Physical characterization of enzymatically modified kraft pulp fibers

ELSEVIER

Journal of Biotechnology 57 (1997) 205-216

Physical

Shawn

characterization of enzymatically kraft pulp fibers

D. Mansfield

a, Ed de Jong ‘, R. Scott Stephens

modified

b, John N. Saddler ‘,*

a Chair of Forest Producls Biotechnology, Department of Wood Science, Faculty of Forestry, University of British Columbia, Vancouver, BC, Canada ’ Materials Technology, Corporate R and D, Weyerhaeuser Company, Federal Way, Washington 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 paper properties were observed. Monitoring pore volume, degree of polymerization, crystallinity, FT-IR spectra, and scanning electron microscopy helped elucidate changes in fiber composition and morphology. Data indicate a decline in intrinsic fiber strength due to an erosion of the fiber surface. Reduction in paper strength resulted from the collective effects of decreased intrinsic fiber strength and the reduction in the degree of polymerization of a large portion of the hemicellulose component of the fibers, as well as fiber defibrillation and fines hydrolysis. 0 1997 Elsevier Science B.V. Keywords:

Scanning

Fiber modification; electron microscopy;

Cellulase; Xylanase; Pore volume; Degree Crystallinity; Douglas-fir; Kraft pulp

1. Introduction During the last few years cellulases and hemicellulases have been evaluated for their ability to beneficially modify pulp and paper characteristics. The various applications that have been investigated include, the deinking of secondary fibers (Prasad, 1993; Elegir et al., 1995; Heise et al.,

* Corresponding author.

FT-IR

spectroscopy;

1996), reduction of refining energy requirements (Freiermuth et al., 1994), enhanced beatability and fibrillation of chemical pulps (NoC et al., 1986; Freiermuth et al., 1994), improvements in the freeness of secondary fibers (Pommier et al., 1990; Bhat et al., 1991), as well as the bleaching of kraft pulps with xylanases and mannanases (Clark et al., 1991; Viikari et al., 1994; Saake et al., 1995; Tolan et al., 1996; Mansfield et al., 1996a). All these 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

of polymerization;

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tions of enzymes over a short incubation period. However, it was apparent that, cellulase treatments generated substantial changes to the fiber characteristics (Mansfield et al., 1996b). It appears that, as well as acting in the traditionally defined endo- or exoglucanase action that has been proposed for cellulose hydrolysis (Wood, 1989) that the cellulases are able to modify both the pulp fibers and paper characteristics in a way that cannot be directly explained by this oversimplified endo/exoglucanase mode of action. Recently, a considerable amount of work has been carried out to try to confirm that the mechanism of xylanase aided bleaching is due to the removal of reprecipitated or lignin carbohydrate complexed (LCC) xylan present on the surfaces of the pulp fibers (Yang and Eriksson, 1992; Kantelinen et al., 1993; Suurnakki et al., 1996a; de Jong et al., 1996). However, the xylanase treatments of different pulp fiber length fractions demonstrated a non-uniform response (Mansfield et al., 1996a), indicating that the composition of the fibers plays a key role in determining the effectiveness of the treatment. In contrast to xylanases, little is know about the enzyme mechanisms involved during the limited initial degradation of pulp fibers by cellulases. Although, significant advances have been made in the understanding of the mechanism by which certain microorganisms degrade hydrogen bondordered cellulose (Withers et al., 1986), the nature of the enzyme interaction and the action and sequence of events which solubilize cellulose have yet to be clearly defined. Furthermore, the extent of the modifications to the fiber morphology by cellulases remains ambiguous. Several groups (Stork et al., 1995; Mansfield et al., 1996b) have indicated that cellulase treatments result in a reduction in strength of the native fibers. Similarly, it has been shown that care must be taken when using xylanases in bleaching of kraft pulps to avoid cellulase contaminated enzyme mixtures which can severely reduce the strength of the pulp (Puls et al., 1990). Gurnagul et al. (1992) demonstrated that treatments of low yield kraft pulp with cellulase lowered the strength drastically, with relatively little change in the degree of polymerization (DP) of the cellulose. These workers

