, (B) roughness, (C) zero-span breaking length and (D) tensile index of handsheets produced from the different fiber length fractions of kraft and mechanical pulps 73 Figure 10 Effects of xylanase treatments on (A) brightness, (B) kappa and (C) absorption coefficient of the fractions of different fiber length 80 Figure 11 Comparison of UV absorbance of xylanase treated reaction filtrates and corresponding controls at (A) 280 nm and (B) 457 nm for each of the different fiber length fractions 84 Figure 12 Analytical size-exclusion chromatographs showing the molecular mass distribution of solubilized material from Douglas-fir kraft pulp: (A) control and (B) following xylanase treatments 86 Xll Page Figure 13 Average effect of xylanase treatment and xylanase treatment followed by peroxide bleaching on handsheet (A) density, (B) roughness, (C) burst index and (D) zero-span breaking length of the fractions of different fiber length fractions of Douglas-fir kraft pulp 89 Figure 14 Pore volume profile of untreated and 5 mg/g pulp cellulase treated Douglas-fir kraft pulp fibers using six different dextran probes 98 Figure 15 Scanning electron micrographs of (A) untreated and (B) 5mg/g pulp cellulase treated Douglas-fir kraft pulp fibers at 2550 x magnification 100 Figure 16 FT-IR difference spectrum of untreated minus 5 mg/g pulp cellulase treated Douglas-fir kraft pulp fibers and (inset) FT-IR absorbance spectrum of untreated Douglas-fir kraft pulp 101 Figure 17 Degree of polymerization of both untreated and 5 mg/g pulp cellulase treated Douglas-fir kraft pulp 106 Figure 18 Crystallinity index of untreated and 5 mg/g pulp cellulase treated Douglas-fir kraft pulp fibers 107 Figure 19 Degree of polymerization of cellobiose dehydrogenase treated Douglas-fir kraft pulp fibers 114 Figure 20 Progressive changes in the degree of polymerization of cellobiose dehydrogenase treated Douglas-fir kraft pulp fibers over time 116 Figure 21 Average change in handsheet (A) density and (B) roughness of cellulase treated 14R and 14R & 28R fiber length fractions of Douglas-fir kraft pulp reconstituted with all other fiber length fractions in the original proportions 128 Figure 22 Average changes in (A) zero-span breaking length, (B) tear index, (C) burst index and (D) tensile index of cellulase treated 14R and 14R & 28R fiber length fractions of Douglas-fir kraft pulp reconstituted with all other fiber length fractions in the original proportions 129 Xlll Page Figure 23 Average change in handsheet (A) density, (B) roughness and (C) pulp freeness of cellulase treated 14R fiber length fraction of Douglas-fir kraft pulp reconstituted with all other fiber length fractions in the original proportions followed by various degrees of refining 133 Figure 24 Average change in (A) zero-span breaking length, (B) tear index, (C) burst index and (D) tensile index of cellulase treated 14R fiber length fraction of Douglas-fir kraft pulp reconstituted with all other fiber length fractions in the original proportions followed by various degrees of refining 135 Figure 25 Average change in handsheet (A) density, (B) roughness and (C) pulp freeness of cellulase treated 14R fiber length fraction of Douglas-fir kraft pulp subject to various degrees of refining, then reconstituted with all other fiber length fractions in original proportions 138 Figure 26 Average change in (A) zero-span breaking length, (B) tear index, (C) burst index and (D) tensile index of cellulase treated 14R fiber length fraction of Douglas-fir kraft pulp subject to various degrees of refining, then reconstituted with all other fiber length fractions in original proportions 139 Figure 27 Effects of industrial fractionation on the (A) fiber length population and (B) fiber lengths distribution of a high-fir market kraft pulp 145 Figure 28 Average change in handsheet (A) density and (B) roughness of cellulase treated rejects fiber fraction followed by recombination with accepts fiber fractions 147 Figure 29 Average change in (A) zero-span breaking length, (B) tear index, (C) burst index and (D) tensile index of cellulase treated rejects fiber fraction followed by recombination with accepts fiber fractions 149 Figure 30 Average change in handsheet (A) density, (B) roughness and (C) pulp freeness of cellulase treated rejects fiber fraction followed by recombination with the accepts fiber fractions and then subject to various degrees of refining 151 Figure 31 Average change in (A) zero-span breaking length, (B) tear index, (C) burst index and (D) tensile index of cellulase treated rejects fiber fraction followed by recombination with the accepts fiber fractions and then subject to various degrees of refining 153 xiv List of abbreviations a alpha AU absorbance units P beta °C degrees celsius CBH cellobiohydrolase CBQ cellobiose:quinone oxidoreductase CDH cellobiose dehydrogenase cm centimeter CMC carboxymethylcellulose CML compound middle lamella CMTP chemi-thermo-mechanical pulp CSF Canadian standard freeness EG endoglucanase EDTA ethylenediaminetetra-acetic acid Da dalton DCPIP 2,6-dichlorophenolindophenol DP degree of polymerization DP N degree of polymerization number average DP W degree of polymerization weight average FAD flavin adenine dinucleotide FPLC fast protein liquid chromatography FT-IR Fourier transformed infrared spectroscopy g gram GPC gel permeation chromatography h hour(s) HPLC high performance liquid chromatography ru international unit XV ISO International Standards Organization kDa kilodalton km kilometer kV kilovolt kW kilowatt L liter LCC lignin-carbohydrate-complex m meter M molar mA milliamp mg milligram min minute(s) ML middle lamella mL milliliter mm millimeter mM . millimolar MOW mixed office waste MW molecular weight nm nanometer o.d. oven dried ox oxidized P peroxide treatment P primary cell wall layer PAD pulsed amperometric detection PAGE polyacrylamide gel electrophoresis PFI papirindustriens forskningsinstitutt ppm parts per million psi pounds per square inch Q chelation Q F volumetric feed flow rate xvi Q R volumetric reject flow rate P v B A relative bonded area R E D reduced rev revolutions RMP refiner mechanical pulp rpm revolutions per minute s second(s) Si first secondary cell wall layer s2 second secondary cell wall layer s3 third secondary cell wall layer SEC size exclusion chromatography SEM scanning electron microscopy SCCM standard cubic centimeter per minute SDS Sodium dodecylsulphate SR Schopper-Riegler index THF tetrahydrofuran ug microgram u.L microliter um micrometer uM micromolar U unit(s) UV ultraviolet light W warty layer w/w weight per weight w/v weight per volume X xylanase treatment XVll Acknowledgments First and foremost, I would like to express my appreciation to my supervisor Dr. Jack Saddler for his support, guidance and most of all friendship. I would also like to thank him for passing on some of the finer points of the "art of science". Thanks are also extended to my committee members Dr. Colette Breuil, Dr. Raj Seth and Dr. Rodger Beatson for their continuing input and direction, and to my industrial mentor Dr. Scott Stephens for his willingness to share his years of experience. I have also benefited enormously from all the members of the Chair of Forest Products Biotechnology group, both past and present, for which I extend many thanks. A special thanks to Dr. Ken Wong and Dr. Ed de Jong for their encouragement and "positive criticism" regarding the project, and for always finding time for the occasional coffee. Funding for this project was gratefully received through scholarships from the Natural Sciences and Engineering Research Council of Canada and a Graduate Research Engineering and Technology award from the Science Council of British Columbia. Industrial support was received from Weyerhaeuser Inc. I would also like to extend a special thanks to Dr. Jurgen Puis and Dr. Bodo Saake of the BFH in Hamburg Germany for hosting me for six weeks. This proved to be an invaluable experience both scientifically and culturally. Many thanks are extended to PAPRICAN (Vancouver) for the use of their equipment and many hours of technical skills generously donated on my behalf. Finally, I extend my thanks to my parents and friends for their encouragement and support through all my academic endeavors, and for that I am truly grateful. 1 Chapter 1 Introduction "The march of invention has clothed mankind with powers of which a century ago the boldest imagination could not have dreamt." Henry George 1.1 General Introduction There have been three noticeable periods of "enlightenment" in our understanding of the role of cellulases in the modification and degradation of lignocellulosics. In the 1950's the pioneering work of Reese and Mandels showed the importance of fungi in degrading cellulose, bringing the genus Trichoderma to preeminence because of the "aggressiveness" of their cellulases. They first introduced the Cx-Cx concept to try to explain how the cellulase system first "opened-up" the cellulose matrix (C^ while the more accessible substrate could then be hydrolyzed by the C x components. The second period of enlightenment showed that, although endoglucanases could act in much the same way as the proposed C x cellulases, exoglucanases did not fill the role of a Cx cellulase, and it appeared that they attacked the cellulose from the non-reducing end to liberate cellobiose. Despite our inability to find an enzyme which could induce "amorphogenesis" to a cellulosic substrate, the proposed synergistic C r C x interaction model was adapted to suggest an endo-exo interaction that resulted in more than just the additive interaction of the component cellulases. 2 In the last ten years the tools of molecular biology have allowed us to determine the amino acid sequence, crystallography and three-dimensional structure of the various cellulase and xylanase enzymes. Rather than classifying these enzymes on the basis of their proposed endo or exo action, this most recent period of enlightenment has allowed us to group cellulase/xylanases on the degree of homology of their binding and catalytic domains. It has been shown that those cellulases exhibiting an exoglucanase mode of action generally have a "tunnel" shaped structure, while the more randomly acting endoglucanases have a more "cleft" shaped catalytic domain [314]. Despite these considerable advances in our understanding of cellulase structure-function we have still not resolved why there is a need for such a multiplicity of "cellulases" or determined the changes that occur to the lignocellulosic structure at the fiber, fibril or elementary microfibril levels. Although a substantial amount of research has been directed at understanding the mechanism(s) of cellulose degradation, the majority of this research has made use of "ideal" model substrates. While these efforts have contributed significantly to our understanding of cellulose saccharification, translating this information to explain the degradation of naturally occurring lignocellulosic has proven an arduous task. Therefore, the traditional endo/exo synergistic model of cellulose degradation may be an oversimplification when considering cellulose degradation of a heterogeneous substrate containing cellulose, hemicellulose and lignin. In retrospect, could the C t enzyme of Reese and Mandels actually exist? Are there other enzymes involved in cellulose degradation? Could enzymes such as cellobiose dehydrogenase (CDH) contribute to the degradation of cellulose by "opening-up" the highly ordered cellulosic matrixes, making them more accessible to the degradative 3 mechanisms of cellulases? It is also likely that other synergistic cooperations must be taken into consideration, such as the relationship between xylanases and cellulases, when considering the degradation or modification of lignocellulosic substrates. As research continues to fmd ways of enhancing our understanding of enzyme-mediated cellulose hydrolysis, various groups have suggested that the selective action of cellulases could also provide a vehicle for achieving specific modifications to pulp fiber properties. To this end we selected Douglas-fir kraft and mechanical pulps as representative substrates to try to evaluate if observed changes in pulp properties such as the tensile, burst, tear or zero-span strengths could be correlated to the proposed mode of action of cellulases and xylanases. Douglas-fir is a desirable species for lumber products because of its strength properties, and accounts for approximately 10 % of the total annual softwood harvested in the province of British Columbia. However, the use of its residual wood chips in thermomechanical pulping is problematic because the high flavonoid content of this species leads to low pulp brightness [152, 233]. More importantly the high fiber coarseness of this pulp yields paper which is relatively rough and weak [264, 265], The.coarseness of fibers from mature Douglas-fir (260-320 \xg/m) is much higher than those from other softwoods such as black spruce and red cedar (100-190 ng/m) [152]. These coarse fibers found in Douglas-fir and other species are stiff, inflexible, bulky, and demonstrate poor inter-fiber bonding properties, yielding paper products which are relatively rough and weak, resulting in a lower fiber content per unit weight [265]. One of the objectives of this thesis was to see if these innate characteristics of 4 Douglas-fir, particularly fiber coarseness, could be selectivity modified by hydrolytic fungal enzymes. Perhaps the surface action of the cellulase enzymes could reduce the undesirable coarse characteristics of these fibers (Figure 1) by an erosion of the outermost cell wall layers, thus enhancing wall collapse. Alternatively, by causing "nicks" or localized areas of weakness, it may be possible to enhance the flexibility of the naturally rigid fiber, which could result in greater fiber conformability and thus increase the relative bonded area of the fiber network. In this way, by coupling the action of cellulases with the traditional pulping technologies, it may be possible to enhance the quality of paper produced from Douglas-fir feedstocks. Cellulase enzymes are already being evaluated for a range of potential applications in the pulp and paper industry, including: fibrillation, reduced refining energy, enzymatic deinking, recycled fiber treatment, and fiber modification. One of the goals of this thesis was to investigate the potential of using fungal enzymes to modify the structure of coarse fibers from Douglas-fir pulp to improve their papermaking properties and thus enhance the value of pulp derived from this species. However, it is also apparent that fundamental research aimed at developing new and novel strategies in the enhancement of fiber properties would not only benefit the Canadian industry, it would also contribute substantially to our understanding of the intricacies of the physical and chemical nature of wood and pulp fibers. 1.2 Cellulases 1.2.1 Cellulolysis Cellulose is the most abundant organic polymer in the biosphere. In its natural environment it will ultimately be synthesized, utilized, degraded and re-enter the carbon 5 Figure 1. Putative mechanism for enzymatic enhancement of fiber collapsibility. The application of hydrolytic enzymes may cause modifications to the surfaces of the fiber, either from the outermost cell wall layers or from the lumen, or both simultaneously, to enhance cell wall collapse during papermaking. 6 cycle via the biological activities of bacteria and fungi. These microorganisms produce a battery of extracellular hydrolytic and oxidative enzymes, which can depolymerize both cellulose and other polymers into more readily utilizable low-molecular weight compounds. These breakdown products then pass through both the cell wall and plasma membrane and serve as energy sources and/or precursors in cell biosynthesis. Early studies using the filamentous fungi, Trichoderma viride and T. koningii [113-115, 237, 238, 337] investigated the capacity of hydrolytic enzymes to degrade cellulose. The complexity of the mechanism of hydrolysis was explained by the C r C x hypothesis. This hypothesis proposed that cellulolytic organisms produce at least two cellulases, the Cx and C x , which acted synergistically to degrade cellulose [238]. The Cx component was considered to be active in the hydrolysis of cellulose which had a high degree of crystallinity, rendering it more susceptible to the catalytic activity of the C x component. Since its inception, a considerable amount of work has been done to elucidate and expand on the C r C x mechanisms of cellulose hydrolysis, most notably the work of Wood and Coughlan [53-56, 337, 340, 342, 346, 347]. The debate surrounding the mechanism of cellulose hydrolysis continues unabated, and various hypotheses concerning the mechanisms of the individual cellulase components have been developed. Although a general mechanism which includes the action of all microbial cellulases (and other enzymes) has not been firmly established, it is generally accepted that the conversion of native cellulose to glucose requires three different types of enzymes. These include a B-l-4-endoglucanase (endoglucanase, EC 3.2.1.4), a B-l-4-exoglucanase (cellobiohydrolase, EC 3.2.1.91) and a B-glucosidase (cellobiase, EC 3.2.1.21). Each one of these components maybe glycosylated, may exist in 7 multiple forms and seems to have a distinct range of activities on different cellulosic substrates. However, other enzymes such as glucohydrolases [236] and cellobiose dehydrogenase [9, 17, 124, 163] may also play a role in cellulose degradation. Cellobiose dehydrogenase (CDH) is an extracellular cellobiose-oxidizing enzyme, which has been shown to be produced by a number of basidiomycete fungi, including both white-rot [12, 131, 243, 321] and a brown-rot fungus [259]. It has been shown that C D H consists of two prosthetic groups, a heme and a flavin adenine dinucleotide ( F A D ) moiety (hemoflavoenzyme). The latter domain is directly involved in both the oxidative and reductive half-reactions, while the former stimulates the reduction of one-electron acceptors 3+ such as cytochrome c and Fe [125, 126]. Cellobiose dehydrogenase has been shown to both enhance the action of cellulases on crystalline cellulose [17] and degrade model wood components such as carboxymethylcellulose, xylan and synthetic lignin [124] through the generation of highly active hydroxyl radicals which participate in Fenton's reactions. Whi le several enzymes such as C D H have been shown to aid in the degradation of cellulose, it has been widely accepted that the hydrolysis of native cellulose by fungal enzyme systems primarily results from the synergistic interaction of (3-1-4-endoglucanases and (3-1-4-exoglucanases (cellobiohydrolase) to yield cellobiose that is subsequently cleaved to glucose by P-glucosidase [343, 344]. The endoglucanases (EGs) act randomly to hydrolyze amorphous cellulose and soluble derivatives of cellulose [77]. This reaction involves the cleavage of B-l-4-glycosidic bonds with little release of reducing sugars. In contrast, cellobiohydrolases (CBHs), which were initially thought to remove cellobiose residues consecutively from the non-reducing end of the cellulose chain, have more recently 8 been shown to attack the cellulose from both the reducing and non-reducing ends of the cellulose chain [127, 228, 229, 314]. Finally, B-glucosidases complete the hydrolytic process by catalyzing the hydrolysis of cellobiose residues to glucose, or by removing glucosyl residues from the non-reducing end of the soluble cellooligosaccharides. Although these definitions are still generally used to group cellulases, recent studies have indicated that the substrate specificities of the various endoglucanases and cellobiohydrolases are considerably more complicated than this oversimplified classification [40-42, 251]. Typically, amorphous cellulose has been reported to be rapidly degraded to cellobiose, while the hydrolysis of crystalline cellulose is slower, and the rate dependent on the degree of polymerization and the crystallinity of the cellulose [338, 347]. The theory proposed by Wood and McCrae [344], was that neither endoglucanases nor exoglucanases acting alone could effect extensive hydrolysis of crystalline cellulose. However, when acting together, the exoglucanases removed cellobiose units from the chain ends provided by the random cleavage of glycosidic linkages by the endoglucanases. The resulting cellobiose units were then degraded to glucose by the B-glucosidase. An extensive investigation into the synergistic action of cellulases by Wood et al, [345-347] using various purified components of the cellulase system of Penicillium pinophilum indicated that multiple forms of the individual components are required in order to initiate extensive cellulose hydrolysis. Thus, the individual isocomponents are believed to have an apparent duplication of function [77]. More recently there have been examples of exo-exo [202] and endo-endo type [179] synergistic cooperation among cellulases. The existence of multiple isocomponents exhibiting duplication of function supports a mechanism of the synergistic action based on the steric considerations resulting from the cleavage of the repeating P-l-4-glycosidic linkages found within the cellulose chains. Therefore, there are two fundamental mechanisms by which glycosidases cleave a P-l-4-glycosidic linkage and these are characterized by the stereochemical outcome of the degradation reaction. If the stereochemistry of the linkage at the anomeric center is inverted in the product (cleaving to yield cx-glucose as a product), then it is an inverting enzyme. If however, the stereochemistry of the linkage at the anomeric center is retained in the product (cleaving to yield P-glucose as a product), then the enzyme is retaining [326]. The basic principles of the mechanisms followed by these enzymes are well established [98, 177, 272, 326, 327]. Inverting enzymes use a single displacement mechanism in which water attacks directly at the anomeric center, displacing the leaving group in a general acid/base catalyzed process via a transition state with considerable oxocarbonium ion character [327]. Retaining enzymes employ a double displacement mechanism involving a covalent glycosyl-enzyme intermediate. The first step involves attack of an enzymatic nucleophile at the anomeric center with general acid-catalyzed displacement of the leaving group to yield a covalent glycosyl-enzyme acylal intermediate. In the second step water attacks the anomeric center of this intermediate in a general base-catalyzed process to yield the product and to release the enzyme in its original protonation state [177, 272, 327]. 1.2.2 Quantification of Cellulase Activity In most cases, the activity of endoglucanases is assayed using a water-soluble 10 carboxymethylcellulose (CMC) or a phosphoric acid-swollen cellulose substrate. The general assay method is quantified by measuring the amount of reducing sugars released from the substrate after interaction with the enzyme [341]. Other methods such as determining the changes in the viscosity of the CMC substrate have also been used to measure endoglucanase activity [341]. Exoglucanases are presumed to differ substantially from the endoglucanases in their substrate specificity, with the former enzymes supposedly capable of solubilizing crystalline cellulose substrates, such as Avicel, filter paper or cotton. Quantification of activity is usually measured by the amount of reducing ends generated by the action of the enzyme [341]. Since Avicel is frequently used as the substrate, the term exoglucanase is often used synonymously with the definition "Avicelase". An alternative method for quantifying exoglucanase activity uses a chromophoric dissacharide derivative and a homologous series of 4-methylumbelliferyl glycosides of cellooligosaccharides. The release of a fluorogenic substrate (phenol production) is measured fluorometrically [305]. While these general assays serve as a means for classification, it is recognized that multiplicity of function does exist within the individual protein structures. Therefore, purity and homogeneity of each component of a cellulase complex is an important criterion for both the determination of substrate specificities of the individual cellulases and the elucidation of molecular mechanisms of enzyme catalysis during cellulose hydrolysis. 1.2.3 Cellulase Activities and its Natural Substrate To try to elucidate the molecular mechanism of fungal breakdown of cellulose in 11 wood, researchers have utilized much simpler model systems, such as evaluating cellulase activities on pure cellulosic substrates and through the use of pure or cloned cellulase enzymes. However, ambiguities in the supramolecular structures of natural cellulosic substrates and problems with enzyme purity make it difficult for us to fully understand the interactions that occur between the enzyme and the substrate at the molecular level. Therefore, the elucidation of the enzymatic mechanisms of degradation of more complexed lignocellulosic has proven to be a challenging subject to investigate. In order to better understand the enzymatic degradation of cellulose, it is important that the general chemical and physical features of the cellulose substrates are accurately understood. As will be discussed later in this chapter, cellulose is usually found in close association with hemicellulose and lignin. In most cases cellulose does not occur alone in a free threadlike chain as is found in the common substrates used in activity quantification, but is usually present in a bundle of fibrillar units with a supramolecular structure consisting of crystalline and amorphous regions [89, 241, 273]. Typically, the crystalline region consists of several sheets of cellulose chains arranged by both intra- and intermolecular hydrogen bonds. Cellulose species are known to exhibit different polymorphs, depending on the forms of cellulose I, II, III, IV, and X, which in some instances also contain sub-classifications. Work continues to try to establish accurate supramolecular structures of different cellulose species in native or modified forms. It is important to know which cellulase specificities are affected by even subtle differences in the stereochemical environment of the surfaces of different cellulose substrates [326, 327]. Thus, the stereochemistry of cellulose chains may prove to be the raison d'etre for both the existence of multiple isozymes of the individual 12 monocomponents of cellulase enzymes and the synergistic effects exhibited by these monocomponents in the enzymatic saccharification of cellulosic substrates. The actions of cellulase enzymes are also affected by the different molecular weights of cellulose substrates and numerous other physiochemical factors. The substrate related factors that influence hydrolysis have not been easy to resolve. The role of the various substrate characteristics has been compounded by the fact that the ultrastructure of cellulose has been a subject of much debate, with still no general agreement as to the arrangement of its microfibrils and their association with other components such as lignin and hemicellulose. The bulk of the research to date has been with pure cellulosic substrates, where associations with other components have not been an issue. While this has been useful, it has not shed much light on the interaction of cellulase enzyme with lignocellulosic substrates as their structures are invariably changed during saccharification [13]. Some of the substrate characteristics which have been investigated to see if they influence enzymatic hydrolysis include: crystallinity, degree of polymerization, particle size and surface area [36, 50, 103, 104, 170, 171, 230, 237, 276, 293]. While some questions may have been partially resolved as to the nature of cellulose itself, it is still not known with any degree of certainty what factors result in the slow hydrolysis rates of lignocellulosic substrates or what causes some substrates to be hydrolyzed faster than others. It seems certain that accessibility plays a key role as lignocellulosic substrates that have been more extensively pretreated tend to be hydrolyzed faster [292]. It has been suggested that this is primarily due to the removal of extraneous substances such as lignin and hemicellulose. Although the exact role of lignin in limiting hydrolysis has been difficult to define, it is 13 probable that one of the most significant limitations is its effect on fiber swelling and its resulting influence on cellulose accessibility [188]. Crystallinity, while originally thought to play a major role in limiting hydrolysis [85, 86] seems to play a less critical role as several workers have shown that the degree of crystallinity of the substrate has no effect on hydrolysis, when all other substrate factors are similar [230]. In some of the studies where crystallinity was suggested to be important, the substrates used were mechanically pretreated lignocellulosic materials where any decrease in crystallinity was invariably accompanied by a decrease in particle size [230]. Thus, it is probable that the rate and extent of saccharification is governed by several factors, of which particle size, surface area, and the degree of polymerization are among the most important. It is apparent that various characteristics within the lignocellulosic substrates can limit both the rate and degree of hydrolysis by cellulases. However, the action of cellulases also alters the inherent characteristics of lignocellulosic substrates as hydrolysis proceeds. These two facts seem to be interrelated and a great deal of research has gone into understanding the contributions that both the substrate and the enzyme make to the total saccharification of the substrate. However, little information is available for situations of limited hydrolysis where the objective is not to completely hydrolyze the substrate for biomass conversion, but rather to harness the specificities of enzymes and direct their attack at specific structural components in order to modify the innate substrate characteristics. Traditionally, natural fibers (pulp) have been characterized by a number of specific parameters, such as fiber length, fiber coarseness (cell wall thickness), particle size, pore volume and water retention values. The strength of these individual fibers is quantified by a 14 zero-span breaking length measurement. Characterization of multiple fibers or a fiber network (i.e. paper) is conducted by measuring properties such as sheet density, roughness, and porosity while the strength of the sheets are quantified by tensile, tear and burst indexes. While a great deal of information has been collected over the years concerning how specific fiber characteristics and modification by different pulping, bleaching or refining treatments to these fibers affect the, resultant paper quality, little information is available concerning what morphological modification cellulase enzymes cause, resulting in the observed changes in fiber/paper properties. For example, how do alteration at the molecular level (i.e. degree of polymerization, crystallinity, sugar composition and birefringence) influence the integrity of fibers at the macroscopic level (zero-span breaking length)? Similarly, what do these alterations in molecular characteristics confer on the resultant paper products, as measured by indexes such as tensile, burst and tear? Is it possible to use the selectivity of enzymes to obtain beneficial effects in papermaking without compromising the strength of fibers? 