Potential of xylanase from thermophilic Bacillus sp. XTR-10 in biobleaching of wood kraft pulp

Potential of xylanase from thermophilic Bacillus sp. XTR-10 in biobleaching of wood kraft pulp

International Biodeterioration & Biodegradation 63 (2009) 1119–1124 Contents lists available at ScienceDirect International Biodeterioration & Biode...

359KB Sizes 5 Downloads 233 Views

International Biodeterioration & Biodegradation 63 (2009) 1119–1124

Contents lists available at ScienceDirect

International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod

Potential of xylanase from thermophilic Bacillus sp. XTR-10 in biobleaching of wood kraft pulp Mahjabeen Saleem a, *, Muhammad Rizwan Tabassum a, Riffat Yasmin b, Muhammad Imran c a

Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore-54590, Pakistan Sheikh Zayed Fedral Postgraduate Medical institute, Lahore-54590, Pakistan c Molecular and Cell Biology laboratory, Department of Physiology and Cell Biology, University of Health Sciences (UHS), Khayaban-e-Jamia Punjab, Lahore-54600, Pakistan b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 June 2009 Received in revised form 19 September 2009 Accepted 20 September 2009 Available online 28 October 2009

An extracellular xylanase produced under optimal conditions by a thermophilic strain of Bacillus sp. XTR10 was evaluated for its potential application in biobleaching of wood kraft pulp. Spectrophotometric analysis showed considerable release of lignin derived compounds and chromophoric material by the xylanase treated pulp samples. Xylanase was found to be effective in the liberation of reducing sugars in the pulp filtrates with increment in enzyme dose and reaction time. Eight hours pretreatment with 40 IU of xylanase/g of dry pulp resulted in 16.2% reduction of kappa number with 25.94% ISO increase in brightness as compared to the control. The same treatment slightly lowered the tensile strength and burst index, however. Enzyme pretreatment of the pulp saved 15% active chlorine charges in single step and 18.7% in multiple steps chemical bleaching with attainment of brightness at the level of the control. These results indicate the potential of enzymatic pretreatment of pulp for reduction in environmental discharge of hazardous waste from the pulp and paper industry. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Bacillus sp. Thermophilic bacteria Xylanase Biobleaching Kraft pulp

1. Introduction Environmental pollution has led the pulp and paper industry to sort out new techniques for the reduction of various pollutants in the effluents emitting from the bleaching plants (Bajpai et al., 1994). One of the choices in this context is the use of xylanase based enzymatic pretreatment of the pulp. This technology allows the development of products and processes capable of reducing the use of hazardous chemicals and ultimately their discharge into the environment. Viikari et al. (1986) first reported the concept of enzyme aided bleaching. It was thought that limited hydrolysis of hemicellulose by hemicellulases may increase the extractability of lignin from kraft pulp during its subsequent chemical bleaching. It is now agreed that enzymatic delignification can substantially improve the brightness of pulp without leading to a loss in its viscosity and strength (Chauvet et al., 1987; Paice et al., 1988). The use of thermostable xylanase is considered particularly important for pulping and bleaching (Subramaniyan and Prema, 2002). After xylanase pretreatment, lower total chlorine charges can be used during chemical bleaching resulting in reduction of chloro organic

* Corresponding author. E-mail address: [email protected] (M. Saleem). 0964-8305/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2009.09.009

discharges (Senior et al., 1992). Li et al. (2005) found 28.3% reduction in chlorine consumption by pretreatment with xylanase from Thermomyces lanuginosus CBS 28.54, while Garg et al. (1998) reported 16% reduction in kappa number for birchwood kraft pulp pretreatment with xylanase from Streptomyces sp. QG-11-3. Atik et al. (2006) observed a 10% increase in ISO brightness of pulp by xylanase pretreatment. ISO (International Organization for Standardization) brightness is the commonly used industry term for the numerical value of reflectance factor of a sample for 457 nm light of specific geometrical characteristics. The objective of this work was to explore the potential of endo1,4-b-D-xylanases (EC 3.2.1.8) produced by Bacillus sp. XTR-10 in the biobleaching of wood kraft pulp and its efficiency to reduce the consumption of bleaching chemicals by the pulp and paper industry. 2. Materials and methods 2.1. Isolation of microbial strain The samples of decaying raw materials were inoculated in 50 ml of modified Han’s medium (Han and Srinivasan, 1968) contained in 250 ml Erlenmeyer flasks. The medium had the composition in g/100 ml: ammonium sulphate 0.1, magnesium sulphate 0.02, dipotassium hydrogen phosphate 0.05, potassium dihydrogenphosphate

M. Saleem et al. / International Biodeterioration & Biodegradation 63 (2009) 1119–1124

0.05, calcium chloride 0.01, yeast extract 0.2, and birchwood xylan (Sigma Chemical Co. USA) 0.5. The pH of the medium was adjusted to 8.0 and then autoclaved at 121  C for 20 min. After inoculation with the collected samples, flasks were incubated at 55  C in a Gallenkamp orbital incubator shaker with shaking at 180 rpm for 24 h. The growth thus obtained was subcultured five times. Serial dilution of 100 ml of the culture broth finally obtained was performed and 100 ml of the sample form each dilution was spread on xylan agar plates consisting of the above mentioned medium containing 1.5% agar. The plates were incubated at 55  C and single colonies thus obtained were plated in duplicate on agar plates containing 0.5% xylan and incubated at 55  C for 24 h. One of the plates was stained with Congo red for 15 min and then destained with 1 M NaC1. The active strains were preserved at 80  C after suspending cells from the growth phase in the culture medium containing 15% glycerol.

