U.S. patent application number 11/620829 was filed with the patent office on 2007-07-05 for secondary liquefaction in ethanol production.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Claus Felby, Christopher Veit.
Application Number | 20070155001 11/620829 |
Document ID | / |
Family ID | 27439829 |
Filed Date | 2007-07-05 |
United States Patent
Application |
20070155001 |
Kind Code |
A1 |
Veit; Christopher ; et
al. |
July 5, 2007 |
Secondary Liquefaction in Ethanol Production
Abstract
The invention relates to a method of producing ethanol by
fermentation, said method comprising a secondary liquefaction step
in the presence of a thermostable acid alpha-amylase or a
thermostable maltogenic acid alpha-amylase.
Inventors: |
Veit; Christopher; (Wake
Forest, NC) ; Felby; Claus; (Vekso, DK) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE
SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes A/S
Bagsvaerd
NC
Novozymes North America, Inc.
Franklinton
|
Family ID: |
27439829 |
Appl. No.: |
11/620829 |
Filed: |
January 8, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10416393 |
May 9, 2003 |
|
|
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PCT/DK01/00737 |
Nov 9, 2001 |
|
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11620829 |
Jan 8, 2007 |
|
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60252213 |
Nov 21, 2000 |
|
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60256015 |
Dec 15, 2000 |
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Current U.S.
Class: |
435/161 ;
435/204; 435/254.21; 435/483 |
Current CPC
Class: |
C12P 19/14 20130101;
Y02E 50/10 20130101; Y02E 50/17 20130101; C12P 7/06 20130101 |
Class at
Publication: |
435/161 ;
435/254.21; 435/483; 435/204 |
International
Class: |
C12P 7/06 20060101
C12P007/06; C12N 9/32 20060101 C12N009/32; C12N 15/74 20060101
C12N015/74; C12N 1/21 20060101 C12N001/21; C12N 1/18 20060101
C12N001/18 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 10, 2000 |
DK |
PA 2000 01676 |
Dec 11, 2000 |
DK |
PA 2000 01854 |
Claims
1-58. (canceled)
59. A method of producing ethanol, comprising; a) liquefaction of a
starch containing material in the presence of an alpha-amylase; b)
jet cooking the liquefied starch, c) a secondary liquefaction in
the presence of a thermostable acid alpha-amylase; wherein the
thermostable acid alpha-amylase maintains more than 90% of its
activity for 1 hour at 70.degree. C. using a DE 12 alpha-amylase
TTC liquefied corn starch at 30% dry substance as substrate, pH
5.5, 0.1 M citrate buffer and 4.3 mM Ca+.sup.2+; d)
saccharification; and e) fermentation to produce ethanol; wherein
steps (a), (b), (c), and (d) are performed in the order (a), (b),
(c), (d), and wherein step (e) is performed simultaneously to or
following step (d).
60. The method of claim 59, further comprising recovering the
ethanol.
61. The method of claim 59, further comprising a
pre-saccharification step which is performed after the secondary
liquefaction in step (c) and before step (d), wherein the
pre-saccharification comprises treating the starch with a
glucoamylase at a temperature in the range of 30-65.degree. C.
62. The method of claim 59, wherein the starch containing material
is selected from the group consisting of tubers, roots, whole
grain, and any combination thereof.
63. The method of claim 59, wherein the starch containing material
is obtained from cereal.
64. The method of claim 59, wherein the starch containing material
is selected from the group consisting of corns, cobs, wheat,
barley, rye, milo, potatoes, and any combination thereof.
65. The method of claim 59, wherein the starch containing material
is whole grain selected from the group consisting of corn, wheat,
barley, and any combination thereof.
66. The method of claim 59, wherein the starch containing material
is whole grain and said method comprises a step of milling the
whole grain before step (a).
67. The method of claim 59, wherein the starch containing material
is obtained by a process comprising milling of whole grain.
68. The method of claim 59, further comprising prior to step (a)
the steps of: i) milling of whole grain; ii) forming a slurry
comprising the milled grain and water to obtain the starch
containing material.
69. The method of claim 68, wherein the milling is a dry milling
step.
70. The method of claim 68, wherein the milling is a wet milling
step.
71. The method of claim 59, wherein the starch-containing material
is a side stream from starch processing.
72. The method of claim 59, further comprising: (f) distillation to
obtain the ethanol; wherein the fermentation in step (e) and the
distillation in step (f) are carried out simultaneously or
separately/sequential.
73. The method of claim 72; wherein the starch containing material
is milled whole grain, said method further comprising the steps of:
(g) separation of whole stillage produced by the distillation in
step (f) into wet grain and thin stillage; and (h) recycling the
thin stillage to the starch containing material prior to step
(a).
74. The method of claim 59, wherein the fermentation in step (e) is
performed using a yeast.
75. The method of claim 59, wherein the fermentation is carried out
in the presence of glucoamylase, phytase and/or protease.
76. The method of claim 75, wherein the protease is an acid
protease, a neutral protease or an alkaline protease.
77. A method for producing ethanol, comprising: a) liquefaction of
a starch containing material in the presence of an alpha-amylase;
b) jet cooking the liquefied starch; c) a secondary liquefaction in
the presence of a thermostable maltogenic acid alpha-amylase;
wherein the thermostable maltogenic acid alpha-amylase maintains
more than 90% of its activity for 1 hour at 70.degree. C. using a
DE 12 alpha-amylase TTC liquefied corn starch at 30% dry substance
as substrate, pH 5.5, 0.1 M citrate buffer and 4.3 mM Ca+.sup.2; d)
saccharifaction; and e) fermentation to produce ethanol; wherein
steps (a), (b), (c) and (d) are performed in the order (a), (b),
(c), (d) and wherein step (e) is performed simultaneously to or
following step (d).
78. The method of claim 77, wherein the thermostable maltogenic
acid alpha-amylase having an amino acid sequence which is at least
95% identical to SEQ ID NO. 1.
79. The method of claim 77, wherein the thermostable maltogenic
acid alpha-amylase is SEQ ID NO: 1.
80. The method of claim 77, further comprising recovering the
ethanol.
81. The method of claim 77, further comprising a
pre-saccharification step which is performed after the secondary
liquefaction in step (c) and before step (d), wherein the
pre-saccharification comprises treating the starch with a
glucoamylase at a temperature in the range of 30-65.degree. C.
82. The method of claim 77, wherein the starch containing material
is selected from the group consisting of tubers, roots, whole
grain, and any combination thereof.
83. The method of claim 77, wherein the starch containing material
is obtained from cereal.
84. The method of claim 77, wherein the starch containing material
is selected from the group consisting of corns, cobs, wheat,
barley, rye, milo, potatoes and any combination thereof.
85. The method of claim 77, wherein the starch containing material
is whole grain selected from the group consisting of corn, wheat,
barley, and any combination thereof.
86. The method of claim 77, wherein the starch containing material
is whole grain and said method comprises a step of milling the
whole grain before step (a).
87. The method of claim 77, wherein the starch containing material
is obtained by a process comprising milling of whole grain.
88. The method of claim 77, further comprising prior to step (a)
the steps of: i) milling of whole grain; ii) forming a slurry
comprising the milled grain and water to obtain the starch
containing material.
89. The method of claim 88, wherein the milling is a dry milling
step.
90. The method of claim 88, wherein the milling is a wet milling
step.
91. The method of claim 77, wherein the starch-containing material
is a side stream from starch processing.
92. The method of claim 77, further comprising: (f) distillation to
obtain the ethanol: wherein the fermentation in step (e) and the
distillation in step (f) are carried out simultaneously or
separately/sequential.
93. The method of claim 92; wherein the starch containing material
is milled whole grain, said method further comprising the steps of:
(g) separation of whole stillage produced by the distillation in
step (f) into wet grain and thin storage; and (h) recycling the
thin stillage to the starch containing material prior to step
(a).
94. The method of claim 77, wherein the fermentation in step (e) is
performed using a yeast.
95. The method of claim 77, wherein the fermentation is carried out
in the presence of glucoamylase, phytase and/or protease.
