U.S. patent application number 14/107481 was filed with the patent office on 2014-05-15 for method of hydrolyzing cellulose slurry using cellulase and flocculent (as amended).
This patent application is currently assigned to Iogen Energy Corporation. The applicant listed for this patent is Iogen Energy Corporation. Invention is credited to Brian Foody, Ziyad Rahme, Jeffrey S. Tolan.
Application Number | 20140134678 14/107481 |
Document ID | / |
Family ID | 36587503 |
Filed Date | 2014-05-15 |
United States Patent
Application |
20140134678 |
Kind Code |
A1 |
Foody; Brian ; et
al. |
May 15, 2014 |
METHOD OF HYDROLYZING CELLULOSE SLURRY USING CELLULASE AND
FLOCCULENT (AS AMENDED)
Abstract
A process for the enzymatic hydrolysis of cellulose to produce a
hydrolysis product from a pre-treated cellulosic feedstock is
provided. The process comprises introducing an aqueous slurry of
the pre-treated cellulosic feedstock at the bottom of a hydrolysis
reactor. Axial dispersion in the reactor is limited by avoiding
mixing and maintaining an average slurry flow velocity of about 0.1
to about 20 feet per hour, such that the undissolved solids flow
upward at a rate slower than that of the liquid. Cellulase enzymes
are added to the aqueous slurry before or during the step of
introducing. An aqueous stream comprising hydrolysis product and
unhydrolyzed solids is removed from the hydrolysis reactor. Also
provided are enzyme compositions which comprise cellulase enzymes
and flocculents for use in the process. In addition, a kit
comprising cellulase enzymes and flocculent is provided.
Inventors: |
Foody; Brian; (Ottawa,
CA) ; Rahme; Ziyad; (Ottawa, CA) ; Tolan;
Jeffrey S.; (Ottawa, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Iogen Energy Corporation |
Ottawa |
|
CA |
|
|
Assignee: |
Iogen Energy Corporation
Ottawa
CA
|
Family ID: |
36587503 |
Appl. No.: |
14/107481 |
Filed: |
December 16, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12762589 |
Apr 19, 2010 |
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14107481 |
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11303424 |
Dec 16, 2005 |
7727746 |
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12762589 |
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60637189 |
Dec 17, 2004 |
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Current U.S.
Class: |
435/99 |
Current CPC
Class: |
C12P 19/04 20130101;
Y02E 50/10 20130101; C12M 45/09 20130101; Y02E 50/17 20130101; C12P
19/02 20130101; Y02E 50/16 20130101; C12P 19/14 20130101 |
Class at
Publication: |
435/99 |
International
Class: |
C12P 19/14 20060101
C12P019/14; C12P 19/02 20060101 C12P019/02 |
Claims
1-68. (canceled)
69. A method of hydrolyzing cellulose to glucose, cellobiose,
glucose oligomers, or a combination thereof, comprising hydrolyzing
a slurry comprising cellulose using cellulase and at least one
flocculent.
70. The method according to claim 69, wherein the flocculent is a
cationic polymer.
71. The method according to claim 69, wherein said cellulase enzyme
is obtained from a plant, fungal or microbial source.
72. The method according to claim 69, wherein the cellulase enzymes
are produced by Aspergillus, Humicola, Trichoderma, Bacillus or
Thermobifida.
73. The method according to claim 69, wherein the at least one
flocculent is mixed with the cellulase enzymes to form an enzyme
composition before addition to the slurry.
74. The method according to claim 73, wherein the enzyme
composition is produced by obtaining cellulase enzymes from a
plant, fungal or microbial source, and combining the cellulase
enzymes with one or more than one flocculent to produce the enzyme
composition.
75. The method according to claim 70, wherein the cellulase enzymes
are produced by Aspergillus, Humicola, Trichoderma, Bacillus or
Thermobifida.
76. The method according to claim 75, wherein the at least one
flocculent is mixed with the cellulase enzymes to form an enzyme
composition before addition to the slurry.
Description
[0001] The present invention relates to processes for the
conversion of cellulosic feedstocks. More specifically, the present
invention relates to processes for enzymatic conversion of
cellulosic feedstocks having improved efficiency.
BACKGROUND OF THE INVENTION
[0002] Fuel ethanol is currently produced from feedstocks such as
cornstarch, sugar cane, and sugar beets. However, the production of
ethanol from these sources cannot expand much further due to
limited farmland suitable for the production of such crops and
competing interests with the human and animal food chain. Finally,
the use of fossil fuels, with the associated release of carbon
dioxide and other products, in the conversion process is a negative
environmental impact of the use of these feedstocks
[0003] The possibility of producing ethanol from
cellulose-containing feedstocks such as agricultural wastes,
grasses, and forestry wastes has received much attention due to the
availability of large amounts of these inexpensive feedstocks, the
desirability to avoid burning or landfilling cellulosic waste
materials, and the cleanliness of ethanol as a fuel compared to
gasoline. In addition, a byproduct of the cellulose conversion
process, lignin, can be used as a fuel to power the cellulose
conversion process, thereby avoiding the use of fossil fuels.
Studies have shown that, taking the entire cycle into account, the
use of ethanol produced from cellulose generates close to nil
greenhouse gases.
[0004] The cellulosic feedstocks that may be used for ethanol
production include (1) agricultural wastes such as corn stover,
wheat straw, barley straw, oat straw, oat hulls, canola straw, and
soybean stover; (2) grasses such as switch grass, miscanthus, cord
grass, and reed canary grass; (3) forestry wastes such as aspen
wood and sawdust; and (4) sugar processing residues such as bagasse
and beet pulp.
[0005] Cellulose consists of a crystalline structure that is very
resistant to breakdown, as is hemicellulose, the second most
prevalent component of cellulosic feedstocks. The conversion of
cellulosic fibers to ethanol requires: 1) liberating cellulose and
hemicellulose from lignin or increasing the accessibility of
cellulose and hemicellulose within the cellulosic feedstock to
cellulase enzymes, 2) depolymerizing hemicellulose and cellulose
carbohydrate polymers to free sugars, and 3) fermenting the mixed
hexose and pentose sugars to ethanol.
[0006] Among well-known methods used to convert cellulose to sugars
is an acid hydrolysis process involving the use of steam and acid
at a temperature, acid concentration and length of time sufficient
to hydrolyze the cellulose to glucose (Grethlein, 1978, J. Appl.
Chem. Biotechnol. 28:296-308). The glucose is then fermented to
ethanol using yeast, and the ethanol is recovered and purified by
distillation.
[0007] An alternative method of cellulose hydrolysis is an acid
prehydrolysis (or pre-treatment) followed by enzymatic hydrolysis.
In this sequence, the cellulosic material is first pre-treated
using the acid hydrolysis process described above, but at milder
temperatures, acid concentration and treatment time. This
pre-treatment process increases the accessibility of cellulose
within the cellulosic fibers for subsequent enzymatic conversion
steps, but results in little conversion of the cellulose to glucose
itself. In the next step, the pre-treated feedstock is adjusted to
an appropriate temperature and pH, then submitted to enzymatic
conversion by cellulase enzymes.
[0008] The hydrolysis of the cellulose, whether by acid or by
cellulase enzymes, is followed by the fermentation of the sugar to
ethanol, which is then recovered by distillation.
[0009] The efficient conversion of cellulose from cellulosic
material into sugars, and the subsequent fermentation of sugars to
ethanol, is faced with a major challenge regarding commercial
viability. In particular, acid prehydrolysis requires large amounts
of acid. For a clean feedstock, such as washed hardwood, the
sulfuric acid demand is 0.5% to 1% of the dry weight of the
feedstock; for agricultural fibers, which can contain high levels
of silica, salts, and alkali potassium compounds from the soil, the
acid demand can be up to about 10-fold higher, reaching 5% to 7% by
weight of feedstock. This adds significant cost to the process. A
second drawback of using large amounts of acids in a prehydrolysis
process is that the acidified feedstock must be neutralized to a pH
between about 4.5 and about 5 prior to enzymatic hydrolysis with
cellulase enzyme. The amount of caustic soda used to neutralize
acidified feedstock is proportional to the amount of acid used to
acidify the feedstock. Thus, high acid usage results in high
caustic soda usage, which further increases the cost of processing
cellulosic feedstock to ethanol. Furthermore, the cost of enzymatic
hydrolysis is high, as cellulose remains resistant to hydrolysis
despite pre-treatment, which increases the enzyme dosage required.
Such increased requirement can be counteracted by increasing the
hydrolysis times (90-200 hours), in turn requiring very large
reactors, which again adds to the overall cost.
[0010] A method of decreasing the enzyme dosage while maintaining
high levels of cellulose conversion is Simultaneous
Saccharification and Fermentation (SSF). In this type of system,
enzymatic hydrolysis is carried out concurrently with yeast
fermentation of glucose to ethanol in a reactor vessel. During SSF,
the yeast removes glucose from the reactor by fermenting it to
ethanol and this decreases inhibition of the cellulase by glucose.
