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Keywords:

  • Bacillus pumilus;
  • bleaching;
  • Cyathus stercoreus;
  • laccase;
  • soda pulp;
  • waste pulp;
  • xylanase

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Aims:  Investigation of waste pulps and soda pulp bleaching with xylanase (X) and laccase mediator system (LMS) alone and in conjunction (one after the other) (XLMS).

Methods and Results:  Soda and different grades of waste pulp fibres [used for making three-layered duplex sheets – top layer (TL), protective layer (PL) and bottom layer (BL)] when pretreated with either xylanase (40·0 IU g−1) or LMS (up to 200·0 U g−1) alone and in combination (one after the other) (XLMS) exhibited an increase in release of reducing sugars [up to 881·0% soda pulp; up to 736·6% (TL), up to 215·7% (PL) and up to 198·0% (BL) waste pulp], reduction in kappa number [up to 17·6% soda pulp; up to 14·0% (TL), up to 25·3% (PL) and up to 10·9% (BL), waste pulp], improvement in brightness [up to 20·4% soda pulp; up to 23·6% (TL), up to 8·6% (PL) and up to 5·0% (BL), waste pulp] when compared with the respective controls. The usage of XLMS along with 15% reduced level of hypochlorite at CEHHXLMS/EHHXLMS bleaching stage reduced kappa number [5·5% soda pulp; 11·4% (TL), 7·9% (PL), waste pulp] and improved brightness [1·0% soda pulp; 0·9% (TL), 1·4% (PL) waste pulp] when compared with the controls. Scanning electron microscopic studies revealed development of cracks, flakes, pores and peeling off the fibres in the enzyme-treated pulp samples. These modifications of the fibre surface during enzymatic bleaching in turn indicated the removal of lignin and derived compounds from the fibre cell wall.

Conclusions:  The work describes synergistic action of xylanase with LMS for bleaching of waste and nonwood pulps for eco-friendly production of paper and thus reveals a new unexploited arena for enzyme-based pulp bleaching.

Significance and Impact of the Study:  The drastic improvement in pulp properties obtained after xylanase and LMS treatment would improve the competitiveness of enzyme–based, environmentally benign processes over chemicals both economically and environmentally.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

The ecological and environmental concerns, market and legislative pressures, wood supply issues, change in agricultural policies and forest land management practices have lead both developed and developing countries to recommend the use of nonwood fibres in paper manufacturing (Roncero et al. 2003; Camarero et al. 2004). The principal advantages of nonwood agricultural fibres (wheat straw, sunflower stalks, flax, hemp, kenaf and jute) are high growth rate, adaptability to various soil types and act as excellent fibres for high-value added paper (Roncero et al. 2003; Sigoillot et al. 2005). Recently developed totally chlorine-free chemical bleaching sequences (using H2O2, O2 and/or O3) often do not confer adequate brightness to these pulps, and Cl2 and ClO2 are being currently used for their bleaching thus alarming environmental concerns (Akin et al. 1996).

Waste pulp, a mixture of pulps obtained from various waste paper types (copy paper, office stationery, printed books, magazines, old news papers, etc.) is de-inked and bleached by chemicals (detergents, fatty acids, dispersants, dithionite, peroxide and chlorine) for making recycled paper and paper board (e.g. duplex sheet which is used for packaging, storage, etc.).

By-products (adsorbable organic halides, chlorinated lignin derivatives, chlorophenols and chlorobiphenyls) generated during conventional bleaching of pulps are toxic, mutagenic, bioaccumulating and are a serious threat to already dwindling eco-systems (Bajpai 1999). Therefore, efforts should be geared towards development of environment-friendly processes for paper manufacturing from nonwood and waste fibres.

To date, biological bleaching of pulp has been approached mainly by the use of lignolytic (Onysko 1993; Bajpai 1999) and hemicellulolytic enzymes (Viikari et al. 1994; Beg et al. 2001; Taneja et al. 2002; Ninawe and Kuhad 2005). Xylanases (EC. 3·2·1·8), a repertoire of hydrolytic enzymes, facilitate the complete hydrolysis of xylan. Xylanases are being used, primarily for the removal of lignin-carbohydrate complex (LCC) that are generated in the kraft process and act as physical barriers to the entry of bleaching chemicals (Paice et al. 1992; Beg et al. 2000).

The laccase (E.C 1·10·3·2) mediator system (LMS) enhances the delignification response of laccase during biobleaching of pulps by oxidation of lignin moieties, oxidative cleavage of side chains, such as cleavage of Cα–Cβ bonds in lignin and selective oxidation and dehydrogenative polymerization of free phenoxy groups in lignin (Sealey et al. 1997; Bajpai 2004).

Biobleaching and bioprocessing of pulps using a combination of xylanase and laccase further broadens the horizon of enzymes in pulp and paper industry. Once modified, hemicellulose(s) is removed by xylanase, the lignin layer is easily available for penetration and degradative action of laccase (Viikari et al. 1994; Bajpai 2004). The exposed lignin moiety thus requires less chlorine for its removal (Kuhad et al. 1997; Bajpai 2004).

In the present work, an attempt has been made to evaluate the differential and synergistic abilities of xylanase from Bacillus pumilus strain MK001 (Sharma et al. 2005) and LMS [laccase from Cyathus stercoreus in conjunction with 4-Hydroxy benzotriazole (HBT)] in prebleaching and poststage treatment of soda and waste pulps.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Chemicals

Guaiacol, birchwood xylan and HBT were purchased from Sigma (St Louis, USA). All other media components and chemicals used were of highest purity grade available commercially. Wheat bran was obtained locally.

Micro-organisms

Bacillus pumilus strain MK001 (Accession no. AY389345) was isolated from sanitary landfill at Bawana, New Delhi and maintained on xylan-agar as described previously (Sharma et al. 2005). Cyathus stercoreus (schw) de Toni 196676 procured from Canadian National Collection of Fungal Cultures, Ottawa, Ontario, Canada, was grown and maintained on malt extract agar as described previously (Vasdev et al. 2005).

