Note: Descriptions are shown in the official language in which they were submitted.
CA 02827951 2013-01-31
Wood Pulp Treatment
Field
The present invention relates to a treatment for mechanical wood pulp that
improves its characteristics during downstream processing.
Background of the Invention:
Wood pulps are generally produced through multistep processes. Initially, logs
can be subjected to grinding in which the logs are forced against a rotating
abrasive
stone which separates the fibers from the log and also the wood cell matrix.
In a refining
process, wood chips are fed between two metal discs, with at least one disc
rotating. In
both cases, essentially all of the constituents of wood are retained in the
pulp that is
eventually produced. Such pulp contains fiber bundles, fiber fragments and
whole
fibers. A lack of uniformity of pulp and constituents and the presence of
lignin in the pulp
give it certain desirable qualities, such as yield, paper bulk and opacity as
well as good
printability. The pulp also has less desirable properties for some paper
types, such as
low strength, relatively coarse surface and a lack of durability.
Chips to be refined can be destructured and impregnated with chemicals or
enzymes prior to further mechanical treatment. This can help increase pulp
quality or
reduce energy consumption. These methods create slightly different pulps and
also vary
with the species of wood, quality of the wood, processing conditions and the
amount of
energy applied. Various forms exist: thermomechanical pulping (TMP), refiner
pulping,
stone groundwood pulping, etc.
In TMP, steam is added to the chips being refined to facilitate pulping and
lower
electricity consumption. Steam is also produced during refining and heat
recovery
systems can help recoup some of the energy cost of the process. The electric
motors
used to operate these refiners require very large amounts of power. The TMP
process
generally involves several refining stages to produce a desirable pulp.
However, only a
small portion of the energy used in each refining stage is actually used to
separate and
develop the fibers. Screening is used after or between refining stages to
separate
adequately refined fibers from longer, coarser fibers. These tougher fibers
are sent to
-1 -
CA 02827951 2013-01-31
"rejects" refiners for further development. Depending on the quality of
refining, the
amount of rejects needing additional refining can be and usually is
significant.
Woody biomass used in these mechanical pulping processes contains cellulose,
hemicelluloses, lignin and extractives in varying amounts throughout the
ultrastructure
of its fibers. These various components act in conjunction to give these
substrates
mechanical strength and resistance to degradation. By selectively removing or
altering
certain components, it is possible to reduce the amount of energy required to
separate
and refine these fibers. The patent literature describes various approaches
using
different enzyme mixtures. For example US Patent Publication No. 2005/0000666,
of
Taylor et al., describes the use of mannanase and xylanase. Certain treatments
have
been found to significantly impact paper strength properties which have
limited their
applications. United States Patent No. 5,865,949, of Pere et al., describes a
process
using an enzyme mixture containing endo-8-glucanase (EG), a limited mannanase
and
cellobiohydrolase (CBH) activity which reduces the negative effects on paper
strength.
United States Patent No. 6,099,688, of Pere et at., describes the use of
isolated
cellobiohydrolase to increase the amount of relative amorphousness of the
cellulose
within the fibers. This process is said to cause even less damage to paper
properties.
Summary
The invention provides a method for preparing e.g., manufacturing a wood pulp.
The pulp is prepared by exposing a mechanical wood pulp to an enzymatic
solution
containing an endoglucanase (EG) and a cellbiohydrolase (CBH), the ratio of
enzymatic
activities of the EG:CBH being at least 3.
It has been found that it is possible to carry out the treatment for an amount
of
time that results in a reduction of energy consumption during subsequent
refining of the
exposed pulp in which the freeness of the pulp (CSF) is reduced by at least
10% in
comparison to the freeness of the same pulp which has not been exposed to the
enzymatic solution while at least maintaining the tensile strength of a
handsheet
produced from the subsequently refined pulp in comparison with a handsheet
produced
from the same pulp which has not been exposed to the enzymatic solution. By
maintaining tensile strength here is meant that the tensile index for the
handsheet of
- 2 -
CA 02827951 2013-01-31
treated material is at least 95% of that of the handsheet from untreated
material, more
preferably at least 96%, 97%, 98% or 99%.
