Physical Characterization of Enzymatic Ally Modified

8/3/2019 Physical Characterization of Enzymatic Ally Modified

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ELSEVIER Journal of Biotechnology 57 (1997) 205-216

Physical characterization of enzymatically modifiedkraft pulp fibersShawn D. Mansfield a, Ed de Jong , R. Scott Stephens b, John N. Saddler ,*

aChair of Forest Producls Biotechnology, Departm ent of W ood Science, Faculty of Forestry, University of British Colum bia,Vancouver, BC, Canada

Mat erials Technology, Corporate R and D, W eyerhaeuser Company, Federal W ay, W ashington DC 98477, USA

Received 15 October 1996; received in revised form 25 March 1997; accepted 2 April 1997

Abstract

Douglas-fir kraft pulps were treated with an enzyme preparation containing both cellulase and xylanase activities.Treatments resulted in the solubilization of 21.1% the xylan and 1.8% of the cellulose. Changes in fiber and paperproperties were observed. Monitoring pore volume, degree of polymerization, crystallinity, FT-IR spectra, andscanning electron microscopy helped elucidate changes in fiber composition and morphology. Data indicate a declinein intrinsic fiber strength due to an erosion of the fiber surface. Reduction in paper strength resulted from thecollective effects of decreased intrinsic fiber strength and the reduction in the degree of polymerization of a largeportion of the hemicellulose component of the fibers, as well as fiber defibrillation and fines hydrolysis. 0 1997Elsevier Science B.V.Keywords: Fiber modification; Cellulase; Xylanase; Pore volume; Degree of polymerization; FT-IR spectroscopy;Scanning electron microscopy; Crystallinity; Douglas-fir; Kraft pulp

1. Introduction

During the last few years cellulases and hemi-cellulases have been evaluated for their ability tobeneficially modify pulp and paper characteristics.The various applications that have been investi-gated include, the deinking of secondary fibers(Prasad, 1993; Elegir et al., 1995; Heise et al.,

* Corresponding author.

1996), reduction of refining energy requirements(Freiermuth et al., 1994), enhanced beatabilityand fibrillation of chemical pulps (NoC et al.,1986; Freiermuth et al., 1994), improvements inthe freeness of secondary fibers (Pommier et al.,1990; Bhat et al., 1991), as well as the bleaching ofkraft pulps with xylanases and mannanases (Clarket al., 1991; Viikari et al., 1994; Saake et al., 1995;Tolan et al., 1996; Mansfield et al., 1996a). Allthese treatments involve relatively low concentra-

0168-1656/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved.PII SOl68-1656(97)00100-4


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206 S.D. Ma nsfield et al. /Journal of Biotechnology 57 (1997) 205-216

tions of enzymes over a short incubation period.However, it was apparent that, cellulase treat-ments generated substantial changes to the fibercharacteristics (Mansfield et al., 1996b). It appearsthat, as well as acting in the traditionally definedendo- or exoglucanase action that has been pro-posed for cellulose hydrolysis (Wood, 1989) thatthe cellulases are able to modify both the pulpfibers and paper characteristics in a way thatcannot be directly explained by this oversimplifiedendo/exoglucanase mode of action.

Recently, a considerable amount of work hasbeen carried out to try to confirm that the mecha-nism of xylanase aided bleaching is due to theremoval of reprecipitated or lignin carbohydratecomplexed (LCC) xylan present on the surfaces ofthe pulp fibers (Yang and Eriksson, 1992; Kante-linen et al., 1993; Suurnakki et al., 1996a; de Jonget al., 1996). However, the xylanase treatments ofdifferent pulp fiber length fractions demonstrateda non-uniform response (Mansfield et al., 1996a),indicating that the composition of the fibers playsa key role in determining the effectiveness of thetreatment.

