Effect of cellulase-assisted refining on the properties of dried andnever-dried eucalyptus pulp

O. García1, A.L. Torres1, J.F. Colom1, F.I.J. Pastor2, P. Díaz2 and T. Vidal1,*1Department of Textile and Paper Engineering, E.T.S.E.I. Terrassa, Polytechnical University of Catalonia,Colon 11, E-08222 Terrassa, Spain; 2Department of Microbiology, Faculty of Biology, University ofBarcelona, Av. Diagonal 645, E-08028 Barcelona, Spain; *Author for correspondence (e-mail:tvidal@etp.upc.es)

Received 24 October 2000; accepted in revised form 21 January 2002

Key words: Biorefining, Cellulase, Enzyme, Eucalyptus pulp, Fibre surface modification, Hornification, Opticalmicroscopy, Paper properties

Abstract

The effect of two different cellulases on the hornification phenomenon, in which drainability (Schopper–Rieglermethod) and mechanical properties diminish when pulps are dried, was studied. The enzyme applications testedincluded a commercial enzyme named ComC (Pergalase A40 from CIBA) and a laboratory enzyme from Paeni-bacillus sp. strain BP-23 named CelB. Industrial never-dried Eucalyptus globulus bleached kraft pulp was splitin two halves and one of them was dried at ambient controlled conditions. We compared enzyme effects on bothpulps (wet pulp and dried pulp) before and after PFI mill refining. Enzyme applications increased drainability(Schopper–Riegler method) and water retention value (WRV) of never-dried bleached pulp, although this did notimply an enhancement of the mechanical properties of paper. Cellulase treatment of dried pulps, by contrast,gave rise to increased drainability and WRV and also to improved mechanical properties. The changes caused bydrying became less significant after enzyme application. Handsheets from CelB-treated dried pulps showed animprovement of tensile and burst indexes while tear decreased. The effect produced by CelB can be considereda biorefining step. In fact, by means of enzyme treatment with CelB the properties of paper manufactured fromdried pulp equalled the properties attained from wet fibres, with the exception of tear index. Changes were alsofound in surface fibre morphology, such as flakes and peeling due to cellulase treatment. The surface modifica-tion of fibres with cellulases gives rise to better bonding properties and a closer structure of paper. The finalconclusion is that treatment with cellulases could compensate the hornification effect and lead to an importantsaving of refining energy. The novel enzyme, CelB, was the most effective in improving paper properties andcounterbalancing the hornification effect caused by drying.

Introduction

Use of enzymes in the pulp and paper industry hasgrown rapidly since the mid-1980s. Although mostareas are still developing, several applications are in-tensively pursued, including the utilisation of en-zymes for drainage enhancement (Oksanen et al.2000), deinking of secondary fibres (Bajpai 1997; Ba-jpai and Bajpai 1998) and modifying inherent fibrecharacteristics (Mansfield et al. 1997; Lumme et al.1999; Vidal et al. 1999).

During the last few years cellulases and hemicel-lulases have been evaluated for their ability to ben-eficially modify pulp and paper characteristics (Kib-blewhite and Clark 1996; España et al. 1998).Enzyme treatments resulted in alterations in the kraftpulp fibre characteristics (Lin et al. 1995; Roncero etal. 2000; Suurnäkki et al. 2000; Torres et al. 2000),which ultimately translated into changes in the paperproperties (Pere et al. 1995; Roncero et al. 1996; Ok-sanen et al. 1997a; Torres et al. 1999). Cellulase treat-ment of dried pulp seems to increase the relative

115Cellulose 9: 115–125, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

bonded area of the fibrous paper network, improvingsome paper properties (Pastor et al. 2001). The extentof the modifications of fibre morphology by the ac-tion of cellulases remains unclear. However, if theenzymes could selectively alter the outermost sur-faces of pulp fibres (Blanco et al. 1998), it is possiblethat inherent characteristics, such as stiffness and col-lapsibility, could be modified to enhance conformabil-ity and inter-fibre bonding (Mansfield et al. 1998).

