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Xylanase prebleaching of kraft pulps derived from three softwood species Nelson, Sandra L. 1995

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X Y L A N A S E PREBLEACHING OF K R A F T PULPS DERIVED F R O M T H R E E SOFTWOOD SPECIES by Sandra L . Nelson B.Sc , Simon Fraser University, 1992 A thesis submitted in partial fulfilment of the requirements for the degree of Master of Science i n the Faculty of Graduate Studies Department of Wood Science We accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH C O L U M B I A Sandra L . N e l s o n © May 1995 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 The University of British Columbia Vancouver, Canada DE-6 (2/88) A B S T R A C T For over a decade, the kraft pulp and paper industry has been challenged to find bleaching sequences that produce bright, high quality pulp without causing detrimental effects on the receiving environment. Historically kraft pulp was bleached with elemental chlorine and other chlorine containing compounds. However, after the discovery of toxic chlorinated organics in bleached kraft mill effluents, industry has tried to limit the use of these chemicals. Pretreatment of kraft pulps with the enzyme xylanase, a hemicellulase that degrades the xylan component of hemicellulose, has been shown to enhance the efficiency of the subsequent bleaching stage. In this study the application of xylanase in a totally chlorine-free (TCF) bleaching sequence was examined using three commercial xylanase preparations and kraft pulps derived from Douglas-fir, western hemlock and cedar woods. The activity of the xylanases Pulpzyme HB, Pulpzyme HC and Irgazyme 40 was compared in order to determine the enzymatic loadings required for the bleaching trials. The kraft pulps were treated with these enzymes (X) in two types of multiple peroxide (P) bleaching sequences (XQPP or QPXP) which included one chelation (Q) stage. The same experiment was repeated with oxygen delignified kraft pulps (OXQPP or OQPXP). The enzyme activity on the pulp was verified by monitoring increases in carbohydrate and UV-absorbing material in the filtrates collected immediately following the xylanase treatment. It was apparent that neither oxygen delignification nor peroxide bleaching inhibited the solubilization of sugar and UV-absorbing materials by the enzymes. Although the xylanases caused a range of brightening effects, direct brightening could be achieved for all of the partially bleached pulps, but not for the kraft brownstocks. In most cases, xylanase treatment directly delignified the brownstock, oxygen delignified and partially bleached pulps. Of the three xylanases, pretreatment with Irgazyme 40 elicited the most consistent bleaching improvements. Kraft pulp derived from cedar reached the highest brightnesses after oxygen delignification for i i both bleaching sequences tested. After xylanase pretreatment there appeared to be no detrimental effect on fiber strength for any of the bleaching trials. The final component of this work examined the toxicity, as monitored by Daphnia magna and Microtox, of the filtrates derived from the pulps after xylanase treatments and peroxide bleaching. The filtrates collected immediately after the xylanase stage were predominantly non toxic, the exceptions being those collected following the OQPX sequence. The toxic response elicited by the combined filtrates was found to be caused primarily by residual peroxide. In modern mills the residual peroxide is neutralized and is therefore not usually a problem. In many cases, xylanase pretreatment, in conjunction with the TCF bleaching sequence used for this study, improved final pulp brightness by 1-1.5 % ISO. Although this improvement in final brightness was variable, optimization of enzyme conditions could further enhance the applicability of xylanase in industry. i i i T A B L E OF C O N T E N T S A B S T R A C T a T A B L E OF C O N T E N T S iv LIST OF TABLES v i i LIST OF FIGURES v i i i LIST OF ABBREVIATIONS X A C K N O W L E D G E M E N T S xi 1. INTRODUCTION I 1.1 Overview 1 1.2 Basic wood composition 3 1.2.1 Cellulose 3 1.2.2 Hemicellulose 3 1.2.3 Lignin 5 1.2.4 Extractives 8 1.2.5 Fiber morphology 9 1.3 The kraft pulping process 9 1.4 Kraft pulp bleaching 13 1.4.1 History of pulp bleaching 13 1.4.2 Environmental concerns 13 1.5 Chlorine-free bleaching of kraft pulps 16 1.5.1 Elemental chlorine-free (ECF) 16 1.5.2 Totally chlorine-free (TCF) 17 1.6 Xylanases 19 1.6.1 Background 19 1.6.2 What are xylanases? 20 1.6.3 Prebleaching reaction mechanism 21 1.6.4 Factors that affect xylanase activity 24 1.6.5 Current and future applications 26 1.7 Research objectives 29 2. M A T E R I A L S A N D M E T H O D S ' ' V 31 2.1 Enzymes 31 2.1.1 Xylanase activity 31 i V 2.1.2 Optimal pH for xylanase activity 32 2.1.3 Other enzyme activities 32 2.2 Kraft pulps 33 2.3 Bleaching conditions 33 2.4 Preparation of pulp and filtrate samples 34 2.4.1 Filtrate samples 36 2.4.2 Pulp samples 36 2.5 Pulp characterization 38 2.5.1 Brightness 38 2.5.2 Kappa number 38 2.5.3 Zero span breaking length 39 2.6 Xylanase filtrate analysis 39 2.6.1 Solubilized sugars 39 2.6.2 UV-absorbing material 39 2.6.3 Residual peroxide 40 2.6.4 Toxicity analysis of filtrates 40 2.6.4.1 Daphnia magna 48 hour LC50 assay 40 2.6.4.2 Microtox 5 minute EC50 assay 42 3. RESULTS A N D DISCUSSION 43 3.1 Enzymatic characterization of three commercial xyalanase preparations 43 3.1.1 Background 43 3.1.2 Xylanase activity 43 3.1.3 Cellulase and mannanase activity 44 3.2 Summary of the effectiveness of three commercial xylanases in a totally chlorine-free bleaching sequence 48 3.2.1 Background 48 3.2.2 The effects of xylanase on kraft pulps derived from two different softwood species 49 3.2.2.1 Pulp brightness 49 3.2.2.2 Change in kappa number 51 3.2.2.3 Materials solubilized during xylanase treatment 51 3.2.2.4 Peroxide consumption during bleaching 52 3.2.3 Comparison of xylanase prebleaching of brownstock and oxygen delignified kraft pulp derived from Douglas-fir 57 3.2.3.1 Pulp brightness 57 V 3.2.3.2 Change in kappa number 58 3.2.3.3 Materials solubilized during xylanase treatment 58 3.2.3.4 Peroxide consumption during bleaching 59 3.2.4 The effects of xylanase on three different oxygen delignified softwood pulps 64 3.2.4.1 Pulp brightness 64 3.2.4.2 Change in kappa number 65 3.2.4.3 Materials solubilized during xylanase treatment 66 3.2.4.4 Peroxide consumption during bleaching 66 3.2.5 Effects of xylanase treatment on pulp brightness, kappa number and the release of carbohydrate and UV-absorbing materials 71 3.2.5.1 Pulp brightness 71 3.2.5.2 Kappa number 72 3.2.5.3 Solubilized materials 73 3.2.6 Comparison of fiber strengths after xylanase treatment of kraft pulps derived from three different wood species 75 3.2.6.1 Background 75 3.2.6.2 Comparisons of fiber strengths 76 3.3 Toxicity profile for peroxide bleaching of softwood kraft pulp that involves xylanase pretreatment 78 3.3.1 Background 78 3.3.2 Toxicity of theTCF bleaching sequence to Daphnia magna and Microtox 79 4. C O N C L U S I O N S 85 5. REFERENCES 89 v i LIST OF TABLES Table 1 Inital kappa numbers and brightness values for brownstock and oxygen 35 delignified kraft pulps Table 2 Percent of each bleaching filtrate used to make combined filtrtaes used 41 for toxicity testing. Table 3 Acute toxicity of the filtrates collected from the different kraft pulps after 81 xylanase treatment, as determined using the LC50 assay for Daphnia magna Table 4 Acute toxicity of the combined filtrates collected from the different kraft 82 pulps after xylanase treatment, as determined using the LC50 assay for Daphnia magna V i i LIST OF FIGURES Figure 1 Chemical components of wood 4 Figure 2 Diminishing structure of cellulose 6 Figure 3 Structure of xylan backbone with associated sidegroups 6 Figure 4 Three major lignin precursors 7 Figure 5 Example of prominent lignin structures in softwood 7 Figure 6 Summary of the kraft pulping process 11 Figure 7 Diagrammatic representation of a lignin-carbohydrate complex 25 Figure 8 Two possible routes to enhance delignification of softwood kraft pulp by 25 xylanases Figure 9 Effect of enzyme treatment on chlorine requirements in a (DC)E0DED bleaching 28 sequence with 50% CIO2 substitution in the CI2 stage Figure 10 Description of bleaching sequences and collection sites for filtrate and 37 pulp samples Figure 11 Comparison of xylanase, cellulase and mannanase activities over a pH 46 range for three commercial xylanase preparations Figure 12 The optimal pH for xylanolytic activity in Pulpzyme HB and 47 Irgazyme 40, in buffered and unbuffered conditions Figure 13 The effect that Pulpzyme HB and Irgazyme 40 have on pulp brightness 53 achieved during peroxide bleaching of kraft pulps derived from Douglas-fir and western hemlock Figure 14 The direct effect that Pulpzyme HB and Irgazyme 40 have on the kappa 54 number achieved during peroxide bleaching of kraft pulps derived from Douglas-fir and western hemlock Figure 15 The direct effect that Pulpzyme HB and Irgazyme 40 have on solubilization of 55 sugars and UV absorbing material during peroxide bleaching of kraft pulps derived from Douglas-fir and western hemlock Figure 16 The effect that Pulpzyme HB and Irgazyme 40 have on peroxide consumption 56 during peroxide bleaching of kraft pulps derived from Douglas-fir and western hemlock . • - -•' Figure 17 The effects the three commercial xylanases have on the pulp brightness during 60 peroxide bleaching of both brownstock and oxygen delignified kraft pulps derived from Douglas-fir v i i i Figure 18 The direct effects the three commercial xylanases have on the kappa 61 number during peroxide bleaching of both brownstock and oxygen delignified kraft pulps derived from Douglas-fir Figure 19 The effects the three commercial xylanases have on the solubilization 62 of sugars and UV absorbing material during peroxide bleaching of both brownstock and oxygen delignified kraft pulps derived from Douglas-fir Figure 20 The effects the three commercial xylanases have on peroxide consumption 63 during peroxide bleaching of both brownstock and oxygen delignified kraft pulps derived from Douglas-fir Figure 21 The effects the three commercial xylanases have on the pulp brightness during 67 peroxide bleaching of oxygen delignified kraft pulps derived from cedar, Douglas-fir and hemlock Figure 22 The direct effects the three commercial xylanases have on the kappa 68 number during peroxide bleaching of oxygen delignified kraft pulps derived from cedar, Douglas-fir and hemlock Figure 23 The effects the three commercial xylanases have on the solubilization of 69 sugars and UV absorbing material during peroxide bleaching of oxygen delignified kraft pulps derived from cedar, Douglas-fir and hemlock Figure 24 The effects the three commercial xylanases have on peroxide consumption during 70 peroxide bleaching of oxygen delignified kraft pulps derived from cedar, Douglas fir and hemlock Figure 25 The effects of treatment with three commercial xylanases on the average 77 zero span breaking lengths of kraft pulps derived from three different wood species Figure 26 Microtox EC50 for filtrates collected from unbleached and partially bleached pulp after the control and xylanase treatments 83 Figure 27 Microtox EC50 for the combined filtrates from the different bleaching 84 sequences i X LIST OF ABBREVIATIONS C l 2 elemental chlorine CIO2 chlorine dioxide CMC carboxymethylcellulose DNS dinitrosalicyclic acid DP degree of polymerization EC50 concentration that causes a particular effect on 50% of a population ECF elemental chlorine-free ISO International Standard Organization LC50 concenration that is lethal to 50% of a population LCC lignin-carbohydrate complex O oxygen delignification P peroxide bleaching stage ppt parts per thousand Q chelation stage TCF totally chlorine-free UV ultra violet X xylanase stage X ACKNOWLED GEMENTS The list of people I would like to thank for making the completion of this thesis possible is overwhelming. I would like to thank my supervisor, Dr. Jack Saddler, for giving me the opportunity to work with such an interesting group of researchers. On the technical side of things, I thank Dr. Ken Wong for being there at all times and answering even the strangest questions that I know I have asked during the past few years. Discussions with Dr. Rodger Beatson concerning the industrial applications of this research were of particular help. The support of Dr. Paul Bicho is also gratefully acknowledged along with everybody else at Forest Products Biotechnology who made my time there not only educational but a lot of fun as well. I also thank everybody who helped me at the Pulp and Paper Research Institute of Canada. Without their experience and the use of their facilities I never would have been able to conduct this research. The financial support from the Science Council of B. C , Energy, Mines and Resources Canada and Canfor Research and Development Centre were all greatly appreciated. I would not have completed this thesis (and maintained my sanity) if it had not been for the support of my friends and family. A special thank you to Mike Baker. He will never know how much help he really was. I thank my parents for not only supporting me during graduate school, but also for encouraging me to go out into the world and accomplish what I wanted to with my life. Finally I would like to thank my daughter Elizabeth, for without her I might not have fully understood how important life really is. X i l . I N T R O D U C T I O N 1.1 O V E R V I E W In 1991 Canada was the world's largest supplier of wood pulp, exporting 26.4 million tonnes (COFI, 1993). The forest industry directly employs 241 000 Canadians, 67 400 of them in the pulp and paper industry. The forest sector represents the single most important contributor towards Canada's trade balance. In addition to wanting high quality and affordability in bleached kraft pulp, the market is also now demanding that the pulp be produced by a process that is more compatible with the receiving environment. Wood pulp is bleached in order to remove residual lignin from the fibers without drastically degrading the cellulose component, which is ultimately responsible for producing a strong paper. During the kraft pulping process the chemical composition of the fibers is modified and the residual lignin remaining in the kraft pulp fibers must be chemically removed. Historically, elemental chlorine and chlorine dioxide were used to bleach kraft pulp. The discovery of toxic chlorinated organics in bleached kraft mill effluent increased public scrutiny and government legislation regulating effluents produced by the pulp and paper industry. The industry has since focused on finding alternative bleaching agents that can replace the use of chlorine in the bleaching sequence. Many alternative bleaching chemicals require a large capital cost to implement and much of the current bleaching research is focused on optimizing the use of these chemicals. Both elemental chlorine-free (ECF) and totally chlorine-free (TCF) bleaching sequences are being analyzed but it is generally believed that a TCF bleaching sequence will be required in order to completely close the mill's bleaching cycle (Reeve, 1982; Korhonen, 1993; Asp, 1994). In the future, individual mills will have to effectively evaluate their options and develop appropriate bleaching sequences using the available bleaching agents (Johnson, 1994; Lachenal and Nguyen-Thy, 1994). 1 In addition to new bleaching chemicals, the pulp and paper industry is looking for bleaching enhancers that, when inserted into the bleaching sequence, improve the final quality of the pulp without requiring a large capital cost. The use of the enzyme xylanase is one type of treatment that has been found to improve the quality of the bleached kraft pulp (Viikari et al., 1986; Tolan and Canovas, 1992; Daneault et al, 1994). It has been shown (Tolan and Canovas, 1992) that in chlorine based bleaching sequences xylanase does not elicit a bleaching effect itself, but requires other chemical bleaching agents in order to elicit an effect. The effectiveness of xylanase as a pretreatment on softwood and hardwood kraft pulps in a TCF bleaching sequence has been assessed in both laboratory and mill trials. These trials have suggested that current xylanase preparations improve the subsequent bleaching stage with variable results. The reason why xylanases are not as effective in TCF bleaching sequences as they are in chlorine based bleaching sequences is not known. Elemental chlorine and chlorine dioxide react with the pulp in very different ways, although the bleaching effects of both these chemicals are greatly enhanced when the pulp is pretreated with xylanase. It has been proposed (Yang and Eriksson, 1992a; Lundgren et al, 1994) that an enhancement of pulp quality with the addition of a xylanase treatment in a TCF bleaching sequence may enable the pulp and paper industry to fulfill current and future market demands. In this introduction, after describing the general composition of wood, the kraft pulping process is examined with emphasis on the characterization of the lignin and hemicellulose fraction of the kraft pulp fiber. After outlining the changes that are being pursued by the industry, who are looking at ECF and TCF bleaching alternatives, the potential of including a xylanase stage is discussed. Factors that affect xylanase "bleaching" on kraft pulps and the possible mechanisms explaining this action are described followed by a discussion of the importance of understanding the bleaching conditions used in conjunction with a xylanase treatment. , ,. -2 1.2 BASIC WOOD COMPOSITION The main components of wood are cellulose, hemicellulose, lignin and extractives (Figure 1). There are quantitative and qualitative differences in these components not only between different tree species, but also between trees of different ages, between trees grown at different sites and even between wood taken from different parts of the same tree. Although there are variations in cellulose content between different softwood species, this fraction should not be modified by xylanase treatment to any great extent. However, the amount and composition of the lignin and hemicellulose fraction after kraft pulping play an important role in creating the complex substrate that the xylanase must act upon. The cellulose component is ultimately responsible for fiber strength and forms the framework on to which the hemicellulose and lignin are deposited. 