The pulp to be treated can be pulp that has been mechanically refined, once,
twice or more prior to the enzymatic treatment. The pulp can be a raw wood
pulp. The
pulp can also be a reject pulp containing a long-fiber fraction that makes it
unsuitable for
e.g., papermaking without further treatment, that can benefit from the
treatment prior to
further processing. Here, "long-fiber fraction" refers to R14 and P14/R30. R14
are fibers
retained on a 14-mesh screen and P14/R30 pass through the 14-mesh screen but
are
retained on a 30 mesh screen.
The reduction in energy can be 5% or more. It can be 6%, 7%, 8%, 9%, 10%,
11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22% or more.
As mentioned, one possible measure of the benefit of treatment can be
determined by processing the treated pulp by further refining and preparation
of
handsheet, and comparing properties of the handsheet with one prepared from
the
same pulp that has not been treated. In the case of tensile strength, such
determination
can be made according to TAPPI standard T 205 sp-06.
In another embodiment, the invention provides a method for producing a wood
pulp, by exposing a wood pulp that has been refined at least once and having a
long-
fiber fraction containing wood fibers having a length of from 1 to 7 mm to an
enzymatic
solution. The pulp can be e.g., screened fraction of a refined pulp. The
exposure time
can be selected to reduce the average fiber length by between 5% and 25%. A
more
likely range of reduction would be between 10% and 20%, and could be about
10%,
about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%,
about 18%, about 19% or about 20%, or up to any of these amounts. This
reduction in
fiber length can also be accompanied by the benefit of a reduction of energy
consumption in a subsequent refining step of the enzymatically treated pulp.
The enzymatic treatment can be part of a larger process such as the
manufacture of cardboard, paper towels, newspaper, hygiene products, etc.
The wood pulp treated in the enzymatic step can have a CSF of greater than 650
ml and be exposed to the enzymatic solution for time sufficient to reduce the
drainability
- 3 -
CA 02827951 2013-01-31
to less than 150. The initial CSF can also be greater than or about 220 ml,
about 250
ml, about 300 ml, about 350 ml, about 400 ml, about 450 ml, about 500 ml,
about 550
ml, or about 600 ml with the drainability of the treated pulp being less than
or about 160
ml, about 170 or about 180 ml.
The enzymatic solution contains at least the aforementioned EG and CBH, and
preferably also contains mannanase (MAN). The activity of the EG relative to
the CBH
is always significantly greater i.e., the ratio of activities of the EG:CBH
are at least 3:1,
but can be at least any of 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, more preferably at
least 10:1, 11:1
or 12:1. The activity of MAN is also greater than CBH, activity ratio MAN:CBH
being at
least 1.5:1, or at least any of 1.6:1, 1.7:1, 1.8:1, 1.9:1 or 2:1.
A measure of the enzymatic activities contained in a pulp treatment solution
is, in
practice, made relative to the substrate being treated. in the case of e.g., a
fraction
containing wood fibers having a length of from 1 to 7 mm, activity can be
determined
based on dry weight measured according to standard T 258 om-06.
The enzymatic activity of the EG is in the range of 0.5 to 25 CMCU per gm of
wood substrate, but can be about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 9, 11, 12,
13, 14, 15, 16,
17, 18, 19, 20, 20, 21, 22, 23, or 24 CMCU per gm of wood substrate. Dry
weight is
measured according to standard T 258 om-06.
The enzymatic activity of the hemicellulase, mannanase is at least 1.5 times
the
activity of the CBH, and is typically at least 0.05 FPU per gm of wood fiber
substrate.
The long-fiber fraction based on dry weight measured according to standard T
258 om-
06.
The enzymatic activity of the CBH, which is always lower than the activities
of the
EG and MAN, as described above, is typically at least 0.05 FPU per gm of the
wood
fiber substrate e.g., long-fiber fraction of the wood pulp being treated,
again based on
dry weight measured according to standard T 258 om-06. Enzymatic activity of
CBH
can be from 0.05 to 10 FPU, but is preferably between 0.1 and 3 FPU/g of wood
on a
dry weight basis.
An embodiment of the invention includes exposing mechanical wood pulp to an
enzymatic solution for a sufficient length of time such that the amount of
fines in a
- 4 -
CA 02827951 2013-01-31
subsequently refined pulp is increased by at least 10% in comparison to
subsequently
refined pulp which has not been exposed to the enzymatic solution. Fines are
measured
according to standard TAPPI T-261. This increase in fines can also be
accompanied by
the benefit of a reduction of energy consumption in a subsequent refining step
of the
enzymatically treated pulp.