1.3 Substrate Wood is a composite of cellulose microfibrils embedded in an amorphous matrix of hemicellulose and lignin. The proportion of these three components varies between hardwoods and softwoods. However, there are also compositional differences between tree species, age, geographical location, and growing conditions [145]. In general, cellulose is the most abundant of these three components, representing 40-45 % of the wood, while the hemicellulose and lignin make up 20-30 % and 15-25 %, respectively [273], 15 1.3.1 Cellulose Cellulose is a linear homopolymer composed of D-glucopyranoside units in the 4 Q conformation and the chain is in a highly extended conformation exhibiting two-fold symmetry (each repeating residues orientation is rotated 180° from its neighbor) with its repeating D-glucopyranoside linked together by P-l-4-glucosidic bonds [273]. Cellulose chains vary in size, ranging between 100-14,000 residues in length, and associate together to form larger marcomolecules [19]. These macromolecules are oriented in a parallel fashion forming highly ordered, crystalline domains interspersed by more disordered, amorphous regions. These chains associate via numerous intra- and intermolecular hydrogen bonding and van der Waals interactions to form rigid and insoluble microfibrils (elementary fibrils). Different types of microfibrils can be distinguished at the ultrastructural level [19, 54, 79]. Sub-elementary microfibrils, which consist of only a few residues (10-20) and exhibit very low levels of crystallinity, can associate and form the elementary microfibril. Microfibrils are composed of approximately 40 residues and are usually of a highly crystalline nature. These microfibrils are normally organized in a parallel fashion to form sheets, with the individual sheets displaying different orientations. Finally, bundles of elementary microfibrils aggregate to form macrofibrils of increasing length which associate to form the fibers characteristic of the plant cell wall (Figure 2). 1.3.2 Hemicellulose Like cellulose most hemicelluloses function as supporting material in the plant cell wall. These polysaccharides play an integral role in defining fiber and pulp characteristics 16 and can significantly influence the interactions between adjacent fibers. Wood hemicelluloses are low-molecular weight polysaccharides, which are associated with the cellulose and lignin of the plant cell walls. Hemicelluloses are heteropolymers constructed from a number or different residues, the most common of which are D-xylose, D-mannose, D-galactose, D-glucose, L-arabinose, D-rhamnose, 4-O-methyl-D-glucuonic acid, D-galacturonic acid and D-glucuronic acid [89, 273]. The complexity and chemical nature of the hemicelluloses varies both between cell types and tree species. In general, hemicelluloses fall into four classes: unbranched chains, such as (l-4)-linked xylans or mannans; helical chains, such as (l-3)-linked xylans; branched chains, such as (l-4)-linked galactoglucomannans; and pectic substances, such as polyrhamnogalacturonic acid. Some hemicelluloses demonstrate substantial degrees of acetylation [273]. Despite the complexity of these polysaccharides, their general structure seems to consist of short chains with branches and side chains folded back towards the main chain by means of hydrogen bonding. This structure undoubtedly facilitates their close interaction with cellulose microfibrils, resulting in a tight association, which gives greater stability and flexibility to the aggregate [247]. While there is some inherent heterogeneity between tree species, the largest differentiation occurs when comparing hardwoods and softwoods. In softwoods the most abundant hemicellulose is glucomannan, consisting of a backbone composed of a B-(l-4)-linked D-glucopyranose and D-mannopyranose unit which is substituted by both D-galactose and acetyl groups in varying proportions. To a much smaller degree a xylan-based hemicellulose can also be found. It is composed of a B-(l-4)-linked D-xylopyranose 17 Figure 2 The molecular architecture of the cellulose molecule showing its relationship to the microfibrils and to the total cell wall (Source: "The Chemistry of Paper", J. C. Roberts, RSC Paperbacks, 1996). 18 backbone, substituted w i t h L-arabinose and 4 -O-methy l -g lucuron ic ac id residues. In contrast, hardwoods exhibit s imi lar classes o f hemicel lu loses, however, the major hemice l lu lose is the x y l a n based o l igomer f o l l owed by the g lucomannan [273]. 1.3.3 L i g n i n L i g n i n forms a matr ix w i t h some o f the polysaccharides w h i c h encrusts the ce l lu los ic mater ia l o f the plant ce l l wa l l . This matr ix aids i n ma inta in ing f iber integrity and structural r ig id ity. However , the exact chemical structure o f l i gn in , as w e l l as its association w i t h the cel lulose and hemice l lu lose o f the plant ce l l w a l l s t i l l remains unclear [146]. These complex h igh-molecu lar we ight molecules f o rm carbon-carbon l inkages between phenylpropane units and are also associated w i t h the hemicel lu lose and cel lu lose f o rm ing l ignin-carbohydrate complexes [89]. L i g n i n is an aromatic, phenol ic based compound fo rmed by random free-radical po l ymer i za t i on o f phenylpropane units [129]. In general three monomer i c units are found w i t h i n l i gn in : coni fery l , s inapyl, and /?-coumaryl a l coho l [89]. The proport ion o f these monomers varies between species and this ratio can be used as a marker fo r species class i f icat ion. In general, depending on the degree o f methoxy lat ion, the aromatic group is either con i fe ry l a l coho l based (guaiacyl l ign in) or s inapy l a l coho l based ( syr ingy l l ignin). The ratio o f gua iacy l to syr ingy l l i gn in can be used to d iscr iminate between softwood and hardwood l i gn in , where softwoods l i gn in is enriched i n gua iacy l l i gn in and contains l itt le or no sy r ingy l l i gn in , w h i l e hardwood l i gn in is composed o f both gua iacy l and sy r ingy l l i gn in [89, 249]. 19 1.3.4 Component Organization and Structure As mentioned earlier cellulose microfibrils are usually encrusted in a lignin-hemicellulose matrix in thin sheets called lamellae, several of which make up a cell wall layer. It is believed that hemicellulose and cellulose are closely associated through hydrogen bonds, while hemicellulose and lignin are covalently linked, forming lignin-carbohydrate complexes [81, 123, 132, 134, 137, 150, 178, 180, 200, 201, 289, 290, 315, 316, 324, 355, 356]. The cell wall consists of several layers varying both in thickness, determined by the number of lamellae, and chemical composition. Generally, the structure of the cell wall is subdivided into the middle lamella, primary wall, secondary wall and the warty layer (Figure 3). The outermost layer, which is highly enriched in lignin, is referred to as the middle lamella. This layer acts as the interface between two adjacent cells. The primary cell wall, which is connected to the middle lamella, is a thin layer which is also highly lignified and contains cellulose microfibrils of irregular orientation. Two adjacent primary walls, connected by the intervening middle lamella, are referred to as the compound middle lamella. Located inside the primary wall is the secondary cell wall, which is subdivided into three independent layers, the S b S2 and S3 layers. The majority of the secondary wall is composed of the S2 layer, which characteristically forms a steep helix of microfibrils around the longitudinal fiber axis. The S2 layer is enclosed by the S! and S3 layers, which lie to the outside and inside, respectively. Both layers are thinner and form flatter helices. Finally the inner most layer of the fiber, located adjacent to the lumen is the warty layer, which is an amorphous layer found in softwoods and some hardwood species [87, 89, 273.]. 20 Figure 3 A simplified structure of a softwood tracheid cell wall, showing the middle lamella (ML), primary wall (P), secondary wall layers (S h S2 and S3) and the warty layer (W). (Source: "Wood Chemistry: Fundamentals and Applications", E. Sjostrom, Academic Press, Inc., 1993) 21 1.4 Fiber Networks in Paper Formation Paper is a layered fibrous network structure and its mechanical, optical and other properties are therefore highly dependent upon the nature of the network. Layering occurs as the pulp fibers lie predominantly in the plane of the sheet and are broadly parallel to each other in the r-direction [274]. At the point of contact between the cellulosic fibers a strong bond is formed once the fibers have been dried. These bonds are formed by hydrogen bonds between the polysaccharides at the fiber surface. Since paper is a heterogeneous material, its mechanical and other properties are not only dependent on the nature of the fiber distribution, but also on the characteristics of bonding between the fibers and the inherent fiber strength itself. Bonding between fibers and also fiber strength are influenced both by the pulping and bleaching conditions employed prior to sheet formation [241]. The nature of the bonds between cellulosic fibers in paper has been the subject of some controversy for many years [89, 241, 274]. Generally, it is accepted that multiple hydrogen bonds form within the bonded area between contacting fibers. Due to the nature of the bond length of hydrogen bonds, which are in the order of a few nanometers, the two surfaces must come into very close contact for bonding to occur [274]. Thus, the flexibility of the fibers in the wet state is an important characteristic and is influenced by the extent of swelling of the fiber cell wall, which can increase the relative bonded area (RBA) between fibers. However, the precise molecular species involved in the hydrogen bonding has not been fully resolved. In general, pure cellulosic surfaces such as those found in cotton or bacterial cellulose exhibit rather poor bonding characteristics, whereas fibers derived from wood sources show much better bonding characteristics [241]. This suggests that the 22 adsorbed polysaccharides of the hemicellulose type may be involved in the formation of inter-fiber hydrogen bonds [51, 294]. Cellulosic fibers are negatively charged, with the ionizable groups on the fibers consisting of carboxyl groups, sulfonic acid groups, phenolic groups or hydroxyl groups. However, under normal papermaking conditions the carboxyl and sulfonic acid groups are the major contributors to the fiber ion-exchange capacity. The acidic groups either originate from the cell wall constituents themselves or are introduced during pulping and/or bleaching of the fibers [175]. In native wood, most of the carboxyl groups stem from the uronic acid residues. They are present as 4-O-methyl-a-D-glucopyransyluronic acid bound to the xylan in hardwood or arabinoxylan in softwood [273]. It is known that the ionic groups are not uniformly distributed throughout the cell walls of the fibers. For softwoods, the glucuronoxylan content is higher in the outer secondary wall and in the inner secondary and tertiary walls than it is in the main secondary wall. The reverse order is found with hardwoods, although, the difference between the amounts found in the outer secondary wall and the main secondary wall is smaller. Sulfonic acid groups are introduced with the sulfite treatment during chemi-(thermo)-mechanical pulping (CTMP) or during sulfite pulping. The introduction of these ionic groups is selective towards the outer cell wall layers [323], The concentration of ionic groups within the fiber can influence the degree of fiber swelling, as well as the surface potential characteristics of the fiber, which regulate the extent of coagulation of the fiber and fines [153]. It has been shown that, for chemical pulps, the pH [136] and the presence of ionic groups during sheet forming affects the strength properties of paper [71]. In earlier work Erikkson and Sjostrom (1968) [74] demonstrated 23 that, by blocking sulfonate and carboxyl groups on high yield pulps, this decreased the swelling properties of the fibers and the resulting paper made from these fibers exhibited inferior paper properties. Scallan and Grignon (1979) [256] demonstrated a linear relationship between the degree of swelling and the tensile strength of paper, where swelling controls the plasticization and conformability of the cell wall and the ability of fibers to form stronger sheets through more extensive bonding. It was also shown that the rate of beating depends on both the pH and the nature of ions present in the pulp fibers, whereby increasing the concentration of multivalent electrolytes deceased the rate of beating [175]. Beating or refining, is a process used to develop pulp suspension characteristics and final sheet properties [274]. Refining enhances fiber swelling and, as a result, the fibers become softer and more flexible. This allows them to conform better in the final sheet, resulting in superior paper properties. In addition, some of the microfibrillar structural components of the cell wall are loosened from the surface, giving rise to a very large increase in the surface area of the fiber [241]. 1.5 Hydrolases in the Pulp and Paper Industry Most of the past work on cellulases has focussed on how these enzymes can degrade cellulose. In the pulp and paper area the goal of hydrolase application is not to degrade the cellulose but rather to use these enzymes to enhance pulp processing or pulp characteristics. However, the vast majority of this work has been very "applied" with most reports describing the results of enzyme treatments in areas such as deinking, dewatering, etc., without much description of the mechanisms involved. One obvious exception is the use of 24 xylanases in biobleaching, where a considerable amount of work has been carried out to explain the substrate changes and enzyme action that results in the enhanced brightness of pulp. As many of the mechanisms involved in applications such as deinking and dewatering, are probably very similar to those required for fiber modification, it is important to review the past work and proposed mechanisms that have been suggested on how hydrolases act on pulp fibers. 1.5.1 Xylanases Traditional methods of pulp bleaching have primarily used chlorine or chlorine based derivatives. In recent years, the bleached pulp industry has been motivated to decrease the use of chlorine-based bleaching compounds for environmental, regulatory, and market reasons, forcing the pulp and paper industry to search for alternative, environmentally benign bleaching processes. The kraft pulping process successfully removes the majority of the lignin, and dissolves and degrades the hemicelluloses leaving the cellulose relatively intact. Kraft pulping results in excellent grades of pulp, however, it also generates large quantities of chromophores which are believed to be composed of a combination of residual lignin and carbohydrate (hemicellulose) degradation products [99, 185, 215, 328, 362]. It is recognized that selective chromophore removal is difficult as they are physically entrapped in and covalently bound to the carbohydrate moieties in the pulp matrix [100, 141, 349]. Xylanases are a unique group of enzymes that specifically degrade the hemicellulose xylan of the pulp. Since xylanases do not directly attack the residual lignin moieties of the 25 pulp, the use of xylanases is an indirect bleaching method. As it has been suggested that the lignin in both wood and pulp are covalently bound to the wood hemicellulose, cleavage of the carbohydrate should enhance lignin removal. Viikari and coworkers [310] demonstrated that cleavage of these bonds by hemicellulolytic enzymes did indeed facilitate lignin removal from both pine and birch kraft pulps. They reported a 25 % reduction in the consumption of active chlorine by their xylanase pretreatment of pine kraft pulp or, for the same charge of active chlorine, more extensive delignification to lower kappa numbers when compared to the reference pulps. This groundbreaking investigation paved the way for several other successes, where xylanases have been shown to modify pulp properties and reduce the chemical requirements for bleaching of both softwood [6, 30, 78, 227, 282, 286, 287, 307, 311, 328, 331, 350] and hardwood kraft pulps [57, 58, 118, 226, 306, 352, 357]. However, the major effects of xylanase treatments are only apparent after subsequent bleaching [296], indicating that the enzyme works by facilitating the reaction mechanisms of the other bleaching chemicals. Xylanases tend to have a greater effect on hardwoods [167], probably because xylan constitutes over 90 % of the hemicellulose in hardwood kraft pulps, but only 50 % of that in softwood pulp [291]. There are several theories as to how xylanases aid the bleaching of kraft pulp. One hypothesis suggests that the xylanases partially hydrolyze the reprecipitated xylan of the pulp without degrading the cellulose portion. As the hydrolysis of the hemicellulose proceeds, a substantial increase in fiber porosity occurs and consequently increases the accessibility of the remaining lignin molecules. Therefore, in subsequent bleaching stages more lignin can be extracted from the pulp fibers reducing the quantities of 26 bleaching chemicals required to obtain the desired paper brightness [142, 165, 234, 261, 271, 296, 297, 306, 308, 312]. Another common hypothesis suggests that the xylan backbone, which has lignin moieties bound to it via lignin-carbohydrate complexes, are hydrolyzed by the action of the enzyme and are consequently made water-soluble. Ultimately, the increased solubility allows for enhanced diffusion away from the pulp fibers [351]. While several groups have demonstrated success with the inclusion of xylanases in the bleaching sequence, others have not met with the same success. This may be a function of the specific enzymes as it has been shown that different hemicellulases, even those produced by the same organism, can display quite different behaviours on pulp fibers [67-69, 105, 107, 331]. These modifications have also demonstrated quite different results with regards to the strength properties of the pulp. It has been reported that with some treatments increases [45], decreases [242], and no change in strength [212, 309] had occurred. Presently, there are several mills worldwide employing xylanases in their bleaching sequences to enhance the bleaching of kraft pulps [298]. Although xylanases are being employed in bleaching applications in the pulp and paper industry, the selectivity of these enzymes, which target the fiber hemicellulose may also provide a means of modifying the structural characteristics of the pulp fibers. 1.5.2 Cellulases The development of enzyme applications in the pulp and paper industry began with studies carried out almost forty years ago which investigated the use of cellulases to facilitate enhanced fiber beating [27] and it has progressed to the stage where numerous commercial 27 applications are currently being evaluated. However, the potential for enzymatic modification of pulp fibers is presently difficult to assess because the information concerning the effects that cellulases have on pulp is rather limited. As has been discussed earlier, almost all of the mechanistic studies of cellulases have been conducted using "ideal" substrates, it is only recently that a greater interest in understanding the affects that cellulase enzymes have on the properties of wood pulp fibers has developed. For example, in the last ten years cellulases have been evaluated for a number of potential applications, including fiber beating, fiber fibrillation, dewatering, decreasing vessel picking, fiber flexibility and more recently deinking. 1.5.2.1 Dewatering During the processing of mechanical pulps and secondary fiber, one parameter that is of primary interest, is the drainage of water from the pulp. It is an important criterion because the subsequent rate of water removal on the paper machine is often the rate-limiting step in mill production capacity [274]. Drainage or pulp freeness, as measured by Canadian Standard Freeness (CSF) or by the Schopper-Riegler index (SR.), is an empirical measure of the ease with which water drains from a pulp suspension. This value is affected by both refining and recycling. The presence of fines and highly fibrillated fibers decreases pulp freeness. Fines have a very large influence on drainage resistance by virtue of their propensity to block up interstices in the fiber network. Freeness depends on a resistance to drainage term, which reflects the specific surface of the pulp fibers and their concentration in the fiber mat [70]. Refining, 28 which is often used to increase pulp strength, produces fines and fibrils and therefore decreases freeness. Freeness can be increased with the use of additives such as cationic starch, which helps the retention of fines by the fibers. An alternative strategy to improving freeness has been the application of hydrolytic enzymes to remove fines and fibrils [95]. Since the initial observation of Fuentes in 1988 [95], many researchers have explored hydrolytic enzymatic treatments as a method for increasing pulp freeness. Most of this past work has shown that treatments with cellulases results in the largest freeness increases [22, 135, 197, 221, 246, 277, 299]. As a result, some of this work has been implemented in both pilot plant trials [222] and mill trials [8]. Cellulases have also been combined with polymers to enhance pulp dewatering and, consequently, increase machine operating speed [252]. Mixtures of cellulases and hemicellulases have also been shown to increase freeness [23, 135, 205, 221], while xylanases and mannanases alone cause no significant change [277]. Having established that the action of cellulases was primarily responsible for the observed increase in freeness, comparative studies were initiated to determine what effects the individual cellulase monocomponents had on freeness [277]. These results indicated that endoglucanases were more effective than cellobiohydrolases. Although the cellobiohydrolases did act synergistically with endoglucanases to enhance freeness, their combined action also resulted in increases in both the amount of sugar liberated and yield loss observed. To try to resolve these negative effects, commercial cellulase enzyme preparations containing a higher level of endoglucanases and little or no cellobiohydrolases are being manufactured, with the intent of improving pulp drainage with no or little yield loss [169]. 29 It was also noted that, if the initial freeness was lower, a greater gain was achievable [22]. Pulps with low freeness have many fines and fibrils and, due to their high specific surface area and accessibility, these fines and fibrils are the primary targets for these enzymes [135]. As these finer materials are hydrolyzed, sugars are produced. However, there has been no direct correlation observed between sugar released and drainage improvement. For example, Pergalase A40, a commercial cellulase greatly improved pulp drainage at minimal hydrolysis, while other cellulase enzymes also improved drainage, but at the cost of significant rates of hydrolysis [22]. It has also been observed that significantly greater increases in freeness were observed with the bleached softwood pulp than was found with the unbleached softwood pulp [23]. This was likely due to the greater accessibility of cellulose in the bleached pulp as lignin in the unbleached pulp probably hindered the accessibility of enzymes. Several theories have been proposed to try to explain why freeness increases with enzyme treatments. Several researchers [37, 170, 221] feel that the enzyme attack involves a peeling mechanism. It has been suggested that in a controlled reaction, fibrils which have a high affinity for water could be peeled away, leaving the fiber less hydrophilic and easier to drain [221]. Another theory hypothesized that a preferential attack on the fines and a cleaning of the fiber surfaces was responsible for the freeness increase [135]. Subsequent work seemed to confirm this possible mechanism as other workers found that fibrils and fiber bundles were cleaved from the surfaces of long fibers, making them appear "cleaner". However, they attributed the increase in drainage to the cleaving of amorphous cellulose on the surface of the fines [277, 278]. All of these proposed mechanisms seem plausible in 30 terms of the enzymes gaining access to the substrates and hydrolyzing away fibrous material. Although enzyme treatments have been successful at increasing pulp freeness, these treatments have generally compromised fiber integrity, as illustrated by decreases in paper strength [22, 277]. The use of an optical microscope showed that, although under extreme conditions the fines disappear, the fibers themselves were attacked [221]. Thus, it is important to control the enzyme reaction, using low doses and short reaction times in order to avoid excessive fiber damage and reduced fiber length. It has also been shown that the hydrolytic reduction of fines can have an adverse effect on mechanical and optical sheet properties of paper derived from mechanical pulp [102, 195, 196]. Thus a goal of current research carried out by various groups is to find a method of increasing pulp freeness while maintaining strength. Enzymatic processes that could increase freeness would be especially useful in mills using recycled fiber. 