Effect of Cellulases and Xylanases on Refining Process and Kraft Pulp Properties
Effect of Cellulases and Xylanases on Refining Process and Kraft Pulp Properties
Kamila Przybysz Buzała 0 1
Piotr Przybysz 0 1
Halina Kalinowska 1
Małgorzata Derkowska 0 1
0 Institute of Papermaking and Printing Technology, Lodz University of Technology , Wolczanska str. 223, 90-924, Lodz , Poland , 2 Institute of Technical Biochemistry, Lodz University of Technology , Stefanowskiego str. 4/10, 90-924, Lodz , Poland
1 Editor: Shihui Yang, National Renewable Energy Laboratory , UNITED STATES
Samples of bleached kraft pine cellulosic pulp, either treated with an enzyme preparation (a Thermomyces lanuginosus xylanase, an Aspergillus sp. cellulase, and a multienzyme preparation NS-22086 containing both these activities) or untreated, were refined in a laboratory PFI mill. The treatment with cellulases contained in the last two preparations significantly improved the pulp's susceptibility to refining (the target freeness value of 30°SR was achieved in a significantly shorter time), increased water retention value (WRV) and fines contents while the weighted average fiber length was significantly reduced. These changes of pulp parameters caused deterioration of paper strength properties. The treatment with the xylanase, which partially hydrolyzed xylan, small amounts of which are associated with cellulose fibers, only slightly loosened the structure of fibers. These subtle changes positively affected the susceptibility of the pulp to refining (refining energy was significantly reduced) and improved the static strength properties of paper. Thus, the treatment of kraft pulps with xylanases may lead to substantial savings of refining energy without negative effects on paper characteristics.
Funding: The work was financially supported by
project LIDER/042/407/L-4/12/NCBR/2013 funded by
National Centre for Research and Development
(NCBiR, Poland). Funder URL: www.ncbr.gov.pl. The
funder had no role in study design, data collection
and analysis, decision to publish, or preparation of
Competing Interests: The authors have declared
that no competing interests exist.
Paper products have been increasingly used for writing and printing as well as for packaging,
sanitary-hygiene and special purposes because of the high functionality, relatively low prices,
biodegradability and many other attractive properties. Furthermore, paper is produced from
renewable biomass feedstocks and waste paper may be either recycled or combusted to
generate energy. These advantages cause that production and usage of various sorts of paper
have been growing. Only in the first decade of XXI century the global use of paper products
increased from 320 milion(mln) tons to 395 milion(mln) tons [
Refining of pulps, consisting primarily of cellulose fibers, is a key step of paper making and
relies on mechanical treatment of their water suspensions, which increases the fibrillation
grade and outer surface area of cellulose fibers that enhances fiber swelling and flexibility as
well as bonding interactions between the fibers. The process name was coined several centuries
ago and does not precisely reflect its character since it suggests reduction of the cellulose fibers
length. Notwithstanding the possible misinterpretation, the term refining is commonly used by
pulp and paper technologists and in literature publications [
]. Refining of paper pulps is
conducted using either conical or disc refiners in order to loosen the structure of cellulose fibers
and this way to produce paper with target properties, meeting end product quality demands.
The principal drawback of currently applied refining technologies is the high unit energy
consumption, usually ranging from 150 to 500 kWh/ton paper and accounting for 30 to 50% of the
total energy used for paper making [
]. For the mean unit energy consumption of 300 kWh
per ton and paper production of 400 mln tons/year, the global refining energy reaches 120
TWh, which is equivalent to incineration of around 24 mln tons of black coal per year.