However, the cellulase enzymes are still inhibited by ethanol. SSF
is typically carried out at temperatures of 35-38.degree. C., which
is a compromise between the 50.degree. C. optimum for cellulase and
the 28.degree. C. optimum for yeast. This intermediate temperature
leads to substandard performance by both the cellulase enzymes and
the yeast. Thus, the inefficient catalysis requires very long
reaction times and very large reaction vessels--both of which are
costly.
[0011] A method for higher volumetric productivity is disclosed in
U.S. Pat. No. 5,258,293 (Lynd). This method utilizes a
lignocellulosic feedstock along with microorganisms that are
continuously introduced into a reaction vessel. Fluid is also
continuously added from the bottom of the reaction vessel, but no
mechanical agitation of the slurry occurs. As the reaction
progresses, the lignocellulosic feedstock being digested tends to
accumulate in a spatially non-homogenous layer while the ethanol
product rises to a top layer, where it is removed. The insoluble
substrate accumulates in a bottom layer and can be withdrawn from
the vessel. This arrangement results in a differential retention of
the fermenting substrate, which allows for increased residence time
in the reactor vessel.
[0012] In another approach, disclosed in U.S. Pat. No. 5,837,506
(Lynd), ethanol is produced using an intermittently agitated,
perpetually fed bioreactor. Lignocellulosic slurry and
microorganisms are added to a reactor; the mixture is then
agitated, either by mechanical means or by fluid recirculation, for
a specific time interval, after which it is allowed to settle.
Ethanol is then removed from a top portion of the reactor,
additional substrate is added, and the cycle continues. In a
similar method, Kleijntjens et al. (1986, Biotechnology Letters,
8:667-672) utilize an upflow reactor to ferment
cellulose-containing substrate in the presence of C. thermocellum.
The substrate slurry settles to form an aggregated fibre bed, which
is accelerated by slow mechanical stirring. Substrate is added
periodically, while liquid is continuously fed to the reactor.
Ethanol product accumulates in a top layer, where it is removed
from the reactor. The methods disclosed in U.S. Pat. No. 5,837,506,
U.S. Pat. No. 5,258,293 and Kleijntjens et al. result in an
increase in the residence time of the feedstock in the reactor
vessel. However, all three methods suffer from the disadvantages of
the SSF process.
[0013] U.S. Pat. No. 5,348,871; U.S. Pat. No. 5,508,183; U.S. Pat.
No. 5,248,484; and U.S. Pat. No. 5,637,502 (Scott) teach a method
to improve the conversion efficiency in enzymatic hydrolysis
through the use of an attritor in association with an agitated
reactor vessel. The agitator produces a high-shear field for size
reduction of solid particles in the cellulosic feedstock, which
constantly provides new surface area for the cellulase enzymes.
Therefore, the reaction efficiency is increased and the enzyme
requirements are decreased. However, the high shear often
inactivates the enzymes. Furthermore, the cost of the attritor
equipment is much greater than the savings due to the decreased
enzyme dosage.
[0014] U.S. Pat. No. 5,888,806 and U.S. Pat. No. 5,733,758 (Nguyen)
teach an alternative approach using a tower hydrolysis reactor
comprising alternating mixed and unmixed zones, thus reducing the
mixing power consumption and cost. The slurry is moved upward in
plug flow through the reactor and is intermittently mixed in the
mixing zones, thus preventing channelling of liquid and ensuring
uniform heat and mass transfer. While the methods disclosed in U.S.
Pat. No. 5,888,806 and U.S. Pat. No. 5,733,758 reduce the shearing
and denaturation of the enzymes, the cost of the mixing equipment
is substantial. Furthermore, the kinetic performance of the enzymes
is no better than can be achieved in a batch hydrolysis mode.
[0015] At present there is much difficulty in the art to attain
high conversion efficiency while maintaining lowered costs.
Increasing hydrolysis times to avoid higher costs of increasing the
enzyme dosage requires larger reactors, which in turn increases
equipment costs. Mixing and intermittent mixing of the feedstock
during hydrolysis can increase enzyme efficiency but equipment
costs will again increase, and shear forces will cause enzyme
denaturation. Other systems compromise the optimal enzyme activity
and reduce the efficiency of the enzymes.
SUMMARY OF THE INVENTION
[0016] The present invention relates to processes for the
conversion of cellulosic feedstocks into products. More
specifically, the present invention relates to processes for the
enzymatic conversion of cellulosic feedstocks having improved
efficiency.
[0017] According to the present invention, there is provided an
upflow settling reactor for enzymatic hydrolysis of cellulose.
[0018] The present invention also provides a process for the
enzymatic hydrolysis of cellulose to produce a hydrolysis product
from a pre-treated cellulosic feedstock, the process
comprising:
[0019] i) providing an aqueous slurry of the pre-treated cellulosic
feedstock, the slurry comprising from about 3% to about 30%
undissolved solids in a liquid, the undissolved solids comprising
at least about 20% cellulose;
[0020] ii) introducing the aqueous slurry at the bottom of a
hydrolysis reactor and limiting axial dispersion in the reactor by
avoiding mixing, and maintaining an average slurry flow velocity of
about 0.1 to about 20 feet per hour, such that the undissolved
solids flow upward at a rate slower than that of the liquid;
[0021] iii) adding cellulase enzymes to the aqueous slurry before
or during the step of introducing (step ii); and iv) removing an
aqueous stream comprising hydrolysis product and unhydrolyzed
solids from the hydrolysis reactor, the hydrolysis product
comprising glucose, cellobiose, glucose oligomers, or a combination
thereof.
[0022] The present invention relates to the process for the
enzymatic hydrolysis of cellulose as defined above, wherein, in the
step of introducing (step ii), the aqueous slurry is introduced at
the bottom of the hydrolysis reactor with a uniform radial
distribution.
[0023] The present invention is directed to the process for the
enzymatic hydrolysis of cellulose as defined above, wherein, in the
step of adding (step iii), one or more than one flocculating
compound is added to the aqueous slurry, separately from, or
together with the cellulase enzymes, or a combination thereof.
Furthermore, the one or more than one flocculating compound may be
added before or during the step of introducing (step ii), or a
combination thereof.
[0024] The present invention pertains to the process for the
enzymatic hydrolysis of cellulose as defined above, wherein, in the
step of providing (step i), the slurry comprises from about 5% to
about 20% by weight undissolved solids, and the undissolved solids
comprise from about 25% to about 70% by weight cellulose.
[0025] The present invention is directed to the process as
described above, wherein the pre-treated cellulosic feedstock is
obtained from wheat straw, oat straw, barley straw, corn stover,
soybean stover, canola straw, sugar cane bagasse, switch grass,
reed canary grass, cord grass, oat hulls, sugar beet pulp or
miscanthus. Furthermore, the pre-treated cellulosic feedstock may
have been subjected to pre-treatment from about 160.degree. C. to
about 280.degree. C. and for about 3 seconds to about 30 minutes at
an acid concentration from about 0% to about 5% prior to enzymatic
hydrolysis. The acid may be selected from the group consisting of
sulfuric acid, sulfurous acid, and sulfur dioxide. Optionally, a
liquid stream comprising sugar may be separated from the
pre-treated cellulosic feedstock prior to the step of introducing
(step ii). The liquid stream may be separated from the feedstock
using processes such as filtration, centrifugation, washing or any
other suitable process as would be known in the art. If washing is
used for the separation, it may be carried out using a suitable
washing medium such as water, a recycled process stream, treated
effluent, or a combination thereof.
[0026] The present invention also provides for the process as
described above, wherein, in the step of adding (step iii), the
cellulase enzyme is added at a dosage from about 1.0 to about 40.0
FPU per gram of cellulose.
[0027] Furthermore, the present invention provides the process as
described above, where, in the step of removing (step iv), at least
a portion of the hydrolysis product stream is separated from the
unhydrolyzed solids by using a clarifier zone at the top of the
unmixed hydrolysis reactor. The hydrolysis product and the
unhydrolyzed solids may be removed from the clarifier zone at
separate locations. Alternatively, at least a portion of the
hydrolysis product stream is separated from the unhydrolyzed solids
using a solids-liquid separator.
[0028] The present invention is directed to the process as
described above, wherein, in the step of adding (step iii), the
cellulase enzymes are chosen to produce glucose, cellobiose,
glucose oligomers, or a combination thereof.
[0029] The present invention also provides for the process as
described above, wherein, in the step of providing (step i), the pH
of the slurry is adjusted from about 4.0 to about 6.0, preferably
from about 4.5 to about 5.5. Furthermore, the temperature may be
from about 45.degree. C. to about 70.degree. C., preferably about
45.degree. C. to about 65.degree. C.
[0030] The present invention relates to the process as described
above, wherein, in the step of adding (step iii), one or more than
one flocculating compound is used. The flocculating compound may be
selected from the group consisting of a cationic polymer, a
non-ionic polymer, an anionic polymer, an amphoteric polymer,
salts, alum, and a combination thereof. Preferably, the one or more
than one flocculating compound is the cationic polymer, for
example, but not limited to, a polyacrylamide. The flocculating
compound may be added at a dosage from about 0.1 to about 4 kg per
tonne solids.
