U.S. patent application number 10/360010 was filed with the patent office on 2003-09-25 for methods for producing ethanol from carbon substrates.
Invention is credited to Lantero, Oreste.
Application Number | 20030180900 10/360010 |
Document ID | / |
Family ID | 27734476 |
Filed Date | 2003-09-25 |
United States Patent
Application |
20030180900 |
Kind Code |
A1 |
Lantero, Oreste |
September 25, 2003 |
Methods for producing ethanol from carbon substrates
Abstract
The present invention provides means for the production of
desired end-products of in vitro and/or in vivo bioconversion of
biomass-based feed stock substrates, including but not limited to
such materials as starch and cellulose. In particularly preferred
embodiments, the methods of the present invention do not require
gelatinization and/or liquefaction of the substrate. In
particularly preferred embodiments, the present invention provides
means for the production of ethanol.
Inventors: |
Lantero, Oreste; (Belvidere,
IL) |
Correspondence
Address: |
Genencor International, Inc.
925 Page Mill Road
Palo Alto
CA
94034-1013
US
|
Family ID: |
27734476 |
Appl. No.: |
10/360010 |
Filed: |
February 6, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60355180 |
Feb 8, 2002 |
|
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Current U.S.
Class: |
435/160 ;
435/161 |
Current CPC
Class: |
Y02E 50/16 20130101;
C12P 7/44 20130101; Y02E 50/10 20130101; C12P 7/20 20130101; Y02E
50/17 20130101; C12P 7/58 20130101; C12P 7/18 20130101; C12P 7/56
20130101; C12P 7/06 20130101; C12P 7/60 20130101; C12P 7/10
20130101 |
Class at
Publication: |
435/160 ;
435/161 |
International
Class: |
C12P 007/16; C12P
007/06 |
Claims
What is claimed is:
1. A method for producing an alcohol as an end-product comprising
the steps of: a) contacting a carbon substrate and at least one
substrate-converting enzyme to produce an intermediate; and b)
contacting said intermediate with at least one
intermediate-converting enzyme, wherein said intermediate is
substantially all converted by said intermediate enzyme to said
alcohol.
2. The method of claim 1, wherein said intermediate-converting
enzyme is a microbial enzyme.
3. The method of claim 2, wherein said intermediate-converting
microbial enzyme is secreted by a microorganism in contact with
said intermediate.
4. The method of claim 1, wherein said substrate-converting enzyme
is a microbial enzyme.
5. The method of claim 4, wherein said substrate-converting
microbial enzyme is secreted by a microorganism in contact with
said substrate.
6. The method of claim 1, wherein said intermediate-converting
enzyme and said substrate-converting enzyme are produced by
microorganisms of the same species.
7. The method of claim 1, wherein said intermediate-converting
enzyme and said substrate-converting enzyme are produced by
microorganisms of the different species.
8. The method of claim 1, wherein concentration level of said
intermediate is maintained at a level below that which triggers
catabolite repression effects upon the conversion of said
intermediate to said end-product.
9. The method of claim 1, wherein concentration level of said
intermediate is maintained at a level below that which triggers
enzymatic inhibition effects upon the conversion of said
intermediate to said end-product.
10. The method of claim 1, wherein said intermediate is converted
to said end-product at a rate sufficient to maintain the
concentration of said at less than 0.25%.
11. The method of claim 1, wherein said substrate is selected from
the group consisting of biomass and starch.
12. The method of claim 11, wherein said biomass comprises corn
solids.
13. The method of claim 1, wherein said intermediate is selected
from the group consisting of hexoses and pentoses.
14. The method of claim 13, wherein said hexose is glucose.
15. The method of claim 1, wherein said contacting said substrate
and substrate-converting enzyme further comprises bioconverting
said substrate to produce said intermediate.
16. The method of claim 1, wherein said alcohol end-product is
ethanol.
17. The method of claim 1, wherein the step of contacting said
substrate and said at least one substrate-converting enzyme further
comprises providing an amount of said substrate-converting enzyme
at a concentration that produces said intermediate at a
concentration that is less than or equal to the amount of said
intermediate converted by said at least one intermediate-converting
enzyme.
18. The method of claim 1, wherein said at least one
substrate-converting enzyme converts at least 50% of said substrate
to said intermediate within 72 hours.