Drying of the fibres results in a hornification phe-nomenon (Jayme 1994), the consequences of whichcan be clearly observed by the deterioration of fibreproperties (Weise 1998; Weise and Paulapuro 1999).Hornification can take place at room temperature andis a feature of chemical pulps. This deterioration ofpaper-making properties of dried pulps is mainly dueto irreversible structural changes of fibres. Removalof water brings adjacent microfibrils into close con-tact and leads to the formation of hydrogen bonds be-tween them. In this way, fibres lose conformabilityand swelling capacity, and consequently a deteriora-tion of pulp properties takes place (Oksanen et al.1997b). Refining improves the bonding characteris-tics of the fibres, increasing strength tests such astensile and burst. However, increased refining alsolowers stock freeness and typically slows down thevelocity of the paper machine (Moran 1996).

In this work, the effect of cellulase treatment onthe properties of dried and never-dried eucalyptuspulp was studied. Eucalyptus bleached pulp wastreated with enzymes and subsequently refined.Drainage properties of pulps and physical propertiesof handsheets were analyzed. The effects of enzymetreatment on the recovery of the properties of horni-fied pulps were also evaluated.

Material and methods

Raw material

Industrial Eucalyptus globulus bleached kraft pulpfrom Torras Papel S.A. mill (Zaragoza, Spain) with50% dryness, 89% ISO brightness and 800 mL g−1

viscosity was used. Half of this pulp was dried at am-bient controlled conditions in darkness (23 °C and50% relative humidity) in order to analyse the effectof drying on the pulp and paper properties. The en-zymes studied were applied in both pulps [dried pulp(dp) and wet pulp (wp)].

Enzymes used

In this study a commercial cellulase enzyme namedComC (CIBA: Pergalase A40) and a novel laboratoryenzyme, from Paenibacillus sp. strain BP-23, namedCelB were used. Pergalase A40 is a mixture of extra-cellular enzymes from Trichoderma longibrachiatum,the main activity of which is on carboxymethylcellu-lose (CMC) (830 IU/g), although it also shows highactivity on filter paper (65 IU/g), Avicel (65 IU/g) andxylan (575 IU/g) (Pommier et al. 1989; Ciba cata-logue). CelB from Paenibacillus sp. strain BP-23 is arecently characterized modular cellulase that containsa catalytic domain of family 9, a carbohydrate-bind-ing module of family III and two fibronectin-like do-mains (Pastor et al. 2001). The enzyme is 103 kDa insize and shows high activity on carboxymethylcellu-lose (CMC) but negligible activity on Avicel, indicat-ing that it is an endoglucanase. CelB preparationtested on pulp is an extract from recombinant Escher-ichia coli cells containing plasmid pC7, harbouringCelB encoding gene. It shows high activity on CMC(35.10 IU/g), but very little activity on Avicel (0.10IU/g) and other polysaccharides, such as xylan (0.12IU/g) or filter paper (0.11 IU/g).

The enzyme treatments were performed in poly-ethylene bags as described previously (Blanco et al.1998). The dose of enzymes used was 2.5CMCase IU g−1 o.d.p. as previously reported (Españaet al. 1998). The conditions of enzyme treatmentswere:

• ComC: 2.5 CMCase IU g−1 o.d.p., pH 4–4.5,consistency 10%, time 60 min and temperature40 °C (CIBA: Pergalase A40)

• CelB: 2.5 CMCase IU g−1 o.d.p., pH 5.5, con-sistency 10%, time 60 min and temperature 53°C (Pastor et al. 2001)

Refining and pulp properties. Standard methodsused

Pulp and paper properties were tested according to thefollowing standard methods, and an indication of thelimit of data repeatability is shown between brackets:water retention value, TAPPI UM 256 and MerkblattIV/33/57 (2%); Schopper–Riegler ISO 5267-1 (4%);viscosity ISO 5351-1 (2.5%). Pulp was refined ac-cording to the PFI method ISO 5264-2. Handsheetswere made according to the standard methods ISO5269-2; density, ISO-534 (2%); permeability, ISO5636/3 (5%); tensile index, ISO1924-1 (4.2%); tear

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index, ISO 1974 (3.5%); burst index, ISO 2758(3.5%). Data treatments are included in the respectivestandard methods.