1.5.2.2 Refining Energy Beating or refining refers to the mechanical treatment of pulps, which generally imparts desirable changes on fibers and enhances their papermaking properties [241, 274]. The importance of beating/refining to the paper manufacture has long been recognized. "There is no operation of the paper mill that requires more careful attention and experienced judgement than that of beating or refining to bring the pulp to the finest possible condition for papermaking.. .for it is here that the paper is really made..." [64]. The main purpose of this process is to enable the paper machine to form a pulp mixture efficiently into a web of paper having the required properties, including parameters such as adequate strength. It is 31 important to note that the effects of this process are highly influenced by pulp type, wood species used, yield, pulping process, drying history, and chemical composition. As well, both the type of equipment and the operating conditions substantially influence the efficiency of refining/beating [43, 274]. A number of alterations in fiber morphology result from the refining action, namely fiber cutting/shortening, compression and curl, internal fibrillation or swelling and external fibrillation (described independently in next section). Fiber shortening/cutting is usually viewed as an undesirable effect of refining, however, sometimes it is desired as it produces short fines for increasing sheet opacity and smoothness of paper derived from mechanical pulp. This effect is observed more frequently in thin-walled springwood fibers, while an unraveling of the S2 layer was more marked for thick-walled summerwood fibers [187]. Low consistency refining can be used to straighten fibers, through the transverse swelling of the fiber wall. These fibers have both good strength and stretch properties. Alternatively, internal fiber changes involve the disruption of bonds within the secondary wall, which allows greater water penetration and swelling to occur. This effect leads to enhanced fiber flexibility and increases in the relative bonded area of the fibers, while consequently increasing paper strength [43]. The importance of this process, to both the strength and optical-properties in paper manufacture, is clear. However, the refining process requires substantial energy input, particularly with mills manufacturing paper containing mechanical pulps. Only a small part of the energy spent in the process is used to separate the fibers, with the major part being transformed to heat. Therefore, the total energy economy of these processes is very poor. 32 For a number of years it has been recognized that hydrolytic enzymes could be used to decrease the energy consumed during the refining process [27, 47, 62] while enhancing external fibrillation (see next section). In 1968, a patent was granted which disclosed a process where cellulases from the white-rot fungus Trametes suaveolens were applied to pulp fibers to reduce the refining or beating time [354]. It has also been shown that a hydrolytic mixture of xylanases and cellulases could reduce the refining energy of secondary fiber [48, 49, 190, 191, 203]. These same researchers have also shown that xylanases can be used to reduce the refining energy required. Biopulping, which involves the direct application of growing fungal cells onto the chips, has also demonstrated substantial reductions in refining energy with concomitant improvements in strength [2-4, 20, 24, 25, 38, 75, 76, 90, 101, 161, 181,216]. Although this technology has demonstrated significant improvements, it has some drawbacks, such as reduced yield and long treatment times (weeks). The advantages that purified enzymes have are, shorter retention times and greater flexibility regarding the site of application during pulp processing. The results obtained by white-rot fungi in biopulping have prompted enzymes other than hydrolases, such as laccases, to be assessed for their potential in reducing refining energy [32, 33]. Since the initial discovery that the direct application of cellulases on pulps could be used to reduce refining energy, great strides have been made [257, 258]. Mill trials have shown that treatments with Pergalase A40 (CLBA, Switzerland) reduced the refining energy (7.5 %) required before papermaking and the resultant improvement in paper quality permitted an increase in machine speed (7 %) [94]. Another group of researchers found that 33 an enzyme treatment in combination with an acrylamide copolymer lead to an increase in freeness [252, 254]. Although the enzyme and polymer when used alone also resulted in an increase, the result was better when they were used in combination, since a lower dose of each could be applied. The use of lower enzyme dosage reduced problems with yield and strength losses. These laboratory results were confirmed by a pilot plant trial, followed by several mill trials [35, 254]. During the mill trials, variations in enzyme doses and the retention times were assessed, while several different points of application were studied to optimize the enzyme performance. The best results were achieved when 0.5 kg/t of enzyme was added before the refiners and an additional 1.5 kg/t of enzyme was added in the machine chest with a residence time of 60 minutes [35]. It was suggested that the addition of the enzyme prior to the refiners could help fibrillate the fibers. This application point resulted in a decrease in the refiner load, which translated to energy savings of approximately 14 %. The freeness gain became evident after retention in the machine chest [35]. More recently a comprehensive study has been conducted in order to assess which component of the cellulases specifically reduces the refining energy required in mechanical pulping [218, 220]. These authors have enzymatically treated the coarse mechanical fibers with monocomponent cellulases (endoglucanases and cellobiohydrolases) and hemicellulases (xylanases and mannanases) from Trichoderma reesei prior to the secondary refining and determined if there was any reduction in energy consumption or improvements in fiber properties. Using a laboratory-scale atmospheric disk refiner, improvements by as much as 20 % over the corresponding control in refining energy consumed, were attained when using cellobiohydrolase I (CBH I). In addition, both the purified mannanase and xylanase 34 demonstrated an approximate 5 % reduction in refining energy. Although similar treatments with an endoglucanase (EG I) also demonstrated reductions in refining energy (10 %), this was achieved at the expense of fiber quality. In contrast to the improvements observed with the other purified enzyme, treatments with cellobiohydrolase II (CBH II) and a mixed cellulase preparation (all monocomponents) demonstrated no effects on energy consumption [218]. Similarly, an evaluation of energy consumption using a low-intensity refiner (wing defibrator), revealed an approximate 40 % reduction in refining energy with CBH I [218]. These impressive results prompted a pilot-scale investigation, where 900 kg of TMP rejects were subjected to CBH I prior to refining. In the two-stage secondary refining an energy savings of 10-15 % was obtained, while maintaining both strength and optical properties comparable to the corresponding controls [218]. These authors suggested that the improved refining properties are due to the ability of CBH I to specifically decrease the crystallinity of the cellulose of the raw material. This reduction is achieved without significant yield loss. A reduction in crystallinity translates into an increased proportion of amorphous material, which has a higher affinity for water. The retention of water by fibers during refining reduces the softening temperature of hemicellulose and lignin between the fibers and simultaneously weakens inter-fiber bonding, consequently improving the separation of the fibers from one another [218, 220]. 1.5.2.3 Fibrillation External fibrillation involves the unraveling of fibrils in the secondary wall so that they protrude from the pulp fiber. These fibrils increase the relative bonded area, leading to 35 stronger inter-fiber bonds and enhanced the cohesiveness of the resultant paper [274]. This phenomenon is normally induced through the refining or beating of the fibers. Although the mechanism of fibrillation is not yet fully understood, by keeping the shear rate low during refining, fibrillation is more likely to occur than is the production of fines [116, 117, 154, 155]. In 1942, microbial hemicellulases from Bacillus and Aspergillus were shown to aid in the refining and hydration of pulp fiber [62]. Later, purified cellulases from Aspergillus niger were successfully used to separate and fibrillate pulp [27]. More recent studies have also indicated that cellulases can facilitate improved fiber fibrillation [94, 144, 204, 277]. However, the principal challenge is to achieve the desired fibrillation without excessive fiber damage. It is also recognized that the extent of exposure to, or overdosing of pulp fiber with, cellulases can result in detrimental reductions in strength. In particular, cellulases have been shown to reduce the degree of polymerization of the cellulose and thereby reduce pulp viscosity. In an attempt to circumvent this problem some researchers have used hemicellulases to obtain similar results [189, 203]. This was achieved by inactivating the cellulase activity by the addition of an inhibitor (HgCl2). This same group of researchers showed that similar results could be achieved by using a cloned xylanase [48, 49]. Electron microscopy studies which hoped to elucidate the mode of enzymatic hydrolysis of cellulosic fibers showed cross-wise fiber breakage at a 200 mg enzyme dose, which reduced the fiber strength substantially [206]. However, smaller enzyme doses showed a gnawing and cracking (20 mg), followed by a peeling of the fiber (100 mg). This suggested that mild enzymatic treatments could enhance the degree of fibrillation by 36 initiating minor disruption in the natural integrity of the fibers, via a surface directed attack of the fiber wall. Ultimately, this action could enhance the degree of fibrillation when the fibers are subsequently subjected to refining^eating stages. 1.5.2.4 Deinking In the last decade there has been an enormous amount of pressure applied to make pulp and paper practices more environmentally friendly. As a result, several innovative processes have come to light. One such initiative has been the application of industrial enzymes for the removal of ink during secondary fiber processing. The use of waste paper in pulp and paper manufacture has increased significantly during this time. For example, in 1992, 72 % of the furnish for German newsprint production came from waste paper [279]. Several investigations have demonstrated that the inclusion of cellulases during deinking resulted in greater brightness and bleachability [158, 207, 208, 232]. Since these earlier reports, several research groups have demonstrated that cellulase enzymes can increase the efficiency of the deinking processes [96, 121, 135, 158, 223-225, 245]. Several different enzyme preparations have been evaluated on a number of different papers and inks. Promising results have been achieved using several classes of waste paper, including: old newsprint [72, 207, 208, 214, 231, 244, 348, 359], sorted white ledger [122, 225], and toner from xerographic and laser printed paper [66, 138-140, 143, 213, 288]. One of the bottlenecks in enzymatic deinking is the heterogeneous nature of the wastepaper supply. While success has been achieved using a single homogeneous wastepaper source, when two or more sources are combined, this application becomes more problematic. Some 37 of the variables include, toner quality and type, the type and amount of sizing and the presence of other contaminants [140, 209]. However, recent developments using enzyme/surfactant combinations have been successfully applied on a variety of wastepapers. Specifically, this approach has been successful in deinking wastepapers composed of mixed office wastepaper, sorted white ledger, sorted colored ledger, computer printout, coated book sections, sulfated bleached sections, groundwood sections and ultraviolet printed papers in various proportions [93, 143], There have been published reports of a mill scale trials using this enzymatic deinking applications, the results from which confirmed the laboratory data [158]. More recently, improved enzyme/surfactant technologies have facilitated an investigation using enzyme-enhanced deinking of mixed office wastes (MOW) in three industrial-scale mill trials (Voith Sulzer pilot plant, Appleton, WI) [120]. Increases in both toner removal and pulp brightness were observed after the application of low levels (0.04 % dry weight) of a commercially available enzyme (Novozyme SP 342, Novo Nordisk) in combination with a surfactant (0.125 % dry weight; BRD 2340, Buckman Laboratory). The enzyme-enhanced deinking trials also displayed improved drainage and preserved fiber integrity, when compared with the control. At the same time, effluent samples from these trials were lower in oxygen demand and toxicity than the effluents from the corresponding control [120]. In general, enzymatic deinking resulted in little or no loss in fiber strength [252-254, 277, 279]. However, the significant hydrolysis of the fines [135, 223-225, 277, 278] has been thought to contribute to the observed reduction in the bondability of the fibers [18, 102, 151, 155, 156, 195, 210, 240]. Although, in most cases, the strength properties have not 38 been compromised, care must be taken not to overload enzyme to the fiber [277]. These authors have found substantial strength reductions when elevated cellulase loadings were used on different types of primary pulps. They found that the strength properties of recycled pulps with a high content of chemical pulp fibers were more drastically reduced when compared to secondary fibers which were primarily composed of mechanical pulps [277]. Mechanistically, improvements in both dewatering and deinking of the various pulps are thought to be accomplished by the peeling of individual fibrils and bundles, which have a high affinity for the surrounding water and ink particles [170]. It appears that cellulase treatments can release ink particles, both bound to the fines and fiber, thereby enhancing the removal of ink by flotation. Therefore, the inclusion of enzymes in these processes results in decreased ink counts, higher brightness, or alternatively, a decrease in the amount of deinking chemicals required [318]. Although, it is clear that cellulases enhance these processes, the mechanical agitation accompanying the enzymatic treatments plays a critical role in the efficiency of ink removal [318, 360, 361]. These claims are consistent with similar findings concerning enzymatic stone washing of cotton fabrics, which indicated that enzymatic treatments in combination with mechanical agitation improve the efficacy of the process [300, 301]. In the textile process, small fiber ends protruding from the yarn are weakened by the action of the enzymes [31, 39], while the simultaneous mechanical action completes the process by releasing the ink from the surface to the fibers [301]. 1.5.2.5 Reduction in Vessel Picking In recent years the pulp and paper industry has seen an influx in the use of tropical 39 hardwood, particularly eucalyptus, as a source of raw material. These trees have the advantage of exceptionally fast growth rates, resulting in shorter rotation times and are useful for many pulp and paper applications, such as tissue paper manufacture. However, they contain vessel elements, which are large and do not fibrillate well during refining. Consequently, they generate paper with rougher surfaces, and during printing these vessels lead to "picking". Picking results in poor ink-fiber contact and results in imperfect images. In addition, during printing, the vessel protrusions can be torn out of the paper mat leaving undesirable and costly voids in the resultant paper. Cellulase technology has been shown to substantially reduce vessel picking in paper generated from tropical hardwoods [133, 302, 303]. These authors have demonstrated that, by treating hardwood fibers with commercial cellulases (0.5 % (w/w)) for 4 hours prior to refining, vessel picking can be reduced by as much as 85 %. At the same time, paper properties such as smoothness and tensile index were improved by these treatments, as was the drainage of the pulps. It has been suggested that the application of cellulases improves both the fibrillation and flexibility of these hardwood fibers [302]. 1.5.2.6 Fiber Modification In paper manufacture, finer fibers are considered to be more desirable for papermaking than coarser fibers, due to the implications of their innate thin cell walls. The most advantageous characteristic of the finer fibers is increased collapsibility, which is attainable without the need for additional chemicals or mechanical pressure to significantly decrease the fiber wall thickness. However, several tree species characteristically possess 40 intrinsically higher coarseness. It has been shown that the specific surface area of bonding can be determined solely by the thickness of the fiber wall, as they are inversely related [264, 265]. Thus, reductions in fiber coarseness would be a valuable goal, since coarseness affects optical (sheet opacity) and mechanical properties (relative bonded area) of the paper [264-266, 269, 270]. One suggested mechanism of cellulose modification is the effective "peeling off of subsequent cell wall layers, which has been discussed earlier in the dewatering and refining sections [23, 37, 170, 221, 222]. At low levels of enzyme loading, it should be possible to weaken the fibers natural integrity, making them more susceptible to collapse. As coarseness is defined as the mass per unit length of fiber [265], one cannot reduce coarseness without getting a yield loss. Scallan and Green [255] have shown that coarseness is proportional to pulp yield and that, as pulp yield decreased, so did fiber coarseness and cell wall thickness. This same principle should also hold true for enzymatic modifications. As the fiber coarseness is decreased enzymatically the yield will also be reduced. Therefore, alterations in paper properties can be facilitated only at the expense of some yield loss. In contrast to the proposed enzymatic "peeling" mechanism [23, 37, 170, 221, 222] which demonstrates concomitant yield loss, other research groups have shown fiber destruction at very low levels of hydrolysis, suggesting that certain critical points in the fibers such as kinks and nodes are targeted [108]. A probable reason for this action is that the regions of damage are points of greater swelling and hence greater accessibility [276]. If this does occur, cellulolytic treatments should cleave bonds, which in turn may alter the integrity of the fiber structure, enhancing fiber swelling and therefore flexibility. Fiber 41 swelling, which is partially a consequence of the supermolecular architecture of a given fiber, may also occur as bonds within the S2 helix are cleaved [84]. In creating greater flexibility, the enzymes may weaken individual fibers to the point where they collapse upon papermaking, therefore, enhancing bonding and ultimately leading to stronger paper. Clearly, either erosion of the fibers or disruptions within the fiber matrix should cause a reduction in the intrinsic fiber strength. However, this may be the tradeoff for enhanced bonding, leading to superior paper strength. Therefore, to achieve beneficial cellulase modifications one must successfully manipulate the balance point between optimum fiber strength and bonding strength. To date, enzymatically enhancing strength properties has proven to be an arduous task, which involves the delicate balance of several parameters. Most of the experimental data has shown decreases in strength properties following cellulase treatments [135, 277]. These strength losses varied with the specific pulp substrates as well as the particular enzymes used. It has been shown that cellulases cause a greater decrease in pulp strength than do treatments with xylanases [135]. It has also been shown that, even small amounts of contaminating cellulases can significantly alter results of xylanase prebleaching. As a result, producers of commercial xylanases often advertise "cellulase-free" products to dispel concern about strength losses [295, 298]. In their attempts to overcome the observed reductions in both strength and yield loss, various researchers have pursued the use of individual cellulase monocomponents, rather than a complete cellulase, which can effectively limit cellulose hydrolysis and the resultant strength degradation [46, 157, 219, 277]. Studies, which have used the individual cellulase 42 components, have suggested that endoglucanases (EG) are more destructive than cellobiohydrolases (CBH). For example, treatment with CBH I showed no major differences in strength properties, while EG II caused severe fiber damage which could not be restored by beating [219]. Other researchers have shown that endoglucanase treatments lowered both fiber and handsheet strength, with the extent of degradation varying for the three different endoglucanases examined [46, 157]. This suggested that the mechanism of enzymatic fiber degradation resulted from the attack of endoglucanases, which loosened accessible regions without breaking the fibers [219]. These weakened fibers would then collapsible more readily during the papermaking process. The resultant handsheets have increased air resistance and density, indicating a greater collapsibility of the fibers [219]. Although greater collapsibility typically increases bonding and enhances strength, in this case, the strength did not increase because the integrity of the individual fibers was greatly compromised, as indicated by significant reductions in zero-span breaking length [219]. While different enzymes and enzyme components have been shown to have varying effects on strength properties, it has also been shown that chemical and mechanical pulps respond differently to these treatments. Researchers have found greater strength losses with kraft pulps than mechanical pulps following enzyme treatment [277]. To date, it has proven difficult to balance all of the experimental parameters and find a compromise between increased fiber flexibility and too much fiber degradation. In enzymatic fiber modification, achieving this balance is the key to success! 43 1.6 Research Approach and Objectives This project was initiated to primarily enhance our understanding as to how microbial extracellular cellulase enzymes interact with pulp substrates. A great deal of time and effort, by a number of research groups, have directed their studies at understanding how fungal and bacterial cellulases degrade their specific substrates. Although these model substrates help elucidate the enzymatic mode of action, they also present additional questions. Do these enzymes act in a similar manner on such a heterogeneous substrate as pulp? Does the presence of other compositional constituents influence the cellulolytic mechanism of enzymatic degradation? Are "multifunctional" enzymes required to act in concert with the cellulase enzymes to achieve the total saccharification? While it is well recognized that cellulase monocomponents act synergistically, is there a similar synergistic relationship between different hydrolases during the saccharification of a heterogeneous lignocellulosic substrate? In an attempt to answer some of these questions, Douglas-fir pulps were chosen as the model lignocellulosic substrate to assess the action of these polysaccharide-degrading enzymes. Utilization of a pulp as a model substrate also served as a second objective, which was to assess whether the specificity of enzymes could enhance pulp fiber characteristics, such as reduced coarseness, to obtain beneficial paper properties. It was hoped that the results of this project would provide some unique insights into how individual cellulases and hemicellulases attack wood fibres and, consequently, enhance our understanding of the roles that various fiber characteristics contribute to paper properties. Commercial cellulases were first evaluated for their ability to modify the 44 characteristics of pulp fibers and changes at the "macroscopic level" were assessed by measuring changes in paper properties. This initial work provided a "proof of concept", demonstrating that beneficial fiber alteration, such as reduced fiber coarseness, could be achieved by the selective action of cellulase enzymes. However, since many commercial cellulases contain high amounts of contaminating xylanase enzymes, studies were also carried out to investigate whether the presence of xylanase enzymes contributed to the modifying action of polysaccharide-degrading enzymes. We also tried to correlate enzymatic modifications of the pulp at both the "molecular" and fiber level, and suggest how the possible enzyme action resulted in alterations to the paper quality. This was achieved by monitoring changes in the ultrastructural characteristics such as degree of polymerization, crystallinity and carbohydrate solubilization, while simultaneously evaluating changes in fiber characteristics, via methods such as pore volume determination, FT-IR spectroscopy and scanning electron microscopy. By examining these various parameters, after selective enzyme treatments, we were able to suggest possible modes of action which resulted in the observed changes in both "macroscopic" paper properties and "microscopic" changes in parameters such as DP and crystallinity. It has been suggested that both the size and mode of action of hydrolytic enzymes influences their degradative capabilities, with the majority of structural modifications of the fiber characteristics occurring at the fiber surfaces. How then is it possible for these relatively large enzymes to penetrate highly order cellulose to disrupt the integrity of the fibrils? Does "amorphogenesis" really occur and does an enzyme component such as the 45 former Cx cellulase really exist? Have we, the "students of cellulases" simply accepted the proposed hypothesis of monocomponent synergism and abandoned the search for contributing components of cellulose degradation? Could oxidoreductases, such as cellobiose dehydrogenase, which have been deemed "enzymes in search of a function" [193], actually be associated with cellulose degradation? We have carried out preliminary work to assess whether these enzymes, which are radical generating enzymes, can facilitate the penetration of other hydrolytic enzymes into the surface. The main goal of this thesis was to ascertain whether hydrolytic enzymes could be used to achieve beneficial changes in pulp properties while obtaining information concerning enzyme-substrate interactions. However, we also carried out some more "applied" work which assessed whether mechanical/physical treatments such as fractionation and refining could be combined with enzymatic treatments to enhance paper characteristics of a Douglas-fir kraft pulp. Hopefully, the results described in the subsequent sections will indicate that I have contributed something to our understanding of the enzymatic modification of pulp in both a fundamental and applied sense. 46 Chapter 2 Methods and Materials 2.1 Pulps Refiner mechanical pulp (RMP) was made by pulping steam pretreated Douglas-fir (Pseudotsuga menziesii) wood chips using a Sprout Waldron refiner under atmospheric pressure. Unbleached, never dried, chemical pulp (kraft) derived from Douglas-fir was obtained from Crofton (Fletcher Challenge, Crofton, BC, Canada). Dried, fully bleached, high-fir (approximately 85 % Douglas-fir), chemical pulp (market kraft) was obtained from Crofton (Fletcher Challenge, Crofton BC, Canada). 2.2 Pulp Composition The lignin and sugar composition of the pulp was determined using sulfuric acid hydrolysates (TAPPI Method T249 cm-85). Each hydrolysate was filtered using a sintered-glass filter of medium coarseness for the gravimetric determination of Klason lignin (acid insoluble lignin) and its absorbance at 205 nm was measured for the quantification of acid soluble lignin (TAPPI Useful Method UM250, 1991). The neutral wood sugar monosaccharide constituents were quantified by anion-exchange chromatography on a CarboPac PA-1 column using a Dionex DX-500 High Performance Liquid Chromatography (HPLC) system (Dionex, Sunnyvale, CA, USA), using fucose as the internal standard. 47 2.3 Laboratory Fractionation The fiber length distribution of the pulps was determined by fractionation using the Bauer-McNett fiber length classifier, collecting the 14R, 28R, 50R, 100R, and 200R fiber length fractions (TAPPI Test Method T233 cm-82). 2.4 Industrial Scale Fractionation Fiber length fractionation was performed with a small industrial pressure screen, a Hooper PSV-2100, in PAPRICAN's pilot plant, Vancouver BC, Canada. The screen rotor used was a four bladed, 1.5-inch foil operated at a constant velocity (1300 rpm), driven by a 30 kW motor. The screen was equipped with pressure transducers, magnetic flow meters, and actuated control valves on the feed, accept and reject streams. Valve positions and motor speeds were controlled by a process computer, which also recorded the pressure and flow information. The pulp was pumped through the pressure screen from-a-10,000 L feed tank. The screen plate used in this study was a 0.085-inch diameter conically drilled, uncontoured (smooth) screen plate with a 15 % open area. The pulp, a dried, fully bleached, high-fir (approximately 85 % Douglas-fir), kraft pulp was separated by a single pass through the screens and split into two streams, an accept and a reject, which were directed into two separate holding tanks. The feed consistency was approximately 1.2 % and the starting freeness was approximately 670 mL CSF. The screen was operated at a target accept flow rate (QF) of 2000 L/min. A constant accepts flow rate and rotor speed, maintains a constant average fluid velocity through and upstream of the screen plate apertures. The reject rate was fixed by targeting a Q R of 500 48 L/min. 2.5 Fiber Analysis Fiber length distributions and fiber coarseness were determined by passing representative samples of more than 20,000 fibers and exact aliquots of approximately 5 mg from each pulp sample through the Kajaani FS-200 (Kajaani, Finland) fiber analyzer, respectively. 2.6 Physical Pulp Testing Handsheets were prepared according to TAPPI Test Method T205-om88, with the white water being recycled to ensure that the fines were included in the resultant handsheet. Pulp freeness was measured at 20°C according to TAPPI Test Method T227 om-94 (Can. Std. Freeness Tester, R. Mitchell Co.). For the determination of tensile index (Model 4202 Universal Testing Instruments, Instron), burst index (Mullen Tester, B.F. Perkins & Sons), tear index (Series 400 Monitor/Tear, Testing Machines Inc.), zero-span breaking length (TroubleShooter, Pulmac Instruments Int.), handsheet roughness (Smoothchek, Sheffield Corp.), handsheet density (Precision Micrometer, Testing Machines Inc.) and handsheet porosity (Porosimeter, Sheffield Corp.), tests were conducted according to TAPPI Test Methods T494 om-88, T231 cm-85, T538 om-88, T220 om-88, and T547 om-88, respectively. 2.7 Optical Pulp Testing Brightness, opacity, scattering coefficient, and absorption coefficient of bleached 49 pulps were determined on 4 g handsheets using a Technibrite TB-1C (Technidyne Corp., New Albany, IN, USA). Light scattering coefficient of unbleached (brownstock) handsheets (1.2 g) was measured at 681 nm (Carl Zeiss, Elrepho). Both sides of the handsheets were read five times and the overall averages were determined. 2.8 Pulp Kappa The residual lignin in pulp samples was determined in duplicate using the microkappa method (TAPPI Useful Methods UM 246, 1991) with 0.5 g dry weight of pulp in 25 mL. Lignin and chromophore content in filtrate samples were analyzed by measuring absorbance at 280 and 457 nm, respectively. These samples were diluted with 50 mM Na-phosphate buffer, pH 7.0, to give absorbance readings between 0.2 and 0.7 AU. 2.9 Pulp Bleaching The bleaching sequence used was XQPP, where X = enzyme treatment, Q = chelation, and P = peroxide. The conditions for the X stage were: starting pH 7.0, no buffer added, 10 % pulp consistency, and 400 nkat xylanase enzyme/g pulp, 50°C for 1 hour. Corresponding control experiments were conducted using equal amounts of heat inactivated enzyme. The pulps were washed at 1 % consistency with de-ionized water after each stage. Each time a pulp was drained the filtrate was passed through the pulp cake three times to collect the fines. For chelation (Q-stage) the pulp was adjusted to pH 5.5 with sulphuric acid and brought to a 3 % consistency. The pulp was then incubated with Na 2EDTA2H 20 (1 % loading based on 50 dry weight of pulp) for 30 min. at 50 °C. For the peroxide bleaching (P-stages) the pulp was brought to a 10 % consistency and bleached with 4 % H 20 2 , 2 % NaOH and 0.05 % MgS04-7H20 for 3 hours at 80 °C. The bleached pulp was adjusted to pH 4.5, thoroughly washed with de-ionized water and used to prepare optical handsheets. 