of Biotechnology 57 (1997) 205-216

concluded that the enzymes preferentially attack structurally irregular zones in the fiber wall, resulting in localized degradation. Previously, we (Mansfield et al., 1996b) and other workers (Jackson et al., 1993; Stork et al., 1995) demonstrated that beneficial changes to pulp fiber properties can be achieved by brief treatments with low concentrations of cellulases. However, this was usually accomplished at the expense of some strength loss (Stork et al., 1995; Mansfield et al., 1996b). The main purpose of this present study was to investigate the nature of changes which occur at both the micro and macro structural level of Douglas-fir kraft pulp fibers during enzymatic treatments with a commercial cellulase. By measuring the amount and type of carbohydrates released, the degree of polymerization, crystallinity, the pore volume and FT-IR spectroscopy of the substrate, as well as visualizing changes in the substrate by scanning electron microscopy we will discuss the observed changes in the fiber morphology. It was apparent that changes to the surface composition of the fibers by enzymatic treatments influenced both the fiber properties and paper strength.

2. Methods and materials 2.1. Pulp composition Unbleached kraft pulp derived from Douglasfir (Pseudotsuga menziesii) was produced at the Crofton mill (Fletcher Challenge), British Columbia, Canada. The lignin and sugar composition of the pulp was determined using sulfuric acid hydrolysates (TAPPI Method T249 cm-85). Each hydrolysate was filtered using a sintered-glass filter of medium coarseness for the gravimetric determination of Klason lignin (acid insoluble lignin), and its absorbance at 205 nm was measured for the quantification of acid soluble lignin (TAPPI Useful Method UM250, 1991). The monosaccharide constituents were quantified by anion-exchange chromatography on a CarboPac PA-l column using a Dionex DX-500 high pressure liquid chromatography (HPLC) system (Dionex, Sunnyvale, CA), using fucose as the internal standard.

S.D. Mansfield et al. /Journal

2.2. Enzyme treatments Novozyme SP342 from Novo Nordisk (Bagsvzrd, Denmark), an enzyme preparation that was derived from Humicola insolens, was used for the enzyme treatments. Its activity on carboxymethylcellulose (2% CMC, Sigma, St. Louis, MO), xylan (1% birchwood xylan, Sigma), and filter paper (No.1 Whatman) was measured using methods described previously (Wood and Bhat, 1988). Proteins in solution were quantified acid protein assay the bicinchonic using (Stoscheck, 1990). The enzyme preparation contained protein 46.7 mg ml-’ and was shown to possess relatively high xylanase activity (2869 IU ml - ‘) as well as cellulase activities (CMCase: 143.5 IU ml-‘; Filter Paper: 9.9 IU ml-‘). Pulp slurries (3% consistency in 50 mM phosphate buffer, pH 7.0) were treated for 1 h at 50°C under continuous agitation (200 rpm) with 5 mg ~ 1 protein of oven-dried fiber. The reactions g were stopped by placing the pulp in a boiling water bath for 15 min. Control pulps were similarly treated with equivalent amounts of heat inactivated enzyme (15 min boiling). The carbohydrates solubilized during the enzymatic treatments were measured by HPLC after a further secondary acid hydrolysis. The absorbance of the filtrates was measured at 280 and 457 nm to quantify the lignin and chromophores leached from the fibers, respectively. 2.3. Pore volume determination The pore volume of control and treated pulps was determined using dextran probes of varying molecular diameters (1.8-56 nm) using a modification of the solute exclusion technique developed by Stone and Scallan (1969). The individual dextran probe solutions (0.5% w/v) were added to the pulp samples, mixed thoroughly and allowed to equilibrate for 5 h with frequent gentle mixing. After equilibration, the pulp samples were allowed to settle and the probe solutions withdrawn and filtered through a sintered-glass funnel. The concentration of the probe solutions was determined refractometrically using a Waters 625 liquid chromatography system equipped with a

