U.S. patent application number 14/109736 was filed with the patent office on 2014-06-26 for process for conversion of granular starch to ethanol.
This patent application is currently assigned to Danisco US Inc.. The applicant listed for this patent is Danisco US Inc.. Invention is credited to Oreste J. LANTERO, Mian LI, Jayarama K. SHETTY.
Application Number | 20140178945 14/109736 |
Document ID | / |
Family ID | 38610259 |
Filed Date | 2014-06-26 |
United States Patent
Application |
20140178945 |
Kind Code |
A1 |
LANTERO; Oreste J. ; et
al. |
June 26, 2014 |
PROCESS FOR CONVERSION OF GRANULAR STARCH TO ETHANOL
Abstract
The present invention concerns a method of producing glucose
from a granular starch substrate comprising, contacting a slurry
comprising granular starch obtained from plant material with an
alpha-amylase at a temperature below the starch gelatinization
temperature of the granular starch to produce oligosaccharides and
hydrolyzing the oligosaccharides to produce a mash comprising at
least 20% glucose and further comprising fermenting the mash to
obtain ethanol.
Inventors: |
LANTERO; Oreste J.;
(Trimble, MO) ; LI; Mian; (Santa Clara, CA)
; SHETTY; Jayarama K.; (Pleasanton, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Danisco US Inc. |
Palo Alto |
CA |
US |
|
|
Assignee: |
Danisco US Inc.
Palo Alto
CA
|
Family ID: |
38610259 |
Appl. No.: |
14/109736 |
Filed: |
December 17, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13113516 |
May 23, 2011 |
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14109736 |
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11447554 |
Jun 6, 2006 |
7968318 |
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13113516 |
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Current U.S.
Class: |
435/99 ;
435/161 |
Current CPC
Class: |
C12P 7/06 20130101; Y02E
50/10 20130101; Y02E 50/17 20130101; C12P 19/14 20130101 |
Class at
Publication: |
435/99 ;
435/161 |
International
Class: |
C12P 7/06 20060101
C12P007/06; C12P 19/14 20060101 C12P019/14 |
Claims
1-31. (canceled)
32. A method of producing a mash comprising contacting a corn
slurry, comprising granular starch obtained by dry milling whole
corn without removing the germ and bran fractions, with an
exogenous a-amylase at a temperature below the starch
gelatinization temperature of the granular starch to produce a mash
comprising at least 30% (w/w) of the soluble starch substrate in
the form of glucose, wherein the contacting is conducted for a
period of 5 minutes to 48 hours at a pH of 3.5 to 7.0, and wherein
no exogenous starch-hydrolysing enzymes other than the
alpha-amylase are added to the corn slurry during the contacting
step.
33. The method according to claim 32, wherein the contacting is
conducted for a period of 2 to 24 hours.
34. The method according to claim 32, wherein the temperature is
45.degree. C. to 70.degree. C.
35. The method according to claim 32, wherein the contacting step
is conducted at a pH of between 4.0 and 6.0.
36. The method according to claim 32, wherein the corn slurry has
20% to 45% dry solids (DS) granular starch.
37. The method according to claim 32, wherein the corn slurry
further comprises thin stillage and/or backset.
38. The method according to claim 32, wherein the mash comprises at
least 50% (w/w) of the soluble starch substrate in the form of
glucose.
39. The method according to claim 32, wherein the mash comprises at
least 50% (w/w) of the soluble starch substrate in the form of
glucose.
40. The method according to claim 39, further comprising fermenting
the mash in a separate reaction vessel in the presence of a
fermenting microorganism and a starch hydrolyzing enzyme at a
temperature of between 10.degree. C. and 40.degree. C. for a period
of time of 10 hours to 250 hours to produce an end product.
41. The method according to claim 40, further comprising recovering
the end product.
42. The method according to claim 41, wherein the end product is an
alcohol.
43. The method according to claim 42, further comprising recovering
a fermentation co-product.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to processes for the
production of an alcohol (e.g., ethanol) from a granular starch
comprising exposing a slurry comprising granular starch from plant
material to an alpha-amylase at a temperature below the
gelatinization temperature of the granular starch followed by
fermentation with a fermenting microorganism.
BACKGROUND OF THE INVENTION
[0002] The commercial viability of producing ethanol as a fuel
source from agricultural crops has generated renewed worldwide
interest due to a variety of reasons that include continued and
increased dependence on limited oil supplies and the fact that
ethanol production is a renewable energy source.
[0003] Alcohol fermentation production processes and particularly
ethanol production processes are generally characterized as wet
milling or dry milling processes. Reference is made to Bothast et
al., 2005, Appl. Microbiol. Biotechnol. 67:19 -25 and THE ALCOHOL
TEXTBOOK, 3.sup.rd Ed (K. A. Jacques et al. Eds) 1999 Nottingham
University Press, UK for a review of these processes.
[0004] In general, the wet milling process involves a series of
soaking (steeping) steps to soften the cereal grain wherein soluble
starch is removed followed by recovery of the germ, fiber (bran)
and gluten (protein). The remaining starch is further processed by
drying, chemical and/or enzyme treatments. The starch may then be
used for alcohol production, high fructose corn syrup or commercial
pure grade starch.
[0005] In general, dry grain milling involves a number of basic
steps, which include: grinding, cooking, liquefaction,
saccharification, fermentation and separation of liquid and solids
to produce alcohol and other co-products. Generally, whole cereal,
such as corn cereal, is ground to a fine particle size and then
mixed with liquid in a slurry tank. The slurry is subjected to high
temperatures in a jet cooker along with liquefying enzymes (e.g.
alpha-amylases) to solublize and hydrolyze the starch in the cereal
to dextrins. The mixture is cooled down and further treated with
saccharifying enzymes (e.g. glucoamylases) to produce fermentable
glucose. The mash containing glucose is then fermented for
approximately 24 to 120 hours in the presence of ethanol producing
microorganisms. The solids in the mash are separated from the
liquid phase and ethanol and useful co-products such as distillers'
grains are obtained.
[0006] Improvements to the above fermentation processes have been
accomplished by combining the saccharification step and
fermentation step in a process referred to as simultaneous
saccharification and fermentation or simultaneous saccharification,
yeast propagation and fermentation. These improved fermentation
processes have advantages over the previously described dry milling
fermentation or even wet milling fermentation processes because
significant sugar concentrations do not develop in the fermenter
thereby avoiding sugar inhibition of yeast growth. In addition,
bacterial growth is reduced due to lack of easily available
glucose. Increased ethanol production may result by use of the
simultaneous saccharification and fermentation processes.
[0007] More recently, fermentation processes have been introduced
which eliminate the cooking step or which reduce the need for
treating cereal grains at high temperatures. These fermentation
processes which are sometimes referred to as no-cook, low
temperature or warm cook, include milling of a cereal grain and
combining the ground cereal grain with liquid to form a slurry
which is then mixed with one or more granular starch hydrolyzing
enzymes and optionally yeast at temperatures below the granular
starch gelatinization temperature to produce ethanol and other
co-products (U.S. Pat. No. 4,514,496, WO 03/066826; WO 04/081193;
WO 04/106533; WO 04/080923 and WO 05/069840).
[0008] While the above mentioned fermentation processes using a
milled grain slurry in combination with granular starch hydrolyzing
enzymes offer certain improvements over previous processes,
additional fermentation process improvements are needed by the
industry for the conversion of granular starch resulting in higher
energy efficiency and high end-product production. The object of
the present invention is to provide improved processes for the
conversion of granular starch into alcohol (e.g. ethanol) and other
end products.
SUMMARY OF THE INVENTION
[0009] The present invention provides processes for producing an
alcohol (e.g. ethanol) from granular starch by contacting the
granular starch with an alpha-amylase and providing suitable
conditions for endogenous plant hydrolytic enzymes, which hydrolyze
solublized starch to produce glucose. The glucose may then be used
as a feedstock in fermentations to produce alcohol.