In contrast to xylanases, little is know aboutthe enzyme mechanisms involved during the lim-ited initial degradation of pulp fibers by cellulases.Although, significant advances have been made inthe understanding of the mechanism by whichcertain microorganisms degrade hydrogen bond-ordered cellulose (Withers et al., 1986), the natureof the enzyme interaction and the action andsequence of events which solubilize cellulose haveyet to be clearly defined. Furthermore, the extentof the modifications to the fiber morphology bycellulases remains ambiguous. Several groups(Stork et al., 1995; Mansfield et al., 1996b) haveindicated that cellulase treatments result in a re-duction in strength of the native fibers. Similarly,it has been shown that care must be taken whenusing xylanases in bleaching of kraft pulps toavoid cellulase contaminated enzyme mixtureswhich can severely reduce the strength of the pulp(Puls et al., 1990). Gurnagul et al. (1992) demon-strated that treatments of low yield kraft pulpwith cellulase lowered the strength drastically,with relatively little change in the degree of poly-merization (DP) of the cellulose. These workers

concluded that the enzymes preferentially attackstructurally irregular zones in the fiber wall, re-sulting in localized degradation.

Previously, we (Mansfield et al., 1996b) andother workers (Jackson et al., 1993; Stork et al.,1995) demonstrated that beneficial changes topulp fiber properties can be achieved by brieftreatments with low concentrations of cellulases.However, this was usually accomplished at theexpense of some strength loss (Stork et al., 1995;Mansfield et al., 1996b). The main purpose of thispresent study was to investigate the nature ofchanges which occur at both the micro and macrostructural level of Douglas-fir kraft pulp fibersduring enzymatic treatments with a commercialcellulase. By measuring the amount and type ofcarbohydrates released, the degree of polymeriza-tion, crystallinity, the pore volume and FT-IRspectroscopy of the substrate, as well as visualiz-ing changes in the substrate by scanning electronmicroscopy we will discuss the observed changesin the fiber morphology. It was apparent thatchanges to the surface composition of the fibersby enzymatic treatments influenced both the fiberproperties and paper strength.

2. Methods and materials2.1. Pulp composition

Unbleached kraft pulp derived from Douglas-fir (Pseudotsuga menziesii) was produced at theCrofton mill (Fletcher Challenge), British Colum-bia, Canada. The lignin and sugar composition ofthe pulp was determined using sulfuric acid hy-drolysates (TAPPI Method T249 cm-85). Eachhydrolysate was filtered using a sintered-glassfilter of medium coarseness for the gravimetricdetermination of Klason lignin (acid insolublelignin), and its absorbance at 205 nm was mea-sured for the quantification of acid soluble lignin(TAPPI Useful Method UM250, 1991). Themonosaccharide constituents were quantified byanion-exchange chromatography on a CarboPacPA-l column using a Dionex DX-500 high pres-sure liquid chromatography (HPLC) system(Dionex, Sunnyvale, CA), using fucose as theinternal standard.


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2.2. Enzyme treatmentsNovozyme SP342 from Novo Nordisk

(Bagsvzrd, Denmark), an enzyme preparationthat was derived from Humicola insolens, wasused for the enzyme treatments. Its activity oncarboxymethylcellulose (2% CMC, Sigma, St.Louis, MO), xylan (1% birchwood xylan, Sigma),and filter paper (No.1 Whatman) was measuredusing methods described previously (Wood andBhat, 1988). Proteins in solution were quantifiedusing the bicinchonic acid protein assay(Stoscheck, 1990). The enzyme preparation con-tained protein 46.7 mg ml- and was shown topossess relatively high xylanase activity (2869 IUml - ) as well as cellulase activities (CMCase:143.5 IU ml-; Filter Paper: 9.9 IU ml-).

Pulp slurries (3% consistency in 50 mM phos-phate buffer, pH 7.0) were treated for 1 h at 50Cunder continuous agitation (200 rpm) with 5 mgg ~ 1 protein of oven-dried fiber. The reactionswere stopped by placing the pulp in a boilingwater bath for 15 min. Control pulps were simi-larly treated with equivalent amounts of heatinactivated enzyme (15 min boiling).