Xylanase activity (U/ml)

1120

75 50 25 0 0

4

pH

8

12

Fig. 2. Effect of pH on xylanase production by Bacillus sp. XTR-10. Bars indicate values for standard deviation (n ¼ 3).

(Sigma Chemical Co. USA) in 50 mM Tris–HCl buffer (pH 8.0) was incubated at 60  C for 10 min. The reducing sugars liberated were estimated as xylose equivalent by DNS method (Ghose, 1987). One unit of the enzyme activity is defined as the amount of enzyme that released 1 mmol of reducing sugars equivalent to xylose per minute under the assay conditions.

2.2. Optimization of culture conditions 2.4. Protein estimation To prepare inoculum, cells from a single active colony were incubated at 55  C in 250 ml Erlenmeyer flask in 20 ml of the above mentioned culture medium containing 0.5% cellobiose instead of xylan. The incubation was carried out until OD of the medium reached 0.6 at 600 nm (1 OD of the biomass is equal to 8  108 cells/ml). Subsequently, 1% of this inoculum was used to inoculate 50 ml of starting fermentation medium having the same composition as above unless otherwise mentioned. Culture conditions were optimized by changing one independent variable at a time while keeping the other variables constant. The culture samples obtained at 2 h time intervals were processed for xylanase activity to determine the optimum incubation time for maximum enzyme production. To further improve the xylanase production, pH was studied in the range of 5.0–10.0 and incubation temperature in the range of 45–65  C. The effect of various inorganic and organic nitrogen sources such as KNO3, NaNO3, urea, peptone and (NH4)2SO4 on enzyme production was studied by varying their amount in the range of 0.5–2.5 mg/ml of the medium. The effect of different carbon sources such as birchwood xylan, oat spelt xylan, cellobiose, sucrose, glucose, xylose and Avicel on xylanase production was studied by keeping their amount in the medium consistent at 0.5%. The use of different natural lignocellulosic materials like wheat straw, rice straw, bagasse, corncobs, lemon peel and banana stalks was also exploited as carbon source for economical production of xylanase. After their grinding and sieving to 20-mesh size, wheat straw, rice straw, bagasse and corncobs were used at 2% consistency while lemon peel and banana stalks were used at 1.5% consistency. The fermentation was carried out at 55  C for 20 h. Following fermentation, 1.0 ml of culture broth was withdrawn and analyzed for protein contents and xylanase activity. 2.3. Xylanase assay

Soluble proteins in the culture broth were measured by the dye binding method of Bradford (1976) using bovine serum albumin (Sigma Chemical Co. USA) as a standard protein. 2.5. Pulp sample Unbleached wood kraft pulp was kindly provided by Packages Ltd. Lahore, Pakistan. 2.6. Biobleaching of kraft pulp For the removal of coloring lignin material, the pulp samples were treated at 5% consistency in a total reaction volume of 50 ml with xylanase ranging 10–50 IU/g of pulp in 50 mM Tris–HCl (pH 8.0). The incubation of the samples was carried out in an orbital incubator shaker at 180 rpm at 55  C for 2 h intervals up to 10 h. After incubation, pulp samples were washed with distilled water and absorbance of the filtrate was measured at 237 nm to estimate the release of lignin derived compounds. The removal of chromophoric material was estimated by absorbance at 465 nm (Wong et al., 1997). Reducing sugars in pulp filtrates released from the enzyme treated pulp were measured at 540 nm by DNS method (Ghose, 1987). The washed pulp samples were either processed for the analysis of their physical and chemical properties or dried in an oven at 55  C to constant weight for their chemical bleaching. The control samples without enzyme were treated under the same conditions. 2.7. Chemical bleaching Chemical bleaching of the pulp was performed in the R&D Labs. of Packages Ltd. Lahore, Pakistan according to the standard TAPPI

0

5 10 15 Incubation time (h)

20

Fig. 1. Effect of incubation period on xylanase production by Bacillus sp. XTR-10. Bars indicate values for standard deviation (n ¼ 3).

75

(U/ml)

75 60 45 30 15 0

Xylanase activity

(U/ml)

Xylanase activity

To determine the xylanase activity, 0.5 ml of appropriately diluted culture supernatant with 0.5 ml of 1% birchwood xylan

50 25 0 0

25

50

75

Temperature (oC) Fig. 3. Effect of temperature on xylanase production by Bacillus sp. XTR-10. Bars indicate values for standard deviation (n ¼ 3).

