Bioresource Technology 169 (2014) 96–102
Contents lists available at ScienceDirect
Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech
Xylanase and laccase based enzymatic kraft pulp bleaching reduces
adsorbable organic halogen (AOX) in bleach effluents: A pilot scale study
Abha Sharma a, Vasanta Vadde Thakur b, Anita Shrivastava a, Rakesh Kumar Jain b,
Rajeev Mohan Mathur b, Rishi Gupta a, Ramesh Chander Kuhad a,⇑
a
b
Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India
Central Pulp and Paper Research Institute, Saharanpur, U.P., India
h i g h l i g h t s
Cost-effective production of xylanase and laccase up to 10 kg substrate.
Sequential enzymatic treatment of pulp proved better than individual treatments.
Enzymatic pre-treatment of pulp reduced 35% ClO2 in ECF bleaching.
Enzyme treatment at pilot scale lowered AOX levels by 34% in effluents.
Enzyme treatment at pilot scale led to reduction in PC No. of pulp by 50%.
a r t i c l e
i n f o
Article history:
Received 17 May 2014
Received in revised form 17 June 2014
Accepted 18 June 2014
Available online 26 June 2014
Keywords:
Xylanase
Laccase
Bleaching
Adsorbable organic halogen
Post color number
a b s t r a c t
In present study, xylanase and laccase were produced in a cost-effective manner up to 10 kg substrate
level and evaluated in elemental chlorine free bleaching of Eucalyptus kraft pulp. Compared to the pulp
pre-bleached with xylanase (15%) or laccase (25%) individually, the ClO2 savings were higher with
sequential treatment of xylanase followed by laccase (35%) at laboratory scale. The sequential enzyme
treatment when applied at pilot scale (50 kg pulp), resulted in improved pulp properties (50% reduced
post color number, 15.71% increased tear index) and reduced AOX levels (34%) in bleach effluents. The
decreased AOX level in effluents will help to meet AOX discharge limits, while improved pulp properties
will be value addition to the paper.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Most of the pulp and paper mills worldwide use chlorine dioxide as elemental chlorine free (ECF) bleaching agent for production
of high quality white pulp (Bajpai, 2012). The high organic content
of wood pulp coupled with chlorine dioxide used in the bleaching
process results in the production of organo-chlorine compounds,
which are finally discharged as bleach effluents in water bodies
(Chaparro et al., 2010). These organo-chlorine compounds (measured as AOX) have been reported to cause genetic and reproductive damages in aquatic as well as terrestrial animals including
humans (Easton et al., 1997). Since these AOX compounds are
⇑ Corresponding author. Tel.: +91 9871509870; fax: +91 11 24115270.
E-mail address: kuhad85@gmail.com (R.C. Kuhad).
http://dx.doi.org/10.1016/j.biortech.2014.06.066
0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
man-made (xenobiotic), microbes have not evolved enzyme systems for their rapid degradation (Thakur, 2004). As a result, many
countries have now set discharge limits for these compounds
(Savant et al., 2006). However, meeting of these discharge limits
will require either end-of-pipe treatment techniques or modification in bleaching technologies of mills. End-of-pipe treatment
techniques include precipitation, biological degradation, and
advanced oxidation processes (Savant et al., 2006). However, use
of these techniques creates new environmental problems like the
need for disposal of waste from treatment facilities (Cerventes
and Pavlostathis, 2006). Therefore, the major interest has been
shifted to develop cost effective and environmentally benign
bleaching technologies for reduced AOX generation. In this aspect,
enzymatic bleaching of pulp with xylanase and laccase offers a
potentially viable option to achieve a clean and green technology
for pulp bleaching (Kuhad et al., 1997).