[0031] The present invention also relates to the process as
described above, wherein the average slurry flow velocity is
between about 0.1 and about 12 feet per hour, more preferably,
between about 0.1 and about 4 feet per hour.
[0032] The present invention also provides an enzyme composition
comprising cellulase enzymes and one or more than one flocculent,
for hydrolyzing cellulose to glucose, cellobiose, glucose
oligomers, or a combination thereof. Preferably, the cellulase
enzymes are produced by Aspergillus, Humicola, Trichoderma,
Bacillus, Thermobifida, or a combination thereof. Furthermore, the
one or more than one flocculating compound may be selected from the
group consisting of a cationic polymer, a non-ionic polymer, an
anionic polymer, an amphoteric polymer, salts, alum, and a
combination thereof. Preferably, the one or more than one
flocculating compound is the cationic polymer, for example a
polyacrylamide.
[0033] The present invention is also directed to a use of an enzyme
composition comprising cellulase enzymes and one or more than one
flocculent for hydrolyzing cellulose to glucose, cellobiose,
glucose oligomers, or a combination thereof.
[0034] The present invention is also directed to a use of an enzyme
composition comprising cellulase enzymes and one or more than one
flocculent for the enzymatic hydrolysis of cellulose to produce a
hydrolysis product from a pre-treated cellulosic feedstock, the use
of the enzyme composition comprising:
[0035] i) providing an aqueous slurry of the pre-treated cellulosic
feedstock, the slurry comprising from about 3% to about 30%
undissolved solids in a liquid, the undissolved solids comprising
at least about 20% cellulose;
[0036] ii) introducing the aqueous slurry at the bottom of a
hydrolysis reactor and limiting axial dispersion in the reactor by
avoiding mixing, and maintaining an average slurry flow velocity of
about 0.1 to about 20 feet per hour, such that the undissolved
solids flow upward at a rate slower than that of the liquid;
[0037] iii) adding the enzyme composition to the aqueous slurry
before or during the step of introducing (step ii); and
[0038] iv) removing an aqueous stream comprising hydrolyzed product
and unhydrolyzed solids from the hydrolysis reactor, the hydrolysis
product comprising glucose, cellobiose, glucose oligomers, or a
combination thereof.
[0039] Preferably, the cellulase enzymes are produced by
Aspergillus, Humicola, Trichoderma, Bacillus, Thermobifida, or a
combination thereof, and the one or more than one flocculating
compound may be selected from the group consisting of a cationic
polymer, a non-ionic polymer, an anionic polymer, an amphoteric
polymer, salts, alum, and a combination thereof. Preferably, the
one or more than one flocculating compound is the cationic polymer,
for example, a polyacrylamide.
[0040] The present invention also provides an enzyme composition
comprising cellulase enzymes and one or more than one flocculent,
for hydrolyzing cellulose to glucose, cellobiose, glucose
oligomers, or a combination thereof, wherein the hydrolysis is
carried out by:
[0041] i) providing an aqueous slurry of the pre-treated cellulosic
feedstock, the slurry comprising from about 3% to about 30%
undissolved solids in a liquid, the undissolved solids comprising
at least about 20% cellulose;
[0042] ii) introducing the aqueous slurry at the bottom of a
hydrolysis reactor, limiting axial dispersion in the reactor by
avoiding mixing, and maintaining an average slurry flow velocity of
about 0.1 to about 20 feet per hour, such that the undissolved
solids flow upward at a rate slower than that of the liquid;
[0043] iii) adding the enzyme composition to the aqueous slurry
before or during the step of introducing (step ii); and
[0044] iv) removing an aqueous stream comprising hydrolysis product
and unhydrolyzed solids from the hydrolysis reactor, the hydrolysis
product comprising glucose, cellobiose, glucose oligomers, or a
combination thereof.
[0045] The present invention provides a kit comprising cellulase
enzymes and one or more than one flocculent and instructions for
hydrolyzing cellulose to produce a hydrolysis product from a
pre-treated cellulosic feedstock, the instructions comprising:
[0046] i) providing an aqueous slurry of the pre-treated cellulosic
feedstock, the slurry comprising from about 3% to about 30%
undissolved solids in a liquid, the undissolved solids comprising
at least about 20% cellulose;
[0047] ii) introducing the aqueous slurry at the bottom of a
hydrolysis reactor and limiting axial dispersion in the reactor by
avoiding mixing, and maintaining an average slurry flow velocity of
about 0.1 to about 20 feet per hour, such that the undissolved
solids flow upward at a rate slower than that of the liquid;
[0048] iii) adding the cellulase enzyme mixture and the one or more
than one flocculent to the aqueous slurry before or during the step
of introducing (step ii); and
[0049] iv) removing an aqueous stream comprising hydrolysis product
and unhydrolyzed solids from the hydrolysis reactor, the hydrolysis
product comprising glucose, cellobiose, glucose oligomers, or a
combination thereof.
[0050] The present invention also provides a method for preparing
an enzyme composition for use in hydrolyzing cellulose to produce a
hydrolysis product from a pre-treated cellulosic feedstock, the
method comprising obtaining one or more than one cellulase enzymes
from a plant, fungal or microbial source, and combining the
cellulase enzymes with one or more than one flocculent to produce
the enzyme composition.
[0051] The present invention also provides the method for preparing
the enzyme composition as described above, wherein the cellulase
enzymes are produced by Aspergillus, Humicola, Trichoderma,
Bacillus and Thermobifida.
[0052] The present invention provides a system for hydrolyzing
cellulose to glucose, cellobiose, glucose oligomers, or a
combination thereof, the system comprising a feedstock slurry
supply line in fluid communication with an input to an upflow
hydrolysis reactor, a solids-liquid separator in fluid
communication with the upflow hydrolysis reactor and comprising a
first output for withdrawing a slurry comprising unhydrolyzed
solids and a second output for withdrawing a stream comprising
hydrolysis product, the hydrolysis product comprising glucose,
cellobiose, glucose oligomers, or a combination thereof, wherein
the feedstock supply line, the upflow hydrolysis reactor, or both
the feedstock supply line and the upflow hydrolysis reactor,
comprise an enzyme composition comprising cellulase enzymes and one
or more than one flocculent.
[0053] The present invention also provides a system as described
above, wherein the feedstock supply line, when in use, comprises a
pre-treated feedstock. The solids-liquid separator may be a
settling tank, a clarifier, a clarifier zone, a centrifuge or a
filter. When the system is in use, cellulase enzymes may be present
at a dosage from about 1.0 to about 40.0 FPU per gram of cellulose
of the pre-treated feedstock and one or more than one flocculating
compound may be present at a dosage from about 0.1 to about 4.0 kg
per tonne of solids of the pre-treated feedstock.
[0054] As described herein, the operation of the hydrolysis of
cellulose within an upflow settling reactor may be enhanced by the
addition of one or more flocculating compounds. The flocculating
compounds increase the size of the cellulosic solids, thereby
increasing the rate of the settling of the cellulosic solids. This
helps to hold the solids in the reactor for a longer period of
time, thereby increasing the degree of conversion of the cellulose.
Furthermore, the process as described herein provides for the
hydrolysis of the feedstock slurry within the hydrolysis reactor in
the absence of mixing, in that no active mixing of the slurry,
through the use of impellers, pumps or the like, within the
hydrolysis tank is required.
[0055] The use of the upflow settling hydrolysis reactor addresses
several of the shortcomings of the prior art. The invention
improves the efficiency of the enzymatic hydrolysis of cellulose.
This results in a higher degree of conversion of the cellulose to
glucose. Alternatively, the upflow settling reactor results in a
lower requirement for cellulase enzymes than conventional
hydrolysis systems. The improved enzymatic hydrolysis is achieved
without the expense of mixing of the slurry within the hydrolysis
reactor, and without adding intense shear to the system. The
improvements associated with the use of an upflow hydrolysis
reactor may be enhanced by using a flocculating compound.
BRIEF DESCRIPTION OF THE DRAWINGS
[0056] These and other features of the invention will become more
apparent from the following description in which reference is made
to the appended drawings wherein:
[0057] FIG. 1 shows a schematic of a system comprising an upflow
reactor that may be used in accordance with an embodiment of the
present invention. FIG. 1A shows the system comprising an upflow
reactor and a settling tank. FIG. 1B shows the upflow reactor of
FIG. 1A. FIG. 1C shows a portion of the system where the upflow
reactor comprises a clarifier zone.
[0058] FIG. 2 shows the undissolved solids content sampled at
various heights within the hydrolysis reactor in the absence of
adding a flocculent.
[0059] FIG. 3 shows the percentage of cellulose conversion measured
by sampling at various heights within the hydrolysis reactor in the
absence of adding a flocculent.
[0060] FIG. 4 shows the undissolved solids content sampled at
various heights within the hydrolysis reactor in the presence of a
flocculent.