96. The method of claim 95, wherein the protease is an acid
protease, a neutral protease or an alkaline protease.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/416,393 filed May 9, 2003, which is a 35 U.S.C. 371 national
application of PCT/DK01/00737 filed Nov. 9, 2001, which claims
priority or the benefit under 35 U.S.C. 119 of Danish application
nos. PA 2000 01676 and PA 2000 01854 filed Nov. 10, 2000 and Dec.
11, 2000, respectively, and U.S. provisional application Nos.
60/252,213 and 60/256,015 filed Nov. 21, 2000 and Dec. 15, 2000,
respectively, the contents of which are fully incorporated herein
by reference,
FIELD OF THE INVENTION
[0002] The invention relates to a process for producing
ethanol.
BACKGROUND OF THE INVENTION
[0003] Ethanol has widespread application as an industrial
chemical, gasoline additive or straight liquid fuel. As a fuel or
fuel additive, ethanol dramatically reduces air emissions while
improving engine performance. As a renewable fuel, ethanol reduces
national dependence on finite and largely foreign fossil fuel
sources while decreasing the net accumulation of carbon dioxide in
the atmosphere. Fermentation processes are used for the production
of ethanol. There are a large number of disclosures concerning
production of alcohol by fermentation, among which are, e.g., U.S.
Pat. No. 5,231,017 and CA 1,143,677. EP 138428 mentions an
Aspergillus niger alpha-amylase preparation for use in liquefaction
in the alcohol industry.
[0004] There is a need for further improvement of ethanol
manufacturing processes.
SUMMARY OF THE INVENTION
[0005] The invention relates to a method of producing ethanol by
fermentation, said method comprising a secondary liquefaction step
in the presence of a thermostable acid alpha-amylase or a
thermostable maltogenic acid alpha-amylase. In particular, is
provided an improved method for production of ethanol based on
whole grain as the starch containing starting material.
[0006] Thus, the invention relates to a method of producing ethanol
from a starch containing material, preferably based on whole grain,
said method comprising the steps of: (a) liquefaction of a starch
containing material in the presence of an alpha-amylase; (b) jet
cooking, (c) liquefaction in the presence of a thermostable acid
alpha-amylase or a thermostable maltogenic acid alpha-amylase, and
(d) saccharification and fermentation to produce ethanol, wherein
steps (a), (b), (c) and (d) are performed in the order (a), (b),
(c), (d).
[0007] The process of the invention may also comprise one or more
additional steps, before, in between and/or after step (a), (b),
(c) and (d), such as, e.g, recovering of the ethanol after step
(d).
[0008] The invention also relates to products obtained or
obtainable by the processes of the invention and to the use of such
products, e.,g., as fuel alcohol or an additive.
[0009] The invention in further aspects relates to use of a
thermostable acid alpha-amylase or a thermostable maltogenic acid
alpha-amylase in a secondary liquefaction step in a process for
production of ethanol, particularly from whole grain.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 illustrates a process flow diagram for the
preparation of ethanol in accordance with one embodiment of the
invention. The primary liquefaction step may be performed by the
presence of the enzyme alpha-amylase in the slurry tank while the
secondary liquefaction step is termed "liquefaction" on the
diagram.
DETAILED DESCRIPTION OF THE INVENTION
Ethanol Production
[0011] The present invention provides a process of producing
ethanol, in particular improvement of the secondary liquefaction
step in a process of producing ethanol from dry milled whole
grain.
[0012] The invention provides a method of producing ethanol by
fermentation, said method comprising a secondary liquefaction step
in the presence of a thermostabte acid alpha-amylase or a
thermostabte maltogenic acid alpha-amylase. A particularly
interesting embodiment relates to a fermentation process of the
invention where the starting material is whole grain which has been
partitioned into finer parts, preferably by dry milling.
[0013] Thus, the invention in one aspect relates to a method of
producing ethanol from a starch containing material, said method
comprising the steps of:
[0014] a) liquefaction of a starch containing material in the
presence of an alpha-amylase;
[0015] b) jet cooking,
[0016] c) liquefaction in the presence of a thermostable acid
alpha-amylase;
[0017] d) saccharification and fermentation to produce ethanol;
wherein steps (a), (b), (c) and (d) are performed in the order (a),
(b), (c), (d).
Raw Material
[0018] In one embodiment, the starch containing material is
selected from the group consisting of: tubers roots and whole
grain; and any combinations of the forgoing. In one embodiment, the
starch containing material is obtained from cereals. The starch
containing material may, e.g., be selected from the groups
consisting of corns, cobs, wheat, barley, cassava, sorghum, rye,
milo and potatoes; or any combination of the forgoing.
[0019] In the ethanol processes of the invention, the starting raw
material is preferably whole grain or at least mainly whole grain.
A wide variety of starch containing whole grain crops may be used
as raw material including: corn (maize), milo, potato, cassava,
sorghum, wheat, and barley.
[0020] Thus, in one embodiment, the starch containing material is
whole grain selected from the group consisting of corn (maize),
milo, potato, cassava, sorghum, wheat, and barley; or any
combinations thereof. In a preferred embodiment, the starch
containing material is whole grain selected from the group
consisting of corn, wheat and barley or any combinations
thereof.
[0021] The raw material may also consist of or comprise a side
stream from starch processing--e.g., C6 carbohydrate containing
process streams that are not suited for production of syrups. In
other embodiments, the raw material does not consist of or comprise
a side stream from starch processing.
Process Steps
[0022] The main process steps of the present invention may in one
embodiment be described as separated into the following main
process stages: milling (when whole grain is used as raw material),
primary liquefaction, heat-treatment as provided by jet-cooking,
secondary liquefaction, saccharification, fermentation,
distillation.
[0023] In a preferred embodiment, the method of the invention
comprises prior to step (a) the steps of: i) dry milling of whole
grain; and ii) forming a slurry comprising the milled grain and
water.
[0024] The individual process steps of alcohol production may be
performed batch wise or as a continuous flow. For the invention
processes where all process steps are performed batch wise, or
processes where all process steps are performed as a continuous
flow, or processes where one or more process step(s) is(are)
performed batch wise and one or more process step(s) is(are)
performed as a continuous flow, are equally contemplated.
[0025] The cascade process is an example of a process where one or
more process step(s) is(are) performed as a continuous flow and as
such contemplated for the invention. For further information on the
cascade process and other ethanol processes consult The Alcohol
Textbook. Ethanol production by fermentation and distillation. Eds.
T. P. Lyons, D. R. Kesall and J. E. Murtagh. Nottingham University
Press, 1995.
Milling
[0026] Thus, in a preferred embodiment of the process of the
invention, the starch containing material is whole grain and the
method comprises a step of milling the whole grain before step (a),
i.e., before the primary liquefaction. In other words, the
invention also encompasses processes of the invention, wherein the
starch containing material is obtainable by a process comprising
milling of whole grain, preferably dry milling, e.g., by hammer or
roller mils. Grinding is also understood as milling.
[0027] In particular embodiments, the process of the invention
further comprises prior to the primary liquefaction step (i.e.,
prior to step (a), the steps of:
[0028] i. milling of whole grain.
[0029] ii. forming a slurry comprising the milled grain and water
to obtain the starch containing material.
[0030] The whole grain is milled in order to open up the structure
and allowing for further processing. Two processes of milling are
normally used in alcohol production; wet and dry milling. The term
"dry milling" denotes milling of the whole grain. In dry milling
the whole kernel is milled and used in the remaining part of the
process. Wet milling gives a good separation of germ and meal
(starch granules and protein) and is with a few exceptions applied
at locations where there is a parallel production of syrups.
[0031] Thus, in a preferred embodiment of the invention, dry
milling is used since the secondary liquefaction step is
advantageously included in dry milling processes for producing
ethanol.
Liquefaction
[0032] In the liquefaction process the starch containing material,
preferably in the form of milled whole grain raw material, is
broken down (hydrolyzed) into maltodextrins (dextrins). In a
preferred embodiment, in the primary liquefaction process of the
invention the starch containing material, preferably in the form of
milled whole grain raw material, is hydrolyzed to a DE (an
abbreviation for dextrose equivalent) higher than 4. DE stands for
"Dextrose equivalents" and is a measure of reducing ends on C6
carbohydrates. Pure glucose has DE of 100. Glucose (also called
dextrose) is a reducing sugar. Whenever an amylase hydrolyzes a
glucose-glucose bond in starch, two new glucose end-groups are
exposed, At least one of these can act as a reducing sugar.