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A. Sharma et al. / Bioresource Technology 169 (2014) 96–102
Xylanase hydrolyzes the re-precipitated xylan of pulp fiber
formed during delignification, rendering the pulp more permeable
and thus facilitate the removal of residual lignin. While, laccase
oxidizes phenolic units and amine compounds in lignin and therefore, allow their easier removal during the subsequent chemical
bleaching stages (Bourbonnais et al., 1997). As a result, use of these
enzymes lower the consumption of chlorine based compounds for
pulp bleaching (Kuhad et al., 1997), thereby reducing AOX generation in bleach effluents (Bajpai, 2012). However, due to high
production cost, most of the studies on enzyme production and
their application in biobleaching are limited to bench scale and
mill scale application of enzymes in pulp bleaching is still in the
developmental stage (Bajpai, 2012). This indicates the need for
the development of efficient and low cost technologies for enzyme
production. The use of solid state fermentation (SSF) for enzyme
production provide significant economic (Osma et al., 2011) and
technical benefits including, high product yields, use of simple
machinery, lesser generation of effluents and lower requirements
for aeration and agitation (Szendefy et al., 2006).
Keeping all this in view, present study focused on enzyme production from Bacillus pumilus (xylanase producer) and Ganoderma
sp. rckk-02 (laccase producer) under SSF using in vitro enzyme
digestion (IVED) approach followed by its scale-up to 10 kg level.
Subsequently, attempt has been made to use these enzymes
(individually and in combination) in bleaching of Eucalyptus kraft
pulp up to pilot scale for reduced AOX levels and improved pulp
properties.
2. Methods
2.1. Chemicals and raw materials
All assay reagents were purchased from Sigma–Aldrich (St.
Louis, MO, USA), while all media components were purchased from
Hi Media Laboratories Pvt. Ltd. (Mumbai, India). The chemicals
used were purchased from Fischer Scientific (Waltham, USA).
Wheat bran was obtained locally. Eucalyptus kraft pulp was
procured from Star Paper Mill, Saharanpur.
2.2. Microorganisms and culture conditions
Xylanase producing bacterium B. pumilus MK001 (accession No.
AY389345) and laccase producing basidiomycetous fungus,
Ganoderma sp. rckk-02 (accession No. AJ749970), our own laboratory isolates were used in the present study. B. pumilus strain
MK001 was maintained on xylan-agar at 37 °C as described by
Kapoor et al. (2007). While, Ganoderma sp. rckk-02 was maintained
on malt extract agar (MEA) at 30 °C as described previously
(Sharma et al., 2005). Pure cultures were stored at 4 °C and
subcultured every fortnight.
inoculated with appropriate volume of fungal pellets to obtain
0.02 g of fungal dry mass/5 g of substrate instead of fungal discs
used by Sharma et al. (2005). The flasks were incubated at 30 °C
for 5 days and the enzyme was extracted as described elsewhere
(Sharma et al., 2005).
2.4. Scale up of enzyme production at pilot scale
The scalability of the enzyme production process was tested
from 250 ml Erlenmeyer flasks to enamel trays of different sizes
containing varied amount of substrate. The large scale production
of enzymes at pilot scale (10 kg level) was carried out in Kozi room
(10 ft 10 ft). At Kozi room, xylanase production was carried out
in batches of 500 g substrate in trays of size 56 41 7.1 cm3,
while the laccase production was carried out in batches of 1000 g
substrate in same sized trays.
2.5. Determining the stability of enzymes in pulp bleaching conditions
The temperature stability of enzymes was determined by incubating the enzyme at 50 °C and the residual enzyme activity was
determined after regular intervals under the respective standard
assays. While, the pH stability was determined by incubating the
enzyme samples in buffer of pH 8.0 at 50 °C and thereafter the
residual activities were determined under the respective standard
assay conditions.
2.6. Enzymatic bleaching of Eucalyptus kraft pulp at bench scale
2.6.1. Optimization of process parameters for enzymatic pretreatment of pulp
Optimum enzyme doses and retention times for enzymatic pretreatment of pulp were decided on the basis of improvement in
final brightness of the pulp in elemental chlorine free (ECF) bleaching sequence [D0E(p)D1D2; D0 – chlorine dioxide stage, E(p) – alkali
extraction with hydrogen peroxide, D1 – chlorine dioxide stage 1,
D2 – chlorine dioxide stage 2] compared with the control pulp.
The control pulp samples were bleached using the ECF sequence
but were not pre-treated with enzymes. The process conditions
used during ECF bleaching sequence are given in Table 1.