19. The method of claim 18, wherein said at least one
substrate-converting enzyme converts at least 90% of said substrate
to said intermediate within 72 hours.
20. The method of claim 19, wherein said at least one
substrate-converting enzyme converts at least 95% of said substrate
to said intermediate within 72 hours.
21. The method of claim 1, wherein said at least one
substrate-converting enzyme and said at least one
intermediate-converting enzyme are obtained from a microorganism
selected from the group consisting of Rhizopus and Aspergillus.
22. A method for producing alcohol as an end-product comprising the
steps of: a) contacting a carbon substrate and at least one
substrate-converting enzyme to produce an intermediate; and b)
contacting said intermediate with at least one
intermediate-converting enzyme, wherein said intermediate is
substantially all converted by said intermediate enzyme to said
alcohol end-product, and wherein the presence of said end-product
does not inhibit the further production of said alcohol
end-product.
23. The method of claim 22, wherein said intermediate-converting
enzyme is a microbial enzyme.
24. The method of claim 22, wherein said intermediate-converting
microbial enzyme is secreted by a microorganism in contact with
said intermediate.
25. The method of claim 22, wherein said substrate-converting
enzyme is a microbial enzyme.
26. The method of claim 22, wherein said substrate-converting
microbial enzyme is secreted by a microorganism in contact with
said substrate.
27. The method of claim 22, wherein said intermediate-converting
enzyme and said substrate-converting enzyme are produced by
microorganisms of the same species.
28. The method of claim 22, wherein said intermediate-converting
enzyme and said substrate-converting enzyme are produced by
microorganisms of the different species.
29. The method of claim 22, wherein said alcohol end-product is
ethanol.
30. A method for producing an alcohol end-product comprising the
steps of: a) contacting a carbon substrate and at least one
substrate-converting enzyme to produce an intermediate; and b)
contacting said intermediate with at least one
intermediate-converting enzyme, wherein said intermediate is
substantially all converted by said intermediate enzyme to said
alcohol end-product, and wherein the presence of said substrate
does not inhibit the further production of said alcohol
end-product.
31. The method of claim 30, wherein said intermediate-converting
enzyme is a microbial enzyme.
32. The method of claim 30, wherein said intermediate-converting
microbial enzyme is secreted by a microorganism in contact with
said intermediate.
33. The method of claim 30, wherein said substrate-converting
enzyme is a microbial enzyme.
34. The method of claim 30, wherein said substrate-converting
microbial enzyme is produced is secreted by a microorganism in
contact with said substrate.
35. The method of claim 30, wherein said intermediate-converting
enzyme and said substrate-converting enzyme are produced by
microorganisms of the same species.
36. The method of claim 30, wherein said intermediate-converting
enzyme and said substrate-converting enzyme are produced by
microorganisms of the different species.
37. The method of claim 36, wherein said alcohol end-product is
ethanol.
Description
[0001] The present application claims priority to U.S. Prov. Patent
Appln. Ser. No. 60/355,180, filed Feb. 8, 2002.
FIELD OF INVENTION
[0002] The present invention provides means for the production of
desired end-products of in vitro and/or in vivo bioconversion of
biomass-based feed stock substrates, including but not limited to
such materials as starch and cellulose. In particularly preferred
embodiments, the methods of the present invention do not require
gelatinization and/or liquefaction of the substrate. In
particularly preferred embodiments, the present invention provides
means for the production of ethanol.
BACKGROUND OF THE INVENTION
[0003] Industrial fermentations predominantly utilize glucose as
feed-stock for the production of proteins, enzymes and chemicals.
These fermentations are usually batch, fed-batch, or continuous,
and operate under conditions that are substrate-limited and/or
designed to produce minimal by-products. As those in the art know,
there are certain critical operating conditions that must be
controlled during fermentation so as to optimize fermentation time,
yield and efficiency.