The carbohydrates solubilized during the enzy-matic treatments were measured by HPLC after afurther secondary acid hydrolysis. The absorbanceof the filtrates was measured at 280 and 457 nm toquantify the lignin and chromophores leachedfrom the fibers, respectively.2.3. Pore volume determination

The pore volume of control and treated pulpswas determined using dextran probes of varyingmolecular diameters (1.8-56 nm) using a modifi-cation of the solute exclusion technique developedby Stone and Scallan (1969). The individual dex-tran probe solutions (0.5% w/v) were added to thepulp samples, mixed thoroughly and allowed toequilibrate for 5 h with frequent gentle mixing.After equilibration, the pulp samples were al-lowed to settle and the probe solutions withdrawnand filtered through a sintered-glass funnel. Theconcentration of the probe solutions was deter-mined refractometrically using a Waters 625 liq-uid chromatography system equipped with a

Waters 410 Differential Refractometer (Millipore,Milford, MA). The concentration of inaccessiblewater was determined as described previously(Gama et al., 1994).2.4. Microscopy

The pulp sample specimens were freeze-driedfrom a water slurry directly onto scanning elec-tron microscope (SEM) sample mounts, whichwere sputter coated with 60:40, Au:Pd. The sam-ple mounts were then observed in a LEO Spec-troscan-360 SEM (Cambridge Instruments, MA)using 10 kV accelerating voltage.2.5. FT-IR spectroscopy

Low grammage handsheets (7.5 g m-*) of con-trol and enzyme treated pulps were made using astandard British handsheet maker, placed in aphosphorus pentoxide desiccator and allowed todry. A standard hole punch was used to remove0.22 mg disks of the handsheets, which were thenplaced on a bed of KC1 powder and analyzed byFT-IR spectroscopy. The FT-IR spectra (256scans, 4 cm - ) were determined by the diffusereflectance method (DRIFT) using a Perkin-Elmer 1600 instrument (Norwalk, CT). The maxi-mal absorbance was less than 1.0 AU for allsamples analyzed. All samples were baseline cor-rected and normalized, the average of eight spec-tra was used as the representative spectrum foreach sample. The second derivative spectra wasused to improve the resolution of certain absorp-tion bands (Michell, 1989).2.6. Degree of polym erizat ion

The molecular weight distribution of both thecontrol and enzyme treated pulps were obtainedby Gel Permeation Chromatography (GPC)analyses of their tricarbanyl derivatives. Carbany-lation of the cellulose was carried out as describedpreviously (Schroeder and Haigh, 1979). The cel-lulose tricarbanylate was recovered by evapora-tion of the reaction solvents (Wood et al., 1986)which was subsequently treated with iso-octane,evaporated to dryness, and solubilized in tetrahy-


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208 S.D. Ma nsfield et al. /Journal of Biotechno1og.v 57 (1997) 205-216drofuran (THF) at concentrations of approxi-mately 0.2 mg ml _ .

The GPC of the tricarbanyl derivatives wascarried out on a Waters 625 liquid chromatogra-phy system (Millipore, Milford, MA). The cellu-lose tricarbanylate samples were filtered through aTeflon membrane (0.45 pm) and analyzed using aseries of 4 TSK-GEL columns (Varian, Sunny-vale, CA., type GlOOO HXL, G3000 HXL, G4000HXL and G6000 HXL with molecular weightcut-offs of 1 x 103, 6 x 104, 4 x lo5 and 4 x lo,respectively). THF was used as the eluting solventat a flow rate of 1 ml min - . The samples in theeluent were detected by a Waters 486 UV spec-trophotometer (Millipore, Milford, MA) at awavelength of 254 nm.