1121

W Xylanase activity (U/ml) he at st ra Ri w ce st ra w Ba ga s Co se Le rnc m ob Ba on s na pe na e l st al ks

71

60 50 40 30 20 10 0

69 67 65 63 61 0

1

2

Concentration (mg/ml) NaNO3

Peptone

(NH4)2SO4

Urea

Fig. 4. Effects of various nitrogen sources on xylanase production by Bacillus sp. XTR10. Standard deviation (S.D) values for xylanase activity ranged from 2.0 to 5.0 U/ml and are not shown to avoid difficulty in reading the figure.

procedures (TAPPI Test Methods, 1996). The enzyme treated pulp samples were bleached in polyethylene bags keeping their consistency at 5%. For single step chemical bleaching, sodium hypochlorite was added into the reaction mixture based on chlorine percentage of oven dry pulp. Bags were placed in water bath at 55  C for 2 h and kneaded after every 15 min. The pulp samples were then washed on 200 mesh screen. In multistage chemical bleaching using CED sequence (C: Chlorination, E: NaOH extraction, D: hypochlorite treatment), chlorination of the enzyme treated pulp samples used at 5% consistency was performed by their incubation with hypochlorite solution (containing chlorine in a ratio of 0.2% of kappa number of oven dry pulp) at 55  C for 1 h. After chlorination, consistency of the pulp samples was made 10% and they were treated with sodium hydroxide (in a ratio of 0.11% of kappa number of oven dry pulp) by their incubation at 70  C for 90 min. After alkali extraction, sodium hypochlorite solution (containing active chlorine in a ratio of 0.06% of kappa number of oven dry pulp) was added to the samples and their incubation was performed at 40  C for 150 min. Samples were filtered and washed on 200 mesh screen.

2.8. Physical and chemical properties of kraft pulp

Bi Soluble proteins (µg/ml) rc hw O oo at d s p xy el lan tx Ce yla llo n bi os Su e cr o G se lu co s Xy e lo se Av ic el

The hand sheets were prepared from the bleached wood kraft pulp by the standard TAPPI methods (TAPPI Test Methods, 1996). The physical and chemical properties of kraft pulp after biobleaching were assessed by determining the kappa number (T236 cm-85), brightness (T452 om-92), tensile strength (T231 cm-96), internal tearing resistance (T414 om-88) and bursting strength (T403 cm-50) using the standard protocols. Three replicates were used for each determination and the units used for all data representation were according to the TAPPI recommendations.

500 400 300 200 100 0

Protein concentration

Xylanase activity (U/ml)

Fig. 5. Effect of mono-, di- and polysaccharides on xylanase production by Bacillus sp. XTR-10. Bars indicate values for standard deviation (n ¼ 3).

Fig. 6. Effect of natural waste carbon sources on xylanase production by Bacillus sp. XTR-10. Bars indicate values for standard deviation (n ¼ 3).

3. Results and discussion Over the years, a variety of lignocellulolytic microorganisms have been isolated and still the list of such microorganisms especially bacteria and fungi is growing day by day. In the present study, a large number of thermophiles isolated from the decaying raw materials collected from suburbs of Lahore, Pakistan, were screened for xylanase activity and a bacterial strain XTR-10 producing the enzyme at maximum quantity was tentatively identified as Bacillus sp. by physical and biochemical reactions using the criteria given in Bergey’s Manual of Systematic Bacteriology (Sneath, 1994). 3.1. Optimization of culture conditions The inoculation of the medium with culture resulted in the maximum xylanase activity of 68 U/ml by Bacillus sp. at 12 h incubation time (Fig. 1). The xylanase activity further increased to 69 U/ml for pH 8.0 (Fig. 2) and to 69.5 U/ml at incubation temperature of 55  C (Fig. 3). Reasonably good amounts of xylanase were also produced at neutral and alkaline pH range. These results for the pH optimization agree with the previous studies conducted by Khanongnuch et al. (1999) and Kansoh and Nagieb (2004). Xylanase activity in the culture medium was further enhanced by the concentration optimization of various nitrogen sources. Although considerable activities were observed at peptone concentration of 1.5 mg/ml and NaNO3 concentration of 1.0 mg/ml, the higher xylanase activity (70.7 U/ml) was observed with the use of (NH4)2SO4 at 1.0 mg/ml concentration (Fig. 4). Khanderparker

1.5

80 70

Reducing sugars ( g/ml)

KNO3

1.2 60 50

0.9

40 0.6

30 20

0.3

OD at 273 nm & 465 nm

Xylanase activity (U/ml)

M. Saleem et al. / International Biodeterioration & Biodegradation 63 (2009) 1119–1124

10 0

0 0

10

20

30

40

50

60

Enzyme dose (IU/g of pulp) Reducing sugars

273 nm

465 nm

Fig. 7. Effect of xylanase dose on biobleaching of wood kraft pulp. Bars indicate values for standard deviation (n ¼ 3).