[0004] Glucose is a natural, carbon based compound that is useful
in a multitude of chemical and biological synthetic applications as
a starting substrate. However, syrups that contain glucose purity
levels of greater than 90% are relatively expensive. In addition,
the presence of high glucose concentrations increases the
susceptibility of the fermentation system to microbial
contamination, thereby resulting in an adverse effect upon the
production efficiency. Another disadvantage is that even the
presence of low to moderate levels of glucose in the fermentation
vat adversely affects the conversion of the glucose to the desired
end product, for example by enzymatic inhibition and/or catabolite
repression, and/or the growth of microorganisms. As a result,
various attempts have been made to reduce the costs of industrial
fermentation, particularly in utilization of substrates that are
less expensive than glucose. However, despite the development of
numerous approaches, there remains a need in the art for
economical, efficiently-utilized substrates for fermentation.
Indeed, there is a great need in the art for methods that utilize a
less expensive starting material than glucose to more efficiently
produce a desired end-product.
SUMMARY OF THE INVENTION
[0005] The present invention provides means for the production of
desired end-products of in vitro and/or in vivo bioconversion of
biomass-based feed stock substrates, including but not limited to
such materials as starch and cellulose. In particularly preferred
embodiments, the methods of the present invention do not require
gelatinization and/or liquefaction of the substrate. In
particularly preferred embodiments, the present invention provides
means for the production of ethanol. In some particularly preferred
embodiments, the present invention provides means for the
production of ethanol directly from granular starch, in which
altered catabolite repression is involved.
[0006] In some embodiments, the present invention provides methods
for producing ethanol in which the glucose concentration of the
conversion medium is maintained at a low concentration, preferably
below the threshold triggering catabolite repression and/or enzyme
inhibition, so as to increase efficiency of the process by avoiding
catabolic repressive and/or enzymatic inhibitive effects of glucose
upon the enzymatic conversion of starch to ethanol.
[0007] In additional embodiments, the present invention provides
methods for producing ethanol comprising the steps of contacting at
least one carbon substrate with at least one substrate converting
enzyme, to produce at least one intermediate, and then contacting
at least one intermediate with at least one intermediate producing
enzyme in a reactor vessel, wherein the at least one intermediate
is substantially all bioconverted an end-product. In some preferred
embodiments, a microorganism is used to achieve this bioconversion.
By maintaining a low concentration of the intermediate in a
conversion medium, the intermediate's catabolite repressive and/or
enzymatic inhibitive effects are altered (e.g., reduced). The
present invention also provides various levels of intermediate
concentration, substrates, intermediates and steps of converting
the intermediate to ethanol.
[0008] The present invention provides methods for producing an
alcohol as an end-product comprising the steps of: contacting a
carbon substrate and at least one substrate-converting enzyme to
produce an intermediate; and contacting the intermediate with at
least one intermediate-converting enzyme, wherein the intermediate
is substantially all converted by the intermediate enzyme to
alcohol. In some preferred embodiments, the intermediate-converting
enzyme is a microbial enzyme. In alternative preferred embodiments,
the intermediate-converting microbial enzyme is secreted by a
microorganism that is in contact with the intermediate. In further
embodiments, substrate-converting enzyme is a microbial enzyme. In
some preferred embodiments, the substrate-converting microbial
enzyme is secreted by a microorganism that is in contact with the
substrate. In still other preferred embodiments, the
intermediate-converting enzyme and the substrate-converting enzyme
are produced by microorganisms of the same species. In alternative
embodiments, the intermediate-converting enzyme and the
substrate-converting enzyme are produced by microorganisms of the
different species. In still further embodiments, the concentration
level of the intermediate is maintained at a level below that which
triggers catabolite repression effects upon the conversion of the
intermediate to the end-product. In additional embodiments,
concentration level of the intermediate is maintained at a level
below that which triggers enzymatic inhibition effects upon the
conversion of the intermediate to the end-product. In some
preferred embodiments, the intermediate is converted to the
end-product at a rate sufficient to maintain the concentration that
is less than 0.25%. In yet other embodiments, the substrate is
selected from the group consisting of biomass and starch. In some
preferred embodiments, the biomass comprises corn solids. In some
particularly preferred embodiments, intermediate is selected from
the group consisting of hexoses and pentoses. In some embodiments,
the hexose is glucose. In some embodiments, the substrate is cooked
prior to its use in the present invention, while in other
embodiments, the substrate is uncooked prior to its use in the
present invention. In yet other embodiments, the step of contacting
the substrate and substrate-converting enzyme further comprises
bioconverting the substrate to produce the intermediate. In most
preferred embodiments, the alcohol end-product is ethanol. In still
further embodiments, the step of contacting the substrate and at
least one substrate-converting enzyme further comprises providing
an amount of the substrate-converting enzyme at a concentration
that produces the intermediate at a concentration that is less than
or equal to the amount of the intermediate converted by at least
one intermediate-converting enzyme. In some additional embodiments,
at least one substrate-converting enzyme converts at least 50% of
the substrate to the intermediate within 72 hours, while in other
embodiments, at least one substrate-converting enzyme converts at
least 90% of the substrate to the intermediate within 72 hours, and
in some preferred embodiments, at least one substrate-converting
enzyme converts at least 95% of the substrate to the intermediate
within 72 hours. In still further embodiments, at least one
substrate-converting enzyme and at least one
intermediate-converting enzyme are obtained from a microorganism
selected from the group consisting of Rhizopus and Aspergillus. In
additional embodiments, the substrate-converting and/or
intermediate-converting enzyme(s) are provided as a cell-free
extract.