1.2.1 Cellulose Cellulose is a linear polysaccharide consisting of D-glucopyranose units, which are linked by fi(l-4)-glucosidic bonds. Cellulose molecules are linear and have a strong tendency to form intra and intermolecular hydrogen bonds. Bundles of cellulose molecules are thus aggregated together in the form of microfibrils. Within these microfibrils there are highly organized (crystalline) regions that alternate with less ordered (amorphous) regions. These microfibrils are packed together to form fibrils which are then bound together to form cellulose fibers (Figure 2). The fibrous structure and strong hydrogen bonds give cellulose high tensile strength and make it insoluble in most solvents (Sjostrom, 1981). 1.2.2 Hemicelluloses Like cellulose, hemicelluloses function as supporting material in the cell walls of plants. However, unlike cellulose, which is a homopolysaccharide, hemicelluloses are branched to various extents and have relatively lower molecular masses. Cellulose can have a degree of 3 21% hardwoods 25% softwoods. Lignin 45%. Wood 2-8% Carbohydrates 35% hardwoods 25% softwoods Extractives terpenes, resin acids fatty acids, phenols, etc. Cellulose glucose Hemicellulose glucose, mannose, galactose, xylose, arabinose Figure 1. Chemical components of wood (adapted from Fengel and Wegener, 1984) 4 polymerization (DP) of 10,000 or more sugar units, while hemicelluloses usually have a DP of around 200. The content and types of hemicellulose in softwoods differ considerably from those in hardwoods, as do the actual sugar constituents. There are several polysaccharides that are considered to be hemicelluloses, such as the xylans, mannans, arabinans, arabinogalactans and glucans. Hemicelluloses are closely associated with cellulose and lignin in the plant cell wall. The nature of the xylan present in the lignocellulosic matrix depends on the plant substrate. In hardwoods, xylans constitute the major hemicellulose, while galactoglucomannans are the major hemicellulose components in softwoods. The xylan backbone is composed of (Hl-4) linked D-xylopyranose units (Figure 3) (Bastawde, 1992). The type and frequency of side chains on the xylan backbone vary between plant species and within each species. Softwood xylans have fewer 4-O-methyl-a-D-glucuronic acid side chains than hardwood xylans. In the case of softwood xylan, one acid group is observed every 9 to 12 D-xylose units, and in hardwood xylan every 5 to 6 D-xylose units. In hardwood xylan, there is usually one acetyl group present for every two xylose residues. Softwood xylan can also contain other residues such as L-arabinofuranose. The ability to modify both cellulose and hemicellulose has become an integral research area as industry searches for new production processes and alternative methods to generate new materials. 1-23 Lignin Lignin is an aromatic polymer which is formed in wood by an enzyme-initiated polymerization of a mixture of different 4-hydroxypropenyl alcohols (Figure 4). The proportions of the three basic alcohols vary with different wood species and even within the same species. These alcohols are linked together in various ways to produce large, amorphous, three dimensional structures. There are many chemical bonds possible within a typical lignin 5 Figure 2. Diminishing structure of cellulose (Smook, 1992) £ -(1-4 J-D-XYLOPYRANOSE LINKAGE H OH D-XYLOPYRANOSE RING CCOH HjOC H OH Ac - ACETYL GROUP 0 a - (1-2 ) -A-O-METYL-D-GLUCURONIC ACID LINKAGE HOHjCY j/H H OH 0 a - ( I — 3 ) - L - A R A Bl NO FURANOSE LINKAGE Figure 3. Structure of xylan backbone with associated sidegroups (Bastawde, 1992) 6 CH,OH i * CH20H 1 CH CH II II CH CH V OH OH p - coumaryl alcohol coniferyl alcohol sinapyl alcohol Figure 4. Three major lignin precursors (Sjostrom, 1981) •OCH, >< 0-0— CH—CH-CH,OH 0 < - C M - C H HOCH, -CH,OH CH,OH '» I \ 0CM> HOCM **M» 0 , e O - ^ - C H—I H - C HxCM —C«| OCX, HC —0 O H —^-CH-CM-CHjOM \ 6 - C « , - C H U c X>H,C H.C0-UvT 0 C^-CH — C H - C M p i i V» o ; e- J "»°T *«««« S*-e"S 0CH' 0 "jP-" CH H*C-CM 9 — C H i CH I C H J O H K-CM V0(-Ci—Q>-0-CrH >;(. 6 HO-^-CHmCH-CMjOH 1 * c HOKjC I C H , O H A -OH.e-CH-c-c^-o r ^-c-ffl^, ' | ^ C H . ? -^HLn.°> v o f V'W <^>c-, H O - Q - C C H - C H - C » I O H I 5 % O.C«hW|{ CM J- HC-Cl ' ' O C H , H C - C M A> WC D C H . I . I - C H H J C »Y „[ - C H I K J i l l CN 0 0 • I hC— CK-CMjCM l»e«»i t-O-CH, o lie * « 1 Figure 5. Examples of prominent lignin structures in softwood (Glasser and Glasser, 1981) 7 structure in addition to extensive branching. Characteristic functional groups such as primary and secondary hydroxyl, etherified and free phenolic hydroxyl, and various types of carbonyl groups can be found (Figure 5). Because of difficulties in isolating native lignin from wood without degradation, the molecular weight of the complex structure is still unknown. Using milled wood lignin, molecular weights of about 20,000 have been found for softwood lignins, whereas lower values have been reported for hardwood lignins. There are varying concentrations of lignin across the cell wall of both softwood and hardwood fibers. The middle lamella contains the highest concentration of lignin of all the cell layers (Sjostrom, 1981). Lignin is responsible for the rigidity of the fiber walls and acts as a bonding agent between the fibers. In wood, lignin is probably chemically linked to hemicelluloses (Yamasaki et ai, 1981). The structural composition of lignin and carbohydrate within a microfibril is still not known. However, one common theory is that the lignin is bound to the hemicellulose which in turn binds the elementary cellulose fibrils together. A simple picture of how the three main building blocks of wood fit together is that cellulose forms the skeleton which is surrounded by hemicelluloses that form a supporting matrix, and everything is glued together with lignin. 1.2.4 Extractives The term "extractives" is normally used for those components of wood that can be extracted by organic solvents such as ethanol, acetone, or dichloromethane. They include compounds such as aliphatics and saturated and unsaturated acids (including resin acids). The distribution of extractives in wood varies greatly depending on the species, place of growth, the age of the tree, and even the location of the wood within the same tree. 8 1.2.5 Fiber morphology Before pulping there are distinct differences in the chemical and physical composition of the different species of wood. During the kraft pulping process these differences are modified. The chemical composition of the wood is changed by decreasing the hemicellulose and lignin content in thge pulp fiber while conserving the cellulose portion. The extractives are solubilized and removed from the fiber. As described in the following section (section 1.3) the reprecipitation of the hemicellulose xylan onto the pulp fiber that occurs during the kraft cook would occur for all wood species. After the kraft cook, the differences between the wood species are not as obvious but characteristics in fiber morphology will be maintained. Each wood species has a certain type of pulp fiber with characteristic differences that can be identified such as: porosity, surface area, fiber wall thickness and length of fiber. These physical characteristics will probably affect not only the way in which the xylan and lignin reprecipitate onto the pulp fiber during the kraft cook, but ultimately how the enzyme xylanase acts on the pulp fiber. 1.3 THE KRAFT PULPING PROCESS In order to make high quality paper, lignin in the raw wood substrate must be removed without destroying the cellulose microfibrils within the wood fibers. During the production of wood pulp, it is desirable to maintain a high quantity of hemicellulose within the fiber to improve yield. Most of the alkali consumption in the pulping process is caused by alkali degradation of the hemicellulose fraction and the extent of delignification is partially controlled by the amount of hemicellulose removed (Browning , 1976). In the kraft pulping process, sodium hydroxide and sodium sulfide are added to the wood chips in the digestor to degrade and dissolve the major portion of the lignin found in the middle lamella. Individual fibers within the wood chips are loosened and separated, and then larger unreacted woody pieces are removed by screening (Figure 6). The filtrate collected after chemical reaction with the wood is called the black liquor. This filtrate contains a high concentration of organic material that can be removed 9 and incinerated for energy. After the organics have been removed from the black liquor the resulting liquor is recycled and can be used in the pulping process again. The kraft pulping process produces higher yields and superior fiber characteristics in comparison to other chemical pulping processes (Fengel and Wegener, 1984). Hemicelluloses are thought to affect the reactivity of residual lignin in kraft pulp by partially covering the lignin and blocking the bleaching chemicals from reacting with it (Clark et al., 1991). During the kraft cook, the hemicellulose fraction of the fiber is extensively modified. The heating period of the cook, when the alkali concentration is comparatively high, dissolves part of the xylan in the pulping liquor (Viikari et al., 1994). The alkali concentration decreases as the cook proceeds, and degraded, short-chain xylan precipitates in a more or less crystalline form on the surface of the cellulose fibers. Most of the xylan side groups are removed during the kraft cook, increasing the tendency of the xylan to crystallize. Although part of the xylan is readsorbed or reprecipitated onto the cellulose, a part remains undissolved at its original position in the fiber. Once the initial dissolution of xylan has occurred, polysaccharide peeling reactions uncover more lignin which can be subsequently released during the kraft cook (Buchert et al., 1993b). The reprecipitation of xylan is followed by the reprecipitation of lignin during the kraft cook. These redeposited compounds have been reported to be chemically linked to each other (Iversen and Wannsrtom, 1986; Yang and Eriksson, 1992b; Daneault et al., 1994). The formation of lignin-carbohydrate complexes (LCCs) could contribute to the difficulty encountered in removing residual lignin from the fibers, even though the major part of the lignin network has been torn apart. New pulping methods may affect the composition and localization of hemicelluloses in the pulp. The extent of reprecipitation of xylan has been suggested to decrease in extended and continuous kraft cooking procedures (Pedersen et al., 1992; Buchert et ai, 1993b). 10 wood chips 1 • digester < white liquor K, + „ blow-tank knots -< knotter wash water • washer screening/cleaning thickening I, h storage-pulp chest CHEMICAL RECOVERY black liquor tall oil flue gas heat i process steam further processing (i.e. chemical bleaching) Figure 6. Summary of the kraft pulping process (adapted from Fengel and Wegener, 1984) 11 There are several reasons why sulfate pulping has become the most common chemical pulping process world-wide. Kraft cooking has a high tolerance for large amounts of extractives as well as portions of decayed wood and bark residues, which allows for considerable variabilities in the wood substrate entering the kraft pulping cycle. Another reason for using the sulfate pulping process is the relatively short cooking times, usually less than two hours for each stage, and the excellent pulp strength properties which make it economically advantageous. Well established processing of the spent liquid, including the recovery of the pulping chemicals, generation of process heat, and the production of valuable by-products such as tall oil and turpentine from pine species make the kraft pulping process economically favorable (Fengel and Wegener, 1984). During the kraft pulping process, lignins in the pulp become darkly colored and if they are not soluble, they are usually redeposited on the pulp fibers during the last stage of cooking (Sjostrom, 1981). These residual lignins darken the pulp and are very difficult to remove without damaging the pulp fibers. Functional groups of partially degraded and modified residual lignin are predominantly responsible for the light absorbing chromophoric components in unbleached pulp. The main purpose of pulp bleaching is to remove color causing components while minimizing losses to pulp quality. Bleaching can be performed either by converting and stabilizing the chromophoric groups (lignin-preserving bleaching), or by removing the lignin from the carbohydrate fraction of the pulp (lignin-removing bleaching). Along with the lignin other compounds such as extractives, ash and some hemicelluloses are also removed. Larger particles such as shives and bark specks may also be partially bleached or completely removed from the pulp mixture during the various bleaching stages. Therefore bleaching can also be regarded as a "purification process" as well as a brightening process (Fengel and Wegener, 1984). After the kraft process, only 3-6% lignin is left in the pulp. Bleaching chemicals are used in order to facilitate the removal of this small percentage of residual lignin left in the pulp after the kraft pulping process. 12 1.4. K R A F T PULP B L E A C H I N G 1.4.1 History of Pulp Bleaching Initially, non-wood libers, such as cotton rags, were gradually brightened with sunlight and/or chemicals such as potash and hypochlorite. The utilization of wood fibers came later but the bleaching was still slow and on a small scale as compared to modern bleaching processes. Towards the end of the nineteenth century, a new period of industrial bleaching of wood pulps began, first with increased use of hypochlorite and then chlorine, in combination with an alkali extraction stage. Larger amounts of much higher quality pulp could be produced when chlorine and chlorine dioxide were introduced into the bleaching process. The pulp and paper industry began to grow and the bleaching of pulps with various types of chlorine-based chemicals was considered the best and cheapest process available. In the past (1950-1980), changes that were of importance to the pulp and paper industry were those that could improve quality, yield, or cost effectiveness during the pulping process. However, during the past decade, vast changes have transpired in response to environmental concerns. There is no longer a "conventional" bleaching process that is used by kraft pulp mills. Bleaching has become a complex and expensive step in the production of market kraft pulp. Many different chemical agents and bleaching sequences are now being used. Currently, individual mills must analyze their options carefully and choose the best bleaching alternatives for their own requirements. The drive towards using fewer chlorine containing compounds means that each mill will become more and more specialized in their own bleaching technology. 1.4.2 Environmental Concerns Currently, the two main environmental concerns of the pulp and paper industry are the discharge of colorants into the receiving waters and the presence of chlorinated organics in bleach-plant effluents and paper products (Brunner and Pulliam, 1993). The main strategies being developed to reduce the environmental impact of bleach-plant effluents include: increased 13 delignification of the pulp before it reaches the bleach plant; the use of alternative bleaching sequences and chemicals; increased effluent treatment; and partially or completely closing the mill cycle. Elemental chlorine and other chlorine containing chemicals are very corrosive. They tend to undergo electrochemical reactions such as the reduction of Cl2 to Cl" (Laliberte and Sharp, 1979; Laliberte and Garner, 1981). The chloride ions react with, and thus slowly degrade, the metal pipes and containers used in the system. The degradative effect is made worse by the recirculation of chlorine containing water in the bleach plant, thus concentrating the chloride ions. Effluents that contain chlorine can not easily be burned or recycled because of their associated impact on the environment. Currently, the filtrate from the final chlorination stage can been recirculated to various dilution points in the chlorination stage, resulting in substantially lower water use and consequently lower volume of effluent discharged into the environment. Organics, released from the kraft pulping process and then chlorinated to varying degrees, were being discharged into the receiving environment in the past. These numerous complex organics were identified as being toxic to their receiving environment (Kringstad and Lindstrom, 1984; Koncel, 1991). Work at the Swedish Pulp and Paper Research Institute and the Pulp and Paper Research Institute of Canada (PAPRICAN) determined that various dioxins, organochlorines that were identified as the most toxic, came from several defoamers and oils, as well as the wood and water used in the pulping process (Strunk et al, 1992). The use of the defoamers and oils was almost immediately stopped and the bulk of the chlorinated dioxins and furans were no longer produced. The amount of organochlorines generated was further reduced by modifying the bleaching conditions and decreasing the use of elemental chlorine. Due to the complexity of the compounds that can be produced in the bleach plant there may be various degrees of impact on the receiving environment. About half of the organochlorine content of bleach plant effluent can be removed by microorganisms in the treatment pond (Fleming, 14 1992). The molecular weight distribution of organochlorines, as determined by ultrafiltration can be divided into two parts (Kringstad and Lindstrom, 1984). The high molecular-weight fraction represents about 70% of the organochlorine in the spent chlorination liquor and about 95% of that in the spent alkali liquor. These large compounds have not been extensively characterized but are probably biologically inactive because they cannot penetrate cell membranes of living organisms. They still constitute an environmental concern because they contain chromophoric structures that are responsible for the high color in the bleach plant effluent. It is also unknown at this time as to whether or not these large molecules degrade into smaller, more biologically active compounds that might have a detrimental effect on various biosystems. It has primarily been the low molecular-weight compounds (<1000 daltons) that researchers have spent most of the last decade analyzing. These smaller compounds are often biologically active and various tests have been developed to help assess their adverse effects on the receiving environment. The reason for the extreme toxicity of many organochlorines is their tendency to accumulate in living tissue, particularly fatty tissue. The detailed effects of bleach kraft mill effluent on the receiving environment are not yet resolved. Although there have been several in-depth studies that monitored the effects of the effluent on various organisms in the surrounding area, conflicting results have been published. The National Swedish Environmental Protection Board (1989) has released a report summarizing the toxic effects of bleached kraft mill effluent. They concluded that chlorinated compounds in bleached kraft mill effluent were responsible for the observed impact on the receiving environment. However, Hall (1991) conducted a study which determined that properly treated pulp mill effluent had no adverse effects on fish growth and reproduction. Furthermore, several papers have reported no differences in the effects on the receiving environment from effluent generated by bleached kraft mills, unbleached kraft mills and mechanical pulp mills (Hodson, 1992; Munkittrick, 1992). Cockram (1993) has reported that groups of scientists in both North America and Scandinavia are becoming more convinced that the cause of the observed effects on fish must be a nonchlorinated compound, possibly originating from the trees themselves. 