Results and discussion

The effect of enzyme treatments with cellulases onthe pulp and paper properties has been evaluated.Dried and wet (never-dried) eucalyptus pulps weretreated with two different cellulase preparations,ComC and CelB, followed by refining using differentPFI revolutions. Pulp and paper properties were de-termined and compared to those of control samplesuntreated with enzymes (original pulp).

Original pulp: comparison of properties of wet pulp(wp) and dried pulp (dp) without enzyme treatment

Figures 1–6 and Table 1 show the differences betweenwet and dry original pulps. The results confirm thegeneral trends quoted in the literature (Jayme 1994;Weise 1998). Comparison of properties of dried andwet pulps, unrefined and refined for the same PFIrevolutions, showed a slight decrease of drainability(Figure 1) and WRV for dried pulps. Drying led to animportant decrease in the WRV of unrefined original

pulp, which was partially recovered at higher revolu-tions (Table 1). WRV values went from 165% (wetpulp) to 158% (dried pulp) at 4500 revolutions (Ta-ble 1). As expected, viscosity variation with dryingwas very small (805 vs. 795 mL g−1).

Regarding paper characteristics, Table 1 shows asmall decrease in density (Figure 2) for dried vs. wetpulps for all degrees of beating studied (0, 3000, 4500and 6000 PFI revolutions). Permeability was alwayshigher in dried pulps. At 3000 revolutions permeabil-ity values for wet and dried pulp were 9.2 �m (Pa s)−1

and 16.7 �m (Pa s)−1, respectively. Tensile and burstindexes decreased in dried pulps (Figures 3 and 4).However, tear index of paper from wet and driedpulps increased with refining up to 3000 revolutions,but decreased at high degrees of refining (4500 and6000 revolutions) (Figure 5).

As stated above, the results shown in Table 1 fullyagree with the previous findings quoted in the litera-ture about the effects of drying on properties of pa-per. Drying decreases the bonding capacity of the fi-bres, giving papers with more bulk (less density) andpermeability, but lower tensile and burst indexes.These consequences of drying and its effects on pa-per are well known by papermakers, especially whendealing with secondary fibres.

Table 1. Pulp and paper properties of original pulp.

Wet pulp Dry pulp

0 3000 4500 6000 0 3000 4500 6000

Pulp properties

Drainability (°SR) 17 30 40 50 16.5 28 36 47

SD 0.48 0.96 1.15 1.00 0.41 0.58 0.58 2.65

Water retention value (%) 123 156 165 170 101 151 158 164

SD 1.24 1.38 1.2 1.8 0.91 1.66 2.14 0.63

Viscosity (mL g−1) 805 – – – 795 – – –

SD 8.49 18.38

Paper properties

Density (g cm−3) 0.56 0.66 0.66 0.70 0.52 0.62 0.65 0.66

SD 0.012 0.016 0.051 0.012 0.006 0.017 0.01 0.01

Permeability (�m (Pa s)−1) 46.0 9.2 5.0 3.1 49.7 16.7 7.4 3.68

SD 0.41 0.22 0.96 0.14 0.26 0.99 0.33 0.17

Tensile index (N m g−1) 33.54 67.67 68.56 77.64 23.70 54.57 62.35 64.25

SD 2.92 5.89 9.5 9.1 3.25 3.93 3.87 7.36

Burst index (kPa m2 g−1) 1.05 4.25 4.25 4.55 0.45 3.00 3.45 3.55

SD 0.08 0.43 0.54 0.34 0.02 0.18 0.34 0.23

Tear index (mN m2 g−1) 8.05 10.6 9.40 9.65 3.75 10.0 10.2 9.80

SD 0.70 1.15 1.15 1.63 2.00 1.53 1.15 1.15

SD: standard deviation.