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57 (1997) 205-216

207

Waters 410 Differential Refractometer (Millipore, Milford, MA). The concentration of inaccessible water was determined as described previously (Gama et al., 1994). 2.4. Microscopy The pulp sample specimens were freeze-dried from a water slurry directly onto scanning electron microscope (SEM) sample mounts, which were sputter coated with 60:40, Au:Pd. The sample mounts were then observed in a LEO Spectroscan-360 SEM (Cambridge Instruments, MA) using 10 kV accelerating voltage. 2.5. FT-IR spectroscopy Low grammage handsheets (7.5 g m-*) of control and enzyme treated pulps were made using a standard British handsheet maker, placed in a phosphorus pentoxide desiccator and allowed to dry. A standard hole punch was used to remove 0.22 mg disks of the handsheets, which were then placed on a bed of KC1 powder and analyzed by FT-IR spectroscopy. The FT-IR spectra (256 scans, 4 cm - ‘) were determined by the diffuse reflectance method (DRIFT) using a PerkinElmer 1600 instrument (Norwalk, CT). The maximal absorbance was less than 1.0 AU for all samples analyzed. All samples were baseline corrected and normalized, the average of eight spectra was used as the representative spectrum for each sample. The second derivative spectra was used to improve the resolution of certain absorption bands (Michell, 1989). 2.6. Degree of polymerization The molecular weight distribution of both the control and enzyme treated pulps were obtained by Gel Permeation Chromatography (GPC) analyses of their tricarbanyl derivatives. Carbanylation of the cellulose was carried out as described previously (Schroeder and Haigh, 1979). The cellulose tricarbanylate was recovered by evaporation 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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drofuran (THF) at concentrations of approximately 0.2 mg ml _ ‘. The GPC of the tricarbanyl derivatives was carried out on a Waters 625 liquid chromatography system (Millipore, Milford, MA). The cellulose tricarbanylate samples were filtered through a Teflon membrane (0.45 pm) and analyzed using a series of 4 TSK-GEL columns (Varian, Sunnyvale, CA., type GlOOO HXL, G3000 HXL, G4000 HXL and G6000 HXL with molecular weight cut-offs of 1 x 103, 6 x 104, 4 x lo5 and 4 x lo’, respectively). THF was used as the eluting solvent at a flow rate of 1 ml min - ‘. The samples in the eluent were detected by a Waters 486 UV spectrophotometer (Millipore, Milford, MA) at a wavelength of 254 nm. The GPC calibration curve was generated from the elution profile of polystyrene standards with narrow molecular weight distributions. Using the Mark-Houwink coefficients previously reported for polystyrene in THF, K, = 1.18 x lop4 and CQ,= 0.74, and for cellulose tricarbanylate in THF, Kc = 2.01 x lop5 and a, = 0.92 the molecular weight of the tricarbanylated cellulose was obtained (Valtasaari and Saarela, 1975). The degree of polymerization (DP) of cellulose was obtained by dividing the molecular weight of the tricarbanylated polymer by the corresponding molecular weight of the tricarbanylated derivative of anhydroglucose (DP = Mwj519). Both the number averages (DP,) and the weight averages (DP,) of the substrates were calculated as described previously (Yau et al., 1979). 2.7. Crystallinity The degree of crystallinity of the pulp samples was obtained by X-ray diffraction. Handsheets (100 g m - ‘) of both control and treated pulps were made and pressed semi-dry at 5000 psi for 5 min. Representative 4.2 x 2.7 cm rectangles were cut from the handsheets, freeze-dried and stored in a phosphorus pentoxide desiccator until analysis. The X-ray diffraction of each sample was recorded using a Siemens diffractometer equipped with a D-5000 rotating anode X-ray generator. The wavelength of the Cu/Kaa radiation source

of Biotechno1og.v 57 (1997) 205-216 was 0.154 nm, and the spectra were obtained at 30 mA with an accelerating voltage of 40 kV. Samples were scanned on the automated diffractometer from 5 to 40” of 20 (Bragg angle), with data acquisition taken at intervals of 0.02” for 1 s. A peak resolution program was used to calculate both the crystallinity index of cellulose and the dimensions of the crystallites (Hindeleh and Johnson, 1978).