2.10 Pulp Chelation For the chelation of metals, pulp samples were maintained at a 2 % consistency and 50 °C, while the pH was adjusted to 5 using H 2S0 4. The sodium salt of EDTA was applied as a concentrated 38 % solution at a charge of 0.6 % on the pulp (oven dry basis) and the chelation was allowed to continue for 30 min. at 50 °C. The pulp was then thoroughly washed with distilled water. The pulp was then analyzed for metals solids by inductively coupled plasma - optical emission spectrophotometry (Analytical Service Laboratory Ltd., Vancouver, BC, Canada). 2.11 Fungal Growth Conditions Phanerochaete chrysosporium BKM F-1767 was maintained on agar plates containing 5 g/L glucose and 3.5 g/L malt extract. Plates were inoculated and grown at 27°C. The growth medium was a modification of Bao et al. [16], containing 2.28 g (NH4)2HP04, 0.5 g MgS04 • 7H20, 0.74 g CaCl2, 0.01 g FeCl3, 0.0158 g NaN03, 6.6 mg ZnS04 • 7H20, 3.8 mg MnS04 • 1H20, 1 mg CoCl2 • 6H20, 0.1 mg Thiamine-HCl, 6.75 g succinate disodium salt hexahydrate and 10 g unquilted cotton cosmetic pads (Safeway, Canada) per litre. P. chrysosporium was pre-grown in 20 mL medium containing 2 % glucose and 0.5 % yeast extract, in 250 mL erlenmeyer 51 flasks at 27 °C for 2 days at 150 rpm. The cultures were then homogenized in a Warning blender for 8 seconds and used to inoculate 250 mL of the succinate medium in 500 mL erlenmeyer flasks. The cultures were grown for 30 days at 27°C. 2.12 Cellobiose Dehydrogenase (CDH) Protein Purification Culture filtrates were collected by vacuum filtration and the extracellular protein present in the supernatant was precipitated with 90 % (NFL^SCv at 0°C. Precipitated protein was collected by centrifugation at 8000 rpm for 2 hours. Between each purification step the protein solution was washed with 10 mM ammonium acetate pH 4.5 and concentrated with an Amicon system using a Diaflo Y M 10 membrane with a 10 kDa cut-off. The concentrated protein was loaded on DEAE-Sepharose CL-6B column (Pharmacia Biotech Inc., Quebec) pre-equilibrated with 10 mM ammonium acetate, pH 4.5 and eluted with 1.5 L of a linear gradient of 10 to 250 mM ammonium acetate, pH 4.5. Only fractions with an A420/A280 nm absorption ratio above 0.03 were pooled, washed and concentrated. Subsequently, concentrated protein was loaded on a Phenyl Superose HR 5/5 (Pharmacia) pre-equilibrated with 0.6 M (NH4)2S04 in 50 mM ammonium acetate, pH 4.0 and eluted with 30 mL of a linear gradient of 0.6 to 0 M (NH4)2S04 in 50 mM ammonium acetate, pH 4.0. Fractions with an A420/A280 nm absorption ratio above 0.1 were pooled, washed and concentrated and applied on a Superose 6 HR 10/30 column (Pharmacia) pre-equilibrated with 50 mM ammonium acetate, pH 5.0. After gel filtration, CDH containing fractions were pooled, concentrated and stored at -80°C before use. 2.13 Cellobiose Dehydrogenase Enzyme Assays Cellobiose dehydrogenase (CDH) activity was assayed by the reduction of cytochrome c 52 at 550 nm (s=28 mM"1 • cm"1). The assay mixture contained 3 mM cellobiose, 20 mM succinate, pH 4.5, 12.5 uM cytochrome c and varying amounts of enzyme preparations making a total of 1 mL. 2,6-Dichlorophenolindophenol (DCPJJ?) was used to measure the combined CDH and CBQ activity at 515 nm (s=6.8 mM 1 • cm'1). The DCPJJ? assay mixture contained 3 mM cellobiose, 20 mM succinate, pH 4.5, 7.5 uM DCPJJ? and varying amounts of enzyme preparations making a total of 1 mL. All assays were performed at 23°C. Enzyme activity, expressed in international units (rU), was equivalent to the reduction of lumol of DCPJJ? per minute. Cytochrome c activity of CDH was found to be equivalent to 3.4 units of the DCPIP activity. 2.14 Commercial Enzyme Characterization Novozyme SP342 from Novo Nordisk (Bagsvaerd, Denmark), an enzyme preparation that was derived from Humicola insolens, was used for all cellulase treatments. Its activity was determined on carboxymethylcellulose (1 % CMC, Sigma, St. Louis, USA), xylan (1 % Birchwood xylan, Sigma), filter paper (No.l Whatman), and mannan (1 % locust bean gum and 1 % guar gum, Sigma) using methods described previously [15, 106, 341]. Proteins in solution were quantified using the bicinchonic acid protein assay [280]. Irgazyme 40S-4X, (Genencor, San Francisco, CA, USA), a commercial xylanase obtained from Trichoderma longibrachiatum was used for the xylanase prebleaching/fiber modification experiments. Its activity on different substrates was determined in accordance to the methods and substrates used for the commercial cellulase. 2.15 Cellulase Treatment of Pulp The pulp slurries (3 % consistency in 50 mM phosphate buffer, pH 7.0) were treated 53 for 1 hour at 50°C under continuous agitation (200 rpm), with a range of enzyme loadings based on the addition of milligrams of protein or CMCase units per gram of oven-dried fiber. The reactions were stopped by placing the pulp samples in a boiling water bath for 15 minutes. Control pulps were similarly treated with equivalent amounts of heat inactivated enzyme (15 min. boiling). The pulps were drained and the filtrates were analyzed for wood sugars released. 2.16 Enzymatic Treatment of Fractionated Kraft Pulp Enzymatic treatments included the treatment of the 14R fraction alone, and treatments of the 14R & 28R fiber length fractions together as described in section 2.15. Following the enzymatic treatments the fiber length fractions were either: 1) recombined with the other fiber length fractions to the same proportions originally present in the unfractionated pulp (this was used to provide a furnish for papermaking or for refining by a PFI laboratory refiner (TAPPI Test Method T248 cm-85), prior to papermaking), or, 2) refined separately and then recombined with the other fiber length fractions to the same proportions originally present in the unfractionated pulp prior to papermaking. It should be noted that these pulps did not include the material that was not retained by the 200R-mesh screen, as this pulp component was lost during fractionation. 2.17 Enzymatic Treatment of Industrially Fractionated Pulp The reject fraction obtained from the pilot plant fractionation was treated as described in section 2.15. Following the treatments the treated reject fraction was recombined with the 54 accept fractions at the same ratio as present in the original unfractionated pulp to form a furnish for papermaking or for refining by a PFI laboratory refiner (TAPPI Test Method T 248 cm-85), which was then subsequently used for papermaking. 2.18 Cellobiose Dehydrogenase (CDH) Pulp Treatment Conditions The 14R fiber length fraction of the unbleached, never-dried Douglas-fir kraft pulp (10 mg/mL) was treated with 0.1 IU/mL of CDH alone in 50 mM sodium acetate buffer, pH 4.5 at 30 °C for 18 hours. Cellobiose dehydrogenase was also supplemented with (final concentration): (a) 20 mM cellobiose, (b) 1.8 mM hydrogen peroxide, (c) 0.2 mM ferric chloride (FeCl3), (d) 20 mM cellobiose +1.8 mM hydrogen peroxide, (e) 20 mM cellobiose + 0.2 mM ferric chloride, (f) 20 mM cellobiose +1.8 mM hydrogen peroxide + 0.2 mM ferric chloride. Controls of the above combinations without added CDH were also investigated, as well as with pulp samples which had previously undergone a chelation step. A time course experiment was conducted using 0.1 IU/mL of CDH in 50 mM sodium acetate buffer, pH 4.5 at 30 °C supplemented with 20 mM cellobiose + 0.2 mM ferric chloride. The reaction were terminated at 1, 2, 6, 12, and 18 hours. The reaction mixtures where inactivated by boiling for 15 minutes. Reaction filtrates were then removed, a portion used directly for monosaccharide and oligosaccharide determination while the remaining portion was freeze-dried and acid hydrolyzed for determination of wood sugars solubilized by the enzymatic treatments (section 2.19). The degree of polymerization of the pulp was determined after tricarbanylation (section 2.24). 55 2.19 Carbohydrate Determination The oligosaccharide constituents and monosaccharide wood sugars (before and after secondary acid hydrolysis) were quantified by high performance anion-exchange chromatography. Monosaccharide sugars were separated on a CarboPac PA-1 column using a Dionex DX-500 High Performance Liquid Chromatography (HPLC) system (Dionex, Sunnyvale, CA, USA) controlled by Peaknet 4.30 software. The column was equilibrated with 10 mM NaOH, and regenerated with 250 mM NaOH. After injecting 20 uL of sample using a SpectraSYSTEM AS3500 autoinjector (Spectra-Physics, Fremont, CA, USA), the sugars were eluted using deionized water at a flow rate of 1 mL/min. The monosaccharides were monitored using a Dionex ED40 electrochemical detector (gold electrode), with parameters set for pulsed amperometric detection of sugars as recommended by the manufacturer. Postcolumn addition of NaOH before the detector was carried out by adding 0.5 M NaOH to the flow stream at a rate of 0.5 mL/min. Oligosaccharide determination was conducted using the same equipment. However, the column was equilibrated with 150 mM NaOH and 50 mM sodium acetate and regenerated with 300 mM NaOH. After injecting 20 uL of sample, the oligosaccharides were eluted using a 50-200 mM gradient of sodium acetate (over 20 min.) at a flow rate of 1 mL/min. The oligosaccharides were monitored using a Dionex ED40 electrochemical detector (gold electrode), with parameters set for pulsed amperometric detection of sugars (as recommended by the manufacturer), without any postcolumn addition. 2.20 Filtrate Analysis Following enzymatic treatments, the reaction filtrates were filtered through glass 56 microfiber filters (Whatman 934-AH). The total reducing sugars were determined using the Nelson-Somogyi method using glucose and xylose as standards, as well as by HPLC, as described in section 2.19. Absorbance of the filtrates in 50 mM phosphate buffer pH 7.0 was measured at 280 nm and 457 nm to evaluate the presence of lignin and chromophores, respectively. 2.21 Size Exclusion Chromatography The molecular mass distribution of solubilized material was analyzed by size-exclusion chromatography. The distribution of lignin was analyzed with Toyopearl HW-55S and HW-50S resins (TosoHaas, Montgomeryville, PA, USA), packed in series in two 0.5 x 20 cm HR columns (Pharmacia, Uppsala, Sweden). These resins have a separation range for dextrans from 0.34 to 40 kDa, which were subsequently used as standards [329]. Elution with 0.3 M NaOH was carried out at 3.6 mL/hour using a Dionex DX 500 HPLC system, and monitored using a Dionex ED40 electrochemical detector and a Dionex AD20 absorbance detector (280 nm, pathlength 6 mm, set at low) in series. Immediately prior to injection, freeze-dried filtrates (2 mL) were resolubilized in 200 uL of 0.3 M NaOH and 20 uL of internal standard (vanillin, 5 mg/mL). The sample was then filtered through a Millipore 0.45 um HV filter (Millipore, Bedford, MA) and 20 uL was injected for chromatographic analysis. 2.22 Pore Volume Determination The pore volume of control and treated pulps was determined using dextran probes of 57 varying molecular diameters (1.8 to 56 nm) using a modification of the solute exclusion technique developed by Stone and Scallan [275]. The individual dextran probe solutions (0.5 % w/v) were added to the pulp samples, mixed thoroughly and allowed to equilibrate for 5 hours with frequent gentle mixing. After equilibration the pulp samples were allowed to settle and the probe solutions withdrawn and filtered through a sintered glass funnel. The concentration of the probe solutions was determined refractometrically using a Waters 625 liquid chromatography system equipped with a Waters 410 Differential Refractometer (Millipore Corp., Milford, Mass, USA). The concentration of inaccessible water was determined as described previously [97]. 2.23 FT-IR Spectroscopy Low grammage handsheets (7.5 g/m ) of control and enzyme treated pulps were made using a standard British handsheet maker, placed in a phosphorus pentoxide desiccator and allowed to dry. A standard hole punch was used to remove 0.22 mg disks of the handsheets, which were then placed on a bed of KC1 powder and analyzed by FT-IR spectroscopy. The FT-IR spectra (256 scans, 4 cm"1) were determined by the diffuse reflectance method (DRIFT) using a Perkin-Elmer 1600 instrument (Norwalk, CT, USA). The maximal absorbance was less than 1.0 AU for all samples analyzed. All samples were baseline corrected and normalized, the average of eight spectra was used as the representative spectrum for each sample. The second derivative spectra was used to improve the resolution of certain absorption bands [182]. 58 2.24 Degree of Polymerization The molecular weight distribution of both the control and enzyme treated pulps were obtained by Gel Permeation Chromatography (GPC) analyses of their tricarbanyl derivatives. Carbanylation of the cellulose was carried out as described previously [260]. The cellulose tricarbanylate was recovered by evaporation of the reaction solvents [333], which was subsequently treated with iso-octane, evaporated to dryness, and solubilized in tetrahydrofuran (THF) at concentrations of approximately 0.2 mg/mL. The GPC of the tricarbanyl derivatives was carried out on a Waters 625 liquid chromatography system (Millipore Corp., Milford, Mass, USA). The cellulose tricarbanylate samples were filtered through a Teflon membrane (0.45 um) and analyzed using a series of 4 TSK-GEL columns (Varian, Sunnyvale, CA., USA. type G1000 HXL, G3000 HXL, G4000 HXL and G6000 HXL with molecular weight cut-offs of lxlO3, 6xl04, 4xl05 and 4xl07, respectively). THF was used as the eluting solvent at a flow rate of 1 mL/min. The samples in the eluent were detected by a Waters 486 UV spectrophotometer (Millipore Corp., Milford, MASS, USA) at a wavelength of 254 nm. The GPC calibration curve was generated from the elution profile of polystyrene standards with narrow molecular weight distributions. Using the Mark-Houwink coefficients previously reported for polystyrene in THF, K p = 1.18xl0"4 and a p = 0.74, and for cellulose tricarbanylate in THF, K c = 2.01xl0"5 and a c = 0.92 the molecular weight of the tricarbanylated cellulose was obtained [304]. The degree of polymerization (DP) of cellulose was obtained by dividing the molecular weight of the tricarbanylated polymer by the corresponding molecular weight of the tricarbanylated derivative of anhydroglucose 59 (DP= MW/519). Both the number averages (DPN) and the weight averages (DPW) of the substrates were calculated as described previously [353]. 2.25 Crystallinity The degree of crystallinity of the pulp samples was obtained by X-ray diffraction. Handsheets (100 g/m ) of both control and treated pulps were made and pressed semi-dry at 5000 psi for 5 min. Representative 4.2 cm x 2.7 cm rectangles were cut from the handsheets, freeze-dried and stored in a phosphorus pentoxide desiccator until analysis. The X-ray diffraction of each sample was recorded using a Siemens diffractometer equipped with a D-5000 rotating anode x-ray generator. The wavelength of the Cu/Kaa radiation source was 0.154 nm, and the spectra were obtained at 30 mA with an accelerating voltage of 40 kV. Samples were scanned on the automated diffractometer from 5 to 40° of 29 (Bragg angle), with data acquisition taken at intervals of 0.02° for 1 s. A peak resolution program was used to calculate both the crystallinity index of cellulose and the dimensions of the crystallites [130]. 2.26 Microscopy The pulp sample specimens were freeze-dried from a water slurry directly onto Scanning Electron Microscope (SEM) sample mounts, which were sputter coated with 60:40, Au:Pd. The sample mounts were then observed in a LEO Spectroscan-360 Scanning Electron Microscope (Cambridge Instruments, Mass. USA) using 10 kV accelerating voltage. Ten representative samples of both enzyme treated and control pulps were 60 analyzed. This work was done by Ron Zarges, Senior Scientist, Weyerhaeuser Company. 2.27 Statistical Analysis All reactions were done in at least duplicate, while the specific quantifications were averages of replicate evaluations. Statistical analysis was conducted using SYSTAT 6.0 for windows. Analysis of variance (ANOVA) was used to determine if there were significant differences between reactions at the 95 % confidence level. While a pair wise comparisons between treatments were done using a Bonferroni adjusted t-test (95 % confidence level). 61 Chapter 3 The Modification of Douglas-fir Pulps by Cellulase Enzymes 3.1 Background As mentioned in the introduction and objective sections, one of the first goals of the research work was to ascertain if, after enzyme treatment, we could obtain beneficial changes to a Douglas-fir pulp while minimizing any detrimental effects that might occur. Earlier work by Stork et al. [277] had shown that the use of cellulases could enhance the processing of recycled fibers. They attributed the enhanced drainage of the recycled pulps to the hydrolysis of amorphous cellulose on the fiber surfaces rather than to the selective hydrolysis of pulp fines. This previous research also demonstrated that limited treatments with low charges of cellulases produced little change in the quality of the recycled fibers, presumably because of a mechanistic peeling of the individual fibrils [170, 277]. The work described in this first chapter examined the potential of using hydrolytic enzymes to enhance the fiber characteristics of chemical and mechanical pulps derived from Douglas-fir, particularly the coarse fractions of these pulps. This initial study was carried out first to ascertain if the reduction of undesirable pulp characteristics, specifically fiber coarseness and stiffness, could be achieved by enzymatic treatments without unacceptable reductions in yield and strength. If this could be achieved we would then look at the possible mechanisms involved in these modifications and determine if we could use other selective enzymes such as xylanases and cellobiose dehydrogenase to obtain beneficial effects while 62 minimizing detrimental effects such as yield or strength losses. 3.2 Results and Discussion 3.2.1 Kraft Pulp As it was expected that higher enzyme dosages would significantly degrade the cellulose fibers, our first step was to establish the enzyme concentrations that would minimize liberation of reducing sugars while maximizing desirable pulp characteristics. The results in Figure 4 A show that the kraft pulp was more readily hydrolyzed than the mechanical pulp. Therefore, to minimize losses in yield and strength, kraft pulp was limited to loadings of no more than 10 mg protein per gram pulp. As enzyme loadings increased, the freeness (CSF) of the kraft pulp increased slightly (Figure 4B ) , while the coarseness slightly decreased (Figure 4C). Since the initial fines component of the kraft pulp was very low (Table 1), the increase in freeness was probably due to a defibrillation of the fibers, which consequently reduced their coarseness. With the kraft pulp, handsheet strength properties such as tensile index, burst index and tear resistance decreased with increasing enzyme loadings (Figures 5A - 5C ) . This decrease in sheet strength was probably due to the damage inflicted on the individual fibers, since the zero-span breaking length was highly affected by enzyme treatment, even at the low enzyme concentrations of 5 mg protein/g oven dry pulp (Figure 5D). The handsheet density of the kraft pulp increased with enzyme loading (Figure 6A). The likely explanation for the increased density is that the cellulase enzymes degrade the fibers to the extent that they collapsed under pressure during handsheet formation. The Figure 4 Average effect of cellulase treatments on (A) hydrolysis yield, (B) pulp freeness and (C) fiber coarseness of kraft and mechanical pulps (n = 2) 64 Figure 5 Average effect of cellulase treatments on (A) tensile index, (B) burst index, (C) tear index and (D) zero-span breaking length of handsheets produced from kraft and mechanical pulps (n = 2) 65 presence of flattened fibers would also explain the observed reduction in handsheet roughness (Figure 6C). Enzyme treatments should also result in smoother fibers and this may partially contribute to the reduced handsheet roughness. Although the porosity data for mechanical pulp could be determined (Figure 6B), no data was obtained for the kraft pulp because the handsheets were too porous for accurate readings to be obtained. Table 1 Percent composition (by weight) of different fiber length fractions of Douglas-fir kraft and mechanical pulps. Fiber Fractions (%)* Pulp 14R 28R 50R 100R 200R Total Fines Mechanical 0.9 13.7 20.6 16.6 12.6 64.4 35.6 Kraft 55.2 21.3 10.8 4.4 1.9 93.6 6.4 * Average of 4 replicates, (+/- 0.15 %). 3.2.2 Mechanical Pulp The freeness of the enzyme-treated mechanical pulp was significantly higher than that of the untreated pulp (Figure 4B), with the maximal effect obtained with a relatively small amount of enzyme (1-5 mg protein/g o.d. pulp). Previously, it was suggested that the preferential hydrolysis of the fines is the likely explanation for this effect [135]. Fiber coarseness decreased progressively with increasing enzyme concentration (Figure 4C), with an enzyme loading of 1 mg protein reducing coarseness by 7 %. While a reduction in fiber coarseness is desirable, it is important that strength loss is 0.55 66 0.50 0.45 rf—• £ 0.40 .o cn 0.35 0.30 0.25 12 10 E O q to 2 3500 3450 3400 3350 - T - 3300 6 3250 d C/5 3200 3150 3100 3050 3000 —t^- RMP Control —±— RMP Treated —•— Kraft Control —•— Kraft Treated _i i i i i ' • 5 10 20 40 Enzyme loading (mg protein/g pulp) Figure 6 Average effect of cellulase treatments on (A) density, (B) porosity and (C) roughness of handsheets produced from kraft and mechanical pulps (n = 2) 67 minimized. A progressive loss in tensile, burst, and tear indexes and zero-span breaking length was apparent with increasing enzyme concentrations (Figures 5A-5D). However, at low. enzyme concentrations (1 mg protein/g o.d. pulp) there was no significant change in the tear index or the zero-span breaking length. At higher enzyme concentrations, the reduction in tensile strength (indicative of inter-fiber bonding), and zero-span breaking length (indicative of fibers strength) suggested that the handsheet strength declined in response to reductions in both the strength of the individual fibers as well as the intrinsic bonding between the fibers. The density of the mechanical pulp handsheets decreased with increasing enzyme concentration (Figure 6A), while the porosity increased (Figure 6B). Although there was a slight decrease in handsheet roughness at low enzyme concentrations (Figure 6C), increasing concentrations did not produce any further decreases in roughness. As the fines constitute a significant portion of the total mechanical pulp (Table 1), a reduction in this component by enzymatic hydrolysis implies that greater amounts of coarse fibers are required to produce handsheets of an equivalent grammage. The reduction in the fines would also lower the amount of "filler" present in the handsheets, thus increasing their porosity and decreasing densities. Concurrent with the hydrolysis of fines, it is possible that fiber defibrillation occurs. This would also contribute to the observed decrease in handsheet density. 3.2.3 Enzymatic Treatments of Fractionated Pulps Previous reports suggested a preferential removal of fines from pulps by enzymatic treatments [135, 222]. This was explained by the higher surface-area-to-weight ratio of the 68 fines, with the increased surface area providing more sites for enzymatic attack. Therefore, both the mechanical and kraft pulps derived from Douglas-fir were fractionated in a fiber length classifier (Table 1), and assessed independently for their response to enzymatic treatments. Our objectives were (a) to both compare the effects that enzyme treatments might have on individual fiber fractions and (b) to investigate the potential of fractionating the pulp and treating only one fraction. The screening results in Table 1 showed that the majority of the kraft pulp was composed of long fibers (14R and 28R), with the shorter fractions (100R and 200R) accounting for only 6.3 % of the total pulp. With the mechanical pulp, there were few long fibers (14R), and a more equal distribution of the other fiber length fractions. The fines, are by definition, the material that was not collected by the 200R fraction screen, accounted for a substantially larger proportion of the mechanical pulp (35.6 %) when compared with the kraft pulp (6.4%). Enzyme concentrations of 5 mg and 20 mg protein/g o.d. pulp were used for the kraft and mechanical pulps respectively, to ensure that similar low levels of hydrolysis were obtained for both pulps. We also had to combine the R14 and R28 mechanical pulp fractions to obtain enough material to form handsheets for physical testing. This combined fraction is referred to as the 28R* fraction. The amount of reducing sugars released as a result of enzymatic hydrolysis was much greater for the unfractionated pulps than it was for any of the individual fractions (Figure 7). This suggested that the fines fraction which was lost during the fractionation process accounted for a large proportion of the hydrolysis in the unfractionated pulp. This is the 69 3.5 3.0 2.5 co co 5 2.0 b >^ 1= 1.5 i—• CD Z_ jo 1.0 0.5 0.0 Mechanical (20mg protein/g pulp) • a £ c g o 3 o cn co CM c o o cd LL o LO o "5 L. Li-CC O o cz o o LL DC O O CM Kraft (5mg protein/g pulp) TJ CD CO c o o CC 3 c c o o • o "«4—* o cd cd LL LL rr cn CO CM o cd k_ LL rr o L O Figure 7 Effects of cellulase treatment on hydrolysis yield for fiber length fractions of mechanical and kraft pulps (error bars indicate standard deviation, n = 4) 70 probable mechanism, as indicated by the fact that the smaller fiber fractions in both pulps released higher quantities of reducing sugars during hydrolysis (Figure 7). 3.2.4 Fractionated Mechanical Pulp It was apparent that the enzyme treatments increased the freeness of the whole mechanical pulp by 39 mL CSF and that of the 100R and 200R fractions by 30 mL CSF (Figure 8A). The longer fiber fractions (28R* and 50R) showed no change in freeness compared with the untreated control. However, a reduction in fiber coarseness with the 50R fiber fraction was apparent (Figure 8B). Coarseness reduction was lower for the shorter fibers (100R & 200R), even though these fractions were hydrolyzed more readily (Figure 7). The coarseness of the 28R* fraction could not be measured as it caused severe blockage of the device used to measure fiber coarseness. Since there was a large reduction in the fiber coarseness of the whole pulp, this suggested that the enzymes hydrolyzed the fines, and defibrillated or "cleaned" the outer surfaces of the fibers. This mechanism has been previously proposed by Oltus et al. [206]. This would partly explain why fiber coarseness decreased for all of the fractions and why the release of reducing sugars is greater with the shorter fiber length fractions. Physical testing of the mechanical pulp samples showed that the fractionated pulp and whole pulp responded similarly to enzyme treatment. The reduction in handsheet density (Figure 9A) and handsheet roughness (Figure 9B) was likely due to the defibrillation of the fibers. The 28R* and 50R roughness values could not be determined as they were outside of the standard scale. The fiber length fractions also showed a small reduction in both fiber 71 strength (measured as zero-span breaking length in figure 9C) and sheet strength (measured as tensile index in figure 9D). The loss of tensile strength in the unfractionated pulp was more substantial than any of the losses observed in the individual fractions. 3.2.5 Fractionated Kraft Pulp As observed earlier (Figure 8A), enzyme treatment increased the freeness of the unfractionated kraft pulp by 16 mL CSF. However, the only fiber length fraction that showed an increase was the 50R fraction. It should be noted that only the 14R, 28R and 50R fractions of the kraft pulp were collected in sufficient quantities (Table 1) to allow us to carry out further enzyme treatment. It was apparent (Figure 8B) that all three of the fiber length fractions and the whole pulp experienced a decrease in fiber coarseness after enzyme treatment. However, the reductions were less dramatic than those observed for the mechanical pulp. The handsheet density increased and the handsheet roughness decreased in all treated fiber length fractions (Figure 9A,B). In both cases, the changes observed for the fiber fractions were comparable with those observed for the whole pulp. It was clear (Figure 9C) that the three fiber fractions experienced a significant drop in zero-span breaking length after enzyme treatment. The strength reduction for the longer fiber length fractions was similar to that observed with the whole pulp. This was expected, since these two fractions (14R and 28R) account for approximately 75 % of the unfractionated kraft pulp. Although the tensile index for the whole kraft pulp declined after enzyme treatment (Figure 9D), the tensile index for all three of the fiber fractions increased, with the longer fibers showing slightly greater improvement. The observed increase in density (Figure 9A) 72 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 Control Figure 8 Average effect of cellulase treatments on (A) pulp freeness and (B) fiber coarseness of the different fiber length fractions (14R, 28R, 50R, 100R, 200R and "W" = whole pulp) of kraft (at 5 mg protein/g pulp) and mechanical (at 20 mg protein/g pulp) pulps (n = 2). 