Despite numerous efforts to improve the energy efficiency of conventional disc and conical
refiners, this parameter has not been reduced enough yet. The energy efficiency of these
refiners ranges from few to over 15% and the majority of refining energy is dissipated as heat,
resulting in pulp warming [
]. Since also alternative refining methods (ultrasounds, cavitation,
steam explosion, freezing etc.) have not given significant energy savings, chemical (e.g.
treatment with bases) and enzymatic methods were proposed to loosen the structure of cellulose
fibers before processing in a refiner. Enzymatic treatment of cellulose fibers enables
degradation of either all types of associated hemicelluloses or only some of them (e.g. xylans). Removal
of these polysaccharides facilitates penetration of water molecules into spaces within cellulose
fibers that may break a part of hydrogen bonds connecting cellulose chains. This in turn
loosens the 3D structure of fibers, which become more flexible and form paper with more compact
structure and high strength . The main objective of application of enzymatic preparations
for pulps treatment is to reduce refining energy and make the process not only less expensive
but also more environmentally friendly [
]. However, enzymatic treatment must not lead to
deterioration of paper properties and therefore selection of suitable enzyme preparations is of
key importance. Other criteria of this selection are enzyme’s cost (must not rise the overall
process costs) and purity since the presence of additional enzymatic activities (e.g. cellulases apart
from xylanases) may negatively affect pulp and paper properties.
In this work, the effect of enzymatic treatment of bleached pine kraft pulp by three different
commercial enzyme preparations (a cellulase, a xylanase and a mixture of cellulases and
xylanases) on the pulp susceptibility to refining and paper quality was studied. The relationship
between the type of glycoside hydrolase (cellulase and/or xylanase) and refining energy
consumption required to achieve pulp freeness value of 30°SR as well as most important pulp and
paper parameters were evaluated.
Materials and Methods
Pulp and its characterization
A bleached pine kraft pulp was kindly donated by one of Polish paper mills. Typically, this
pulp is used for production of high quality graphic papers. The pulp had a form of air-dried
sheets. The following properties of the unbeaten pulp were determined:
dry matter content– 96%, the standard deviation of results of four measurements was 0.1%,
water retention value (WRV)– 93%, (according to the ISO 23714:2014 standard), the
standard deviation of results of four measurements was 0.8%,
weighted average fiber length– 1.76 mm (according to the ISO 16065–2:2014 standard), the
measurements were performed in triplicate and the standard deviation was 0.01 mm,
polymerization degree– 1250 (according to the ISO 5351:2012 standard), the measurements
were performed in triplicate and the standard deviation was 40.
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Enzymes and chemicals
A commercial multienzyme (a mixture of cellulases, xylanases and other glycoside hydrolases)
preparation NS-22086 was kindly donated by Novozymes A/S (Denmark). Preparations of
cellulase from Aspergillus sp. and xylanase from Thermomyces lanuginosus were purchased from
Sigma-Aldrich (USA). All other chemicals were analytical grade and procured from either
Sigma-Aldrich (USA) or POCh (Poland).
Activities of cellulases and xylanases were assayed by the 3,5-dinitrosalicylic acid (DNS)
] at pH 5.0 and 50°C for 0.5% carboxymethylcellulose (CMC) and 0.5% birch xylan,
respectively (reaction time of 5 min). Activities of both the glycosidases were expressed as
micromoles of reducing sugars released from the polysaccharide substrates in 1 min (U). The
filter paper activity was determined at pH 5.0 and 50°C according to Adney and Baker [
expressed in FPU.
Enzymatic treatments of pulps
Samples of the pulp (22.5 g dry weight) were soaked in water for 24 h before their treatment
with enzymes. Then these samples were disintegrated in a laboratory propeller pulp
disintegrator (type R1 from Labormeks, Poland) according to the standard ISO 5263–1:2006 and treated
with the three listed above enzyme preparations, used separately. After addition of the enzyme
preparation to the pulp, the mixture was incubated in a water bath (at 50°C) with shaking (60
rpm) for either 30 minutes (for the Aspergillus sp. cellulase and preparation NS 22086) or 60
minutes (for the T. lanuginosus xylanase). Then the enzymes were inactivated by 10 minute
incubation of enzyme-treated pulp in a boiling water bath and the pulp was cooled and filtered
through a mesh sieve no. 200. The filtrates were analyzed for glucose concentration using a
commercial GOD-POD enzymatic analytical kit (Biomaxima, Poland), to determine the extent
of enzymatic pulp degradation.