In another embodiment, the invention includes exposing mechanical wood pulp
to an enzymatic solution for a sufficient length of time such that handsheet
density of a
handsheet produced from said subsequently refined pulp is increased by at
least 5% in
comparison to the handsheet density of a handsheet produced from the same pulp
which has not been exposed to the enzymatic solution. Handsheet density is
determined according to standard TAPPI T 220 sp-06. This comparative increase
in
handsheet density can also be accompanied by the benefit of a reduction of
energy
consumption in a subsequent refining step of the enzymatically treated pulp.
According to another embodiment, mechanical wood pulp is exposed to the
enzymatic solution for a length of time selected to preclude the change in
tear index of a
handsheet produced from said subsequently refined pulp to no more than a
decrease of
15% in comparison to the tear index of a handsheet produced from the same pulp
which
has not been exposed to the enzymatic solution. By this is meant that the tear
index of a
handsheet can increase or be the same, but if it decreases, it decreases no
more than
15% with respect to the comparative sheet. Tear index of a handsheet is
determined
according to standard TAPPI T 414 om-12.
In yet another embodiment, a mechanical wood pulp is exposed to an enzymatic
solution for a length of time selected such that brightness of subsequently
refined pulp
is at least maintained in comparison to subsequently refined pulp which has
not been
exposed to the enzymatic solution. Brightness (ISO) is determined according to
standard TAPPI T 452 om-08. This maintenance of optical brightness can also be
accompanied by the benefit of a reduction of energy consumption in a
subsequent
refining step of the enzymatically treated pulp.
The method of the invention has been demonstrated with the softwood Black
Spruce, Picea mariana. Suitable wood fibers contain between 38 and 52% by
weight
- 5 -
CA 02827951 2013-01-31
cellulose, between 20 and 30% by weight lignin, between 20 and 30% by weight
hemicelluloses (hemicellulose typically being from 15 to 20% mannans by total
weight
of the wood chips and from 15 to 20% xylans by total weight of the wood
chips).
The invention includes a method for producing a paper product that includes
the
steps of: (a) introducing mechanical wood pulp into a vessel; (b) introducing
into the
vessel an enzymatic solution comprising an endoglucanase (EG), a
cellbiohydrolase
(CBH) and a mannanase (MAN) wherein the ratio of enzymatic activity of EG:CBH
is at
least 3, and the ratio of enzymatic activity of MAN:CBH is at least 1.5; (c)
waiting a
length of time sufficient for the freeness of the pulp to be reduced to a
selected level of
freeness of fibers in the pulp; and (d) making the paper product with the pulp
produced,
the paper having a tensile strength at least as great as paper produced from
the
mechanical wood pulp by the same method without exposure to said enzymatic
solution.
The invention includes a method of manufacturing a wood pulp that includes the
step of: exposing a mechanical wood pulp to an enzymatic solution comprising
an
endoglucanase (EG), a cellbiohydrolase (CBH) and a mannanase (MAN) wherein the
ratio of enzymatic activity of EG:CBH is at least 3, and the ratio of
enzymatic activity of
MAN:CBH is at least 1.5, for a sufficient amount of time to reduce energy
consumption
during subsequent refining of the exposed pulp in comparison to energy
consumption
during refining of the same pulp which has not been exposed to the enzymatic
solution
while at least maintaining the tensile strength of a handsheet produced from
said
subsequently refined pulp in comparison with a handsheet produced from the
same
pulp which has not been exposed to the enzymatic solution, the tensile
strength being
determined according to TAPPI standard T 205 sp-06.
The present invention thus relates to methods for reducing the amount of
energy
required to refine reject pulp by treating said pulp with a solution
containing enzymes
and preferably some stabilizer compounds. Stabilizer agents and surfactants
containing
mainly propylene glycol, glycerol, sorbitol and to a lesser degree proxel,
potassium
sorbate and ethoxylated fatty alcohols can be used. The enzymatic treatment
can be
carried out at process temperatures of from 20 C. to 80 C., for example
between 40 C.