Enzyme production

Xylanase production was carried out under solid state fermentation (SSF) as described previously (Sharma et al. 2005). Air-dried wheat bran (700·0 g), moistened with mineral salt solution, was spread in separately sterilized enamel trays (780 × 510 × 80 mm) to about 1·0 cm thick. The trays were covered with two layers of muslin cloth with cotton sandwiched between them and sterilized at 121°C (15 psi) for 30 min. The substrate was inoculated with 10·0% (v/v) of seed culture of B. pumilus strain MK001 containing 106 CFU ml−1 and incubated for 7 days at 37 ± 1°C in a 99% relative humidity chamber. The xylanase was harvested by adding 0·1 mol l−1 citrate-phosphate buffer pH 6·0 in a similar way as described elsewhere (Sharma et al. 2005).

Laccase production from C. stercoreus was carried out in a 13·5-l air-lift fermentor (Biostat-B; Sartorius India, Bangalore, India) with 5·0 l malt extract broth (MEB), inoculated with 10·0% (v/v) of 6-day-old inoculum. The temperature was maintained at 30°C by circulation of temperature-controlled water. Air was supplied to the fermentor in a continuous way at a rate of 10·0 l min−1 and pH (5·0) was left uncontrolled. The fermentation was carried out for a period of 96 h. Xylanase and laccase were concentrated in Pellicon ultra filtration system using a 10-kDa cassette (Millipore, Bedford, USA).

Enzyme assays

The xylanase activity was determined by measuring the release of reducing sugars from birch wood xylan (1·0% w/v) by dinitrosalicyclic acid method (Miller 1959). One unit of xylanase was defined as an amount of the enzyme required to release 1 μmol of xylose from birch wood xylan in 1 min under the standard assay conditions (60°C, pH 6·0) as described elsewhere (Sharma et al. 2005). Guaiacol was used as substrate for assaying laccase activity as described previously (Vasdev et al. 2005). One unit (U) of laccase was defined as the change in absorbance of 0·01 ml−1 min−1 at 470 nm.

Biobleaching of soda and waste pulps

Pulp samples

All bleaching experiments were carried out at Bindlas Duplex Ltd, Muzaffarnagar, Uttar pradesh, India. The soda pulp was composed of (% w/v): wheat straw (Triticum astivum), 78·8; sarkanda (Saccharum spontaneum), 10·6; and candy (Eragrostis sp.), 10·6. The ingredients were cooked with alkali at temperature ranging from 165–175°C for 30 min at a pressure of 7·0–7·5 kg m−3. Waste pulps of three different grades were used – grade I: old notebooks paper used in educational institutes, white ledger, coloured ledger; grade II: brown-colour grocery and food bags, telephone directory and glue-bound book without a hard cover; and grade III: unbound newsprint materials. The different grades of waste pulp were used for making different layers of duplex sheet [top layer (TL), grade I; protective layer (PL), grade II; bottom layer (BL), grade III]. Grade II and grade III pulps used in the present investigation were obtained after deinking stage from the paper mill. All the studies were performed at 10·0% pulp consistency (% consistency = mass of oven dried pulp)/(mass of oven dried pulp + mass of water present), pH 7·5–9·0 at 50°C, unless otherwise mentioned. Pulp samples were thoroughly washed with tap water before use.

Xylanase and LMS aided prebleaching of soda and waste pulps

Xylanase from B. pumilus strain MK001 at a dose of 40·0 IU g−1 moisture-free pulp was used for treating 10·0 g moisture-free soda and waste pulps for 4 h in sealed polythene bags at 50°C. Laccase with 0·002 mol l−1 HBT (mediator) was applied to soda (200·0 U g−1) and waste pulps (150·0 U g−1) in a preheated pressure vessel maintained at 50°C. The resulting mixture was stirred for 3 min and thereafter, pressure vessel was sealed and the reactants were stirred for variable time intervals up to 2·0 h at 50°C, 10-bar O2 pressure. The soda and waste pulps were also given a combined biotreatment (one after the other) at 50°C with xylanase (200·0 IU g−1) and LMS (soda 200·0 U g−1; waste 150·0 U g−1 HBT 0·002 mol l−1) [xylanase followed by laccase (XLMS), laccase followed by xylanase (LMSX)]. After the experiments, the pulp samples were washed with distilled water, suction-dried and stored in a plastic bag for analysis of pulp.

Xylanase and LMS aided post-treatment of soda and waste pulps

The soda and waste pulp samples were bleached using different multistage bleaching sequences viz. CEHH sequence (C, chlorination; E, NaOH extraction; H, hypochlorite treatment) and EHH sequence (E, NaOH extraction; H, hypochlorite treatment), respectively. The pulps obtained at various stages of chemical bleaching were given a post-treatment at 50°C with xylanase and LMS, individually and in combination (one after the other) (XLMS). After the experiments, the pulp samples were washed with distilled water, filtered, suction-dried and stored in a plastic bag for determination of pulp properties.

Physical and chemical characterization of soda and waste pulps

Hand sheets were made from treated pulps and were air dried in a room with standardized light and humidity following Technical Association of Pulp and Paper Industry (TAPPI) recommendations. The physical and chemical characterization of pulps were carried out according to the standard methods of TAPPI (Anon 1991): kappa number (T 236 cm-85), brightness (T 452 om-92), viscosity (T 230 om-89), burst factor (T 403 cm-50), tear index (T 231 cm-96) and release of phenolic compounds (A237nm) and hydrophobic material (A465nm) as per method described elsewhere (Gupta et al. 2000) and release of reducing sugars following the method of Miller (1959).

Scanning electron microscopy

The fibres were washed thrice in deionized H2O and transferred in 2·0% (v/v) glutaraldehyde in 0·1 mol l−1 phosphate buffer, pH 7·2 for 1 h. The fibres were separated from glutaraldehyde and washed thrice with the same buffer and were gradually dehydrated with ethanol gradient between 30% and 90% and finally suspended in 100·0% ethanol. The fibres were then immersed in hexamethyl-disilazane for 5 min and kept in a desiccator under low vacuum. The fibres were placed on the stubs mounted with silver tape and sputter coated with gold using fine coat, JEOL ion sputter, Model JFC-1100. The samples were examined and photographed at 20 kV under scanning electron microscope (Model JSM 6100, JEOL) at various magnifications.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Enzyme production

Bacillus pumilus strain MK001, when grown on wheat bran under SSF conditions, produced 44 000·0 IU g−1 dry wheat bran of xylanase after 168 h of incubation at 37°C. Cyathus stercoreus, when grown under submerged fermentation conditions in a airlift bioreactor (13·5 l), produced 9·0 U ml−1 of laccase after 96 h of incubation at 30°C.