15 Chlorine was used to bleach pulp, but the discovery of toxic chlorinated organics within kraft mill effluent prompted the pulp and paper industry to find alternative bleaching agents that did not use chlorine. It has since been discovered that there may be other, undetermined, components generated during the kraft pulping process that may contribute to the toxic effects previously observed. However, government legislation and public pressure is still motivating industry to decrease or even eliminate the use of chlorinated chemicals in the production of high quality bleached kraft pulp. 1.5 CHLORINE-FREE B L E A C H I N G OF K R A F T PULPS Current market demands of the pulp and paper industry insist that paper be produced with little or no chlorine (Beaton, 1994). Research in bleaching is concentrating on finding new procedures and chemicals that can delignify and bleach the pulp, thereby facilitating the elimination of elemental chlorine and decreasing the amounts of other chlorinated bleaching agents required (McDonough, 1995). Bleaching alternatives are necessary in order to eliminate the use of chlorine which will then decrease the organochlorine content of the bleach kraft mill effluent, increasing the possibility of closing the mill discharge system (Glowacki, 1994). 1.5.1 Elemental Chlorine-Free (ECF) Elemental chlorine (CI2) will probably only be used for very sensitive bleaching operations in the future, such as the production of photographic paper. As of mid 1992, 20 of the 47 bleached kraft mills in Canada could operate at full production without using any elemental chlorine, and eight more could do so at reduced production rates (Williamson, 1992). There has been no elemental chlorine used in Swedish mills since the end of 1992, and Finland will be following this policy soon. In an elemental chlorine-free bleaching sequence there may be other chlorine containing compounds present. These chemicals do not usually produce the amount nor the types of organochlorines that are produced when elemental chlorine is used. 16 Chlorine dioxide is the main chemical that has been used to substitute for elemental chlorine and it is believed to be the most efficient bleaching agent available at this time. Not only does CIG7 produce fewer organochlorines than elemental chlorine, it also appears to degrade phenolic residues in the pulp and it is very effective at removing bacteria and fungi that may be present in the pulp (Croon, 1993). Toxic bioaccumulation and chronic effects were not evident from a study in which effluent from 100% chlorine dioxide bleached pulp was tested on fish and Crustacea (Haglind et al, 1991). 1.5.2 Totally Chlorine-Free (TCF) In the search for a compound that will help the bleaching industry eliminate the use of chlorinated bleaching compounds, Wearing (1993) indicated that it is important to ensure that new bleaching technologies are environmentally sound and represent an improvement over current technology. Effluent generated from new bleaching sequences should be easily treated and disposed in a way that will meet any present or proposed government legislation, and they should improve the chances of a closed mill system being developed. Totally chlorine-free (TCF) bleaching means that there are no chlorine-containing compounds used in the bleaching sequence. The production of TCF paper is currently one of the most intensely researched areas of pulp and paper technology and it is the subject of several controversies. A considerable amount of effort is being spent on trying to find chlorine-free bleaching agents that are affordable and effective. In the search for the perfect bleaching alternative, the driving forces which often conflict with one another include: the effectiveness of the new bleaching technologies; the quality of the final pulp and paper; production costs; selling prices; consumer demands; and pressure from environmental groups (Cockram, 1993). Most of the TCF pulp and paper on the market today is a compromise. Totally chlorine-free pulps derived from hardwood, such as birch and eucalyptus, have been produced but those from softwoods pulps are still problematic. High brightness has been achieved in softwood pulps but often with a loss of yield and strength. It appears that newer mills will be able to produce TCF pulp with only a 17 slight increase in cost (due to the use of more expensive bleaching chemicals) but older mills will face higher costs due to the necessity of retro-fitting equipment (Cockram, 1993). Many paper producers are not convinced that the environmental benefits of TCF bleaching, compared with ECF bleaching, justify the additional costs and quality compromises currently involved. Although industry is actively trying to decrease chlorine consumption, it may be impossible to completely eliminate organochlorine generation. Berry (1993) states that with process water alone, 0.1 kg of chlorine per ton of pulp is taken in. Wood naturally contains enough chlorine to generate approximately 0.1 g of organochlorine compounds per ton of pulp. The market for chlorine-free pulp is growing and producers have been attempting to fill these demands using chlorine-free chemicals (Koncel, 1991; Cox, 1992). The two chlorine-free alternatives used for this study were oxygen delignification and hydrogen peroxide. Oxygen delignification uses relatively high temperatures and pressures, and requires a substantial capital cost to implement within the bleach plant operations. However, the worldwide production capacity of oxygen delignified pulp has exhibited dramatic growth during the last several years, and oxygen delignification is considered a prerequisite for the production of TCF pulp (Johnson, 1993). The brightening effects of hydrogen peroxide have been recognized for years. However, the effectiveness of peroxide as a bleaching agent has been limited by its degradative effects on pulp strength (van Lierop et al, 1993). Current research has focused on improving the action of peroxide on the pulp without damaging the pulp fiber. For this study, the enzyme xylanase was used in conjunction with oxygen delignification and multiple peroxide stages in order to determine if enzymatic pretreatment of the kraft pulp could facilitate subsequent bleaching stages. 18 1.6 X Y L A N A S E S 1.6.1 Background Recently, a lot of attention has been given to the bleach boosting effect that the enzyme xylanase has in the chemical bleaching of both softwood and hardwood kraft pulps. Hemicellulases have previously been tested for the removal of hemicellulose from pulp in order to facilitate the production of dissolving pulp (Paice and Jurasek, 1984). The authors concluded that a xylanase mixture was not able to remove enough of the hemicellulose. Since this work, Viikari et al. (1986) and Kantelinen et al. (1988) introduced the idea of using xylanase to decrease the amounts of chlorinated chemicals required to bleach kraft pulp. It has been found that xylanases are the most effective hemicellulases for enhancing the extractability of lignins by conventional bleaching chemicals (Viikari et al. ,1986; 1987; Kantelinen et al. ,1988). Several reviews have summarized the current status of xylanases being studied for use in the pulp and paper industry (Bajpai and Bajpai, 1992; Lavielle, 1993; Wong and Saddler, 1993; Viikari et al, 1994; Daneault et al, 1994; Tolan, 1995). For environmental reasons, the possibility of reducing the use of elemental chlorine in pulp bleaching has pushed research and development of xylanases forward. However, it was soon realized that, when combined with chlorine-free chemicals such as hydrogen peroxide, xylanase treatment was not able to boost pulp brightness to the same extent achieved by chemicals containing chlorine. Many researchers believe that a greater understanding of the mechanism of xylanase bleaching will improve the applicability of these enzymes in a TCF bleaching sequence. It has also become apparent that conditions within the pulp matrix complicate xylanase action when compared to those with the isolated substrates often used in the laboratory. Research is currently focusing on the optimization of xylanase action on the kraft pulp in order to further reduce the chemicals required to bleach the pulp. Because the pulp and paper industry has been forced to consider many new bleaching alternatives, the potential of 19 using xylanases without requiring significant capital expenditure has made them worthy of study. 1.6.2 What are Xylanases? Hemicelluloses are closely associated with cellulose and lignin in the plant cell wall (Section 1.2.2). Of the polysaccharides classified as hemicelluloses, the most important, and abundant, are xylans and mannans. The ability to modify these two hemicelluloses with enzymes has become an intensive area for research as industry searches for new pulp production processes and alternative methods to bleach chemical pulp. Enzymes are highly specific protein catalysts that, in small amounts, speed up reactions that may or may not already be occurring within a reaction mixture. Xylanases are one type of hemicellulase required by many microorganisms to degrade plant biomass. Hemicellulases, responsible for the degradation of hemicelluloses, are generally classified according to the substrate that they degrade. Collectively, hemicellulases, pectinases, amylases and cellulases are referred to as glycan hydrolases (EC 3.2.1). Xylanase, the enzyme responsible for xylan breakdown, occurs in both prokaryotes and eukaryotes (Dekker and Richards, 1976). Extracellular and intracellular xylanases from various bacterial and fungal sources have been extensively studied (Reilly, 1981; Woodward, 1984; Dekker, 1985; Bastawde, 1992). Endo-P-(l-4)-D-xylanase acts randomly on xylan to produce xylo-oligosaccharides of various chain lengths. Exo-P-(l-4)-D-xylanase removes single xylose units from the non-reducing end of the xylan chain. P-xylosidase (or xylobiase) hydrolyses disaccharides like xylobiose and xylo-oligosaccharides with decreasing specific affinity. Of these three types of enzymes, the endoxylanase appears to be the most important to the pulp and paper industry to date (Bastawde, 1992; Kantelinen et al., 1993). Two general methods are currently being used for the production of xylanases. Batches of a particular microorganism that produces large amounts of extracellular xylanases, such as the 20 fungus Trichoderma sp., are grown, and the filtrates collected and partially purified by gel filtration. Unfortunately, it is extremely difficult to remove all of the other contaminating hydrolytic enzymes, such as cellulase, that are also produced by most microorganisms using this method. The second method involves the cloning of the gene encoding a xylanase from one microorganism. This method is particularly successful for the production of cellulase-free xylanases required by the pulp and paper industry. There are, however, researchers who believe that by removing the other enzymes from the native xylanase preparation, additive and synergistic activities that may otherwise improve the overall bleach boosting results are decreased, if not completely eliminated. There are few microorganisms that are known to produce a single type of xylanase. A multiplicity of xylanases, especially endoxylanases, has been reported for bacteria, moulds and yeasts (Bastawde, 1992). The stability and activity of the different xylanases often depends on factors such as pH, temperature, ion concentration, and variability within the substrate itself. Xylanases appear to have a wide range of pH and temperature optima, from 4-7 and 37-80°C, respectively (Bastawde, 1992). Most of the characterized xylanases presented by Bastawde (1992) remained active within several pH units above and below the reported pH optimum for each xylanase. 1.63 Prebleaching Reaction Mechanism Xylans go through complex transformations during the kraft pulping process which ultimately affect the mechanism of xylanase action. Native hardwood and softwood xylans differ in the degree of polymerization of their backbone. After the kraft cook the relative amount of glucomannan compared with xylan is lower in the pulp than in the original wood (Sjostrom, 1981). This is thought to be one reason why xylanases may be more effective at enhancing delignification than other hemicellulases (Kantelinen et ai, 1993). As previously discussed (section 1.3), uronic acids in xylan are rapidly lost during the alkali kraft pulping process. The xylan therefore becomes more crystalline and less soluble, and therefore 21 reprecipitates onto the pulp fibers. This fraction may also penetrate into less accessible portions of the cellulose (Browning, 1976). The precise mechanism by which xylanase enhances kraft pulp bleaching is not known but several theories have been suggested (Wong and Saddler, 1992): 1. Disruption of lignin-carbohydrate complexes (LCC) to produce smaller lignin molecules, which should be easier to remove by the subsequent bleaching sequence (Figure 7) (Tolan and Canovas, 1992). 2. Removal of reprecipitated xylan on the fiber surface that entraps residual lignin or blocks penetration of bleaching chemicals (Kantelinen et al, 1991, 1993). 3.. Solubilization of chromophores formed within the xylan during the kraft cooking process (Patel et al, 1993). 4. Disruption of interactions between xylan and cellulose which results in a swelling of the fiber matrix that facilitates lignin removal (Buchert et al, 1993b). Xylanase action for prebleaching of kraft pulps may involve one or more of the above mechanisms. It is difficult to test the validity of these mechanisms because residual lignin and reprecipitated xylan constitute a small percent of the pulp weight (Meller, 1965). The effect of xylanase action on molecular weight distribution has been used to examine the occurrence of lignin-carbohydrate complexes. Kantelinen et al. (1993) found that partial removal of surface xylan from the pulp increased the amount and molecular mass of lignin extracted by alkali. The average molecular weight of LCCs has been found to increase after treatment with xylanases (Yang and Eriksson, 1992). The authors also reported that hardwood and softwood kraft pulps treated with low molecular weight xylanase leached greater amounts of LCCs than high molecular weight enzyme fractions. This may indicate that the action of the larger xylanase is hindered by some portion of the pulp fiber matrix. - The role that swelling of the fiber plays on the action of xylanases has been examined by Buchert et al. (1993b). In this research it was determined that the greater the negative surface charge, the more swollen the pulp fiber, and the 22 less the substrate was hydrolyzed. These parameters could be manipulated in order to optimize the action of the xylanases within the pulp matrix. Figure 7 and Figure 8 represent diagrammatic representations of a possible lignin-carbohydrate complex and possible modes of action for xylanase on reprecipitated xylan within the kraft pulp fiber respectively. In the past it has been assumed that pulp color was only caused by residual lignin, but recent studies (Ziobro, 1990a, 1990b) implicate carbohydrate degradation products as a possible source of color. It appears that sugars cooked under kraft conditions give rise to UV-absorbing materials with absorption spectra similar to those in kraft liquors. These studies do not provide direct characterization of the chromophores present in kraft pulp because they were carried out on isolated sugars, which are not as complex as the matrix formed during sulfate pulping of cellulose, hemicellulose and lignin. Chromophores have been found to be released during xylanase treatment of kraft pulp (Patel et al., 1993). It was also found that different xylanase preparations had varying abilities for releasing chromophoric materal. The authors concluded that it was necessary to determine the xylanase action responsible for facilitating kraft pulp bleaching in order to optimize the conditions required for xylanase treatment. During xylanase prebleaching there is a definite increase in the availability of the lignin to the bleaching reactions indicating that the xylan in the kraft fiber matrix is being modified in some way. The degree of polymerization (DP) of xylan decreases significantly after treatment with xylanase but only a small amount of xylan is actually removed (Paice et aI.,T992; Patel et al., 1993). It has been proposed that the shortening of the xylan chain and/or the redeposition of the xylan onto different areas of the fiber was able to increase the freedom for lignin movement and diffusion out of the hemicellulose. This could explain how partial hydrolysis of xylan improves lignin removal during the subsequent chlorination and alkali stages. 