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Effect of cellulase-assisted refining on the propertiesof never-dried eucalyptus pulp (wp)

Unrefined pulpPulp properties, drainability and WRV (Tables 1–3)showed similar values in original and enzymaticallytreated pulps (ComC and CelB). Viscosity wasslightly modified in the case of CelB (760 vs. 805mL g−1), but there was a higher reduction in the caseof ComC (730 vs. 805 mL g−1). Paper properties ofunrefined pulps were not significantly affected by en-zyme treatment. In this way, density was similar inall samples, while permeability was slightly lower inenzyme-treated pulps. Tensile and burst indexes ofCelB- or ComC-treated pulps were slightly higher

than those of original pulps, while tear index wasslightly lower in enzyme-treated pulps. These resultsare in agreement with previous reports (Blanco et al.1998; España et al. 1998).

Refined pulpDrainability and WRV of all wet pulps increased withrefining (Figure 1), but the increase detected in en-zyme-treated pulps was higher than that found in pulpuntreated with enzymes, and this agrees with Oksanenet al. (1997a). In general, the order of increase wasComC pulp>CelB pulp>original pulp. However, mostpaper properties were diminished by enzyme treat-ment. Tensile and burst indexes increased with refin-ing but the increase was smaller in enzyme-treated

Figure 1. Drainability (°SR) versus PFI-refining revolutions.

Figure 2. Density versus permeability.

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pulps. Permeability decreased with refining, and thedecrease was higher in enzyme-treated pulps. On theother hand, density was similar in all cases. In fact,permeability as well as tensile, burst and tear indexeswere always higher in original pulp.

The results found show that increases in drainabil-ity (Schopper–Riegler) and WRV, implying lessdrainage in the paper machine wire, do not necessar-ily imply improvement in paper properties. These re-sults indicate that treatment with cellulases in wetpulps (never-dried) does not result in improvementsin paper properties. Similar results have been reportedpreviously (Pere et al. 1995; Oksanen et al. 1997a).

Effect of cellulase-assisted refining on the propertiesof dried eucalyptus pulp (dp)

Unrefined pulpThe tendency of drainability, WRV, viscosity, densityand permeability of all dried pulp samples was simi-lar to those of never-dried pulps (Tables 1–3). How-ever, tensile and burst indexes were significantlyhigher in the two enzyme-treated pulps (Figures 3 and4), and this agrees with España et al. (1998) andBlanco et al. (1998). Tear index of enzyme-treatedpulps was notably higher than that of pulps untreatedwith enzyme (Figure 5).

Figure 3. Tensile index versus PFI-refining revolutions.

Figure 4. Burst index versus PFI-refining revolutions.

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Refined pulpDevelopment of drainability value (°SR) during refin-ing of original and enzyme-treated dried pulps isshown in Figure 1. Similarly to what was found forwet pulps, for the same mechanical refining action(PFI revolutions), the use of enzymes gave rise to in-crease of drainability values (°SR). However, the ex-tent of increase was much higher in the case of driedpulps. A comparison of mechanical properties of re-fined dried pulps showed different results to thosefound for wet pulps. ComC treatment gave rise to anincrease in tensile and burst indexes of dried pulpsrefined at 3000 revolutions, while when pulps wererefined at 4500 or 6000 revolutions, these indexeswere not significantly modified (Figures 3 and 4). On

the contrary, application of CelB to dried pulps gaverise to an increase in tensile and burst indexes for allPFI revolutions applied. Tensile and burst values ofrefined CelB-treated dried pulps were much higherthan those of refined dried original pulp, and similarto those of refined non enzyme-treated wet pulp (Fig-ures 3 and 4). When the influence of enzyme treat-ment on other parameters of refined pulps was ana-lyzed, it was found that enzyme treatment loweredtear index, being the lowest values reached by ComCpulp (Figure 5). Enzyme treatment of dried pulps in-creased the density of paper slightly, but gave rise toan important decrease in permeability (Figure 2, Ta-bles 1–3).