3. Results and discussion In previous work (Mansfield et al., 1996b) we assessed a cellulase enzyme preparation for its potential to enhance the fiber characteristics of pulps derived from Douglas-fir wood chips. These preliminary results indicated that the enzymatic effects on the pulp properties were highly dependent on the dosage. For example, an enzyme charge of 5 mg protein per gram of pulp, which corresponded to a 3.2% hydrolysis of the pulp, produced a 12.7% reduction in paper strength (tensile index) and a 35.2% loss in fiber strength (zero-span breaking length). Although the enzyme treatments resulted in a considerable loss in both the paper and fiber strength, we wanted to determine which modifications to the fiber structure could be correlated with these strength changes. 3.1. Carbohydrate

solubilization

A more thorough investigation by HPLC analysis indicated which specific carbohydrates were solubilized by the enzyme treatments after 3.2% of the pulp was hydrolyzed (Table 1). It was apparent that the enzyme treatment liberated almost equivalent amounts of both glucose (229 mg) and xylose (226 mg), which corresponds to 1.8 and 21 .l% hydrolysis, respectively, of the total amount of cellulose and xylan that was originally available in the pulp. Previously it was proposed that, at the end of the kraft cooking process,, debranched xylan redeposits on the surface of the fibers (Yllner et al., 1957), and that this redeposited xylan is the primary substrate for xylanases in the prebleaching process (Kantelinen et al., 1993). However, Suur-

S.D. Mansfield Table 1 Composition Fraction Pulp (%) Filtrate” %Solubilized

of the Douglas-fir Arabinose 0.41 21.01 28.8

et al. /Journal

pulp and filtrates Galactose 0.56 0 0

N/A, not assessed. a Values represent total mg of carbohydrate

obtained

Glucose

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Xylose

Mannose

57 (1997) 205-216

209

treatment Klason

lignin

Acid-soluble

lignin

Recovery

12.56

5.95

6.66

4.91

0.49

91.54

229.39 1.8

225.83 21.1

16.09 1.3

N/A N/A

0.08 16.3

N/A N/A

liberated

from enzyme

treated

minus

control

filtrates

of 18 g pulp samples.

nakki et al. (1996b) have recently questioned the importance of redeposition when pine kraft pulps were treated with xylanases. Our results seem to indicate that the debranched xylan is not the major substrate for the xylanases, as the arabinose/xylose ratio in the filtrates was increased when compared to that found in the pulp (Table 1). Therefore, it is possible that the concerted hydrolysis by both the cellulase and xylanase enzymes liberated both glucose and the more highly substituted xylan found in the inner fiber wall (Suurnakki et al., 1996~). This would account for the large proportion of the arabinose hydrolyzed by the enzymatic treatments. A 5- and 3.5fold increase in the absorbances at 280 and 457 nm was observed after enzyme treatment, respectively. These increases were probably due to the liberation of lignin and coloured compounds by the action of the xylanases on the pulp fibers. Although the cellulases have been shown to be rather ineffective in releasing coloured materials from kraft pulps (Buchert et al., 1994), they undoubtedly hydrolyze the available surface cellulose and continue to work in concert with the xylanases resulting in the hydrolysis of some of the internal structural polysaccharides.

al., 1988). It has also been used in biobleaching experiments, where the removal of hemicellulose and its associated lignin by xylanase treatment resulted in an increase in the pore volume of the fibers (Yu et al., 1994) and a slight increase in the median pore width (Suurnakki, 1996d). In this work we have used pore volume determination to investigate the changes that occurred as a result of the enzymatic treatments to the Douglas-fir pulp fibers. As indicated previously, a substantial amount of the hemicellulose component of the pulp was hydrolyzed after enzyme treatment, suggesting that there might be a corresponding increase in the pore volume of the fibers. However, we found that the cumulative pore volume (inaccessible water) of the fibers was significantly reduced by these enzyme treatments (Fig. 1). The profile exhibited by the enzyme treatments not only demonstrated a substantial reduction in the pore volume for each of the dextran probes used, it also showed a reduction in the fiber saturation point (as measured by the 56 nm probe). However, there was an increase in the median pore width of the enzyme treated pulps.