Pulp studies

Xylanase aided pulp bleaching

Xylanase prebleaching resulted in 12·0% reduction in kappa number and 880·0% increase in release of reducing sugars in case of soda pulp (Table 1). However, in waste pulps 12·0%, 22·2% and 9·0% reduction in kappa number and 700·0%, 210·0% and 190·0% increase in release of reducing sugars were observed for TL, PL and BL, respectively (Table 1). The release of phenolic (A237nm) and hydrophobic compounds (A465nm) were in correlation with release of reducing sugar (Table 1). The analysis of paper properties revealed that when compared with control pulp, hand sheets made of xylanase prebleached soda or waste pulps showed increased brightness (18·1% soda pulp; up to 19·0% waste pulps), opacity (25·0% soda pulp; up to 10·6% waste pulp), tear index (48·7% soda pulp; up to 10·7% waste pulp) and burst factor (62·5% soda pulp; up to 55·5% waste pulp) (Tables 2 and 3). However, no significant changes in viscosity were observed in either soda or waste pulp treated with xylanase when compared with respective control.

Table 1.   Optimization of pulp prebleaching by xylanase (X), laccase mediator system (LMS) and X followed by LMS (XLMS)
Bleaching stage  Pulp properties
Kappa numberReducing sugars (mg g−1 pulp)Absorbance (A237nm)Absorbance (A465nm)
Soda pulpWaste pulpSoda pulpWaste pulpSoda pulpWaste pulpSoda pulpWaste pulp
TLPLBLTLPLBLTLPLBLTLPLBL
  1. Values are means of five replicates. The standard deviation was within 10%.

  2. X, xylanase treatment [IU g−1 moisture-free pulp (50°C for 4 h; pH 8·5–9·0)]; LMS, laccase treatment [U g−1 moisture-free pulp in conjunction with HBT (4-Hydroxy benzotriazole) (mol l−1) (50°C for 2 h; pH 7·5–8·0)]. TL, top layer; PL, protective layer; BL, bottom layer.

Untreated (control)  18·25·009·0011·001·430·300·700·500·1430·0400·1200·2000·0320·0500·0180·080
X16·04·407·0010·0012·602·101·500·981·1300·2500·8050·4300·0980·1500·0310·120
LMS15·54·506·8010·001·480·380·790·573·6200·3850·6780·7200·4200·3300·2220·200
XLMS 
X (IU g−1)LMS                
Laccase (U g−1)HBT (mol l−1)                
20·0100·00·00116·54·77·910·05·541·30·900·651·8540·1850·4230·5890·3240·2400·1560·142
0·00217·64·98·610·65·531·30·970·661·0120·1230·3010·4150·2010·1540·1400·129
0·00518·15·08·911·05·501·30·980·670·5100·0800·1350·2200·0900·0700·0400·087
40·0150·00·00117·14·98·910·57·001·91·460·941·5230·1300·3210·3230·2980·1640·1000·111
0·00216·34·36·79·87·102·11·510·992·8780·3900·8500·7300·4150·3400·2400·210
0·00517·54·77·910·07·122·11·480·950·8720·1870·4570·4670·1540·2290·1250·129
40·0200·00·00117·54·98·310·612·481·81·460·931·2980·1000·2790·3110·1990·1230·0900·105
0·00215·04·57·09·912·602·01·500·983·6800·3820·7010·7220·4500·3350·2340·197
0·00516·74·68·010·012·492·21·470·962·1450·1980·5110·5470·2870·2500·1250·135
60·0250·00·00117·95·09·011·012·622·11·511·000·9870·0500·2230·3000·1260·1100·0870·100
0·00215·74·57·09·912·602·21·501·003·2150·3790·6760·7110·4210·3270·2290·197
0·00516·14·67·69·912·612·21·521·112·0200·2230·5470·6110·3330·2670·1230·154
Table 2.   Effect of xylanase and laccase bleaching on the physical properties of soda pulp after prebleaching and post-treatment
Bleaching stagesPulp properties
Brightness (% ISO)Tear index (Nm3 g−1)Burst factor (KPam2 g−1)Opacity (%)Viscosity (g cm.s−1)
  1. The values are means of 5 replicates. Standard deviation was within 10%.

  2. X, xylanase treatment with 40·0 IU g−1 moisture-free pulp (50°C for 4 h; pH 8·5–9·0); LMS, laccase treatment with 200·0 U g−1 moisture-free pulp in conjunction with 0·002 mol l−1 HBT (4-Hydroxy benzotriazole) (50°C for 2 h; pH 7·5–8·0); C, chlorine treatment with 4·5% Cl2 (50°C for 45 min; pH 4·5–5·0); H*, hypochlorite treatment with 6·0% hypochlorite (40°C for 150 min; pH 8·5–9·0); H$, hypochlorite II treatment with 4·5% hypochlorite (40°C for 90 min; pH 8·5–9·0); H#, hypochlorite II treatment with 3·8% hypochlorite (40°C for 90 min; pH 8·5–9·0).

Untreated control22·08·20·840·00·32
X26·012·21·350·00·32
LMS25·510·01·049·00·31
X LMS26·512·41·451·30·31
C48·012·01·465·40·30
CX50·012·71·468·60·30
C LMS50·012·51·467·20·29
CX LMS50·612·81·568·70·29
CEH*H$70·413·21·587·00·28
CEH*H# X70·513·61·587·20·29
CEH*H# LMS70·413·41·587·10·28
CEH*H# X LMS71·214·01·587·60·28
Table 3.   Effect of xylanase and laccase bleaching on the physical properties of waste pulp after prebleaching and post-treatment
Bleaching stagesPulp properties
Brightness (% ISO)Tear index (Nm3g−1)Burst factor (KPam2g−1)Opacity (%)
TLPLBLTLPLBLTLPLBLTLPLBL
  1. The values are means of five replicates. Standard deviation was within 10%.