Therefore the degree of hydrolysis can be measured as an increase
in reducing sugars. The value obtained is compared to a standard
curve based on pure glucose--hence the term dextrose equivalent.
The DE may, e.g., be measured using Fehlings liquid by forming a
copper complex with the starch using pure glucose as a reference,
which subsequently is quantified through iodometric titration. In
other words, DE (dextrose equivalent is defined as the amount of
reducing carbohydrate (measured as dextrose-equivalents) in a
sample expressed as w/w % of the total amount of dissolved dry
matter. It may also be measured by the neocuproine assay (Dygert,
Li Floridana, 1965, Anal. Biochem. No 368). The principle of the
neocuproine assay is that CuSO.sub.4 is added to the sample,
Cu.sup.2+ is reduced by the reducing sugar and the formed
neocuproine complex is measured at 450 nm.
[0033] The hydrolysis may be carried out by acid treatment or
enzymatically. The liquefaction is preferably carried out by
enzymatic treatment, preferably an alpha-amylase treatment. In one
embodiment, the liquefaction is carried out by preparing a slurry
comprising milled raw material, preferably milled whole grain, and
water, heating the slurry to between 60-95.degree. C., preferably
80-85.degree. C., and the enzyme(s) is (are) added to initiate
liquefaction (thinning). This is also termed the "primary
liquefaction", ie., it occurs before the process step of
jet-cooking (step (b)). The liquefaction in the process of the
invention is performed at any conditions ie., e.g., pH, temperature
and time) found suitable for the enzyme in question. Within the
scope is a method of the invention, wherein the liquefaction in
step (a) is performed at 60-95.degree. C. for 10-120 min,
preferably at 75-90.degree. C. for 15-40 min. In one embodiment,
the liquefaction in step (a) is performed at a pH in the range of
about pH 4-7, preferably pH about 4.5-6.5. The pH of the slurry may
by adjusted or not, depending on the properties of the enzyme(s)
used. Thus, in one embodiment the pH is adjusted, e.g., about 1
unit upwards, e.g., by adding NH.sub.3. The adjusting of pH is
advantageously done at the time when the alpha-amylase is added. In
a preferred embodiment, the pH is not adjusted and the
alpha-amylase has a corresponding suitable pH-activity profile,
such as being active at a pH about 4.
[0034] After the primary liquefaction step, the slurry is
preferably jet-cooked at appropriate conditions to further
gelatinize the starch, such as, e.g., at a temperature between
95-140.degree. C., preferably 105-125.degree. C. to ensure the
gelanitization. In one embodiment, the jet-cooking in step (b) is
performed under conditions 1-10 min, 105-150.degree. C. and e.g.,
pH 4-7; preferably for 1-5 min, 105-120.degree. C. and e.g., pH
4.5-6; such as, e.g., about 5 min, about 105.degree. C., and e.g.,
pH about 5.0. As used herein, generally, the term jet-cooking also
covers any other method which can be used to obtain a similar
result.
[0035] Then the slurry is preferably cooled, eg., to about
60-95.degree. C. and more enzyme(s) is (are) added to obtain the
final hydrolysis; the later is termed "secondary liquefaction",
i.e., liquefaction after jet-cooking which by the process of the
invention is obtained by addition of at least a thermostable acid
alpha-amylase or a thermostable maltogenic acid alpha-amylase.
[0036] The secondary liquefaction in step (c) is performed at
suitable conditions (pH, temperature and process time). The
secondary liquefaction in step (c) may e.g., be performed at
60-95.degree. C. for 10-120 min, preferably at 70-85.degree. C. for
15-80 min and at pH 4.5-6.5. In one embodiment, the pH is not
adjusted for the secondary liquefaction. In preferred embodiment,
the pH during the secondary liquefaction is at most about 5.
[0037] In one preferred embodiment, in the secondary liquefaction
step in the method of the invention the starch containing material,
e.g., obtained from dry milled whole grain, is hydrolyzed to a DE
in the range of about 5-15, e.g., 8-15, 8-14, such as, such as a DE
in the range about 10-14., e.g., about 10-12.
[0038] The liquefaction process (both the primary and the secondary
liquefaction process) is carried out at a suitable pH, e.g., at a
pH in the range 4.5-6.5, such as at a pH between about 5 and about
6.
[0039] Milled and liquefied whole grain are also known as mash.
[0040] Saccharification
[0041] To produce low molecular sugars DP.sub.1-2 that can be
metabolized by yeast, the maltodextrin from the liquefaction is
preferably further hydrolyzed; this is also termed
"saccharification". The hydrolysis may be done enzymatically by the
presence of a glucoamylase. An alpha-glucosidase and/or an acid
alpha-amylase may also be present in addition to the
glucoamylase.
[0042] A full saccharification step may last up to 72 hours.
However, the saccharification and fermentation (SSF) may be
combined, and in some embodiments of the invention a
pre-saccharification step of 1-4 hours may be included.
Pre-saccharification is carried out at any suitable process
conditions. In a preferred embodiment, the pre-saccharification is
carried out at temperatures from 30-65.degree. C. such as around
60.degree. C., and at, e.g., a pH in the range between 4-5,
especially around pH 4.5.
[0043] Thus in one embodiment, the method of the invention may
further comprise a pre-saccharification step, as described herein,
which is performed after the secondary liquefaction step (c) and
before step (d).
[0044] In other embodiments, the process of the invention does not
comprise a pre-saccharification and the saccharification is
essentially only performed during fermentation, e.g., by the
presence of a glucoamylase and optionally phytase.
Fermentation
[0045] The microorganism used for the fermentation is added to the
mash and the fermentation is ongoing until the desired amount of
ethanol is produced; this may, e.g., be for 24-96 hours, such as
35-60 hours. The temperature and pH during fermentation is at a
temperature and pH suitable for the microorganism in question, such
as, e.g, in the range about 26-34.degree. C., e.g., about
32.degree. C., and at a pH, e.g., in the range about pH 3-6, e.g.,
about pH 4-5.
[0046] In a preferred embodiment, a simultaneous saccharification
and fermentation (SSF) process is employed where there is no
holding stage for the saccharification, meaning that yeast and
saccharification enzyme is added essentially together. In one
embodiment, when doing SSF is introduced a pre-saccharification
step at a temperature above 50.degree. C., just prior to the
fermentation.
[0047] In one embodiment, the fermentation is carried out in the
presence of glucoamylase and/or protease.
[0048] In a further embodiment, the addition of a thermostable acid
alpha-amylase or a thermostable maltogenic acid alpha-amylase in
the secondary liquefaction step in the process of the invention may
make it possible to substitute the presence of glucoamylase
activity in the fermentation step. Thus, one embodiment relates to
a process of the invention for the production of ethanol, without
addition of glucoamylase in the fermentation step or prior to the
fermentation step.
Distillation
[0049] The method of the invention may further comprise recovering
of the ethanol; hence the alcohol may be separated from the
fermented material and purified. Following the fermentation the
mash may be distilled to extract the ethanol. Ethanol with a purity
of up to, e.g., about 96 vol. % ethanol can be obtained by the
process of the invention.
[0050] Thus, in one embodiment, the method of the invention further
comprises the step of: (e) distillation to obtain the ethanol. The
fermentation in step (d) and the distillation in step (e) may be
carried out simultaneously and/or separately/sequentially;
optionally followed by one or more process steps for further
refinement of the ethanol.
By-Products from Distillation and Recycling:
[0051] In one embodiment of the process of the invention, the
aqueous by-product ("Whole Stillage", cf. FIG. 1) from the
distillation process is separated into two fractions, for instance
by centrifugation: 1) "Wet Grain" (solid phase, see FIG. 1), and 2)
"Thin Stillage" (Supernatant, see FIG. 1).
[0052] In one embodiment, the starch containing material entering
the process of the invention is dry milled whole grain, and the
method of the invention comprising steps (a), (b), (c), (d), and
(e) further comprises the steps of:
[0053] (f) separation of Whole Stillage produced by of the
distillation in step (e), into wet grain and Thin stillage; and
[0054] (g) recycling Thin stillage to the starch containing
material prior to the primary liquefaction of step (a).
[0055] In one embodiment, in the process of the invention, the Thin
Stillage (cf. FIG. 1) is recycled to the milled whole grain
slurry.