23 1.6.4 Factors That Affect Xylanase Activity Enzymes are derived from biological organisms and they tend to require relatively specific conditions for optimal activity. It is possible to formulate an optimal mixture of xylanases that could completely hydrolyze the xylan in an isolated substrate (Viikari et ai, 1994). Xylanases have been used for the selective removal of xylan from pulps but several factors often limit the action of the enzyme on the substrate (Senior et al., 1990). When hemicelluloses are bound to the pulp fiber, other factors such as porosity, specific surface area, charge of the fibers and distribution within the substrate tend to affect the efficiency of the enzymatic hydrolysis. The inaccessibility of the substrate to the xylanase may be due to physical limitations and the influence of lignin-carbohydrate linkages on xylan hydrolysis. Other inhibiting factors include, difficulty in hydrolyzing the xylan due to its branched nature, thermal instability of the enzyme preparations, non-selective adsorption of xylanases onto the cellulose and lignin components of the pulps, and the inactivation or inhibition of the xylanase by materials present in the pulp. The adsorption and/or inactivation of xylanase would reduce the concentration of the enzyme available for the hydrolysis of xylan. Senior et al (1990) determined that the incubation of xylanase with unbleached pulps resulted in a significant loss of activity that was mainly due to the presence of leachable materials from the pulp. They also reported that non-selective adsorption of the xylanases onto components of the pulp other than xylan severely limited the effective concentration of the enzyme. Most enzymes are sensitive to both temperature and pH extremes. A bleach plant may have difficulty adjusting to and maintaining optimal conditions for enzyme activity, although this has not been found to be a serious problem in modern mills. Current research is being conducted to develop xylanases that are active at higher temperatures (Simpson et al., 1991) and broader pH ranges (Daneault et ai, 1994). Recently, the activity of a commercially available xylanase was tested at various pHs and temperatures and there appeared to be an increase in pulp brightness for a wide range of conditions (Lahrinen ct ai, 1993). 24 B Cellulose Glucomannan Xylan Residual lionin Glucomannan - Cellulose Figure 8. Two possible routes to enhance delignification of softwood kraft pulp by xylanases. Enzyme hydrolysis sites are shown by "X". Diagram A indicates hydrolysis of adsorbed xylan which exposes residual lignin to bleaching chemicals. Diagram B shows cleavage of LCC bonds. (Daneault et al, 1994) 25 In order to achieve the highest brightness possible from a TCF bleaching sequence the action of the xylanase treatment must be optimized. One TCF bleaching sequence that has already been used in industry is oxygen delignification (O) in conjunction with multiple peroxide stages (P). This sequence has been able to produce pulp brightnesses of around 80 for softwoods and 85 for hardwoods, without compromising pulp strength. If a xylanase treatment could enhance these final brightnesses then it would be worthwhile inserting a xylanase treatment into the bleaching sequence. There are several conditions for kraft pulp bleaching that require close monitoring if it is being used in conjunction with xylanases. Important conditions, other than the pH and temperature of the pulp prior to and during the xylanase stage, include the duration of the xylanase stage, the presence of ions in the pulp, and the extent of pulp washing before and after the xylanase stage. If any of these conditions are not appropriately set then the action of the xylanase will not be as efficient as it could otherwise be. 1.6.5 Current and Future Applications Xylanase treatment of kraft pulps prior to chemical bleaching requires minimal capital cost, and if the conditions during enzyme treatment are optimized they may offer a viable solution to the pulp and paper industry. In a chlorinated bleaching sequence the major effects of xylanase treatment are only apparent after subsequent bleaching (Tolan and Canovas, 1992). When pulps of the same initial lignin content were treated, xylanase treated pulps required less chlorinated bleaching chemicals to attain the same brightness as control pulps (Figure 9). The usefulness of xylanase for enhancing pulp bleaching with chlorine dioxide or peroxide has also been studied (Elm et ai, 1993; van Lierop, 1993). Many laboratory and mill trials have been conducted in order to determine the best way to use xylanase. The original purpose in using xylanases inpulp bleaching was to reduce the use of elemental chlorine (CI2), and thus reduce the environmental impact of effluent discharged from the bleach plant. In an experiment using a conventional chlorine based bleaching sequence, 26 Liebergott et al. (1991a) showed that treating a kraft brownstock with xylanase can decrease the amount of chlorine required to remove the same amount of lignin, by about 30 %. Other trials using chlorine based bleaching have also found similar improvements in pulp bleaching after xylanase treatment (Lavielle et al. 1992; Turner et al., 1992; Scott et ai, 1993; Tolan, 1995). In a chlorine-free sequence using O2 delignification and multiple peroxide steps, the best result was a 1 - 2 % brightness gain (van Lierop et al., 1993). An extended trial run was performed at the Metsa-Bothnia mill in Kaskinen, Finland, and it produced a brightness of 75-80% for softwood pulp and 80-85% for hardwood (birch) pulp. These kraft pulps were produced by extended cooking, prebleached with xylanase and chemically bleached with oxygen and peroxide. Pulp properties were found to be similar to the untreated controls and color reversion in peroxide bleached pulp affected brightness over time (Malinen, 1992). This reduction in brightness was not necessarily caused by the xylanase treatments but by the TCF bleaching sequence itself (Malinen, 1992). Ozone has attracted a lot of attention recently as a very effective TCF bleaching chemical. The Enzone process (Yang et ai, 1994) uses oxygen, ozone, xylanase and peroxide in order to produce a kraft pulp with high brightness. This process is able to produce pulps with good brightness stability and similar strength properties to conventionally bleached pulps. Other research has found that xylanases in conjunction with ozone and chlorine dioxide improved ozone bleaching effectiveness by about 25 % (Allison and Clark, 1994). Xylanases appear to offer one alternative for the pulp and paper industry to improve the brightness of kraft pulp without requiring significant capital costs. 27 6.5 7.0 7.5 8.0 6.5 9.0 9.5 10.0 TOTAL ACTIVE CHLORINE, % Figure 9. Effect of enzyme treatment on chlorine requirements in a (DC)EDDED bleaching sequence with 50% GO"2 substitution in the Q2 stage (Tolan and Canovas, 1992) 28 1.7 RESEARCH OBJECTIVES There are many questions that can be posed on the effectiveness of xylanase treatment in a TCF bleaching sequence. Do different commercial xylanase preparations have similar effects on kraft pulp? Does oxygen delignification and/or peroxide affect xylanase action on the kraft pulps? Is there a difference in final brightness of the pulps if the xylanase treatment is inserted before the kraft pulps are bleached in any way? Are there any differences in activity among commercial xylanases when tested on pulps derived from different wood species? In an attempt to address these questions, a study was designed that looked at these parameters. The main research objective for this study was to determine the effectiveness of three commercial xylanase preparations in a TCF bleaching sequence. The basic TCF sequence consisted of a chelation stage to remove any contaminating metal ions followed by two separate hydrogen peroxide stages. The xylanase treatment was inserted at two different points in the bleaching sequence, before the first peroxide stage and between the two peroxide stages. After testing the effects that the different xylanases have on the pulp brownstocks, the original pulps were oxygen delignified and the peroxide bleaching trials were repeated. Xylanase activity was monitored by analysis of carbohydrate and UV-absorbing material released during the enzyme treatment. Xylanase effectiveness was measured by improvements in pulp brightness and by delignification immediately following treatment with xylanase. The results from the brownstock and oxygen delignified pulps were compared in an attempt to identify any differences in xylanase effectiveness for the two enzyme insertion sites used. For this study, kraft pulps, derived from several different wood species, were used in order to determine if the xylanases have similar activities on different wood substrates. An effort was made to determine any effects that the xylanase treatment had for any of the wood species or bleaching sequences on individual fiber strength. Comparisons between wood species and xylanase insertion sites were also made for fiber strength, solubilized materials and residual 29 peroxide. During the course of my research, a preliminary study was designed to identify the effects that this alternative bleaching sequence may have on the receiving environment. Filtrates generated by the TCF bleaching sequence were measured for toxicity using two different assays, one for fresh water and one representing the marine environment. In this way we hoped to obtain a general indication of overall toxicity of the effluents which included a xylanase bleaching stage. 30 2. MATERIALS AND METHODS 2.1. ENZYMES The three commercial xylanases used were Pulpzymes HB and HC (Novo Nordisk, Denmark) and Irgazyme 40S-4X (formerly Albazyme; Genencor, USA). The Pulpzyme HB preparation is derived from a strain of bacterial origin, which contains an endo-l,4-p-D-xylanase with virtually no cellulase activity. Pulpzyme HC contains an endo-l,4-P-D-xylanase obtained from submerged fermentation of a selected strain of Bacillus. Irgazyme 40S-4X contains a xylanase concentrated from Trichoderma longibrachiatum (formerly T. reesei). 2.1.1 Xylanase Activity Xylanase activity was determined using 50 mM Tris buffer at pH's 6, 7 and 8. Initially, serial dilutions were used to determine the range of activity for each enzyme. A more narrow set of dilutions was then used to quantify the actual activities of each xylanase. All enzyme activities were done in replicate. The same assays were then conducted using an unbuffered, pH adjusted, system in order to determine the activity of each xylanase in water. The activity determined in buffer at pH 7 was used to determine the enzyme loading for the bleaching experiments. Xylanase activity was expressed in nkatals, which represents the release of 1 nmol of xylose equivalents from the xylan substrate per second (nmol/s). The DNS xylanase assay was used to determine xylanase activity (Bailey et al, 1992). This assay is used to detect reducing sugars, such as xylose, that are released during an enzyme reaction on a specific substrate. Representative standards are run concurrently in order to determine relative carbohydrate concentrations. Each enzyme dilution was made up using either buffer or water and stored on ice. Reaction tubes containing 900 uL of appropriately pH adjusted 1 % birchwood xylan (Sigma Chemical, St. Louis, USA) were brought to 50 °C in a water bath. To start the reaction, 100 uL of diluted enzyme were added to the prewarmed substrate. After 31 exactly 5 minutes the reaction was stopped by adding 1.5 mL of the DNS solution. Enzyme blanks (100 uL of highest enzyme dilution) and xylose standards (100 uL containing 0-1.0 umol xylose) were added to incubated substrate after the DNS was added. All of the test tubes were then placed in a boiling water bath for 5 minutes and then cooled in a water bath. The absorbance of each reaction mixture was measured using a Milton Roy 1001 plus spectrophotometer (Rochester, New York) at a wavelength of 540 nm using acryl-cuvettes with a 1 cm path length. 2.1.2 Optimal pH for Xylanase Activity The DNS xylanase assay was modified to determine optimal pH for xylanase activity by using a broad range buffer (0.05-0.05-0.1 M acetic acid-Mes-Tris). All pH adjustments were carried out using sulfuric acid (H2SO4) or sodium hydroxide (NaOH) immediately before the reaction. The pH's tested ranged from 4.0 to 11.0. Enzyme blanks were run concurrently for each pH tested. The optimal pH was also determined in an unbuffered system where there tended to be a pH shift in the substrate towards pH 7 over the course of the reaction. In these experiments, the final pH of the reaction was used for evaluating optimal pH for the activity of each of the xylanases. Optimal pH assays in unbuffered xylan substrate were carried out twice. 2.1.3 Other Enzyme Activities Cellulase and mannanase activities were assayed as above except that 1% carboxymethyl-cellulose (CMC) (medium viscosity; Sigma Chemical, St. Louis, USA) and 1% galactomannan (low viscosity, derived from carob bean; Megazyme, Sydney, Australia) were used, respectively. Optimal pH ranges for Irgazyme 40 and Pulpzyme HB were also determined for these substrates. Much higher enzyme concentrations (lOOx more concentrated than those used for the xylanase assay) were required to observe the small amount of activity present. 32 2.2 K R A F T PULPS Unbleached kraft pulps derived from Douglas-fir (Pseudotsuga menzesii) and Western red cedar (Thuja plicata) were produced at the Crofton mill (Fletcher Challenge) in British Columbia which uses a batch cooking process. Unbleached kraft pulp was produced from Western hemlock (Tsuga heterophylla), using a modified continuous cook process, by the Howe Sound Pulp & Paper mill located in Port Mellon, British Columbia. These three pulps were selected for this research because they are all found in British Columbia, and are often used by the pulp and paper industry as a wood fiber source. Because these pulps were provided by commercially operating mills, there may be some question as to their exact wood species content. Although it as been assumed that the pulps used for this research were derived from the individual wood species it is possible that the Western hemlock pulp is a mixture of hemlock and true fir (Abies) that is commonly produced on the West Coast. Initial kappa numbers and brightnesses for these three pulps are shown in Table 1. 2.3 B L E A C H I N G CONDITIONS Two different bleaching sequences, XQPP and QPXP, were carried out on the brownstock and oxygen delignified kraft pulps. All of the bleaching sequences (except oxygen delignification) were done in heat sealable polyester bags in an appropriately heated water bath. All pH adjustments were done using a 1 % pulp slurry in deionized water adjusted to the appropriate pH with stirring for 1 hour. The conditions for the xylanase treatment (X) were start pH 7, no buffer, 10 % pulp consistency, 200 nkat xylanase/g pulp, 50 °C, for 1 h. The conditions for chelation (Q) were, start pH 5.5, 3 % consistency, 1 % Na2-EDTA-2H20,50 °C, for 0.5 h. Those for peroxide bleaching (P) were, 10 % consistency, 0.05 % MgS04,2 % NaOH, 2 % H 2 0 2 ,80 °C, 3 h. Oxygen delignification (O) was conducted using a Mark IV High Intensity Quantum Mixer/Reactor (Quantum Technologies Inc., Twinsburg, Ohio). The mixer settings for each reaction were 0.5 second ramp time and 5 second mix time in manual mode. The 5 second mixes 33 were done manually every 15 minutes starting at the beginning of the reaction (4 mixes in total). For each oxygen delignification run, 240 g dry weight equivalent of the pulp was placed in the full size bowl (4 L). The conditions used were 10 % consistency, 0.3 % MgS04-7H20,1.5 % NaOH, 70 psi 0 2 , 90 °C, 1 h. For the peroxide stage the concentration of peroxide added to the pulp was determined in triplicate by iodiometric titration as follows. An approximate 3 % peroxide solution was made by volumetrically diluting a 30 % solution 10 fold. One mL of this diluted peroxide stock solution was then added to 300 mL Erlenmeyer flasks containing 50 mL water and 10 mL 10 % H2SO4. Exactly 2.00 mL of 10 % potassium iodide (KI) was added to each flask. Three to five drops of aluminum molybdate was added to each flask prior to titration with 0.100 N thiosulphate (Na2S203), using starch as an indicator. Tine 3% peroxide stock was stored on ice in a capped volumetric flask prior to dilution for each bleaching reaction (2 % on pulp basis). 2.4 PREPARATION OF PULP AND FILTRATE SAMPLES Figure 10 describes the different xylanase insertion sites and the filtrate and pulp sample collection sites that were carried out for this study. Prior to conducting the bleaching trials, the activities of the two xylanases, Pulpzyme HB and Irgazyme 40, were tested after chelating with EDTA on Douglas-fir kraft pulp in order to ensure that these enzymes were still active following chelation under our experimental conditions. Chelation had no effect on the xylanase activity of these two enzymes. Pulpzyme HB and Irgazyme 40 were also applied to Douglas-fir kraft pulp after chelation to identify any effects that the two enzymes may have on the following peroxide stage. There appeared to be no effect on peroxide bleaching from these two enzymes even if the pulps were not washed between the xyalanse and peroxide stage. 34 Table 1. Initial kappa number and brightness values for brownstock and oxygen delignified kraft pulps. Two oxygen delignification trials were run for each pulp type and the average values with respective ranges for the resulting kappa number and brightness values are shown. Brightness (% ISO) kappa number Douglas-fir Brownstock 27.9 23.8 Oxygen delignified 35.8 ± 0.5 12.1 ± 0.3 Western hemlock Brownstock 27.7 22.0 Oxygen delignified 31.7 ±1.4 11.8 ±0.4 Western red cedar Brownstock 28.2 23.4 Oxygen delignified 34.5 ±1.1 12.0 ±0.8 35 2.4.1 Filtrate Samples Filtrate samples were taken after the enzyme stage and each bleaching stage. Approximately 100 mL of filtrate from the xylanase stage were collected and divided into two, 50 mL samples, which were used for subsequent toxicity analysis and other characterizations. The 50 mL portion of the xylanase filtrate used for total sugar and UV-absorbing material determination was boiled for 10 minutes in order to ensure enzyme inactivation. The final pH of unboiled enzyme filtrates was measured immediately after each enzyme sequence. Following the chelation and peroxide stages, about 100 mL and 50 mL of the filtrates were collected, respectively. From the peroxide filtrates, 25 mL were titrated to determine the level of residual peroxide. Filtrates from all the bleaching stages were stored at 4 °C until testing. Those filtrates used for toxicity tests were stored without an air space. Each replicate experiment had three treatments: one for each of the three enzymes, and one control. The control was treated in parallel and received water instead of enzyme during the enzyme stage. 2.4.2 Pulp Samples Pulp samples were collected after the xylanase stage and after the two peroxide stages. After each bleaching stage the filtrate was collected and the pulp cake was mixed at 1 % consistency in water, drained, and the wash water passed through the pulp cake to collect the fines. This was done to ensure proper washing between stages (especially following the chelation stage). The pulp samples collected after a peroxide stage were adjusted to pH 4.5 in a 1 % pulp slurry using H2SO4 and then drained. The pulp samples were stored in the dark at 4 °C until testing. 