1122

M. Saleem et al. / International Biodeterioration & Biodegradation 63 (2009) 1119–1124

70

1.5

60

50 0.9

40

30

0.6

20

OD at 273 nm & 465 nm

Reducing sugars ( g/ml)

1.2

0.3 10

0

0 0

2

4

6

8

10

12

Incubation time (h) Reducing sugar

273 nm

465 nm

Fig. 8. Effect of incubation period on biobleaching of wood kraft pulp. Bars indicate values for standard deviation (n ¼ 3).

and Bhosle (2006) obtained the highest xylanase production by Enterobacter sp. when the medium was supplemented with peptone (0.45 mg/ml), yeast extract (2 mg/ml) and (NH4)2SO4 (1.25 mg/ml). Szendefy et al. (2006) tested inexpensive organic nitrogen sources along with inorganic nitrogen sources and obtained the highest enzyme yield by Aspergillus niger with the use of ammonium nitrate and corn steep liquor in the presence of eucalyptus pulp. The production of xylanase by the isolate was also studied in the presence of mono-, di- and polysaccharides. The strain produced substantial amounts of xylanase activity with several carbon sources, although the highest level of xylanase production was achieved at 55  C after 12 h fermentation in the presence of birchwood xylan as a sole carbon source (Fig. 5). Khanderparker and Bhosle (2006) also produced the maximum amounts of xylanase in the presence of birchwood xylan, but the fermentation time period was relatively higher (48 h). Rawashdeh et al. (2005) achieved maximal xylanase activity by Streptomyces sp. at 4th day of fermentation with the use of 0.5% xylan alone as well as in combination with 0.2% carboxymethyl cellulose. However, they observed the suppression of enzyme production in the presence of xylose, arabinose and glucose. In the present study, 50.2 U/ml xylanase activity was produced by Bacillus sp. XTR-10 in the presence of xylose. Thomson (1993) studied the role of low molecular mass hydrolysis products of xylan like xylose, xylooligosaccharides and heterodisaccharides in the regulatory pathway of xylanase synthesis and observed that the soluble and hydrolysable sugars like glucose and sucrose did not significantly enhance the enzyme production. The detection of cellulase activities in the culture broth

is an essential prerequisite for the applications of xylanases in pulp bleaching (Paice and Jurasek, 1984), because xylanases are only useful if the pulp cellulose fibers are not damaged by the cellulases secreted in the medium (Viikari et al., 1993). The Bacillus sp. XTR-10 secreted elevated levels of xylanase activity with very small amount of cellulase activity in the culture broth. Singh et al. (2003) have presented the production of cellulase free xylanase whereas the synergistic effect of xylanase and other enzymes present in the crude broth, such as mannanase and arabinase, on delignification efficiency has been reported by Duarte et al. (2003). Various waste raw materials could be utilized for economical production of fermentable sugars on large scale (Techapun et al., 2002). Among the natural and food waste materials used, the maximum levels of xylanase activity were obtained at 55  C after 20 h incubation in the presence of 2.0% wheat straw as the only carbon source. Significant amounts of xylanase were also produced in the presence of other raw materials (Fig. 6). The time lag for induction of xylanase is reported to be 18 h when Humicola grisea var. thermoidea was grown in the presence of banana stalk as substrate (Lucena-Neto and Ferreira-Filho, 2004). In another study, Li et al. (2006) reported the highest xylanase production on corncob followed by wheat straw and comparatively lower levels of enzyme activity were observed on wheat straw, wheat bran and other lignocellulosic materials. Seyis and Aksoz (2005) analyzed the effect of various natural wastes as carbon source and potential alternatives of organic nitrogen source (cotton leaf and soybean residue wastes) on xylanase production by Trichoderma harzianum 1073 D3 and found them environmentally and economically sound. Xylanase production using banana waste as carbon source is also described by Reddy et al. (2003). Because of the low fermentation cost and increased environmental protection, the natural substrates of lignocellulosic origin are highly appreciated for xylanase production in large quantities required for commercial purposes (Kuhad and Singh, 1993; Haltrich et al., 1996).

3.2. Biobleaching of kraft pulp Operating biotechnological process at elevated temperature is highly appreciated because it reduces the risk of contamination, increases the rate of reaction, decreases viscosity and provides high percentage of yield (Krahe et al., 1996). Pulp is bleached at high temperature and basic pH, therefore, the enzyme with high thermostability and activity in alkaline pH range are gaining great interest in paper and pulp industry (Jacques et al., 2000). Commercial enzymes used in paper and pulp industry have pH and temperature optima ranging from 3 to 8 and 30 to 75  C, respectively (Viikari et al., 1994). The xylanase produced by Bacillus sp. XTR-10 showed the pH and thermal stability within these limits, therefore, it can be used as a potential bleaching agent for pulp bleaching. During kraft pulping, approximately 90% of wood lignin is solubilized in the cooking liquor. Remaining lignin is responsible for the brown color of kraft pulp. The classical method for the