For optimization of xylanase dose, unbleached hardwood pulp
(10% wv 1 consistency) was pre-treated with different xylanase
doses [10–50 IUg 1 oven dried pulp (odp)] at 50 °C and pH 8.0
for 120 min. While, the effect of laccase dose on pulp bleaching
was studied by pre-treating pulp with varying laccase doses
(40–100 IUg 1 oven dried pulp) in presence of 0.1% ww 1 of HBT
at 50 °C and pH 8.0 for 240 min. The effect of retention time on
the efficiency of pulp pre-treatment with xylanase (X) and laccase
mediator system (LMS) was studied by incubating pulp samples
with the optimized doses of each enzyme for varying retention
times (30–300 min.) at 50 °C and pH 8.0.
2.3. Enzyme production at bench scale
Xylanase production from B. pumilus MK001 was carried out
under solid state fermentation (SSF) conditions as described earlier
(Kapoor et al., 2007). While, laccase production from Ganoderma
sp. rckk-02 was carried out following the IVED approach as given
by Sharma et al. (2005). In IVED approach, wheat bran fermented
with B. pumilus MK001 from which xylanase has been extracted
was washed, sterilized and used as substrate for laccase production
by Ganoderma sp. rckk-02 under SSF. Each 250 ml Erlenmeyer flask
containing 5.0 g of in vitro xylanase digested (IVXD) wheat bran
was moistened with mineral salt solution containing (gL 1):
Ca(NO3)2, 0.5; KH2PO4, 0.5 and MgSO47H2O, 0.5 (pH 5.4) to obtain
substrate to moisture ratio of 1:3. The flasks were autoclaved and
Table 1
Process conditions applied during chemical bleaching of pulp.
Particulars
*
Temperature, °C
Consistency, %
Treatment time (min)
pH
50
3.0
45
2.0
D0 stage
**
E(p) stage
70
10.0
60
11.0
#
D1 stage
80
10.0
180
3.0
##
D2 stage
80
10.0
180
3.0
*
D0 is the chlorine dioxide stage with 28 ClO2 Kg tp 1 (Kilograms per ton of
pulp).
**
E(p) is alkali extraction with hydrogen peroxide with 20 NaOH Kg tp 1 and
10 Kg tp 1 H2O2.
#
D1 is the chlorine dioxide stage 1 with 11 ClO2 Kg tp 1.
##
D2 D1 is the chlorine dioxide stage 2 with 5.0 ClO2 Kg tp 1.
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A. Sharma et al. / Bioresource Technology 169 (2014) 96–102
2.6.2. Enzymatic pre-treatment of pulp followed by ECF bleaching for
reduced chlorine dioxide demand
The Eucalyptus kraft pulp was treated with xylanase and laccase individually (X or LMS) as well as in combination (X + LMS)
using the optimized enzyme dose for optimized time period. After
the enzymatic treatment, pulps were subjected to ECF bleaching
sequence [D0E(p)D1D2] with reduced chlorine dioxide demand in
the D0 stage (0%, 10%, 20%, 30% and 40%) and the final pulp
brightness was measured. In the control experiment, enzyme
was replaced with water and the pulp was bleached in the ECF
bleaching sequence.
2.7. Enzymatic bleaching of Eucalyptus kraft pulp at pilot scale
Based on the laboratory studies, pilot plant trial for enzymatic
bleaching of 50 kg Eucalyptus kraft pulp was carried out in the
pulp tower of the pilot plant at Central Paper and Pulp Research
Institute (CPPRI), Saharanpur. In the control run, enzymes were
replaced with water and the conditions used for bleaching were
same as given in Table 1. While, test was run with the pulp
pre-treated sequentially with X + LMS followed by D0E(p)D1D2
chemical bleaching sequence with 35% reduced ClO2 in the D0
Stage. Enzymes were added to the pulp after sufficient dilution
and mixed properly by agitator. After each bleaching stage, the
pulp was washed thoroughly by passing through belt washer and
the final pulps (both enzyme treated and untreated) were characterized for brightness, post color number (PC No.), tensile index,
burst index and tear index. The AOX, BOD and COD levels in the
final bleach effluents of both treated and untreated pulps were also
analyzed.