36 kraft pulp brown stock 1 xylanase (X) oxygen de - • xylanase • (X) ignification (0) chelation (Q) peroxide (P) — • peroxide (P) pulp samples brightness - after each stage except Q. kappa number - after X zero span breaking length - after X solubilized materials - after X - total sugars filtrate samples s : - UV-absorbing material residual peroxide - after P toxicity - after each stage Figure 10. Description of bleaching sequences and collection sites for filtrate and pulp samples 37 2.5 PULP CHARACTERIZATION 2.5.1 Brightness Pulp brightness was determined using 4 g handsheets made according to CPPA standard C.5. The type of apparatus used for determining the brightness of the handsheets was a Technibrite Micro TB-1C made by the Technidyne Corp. (New Albany, Indiana). All physical tests were carried out at standard conditions and atmosphere (temperature of 23.0 ± 1.0 °C and relative humidity of 50.0 ± 2.0 %). 2.5.2 Kappa Number Residual lignin in the pulp samples collected immediately after the enzyme stage was measured using the microkappa method (TAPPI Useful Method UM 246, 1991). The 4 g handsheet made for brightness determination was used to determine the kappa number. Approximately 0.3-0.55 g of each handsheet was required for the test, with the higher amount required for the partially bleached pulp samples. Samples for dry weight determination were measured out concurrently with samples used for kappa number determination. For disintegration, 50 mL deionized water was used initially and then another 30 mL total was used for three small washes in order to remove all the fibers from the stainless steel blender. Disintegrated pulp and washes were placed in 300 mL erlenmyer flasks and 10 mL of 4 N H2SO4 was added. Water blanks consisted of 80 mL deionized water and 10 mL 4 N H2SO4. Two water blanks were used to bracket each series of eight pulp samples. The flasks were placed in a 25.0 ± 0.1 °C shaking water bath and allowed to come to temperature. The reaction was started by adding 10.00 mL of 0.100 N potassium permanganate (KMnG*4) and stopped after 10 minutes by adding 2.00 mL of 1.0 N KI. Each flask was titrated with 0.100 N Na2S203 using starch as an indicator. Kappa numbers were calculated using TAPPI Classical Method T 236 cm-85. 38 2.5.3 Zero Span Breaking Length Physical testing handsheets weighing 1.2 g were prepared following Tappi standard TAPPI T 205 using 1500 revolutions in a British disintegrater (Pointe Claire, Quebec). Before any handsheet was used it was first placed on a back lit table to detect defects in the handsheet that should be excluded from the test. From these handsheets, 5 squares (2.5 cm x 2.5 cm), were cut at different angles. The type of zero span apparatus used for the measurements was a Trouble Shooter (Pulmac Instruments, LaSalle, Quebec). For each different pulp, 5 tests were conducted in a humidity and temperature controlled environment. After the test, each sample square was oven dried and the average moisture-free basis weight of the sample in grams per square meter (g/m^) was determined. The calculated zero span breaking lengths for each sample were presented in kilometers (km) 2.6 X Y L A N A S E FILTRATE A N A L Y S E S 2.6.1 Solubilized Sugars The phenol-sulfuric acid method (Dubois et al, 1956) was used to determine the total sugars solubilized by each enzyme. For this assay the boiled enzyme filtrates were diluted four fold by mixing 50 uL of each sample in 150 uL deionized water. Standards contained 0-0.20 umol xylose. One of the replicates was spiked with 0.04 umol xylose in order to evaluate the occurrence of any assay interference. A water blank was used to zero the spectrophotometer set at a wavelength of 490 nm (cell path of 1 cm). An additional blank for each sample was also prepared by omitting the phenol in the reaction. 2.6.2 UV Absorbing Materials To measure the material absorbing at 205 and 280 nm, the filtrate was diluted using deionized water, to give an absorbance ranging from 0.3-0.7 for a path length of 1 cm. The 39 "relative absorbance" was calculated by multiplying the measured absorbance by the dilution factor and then subtracting the enzyme blank. 2.6.3 Residual Peroxide For each sample, 25 mL of filtrate was added to 50 mL deionized water and 10 mL 10% H2SO4 in a 300 mL flask, and subsequently reacted with 2.00 mL 10 % KI. To this mixture several drops of 10 % aluminum molybdate was added. Each filtrate was then titrated with 0.100 N thiosulphate, using starch as an indicator. 2.6.4 Toxicity Analysis of the Filtrates ' The unboiled xylanase filtrate was tested for toxicity. The combined filtrates were obtained by mixing the filtrates collected from each bleaching stage in volumes proportional to those used during each stage (Table 2). For the combined filtrates a final volume of 25 mL was used for all the toxicity tests. 2.6.4.1 Daphnia magna 48 Hour LC50 Assay Tests were conducted using 150 ml beakers that were well rinsed with deionized water and then with aerated Daphnia water. The aerated Daphnia water was prepared with deionized water brought to a moderate hardness of 80-100 mg/L CaCC>3 by adding sodium carbonate, calcium sulfate, magnesium sulfate and potassium chloride. Only Daphnia magna neonates, under 48 hours old, that had not been fed for 12 hours were used for the assay. The pH and dissolved oxygen content of all samples were measured before and after the 48 hour assay. Prior to testing, samples were allowed to warm to room temperature. The combined filtrate samples were also adjusted to approximately pH 7 with HC1 prior to testing. The undiluted xylanase filtrate (50 mL) was first tested for toxicity by using 5 neonate Daphnia. 40 Table 2 Percent of each bleaching filtrate used to make combined filtrates used for toxicity testing. Bleaching Stage Pulp consistency Water in bleaching stage % of combined filtrate Enzyme (X) 10% 9 ml/g pulp 15.8% Chelation (Q) 3% 32.3 ml/g pulp 52.6% Peroxide (PI) 10% 9 ml/g pulp 15.8% Peroxide (P2) 10% 9 ml/g pulp 15.8% 41 If any mortalities occurred in the undiluted filtrate (100%) then the samples were diluted using aerated hardened water to the logarithmic concentrations suggested by Environment Canada (EPS, 1990); those being 56% and 32%. The same hardened water was used for the controls. Combined filtrates were tested after being diluted to 5.6%, 3.2% and 1%. All procedures used for the Daphnia magna assay were done in accordance with Environment Canada standards. The xylanase filtrates were reported as having LC50's greater or less than 100% (i.e. nontoxic or slightly toxic). 2.6.4.2 Microtox 5 Minute EC50 Assay For each sample, 5 mL of the filtrate were adjusted to 2% saline using analytical grade sodium chloride (NaCl). The samples were then stored in capped 5 mL test tubes at 4 °C until testing. The methods used for the Microtox™ assay followed the manual published by Environment Canada (EPS, 1992) as well as the manual provided by Microbics (Microbics, 1992). A 2% NaCl solution made by adding 10 grams NaCl to 500 mL deionized water was used for all dilutions and the controls. Microtox™ reagent, consisting of freeze dried photoluminescent bacteria, was kept at -80 °C until needed. A 10-50 uL repeater pipette was used to dispense 10 uL of the Microtox reagent into each standard Microtox™ glass cuvette. The dilution series used was 50%, 25%, 12.5% and 6.25% for the xylanase filtrates, and 12.5%, 6.25%, 3.125% and 1.563% for the combined filtrates. The results for this 5 minute EC50 assay were analyzed using the calculations described by Microbics (Microbics, 1992). 42 3. RESULTS AND DISCUSSION 3.1 E N Z Y M A T I C C H A R A C T E R I Z A T I O N O F T H R E E C O M M E R C I A L X Y L A N A S E PREPARATIONS 3.1.1 Background Before testing their effectiveness on kraft pulp, the three commercial xylanases were characterized for their xylanase, mannanase and cellulase activities. Initially, Irgazyme 40, Pulpzyme HB and Pulpzyme HC were tested over a dilution range to determine xylanase activity in a buffered system using 1% birchwood xylan. This same assay was also repeated, in an unbuffered solution. An appropriate dilution was chosen for each enzyme to evaluate the optimal pH for xylanase activity. Irgazyme 40 and Pulpzyme HB were also tested for mannanase and cellulase activity over the same pH range, but 100 times more enzyme was required to observe any hydrolysis of these two substrates. Pulpzyme HC is marketed as having negligible contaminating cellulase, therefore, cellulase activity was only determined at pH's 6, 7, and 8 in buffered and unbuffered systems. This determination is important as the presence of cellulase can cause significant damage to the pulp fibers, decreasing the strength and quality of the final pulp by destroying the long cellulose chains (Bajpai et al, 1993). 3.1.2 Xylanase Activity Using the activity assays with birchwood xylan as the substrate, Irgazyme 40 was found to have the most concentrated xylanase activity (0.358 nkat/nl), followed by Pulpzyme HB (0.259 nkat/nl) and Pulpzyme HC (0.095 nkat/nl). For all three enzymes, xylanase activities in the unbuffered conditions were slightly greater than those in the buffered conditions. It is probably not correct to directly compare the activity on isolated xylan to that of the xylan within the pulp fiber which is closely associated with lignin, cellulose and other hemicelluloses in the fiber matrix. For this reason, it is difficult to assume that the activity determined for the xylanases on 43 the isolated substrate is the same as that achieved on the pulp. Furthermore, there is often a shift in the pH of the pulp mixture during the 1 hour xylanase treatment that is not seen during the 5 minute DNS assay using a buffered system to determine enzyme activity. It is probable that a shift of 1 pH unit will affect the activity of the xylanase over the 1 hour reaction time. Although it is recognized that there are drawbacks to using an isolated xylan substrate as a means of determining enzyme activity, it is generally accepted that this is one way to standardize the xylanase loadings used for laboratory studies. The three commercial enzymes have different optimal pH's for xylanase activity (Figure 11). Pulpzyme HC was found to have an optimal pH of about 8 for xylanase activity on buffered isolated birchwood xylan at 50°C. The optimal pH for xylanase activity in Pulpzyme HB and Irgazyme 40 activities in a buffered solution were 6.5 and 4-6, respectively. These activities appeared to shift upwards by 0.5-1 pH units when the buffer was absent (Figure 12). Buchert et al., (1992) noticed that the optimal pH for activity of a xylanase shifts up when tested on fiber bound xylan (i. e. pulp), in comparison to values obtained with isolated xylan (such as birchwood xylan). The improvement in xylanase activity for Irgazyme 40 at pH 4 could be due to the presence of more than one xylanase active site within the enzyme preparation. After determining the optimal pH ranges, it was decided that pH 7 would be the most representative for studying the three commercial enzymes. For the bleaching trials the xylanase loading of 200 nkat/g pulp was calculated using values determined in pH 7 buffer. The three xylanases were relatively active from pH 6-8, which allowed for some shifting of the pH without significant inactivation of the enzyme. 3.1.3 Cellulase and Mannanase Activity At the enzyme dilutions used for the xylanase assays, there was negligible cellulase and mannanase activity in both the Irgazyme 40 and. Pulpzyme HB preparations. To try to ensure that there were no detrimental effects due to contaminating enzyme activities, Pulpzyme HB and Irgazyme 40 were added to 1 % CMC and 1 % galactomannan solutions at concentrations 50 and 100 44 times higher, respectively, than would normally be added to the pulp. Negligible activities were again detected (Figure 11). Pulpzyme HC had no cellulase activity when loaded at full strength onto the CMC substrate for 5 minutes under the conditions described for the DNS assay. Cellulase is usually considered to be destructive to the pulp fiber and minimal cellulose degradation would be desirable. Although, some cellulase activity may enhance the physical properties of the pulp such as porosity and freeness, there is probably a corresponding decrease in strength. These effects appear to be dose related therefore it may be possible to determine an optimal cellulase loading that enhances final pulp quality without compromising pulp strength (pers. comm. Shawn Mansfield, PhD student, UBC). Controversy exists concerning the effects that different enzymes can have on kraft pulp fibers. Paice et al. (1992) found that xylanases are much more effective than mannanases in enhancing the bleaching of both hardwood and softwood kraft pulps. However, it has been suggested that mannanase may enhance xylanase action on pulp in a synergistic (Yang and Eriksson, 1992b) or additive (Buchert et al., 1993) manner. This initial characterization of the three commercial xylanase preparations provided relevant information on the possible effects that these enzymes may have on a more complex substrate such as kraft pulp. In the following section these three enzymes are loaded onto kraft pulps derived from three different wood species and assessed for possible enhancement of TCF bleaching. As previously discussed (section 1.3), the kraft pulping process causes significant changes within the pulp fiber matrix. The hemicellulose within the kraft pulp is chemically and structurally modified and this change may affect the action of the xylanase on the xylan fraction. By looking at the different kraft pulps, and different bleaching sequences, a more detailed understanding of the effectiveness of these three xylanases on kraft pulp should be achieved. 45 pH t>0 « re O 3 3 t>0_ £ o 5 a OS 1.4 1.2 - -1 - -0.8 0.6 0.4 0.2 Irgazyme 40 (1/1000) Pulpzyme HB (1/500) 10 1.4 • oi « > S 3 c t r o> o> tsO oi « « 3 C C 60 " G _ •S ° 3 g 13 a. 0 01 1.2 • 1 • 0.8 • 0.6-0.4 • 0.2 -Irgazyme 40 (1/1000) Pulpzyme HB (1/500) • — • — » 10 Figure 11. Comparison of xylanase, cellulase and mannanase activities over a pH range for three commercial xylanase preparations. The different enzyme dilutions used for the different substrates are shown in parenthesis. 46 Figure 12. The optimal pH for xylanolyric activity in Pulpzyme HB and Irgazyme 40, in buffered and unbuffered conditions. When no buffer was used, the final pH was monitored. When present, error bars show the range from replicates. 47 3.2 T H E EFFECTIVENESS OF C O M M E R C I A L XYLANASES IN PEROXIDE B L E A C H I N G OF K R A F T PULP 3.2.1 Background The effectiveness of xylanase pretreatment in bleaching sequences with elemental chlorine (Tolan and Canovas, 1992; Senior et al, 1992) and chlorine dioxide (Turner et al., 1992; Scott et ai, 1993; Allison et ai, 1993) has been well documented. As previously mentioned (section 1.4.2), the pulp and paper industry is continuously being pressured to use more environmentally friendly bleaching sequences that include little or no chlorine containing compounds for the production of bleached kraft pulp. There have only been a few references that discuss the effectiveness of xylanases in a TCF bleaching sequence (Ragaukas et al., 1994; Yang et ah, 1994; Allison and Clark, 1994). As part of the worldwide, ongoing, evaluation of the potential for xylanase prebleaching in the pulp and paper industry it was decided to focus on the effectiveness of several commercial xylanases in a peroxide based TCF bleaching sequence. There were three main objectives to the initial part of this work. The first section dealt with the effects that xylanase had on two different kraft brownstocks, derived from Douglas-fir and hemlock wood chips. The pulp was either directly treated with xylanase and then bleached using a chelation stage followed by two peroxide stages (XQPP), or partially bleached with peroxide, treated with xylanase and then a final peroxide stage (QPXP). Results from the two different xylanase insertion sites were compared to see if partial bleaching of the pulp with a chelation stage and peroxide changed the effectiveness of the xylanase treatment. In the past, research has looked at the effects of xylanase on pulp derived from one particular wood species, or on pulp derived from a mixture of wood species that were commonly produced by a commercial pulp mill. Previous results indicate that the xylanase preparations currently available are able to improve the brightness of the pulp slightly, without damaging strength quality. Prior to the work carried out for this thesis, 48 there had not yet been a direct comparison of the effectiveness of different xylanases on several pulps derived from defined wood species. The second section describes a comparison between the effectiveness of xylanase on brownstock and oxygen delignified kraft pulp derived from Douglas-fir. Most of the protocol used for the brownstock were taken from the first section, and the same two enzyme insertion sites were used. This experiment was done in order to determine if the three commercial xylanases were as effective on an oxygen delignified kraft pulp as on the previously tested brownstock. The final section compares the effectiveness of xylanase pretreatment on oxygen delignified kraft pulps derived from three different wood species. The results are also compared to those obtained for the different brownstocks. Related research is also discussed in order to evaluate the overall effectiveness of xylanase treatment in a peroxide based TCF bleaching sequence. 3.2.2 The Effects of Xylanase on Brownstock Pulps Derived from Two Different Softwood Species 3.2.2.2 Pulp Brightness This first bleaching experiment was carried out on brownstocks derived from Douglas-fir and hemlock. The final pH's of pulp treatments using Irgazyme 40 and Pulpzyme HB were similar for the same experiment. It therefore appeared that the xylanase treatments were carried out with similar activities of the two xylanase preparations. However, the activity was assayed using an isolated xylan so that it does not necessarily correspond to the possible effects on pulps. Under the defined conditions, the two xylanases differed in both their ability to enhance peroxide bleaching of kraft pulps derived from the two wood species examined, and in their effectiveness when added at two insertion sites in the bleaching sequence (Nelson et ai, 1995a). 