- 6 -
CA 02827951 2013-01-31
and 60 C. The enzymatic treatment can be carried out at a pH of from about 2
to about
10. The treatment time can be from 30 minutes to 10 hours. Other temperatures,
pHs
and or times can be used.
It is possible to maintain tensile strength although some loss of tear
strength of
refined pulp and resultant paper products was observed.
The enzyme solution preferably possesses the following relative activities:
the
EG should have a 10 fold greater activity than the CBH and the mannanase
should
have a 2 fold greater activity than the CBH. This enzyme solution is available
commercially from Novozymes under the name Celluclast 1.5LTm.
Methods of refining pulp with lower energy requirements to obtain a desirable
degree of refining are set forth herein. Methods for refining the pulp wherein
the refining
process includes treatment of the pulp with a complex enzyme mixture are
presented,
wherein the resultant pulp and/or paper products have maintained tensile
strength,
improved optical properties and slightly reduced tear index as compared to
untreated
pulps or products therewith.
Pulp and paper products made therefrom having maintained tensile strength,
improved optical properties and slightly reduced tear strength are provided.
Pulp and
papers made therefrom which require less energy to produce are provided.
It is to be understood that both the foregoing general description and the
following detailed description are exemplary and explanatory only and are only
intended
to provide a further explanation of the present invention as claimed
Brief Description of the Drawings:
Embodiments illustrating the invention and establishing feasibility of various
aspects thereof are described below with reference to the accompanying
drawings, in
which:
Figure 1 is a graph showing the amount of sugars released per gram of oven
dried pulp (ODP) into the liquor after a 1 hour enzyme hydrolysis at different
dosages.
Based on these results dosages (5 and 10FPU/g ODP) were chosen for refining
trials;
- 7 -
CA 02827951 2013-01-31
Figure 2 is a bar graph showing the freeness of pulps obtained after the
enzymatically treated pulps were refined under the same conditions of feed
speed, plate
gap and consistency;
Figure 3 is a plot showing percent decrease in fiber length with dosage, after
enzymatically treated pulps were refined;
Figure 4 is a plot showing percent increase in fines with dosage, after
enzymatically treated pulps were refined;
Figure 5 is a plot showing handsheet density as function of enzymatic loading,
of
handsheets made from enzymatically treated refined pulps;
Figure 6 is a plot showing tear strength as a function of enzymatic loading,
of
handhseets made from enzymatically treated refined pulps;
Figure 7 is a plot showing tensile strength as a function of enzymatic
loading, of
handsheets made from enzymatically treated refined pulps; and
Figure 8 is a plot showing brightness as a function of enzymatic loading of
handsheets made from enzymatically treated refined pulps.
Detailed Description
The present invention relates to a method of refining pulp, wherein the method
includes the use of an enzyme mixture containing celluiases and hemicellulase.
Treatment with this solution following primary defibering and selective
screening prior to
secondary reject or post refining can reduce the energy required to reach a
given
degree of refining. This enzyme mixture is to contain a significant EG
activity, a marked
mannanase activity and a CBH activity that is lower than the first two but not
negligible.
As used herein, an endo-13-glucanase is preferably a cellulase classified as
EC
3.2.1.6 ¨ endo-1,3(4)-13 -glucanase. This enzyme is preferably capable of
endohydrolysis of 1,3- or 1,4-linkages in 13 -D-glucans when the glucose
residue whose
reducing group is involved in the linkage to be hydrolysed is itself
substituted at C-3.
This hydrolysis cleaves the 0-glycosyl bond of the cellulose backbone.
As used herein, a "mannanase" is preferably a hemicellulase classified as EC
3.2.'1.78, and called endo-1,4- f -mannosidase. Mannanase includes f3 -
mannanase,
endo-1,4-mannanase, and galactomannanase. Mannase is preferably capable of
- 8 -
CA 02827951 2013-01-31
catalyzing the hydrolysis of 1,4- p -D-mannosidic linkages in mannans,
including
glucomannans, galactomannans and galactoglucomannans. Mannans are
polysaccharides primarily or entirely composed of D-mannose units.