73 Control Control Figure 9 Average effect of cellulase treatments on (A) density, (B) roughness, (C) zero-span breaking length and (D) tensile index of handsheets produced from the different fiber length fractions (14R, 28R, 50R, 100R, 200R and "W" = whole pulp) of kraft (at 5 mg protein/g pulp) and mechanical (at 20 mg protein/g pulp) pulps (n = 2) 74 suggested that the fibers had collapsed and flattened. Fibers that are flat would be expected to have more surface area available for bonding, and this enhanced fiber-fiber contact would increase the tensile index. It was apparent (Figure 9) that the shorter fiber fractions produced denser handsheets and had higher tensile indexes, indicating a tighter fibrous network with closer fiber-fiber contact. This proposed mechanism has been reported earlier [162]. The tensile index for the unfractionated pulp was slightly greater than the value obtained for the 50R fiber fraction and substantially greater than the 14R fiber fraction. However, approximately 55 % of the unfractionated pulp was contained in the 14R fraction. These results highlight the importance of the smaller fiber fractions in forming a well bound, strong paper product. The probable explanation for why the unfractionated pulp did not exhibit a similar enhancement in tensile index is that the fines, which constitute 6.4 % of the unfractionated pulp were preferentially hydrolyzed by the enzyme (Figure 7). The positive effect of fiber flattening was thus overshadowed by the removal of the fines. 3.3 Conclusion The enzyme treatments reduced the coarseness of fibers in both the Douglas-fir mechanical and kraft pulps. Coarseness reduction was greater for the longer and coarser fiber length fractions, suggesting that the reduced coarseness observed for the whole unfractionated pulp was at least partly the result of the reduced coarseness of the larger fibers. Enzyme treatments affected the strength of each of the whole pulps (mechanical and kraft) and the individual fiber length fractions. The kraft pulp fibers showed a higher degree of susceptibility to strength loss (measured by zero-span breaking length), probably because 75 of the greater accessibility and higher cellulose content of these fibers. The observed strength loss in the mechanical pulp seemed to be more a function of fines removal. The enzymatic treatments could be used to alter the physical characteristics of paper products made from Douglas-fir, such as fiber coarseness, which resulted in improvements in the density and smoothness of kraft pulp handsheets. Individual fiber fractions generally showed the same response to treatments as the whole pulp. The one significant exception was the tensile index of fiber length fractions of kraft pulp. In this case, the enzyme treatments increased the tensile index of the different fiber fractions, while the whole pulp showed a slight decline in tensile index. This initial investigation clearly demonstrated that selectivity of enzymatic applications could indeed alter the fiber characteristics of Douglas-fir pulps, resulting in some beneficial changes in paper properties. However, the beneficial modifications were usually accompanied by both reduced paper strength and yield losses. This initial study used a commercial cellulase, which contained high levels of both cellulase and xylanase activity. One possible way to circumvent these latter detrimental effects is by utilizing the selectivity of other carbohydrate degrading enzymes such as xylanases and/or cellobiose dehydrogenase. Another possible way to overcome the losses in yield may be to selectively treat only the longer, coarser fibers enzymatically, as the shorter, finer fibers demonstrate increase degrees of hydrolysis. These selectively treated fibers could then be subject to mechanical refining and assessed for their papermaking potential. These possible applications were studied in subsequent work reported in the following chapters. 76 Chapter 4 The Potential of Xylanases to Modify Douglas-fir Kraft Pulp 4.1 Background In the previous chapter we had used a mixed commercial cellulase preparation, which also included a substantial amount of xylanase activity, to try to enhance the characteristics of Douglas-fir pulp fibers. One possible way of obtaining beneficial fiber changes while minimizing detrimental effects such as yield and strength losses may be to use a cellulase-free xylanase that primarily acts on the hemicellulose component of the pulp while leaving the cellulose fraction largely untouched. Currently there are several kraft mills routinely using xylanases in their bleaching regimes to produce elemental-chlorine-free and total-chlorine-free pulps [298]. However, there is still considerable debate regarding the actual mechanisms by which xylanase treatments enhance pulp bleaching. For example, xylanases have been shown to release considerable amounts of UV-absorbing material from both softwoods and hardwoods [67]. It has also been suggested that it is primarily the hydrolysis of xylan or lignin-bound xylan on the surface of pulp fibers that enhances the removal of lignin in subsequent bleaching steps [148, 287]. This proposed mechanism of xylanase prebleaching implies that the target substrates for the enzyme are not uniformly distributed across the fiber wall and occur principally on the surface of the pulp fibers. Several investigations, based on the mechanical separation of kraft pulp fibers, add support to this theory as they indicate that kraft fibers 77 exhibit uneven lignin distribution [119]. These lignin-rich fibers adversely affect the bleaching process and may be differentially distributed among fibers. Therefore, it is possible that fractions of certain fiber lengths play key roles in the xylanase prebleaching mechanism. It has also been suggested that, as a result of the higher surface-area-to-weight ratio exhibited by fines, hydrolytic enzymes preferentially attacked this fiber fraction [135]. We have also found that the fractions of shorter fiber length were more readily hydrolyzed by cellulase treatments (chapter 3). In addition, it has been established that the shorter fibers and primary fines are enriched in lignin [176] and therefore may contribute significantly to pulp colour. As our previous work with cellulase enzymes had indicated that the fractions of shorter fiber length were preferentially attacked by these enzymes, we thought it worthwhile to investigate whether the action of xylanases was also influenced by the dimensions and composition of the different fractions. Since hemicelluloses also contribute to fiber strength, the partial hydrolysis of xylan will not only affect the bleaching of pulp fibers, it will also result in some modifications to pulp fiber optical and physical properties. Previously we found (previous chapter) that similar changes in the strength and papermaking properties of Douglas-fir pulps were obtained after cellulase treatments of the fractions of different fiber length. This was indicative of the large proportion of cellulose that was accessible to the cellulase enzymes, regardless of the fiber length fraction that was used. Significantly lower amounts of xylan are present in these fractions and thus will greatly influence the ability of the xylanases to modify any fiber characteristics. Xylanase treatments have also been shown to be quite substrate specific, with the xylan component 78 selectively hydrolyzed while the cellulose component of the fiber remained relatively unchanged [248]. Most commercial xylanases that have been used for prebleaching are essentially free of cellulase activity. However, commercial cellulases have been shown to contain relatively high xylanase activity, which may play a role in the changes to fiber characteristics that have been observed. Thus, it would be useful to determine whether certain fiber modifications could be achieved solely by the action of the xylanases or whether cellulase activities are required. In the work reported in this chapter, we examined the response of fractions of distinct fiber length to xylanase treatment. In addition, we have investigated whether the modifications to pulps by xylanase pretreatments are a function of the selected fiber length or the result of cumulative changes occurring on all fiber fractions of a pulp. 4.2 Results and Discussion 4.2.1 Optical Properties Analysis of optical handsheets immediately following enzyme treatment indicated that there was no direct brightening of the pulp by the enzyme. After peroxide bleaching, the brightness of the control pulps was between 66 % and 69 % ISO (International Standardization Organization) for all the fractions other than the 200R fractions, which had a brightness of 49 % ISO (Figure 10A). After xylanase treatment, an enhancement in the final pulp brightness was attained with fractions of all fiber lengths, with the increase ranging from 0.4% to 3.3 % ISO. Previous work by Nelson et al. [199] indicated that softwood pulps treated with 79 xylanases do not exhibit direct brightness gains, in that there was no increase in brightness immediately after the enzyme stage. However, following subsequent peroxide bleaching stages, the xylanase-treated pulps demonstrated increased paper brightness, suggesting that xylanases exhibit a bleach-boosting effect. Our present results support these previous findings and provide strong evidence that the individual fractions of different fiber length respond differently to xylanase prebleaching. The smallest and largest gains in brightness were achieved with the 14R and 200R fractions respectively. The improvement in the brightness of the whole pulp was substantially greater than that obtained with the 14R fractions, although it was not as great as that obtained with any of the other fractions. A corresponding trend was observed when the kappa numbers of the handsheets were measured immediately following enzyme treatment. The 200R fraction had a substantially higher residual lignin content than any of the other fractions (Figure 10B). However, it exhibited the largest decrease in kappa number as a result of xylanase treatment, while the 14R fraction exhibited the smallest. The absorption, opacity and scattering coefficients of the bleached pulps were measured to examine the mechanism by which xylanases enhance pulp brightness. The opacity of the handsheets increased consistently with decreasing fiber lengths (from 91 % to 98 % ISO), with the whole pulp having an opacity value of 93 % ISO. No significant differences were observed between the pulps that received enzyme treatment and the corresponding control pulps. The scattering coefficient of the handsheets also demonstrated little difference between the control and treated pulps. Only the absorption coefficient indicated a difference among the fractions of different fiber length in response to the enzyme 80 ^ 3.0 E 2.5 r-c CD o 2.0 1.5 CD o O c o Q. 1.0 O w < 0.5 0.0 Xylanase Treated F// 7] Control Figure 10 Effects of xylanase treatments on (A) brightness, (B) kappa and (C) absorption coefficient of the fractions of different fiber length (error bars indicate range, n = 2) 81 treatment (Fig IOC), with the 200R fraction demonstrating a substantially higher drop. The absorption coefficient of the 200R fraction was also much higher than that of all the other fractions. 4.2.2 Chemical Composition of the Fiber Length Fractions The initial characterization of the unfractionated pulp indicated that the majority of the pulp was composed of longer fibers (14R), with 55 % of the original material present in the longest fiber length fraction (Table 1). There was a consistent decrease in the amount of fibers found within each of the shorter fiber length fractions. Only 6.4 % of the pulp could not be accounted for by the summation of the fiber fractions retained by the 14R through to the 200R screens. Lignin analysis of the various fractions revealed that the shorter fiber length fractions showed the highest concentration of both acid-soluble and acid-insoluble (Klason) lignin (Table 2). Carbohydrate analysis suggested that the shorter fibers also contained a higher proportion of xylose and galactose, while the quantity of glucose and mannose tended to decrease accordingly. There was no apparent trend in arabinose levels among the different fiber length fractions. Fiber composition seems to play a key role in the bleach-boosting effect obtained by xylanase treatments of the fractions of shorter fiber length. The shorter fibers were found to contain a higher percentage of lignaceous material, confirming previous reports that fractions of shorter fiber length and that the primary fines fraction from kraft pulps are highly enriched in lignin [176]. The increased lignin content of the shorter fiber length fractions is 82 Table 2 The composition (percent by weight) of different fiber length fractions of Douglas-fir kraft pulp. Pulp Arabinose Fraction Galactose Glucose Xylose Mannose Klason Lignin Acid-soluble Lignin 14R 0.41 0.54 74.94 5.47 6.26 3.88 0.36 28R 0.46 0.46 73.81 6.32 6.26 4.01 0.37 50R 0.46 0.49 72.76 6.30 6.14 4.38 0.38 100R 0.44 0.56 73.23 6.71 5.76 4.73 0.38 20'OR 0.44 0.65 72.50 6.98 5.89 5.61 0.43 Whole Pulp 0.41 0.53 74.27 5.75 6.39 4.40 0.36 * Average of 3 replicates (+/- 0.06). probably a function of certain morphological entities found within the pulp fibers, such as ray cells and fragments of the primary cell wall, which have both been shown to have a substantially higher lignin content [250, 319]. The shorter fibers, when compared to the longer fiber lengths, also contained a higher proportion of xylose and a lower proportion of glucose. However, it is difficult to say whether this is an innate characteristic of the fibers or a result of the redeposition of xylan on the fibers during the final stages of the kraft cook. As the smaller fibers have a larger surface-area-to-weight ratio, uniform redeposition of xylan on the surface of all fractions [335] would be expected to result in a higher percentage of xylan per unit weight of shorter fibers. Since the arabinose content was similar for all fiber length fractions, a decreasing arabinose to xylose ratio was observed with decreasing fiber lengths. This would agree with the suggestion that the shorter fibers have relatively more redeposited xylan per unit weight, owing to the loss of the substituted arabinose from 83 the xylan backbone [356]. 4.2.3 Solubilized Material The hydrolysis resulting from xylanase treatments of the pulp was estimated by measuring the reducing sugars liberated into the reaction filtrates. The amount of reducing sugars leached from the fibers during control treatments were very low (approx. 0.004 % of the total pulp), while the amount of material leached from the 200R fraction was slightly higher (0.009 %). The net hydrolysis was also low, ranging from 0.039 % to 0.050 % of the total pulp. These data indicated that net hydrolysis was lowest for the longest fiber lengths (14R) and highest for the shortest fiber lengths (200R). Hydrolysis of the whole pulp was slightly greater than that observed in the 14R fractions while not as great as the rates of hydrolysis obtained with any of the other fractions. The UV absorbance of the reaction filtrates at 280 nm was used to evaluate the amount of lignin-like material solubilized from the fibers. Some lignaceous material was released from both the treated and control fibers (Figure 11 A). It was also evident that, even in the absence of enzyme treatments, more lignin was liberated from the fractions of shorter fiber length. Xylanase treatment resulted in an increased solubilization of lignin-like material, with similar amounts released for all of the fractions other than the 200R fraction, where almost double the amount of material was released. The reaction filtrates were also analyzed for the presence of chromophores absorbing at 457 nm. This is the same wavelength used for measuring pulp brightness. Similar results to those obtained at 280 nm indicated that chromophores were leached from the pulp during 84 Figure 11 Comparison of U V absorbance of xylanase treated reaction filtrates and corresponding controls at (A) 280 nm and (B) 457 nm for each of the different fiber length fractions (error bars indicate range, n = 2) 85 the control treatments and the amounts released increased with prior enzyme treatment (Figure 1 IB). However, there were differences as both the 100R and 200R fractions released substantially more chromophores after enzyme treatment. It seemed probable that the quantity of coloured material released is a function of the size of the fibers. We next determined the molecular mass distribution of material absorbing at 280 nm (Figure 12A and 12B). The values obtained for both the control and treated samples of the 28R and 100R fractions were comparable to the 50R fraction. All control pulps released significant amounts of low-molecular-mass compounds (1000 Da), while xylanase treatments increased the overall liberation, as well as, the proportion of higher-molecular-mass compounds. Only the 200R fraction demonstrated a substantial increase in the release of relatively high-molecular-mass (> 10,000 Da) compounds. A similar trend was observed when the pulsed amperometric signal (PAD) was used as a selective detection method for sugars, where size-exclusion chromatography indicated that the control samples released only material of low-molecular-mass (Figure 12A). However, after xylanase treatment, all of the fractions showed a significant increase in the release of high-molecular-mass compounds released, with the largest signal corresponding to the 200R fraction (Figure 12B). The liberation of coloured compounds, presumably lignin, by xylanase treatments is partially supported by the observed reduction in kappa numbers for all fractions. However, the nature of the solubilized chromophore warrants further consideration in order to evaluate their contribution to the bleach-boosting effect. In past work, Ziobro [362] implicated carbohydrate degradation products as a source of coloration in pulp effluents, while Gellerstedt and Li [99] have implicated their contribution to kappa number. The increased release of chromophores from the short fibers during enzyme treatment seemed to correlate 86 Figure 12 Analytical size-exclusion chromatographs showing the molecular mass distribution of solubilized material from Douglas-fir kraft pulp: (A) control and (B) following xylanase treatments 87 to an increased enhancement in pulp brightness after bleaching. For the short fibers, the improvement in final brightness could be attributed to a measurable decrease in absorption. Allison and Graham [7] demonstrated that the fines fraction of radiata pine thermomechanical pulp had a much higher absorption coefficient than did the other fiber fractions. They also showed that, following peroxide bleaching, which removes a portion of the chromophores, a reduction in the absorption coefficient was evident. It appears that the ability of xylanase to decrease absorption coefficient further plays an important role in the xylanase prebleaching phenomenon. However, it is unclear how the smaller bleach-boosting effects were obtained in the other fractions of different fiber length without any observable changes in absorption, opacity or scattering coefficient. 4.2.4 Papermaking Properties Only the 14R, 28R and 50R fractions of the kraft pulp could be collected in sufficient quantities to allow an effective evaluation of papermaking properties. Physical tests were conducted both immediately after xylanase treatment and after subsequent peroxide bleaching. The results of handsheet density measurements (Figure 13A) indicated that the xylanase treatment increased the density for all the fractions tested, while the whole pulp remained unchanged. The density of the handsheets from the corresponding bleached fibers indicated that the subsequent peroxide stages further enhanced the density of the handsheets, while maintaining the increased density of the enzyme treated pulps. Both xylanase treatment and peroxide bleaching decreased the roughness of the handsheets (Figure 13B), while the roughness of all the bleached fractions was lower than that of the corresponding 88 unbleached fractions. The physical strength properties of the handsheets indicated that, in general, the xylanase treatment resulted in very little loss of strength in the resultant paper products. With the exception of the handsheets derived from the unbleached 50R fraction, which demonstrated a slight increase in burst index, all of the other fractions of different fiber length displayed little or no change in burst index (Figure 13C). Subsequent analysis of the bleached pulp indicated that its strength was higher, with the enzyme treatment perhaps increasing the burst index slightly in some cases. However, analysis of both tensile and tear indexes indicated no observable differences in strength between the control and treated pulps. In contrast, the strength of the individual fibers, as measured by zero-span breaking length, decreased as a result of xylanase treatment by 0.3 km to 0.5 km (Figure 13D). In general, peroxide bleaching resulted in further losses in the strength of individual fibers. Previously it was suggested that hemicelluloses contribute to both fiber morphology and the physics of the paper produced [149]. However, there have been mixed opinions as to what effects the partial removal of hemicelluloses has on the paper and fiber strength. For example, xylanase treatments have been reported to reduce the strength of paper [242], to improve certain strength parameters [45], and to have no effect [212, 309]. Our results indicate that the strength properties of the individual fibers, as measured by zero-span breaking length, decreased as a result of treatment with a commercial xylanase. This did not appear to be due to the extremely small amount of cellulases contaminating this enzyme preparation [199], as considerably higher cellulase activities were required to obtain significant reductions in the strength of Douglas-fir kraft pulp (chapter 3). In contrast to the 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 C o n t r o 1 Control Figure 13 Average effect of xylanase treatment and xylanase treatment followed by peroxide bleaching on handsheet (A) density, (B) roughness, (C) burst index and (D) zero-span breaking length of the fractions of different fiber length fractions (14R, 28R, 50R, 100R, 200R and "W" = whole pulp) of Douglas-fir kraft pulp (n = 2) 90 previously observed decrease in fiber strength, after xylanase treatment, the fiber-fiber bonding and therefore paper strength remained relatively unchanged. Xylanase treatment also resulted in a slight increase in handsheet density. This was probably due to xylanase attack on the surface of the fiber, which liberates a portion of the stiff, inflexible redeposited lignin. Similarly, the decrease in handsheet roughness could be a result of the more densely packed network of fibers in handsheets. However, these fiber modifications were very small when compared to the modifications previously obtained with commercial cellulases (previous chapter). In general, the corresponding bleached pulps demonstrated similar changes in physical properties after enzyme treatments. However, they show that bleaching usually has a greater effect on fiber properties. Previous work [155] has shown that fibers that have been bleached possess a greater degree of flexibility when compared to fibers of the same origin which have not been bleached or have been dried. Thus it is possible that the different fiber length fractions formed handsheets with increased densities after bleaching and this resulted in lower handsheet roughness and greater paper strength properties. 4.3 Conclusion It was apparent that enzymatic treatments with a commercial xylanase caused a bleach-boosting effect on all the Douglas-fir pulp fiber length fractions, which resulted in a significant enhancement in pulp brightness. However, the mechanism involved does not appear to be consistent for all of the different fiber lengths. In contrast to cellulase treatments (chapter 3), which seemed to act in a similar fashion on all the fiber length 91 fractions, the xylanase treatments seem to be slightly influenced by compositional differences within the fibers. For example, the greater increase in brightness obtained with the shorter fiber length fractions may be related to a reduction in their higher lignin composition and optical properties such as the absorption coefficient. The physical properties of handsheets derived from individual fiber length fractions generally respond in a similar manner when subjected to xylanase prebleaching. However, the modifications were only minor, even at a relatively high xylanase loading. These results suggest that, although xylanases do contribute to modifications in fiber properties, the substantial alterations to the fibers that resulted during the application of a commercial cellulase (chapter 3), (which contains both high cellulase and xylanase activities) were primarily due to the action of cellulases. However, the independent contributions of both these enzyme activities to fiber modification could not be clearly differentiated. It would be invaluable if we could determine what changes occurred at the "fiber" and "microfibril" level as a result of these enzyme treatments. For example, the various mechanisms that the xylanase could be involved in, such as the removal of the xylan on the surface of the fibers decreasing inter-fiber bonding, could perhaps be correlated with possible changes in the degree of polymerization (DP) of the xylan component of the pulp. Thus, in the next chapter we describe our attempts to compare the observed changes in pulp characteristics after cellulase/xylanase treatments with changes to "molecular" properties such as the DP, pore volume and crystallinity of the pulp. In this way we hope to interrelate the observed changes in pulp characteristics such as coarseness, strength, freeness, with the more traditional enzyme characterization methods such as sugars released and pore volume 92 determination, to see if this could explain the mode of fiber modification carried out by these hydrolytic enzymes. 93 Chapter 5 Physical Characterization of Enzymatically Modified Kraft Pulp Fibers 5.1 Background In my earlier work it was apparent that cellulase treatments generated substantial changes to the fiber characteristics (chapter 3). It appears that, as well as acting synergistically in the traditionally defined endo/exoglucanase mechanisms proposed for cellulose hydrolysis [339], that the cellulases are able to modify both the pulp fibers and paper characteristics in a way that cannot be directly explained by this over simplified endo/exoglucanase mode of action. Recently, a considerable amount of work has been carried out to try to confirm that the mechanism of xylanase aided bleaching is due to the removal of reprecipitated or lignin carbohydrate complexed (LCC) xylan present on the surfaces of the pulp fibers [60, 148, 285, 351]. However, the xylanase treatments of different pulp fiber length fractions demonstrated a non-uniform response (chapter 4), indicating that the composition of the fibers plays a key role in determining the effectiveness of the treatment. Although we have come part way to understanding the mechanisms of xylanases, little is know about the enzyme mechanisms involved during the limited initial degradation of pulp fibers by cellulases. While significant advances have been made in clarifying the mechanism by which certain microorganisms degrade hydrogen bond-ordered cellulose [326], the nature of the enzyme interaction and the action and sequence of events which 94 solubilize cellulose have yet to be clearly defined. At the same time the actual mechanism by which enzymes are able to modify fiber morphology remains ambiguous. Several groups [135, 277, 279] have indicated that cellulase treatments result in a reduction in strength of the native fibers (chapter 3). Similarly, it has been shown that care must be taken when using xylanases in bleaching of kraft pulps to avoid cellulase contaminated enzyme mixtures which can severely reduce the strength of the pulp [226]. Gurnagul and co-workers [108] demonstrated that treatments of low yield kraft pulp with cellulases lowered the strength drastically, with relatively little change in the degree of polymerization (DP) of the cellulose. These workers concluded that the enzymes preferentially attack structurally irregular zones in the fiber wall, resulting in localized degradation. Previously, we (chapter 3) and other workers [135, 277] demonstrated that beneficial changes to pulp fiber properties can be achieved by brief treatments with low concentrations of cellulases. However, this was usually accomplished at the expense of some strength and yield loss. Thus, the main purpose of the work carried out in this chapter was to investigate the nature of changes which occur at both the micro and macro structural level of Douglas-fir kraft pulp fibers during enzymatic treatments with a commercial cellulase. By measuring the amount and type of carbohydrates released, the degree of polymerization, crystallinity, the pore volume and FT-IR spectroscopy of the substrate, as well as visualizing changes in the substrate by scanning electron microscopy we hoped to elucidate the mechanism by which the cellulase enzyme changed the fiber morphology which resulted in the changes in paper properties described in the previous chapters. It was apparent that changes to the surface composition of the fibers by enzymatic treatments influenced both the fiber properties and 95 paper strength. 5.2 Results and Discussion As indicated earlier, (chapter 3) the enzymatic effects on the pulp properties are highly dependent on the enzyme dosage that is used. For example, an enzyme charge of 5 mg protein per gram of pulp, which corresponded to a 3.2 % hydrolysis of the pulp, produced a 12.7 % reduction in paper strength (tensile index) and a 35.2 % loss in fiber strength (zero-span breaking length). Although the enzyme treatments resulted in a considerable loss in both the paper and fiber strength, we wanted to determine which modifications to the fiber structure could be correlated with these strength changes. 