[0061] FIG. 5 shows the amount of converted cellulose sampled at
various heights within the hydrolysis reactor in the presence of
adding a flocculent.
[0062] FIG. 6 shows an increase in efficiency in cellulose
conversion in the product leaving the hydrolysis reactor in the
presence of added flocculent compared to the absence of
flocculent.
DESCRIPTION OF PREFERRED EMBODIMENT
[0063] The present invention relates to processes for the
conversion of cellulosic feedstocks into products. More
specifically, the present invention relates to processes having
improved efficiency for enzymatic conversion of cellulosic
feedstocks.
[0064] The following description is of a preferred embodiment by
way of example only and without limitation to the combination of
features necessary for carrying the invention into effect.
[0065] The invention relates to a process for the enzymatic
conversion of cellulose to break down products, for example, but
not limited to, glucose, cellobiose, glucose oligomers, or a
combination thereof. In an aspect of the invention, the process
involves pumping an aqueous cellulose slurry with cellulase upward
in an unmixed hydrolysis reactor. The upward velocity of the slurry
is slow, such that the solid particles, which are denser than the
bulk slurry, tend to flow upward more slowly than the liquor. It is
well established that cellulase enzymes bind tightly and
preferentially to cellulose. The slow upward flow of the
cellulose-containing solid particles retains the
cellulose-containing solids and the bound cellulase enzyme in the
reactor for a longer time than the liquid. The retention of
cellulose and bound cellulase increases the conversion of cellulose
to products, for example, glucose. Near the top of the reactor, the
aqueous product or sugar stream and the unhydrolyzed solids are
withdrawn. If the product is glucose, the aqueous sugar stream is
withdrawn for fermentation to ethanol and other further processing.
The process as described herein achieves a longer cellulose
hydrolysis time within a smaller reactor than would otherwise be
required for plug flow of the liquid and solids. Alternatively, the
process as described herein achieves a higher cellulose conversion
with less cellulase enzyme than would otherwise be required.
[0066] The present invention provides a process for the enzymatic
hydrolysis of cellulose to produce a hydrolysis product from a
pre-treated cellulosic feedstock, the process comprising:
[0067] i) providing an aqueous slurry of the pre-treated cellulosic
feedstock, the slurry comprising from about 3% to about 30%
undissolved solids in a liquid, the undissolved solids comprising
at least about 20% cellulose;
[0068] ii) introducing the aqueous slurry at the bottom of a
hydrolysis reactor and limiting axial dispersion in the reactor by
avoiding mixing, and maintaining an average slurry flow velocity of
about 0.1 to about 20 feet per hour, such that the undissolved
solids flow upward at a rate slower than that of the liquid;
[0069] iii) adding cellulase enzymes to the aqueous slurry before
or during the step of introducing (step ii); and
[0070] iv) removing an aqueous stream comprising hydrolysis product
and unhydrolyzed solids from the hydrolysis reactor, the hydrolysis
product comprising glucose, cellobiose, glucose oligomers, or a
combination thereof.
[0071] Furthermore, in the step of adding (step iii), a flocculent
may also be added to the slurry, either directly to the slurry, or
along with the cellulase enzymes being added to the slurry.
[0072] The glucose may then be used for further processing to
produce a product of interest, for example, but not limited to,
ethanol.
[0073] Even though the upflow settling reactor, and process as
described herein are suited to the enzymatic conversion of
cellulose to glucose, this reactor and associated process may also
be used to convert cellulose to other products, including, but not
limited to, cellobiose (preferably if the enzyme,
.beta.-glucosidase (.beta.G), is omitted from the cellulase) and
glucose oligomers (preferably if the cellobiohydrolase enzymes
(CBH) and PG are omitted from the cellulase). To further exemplify
the present invention, the process for converting cellulose to
glucose is described. However, it is to be understood that that
this process may be used for the production of alternate products
by incorporating different cellulase enzyme mixtures during
hydrolysis of the feedstock.
[0074] By the term "cellulosic feedstock" or "cellulosic material",
it is meant any type of biomass comprising cellulose such as, but
not limited to, non-woody plant biomass, agricultural wastes and
forestry residues and sugar-processing residues. For example, the
cellulosic feedstock can include, but is not limited to, grasses,
such as switch grass, cord grass, rye grass, miscanthus, or a
combination thereof; sugar-processing residues such as, but not
limited to, sugar cane bagasse and sugar beet pulp; agricultural
wastes such as, but not limited to, soybean stover, corn stover,
oat straw, rice straw, rice hulls, barley straw, corn cobs, wheat
straw, canola straw, oat hulls, and corn fiber; and forestry
wastes, such as, but not limited to, recycled wood pulp fiber,
sawdust, hardwood, softwood, or any combination thereof. Further,
the cellulosic feedstock may comprise cellulosic waste or forestry
waste materials such as, but not limited to, newsprint, cardboard
and the like. Cellulosic feedstock may comprise one species of
fiber or, alternatively, cellulosic feedstock may comprise a
mixture of fibers that originate from different cellulosic
feedstocks. Wheat straw, barley straw, corn stover, soybean stover,
canola straw, switch grass, reed canary grass, sugar cane bagasse,
cord grass, oat hulls, sugar beet pulp and miscanthus are
particularly advantageous as cellulosic feedstocks due to their
widespread availability and low cost.
[0075] In principle, any material that contains a substantial
amount of cellulose is suitable for the process of the present
invention. In practice, the cellulosic material comprises cellulose
in an amount greater than about 20% (w/w) to produce a significant
amount of glucose. The cellulosic material can be of higher
cellulose content, for example at least about 30% (w/w), 35% (w/w),
40% (w/w) or more. Therefore, the cellulosic material may comprise
from about 20% to about 70% (w/w) cellulose, or from 25% to about
70% (w/w) cellulose, or about 35% to about 70% (w/w) cellulose, or
more, or any amount therebetween, for example, but not limited to,
20, 22, 24, 25, 26, 28, 30, 32, 34, 35, 36, 38, 40, 42, 44, 46, 48,
50, 52, 54, 56, 58, 60, 62, 64, 66, 68 and 70% (w/w) cellulose.
[0076] The present invention may be practiced with a natural
cellulosic feedstock or a cellulosic material that has been
processed or pre-treated. Processing and pre-treatment methods are
intended to deliver a sufficient combination of mechanical and
chemical action so as to disrupt the fiber structure and increase
the surface area of feedstock accessible to cellulase enzymes.
Mechanical action typically includes, but is not limited to, the
use of pressure, grinding, milling, agitation, shredding,
compression/expansion, or other types of mechanical action.
Chemical action can include, but is not limited to, the use of heat
(often steam), acid, and solvents. Several chemical and mechanical
pre-treatment methods are well known in the art.
[0077] One approach to pre-treatment of the feedstock is steam
explosion, using the process conditions described in U.S. Pat. No.
4,461,648, and U.S. Pat. No. 4,237,226 (which are herein
incorporated by reference). In this process, lignocellulosic
biomass is loaded into a steam gun in the presence of 0% to 5%
(v/v), or any amount therebetween, sulfuric acid or any other
suitable acid. The steam gun is then filled rapidly with steam to a
temperature of about 160.degree. C. to about 280.degree. C., or any
amount therebetween, and held at high pressure for a cooking time
of between about 3 seconds to about 30 minutes, or any amount
therebetween. The vessel is then rapidly depressurized to expel the
pre-treated biomass. Any parameters known in the prior art to
effect steam explosion pre-treatments such as, but not limited to,
those described in Foody, et al., (Final Report, Optimization of
Steam Explosion Pre-treatment, U.S. DEPARTMENT OF ENERGY REPORT
ET230501, April 1980; which is herein incorporated by reference)
may be used in the method of the present invention. The conditions
chosen for steam explosion will depend upon the nature of the
feedstock and the desired degree of susceptibility to enzymes.
However, other methods that are known within the art may be used as
required for preparation of a pre-treated feedstock, for example,
but not limited to, those disclosed in U.S. Pat. No. 5,846,787
(Ladisch), U.S. Pat. No. 5,198,074 (Villavicencio), U.S. Pat. No.
4,857,145 (Villavicencio), or U.S. Pat. No. 4,556,430 (Converse;
which are incorporated herein by reference), ammonia freeze
explosion (U.S. Pat. No. 5,171,592, Holtzapple) and concentrated
alkali treatment.
[0078] Regardless of whether a pre-treatment step is performed, the
cellulosic feedstock may optionally be washed with water, or
leached with water, for example, as disclosed in WO 02/070753
(Griffin et al., which is incorporated herein by reference) prior
to enzymatic hydrolysis. Washing of pre-treated cellulosic
feedstock can remove inhibitors of cellulase enzymes such as
dissolved sugars and sugar degradation products, dissolved lignin
and phenolic compounds, and other organic compounds in the system.
The concentration of cellulose within washed pre-treated feedstock
typically increases, for example up to levels of about 50%-70%.
