Biotechnology in the Pulp and Paper Industry L. Viikari and R. Lantto (Editors) �9 2002 Elsevier Science B.V. All rights reserved. 247

Evaluat ion of the b leach-enhancing effect of xylanases on bagasse pulp

Sadhvir Bissoon a'b, Suren Singh a and Lew Christov b'c

aDepartment of Biological Sciences, ML Sultan Technikon, Box 1334, Durban 4000, South Africa

bSappi Biotechnology Laboratory, Department of Microbiology and Biochemistry, University of the Free State, PO Box 339, Bloemfontein 9300, South Africa

CSappi Management Services, PO Box 3252, Springs 1560, South Africa

Until recently enzymatic bleaching of non-woody materials has received scant attention. In this study three commercial xylanases were evaluated in both ECF and TCF bleaching to enhance the bleachability of bagasse soda pulp. The enzyme pretreatment was carried out at various charges under optimized conditions. In both bleaching sequences, Xylanase P (Iogen Corp.) was the most efficient bleach-enhancing enzyme, when compared on an equal enzyme charge on pulp (as U/g) basis. This enzyme was able to improve brightness of DED bleached bagasse pulp by up to 8.2 brightness points or alternatively reduce the consumption of chlorine dioxide by 33%. Ecopulp TX-200C (R6hm Enzyme) and Cartazyme NS-10 (Clariant) were equally efficient in the ECF bleaching of bagasse pulp producing chlorine dioxide savings of 20%. In all instances, however, the biobleaching effect induced due to xylanase pretreatment was dependent on both the enzyme and chlorine dioxide charges used. At similar brightness levels, the TCF biobleaching in sequence X-OqPaP was less efficient than the ECF DED biobleaching. Mechanical pretreatment of the pulp prior to the ECF biobleaching slightly increased final brightness, indicating "activation" of pulp to facilitate enzyme and chemical bleaching. Xylanase pretreatment had an insignificant effect on the physical properties of the ECF bleached pulp.

1. INTRODUCTION

Growing concerns on environmental issues have prompted governments to develop stringent regulations against polluting processes. The use of chlorine and chlorine chemicals during the bleaching process results in the formation of chlorinated organic substances, some of which are toxic, mutagenic and non-biodegradable (1).

Xylanases hydrolyze the xylans redeposited on pulp fibers during kraft pulping. This is thought to facilitate the diffusion of lignin fragments degraded and solubilized during subsequent bleaching (2).

It has been already demonstrated that xylanases can enhance the bleaching of kraft pulps on a large scale without major capital investments (3). Xylanases can provide a cost effective way to reduce the use of chlorine-containing compounds and other bleaching chemicals.

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There are reports of 8-15% savings of the total amount of chemicals across the bleach plant and a decrease in the adsorbable organic halogen levels of 12-25% in the bleach effluent (1). Increased tear strength and pulp throughput have also been reported (4).

Several commercially available xylanase preparations, most of which are active at slightly acidic or neutral pH, have been investigated in pulp bleaching. Pulpzyme HA (Novo Nordisk, Denmark), produced by Trichoderma reesei, was the first commercial xylanase to be used in biobleaching. It achieved a 20% kappa number decrease of oxygen-delignified birch kraft pulp (5). Cartazyme (Clariant) also improved the brightness of kraft pulps (6). The effectiveness of xylanases have been well documented for wood pulps, however, very little attention has been given to the application of xylanases to non-woody materials such as bagasse pulp. The aim of this study was to evaluate and compare the bleaching potential of three commercial xylanase preparations on bagasse soda pulp.

2. MATERIALS AND METHODS

2.1. Pulp Unbleached bagasse soda pulp was obtained from Sappi Stanger, South Africa. The pulp

was thoroughly washed with distilled water until a neutral pH of the wash waters was attained. The washed pulp had a moisture content of 84% (w/w) and it was stored in sealed plastic bags at 4~ until used.

2.2. Enzymes and enzyme assays Three commercial enzymes, Xylanase P (Iogen Corp., Canada), Ecopulp TX-200C (R6hm

Enzyme, Finland) and Cartazyme NS-10 (Clariant, UK), were evaluated in bleaching of bagasse pulp. All three xylanases belonged to family 11 of glycosyl hydrolase enzymes and were produced from genetically modified Trichoderma reesei (Xylanase P and Ecopulp TX- 200C) or Bacillus strains (Cartazyme NS-10).