5.2.1 Carbohydrate Solubilization A more thorough investigation by HPLC analysis indicated which specific carbohydrates were solubilized by the enzyme treatments after 3.2 % of the pulp was hydrolyzed (Table 3). It was apparent that the enzyme treatment liberated almost equivalent amounts of both glucose (229 mg) and xylose (226 mg), which corresponded to 1.8 and 21.1 % hydrolysis respectively, of the total amount of cellulose and xylan that was originally available in the pulp. It has been proposed that, at the end of the kraft cooking process, debranched xylan redeposits on the surface of the fibers [356], and that this redeposited xylan is the primary substrate for xylanases in the prebleaching process [148]. However, Suurnakki and co-workers [284] have recently questioned the importance of redeposition when pine kraft pulps 96 were treated with xylanases and instead suggested that it may be the innate hemicellulose that is the target substrate. Our results seem to indicate that the debranched xylan is not the major substrate for the xylanases, as the arabinose/xylose ratio in the filtrates was increased when compared to that found in the pulp (Table 3). Therefore, it is possible that the concerted hydrolysis by both the cellulase and xylanase enzymes liberated both glucose and the more highly substituted xylan found in the inner fiber wall [283]. This would account for the large proportion of the arabinose hydrolyzed by the enzymatic treatments. Table 3 Composition of the Douglas-fir kraft pulp and filtrates obtained after a 5 mg/g pulp cellulase treatment. Fraction Arabinose Galactose Glucose Xylose Mannose Klason Acid-Lignin soluble Lignin Pulp (%) 0.41 0.56 72.56 5.95 6.66 4.91 0.49 Filtrate* 21.01 0 229.39 225.83 16.09 N/A 0.08 % Solubilized 28.8 0 1.8 21.1 1.3 N/A 16.3 * Values represent total mg of carbohydrate liberated from enzyme treated minus control filtrates of 18 g pulp samples. Average of 3 replicates (+/- 0.12). N/A represents not assessed. A 5 and 3.5 fold increase in the absorbance at 280 nm and 457 nm respectively, was observed after enzyme treatment. These increases were probably due to the liberation of lignin and coloured compounds by the action of the xylanases on the pulp fibers. Although the cellulases have been shown to be rather ineffective in releasing coloured materials from kraft pulps [29], they undoubtedly hydrolyzed the available surface cellulose and continued 97 to work in concert with the xylanases resulting in the hydrolysis of some of the internal structural polysaccharides. 5.2.2 Pore Volume Determination The solute exclusion technique of Stone and Scallan [275] has been a valuable tool for characterizing the ultrastructural morphology of fibers after various physical and chemical pretreatments. It has been successfully used to demonstrate the importance of fiber porosity during the enzymatic hydrolysis of various cellulosic substrates [103, 317, 330]. It has also been used in biobleaching experiments, where the removal of hemicellulose and its associated lignin by xylanase treatments resulted in an increase in the pore volume of the fibers [358] and a slight increase in the median pore width [281]. In this work we have used pore volume determination to investigate the changes that occurred as a result of the enzymatic treatments to the Douglas-fir pulp fibers. As indicated previously, a substantial amount of the hemicellulose component of the pulp was hydrolyzed after enzyme treatment, suggesting that there might be a corresponding increase in the pore volume of the fibers. However, we found that the cumulative pore volume (inaccessible water) of the fibers was significantly reduced by these enzyme treatments (Figure 14). The profile exhibited by the enzyme treatments not only demonstrated a substantial reduction in the pore volume for each of the dextran probes used, it also showed a reduction in the fiber saturation point (as measured by the 56 nm probe). However, there was an increase in the median pore width of the enzyme treated pulps. Figure 14 Pore volume profile of untreated and 5 mg/g pulp cellulase treated Douglas-fir kraft pulp fibers using six different dextran probes (error bars indicate range, n = 2) 99 5.2.3 Scanning Electron Microscopy Scanning electron microscopy indicated a qualitative change in the outermost fiber surface of the enzymatically treated pulp compared to control pulps (Figure 15). However, the electron micrographs did not reveal any discernible alterations to the fiber surfaces (i.e. cleavages or pit enlargements) other than the erosion of surface material. Further cross-sectional electron micrographs (data not shown) did not indicate any changes to the internal morphology of the fibers. As suggested previously [170, 222], it would appear that the concerted action of these enzymes may ultimately remove subsequent "layers" from the fiber surface, resulting in a "polishing or cleaning" of the fibers. 5.2.4 FT- IR Spectroscopy All the FT-IR spectroscopy data indicated that the enzyme treatment had removed substantial amounts of xylan from the fibers, while the cellulose appeared to be relatively unchanged. The DRIFT spectrum of untreated Douglas-fir kraft pulp, which was the average of 8 separate disk measurements (Figure 16, inset), showed characteristic cellulose peaks around 1000-1200 cm"1 [28, 182]. The relative high absorbance at 1045-1050 cm"1 and the bands at 1460, 1250, 811 cm"1 indicated the presence of some hemicellulose, while the weak absorption band at 1512 cm"1 showed that only a small amount of lignin was still present [174, 182, 332]. Although the spectrum from the enzyme treated pulp initially appeared to be comparable with the untreated sample, subtraction of the enzyme treated spectrum from the profile of the control revealed some interesting differences (Figure 16). The major difference was shown to be around 1045-1055 cm"1, which corresponds to the native xylan spectra at 1045-1058 cm"1 [88, 182]. These results confirmed that the total amount of xylan in the sample had been reduced by the 100 Figure 15 Scanning electron micrographs of (A) untreated and (B) 5 mg/g pulp cellulase treated Douglas-fir kraft pulp fibers at 2550 x magnification. 101 1190 —J i i l—i i i i l i i i i l i i i i I i i i i I i i i • i » i i i I • • • • 1 4000 3500 3000 2500 2000 1500 1000 500 Wavelength (nm) Figure 16 FT-IR difference spectrum of untreated minus 5 mg/g pulp cellulase treated Douglas-fir kraft pulp fibers and (inset) FT-IR absorbance spectrum of untreated Douglas-fir kraft pulp (n = 8) 102 enzyme treatment. The 895 cm"1 band which is characteristic for B-linkages, especially in hemicelluloses [182], was also reduced after enzyme treatment. However, the 811 cm"1 band, which is characteristic of galactoglucomannan [88], was unaltered by enzyme treatment. This was in good agreement with the chemical analysis of the filtrates (Table 3). Other major differences were seen at higher wavelengths. The band at 1106 cm"1 could be ascribed to the antisymmetric ring stretch, the band near 1160-1170 cm"1 was representative of the antisymmetric bridge stretching of C-O-C groups in cellulose and hemicellulose [182], and the band at 1315-1317 cm"1 could be ascribed to CH2-wagging vibrations in cellulose and hemicellulose [174]. There was also a substantial reduction in the band around 1590-1600 cm"1 which has been attributed to COO1"-groups in glucuronoxylan after salt formation [184, 332]. While the bands at 3300 cm"1 are representative of OH vibrations with intermolecular H-bonds. Our data were collected using uniform disks obtained from 7.5 g/m handsheets placed on top of beds of KC1. It has been suggested that this procedure gives comparable results with spectra of kraft fibers diluted directly in KBr [183]. The advantage of the former technique is the reproducibility that can be achieved within each sample treatment. Although it was apparent that the enzyme treatments caused substantial hydrolysis of the xylan fraction and a polishing of the fibers, no major changes in the cellulose moiety were observed. 5.2.5 Degree of Polymerization The importance of the hemicellulose component in papermaking has been well documented [51, 294]. Pulps which contain a higher concentration of hemicellulose, up to a certain maximum, exhibit greater strength. Similarly, those fibers which contain a higher 103 proportion of glucomannan (hexosan), with respect to other hemicellulose carbohydrates, tend to show enhanced adhesive capabilities and result in higher paper strength [51, 294]. However, in addition to the quantity, chemical structure and distribution of the hemicellulose, the degree of polymerization of this fraction plays an integral role in determining the final paper strength [73]. It was apparent (Table 3) that a large percentage of the hemicellulose within these pulp samples had been hydrolyzed. Therefore, it is probable that, as well as the removing the fines and fiber defibrillation, the reduction in available hemicellulose plays a substantial role in the previously observed reduction in paper strength (chapter 3). The subsequent determination of the molecular weight distribution of the cellulose tricarbanylate by size exclusion chromatography indicated that, although there was no substantial change in the degree of polymerization of the cellulose component, the higher molecular weight hemicellulose component was reduced (Figure 17). This shift in molecular weight distribution after xylanase treatments has previously been documented for various pulp types and treatments [186, 189]. Therefore, it is possible that both the removal of hemicellulose and the concomitant reduction in the degree of polymerization of the residual hemicellulose may have contributed to the reduced paper strength. The role of hemicellulose in intrinsic fiber strength is still a topic of some debate. Opinions differ on the effect of partial removal of hemicelluloses on the fiber strength. For example, xylanase treatments have been reported to cause a rapid reduction, and then a leveling off in zero-span breaking length [203]. This reduction was further correlated to the diminishing viscosity of the dissolving pulps, and the authors concluded that the hydrolysis of the xylan macromolecules played an important role in fiber wall cohesion [203]. In contrast, Paice and co-workers [212] observed a marked decrease in the degree of 104 25000 Degree of Polymerization Figure 17 Degree of polymerization of both untreated and 5 mg/g pulp cellulase treated Douglas-fir kraft pulp (n = 6) 105 polymerization of the xylan component of a kraft pulp after xylanase treatment, even though the pentosan content was only reduced by approximately 10 % of its original composition. This work also indicated that the fiber strength was generally unaltered by the change in degree of polymerization of the xylan. In earlier work we found that treatments of pulp with a xylanase decreased the zero-span breaking length by approximately 2 % (chapter 4). Subsequent work indicated that the degree of polymerization of xylan had been altered, mimicking the changes shown in Figure 17. This seems to confirm the observations of Paice et al. [212] that changes in the degree of polymerization of the xylan had little or no effect on the intrinsic fiber strength. Therefore, it appears that the observed reduction in fiber strength must be a result of a modification in the fibers' cellulose component, rather than any effect resulting from xylan depolymerization. Previously Page et al. [211] carried out a comprehensive assessment of the strength and chemical composition of wood pulp fibers. They concluded that, in pulps with small fibril angles, the fiber strength is directly proportional to the cellulose component of pulps with a less than 80 % cellulose content. Our work indicated that the 35.2 % reduction in intrinsic fiber strength, resulting from enzyme treatment, occurred without any change in the degree of polymerization of the cellulose. These results concur with those reported previously [108, 212], which concluded that the enzymes preferentially attack structurally irregular zones such as at kinks and nodes in the fiber wall. This localized attack resulted in a reduction in the intrinsic fiber strength. However, in addition to this localized attack, our results suggest that the cellulase enzymes act in a manner which consecutively remove the outermost layers of the fiber wall, resulting in a surface "polishing" effect. 106 5.2.6 Crystallinity The x-ray diffractograms of enzyme treated and control pulps indicated that there was no discernible difference in the degree of crystallinity (Figure 18) of the cellulose component of the two samples (76.47 and 76.80 % crystalline for the control and enzyme treated samples, respectively). This suggested that the cellulase enzymes acted in a manner which removed cellulose molecules without interrupting the integrity of the remaining molecules. This again alludes to the removal of surface cellulose. 5.3 Conclusion Although a portion of the cellulosic component of the fibers had been hydrolyzed, the degree of polymerization as measured by gel permeation chromatography and the crystallinity of the substrate were unaltered. However, the substantial reduction in the pore volume of the fibers indicated that the enzymatic treatments had eroded the surfaces of the fibers. It is probable that, in conjunction with the localized attack at structural irregularities [108], the reduced fiber strength observed with enzymatic treatments is directly related to the removal of surface material from the fibers, as evidenced by the electron micrographs of the treated fibers. The compromised paper strength appeared to be the result of this reduction in intrinsic fiber strength and the removal of hemicellulose from the fiber. Carbohydrate analyses, degree of polymerization measurements and FT-IR spectra, all indicated that the hemicellulose had been substantially depolymerized and solubilized by the enzymatic treatments. This investigation suggests that the results obtained by the action of the commercial hydrolases (chapter 3 & 4) are directed at the surface of the fibers, resulting in an erosion of the surface material. Although these actions have generated some beneficial changes in fiber 107 800 600 1 Control (Crl = 76.47%) / \ 1 Enzyme Treated 400 I \ I \ (Crl = 76.80%) 200 T i 1 _.i i i i i i i i I " V " ~ T " I i ~ V — 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 2 Theta Figure 18 CrystaUinity index of untreated and 5 mg/g pulp cellulase treated Douglas-fir kraft pulp fibers (n = 3) 108 quality (i.e. enhanced fiber collapsibility as measured by handsheet density), they are achieved at the expense of both fiber strength and yield losses. The key to enhancing fiber collapsibility and/or flexibility without significant strength losses may be by achieving an "amorphogenesis" of the pulp fibers. This may occur by achieving an "opening-up" of the fiber matrix, subsequently resulting in a delamination of the pulp fibers, in the same way that is achieved by pulp refining. However, we want to achieve this without the fiber shortening that traditionally occurs during this refining process. It is possible that this goal may be achieved by harnessing the specificities of other microbial enzymes which employ an alternate mode of carbohydrate degradation, such as the radical generating enzymes. One such group is the oxidoreductases, which may be associated with the hydrolases in carbohydrate degradation. Specifically, cellobiose dehydrogenase (CDH), which is a hydroxyl generating enzyme and contains a cellulose binding domain [239]. Therefore, the use of this enzyme allows for the generation of a very active agent which can be directed at the cellulose constituents of the pulp fibers. The possible use of this enzyme in pulp modification was studied and is described in the next chapter. 109 Chapter 6 Changes in Cellulose Ultrastructure using Cellobiose Dehydrogenase (CDH) 6.1 Background Generally, hydrolytic enzymes such as cellulases, xylanases and mannanases are thought to be primarily responsible for the degradation of the carbohydrate moieties, while oxidative enzymes such as laccases, lignin peroxidase and manganese peroxidase, in combination with low molecular weight mediators have been shown to be primarily involved in lignin biodegradation [59]. Although many of the wood-degrading fungi can be grouped into categories such as white-rot, brown-rot or soft-rot fungi, this delineation does not readily carry over into the specific enzymes produced by these organisms involved in the degradation of the different wood components. While there has been a great deal of progress made in characterizing many of the enzymes involved in the degradation of the native wood constituents, there is still the apprehension that some enzymes may be multifunctional and cause modifications to more than one or all of the wood components. One such group of enzymes are the extracellular cellobiose-oxidizing enzymes cellobiose dehydrogenase (CDH) and cellobiose:quinone oxidoreductase (CBQ), which have been shown to be produced by a number of basidiomycete fungi, including both white-rot [12, 131, 243, 321] and a brown-rot fungus [259]. This class of enzymes has also been isolated from the culture filtrates of non-ligninolytic, cellulolytic microorganisms such as 110 Monales sitophila [61], Chaetomium cellulolyticium [83], Sporotrichium thermophile [34] and bacterial strains [173]. Cellobiose dehydrogenase is an enzyme consisting of two prosthetic groups, a heme and a flavin adenine dinucleotide (FAD) moiety (hemoflavoenzyme). The latter domain is directly involved in both the oxidative and reductive half-reactions, while the former stimulates the reduction of one-electron acceptors such as cytochrome c and Fe 3 + [125, 126]. Recently, the size and shape of this enzyme have been determined by small-angle X-ray scattering [172], while the cDNA of Phanerochaete chrysosporhim CDH has been successfully cloned and sequenced [235]. It has also been shown that the proteolysis of CDH both in vitro [126] and by the action of isolated proteases [65, 110] results in the generation of a second active enzyme, CBQ [334]. Cellobiose:quinone oxidoreductase is a flavoenzyme which, in the presence of cellobiose can reduce compounds such as quinones and phenoxy radicals [9]. Although these enzymes were originally isolated and characterized more than 20 years ago [321, 322], their role in wood decomposition has yet to be clearly defined. Several putative functional roles for this enzyme have been suggested: CDH acting as a vehicle to generate radicals as antibacterial agents [325]; the reduction of quinones as a defence mechanism against toxins [193]; CDH acting as a regulatory enzyme preventing the repolymerization of lignin radicals generated by other oxidative enzymes [9, 80]; and the generation of highly active hydroxyl radicals which participate in Fenton's reactions to degrade cellulose, xylan and lignin [124, 336]. However, as Morpeth observed several years ago "cellobiose oxidoreductases are enzymes in search of a function" [193]. I l l As we had previously used Douglas-fir kraft pulps to assess the action of various cellulases and xylanases (chapter 3 & 4) we thought this would be an effective substrate to evaluate what changes, if any, CDH caused to the carbohydrate constituents of the pulp. In an attempt to create a homogeneous substrate, the pulp was fractionated and only the longest fiber length fraction (14R) was used. This eliminated some of the variability that can arise in treating whole pulps. Although CDH has been shown to both enhance the action of cellulases on crystalline cellulose [17] and degrade model wood components such as carboxymethylcellulose, xylan and synthetic lignin [124], there have been few studies to date on its direct role in cellulose modification in substrates such as pulp. In this chapter, we describe how we isolated CDH from the white-rot fungus Phanerochaete chrysosporium and. carried out preliminary work which assessed the enzymes ability to modify the degree of polymerization (DP) of a Douglas-fir kraft pulp in the absence or presence of various co-factors. 6.2 Results and Discussion To date, much of the work related to cellobiose dehydrogenases has focused on their interaction with ligninolytic enzymes and their action on lignin-related compounds [10, 243]. It has been suggested that CDH may be one of the components of the fungal enzymatic machinery involved in the depolymerization-repolymerization of lignin-related compounds by a variety of fungi [9]. This is most likely directly related to the oxidoreductive nature of this enzyme. However, other workers [17], have also shown that CDH enhanced the cellulolytic degradation of crystalline cellulose. These authors indicated that the 112 supplementation of Trichoderma cellulases with CDH increased the substrate hydrolysis by approximately 20 % over the non-CDH-supplemented reaction. More recently it has been demonstrated that CDH can degrade or modify cellulose and xylan derivatives, as well as synthetic lignin [124]. Previous work has also shown that cellobiose dehydrogenase initiates a two-electron oxidation of cellodextrins, generating the corresponding lactone [128]. Cellobiose was found to be the substrate of choice, with cellodextrins of increasing DP demonstrating reduced activity. Meanwhile limited activity was observed when glucose was used as the substrate [125]. The reductive half-reactions (see reaction scheme below) include a preferential single electron reduction of Fe(III) to Fe(II) [52], while in situations of limited Fe(III), CDH can reduce 0 2 to generate H 2 0 2 directly [320, 321] or via the production of superoxide which can subsequently react with Fe(II) [336]. Furthermore, autooxidation of Fe(II) can result in the generation of hydrogen peroxide [336]. Together hydrogen peroxide and reduced iron undergo Fenton's chemistry generating hydroxyl radicals, which then can actively be involved in the attack on localized substrates [14, 112, 159, 160]. C D H o x + Cellobiose => CDHRED + Cellobiono-l,5-lactone (1) 2CDHRED + 2Fe3+ => 2CDH o x + 2Fe2+ (2) CDHREU + OJ => CDHox + H 2 0 2 (3) 2Fe2+ + 02+ 2H+ => 2Fe3+ + H 2 0 2 (4) Fe 2 + +H 2 0 2 => Fe 3 + + OH" + OH (5) The potential role of these various components encouraged us to assess the effect of 113 different combinations of CDH, cellobiose, iron and hydrogen peroxide on the molecular weight distribution of the 14R fraction of the Douglas-fir kraft pulp (Figure 19, Table 4). All control combinations without CDH (data not shown) gave profiles similar to that of the original sample in buffer (DPN = 341, DP W = 2,337) with the exception of the reaction mixtures containing both hydrogen peroxide and ferric chloride (line 2, Figure 19). This combination generates Fenton's reagent and results in a reduction in the DP of the pulp cellulose even in the absence of CDH. The addition of CDH alone to the pulp (line 3, Figure 19) resulted in little change in the DP (DPN = 334, DP W = 2,279). However, with the addition of excess cellobiose, the DP of the pulp was reduced substantially. It was apparent that the combination of CDH and cellobiose (line 4, Figure 19) could depolymerize the cellulose marginally more than the reaction mixture lacking CDH but capable of generating Fenton's chemistry (line 2, Figure 19). The addition of CDH to the reaction mixture containing the individual components of Fenton's reagent in the absence of cellobiose (line 5, Figure 19) caused a reduction in the DP of the cellulose even greater than that exhibited by the ferric chloride and hydrogen peroxide alone (line 2, Figure 19). This result confirmed that the CDH could modify the substrate without the addition of easily oxidizable substrates such as cellobiose [164]. However, when the pulp samples were first subjected to chelation and then treated with CDH without the supplementation of iron, the ability of CDH to modify the DP of the pulp cellulose was substantially reduced (DPN = 331, DP W = 2,254). The greatest change in DP of the cellulose occurred when the cellobiose and ferric chloride were added to the CDH. The mixture containing CDH, cellobiose, iron and hydrogen 114 10 100 1000 10000 Degree of Polymerization Figure 19 Degree of polymerization of cellobiose dehydrogenase treated Douglas-fir kraft pulp fibers. Pulp (10 mg/mL) was incubated with combinations of 0.1 IU CDH/mL, 20 mM cellobiose (C), 1.8 m M H 2 0 2 (H), and 0.2 mMFeCl 3 (F) (n = 3) 115 peroxide demonstrated only slightly more depolymerization than the corresponding control lacking the enzyme. In all cases, the attack seems to be directed at the higher-molecular-mass (DPs of 2000-5000) cellulose component of the pulp, as indicated by the absolute values of DP N and DP W for the different reaction scenarios (Table 4). These values clearly indicate that the depolymerization of pulp cellulose is substantially enhanced by the addition of CDH. Having established that the CDH supplemented with cellobiose and ferric chloride resulted in the greatest changes in cellulose morphology, the nature of the reaction was followed by monitoring the changes in DP through a time-controlled experiment (Figure 20, Table 4). As the reaction proceeded, the enzyme complex continued to react with the cellulosic material of higher DP (5000), generating molecules of a lower DP and producing a new maximal peak at approximately a DP of 1000-2000 (Figure 20, Table 4). A subsequent treatment with twice as much CDH enzyme resulted in a further reduction in the average DP (DPN = 193, DP W = 1558), as indicated by the shift in the shoulder of material found between DPs of 200 and 800 (Figure 20, Table 4). The supplementation of additional cellobiose, by a 20 mM cellobiose spike at hour 9 of an 18 hour reaction, had no effect on the DP (data not shown). Our results clearly indicate that CDH can depolymerize wood-derived cellulose. In vivo, an even more efficient depolymerization may be possible as an accumulation of hydrogen peroxide may result from the autooxidization of iron complexes such as Fe(II) oxalate [336], and/or the slow continuous generation of small amounts of hydrogen peroxide by extracellular oxidases [59]. 116 10 100 1000 10000 Degree of Polymerization Figure 20 Progressive changes in the degree of polymerization of cellobiose dehydrogenase treated Douglas-fir kraft pulp fibers over time. Pulps were supplemented with 0.1 and 0.2 IU CDH/mL, 20 mM cellobiose, and 0.2 mM FeCl 3 (n = 3) 117 Table 4 Average DP N and DPw values of cellobiose dehydrogenase treated Douglas-fir kraft pulp. Reaction conditions included combinations of cellobiose dehydrogenase (CDH, 0.1 Reaction Conditions D P N a DP • a N C 341 (4) 2337 (11) C + F + H 301 (4) 2093 (4) C + H 334 (6) 2279 (7) C + CDH (0.1 IU/mL, 18 hr.) 295 (5) 2069 (8) F + H + CDH (0.1 IU/mL, 18 hr.) 293 (4) 2047 (6) C + H + CDH (0.1 IU/mL, 18 hr.) 290 (5) 1987 (7) C + F + CDH (0.1 IU/mL, 1 hr.) 326 (3) 2307 (5). C + F + CDH (0.1 IU/mL, 2 hr.) 307 (4) 2243 (8) C + F + CDH (0.1 IU/mL, 6 hr.) 293 (3) 2190 (10) C + F + CDH (0,1 IU/mL, 12 hr.) 282 (6) 2086 (6) C + F + CDH (0.1 IU/mL, 18 hr.) 262 (9) 1832 (15) C + F + CDH (0.2 IU/mL, 18 hr.) 193 (8) 1588 (21) C + F + H + CDH (0.1 IU/mL, 18 hr.) 299 (12) 2201 (14) Values in brackets indicate standard deviation (n = 4). The attack on the natural woody substrate seems to be directed at the cellulose of higher DP, generating molecules of cellulosic material of smaller size. This is indicated by the appearance of the shoulders in the lower DP ranges of the chromatographs. Recently, Ander et al. [11] found, through the use of polarized light micrographs, that the attack by CDH on pine holocellulose fiber seemed to be directed at specific sites of compression. These "nodes," whose origin are still under investigation, appear approximately every 100-200 urn. It would be interesting to see whether these "nodes" are enriched in iron or other transition metals, as concentrated deposits in these regions would support the localized attack 118 observed previously [11]. High performance anion-exchange chromatography was subsequently used to determine if any oligosaccharides were liberated into the filtrates during the reactions (Table 5). For all of the controls, no sugars other than those containing added cellobiose were detected. However, in the reactions containing CDH and additional components, cellobionic acid was detected after 18 hours incubation. Cellobionic acid is generated during HPLC analysis by base hydrolysis of the cellobionolactone found within the actual reaction filtrates. Maximum amounts of cellobionic acid (cellobionolactone) were generated when both cellobiose and ferric chloride accompanied the CDH (Table 5). Cellobionic acid (cellobionolactone) was also produced when only CDH and cellobiose were present, but not to the same extent as was observed when iron was also present. Supplementation with hydrogen peroxide demonstrated a reduced level of cellobionic acid (cellobionolactone) generation, which was even more pronounced when both hydrogen peroxide and iron were present. In conjunction with the generation of cellobionolactone from cellobiose by these different reaction scenarios, the generation of glucose and to a lesser extent arabinose was also observed (Table 5). No other neutral wood sugars (galactose, xylose or mannose) were . produced. Small amounts of glucose were also present in the reaction filtrates supplemented with only cellobiose, indicating a minor contamination of the cellobiose with glucose. When the optimal reaction mixture of CDH supplemented with only cellobiose and ferric chloride was used, it was apparent that, as the reaction proceeded over an 18 hours incubation period, both glucose and cellobionolactone were generated while the cellobiose was slowly degraded (Table 6). Similarly, using twice as much CDH resulted in an 119 Table 5 Polysacchar ides l iberated (mg/mL) by cel lobiose dehydrogenase after 18 hours incubation. React ion conditions inc luded combinations o f ce l lob iose dehydrogenase ( C D H , 0.1 IU/mL), 20 m M cel lobiose (C), 1.8 m M H 2 0 2 (H) and 0.2 m M F e C l 3 (F). React ion Condit ions Glucose a Arabinose a Ce l l ob io se a Ce l lob ion ic A c i d a C 0.022 (0.002) 0.000 6.799 (0.020) 0.000 C + F e 0.023 (0.002) 0.000 6.811 (0.013) 0.000 C + F + H 0.114 (0.001) 0.006 (0.001) 6.772 (0.019) 0.000 C + C D H 0.157 (0.006) 0.008 (0.001) 6.046 (0.009) 1.285 (0.009) C+ H + C D H 0.225 (0.010) 0.013 (0.002) 6.188 (0.022) 0.893 (0.017) C + F + C D H 0.171 (0.005) 0.008 (0.001) 5.252 (0.016) 1.999 (0.024) C + F + H + C D H a T i * . . . . . 