[0079] The cellulosic material is slurried in a liquid at a
concentration that is thick and can still be pumped. For example,
but without wishing to be limiting, the liquid may be water, a
recycled process stream or treated effluent. The concentration of
cellulosic feedstock in the slurry depends on the material, but may
be between about 3% to about 30% (w/w) undissolved solids, or any
concentration therebetween, for example, from about 5% to about
20%, or from about 10% to about 20% undissolved solids, or any
amount therebetween. For example, the concentration of cellulosic
feedstock in the slurry may be 3, 5, 6, 8, 10, 12, 14, 16, 18, 20,
22, 24, 26, 28 or 30% undissolved solids (w/w). As is well known in
the art, the concentration of suspended or undissolved solids can
be determined by filtering a sample of the slurry using glass
microfiber filter paper, washing the filter cake with water, and
drying the cake overnight at 105.degree. C.
[0080] The pH of the slurry is generally adjusted to within the
range of optimum pH for the cellulase enzymes used. Generally, the
pH of the slurry is adjusted to within the range of about 3.0 to
about 7.0, or about 4.0 to about 6.0, or any pH therebetween,
preferably within the range of about 4.5 to about 5.5. For example,
the pH may be about 3.0, 3.5, 4.0, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,
5.1, 5.2, 5.3, 5.4, 5.5, 6.0, 6.5 or 7.0. The pH of the slurry may
be adjusted using any suitable acid or base known in the art. For
example, sodium hydroxide, ammonia, ammonium hydroxide, potassium
hydroxide or other suitable base (if the slurry is acidic), or
sulfuric acid, or other suitable acid (if the slurry is alkaline),
may be used. However, the pH of the slurry can be higher or lower
than about 4.5 to 5.5 if the cellulase enzymes used are
alkalophilic or acidophilic, respectively. The pH of the slurry
should be adjusted to within the range of optimum pH for the
enzymes used.
[0081] The temperature of the slurry is adjusted to the point that
is within the optimum range for the activity of the cellulase
enzymes. Generally, a temperature of about 45.degree. C. to about
70.degree. C., or about 45.degree. C. to about 65.degree. C., or
any temperature therebetween, is suitable for most cellulase
enzymes. For example, the temperature of the slurry may be adjusted
to about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70.degree. C.
However, the temperature of the slurry may be higher for
thermophilic cellulase enzymes.
[0082] Cellulase enzymes are then added to the slurry. By the term
"cellulase enzymes", "cellulase", or "enzymes", it is meant enzymes
that catalyse the hydrolysis of cellulose to products such as
glucose, cellobiose, and other cellooligosaccharides. Cellulase is
a generic term denoting a multienzyme mixture comprising
exo-cellobiohydrolases (CBH), endoglucanases (EG) and
.beta.-glucosidases (.beta.G) that can be produced by a number of
plants and microorganisms. The process of the present invention can
be carried out with any type of cellulase enzymes, regardless of
their source; however, microbial cellulases are generally available
at lower cost than those of plants. Among the most widely studied,
characterized, and commercially produced cellulases are those
obtained from fungi of the genera Aspergillus, Humicola, and
Trichoderma, and from the bacteria of the genera Bacillus and
Thermobifida. Cellulase produced by the filamentous fungi
Trichoderma longibrachiatum comprises at least two
cellobiohydrolase enzymes termed CBHI and CBHII and at least 4 EG
enzymes.
[0083] Cellulase enzymes work synergistically to degrade cellulose
to glucose. CBHI and CBHII generally act on the ends of the glucose
polymers in cellulose microfibrils liberating cellobiose (Teleman
et al. 1995, European J. Biochem 231:250-258), while the
endoglucanases act at random locations on the cellulose. Together
these enzymes hydrolyse cellulose to smaller cello-oligosaccharides
such as cellobiose. Cellobiose is hydrolysed to glucose by
.beta.-glucosidase.
[0084] The cellulase enzyme dosage added to the slurry is chosen to
achieve a sufficiently high level of cellulose conversion without
overdosing. For example, an appropriate cellulase dosage can be
about 1.0 to about 40.0 FPU per gram of cellulose, or any amount
therebetween. For example, the cellulase dosage may be about 1.0,
3.0, 5.0, 8.0, 10.0, 12.0, 15.0, 18.0, 20.0, 22.0, 25.0, 28.0,
30.0, 32.0, 35.0, 38.0 or 40.0 FPU per gram, or any amount
therebetween. The FPU (Filter Paper Unit) is a standard measurement
familiar to those skilled in the art and is defined and measured
according to Ghose (1987, Pure and Appl. Chem. 59:257-268). For
complete conversion to glucose, it is preferred that the cellulase
contain an adequate quantity of .beta.-glucosidase (cellobiase)
activity. The dosage level of .beta.-glucosidase is about 5 to
about 600 .beta.-glucosidase units per gram of cellulose, or any
amount therebetween. A typical dosage level of .beta.-glucosidase
is about 10 to about 400 .beta.-glucosidase units per gram of
cellulose, or any amount therebetween; for example, the dosage may
be 10, 12, 15, 17, 20, 22, 25, 27, 30, 32, 35, 37, 40, 42, 45, 47,
50, 52, 55, 57, 60, 62, 65, 67, 70, 72, 75, 77, 80, 82, 85, 87, 90,
92, 95, 97, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300,
320, 340, 360, 380 and 400 .beta.-glucosidase units per gram of
cellulose, or any amount therebetween. The .beta.-glucosidase unit
is measured according to the method of Ghose (1987, Pure and Appl.
Chem. 59:257-268).
[0085] The cellulase enzymes may be handled in an aqueous solution,
as a powder or as a granulate. The enzymes may be added to the
slurry at any point prior to its entry into the reaction vessel
(also referred to as a hydrolysis tower or hydrolysis reactor; 110
or 110', FIG. 1). For example, but without wishing to be limiting,
the cellulase enzymes may be added to the slurry immediately prior
to entering the hydrolysis tower. The enzymes may be mixed into the
slurry using mixing equipment that is familiar to those of skill in
the art. In a non-limiting example, a small make-up tank (90, FIG.
1A) located upstream of the main hydrolysis reactor (110 or 110')
may be used for adding the enzymes to the slurry, adjusting the pH
and achieving the desired temperature of the slurry.
[0086] With reference to FIG. 1A, the feedstock 10 is pre-treated
as described above. This stream is cooled using a heat exchanger 20
that exchanges against product stream 30 or other suitable fluid.
The slurry 40 may then be further cooled using a second fluid, for
example cold water 45, at heat exchanger 50. The slurry may then be
pumped into a hydrolysis make-up tank 90, along with cellulase
enzymes 70 and ammonium hydroxide 80, to adjust the pH. In this
example, the contents of the hydrolysis make-up tank 90 are mixed
and pumped out of the make-up tank 90, along pipe 100, to the
hydrolysis tank 110. However, the cellulase enzymes may be mixed
with the feedstock elsewhere, for example, at 190, 195 or 197 or
within a line that feeds the hydrolysis reactor, including, but not
limited to, line 10, 40 or 100, or a combination thereof.
[0087] By the term "hydrolysis tower", "upflow hydrolysis reactor",
"hydrolysis reactor" "hydrolysis tank", or "upflow settling
reactor", it is meant a reaction vessel (tower) of appropriate
construction to accommodate the hydrolysis of cellulosic slurry by
cellulase enzymes, for example 110 (FIG. 1B) or 110' (FIG. 1C). The
hydrolysis tank may be jacketed with insulation, steam, hot water,
electrical heat tracing, or other heat source to maintain the
desired temperature. In the present application, the hydrolysis
reactor is an unmixed hydrolysis reactor, in the sense that no
mixing of the reactor contents takes place during the hydrolysis
reaction. As set out below, some small amount of localized mixing
of the reactor contents may occur due to the small amount of power
input associated with the addition and withdrawal of solids and
liquids from the system. The slurry and cellulase mixture may enter
the upflow settling reactor directly at the bottom and be pumped
upward in the hydrolysis tower; alternatively, the slurry can be
pumped downward through a pipe located in the centre (e.g. 105) of
the reactor and emerge at the bottom of the reactor to flow upward,
surrounding the pipe. The latter configuration is advantageous in
that heat from the slurry can be captured in the hydrolysis
reactor. Once the slurry reaches the bottom of the hydrolysis tank
110 or 110', the slurry moves upward and is dispersed across the
width of the hydrolysis tank; axial dispersion (i.e. dispersion
along the height of the tank) is minimized by avoiding mixing. The
slurry flow velocity is chosen such that the liquid component of
the slurry flows upward at a rate faster than that of the
undissolved solids. The hydrolysis tower is designed such that the
contents of the slurry are relatively uniform in the radial
direction, at any given height. The uniform distribution may be
achieved using distributors (e.g. 120), or other equipment well
known in the art. This may include fractal distributors (for
example, Rohn Haas Advanced Amerpack.TM. system manufactured by
Amalgamated Research Inc.) or a rotating wand. For example, in FIG.