As used herein, a cellobiohydrolase is preferably a cellulase classified as EC
3.2.1.91 and called cellulose 1,4- p -cellobiosidase (non-reducing end). This
enzyme
produces the hydrolysis of (1--+4)-8-D-glucosidic linkages in cellulose and
cellotetraose,
releasing cellobiose from the non-reducing ends of the chains
EG activity can be determined following the carboxymethyl cellulose (CMC)
method described in Measurement of Cellulase Activities by T.K. Ghose (Pure &
Appl.
Chem. Vol 69, No. 2, pp. 257-268, 1987). The amount of reducing sugars
released from
enzymatic hydrolysis of a 2% solution of a well characterized CMC is used to
determine
the enzymes EG activity. Sugar concentration is determined by the well known
DNS
method described by G.L. Miller (Analytical Chem., No. 31, p.426, 1959).
CBH activity can be determined following the filter paper assay method
described
in Measurement of Celluiase Activities by T.K. Ghose (Pure & Appl. Chem. Vol
69, No.
2, pp. 257-268, 1987). The amount of reducing sugars released from enzymatic
hydrolysis of Whatman No. 1 filter paper strip of known size is used to
determine the
enzyme's CBH activity. Sugar concentration is determined by the well known DNS
method described by G.L. Miller (Analytical Chem., No. 31, p.426, 1959).
Mannanase activity can be determined following the method describer by M.
Ratto and K. Poutanen (Biotechnology Letters, No 9, pp-661-664, 1988). The
amount of
reducing sugars released from enzymatic hydrolysis of a 0.5% solution of
locust bean
gum is used to determine the enzymes mannanase activity. Sugar concentration
is
determined by the well known DNS method described by G.L. Miller (Analytical
Chem.,
No. 31, p.426, 1959).
An enzyme solution containing EG, CBH and mannanase activities in the correct
ratios is commercially available from Novozymesa under the name Celluclast
1.5LTm.
This solution contains between 40mg and 50mg of total protein per millilitre
of solution.
When kept at between 0 C and 25 C, the solution is stable and its activity is
maintained
- 9 -
CA 02827951 2013-01-31
for about 18 months. Storage at higher temperatures will reduce this effective
storage
time.
The enzyme solution can vary slightly in ratio of activities which still give
the
desired energy reductions and paper qualities. The amount of total protein in
the correct
ratio should be between 0.02kg and 10kg per metric ton of oven dried wood.
This
amount of total protein can vary depending on the type of woody substrate
being used,
for example virgin hardwood kraft, virgin softwood kraft, recycled groundwood,
refiner
groundwood, pressurized refiner groundwood, thermomechanical,
chemithermomechanical or a mixture thereof; or the species of wood which makes
up
this substrate, for example Populus sp., Acer sp., Picea sp., Abies sp., Pinus
sp.,
Conium sp., etc.
The pulp of the present invention can be treated with one or more other
components, including polymers such as anionic and non-ionic polymers, clays,
other
fillers, dyes, pigments, defoamers, microbiocides, pH adjusting agents such as
alum or
hydrochloric acid, other enzymes, and other conventional papermaking or
processing
additives. These additives can be added before, during or after introduction
of the
enzyme solution. The enzyme solution can be added, and is preferably added to
the
papermaking pulp before the addition of coagulants, flocculants, fillers and
other
conventional and non-conventional papermaking additives, including additional
enzymes.
The pulp can be any conventional softwood or hardwood species used in
mechanical pulp production, such as spruce, fir, hemlock, aspen, acacia,
birch, beech,
eucalyptus, oak and other softwood and hardwood species. The pulp can contain
cellulose fibers in an aqueous medium at a concentration of at least 35% by
weight
based on the oven dried solids content of the pulp. The pulp can be, for
example, virgin
pulp (e.g. spruce, fir, pine, eucalyptus, and include virgin hardwood or
virgin softwood),
hardwood kraft, softwood kraft, recycled groundwood, refiner groundwood,
pressurized
refiner groundwood, thermomechanical, chemithermomechanical or mixtures
thereof.
According to various embodiments, the papermaking system can include a
primary refiner, a secondary refiner, a screen, a mixer, a latency and/or
blend chest,
-10-
CA 02827951 2013-01-31
and papermaking equipment, for example, screens. The papermaking system can
also
include metering devices for providing a suitable concentration of the enzyme
composition or other additives to the flow of pulp. Valving, pumps, and
metering
equipment as known to those skilled in the art can also be used for
introducing various
additives described herein to the pulp.