[0009] In further preferred embodiments, the contacting steps take
place in a reaction vessel, including but not limited to vats,
bottles, flasks, bags, bioreactors, and any other receptacle
suitable for conducting the methods of the present invention.
[0010] The present invention further provides methods for producing
alcohol as an end-product comprising the steps of contacting a
carbon substrate and at least one substrate-converting enzyme to
produce an intermediate; and contacting the intermediate with at
least one intermediate-converting enzyme, wherein the intermediate
is substantially all converted by the intermediate enzyme to the
alcohol end-product, and wherein the presence of the end-product
does not inhibit the further production of the alcohol end-product.
In some preferred embodiments, the intermediate-converting enzyme
is a microbial enzyme. In alternative preferred embodiments, the
intermediate-converting microbial enzyme is secreted by a
microorganism that is in contact with the intermediate. In further
preferred embodiments, the substrate-converting enzyme is a
microbial enzyme. In still further embodiments, the
substrate-converting microbial enzyme is secreted by a
microorganism that is in contact with the substrate. In some
embodiments, intermediate-converting enzyme and the
substrate-converting enzyme are produced by microorganisms of the
same species, while in other embodiments, intermediate-converting
enzyme and the substrate-converting enzyme are produced by
microorganisms of the different species. In yet other embodiments,
the substrate is selected from the group consisting of biomass and
starch. In some preferred embodiments, the biomass comprises corn
solids. In some particularly preferred embodiments, intermediate is
selected from the group consisting of hexoses and pentoses. In some
embodiments, the hexose is glucose. In some embodiments, the
substrate is cooked prior to its use in the present invention,
while in other embodiments, the substrate is uncooked prior to its
use in the present invention. In some particularly preferred
embodiments, the alcohol end-product is ethanol. In still further
embodiments, the step of contacting the substrate and at least one
substrate-converting enzyme further comprises providing an amount
of the substrate-converting enzyme at a concentration that produces
the intermediate at a concentration that is less than or equal to
the amount of the intermediate converted by at least one
intermediate-converting enzyme. In some additional embodiments, at
least one substrate-converting enzyme converts at least 50% of the
substrate to the intermediate within 72 hours, while in other
embodiments, at least one substrate-converting enzyme converts at
least 90% of the substrate to the intermediate within 72 hours, and
in some preferred embodiments, at least one substrate-converting
enzyme converts at least 95% of the substrate to the intermediate
within 72 hours. In still further embodiments, at least one
substrate-converting enzyme and at least one
intermediate-converting enzyme are obtained from a microorganism
selected from the group consisting of Rhizopus and Aspergillus. In
additional embodiments, the substrate-converting and/or
intermediate-converting enzyme(s) are provided as a cell-free
extract. In further preferred embodiments, the contacting steps
take place in a reaction vessel, including but not limited to vats,
bottles, flasks, bags, bioreactors, and any other receptacle
suitable for conducting the methods of the present invention.