1B, a vertical control shaft supports a pair of cantilevered truss
arms at the bottom of the reactor 110 and another pair at the top
of the reactor. The arms incorporate a header system with nozzles
to distribute the product at the bottom of the vessel and to
collect it at the top. A two-port rotary joint is used for the feed
and the discharge from the central shaft. The distribution of the
slurry within the hydrolysis tank is achieved in the absence of
active mixing by impellers or pumps.
[0088] FIG. 1C shows an alternate hydrolysis tower 110' comprising
a clarifier zone 135 positioned at the top of the tank. As
described in more detail below, the majority of the solids are
withdrawn at the top of zone 130. Excess clarified liquid continues
to flow upward in zone 135 and is withdrawn as clear liquid.
[0089] The hydrolysis reactor 110 or 110' may be of any dimensions
that will maintain a relatively uniform slurry concentration across
the reactor, as described. Without wishing to be limiting, the
hydrolysis tower may have a diameter of between about 10 feet to
about 130 feet (3 to 40 m), or any amount therebetween; for
example, the diameter of the reactor may be 10, 15, 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115,
120, 125, or 130 feet, or any amount therebetween. The height of
the hydrolysis reactor can be of any height, provided that the
reactor achieves the purposes herein described. Without wishing to
be limiting, the reactor height may be of about 5 to about 75 feet
or about 5 to about 65 feet (1.5 to 23 m), or any amount
therebetween, preferably from about 20 to about 65 feet; for
example, the reactor height may be about 5, 7.5, 10, 12.5, 15,
17.5, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5,
50, 52.5, 55, 57.5, 60, 62.5, or 65 feet. The height-to-diameter
ratio of the hydrolysis reactor may be between about 0.5 to about
10, or any ratio therebetween; preferably the height-to-diameter
ratio is from about 0.5 to about 3. The overall size of the reactor
should be chosen so as to avoid placing an undue burden on the
foundation supporting the reactor when it is filled with water. In
a non-limiting example, a hydrolysis reactor having a diameter of
110 feet and height of 65 feet would have a volume of 4.60 million
gallons.
[0090] The slurry is pumped upward into the reactor 110 or 110' at
an average slurry flow velocity that allows the liquid to flow
uniformly up the reactor while the cellulose-containing particles,
which are denser than the liquid, flow up the reactor more slowly
than the liquid, settle, and pack to some solids concentration that
is higher than the feed solids concentration. The "average flow
velocity" of the slurry or "slurry flow velocity" is the height of
the hydrolysis reactor divided by the nominal slurry residence
time, based on the reactor volume and the slurry flow rate to the
reactor. For example, a slurry feed rate of 10,000 gallons per hour
to a 120,000 gallon hydrolysis reactor that is 30 feet tall has a
nominal slurry residence time of 120,000 gallons/(10,000
gallons/hr)=12 hours and an average slurry flow velocity of 30
ft/12 hr=2.5 ft/hr. The average flow velocity is selected to permit
solids to flow upward at a slower rate than the average slurry flow
velocity. This permits the solids to have a longer average
residence time in the hydrolysis reactor than the nominal slurry
residence time. This differs from slurry plug flow reactors in
which the flow velocity and the retention time of the liquid and
solids in the reactor are substantially the same. The flow velocity
at which this is achieved will be dependent on the feedstock and
the size of the solid particles in the slurry, as well as the
presence of any added flocculent. The use of a flocculent may
permit the use of a higher average flow velocity. Generally, the
average flow velocity is about 0.1 to about 20 feet per hour, or
any velocity therebetween. Preferably, the average flow velocity is
between about 0.1 to about 12 feet per hour, or any amount
therebetween. More preferably, the average flow velocity is between
about 0.1 to about 4.0 feet per hour, or any amount therebetween.
For example, the average slurry flow velocity may be about 0.1,
0.2, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0,
5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0,
11.5, 12.0 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5,
17.0, 17.5, 18.0, 18.5, 19.0, 19.5, or 20.0 feet per hour. The
nominal slurry residence time in the hydrolysis reactor is
typically 4-120 hours, preferably 12-100 hours and most preferably
20-100 hours.
[0091] Using the method of the present invention, the properties of
the slurry will change as hydrolysis of cellulose proceeds. Without
wishing to be bound by theory, during the hydrolysis reaction, the
cellulase enzymes bind to the cellulose and therefore remain bound
to the cellulose-containing solid particles in the slurry. The
average upward velocity of the slurry is slow, such that the solid
particles, which are denser than the bulk slurry, tend to flow
upward more slowly than the liquor. The slow upward flow of the
cellulose-containing solid particles retains the
cellulose-containing solids and the bound cellulase enzyme in the
reactor for a longer time than the liquid. As the bound enzymes
digest the cellulose and release glucose into solution, the amount
of cellulose, and the density of the solid particles, changes.
Depending on the altered density, smaller particles will flow
upward with the liquid or settle to the bottom of the reactor. Due
to the differential retention of the cellulose-containing particles
relative to the liquor, the concentration of cellulose will
decrease from the bottom to the top of the hydrolysis reactor while
the concentration of glucose will increase from the bottom to the
top of reactor. The decrease in the concentration of cellulose, and
an increase in the concentration of glucose, occurs in the
"hydrolysis zone" 130 of the hydrolysis reactor 110 (see FIGS. 1A
and 1B). The aqueous sugar stream, the unhydrolyzed solids, and any
nearby cellulose-containing particles are withdrawn 150 near the
top of the hydrolysis zone 140 of the reactor 110. At least a
portion of the solids are then separated from the glucose stream,
for example using a solids-liquid separator, for example, settling
tank 160 and the product stream 30 is sent for fermentation to
ethanol and other further processing (170). The longer retention of
cellulose within the hydrolysis tower increases the extent of
conversion of the cellulose to glucose, thereby achieving a longer
cellulose hydrolysis time with a smaller reactor than would be
achieved with a mixed reactor. Alternatively, a higher cellulose
conversion is achieved with a lower enzyme dosage than would be
required otherwise.
[0092] By the term "unhydrolyzed solids" or "unconverted solids",
it is meant cellulose that is not digested by the cellulase enzyme,
as well as non-cellulosic, or other, materials that are inert to
cellulase, present in the feedstock. For example, but without
wishing to be limiting in any manner, the unconverted solids may
comprise lignin, silica or other solid material. As the cellulose
in the feedstock is hydrolyzed, the concentration of unconverted
solids within the cellulose-containing solid particles increases.
Depending on the density and particle size, the unconverted solids
may be removed with the products at 150 or settle to the bottom in
a sediment or sludge 180. If a sludge layer forms at the bottom of
the reactor due to very heavy particles, any means known in the art
may be employed to remove the sludge or sediment. In a non-limiting
example, a scraper may be used to remove the sludge. In a further
example, the bottom of the reactor may be tapered to provide a path
in which the heaviest solids may settle, and be removed (e.g. line
167) and sent for lignin processing 165.
[0093] The aqueous glucose, unconverted solids and other particles
that are found near the top 140 of the hydrolysis reactor 110 can
be removed as a stream 150. Following withdrawal from the top of
the reactor, at least a portion of the unconverted solids may be
separated from the soluble sugar stream. Removal of the unconverted
solids can be accomplished using a solids-liquid separator, for
example by filtration (for example, a filter press, belt filter,
drum filter, vacuum filter or membrane filter), centrifugation,
settling, for example, settling tank 160, an inclined settler (for
example, as disclosed in Knutsen and Davis, 2002, Appl., Biochem.
Biotech., 98-100:1161-1172 and Mores et al., 2001, Appl. Biochem.
Biotech., 91-93:297-309, both of which are incorporated herein by
reference), a clarifier, or any other suitable process as would be
known in the art. The clarifier may comprise a number of inclined
plates to facilitate the separation of the solids and liquid or
other features that are known in the art of solids-liquid
separation. The soluble glucose, essentially free of undissolved
solids, is then suitable for fermentation to ethanol (170). The
unconverted solids are primarily lignin, which can be burned and
used as fuel for the plant.
[0094] Alternatively, the aqueous glucose stream is withdrawn at a
location separate from the withdrawal of unconverted solids. An
alternative method for separating the unconverted solids from the
glucose is to use the reactor 110' in FIG. 1C with a hydrolysis
zone 130 extending from the bottom of the hydrolysis tower to a
level about 65% to about 85% of the way up, and a "clarifier zone"
135 directly above the hydrolysis zone. The hydrolysis stream is
pumped from the top portion of the hydrolysis zone 130 into the
clarifier zone 135. A majority of the solids are removed at the top
of the hydrolysis zone 130. For example, but not wishing to be
limiting in any manner, a horizontal wand with nozzles is passed
back and forth across the top of the reactor at time intervals, and
the solids-rich stream is withdrawn into the wand and pumped out of
the reactor (162). Excess clarified liquid continues to flow upward
into clarifier zone 135. In the clarifier zone 135, the solids
generally settle to the level of the wand, while the aqueous sugar
stream, essentially free of solids, is removed (30) from the top.