2.8. Analytical procedures
The xylanase activity was estimated by measuring the release of
xylose from birch wood xylan (1.0% wv 1) following the method
described elsewhere (Kapoor et al., 2007). Laccase activity was
determined using guaiacol as substrate according to the method
described previously (Sharma et al., 2005). The physical and
chemical characterization of pulp and effluents was carried out
according to the standard test methods of Technical Association
of the Pulp and Paper Industry (TAPPI, 1992), International
Standards Organization (ISO) and methods of American Public
Health Association (APHA, 1992).
3. Results and discussion
3.1. Enzyme production
3.1.1. Xylanase production at bench scale
B. pumilus MK001 produced high titers of xylanase
[47,100 ± 2129 IUg 1ds (dry substrate)] when grown under SSF
on 5 g wheat bran. The higher production of xylanase by B. pumilus
MK001 is because various nutritional and environmental conditions that affect xylanase production from the strain have been
studied in detail by our group earlier (Kapoor et al., 2007;
Kapoor et al., 2008). The bacterium grows luxuriantly on wheat
bran and produces comparatively high levels of xylanase (Kapoor
et al., 2008) than many other earlier reports. Banu and Ingale
(2011) reported 1324.24 IUg 1ds of xylanase from B. pumilus
AB-1 grown on 10 g wheat bran under SSF. While, Kamble and
Jadhav (2012) reported 910.45 IUg 1ds of xylanase produced from
Bacillus sp. grown on 10 g wheat bran under SSF. The relatively
higher xylanase production by B. pumilus MK001 shows promise
of offering great potential in production of xylanase for various
biotechnological applications.
3.1.2. Laccase production at bench scale
The production of laccase from Ganoderma sp. rckk-02 was
tested on fresh as well as on IVXD wheat bran. It was observed that
the laccase production on IVXD bran (9189 ± 610 IUg 1ds) was
3-fold higher than that obtained on untreated bran
(2876 ± 234 IUg 1ds). The re-utilization of substrate will make
the enzyme production process cost-effective and will also reduce
the problem of solid disposal. Sharma et al. (2005) hypothesized
that an increase in laccase production on IVXD waste wheat bran
under SSF is due to the action of enzymes secreted by the bacterium that cause degradation of the lignocellulosics present in
wheat bran into some aromatic compounds, which act as inducers
for laccase production from the fungus. Interestingly, the laccase
titers (9189 ± 610 IUg 1ds) obtained in the present study were
5.0-fold higher than our earlier report (Sharma et al., 2005). The
difference in enzyme tires might be due to the use of fungal pellets
as inoculum in place of fungal discs. Fungal pellets used as inoculum can prove to be advantageous as compared to fungal discs in
terms of uniform mixing and larger surface area for better mass
and oxygen transfer in SSF (Shrivastsva et al., 2011).
3.1.3. Scale-up of xylanase and laccase production at pilot scale
Scale-up experiments are very important for transferring a
laboratory scale process to commercial application (Lonsane
et al., 1990). Unsuccessful scale-up experiments lead to wasted
time and energy spent on laboratory work. The scale up of enzyme
production in present study was performed in enamel trays in Kozi
room, which is a specially designed fermentation facility equipped
with temperature and humidity control under circulating air. Trays
are arranged one above the other inside the Kozi room with suitable gap between them (Bharghav et al., 2008). An advantage of
using trays for enzyme production is that by increasing the number
of trays in Kozi room, scale up for SSF becomes easier.
During the scale up of xylanase production from B. pumilus
MK001 in enamel trays of different sizes, highest xylanase
production (44,600 ± 1987 IUg 1ds) was achieved in 500 g wheat
bran in tray of size 56 41 7.1 cm3, which was very close to that
obtained at flask level (Table 2). However, on increasing the substrate amount to 1000 g and 1200 g wheat bran in tray of same
size, a significant decline in enzyme production was observed
(Table 2). Decrease in enzyme production on increasing the
substrate amount in same sized tray may be due to reduction in
oxygen content required for the growth of bacteria and nonmaintenance of nutritional and fermentation conditions required
in scale-up experiments (Battan et al., 2007). Therefore, the large
scale xylanase production up to 10.0 kg substrate level was carried
out in batches of 500 g wheat bran contained in trays of size
56 41 7.1 cm3, incubated in Kozi room (Table 2). While, on
scaling up laccase production from Ganoderma sp. rckk-02, highest
laccase titers (11,567 ± 629 IUg 1ds) were obtained on 1000 g
IVXD wheat bran in enamel trays of size 56 1 7.1 cm3 (Table 2).