49 Differences between ivood species The initial brightness of the brownstock from Douglas-fir was 1 % ISO higher than that from western hemlock and the final brightness of the former was 4 % ISO higher than the latter (Figure 13). It would appear that Douglas-fir was more readily bleached with the same charge of peroxide. However, this species showed a lower response to xylanase treatment. Enzymatic enhancement of its bleaching was observed in only one case, when the brownstock was treated with Irgazyme 40. For hemlock, all four cases showed a brightness increase that ranged from 0.9-2.2 % ISO. This is comparable to the results obtained in a TCF mill trial carried out at the Korsnas mill where a xylanase treatment of the brownstock resulted in a brightness enhancement of 2 % ISO (Lundgren et ai, 1994). For the brownstocks from both wood species, there was no immediate increase in brightness after xylanase treatment. In these cases xylanase treatment only enhanced subsequent chemical bleaching. However, the enzymes did directly increase the brightness of pulp that had been partially bleached with one peroxide stage (QPX). This brightness increase ranged from 0.8-1.4 % ISO in three of the four cases. With western hemlock, this direct brightening corresponded to an enhancement of the final pulp brightness. Differences between insertion sites One difference between the XQPP and QPXP sequences was that the xylanase could directly brighten the partially bleached pulp in the latter sequence (Figure 13). However, this enhancement of pulp bleaching did not translate to a superior final brightness. Indeed, the final brightness achieved with the QPXP sequence was consistently lower than those seen for the XQPP sequence. In the case of Douglas-fir, this may be due to the absence of the extra wash before chelation. However, all hemlock pulps showed a'similar brightness after the first peroxide stage, and subsequent neutralization appeared to reduce the brightness gain in the second peroxide stage. It is possible that metal ions are modifying the bleaching of the pulp, as well as the action 50 that xylanase has on kraft pulp (Buchert et al., 1993b). Transition metal ions such as iron, manganese and copper greatly accelerate the decomposition of the active component responsible for the brightening action of peroxide (van Lierop et al., 1994). Metal ions occurring naturally in the wood are the main source, with secondary contamination arising from process water and equipment. Differences between enzymes The two xylanase preparations (Pulpzyme HB and Irgazyme 40) were similar in that neither could directly brighten the brownstock. Irgazyme 40 appeared to be the more reliable enzyme because it helped to generate the highest brightness in both kraft pulps (Figure 13). Pulpzyme HB enhanced the final brightness of kraft pulp from hemlock but not that from Douglas-fir. The final pH's for the enzyme stage (Douglas-fir - XQPP: 6.5-7.2; QPXP: 7.3-7.7; hemlock - XQPP: 7.8-8.4; QPXP: 8.4-8.7) were within 0.4 pH units in each experiment. 3.2.2.2 Change in Kappa Number When measured immediately after enzyme treatment, the kappa number of the pulps showed a decrease of 0.4-1.2 pts in seven of the eight cases (Figure 14). It appears that xylanase treatment often facilitates lignin solubilization. Irgazyme 40 consistently had the greater effect. However, direct delignification did not necessarily correspond to an enhancement of final pulp brightness. The decrease in lignin content appeared to be independent of the bleaching sequence used, showing a similar magnitude after the first peroxide stage had decreased the kappa number by about 10 pts. 3.2.2.3 Materials Solubilized During Xylanase Treatment The solubilization of sugars and UV-absorbing material from pulp confirmed that the enzymes were active in all experiments (Figure 15). Irgazyme 40 appeared to have a greater effect on solubilization of these materials. This was not expected due to the high final pH that 51 results in most treatments and the lower pH optimum that was previously determined for this enzyme preparation (Figure 11). The partially bleached pulps also released more UV-absorbing material during xylanase treatment, even though less material was released during the control treatment. The solubilization of UV-absorbing material may be related to the direct delignification observed in these pulps. However, there appeared to be no correlation between the consistent increase in solubilized materials after xylanase treatment and pulp brightness, which did not increase for all treatments. 3.2.2.4 Peroxide Consumption During Bleaching Although xylanase treatment increased peroxide consumption in many cases (Figure 16), it did not necessarily lead to higher pulp brightness. Even when comparing the control pulps, high peroxide consumption appeared to be related to lower brightness, suggesting that it may be the result of peroxide instability. Peroxide stability has been shown to be greatly dependent on the efficiency of metal ion removal prior to peroxide bleaching (van Lierop et al., 1993). If the metal ions are not removed then they may react with the peroxide making it unavailable for pulp bleaching. This may be one reason for the variable residual peroxide values between treatments. For most of the bleaching sequences in this experiment it appeared that Irgazyme 40 increased peroxide consumption more than Pulpzyme HB. 52 Douglas-fir-XQPP Douglas-fir-QPXP stage control J X 28.8 ± 0.1 t 0 XQP 52.6 ± 0.3 L D XQPP 65.6±0.9 SlSflg control QP 49.6 ± 0.1 (3 QPX 49.3 ± 0.0 • QPXP 63.1 ±1.1 hemlock - XQPP hemlock-QPXP S> 3 h c ro stage control 1 X 27.5 ± 0.2 t • XQP 49.4 ± 0.8 • XQPP 61.8 ±1.1 stage control QP 49.9 ± 0.7 • QPX 50.4 ± 0.4 • QPXP 58.6 ±1.7 Pulpzyme HB Irgazyme 40 Enzyme Pulpzyme HB Irgazyme 40 Figure 13. The effect that Pulpzyme HB and Irgazyme 40 have on the pulp brightness achieved during peroxide bleaching of kraft pulps derived from Douglas-fir and western hemlock. The enzyme treatment was carried out on the brownstock (XQPP) and the partially bleached pulp (QPXP). Pulp brightness after each bleaching stage was compared to the corresponding control pulp. The bars show the range from two replicate experiments. 53 S> 0-5 c CO 5 0 0 H -0.5 -1.0 -1.5 -2.0 -2.5 species conlrol | Douglas-fir 122 ± 0.2 t U hemlock 13.6 ±1.3 QPXi Pulpzyme HB Enzyme Irgazyme 40 Figure 14. The direct effect that Pulpzyme HB and Irgazyme 40 have on the kappa number achieved during peroxide bleaching of kraft pulps derived from Douglas-fir and western hemlock. The enzyme treatment was carried out on the brownstock (XQPP) and the partially bleached pulp (QPXP). Pulp brightness after each bleaching stage was compared to the corresponding control pulp. The bars show the range from two replicate experiments. 54 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Douglas-fir - X Douglas-fir - QPX control F- • sugars 0.37 ± 0.13 0 A280 1.6 ± 0 . 2 Eg A205 7.0+ 1.1 hemlock - X control • sugars 0.33 ± 0.04 0 A280 4.6 ± 0.5 gjj A205 16.5 + 03 conlrol t- Q sugars 0.41 ± 0.04 • A280 0.4 ± 0.0 gg A205 1.8 ± 0 . 4 hemlock-QPX control • sugars 033 ± 0.03 0 A260 0.3 ± 0 . 1 E | A205 1.6 ± 0 . 6 Pulpzyme HB Irgazyme 40 Pulpzyme HB Enzyme Irgazyme 40 30 -3 io Figure 15. The direct effect that Pulpzyme HB and Irgazyme 40 have on the solubilization of sugars and UV-absorbing materials released during peroxide bleaching of kraft pulps derived from Douglas-fir and western hemlock. The enzyme treatment was carried out on the brownstock (XQPP) and the partially bleached pulp (QPXP). The amount of solubilized materials released after each bleaching stage is compared to the corresponding control pulp. The bars show the range from two replicate experiments. 55 J Douglas-fir - XQPP Douglas-fir-QPXP • Slaflfi control 79 ± 6 71 ±14 T 1 LE3 P, • P. Slaofi control 95 ± 2 85±11 hemlock - XQPP hemlock-QPXP Pulpzyme HB Irgazyme 40 Pulpzyme HB Enzyme Irgazyme 40 Figure 16. The effect that Pulpzyme HB and Irgazyme 40 have on peroxide consumption during peroxide bleaching of kraft pulps derived from Douglas-fir and western hemlock. The enzyme treatment was carried out on the brownstock (XQPP) and the partially bleached pulp (QPXP). Residual peroxide after each bleaching stage was measured, and those results for enzyme treatment are compared to those for the corresponding control pulps. The bars show the range from two replicate experiments. 56 3.2.3 Comparison of Xylanase Prebleaching of Brownstock and Oxygen Delignified Kraft Pulp Derived from Douglas-fir 3.2.3.2 Pulp Brightness The three xylanases (Pulpzyme HB, Pulpzyme H C and Irgazyme 40) varied in their ability to improve the brightness of kraft pulp derived from Douglas-fir. Oxygen delignification did not appear to diminish the effectiveness of the xylanase pretreatment whether it was inserted before or after the first peroxide stage (Nelson et al, 1995b). Comparison between brownstock and oxygen delignified kraft pulps For both the brownstock and the oxygen delignified kraft pulp, the partially bleached pulp (OQPX and QPX) showed a direct brightening effect after the xylanase treatment (Figure 17). It also appeared that the addition of a peroxide stage before xylanase treatment may cause a more consistent brightness gain for oxygen delignified Douglas-fir pulp. When the xylanase treatment directly followed oxygen delignification (OX), only Irgazyme 40 improved pulp brightness. Irgazyme 40 was also the only enzyme that enhanced pulp brightness when applied to the brownstock prior to any peroxide bleaching (XQPP). Comparison between insertion sites For both insertion sites, oxygen delignification led to an increase in final brightness of about 6 % ISO. After each peroxide stage, the brownstock and oxygen delignified pulps bleached with XQPP were consistently 2 % ISO brighter than those bleached with QPXP (Figure 17). It appeared that all three xylanases cause a greater direct brightening effect when inserted after one peroxide stage (QPX). Direct brightening of the oxygen delignified pulp was also observed for the two enzyme insertion sites. These results indicate that partial removal of lignin by both oxygen delignification and peroxide seem to enhance the direct brightening effect that xylanase has on a kraft pulp derived from Douglas-fir. 57 Comparison of enzymes Under our experimental conditions, the three xylanases showed variable capabilities of enhancing peroxide bleaching of both the brownstock and the oxygen delignified kraft pulp (Figure 17). The highest brightness, 73.3 % ISO, was reached using the OXQPP sequence where Irgazyme 40 enhanced brightness by 1.6 % ISO. Another study has also indicated that xylanase can enhance an OQP bleaching sequence by increasing pulp brightness by about 1 % ISO (van Lierop et ai, 1994). 3.2.3.2 Change in Kappa Number With the exception of the oxygen delignified (OX) and the peroxide bleached (QPX) pulps treated with Pulpzyme HB, xylanase treatment directly decreased the kappa number of both the brownstock and the oxygen delignified pulps by more than 0.5 points (Figure 18). Pulpzyme HC and Irgazyme 40 appeared to be the more effective enzymes in lowering the kappa number of both brownstock and oxygen delignified kraft pulp derived from Douglas-fir. All three enzymes appeared to be able to directly delignify the pulp when inserted after the first peroxide stage (QPX), even after the pulp was first oxygen delignified. 3.2.33 Materials Solubilized During Xylanase Treatment The solubilization of sugars and UV-absorbing material from the pulp during the xylanase treatment is shown in Figure 19. The net increase in these materials confirmed that the enzymes were active during pulp treatment. In most cases, the enzymes solubilized more UV-absorbing material from the oxygen delignified and peroxide bleached pulps than from the corresponding brownstock. The increase in UV absorbance of the xylanase filtrate may be indicating an enhanced release of lignin-like material during the xylanase treatment. In all cases there was a corresponding increase in solubilized sugars. -58 3.2.3.4 Peroxide Consumption During Bleaching After measuring the residual peroxide after each P stage it was apparent that whether or not the xylanase treatment was inserted before the P| stage, more peroxide was consumed during P| for the brownstock than the corresponding oxygen delignified pulp (Figure 20). Furthermore, with or without oxygen delignification, it appeared that the QPXP sequence consumed a greater amount of peroxide than the XQPP sequence. It is possible that the enzyme preparations contain something (i. e. metal ions) that may be inhibiting peroxide bleaching, or that adjustment to pH 7 after the first peroxide stage may cause the release of something that inhibits the second peroxide stage. 59 stage control "11 X 28.8 ±0.1 E3 XQP 52.6 ± 0.3 . • XQPP 65.6 ±0.9 XQPP 1— Slaflfi control QP 49.6 ± 0.1 [3 QPX 49.3 ± 0.0 . f j QPXP 63.1 ±1.1 Q P X P . • 1 staoe control ! M OX 35.8 ± 0.7 E3 OXQP 63.0 ± 0.7 F-D OXQPP 71.7 ±1.0 OXQPP J stage control OQP 61.1 ±0.9 Q OQPX 61.4 ±1.3 • OQPXP 69.8 ±1.2 OQPXP. Pulpzyme HB Pulpzyme HC Irgazyme 40 Enzyme Pulpzyme HB Pulpzyme HC Irgazyme 40 Figure 17. The effects the three commercial xylanases have on the pulp brightness during peroxide bleaching of kraft pulps derived from Douglas-fir. The enzyme treatment was carried out on the brownstock and the oxygen delignified kraft pulp before (XQPP) and after (QPXP) the first peroxide stage. Each bleaching sequence is identified at the upper right corner of each set of histograms. Pulp brightness after each stage is compared to the corresponding control pulp. The bars show the range from two replicate experiments. Only one experiment was performed with Pulpzyme HC for the OXQPP sequence. 60 QJ S3 E 3 Z CO CL CL CO CD c CO .c O 03 0.5 0.0 -0.5 -1.0 -1.5 -2.0 -2.5 0.5 0.0 -0.5 -1.0 -1.5 -2.0 -2.5 x/ox m i Slafls control ' ^ X 23.3 ± 0.4 [gj OX 12.0 ± 0.4 I QPX/OQPX ; sJaaa control ~M QPX 1 2 7 ± 0 . 2 • OQPX 7.4 ± 0.9 Pulpzyme HB Pulpzyme HC Irgazyme 40 Enzyme Figure 18. The direct effects that three commercial xylanases have on the kappa number of kraft pulps derived from Douglas-fir. The enzyme treatment was carried out on the brownstock and the oxygen delignified kraft pulp before (XQPP) and after (QPXP) the first peroxide stage. Each bleaching sequence is identified at the upper right corner of each set of histograms. The kappa number after the xylanase treatment is compared to the corresponding control pulp. The bars show the range from two replicate experiments. Only one experiment was performed with Pulpzyme HC for the OXQPP sequence. 61 Q . Q . E c o co CM T J CO N 15 _2 o 05 D ) 4.0 — 1 X 1 3.0 o c (0 .a o </> < > 2.0 1.0 0.0 control • sugars 0.37 ±0.13 jg A280 1.6 ±0.2 M A205 7.0 ±1.1 • sugars 0.41 ±0.04 E3 A280 0.4 ±0.0 ra A205 1.8 ±0.4 QPX I control • sugars 0.68 ±0.02 H A280 0.9 ±0.4 ^ A205 4.8 ±0.4 20 CD O rr 4 1 0 | (D OQPX 3 S c < > 20 S" q cr w 3 O (D 10 ro o tn 3 3 Pulpzyme HB Pulpzyme H C Irgazyme 40 Enzyme Pulpzyme HB Pulpzyme H C Irgazyme 40 Figure 19. The effects that the three commercial xylanases have on the solubilization of sugars and UV-absorbing material during peroxide bleaching of kraft pulps derived from Douglas-fir. The enzyme treatment was carried out on the brownstock and the oxygen delignified kraft pulp before (XQPP) and after (QPXP) the first peroxide stage. Each bleaching sequence is identified at the upper right corner of each set of histograms. Results after the xylanase treatment were compared to the corresponding control filtrates. The bars show the range from two replicate experiments. Only one experiment was performed with Pulpzyme HC for the OXQPP sequence. 62 25 20 ion 15 a. E 10 3 Vt 5 o O 0 CD T3 'x .5 8 CO -10 c *™~ 25 0} cn 20 c CO .c O 15 *-» CD z 10 Slaaa control . E3 P, 79 ± 6 • P, 71 ± 14 5 0 -5 -10 slafls. £2ni£oi P, 46 ± 1 • p. 67 ±18 XQPP T 1 OXQPP stage control E3 P, 60 ± 5 • P. 86±14 OQPXP 3 Pulpzyme HB Pulpzyme H C Irgazyme 40 Pulpzyme HB Pulpzyme H C Irgazyme 40 Enzyme Figure 20. The effects that the three commercial xylanases have on peroxide consumption during peroxide bleaching of kraft pulps derived from Douglas-fir. The enzyme treatment was carried out on the brownstock and the oxygen delignified kraft pulp before (XQPP) and after (QPXP) the first peroxide stage. Each bleaching sequence is identified at the upper right corner of each set of histograms. Residual peroxide after the xylanase treatment following each P stage was compared to the corresponding control P filtrates. The bars show the range from two replicate experiments. Only one experiment was performed with Pulpzyme HC for the OXQPP sequence. 63 3.2.4 The Effects of Xylanase on Three Different Oxygen Delignified Softwood Pulps 3.2.4.2 Pulp Brightness Comparison between wood species One of the objectives of this study was to determine if there were any differences in the effectiveness of xylanase treatment on the three different kraft pulps. Under the conditions used, pulp derived from cedar consistently reached the highest brightness level of 73.0% ISO. This final brightness was at least 1 and 3% ISO higher than the those reached with pulp derived from Douglas-fir and hemlock respectively (Figure 21). The second peroxide stage increased the final pulp brightness 8.3-10 % ISO. There were few differences in the three xylanases effectiveness towards the three kraft pulps, although treatment with Irgazyme 40 usually enhanced final pulp brightness the most. Cedar pulps appeared to respond more consistantly to xylanase treatment, with an enhancement of pulp brightness seen in all but one case. Comparison between insertion sites Another objective for this study was to identify the effectiveness of a xylanase treatment after partially bleaching the oxygen delignified kraft pulps with peroxide. An increase in brightness was seen after the xylanase stage for both insertion sites and for each of the three wood species. This direct brightening effect was more pronounced when the xylanase was inserted after one peroxide stage (1.1-1.7 % ISO) than before peroxide bleaching (0.4-0.8 % ISO). For cedar pulps the direct brightness gain in the OXQPP sequence enhanced the final brightness increase by 1-1.5 % ISO (Figure 21). In the OQPXP bleaching sequence, the gain in final brightness was achieved during direct brightening. This was true for all three wood species. With the exception of pulp derived from Douglas-fir, similar levels of final pulp brightness were reached for the two bleaching sequences tested. For the pulps derived from Douglas-fir and hemlock, the bleach boosting effect was more consistently observed in the OQPXP sequence (Figure 21). 