The xylanase activities were determined according to the method of Bailey et al. (7) using a 1% birchwood xylan solution. Reducing sugars (RS) were assayed by adding 1.5 ml DNS reagent to the reaction tube, boiling for 5 min, cooling, and measuring the absorbance against the reagent at 540 nm. One unit of enzyme activity was defined as the amount of enzyme that catalyzes the release of one l.tmole of xylose equivalents per minute under the assay conditions.

2.3. Xylanase treatment of pulp Washed unbleached bagasse pulp was treated at a charge of 5 U xylanase/g pulp under

various pH (4.0-9.0) and temperature (40-80~ conditions for 1 h. The optimum conditions for the use of each enzyme preparation (Table 1) were determined based on the release of RS from pulp (data not shown).

Table 1. Conditions used for enzyme treatment of bagasse pulp Enzyme Temperature (~ pH Time (h) Xylanase P 60~ 5.0 2 10 Ecopulp TX-200C 65~ 7.0 2 10 Cartazyme NS- 10 60~ 8.0 2 10

Consistency (%)

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Washed unbleached bagasse pulp was treated at various xylanase charges (1, 5 and 10 U/g) under the optimum pH and temperature of each enzyme and 10% pulp consistency for 2 h. Controls were run under the same conditions however using boiled enzymes. After the incubation period, the pulp was filtered and washed. Pulp and enzyme filtrates were retained for further analyses and use.

2.4. Mechanical treatment of pulp Prior to use of xylanase at a charge of 1 U/g, washed unbleached bagasse pulp was

pretreated mechanically using a pulp homogenizer (Heidolph, Germany). Pretreatment was carried out at 26 000 rpm, room temperature and 1.5% pulp consistency for 15 rain.

2.5. Analyses of pulp filtrates The enzyme mediated release of ultraviolet (UV) absorbing and chromophoric material

from pulp was monitored in filtrates by measuring the absorbance at 280 nm and 465 nm, respectively (8). The amount of RS released from pulp was determined spectrophotometrically at 540 nm according to the DNS method (9).

2.6. Chemical bleaching of bagasse pulp Xylanase pretreated pulp underwent chemical bleaching. The conditions for the elemental

chlorine-free (ECF) bleaching are summarized in Table 2. The charges of chlorine dioxide were based on a kappa factor (kf) of 0.3, 0.4, 0.5, 0.6 and 0.75. The conditions for the totally chlorine-free (TCF) bleaching are shown in Table 3.

Table 2. Conditions used for ECF bleaching of bagasse pulp -Bleaching step " Charge (%) T (~ Time Consistency pH

(h) (%) Chlorine dioxide (D1) kappa no x kf 70 1 10 3.0 Alkali extraction (E) 2.5% NaOH 70 2 10 - Chlorine dioxide (D2) 0.5 x D1 70 3 .... 10 4.0

Table 3. Conditions used for TCF bleaching of bagasse pulp .... Bleacifing step Charge (%) T (~ Time Consistency pH

(h) (%) Oxygen (O) 1.2% NaOH" 400 kPa 02 95 1 10 >10.0 Chelated peracetic acid (qPa) 0.3% Pa; 0.3% H202 75 2 10 8.5 Hydrogen peroxide (P) 3% H202; 1.8% NaOH 75 3 10 11.0

2.7. Pulp properties The kappa number and physical properties of bagasse pulp were determined according to

the Tappi Test Methods. Brightness was measured using an Elrepho photoelectric reflectance photometer (Carl Zeiss, Germany). The kappa number and brightness of unbleached bagasse pulp were 8.7 and 39.3%, respectively. All analyses were carried out in duplicate.

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3. RESULTS AND DISCUSSION

3.1. Analyses of enzyme filtrates Xylanase P was most effective in releasing RS, UV and visible light absorbing material

from bagasse pulp while Ecopulp TX-200C and Cartazyme NS-10 followed in a decreasing order of efficiency (Table 4). The amount of UV absorbing material detected in the filtrates was significantly greater than that of the visible light absorbing material. Overall, the concentration of material released from pulp increased with increasing enzyme charges. Xylanase P at a charge of 10 U/g was able to release 62% and 69% more UV and visible light absorbing material, respectively, and 2.14 times more RS than the control.