  2. TL, top layer; PL, protective layer; BL, bottom layer; ND, not done; X, xylanase treatment with 40·0 IU g−1 moisture-free pulp (50°C for 4 h; pH 8·5–9·0); LMS, laccase treatment with 200·0 U g−1 moisture-free pulp in conjunction with 0·002 mol l−1 HBT (4-Hydroxy benzotriazole) (50°C for 1·5 h; pH 7·5–8·0); C, chlorine treatment with 4·5% Cl2 (50°C for 45 min; pH 4·5–5·0); H*, hypochlorite treatment I with 3·0% hypochlorite (40°C for 150 min; pH 8·5–9·0); H$, hypochlorite treatment II with 1·5% hypochlorite (40°C for 90 min; pH 8·5–9·0); H# hypochlorite II treatment with 1·35% hypochlorite (40°C for 90 min; pH 8·5–9·0).

Untreated (control)55·052·044·04·62·802·200·60·40·466·066·055·0
X65·556·546·04·83·102·210·80·70·673·070·057·0
LMS66·056·045·54·82·902·210·80·70·673·069·056·5
X LMS68·057·046·24·83·002·210·90·90·674·071·057·2
E60·555·046·04·83·902·330·90·80·570·068·056·0
EX67·057·548·25·04·102·381·21·10·673·270·557·0
E LMS67·057·547·65·14·002·371·31·00·673·570·057·0
EX LMS67·559·050·05·24·152·561·31·10·674·371·557·0
EH*H$67·056·0ND5·44·00ND1·10·9ND82·070·0ND
EH*H# X67·556·2ND5·54·15ND1·31·2ND84·071·0ND
EH*H# LMS67·456·2ND5·44·10ND1·31·1ND83·571·0ND
EH*H# X LMS68·157·0ND5·64·12ND1·31·2ND84·571·5ND

The overall aim of consecutive usage of xylanase along with chemicals in CEHH and EHH bleaching of soda and waste pulps was to decrease the hypochlorite charge for pulp bleaching, all the while increasing pulp brightness. The pH of the pulp at various stages in either CEHH or EHH sequences was between 4·0–9·0, at which xylanase from B. pumilus strain MK001 is active and for this reason no pH adjustment was required prior to testing.

In case of soda pulp, addition of xylanase at various stages of bleaching resulted in increase in release of reducing sugars (3·0–25·0%), phenolic moieties (8·9–53·7%), hydrophobic compounds (3·3–55·0%) and reduction in kappa number (0·7–4·1%) when compared with respective controls (Table 4). The analysis of physical properties, as given in Table 2, clearly shows increase in brightness (up to 4·1%), tear index (up to 5·8%) and opacity (up to 4·8%) after xylanase addition at various stages of pulp bleaching. Moreover, it was also demonstrated at CEHHX stage that brightness improvements obtained with xylanase post-treatment could be translated into savings of hypochlorite by 15·0% (Table 2).

Table 4.   Effect of xylanase and laccase bleaching on the physicochemical properties of soda pulp obtained after prebleaching and post-treatment
Bleaching stagePulp properties
Kappa numberReducing sugars (mg g−1 pulp)Absorbance (A237nm)Absorbance (A465nm)
  1. The values are means of five replicates. Standard deviation was within 10%.

  2. X, xylanase treatment with 40·0 IU g−1moisture free pulp (50°C for 4 h; pH 8·5–9·0); LMS, laccase treatment with 200·0 U g−1 moisture free pulp in conjunction with 0·002 mol l−1 HBT (4-Hydroxy benzotriazole) (50°C for 2 h; pH 7·5–8·0); C, chlorine treatment with 4·5% Cl2 (50°C for 45 min; pH 4·5–5·0); E, caustic extraction with 0·8% NaOH (55°C for 90 min; pH 9·0–9·5); H*, hypochlorite treatment with 6·0% hypochlorite (40°C for 150 min; pH 8·5–9·0); H$, hypochlorite II treatment with 4·5% hypochlorite (40°C for 90 min; pH 8·5–9·0), H#, hypochlorite II treatment with 3·8% hypochlorite (40°C for 90 min; pH 8·5–9·0).

Untreated (control)18·21·430·1430·032
C14·410·201·2020·059
CX14·310·501·3100·082
C LMS14·310·292·4900·380
CX LMS13·810·512·5220·390
CE10·03·200·5120·039
CEX9·603·500·6920·052
CE LMS9·203·270·9800·200
CEX LMS9·103·521·0200·210
CEH*8·521·200·2210·059
CEH*X8·251·320·2790·061
CEH* LMS7·921·260·7700·140
CEH*X LMS7·851·300·7700·147
CEH*H$7·200·800·0930·029
CEH*H#X6·901·000·1430·045
CEH*H#LMS7·000·820·1800·098
CEH*H#X LMS6·800·980·1980·098

The xylanase post-treatment in EHH sequence in waste pulps provided a significant bleaching effect with regard to delignification and brightness gains. Xylanase-treated pulps exhibited kappa number reduction [up to 13·0% (TL), 15·7% (PL), 4·3% (BL)], increase in reducing sugars [up to 166·0% (TL), 200·0% (PL), 100·0% (BL)], increase in release of phenolics [88·8% (TL), 350·0% (PL), 555·0% (BL)] and hydrophobic compounds [up to 290·0% (TL), 75·0% (PL), 66·6% (BL)] (Table 5). The values for tear index, brightness, burst factor and opacity obtained for various grades of waste pulp were also better than those of control pulp (Table 3). Moreover, the EHHX stage yielded a pulp with similar brightness with a smaller dose of hypochlorite (1·35%). This pulp exhibited a brightness of 67·5% ISO (TL) and 56·2% ISO (PL), while the control pulp exhibited a brightness of 67·0% ISO (TL) and 56·0% ISO (PL) with 1·5% hypochlorite (Table 3). This clearly shows that xylanase action resulted in reduction of chemical consumption. The better values of tear index during xylanase post-treatment indicated that cellulose microfibrils were not disrupted inside the fibres and illustrate the selectivity of xylanase.

Table 5.   Effect of xylanase and laccase bleaching on the physicochemical properties of waste pulp obtained after prebleaching and post-treatment
Bleaching stagePulp properties
Kappa numberReducing sugars (mg g−1 pulp) Absorbance (A237nm) Absorbance (A465nm)
TLPLBLTLPLBLTLPLBLTLPLBL
  1. The values are mean of five replicates. Standard deviation was within 10%.