3.2. Pore volume determination

Scanning electron microscopy, indicated a qualitative change in the outermost fiber surface of the enzymatically treated pulp compared to control pulps (Fig. 2). However, the electron micrographs did not reveal any discernible alterations to the fiber surfaces (i.e. cleavages or pit enlargements) other than the erosion of surface material. Further cross-sectional electron micrographs (data not shown) did not indicate any changes to the internal morphology of the fibers. It would appear

The solute exclusion technique of Stone and Scallan (1969) has been a valuable tool for characterizing the ultrastructural morphology of fibers after various physical and chemical pretreatments. It has been successfully used to demonstrate the importance of fiber porosity during the enzymatic hydrolysis of various cellulosic substrates (Grethlein, 1985; Weimer and Weston, 1985; Wong et

3.3. Scanning electron microscopy

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1.2

w A

Control Enzyme Treated

0.0 1

100

10

Molecular diameter (nm) Fig. 1. Pore volume

profile of untreated

and enzymatically

treated

that the concerted action of these enzymes ultimately removes subsequent ‘layers’ from the fiber surface, resulting in a ‘polishing or cleaning’ of the fibers, as has been suggested previously (Lee et al., 1983; Pommier et al., 1990). 3.4. FT-IR

spectroscopy

All the FT-IR spectroscopy data indicated that the enzyme treatment had removed substantial amounts of xylan from the fibers, while the cellulose appeared to be relatively unchanged. The DRIFT spectrum of untreated Douglas-fir kraft pulp, which was the average of eight separate disk measurements (Fig. 3, inset), showed characteristic cellulose peaks around lOOO- 1200 cm ~ ’ (Michell, 1989; Bouchard and Douek, 1993). The relative high absorbance at 10455 1050 cm -- ’ and the bands at 1460, 1250, 811 cm -. ’ indicated the presence of some hemicellulose, while the weak absorption band at 1512 cm - ’ shows that only a small amount of lignin was still present (Liang et al., 1960; Michell, 1989; Wong et al., 1996). Although the spectrum from the enzyme treated pulp initially appeared to be comparable with the untreated sample, subtraction of the enzyme

Douglas-fir

kraft

pulp fibers using six different

dextran

probes.

treated spectrum from the profile of the control revealed some interesting differences (Fig. 3). The major difference was shown to be around 10451055 cm ~ ‘, which corresponds to the native xylan spectra at 104551058 cm- ’ (Michell, 1989; Fengel, 1992). These results confirmed that the total amount of xylan in the sample had been reduced by the enzyme treatment. The 895 cm- ’ band which is characteristic for b-linkages, especially in hemicelluloses (Michell, 1989) was also reduced after enzyme treatment. However, the 811 cm ~ ’ band which is characteristic of galactoglucomannan (Fengel, 1992) was unaltered by enzyme treatment. This was in good agreement with the chemical analysis of the filtrates (Table 1). Other major differences were seen at higher wavelengths. The band at 1106 cm ~ ’ could be ascribed to the antisymmetric ring stretch, the band near 11601170 cm ~ ’ was representative of the antisymmetric bridge stretching of CO-C groups in cellulose and hemicellulose (Michell, 1989), and the band at 13 15 - 13 17 cm ~ ’ could be ascribed to CH,-wagging vibrations in cellulose and hemicellulose (Gang et al., 1960). There was also a substantial reduction in the band around 15901600 cm ~ ’ which has been attributed to COO’ -

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Fig. 2. Scanning

electron

micrographs

of (A) untreated

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and (B) enzyme

groups in glucuronoxylan after salt formation (Michell et al., 1965; Wong et al., 1996). While the bands at 3300 cm - ’ are representative of OH vibrations with intermolecular H-bonds. Our data were collected using uniform disks obtained from 7.5 g m -* handsheets placed on top of beds of KCl. It has been suggested that this procedure gives comparable results with spectra

treated

kraft

211

pulp fibers at 2550 x magnification

of kraft fibers diluted directly in KBr (Michell, 1991). The advantage of the former technique is the reproducibility that can be achieved within each sample treatment. Although it was apparent that the enzyme treatments caused substantial hydrolysis of the xylan fraction and a polishing of the fibers, no major changes in the cellulose moiety were observed.

S.D. Mansfieldet al. /Journal of Biotechnology57 (1997) 205-216

212

0.08

0.06

4000

3500

3000

2500

2000

1500

1000

500

Wavelength (run) Fig. 3. FT-IR difference spectrum of untreated minus enzymatically absorbance spectrum of untreated Douglas-fir kraft pulp.