[0010] In one aspect, the invention relates to a process of
producing glucose from a granular starch substrate comprising:
[0011] a) contacting a slurry comprising granular starch obtained
from plant material with an alpha-amylase at a temperature below
the starch gelatinization temperature of the granular starch to
produce oligosaccharides and allowing endogenous plant carbohydrate
hydrolyzing enzymes to hydrolyze the oligosaccharides, and [0012]
b) producing a mash comprising at least 10% glucose.
[0013] In a further embodiment of this aspect, the mash is
fermented in the presence of a fermenting microorganism and starch
hydrolyzing enzymes at a temperature of between 10.degree. C. and
40.degree. C. for a period of time of 10 hours to 250 hours to
produce alcohol, particualrly ethanol.
[0014] In another aspect, the invention relates to a process for
producing ethanol comprising: [0015] a) contacting a slurry
comprising granular starch with an alpha-amylase capable of
solublizing granular starch, wherein said contacting is at a pH of
3.5 to 7.0; at a temperature below the starch gelatinization
temperature of the granular starch; and for a period of 5 minutes
to 24 hours and obtaining a mash substrate comprising greater than
20% glucose, and [0016] b) fermenting the substrate in the presence
of a fermenting microorganism and a starch hydrolyzing enzyme at a
temperature of between 10.degree. C. and 40.degree. C. for a period
of 10 hours to 250 hours to produce ethanol. In further embodiments
of either aspect described above, the process includes recovering
the ethanol. In yet further embodiments of the described aspects,
the process may include additional steps not specified which are
performed prior to, during or after the enumerated steps.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is a general schematic diagram that illustrates an
embodiment of the invention wherein the slurry comprising a milled
grain containing granular starch and having a DS of 20 to 40% is
contacted with an alpha-amylase at a temperature between 55.degree.
C. to 70.degree. C. and a pH of 4.0 to 6.0 for 2 to 24 hours. The
resulting mash comprising glucose is transferred to a fermentor and
fermented at pH 3.0 to 5.0 at a temperature of 25.degree. C. to
35.degree. C. for 24 to 72 hours in the presence of yeast,
nutrients, acid and starch hydrolyzing enzymes to produce
ethanol.
DETAILED DESCRIPTION OF THE INVENTION
[0018] Unless defined otherwise herein, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs.
[0019] Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present invention, the preferred methods and materials are
described.
[0020] The invention will now be described in detail by way of
reference only using the following definitions and examples. All
patents and publications, including all sequences disclosed within
such patents and publications, referred to herein are expressly
incorporated by reference.
Definitions:
[0021] The term "fermentation" refers to the enzymatic and
anaerobic breakdown of organic substances by microorganisms to
produce simpler organic compounds. While fermentation occurs under
anaerobic conditions it is not intended that the term be solely
limited to strict anaerobic conditions, as fermentation also occurs
in the presence of oxygen.
[0022] As used herein the term "starch" refers to any material
comprised of the complex polysaccharide carbohydrates of plants,
comprised of amylose and amylopectin with the formula
(C.sub.6H.sub.10O.sub.5).sub.x, wherein x can be any number.
[0023] The term "granular starch" refers to raw (uncooked) starch,
that is starch in its natural form found in plant material (e.g.
grains and tubers).
[0024] As used herein the term "dry solids content (DS)" refers to
the total solids of a slurry in % on a dry weight basis.
[0025] The term "slurry" refers to an aqueous mixture comprising
insoluble solids, (e.g. granular starch).
[0026] The term "dextrins" refers to short chain polymers of
glucose (e.g. 2 to 10 units).
[0027] The term "oligosaccharides" refers to any compound having 2
to 10 monosaccharide units joined in glycosidic linkages. These
short chain polymers of simple sugars include dextrins.
[0028] The term "soluble starch" refers to starch which results
from the hydrolysis of insoluble starch (e.g. granular starch).
[0029] The term "mash" refers to a mixture of a fermentable
substrate in liquid used in the production of a fermented product
and is used to refer to any stage of the fermentation from the
initial mixing of the fermentable substrate with one or more starch
hydrolyzing enzymes and fermenting organisms through the completion
of the fermentation run.
[0030] The terms "saccharifying enzyme" and "starch hydrolyzing
enzymes" refer to any enzyme that is capable of converting starch
to mono- or oligosaccharides (e.g. a hexose or pentose).
[0031] The terms "granular starch hydrolyzing (GSH) enzyme" and
"enzymes having granular starch hydrolyzing (GSH) activity" refer
to enzymes, which have the ability to hydrolyze starch in granular
form.
[0032] The term "hydrolysis of starch" refers to the cleavage of
glucosidic bonds with the addition of water molecules.
[0033] The term "alpha-amylase (e.g., E.C. class 3.2.1.1)" refers
to enzymes that catalyze the hydrolysis of alpha-1,4-glucosidic
linkages. These enzymes have also been described as those effecting
the exo or endohydrolysis of 1,4-.alpha.-D-glucosidic linkages in
polysaccharides containing 1,4-.alpha.-linked D-glucose units.
[0034] The term "gelatinization" means solubilization of a starch
molecule by cooking to form a viscous suspension.
[0035] The term "gelatinization temperature" refers to the lowest
temperature at which gelatinization of a starch containing
substrate begins. The exact temperature of gelatinization depends
on the specific starch and may vary depending on factors such as
plant species and environmental and growth conditions.
[0036] The term "below the gelatinization temperature" refers to a
temperature that is less than the gelatinization temperature.
[0037] The term "glucoamylase" refers to the amyloglucosidase class
of enzymes (e.g., E.C.3.2.1.3, glucoamylase, 1,4-alpha-D-glucan
glucohydrolase). These are exo-acting enzymes, which release
glucosyl residues from the non-reducing ends of amylose and
amylopectin molecules. The enzymes also hydrolyzes alpha-1,6 and
alpha-1,3 linkages although at much slower rate than alpha-1,4
linkages.
[0038] The phrase "simultaneous saccharification and fermentation
(SSF)" refers to a process in the production of end products in
which a fermenting organism, such as an ethanol producing
microorganism, and at least one enzyme, such as a saccharifying
enzyme are combined in the same process step in the same
vessel.
[0039] The term "saccharification" refers to enzymatic conversion
of a directly unusable polysaccharide to a mono- or oligosaccharide
for fermentative conversion to an end product.
[0040] The term "milling" refers to the breakdown of cereal grains
to smaller particles. In some embodiments the term is used
interchangeably with grinding.
[0041] The term "dry milling" refers to the milling of dry whole
grain, wherein fractions of the grain such as the germ and bran
have not been purposely removed.
[0042] The term "liquefaction" refers to the stage in starch
conversion in which gelatinized starch is hydrolyzed to give low
molecular weight soluble dextrins.
[0043] The term "thin-stillage" refers to the resulting liquid
portion of a fermentation which contains dissolved material and
suspended fine particles and which is separated from the solid
portion resulting from the fermentation. Recycled thin-stillage in
industrial fermentation processes is frequently referred to as
"back-set".
[0044] The term "vessel" includes but is not limited to tanks,
vats, bottles, flasks, bags, bioreactors and the like. In one
embodiment, the term refers to any receptacle suitable for
conducting the saccharification and/or fermentation processes
encompassed by the invention.
[0045] The term "end product" refers to any carbon-source derived
product which is enzymatically converted from a fermentable
substrate. In some preferred embodiments, the end product is an
alcohol, such as ethanol.
[0046] As used herein the term "fermenting organism" refers to any
microorganism or cell which is suitable for use in fermentation for
directly or indirectly producing an end product.
[0047] As used herein the term "ethanol producer" or ethanol
producing microorganism" refers to a fermenting organism that is
capable of producing ethanol from a mono- or oligosaccharide.
[0048] The term "derived" encompasses the terms "originated from",
"obtained" or "obtainable from", and "isolated from" and in some
embodiments as used herein means that a polypeptide encoded by the
nucleotide sequence is produced from a cell in which the nucleotide
is naturally present or in which the nucleotide has been
inserted.