  2. ND, not done; TL, top layer; PL, protective layer; BL, bottom layer; X, xylanase treatment with 40·0 IU g−1 moisture-free pulp (50°C for 4 h; pH 8·5–9·0); LMS, laccase treatment with 150·0 U g−1 moisture-free pulp in conjunction with 0·002 mol l−1HBT (4-Hydroxy benzotriazole) (50°C for 1·5 h; pH 7·5–8·0); E, caustic extraction with 0·8% NaOH (55°C for 90 min; pH 9·0–9·5); H*, hypochlorite treatment I with 3·0% hypochlorite (40°C for 150 min; pH 8·5–9·0); H$, hypochlorite treatment II with 1·5% hypochlorite (40°C for 90 min; pH 8·5–9·0); H#, hypochlorite II treatment with 1·35% hypochlorite (40°C for 90 min; pH 8·5–9·0).

Untreated (control)5·009·0011·000·300·700·500·0400·1200·2000·0500·0180·080
E4·605·709·201·501·200·800·1000·5000·3000·0900·0500·090
EX4·004·808·802·903·001·600·3900·8300·5000·1000·0600·140
ELMS3·904·608·501·561·240·830·4500·9000·7800·3100·1600·130
EXLMS3·504·508·402·953·051·550·4800·9500·8000·3200·1650·140
EH*3·004·50ND0·050·10ND0·0500·200ND0·0100·060ND
EH*X2·804·30ND0·101·30ND0·1000·350ND0·0340·090ND
EH*LMS2·604·20ND0·050·14ND0·1800·360ND0·0200·120ND
EH*XLMS2·504·15ND0·101·32ND0·1820·365ND0·0300·150ND
EH*H$2·203·80ND0·030·03ND0·0900·090ND0·0090·020ND
EH*H#X2·003·60ND0·080·09ND0·1100·150ND0·0170·090ND
EH*H#LMS2·103·60ND0·040·03ND0·1700·160ND0·0180·050ND
EH*H#X LMS1·953·50ND0·070·08ND0·1900·300ND0·0200·095ND
Laccase mediator system (LMS) aided pulp bleaching

The extent of delignification after LMS prebleaching of soda and waste pulps was evident through reduction in kappa number [14·8% (soda pulp) and 10·0% (TL), 24·4% (PL), 9·0% (BL) (waste pulp)], increase in release of phenolics (A237nm) [(2431·0% (soda pulp); 962·0% (TL), 465·0% (PL), 260·0% (BL) (waste pulp)] and hydrophobic compounds (A465nm) [92·38% (soda pulp); 560·0% (TL), 1133·0% (PL), 150·0% (BL) (waste pulp)] (Table 1). There was negligible rise in reducing sugars from soda and waste pulps after LMS pretreatment, indicating nonsusceptibility of the laccase preparation towards hemicellulosic residues present in pulp fibres.

The analysis of physical properties of hand sheets made from LMS prebleached soda and waste pulps indicated intensive fibre modification which allowed considerable improvement in pulp properties [Brightness 16·0%, (soda pulp); 20·0% TL, 7·7% PL, 3·4% BL (waste pulp); Tear index 22·0% (soda pulp) 4·3% TL, 3·6% PL, 0·4% BL (waste pulp); burst factor 25·0% (soda pulp) 33·3% TL, 55·5% PL, 50·0% BL (waste pulp), opacity 22·5% (soda pulp) 10·6% TL, 4·5% PL, 2·7% BL (waste pulp)] (Tables 2 and 3).

In case of soda pulps, addition of LMS at various stages of bleaching showed that CLMS stage (described in Table 4) was characterized by higher release of phenolic moieties at A237nm (2431·5%), hydrophobic compounds at A465nm (544·1%) and decrease in kappa number (8·3%). This could be due to the fact that pH of chlorine-bleached pulp is near to 4·0–5·0, which is the optimum range for maximum activity of laccase used in the present study. On the contrary, the CELMS and CEHLMS stages showed a lesser reduction in kappa number and lesser increase in A237nm and A465nm. The analysis of physical properties in soda pulp as given in Table 2, clearly shows maximum increase in brightness (4·1%), tear index (4·2%) and opacity (2·7%) at CLMS stage. It was also demonstrated at CEHHLMS stage that similar brightness could be obtained with LMS post-treatment leading to hypochlorite savings by 15·0% (Table 2).

The application of LMS post-treatment in EHH sequence-based bleaching of waste pulps showed differential behaviour in terms of bleaching parameters towards different grades of pulps. However, irrespective of the bleaching stages, the LMS-based postbleaching benefits were more pronounced after alkali extraction treatment (ELMS) [reduction in kappa number 15·2% (TL), 19·3% (PL), 7·6% (BL); increase in release of phenolic A237nm 350·0% (TL), 80·0% (PL), 160·0% (BL) and hydrophobic compounds A465nm 244·0% (TL), 220·0% (PL), 44·4% (BL)] when compared with other bleaching stages (Table 5). Other postbleaching benefits viz. increase in brightness [(10·7% (TL), 4·5% (PL), 3·4% (BL), tear factor 6·25% (TL), 2·6% (PL), 1·7% (BL), burst factor 44·4% (TL), 25·0% (PL), 20·0% (BL)] also followed a similar trend (Table 3). Our results also revealed that LMS in a similar fashion to xylanase, when applied at a 15·0% reduced level of hypochlorite at EHHLMS stage gave more or less similar brightness when compared with respective controls (Table 3).