[0056] The Wet Grain fraction may be dried, typically in a drum
dryer. The dried product is referred to as "Distillers Dried
Grains" (see FIG. 1), and can be used, e.g., as animal feed.
[0057] The Thin Stillage fraction may be evaporated providing two
fractions (see FIG. 1):
[0058] (i) a Condensate fraction of 4-6% DS (mainly of starch,
proteins, and cell wall components), and
[0059] (ii) a Syrup fraction, mainly consisting of limit dextrins
and non fermentable sugars, which may be introduced into a dryer
together with the Wet Grains (from the Whole Stillage separation
step) to provide a product referred to as "Distillers Dried Grain",
which also can be used as animal feed.
[0060] "Thin Stillage" is the term used for the supernatant of the
centrifugation of the Whole Stillage (see FIG. 1). Typically, the
Thin Stillage contains 4-6% DS (mainly starch and proteins) and has
a temperature of about 60-90.degree. C.
[0061] In another embodiment Thin Stillage is not recycled, but the
condensate stream of evaporated Thin Stillage is recycled to the
slurry containing the milled whole grain to be jet cooked.
[0062] One embodiment of the invention relates to a method of
producing ethanol, said method comprising the steps of:
[0063] a) primary liquefaction of a starch containing material in
the presence of alpha-amylase activity;
[0064] b) jet cooking the material of step(a);
[0065] c) secondary liquefaction of the material of step (b) in the
presence of a thermostable acid alpha-amylase or a thermostable
maltogenic acid alpha-amylase; and
[0066] d) saccharification and fermentation to produce ethanol;
wherein steps (a) (b), (c) and (d) are performed in the order (a),
(b), (c), (d).
[0067] Optionally the saccharification and fermentation may be
performed in separate steps. Thus the invention also relates to a
method of producing ethanol, said method comprising the steps
of:
[0068] a) primary liquefaction of a starch containing material in
the presence of alpha-amylase activity,
[0069] b) jet cooking the material of step(a);
[0070] c) secondary liquefaction of the material of step (b) in the
presence of a thermostable acid alpha-amylase or a thermostable
maltogenic acid alpha-amylase, and
[0071] d) saccharification;
[0072] e) fermentation to produce ethanol;
wherein steps (a), (b), (c) and (d) are performed in the order (a),
(b), (c), (d) and wherein (e) is performed simultaneously to or
following (d).
[0073] The invention in also relates to a method of producing
ethanol, said method comprising the steps of:
[0074] a) dry milling of whole grain;
[0075] b) forming a slurry comprising the milled grain and
water;
[0076] c) liquefaction in the presence of an alpha-amylase;
[0077] d) jet cooking;
[0078] e) liquefaction in the presence of a thermostable acid
alpha-amylase or a thermostable maltogenic acid alpha-amylase;
[0079] f) saccharification in the presence of phytase and/or
glucoamylase and fermentation to produce ethanol;
[0080] g) distillation;
optionally followed by one or more process steps for further
refinement of the ethanol;
wherein steps (a), (b), (c), (d), (e) and (f) are performed in the
order (a), (b), (c), (d), (e), (f); and
wherein step (g) is performed simultaneously with step (f) and/or
after step (f).
[0081] In a further embodiment, the method of the invention for
producing ethanol may comprise the following steps:
[0082] a) milling whole grain;
[0083] b) making a slurry comprising the milled whole grain and
water;
[0084] c) liquefying in the presence of an alpha-amylase;
[0085] d) saccharifying in the presence of a glucoamylase and
fermenting using a microorganism,
[0086] f) distillation of the fermented material, providing two
streams 1) alcohol and 2) Whole Stillage;
[0087] (g1) recovering alcohol for further refinement;
optionally,
[0088] (g2) separating the Whole Stillage into two fractions of, 1)
Wet Grain, and 2) Thin Stillage;
[0089] (h1) the whole grain fraction is dried to provide a protein
containing product, and optionally
[0090] (h2) the Thin Stillage is evaporated providing two streams:
1) condensate stream and 2) syrup;
wherein the Thin Stillage and optionally the condensate from step
(h2) is recycled to step (b) with or without further treatment.
Enzyme Activities
Alpha-Amylase
[0091] The "primary liquefaction" is preferably performed in the
presence of an alpha-amylase, e.g., derived from a micro-organism
or a plant. Preferred alpha-amylases are of fungal or bacterial
origin Bacillus alpha-amylases (often referred to as "Termamyl-like
alpha-amylases"), variant and hybrids thereof, are specifically
contemplated according to the invention. Well-known Termamyl-like
alpha-amylases include alpha-amylase derived from a strain of B.
licheniformis (commercially available as Termamyl.TM.), B.
amyloliquefaciens, and B. stearothermophilus alpha-amylase. Other
Termamyl-like alpha-amylases include alpha-amylase derived from a
strain of the Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 or
DSM 9375, all of which are described in detail in WO 95/26397, and
the alpha-amylase described by Tsukamoto et al., Biochemical and
Biophysical Research Communications, 1988, 151: 25-31. In the
context of the present invention a Termamyl-like alpha-amylase is
an alpha-amylase as defined in WO 99/19467 on page 3, line 18 to
page 6, line 27. Contemplated variants and hybrids are described in
WO 96/23874, WO 97/41213, and WO 99/19467, and include the Bacillus
stearothermophilus alpha-amylase (BSG alpha-amylase) variant,
alpha-amylase TTC, having the following mutations
delta(181-182)+N193F (also denoted I181*+G182*+N193F) compared to
the wild-type amino acid sequence set forth in SEQ ID NO: 3
disclosed in WO 99/19467. Contemplated alpha-amylases derived from
a strain of Aspergillus includes Aspergillus oryzae and Aspergillus
niger alpha-amylases.
[0092] Commercial alpha-amylase products and products containing
alpha-amylases include TERMAMYL.TM. SC, FUNGAMYL.TM., LIQUOZYME.TM.
SC and SAN.TM. SUPER, (Novozymes A/S, Denmark) and DEX-LO.TM.,
SPEZYME.TM. AA, and SPEZYME.TM. DELTA AA (from Genencor Int.).
[0093] Other contemplated alpha-amylases are the KSM-K36
alpha-amylase disclosed in EP 1,022,334 and deposited as FERM BP
6945, and the KSM-K38 alpha-amylases disclosed in EP 1,022,334, and
deposited as FERM BP-6946. Also variants thereof are contemplated,
in particular the variants disclosed in Danish patent application
no. PA 2000 11533 (from Novozymes A/S.
[0094] The "secondary liquefaction" is performed in the presence of
an alpha-amylase, in particular a thermostable acid alpha-amylase
or a thermostable maltogenic acid alpha-amylase as described herein
for use in the secondary liquefaction step in the process of the
invention. The alpha-amylase is preferably derived from a
micro-organism, including fungal and bacterial, or derived from a
plant. Preferred thermostabte acid alpha-amylases are of bacterial
origin. Preferred thermostable maltogenic acid alpha-amylases are
of fungal orgin.
[0095] It is understood that enzymes are added in an effective
amount for the actual conditions (temperature, pH) of the process,
e.g., that the thermostable acid alpha-amylase is added in an
amount effective in step (c).
[0096] In further embodiments of the process of the invention, in
step (c) apart from the addition of the thermostable acid
alpha-amylase is also added an alpha-amylase which is not a
thermostable acid alpha-amylase.
[0097] The term "thermostable" in the context of a thermostable
acid alpha-amylase means in one embodiment that the enzyme is
active up to 90.degree. C. at pH 5.0 using a 0.1 M citrate buffer
and 4.3 mM Ca.sup.2+.
[0098] The thermostable acid alpha-amylase should have activity at
the pH present during the liquefaction and fermentation, such as,
e.g., at a pH in the range pH 2.5-5.5 using a 0.1 M citrate buffer
and 4.3 mM Ca.sup.2+. The enzyme should preferably at least be
active in the range at pH 3-5. It is understood that the enzyme may
also be active outside the pH ranges mentioned.