According to one embodiment, the enzyme solution can be added to the pulp
after the pulp leaves the first refiner (also known as the primary refiner)
during the
refining process. For example, the enzyme solution can be added before the
second
refiner (also known as the secondary refiner), after the second refiner,
before the
screen, after the screen, before the mixer, after the mixer, before the
latency and/or
blend chest, to the latency and/or blend chest. For example, the enzyme
solution can be
added after the second refiner, between the screen and the mixer, or after the
mixer.
Other additives as described can be added to the papermaking system as known
to
those skilled in the art.
The pulp can be treated with the enzyme solution when the pulp is at a
temperature of from 100C. to about 75 C., from about 30 C. to about 70 C,, or
from
about 40 C. to about 60 C. The pulp can be at a pH of from 2 to 10, from about
4 to 7,
or from 4.5 to 5.5. A treatment time can be from 10 minutes to about 10 hours,
from
about 30 minutes to about 5 hours or from 1 hours to 2 hours.
Various ranges of components such as enzymatic activities, times, and values
of
such are described herein. It is to be understood that additional combinations
of such
ranges and values are also disclosed by such descriptions. As a general
example, a
range of from 2 to 5 describes values of about 2 and about 5; values of about
2, 3, 4
and 5 describes ranges of 2 to 5, 3 to 4, 2 to 4, etc.
The enzyme treatment is carried out during the refining process, but before
completion of the refining process. The enzyme treatment is carried out on
"coarse
pulp". A "coarse pulp" refers to a woody material used as the raw material of
the
mechanical pulp, which has been subjected to at least one mechanical refining
process
step. The term coarse pulp therefore encompasses, e.g. once refiner or ground
pulp,
twice refined or ground pulp, the reject pulp and/or long fiber fractions, and
- 11 -
CA 02827951 2013-01-31
combinations thereof. Preferably, the enzyme treatment is carried out on once
refined or
ground pulp or the reject pulp. More preferably the enzyme solution is carried
out on
once refined or ground pulp, a screened long fiber pulp fraction and the
reject pulp.
In another embodiment, the enzyme solution can be added at the latency chest
in a refining operation. As an example, the enzyme solution can be added after
screening and in the feedline before the latency chest. In this embodiment,
the
screened pulp is directed to a latency chest prior to a reject refiner. The
pulp is then
refined to desired specifications before being returned to the papermaking
system
stream.
The introduction of the enzyme solution can be made at one or more points and
the introduction can be continuous, semi-continuous, batch, or combinations
thereof.
According to various embodiments, the consistency of the pulp can be less than
20%, from about 1% to '15%, or from about 4% to 10%.
A pulp processed as described herein can exhibit maintained tensile strength,
while suffering some loss of tear strength. Paper products made from the pulp
also
maintain tensile strength while losing some tear strength. The addition of the
enzyme
solution creates fiber weaknesses which allow the formation of shorter fibers
but also
enhance fiber fibrillation which is why tear is affected while tensile
strength is
maintained. Fines production increases, thus lowering freeness at a given
specific
energy of refining SEC. The addition of the enzyme solution to coarse pulp
reduces the
amount of SEC needed to obtain a desired level of freeness.
A pulp produced by the methods described herein can be used in the production
of paper products, including, for example, cardboard, paper towels, newspaper,
and
hygiene products. The methods described herein can also be suitable for
textile
manufacturing.
EXAMPLES
Example 1 - Enzymatic activities
The commercial enzyme product, Celluclast 1.5LTm, was tested for several
enzymatic activities and was found to have several different types of
activities. Table 1
list all relevant and significantly measurable activities and protein
concentration.
-12-
CA 02827951 2013-01-31
Carboxymethyl cellulase (CMC) activity, equivalent to endo-p-glucanase
activity,
was determined following the CMC method described in Measurement of Cellulose
Activities by T.K. Ghose (Pure & Appl. Chem. Vol 69, No. 2, pp. 257-268,
1987). The
amount of reducing sugars released from enzymatic hydrolysis of a 2% solution
of a
well characterized CMC during a 30.0 minute hydrolysis at pH 4.8 and 50 C is
used to
determine the enzymes EG activity. Sugar concentration is determined by the
well
known 3,5-dinitrosalicylic acid (DNS) solution method described by G.L. Miller
(Analytical Chem., No. 31, p.426, 1959). The addition of the DNS solution to
the
hydrolysis filtrate stops the reaction. The mixture was boiled for 5.0 minutes
to allow for
color formation. After cooling, the absorbency is measured at 540nm and the
concentration is determined against a standard curve.