While, contrary to the results obtained for scale-up of xylanase
production from B. pumilus MK001 under SSF, where enzyme
production declined on 1000 g wheat bran in tray of size
56 41 7.1 cm3, laccase production from Ganoderma sp. rckk-02
increased on 1000 g wheat bran contained in same sized tray. This
could be because of difference in the oxygen requirements of
bacteria and fungus for enzyme production (Grahl et al., 2012).
On further increasing the substrate amount to 1200 g in trays of
same size, laccase production declined substantially (Table 2),
which could be due to drastic reduction in effective aeration
required for fungal growth and metabolism. Therefore, pilot scale
laccase production up to 10.0 kg substrate was carried out in
batches of 1000 g wheat bran contained in trays of size
56 41 7.1 cm3 at Kozi room (Table 1). Interestingly, as compared to the flasks, fermentation in trays resulted in higher laccase
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A. Sharma et al. / Bioresource Technology 169 (2014) 96–102
Table 2
Scale up of xylanase and laccase production from B. pumilus MK001 and Ganoderma sp. rckk-02, respectively under SSF.
Amount of wheat bran (grams)
Flask/tray
Xylanase IU g
5
10
50
100
200
500
1000
1200
10,000
250 ml Flask
500 ml Flask
25.0 21.5 4.0 cm3 tray
32.2 30.1 5.0 cm3 tray
42.0 30.1 6.0 cm3 tray
56 41 7.1 cm3 tray
56 41 7.1 cm3 tray
56 41 7.1 cm3 tray
Kozi room
47,100 ± 2129
47,000 ± 2067
44,157 ± 2098
45,023 ± 1954
42,194 ± 1985
44,600 ± 1987
38,600 ± 1987
25,109 ± 1519
45,610 ± 1875
yields, which may be attributed to the larger surface area in tray
configurations for the fungal growth leading to better transfer of
oxygen and nutrients (Singhania et al., 2009). Dhillon et al.
(2012) also observed increased laccase production from Tinea
versicolor in plastic trays than in flasks.
This is the first report showing successful production of
xylanase and laccase under SSF up to 10 kg substrate in Kozi room.
Earlier, Battan et al. (2007) showed scale up of xylanase production
under SSF from B. pumilus ASH up to 300 g substrate level. While,
Dhillon et al. (2012) scaled up laccase production from T. versicolor
up to 400 g substrate under SSF.
3.2. Determining the stability of enzymes in pulp bleaching conditions
The optimum incubation time for efficient pulp bleaching with
xylanase usually range from 120–180 min (Brijilall et al., 2011)
while, it ranges from 120–240 min with laccase (Valls et al.,
2010a). Therefore, for enzymatic bleaching of pulp it is a prerequisite to use enzymes which are stable at 50 °C and pH 8.0–8.5
(conditions prevalent in the paper mills) for the said incubation
time periods. In the present work, the xylanase produced from
B. pumilus MK001 retained 88% of its maximum activity at 50 °C
after 180 min (Fig. 1). While, the laccase from Ganoderma sp.
rkk-02 retained 72% of its maximum activity at 50 °C after
240 min (Fig. 1). Guimaraes et al. (2013) used xylanase from
Aspergillus niger which retained 85% of its activity at 50 °C after
120 min for pulp bleaching.
On studying the pH stability of the enzymes, 65% (xylanase) and
50% (laccase) of their maximum activities are retained at pH 8.0
after 180 min and 240 min, respectively (Fig. 1). PeixotoNogueira et al. (2009) applied xylanase from Aspergillus fumigatus
for pulp bleaching, which retained 70% of its activity at pH 8.0
for 60 min. While, most of the laccases used so far for pulp bleaching are stable mainly in acidic pH range (Kapoor et al., 2007; Fillat
Fig. 1. Temperature and pH stability of xylanase and laccase at 50 °C and pH 8.0.
