[Home] [APPW 2004] [Journal papers]


Adilson R. Gonçalves, Denise S. Ruzene, Regina Y. Moriya and Luis R. M. Oliveria

Presented at the 59th Appita Conference, Auckland, New Zealand, 16-19 May 2005



Alternative pulping and bleaching systems have been studied by several research groups around the world. Different lignocellulosics were evaluated as raw material for these studies. In our group we combined organosolv pulping (acetosolv and ethanol/water) of sugarcane bagasse and straw with enzymatic bleaching with xylanases. The viscosity of the pulps ranged from 9-19 cP and kappa number was lesser than 30. Xylanase from different sources was applied to the pulp, with varying dosage and time. The pulps showed a lower kappa number even after 4 h of treatment. Reusing of the xylanase was evaluated and the activity and viscosity of pulps were maintained after 2 or 3 reuses. Viscosity was used as a control parameter since the obtained pulps should be used as cellulose derivatives. Pulps were also analyzed for brightness and holocellulose and α-cellulose contents.



Environmental and legislative pressures have forced the pulp and paper industry to modify its pulping, bleaching and effluent treatment technologies to reduce the environmental impact of mill effluents (Bajpai et al., 1994). These pressures have also led to the design of many techniques aimed at improving the pulping process and reducing the lignin content of the pulp entering the bleach plant.

The cooking process generates large amounts of concentrated wastewater especially that from sulphite and sulphate processes. One solution to this problem is the use of organic solvents. Although the effectiveness of such solvents has been known for a long time, they have only recently started to be used for this purpose at pilot- and small-scale industrial plants (Jiménez et al., 1997; Beg et al., 2001). Organosolv processes have been applied with varied success to hard- and softwood and also, to a lesser extent, to nonwood materials. Agricultural fibres constitute an alternative to wood as a raw material for making pulp on account of their high growth rate and adaptability to various soil types. In Brazil where sugarcane is an abundantly grown crop, bagasse is available for paper making, besides other non-wood materials such as wheat straw and various other agricultural residues. The interest in agricultural fibres promoted research in this area, regarding their potential as raw material for cooking (Seisto et al., 1997; Jiménez et al., 1998; Usta et al., 1999).

In recent years scientific studies have been directed towards the development of environmentally clean and non-toxic bleaching methods for kraft pulp production (Cubbin, 1994). Some of the problems encountered with the use of chlorine containing bleaching agents are associated with corrosion of storage tanks, formation of toxic and/or mutagenic organic chlorinated compounds and increased chloride and AOX levels in bleach plant effluents (Viikari et al., 1994). In response to environmental concerns and stringent emission standards, a new improvement in this field is the inclusion of an enzyme stage in the bleaching process. A significant decrease in the quantity of chlorine-containing reagents has been achieved in this way, pulp bleaching has been made easier in subsequent stages and pulp brightness has been improved. Such techniques using xylan specific enzyme (xylanase) have found their industrial application in a number of plants in Scandinavia and North America (Yean et al., 1995).

The mechanism of the enzyme action in the pulp is the removal of the redeposited xylan on fibres and the rupture of the lignin-carbohydrate complex. The redeposited xylan on the pulp surface covers the residual lignin making it inaccessible to the bleaching reagents. The xylanase hydrolyzes part of this xylan permitting better access of the bleaching reagents to the residual lignin, making the removal of this lignin easier (Wong et al., 1997; Farrel et al., 1996; Young and Akthar, 1998). The lignin-carbohydrate complex theory assumes that there is an union between lignin and polyoses in pulp that restricts the removal of residual lignin. The xylan bond cleavage by xylanase separates the lignin-carbohydrate linkages improving the access of the bleaching reagents and facilitating the lignin removal in the subsequent bleaching chemical sequences (Wong et al., 1997; Young and Akthar, 1998).

Most of the published studies on xylanase bleaching were focused on either hardwood or softwood pulps (Shah et al., 1999). The bleach boosting effects of xylanase on kraft pulps (Hortling et al., 1994) and sulphite pulps (Christov and Prior, 1995) are well documented. However, literature reports on the enzymic pretreatment of bagasse pulp are limited (Shah et al., 1999; Bissoon, 1998).

The cost of ethanol production and consumption has been admitted as the barrier for the economical conversion of biomass (Nguyen and Saddler, 1991). Several strategies have been used to increase the efficiency and reduce the costs of the components of the process. A successful strategy is to enhance the enzyme productivity by fungi and to reuse the enzymes several times in the process (Greeg and Saddler, 1996).