Combined xylanase and LMS aided pulp bleaching

The present work has revealed synergism between xylanase and LMS for pulp bleaching. The combined prebleaching (one after the other) (XLMS) of soda and waste pulps with xylanase (200·0 IU g−1) and LMS (soda 200·0 U g−1; waste 150·0 U g−1 HBT 0·002 mol l−1) resulted in kappa number reduction up to [3·2% (soda pulp); 4·5% (TL), 1·1% (PL) and 2·0% (BL), (waste pulp)], increase in release of phenolic moieties up to [1·6% (soda pulp); 1·3% (TL), 5·6% (PL) and 1·4% (BL), (waste pulp)] and hydrophobic compounds up to [7·1% (soda pulp); 3·0% (TL), 8·1% (PL) and 5·0% (BL), (waste pulp)] when compared with either xylanase or LMS prebleaching (Table 1). However, no additive or synergistic effect for enhanced release of reducing sugars was observed. Among physical properties, improvement in brightness [up to 6·2% (soda pulp); 3·0% (TL), 0·88% (PL), and 0·43% (BL), (waste pulp)], burst factor [up to 1·6% (soda pulp); 12·5% (TL), 28·5% (PL), (waste pulp)] when compared with either xylanase or LMS prebleaching were also observed, but no noticeable difference in opacity and tear index were observed (Tables 2 and 3). No cooperation between xylanase and LMS in terms of bleaching benefits [Kappa number: soda pulp; 15·6 waste pulp 4·50 (TL), 6·79 (PL), 10·0 (BL) reducing sugars: soda pulp 1·45; waste pulp 0·35 (TL), 0·08 (PL), 0·07 (BL) A237nm: soda pulp 0·410; waste pulp 0·380 (TL), 0·670 (PL), 0·700 (BL), A465nm: soda pulp 3·550; waste pulp 0·320 (TL), 0·220 (PL), 0·198 (BL),] on either soda or waste pulps was observed when LMS prebleaching of the pulp was followed by xylanase prebleaching (LMSX).

The action of XLMS at various stages of CEHH bleaching sequence of soda pulps also revealed additive effect for improvement in pulp characteristics. In soda pulp, decrease in kappa number (up to 3·5%), increase in release of phenolic A237 (up to 4·1%) and hydrophobic A465 (up to 5·0%) compounds, increase in brightness (up to 1·28%), tear index (up to 0·78%), burst factor (up to 7·1%) and opacity (up to 0·14%) were observed at various bleaching stages when compared with xylanase or LMS used alone (Tables 2 and 4).

In waste pulps, the maximum XLMS-based post-treatment effects were observed after alkali extraction stage where increase in brightness [0·7% (TL), 2·6% (PL), 1·65% (BL)], tear index [2·0% (TL), 1·2% (PL), 7·6% (BL)] and opacity [1·1% (TL), 1·41% (PL)] were observed apart from reduction in kappa number [10·2% (TL), 2·2% (PL), 1·2% (BL)], increase in phenolic and hydrophobic compounds [6·6% (TL), 5·5% (PL), 7·7% (BL)] when compared with either xylanase or LMS post-treatment (Tables 3 and 5).

A gain in brightness of 1·0% (soda pulp), 0·9% (waste pulp, TL) and 1·4% (waste pulp, PL) was observed using XLMS with 15·0% lower level of hypochlorite at CEHHXLMS and EHHXLMS stages (Tables 2 and 3).

Scanning electron microscopy

Soda pulp

The fibres in the raw soda pulp were uniform and straight, intact with a smooth, silky surface and bear an appearance of compactness (Fig. 1a). In addition, they showed no sign of external fibrillation or formation of fibrils. The soda pulp fibres either treated with xylanase (Fig. 1b) or laccase (Fig. 1c) were less straight and their surface was rough and striated. However, no marked visible differences were observed between fibres pretreated with either xylanase or laccase as both the enzymes caused greater porosity, swelling and separation of pulp microfibrils and pulp fibres when compared with smooth surfaces of untreated pulp. When XLMS-treated fibres were subsequently treated with 3·8% hypochlorite (CEHHXLMS), there was a higher degree of swelling, separation and loss in compactness in the pulp fibres as shown in Fig. 1d.

image

Figure 1.  Scanning electron micrographs of pulp fibres: X, xylanase; LMS, laccase mediator system; TL, top layer; PL, protective layer; BL, bottom layer; XLMS (xylanase followed by laccase)*, 3·8% hypochlorite-treated soda pulp; XLMS#, 1·35% hypochlorite-treated waste pulp.

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Waste pulp

Scanning electron microscopy (SEM) photographs of raw waste pulp fibres (Fig. 1e,i,m) revealed a high degree of damage where various lamellae of the secondary wall can be seen. Scanning electron micrograph also showed that they were severely fractured along the cell axis. Moreover, lack of fibrillation suggests lower bond area and consequently diminished bondability. Irrespective of the mode of action of the enzymes tested, SEM photographs of enzyme treated, X [Fig. 1f(TL), j(PL), n(BL)] LMS [Fig. 1g(TL), k(PL), o(BL)], and XLMS [Fig. 1h(TL), l(PL), p(BL)], waste pulps reveal overall loosening of cross linking with in different fibre walls, thereby allowing partial separation of constituents fibrils at the fibre surface.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

The principal objective of this work was to test and compare a hydrolase (xylanase) and oxido-reductase (laccase) to enhance the bleachability of soda and waste pulps. Moreover, the bleaching efficiency of both the enzymes together has also been evaluated.

Xylanase aided prebleaching of soda and waste pulps

Bacillus pumilus strain MK001 exhibited good xylanase with negligible cellulase activity, when grown on wheat bran, an inexpensive lignocellulosic substrate, under (SSF).

The xylanase aided prebleaching, and post-treatment of soda and different grades of waste pulps was carried out. In xylanase prebleaching studies, the maximum quantity of chromophores, hydrophobic compounds and reducing sugars were released at a dosage of 40·0 IU g−1 from both soda and waste pulps. The correlation between the release of chromophores (A237nm) and hydrophobic compounds (A465nm) and the reduction in kappa number coupled with release of reducing sugars suggested the dissociation of LCC (Gupta et al. 2000) by xylanase attack on lignin-carbohydrate linkages such as ether or glycosidic linkages, which lead to increased solubility of lignin (Zhao et al. 2006). The crude xylanase was used at 10·0% pulp consistency throughout our studies as it ensured optimal contact between the enzyme and the pulp because of reduced volume of the liquid phase, facilitating xylanase adsorption to pulp and the sequential attack on hemicellulosic fraction. Higher pulp consistencies are known to keep the enzyme away from physically reaching further substrates (Haarhoff et al. 1999).