Table 1 Effect of enzyme dose on physicochemical properties of 5% wood kraft pulp treated for 8 h at 55  C in 50 mM Tris (pH 8.0). Enzyme dose IU

Kappa number

%Decrease in kappa number

Brightness %ISO

Increase in brightness %ISO

Burst index KNg1

Tear index mNm2 g1

Tensile strength Nmg1

Control 10 20 30 40 50

21.6  21.1  20.7  19.4  18.1  18.0 

– 2.31 4.1 10.18 16.20 16.66

23.9 25.9 27.4 28.6 30.1 30.2

– 8.36 14.64 19.66 25.94 26.35

4.45 4.34 4.28 4.18 4.15 4.11

14.80 14.92 14.98 15.15 15.19 15.26

71.7 71.2 71.0 70.8 70.7 70.6

0.58 0.57 0.59 0.52 0.56 0.60

     

1.18 1.16 1.19 1.20 1.18 1.17

     

0.16 0.18 0.12 0.19 0.15 0.11

     

0.35 0.38 0.32 0.39 0.34 0.37

     

1.75 1.73 1.79 1.71 1.78 1.70

M. Saleem et al. / International Biodeterioration & Biodegradation 63 (2009) 1119–1124

1123

Table 2 Effect of incubation period on physicochemical properties of 5% wood kraft pulp treated with xylanase (40 IU/g dry pulp) at 55  C in 50 mM Tris (pH 8.0). Incubation time (h)

Kappa number

%Decrease in kappa number

Brightness %ISO

Increase in brightness %ISO

Burst index KNg1

Tear index mNm2 g1

Tensile strength Nmg1

Control 2 4 6 8 10

21.5 21.0 20.5 19.1 18.0 17.9

– 2.32 4.65 11.62 16.27 16.74

23.5 25.1 26.7 27.6 29.7 29.9

– 6.80 13.61 17.44 26.38 27.23

4.51 4.43 4.39 4.28 4.19 4.14

14.63 14.82 15.01 15.13 15.22 15.31

71.51 71.41 71.11 70.78 70.65 70.59

     

0.60 0.59 0.58 0.52 0.55 0.53

     

1.18 1.20 1.19 1.16 1.19 1.18

removal of lignin is the addition of chlorine based bleaching agents to the pulp. The efficacy of chlorine based chemical bleaching of pulp is evident; however, such treatment is responsible for the production of toxic and mutagenic chloro organic compounds (Stepanova et al., 2000; Pokhrel and Viraraghavan, 2004). When wood kraft pulp was treated with cell free filtrates of xylanase from Bacillus sp. XTR-10, the release of color and reducing sugars from pulp increased with the increase in enzyme dose (Fig. 7) and incubation time (Fig. 8). The maximum liberation of these materials occurred at 40 IU xylanase dose/g of pulp by 8 h incubation. However, lower further increment in the liberation was also observed by increasing the xylanase dose to 50 IU and incubation time to 10 h. The relationship between chromophore and reducing sugars released from pulp may be an appropriate way to demonstrate the enzyme effectiveness in the pulp bleaching process (Eligir et al., 1995). The release of chromophores (237 nm), hydrophobic compounds (465 nm), reducing sugars (540 nm) and reduction of kappa number demonstrated the significant dissociation of lignin carbohydrate complex from the cellulose fibers. Xylanase breaks the hemicellulose chains that are responsible for the close adherence of lignin to cellulose network. There is thus a reduced need for bleach to remove loosened lignin (Sunnarki et al., 1997). Li et al. (2005) reported the release of the most of UV light absorbing and visible light absorbing material from the pulp within 2 h of the enzyme treatment. The relationship between the xylanase dose and the release of color compounds is also reported by Kulkarni and Rao (1996) and Khanderparker and Bhosle (2007). The highest decreases of kappa number (16.2%) were found when the pulp was treated with 40 IU/g of pulp at 5% consistency (pH 8.0) for 8 h at 55  C. However, further increased amount of xylanase under optimal conditions did not improve the outcome. Khanderparker and Bhosle (2007) reported 20% reduction in kappa number with the optimized dose (20 IU/g pulp used at 6% consistency) of xylanase from Arthrobacter sp. Higher enzyme dose or longer periods of incubation did not enhance the extent of biobleaching significantly. While Gupta et al. (2000) reported 25%

Brightness (%ISO)

75 60 45 30 15

     

0.19 0.15 0.18 0.17 0.13 0.14

     

0.38 0.40 0.36 0.39 0.35 0.37

     