1
ds
Laccase IU g
1
ds
9189 ± 610
9192 ± 580
9198 ± 550
9456 ± 623
9564 ± 589
9,700 ± 590
11,567 ± 629
7299 ± 600
11,589 ± 556
and Roncero, 2009; Valls et al., 2010a,b; Martin-Sampedro et al.,
2012; Ravalason et al., 2012), requiring addition of acid to bring
down the pH of the pulp in mills and hence making the commercial
implementation of the bioprocess difficult. Therefore, xylanase
from B. pumilus MK001 and laccase from Ganoderma sp. rckk-02
are amenable for their use in pulp bleaching as considerable% of
their activities are retained at 50 °C and pH 8.0 for 180 and
240 min, respectively.
3.3. Enzymatic bleaching of Eucalyptus kraft pulp at bench scale
3.3.1. Optimization of xylanase pre-treatment of pulp [XD0E(p)D1D2]
Optimization of xylanase dose for pretreatment of hardwood
pulp revealed that pulp brightness increased with increase in
enzyme concentration (Table 3). However, at higher enzyme doses
(>30 IUg 1 odp), pulp yield decreased substantially (Table 3).
Hence, xylanase dose of 30 IUg 1 odp was considered as optimum
for pre-treatment of pulp resulting in brightness improvement by
1.8 units, while maintaining pulp yield (Table 3). Battan et al.
(2007) also concluded that higher xylanase dose did not enhance
the extent of biobleaching significantly but affected the pulp
strength. Similarly, Saleem et al. (2009) reported that xylanase
pre-treatment of kraft pulp at higher enzyme dose decreased
bonding of the pulp fiber. While, among different incubation time
periods tested (Fig. 2), the pulp when treated with xylanase dose of
30 IUg 1 odp for 150 min brought about maximum improvement
in brightness (2.0 Units). On increasing the incubation time after
150 min did not enhance the pulp brightness significantly
(Fig. 2). Similar to our results, Brijilall et al. (2011) observed that
treatment of pulp with xylanase dose of 50 IUg 1 odp for
180 min. increased pulp brightness by 2.1 units.
3.3.2. Optimization of LMS pre-treatment of pulp [(LMS)D0E(p)D1D2]
Laccase alone has a very limited effect on lignin degradation
because of (a) its large size, (Morozova et al., 2007) and (b) its
low redox potential (Bourbonnais et al., 1997). While, combination
of the enzyme with low molecular weight redox mediators lead to
higher rates and yields of transformation of lignin (Morozova et al.,
2007). Redox mediators migrate into the aromatic structure of the
lignin and accelerate the rate of its oxidation by shuttling electrons
between lignin and laccase. As a result, laccase is mainly applied
for pulp bleaching in conjunction with synthetic mediators. In
the present study, it was observed that the bleaching efficiency
of laccase from Ganoderma sp. rckk-02 varied with the nature of
mediator and the enzyme showed better bleach response in presence of HBT than with ABTS (data not shown). Hence, in subsequent experiments of pulp biobleaching, 0.1% ww 1 HBT was
used as laccase mediator. Among varied dosages of laccase used,
the pulp brightness increased with increase in laccase dose up to
60 IUg 1 odp and remained almost constant thereafter (Table 4).
However, the optimum incubation time for LMS treatment
obtained was 240 min. with improvement in pulp brightness by
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A. Sharma et al. / Bioresource Technology 169 (2014) 96–102
Table 3
Effect of enzyme dose on xylanase pre-treatment of pulp.
Xylanase dose (IUg
1
odp)
10.0
20.0
30.0
40.0
50.0
Control
Brightness, % ISO
Brightness improvement unit
Pulp yield (%)
81.82 ± 0.16
82.21 ± 0.14
82.80 ± 0.12
83.0 ± 0.13
83.05 ± 0.12
81.0 ± 0.11
0.82
1.21
1.80
2.0
2.05
–
99.8 ± 0.10
98.6 ± 0.26
97.5 ± 0.42
85.3 ± 0.76
80.1 ± 0.81
100
Fig. 2. Effect of retention time on enzyme pre-treatment of pulp.