Mannanase activity was determined following the method described by M. Ratto
and K. Poutanen (Biotechnology Letters, No 9, pp-661-664, 1988). The amount of
reducing sugars released from enzymatic hydrolysis of a 0.5% solution of
locust bean
gum during a 30.0 minute hydrolysis at pH 4.8 and 50 C is used to determine
mannanase activity. Sugar concentration is determined by the well known DNS
method
described by G.L. Miller (Analytical Chem., No. 31, p.426, 1959) and described
thoroughly above.
Filter paper activity, equivalent to CBH activity, was determined following
the filter
paper assay method described in Measurement of Cellulose Activities by T.K.
Ghose
(Pure & Appl. Chem. Vol 69, No. 2, pp. 257-268, 1987). This method uses the
amount
of reducing sugars released from enzymatic hydrolysis of Whatman No. 1 filter
paper
strip of known size during a 30.0 minute hydrolysis at pH 4.8 and 50 C to
determine the
enzymes CBH activity. Sugar concentration is determined by the well known DNS
method described by G.L. Miller (Analytical Chem., No. 31, p.426, 1959) and
described
thoroughly above.
Protein concentration was determined using the Bradford assay. Bradford assay
kits purchased from Sigma-Aldrich were used. This well known method uses the
binding
of protein with a solution of Coomassie Blue which allows colorimetric
determination of
-13-
CA 02827951 2013-01-31
protein concentration based on a standard curve produced using bovine serum
albumin.
Absorbency is measured at 595nm.
TABLE 1: Measured parameters of Celluclast 1.5LTM
Parameter Value Unit
Endo-6-glucanase 1860 CMC/m1
Mannanase activity 285 1U/m1
Cellobiohydrolase 150 FPU/ml
Total protein 43 .4 mg/ml
Example 2 - Sugars released
The enzyme solution was added to a TMP reject pulp (5 g ODP) using the
solution's filter paper activity as a dosage indicator. Several dosages (5 and
10 FPU/g
ODP), chosen based on reducing sugar results, and a control were done in
duplicate
and measured in duplicate for a total of four data sets. Hydrolysis was
carried out at a
consistency of 10%, a temperature of 50 C and a time of 1 hour. After which,
the
samples were filtered and the filtrate was treated using the well known 3,5-
dinitrosalicylic acid (DNS) solution method described by GI. Miller
(Analytical Chem.,
No. 31, p.426, '1959). The addition of the DNS solution to the hydrolysis
filtrate stops the
reaction. The mixture was boiled for 5.0 minutes to allow for color formation.
After
cooling, the absorbency is measured at 540nm and the concentration is
determined
against a standard curve. This is shown in Figure 1 from the data in Table 2.
TABLE 2: Sugars released during bench-scale Celluclast 1.5LTm trials
Enzyme dosage Sugars released into liquor Standard deviation
(FPU/g oven dried PIP) (mq/q ODP) (mg/g ODP)
0.54 0.01
1.0 6.13 0.06
2.0 9.79 0.11
3.0 12.74 0.16
4.0 14.15 0.19
5.0 16.62 0.03
10.0 22.31 0.05
-14-
CA 02827951 2013-01-31
Example 3 - Freeness
The enzyme solution was added to a TMP reject pulp (200 g ODP) using the
solution's filter paper activity as a dosage indicator. Two dosages (5 and 10
FPU/g
ODP), chosen based on reducing sugar results, and a control were done in
duplicate.
Hydrolysis was carried out at a consistency of 4%, a temperature of 50 C and a
time of
1 hour. After this treatment, pulp was dewatered to 20% consistency and
refined in a
KRK refiner with a disc gap of 0.10mm. Refined pulp was collected and moisture
was
checked prior to measuring Canadian Standard Freeness (CSF). Results are shown
in
the Table 3 and Figure 2.