The GPC calibration curve was generated fromthe elution profile of polystyrene standards withnarrow molecular weight distributions. Using theMark-Houwink coefficients previously reportedfor polystyrene in THF, K, = 1.18 x lop4 andCQ, 0.74, and for cellulose tricarbanylate in THF,Kc = 2.01 x lop5 and a, = 0.92 the molecularweight of the tricarbanylated cellulose was ob-tained (Valtasaari and Saarela, 1975). The degreeof polymerization (DP) of cellulose was obtainedby dividing the molecular weight of the tricar-banylated polymer by the corresponding molecu-lar weight of the tricarbanylated derivative ofanhydroglucose (DP = Mwj519). Both the num-ber averages (DP,) and the weight averages(DP,) of the substrates were calculated as de-scribed previously (Yau et al., 1979).2.7. Crystallinity

The degree of crystallinity of the pulp sampleswas obtained by X-ray diffraction. Handsheets(100 g m - ) of both control and treated pulpswere made and pressed semi-dry at 5000 psi for 5min. Representative 4.2 x 2.7 cm rectangles werecut from the handsheets, freeze-dried and storedin a phosphorus pentoxide desiccator until analy-sis.

The X-ray diffraction of each sample wasrecorded using a Siemens diffractometer equippedwith a D-5000 rotating anode X-ray generator.The wavelength of the Cu/Kaa radiation source

was 0.154 nm, and the spectra were obtained at 30mA with an accelerating voltage of 40 kV. Sam-ples were scanned on the automated diffractome-ter from 5 to 40 of 20 (Bragg angle), with dataacquisition taken at intervals of 0.02 for 1 s. Apeak resolution program was used to calculateboth the crystallinity index of cellulose and thedimensions of the crystallites (Hindeleh and John-son, 1978).

3. Results and discussionIn previous work (Mansfield et al., 1996b) we

assessed a cellulase enzyme preparation for itspotential to enhance the fiber characteristics ofpulps derived from Douglas-fir wood chips. Thesepreliminary results indicated that the enzymaticeffects on the pulp properties were highly depen-dent on the dosage. For example, an enzymecharge of 5 mg protein per gram of pulp, whichcorresponded to a 3.2% hydrolysis of the pulp,produced a 12.7% reduction in paper strength(tensile index) and a 35.2% loss in fiber strength(zero-span breaking length). Although the enzymetreatments resulted in a considerable loss in boththe paper and fiber strength, we wanted to deter-mine which modifications to the fiber structurecould be correlated with these strength changes.3.1. Carbohydrate solubilization

A more thorough investigation by HPLC anal-ysis indicated which specific carbohydrates weresolubilized by the enzyme treatments after 3.2% ofthe pulp was hydrolyzed (Table 1). It was appar-ent that the enzyme treatment liberated almostequivalent amounts of both glucose (229 mg) andxylose (226 mg), which corresponds to 1.8 and21 .l% hydrolysis, respectively, of the totalamount of cellulose and xylan that was originallyavailable in the pulp.

Previously it was proposed that, at the end ofthe kraft cooking process,, debranched xylan rede-posits on the surface of the fibers (Yllner et al.,1957), and that this redeposited xylan is the pri-mary substrate for xylanases in the prebleachingprocess (Kantelinen et al., 1993). However, Suur-


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Table 1Composition of the Douglas-fir pulp and filtrates obtained after enzymatic treatmentFraction Arabinose Galactose Glucose Xylose Mannose Klason lignin Acid-soluble lignin RecoveryPulp (%) 0.41 0.56 12.56 5.95 6.66 4.91 0.49 91.54Filtrate 21.01 0 229.39 225.83 16.09 N/A 0.08 N/A%Solubilized 28.8 0 1.8 21.1 1.3 N/A 16.3 N/AN/A, not assessed.a Values represent total mg of carbohydrate liberated from enzyme treated minus control filtrates of 18 g pulp samples.

nakki et al. (1996b) have recently questioned theimportance of redeposition when pine kraft pulpswere treated with xylanases. Our results seem toindicate that the debranched xylan is not themajor substrate for the xylanases, as the ara-binose/xylose ratio in the filtrates was increasedwhen compared to that found in the pulp (Table1). Therefore, it is possible that the concertedhydrolysis by both the cellulase and xylanase en-zymes liberated both glucose and the more highlysubstituted xylan found in the inner fiber wall(Suurnakki et al., 1996~). This would account forthe large proportion of the arabinose hydrolyzedby the enzymatic treatments.