The clarifier zone may comprise a number of inclined plates to
facilitate the separation of the solids and liquid and other
features that are known in the art of solids-liquid separation. The
unconverted solids (or unhydrolyzed solids) may be transferred to a
solids-liquid separator to separate at least a portion of the
hydrolysis product from the unhydrolyzed solids.
[0095] If so desired, the cellulose-containing solids obtained by
separation from the glucose stream can be recycled back into the
upflow settling reactor, or the hydrolysis zone, with the incoming
feedstock for further conversion to glucose.
[0096] It should be appreciated that some small amount of localized
mixing of the reactor contents may occur due to the small amount of
power input associated with the addition and withdrawal of solids
and liquids from the system. For example, localized mixing may
occur due to the action of the distributors 120, wands or pump(s)
that feed the slurry into the hydrolysis reactor. For best
operation, the power required for carrying out addition and
withdrawal of solids and liquids associated with the operation of
the hydrolysis reactor does not exceed 0.1 HP/1000 gal. The power
associated with these upflow reactor functions may be between 0.001
and 0.1 HP/1000 gal, or any range therebetween. For example, the
power associated with these upflow reactor functions may be 0.001,
0.003, 0.008, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09
or 0.1 HP/1000 gal. There are no impellers, agitators, eductors, or
other equipment in the reactor specifically designed to mix the
slurry.
[0097] If more than one hydrolysis reactor is employed, the
reactors may be run in a series of two or more than two reactors,
in which case the outlet of a first reactor feeds the inlet of a
second reactor. Alternatively, the reactors may be run in parallel.
Furthermore, some of the reactors in the sequence may be run in
series, while others may be run in parallel.
[0098] It should also be appreciated that one or more other reactor
types in addition to the upflow reactor may be utilized, such as
one or more batch or continuous stirred reactors. In a non-limiting
example, the outlet of a continuous stirred reactor feeds the inlet
of an upflow reactor. As would be apparent to one of skill in the
art, other combinations of reactor types may be used in the present
invention.
[0099] In an alternative aspect of the present invention,
flocculating compounds may be added to the slurry to enhance the
efficiency of the present invention. Flocculating compounds are
typically polymers that are cationic, nonionic, anionic, or
amphoteric (containing a mixture of charged groups), or salts such
as alum. Without wishing to be bound by theory, flocculating
compounds serve to aggregate the solids within the hydrolysis
reactor to ensure a more complete exposure to the enzyme
mixture.
[0100] In practising the present invention, one flocculating
compound, or a mixture containing more than one flocculating
compound, may be used. The flocculent may be provided in any
suitable form for addition to the slurry; for example, the
flocculent may be a powder, a liquid, or a dispersion; for example,
the dispersion may be a flocculent slurried in oil or an aqueous
solution. A non-limiting example of a suitable flocculent is a
cationic polymer, more specifically, a polyacrylamide. Such
flocculants include, but are not limited to, CA4500 (SNF
Floerger.RTM., France) and Zetag.RTM. 7651 (Ciba.RTM. Specialty
Chemicals, Canada).
[0101] The amount of flocculent used will be determined by the
amount necessary to aggregate the solids in the upflow reactor. A
person of skill in the art will be able to determine the amount of
flocculent to add that would aid in solids-aggregation, without the
addition of undesirable cost to the overall process. For example,
but without wishing to be limiting, the amount of flocculent added
may be an amount in the range of about 0.1 to about 4.0 kg per
tonne solids or any amount therebetween, or about 0.5 to about 2.0
kg per tonne solids, or any amount therebetween. For example, the
amount of flocculent added may be about 0.1, 0.5, 0.75, 1.0, 1.25,
1.5, 1.75, 2.0, 2.5, 3.0, 3.5, or 4.0 kg per tonne solids.
[0102] Flocculating compounds may be added directly to the slurry,
or dispersed and diluted in water prior to addition to the slurry.
The flocculating compounds can also be mixed with the cellulase
enzymes before addition to the slurry. Dispersion of the flocculent
may help ensure uniform application of the flocculent in the
system. In a non-limiting example of the present invention, the
flocculent may be dispersed in water at a concentration of about
0.01% to about 25% by weight, or any amount therebetween; for
example, the concentration of flocculent may be about 0.01, 0.02,
0.04, 0.06, 0.08, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5,
1.0, 2.0, 3.0, 4.0, 5.0, 7.0, 10.0, 12.0, 14.0, 16.0, 18.0, 20.0,
22.0, or 25.0% by weight. The flocculent may be dispersed at this
concentration by mixing for an appropriate amount of time, for
example about 1 minute to about 1 hour. The dispersed flocculent
may then be added directly to the cellulose slurry, or be further
diluted in water to a concentration of about 0.01% to about 1.0%,
by weight, or any amount therebetween, prior to addition to the
hydrolysis reactor. For example, the concentration of the further
diluted flocculent may be 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07,
0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0% by
weight.
[0103] The present invention therefore provides a system for
hydrolyzing cellulose to glucose, cellobiose, glucose oligomers, or
a combination thereof, the system comprising a feedstock slurry
supply line (e.g. 10, 40, 100) in fluid communication with an input
to an upflow hydrolysis reactor (e.g. 110 or 110'), a solids-liquid
separator (e.g. 160 or 135) in fluid communication with the upflow
hydrolysis reactor and comprising a first output for withdrawing a
slurry comprising unhydrolyzed solids and a second output for
withdrawing a stream comprising hydrolysis product, the hydrolysis
product comprising glucose, cellobiose, glucose oligomers, or a
combination thereof, wherein the feedstock supply line, the upflow
hydrolysis reactor, or both the feedstock supply line and the
upflow hydrolysis reactor, comprise an enzyme composition
comprising cellulase enzymes and one or more than one flocculent.
Preferably, the feedstock supply line comprises a pre-treated
feedstock prepared as outlined above. Furthermore, the cellulase
enzymes within the system are present at a dosage from about 1.0 to
about 40.0 FPU per gram of cellulose of the pre-treated feedstock,
and the one or more than one flocculating compound is present at a
dosage from about 0.1 to about 4.0 kg per tonne solids of the
pre-treated feedstock.
[0104] The present invention also pertains to a use of an enzyme
composition comprising cellulase enzymes and one or more than one
flocculent, for hydrolyzing cellulose to glucose, cellobiose,
glucose oligomers, or a combination thereof, as described herein.
As this system results in about 60 to about 98% conversion of the
cellulose to glucose, the glucose may be used for the production of
ethanol. Preferably, the cellulase enzymes are produced by
Aspergillus, Humicola, Trichoderma, Bacillus, Thermobifida, or a
combination thereof. The one or more than one flocculating compound
may be selected from the group consisting of a cationic polymer, a
non-ionic polymer, an anionic polymer, an amphoteric polymer,
salts, alum, and a combination thereof. Preferably, the one or more
than one flocculating compound is the cationic polymer, for example
a polyacrylamide. The cellulase enzymes and one or more than one
flocculent as described herein may also be used for the preparation
of an enzyme composition for hydrolyzing cellulose to glucose,
cellobiose, glucose oligomers, or a combination thereof. Therefore,
the present invention also provides a use of an enzyme composition
comprising cellulase enzymes, and one or more than one flocculent,
for hydrolyzing cellulose to glucose, cellobiose, glucose
oligomers, or a combination thereof, for the production of
ethanol.
[0105] The flocculent, or the diluted flocculent may be added prior
to the point of enzyme addition (195; FIG. 1A) to the slurry, at
the point of enzyme addition (190), after the point of enzyme
addition (197), or a combination of these locations. Furthermore,
the flocculent may be added to the slurry at or near the bottom of
the hydrolysis reactor (192; FIGS. 1A, 1B and 1C), in the
midsection of the hydrolysis reactor (191), at the top of the
reactor (193), at a location outside the reactor (195, 197), or a
combination of these locations. In a non-limiting example,
flocculent may be added at the bottom (192) and in the midsection
(191) of the reactor. If the flocculent inhibits or negatively
impacts on enzyme activity, then the flocculent should not be in
direct contact with the enzyme in a manner that might be
deleterious to the enzyme prior to addition to the slurry or
hydrolysis tank.
[0106] The flocculent may be added by pumping through a series of
valves and elbows. Such a configuration may improve mixing of the
flocculent with the slurry and prevent backing up of the mixture
into the system. In an alternate example, an in-line mixer can be
used to impart turbulence to the flocculent and thereby enhance the
dispersion. Alternatively, the flocculent may be added to the
slurry (e.g. at 190) in the make-up tank 90, then pumped to the
hydrolysis reactor 110 or 110' along with the slurry and cellulase
enzymes 40.
[0107] Once in the hydrolysis reactor 110 or 110', the flocculent
proceeds to bind to the solid particles and aid in the aggregation
in the hydrolysis zone 130. Without wishing to be bound by theory,
this helps prevent the cellulose-containing particles from being
removed out the top of the reactor, from settling in the sludge
layer by keeping them suspended in the reactor, or a combination
thereof. Binding the solid particles allows greater residence time
for the cellulose-containing particles within the hydrolysis zone
(130) and results in a more complete and efficient digestion of
cellulose by the enzymes. At the top of the upflow reactor 140 the
flocculent is primarily bound to the unconverted solids, and exits
the reactor (150; FIG. 1A) with the particles.