[0099] Examples of thermostable acid alpha-amylases as used herein
are the alpha-amylases selected from the group consisting of LE399;
the Aspergillus oryzae TAKA alpha-amylase (EP 238 023); the
Aspergillus niger alpha-amylase disclosed in EP 383,779 B2 (section
[0037] (see also the cloning of the A. niger gene in Example 1);
the Aspergillus niger alpha-amylase disclosed in Example 1 of EP
140,410; Commercial fungal alpha-amylases FUNGAMYL.RTM. (Novozymes
A/S); and Clarase.TM. (from Genencor Int., USA), the latter both
derived from Aspergillus.
[0100] LE399 is a hybrid alpha-amylase. Specifically, LE399
comprises the 445 C-terminal amino acid residues of the Bacillus
licheniformis alpha-amylase (shown in SEQ to NO: 4 of WO 99/19467)
and the 37 N-terminal amino acid residues of the alpha-amylase
derived from Bacillus amyloliquefaciens (shown in SEQ ID NO: 5 of
WO 99/19467), with the following substitution:
G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S (using the
numbering in SEQ ID NO: 4 of WO 99/19467).
[0101] By the expression "secondary liquefaction in the presence of
a thermostable acid alpha-amylase" is understood as liquefaction in
the secondary liquefaction step in the process of the invention by
treatment with an effective amount of a thermostable acid
alpha-amylase" as defined herein.
[0102] The thermal/pH stability may be tested using, e.g., the
following method: 950 micro liter 0.1 M Citrate+4.3 mM Ca.sup.2+
buffer is incubated for 1 hour at 60.degree. C. 50 micro liters
enzyme in buffer (4 AFAU/ml) is added. 2.times.40 micro liter
samples are taken at 0 and 60 minutes and chilled on ice. The
activity (AFAU/ml) measured before incubation (0 minutes) is used
as reference (100%). The decline in percent is calculated as a
function of the incubation time. To determine the Thermal stability
the test is repeated using different temperatures, for instance 50,
60, 70, 80 and 90.degree. C. To determine the pH stability the test
is repeated using different pH's, for instance, pH 2.5; 3; 3.5; 4;
4.5; 5.
[0103] An example of an alpha-amylase, in particular a thermostabte
maltogenic acid alpha-amylase, used in the process of the invention
is the alpha-amylase having the amino acid sequence set forth in
SEQ ID NO: 1 (also named SP288) and variants thereof having one or
more amino acid residues which have been deleted, substituted
and/or inserted compared to the amino acid sequence of SEQ ID NO:
1; which variants have alpha-amylase activity, preferably being a
thermostable maltogenic acid alpha-amylase.
[0104] Thus, the alpha-amylase used in the secondary liquefaction
step in the process of the invention, may, e.g., be an
alpha-amylase, in particular a thermostable maltogenic acid
alpha-amylase, having an amino acid sequence which has at least 70%
identity to SEQ ID NO: 1 preferably at least 75%, 80%, 85% or at
least 90%, e.g., at least 95%, 97%, 98%, or at least 99% identity
to SEQ ID NO: 1. In the present context, the degree of identity
between two amino acid sequences is described by the parameter
"identity" given in %. For purposes of the present invention, the
degree of identity between two amino acid sequences is preferably
determined by the Clustal method (Higgins, 1989, CABIOS 5: 151-153)
using the LASERGENE.TM. MEGALIGN.TM. software (DNASTAR, inc.,
Madison, Wis.) with an identity table and the following multiple
alignment parameters: Gap penalty of 10, and gap length penalty of
10. Pairwise alignment parameters were Ktuple=1, gap penalty=3,
windows=5, and diagonals=5].
[0105] Thus, the thermostable maltogenic acid alpha-amylase used in
the process of the invention may, e.g., also be an alpha-amylase,
in particular a thermostable maltogenic acid alpha-amylase, having
an amino acid sequence which is a fragment of SEQ ID NO: 1. When
using the term "alpha-amylase" or "thermostable maltogenic acid
alpha-amylase" in the context of variants and fragments of, e.g.,
SEQ ID NO: 1, it is to be understood that the enzyme is capable of
being enzymatically active. When used herein, a "fragment" of SEQ
ID NO: 1 is a polypeptide having one or more amino acids deleted
from the amino and/or carboxyl terminus of this amino acid
sequence. Preferably, a fragment contains at least 50 amino acid
residues or at least 100 amino acid residues.
[0106] The enzyme given by SEQ ID NO: 1 is also disclosed in Boel
E. et al., "Calcium binding in alpha-amylases: an X-ray diffraction
study at 2.1-A resolution of two enzymes from Aspergillus".
Biochemistry, 1980, 29:6244-6249, e.g., in table 1 and under
"Material and Methods" of the same.
[0107] Other examples of alpha-amylases which may be used in the
secondary liquefaction step in the process of the invention, is the
alpha-amylase disclosed in Agric. Biol. Chem., 1979, 43:1165-1171
by Guy-Jean Moulin and Pierre Galzy.
[0108] It is understood that the enzyme are added in an effective
amount for the actual conditions (temperature, pH) of the process,
e.g., that the thermostable maltogenic acid alpha-amylase is added
in an amount effective in step (c).
[0109] In further embodiments of the process of the invention, in
the secondary liquefaction step (c) apart from the addition of a
thermostable maltogenic acid alpha-amylase (e.g., the alpha-amylase
of SEQ ID NO: 1 and variants thereof as described herein) is also
added an alpha-amylase which is not a thermostable maltogenic acid
alpha-amylase as defined herein, such as e.g., the alpha-amylase
TTC.
[0110] The thermostable maltogenic acid alpha-amylase should have
activity at the pH present during the liquefaction and
fermentation; such as e.g., at a pH in the range pH 2.5-5.5 using a
0.1 M citrate buffer and 4.3 mM Ca.sup.2+, a substrate consisting
of DE 12 alpha-amylase TTC liquefied corn starch at 30% dry
substance. The enzyme should preferably at least be active in the
range at pH 3-5, preferably at least pH 2.5-5. It is understood
that the enzyme may also be active outside the pH ranges
mentioned.
[0111] The term "maltogenic" in the context of the invention, means
that the enzyme is capable of releasing a relatively high amount of
.alpha.-maltose as a product of its enzymatic activity.
[0112] In a particular interesting embodiment, the term
"maltogenic" means that the enzyme using a DE 12 alpha-amylase TTC
liquefied corn starch at 30% dry substance at 60.degree. C., pH 4.5
and dosing the enzyme at 1 AFAU/g dry substance, the enzyme will in
24 hours catalyze the formation of at least 15%, or at least 20%,
at least 25%, at least 30 w/w maltose as based on the total amount
of starch. The maltose content may for instance be measured by with
HPLC as known by the person skilled in the art.
[0113] The term "DE 12 alpha-amylase TTC liquefied corn starch" in
this context means that the substrate used for testing the
maltogenicity of the alpha-amylase enzyme, is corn starch liquefied
to a DE of 12 with alpha-amylase TTC.
[0114] The term "thermostable" means that the enzyme is relatively
stable at higher temperatures. In one embodiment, the enzyme will
maintain more than 90% of its activity for 1 hour at 70.degree. C.
using a DE 12 alpha-amylase TTC liquefied corn starch at 30% dry
substance as substrate, pH 5.5, 0.1 M citrate buffer and 4.3 mM
Ca.sup.2+.
[0115] The term "acid" means that the enzyme is relatively stable
at low pH. In one embodiment, the enzyme will maintain more than
70% of its activity in the range from pH 3.5-5.0 (e.g., at pH 4),
or preferably in the range from pH 3.8-4.7 (e.g., at pH 4.2) at the
conditions: substrate DE 12 alpha-amylase TTC liquefied corn starch
at 30% dry substance, temperature of 40.degree. C., 0.1 M citrate
buffer and 4.3 mM Ca.sup.2+.
[0116] In one embodiment, the pH window (profile) of the enzyme
used in the secondary liquefaction step in the process of the
invention is as follows: the maximum activity of the enzyme is
found at approximately pH 4.2 and/or the enzyme will maintain more
than 70% of its activity in the range from pH 3.5-5.0 at the
conditions; substrate is DE 12 alpha-amylase TTC liquefied corn
starch at 30% dry substance, temperature of 40.degree. C., 0.1 M
citrate buffer and 4.3 mM Ca.sup.2+.