1.70 1.76 1.72 1.75 1.73 1.71

reduction in kappa number with the optimum xylanase dose of 1.8 IU/g of moisture free eucalyptus kraft pulp at 50  C for 4 h. Xylanase treatment greatly improved and enhanced the brightness of wood pulp at all enzyme charges (10–50 IU/g) as shown in Table 1. About 26% ISO increment in brightness was observed when pulp was treated at the charge of 40 IU/g and further increment in enzyme dose to 50 IU did not improve the brightness of pulp significantly. Xylanase from Bacillus sp. NCIM-59 increased the brightness of bagasse pulp by 2.5% (Kulkarni and Rao, 1996). Improvement in brightness and reduction in kappa number of biobleached pulp with increase in enzyme dose suggests the reduction in the requirement of chlorinated chemicals during bleaching process of pulp. At the same time, different strength and texture properties of pulp may be improved. Xylanase treatment showed a slight decrease in tensile strength and burst index than the control. Garg et al. (1998) has also described the similar impact of xylanase treatment on tensile strength and burst index properties of kraft pulp. In another study, Gupta et al. (2000) reported the increase in burst factor and tensile strength indicating biobleaching treatment could facilitate the increase in pulp fibrillation, water retention and restoration of fibers bonding. Enzyme treatment of kraft pulp improved the fiber average length that contributed the increased tear index but decreased the bonding of the fiber. The incubation of the pulp with 40 IU xylanase dose/g of pulp showed a slight increase in brightness by increasing the time to 10 h (Table 2), therefore, to keep the enzyme reacting with kraft pulp for longer time period was no more required (Montiel et al., 2002). 3.3. Chemical bleaching of kraft pulp pretreated with xylanase The criteria for evaluating the biological prebleaching efficiency of enzyme is to attain higher final brightness of pulp in chemical bleaching with the diminished use of toxic chlorinated compounds (Viikari et al., 1994; Clarke et al., 2000). The single step chemical bleaching of the enzyme treated pulp samples with chlorine charges ranging 7–10% showed the maximum 70.6% brightness with the use of 8.5% active chlorine charges (Fig. 9). The unbleached 5 g oven dry straw pulp consumed 10% active chlorine to give 70.5% brightness. Thus, enzyme pretreatment saved 15% chlorine charges to reach the brightness at the control level. In some other studies, pretreatment of pulp with xylanases of different microbial origins resulted in chlorine saving by 20% (Dhillon and Khanna, 2000), 0.3% (Duarte et al., 2003) and 28% (Li et al., 2005). In multistage chemical bleaching, the control pulp used 4.32% chlorine charges to obtain 70.0% ISO brightness. The use of active chlorine charges was Table 3 Three stage (XCED) bleaching of 5% wood kraft pulp treated with xylanase (40 IU/g dry pulp) at 55  C for 8 h in 50 mM Tris (pH 8.0).

0 7

7.5

8

8.5

9

10

Chlorine charges (g/100g of pulp) Fig. 9. Increase in brightness of xylanase treated wood kraft pulp after bleaching with chlorine charges. Bars indicate values for standard deviation (n ¼ 3).

Pulp

Chlorine charges

NaOH charges

Hypo charges

Brightness %ISO

Control XCED I XCED II XCED III

4.32% 3.88% 3.70% 3.55%

2.13% 2.13% 2.13% 2.13%

1.29% 1.18% 1.18% 1.18%

70.0 63.5 67.8 70.2

   