In the ethanol production process from lignocellulosics, the enzyme recycle plays an important role in the reduction of costs. Ramos and Saddler (1994) reported that recovery and recycle of the β-Glucosidase enzyme can reach 70% of the original protein added in the first hydrolysis reaction after seven hydrolysis cycles. Greeg et al. (1998) showed that enzyme recycle using hydrolysis reactors for two cycles decreased the production ethanol cost by 12%.

Despite several studies in this area, the reuse of the enzyme in the pulp biobleaching process has not yet been investigated.


Acetosolv pulping

Acetosolv pulping of sugarcane bagasse was carried out with acetic acid 93% (v/v) and HCl as catalyst. The bagasse/solvent ratio was 1:14 (w/v). The temperature of pulping was 110°C (temperature of solvent mixture) for 2 h, according to Benar (1992). Acetosolv pulping of sugarcane straw was carried out with acetic acid 93% (v/v) and HCl as catalyst. The straw/solvent ratio was 1:30 (w/v). The temperature of pulping was 110°C for 4 h. The pulps were washed with acid acetic 93% (v/v) and thoroughly washed with water until the wash water attained a neutral pH.

Ethanol/water pulping

Pulping of depithed sugarcane bagasse with ethanol/water 1:1 (v/v) mixture was carried out in a closed and pressurized vessel. The pulping was evaluated using H2SO4 in a concentration between 0.01 and 0.05 mol.L-1 for 0.5 - 3.0 h and NaOH (5 -10% base bagasse) for 3.0 h. Ethanol/water pulping of sugarcane straw was performed in a 200-mL closed vessel, using ethanol/water mixture 1:1 (v/v), bagasse to solvent ratio of 1:1 (m/v) and 2.5 h cooking time, according to Gonçalves and Ruzene (2003). The pulps were filtered and washed with 2500 mL ethanol/water.

Xylanase assay

The xylanases used in this work were: xylanase from Thermomyces lanuginosus IOC-4145, optimal pH 6.0 and temperature of 75ºC, with a molecular weight of 21 kDa, supplied by Damaso et al., (2000); xylanase from Humicola grisea diluted in citrate buffer, pH4.8, pI 8.0, with 4641 UI.L-1 activity and molecular weight of 25kDa, supplied by Faria et al., (2002); Bacillus pumilus xylanase diluted in glycine-NaOH buffer, pH 8.5 with 550 UI.L-1 activity, supplied by Duarte et al., (1999) and Cartazyme HS 10 (Sandoz Chemicals Ltd., Birmingham, UK) with declared activity 10,000 Ug-1, pH 3.0-5.0 at 35-55°C.

The xylanases of different sources were assayed as described by Bailey (1992) by incubating the diluted enzyme solution (suitable buffer) at 50ºC for 5 min using a substrate solution of 1% (w/v) birchwood xylan (Roth, Karlsrule, Germany). One unit of xylanase activity was defined as that amount of enzyme that catalyses the release of one μmol of xylose equivalents per minute of reaction.

Xylanase pretreatment of pulp

Samples of acetosolv or ethanol/water pulp with 3% consistency were incubated in Erlenmeyer flasks in a shaker at 50°C, for 4 h, 8 h and 12 h. A set of sample was incubated with 18 UI xylanase and another set was incubated with the same charge of commercial enzyme Cartazyme. After incubation, the pulp was filtered through a Büchner funnel and the pulp was washed thoroughly with distilled water. The wet enzyme-pretreated bagasse pulp (3 g of dry weight equivalent) was placed in an Erlenmeyer flask and treated with 2% NaOH at 60°C for 1 h. The pulp was filtered and washed with distilled water.

A set of experiments was performed using acetosolv bagasse pulps. The enzymatic reuse was carried out three times, the first utilizing water as solvent and 18 UI/g dry pulp; in the second and in the third 36 UI/g dry pulp were used, in agreement with Ruzene and Gonçalves (2003). The second reuse was carried out utilizing distilled water and the third one using acetate buffer 0.05M pH 5.5 as solvent.

In all cases, control pulp treatment was performed without enzyme in the same conditions described above and treatments pulps were made in triplicate.