Figure 5. Tear index versus tensile index.

Figure 6. Density versus tensile index.

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The results show that CelB causes an importantimprovement of bonding properties, tensile and burstindexes; but it results in a dramatic decrease in per-

meability (Figure 2). These results confirm previousresults (España et al. 1998; Torres et al. 1999; Pastoret al. 2001) showing that enzymes can improve the

Table 2. Pulp and paper properties of ComC-treated pulp.

Wet pulp Dry pulp

0 3000 4500 6000 0 3000 4500 6000

Pulp properties

Drainability (°SR) 18.5 36 50 55 18 54 70 81

SD 0 0 1.15 0.58 0.29 1.41 2.00 1.77

Water retention value (%) 123 165 183 187 106 179 202 211

SD 0.72 1.55 2.45 0.87 1.58 0.64 3.34 1.08

Viscosity (mL g−1) 730 – – – 700 – – –

SD 2.83 15.56

Paper properties

Density (g cm−3) 0.55 0.67 0.70 0.72 0.51 0.70 0.74 0.80

SD 0.004 0.009 0.015 0.018 0.008 0.010 0.009 0.016

Permeability (�m (Pa s)−1) 42.3 6.0 2.3 1.40 47.9 1.68 0.50 0.24

SD 0.89 0.31 0.12 0.05 1.69 0.04 0.02 0.02

Tensile index (N m g−1) 33.59 65.35 66.40 73.52 26.21 58.55 60.66 62.84

SD 2.33 5.41 6.09 4.62 1.73 4.32 4.36 4.38

Burst index (kPa m2 g−1) 1.25 3.90 3.40 4.40 0.75 3.30 3.50 3.65

SD 0.06 0.16 0.35 0.22 0.08 0.21 0.22 0.16

Tear index (mN m2 g−1) 7.90 8.40 8.10 7.95 7.40 5.75 5.10 4.85

SD 0.57 0.95 2.00 2.38 1.00 1.15 0.58 0.58

SD: standard deviation.

Table 3. Pulp and paper properties of CelB-treated pulp.

Wet pulp Dry pulp

0 3000 4500 6000 0 3000 4500 6000

Pulp properties

Drainability (°SR) 17.5 32 43 62 17.5 42 59 73

SD 0.5 0.29 0.00 0.00 0.71 1.00 2.36 2.52

Water retention value (%) 125 160 170 179 104 165 177 190

SD 0.59 1.03 1.15 1.99 1.25 2.33 0.51 2.71

Viscosity (mL g−1) 760 – – – 800 – – –

SD 5.66 11.31

Paper properties

Density (g cm−3) 0.55 0.65 0.69 0.72 0.51 0.64 0.69 0.73

SD 0.007 0.014 0.014 0.009 0.008 0.008 0.013 0.007

Permeability (�m (Pa s)−1) 41.7 8.0 3.2 1.3 44.4 4.4 1.29 0.44

SD 0.69 0.31 0.12 0.07 1.12 0.24 0.06 0.01

Tensile index (N m g−1) 36.83 65.45 69.73 73.97 27.95 67.92 73.89 77.66

SD 2.71 5.68 6.35 7.19 3.53 5.06 6.67 7.21

Burst index (kPa m2 g−1) 1.45 3.70 3.95 4.35 0.85 3.80 4.30 4.85

SD 0.10 0.38 0.29 0.24 0.05 0.38 0.21 0.24

Tear index (mN m2 g−1) 7.45 8.80 8.15 7.60 7.15 8.35 7.90 7.40

SD 2.38 0.81 0.81 2.21 0.58 0.58 0.58 0.58

SD: standard deviation.

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refining effect on the fibres when pulps have beendried.