Pulp refining and freeness measurements
After inactivation of the enzymes, the pulps were refined using a PFI laboratory mill at 3.4 kG
and 1440 rpm, according to the standard ISO 5264–2:2011. The process was accomplished
when the pulp freeness value was above 30°SR (its duration was measured with precision of 1
second to calculate the number of mill revolutions). Then, samples of the refined pulp were
transferred from the mill to a mixer.
Pulp quality analysis
The refined pulps were placed in the mixer and the concentration of their suspensions was
adjusted to 0.25%. The thoroughly mixed pulp samples were analyzed for the: freeness
(according to the standard ISO 5267–1:2002), weighted average fiber length and WRV (according to
standards ISO 16065–2:2014 and ISO 23714:2014, respectively) and then were used to produce
handsheets of paper.
Changes in the appearance of fibers caused by the action of cellulolytic and xylanolytic
enzymes were observed using an electron scanning microscope S-4700 Hitachi SEM/EDS at
3 / 14
Paper production and properties
Handsheets of paper were produced with the use of a standard laboratory Rapid-Köthen class
sheet former, according to the standard ISO 5269–2:2007.
The following paper properties were measured after conditioning of the handsheets in
standard conditions, in compliance with ISO 187:1990 (at air relative humidity φ of 50% and
temperature of 23°C):
grammage—according to ISO 536:2012, only paper sheets of grammage between 74 and 76
g/m2 were accepted for further investigation,
bulk density—according to PN-EN ISO 534:2012, twelve measurements were made,
breaking length—according to PN-EN ISO 1924–2:2010, the twelve measurements were
tear resistance—according to PN-EN ISO 1974:2012, the twelve measurements were made.
The complete information related to the experimental data and standard deviation is
enclosed in data set available at datadryad.org web page.
The applied enzymatic preparations showed activities of both cellulases and xylanases
(Table 1). Even the preparation of T. lanuginosus xylanase displayed the cellulolytic activity for
CMC and filter paper. The multienzyme preparation NS-22086 was characterized by the
highest activity of cellulases per mass unit (80.58 U/ml for CMC and 112.12 FPU/ml). Its
xylanolytic activity was also relatively high (192.48 U/ml for birch xylan). The action of the latter
preparation on various cellulosic pulps and lignocellulosic materials was characterized in our
former studies [
]. HPLC analyses of hydrolysates produced using this preparation
showed that it contains endo-1,4-glucanase, cellobiohydrolase, beta-glucosidase, endo- and
exo-type xylanases and other hemicellulases as well as pectinases.
The optimal dose of enzymatic preparation and time of pretreatment were selected based
on the results of preliminary experiments that are presented in Table 2. The optimal dose of
enzyme was selected taking into consideration the maximal tear resistance of paper produced
from the pretreated pulp.
Based on these results, the dose of NS-22086 preparation was set at 2.1 μl/1 g d.w. pulp to
minimize the extent of cellulose hydrolysis. This dose corresponded to 0.17 U of cellulase
activity for CMC (0.24 FPU) and 0.40 U of xylanase activity per 1 g d.w. pulp. Also doses of the two
other enzyme preparations, which were characterized by the higher purity (and price) than the
NS-22086 as well as different ratios of cellulase: xylanase activities were relatively low to
minimize cellulose hydrolysis.
The doses of the three enzyme preparations added to the pulp were as follows:
4 / 14
Thus the dose of cellulase from Aspergillus sp. corresponded to 24.9 x 10−3 U of cellulase
activity for CMC (20.6 x 10−3 FPU) and 49.1 x 10−3 U of xylanase activity per 1 g d.w. pulp,
which was around 10-fold lower compared to the activities contained in the dose of NS-22086
preparation. The dose of xylanase from T. lanuginosus corresponded to 0.76 U of cellulase
activity for CMC (2.44 FPU) and 149.2 U of xylanase activity per 1 g d.w. pulp. The latter
activity was around 47-fold higher compared to the activity of xylanases contained in the dose of
NS-22086 preparation while the activities of cellulases were only around 1.8 (for CMC) and
0.8-fold (for filter paper) higher.