A 5- and 3.5fold increase in the absorbances at280 and 457 nm was observed after enzyme treat-ment, respectively. These increases were probablydue to the liberation of lignin and coloured com-pounds by the action of the xylanases on the pulpfibers. Although the cellulases have been shown tobe rather ineffective in releasing coloured materi-als from kraft pulps (Buchert et al., 1994), theyundoubtedly hydrolyze the available surface cellu-lose and continue to work in concert with thexylanases resulting in the hydrolysis of some ofthe internal structural polysaccharides.3.2. Pore volum e determi na tion

The solute exclusion technique of Stone andScallan (1969) has been a valuable tool for char-acterizing the ultrastructural morphology of fibersafter various physical and chemical pretreatments.It has been successfully used to demonstrate theimportance of fiber porosity during the enzymatichydrolysis of various cellulosic substrates (Greth-lein, 1985; Weimer and Weston, 1985; Wong et

al., 1988). It has also been used in biobleachingexperiments, where the removal of hemicelluloseand its associated lignin by xylanase treatmentresulted in an increase in the pore volume of thefibers (Yu et al., 1994) and a slight increase in themedian pore width (Suurnakki, 1996d). In thiswork we have used pore volume determination toinvestigate the changes that occurred as a result ofthe enzymatic treatments to the Douglas-fir pulpfibers. As indicated previously, a substantialamount of the hemicellulose component of thepulp was hydrolyzed after enzyme treatment, sug-gesting that there might be a corresponding in-crease in the pore volume of the fibers. However,we found that the cumulative pore volume (inac-cessible water) of the fibers was significantly re-duced by these enzyme treatments (Fig. 1). Theprofile exhibited by the enzyme treatments notonly demonstrated a substantial reduction in thepore volume for each of the dextran probes used,it also showed a reduction in the fiber saturationpoint (as measured by the 56 nm probe). How-ever, there was an increase in the median porewidth of the enzyme treated pulps.3.3. Scanning electron microscopy

Scanning electron microscopy, indicated a qual-itative change in the outermost fiber surface of theenzymatically treated pulp compared to controlpulps (Fig. 2). However, the electron micrographsdid not reveal any discernible alterations to thefiber surfaces (i.e. cleavages or pit enlargements)other than the erosion of surface material. Fur-ther cross-sectional electron micrographs (datanot shown) did not indicate any changes to theinternal morphology of the fibers. It would appear


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210 S.D. Mumfield et al. /Journal of Biotechnolofiy 57 (1997) 205-216

1.2

w ControlA Enzyme Treated

0.01 10 100Molecular diameter (nm)

Fig. 1. Pore volume profile of untreated and enzymatically treated Douglas-fir kraft pulp fibers using six different dextran probes.

that the concerted action of these enzymes ulti-mately removes subsequent layers from the fibersurface, resulting in a polishing or cleaning ofthe fibers, as has been suggested previously (Lee etal., 1983; Pommier et al., 1990).3.4. FT-IR spectroscopy

All the FT-IR spectroscopy data indicated thatthe enzyme treatment had removed substantialamounts of xylan from the fibers, while the cellu-lose appeared to be relatively unchanged. TheDRIFT spectrum of untreated Douglas-fir kraftpulp, which was the average of eight separate diskmeasurements (Fig. 3, inset), showed characteris-tic cellulose peaks around lOOO- 1200 cm ~ (Michell, 1989; Bouchard and Douek, 1993). Therelative high absorbance at 10455 1050 cm -- andthe bands at 1460, 1250, 811 cm -. indicated thepresence of some hemicellulose, while the weakabsorption band at 1512 cm - shows that only asmall amount of lignin was still present (Liang etal., 1960; Michell, 1989; Wong et al., 1996). Al-though the spectrum from the enzyme treatedpulp initially appeared to be comparable with theuntreated sample, subtraction of the enzyme