[0049] The term "heterologous" with reference to a protein or
polynucleotide refers to a protein or polynucleotide that does not
naturally occur in a host cell.
[0050] The term "endogenous" with reference to a protein or
polynucleotide refers to a protein or polynucleotide that does
naturally occur in a host cell.
[0051] The phrase "endogenous plant hydrolytic enzymes capable of
hydrolyzing soluble starch" refers to hydrolytic enzymes that are
expressed and produced in a plant and may be produced by the
expression of endogenous or heterologous genes.
[0052] The term "enzymatic conversion" in general refers to the
modification of a substrate by enzyme action. The term as used
herein also refers to the modification of a fermentable substrate,
such as a granular starch containing substrate by the action of an
enzyme.
[0053] The terms "recovered", "isolated", and "separated" as used
herein refer to a compound, protein, cell, nucleic acid or amino
acid that is removed from at least one component with which it is
naturally associated.
[0054] As used herein the term "enzyme unit" refers to the amount
of enzyme that produces 1 micromole of product per minute under the
specified conditions of the assay. For example, in one embodiment,
the term "glucoamylase activity unit" (GAU) is defined as the
amount of enzyme required to produce 1 g of glucose per hour from
soluble starch substrate (4% DS) under assay conditions of
60.degree. C. and pH 4.2.
[0055] The term "yield" refers to the amount of end product
produced using the methods of the present invention. In some
embodiments, the term refers to the volume of the end product and
in other embodiments, the term refers to the concentration of the
end product.
[0056] The term "DE" or "dextrose equivalent" is an industry
standard for measuring the concentration of total reducing sugars,
calculated as D-glucose on a dry weight basis. Unhydrolyzed
granular starch has a DE that is essentially 0 and D-glucose has a
DE of 100. An instructive method for determining the DE of a slurry
or solution is described in Schroorl's method (Fehling's assay
titration).
[0057] As used herein the term "comprising" and its cognates are
used in their inclusive sense; that is, equivalent to the term
"including" and its corresponding cognates.
[0058] "A", "an" and "the" include plural references unless the
context clearly dictates otherwise.
[0059] Numeric ranges are inclusive of the numbers defining the
range.
[0060] The headings provided herein are not limitations of the
various aspects or embodiments of the invention, which can be had
by reference to the specification as a whole.
Embodiments of the Invention
(A) Raw Materials:
Granular Starch
[0061] Granular starch may be obtained from plant material
including but not limited to wheat, corn, rye, sorghum (milo),
rice, millet, barley, triticale, cassava (tapioca), potato, sweet
potato, sugar beets, sugarcane, and legumes such as soybean and
peas. Preferred plant material includes corn, barley, wheat, rice,
milo and combinations thereof. Particualrly preferred plant
material is obtained from corn (Zea mays). Plant material may
include hybrid varieties and genetically modified varieties (e.g.
transgenic corn, barley or soybeans comprising heterologous genes).
Any part of the plant may be used to provide granular starch
including but not limited to plant parts such as leaves, stems,
hulls, husks, tubers, cobs, grains and the like. In some
embodiments, essentially the entire plant may be used, for example,
the entire corn stover may be used. In one embodiment, whole grain
may be used as a source of granular starch. Preferred whole grains
include corn, wheat, rye, barley, sorghum and combinations thereof.
In other embodiments, granular starch may be obtained from
fractionated cereal grains including fiber, endosperm and/or germ
components. Methods for fractionating plant material such as corn
and wheat are known in the art. In some embodiments, plant material
obtained from different sources may be mixed together to obtain
granular starch used in the processes of the invention (e.g. corn
and milo or corn and barley).
[0062] In some embodiments, plant material comprising granular
starch may be prepared by means such as milling. In particular,
means of milling whole cereal grains are well known and include the
use of hammer mills and roller mills.
Alpha-Amylases
[0063] In some of the embodiments encompassed by the invention, the
alpha-amylase is a microbial enzyme having an E.C. number, E.C.
3.2.1.1-3 and in particular E.C. 3.2.1.1. Any suitable
alpha-amylase may be used in the present process. In some
embodiments, the alpha-amylase is derived from a bacterial strain
and in other embodiments the alpha-amylase is derived from a fungal
strain. In further embodiments, the preferred alpha-amylase is a
bacterial alpha-amylase. In other embodiments, the alpha-amylase is
an acid stable alpha-amylase. Suitable alpha-amylases may be
naturally occurring as well as recombinant (hybrid and variants)
and mutant alpha-amylases (WO 99/19467 and WO 97/41213). In
particularly preferred embodiments, the alpha-amylase is derived
from a Bacillus species. Preferred Bacillus species include B.
subtilis, B. stearothermophilus, B. lentus, B. licheniformis, B.
coagulans, and B. amyloliquefaciens (U.S. Pat. No. 5,093,257; U.S.
Pat. No. 5,763,385; U.S. Pat. No. 5,824,532; U.S. Pat. No.
5,958,739; U.S. Pat. No. 6,008,026, U.S. Pat. No. 6,361,809; U.S.
Pat. No. 6,867,031; WO 96/23874; WO 96/39528 and WO 05/001064).
Particularly preferred alpha-amylases are derived from Bacillus
strains B. stearothermophilus, B. amyloliquefaciens and B.
licheniformis ((U.S. Pat. No. 6,187,576; U.S. Pat. No. 6,093,562;
U.S. Pat. No. 5,958,739; US 2006/0014265 and WO 99/19467).
[0064] Most preferred alpha-amylases are amylases derived from B.
stearothermophilus and B. licheniformis including wild-type, hybrid
and variant alpha-amylase enzymes. See Suzuki et al., (1989) J.
Biol. Chem. 264:18933-18938 and US 2006/0014265, particularly SEQ
ID NOs: 3, 4 and 16. Reference is also made to strains having
American Type Culture Collection (ATCC) numbers--ATCC 39709; ATCC
11945; ATCC 6598; ATCC 6634; ATCC 8480; ATCC 9945A and NCIB
8059.
[0065] In addition to the bacterial alpha-amylases, fungal
alpha-amylases are contemplated for use in the processes of the
invention. Suitable fungal alpha-amylases are derived from
filamentous fungal strains such as Aspergillus, such as A. oryzae
and A. niger (e.g. FUNGAMYL and CLARASE L), and Trichoderma,
Rhizopus, Mucor, and Penicillium.
[0066] Commercially available alpha-amylases contemplated for use
in the methods of the invention include; SPEZYME AA; SPEZYME FRED;
SPEZYME ETHYL; GZYME G997; CLARASE L (Genencor International Inc.);
TERMAMYL 120-L, LC, SC and SUPRA (Novozymes Biotech); LIQUOZYME X
and SAN SUPER (Novozymes A/S) and ULTRA THIN (/Valley
Research).
Plant Enzymes
[0067] Plants have naturally occurring starch degrading enzymes
such as alpha-amylases (EC 3.1.1.1); beta-amylases (EC 3.1.1.2),
amyloglucosidases (glucoamylase) (EC 3.1.1.3) and starch
phosphorylases (EC 2.4.1.1). In addition, plants may have been
genetically engineered to include heterologous genes encoding
starch degrading enzymes, such as amylases, glucoamylase and others
(WO 03/018766 and WO 05/096804). Endogenous starch degrading plant
enzymes, whether naturally occurring or expressed from an
introduced polynucleotide, with exposure to elevated temperatures,
such as the temperatures of jet cooking or even temperatures above
the gelatinization temperature of granular starch will become
inactivated. However, at temperatures conducted in the present
process, it is believed that the endogenous starch degrading
enzymes are not inactivated and actually contribute to the
hydrolysis of granular starch. Although not bound to theory, the
inventors believe that the alpha-amylase provided in the contacting
step modifies the granular starch structure of the plant material
allowing for the production of oligosaccharides including dextrins.