Table 4. Analyses of enzyme filtrates following xylanase pretreatment of bagasse pulp Enzyme Charge (U/g) A280 nm m465nm RS (mg/g)

1 4.354 0.198 0.345 Xylanase P 5 4.503 0.210 0.421

10 4.636 0.240 0.587 1 4.194 0.186 0.304

Ecopulp TX-200C 5 4.369 0.195 0.367 10 4.495 0.223 0.453 1 3.956 0.184 0.296

Cartazyme NS-10 5 4.212 0.201 0.355 10 4.305 0.219 0.395

Control 0 2.864 0.151 0.187

3.2. Impact of xylanase pretreatment on kappa number and brightness of bagasse pulp Xylanase pretreatment at a charge of 1 U/g appeared to have very little effect on the kappa

number of bagasse pulp (Table 5). An increase in the enzyme charge to 10 U/g produced a slight decrease in kappa number with reductions of 6%, 7 % and 12% achieved with Cartazyme NS-10, Ecopulp TX-200C and Xylanase P, respectively. All three enzymes induced a direct brightening effect on bagasse pulp when used at all three charges. At 10 U/g, Xylanase P was the superior enzyme increasing the brightness by 2.2 brightness points while Cartazyme NS-10 and Ecopulp TX-200C caused brightness increases of 1.4 and 1.8 points over control, respectively.

Table 5. Impact of xylanase pretreatment on kappa number and brightness of bagasse pulp Enzyme Charge (U/g) Kappa no reduction (%) Brightness increase (points)

1 5 0.6 Xylanase P 5 8 1.3

10 12 2.2 1 0 0.4

Ecopulp TX-200C 5 5 1.0 10 7 1.8 1 0 0.0

Cartazyme NS- 10 5 0 0.6 10 6 1.4

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3.3. ECF biobleaching of bagasse pulp The effect of xylanase pretreatments was analyzed under varying conditions of chlorine

dioxide charges. Xylanase P appeared as the most efficient enzyme to enhance bleachability of bagasse pulp. The biobleaching effect induced by this enzyme was between 0.3 and 1.6 points brightness gain over control at a kf of 0.75 (Fig. !) and between 4.9 and 8.2 points

. = =

~ p...~

o = . q

1.5

0.5

Xylanase P Ecopulp TX-200C

Enzyme

B5U/g I �9 10 U/g I

Cartazyme NS 10

Figure 1. X-DED bleaching of bagasse pulp (kf of 0.75)

when pulp was bleached at a kf of 0.3 (Fig. 2). This clearly indicates that the biobleaching effect due to xylanase pretreatment is dependent on both the enzyme and chlorine dioxide charges used. Brightness of bleached pulp progressively increased with the increase of the enzymes charges. However, as the chlorine dioxide charges increased, the bleach-enhancing efficiency of the xylanases decreased.

Using Xylanase P, the bagasse pulp could be bleached with a kf of 0.5 to the same brightness (83.8%) as the control bleached with a kf of 0.75 without enzyme (Fig. 3).

9

= 7.5 . p...~

~" 6

�9 "~ 4.5

3

,~ 1.5

" " 0

Xylanase P Ecopulp TX-200C Cartazyme NS 10

Enzyme

Figure 2. X-DED bleaching of bagasse pulp (kf of 0.3)

This represents a reduction in the chlorine dioxide consumption of 33.3%. The brightness gain induced by both Ecopulp TX-200C and Cartazyme NS-10, on the other hand, could be

252

2

15 ~

�9 =- 0.5

0 r ~ ID

- - 0 . 5

�9 r. - 1

-1.5

- 2

0.75 0.6 0.5 0.4

Kappa factor (kf)

Figure 3. Reduction of chlorine dioxide consumption following X-DED bleaching using Xylanase P (10 U/g)

translated into chlorine dioxide savings of 20% (data not shown). Mechanical pretreatment of pulp prior to xylanase (1 U/g) rendered the pulp even more

susceptible to bleaching. A notable increase in brightness was observed after final bleaching of the mechanically pretreated pulp compared to the pulp bleached without pretreatment (Fig. 4). These results indicate that mechanical pretreatment did "activate" the pulp to a degree thereby enhancing the bleaching effect of the enzymes and the chemicals.