0.175 (0.003) 0.010 (0.001) 6.389 (0.011) 0.463 (0.008) Va lues i n brackets indicate standard deviation (n = 4). approximate proport ional twofo ld increase in the amount o f g lucose and lactone generated, w h i l e deplet ing the supplemented cellobiose. Bo th arabinose and glucose were generated as the react ion proceeded and the addition o f twice as m u c h C D H also generated approximately tw ice as m u c h o f these two sugars (Table 6). T o examine whether the action o f C D H l iberated any sugars f r om the pulp, the reaction filtrates were acid hydro l yzed and subsequently analyzed for the possible presence o f w o o d sugars. An ion-exchange chromatography indicated that no neutral wood sugars were l iberated. Prev ious studies us ing ! H nuclear magnetic resonance spectroscopy indicated that C D H does select ively convert P-D-cellobiose to its ce l lob iono- l ,5 - lac tone derivative [128]. Our present study us ing H P L C methods conf i rmed these f indings, and demonstrated the rapid product ion o f cel lobionolactone. Prolonged incubat ion t imes resulted i n increased cel lobionolactone generation, w i t h the simultaneous generation o f smaller amounts o f 120 Table 6 Polysaccharides liberated (mg/mL) by cellobiose dehydrogenase (CDH, 0.1 IU/mL or 0.2 IU/mL) supplemented with 20 mM cellobiose and 0.2 mM FeCl 3 over time. Reaction Glucosea Arabinose a Cellobiosea Cellobionic Conditions Acid a 1 hr. 0.028 (0.001) 0.000 6.454 (0.011) 0.401 (0.009) 2hr. 0.041 (0.003) 0.000 6.194 (0.019) 0.532 (0.022) 6hr. 0.091 (0.004) 0.002 (0.001) 6.001 (0.023) 1.105 (0.019) 12 hr. 0.143 (0.008) 0.005 (0.001) 5.655 (0.011) 1.663 (0.031) 18 hr. 0.173 (0.003) 0.009 (0.001) 5.191 (0.017) 2.081 (0.028) 18 hr. (0.2 IU/mL) 0.273 (0.010) 0.018 (0.002) 3.108 (0.044) 3.995 (0.016) Values in brackets indicate standard deviation (n = 4). glucose. Similarly, the addition of CDH and cellobiose alone to the pulp without either hydrogen peroxide or iron generated substantial amounts of the cellobionolactone, indicating that the iron content of the pulp (67 ppm) or other transition metals such as cobalt or copper (4 or 228 ppm, respectively) was high enough for the putative reaction to proceed. Furthermore, treating the pulp with an EDTA chelation step effectively reduced the depolymerizing action of CDH. It is also possible that other reducible substrates, such as quinones are present. In the situations when the reaction mixture was supplemented with hydrogen peroxide, reduced amounts of lactone were generated. These results are contrary to those found by Henriksson et al. [124], who reported increased degradation of both xylan and cellulose when CDH was incubated in the presence of cellobiose, iron and hydrogen peroxide. However, our reaction conditions differed not in iron concentration but in source. These previous workers [124] used ferric cyanide, while we used ferric acetate. It has been shown that ferric acetate participates much more readily in Fenton's chemistry than does 121 ferric cyanide [336], ultimately generating hydroxyl radicals. These radicals have a high and indiscriminate reactivity and a very short half-life. It has been postulated that enzyme damage can occur when the Fenton's reaction takes place in close proximity to the enzyme [336]. This is likely the case when additional hydrogen peroxide was added to this reaction mixture, while in the reaction mixtures not supplemented with hydrogen peroxide this compound can readily be generated by the action of CDH and the supplemented iron acetate (see reaction scheme). The appearance of arabinose in the acid hydrolysates of the reaction filtrates was rather surprising, as no xylose was detected. Since the majority of the arabinose found in pulp is directly associated with the xylan backbone, and no xylose was liberated, this observation suggested that the arabinose did not result from the selective liberation of a neutral wood sugars from the pulp but was rather a degradation product of the supplemented cellobiose. This assumption was confirmed when arabinose was detected in reaction filtrates which mimicked the pulp treatments but lacked pulp as a substrate. Maximal arabinose was detected in samples which had been supplemented with hydrogen peroxide, suggesting that Fenton's chemistry was directly involved in arabinose generation. Previously it had been reported that the products of cellobiose degradation under Fenton's chemistry include both glucose and arabinose as well as other organic acids [147]. Thus, it is highly likely that the observed cellulose depolymerization was directly related to the action of hydroxyl radicals generated by this enzyme. The breakdown products of cellobiose were also observed when only cellobiose and CDH were added to pulp, suggesting that the concentration of transition metals within the pulp was enough to initiate Fenton's type reactions. However, this did not 122 occur to the extent that was exhibited when iron was added to the reaction mixture, and was reduced substantially when the pulps were first subject to a chelation step. This was further supported by the fact that CDH and cellobiose alone could depolymerize cellulose to a greater extent than was observed with just the addition of iron and hydrogen peroxide. 6.3 Conclusion Most of the past work on CDH has primarily investigated its action on lignin or lignin-related compounds, implying that this enzyme may have a primary role in lignin degradation. However, the fact that this enzyme contains a cellulose binding domain [239], is produced during primary metabolism with cellulose as a substrate [192], and demonstrates the ability to degrade both native cellulose and cellulose and xylan derivatives suggested that this enzyme may actually be more closely associated with the cellulases rather than the ligninases, while it functions as a dehydrogenase. Recent work by Ander et al. [11] supports this suggestion, as these workers found that the attack by CDH on pine holocellulose was more pronounced when supplemented with exoglucanases, compared to endoglucanases. As the natural iron content of the pulp (20-100 ppm) [166] seems to support enzyme activity and the generation of cellobiose occurs by the action of accompanying cellulases (exoglucanases), it is probable that all of the required co-factors will be present in the natural environment. Further evidence for its close association with the cellulases is that CDH has been found in non-ligninolytic fungi such as Monilia sp. [61], Sporotrichium thermophile [34], Chaetomium cellulolyticum [83] and in bacteria [173], which all possess a complete cellulase system and no ligninolytic enzymes. It is also known that cellobionolactone, which 123 is generated by CDH, can act as an inducer of cellulolytic enzymes while inhibiting B-glucosidases. By actively competing for the available cellobiose with the B-glucosidases, this may limit the actual amount of degradation that occurs naturally. It is worth noting that all the white-rot fungi and the single strain of brown-rot fungus (Coniophora puteana) from which CDH have been purified to date contain a complete cellulase system. Other brown-rot fungi, which are not members of the Coniophoraceae, do not possess a complete cellulase system, as they lack exoglucanases, and these fungi have yet to demonstrate the existence of CDH as part of their enzymatic machinery. Therefore, the generation of cellobiose by exoglucanases and the natural iron content in woody material complete the requirements for active CDH enzyme, which could then act in concert with the cellulolytic enzymes to degrade the carbohydrate moieties while also causing modifications to the lignin. In this first part of the work we were interested in assessing whether a "crude" cellulase preparation could improve fiber characteristics and determining whether purified enzymes such as CDH or xylanase could selectively modify characteristics such as DP or pore volume. At the same time we were also interested in knowing whether enzymatic treatments could be combined with mechanical treatments such as fractionation or refining to further improve pulp and paper properties. In the remaining chapters of the thesis, laboratory and industrial scale studies aimed at combining enzymatic and mechanical treatments are described. 124 Chapter 7 Improvements in the Paper Properties of Douglas-fir Kraft Pulp by Cellulase Treatments of Different Fiber Length Fractions 7.1 Background In the earlier chapters (chapters 3 & 4), commercial hydrolytic enzymes were assessed for their ability to beneficially enhance the fiber characteristics of chemical pulps derived from Douglas-fir. Its was clear from this work that, a cellulase preparation, possessing high xylanase activity, could significantly alter the fiber characteristic, while a relatively pure xylanase only resulted in marginal changes. As mentioned in the introduction, various theories have been proposed to explain the enzyme mechanism involved in enzymatic fiber modification, with the predominant suggestion being that the fibrils and fiber bundles are attacked on the surface, peeling off subsequent layers and eventually leading to disintegration of the fibers [37, 170]. If these enzymes could selectively act on the fiber walls, by attacking the S2 layer, the lumen or both simultaneously, it is possible that the stiff, inflexible nature of the coarser fibers could be modified to enhance collapsibility and inter-fiber bonding. It was apparent from our earlier work (chapter 3) that, although it was possible to enhance some paper properties, it was often obtained at the expense of strength loss [277]. Furthermore, our work and the work of others [135], indicated that cellulase treatments of pulps often resulted in the preferential hydrolysis of the fines. These observed reductions in paper strength are thought to be due to the collective effects of decreased intrinsic fiber 125 strength as well as fiber defibrillation and fines hydrolysis (chapter 5). It was also apparent from our earlier work that enzyme treatment of the individual fiber length fractions and the unfractionated pulp increased the handsheet densities, while the handsheet roughness was decreased or remained relatively unchanged. However, after cellulase treatments, all of the fiber length fractions (14R, 28R and 50R) showed significant reductions in both zero-span breaking length and burst index. It was expected that the strength reductions of the longer fiber length fractions would be similar to those observed with the original pulp, as these fraction (14R and 28R) accounted for over 75 % of the unfractionated kraft pulp (Table 1). However, it was found that, after cellulase treatment the tensile index for the unfractionated kraft pulp decreased, while the three longer fiber length fractions showed significant improvements. This increase combined with the similar increase in handsheet density suggested that the fibers had collapsed and flattened. It could be anticipated that flatter fibers would exhibit a greater surface area available for bonding and that this enhanced inter-fiber contact would increase the tensile index. With the heat inactivated enzyme control pulps the tensile index for the unfractionated pulp was slightly greater than that demonstrated by the 50R fiber length fraction and substantially greater than the 14R fiber length fraction. However, approximately 55 % of the unfractionated pulp was contained in the 14R fraction. This confirmed previous observations regarding the importance of the smaller fiber length fractions in the formation of well-bonded, strong paper products [265]. Therefore, it is possible that the unfractionated pulp did not exhibit a similar increase in the tensile index because the finer, thinner walled fibers remaining in the unfractionated pulp were preferentially hydrolyzed by the cellulase enzymes. As a result, the 126 positive effect of fiber flattening of the longer, coarser fibers was overshadowed by fiber defibrillation and the removal of the fines by enzymatic hydrolysis. One possible way of circumventing the reduction in paper strength observed during the application of cellulases to pulp fiber during paper manufacture is to physically separate out the finer, thinner walled fibers, which seem to be more susceptible to enzymatic degradation, from the coarser fibers. This can be accomplished by the use of existing fractionation technology, followed by the selective treatment of the coarser fibers which are then finally recombined with the finer fiber fractions. In the work reported in this chapter we investigated the potential of selectively treating specific fiber length fractions with a commercial cellulase mixture, recombining the treated fiber length fractions with untreated fiber length fractions in an attempt to improve the quality of the paper made from a Douglas-fir feedstock. 7.2 Results and Discussion 7.2.1 Selective Treatments and Reconstitution In an attempt to ascertain if the damage endured by the finer fibers overshadows the enzymatically improved fiber characteristics of the longer, coarser fibers, a Douglas-fir kraft pulp was fractionated in a fiber-length classifier. This confirmed earlier work which showed that the majority of the pulp was composed of the 14R and 28R fiber length fractions (>76 %) (Table 1). After fractionation the longer fiber length fractions (14R alone and 14R & 28R combined) were treated at various enzyme loadings with a commercial cellulase. The treated fibers were then reconstituted with the other fiber length fractions in the same 127 proportions as were originally determined by Bauer-McNett fractionation. However, the fines (that material not retained by 200R mesh) were excluded from the reconstitution. Substantial improvements in handsheet density (Figure 21 A) were achieved and it was apparent that the observed improvements were again dose dependent, with increasing enzyme loadings resulting in a corresponding reduction in handsheet roughness (Figure 2IB). It is probable that the cellulase enzymes attacked and weakened the pulp fibers so that the physical pressures applied during handsheet formation resulted in their greater collapse. This would also explain the observed reduction in handsheet roughness as the fibers flattened to form a smoother surface. Although there did not appear to be any advantage to treating either the 14R fraction alone, or the combined 14R & 28R fractions when handsheet density or roughness were compared, clear differences were apparent when the strength properties of the resultant paper were evaluated (Figure 22). The wet zero-span values indicated that enzyme treatments resulted in a significant reduction in fiber strength even at relatively low cellulase loadings (Figure 22A), with increasing dosages compromising the fiber strength proportionally. The tear index (Figure 22B), which is directly influence by the intrinsic fiber strength of pulp fibers, was also reduced, again demonstrating a dose dependent trend. It is likely that the reduction in tear strength is a function of reduced fiber strength and an enhancement in inter-fiber bonding, both of which can adversely affect the tear index [267, 268]. It was evident that the independent treatment of the 14R fraction from the 28R fraction did not result in as great a loss in strength, suggesting that the inclusion of fibers of shorter fiber length, which generally possess thinner cell walls, may be more susceptible to enzyme mediated damage. 128/ Figure 21 Average change in handsheet (A) density and (B) roughness of cellulase treated 14R and 14R & 28R fiber length fractions of Douglas-fir kraft pulp reconstituted with all other fiber length fractions in the original proportions (error bars indicate standard deviation, n = 3) 129 E 12 10 V zL_ f H 14R&28R Id p i 1 1 1 1 1 12 E E 10 2L2 B 4^ 2.5 E cd G L 2.0 1.5 c o O O o O o co O :> o C 1 p ; £| 1 1 i 1 1 A 26 o o 2 4 V E 22 20 18 Id c: o O i f ] 22 i £ 1 2 o o CM O o co O o 1 £1 o o LO Figure 22 Average change in (A) zero-span breaking length, (B) tear index, (C) burst index and (D) tensile index of cellulase treated 14R and 14R & 28R fiber length fractions of Douglas-fir kraft pulp reconstituted with all other fiber length fractions in the original proportions (error bars indicate standard deviation, n = 3) 130 Analysis of the reaction filtrates (Table 7) supported this hypothesis, as the enzymatic treatments of the combined 14R & 28R fractions resulted in a greater degree of hydrolysis when compared to the treatments of the 14R fraction alone. This inferred that shorter, finer fibers (28R fiber length fraction) in the reaction treatments are hydrolyzed to a greater extent than when the reaction treatments include only the 14R fiber length fraction. Our previous work showed that, at similar enzyme loadings, the smaller the fiber length fractions treated with a cellulase preparation, the greater the degree of hydrolysis (chapter 3). Table 7 Hydrolysis and yield loss resulting from different cellulase treatments of the specific fiber length fractions of Douglas-fir kraft pulp separated by Bauer-McNett fractionation. 1 CMC a 2CMC a 3 CMC a 4 CMC a 5 CMC a 14R % Hydrolysis* 1.12 (.03) 1.23 (.09) 1.59 (.10) 2.01 (.09) 2.43 (.11) Yield Loss (%)** 0.62 " 0.68 0.88 1.11 1.34 14R& 28R % Hydrolysis* 1.22 (.05) 1.74 (.08) 2.12 (.06) 2.49 (.12) 2.92 (.13) Yield Loss (%)** a -. r i • , . . . . 0.93 1.33 1.62 1.91 2.23 Values in brackets indicate standard deviation (n = 4). * Percentage of specified fiber which has been hydrolyzed by the various enzyme treatments. **Percentage of fiber feedstock loss as a result of the specified treatment. Although both the wet zero-span and the tear index were adversely affected by the enzymatic treatments of the longer fiber length fractions, other paper parameters such as the burst and tensile indexes (Figures 22C,D) were improved substantially. These two paper properties were positively influenced by increasing enzyme loading, until a maximum was achieved (4 CMC units). At higher enzyme loadings a decrease in strength was observed. However, at these upper loadings, these strength properties where still significantly greater than those obtained with the heat inactivated controls. It was again apparent that, by treating the 14R fraction alone, it was possible to achieve substantially greater improvements than 131 when treating the combination of the 14R and 28R fractions. It was also clear that the maximal enzyme dosage that could be used before the enhanced strength properties were compromised was much lower when the treatments included the 28R fraction fibers (2 CMC vs. 4 CMC units of enzyme). This suggested that the 28R fiber length fraction of the pulp was much more susceptible to damage than was the longer 14R fiber length fraction. As a result, the beneficial improvements in fiber collapsibility/flexibility made possible by enzyme treatment of the 14R fractions were eclipsed when the 28R fiber length fraction was included. As a consequence, the enzyme charge required to make the largest modification in the most "undesirable" fraction (14R) resulted in detrimental changes to the other fiber length fractions. 7.2.2 Selective Treatments, Reconstitution and Refining Having established that it was possible to achieve substantial improvements in paper properties by selectively treating the longer fiber length fractions and reconstituting them back to the remaining fiber length fractions in the original proportions, we wanted to see how these fibers would then respond to a refining stage. It was clear from our initial investigations that the inclusion of the 28R fraction in the treatments did not yield as great an improvement as did the treatment of the 14R fiber length fractions alone. Therefore, to investigate the response of the treated fiber to refining, only the 14R fibers were enzymatically treated, reconstituted with the fiber length fractions, and then all the fibers were subject to various degrees of refining. The fibers were again treated at a range of 132 enzyme loadings in order to determine the optimal enzyme charge required to obtain improvements in combination with a refining stage. The sheet properties that were obtained were compared on the basis of a similar level of beating so that an equivalent comparison could be made. Standard handsheet properties were determined and analyzed to identify gross differences between pulp furnishes consisting of enzymatically treated 14R fraction fibers and those that were subject to control treatments (Figure 23). The handsheet densities of the pulps containing enzymatically treated 14R fibers were all greater than their corresponding controls. It was clear that the pulps containing enzymatically treated fibers formed denser handsheets at all levels of beating (Figure 23 A). The extent to which improvements in sheet density occur were a function of enzyme charge, with the larger enzyme loadings resulting in increased sheet densities. The increased densities also corresponded to the observed reductions in handsheet roughness, with the sheets which demonstrated the largest improvements in density also exhibiting the most pronounced reductions in sheet roughness (Figure 23B). The freeness values of the unbeaten pulps were relatively unchanged. However, beating resulted in a decrease in freeness (Figure 23C). Freeness reductions were slightly quicker for those pulps which contained enzyme treated fibers, up to 3000 PFI revolutions. The extent to which the freeness dropped was again a function of the enzyme charge. The changes in freeness of the enzymatically treated fibers were more extensive for the 6000 revolution refining stage. While it is possible to conclude that the enzyme treatments enhance fiber collapsibility and flexibility, as shown by the improvements in sheet density of the unbeaten fibers, the observed changes in pulp 0.65 133 3280 3240 3200 O 3160 O CO 3120 3080 3040 3000 800 3000 6000 PFI Revolutions Figure 23 Average change in handsheet (A) density, (B) roughness and (C) pulp freeness of cellulase treated 14R fiber length fraction of Douglas-fir kraft pulp reconstituted with all other fiber length fractions in the original proportions followed by various degrees of refining (error bars indicate standard deviation, n = 3) 134 freeness of the beaten fibers undoubtedly contributed to the corresponding improvements in some of the paper properties. The alterations in fiber strength, as indicated by wet zero-span measurements, demonstrated that all enzymatic treatments had reduced the intrinsic fiber strength in a dose dependent fashion. Regardless of the degree of beating, those pulps which contained enzymatically treated fibers showed reduced fiber strength (Figure 24A) and tear index (Figure 24B). However, the greater degree of inter-fiber bonding obtained with the pulps containing enzymatically treated fibers was reflected in the burst and tensile indexes (Figure 24C,D). The burst index indicated that at 0 and 1000 revolutions all treatments were greater than the corresponding controls, with the 1 CMC treatment being optimal (24 % improvement). However, at 3000 revolutions only the 1 CMC treatment was slightly greater (3 %) than the control, with further refining resulting in a reduction in the burst index. A similar trend was shown for in the tensile index (Figure 24D), where again at 1000 revolutions the 1 CMC treatment resulted in the greatest improvement (17 % ) , while maintaining an approximately 5 % increase in tensile strength at 3000 revolutions than did the control. However, as seen with the burst index, refining past 3000 revolutions resulted in decreased strength when compared to the corresponding controls. To achieve significant improvements in paper properties an enzyme charge of at least 4 CMC units per gram oven dried fiber was required for unrefined fibers. At this enzyme loading approximately 2 % of the 14R fiber length fraction was hydrolyzed by the enzyme, which is equivalent to an approximate 1.1 % loss in yield. In contrast, the enzyme loading required to obtain optimal improvement in the paper quality derived from the Douglas-fir feedstock, when a 135 PFI Revolutions PFI Revolutions Figure 24 Average change in (A) zero-span breaking length, (B) tear index, (C) burst index and (D) tensile index of cellulase treated 14R fiber length fraction of Douglas-fir kraft pulp reconstituted with all other fiber length fractions in the original proportions followed by various degrees of refining (error bars indicate standard deviation, n = 3) 136 refining stage is included is much less (1 CMC units). At this enzyme loading, approximately 1.1 % of the 14R fraction is hydrolyzed, this inturn translates into only a 0.6 % loss in total feedstock (Table 7). 7.2.3 Selective Treatments, Refining and Reconstitution The selective treatment of the longer fiber length fraction followed by reconstitution of the pulp in their original proportions, generated significant improvements in the surface and strength properties of the resultant paper, both with and without subsequent refining of the reconstituted pulp. It was apparent that the enzyme dose required for the treatment of the 14R fractions, when it was combined with refining, was far less than that required to achieve improvements in paper properties without refining. At an enzyme dose of 1 CMC unit per gram of dry fiber, beneficial changes in density, smoothness, burst and tensile were achieved at the expense of intrinsic fiber strength. Having determined the optimal charge for improvements when subsequent refining was included, we next wanted to ascertain whether similar improvements could be obtained by enzymatically treating the 14R fraction, refining it alone, and then reconstituting it back in the original proportion with the other unrefined fiber length fractions (Figure 25). Following this approach the pulp properties where compare against the controls at a single enzyme charge of 1 CMC unit/g pulp, which proved to be optimal using the earlier approach. It was apparent that handsheet density (Figure 25A) increased while handsheet roughness (Figure 25B) and pulp freeness (Figure 25C) decreased. However, the increases in the sheet densities were not as significant as was obtained when all of the pulp fractions where refined. 137 Correspondingly, the decrease in freeness was not as significant as when all of the fractions were refined, indicating that not as many fines were generated by this approach. This factor alone had a major influence on the sheet densities, and could be explained by the fact that approximately 45 % of the pulp was not refined. The change in fiber strength, as indicated by wet zero-span, indicated that the enzyme treatment of the individual fraction had reduced the intrinsic fiber strength of the combined pulp to the same degree as was obtained when the whole pulp had been refined (Figure 26A). This was expected, as in both cases, it is only the 14R fraction which is subject to the enzymatic treatments. Tear index (Figure 26B) demonstrated similar trends and the absolute values of this index were again comparable to the previous approach, when all of the pulp feedstock was refined. As was found previously, the burst (Figure 26C) and tensile (Figure 26D) indexes both demonstrated improvements over the corresponding controls. However, the improvements were not as significant with both trends demonstrating maximal improvements of approximately 7 % over the controls at 1000 PFI revolutions of refining. Using the former approach at 1000 PFI revolutions, improvements as great as 24 % and 17 % were attained in burst and tensile indexes, respectively. It is clear that the improvements obtained by the selective treatment of the longer fiber length fractions of a pulp, combined with refining stages, were greater when the entire pulp was refined, as compared to refining only the enzymatically treated longer fraction. The reasons for the difference in degree of enhanced paper quality between these two different, but related approaches are multifaceted. In both cases the enzymatic treatments of the 14R fraction results in modifications to these fibers, which enhances fiber 138 E o 0.55 0.50 0.45 0.40 3300 3200 3100 O 3000 O CO 2900 2800 •— Control •— 1 CMC 2700 I—i B 750 700 650 600 550 500 3000 6000 PFI Revolutions Figure 25 Average change in handsheet (A) density, (B) roughness and (C) pulp freeness of cellulase treated 14R fiber length fraction of Douglas-fir kraft pulp subject to various degrees of refining, then reconstituted with all other fiber length fractions in original proportions (error bars indicate standard deviation, n = 3) 139 50 40 TO E 30 20 3000 PFI Revolutions o — Control 1 CMC 3000 PFI Revolutions 6000 Figure 26 Average change in (A) zero-span breaking length, (B) tear index, (C) burst index and (D) tensile index of cellulase treated 14R fiber length fraction of Douglas-fir kraft pulp subject to various degrees of refining, then reconstituted with all other fiber length fractions in original proportions (error bars indicate standard deviation, n = 3) 140 collapsibility/flexibility. However, during the refining of all fractions of the pulp, the degree of fibrillation and the generation of fines is greatly elevated. As a result there is a significant improvement in the relative bonded area and the amount of filler. Therefore, both the strength and physical properties of the paper are improved. 