[0117] In one embodiment the temperature window (profile) of the
alpha-amylase enzyme used in the secondary liquefaction step in the
process of the invention is as follows; the enzyme will maintain
more than 80% of its activity for 15 min in the range from
50-80.degree. C. using a DE 12 alpha-amylase TTC liquefied corn
starch at 30% dry substance as substrate, pH 5.5, 0.1 M citrate
buffer and 4.3 mM Ca.sup.2+.
[0118] The alpha-amylase enzyme used in the secondary liquefaction
step in the process of the invention may catalyze the hydrolysis of
beta-cyclodextrins which is one of the characteristics of the
enzyme having the amino acid sequence of SEQ ID NO: 1.
[0119] By the expression "secondary liquefaction in the presence of
a thermostable maltogenic acid alpha-amylase" is understood
liquefaction in the secondary liquefaction step by treatment with
an effective amount of a thermostable maltogenic acid alpha-amylase
as defined herein. The alpha-amylase used in the secondary
liquefaction is preferably a thermostable maltogenic acid
alpha-amylase. The term "thermostable maltogenic acid
alpha-amylase", means that the alpha-amylase is both thermostable,
acid and maltogenic as defined herein. In one embodiment, the
alpha-amylase is at least thermostable and acid as defined herein,
optionally being maltogenic as defined herein.
[0120] The thermostable maltogenic acid alpha-amylase may be
employed in the primary liquefaction step; however, the maximum
effect is obtained if the enzyme is added the secondary
liquefaction step.
Enzyme Activities used During Saccharification or SSF
Glucoamylase
[0121] The saccharification step or the simultaneous
saccharification and fermentation step (SSF) may be carried out in
the presence of a glucoamylase. The glucoamylase may be of any
origin, e.g., derived from a microorganism or a plant. Preferred is
glucoamylase of fungal or bacterial origin selected from the group
consisting of Aspergillus niger glucoamylase, in particular A.
niger G1 or G2 glucoamylase (Boel et al., 1984, EMBO J. 3 (5):
1097-1102), or variants thereof, such as disclosed in WO 92/00381
and WO 00/04136: the A. awamori glucoamylase (WO 84/02921), A.
oryzae (Agric. Biol. Chem., 1991, 55 (4): 941-949), or variants or
fragments thereof.
[0122] Other contemplated Aspergillus glucoamylase variants include
variants to enhance the thermal stability: G137A and G139A (Chen et
al., 1996, Prot. Engng. 9: 499-505); D257E and D293E/Q (Chen et
al., 1995, Prot. Engng. 8: 575-582); N182 (Chen et al., 1994,
Biochem. J. 301: 275-281); disulphide bonds, A246C (Fierobe et al.,
1996, Biochemistry, 35: 8698-8704; and introduction of Pro residues
in position A435 and S436 (Li et al., 1997, Protein Engng. 10:
1199-1204. Furthermore, Clark Ford presented a paper on Oct. 17,
1997, ENZYME ENGINEERING 14, Beijing/China Oct. 12-17, 1997,
Abstract number: Abstract book p. 0-61. The abstract suggests
mutations in positions G137A, N20C/A27C, and S30P in an Aspergillus
awamori glucoamylase to improve the thermal stability. Other
glucoamylases include Talaromyces glucoamylases, in particular
derived from Talaromyces emersonii (WO 99/28448), Talaromyces
leycettanus (U.S. Pat. No. Re. 32,153), Talaromyces duponti,
Talaromyces thermopiles (U.S. Pat. No. 4,587,215). Bacterial
glucoamylases contemplated include glucoamylases from the genus
Clostridium, in particular C. thermoamylolyticum (EP 135,138), and
C. thermohydrosulfuricum (WO 86/01831).
[0123] Commercial products include SAN.TM. SUPER.TM. and AMG.TM. E
(from Novozymes A/S).
Protease
[0124] Addition of protease(s) in the saccharification step, the
SSF step and/or the fermentation step increase(s) the FAN (Free
amino nitrogen) level and increase the rate of metabolism of the
yeast and further gives higher fermentation efficiency.
[0125] Suitable proteases include microbial proteases, such as
fungal and bacterial proteases. Preferred proteases are acidic
proteases, i.e., proteases characterized by the ability to
hydrolyze proteins under acidic conditions below pH 7.
[0126] In a preferred embodiment, the protease is selected from the
group of fungal proteases, such as e.g., an acid fungal protease
derived from a strain of Aspergillus.
[0127] Suitable acid fungal proteases include fungal proteases
derived from Aspergillus, Mucor, Rhizopus, Candida, Coriolus,
Endothia, Enthomophtra, Irpex, Penicillium, Sclerotiumand
Torulopsis. Especially contemplated are proteases derived from
Aspergillus niger (see, e.g., Koaze et al., 1964, Agr. Biol. Chem.
Japan, 28: 216), Aspergillus saitoi (see, e.g., Yoshida, 1954, J.
Agr. Chem. Soc. Japan, 28:66), Aspergillus awamori (Hayashida et
as., 1977, Agric. Biol. Chem., 42(5): 927-933, Aspergillus
aculeatus (WO 95/02044), or Aspergillus oryzae, such as the pepA
protease; and acidic proteases from Mucor pusillus or Mucor
miehei.
[0128] Also contemplated are neutral or alkaline proteases, such as
a protease derived from a strain of Bacillus. Bacterial proteases,
which are not acidic proteases, include the commercially available
products Alcalase.RTM. and Neutrase.RTM. (available from Novozymes
A/S.
Additional Enzymes:
[0129] One or more additional enzymes may also be used during
saccharification/pre-saccharification or SSF. Additional enzymes
include e.g., pullulanase and/or phytase. Thus, in one embodiment,
a glucoamylase and/or phytase is added in order to promote the
fermentation.
Phytase:
[0130] The phytase used according to the invention may be any
enzyme capable of effecting the liberation of inorganic phosphate
from phytic acid (myo-inositol hexakisphosphate) or from any salt
thereof (phytates). Phytases can be classified according to their
specificity in the initial hydrolysis step, viz. according to which
phosphate-ester group is hydrolyzed first. The phytase to be used
in the invention may have any phytase specificity, e.g., a
3-phytase (EC 3.1.3.8), a 6-phytase (EC 3.1.3.26) or a
5-phytase.
[0131] A suitable dosage of the phytase is e.g., in the range
5,000-250,000 FYT/g DS, particularly 10,000-100,000 FYT/g DS.
[0132] The phytase activity may be determined FYT units, one FYT
being the amount of enzyme that liberates 1 micromole inorganic
ortho-phosphate per min. under the following conditions: pH 5.5,
temperature 37.degree. C.; substrate: sodium phytate
(C.sub.6H.sub.6CO.sub.24P.sub.6Na.sub.12) at a concentration of
0.0050 mole/I.
[0133] The phytase may be of any origin, such as, e.g., microbial,
such as, e g., derived from a strain of Peniophra lycii or
Aspergillus oryzae. It may be produced recombinantly or
non-recombinantly. The phytase may be derived e.g., from plants or
microorganisms, such as bacteria or fungi, e.g., yeast or
filamentous fungi.
[0134] The plant phytase may be from wheat-bran, maize, soy bean or
lily pollen. Suitable plant phytases are described in Thomlinson et
al, 1962, Biochemistry, 1: 166-171; Barrientos et al., 1994, Plant.
Physiol., 106: 1489-1495; WO 98/05785; WO 98/20139.
[0135] A bacterial phytase may be from genus Bacillus, Pseudomonas
or Escherichia, specifically the species B. subtilis or E. coli.
Suitable bacterial phytases are described in Paver and Jagannathan,
1982, Journal of Bacteriology 151:1102-1108; Cosgrove, 1970,
Australian Journal of Biological Sciences 23:1207-1220; Greiner et
al., 1993, Arch. Biochem. Biophys., 303, 107-113: WO 98/06856; WO
97/33976; WO 97/48812.
[0136] A yeast phytase or myo-inositol monophosphatase may be
derived from genus Saccharomyces or Schwanniomyces, specifically
species Saccharomyces cerevisiae or Schwanniomyces occidentalis.
