Enzymatic treatments of paper surface using laccase,
hydrophobic compounds and lignin
Cusola, Oriol: Ph.D Candidate, UPC, Catalunya, Spain [email protected]
Roncero, M.Blanca: Full Professor, UPC, Catalunya, Spain [email protected]
Garcia-Ubasart, Jordi: Ph.D Candidate, UPC, Catalunya, Spain [email protected]
Valls, Cristina: Full Professor, UPC, Catalunya, Spain [email protected]
Abstract
This study demonstrates the potential of applying surface enzymatic treatments on
finished paper sheets to improve their physical characteristics. Research studies have recently
reported that is possible to internally size paper using enzymatic treatments, applied in mass.
In the present work an innovative method for achieving enzymatic surface sizing on finished
paper sheets is presented. A commercial filter paper and handmade eucalyptus sheets were
surface sized with laccase derived from Trametes Villosa, Lauryl Gallate (LG) as hydrophobic
compound, and soluble lignin. Moreover, this study shows the positive influence of sulfonated
lignin since it acts as a dispersant, improving the homogeneous distribution of the LG along the
paper sheet.
Keywords: Laccase, surface sizing, hydrophobicity, cellulosic fibers, Lauryl Gallate, dispersant,
lignin.
Introduction
The absorption of liquids into the structure of paper is a key factor for the end-use
performance of the paper products (e.g. paper cups, paper bags, packaging boxes or liquid
containers) and for the runnability of papermaking processes as well, e.g. in size press or
printing (1, 2). Papermakers carry out processes to reduce the rate of liquid absorption into the
paper structure by treating the stock with hydrophobic substances (3). In papermaking process,
cellulosic fibers are placed in an aqueous suspension before forming the paper sheet. At this
very moment several additives are added to the suspension to confer extra properties to paper.
Enzymes appear as an effective alternative to chemicals used traditionally in the paper
industry, since they need milder treatment conditions, produce less damages to the fibers, are
derived from renewable resources, and are biodegradable (4). They act as biological catalysts
in biochemical reactions of all living organisms, but meanwhile have also been established as
reliable and convenient processing aids in many technical industries. As all catalysts, a
biochemical catalyst like an enzyme is defined as a reaction partner who increases the rate or
velocity of a chemical reaction without itself being changed in the overall process (5).
Laccases are enzymes which belong to the so-called blue-cooper family of oxidases.
Research studies have demonstrated that laccases, and LMS (Laccase-mediator system) can
be used in delignification (6) and bleaching processes (7, 8) in pulp industry. Likewise, other
studies have shown the possibility functionalizing fibers using the LMS system (9, 10), or affect
the chemical and refining properties of the pulps (11).
Capability to carry out enzymatic treatments (traditionally performed on the paper pulp)
on the surface of finished paper sheets is evaluated in this work. Moreover, the present study
tests the performance of sulfonated lignin as a dispersant (12, 13), in order to improve the
distribution of the Lauryl Gallate (LG) along the paper sheet. The study highlights the possibility
of applying enzymatic treatments in elsewhere of the paper mill.
Materials & Methods
Paper, enzyme and chemicals
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5th International Colloquium on Eucalyptus Pulp, May 9-12, 2011. Porto Seguro, Bahia, Brazil.
Filter paper sheets (73 g/m ) were purchased from FILTERLAB (Sant Pere de
Riudebitlles, Barcelona – Spain). The enzyme used in this work was a laccase from Trametes
villosa with an activity of 588U/mL, supplied by Novozymes. Lauryl-Gallate compound was used
in the laccase treatments as hydrophobic compound. Soluble sulfonated kraft lignin
(Polymerisation degree 401) was supplied by Borreegard, and used as received.
2
Paper and laccase-mediator treatments
Paper filter sheets were cutted into 4cm diameter circular sheets. The enzymatic
treatments were performed in an agitated bath, using 250ml beakers. The treatment conditions
were those reported on previous studies by Garcia-Ubasart et.al (14) by means of treating the
pulp stock. Treatments were performed at 3% consistency in 50mM sodium tartrate buffered at
1
ph 4, 4%(w/w) LG, 4%(w/w) lignin, and 40U/g (odp ) laccase. The reaction was stopped by
washing the beakers with cold water, removing the paper sheets from the beakers, and
extending them onto blotting paper. A control treatment was performed at the same conditions
of the enzymatic treatment but without the presence of enzyme. The results were compared to
initial paper sheets.
UV-VIS spectroscopy
The oxidation of LG, lignin, and LG+lignin compounds caused by laccase was analyzed
by UV-visible spectroscopy, using a Thermo Scientific Evolution 600 spectrophotometer. The
1ml reference quartz cubette was filled with 50mM sodium tartrate buffered at ph 4, and 40U/g
1
(odp ) laccase. Effluents resulting from treatments were placed in the 1mL quartz cubette
destined to measure. Residual laccase activity was also measured. One activity unit was
defined as the amount of laccase transforming 1 µmol/min ABTS to its cation radical (ε436 nm =
-1
-1
29,300 M cm ) in 0.1 M sodium acetate buffer (pH 5) at 25ºC.
Water drop tests
Water drop tests (WDT) were performed for each treated paper specimen according to
tappi standard T835 om-08. Before performing the WDT, sheets were conditioned according to
ISO 187. Fifteen measures were performed for each treated paper specimen.