Refining response of never-dried vs. driedenzyme-treated eucalyptus pulp

ComC-treated pulpComC dried pulp has a larger drainability value (°SR)and WRV (Table 2) values than ComC wet pulp. Thisbehaviour is opposite to that of original pulp, whichshows a decrease of drainability and WRV with dry-ing (Figure 1, Table 1). On the contrary, paper prop-erties such as tensile index and burst index (Table 2)showed lower values for dry pulp than for wet pulp,similarly to what was found with original pulp (Fig-ures 3–5, Table 1). However, with drying, tear de-creased (Table 2) in contrast to original pulp (Ta-ble 1), for which tear has a tendency to increase.Density increased with drying, while original pulpshowed a decrease. Regarding permeability, ComC-treated dry pulps showed lower values than wet pulps(Table 2), in contrast to original pulp (Figure 2) inwhich there was an increase of permeability com-pared to dried pulps.

The observed high increase in drainability andWRV of ComC dried pulp translates, as expected, intoan increase in density and a decrease in permeability(Table 2). However, the decrease in tensile and burstindexes found (4500 and 6000 revolutions) is not con-sistent with the increase in WRV because a well-known positive correlation exists between WRV andbonding properties. These results agree with those ob-tained by Oksanen et al. (1997a), who determineddrainability in terms of °SR value as a measure ofpulp drainage.

CelB-treated pulpCelB-dried pulp had a larger drainability value (°SR)and WRV than the wet pulp (Table 3). For example,WRV increased from 170% (wp) to 177% (dp) at4500 revolutions. Opposite to what was found forComC pulps, tensile and burst indexes of CelB-treated dry pulp increased with refining to a notablyhigher extent than was the case for wet pulp. Thevalues obtained for dry pulp refined at 4500 revolu-tions (tensile index: 73.89 N m g−1; burst index: 4.30kPa m2 g−1) are even higher than those obtained foruntreated wet pulp refined to the same degree (68.56N m g−1 and 4.25 kPa m2 g−1). Treatment of dry pulpwith CelB seems to compensate the deterioration ofpaper properties caused by hornification. Tear index

of CelB-treated pulps is lower than that of the origi-nal pulps, showing that CelB dry pulps have the low-est value. Viscosity of dry pulp (800 mL g−1) wasslightly higher than that of wet pulp (760 mL g−1).Dry pulp permeability decreases more with refiningthan that of wet pulp, while density is similar in bothpulps. From these results we can suggest that CelBapplication has a ‘biorefining’ effect.

Biorefining

Conventional refining compacts sheets (increasesdensity) as a response to mechanical energy. This iscorrelated with a decrease in permeability and an in-crease in physical properties that are related to fibrebonding (e.g. tensile strength), while tear index de-creases.

One of the most striking results of the applicationof CelB to dried pulp is that, for a given sheet den-sity, lower permeability and a higher tensile index areobtained (Figures 2 and 6). An interesting accom-plishment of the application of CelB in dried pulp isthe reduced refining energy requirement to achieve acertain value of physical properties compared withuntreated dried pulp. The use of the CelB enzyme atthe studied doses can lead to significant refining en-ergy savings when processing market pulp (Figure 3).As an example, CelB-dried pulp refined at 3000 rev-olutions achieves a higher tensile index (67.92N m g−1) than dried original pulp processed at 6000revolutions (64.25 N m g−1), and it is similar to wetoriginal pulp refined at 3000 revolutions (67.67N m g−1). These results indicate that CelB improvesbonding properties of fibers. A possible reason is thatvoids between the fibres have been filled with cellu-losic material (debris, fibrils) which bonds the fibresto each other. This cellulosic material, responsible fora better bonding, probably hinders the passage of air.Thus, permeability drops without decreasing thethickness of paper, and density is maintained. Thisphenomenon could be considered an enzyme-boostedmechanical refining: ‘biorefining’.

A schematic presentation for mechanical refiningand biorefining is proposed in Figure 7. In mechani-cal refining the fibres collapse, bulk and permeabilityof paper decrease, and this yields better bondingproperties. Consequently, the tensile index increases.In the proposed enzyme-boosted refining, for a givendensity, permeability of paper decreases and tensileindex increases compared to conventional refining.