6 4 Comparison between enzymes The bleach boosting effects of the three commercial enzymes differed considerably. Irgazyme 40 was the only xylanase that consistently improved the final brightness of the three types of kraft pulps tested. However, all three enzymes improved the final brightness of cedar pulps when inserted after the oxygen delignification stage (OX). Pulpzyme HC appeared to be the least effective at improving the final brightness of the three kraft pulps tested. When inserted after one peroxide stage, Irgazyme 40 and Pulpzyme HB consistantly improved final brightness of the three pulps by about 1% ISO (Figure 21). Neither Pulpzyme HB nor Pulpzyme HC were effective in enhancing the final brightness of pulp bleached with the OXQPP sequence. All three xylanases were effective in directly brightening the pulp. The final pH's for the enzyme stage (Douglas-fir - OXQPP: 6.6-7.6; OQPXP: 7.1-7.6; hemlock - OXQPP: 6.8-7.6; OQPXP: 7.2-7.3; cedar - OXQPP: 7.6-7.8; OQPXP: 7.0-7.2) were within 0.2 pH units in each experiment. All three enzymes should have been active within this range. 3.2.4.2 Change in Kappa Number The kappa numbers for the oxygen delignified pulps derived from cedar, hemlock and Douglas-fir were quite similar with values of 11.3, 11.3 and 12.0 respectively (Figure 22). The corresponding partially bleached pulps (OQPX) had kappa numbers of 5.9, 7.5 and 7.4, respectively. For the oxygen delignified pulps, Irgazyme 40 was the only xylanase that elicited a direct delignification effect on all three pulps. Although cedar pulp was the only oxygen delignified kraft pulp that was directly delignified by all three xylanases, all three partially bleached pulps were directly delignified (Figure 22). For the hemlock pulp, a xylanase treatment after the first peroxide stage (OQPX) appeared to result in a more substantial drop in kappa number than that inserted before the peroxide stage (OX). This trend was also seen for the Douglas-fir pulps, however, there was less difference between the two types of pulps. Direct delignification did not consistently lead to a gain in final pulp brightness. 65 3.2.4.3 Materials Solubilized During Xylanase Treatment Compared to the control pulps, there was an increase in the solubilized materials found after enzyme treatment for all three xylanase preparations (Figure 23). For the materials that absorbed at 205 nm, the control pulp derived from cedar showed the largest difference between the oxygen delignified (OX) and the partially bleached pulps (OQPX), with values of 0.9 and 6.2, respectively. Although the OQPX pulp released large amounts of material that absorbed at 205 nm after control treatments, the addition of xylanase was still able to increase the solubilization of this material. The solubilization of total sugars varied greatly between wood species and xylanase treatments with amounts ranging from less than 1 ppt for oxygen delignified Douglas-fir after treatment with Pulpzyme HB, to greater than 4 ppt for the same pulp treated with Irgazyme 40. 3.2.4.4 Peroxide Consumption During Bleaching The net change in peroxide consumption varied greatly among the different enzymes and wood species (Figure 24). No pattern was seen among the different enzymes but it appeared that oxygen delignified kraft pulp derived from cedar consumed the least amount of peroxide, particularly during the first peroxide stage. It was originally thought that the presence of residual peroxide would indicate that more than enough peroxide was added to bleach the pulp. For all of the bleaching experiments in this section it appeared that higher residual peroxide did not necessarily lead to greater improvement in pulp brightness as a result of enzyme treatment. 66 Cedar - OXQPP Cedar - OQPXP A-sjarjfi control H OX 37.8 ± 0.4 0 OXQP 64.4 ± 0.4 • OXQPP 72.8 ±1.1 I £lafl£ control OQP 64.5 ± 0.7 . 0 OQPX 64.7 ± 0.9 • OQPXP 73.0 ± 0.8 o oo 3 —" in t/> cu 2 c JE D ) 1 m CL 0 0_ c CD -1 CO c CO x: -2 O CD z Douglas-fir - OXQPP Douglas-fir - OQPXP stage control H OX 35.8 ±0.7 H OXQP 63.0±0.7 • OXQPP 71.7 ±1.0 7^ Sl2fl£ control OQP 61.1 ±0 .9 . [3 OQPX 61.4 ± 1.3 • OQPXP 69.811.2 Hemlock-OXQPP Hemlock-OQPXP t ± L stage control ^ O X 34.610.7 0 OXQP 60.711.9 • OXQPP 69.2 12.3 I 7 7 * stage control OQP 58.815.6 0 OQPX 58.615.7' • OQPXP 69.412.1 Pulpzyme HB Pulpzyme H C Irgazyme 40 Enzyme Pulpzyme HB Pulpzyme H C Irgazyme 40 Figure 21. The effects that three commercial xylanases have on the pulp brightness during peroxide bleaching of oxygen delignified kraft pulps derived from cedar, Douglas-fir and hemlock. Each bleaching sequence is identified at the upper left corner of each set of histograms. Pulp brightness after each stage is compared to the corresponding control pulp. The error bars show the range from two replicate experiments. Only one experiment was performed for Douglas-fir with Pulpzyme HC for the OXQPP sequence 67 .E -2.5 CO c 03 0.5 0.0 0 1 -0.5 -1.0 -1.5 -2.0 -2.5 species control \-m Cedar 5.9 ± 0.2 H hemlock 7.5 ± 1.3 • Douglas-fir 7,4 ± 0.9 OQPX: Pulpzyme HB Pulpzyme HC Irgazyme 40 Enzyme Figure 22. The direct effects that three commercial xylanases have on the kappa number during peroxide bleaching of oxygen delignified kraft pulps derived from cedar, Douglas-fir and hemlock. Each bleaching sequence is identified at the upper right corner of each set of histograms. Kappa numbers after the xylanase stage are compared to the corresponding control pulps. The error bars show the range from two replicate experiments. Only one experiment was performed for Douglas-fir with Pulpzyme HC for the OXQPP sequence . 68 Cedar - OX Cedar - OQPX 4.0 3.0 h oojilroj " • sugars 0.21 ± 0.09 H A280 02 ± 0.0 A20S 0.9 ± 0.0 2.0 1.0 0.0 irr. Douglas-fir - OX Douglas-fir - OQPX control " • sugars 0.61 ± 0.10 [3 A280 0.3 ± 0.1 M A205 1.4 1 0.0 control sugars 0.68 ± 0.01 03 A280 0.9 ± 0.4 S A205 4.8 ± 0.4" 40 30 20 10 < Hemlock - OX 4.0 h conlrol • sugars 0.53 ± 0 . 0 2 {3 A280 0.6 ± 0.0 Ei A205 1.9± 0.1 r i i rh Hh Hemlock - O Q P X conlrol " • sugars 0.46 ± 0 . 0 3 0 A280 " " " " m A205 0.6 ± 02 2.7 + 0.7 f t , nh i t 40 30 20 10 Pulpzyme HB Pulpzyme HC Irgazyme 40 Pulpzyme HB Pulpzyme HC Irgazyme 40 Enzyme Figure 23. The effects the three commercial xylanases have on the solubilization of sugars and UV-absorbing material during peroxide bleaching of oxygen delignified kraft pulps derived from cedar, Douglas-fir and hemlock. Each bleaching sequence is identified at the upper left corner of each set of histograms. Results from each sequence are compared to the corresponding control filtrates. The error bars show the range from two replicate experiments. Only one experiment was performed for Douglas-fir with Pulpzyme HC for the OXQPP sequence. , 69 25 20 15 10 5 0 -5 -10 Cedar - OXQPP Slaflfl control -E3 P 3 9 ± 0 • P, 52±13 Cedar - OQPXP P E 3 P. • P. r Slaaa control 35 ±17 58±14 C L E o O CD T J CD O -25 20 15 10 5 0 -5 10 Douglas-fir - OXQPP Douglas-fir - OQPXP siaas. caniLoi . • 46 ± 1 • P. 67±18 • 0 P, E D P control 60 ± 5 86 ±14 25 20 15 10 5 0 -5 -10 Hemlock - OXQPP stage control E3 P, 69±21 • P, 59±35 FES Hemlock-OQPXP Slaas control t-E3 P 73±24 h • P, 57±12 Pulpzyme HB Pulpzyme H C Irgazyme 40 Enzyme Pulpzyme HB Pulpzyme H C Irgazyme 40 Figure 24. The effects that three commercial xylanases have on peroxide consumption during peroxide bleaching of oxygen delignified kraft pulps Each bleaching sequence is identified at the upper left corner of each set of histograms. Residual peroxide after xylanase treatment for each P stage is compared to the corresponding control filtrates. The error bars show the range from two replicate experiments. Only one experiment was performed for Douglas-fir with Pulpzyme H C for the OXQPP sequence . 70 3.2.5 Effects of Xylanase Treatment on Pulp Brightness, Kappa Number and the Release of Carbohydrate and UV-Absorbing Materials 3.2.5.1 Pulp Brightness In a similar fashion to the response previously observed with the brownstock pulps (Section 3.2.2.1), the oxygen delignified pulps responded to certain xylanases under the described conditions by showing a brightness gain of 1.1-1.6 % ISO. These results indicate that oxygen delignification and peroxide bleaching do not adversely affect the brightness gain that can be achieved by the xylanase treatment. Other work with a mixed softwood kraft pulp also showed that the addition of a xylanase stage in an OXQP sequence was able to increase the final brightness of 63.1% by about 1.5 % ISO (van Lierop et al., 1993). This is in agreement with the results presented in the previous section (3.2.4.1) where pulp brightnesses ranging from 60.7-64.4% ISO were achieved after an OQP sequence, with the enzyme treatment providing a similar brightness gain. In other research, the accessibility of the substrate was studied by repeatedly bleaching a pine kraft pulp with a series of xylanase and peroxide stages (Viikari et al., 1990). These workers determined that a xylanase loading of 30 nkat/g of pulp, prior to each repeated peroxide stage, could substantially improve the brightness of the softwood kraft pulp after each P stage. Although the greatest brightness gain was achieved after the first cycle, the brightness improvement carried through the following cycles indicated that only part of the xylan could be hydrolyzed by the xylanase treatment (Viikari et al, 1990). The results presented in this thesis also indicate that a xylanase pretreatment is able to improve pulp brightness even after substantial bleaching. In addition to the improvements in pulp.brightness achieved with xylanase treatment in the laboratory, results from industrial scale mill trials have been promising. A successful TCF mill trial on a softwood kraft pulp was reported at the Kornas mill in Sweden (Lundgren et al, 71 1994). Pretreatment with a thermostable and alkaline stable xylanase provided a brightness gain of 2 % to achieve 78 % ISO and a drop of 1.5 kappa units. The bleaching sequence used for this trial was very similar to that used in my work except that there were double oxygen delignification stages and chelation stages (OOQQPP). The xylanase treatment was inserted before the first chelation stage. During the TCF mill trial there was a 20% hydrogen peroxide savings and strength properties were similar to those achieved with 100 % chlorine dioxide bleached pulp. 3.2.5.2 Kappa Number Xylanase treatment on the brownstock (X), with initial kappa numbers of 21.4-23.3 caused less than a 1.2 point decrease in kappa number (section 3.2.2.2). The pulps after one peroxide stage, which had kappa numbers of about 13, also showed a similar decrease in kappa number after xylanase treatment. Oxygen delignification of the brownstock pulps resulted in kappa numbers of 11.3-12.0 (section 3.2.4.2), and after treatment with Pulpzyme HC and Irgazyme 40 both caused a decrease in kappa number. Xylanase treatment of the oxygen delignified pulp after partial bleaching (OQPX) resulted in the greatest decrease in kappa number for all of the sequences tested. These pulps had the lowest initial kappa numbers. However, following the xylanase treatment, average decreases in final kappa numbers ranged from 0.4 to almost 1.5. It appeared that even after substantial bleaching, xylanase treatment on softwood kraft pulps still elicited a drop in kappa number. This is in contrast to a previous report that, the lower the kappa number, the smaller the effect of the xylanase treatment (Kantelinen et ai, 1993). The results presented in this thesis indicate that xylanase treatment can enhance delignification when inserted before or after a peroxide stage for both the brownstock and oxygen delignified kraft pulps. The decrease in kappa number and the improvement in brightness obtained directly after the xylanase treatments^  (OX pr OQPX) indicate that the xylanase is removing some chromophoric material from the pulp. As discussed previously (section 1.6.3), the mechanism for xylanase activity on kraft pulp has not been completely determined as of yet. 72 Many workers believe that hemicelluloses remaining in the pulp are closely associated with lignin. For example, recent work has suggested that lignin-carbohydrate complexes (LCC) may be present in the pulp and contribute to the difficulty of removing lignin from the pulp fibers (Yang and Eriksson, 1992; Ragauskas et al, 1994). It is therefore possible that xylanases attack xylans associated with the LCCs and thus facilitate the removal of chromophoric material, not only directly after the enzyme treatment, but also after subsequent chemical bleaching. 3.2.5.3 Solubilized Materials It has been suggested that extensive hydrolysis of the hemicellulose fraction of the pulp is not necessary for the bleach boosting effect because there appears to be no correlation between the amount of hydrolysis and the increase in bleachability (Clark et al, 1991). It would seem that, for my experiments, enough of each enzyme was loaded because direct brightening and delignification could be observed, with a corresponding solubilization of sugars and UV-absorbing materials from the fiber. More material was solubilized from the oxygen delignified and partially bleached pulps than from the brownstock. This would seem to indicate that oxygen delignification and partially bleaching of the pulp with peroxide does not adversely effect the activity of the xylanase on the pulp when compared to the brownstock. Indeed, accessability may have been improved with bleaching. The effects that different fiber morphology's have on xylanase activity is not clear. As previously described (section 1.2.5) the kraft pulps derived from the different wood species would probably have similar amounts of reprecipitated xylan and lignin on the fiber surface, but the fiber itself would have different physical characteristics. Application of a xylanase would initially enhance solubilization of the carbohydrate and lignin material on the surface of the fiber. However, differences in the porosity and surface area of the fibers would probably affect xylanase activity. There is also the possibility, that,cell wall thickness and length would ultimately affect the activity of the xylanase on the different species of pulp fiber. 73 The results presented confirm that the three commercial xylanases modify the kraft pulp fiber to various extents, causing the release of sugars and chromophoric material. However, if the pulp fiber is substantially weakened by the xylanase treatment, then it will not be considered an appropriate bleaching alternative for the industry. In order to determine the extent of enzymatic modification of fiber strength, zero span breaking length of the pulp was measured and the results are presented in the next section. 74 3.2.6 Comparison of Fiber Strengths After Xylanase Treatment of Kraft Pulps Derived from Three Different Wood Species 3.2.6.1 Background In the search for new bleaching alternatives it is imperative that the strength of the pulp not be sacrificed. For this research, the zero span breaking length values for the different bleaching sequences not only gave an indication of the xylanase effects but they also gave an indication of the applicability of the peroxide based TCF sequence that was used. The latter was thought to be important as peroxide has become a common chlorine-free bleaching alternative because of its relatively simple implementation. However, its aggressive action towards pulp is also well known (AxegSrd et al, 1992), and peroxide loaded onto a pulp under improper conditions will not have the desired brightness effects and it may actually destroy the strength of the fibers by attacking the cellulose component. The addition of an oxygen delignification stage greatly enhances peroxide bleaching, but like peroxide, generates destructive hydroxyl radicals that can attack the cellulose in the fiber if conditions are not monitored. The zero span breaking length method was used to obtain an indication of the average strength of the longitudinal structure of individual fibers in the handsheets produced from the pulp subsamples collected after the xylanase treatment (Tappi standard TAPPI T 205, 1985). This test is an index of the breaking length (or tensile strength) of a pulp beaten to its maximum value, and it is an excellent measure of the "maximum strength" of the pulp. The removal of hemicelluloses during xylanase treatment on a kraft pulp is not expected to decrease the zero span breaking length of the fibers. However if there is substantial cellulase activity, the strength of the cellulose chains within the fiber will be compromised, thereby decreasing the zero span breaking length (Paice et al, 1992). Pulp samples collected after the xylanase stage were tested in order to determine any effects that xylanase, oxygen delignification and peroxide had on the fiber strength of the three different wood species analyzed. 75 3.2.6.2 Comparison of Fiber Strengths Although there was considerable variability in the results obtained from the five tests conducted for each pulp sample, most of the average results obtained for the xylanase treated pulp were within 5 % of the values obtained for the corresponding control pulps (Figure 25). There were only two enzyme-treated samples that differed by more than 10 % from their corresponding controls. These were the hemlock pulp treated with Pulpzyme HB in the QPXP sequence and the Douglas-fir pulp treated with Irgazyme 40 in the OQPXP sequence. Even in these two cases, the standard deviations of the measurements were large enough as to suggest that there was no significant difference from the corresponding samples and controls. This was true even for Irgazyme 40, which was found to have a small amount of cellulase activity (section 3.1.3). Xylanase treatment can affect different parameters within the pulp matrix. Due to reprecipitation, xylan is concentrated on the outer surfaces of the microfibrils. The removal of these reprecipitated xylans appears to cause a reduction in interfiber bonding. However, improvement in flexibility and swelling of the individual fibers has been obtained (Noe et al, 1986; Viikari et al., 1994). Although there appears to be a decrease in the degree of polymerization (DP) of the xylan component of the pulp after xylanase treatment the strength of the fibers remains intact (Paice et al, 1992). It has been proposed that the drop in xylan DP is due to the removal of small amounts of xylan which are covalently bound to lignin fragments (Paice et al, 1992; Patel et al, 1993). The strength of the individual fibers is not compromised because the cellulose component of the fiber is not compromised by xylanase treatment. 