Results & Discussion
Oxidation of LG and lignin caused by laccase was monitored via the absorbance changes
on the spectrum at different times (5’, 10’, 15’, 30’, 45’, 60’ and 75’). The oxidation measures
were also performed with the presence/absence of paper sheets for comparison. Figure 1
shows the UV-visible spectra of LG and lignin molecules alone before enzymatic treatments; LG
presents two absorbance peaks at 220 and 275nm, while lignin presents two absorbance peaks
located at 210 and 280nm. LG spectra experienced significant changes as a result of the
enzymatic treatments; only one LG peak at 240nm was observed after reaction, and reaction
kinetics showed significant absorbance variations for all tested reaction times. Lignin retained
the original peaks and shape after treatments, and very slight changes in absorbance levels
were observed on lignin after reaction. The presence of cellulose sheets on the enzymatic
reaction seemed to preserve lignin from oxidation and caused variations on the shape of UVvisible spectra of the LG. An idea on the coupling of the LG to the cellulosic fibers was reported
by Cadena et al. (10).
As a result of the enzymatic treatments with the LG alone, the filter paper and eucalyptus
sheets showed dark spots in certain regions due to the oxidation of the LG. In order to increase
the LG dispersion sulfonated lignin was added as natural surfactant to the treatment solution.
Figure 2 shows the difference between the treatments performed without and using sulfonated
lignin as surfactant; the addition of lignin improved the spreading of the LG and more
homogeneous sheets were obtained.
1
odp means oven died pulp
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5th International Colloquium on Eucalyptus Pulp, May 9-12, 2011. Porto Seguro, Bahia, Brazil.
2
1,8
Lauryl Gallate
1,6
Lignin
Abs.
1,4
1,2
1
0,8
0,6
0,4
0,2
600
550
500
450
400
350
300
250
200
0
nm
Figure 1. UV-visible spectra for LG (continuous line) and lignin (dashed line).
a
b
Figure 2. 20x images of filter paper sheets surface after enzymatic treatment without the
addition (a), and adding (b) sulfonated lignin to the treatment solution.
The evolution on the enzymatic activity corresponding to the oxidation of the LG and
lignin was monitored by measuring the residual enzymatic activity of effluents resulting from
each reaction time (5’, 10’, 15’, 30’, 45’, 60’ and 75’). The enzymatic activity decreased
gradually to 0 after 75 minutes of treatment with LG alone. On the contrary, when treatments
were performed by using lignin alone, activity of laccase was only reduced by half after 75
minutes. The residual enzymatic activity was also reduced by half after 75 minutes for the
treatments containing LG and lignin on the reaction solution. This means that lignin besides
acting as a LG dispersant, also acted as a preservative of the enzyme activity.
Hydrophobic behavior of treated sheets was studied by Tappi normalized WDT. WDT is a
simple method involving placing a drop of deionized water on the surface of the paper and
recording the time needed for complete absorption, which is signaled by vanishing of the
specular gloss of the water drop. Measures concerning hydrophobicity behavior were performed
on filter paper; the initial absorption time of non-treated filter paper sheets was 4±0,85s.
Treatment time was increased until a stabilization of the hydrophobic behavior was observed on
paper sheets. As it is shown in Table 1, slight but gradual increase on hydrophobicity is
observed after 1h of treatment. A sharp increase on the hydrophobic behavior is observed
between 3 and 4 hours. Absorption time reaches stabilization after 4 hours of treatment in a
level greater than 2300s (~40minutes for the absorption of the water drop).
Reaction time
Absorption time
minutes
hours
seconds
stdv.
0
5
10
15
30
45
60
75
90
105
120
135
150
240
390
1359
0,0
0,1
0,2
0,3
0,5
0,8
1,0
1,3
1,5
1,8
2,0
2,3
2,5
4,0
6,5
22,7
7
7
7
9
15
31
91
93
326
415
445
529
609
3006
3003
2486
2
1
1
2
1
6
13
18
59
55
76
98
73
561
626
150
Table 1. Evolution of the hydrophobic behavior of filter paper sheets with the increasing
treatment time.
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5th International Colloquium on Eucalyptus Pulp, May 9-12, 2011. Porto Seguro, Bahia, Brazil.
Conclusions
This study demonstrated the potential of applying surface enzymatic treatments on
finished paper sheets to improve their hydrophobic characteristics, with the positive influence of
sulfonated lignin acting as dispersing agent and enzymatic activity preserver. It was clearly
demonstrated the oxidation suffered by the LG due to the enzyme, and the slight oxidation
suffered by lignin. Furthermore, the study highlighted the high level of hydrophobic behavior
assessed when reaction time was optimized.
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5th International Colloquium on Eucalyptus Pulp, May 9-12, 2011. Porto Seguro, Bahia, Brazil.
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Acknowledgements
The authors are especially grateful to the integrated project from sixth framework program
BIORENEW (NMP2-CT-2006-026456), as well as FUNCICEL project (CTQ2009-12904) and
BIOFIBRECELL project (CTQ2010-20238-CO3-01) within the framework of the Spanish’s
MICINN.
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5th International Colloquium on Eucalyptus Pulp, May 9-12, 2011. Porto Seguro, Bahia, Brazil.