The oligosaccharides are further degraded at the temperatures
encompassed by the contacting step (e.g. 45.degree. C. to
70.degree. C.) by plant starch degrading enzymes. The plant starch
degrading enzymes act on the partially hydrolyzed starch to produce
glucose. While exogenous sources of glucoamylases may be included
in the contacting step, the addition of exogenous glucoamylase is
not required to provide glucose, which is then optionally used as a
feedstock for alcohol fermentation. Therefore in one embodiment,
the contacting step of the invention does not include the addition
of glucoamylases derived from microbial sources. However, the
addition of glucoamylases and/or other enzymes such as phytases and
proteases may increase the production of solublized granular
starch.
Fermenting Organisms
[0068] Examples of fermenting organisms are ethanologenic
microorganisms or ethanol producing microorganisms such as
ethanologenic bacteria which express alcohol dehydrogenase and
pyruvate dehydrogenase and which can be obtained from Zymomonas
moblis (See e.g. U.S. Pat. No. 5,000,000; U.S. Pat. No. 5,028,539,
U.S. Pat. No. 5,424,202; U.S. Pat. No. 5,514,583 and U.S. Pat. No.
5,554,520). In additional embodiments, the ethanologenic
microorganisms express xylose reductase and xylitol dehydrogenase,
enzymes that convert xylose to xylulose. In further embodiments,
xylose isomerase is used to convert xylose to xylulose. In
particularly preferred embodiments, a microorganism capable of
fermenting both pentoses and hexoses to ethanol are utilized. For
example, in some embodiments the microorganism may be a natural or
non-genetically engineered microorganism or in other embodiments
the microorganism may be a recombinant microorganism. For example,
in some embodiments the preferred fermenting microorganisms include
bacterial strains from Bacillus, Lactobacillus, E. coli, Erwinia,
Pantoea (e.g., P. citrea) and Klebsiella (e.g. K. oxytoca). (See
e.g. U.S. Pat. No. 5,028,539, U.S. Pat. No. 5,424,202 and WO
95/13362).
[0069] In further preferred embodiments, the ethanol-producing
microorganism is a fungal microorganism, such as a yeast and
specifically Saccharomyces such as strains of S. cerevisiae (U.S.
Pat. No. 4,316,956). A variety of S. cerevisiae are commercially
available and these include but are not limited to FALI
(Fleischmann's Yeast), SUPERSTART (Alltech), FERMIOL (DSM
Specialties), RED STAR (Lesaffre) and Angel alcohol yeast (Angel
Yeast Company, China).
Secondary Enzymes
[0070] While in one embodiment, it is contemplated that additional
starch hydrolyzing enzymes are not needed, and therefore not
included in the contacting step, additional enzymes may be included
in both the contacting step and fermenting step encompassed by the
invention. In some embodiments, these enzymes will be included as a
secondary enzyme in the contacting step, which comprises contacting
the granular starch slurry with an alpha-amylase and one or more
secondary enzymes. In other embodiments, the additional enzymes
will be included in the fermentation step along with yeast and
other components.
[0071] In some embodiments during the contacting step with the
alpha-amylase, the secondary enzyme may include a glucoamylase,
granular starch hydrolyzing enzymes, a protease, a phytase, a
cellulase, a hemicellulases, a pullulanase, a xylanase, a lipase, a
cutinase, a pectinase, a beta-glucanase, a beta amylase, a
cyclodextrin transglycosyltransferase and combinations thereof. In
some preferred embodiments, the contacting step will include a
combination of an alpha-amylase, a phytase and optionally a
protease. In other embodiments, the contacting step will include a
combination of an alpha-amylase, a glucoamylase and optionally a
protease. In yet other embodiments, the contacting step will
include a combination of an alpha-amylase, a glucoamylase, a
phytase and optionally a protease.
[0072] Glucoamylases (GA) (E.C. 3.2.1.3.) may be derived from the
heterologous or endogenous protein expression of bacteria, plants
and fungi sources. Preferred glucoamylases useful in the
compositions and methods of the invention are produced by several
strains of filamentous fungi and yeast. In particular,
glucoamylases secreted from strains of Aspergillus and Trichoderma
are commercially important. Suitable glucoamylases include
naturally occurring wild-type glucoamylases as well as variant and
genetically engineered mutant glucoamylases. The following
glucoamylases are nonlimiting examples of glucoamylases that may be
used in the process encompassed by the invention. Aspergillus niger
G1 and G2 glucoamylase (Boel et al., (1984) EMBO J. 3:1097-1102; WO
92/00381, WO 00/04136 and U.S. Pat. No. 6,352,851); Aspergillus
awamori glucoamylases (WO 84/02921); Aspergillus oryzae
glucoamylases (Hata et al., (1991) Agric. Biol. Chem. 55:941-949)
and Aspergillus shirousami. (See Chen et al., (1996) Prot. Eng.
9:499-505; Chen et al. (1995) Prot. Eng. 8:575-582; and Chen et
al., (1994) Biochem J. 302:275-281).
[0073] Glucoamylases are also obtained from strains of Talaromyces
such as those derived from T. emersonii, T. leycettanus, T. duponti
and T. thermophilus (WO 99/28488; U.S. Pat. No. RE: 32,153; U.S.
Pat. No. 4,587,215); strains of Trichoderma, such as T. reesei and
particularly glucoamylases having at least 80%, 85%, 90% and 95%
sequence identity to SEQ ID NO: 4 disclosed in US Pat. Pub. No.
2006-0094080; strains of Rhizopus, such as R. niveus and R. oryzae;
strains of Mucor and strains of Humicola, such as H. grisea (See,
Boel et al., (1984) EMBO J. 3:1097-1102; WO 92/00381; WO 00/04136;
Chen et al., (1996) Prot. Eng. 9:499-505; Taylor et al., (1978)
Carbohydrate Res. 61:301-308; U.S. Pat. No. 4,514,496; U.S. Pat.
No. 4,092,434; and Jensen et al., (1988) Can. J. Microbiol.
34:218-223). Other glucoamylases useful in the present invention
include those obtained from Athelia rolfsii and variants thereof
(WO 04/111218).
[0074] Enzymes having glucoamylase activity used commercially are
produced for example, from Aspergillus niger (trade name
DISTILLASE, OPTIDEX L-400 and G ZYME G990 4X from Genencor
International Inc.) or Rhizopus species (trade name CU.CONC from
Shin Nihon Chemicals, Japan). Also the commercial digestive enzyme,
trade name GLUCZYME from Amano Pharmaceuticals, Japan (Takahashi et
al., (1985) J. Biochem. 98:663-671). Additional enzymes include
three forms of glucoamylase (E.C.3.2.1.3) of a Rhizopus sp., namely
"Gluc1" (MW 74,000), "Gluc2" (MW 58,600) and "Gluc3" (MW 61,400).
Also the enzyme preparation GC480 (Genencor International Inc.)
finds use in the invention.
[0075] Granular starch hydrolyzing enzymes (GSHEs) are able to
hydrolyze granular starch, and these enzymes have been recovered
from fungal, bacterial and plant cells such as Bacillus sp.,
Penicillium sp., Humicola sp., Trichoderma sp. Aspergillus sp.
Mucor sp. and Rhizopus sp. In one embodiment, a particular group of
enzymes having GSH activity include enzymes having glucoamylase
activity and/or alpha-amylase activity (See, Tosi et al., (1993)
Can. J. Microbiol. 39:846-855). A Rhizopus oryzae GSHE has been
described in Ashikari et al., (1986) Agric. Biol. Chem. 50:957-964
and U.S. Pat. No. 4,863,864. A Humicola grisea GSHE has been
described in Allison et al., (1992) Curr. Genet. 21:225-229; WO
05/052148 and European Patent No. 171218. An Aspergillus awamori
var. kawachi GSHE has been described by Hayashida et al., (1989)
Agric. Biol. Chem 53:923-929. An Aspergillus shirousami GSHE has
been described by Shibuya et al., (1990) Agric. Biol. Chem.
54:1905-1914.
[0076] In one embodiment, a GSHE may have glucoamylase activity and
is derived from a strain of Humicola grisea, particularly a strain
of Humicola grisea var. thermoidea (see, U.S. Pat. No. 4,618,579).