One of the products of pulp treatment by the three enzyme preparations was glucose. Its
concentrations in the filtrates of enzyme-treated pulp were relatively low, of 0.016, 0.061 and
0.182 mg/ml for the Aspergillus sp. cellulase, T. lanuginosus xylanase and NS-22086
preparation, respectively. Thus even the xylanase preparation released some glucose from the pulp.
Impact of enzymatic and mechanical treatments on fibre morphology
Pulp freeness is the relatively easily measurable parameter, characterizing refining level and
negatively correlated with the pulp dewatering ability, which decreases with the increase in
freeness. Because the yield of paper production is diminished when dewatering ability is too
low, in industrial conditions freeness values should not be higher than 30°SR.
The impact of enzymatic preparations used for pulp treatment on the dynamics of freeness
growth during refining is presented in Fig 1A. These results provide evidence that the
controlled pulp digestion with the three enzymatic preparations significantly reduced the
number of revolutions of the PFI mill, which were necessary to achieve the freeness of 30°SR.
This means that the target refining level was achieved in a shorter time. Thus, modification
of fiber characteristics by these enzyme preparations significantly reduced refining energy
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Fig 1. Effects of enzyme treatment and PFI revolutions number on pulp freeness (a), water retention
value (b) and fines content (c).
6 / 14
The numbers of mill revolutions required to achieve the pulp freeness value of 30°SR are
compared in Table 3. The effect of enzymatic pulp treatment with Aspergillus sp. cellulase on
pulp freeness was stronger than in case of NS-22086 preparation and T. lanuginosus xylanase
(the weakest impact despite the highest cellulolytic and xylanolytic activities used per 1 g d.w.
pulp). However, apart from the refined pulp freeness also other parameters such as WRV,
average fiber length and fines content decide of paper properties.
The data shown in Fig 1B demonstrate that the treatments with the Aspergillus sp. cellulase
and preparation NS-22086, which contains enzymes hydrolyzing cellulose and xylan, caused
that the swelling ability (expressed as WRV) grew significantly faster during refining,
compared to the untreated pulp. The influence of T. lanuginosus xylanase on the growth of this
parameter was not so strong. Also the level of fines in the refined pulp was only slightly affected
by this enzyme, and interestingly, at the end of pulp refining the percentage of fines was lower
the pulp processed with the xylanase than in the untreated pulp (12.8% d.w. versus 13.4% d.w.,
The modification of fibers structure by Aspergillus sp. cellulase caused that the percentage of
fines was above 70% after around 2000 revolutions of the PFI mill and the resulting refined
pulp was useless for paper production. At the freeness of 30°SR the pulp treated with the latter
preparation contained as much as 54.5% fines while that treated with NS-22086 preparation
contained around twice more fines than the untreated pulp (23.8 versus 13.4% d.w.).
The positive effect of pulp treatment with Aspergillus sp. cellulase and NS-22086
preparation before refining on the growth of pulp freeness is in compliance with results reported
among others by Cui et al. [
]. Based on experimental results and literature data these authors
concluded that the impact of various enzyme preparations on pulp and paper parameters
depended on a balance between activities of cellulases and hemicellulases. Our study revealed
that pulp treatment with the three enzyme preparations caused the rise in WRV of the pulp
during refining and that the impact of T. lanuginosus xylanase was weaker than the influence
of the two cellulases. However, at the end of refining (at the freeness of 30°SR) this parameter
was the highest for the untreated pulp (Table 4) while in the study of Liu et al. [
T. lanuginosus xylanase NS-22086
Aspergillus sp. cellulase
7 / 14
Fig 2. The effect of enzyme preparations and number of PFI mill revolutions on the weighted average
increased significantly due to the pulp treatment with xylanase before refining. However, also
these authors observed the decrease in fines content (in our study their percentage decreased
from 13.4 to 12.8%).