Suitable yeast phytases are described in Nayini et al., 1984,
Lebensmittel Wissenschaft und Technologie 17:24-26; Wodzinski et
al., Adv. Appl. Microbiol., 42: 263-303; AU-A-24840/95;
[0137] Phytases from filamentous fungi may be derived from the
fungal phylum of Ascomycota (ascomycetes) or the phylum
Basidiomycota, e.g., the genus Aspergillus, Thermomyces (also
called Humicola), Myceliophthora, Manascus, Penicillium,
Peniophora, Agrocybe, Paxillus, or Trametes, specifically the
species Aspergillus terreus, Aspergillus niger, Aspergillus niger
var. awamori, Aspergillus ficuum, T. lanuginosus (also known as H.
lanuginosa), Myceliophthora thermophila, Peniophora lycii, Agrocybe
pediades, Manascus anka, Paxillus involtus, or Trametes pubescens.
Suitable fungal phytases are described in Yamada et al., 1986,
Agric. Biol. Chem. 322:1275-1282; Piddington et al., 1993, Gene
133:55-62, EP 684,313; EP 0 420 358; EP 0 684 313; WO 98/28408; WO
98/28409, JP 7-67635; WO 98/44125; WO 97/38096; WO 98/13480.
[0138] Modified phytases or phytase variants are obtainable by
methods known in the art, in particular by the methods disclosed in
EP 897010; EP 897985; WO 99/49022; WO 99/48330.
Microorganism Used for Fermentation
[0139] Preferably microorganisms are used for the fermentation in
step (d). The microorganism may be a fungal organism, such as
yeast, or bacteria. Suitable bacteria may, e.g., be Zymomonas
species, such as Zymomonas mobilis and E. coli. Examples of
filamentous fungi include strains of Penicillium species. Preferred
organisms for ethanol production are yeasts, such as, e.g., Pichia
or Saccharomyces. Preferred yeast according to the invention is
Saccharomyces species, in particular Saccharomyces cerevisiae or
bakers yeast.
Use of the Products Produced by the Method of the Invention
[0140] The ethanol obtained by the process of the invention may be
used as, e.g., fuel ethanol; drinking ethanol, ie., potable neutral
spirits, or industrial ethanol, including fuel additive.
[0141] The invention in further aspect relates to use of a
thermostable acid alpha-amylase or a thermostable maltogenic acid
alpha-amylase in the secondary liquefaction step in a process for
production of ethanol; included is use of a thermostable acid
alpha-amylase or a thermostable maltogenic acid alpha-amylase in
the secondary liquefaction step in the processes of the invention
disclosed herein.
ADVANTAGES OF THE PROCESS OF THE INVENTION
[0142] By employing the thermostable acid alpha amylase of the
invention in the secondary liquefaction step, the process of the
invention provides an improved process of producing ethanol. By the
process of the invention the overall yield and/or process economy
is increased. The process of the invention may make possible a
lowering of the fermentation time. Further, the process of the
invention may enhance the fermentation efficiency, e.g, by reducing
the residual starch otherwise left over in the fermentation.
Furthermore, the process of the invention may reduce or eliminate
the need for a pre-saccharification step.
[0143] By employing the thermostable maltogenic acid alpha amylase
of the invention in the secondary liquefaction step, the process of
the invention provides an improved process of producing ethanol. By
the process of the invention the overall yield and/or process
economy is increased. The described thermostable maltogenic acid
alpha-amylase will, when used in the secondary liquefaction,
produce a higher number of fermentable sugars (maltose) as compared
to the non-maltogenic alpha-amylases presently employed. This
reduces the fermentation time and/or the dosage of glucoamylase
enzyme which is required to form fermentable sugars. Also as
molecules of a lower molecular weight are formed the viscosity will
be reduced as compared to non-maltogenic alpha-amylases. Reduced
viscosity is desired in e.g., heat exchangers and dryers.
Furthermore, the thermostable maltogenic acid alpha-amylase by
being active during fermentation conditions, and since this enzyme
has an endo-breakdown mechanism it will in combination with the
glucoamylase which is an exo-enzyme enable a more efficient
hydrolysis of the starch during fermentation. Thus the process of
the invention may make possible a lowering of the fermentation
time. The process of the invention may enhance the fermentation
efficiency, e.g., by reducing the residual starch otherwise left
over in the fermentation. Furthermore, the process of the invention
may reduce or eliminate the need for a pre-saccharification
step.
Material & Methods
Determination of Viscosity
[0144] The mash is heated to a temperature of 50-70.degree. C.,
depending on the treatment. Following treatment viscosity is
measured using a Haake VT02 rotation based viscosimeter. The unit
of viscosity is centipois (cps), which is proportionally related to
the viscosity level.
Determination of Alpha-Amylase Activity (KNU)
[0145] The KNU is used to measure bacterial alpha-amylases with a
high pH optima.
Phadebas Assay
[0146] Alpha-amylase activity is determined by a method employing
Phadebas.RTM. tablets as substrate. Phadebas tablets (Phadebas.RTM.
Amylase Test, supplied by Pharmacia Diagnostic) contain a
cross-linked insoluble blue-colored starch polymer, which has been
mixed with bovine serum albumin and a buffer substance and
tabletted.
[0147] For every single measurement one tablet is suspended in a
tube containing 5 ml 50 mM Britton-Robinson buffer (50 mM acetic
acid, 50 mM phosphoric acid, 50 mM boric acid, 0.1 mM CaCl.sub.2,
pH adjusted to the value of interest with NaOH). The test is
performed in a water bath at the temperature of interest. The
alpha-amylase to be tested is diluted in x ml of 60 mM
Britton-Robinson buffer. 1 ml of this alpha-amylase solution is
added to the 5 ml 50 mM Britton-Robinson buffer. The starch is
hydrolyzed by the alpha-amylase giving soluble blue fragments. The
absorbance of the resulting blue solution, measured
spectrophotometrically at 620 nm, is a function of the
alpha-amylase activity.
[0148] It is important that the measured 620 nm absorbance after 10
or 15 minutes of incubation (testing time) is in the range of 0.2
to 2.0 absorbance units at 620 nm. In this absorbance range there
is linearity between activity and absorbance (Lambert-Beer law).
The dilution of the enzyme must therefore be adjusted to fit this
criterion. Under a specified set of conditions (temp., pH, reaction
time, buffer conditions) 1 mg of a given alpha-amylase will
hydrolyze a certain amount of substrate and a blue color will be
produced. The color intensity is measured at 620 nm. The measured
absorbance is directly proportional to the specific activity
(activity/mg of pure alpha-amylase protein) of the alpha-amylase in
question under the given set of conditions.
Alternative Method
[0149] Alpha-amylase activity is determined by a method employing
the PNP-G7 substrate. PNP-G7 which is an abbreviation for
p-nitrophenyl-alpha,D-maltoheptaoside is a blocked oligosaccharide
which can be cleaved by an endo-amylase. Following the cleavage,
the alpha-glucosidase included in the kit digest the substrate to
liberate a free PNP molecule which has a yellow color and thus can
be measured by visible spectophometry at lambda=405 nm (400-420
nm). Kits containing PNP-G7 substrate and alpha-glucosidase is
manufactured by Boehringer-Mannheim (cat. No. 1054635).
[0150] To prepare the substrate one bottle of substrate (BM
1442309) is added to 5 ml buffer (BM1442309). To prepare the
alpha-glucosidase one bottle of alpha-glucosidase (BM 1462309) is
added to 45 ml buffer (BM1442309). The working solution is made by
mixing 5 ml alpha-glucosidase solution with 0.5 ml substrate.
[0151] The assay is performed by transforming 20 micro I enzyme
solution to a 96 well microtitre plate and incubating at 25.degree.
C., 200 micro I working solution, 25.degree. C. is added. The
solution is mixed and pre-incubated 1 minute and absorption is
measured every 15 sec. over 3 minutes at OD 405 nm.
[0152] The slope of the time dependent absorption-curve is directly
proportional to the specific activity (activity per mg enzyme) of
the alpha-amylase in question under the given set of
conditions.
Determination of FAU Activity
[0153] One Fungal Alpha-Amylase Unit (FAU) is defined as the amount
of enzyme, which breaks down 5.26 g starch (Merck Amylum solubile
Erg. B.6, Batch 9947275) per hour based upon the following standard
conditions: [0154] Substrate Soluble starch [0155] Temperature
37.degree. C. [0156] pH 4.7 [0157] Reaction time 7-20 minutes
Determination of Acid Alpha-Amylase Activity (AFAU)
[0158] Acid alpha-amylase activity is measured in AFAU (Acid Fungal
Alpha-amylase Units), which are determined relative to an enzyme
standard.