[0011] The present invention further provides methods for producing
an alcohol end-product comprising the steps of: contacting a carbon
substrate and at least one substrate-converting enzyme to produce
an intermediate; and contacting the intermediate with at least one
intermediate-converting enzyme, wherein the intermediate is
substantially all converted by the intermediate enzyme to the
alcohol end-product, and wherein the presence of the substrate does
not inhibit the further production of the alcohol end-product. In
some preferred embodiments, the intermediate-converting enzyme is a
microbial enzyme. In alternative preferred embodiments, the
intermediate-converting microbial enzyme is secreted by a
microorganism that is in contact with the intermediate. In further
preferred embodiments, the substrate-converting enzyme is a
microbial enzyme. In still further embodiments, the
substrate-converting microbial enzyme is secreted by a
microorganism that is in contact with the substrate. In some
embodiments, intermediate-converting enzyme and the
substrate-converting enzyme are produced by microorganisms of the
same species, while in other embodiments, intermediate-converting
enzyme and the substrate-converting enzyme are produced by
microorganisms of the different species. In yet other embodiments,
the substrate is selected from the group consisting of biomass and
starch. In some preferred embodiments, the biomass comprises corn
solids. In some particularly preferred embodiments, intermediate is
selected from the group consisting of hexoses and pentoses. In some
embodiments, the hexose is glucose. In some embodiments, the
substrate is cooked prior to its use in the present invention,
while in other embodiments, the substrate is uncooked prior to its
use in the present invention. In some particularly preferred
embodiments, the alcohol end-product is ethanol. In still further
embodiments, the step of contacting the substrate and at least one
substrate-converting enzyme further comprises providing an amount
of the substrate-converting enzyme at a concentration that produces
the intermediate at a concentration that is less than or equal to
the amount of the intermediate converted by at least one
intermediate-converting enzyme. In some additional embodiments, at
least one substrate-converting enzyme converts at least 50% of the
substrate to the intermediate within 72 hours, while in other
embodiments, at least one substrate-converting enzyme converts at
least 90% of the substrate to the intermediate within 72 hours, and
in some preferred embodiments, at least one substrate-converting
enzyme converts at least 95% of the substrate to the intermediate
within 72 hours. In still further embodiments, at least one
substrate-converting enzyme and at least one
intermediate-converting enzyme are obtained from a microorganism
selected from the group consisting of Rhizopus and Aspergillus. In
additional embodiments, the substrate-converting and/or
intermediate-converting enzyme(s) are provided as a cell-free
extract. In further preferred embodiments, the contacting steps
take place in a reaction vessel, including but not limited to vats,
bottles, flasks, bags, bioreactors, and any other receptacle
suitable for conducting the methods of the present invention.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIG. 1 provides a graph showing the ethanol results for the
experiments described in Example 1.
[0013] FIG. 2, Panels A, B and C provide graphs showing the ethanol
results from uncooked ground corn fermentation using M1 (Panel A),
CU (Panel B), and M1 with DISTILLASE.RTM. (Panel C).
[0014] FIG. 3, Panels A, B and C provide graphs showing the ethanol
results obtained in the experiments described in Example 3.
[0015] FIG. 4 shows the response of ethanol to the amount of
stillage added in both types of mashes.
[0016] FIG. 5 shows the glucose profile after 72 hour of
fermentation as described in Example 4.
[0017] FIG. 6 is a plot of the disaccharides after 72 hours of
fermentation with respect to stillage added (See, Example 4).
[0018] FIG. 7 shows the levels of the higher sugars (i.e.,
oligosaccharides greater than disaccharides) (See, Example 4).
[0019] FIG. 8 shows the lactic acid level after 72 hours of
fermentation (See, Example 4).
[0020] FIG. 9 summarizes the glycerol levels after 72 hours of
fermentation (See, Example 4).
DESCRIPTION OF THE INVENTION
[0021] The present invention provides means for the production of
desired end-products of in vitro and/or in vivo bioconversion of
biomass-based feed stock substrates, including but not limited to
such materials as starch and cellulose. In particularly preferred
embodiments, the methods of the present invention do not require
gelatinization and/or liquefaction of the substrate. In
particularly preferred embodiments, the present invention provides
means for the production of ethanol. In some particularly preferred
embodiments, the present invention provides means for the
production of ethanol directly from granular starch, in which
altered catabolite repression is involved.
[0022] In particular, the present invention provides means for
making ethanol in a manner that is characterized by having altered
levels of catabolite repression and enzymatic inhibition, thus
increasing the process efficiency. The methods of the present
invention comprise the steps of contacting a carbon substrate and a
substrate converting enzyme to produce an intermediate; and
contacting the intermediate with an intermediate producing enzyme
in a reactor vessel, wherein the intermediate is substantially all
bioconverted by an end-product producing microorganism. By
maintaining a low concentration of the intermediate in a conversion
medium, the catabolite repressive or enzymatic inhibitive effects
of the intermediate on the process are altered.