2.5 units (Fig. 2). In a study by Fillat and Roncero, (2009), laccase
dose of 20 IUg 1 odp caused maximum improvement in pulp
brightness after 960 min.
3.3.3. Bleaching studies of xylanase and LMS treated pulp for reduced
chlorine dioxide demand
Under the standard conditions of ECF bleaching [D0E(p)D1D2],
the brightness of pulp obtained was 81.0% ISO. However, it was
possible to obtain the same brightness of pulp pre-bleached with
xylanase using 15% lesser chlorine dioxide in the D0 stage
(Fig. 3). While, LMS treated pulps required 25% lesser chlorine
dioxide in the D0 stage to achieve same pulp brightness as that
of untreated pulp (Fig. 3). The overall effect of xylanase in bleaching is due to the disruptive action of xylanase on xylan chain, interrupting the lignin-carbohydrate bonds and thus enhancing
removal of lignin in the subsequent chemical bleaching stages
and thereby, reducing the requirement of chlorine dioxide used
for the same purpose (Kapoor et al., 2007). While, the bleaching
effect of LMS can be explained by the oxidative action of the
enzyme mediator system on lignin content of the pulp, facilitating
its easier extraction and thereby, reducing the requirement of chlorine dioxide in the subsequent chemical bleaching stages (Bajpai,
2012). Interestingly, sequential treatment of pulp with xylanase
followed by LMS (X + LMS), saved 35% chlorine dioxide in the D0
stage of ECF bleaching to achieve targeted brightness of 81% ISO
(Fig. 3). This is because xylanase eliminates xylan present on the
Fig. 3. Bleaching of enzyme treated and untreated pulps for reduced chlorine
dioxide demand.
fiber surface, thereby favoring enzyme and/or chemical access to
previously inaccessible lignin (Martin-Sampedro et al., 2012).
The results obtained in the present study are better with those
reported in literature. Lin et al. (2013) reported reduction in chlorine consumption by 10% for bleaching of xylanase treated pulps to
achieve similar strength and optical properties of pulp as that of
untreated pulp. Ravalason et al. (2012) achieved 19% ClO2 savings
for bleaching of LMS treated pulp to obtain same targeted pulp
brightness as of the control pulp. On the other hand, Valls et al.
(2010b) studied the effect of using LMS treatment of pulp after
xylanase stage and observed improvement in pulp brightness in
the sequential treatment of pulp than with individual treatments.
Similarly, Martin-Sampedro et al. (2012) showed better colorimetric properties of pulp given sequential xylanase and laccase
pre-treatment than with the pulp given individual treatments.
3.4. Enzymatic bleaching of Eucalyptus kraft pulp at pilot scale
During the pilot plant trials of pulp bleaching, the enzymatically
(X + LMS) pre-treated and untreated pulps were compared on the
basis of their optical properties, fiber strength and environmentally
benign nature. It was observed that although the enzyme
prebleached pulp was treated with 35% lesser chlorine dioxide,
no significant difference in the brightness of the pulp was observed
(Table 5). Interestingly, the Post color number (PC No.) was
Table 4
Effect of enzyme dose on LMS pre-treatment of pulp.
Laccase dose (IUg
40.0
50.0
60.0
70.0
80.0
90.0
100.0
Control
1
odp)
Brightness (% ISO)
Brightness improvement unit
Pulp yield (%)
81.80 ± 0.11
82.93 ± 0.14
83.50 ± 0.12
83.54 ± 0.09
83.58 ± 0.10
83.61 ± 0.12
83.63 ± 0.13
81.00 ± 0.13
0.80
1.93
2.50
2.54
2.58
2.61
2.63
–
99.6 ± 0.21
99.4 ± 0.24
99.0 ± 0.36
98.5 ± 0.35
98.2 ± 0.38
98.0 ± 0.40
98.0 ± 0.52
100
A. Sharma et al. / Bioresource Technology 169 (2014) 96–102
Table 5
Optical and strength properties of enzyme treated and untreated pulps at pilot scale.
Particular
Control pulp
X + LMS treated pulp
Brightness (% ISO)
Burst index (kPa m2g 1)
Tensile index (Nm g 1)
Tear index (Nm m2g 1)
PC No.