TABLE 3: Freeness of pulp treated with Celluclast '1.5L.TM trials before
refining
Enzyme dosage
Average CSF (m1) Standard devi
(FPU/g oven dried pulp) ation (m1)
Control (0 FPU/g ODP) 220 14
5 179 6
178 0
10 Example 4 - Energy savings
The enzyme solution was added to a TMP reject pulp (200 g ODP) using the
solution's filter paper activity as a dosage indicator. Two dosages (5 and 10
FPU/g
ODP), chosen based on reducing sugar results, and a control were done in
duplicate.
Hydrolysis was carried out at a consistency of 4%, a temperature of 50 C and a
time of
1 hour. After this treatment, pulp was dewatered to 20% consistency and
refined in a
KRK refiner with a disc gap of 0.10mm. Energy consumption was monitored with
an
online monitor and networked computer. Results are shown in Table 4.
- 15 -
CA 02827951 2013-01-31
TABLE 4: Specific Energy Consumption needed to refine pulp treated with
Celluclast 1.5LTm to approximately 200m1 freeness
Enzyme loading Meter reading Net SEC* Average SEC Energy Saving
fFPU/g) (kWh) (kWh/t) (kWh/t) fok)
o 0.503 1892.2
1962.2 0
0 0.531 2032.2
5.0 , 0.462 1687.2
1702.2 -13.5
5.0 0.468 , 1717.2
10.0 0.425 1502.2
1524.7 -22.3
10.0 0.434 1547.2
* No-load energy consumption (3 minutes of warm-up energy was calculated to be
0.12466 kWh) was subtracted from the meter reading to give the net energy
consumption
Example 5 - Fiber properties
The enzyme solution was added to a TMP reject pulp (200 g ODP) using the
solution's filter paper activity as a dosage indicator. Two dosages (5 and 10
FPU/g
ODP), chosen based on reducing sugar results, and a control were done in
duplicate.
Hydrolysis was carried out at a consistency of 4%, a temperature of 50 C and a
time of
1 hour. After this treatment, pulp was dewatered to 20% consistency and
refined in a
KRK refiner with a disc gap of 0.10mm. Energy consumption was monitored with
an
online monitor and networked computer. Refined pulp was collected and moisture
was
checked prior to testing fiber properties with a Fiber Quality Analyzer.
Results are
shown in Table 5 and in Figures 3 and 4.
TABLE 5: Some fiber properties of pulp treated with Celluclast1.5Lrm and
refined to approximately 200m1 freeness
Enzyme loading Mean length weighted fiber Mean length weighted
fines
(FPU/g oven dried pulp) length (mm) percent
(%)
Control (0 FPU/g ODP) 1.202 0.035 12.63 0.82
0.997+0.030 14.29 0.39
10 0.882+0.024 16.43 0.56
- 16 -
CA 02827951 2013-01-31
Example 6 - Handsheet properties
The enzyme solution was added to a TMP reject pulp (200 g ODP) using the
solution's filter paper activity as a dosage indicator. Two dosages (5 and 10
FPU/g
ODP), chosen based on reducing sugar results, and a control were done in
duplicate.
Hydrolysis was carried out at a consistency of 4%, a temperature of 50 C and a
time of
1 hour. After this treatment, pulp was dewatered to 20% consistency and
refined in a
KRK refiner with a disc gap of 0.10mm. Energy consumption was monitored with
an
online monitor and networked computer. Refined pulp was collected and moisture
was
checked prior to preparing handsheets following TAPP! standard T 205 sp-06.
Results
are shown in Table 6 and in Figures 5, 6, 7 and 8.
TABLE 6: Handsheet properties of paper made from pulp treated with
Celluclast 1.5LTm and refined to approximately 200m1 freeness
Enzyme loading
Mean density Mean Tear
Mean Tensile Mean Brightness
f FPU/g ovenIndex
(g/cm3) Index (N*
dried pulp (mN m/g) (ISO)
) 4-7-77i /P)
Control (0 FPU/g
0.47+0.02 7.71+0.11 34.33+0.99 47.63+1.66
ODP)
5 0.52+0.01 6.62+0.20 33.39+0.54 51.62+0.22
10 0.53+0.02 5.43+0.17 33.12+1.20 51.85+0.91
All patents, applications and publications mentioned above and throughout this
application are incorporated in their entirety by reference herein.
- 17 -