Another parameter characterizing the refining outcomes is the weighted average length of
fibers, which for the untreated pulp was decreased from 1.76 to 1.66 mm after 5200 revolutions
of PFI mill (Fig 2). The decrease in the length of fibers after pulp treatment with the xylanase
from T. lanuginosus was almost the same (from 1.76 to 1.65 mm) while the Aspergillus sp.
cellulase and multienzyme preparation NS-22086 caused the significant decrease in this
parameter (to 1.04 and 1.34 mm at the end of refining, respectively).
These results are consistent with findings of Znidarsic-Plazl et al. [
] who observed that
pulp treatment with multienzyme preparations caused the significant decrease in the average
Effect of enzyme treatments on physical properties of handsheets
The presented above changes in the pulp characteristics caused by partial enzymatic
degradation of its components were found to affect properties of paper derived from these pulps. Static
paper strength properties are characterized by breaking length. The data presented in Fig 3A
demonstrate that pulp treatments with T. lanuginosus xylanase and NS-22086 preparation
increased the maximum value of this parameter from around 8000 m (for the untreated pulp)
to around 9700 m and 8200 m, respectively while the treatment with cellulase from Aspergillus
sp. significantly decreased its value to 6500 m.
The tear resistance of paper characterizes its dynamic strength properties. The results
shown in Fig 3B prove that this parameter was reduced by the treatment with each of the three
enzymatic preparations (by around 8% for the T. lanuginosus xylanase, and by around 60% for
the Aspergillus sp. cellulase and NS-22086 preparation).
The effect of pulp treatment with cellulases on values of breaking length and tear resistance
of paper produced from these pulps, observed in our study, is consistent with results reported
8 / 14
Fig 3. Effects of enzyme treatment and PFI mill revolutions number on breaking length (a) and tear
resistance (b) of paper.
by Garcia-Ubasart et al. [
] who also observed that the breaking length was positively
correlated with the refining level.
The ratio of tear resistance to breaking length is the basic parameter characterizing the
strength properties of paper. The data presented in Fig 4 provide evidence that pulp treatment
with the T. lanuginosus xylanase significantly increased this parameter while the two
cellulolytic preparations reduced it, which was also reported by Garcia-Ubasart et al. [
The impact of the three enzyme preparations on pulp and paper parameters is presented in
Table 4 (the refined pulp freeness was 30°SR in each case).
The data collected in Table 4 demonstrate that pulp processing with the tested cellulases
and xylanases significantly reduced the time (the number of mill revolutions) during which
pulp freeness reached 30°SR, however the action of cellulases on the fibers caused significant
deterioration of paper strength properties (reflected by strongly reduced tear resistance and in
case of Aspergillus sp. cellulase—also breaking length). These properties were not worsened
after pulp treatment with the xylanase preparation (despite 8% decrease in tear resistance).
9 / 14
Fig 4. The dependence of tear resistance: breaking length ratio on enzymatic pulp treatment.
The impact of pulp enzymatic treatment on refining—microscopic
The action of the three enzyme preparations on the pulp was also observed by means of
electron microscopy and the microscopic images of enzyme treated and untreated fibers are
presented in Fig 5
These images demonstrate that the two preparations of cellulases (from Aspergillus sp. and
NS-22086) significantly modified the structure of fibers. The damage of cellulose fibers by
cellulases was also reported by Garcia-Ubasart et al. [
]. The T. lanuginosus xylanase only
slightly modified the surface of the pulp fibers, which was consistent with images presented by
Liu et al. [
Enzymatic preparations, particularly those displaying the high cellulolytic and xylanolytic
activities, have been increasingly used for bioconversion of lignocellulosic materials to biofuels
and numerous valuable chemicals as well as in papermaking. In the latter case, enzymes are
used primarily for pulp bleaching, deinking of waste paper and waste water treatment [
Enzymes were also found to reduce the pulp refining energy and improve paper strength
properties. Studies of many authors focus on enzymatic pulp treatment performed to reduce
refining energy and make paper manufacturing more environmentally friendly [
]. In this work,
three different preparations such as T. lanuginosus xylanase, Aspergillus sp. cellulase and the
multienzyme preparation NS-22086 (containing both cellulases and xylanases) were tested in
terms of their influence on pulp and paper properties and potential to reduce energy of pulp
refining. The most appropriate of them, in terms of refining energy reduction and maintenance
of paper strength properties, was the xylanase preparation.