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The positive performance of CelB on pulp modi-fication is not well understood. Several reports showthat treatment with endoglucanases can increase thebeatability of never-dried pulps but diminishesstrength properties (Pere et al. 1995; Oksanen et al.1997a). However, selective treatment of the long-fi-ber fraction of a furnish with cellulases can increasehandsheet properties (Mansfield et al. 1998). The ben-eficial effect of CelB can result from the fact thatpulps were dry or from distinctive molecular featuresof CelB. The enzyme has a catalytic domain of fam-ily 9 and a carbohydrate-binding module of familyIII. The presence of carbohydrate-binding modules inendoglucanases does not seem to have a significantinfluence on strength properties, although it can in-crease pulp beatability (Suurnäkki et al. 2000). How-ever, it has been reported that endoglucanases candiffer widely in their hydrolytic action on pulps (Pereet al. 1995) and this could be correlated to the cata-lytic domain family. The occurrence of a family 9catalytic domain can give enzymes of this group anas yet unidentified differential mode of action onpulps, that would be responsible for the beneficial fi-bre modification found.

Microscopy

Microscopic techniques can contribute to a better un-derstanding of the effect of enzymes on paper prop-erties. Sheets from initial and CelB-treated driedpulps, refined at 6000 revolutions, were compared inorder to assess the action of CelB on eucalyptus fibremorphology (Figures 8 and 9). Microphotographsshow that CelB-treated refined pulps show greatersurface modification than pulps refined without en-zyme pre-treatment. Changes in fibre surface mor-phology, such as flakes and peeling due to the enzyme

treatment, are shown by optical microscopy (Fig-ure 9). This fibre modification probably increasesbonding properties, yielding pulps of higher strength.The decrease in permeability previously mentionedindicates a more closed paper structure, which is inaccordance with the results observed by microscopy.These results are in good agreement with previouswork (Blanco et al. 1998; España et al. 1998).

Conclusions

The comparison of properties of wet and dried un-treated Eucalyptus globulus pulp shows results inagreement with those previously reported. It has beenconfirmed that dried pulp, due to the hornification ef-fect, shows a loss of mechanical properties: burst andtensile indexes decrease, while tear index for highrevolutions of PFI mill is similar. Permeability was

Figure 7. Schematic presentation of mechanical refining and biore-fining.

Figure 8. Microphotography of original dried pulp refined at 6000PFI revolutions.

Figure 9. Microphotography of CelB-treated dried pulp refined at6000 PFI revolutions.

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always higher in dried pulps. This is consistent withthe loss of bonding capacity due to the hornificationeffect.

Enzyme treatment with cellulases prior to refininghad no significant effect on properties of paper manu-factured with never-dried bleached pulp. It has an in-fluence on drainability (Schopper–Riegler method)and water retention value, but mechanical propertiesof paper did not improve.

On the contrary, the effect produced by the novelenzyme CelB on dried pulps can be considered abiorefining step. When dried pulps were treated withCelB, burst and tensile properties increased signifi-cantly, reaching values similar to those obtained inwet pulps. The use of CelB can give rise to importantsavings of refining energy due to the fact that strengthproperties can be developed with less refining energyrequirements. Enzyme treatment of dried pulps pro-duced a dramatic decrease in permeability. This isconsistent with microscopic analysis, which showeda strong increase in fibrillation, producing flakes anddebris when applying cellulases.

Treatment of dried eucalyptus pulp with CelBcounterbalanced the hornification effect, improvingthe bonding capacity of dried pulp. The different per-formance of CelB compared to ComC can result fromsome distinctive molecular feature of CelB, like en-zyme family or mode of action on cellulose, thatcould be responsible for the found increase instrength. The use of certain cellulases could be verypromising for paper makers who use secondary fibres,due to the possibility of compensating the hornifica-tion effect.

Acknowledgements

This work was supported by the Scientific and Tech-nological Research Council (CICYT, Spain) grantNos PPQ2001-2161-C02-01-02 and 2002FI 00556.

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