[0159] The standard used is AMG 300 L (from Novozymes A/S,
glucoamylase wildtype Aspergillus niger G1, also disclosed in Boel
et al., 1984, EMBO J. 3 (5): 1097-1102) and WO 92/00381). The
neutral alpha-amylase in this AMG falls after storage at room
temperature for 3 weeks from approx. 1 FAU/mL to below 0.05
FAU/mL.
[0160] The acid alpha-amylase activity in this AMG standard is
determined in accordance with the following description. In this
method, 1 AFAU is defined as the amount of enzyme, which degrades
5.260 mg starch dry matter per hour under standard conditions.
[0161] Iodine forms a blue complex with starch but not with its
degradation products. The intensity of color is therefore directly
proportional to the concentration of starch. Amylase activity is
determined using reverse colorimetry as a reduction in the
concentration of starch under specified analytic conditions.
##STR1## Standard Conditions/Reaction Conditions: (Per Minute)
[0162] Substrate, Starch, approx. 0.17 g/L [0163] Buffer: Citate,
approx. 0.03 M [0164] Iodine (I.sub.2): 0.03 g/L [0165] CaCl.sub.2:
1.85 mm [0166] pH: 2.50.+-.0.05 [0167] Incubation temperature:
40.degree. C. [0168] Reaction time: 23 seconds [0169] Wavelength:
lambda=590 nm [0170] Enzyme concentration: 0.025 AFAU/mL [0171]
Enzyme working range, 0.01-0.04 AFAU/mL
[0172] If further details are preferred these can be found in
EB-SM-0259.02/01 available on request from Novozymes A/S, and
incorporated by reference
EXAMPLES
Example 1
Secondary Liquefaction Using a Thermostable Acidic Alpha
Amylase
[0173] 400 mL of a ground corn slurry was liquefied by a bacterial
alpha-amylase and jet cooked at 105.degree. C. for 5 min the
resulting corn mash had 30% dry substance, DE 7 and pH 5.0. The
mash was heated to 80.degree. C. and the viscosity was measured to
500 CPS using a VT 180 viscosimeter.
[0174] The mash was treated with a thermostable acidic alpha
amylase from Aspergillus niger. The enzyme loading was 0.25 AFAU/g
of dry matter, with 1 AFAU defined as the amount of enzyme that
under standard conditions (37.degree. C., pH 2.5 in 0.01 M acetate
buffer) hydrolyzes 5.25 g starch so that the hydrolyzed starch is
only slightly colored by addition of iodine-potassium-iodide.
[0175] After 30 min the viscosity and DE value were measured to 350
CPS and DE 12, which shows that a final liquefaction of the corn
mash was obtained.
Example 2
Secondary Liquefaction Using a Thermostable Maltogenic Acidic Alpha
Amylase
[0176] 400 mL of a ground corn slurry was liquefied by
alpha-amylase TTC and jet cooked at 105.degree. C. for 5 min; the
resulting corn mash had 30% dry substance, DE 7 and pH 5.0. The
mash was heated to 80.degree. C. and the viscosity was measured to
500 CPS using a VT 180 viscosimeter.
[0177] The mash was treated with a thermostable maltogenic acidic
alpha amylase from Aspergillus niger having the amino acid sequence
disclosed in SEQ ID NO: 1. The enzyme loading was 0.25 AFAU/g of
dry matter, with 1 AFAU defined as the amount of enzyme which under
standard conditions (37.degree. C., pH 2.5 in 0.01 M acetate
buffer) hydrolyzes 5.25 g starch so that the hydrolyzed starch is
only slightly colored by addition of iodine-potassium-iodide.
[0178] After 30 min the viscosity and DE value were measured to 350
CPS and DE 12, which shows that a final liquefaction of the corn
mash was obtained.
Sequence CWU 1
1
1 1 484 PRT Aspergillus niger misc_feature SEQ ID NO1 1 Leu Ser Ala
Ala Ser Trp Arg Thr Gln Ser Ile Tyr Phe Leu Leu Thr 1 5 10 15 Asp
Arg Phe Gly Arg Thr Asp Asn Ser Thr Thr Ala Thr Cys Asn Thr 20 25
30 Gly Asn Glu Ile Tyr Cys Gly Gly Ser Trp Gln Gly Ile Ile Asp His
35 40 45 Leu Asp Tyr Ile Glu Gly Met Gly Phe Thr Ala Ile Trp Ile
Ser Pro 50 55 60 Ile Thr Glu Gln Leu Pro Gln Asp Thr Ala Asp Gly
Glu Ala Tyr His 65 70 75 80 Gly Tyr Trp Gln Gln Lys Ile Tyr Asp Val
Asn Ser Asn Phe Gly Thr 85 90 95 Ala Asp Asn Leu Lys Ser Leu Ser
Asp Ala Leu His Ala Arg Gly Met 100 105 110 Tyr Leu Met Val Asp Val
Val Pro Asp His Met Gly Tyr Ala Gly Asn 115 120 125 Gly Asn Asp Val
Asp Tyr Ser Val Phe Asp Pro Phe Asp Ser Ser Ser 130 135 140 Tyr Phe
His Pro Tyr Cys Leu Ile Thr Asp Trp Asp Asn Leu Thr Met 145 150 155
160 Val Glu Asp Cys Trp Glu Gly Asp Thr Ile Val Ser Leu Pro Asp Leu
165 170 175 Asp Thr Thr Glu Thr Ala Val Arg Thr Ile Trp Tyr Asp Trp
Val Ala 180 185 190 Asp Leu Val Ser Asn Tyr Ser Val Asp Gly Leu Arg
Ile Asp Ser Val 195 200 205 Leu Glu Val Gln Pro Asp Phe Phe Pro Gly
Tyr Asn Lys Ala Ser Gly 210 215 220 Val Tyr Cys Val Gly Glu Ile Asp
Asn Gly Asn Pro Ala Ser Asp Cys 225 230 235 240 Pro Tyr Gln Lys Val
Leu Asp Gly Val Leu Asn Tyr Pro Ile Tyr Trp 245 250 255 Gln Leu Leu
Tyr Ala Phe Glu Ser Ser Ser Gly Ser Ile Ser Asn Leu 260 265 270 Tyr
Asn Met Ile Lys Ser Val Ala Ser Asp Cys Ser Asp Pro Thr Leu 275 280
285 Leu Gly Asn Phe Ile Glu Asn His Asp Asn Pro Arg Phe Ala Lys Tyr
290 295 300 Thr Ser Asp Tyr Ser Gln Ala Lys Asn Val Leu Ser Tyr Ile
Phe Leu 305 310 315 320 Ser Asp Gly Ile Pro Ile Val Tyr Ala Gly Glu
Glu Gln His Tyr Ala 325 330 335 Gly Gly Lys Val Pro Tyr Asn Arg Glu
Ala Thr Trp Leu Ser Gly Tyr 340 345 350 Asp Thr Ser Ala Glu Leu Tyr
Thr Trp Ile Ala Thr Thr Asn Ala Ile 355 360 365 Arg Lys Leu Ala Ile
Ala Ala Asp Ser Ala Tyr Ile Thr Tyr Ala Asn 370 375 380 Asp Ala Phe
Tyr Thr Asp Ser Asn Thr Ile Ala Met Ala Lys Gly Thr 385 390 395 400
Ser Gly Ser Gln Val Ile Thr Val Leu Ser Asn Lys Gly Ser Ser Gly 405
410 415 Ser Ser Tyr Thr Leu Thr Leu Ser Gly Ser Gly Tyr Thr Ser Gly
Thr 420 425 430 Lys Leu Ile Glu Ala Tyr Thr Cys Thr Ser Val Thr Val
Asp Ser Ser 435 440 445 Gly Asp Ile Pro Val Pro Met Ala Ser Gly Leu
Pro Arg Val Leu Leu 450 455 460 Pro Ala Ser Val Val Asp Ser Ser Ser
Leu Cys Gly Gly Ser Gly Arg 465 470 475 480 Leu Tyr Val Glu
* * * * *