7.3 Conclusion Our previous work using cellulase had indicated that, although some beneficial improvements in paper properties could be obtained, this was usually achieved at the expense of a decrease in both paper and fiber strength, as well as a loss in yield. It was apparent that these detrimental effects could be curtailed by physically separating the fiber and then selectively treating individual fiber length fractions with cellulases. Although the strength properties were generally improved, the tear index and wet zero-span values were compromised. However, these losses were not detrimental to the quality of the other paper properties. By using this combined physical/enzymatic approach strength improvements as large as 35 % in burst index and 25 % in tensile index could be obtained. Significant improvements in sheet density and smoothness were also obtained without the need for subsequent refining. These substantial improvements in paper properties could also be maintained when the enzymatic treatments were followed by refining, with the additional advantage of lower amounts of enzyme being required. Thus, fiber fractionation, followed by selective cellulase treatments of the longer fibers can improve paper properties of unrefined pulps, improve the reinforcing strength of refined fibers when compared to the traditional pulping methods at set degree of refining, or reduce the amount of refining 141 required to achieve a specified strength parameter. Although these improvements were achieved at the expense of fiber strength, this trade-off may be required to obtain beneficial gains in other pulp characteristics. Therefore, it is possible that the key to enzymatic fiber modification is achieving a compromise between the degradation of individual fibers and the enhanced fiber flexibility/co 11 apsibi 1 ity gained through these treatments, resulting in enhanced inter-fiber bonding. This compromise has a sensitive balance point, which is affected both by the enzyme loading and the nature of the pulp which is being treated. To further pursue this possible application of the combined enzymatic and physical fractionation/refining of Douglas-fir derived kraft pulp we next assessed the potential of this approach using industrial scale fractionation. 142 Chapter 8 Enhancement of Pulp Properties by Industrial Fractionation of Kraft Pulp Followed by Cellulases Treatments 8.1 Background As mentioned earlier, fiber fractionation generally involves the mechanical separation of certain classes of fibers from a mixture to produce at least two fractions containing fibers with different properties. One stream, the "finer fiber fraction", contains the shorter, smaller fibers. While the other stream, the "long fiber fraction", consists of longer, coarser fibers, bundles and splinters [168]. Routine use of fiber fractionation technology has traditionally been employed in conjunction with the mechanical pulping processes to improve the quality of printing papers. Progress in this field has been primarily driven by the continued demand for high quality mechanical pulps with superior surface properties required for high grade printing papers [313]. During fiber fractionation, the longer fiber length fractions are separated and subjected to additional refining. This significantly enhances the quality of the resulting paper products. Fractionation technologies are also being employed or considered in a number of other pulping applications, including the processing of secondary fibers where fractionation can separate the secondary fiber feed to produce several grades of paper [262]. Similarly, fiber fractionation is routinely used in the production of linerboard and in the processing of corrugated containers and paperboards [26, 44, 111, 263]. Other applications which have been investigated, include the use of the fractionation for the production of grocery bags from kraft pulp [63] and the separation of mixed office waste and printing and writing 143 papers [1, 5, 82, 91, 92, 109, 168, 194, 217]. In the previous chapter we demonstrated that, by using a combined mechanical separation/enzymatic approach, the strength properties of unrefined Douglas-fir sheets were significantly improved, compared to their corresponding controls (chapter 7). These improvements were also maintained when these pulp fibers were subject to refining. Having established that laboratory fractionation, via a Bauer-McNett fiber length classifier, followed by cellulase treatment of the longer fiber length fraction could enhance the quality of paper derived from a Douglas-fir kraft pulp, we wanted to determine if similar improvements could be achieved using industrial fractionation of a market pulp. 8.2 Results and Discussion In the previous chapter (chapter 7) we demonstrated that it is possible to improve the quality of paper generated from Douglas-fir kraft pulp by selectively treating the longer fiber length fractions of the pulp with a commercial cellulase. This process made use of the Bauer-McNett fiber length classifier to separate out the different fibers length fractions, selectively treating the 14R fraction, and then recombining the treated 14R fraction with the other fiber length fractions in their original proportions. Although this process revealed some significant improvements in the resulting paper, the Bauer-McNett separation process is a relatively "clean" separation in comparison to what is usually achieved industrially. Therefore, we wanted to determine if industrial fraction followed by the selective treatment of the reject (longer fiber) fraction could accomplish similar beneficial improvements as was seen with laboratory refining. 144 8.2.1 Fiber Separation The original feed pulp, a dried, fully bleached high-fir kraft pulp, was subject to a single pass through a small industrial pressure screen in order to obtain two streams of pulp, the accepts (smaller fiber lengths) and the rejects (longer fiber lengths). The accept stream contained fewer of the long fibers found in the feed pulp and more shorter fibers and fines, while the reject stream was enriched with long fibers (Figure 27A). While this distribution plot clearly indicated that the fractionation process had enriched the reject stream with longer fibers, it appeared that a significant proportion of fibers of smaller length (0-1 mm) still remained in this pulp stream. However, when the ensuing fiber length fractions of these same streams were compared by Bauer-McNett (Figure 27B), it was clear that the fractionation process had enriched the reject steam with longer fibers (containing approximately 82 % 14R fraction), while it substantially reduced the proportions of finer fibers in this stream. The opposite was true for the accept stream. 8.2.2 Cellulase Treatment of the Rejects In the previous investigation we had treated the 14R fraction of a brownstock Douglas-fir pulp at a variety of enzyme loadings to establish the optimal dosage needed to achieve an improvement in the unrefined paper properties. These same loadings could not be applied in this scenario, primarily because of differences in the feedstock that was used. The feedstock used in the previous investigation was a 100 % Douglas-fir kraft pulp, while the feedstock used for the industrial fractionation was a high-fir (85 % Douglas-fir) market pulp. In the laboratory based fractionation study (chapter 7) the pulp was unbleached, while 145 1-2 2-3 3-4 4-5 Fiber Length (mm) >5 ] Feed Pulp Accepts f Z 3 Rejects B 14R 28R 50R 100R 200R Fines Fiber Length Distribution Figure 27 Effects of industrial fractionation on the (A) fiber length population and (B) fiber lengths distribution of a high-frr market kraft pulp 146 in this case the pulp was a fully bleached pulp. It is well recognized that bleached pulps react differently to unbleached pulps when treated with hydrolytic enzymes [46, 157]. Another factor which probably affected the efficiency of the enzymatic treatment is that the market pulp had been dried, while the brownstock used in the previous study was a never-dried pulp. Drying has been shown to affect the pore-size distribution of fibers which alters the interaction of the cellulase enzymes with the substrate [276], limiting the accessibility of the enzyme to the fibers [21, 85, 103, 198]. In work carried out in the previous chapter it was clear that, although beneficial modification did occur when treating a combination of the 14R & 28R fractions, the improvements were not as substantial and the maximal improvements were attained at lower enzyme loadings, than those obtained when treating the 14R fiber length fraction alone. To try to resolve some of these concerns, we subjected the reject stream to a variety of different enzyme loadings, recombined the treated reject fibers with the accept fibers in their original ratios and compared the resulting handsheet properties to control (similar conditions with heat inactivated enzyme) treatments. In addition, handsheets made from the original pulp (feed pulp) which had not been fractionated or enzyme treated, were used to determine any changes in sheet properties which resulted from just the mechanical processing (separation) of the fibers. From the analysis of the non-destructive handsheet properties it was clear that substantial improvements in handsheet density (Figure 28A) could be achieved and that the observed improvements were dose dependent, similar to the results obtained with the brownstock Douglas-fir pulp. It was also apparent that the separation of the fiber by fractionation followed by their recombination had slightly altered (reduced) the density of 147 0.51 0.50 0.49 E o 0.48 0.47 0.46 0.45 3 2 5 0 3 2 2 5 O O CO 3 2 0 0 3 1 7 5 B CD Q) LL o O O o LO d O O o o CM o o co Figure 28 Average change in handsheet (A) density and (B) roughness of cellulase treated rejects fiber fraction followed by recombination with accepts fiber fractions (error bars indicate standard deviation, n - 3) 148 the sheets. However, the application of cellulase to the reject fiber, followed by recombination resulted in handsheets with significantly elevated densities over both the control and original pulp handsheets. The smoothness measurements of the handsheets (Figure 28B) indicated that improvements in this paper property were also attained by enzyme treatments, and that by increasing the enzyme loading, corresponding reductions in roughness were evident. In order to assess what degree of fiber degradation had occurred as a result of the cellulase treatments, the intrinsic fiber strength was determined by wet zero-span measurements (Figure 29A). These measurements demonstrated that significant reductions in fiber strength had occurred even at low enzyme loadings (1 CMC) and that by increasing the enzyme concentration even greater reductions were obtained. The tear index, which is directly influenced by intrinsic fiber strength [264, 265, 267, 270], demonstrated a similar trend (Figure 29B) to what was observed with the wet zero-span measurements. In contrast to both fiber strength and tear index, which showed reductions when the reject fibers were cellulase treated, both burst and tensile indexes (Figure 29C,D) were improved by these same treatments. Maximal improvements in both burst and tensile indexes were achieved at 2 CMC units enzyme loading, resulting in a 15 % and 14 % increase respectively, over the heat inactivated controls. These improvements were not as substantial as were the improvements obtained previously with the treatment of the 14R fraction of the Douglas-fir brownstock pulp (chapter 7). However, they do mimic the enhancements in paper properties obtained when treating the combined 14R & 28R fractions. Similarly, the greatest improvements obtained in the present study were achieved at the same level of enzyme loading while the 149 12 ^ 10 16 Z E 12 B 3.00 2.75 2.50 2.25 T3 CD CD L L C o O O o LO d O O o o CM o o co 4 0 38 E Z 36 3 4 32 T3 CD CD UL •J^  : c o O O o LO d O O o o CM O o co Figure 29 index and Average change in (A) zero-span breaking length, ( B ) tear index, ( C ) burst ( D ) tensile index of cellulase treated rejects fiber fraction followed by recombination with accepts fiber fractions (error bars indicated standard deviation, n = 3) 150 same trend in property enhancement over the range of enzyme treatments was also observed. 8.2.3 Cellulase Treatment of the Rejects Followed by Refining Having established that significant beneficial modifications in paper properties could be obtained by treating the reject stream of an industrially fractionated feedstock, we wanted to investigate what response these same fibers would have when subject to refining. In order to determined the effects of refining, the reject stream was exposed to range of enzyme loadings, recombined with the accept stream and then subjected to 1000, 2000 and 3000 revolutions on a PFI laboratory refiner. For the refming experiments cellulase loadings of 0.25, 0.5 and 0.75 CMC units per gram of over dried fiber were applied to the pulp (rejects). This resulted in approximately 0.42, 0.56 and 0.77 % hydrolysis of the fiber respectively (Table 8), which was equivalent to a yield loss of approximately 0.17, 0.22 and 0.3 % respectively of feedstock for papermaking. Examination of the handsheet properties over this refining curve indicated that, at these enzyme loadings, improvements in both sheet densities and smoothness were attained (Figure 30A,B). An increased in enzyme charge resulted in increases in sheet density while, with these same sheets, the roughness was reduced. In both cases the extent of the improvements were dose dependent. Although it is well known that the refining of pulp results in reduced freeness, the application of cellulase enzyme to the fibers reduced the freeness of the pulp even further (Figure 30C) when compared to the corresponding controls. Again, the extent to which the freeness of the pulp was altered was directly related to the enzyme dosage applied to the pulp fibers. 151 E o 0.60 • A 0.58 0.56 0.54 0.52 // —•— Control 0.50 // — • — 0.25 CMC // — A — 0 . 5 CMC 0.48 // — r — 0.75 CMC 0.46 i i 1 i 3 2 5 0 3 2 0 0 g 3 1 5 0 CO 3 1 0 0 3 0 5 0 6 8 0 640 V 600 560 520 480 1000 2 0 0 0 PFI Revolutions 3 0 0 0 Figure 30 Average change in handsheet (A) density, (B) roughness and (C) pulp freeness of cellulase treated rejects fiber fraction followed by recombination with the accepts fiber fractions and then subject to various degrees of refining (error bars indicate standard deviation, n = 3) 152 Table 8 Hydrolysis and yield losses of cellulase treated reject fibers collected by the industrial fractionation of a high-fir market kraft pulp 0.25 0.5 0.75 1 2 3 4 CMC a CMC a CMC a CMC a CMC a CMC a CMC a % Hydrolysis of Rejects* 0.42 0.56 0.77 0.87 1.31 1.71 1.83 (.04) (.08) (.06) (.07) (.07) (.06) (.02) Yield Loss (%)** 0.17 0.22 0.30 0.34 0.52 0.68 0.73 a Values in brackets indicate standard deviation (n = 4). * Percentage of specified fiber which has been hydrolyzed by the various enzyme treatments. **Percentage of fiber feedstock loss as a result of the specified treatment. It was clear from the values obtained with the unrefined fibers that cellulase treatment could cause significant alteration to the intrinsic fiber strength, even at low enzyme loadings (Figure 28A). In order to assess the effects of refining, enzyme charges were even further reduced (Figure 31 A). At these loadings it was apparent the fiber strength was also substantially compromised and that the refining action improved the strength of the control fiber slightly more at each level of refining, as did it with each of the different sets of enzymatically treated fibers. However, the losses in strength endured by the fibers as a result of enzyme treatments could not be recovered by refining. As was previously found with the unrefined fibers, which had shown a reduction in tear index, the refined fibers also demonstrated a reduction (Figure 3 IB), which again correlated well with the observed reductions in intrinsic fiber strength. Although the greatest improvements in both sheet density and smoothness were obtained with an enzyme charge of 0.75 CMC, at this loading all the strength parameters of the paper were compromised, including both the burst index (Figure 31C) and tensile index (Figure 3 ID). However, the 0.25 CMC unit charge slightly improved the burst strength 153 13.5 E 3 0 0 0 1000 2000 PFI Revolutions 80 70 CD g 60 5 0 40 3 0 0 0 3 0 — a — Control — 0 . 2 5 CMC 0.5 CMC — T — 0.75 CMC 1000 2 0 0 0 PFI Revolutions 3000 Figure 31 Average change in (A) zero-span breaking length, (B) tear index, (C) burst index and (D) tensile index of cellulase treated rejects fiber fraction followed by recombination with the accepts fiber fractions and then subject to various degrees of refining (error bars indicate standard deviation, n = 3) 154 (Figure 31C) of the paper, with the greatest improvement (3 %) over the corresponding control occurring after 2000 revolutions of refining. The tensile strength (Figure 3 ID) was only negatively affected by the 0.75 CMC unit enzyme charge, after 2000 revolutions refining, at which point paper derived from the enzyme treated fibers was weaker than the control paper. At the lower enzyme loadings, a significant increase in the tensile strength of the paper was obtained, with the largest improvement (10 %) occurring at an enzyme loading of 0.5 CMC units and 1000 revolutions refining. This enzyme charge maintained improved tensile strength over the control pulp throughout entire refining curve, demonstrating an approximately 4 % strength improvement after 3000 revolutions of refining. It is fair to conclude that the enzyme treatments of the longer, coarser fraction (reject fibers) enhance fiber collapsibility and flexibility as evidenced by the improvements in the sheet density of the unbeaten fibers. Increases in density implies that these treatments increased the relative bonded area within the sheets and, as a result, improved the general strength of the paper. The exception is tear strength, which is adversely affected by increases in relative bonded area. Unfortunately these improvements were obtained at the expense of fiber strength, which again adversely affected the tear index [264, 265, 270], These beneficial modifications were maintained when the treated fibers were subsequently subject to refining stages. However, at a much reduced enzyme charge. While enhanced fiber collapsibility/flexibility can be used to explain the improved paper properties of the unrefined fibers, the observed reductions in the pulp freeness of the refined fibers undoubtedly contributed to the corresponding improvements in some of the paper properties. In the previous chapter we had shown that the detrimental effects that cellulase 155 treatments had on paper strength properties could be curtailed by first physically separating the fiber and then selectively treating a specific fiber length fraction. It was also concluded that treating the 14R fiber length fractions alone, generated substantially greater improvements in paper properties than did the enzymatic treatment of multiple fiber length fractions. In this study we assessed the potential of this approach using industrial fractionation technology. It was apparent that enhanced paper properties could still be achieved using this approach, although not to the same extent that was obtained when using laboratory fiber separation technology. However, the results obtained using the industrial fractionation process were very similar to those obtained when treating the combined 14R & 28R fractions of the Douglas-fir brownstock pulp, which generated improvements in paper properties, but not to the same extent as when only the 14R fraction was treated alone. While the industrial fractionation processes substantially enriched the reject stream with longer, coarser fibers, it by no means generated a homogenous stream of pulp fibers. This would suggest that the bottleneck in this application would be the efficiency of the fractionation process, as this maybe the key to successfully achieving far superior paper products via cellulase treatments. Therefore, improvements in the efficiency of the separation process should also positively enhance the degree of modification in the resulting paper products. The desired efficiencies in fractionation may be achievable by subjecting the reject stream to a second pass through the pressure screens, including a second screen of different hole diameter in series during the fractionation step, or by using a different screen type. It is also recognized that different feedstocks may require different fractionation technologies. 156 8.3 Conclusion A l t h o u g h some shortcomings exist when us ing industr ia l f ract ionat ion, improvements by as m u c h as a 10 % increase in tensile strength, cou ld be achieved w h e n us ing the comb ined mechan ica l separat ion/enzymatic approach. T h e u t i l i za t ion o f this technology offers several benef i ts, such as the improvements i n re in fo rc ing strength that are attainable or the reduced re f in ing energy required to attain a set strength (tensile) parameter. The integrat ion o f an enzyme treatment stage w i th re f in ing m a y also prov ide a means o f incorporat ing feedstocks such as Doug las- f i r , that have t rad i t ional ly been considered less than desirable for papermak ing (because o f their innate h igh coarseness), into rout ine paper manufacture. O u r in i t ia l invest igat ion w i th cellulases (chapter 3) c lear ly demonstrated that cel lu lase treatments cou ld benef ic ia l l y improve some o f the resultant paper propert ies, such as sheet density and smoothness. A l t hough this was achieved at the expense o f both strength and y ie ld losses, the ind iv idua l f iber length fractions o f Doug las - f i r kraft pulp demonstrated improvements in tensi le strength. Th is latter result p rompted an invest igat ion w h i c h comb ined the use o f enzymat ic and fract ionation technologies to spec i f i ca l l y target the longer, coarser f ibers (chapter 7). Th is approach resulted in s ign i f icant improvements i n the strength o f paper der ived f rom Doug las - f i r kraft pu lp, as w e l l as reduc ing both y i e l d losses and the amount o f enzyme required for enzymat ic treatments. W h e n this appl icat ions was tested us ing industr ia l f ract ionat ion technologies and market pulp f ibers, s ign i f icant improvements in paper properties, inc lud ing tensile strength, were achieved. 157 General Conclusions and Future Work Conclusions The overall goal of the proposed work was to investigate the potential of using fungal enzymes to modify the structure of pulp fibers. The working hypothesis was that, by using the selectivity of the various hydrolytic enzymes produced by wood-degrading fungi, it may be possible to obtain beneficial changes to the physical characteristics of pulp, such as reduced fiber coarseness. At the same time, it was hoped that the research would also provide some unique insights into how individual cellulases and hemicellulases modify pulp fibers while enhancing our understanding of the roles that various fiber characteristics contribute to paper properties. It was clear that enzymatic treatments could be used to alter the physical characteristics of Douglas-fir pulps, such as fiber coarseness, while improving in the density and smoothness of kraft pulp handsheets. The preliminary work clearly demonstrated that the selectivity of enzymatic applications could indeed alter the fiber characteristics of Douglas-fir pulps, resulting in some beneficial changes in paper properties. However, the beneficial modifications were accompanied by both reduced paper strength and yield losses. This initial study used a commercial cellulase, which contained high levels of both cellulase and xylanase activity. In an attempt to circumvent the detrimental effects resulting from the cellulase treatments, the selectivity of other carbohydrate degrading enzymes such as xylanases and cellobiose dehydrogenase were assessed for their ability to modify kraft pulp characteristics. 158 In contrast to the cellulase treatments, which seemed to act in a similar fashion on all the different fiber length fractions, the xylanase treatments seem to be slightly influenced by compositional differences in the fibers. The physical properties of handsheets derived from individual fiber length fractions generally respond in a similar manner when subjected to xylanase prebleaching. However, the modifications were only minor, even at relatively high xylanase loadings. These data suggest that, although xylanases do contribute to modifications in fiber properties, the substantial alterations that resulted during the application of a commercial cellulase, which contains both high cellulase and xylanase activities, are primarily due to the cellulase activity. Further work which looked at the ultrastructural and macroscopic fibrillar changes within the fibers implied that, the decreased fiber strength which often resulted from enzymatic treatments was related to modifications on the surface of the fibers. It appeared that the compromised paper strength was a result of a reduction in intrinsic fiber strength and the removal of hemicellulose from the fiber, in combination with fines removal and fiber defibrillation. Cellobiose dehydrogenase clearly demonstrated the ability to modify the carbohydrate moieties of pulp fibers by substantially depolymerizing the cellulosic component of the fibers, through the generation of hydroxyl radicals. It is possible that CDH is used to disrupt the highly ordered crystalline nature of cellulose, allowing for cellulase components to penetrate past the outer surfaces and initiate an "amorphogenesis" or fiber delamination type action. The ability of cellulase treatments to reduce the coarseness of pulp fibers, and improve the strength of the resultant paper, suggested that there might be a direct application for enzyme treatments of pulps. This was accomplished by directing the action of cellulase enzymes at only 159 the longer, coarser fibers, without the inclusion of finer material. Using this approach, substantial improvements in the resultant paper strength were obtained with the tensile and burst indexes showing a respective 24 % and 35 % increase, over their corresponding controls. These significant improvements were maintained when the fibers were subsequently subject to refining, demonstrating a respective 17 % and 24 % increases in tensile and burst indexes after 1000 PFI revolutions. The improvements achieved at the laboratory scale were also obtained when using industrial fractionation technology and a market pulp. Thus, significant improvements in both paper and strength properties could be achieved when using a combined enzymatic/fractionation approach. Future Work Although this thesis has addressed some aspects of enzymatic fiber modification, many questions remain unanswered, from both an applied and a fundamental point of view. Hopefully, the following suggestions may be useful in guiding the direction of any further research in the field of "enzymatic fiber modification". Applied Studies • It became apparent from this research that it is possible to enzymatically modify pulp fibers to improve the resultant paper characteristics. The improvements in paper properties were achieved and optimized using a single commercial enzyme preparation. Although improvements were observed with this enzyme preparation, other sources of enzyme such as monocomponents or defined "cocktails" should be 160 considered and evaluated for their ability to modify pulp feedstocks. One of the applied objectives of this thesis was to ascertain whether some of the inherent "undesirable" fiber characteristic of Douglas-fir pulps could be selectively modified to improve its resultant paper properties. This objective was met using a single enzyme preparation and Douglas-fir as the sole pulp feedstock. Having established that beneficial modifications did occur using Douglas-fir as a substrate, it would be interesting to see if beneficial changes can also be obtained when using other pulp as a substrate which possess similar fiber characteristics, such as Southern Yellow Pine, Jack Pine or radiata pine. The observed modifications to fiber properties were attained at the expense of minor yield losses due to carbohydrate hydrolysis. Previously in the literature it was suggested that the enzymatic component(s) of cellulase preparations that significantly alter pulp properties are the endoglucanases. It was also implied that the degree and rate of hydrolysis is influenced by the concerted action of both endoglucanases and cellobiohydrolases in cellulase preparations. Therefore, it may be possible to obtain beneficial fiber modifications using only an endoglucanase and subsequently reduce the amount of yield loss due to carbohydrate solubilization by hydrolysis. During the course of this study it was shown that small amounts of cellobiose dehydrogenase could modify the ultrastructure of the pulp carbohydrate moieties. This enzyme resulted in the generation of hydroxyl radicals, which are small enough to penetrate into the fiber and disrupt the internal integrity of the fibers (as shown by the significant changes in cellulose DP). This enzymatic mechanism may "open-up" 161 the fiber and initiate "amorphogenesis". A synergistic type study using cellobiose dehydrogenase in combination with cellulase monocomponents should be conducted. The use of CDH in combination with the cellulase preparations may facilitate the penetration of cellulases into the fiber and could result in significant alterations to fiber properties. Fundamental Studies • Although the analyses conducted during this research helped us understand what alterations in fiber morphology had resulted from the specific enzyme treatments they did not conclusively indicate which enzyme component was responsible for the observed changes. 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