Analysis and chemical composition of the pulps

Kappa number and viscosity of the pulps were determined by standard methods(TAPPI, 1985;1992). One gram of dry pulp or 2 g of bagasse/straw was treated with 10 mL of 72% H2SO4 with stirring at 45°C for 7 min. The reaction was interrupted by adding 25 mL of distilled water, the mixture was transferred to a 250-mL Erlenmeyer flask, and the volume made up to 140 mL. The flask was autoclaved for 30 min at 1.05 bar for the complete hydrolysis of oligomers. The mixture was filtered and the filtrate (hydrolysate) made up to 250 mL. A 20-mL sample of the hydrolysate was diluted to 25 mL and the pH adjusted to 2.0 with 2 mol.L-1 NaOH. After filtration in a Sep-Pak C18 Cartridge to remove aromatic compounds, the hydrolysate was analyzed in an Aminex HPX-87H columm (300 x 7.8 mm) (Bio-Rad) at 45°C by using a Shimadzu chromatograph and refraction-index detector. The mobile phase was 0.005 mol/L of H2SO4 at 0.6 mL.min-1. Sugar concentrations reported as xylan and glucan were determined using calibration curves of pure compounds. Lignin was determined by gravimetric analysis (Rocha, 2000).

Determination of brightnesss

The brightness of pulps was determined in agreement with TAPPI 452 om-98. Samples were prepared following TAPPI 218 sp-97: 3 g of pulp (dry base) was disaggregated by 5 min under pH 5.5 and 0.3% consistence. The pulp suspension was filtered in a Buchner funnel. After filtration, the funnel was inverted and the pulp was liberated using air flow. The leaf formed was pressed (10-12 kgf/cm2) during 90 s and put to dry in darkness. After one day, the leaves with 310-315 g.m-2 were analyzed in a Photovolt 577 equipment. The reflection percentage was determined at five different points and results were presented as mean values.

Determination of holocellulose

Samples of 5 g of dry pulp (with known moisture) were transferred to 250-mL Erlenmeyer flasks with 160 mL of distilled water, 0.5 mL of acetic acid and 1.5 g of sodium chlorite. The samples were heated in a waterbath at 70-80°C with agitation at each 10 min. After 60 min reaction, 0.5 mL of acetic acid and 1.5 g of sodium chlorite were added. The addition was repeated at each 60 min until the final time of 4 h of reaction. At the end of the fourth hour the Erlenmeyer flask was put in an icebath reaching 10°C and the samples were filtered in crucibles of porosity 2. The residue was washed with 1.6 L of hot distilled water under suction. Subsequently, samples were washed with acetone and dried at room temperature.

Determination of α- cellulose

Samples of 1 g of holocellulose (with known humidity) were transferred to 150ml beakers and put in a water-bath at 20°C. Then 11.8 mL of 17.5% NaOH solution was added with gentle stirring: 5 mL after 1 min, 3.4 mL after 45 sec, 3.4 mL after 15 sec. The samples were left for 3 min and then 13.6 mL of 17.5% NaOH was added:  3.4 mL followed by stirring for 10 min; during those 10 min was added another 3.4 mL after 2.5 min; 3.4 mL after 5.0 min and 3.4 mL after 7.5 min. The samples were covered and left for 30 min at 20°C. After that time, 33.4 mL of distilled water were added, and the mixture left for 30 min more at 20°C. The samples were filtered in crucibles of porosity 2, washed with 8 mL of 8.3% NaOH and washed with 400 mL of distilled water. The volume of crucible was made up with acetic acid 2 mol.L-1 and left to rest for 3 min. The samples were filtered to remove the acetic acid, washed with 3 L of distilled water at room temperature and dried overnight.


The sugarcane bagasse and sugarcane straw are lignocellulosic material of different chemical composition, as observed in tables 1 and 2. The straw showed 41% less glucan than bagasse and showed 28% more xylan and 30% more lignin than bagasse. Ethanol/water and acetosolv pulps of sugarcane bagasse showed similar viscosity to that of ethanol/water and acetosolv straw pulps (viscosity 7-9 cP). Kappa numbers of the ethanol/water and acetosolv bagasse pulps are similar to that of straw pulps (kappa number 52-59); only acetosolv bagasse pulp and acetosolv straw pulp showed different kappa numbers. The kappa number of acetosolv straw pulp was less than that of acetosolv bagasse pulp.