In some preferred embodiments, the Humicola enzyme having GSH
activity will have at least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%
and 99% sequence identity to the amino acid sequence of SEQ ID NO:
3 of WO 05/052148.
[0077] In another embodiment, a GSHE may have glucoamylase activity
and is derived from a strain of Aspergillus awamori, particularly a
strain of A. awamori var. kawachi. In some preferred embodiments,
the A. awamori var. kawachi enzyme having GSH activity will have at
least 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% and 99% sequence
identity to the amino acid sequence of SEQ ID NO: 6 of WO
05/052148.
[0078] In another embodiment, a GSHE may have glucoamylase activity
and is derived from a strain of Rhizopus, such as R. niveus or R.
oryzae. The enzyme derived from the Koji strain R. niveus is sold
under the trade name "CU CONC or the enzyme from Rhizopus sold
under the trade name GLUZYME.
[0079] Another useful GSHE having glucoamylase activity is
SPIRIZYME Plus (Novozymes A/S), which also includes acid fungal
amylase activity.
[0080] In another embodiment, a GSHE may have alpha-amylase
activity and is derived from a strain of Aspergillus such as a
strain of A. awamori, A. niger, A. oryzae, or A. kawachi and
particularly a strain of A. kawachi.
[0081] In some preferred embodiments, the A. kawachi enzyme having
GSH activity will have at least 85%, 90%, 92%, 94%, 95%, 96%, 97%,
98% and 99% sequence identity to the amino acid sequence of SEQ ID
NO: 3 of WO 05/118800 and WO 05/003311.
[0082] In some embodiments, the enzyme having GSH activity is a
hybrid enzyme, for example one containing a catalytic domain of an
alpha-amylase such as a catalytic domain of an Aspergillus niger
alpha-amylase, an Aspergillus oryzae alpha-amylase or an
Aspergillus kawachi alpha-amylase and a starch binding domain of a
different fungal alpha-amylase or glucoamylase, such as an
Aspergillus kawachi or a Humicola grisea starch binding domain. In
other embodiments, the hybrid enzyme having GSH activity may
include a catalytic domain of a glucoamylase, such as a catalytic
domain of an Aspergillus sp., a Talaromyces sp., an Althea sp., a
Trichoderma sp. or a Rhizopus sp. and a starch binding domain of a
different glucoamylase or an alpha-amylase. Some hybrid enzymes
having GSH activity are disclosed in WO 05/003311, WO 05/045018;
Shibuya et al., (1992) Biosci. Biotech. Biochem 56: 1674-1675 and
Cornett et al., (2003) Protein Engineering 16:521-520.
[0083] Suitable proteases include microbial proteases, such as
fungal and bacterial proteases, for example, acid fungal proteases
such as NSP24 and also GC106 (Genencor International Inc.).
Preferred fungal proteases are derived from strains of Aspergillus
(e.g. proteases from A. niger and A. oryzae), Mucor (e.g. M.
miehei), Trichoderma, Rhizopus, and Candida. Preferred bacterial
proteases are derived from strains of Bacillus such as B.
amyloliquefaciens. Proteases added to the fermentation may increase
the free amino nitrogen level and increase the rate of metabolism
of the yeast and further give higher fermentation efficiency.
[0084] Enzymes that may be used in the methods of the invention
include beta-amylases (E.C. 3.2.1.2). These are exo-acting
maltogenic amylases, which catalyze the hydrolysis of
1,4-alpha-glucosidic linkages in amylose, amylopectin and related
glucose polymers. Commercial beta-amylases are available from
Genencor International Inc., and examples include SPEZYME BBA and
OPTIMALT BBA.
[0085] Cellulases (E.C. 3.2.1.4) such as endo-glucanases may be
used in the methods of the invention. Examples of cellulases
include cellulases from filamentous fungus such as Trichoderma,
Humicola, Fusarium, and Aspergillus. Commercially cellulases are
available as SPEZYME CP and LAMINEX (Genencor International, Inc)
and CELLUZYME and ULTRAFLO (Novozymes A/S).
[0086] Xylanases useful in the methods of the invention may be from
bacterial or fungal sources, such as Aspergillus, Trichoderma,
Neurospora, and Fusarium. Commercial preparations include SPEZYME
CP and LAMINEX (Genencor International, Inc.) and ULTRAFLO
(Novozymes A/S).
[0087] A number of bacterial and fungal phytases (E.C. 3.1.3.8 and
3.1.3.26) are known and in some embodiments the addition of
phytases are particularly useful in the methods. Yeast phytases may
be derived from strains of Saccharomyces (e.g. S. cerevisiae) and
Schwanniomyces (e.g. S. occidentalis) (Wodzinski et al., Adv.
Apple. Microbiol., 42:263-303). Other fungal phytases have been
described in the literature and reference is made to Wyss et al.,
(1999) Appl. Environ Microbiol. 65:367-373; Berka et al., (1998)
Appl. Environ. Microbiol. 64: 4423-4427; Yamada et al., (1986)
Agric. Biol. Chem. 322:1275-1282; PCT Publication Nos. WO 98/28408;
WO 98/28409; WO 97/38096 and WO 9844125; and U.S. Pat. Nos.
6,734,004; 6,350,602; and 5,863,533). Fungal phytases have been
derived from Aspergillus (e.g. A. niger, A. awamori, A. terreus, A.
oryzea and A. fumigatus); Thermomyces (Humicola) lanuginousus;
Fusarium (F. javanicum and F. versillibodes). Bacterial phytases
may also find use in the invention (Greiner R. et al. (1993) Arch.
Biochem. Biophys. 303: 107-113; Yoon S. J. et al. (1996) Enzyme and
Microbial Technol. 18: 449-454; and WO 06/043178).
[0088] Commercially available phytases which may be used according
to the invention include PHYZYME XP 5000 (Danisco A/S); FINASE
(Altech); GC 491; FINASE, SPEZYME HPA (Genencor), BIO-FEED PHYTASE
and PHYTASE NOVO (Novozymes) and NATUPHOS (DSM).
[0089] One skilled in the art can readily determine the effective
amount of the enzymes which may be used in the process steps
encompassed by the invention.
(B) Process Steps
[0090] The granular starch (e.g. milled cereal grain) to be
processed is mixed with an aqueous solution to obtain a slurry. The
aqueous solution may be obtained, for example from water, thin
stillage and/or backset.
[0091] A slurry may have a DS of between 5-60%; 10-50%; 15-45%;
15-30%; 20-45%; 20-30% and also 25-40%. The contacting step with an
alpha-amylase is conducted at a pH range of 3.5 to 7.0; also at a
pH range of 3.5 to 6.5; preferably at a pH range of 4.0 to 6.0 and
more preferably at a pH range of 4.5 to 5.5. The slurry is held in
contact with the alpha-amylase at a temperature below the starch
gelatinization temperature of the granular starch. In some
embodiments, this temperature is held between 45.degree. C. and
70.degree. C.; in other embodiments, the temperature is held
between 50.degree. C. and 70.degree. C.; between 55.degree. C. and
70.degree. C.; between 60.degree. C. and 70.degree. C., between
60.degree. C. and 65.degree. C.; between 55.degree. C. and
65.degree. C. and between 55.degree. C. and 68.degree. C. In
further embodiments, the temperature is at least 45.degree. C.,
48.degree. C., 50.degree. C., 53.degree. C., 55.degree. C.,
58.degree. C., 60.degree. C., 63.degree. C., 65.degree. C. and
68.degree. C. In other embodiments, the temperature is not greater
than 65.degree. C., 68.degree. C., 70.degree. C., 73.degree. C.,
75.degree. C. and 80.degree. C.
[0092] The initial starch gelatinization temperature ranges for a
number of granular starches which may be used in accordance with
the processes herein include barley (52.degree. C. to 59.degree.