3.4. TCF biobleaching of bagasse pulp Application of xylanases to the sequence OqPaP revealed that the TCF biobleaching was

less efficient than ECF. Using Xylanase P, brightness was improved by up to 2.3 points (Fig. 5) to a final brightness of up to 71.9%. For comparison, using Xylanase P in the ECF bleaching, a brightness increase of 8.2 points was gained to produce a final brightness of

0 8 e~

0.r

0.4- r ~ r ~

~= 0.2- -I,U

O- Xylanase P

I"! Mechanical pretreatment

1 No pretreatment

Ecopulp TX-200C Cartazyme NS 10

Enzyme Figure 4. Impact of mechanical pretreatment on X-DED bleaching of bagasse pulp (1 U/g; kf of 0.75).

253

76.1% (Fig. 2). Ecopulp TX-200C and Cartazyme NS-10, when used at 10 U/g, brightened pulp by 2.1 and 2.0 points, respectively (Fig. 5).

.

-~ 2.5- .=,

~, 2- ~

1.5-

1

0.5 o~ , , q

0 Xylanase P Ecopulp TX-200C

Enzyme

171 U/g

i15 U/g

n 10 U/g

Cartazyme NS 10

Figure 5. TCF biobleaching of bagasse pulp in sequence X-OqPaP

3.5. Physiea| properties Analysis of the physical properties of bagasse pulp indicated minimal changes of the

enzyme treated pulp compared to the control (Table 6). Xylanase P and Ecopulp TX-200C, however, did increase the tensile strength of the paper with a corresponding increase in the amount of energy required to rupture the test strip. The percentage stretch of the paper strips was slightly lower with the enzyme treated samples. The stretchability values obtained with Cartazyme NS-10 were lower than the other xylanases under study. All enzymes did slightly decrease the viscosity of the pulps. However, there were no significant changes in the physical properties of bagasse pulp with the incorporation of xylanases into the bleach sequence.

Table 6. Effect of xylanase pretreatment (10 U/g) on the physical properties of X-DED bleached bagasse pulp (kf of 0.75)

Tensile strength Stretch TEA Viscosity Enzyme (N/15mm) (%) (J/m 2) (mPas) Control 13.76 2.72 13.83 48.4 Xylanase P 14.12 2.55 14.56 46.9 Ecopulp TX-200C 13.87 2.58 14.03 47.2 Cartazyme NS- 10 13.56 2.31 13.65 47.3

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4. CONCLUSIONS

Comparison of enzyme efficiency in this work was made based on equal activity unit enzyme charges on pulp (U/g) rather than equal enzyme costs, therefore conclusions drawn herein do not represent a commercial evaluation of enzyme products or enzyme manufacturers. With respect to the above, Xylanase P, when compared to Ecopulp TX-200C and Cartazyme NS-10 at equal enzyme doses as U/g, appeared as the most efficient bleach- enhancing enzyme on bagasse pulp in both ECF (DED) and TCF (OqPaP) bleaching. In DED bleaching (kf of 0.75), a brightness gain of 1.6 points over control could be attained using Xylanase P. Overall, Ecopulp TX-200C and Cartazyme NS-10 followed Xylanase P in order of decreasing biobleaching efficiency. Xylanase P was able to reduce the use of chlorine dioxide by 33% as compared to 20% reduction of the chlorine dioxide consumption achieved with the other two enzyme preparations. The biobleaching effect induced due to xylanase pretreatment depended on both the enzyme and chlorine dioxide charges used. Overall, use of high kappa factors were required for a satisfactory bleaching which could be due to a low initial brightness and a relatively low brightness ceiling of bagasse pulp. Mechanical pretreatment of the pulp prior to ECF biobleaching did produce a slight increase in final brightness, indicating "activation" of pulp to enzyme and chemical bleaching. At similar brightness levels, the TCF biobleaching in sequenceX-OqPaP appeared as less efficient than the ECF biobleaching in terms of biobleaching effect. Examination of the physical properties of the paper strips revealed insignificant differences between the control and the xylanase treated samples following ECF bleaching.

ACKNOWLEDGEMENTS

We acknowledge the National Research Foundation, ML Sultan Tecknikon Research Fund and Sappi Management Services for financial support, Sappi Management Services for giving permission to publish this work and Sappi Biotechnology Laboratory for excellent technical assistance.

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