1.20 1.21 1.20 1.22

1124

M. Saleem et al. / International Biodeterioration & Biodegradation 63 (2009) 1119–1124

reduced at every bleaching stage of the biopulp and brightness at the level of the control was achieved using 3.55% chlorine charges in the final stage (Table 3). Thus, 18.7% active chlorine charges were saved to achieve brightness at the level of the control. Garg et al. (1998) carried out the multistage (CEDED) chemical bleaching of the pulp pretreated with low levels of xylanase from Streptomyces thermoviolaceus and observed 30–35% chlorine saving to obtain pulp brightness at the level of the control. Georis et al. (2000) also performed the multistage (CEDED) bleaching of the enzyme treated pulp and observed 17.4 mg chlorine saving per g of hardwood pulp and 8.6 mg per g of softwood pulp. As the chlorine saving in single step (15%) and multistage (18.7%) chemical bleaching is comparable, the single step chlorine bleaching of the enzyme treated pulp may suitably be considered for the management of cost, time and risk related to the bleaching process. The use of xylanases in the pulp and paper industry will open the door for the use of other enzymes such as lipases for reducing pitch, triglycerides and resin acids, and for other biotechnological developments such as biobleaching and biopulping using fungi (Bar-Lev et al., 1982; Fujita et al., 1992). References Atik, C., Immoglu, S., Bermer, H., 2006. Impact of xylanase pretreatment as peroxide stage of biocraft pulp. International Biodeterioration and Biodegradation 58, 22–26. Bajpai, P., Bhardwaj, N.K., Bajpai, P.K., Jauhari, B.M., 1994. The impact of xylanases on bleaching of eucalyptus kraft pulp. Journal of Biotechnology 38, 1–6. Bar-Lev, S.S., Kirkand, T.K., Chang, H.M., 1982. Fungal treatment can reduce energy requirement for secondary refining of TMP. TAPPI Journal 65, 111–113. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Analytical Biochemistry 72, 248–254. Chauvet, J.M., Comfat, T., Noe, P., 1987. Assistance in bleaching of never dried pulp by the use of xylanases, consequences on pulp properties. In: Proceedings on Wood and Pulping Chemistry, Paris 2, pp. 3225–3227. Clarke, J.H., Davidson, K., Rixon, J.E., Halstead, J.R., Fransen, M.P., Gilbert, H.J., Hazlewood, G.P., 2000. A comparison of enzyme-aided bleaching of softwood paper pulp using combinations of xylanase, mannanase and b-galactosidase. Applied Microbiology and Biotechnology 53, 661–667. Dhillon, A., Khanna, S., 2000. Production of a thermostable alkali tolerant xylanase from Bacillus circulans AB16 grown on wheat straw. World Journal of Microbiology and Biotechnology 27, 325–327. Duarte, M., Silva, E., Games, I., Ponezi, A., Portugal, E., Vicente, J., Davanzo, E., 2003. Xylan hydrolyzing enzyme system from Bacillus pumilus CBMA I0008 and its effect on eucalyptus grandis kraft pulp for pulp bleaching improvement. Bioresource Technology 88, 9–15. Eligir, G., Sykes, M., Jeffries, T.W., 1995. Differential and synergistic action of Streptomyces endoxylanases in prebleaching of kraft pulp. Enzyme and Microbial Technology 17, 954–959. Fujita, Y., Awaji, H., Taneda, H., Matsukura, M., Hata, K., Shimoto, H., Sharyo, M., Sakaguchi, H., Gibson, K., 1992. Recent advances in enzymatic pitch control. TAPPI Journal 75, 117–122. Garg, A.P., Roberts, J.C., McCarthy, A.J., 1998. Bleach boosting effect of cellulase-free xylanase of Streptomyces thermoviolaceus and its comparison with two commercial enzyme preparations on birchwood kraft pulp. Enzyme and Microbial Technology 22, 594–598. Georis, J., Giannotta, F., Buyl, E.D., Granier, B., Frere, J.M., 2000. Purification and properties of three endo-b-1,4-xylanases produced by Streptomyces sp. strain S38 which differ in their ability to enhance the bleaching of kraft pulps. Enzyme and Microbial Technology 26, 178–186. Ghose, T.K., 1987. Measurement of cellulase activities. Pure and Applied Chemistry 59, 257–268. Gupta, G., Bhushan, B., Hoondal, G.S., 2000. Isolation, purification and characterization of xylanase from Staphylococcus sp. SG-13 and its application in biobleaching of kraft pulp. Journal of Applied Microbiology 88, 325–334. Haltrich, D., Nidetzky, B., Kulbe, K.D., Steiner, W., Zupancic, S., 1996. Production of fungal xylanases. Bioresource Technology 58, 137–161. Han, Y.W., Srinivasan, V.R., 1968. Isolation and characterization of cellulose utilizing bacterium. Applied Microbiology 16, 1140–1145. Jacques, G., Frederic, D.L., Joste, L.B., Viviane, B., Bart, D., Fabrizio, G., Benoit, G., Jean– Marie, F., 2000. An additional aromatic interaction improve the thermostability and thermophilicity of a mesophilic family 11 xylanase; structural basis and molecular study. Protein Science 9, 466–475. Kansoh, A.L., Nagieb, Z.A., 2004. Xylanase and mannanase enzymes from Streptomyces galbus NR and their use in biobleaching of softwood draft pulp. Antonie Van Leeuwenhoek 85, 103–114.