C.), wheat (58.degree. C. to 64.degree. C.), rye (57.degree. C. to
70.degree. C.), corn (62.degree. C. to 72.degree. C.), high amylose
corn (67.degree. C. to 80.degree. C.), rice (68.degree. C. to
77.degree. C.), sorghum (68.degree. C. to 77.degree. C.), potato
(58.degree. C. to 68.degree. C.), tapioca (59.degree. C. to
69.degree. C.) and sweet potato (58.degree. C. to 72.degree. C.).
(J.J.M. Swinkels pg 32-38 in STARCH CONVERSION TECHNOLOGY, Eds Van
Beynum et al., (1985) Marcel Dekker Inc. New York and The Alcohol
Textbook 3.sup.rd ED. A Reference for the Beverage, Fuel and
Industrial Alcohol Industries, Eds Jacques et al., (1999)
Nottingham University Press, UK).
[0093] In the contacting step, the slurry may be held in contact
with the alpha-amylase for a period of 5 minutes to 48 hours; and
also for a period of 5 minutes to 24 hours. In some embodiments the
period of time is between 15 minutes and 12 hours, 15 minutes and 6
hours, 15 minutes and 4 hours and also 30 minutes and 2 hours.
[0094] The effective concentration of the alpha-amylase used in the
contacting step will vary according to the specific process
conditions and granular starch used. However, in general the amount
of alpha-amylase used will be in the range of 0.001 to 50 AAU/g DS,
0.01 to 30 AAU/g DS, 0.01 to 10 AAU/g DS and also 0.05 to 5.0 AAU/g
DS.
[0095] In some embodiments, the effective dose of an alpha-amylase
in the contacting step and/or fermentation step will be 0.01 to 15
SSU/g DS; also 0.05 to 10 SSU/g DS; also 0.1 to 10 SSU/g DS; and
0.5 to 5 SSU/g DS.
[0096] In some embodiments, the effective dose of a glucoamylase
for the contacting step and/or the fermentation step will be in the
range of 0.01 to 15 GAU/g DS; also 0.05 to 10 GAU/g DS; also 0.1 to
10 GAU/g DS and even 0.5 to 5 GAU/g DS.
[0097] In some embodiments, the effective dose of a phytase to be
used in the contacting step and/or fermentation step will be in the
range of 0.001 to 15 FTU/g DS; also 0.005 to 10 FTU/g DS; and also
0.05 to 5 FTU/g DS. One phytase unit (FTU) is the amount of enzyme,
which liberates 1 micromole inorganic phosphorus per minute from
sodium phytate, 0.0051 moles/ liter, at 37.degree. C. and at pH
5.0.
[0098] In some embodiments, the effective dose of a protease to be
used in the contacting step and/or fermentation step will be in the
range of 0.01 to 15 SAPU/g DS; also 0.01 to 10 SAPU/g DS; and also
0.05 to 5 SAPU/g DS. SAPU refers a spectrophotometric acid protease
unit, wherein 1 SAPU is the amount of protease enzyme activity that
liberates one micromole of tyrosine per minute from a casein
substrate under conditions of the assay.
[0099] During the contacting step between 25-90% of the granular
starch is solublized to produce oligosaccharides comprising
dextrin. In some embodiments, greater than 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% and 90% of the granular
starch is solublized.
[0100] After contacting the granular starch with the alpha-amylase
for a period of time as indicated above, a soluble starch substrate
(mash) is obtained which comprises greater than 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% and 80%
glucose.
[0101] In some preferred embodiments of the contacting step, a
slurry comprising granular corn starch having a DS of 20-40% is
contacted with an alpha-amylase derived from Bacillus
stearothermophilus or Bacillus licheniformis for 1 to 6 hours at a
temperature between 55.degree. C. to 70.degree. C. to obtain a
soluble starch substrate comprising at least 30% glucose. In other
preferred embodiments of the contacting step, a slurry comprising
granular milo starch having a DS of 20-40% is contacted with an
alpha-amylase derived from Bacillus stearothermophilus or Bacillus
licheniformis for 1 to 6 hours at a temperature between 55.degree.
C. to 70.degree. C. to obtain a soluble starch substrate comprising
at least 50% glucose.
[0102] After the contacting step which results in the production of
a mash comprising glucose, the mash is subjected to fermentation
with a fermenting microorganism (e.g. an ethanol-producing
microorganism).
[0103] However, prior to subjecting the mash including at least 10%
glucose to fermentation, the mash may be further exposed to an
aqueous solution comprising, for example backset and/or corn steep
and adjusted to a pH in the range of pH 3.0 to 6.0; pH 3.5 to 5.5,
or pH 4.0 to 5.5. In this embodiment of the invention, the % DS of
the mash may be diluted. For example, the DS of the diluted mash
maybe between 5 to 35%; 5 to 30%; 5 to 25%; 5 to 20%; 5 to 20%; 5
to 15%; and 5 to 10% less than the % DS of the slurry in the
contacting step. In one non-limiting example, if the % DS of the
slurry in the contacting step is approximately 32% and the mash is
further exposed to a diluting aqueous solution which dilutes the DS
between 5 to 10%, the DS of the mash to be fermented will be
between 22% and 27%. In some preferred embodiments, if the DS of
the contacting slurry is between 30 to 35%, the DS of the diluted
slurry will be about 20 to 30%.
[0104] In a preferred embodiment, the mash comprising at least 10%
glucose is then subjected to fermentation processes using
fermenting microorganisms as described above. These fermentation
processes are described in The Alcohol Textbook 3.sup.rd ED, A
Reference for the Beverage, Fuel and Industrial Alcohol Industries,
Eds Jacques et al., (1999) Nottingham University Press, UK.
[0105] In some preferred embodiments, the mash is fermented with a
yeast at temperatures in the range of 15 to 40.degree. C. and also
25 to 35.degree. C.; at a pH range of pH 3.0 to 6.5; also pH 3.0 to
6.0; pH 3.0 to 5.5, pH 3.5 to 5.0 and also pH 3.5 to 4.5 for a
period of time of 12 to 240 hours, preferably 12 to 120 and more
preferably from 24 to 90 hours to produce an alcohol product,
preferably ethanol.
[0106] Yeast cells are generally supplied in amounts of 10.sup.4 to
10.sup.12, and preferably from 10.sup.7 to 10.sup.10 viable yeast
count per ml of fermentation broth. The fermentation will include
in addition to a fermenting microorganisms (e.g. yeast) nutrients,
optionally acid and additional enzymes.
[0107] In one preferred embodiment, the contacting step is
conducted in a separate vessel from the fermenting step. It is also
contemplated that the contacting step and fermenting step may be
conducted in a SSF process in the same vessel.
[0108] In some embodiments, in addition to the raw materials
described above, fermentation media will contain supplements
including but not limited to vitamins (e.g. biotin, folic acid,
nicotinic acid, riboflavin), cofactors, and macro and
micro-nutrients and salts (e.g. (NH4).sub.2SO.sub.4;
K.sub.2HPO.sub.4; NaCl; MgSO.sub.4; H.sub.3BO.sub.3; ZnCl.sub.2;
and CaCl.sub.2).
[0109] Additional enzymes to be included in the fermentation step
may be the same or different from the enzymes used in the
contacting step. In some embodiments, the enzyme will include
alpha-amylases and glucoamylases, including granular starch
hydrolyzing enzymes. In some preferred embodiments, the
glucoamylase and alpha-amylase may occur in a blend. Particualrly
preferred enzyme blends include STARGEN 001 (Genencor International
Inc.), which is a blend of an alpha-amylase from A. kawachi and a
glucoamylase from A. niger. In some preferred embodiments, the
glucoamylase will be derived from a Trichoderma reesei
glucoamylase, a Athelia rolfi glucoamylase, a Talaromyces
glucoamylase, a Aspergillus glucoamylase and hybrid and variants
glucoamylase derived there from. In some preferred embodiments, the
enzyme is selected from a cellulase, a phytase and a protease.
Recovery of Alcohol and Other End Products
[0110] The preferred end product of the instant fermentation
process is an alcohol product, preferably ethanol. The end product
produced according to the process may be separated and/or purified
from the fermentation media. Methods for separation and
purification are known, for example by subjecting the media to
extraction, distillation and column chromatography. In some
embodiments, the end product is identified directly by submitting
the media to high-pressure liquid chromatography (HPLC)
analysis.