3.5. Degree

of polymerization

The importance of the hemicellulose component in papermaking has been well documented. Pulps which contain a higher concentration of hemicellulose, up to a certain maximum, exhibit greater strength. Furthermore, those fibers which contain a higher proportion of glucomannan (hexosan), with respect to other hemicellulose carbohydrates, tend to show enhanced adhesive capabilities and result in higher paper strength (Cottrall, 1950; Thompson et al., 1953). However, in addition to the quantity, chemical structure and distribution of the hemicellulose, the degree of polymerization of this fraction plays an intricate role in determining the final paper strength (Eremeeva et al., 1995). It was apparent (Table 1) that a large percentage of the hemicellulose within these pulp samples had been hydrolyzed. Therefore, it is probable that, as well as the removal of the fines and fiber defibrillation, the reduction in available hemicellu-

treated

Douglas-fir

kraft

pulp

fibers

and

(inset)

FT-IR

lose plays a substantial role in the previously observed reduction in paper strength (Mansfield et al., 1996b). The subsequent determination of the molecular weight distribution of the cellulose tricarbanylate by size exclusion chromatography indicated that, although there was no substantial change in the degree of polymerization of the cellulose component, the higher molecular weight hemicellulose component was reduced (Fig. 4). This shift in molecular weight distribution after xylanase treatments has previously been documented for various pulp types and treatments (Mora et al., 1986; Miller et al., 1991). Therefore, it is possible that both the removal of hemicellulose and the concomitant reduction in the degree of polymerization of the residual hemicellulose may have contributed to the reduced paper strength. The role of hemicellulose in intrinsic fiber strength is still a topic of some debate. Opinions differ on the effect of partial removal of hemicelluloses on the fiber strength. For example, xy-

S.D. Mansfield

et al. /Journal

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57 (1997) 205-216

213

20000

15000

10000

5000

0

100

1000

Degree of Polymerization Fig. 4. Degree

of polymerization

of both untreated

lanase treatments have been reported to cause a rapid reduction, and then a leveling off in zerospan breaking length. This reduction was further correlated to the diminishing viscosity of the dissolving pulps, and the authors concluded that the hydrolysis of the xylan macromolecules played an important role in fiber wall cohesion (No& et al., 1986). In contrast, Paice et al. (1992) observed a marked decrease in the degree of polymerization of the xylan component of a kraft pulp after xylanase treatment, even though the pentosan content was only reduced by approximately 10% of its original composition. Their work also indicated that the fiber strength was generally unaltered by the change in degree of polymerization of the xylan. In earlier work we found that treatments of pulp with a xylanase decreased the zerospan breaking length by approximately 2% (Mansfield et al., 1996a). Subsequent work indicated that the degree of polymerization of xylan had been altered, mimicking the changes shown in Fig. 4. This seems to confirm the observations of Paice et al. (1992) that changes in the degree of polymerization of the xylan have little or no effect on the intrinsic fiber strength.

and enzymatically

treated

Douglas-fir

kraft

pulp

Therefore, it appears that the observed reduction in fiber strength must be a result of a modification in the fibers’ cellulose component, rather than any effect resulting from xylan removal. Previously Page et al. (1985) carried out a comprehensive assessment of the strength and chemical composition of wood pulp fibers, which concluded that, in pulps with small fibril angles, the fiber strength is directly proportional to the cellulose component of pulps with a less than 80% cellulose content. Our work indicated that the 35.2% reduction in intrinsic fiber strength, resulting from enzyme treatment, occurred without any change in the degree of polymerization of the cellulose. These results concur with those reported previously (Gurnagul et al., 1992; Paice et al., 1992), which concluded that the enzymes preferentially attack structurally irregular zones such as at kinks and nodes in the fiber wall. This localized attack resulted in a reduction in the intrinsic fiber strength. However, in addition to this localized attack, our results suggest that the cellulase enzymes act in a manner which consecutively remove the outer most layers of the fiber wall, ultimately reducing the thickness of the cell walls.

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3.6. Crystdinity The X-ray diffractograms of enzyme treated and control pulps indicated that there was no discernible difference in the degree of crystallinity of the cellulose component of the two samples (76.47 and 76.80% crystalline for the control and enzyme treated samples, respectively). This suggests that the cellulase enzymes acted in a manner which removed cellulose molecules without interrupting the integrity of the remaining molecules. This again alludes to the removal of surface cellulose.