Khanderparker, R.D.S., Bhosle, N.B., 2006. Isolation, purification and characterization of the xylanase produced by Arthrobacter sp. MTCC 5214 when grown in solid-state fermentation. Enzyme and Microbial Technology 39, 732–742. Khanderparker, R., Bhosle, N.B., 2007. Application of thermophilic xylanase from Arthrobacter sp. MTCC 5214 in biobleaching of kraft pulp. Bioresource Technology 98, 897–903. Khanongnuch, C., Lumyong, So., Ooi, T., Kinoshita, S., 1999. A non-cellulase production strains of Bacillus subtilis and its potential use in pulp biobleaching. Biotechnology Letters 21, 61–63. Krahe, M., Antranikian, G., Markl, H., 1996. Fermentation of extremophilic microorganisms. FEMS Microbiology Reviews 18, 271–285. Kuhad, R.C., Singh, A., 1993. Lignocellulose biotechnology: their properties and application. Critical Reviews in Biotechnology 13, 51–72. Kulkarni, N., Rao, M., 1996. Application of xylanase from alkaliphilic thermophilic Bacillus sp. NCIM 59 in biobleaching of bagasse pulp. Journal of Biotechnology 51, 167–173. Li, L., Tian, H., Cheng, Y., Jiang, Z., Yang, S., 2006. Purification and characterization of a thermostable cellulose-free xylanase from the newly isolated Paecilomyces thermophila. Enzyme and Microbial Technology 38, 780–787. Li, X.T., Jiang, Z.Q., Li, L.T., Yang, S.Q., Feng, W.Y., Fan, J.Y., Kusakahe, I., 2005. Characterization of a cellulase free neutral xylanase from Thermomyces lanuginosus CBS 288.54 and its biobleaching effect on wheat straw pulp. Bioresource Technology 96, 1370–1379. Lucena-Neto, S.A., Ferreira-Filho, E.X., 2004. Purification and characterization of a new xylanase from Humicola grisea Var. thermoidea. Brazilian Journal of Microbiology 35, 86–90. Montiel, M.D., Hernandez, M., Rodriguez, J., Arias, M.E., 2002. Evaluation of an endo beta mannanase produced by Streptomyces ipomoea CECT 3341 for the biobleaching of pine kraft pulps. Applied Microbiology and Biotechnology 58, 67–72. Paice, M.G., Jurasek, L., 1984. Removing hemicellulose from pulps by specific enzymic hydrolysis. Journal of Wood Chemistry and Technology 4, 187–198. Paice, M.G., Bernier Jr., R., Jurasek, L., 1988. Viscosity enhancing bleaching of hardwood kraft pulp with xylanase from a cloned gene. Biotechnology and Bioengineering 32, 235–239. Pokhrel, D., Viraraghavan, T., 2004. Treatment of pulp and paper mill wastewater – a review. Science of the Total Environment 333, 37–58. Rawashdeh, R., Saadoun, I., Mahasneh, A., 2005. Effect of cultural conditions on xylanase production by Streptomyces sp. (strain Ib 24D) and its potential to utilize tomato pomace. African Journal of Biotechnology 4, 251–255. Reddy, G.V., Babu, P.R., Komaraih, P., Roy, K.R.R.M., Kothari, I.L., 2003. Utilization of banana waste for the production of lignolytic and cellulolytic enzymes by solid substrate fermentation using two Pleurotus species. Process Biochemistry 38, 1457–1462. Senior, D.J., Hamilton, J., Bernier, R.L., Monoir, J.R., 1992. Reduction in chlorine use during bleaching of kraft pulp following xylanase treatment. TAPPI Journal 75, 125–130. Seyis, I., Aksoz, N., 2005. Xylanase production form Trichoderma harzianum 1073 D3 with alternative carbon and nitrogen sources. Food Technology and Biotechnology 43, 37–40. Singh, S., Madlala, A.M., Prior, B.A., 2003. Thermomyces lanuginosus: properties of strains and their hemicellulases. FEMS Microbiology Reviews 27, 3–16. Sneath, P.H.A., 1994. Bergey’s manual of systematic bacteriology. In: Hensyl, W.M. (Ed.), nineth ed. Williams and Wilkins, Philadelphia. Stepanova, L.I., Lindstrom-Seppa, P., Hanninen, O.O.P., Kotelevsev, S.V., Glaser, V.M., Novikov, C.N., 2000. Lake Baikal: biomonitoring of pulp and paper mill waste water. Aquatic Ecosystem Health and Management 3, 259–269. Subramaniyan, S., Prema, P., 2002. Biotechnology of microbial xylanases: enzymology, molecular biology and applications. Critical Reviews in Biotechnology 22, 33–64. Sunnarki, A., Tenkanen, M., Buchert, J., Viikari, L., 1997. Hemicellulases in bleaching of chemical pulp. Advances in Biochemical Engineering/Biotechnology 57, 262–287. Szendefy, J., Szakacs, G., Christopher, L., 2006. Potential of solid-state fermentation enzymes of Aspergillus oryzae in biobleaching of paper pulp. Enzyme and Microbial Technology 39, 1354–1360. TAPPI Test Methods, 1996. Technical Association of the Pulp and Paper Industry. TAPPI Press, Atlanta, GA. Techapun, C., Sinsuwongwat, S., Watanabe, M., Sasaki, K., Poosaran, N., 2002. Production of cellulose-free xylanase by a thermotolerant Streptomyces sp. grown on agricultural waste and media optimization using mixture design and Plackett– Burman experimental design methods. Biotechnology 24, 1437–1442. Thomson, J.A., 1993. Molecular biology of xylan degradation. FEMS Microbiology Reviews 104, 65–82. Viikari, I., Ranua, M., Kantelinen, A., Sundquist, J., Lino, M., 1986. Bleaching with enzyme. In: International Conference on Biotechnology in the Pulp and Paper Industry, Stockholm, Sweden, third ed., pp. 67–69. Viikari, L., Tenkanen, M., Buchert, J., Ratto, Mo., Bailey, M., Siikaho, M., Linko, M., 1993. Hemicellulases for industrial applications. In: Saddler, J.N. (Ed.), Bioconversion of Forest and Agricultural Plant Residues. C.A.B. International, Wallingford, England, pp. 131–182. Viikari, L., Kantelinen, A., Sundquist, J., Linko, M., 1994. Xylanases in bleaching: from an idea to the industry. FEMS Microbiology Reviews 13, 335–350. Wong, K.K.Y., Jong, E.D., Saddler, J.N., Allison, R.W., 1997. Mechanism of xylanase aided bleaching of kraft pulp. Part 1: process parameters. Appita Journal 50, 415–422.