The effect of xylanases on pulp refining was also studied by Liu et al. [
], Dickson et al.
] and Batalham et al. [
]. Differences between the published experimental results might be
caused by different enzyme: substrate ratios as well as pH and temperature conditions. Our
10 / 14
Fig 5. Microscopic images (electron scanning microscope, 50 μm bar enables estimation of the
fibres’ size) illustrating the effect of enzymatic pulp treatment on the appearance of fibres before and
results are to some extent consistent with findings of Liu et al. [
] who observed increased
breaking length and tear resistance of paper while our experiments showed that the first
parameter was increased by 20% while the second was reduced by 8% due to the pulp treatment
with the xylanase. On the contrary, Batalha et al. [
] found that values of both these
parameters were decreased in consequence of pulp treatment with xylanase.
Action of the multienzyme preparation NS-22086, showing xylanolytic and cellulolytic
activities caused that the time of refining was longer than in case of Aspergillus sp. cellulase and
11 / 14
shorter than after the treatment with T. lanuginosus xylanase. The paper strength properties
were better after pulp treatment with NS-22086 preparation than after the treatment with
cellulase and worse than after the treatment with xylanase. Also Ko et al. [
], Zhang et al. [
Kim et al. [
] and Lecourt et al. [
] observed that cellulases damaged the fibers, which caused
deterioration of paper strength properties, particularly the tear resistance.
Cui et al. [
] postulated that none of industrial enzyme preparations enables both to reduce
refining time and maintain or improve paper properties. The same conclusions were presented
by Garcia et al. [
], Gil et al. [
], Kim et al. [
] and Oksanen et al. [
This study showed that pulps should be treated with preparations characterized by the high
xylanolytic activity since the xylanase from T. lanuginosus reduced the refining time required
to reach freeness of 30°SR by around 20% and increased the paper breaking length by around
20%. The only disadvantage caused by the treatment with this enzyme was the 8% decrease in
Unfortunately, the majority of industrial preparations of xylanases contain also cellulolytic
enzymes and pure xylanases are relatively expensive. This study provides evidence that the
availability of purified xylanases is of key importance for paper making because pulp treatment
with these enzymes may give rise to refining energy savings and improved paper strength
The xylanase from T. lanuginosus outperformed two preparations of cellulases (Aspergillus sp.
cellulase and the multienzyme preparation NS-22086, containing also xylanases) when used
for kraft pine pulp treatment before refining. All three enzyme preparations significantly
reduced the refining energy because the freeness of 30°SR was achieved in a significantly
shorter time compared to the untreated pulp. However, partial cellulose degradation by the
two preparations of cellulases increased fines contents and reduced the weighted average fiber
length that caused deterioration of paper strength properties. The xylanase from T. lanuginosus
partially removed xylan, associated with cellulose fibers, and only slightly loosened their
structure and reduced the weighted average fiber length. The removal of xylan not only increased
the susceptibility of the pulp to refining but also improved the static strength properties of
paper. Thus, the treatment of kraft pulps with robust, cellulase-free preparations of xylanases
may not only reduce refining energy but also facilitate production of paper with desirable
The work was financially supported by project LIDER/042/407/L-4/12/NCBR/2013 funded by
National Centre for Research and Development (NCBiR, Poland).
Conceptualization: PP KPB HK.
Data curation: PP.
Formal analysis: PP.
Funding acquisition: PP.
Investigation: KPB MD.
Methodology: KPB HK.
12 / 14
Project administration: PP.
Supervision: PP HK.
Validation: PP HK KPB.
Writing – original draft: KPB.
Writing – review & editing: HK.
13 / 14
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