[0108] It is contemplated that glucose produced by the hydrolysis
of cellulose from the pre-treated feedstock that leaves the reactor
110 or 110' through line 30 may be fermented to ethanol (170).
Fermentation of glucose and other sugars to ethanol may be
performed by conventional processes known to those skilled in the
art, and may be effected by a variety of microorganisms including
yeast and bacteria or genetically modified microorganisms, for
example, but not limited those described in WO 95/13362, WO
97/42307, or Alcohol production from Cellulosic Biomass: The Iogen
Process (in: The Alcohol Textbook, Nottingham University Press,
2000; which are herein incorporated by reference). Ethanol
production and recovery are performed using well-established
processes known to one of skill in the art in the alcohol
industry.
[0109] As indicated previously, the upflow settling reactor is
suited to the enzymatic conversion of cellulose to glucose.
However, this type of system can be used to convert cellulose to
other products, including cellobiose (preferably if .beta.G is
omitted from the cellulase) and glucose oligomers (preferably if
CBH and .beta.G are omitted from the cellulase).
[0110] The method of the present invention increases the length of
time that the solids are in the reactor, which increases the
contact time between the cellulase enzymes and cellulose because
the enzymes remain bound to the cellulose. This in turn increases
the efficiency of the hydrolysis process by increasing the degree
of cellulose conversion obtained in a hydrolysis reactor of a given
size. Thus, the costs of enzymatic hydrolysis are minimized.
Furthermore, the upflow settling method does not require agitation
within the reactor, saving the power and equipment costs associated
with mixing.
[0111] The above description is not intended to limit the claimed
invention in any manner. Furthermore, the discussed combination of
features might not be absolutely necessary for the inventive
solution. The present invention will be further illustrated in the
following examples. However, it is to be understood that these
examples are for illustrative purpose only, and should not be used
to limit the scope of the present invention in any manner.
EXAMPLES
Example 1
Large Scale Upflow Hydrolysis Reactor
[0112] Pre-treated wheat straw is prepared using the method of U.S.
Pat. No. 4,461,648 (Foody, which is incorporated herein by
reference). The pre-treated material is an aqueous slurry of 7.8%
undissolved solids, at a temperature of 70.degree. C., and a mass
flow rate of 553 t/hr. This aqueous slurry is cooled to 60.degree.
C. in a heat exchanger (20), against the product stream (30). The
60.degree. C. slurry is then cooled to a final temperature of
50.degree. C. by cold water at heat exchanger (45). The slurry is
pumped into the hydrolysis make-up tank (90; volume 86,000 gallons)
along with cellulase enzymes (70; 5 FPU/g cellulose) and ammonia
(80; 1200 grams per tonne wet slurry), to adjust the pH to 4.5 to
5.0. The contents of the hydrolysis make-up tank are mixed for a
residence time of 40 minutes and then the combined stream (100) is
pumped out of the make-up tank and down a pipe through the middle
(105) of the hydrolysis tank (110). The hydrolysis tank is jacketed
with 15 psig steam used to maintain 50.degree. C. At the bottom of
the hydrolysis tank, the slurry is pumped upward and dispersed
(120) across the width of the hydrolysis tank.
[0113] The hydrolysis tank has a volume of 4.6 million gallons. The
liquid flows upward in the hydrolysis tank faster than the solids,
which settle to a concentration of about 10% throughout the tank.
The cellulose is held up in the tank 188 hrs, which is long enough
to convert 92% of the cellulose to glucose. The aqueous sugar
stream and the unhydrolyzed solids (150) flow out of the top of the
tank and are pumped to the settling tank (160). In the settling
tank, which had a volume of 1.12 million gallons, the solids settle
to the bottom to a concentration of 10% and are pumped out via line
162 to a lignin filter press 165 to recover sugar from the solids
by pressing and washing. The sugar from this stream is combined
with the sugar stream from the top of the settler, which is then
sent for fermentation to ethanol (170).
Example 2
Upflow Hydrolysis with Trichoderma Cellulase Enzyme
[0114] Wheat straw was pre-treated using the method of U.S. Pat.
No. 4,461,648 (Foody, which is incorporated herein by reference).
The pre-treated material was slurried in water at a concentration
of 3.7% undissolved solids and the pH was adjusted to 5.5 with 30%
sodium hydroxide. The undissolved solids were 55% cellulose. The
slurry was pumped at a rate of 40 liters per minute into the bottom
of a vertical hydrolysis reactor (110; FIG. 1C). This corresponds
to an upward flow velocity of 0.7 feet/hr. The tower volume was
144,700 liters, of which the hydrolysis zone (130) was the lower
115,000 liters (a height of 34.4 feet) and the top 29,700 liters
was a clarifier (135). The diameter of the tower was 3.8 meters and
the height was 13.5 meters (44.3 feet). The temperature of the
slurry was 55.degree. C. upon entry in the reactor, and gradually
decreased to 50.degree. C. near the top of the reactor. The
cellulase enzyme, obtained from Trichoderma (available from Iogen
Bioproducts, Ottawa) was added to the slurry in the hydrolysis make
up tank (90; FIG. 1A) at a dosage of 36 FPU per gram cellulose,
added to the line before entering the tower as in Example 1.
[0115] The slurry containing pre-treated solids and cellulase was
pumped into the bottom of the tower 105 and hydrolysis took place
as the slurry flowed up the tower. At the top of the hydrolysis
zone, which was 79% of the volume of the tower, the slurry was
transferred to a clarifier zone 135. A stream containing settled
solids was withdrawn (162; FIG. 1C) at the interface of the
hydrolysis zone and the clarifier zone. A second stream 30
containing glucose in the aqueous phase with little undissolved
solids was collected at the top of the clarifier zone.
[0116] The solids profile in the reactor over the course of the run
is shown in FIG. 2. At a level of 1 foot above the bottom, the
undissolved solids settled to a concentration of 8% to 14%, by
weight. This was significantly more concentrated than the feed
concentration of 3.7% undissolved solids. At points higher than
this, the solids concentration was lower.
[0117] The cellulose conversion profile is shown in FIG. 3. The
degree of cellulose conversion was 73% to 83% early in the run, and
by the end was 95% at the heights of 15 feet and 24 feet. The
glucose concentration in the stream flowing out of the upflow
reactor was 25 g/L. This represents a good level of cellulose
conversion and glucose production obtained without mixing a
reactor, providing shear, or otherwise moving the material beyond
pumping it slowly up the tower.
Example 3
Upflow Hydrolysis with Trichoderma Cellulase Enzyme in Presence of
Flocculating Compound
[0118] A hydrolysis of pre-treated wheat straw was carried out as
described in Example 2 with 4.4% undissolved solids, except that a
flocculent was added to improve the settling of the solids. A
cationic polymer, CA4500 (SNF Floerger.RTM., France), was added at
a dosage of 2 kg per tonne undissolved solids and dispersed inline
upon addition after the point of enzyme addition to the slurry.
[0119] The solids profile in the reactor over the course of the run
is shown in FIG. 4. At a level of 1 foot above the bottom, the
undissolved solids settled to a concentration of 6% to 10%, by
weight, similar to that observed in Example 2, and was
significantly more concentrated than the feed concentration of 4.4%
undissolved solids. At points higher than this within the reactor,
the solids concentration was 5.5% to 8%. This indicated the
flocculent was effective at aggregating and settling the
solids.
[0120] The cellulose conversion profile is shown in FIG. 5. The
degree of cellulose conversion was 65% to 85% early in the run, and
by the end was 85% to 92% at the heights of 15 feet and 24 feet.
The glucose concentration in the stream flowing out of the reactor
was 27 g/L. This represents a good level of cellulose conversion
obtained without mixing a reactor, providing shear, or otherwise
moving the material beyond pumping it slowly up the tower.
Example 4
Cellulose Conversion at Various Enzyme Levels in Presence and
Absence of Flocculating Compound
[0121] The hydrolysis reactions described in Examples 2 and 3
showed a similar final glucose concentration, which may be due to
the presence of an excess of enzymes. In order to determine the
effect of flocculent on hydrolysis efficiency, hydrolysis was
performed in the presence and absence of flocculent at various
enzyme dosages.
[0122] A hydrolysis of pre-treated wheat straw is carried out as
described in Examples 2 and 3, except that the amount of cellulase
enzyme added is varied. A hydrolysis with 8, 12, 16, 20, 24, 28,
32, and 36 FPU enzyme is carried out in the presence and absence of
flocculent for 48 hours. The cellulose conversion (in %) is
measured and shown in FIG. 6.
[0123] As shown in FIG. 6, at lower enzyme dosages, the use of
flocculent results in increased cellulose conversion. This
represents an overall saving in the cost of cellulose
conversion.
* * * * *