[0111] In further embodiments, the mash may be separated by for
example centrifugation into the liquid phase and solids phase and
end products such as alcohol and solids recovered. The alcohol may
be recovered by means such as distillation and molecular sieve
dehydration or ultra filtration.
[0112] In some embodiments, the yield of ethanol will be greater
than 8%, 10%, 12%, 14%, 16% and 18% by volume. The ethanol obtained
according to process of the invention may be used as a fuel
ethanol, potable ethanol or industrial ethanol.
[0113] In further embodiments, the end product may include the
fermentation co-products such as distillers dried grains (DDG) and
distiller's dried grain plus solubles (DDGS), which may be used as
an animal feed.
Experimental
[0114] The following examples are provided in order to demonstrate
and further illustrate certain preferred embodiments and aspects of
the present invention and are not to be construed as limiting the
scope thereof. Indeed, it is contemplated that these teachings will
find use in further optimizing the process systems described
herein.
[0115] In the disclosure and experimental section which follows,
the following abbreviations apply: GA (glucoamylase); wt % (weight
percent); .degree. C. (degrees Centigrade); H.sub.2O (water);
dH.sub.2O (deionized water); dIH.sub.2O (deionized water, Milli-Q
filtration); g or gm (grams); .mu.g (micrograms); mg (milligrams);
kg (kilograms); .mu.L (microliters); ml and mL (milliliters); mm
(millimeters); .mu.m (micrometer); M (molar); mM (millimolar);
.mu.M (micromolar); U (units); MW (molecular weight); sec
(seconds); min(s) (minute/minutes); hr(s) (hour/hours); DO
(dissolved oxygen); W/V (weight to volume); W/W (weight to weight);
V/V (volume to volume); Genencor (Genencor International, Inc.,
Palo Alto, Calif.); MT (Metric ton); and ETOH (ethanol).
The following enzyme preparations were used in the examples
below:
[0116] SPEZYME Ethyl (available from Genencor)--a bacterial
alpha-amylase obtained from a genetically modified strain of
Bacillus licheniformis.
[0117] GC100--an experimental bacterial alpha-amylase disclosed in
US 2006/0014265.
[0118] Humicola grisea glucoamylase (HGA) having the amino acid
sequence disclosed as SEQ ID NO: 3 of WO 2005/052148.
[0119] STARGEN 001 (available from Genencor)--a blend of
Aspergillus niger glucoamylase and Aspergillus kawachi
alpha-amylase.
The following assays were used in the examples below:
[0120] The activity of alpha-amylase is expressed as alpha amylase
units (AAU) and enzyme activity was determined by the rate of
starch hydrolysis, as reflected in the rate of decrease of
iodine-staining capacity, which was measured
spectrophotometrically. One AAU of bacterial alpha-amylase activity
is the amount of enzyme required to hydrolyze 10 mg of starch per
min under standardized conditions.
[0121] Alpha-amylase activity made also determined as soluble
starch unit (SSU) and is based on the degree of hydrolysis of
soluble potato starch substrate (4% DS) by an aliquot of the enzyme
sample at pH 4.5, 50.degree. C. The reducing sugar content is
measured using the DNS method as described in Miller, G. L. (1959)
Anal. Chem. 31:426-428. One unit of the enzyme activity (SSU) is
equivalent to the reducing power of lmg of glucose released per
minute at the specific incubation conditions.
[0122] Glucoamylase activity was measured using a well-known assay
which is based on the ability of glucoamylase to catalyze the
hydrolysis of p-nitrophenyl-alpha-D-glucopyranoside (PNPG) to
glucose and p-nitrophenol. At an alkaline pH, the nitrophenol;
forms a yellow color that is proportional to glucoamylase activity
and is monitored at 400 nm and compared against an enzyme standard
measured as a GAU.
[0123] One "Glucoamylase Activity Unit" (GAU) is the amount of
enzyme that will produce 1 gm of reducing sugar, calculated as
glucose per hour from a soluble starch substrate (4% DS) at pH 4.2
and 60.degree. C.
[0124] Brix, the measurement of total solublizied solid content at
a given temperature was determined by measurement with a
Refractometer.
[0125] Determination of total starch content: The enzyme-enzyme
starch liquefaction and saccharification process was used to
determine the total starch content. In a typical analysis, 2 g of
dry sample was taken in a 100 ml Kohlraucsh flask and 45 ml of MOPS
buffer, pH 7.0 was added. The slurry was well stirred for 30 min.
SPEZYME FRED (1:50 diluted in water) (Genencor), 1.0 ml was added
and heated to boiling for 3-5 min. The flask was placed in an
autoclave maintained at 121.degree. C. for 15 min. After
autoclaving the flask was placed in a water bath at 95.degree. C.
and 1 ml of 1:50 diluted SPEZYME FRED was added and incubated for
45 min. The pH was adjusted to pH 4.2 and the temperature was
reduced to 60.degree. C. This was followed by addition of 20 ml
acetate buffer, pH 4.2. Saccharification was carried out by adding
1.0 ml of 1:100 diluted OPTIDEX L-400 (Genencor) and the incubation
was continued for 18 hr at 60.degree. C. The enzyme reaction was
terminated by heating at 95.degree. C. for 10 min. The total sugar
composition was determined by HPLC analysis using glucose as a
standard. The soluble starch hydrolysate from water extraction of a
sample at room temperature without enzymatic treatment was
subtracted from the total sugar.
[0126] Determination of the % solubilized solids--a 7 ml sample was
placed in a small screw cap test tube (pH adjusted to 5.0 to 6.0)
and 0.007 ml SPEZYME Fred was added to the tube. The test tube was
placed in a boiling water bath for 10 min and gently mixed at
various times during the incubation. After 10 min the tube was
removed and placed in a 80.degree. C. water bath for 1 hr. The tube
was cooled and centrifuges. The Brix of the supernatant was
determined and compared to a control sample. The % solubilized
solids=control Brix.times.100/sample Brix.
[0127] Ethanol and carbohydrate determinations of the samples were
determined using the HPLC method as follows:
[0128] a 1.5 mL Eppendorf centrifuge tube was filled with fermentor
mash and cooled on ice for 10 min; the sample tube was centrifuged
for 1 min in an Eppendorf table top centrifuge; a 0.5 mL sample of
the supernatant was transferred to a test tube containing 0.05 mL
of 1.1N H.sub.2SO.sub.4 and allowed to stand for 5 min; 5.0 mL of
water was added to the test tube and then the sample was filtered
into a HPLC vial through 0.2 .mu.m Nylon Syringe Filter; and run on
HPLC. The HPLC conditions included:
[0129] Ethanol System: Column: Phenomenex Rezex Organic Acid Column
(RHM-Monosaccharide) #00H 0132-KO (Equivalent to Bio-Rad 87H);
Column Temperature: 60.degree. C.; Mobile Phase: 0.01 N
H.sub.2SO.sub.4; Flow Rate: 0.6 mL/min; Detector: RI; and Injection
Volume: 20 .mu.L.
[0130] Carbohydrate System: Column: Phenomenex Rezex Carbohydrate
(RCM-Monosaccharide) #00H-0130-KO (Equivalent to Bio-Rad 87H);
Column Temperature: 70.degree. C. ; Mobile Phase: Nanopure DI
H.sub.2O; Flow Rate: 0.8 mL/min; Detector: RI; Injection Volume: 10
.mu.L (3% DS material).
[0131] The column separated based on the molecular weight of the
saccharides, which are designated as DP1 (glucose); DP2
(disaccharides); DP3 (trisaccharides) and DP>3 (oligosaccharide
sugars having a degree of polymerization greater than 3).