The varied optimal doses and reaction periods for pulp bleaching by xylanases from different micro-organisms have been reported (Beg et al. 2001). It may be because of the differences in the enzyme characteristics and pulping conditions. Recently, xylanase I and xylanase II from Aspergillus caespitosus at an enzyme dosage of 10 IU g−1 dry pulp reduced kappa number by 12·6% and 1·7%, respectively, and also released large amounts of 237 and 465 nm absorbing materials, when compared with controls (Sandrim et al. 2005).

The xylanase from B. pumilus strain MK001 when used at various stages in CEHH and EHH bleaching sequences of soda and waste pulps, respectively, caused an increase in biobleaching benefits at all stages but the hypochlorite (H) stage in soda pulp and alkaline extraction (E) stage in waste pulps recorded maximum reduction in kappa number, increase in release of reducing sugars, chromophoric and hydrophobic compounds when compared with respective controls. Moreover, xylanase treatment of soda and waste pulps resulted in 15·0% reduction in hypochlorite consumption and achieved the same parameters of delignification and brightness as present in control. Similarly, Zhao et al. (2006) reported reduction in chlorine charge by 20–30%, or increase in final brightness by approximately 4–5% ISO, along with improved pulp strength properties after xylanase application. Beg et al. (2000) have reported that when xylanase-treated pulp was subsequently treated with 4·5% chlorine it resulted in reduction of kappa number by 25% and enhanced brightness by 20%.

The differences in the level of bleaching benefits (reduction of kappa number, release of reducing sugars etc.) obtained during pulp prebleaching and post-treatment by xylanase may be due to different target substrates in pre- and post-treated pulps. In prebleaching, the substrate is highly modified xylan that has reprecipitated back on fibre surfaces at the end of the kraft cook (Kantelinen et al. 1993) and is more affected with bleach boosting. However, during post-treatment, substrate is either native xylan or xylan modified by bleaching liquors (Buchert et al. 1994) and is more associated with direct brightening. The overall xylanase action in bleaching is by disruption of xylan chain by the enzyme, which interrupts lignin-carbohydrate bonds facilitate easier removal of solubilized lignin in bleaching (Paice et al. 1992).

The effect of xylanase prebleaching and post-treatment on the bonding of fibres in standard hand sheets can be deduced by comparison of physical properties. The brightness and opacity increased considerably in xylanase prebleaching and also to certain extent at every stage of enzyme aided CEHH and EHH sequences in soda and waste pulps, respectively. The increased burst factor (a measure of the degree of the interfibre bonding) and tear index by xylanase prebleaching and post-treatment indicate an increase in pulp fibrillation, water retention and restoration of bonding in fibres (Beg et al. 2000). Madlala et al. (2001) have reported that chlorine bleached, alkali-extracted baggasse and postoxygen kraft pulps, pretreated with xylanase, gained over five brightness points over control and produced chlorine dioxide savings of 30·0%. Viscosity was not affected by the use of xylanase. In some cases a slight increase in viscosity was observed. This could be explained by the loss of the lower molecular weight material (Vidal et al. 1997).

LMS aided bleaching of soda and waste pulps

Cyathus stercoreus produced laccase maximally after 96 h of incubation in airlift bioreactor. During fermentation, pellets of regular size were produced, which maintained their shape over the course of fermentation without formation of mycelial agglomerates. This avoided bed clogging, which can hinder mass and oxygen transfer rate (Rancano et al. 2003).

To make the laccase-HBT system (LMS) compatible with industrial bleaching processes, variables like enzyme-mediator (0·001–0·005 mol l−1) dosage, pulp consistency and reaction time were optimized. Good levels of delignifying and depolymerizing activity of the laccase-HBT system were observed in LMS-prebleaching (Laccase dose 150·0–200·0 U g−1 moisture-free pulp along with 0·002 mol l−1 HBT) of soda and waste pulps through release of UV-absorbing material and chromophoric substances in enzyme-treated pulps. Sigoillot et al. (2005) observed strong delignification of nonwood pulps when laccase treatment was performed in the presence of HBT (15–40 mg g−1 moisture-free pulp).

Bourbonnais et al. (1997) reported LMS bleaching of kraft pulp in which the reaction was performed for 2 h at 60°C, pH 5·0 with 300 kPa of O2, 1% ABTS and 5 U g−1 of laccase on 10% w/v pulp. After LMS treatment followed by alkaline extraction, the extent of delignification varied from 25% to 40% with various kraft pulps and was over 50% with a sulfite pulp. By repeating the treatment with laccase-ABTS and alkaline extraction, the kappa number of a softwood (SW) kraft pulp was decreased by 55%.

To elucidate whether laccase from C. stercoreus in conjunction with HBT is able to catalyze the oxidation of substrates in CEHH- and EHH-bleached soda and waste pulp, respectively, LMS at optimized dosage was added at various stages during pulp post-treatment. The LMS post-treatment of soda and waste pulps was most effective at alkaline extraction stage in comparison to other bleaching stages. This may be due to lack of suitable substrate or change in chemical composition of lignin at other stages, as some of the phenolic structures are opened because of oxidation by bleaching chemicals (Gierer 1985). It has also been reported that both the initial lignin content and chemical structure of the pulp significantly influence mediator-aided laccase delignification and subsequent final bleaching (Poppius-Levlin et al. 1997; Bajpai 2004). These observations could explain different reactivity of LMS towards soda and waste pulp in terms of bleaching benefits.

Sealey et al. (1997) reported that with oxygen-reinforced alkaline extraction, laccase-HBT biobleaching could obtain over 70% delignification in one stage. Chandra et al. (2001) have reported that the bleaching of high kappa kraft pulps with a laccase-mediator system provided 42·6–61·1% delignification following an E + P stage, when violuric acid was used as the mediator.

The possible mechanism responsible for delignification of the pulp by LMS is the occurrence of redox cycles in which oxygen oxidizes the reduced form of laccase to the native laccase species (Laccase-oxidized), which in turn oxidizes a mediator to produce oxidized mediator species (Mediator-oxidized) and the original laccase. Lignin moieties in the residual lignin in pulp then undergo oxidation by the mediator-oxidized species, resulting in degradation and dehydrogenetive polymerization of lignin (Lignin-oxidized) and reduction of mediator (Call and Mücke 1997).