treated spectrum from the profile of the controlrevealed some interesting differences (Fig. 3). Themajor difference was shown to be around 1045-1055 cm ~ , which corresponds to the native xylanspectra at 104551058 cm- (Michell, 1989; Fen-gel, 1992). These results confirmed that the totalamount of xylan in the sample had been reducedby the enzyme treatment. The 895 cm- bandwhich is characteristic for b-linkages, especially inhemicelluloses (Michell, 1989) was also reducedafter enzyme treatment. However, the 811 cm ~ band which is characteristic of galactoglucoman-nan (Fengel, 1992) was unaltered by enzyme treat-ment. This was in good agreement with thechemical analysis of the filtrates (Table 1). Othermajor differences were seen at higher wavelengths.The band at 1106 cm ~ could be ascribed to theantisymmetric ring stretch, the band near 1160-1170 cm ~ was representative of the antisymmet-ric bridge stretching of CO-C groups incellulose and hemicellulose (Michell, 1989), andthe band at 13 15 - 13 17 cm ~ could be ascribed toCH,-wagging vibrations in cellulose and hemicel-lulose (Gang et al., 1960). There was also asubstantial reduction in the band around 1590-1600 cm ~ which has been attributed to COO -


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S.D. Ma nsfield et al. /Journal of Biot echnology 57 (1997) 205-216 211

Fig. 2. Scanning electron micrographs of (A) untreated and (B) enzyme treated kraft pulp fibers at 2550 x magnification

groups in glucuronoxylan after salt formation(Michell et al., 1965; Wong et al., 1996). While thebands at 3300 cm - are representative of OHvibrations with intermolecular H-bonds.

Our data were collected using uniform disksobtained from 7.5 g m -* handsheets placed ontop of beds of KCl. It has been suggested that thisprocedure gives comparable results with spectra

of kraft fibers diluted directly in KBr (Michell,1991). The advantage of the former technique isthe reproducibility that can be achieved withineach sample treatment. Although it was apparentthat the enzyme treatments caused substantialhydrolysis of the xylan fraction and a polishing ofthe fibers, no major changes in the cellulose moi-ety were observed.


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0.08

0.06

4000 3500 3000 2500 2000 1500 1000 500Wavelength (run)

Fig. 3. FT-IR difference spectrum of untreated minus enzymatically treated Douglas-fir kraft pulp fibers and (inset) FT-IRabsorbance spectrum of untreated Douglas-fir kraft pulp.

3.5. Degree of polym erizat ionThe importance of the hemicellulose compo-

nent in papermaking has been well documented.Pulps which contain a higher concentration ofhemicellulose, up to a certain maximum, exhibitgreater strength. Furthermore, those fibers whichcontain a higher proportion of glucomannan(hexosan), with respect to other hemicellulose car-bohydrates, tend to show enhanced adhesive ca-pabilities and result in higher paper strength(Cottrall, 1950; Thompson et al., 1953). However,in addition to the quantity, chemical structure anddistribution of the hemicellulose, the degree ofpolymerization of this fraction plays an intricaterole in determining the final paper strength (Ere-meeva et al., 1995).

It was apparent (Table 1) that a large percent-age of the hemicellulose within these pulp sampleshad been hydrolyzed. Therefore, it is probablethat, as well as the removal of the fines and fiberdefibrillation, the reduction in available hemicellu-

lose plays a substantial role in the previouslyobserved reduction in paper strength (Mansfieldet al., 1996b).

The subsequent determination of the molecularweight distribution of the cellulose tricarbanylateby size exclusion chromatography indicated that,although there was no substantial change in thedegree of polymerization of the cellulose compo-nent, the higher molecular weight hemicellulosecomponent was reduced (Fig. 4). This shift inmolecular weight distribution after xylanase treat-ments has previously been documented for vari-ous pulp types and treatments (Mora et al., 1986;Miller et al., 1991). Therefore, it is possible thatboth the removal of hemicellulose and the con-comitant reduction in the degree of polymeriza-tion of the residual hemicellulose may havecontributed to the reduced paper strength.