81.08 ± 0.21
6.21 ± 0.10
88.0 ± 1.23
7.08 ± 0.31
3.01 ± 0.21
81.11 ± 0.18
6.16 ± 0.09
87.21 ± 1.81
8.10 ± 0.22
1.50 ± 0.17
Table 6
Characterization of bleach effluents of enzyme treated and untreated pulps at pilot
scale.
Parameter
BOD
COD
AOX
AOX
(Kg tp 1)
(Kg tp 1)
(Kg tp 1)
(% reduction)
Control
X + LMS treated pulp
28.96 ± 0.33
39.56 ± 0.31
0.61 ± 0.006
–
31.30 ± 0.36
58.12 ± 0.25
0.40 ± 0.003
34.42
reduced by 50% in the enzyme pre-bleached pulp (Table 5). The
reduction in PC No. can be explained by the fact that xylanase
increases access of the LMS to hexauronic acids, thereby facilitating
their removal (Valls et al., 2010b). Hexauronic acids are formed
during alkaline cooking of wood pulp and cause brightness reversion in pulps thereby, increasing PC No. and consumption of
bleaching chemicals (Valls et al., 2010a). Thus, the paper made
from pulp with lower PC No. will be more stable in terms of brightness. Similarly, the fiber properties of enzyme prebleached pulp
were at par with the control with higher tear index (Table 5).
The tear index of enzyme treated pulp was increased by 15.71%
than that of the untreated pulp (Table 5). Moldes and Vidal,
(2008) also showed 15% higher tear index in LMS treated pulp.
Lin et al. (2013) reported similar mechanical properties of pulp
after xylanase treatment.
While, most importantly, the reduction in chlorine dioxide
demand in the enzymatic pulp bleaching lowered the concentration of AOX by 34% in the final bleach effluents (Table 6). This is
because the proportion of chlorine atoms applied during bleaching
ends up as AOX in bleach effluents (Bajpai, 2012). The lowering of
AOX levels in the effluents will help the mills to meet out the discharge norms set by environmental protection agencies as well as
to address the public concern over the release of toxic organochlorine compounds in water bodies. To the best of our knowledge,
no research on effluents from sequential enzymatic treatment
(X + LMS) of pulp with respect to AOX reduction has been conducted previously. Further, as these AOX compounds are toxic to
the growth of aquatic aerobic micro-organisms (Chaparro et al.,
2010), decrease in their levels also increased the biodegradability
of organic matter present in the effluents. As a result, increased
BOD levels (13.98%) are obtained in effluents from enzyme treated
pulps (Table 6). While, the higher chemical oxygen demand (COD)
values (26.39%) in effluents generated from enzyme treated pulps
(Table 6) is due to the release of products by oxidation of lignin
by LMS and hydrolysis of xylan by xylanase. Monje et al. (2010)
reported increased COD and BOD values in bleach effluents
generated after LMS treatment. While, Singh et al. (2010) observed
increased COD values in effluents obtained from xylanase treated
pulp.
4. Conclusion
A sequential treatment of xylanase and laccase for pulp bleaching was found better than the individual treatments in terms of
chlorine dioxide savings and eventually reduced AOX levels in
101
the final bleach effluents along with improved physical properties
of paper. Moreover, cost-effective production of xylanase and
laccase using a recent approach (IVED) to economize enzyme
production process is of commercial significance and thus paving
way for adoption of enzyme based technologies in pulp bleaching
by paper mills.
Acknowledgements
All the authors acknowledge financial support from Department
of Biotechnology (DBT), Ministry of Science and Technology, Govt.
of India and University of Delhi South Campus, New Delhi and
CPPRI, Saharanpur for providing necessary research facilities.
Author AS acknowledges Council of Scientific and Industrial
Research (CSIR), Human Resource Development group, Govt. of
India for providing senior research fellowship (SRF) while, RG
would like to acknowledge Department of Science and Technology
(DST), Govt. of India for financial support. Authors are thankful to
Star paper mill, Saharanpur for providing pulp samples and Ms.
Urvashi Kuhad, Research scholar, Department of Modern Indian
Languages and Comparative Literature, University of Delhi for
editing the manuscript.
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