76 Cedar 15.3 14.8 OXQPP H Pulpzyme HB H Pulpzyme HC f~1 Irgazyme 40 OQPXP Douglas-fir 16.8 16.5 16.3 14.6 XQPP QPXP OXQPP OQPXP hemlock 17.0 16.6 15.0 14.2 si 1 I I 1 1 I 1 1 i XQPP QPXP OXQPP OQPXP Bleaching sequence Figure 25 The effects of treatment with three commercial xylanases on the average zero span breaking lengths of kraft pulps derived from three different wood species. The average values for the control pulps in each bleaching sequence are listed at the top of each graph and the histograms represent the difference from the controls. The bars represent the standard deviation determined from five samples. 77 3.3 TOXICITY PROFILE FOR PEROXIDE B L E A C H I N G OF S O F T W O O D K R A F T PULPS T H A T INCLUDE A XYLANASE S T A G E 33.1 Background Axegard et al. (1993) have produced an excellent summary of the current literature on pulp bleaching and the environment. They concluded that it was very difficult to make any generalizations about the environmental impact caused by effluent from pulp mills. The main problems were that each pulp mill is unique with respect to the raw material, process technology, effluent treatment used as well as the conditions and flora/fauna in the receiving environment. Many researchers are also considering the possibility that the current toxicity of bleached kraft mill effluent may be caused by natural components in the wood itself. Axegard et al. (1993) concluded that there is insufficient data concerning mill effluents produced from alternative ECF and TCF bleaching, and their effects on biological systems. It has been shown that xylanase treatment may increase the biological and chemical oxygen demands of the mill effluents which may adversely affect the receiving environment (Senior and Hamilton, 1992). However, mill trials using xylanases in conjunction with peroxide have been able to decrease chemical loading which may decrease effluent toxicity (Lundgren et al., 1994). Previously O'Connor et al (1993) showed that peroxide based TCF bleaching sequences elicited the highest chronic toxicity towards Ceriodaphnia when compared to bleaching sequences that use chlorine dioxide. In other research an OQP bleaching sequence was shown to be non toxic to Daphnia magna as long as the residual peroxide was destroyed prior to testing (van Lierop et al., 1993). At this time little is known about the effects that xylanase may have on the toxicity of an effluent produced from a TCF bleaching sequence. The quality of the pulp that is produced using the'new bleaching agents is always closely monitored. It is apparent that new bleaching sequences should have less of an effect on the environment than the chlorinated sequences used in the past. Previously it was found that the use 78 of a xylanase treatment in an elemental chlorine based bleaching sequence decreased the amount of chlorine required, thereby decreasing the amount of organochlorine discharged into the receiving waters (Clark et al., 1991; Senior et al, 1992; Tolan and Canovas, 1992; Turner et ai, 1992; Allison et al, 1993; Tolan, 1993). Xylanases have also been found to decrease the amount of chlorine dioxide required to fully bleach kraft pulp (Yang et al, 1992; Elm et al, 1993; Lahrinen et al, 1993; Scott et al, 1993; Jean et al, 1994; Suurnakki et al, 1994). It would be important to consider the effects that xylanase treatment has on the toxicity of effluent generated by a TCF bleaching sequence. The filtrates generated during (O)QPXP and (O)XQPP bleaching sequences were analyzed for acute toxicity using Daphnia magna and Microtox assays (Nelson et al, 1994). These two acute toxicity methods were used in order to get a general idea of the overall toxicity of the TCF effluents generated during my bleaching trials. The fresh water amphipod Daphnia magna has been used by industry and government agencies for many years as a relatively quick assay for acute toxicity determination in a fresh water environment. Microtox, an assay that utilizes a marine bacterium, has recently become a rapid test for indicating acute toxicity (McCubbin, 1984). The Microtox test is currently being implemented into many industrial applications, including the pulp and paper industry, because of its speed and sensitivity to environmental contaminents. These assays were conducted on filtrates collected immediately following the xylanase treatment as well as on a combination of the filtrates collected from the complete bleaching sequences. The filtrates collected from the control pulps gave an indication of the toxicity due to wood species and oxygen delignification. 3.3.2 Toxicity of the TCF Bleaching Sequence to Daphnia magna and Microtox™ There was no change in the toxicity of the filtrates collected immediately after xylanase treatment using the 48 hour LC50 assay for Daphnia magna as compared to the controls. The results presented in Table 3 illustrate the difference in toxicities for each wood species and each xylanase insertion site tested. The three filtrates which were found to be non toxic were collected 79 from the brownstock and oxygen delignified pulp derived from Douglas-fir and from the oxygen delignified hemlock pulp prior to the first P stage. For all three wood species, xylanase filtrates from the OQPX sequence were found to be the most toxic to Daphnia magna, with LC50 values between 56-100%. The enzyme filtrate collected from the oxygen delignified cedar pulp also elicited a toxic response at the 56 % dilution. There appeared to be no change in the toxicity of the combined filtrates after xylanase treatment when they were compared to the corresponding controls (Table 4). Most of the filtrates collected after xylanase treatment of the brownstock and oxygen delignified pulps showed very little toxicity to Microtox (Figure 26). Combined filtrates (Figure 27) were consistently more toxic than the filtrates collected immediately following the xylanase stage (Figure 26). It appears that the low concentrations of residual peroxide present in the combined filtrates were toxic to Daphnia magna. The Microtox assay appeared to be slightly less sensitive to the concentrations of peroxide present in the combined filtrates than Daphnia magna. There was very little difference in toxicity between wood species. Although the filtrates containing peroxide were pH adjusted to 7 prior to testing, peroxide is toxic to Daphnia magna below 0.01 mg/L. Residual peroxide ranged from 0.06-0.37 mg/L for the bleaching sequences tested. Oxygen delignification may slightly decrease overall toxicity to both Daphnia magna (Table 4) and Microtox (Figure 26). The one exception being for the partially bleached Douglas-fir. None of the xylanase filtrates were found to be more toxic than the water controls. In many cases the enzyme filtrates collected from the partially bleached pulps (QPX and OQPX) were more toxic to Daphnia magna than those collected from the corresponding unbleached pulps (X and OX). This may indicate that peroxide modifies the pulp, and changes the composition of the materials solubilized during the xylanase treatment. It seems unlikely that there could be any residual peroxide left in the pulp during the enzyme treatment because the pulp was washed after the peroxide stage and then pH adjusted to 7, and both steps required a 1 % wash. 80 Table 3. Acute toxicity of the filtrates collected from different pulps after the xylanase stage, as determined using the LC50 for Daphnia magna. Results for filtrates collected from controls and corresponding xylanase treatments were the same. Undiluted filtrates were initially tested for toxicity at 100%. If greater than 50% mortality was found then the samples were diluted to 56% using aerated water that had been adjusted to a medium hardness. Species & toxicity range Bleaching sequence X OX OPX OOPX Douglas-fir Non toxic X X - --100% - - X -56-100% - - - X hemlock Non toxic X - --100% X - X -56-100% - - - X cedar Non toxic rd - rd --100% - -56-100% X X X = LC50 falls in this toxicity range nd = not determined -100% = 50% mortality for undiluted sample, therefore 56% dilution was tested but found to be non toxic. 81 Table 4. Acute toxicity of the combined filtrates collected from different pulps, as determined using the LC50 for Daphnia magna. Results for filtrates collected from controls and corresponding xylanase treatments were the same. Filtrates were initially tested for toxicity at 5.6%. If greater than 50% mortality was found then the samples were tested at 1.0% using aerated water that had been adjusted to a medium hardness for dilutions. Species & toxicity range XOPP Bleaching sequence OXOPP OPXP OOPXP Douglas-fir >5.6% - - X -1.0%-5.6% X X - X hemlock >5.6% _ X X X 1.0%-5.6% X - - -cedar >5.6% nd - nd -1.0%-5.6% X X X = LC50 falls in this toxicity range nd = not determined >5.6% = no mortalities at 5.6% but the LC50 is expected to be less than 10% (preliminary Daphnia assays determined the LC50 to be between 1.0-10%) 82 0) o X OX X OX OX as c: o QPX OQPX QPX OQPX OQPX hemlock Douglas-fir cedar Figure 26. Microtox™ EC50 for filtrates collected from unbleached and partially bleached pulp after the control and xylanase treatments. The bars represent the minimum and maximum values found for the average of the three xylanase treatments. Serial dilutions starting with filtrate concentrations of 50% were used for determining the EC50. 83 ^ c o n t r o l • e n z y m e XQPP OXQPP XQPP OXQPP OXQPP o 100 a o LU 10 I QPXP OQPXP hemlock QPXP OQPXP Douglas-fir JJ OQPXP cedar Figure 27. Microtox™ EC50 for the combined filtrates from the different bleaching sequences. The bars represent the minimum and maximum values found for the average of the three xylanase treatments. Serial dilutions starting with filtrate concentrations of 50% were used for determining the EC50. ... -84 4. CONCLUSIONS In most cases, xylanase pretreatment enhanced the brightness of kraft pulp bleached using a peroxide based TCF sequence. The three commercial xylanases could improve the pulp bleachability of brownstock and oxygen delignified pulps to varying extents, whether the enzymes were inserted before or after a peroxide stage. There appeared to be little difference in the ability of the xylanases to brighten the kraft pulps derived from the three different wood species, although oxygen delignified cedar pulp reached the highest brightness in these experiments. However, Douglas-fir pulp did not seem to respond as well with Pulpzyme HB and Pulpzyme HC. Of the three enzymes tested, treatment with Irgazyme 40 resulted in the most consistent improvement in pulp bleachability. However, all three enzymes enhanced carbohydrate solubilization and increased the release of UV absorbing material from the pulps. There was also a direct delignifying effect in nearly all of the bleaching trials. There may be several explanations why Irgazyme 40 was the most successful bleach booster of the three enzymes tested. As the amount of enzyme loaded onto the pulp for each treatment was determined using an isolated xylan from birchwood, the activity calculated for each xylanase using this purified substrate may not be the same as that action that results on the complex kraft pulp matrix. The repreci pita ted xylan within the kraft pulp has not only been chemically modified, it is also closely associated with lignin and other hemicelluloses that probably affect the action of the xylanase. It is also not clear what constitutes the target substrates during xylanase prebleaching, whether they are lignin-carbohydrate complexes or xylan associated with sugar derived chromophores or a combination. Differences in the physical characteristics of the pulp fibers derived from the different wood species could be another factor that would affect xylanase activity. The activity of the xylanase is probably affected by the porosity and surface area of the kraft pulp fiber because these characteristics will affect the position of the reprecipitated xylan. Although xylanase 85 treatment is thought to initially solubilize reprecipitated xylan on the surface of the pulp fiber, the availability of this substrate will affect the enzymes's activity. Differences in kraft fiber morphology between the wood species would also affect the other xylanase mechanisms that have been suggested, such as disruption of LCCs and release of chromophoric xylan because of their availability to the enzyme. Differences in the composition of the three commercial xylanase preparations may also affect their action on kraft pulps. Pulpzyme HB and Pulpzyme HC are prepared using cloned genes which enable the production of xylanases free of contaminating cellulases and hemicellulases. Irgazyme 40 is prepared by bulk concentration of the xylanase fraction present in a complex system of fungal enzymes produced by a particular fungus. Because Irgazyme 40 is not a completely purified xylanase there may be other enzymes present in the mixture that facilitate chemical bleaching or enhance the action of the xylanase itself. This might explain the greater reliability of Irgazyme 40 when used for bleach boosting. Further research, using different types of xylanase preparations, could help to expand the full potential of xylanase prebleaching. It is possible that there are, as of yet, unidentified components within the enzyme system which may contribute to the enhancement of pulp bleaching. Previous research (Yang and Eriksson, 1992; Buchert et al, 1993; Kantelinen et al., 1993) has indicated that other hydrolytic enzymes such as mannanases and oxidative enzymes such as laccases may facilitate xylanase action by working on the pulp fiber together, and synergistically improving the subsequent bleaching stages. The results from the research presented in this thesis clearly demonstrate the ability of all the three xylanases to increase both the solubilization of carbohydrates and the release of UV absorbing material from kraft pulps. It was apparent that neither oxygen delignification nor peroxide bleaching inhibited these actions. In fact, in many cases, there appeared to be an increase in xylanase activity as measured by an increase solubilization of carbohydrates and UV absorbing material from the partially bleached pulps. Direct improvement in pulp brightness, by xylanase treatment, was seen after oxygen delignification and one peroxide stage. This may 86 indicate that partially bleaching the pulps improves the action of the xylanase on the fiber matrix, or that the effect on pulp brightness is more easily detected. Although xylanase directly delignifies and brightens the partially bleached pulp, the enzyme does not appear to enhance the final quality of the pulp to the same extent as that of the brownstock. This result differs from what was seen with chlorine bleaching, where xylanase pretreatment appeared to have little direct effect on the pulp but greatly enhances the capacity of the subsequent bleaching stage (Tolan and Canovas, 1992). Direct delignification and brightening caused by the xylanase treatment following oxygen delignification, peroxide bleaching or both suggests that these two TCF bleaching methods may enable the xylanase to act more effectively on the pulp fiber when compared to the brownstock pulps. This improvement in effectiveness may be caused by increased fiber swelling or changes in the arrangement of lignin and hemicellulose on the fiber exterior that ultimately improves accessibility of the enzyme. Although all three commercial xylanases increased the solubilization of carbohydrates and UV-absorbing materials, which indicated enzymatic activity, the final brightness of the pulp was not always improved. Similar results (Clarke et al., 1991), also showed no clear correlation between the hydrolysis of the kraft fiber by the xylanase treatment and the increase in bleachability. Although one of the objectives of this thesis was to test the effectiveness of three commercial xylanases on kraft pulps derived from three different wood species, the results have also provided some preliminary information on the possible mechanisms that may be involved in xylanase treatment. The direct brightening and direct delignifying effects observed following the xylanase treatment should provide some information on the substrate hydrolyzed by the enzymes. This information may improve our understanding of why the xylanase treatment is more efficient after oxygen delignification and peroxide bleaching, and-why Irgazyme 40 is more consistently effective in pulp treatment. Analysis of the molecular weight distribution of the components in 87 these filtrates could also be used to characterize the organic molecules being released by the different xylanases at the two insertion sites in the bleaching trials. The bleaching conditions used for this study were kept as similar as possible to those used in industry. It is also important to note that there appeared to be no detrimental affects on individual fiber strength after xylanase treatment for any of the bleaching trials. Therefore at the xylanase loadings used for these bleaching trials, increased delignification and bleachability was observed in many cases with no reduction in fiber strength. From the results presented in my study, it can be concluded that xylanase application can be useful in a TCF bleaching sequence. There was an enhancement of pulp brightness with no substantial loss in fiber strength. This could be achieved using different commercial xylanases and pulps derived from different softwood species. The enzyme treatment did not influence effluent toxicity significantly. The optimization of bleaching conditions and enzyme loadings (if cellulase activity is present) will be important factors when implementing xylanase pretreatment in the mill. A greater understanding of the mechanism of xylanase on kraft pulps should also facilitate the use of these enzymes by improving their activity. The elucidation of possible unknown contaminants in some enzyme mixtures, and/or synergistic or additive effects that other enzymes might have on this system, may enable xylanase to become more effectively used for enhancing the bleachability of kraft pulps, especially in conjunction with TCF bleaching sequences. From the results presented in this thesis, it would appear that there are few differences in the effectiveness of the three commercial xylanases, inserted into the TCF bleaching sequence, for improving the bleachability of the kraft pulps derived from three different wood species. 88 5. R E F E R E N C E S Allison, R. W., T. A. Clark and S. H. Wrathall, 1993. Pretreatment of radiata pine kraft pulp with a thermophilic enzyme Part 1. Effect on conventional bleaching. Appita. 46(4): 269 Allison, R. W. and T. A. Clark, 1994. Effect of enzyme pretreatment on ozone bleaching. Tappi T. 77(7): 127 Asp, M. 1994. Bleaching in the "new" and "old" world. 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