Information about usage of lignin-oxidizing enzymes on soda and waste pulp bleaching is scanty. The prebleached and post-LMS-treated soda and waste pulps did not showed any major change in viscosity when compared with control. This may be due to the specific action of lignin oxidizing enzymes viz. laccase on lignin and hence these enzymes do not affect the viscosity, strength properties and yield of the pulp (Bajpai 2004). Bourbonnais and Paice (1992) have reported that the treatment of kraft pulp with laccase and ABTS did not affect the pulp viscosity and zero span breaking length.

The physical properties viz. brightness, tear index, burst factor and opacity deduced from hand sheets prepared from LMS-treated soda and waste pulps exhibited considerable improvements when compared with respective controls. Camarero et al. (2004) have shown significant improvement by laccase treatment in the tear strengths, which were 14–17% higher than those of the reference tensile index 70 Nm g−1.

Xylanase and LMS aided bleaching of soda and waste pulps

The cumulative action of XLMS on soda and waste pulps indicated a cooperative interaction between xylanase and LMS for pulp bleaching as evident by higher release of reducing sugars, phenolic moieties and hydrophobic compounds along with increased brightness, tear index and opacity when compared with either xylanase or LMS alone. Maximum synergism between xylanase and LMS in XLMS was observed at enzyme dosages, xylanase (40·0 IU g−1) and LMS (soda 200·0 U g−1; waste 150·0 U g−1 HBT 0·002 mol l−1). No difference between xylanase and LMS dosages was observed for achieving maximum bleaching benefits in soda and waste pulps, when applied individually (X, LMS) or in combination (one after the other) (XLMS). This might be due to the fact that xylanase and LMS have totally different target substrates and mode of action on pulp fibres. The enhanced bleaching effects of XLMS could be because of degradation of xylan, which is sandwiched between the lignin and cellulose layers, by xylanase (Suurnakki et al. 1997; Beg et al. 2000), which eventually might have enhanced accessibility of LMS towards lignin in the pulp fibres leading to decreased diffusion resistance to the outward movement of the degraded lignin fragments and allowed the removal of the less degraded lignin fragments from the fibre wall (Bajpai 2004). Our results on optimization of HBT dose in XLMS indicated that higher levels of HBT considerably reduced bleaching benefits. This may be due to the fact that higher concentrations of HBT lead to inactivation of laccase (Ibarra et al. 2006).

The XLMS treatment showed that, in addition to the positive affects on the various bleaching parameters mentioned above, 15·0% less hypochlorite was required to obtain higher brightness up to 1·4% (when compared with control) in CEHH and EHH stages in soda and waste pulps, respectively. This may contribute to better paper quality as well as to reduced chlorinated residues in the waste treatment system (Bajpai 2004).

Cooperative interactions between different enzyme systems for pulp bleaching have been reported in literature. Herpoël et al. (2002) reported treatment of wheat straw chemical pulp by combining xylanases and LMS followed by alkaline extraction resulting in a final brightness of 69·0% ISO. Treating SW kraft pulp fibres with xylanase followed by laccase provided a collective 25·0% and 46·0% increase in dry and wet tensile strength properties, respectively (Chandra and Ragauskas 2005).

No cooperation/synergy between xylanase and LMS was observed when LMS prebleaching of the pulp was followed by xylanase prebleaching. The values of various bleaching parameters like kappa number, release of reducing sugars, phenolic and hydrophobic compounds were more or less similar to values obtained with LMS treatment alone. This could be due to suppression of xylanase activity by the presence of ferulic, caffeic, sinapic, p-coumaric, benzoic and vanillic acids released by the action of LMS on lignocellulosic fraction of the pulp fibre (Gamble et al. 2000). Our results are in accordance with Milagres et al. (1995) as they reported that treating hardwood kraft pulp sequentially with laccase (L) and xylanase (X) in sequence order L–L–X, L–X–L did not have much effect on the lignin content (12·9% delignification).

It is interesting to note that average values of chromophores, hydrophobic compounds and reduction in kappa number obtained in either XLMS or xylanase/LMS-treated pulps were always higher in soda pulps when compared with waste pulps. This is due to the fact that waste pulp comprises of various grades of paper that has been previously bleached either partially (Grade II, brown colour grocery, glue-bound book without a hard cover, etc.) or in some cases fully (grade I, old notebooks paper, etc.) and thus have on average lower level of lignin. Furthermore, the bleaching ability of xylanase and laccase were more pronounced during pulp prebleaching when compared with post-treatment because of target substrate modification apart from reaction conditions employed during the latter. The varied differences obtained in the physical and chemical properties of different grades of waste pulps and soda pulp might be due to the difference between substrate distribution with in the matrix, specific surface area and the charge of the fibres comprising soda and waste pulps.

Scanning electron microscopy

The photomicrographs showed significant changes on the fibre surface of xylanase and LMS-treated pulps as a result of xylan hydrolysis and lignin removal, respectively. No such effect was observed in untreated pulps. In soda pulps, xylanase and LMS-treated fibre surface appeared to be stripped and a delamination of the cell wall was observed. Moreover, xylanase treatment seems to be effective in opening the closed cell-wall pores of the soda pulps.

In waste pulps, changes in fibre surface morphology such as cracks, flakes, filaments and peeling were observed in enzyme-treated pulps. These crack and pores in both waste and soda pulps might have facilitated the diffusion out through the fibre cell wall of the larger lignin macromolecules. Roncero et al. (2000) also showed similar surface changes in enzyme-treated fibres that were not apparent in untreated fibres.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

Nonwood plant fibres and recycled waste pulp appears to be suitable alternatives not only for coping up with the steady increase in paper demand, which continues to grow at a dramatic rate, but also for sustainable production of pulp and paper with natural ecological balance. Our results indicates that both xylanase and LMS treatments alone and in combination (one after the other) may serve as eco-friendly alternatives for improving pulp properties of waste paper and nonwoods pulps.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References

The authors are thankful to Mr Pankaj Aggarwal, Managing Director, Bindlas Duplex Ltd, Uttar Pradesh, India, for providing facilities and support for conducting bleaching experiments at their paper mill. The authors also acknowledge the research grant from Department of Biotechnology, India. M.K is grateful to the Council of Scientific and Industrial Research for grant of senior research fellowship. The technical assistance provided by Mr Rajesh Pathania, AIIMS, New Delhi for scanning electron microscopic studies is also gratefully acknowledged.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgements
  9. References
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