Figure 1 shows the viscosity of ethanol/water and acetosolv bagasse pulps treated with xylanase by different microbiological sources. The higher viscosity (18.5 cP) was shown by ethanol/water bagasse pulp treated with Cartazyme in 4 h of treatment. The second higher viscosity (14.5 cp) was for B. pumilus-treated pulps in 8 h of treatment. Compared with control pulp (pulp treated without enzyme) this represented a 15% increase.

pulping sugarcane table 1 and 2


Ethanol/water and acetosolv pulping are different methods of delignification. Therefore the pulps showed different properties. Acetosolv bagasse pulps showed lower viscosity than ethanol/water pulps. The acetosolv pulps treated with xylanase of different sources displayed similar viscosity (figure 1B).

The ethanol/water bagasse pulp treated with xylanase by T. lanuginosus showed kappa number 30, in 4 h of treatment: compared with control pulps (kappa number 52) it represented a 42% decrease. The pulps treated with xylanase by T. lanuginosus followed by alkaline extraction gave kappa number 4 (the lower kappa number) (figure 2 A). The acetosolv pulps treated with different sources by xylanase gave kappa number 30. The enzymatic treatment followed by alkaline extraction furnished acetosolv bagasse pulps with similar kappa number, considering the standard deviation (figure 2 B).

Data obtained of the kappa number and viscosity after enzyme reuse are in table 3 and the enzymatic activity values in table 4.

pulping sugarcane fig 1

pulping sugarcane table 3


A maintenance and even an increase of the pulp viscosity was observed, while kappa number suffered an increase. With the enzymatic activity values there was an abrupt fall in the first treatment; in the second this decrease was lower (only 50%).

pulping sugarcane fig 2

Fig 2 Kappa number of organosolv sugarcane bagasse
pulps treated with xylanase of different microbiological

pulping sugarcane table 4


Ethanol/water pulping using higher amounts of bagasse was performed in acidic and alkaline media using the best conditions found by the analysis of kappa number, viscosity and chemical composition. After pulping, the pulps were washed with tap water until the washing water became colourless, due to the high amounts of ethanol/water mixture needed.

The results of ethanol/water pulping of sugarcane bagasse in acid and alkaline conditions are shown in table 5. When washing the pulp with water the results were very close to those obtained with the ethanol/water mixture. The pH for the ethanol/water pulps in acid and alkaline conditions, obtained after the washing with water was 6.5 and 7.5, respectively. Initial and pH values did not suffer significant alterations with change of washing method. With the change of the method of washing of the pulp, the total yield in acid and alkaline conditions suffered reduction of only 3.8% and 2.8%, respectively. Viscosity was reduced by 28% in the acid condition due to higher degradation of fibres. In alkaline condition the viscosity reduction was 22%.

In acid conditions the kappa number was reduced by 2.4% and it suffered an increase of 25.5% in alkaline conditions, showing that the reprecipitation of lignin occurred after the washing of the pulp with water. The lignin in the pulps in acid and alkaline conditions can be removed in the subsequent bleaching stages.

Table 6 gives the results of the chemical composition of the pulps expressed as glucan, xylan, total lignin (%) and xylan/glucan ratio in acid and alkaline conditions. The mass balance did not reach 100% due to the presence of other components such as low molecular-weight compounds and extractives. The most abundant component of the pulps obtained from sugarcane bagasse using pulping process ethanol/water/H2SO4 and ethanol/water/NaOH, was cellulose (glucan) with 75.9 ± 3.2% and 55.3 ± 0.6%, respectively . Acid and alkaline conditions degrade lignin and hemicelulloses (xylan) preserving the cellulose.

pulping sugarcane table 5 and 6


For the acid conditions, the amount of glucan was reduced by 38% and in alkaline condition it increased by 4.5%. The amount of xylan also reduced by 48% in acid conditions and 15.7 % in alkaline conditions. The total lignin in acidic condition did not suffer alteration and in alkaline conditions it showed an increase of 29%. The increase of the amount of lignin must have occurred due to lignin precipitation on fibres after washing of the pulp with water.

Table 7 shows the holocellulose (%), α- cellulose (%) and brightness of ethanol/water pulps in acid and alkaline conditions.

The holocellulose value is the sum of hemicellulose and cellulose present in the pulp, reaching 90%. Value of α-cellulose was 70% while the brightness for acid and alkaline condition was 30.5 ± 1.2% and 25.0 ± 1.2%, respectively. Faria (1994) found brightness of 35.4% for NaOH pulps of bagasse, showing that the results were close to that obtained by the literature.

pulping sugarcane table 7



The organosolv bagasse pulps obtained in this work should be used as cellulose derivatives. Xylanases obtained from different microbial sources showed similar bleaching results to commercial xylanase. The results suggest the possibility of enzyme reuse, maintaining pulp viscosity.