EXAMPLE 1
Solubilization and Ethanol Production from Granular Starch of Whole
Ground Corn and Fractionated Corn
[0132] This experiment was run on three different corn granular
starch substrates (A) 370 g of whole ground corn having a moisture
content of 13.3%; (B) 354.2 g corn endosperm having a moisture
content of 9.2%; and (C) refined corn starch obtained from having a
moisture content of 11.8%. Each substrate was weighed and
transferred to a stainless steel vessel to make a final 1000 g
slurry with water corresponding to 32% DS.
[0133] The pH of the slurry was adjusted to pH 5.5 using 6N
H.sub.2SO.sub.4. GC100 (4.0 AAU/g DS) was added. The temperature
was maintained at 60.degree. C. During the incubation the slurry
was gently stirred with an overhead mixer. After time internals of
2, 4, 6, 12 and 24 hours, the BRIX, % solublilzed starch and sugar
compositions (% W/W) were determined, and the results are
illustrated in Table 1. At 24 hours, 79.1%, 71.1% and 60.0% of the
granular starch from whole ground corn, endosperm and refined sugar
was solubilized during the contacting step respectively. The %
glucose of the hydrolyzate at 24 hours was 65.22% for whole ground
corn, 49.64% for endosperm and only 5.79% for refined starch.
TABLE-US-00001 TABLE 1 % Starch Solubi- % % % Grain Time BRIX lized
Glucose DP2 DP3 DP >3 Whole 2 11.5 31.60 21.36 13.48 33.56 corn
4 14.0 37.56 24.31 13.65 24.48 6 16.3 41.33 25.42 13.36 19.88 12
18.2 46.23 26.18 12.63 14.96 24 20.0 79.1 65.22 22.50 7.36 4.92
Endosperm 2 13.4 27.12 13.85 8.88 50.15 4 16.1 33.01 16.45 9.39
41.15 6 17.5 36.56 18.18 9.59 35.67 12 21.7 47.43 20.98 9.65 21.94
24 22.9 71.1 49.64 21.33 9.52 19.51 Refined 2 14.4 1.40 10.37 15.27
72.96 Cornstarch 4 16.8 2.34 11.60 15.22 70.84 6 17.8 5.14 12.27
15.13 67.45 12 19.1 4.94 13.21 15.62 66.22 24 20.9 60.0 5.79 14.40
17.42 62.39
[0134] After 24 hours, using the samples from whole ground corn
(32% DS), yeast fermentations were conducted at pH 4.2, 32.degree.
C. in the presence of 400 ppm urea; Red Star Red yeast (Fermentus);
STARGEN 001; and 0.1 SAPU/g DS protease in a 125 ml flask. HPLC
data are illustrated in Table 2.
TABLE-US-00002 TABLE 2 STARGEN 001 % V/V ETOH % V/V ETOH % V/V ETOH
GAU/g 24 hrs 48 hrs 72 hrs 0.1 10.11 11.74 13.64 0.2 10.36 12.19
14.62 0.4 10.83 12.73 15.35
EXAMPLE 2
Solubilization and Ethanol Production from Milo Granular Starch
[0135] Two pretreatments were run: (A) 160 g of whole ground milo
having a moisture content of 11.6% and a total starch content of
53.3% was weighed and transferred to a stainless steel vessel
containing 340 g water. The pH of the slurry was adjusted to pH 5.5
using 6 N sulphuric acid. SPEZYME Ethyl (1.0 AAU/g DS) was added.
The temperature was maintained at 62.degree. C. and 32% DS. (B) HGA
was included in the pretreatment as described above in (A) at the
equivalent of 0.1 GAU HGA/g DS. The % solublized solids and %
glucose are presented in Table 3.
TABLE-US-00003 TABLE 3 % Time % DP1 % % % solublized Enzyme (hrs)
(glucose) DP2 DP3 DP >3 starch SPEZYME 2 57.71 24.60 10.42 7.27
Ethyl 4 64.41 22.50 9.13 3.95 6 67.16 21.83 8.16 2.85 24 86.50 9.91
2.95 0.63 54.7 SPEZYME 2 84.34 9.48 1.47 4.72 Ethyl + 4 87.72 8.07
1.18 3.03 HGA 6 88.91 7.68 1.03 2.37 24 93.10 5.26 0.59 1.05
69.3
[0136] The feedstock (mash) from the HGA pretreatment described
above was evaluated under regular yeast fermentation conditions
(e.g. Red Star Yeast, pH 4.2, 32.degree. C. in the presence of 400
ppm urea; STARGEN 001 and 0.05 SAPU/g DS) in a 125 ml flask. HPLC
results are illustrated in Table 4.
TABLE-US-00004 TABLE 4 GAU/g % V/V Ethanol % V/V Ethanol
Pre-treatment STARGEN 001 24 hrs 48 hrs SPEZYME 0.1 10.02 11.98
Ethyl + HGA 0.2 10.22 12.75
EXAMPLE 3
Effect of Temperature on Glucose Production from Whole Ground
Milo
[0137] Incubation of a 30% ds aqueous slurry of whole ground milo
at pH 5.5 containing GC100 (4.0 AAU/g DS) was carried out at
60.degree. C., 65.degree. C. and 70.degree. C. After 6 hours of the
incubation, the samples were withdrawn and centrifuged to separate
the insolubles. The Brix and HPLC composition of the clear
supernatant was measured. The % solubilized starch and % glucose
were determined and the results are illustrated in Table 5.
TABLE-US-00005 TABLE 5 % % W/W Solubilized DP1 % W/W % W/W % W/W
.degree. C. Starch (Glucose) DP2 DP3 DP > 3 60 69.9 52.48 27.07
12.08 8.36 65 68.2 52.61 23.42 10.41 13.56 70 61.9 36.49 18.98
10.27 34.26
[0138] As the incubation temperature increased from 60.degree. C.
to 70.degree. C. during the contacting step with whole ground milo,
the solubilization of starch and the glucose content decreased.
This suggests that the alpha-amylase may be inactivated at
70.degree. C. More than 50% of the solubilized starch was
hydrolyzed to glucose at 65.degree. C. suggesting the endogenous
plant starch hydrolyzing enzymes are capable of hydrolyzing the
soluble oligosaccharides into glucose.
[0139] The feedstock from the pretreatment described above at
65.degree. C. was evaluated under regular fermentation conditions
as essentially described in example 2; except the % DS was 30. The
results are illustrated in Table 6.
TABLE-US-00006 TABLE 6 STARGEN 001 % V/V ETOH % V/V ETOH % V/V ETOH
(GAU/g DS) 24 hrs 48 hrs 72 hrs 0.1 9.19 11.00 11.29 0.2 9.32 11.07
12.80 0.4 9.61 11.47 13.32
EXAMPLE 4
Solubilization and Ethanol Production from Rice Granular Starch
[0140] Rice grain (116 g) having a starch content of 81.5%; a
moisture content of 14% and a particle size that passes through a
30 mesh screen was mixed with 284 g of water to make a 25% DS
slurry. GC100 (4.0 AAU/g DS) was added to the slurry. The
temperature was maintained at 65.degree. C. and pH adjusted to pH
5.5. The Brix was measured at 2, 4 6, and 24 hrs. The % soluble
starch and % glucose were determined and the results are
illustrated in Table 7.
TABLE-US-00007 TABLE 7 % % W/V Time solubilized DP1 % W/V % W/V %
W/V hrs Brix starch (glucose) DP2 DP3 .DP3 1 11 39.2 27.5 19.8 12.7
39.6 2 12.5 44.6 31.2 22.2 13.4 32.6 4 14.8 52.8 33.4 24.0 13.8
28.7 6 16.2 57.5 33.8 24.4 13.9 27.7 24 16.4 58.4 36 26.2 14.6
23.2
[0141] After 24 hours, yeast fermentations were conducted at pH
4.2, 30.degree. C. in the presence of 0.75 GAU/g DS STARGEN 001,
400 ppm urea, and Angel yeast (Jiangxi, China) at 0.4%. HPLC
samples were taken at 24, 48 and 67 hrs (Table 8).
TABLE-US-00008 TABLE 8 Time % W/V % V/V hrs glucose ETOH 24 2.15
6.62 48 0.32 10.4 67 0.35 12.0
* * * * *