4. Conclusions Although a portion of the cellulosic component of the fibers had been hydrolyzed, the degree of polymerization as measured by gel permeation chromatography and crystallinity of the substrate were unaltered. However, the substantial reduction in the pore volume of the fibers indicated that the enzymatic treatments had eroded the surfaces of the fibers. It is probable that, in conjunction with the localized attack at structural irregularities (Gurnagul et al., 1992), the reduced fiber strength observed with enzymatic treatments is directly related to the removal of surface material from the fibers, as evidenced by the electron micrographs of the treated fibers. The compromised paper strength appeared to be the result of this reduction in intrinsic fiber strength and the removal of hemicellulose from the fiber. Carbohyanalyses, of polymerization drate degree measurements and FT-IR spectra, all indicated that the hemicellulose had been substantially depolymerized and solubilized by the enzymatic treatments.

Acknowledgements We 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: MiWeyerhaeuser Company for his crostructure,

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skillful preparation crographs.

of the Scanning

Electron

Mi-

References Bhat, G.R., Heitmann, J.A., Joyce, T., 1991. Novel techniques for enhancing the strength of secondary fiber. Tappi J. 74 (IO), 151l157. Bouchard, J., Douek, M., 1993. Structural and concentrational effects on the diffuse reflectance FT-IR spectra of cellulose, lignin and pulp. J. Wood Chem. Technol. 13, 481-499. Buchert, J., Ranua, M., Siika-aho, M., Pere, J., Viikari, L., 1994. Trichoderma reesei cellulases in the bleaching of kraft pulp. Appl. Microbial. Biotechnol. 40, 941-945. Clark, T., Steward, D., Bruce, M.E., McDonald, A.G., Singh, A.P., Senior, D.J., 1991. Improved bleachability of radiata pine kraft pulps following treatment with hemicellulolytic enzymes. Appita J. 44, 3899404. Cottrall, L.G., 1950. The influence of hemicelluloses in wood pulp fibers on their papermaking properties. Tappi J. 33, 471-480. de Jong, E., Wong, K.K.Y., Martin, L.A., Mansfield, SD., Gama, F.M., Saddler, J.N., 1996. Molecular mass distribution of materials solubilized by xylanase treatment of Douglas-fir kraft pulp. ACS Symp. Ser. 655, 4462. Elegir, G., Sykes, M., Jeffries, T.W., 1995. Differential and synergistic action of Streptomyces endoxylanases in prebleaching of kraft pulps. Enzyme Microb. Technol. 17, 9544959. Eremeeva, T., Bykova, T., Treimanis, A., 1995. The accessibility and the properties of wood pulp residual hemicelluloses and lignin in different fiber wall layers. 8th Intl. Symp. Wood Pulping Chem., Vol. III, Helsinki, Finland, pp. 2255230. Fengel, D., 1992. Moglichkeiten und Grenzen der FTIR-Spektroskopie bei der Charakterisierung von Cellulose. Teil 3. Einfluss von Begleitstoffen auf das IR- Spektrum von Cellulose. Das Papier 46 (I), 7- 1 f Freiermuth, B., Garrett, M., Jokinen, O., 1994. The use of enzymes in the production of release paper. Paper Technol. 35 (3) 21-23. Gama, F.M., Mota, M., Teixeira, J.A., 1994. Cellulose morphology and enzymatic reactivity: A modified solute exclusion technique. Biotechnol. Bioeng. 43, 381-387. Grethlein, H.E., 1985. The effect of pore size distribution on the rate of enzymatic hydrolysis of cellulosic substrates. Bio/Technol. 2, 155-159. Gurnagul, N., Page, D.H., Paice, M.G., 1992. The effect of cellulose degradation on the strength of wood pulp fibers. Nordic Pulp Paper Res. J. 7 (3), 152-154. Heise, O.U., Unwin, J.P., Klungness, J.H., Finerdn, W.G. Jr., Sykes, M., Abubakr, S., 1996. Industrial scale-up of enzyme-enhanced deinking of non-impact printed toners. Tappi J. 79 (3) 207-212.

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