This work was supported by Brazilian agencies: FAPESP, CNPq and CAPES.


Bailey, M.J., Biely, P. and Pourtanen, K. - J. Biotechnol. 23:257(1992).

Bajpai, P., Bhardwaj, N.K., Bajpai, P.K. and Jauhri, M.B. - J. Biotechnol. 38:1(1994).

Beg, Q.K., Kapoor, M., Mahajan, L. and Hoondal, G.S. - Appl. Microbiol. Biotechnol. 56:326(2001).

Benar, P. Polpação acetosolv de bagaço de cana e madeira de eucalipto: MSc thesis, UNICAMP/Instituto de Química, Campinas, Brazil (1992).

Bissoon, S. - β-Xylanase pretreatment of bagasse pulp. MSc Thesis. University of Durban Westville, Durban. South Africa (1998).

Christov, L.P., Prior, B.A. - Biotechnol Lett. 17:821(1995).

Cubbin, M. - Pulp Paper Can. 95:12(1994).

Damaso, M.C.T., Andrade, C.M.M.C. and Pereira Jr, N. - Appl. Bioch. Biotech. 84-86:821 (2000).

Duarte, M.C.T., Portugal, E.P., Ponezi, A.N, Bim, M.A and Tagliari, C.V. - Bioresource Technol. 68:49(1999).

Faria, L.F.F., Ms Thesis, FAENQUIL, Lorena-SP, Brazil (1994).

Faria, F.P., Czifersky, A., Nevalainen, H., Azevedo, O., Gibbs, M., Berquist, P.L. and Teo, V.S.J. - Appl. Biochnol. Biotech. 98-100:78(2002).

Gonçalves, A.R. and Ruzene, S. - Appl. Biochem. Biotechnol. 105-108:769(2003).

Hortling, B., Korhonen, M., Buchert, J., Sundquist J., Viikari, L. - Holzforschung 56:245(1994).

Jiménez, L., Maestre, F., Pérez, I. - Afinidade 44(467):45 (1997).

Jiménez, L., de la Torre, M.J., Maestre, F., Ferrer, J.L., Pérez, L. - Holzforschung 52(2):191(1998).

Rocha, G.J.M. PhD thesis, São Carlos/ Universidade de São Paulo, Brazil, (2000).

Shah, A K., Sidid, S.S., Ahmed, A .Rele, M. V. - Bioresource Technol. 68:133(1999).

Seisto, A ., Poppius-Levlin, K., Jousimaa, T. - Tappi J. 80(10):235(1997).

TAPPI Standard Methods T. 236 cm-85 (1985).

TAPPI Standard Methods T. 230 om-82 (1992).

Usta, M., Eroglu, H., Karaoglu, C. – Cell. Chem. Technol. 33(1-2):91(1999).

Viikari, L., Kantelinen, A., Sundquist, J., Linko, M. - FEMS Microbiol Rev. 13:335(1994).

Yean, P., Hamilton, G., Senior, D.J. - Pulp Paper Can. 98:126(1995).

Wong, K.K.Y., Jong, E.D., Saddler, J.N., Allison, R. W. - Appita J. 50:5(1997).

Farrel, R.L., Viikari, L., Senior, D. - 365-375 The technology of chemical pulp bleaching: enzyme treatments of pulp. In DENCE, C.W., REEVE, D.W. Pulp Bleaching: Principles and Practice. Atlanta: Tappi Press (1996).

Young, R.A., Akthar, M. - 5-69 Developments in organosolv pulping – an overview. In: Environmentally Friendly Technologies for the Pulp and Paper Industry. New York: Jonh Wiley & Sons (1998).

Nguyen, Q.A., Saddler, J.N. - Biores. Technol. 35:275(1991)

Greeg, D.J., Saddler, J.N. - Biotechnology and Bioengineering, 51:375(1996).

Ramos, L.P., Saddler, J.N. - Appl. Biochem. Biotechnol. 45/46:193(1994).

Greeg, D.J., Boussaid, A., Saddler, J.N. - Bioresource Technology 63:7(1998).


Authors' contact details

Departamento de Biotecnologia – FAENQUIL Cx. Postal 116, CEP 12.600-970 Lorena-SP, Brazil

E-mail: adilson@debiq.faenquil.br