The role of hemicellulose in intrinsic fiberstrength is still a topic of some debate. Opinionsdiffer on the effect of partial removal of hemicel-luloses on the fiber strength. For example, xy-


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2 0 0 0 0

1 5 0 0 0

1 0 0 0 0

5 0 0 0

01 0 0 1 0 0 0

Degree of PolymerizationFig. 4. Degree of polymerization of both untreated and enzymatically treated Douglas-fir kraft pulp

lanase treatments have been reported to cause a Therefore, it appears that the observed reduc-rapid reduction, and then a leveling off in zero- tion in fiber strength must be a result of a modifi-span breaking length. This reduction was further cation in the fibers cellulose component, rathercorrelated to the diminishing viscosity of the dis- than any effect resulting from xylan removal.solving pulps, and the authors concluded that the Previously Page et al. (1985) carried out a com-hydrolysis of the xylan macromolecules played an prehensive assessment of the strength and chemi-important role in fiber wall cohesion (No& et al., cal composition of wood pulp fibers, which1986). In contrast, Paice et al. (1992) observed a concluded that, in pulps with small fibril angles,marked decrease in the degree of polymerization the fiber strength is directly proportional to theof the xylan component of a kraft pulp after cellulose component of pulps with a less than 80%xylanase treatment, even though the pentosan cellulose content. Our work indicated that thecontent was only reduced by approximately 10% 35.2% reduction in intrinsic fiber strength, result-of its original composition. Their work also indi- ing from enzyme treatment, occurred without anycated that the fiber strength was generally unal- change in the degree of polymerization of thetered by the change in degree of polymerization of cellulose. These results concur with those reportedthe xylan. In earlier work we found that treat- previously (Gurnagul et al., 1992; Paice et al.,ments of pulp with a xylanase decreased the zero- 1992), which concluded that the enzymes prefer-span breaking length by approximately 2% entially attack structurally irregular zones such as(Mansfield et al., 1996a). Subsequent work indi- at kinks and nodes in the fiber wall. This localizedcated that the degree of polymerization of xylan attack resulted in a reduction in the intrinsic fiberhad been altered, mimicking the changes shown in strength. However, in addition to this localizedFig. 4. This seems to confirm the observations of attack, our results suggest that the cellulase en-Paice et al. (1992) that changes in the degree of zymes act in a manner which consecutively re-polymerization of the xylan have little or no effect move the outer most layers of the fiber wall,on the intrinsic fiber strength. ultimately reducing the thickness of the cell walls.


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3.6. CrystdinityThe X-ray diffractograms of enzyme treated

and control pulps indicated that there was nodiscernible difference in the degree of crystallinityof the cellulose component of the two samples(76.47 and 76.80% crystalline for the control andenzyme treated samples, respectively). This sug-gests that the cellulase enzymes acted in a mannerwhich removed cellulose molecules without inter-rupting the integrity of the remaining molecules.This again alludes to the removal of surface cellu-lose.

4. ConclusionsAlthough a portion of the cellulosic componentof the fibers had been hydrolyzed, the degree of

polymerization as measured by gel permeationchromatography and crystallinity of the substratewere unaltered. However, the substantial reduc-tion in the pore volume of the fibers indicated thatthe enzymatic treatments had eroded the surfacesof the fibers. It is probable that, in conjunctionwith the localized attack at structural irregulari-ties (Gurnagul et al., 1992), the reduced fiberstrength observed with enzymatic treatments isdirectly related to the removal of surface materialfrom the fibers, as evidenced by the electron mi-crographs of the treated fibers. The compromisedpaper strength appeared to be the result of thisreduction in intrinsic fiber strength and the re-moval of hemicellulose from the fiber. Carbohy-drate analyses, degree of polymerizationmeasurements and FT-IR spectra, all indicatedthat the hemicellulose had been substantially de-polymerized and solubilized by the enzymatictreatments.

AcknowledgementsWe would like to thank NSERC Canada and

Weyerhaeuser for a scholarship held by S.Mansfield. We are also grateful to Ron Zarges,Senior Scientist, Analysis and Testing: Mi-crostructure, Weyerhaeuser Company for his

skillful preparation of the Scanning Electron Mi-crographs.

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Physical Characterization of Enzymatic Ally Modified

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