[0023] The present invention also provides methods in which
starches or biomass and hydrolyzing enzymes are used to convert
starch or cellulose to glucose. In addition, the present invention
provides methods in which these substrates are provided at such a
rate that the conversion of starch to glucose matches the glucose
feed rate required for the respective fermentative product
formation. Thus, the present invention provides key glucose-limited
fermentative conditions, as well as avoiding many of the metabolic
regulations and inhibitions.
[0024] In some preferred embodiments, the present invention
provides means for making desired end-products, in which a
continuous supply of glucose is provided under controlled rate
conditions, providing such benefits as reduced raw material cost,
lower viscosity, improved oxygen transfer for metabolic efficiency,
improved bioconversion efficiency, higher yields, altered levels of
catabolite repression and enzymatic inhibition, and lowered overall
manufacturing costs.
[0025] As indicated above, there is a great need in the art for
methods in which less expensive starting materials than glucose are
used to efficiently produce a desired end-product. As described in
greater detail herein, the present invention provides methods
involving such substrates, including starch (e.g., corn and wheat
starch) and biomass.
[0026] Starch is a plant-based fermentation carbon source. Corn
starch and wheat starch are carbon sources that are much cheaper
than glucose carbon feedstock for fermentation. Conversion of
liquefied starch to glucose is known in the art and is generally
carried out using enzymes such alpha-amylase, pullulanase, and
glucoamylase. A large number of processes have been described for
converting liquefied starch to the monosaccharide, glucose. Glucose
has value in itself, and also as a precursor for other saccharides
such as fructose. In addition, glucose may also be fermented to
ethanol or other fermentation products. However the ability of the
enzymatic conversion of a first carbon source to the intermediate,
especially glucose, may be impaired by the presence of the
intermediate.
[0027] For example, the typical methods used in Japanese sake
brewing and alcoholic production use starch without cooking.
However, these techniques require some special operations such as
acidification of mash (pH 3.5), which prevents contamination of
harmful microorganisms. Furthermore, these methods require a longer
period of the time for the saccharification and fermentation than
the present invention. In addition, these methods require complex
process steps such as dialysis of a fermented broth and are too
cumbersome to utilize in the general production of products via
fermentation.
[0028] The use of soluble dextrins and glucose as feed-stock in
fermentations have various drawbacks, including high processing
cost, problems associated with viscosity and oxygen transfer. In
addition, in comparison to the present invention, these methods
produce lower yields of the desired products and more problems
associated with the formation of by-products. Indeed, the costs of
converting starch or biomass to dextrins are substantial and
involve high energy input, separate reactor tanks, more time, a
detailed bioprocess operation, incomplete saccharification,
back-reaction, and enzymes associated with the typical
pre-fermentation saccharification step. These problems have led to
a number of attempts to provide methods for conversion directly to
starch within one reaction vessel or container and at lower
temperatures. Biotransformation of a carbohydrate source to
1,3-propanediol in mixed cultures is described in U.S. Pat. No.
5,599,689 (Haynie, et al.). The method described by Haynie et al.,
involves mixing a glycerol (i.e., an intermediate) producing
organism with a diol producing organism (i.e., an end-product),
contacting the mixed culture medium with a carbon substrate and
incubating the mixed culture medium to produce the desired
end-product, 1,3-propanediol. In U.S. Pat. No. 4,514,496, Yoshizuma
describes methods that involve maintaining the concentration of the
material in the slurry relative the mashing liquid to produce
alcohol by fermentation without cooking (i.e., without high
temperature liquefaction before saccharization. Nonetheless, these
methods lack the efficiency and economical advantages provided by
the present invention.
[0029] The present invention provides methods for producing
end-products, including organic acids (e.g., gluconic acid,
ascorbic acid intermediates, succinic acid, citric acid, acetic
acid, gluconic acid, and lactic acid), amino acids, antibiotics,
enzymes and organic solvents (e.g., 1,3-propanediol, butanediol,
and acetone), glycerol, ethanol are provided. In some preferred
embodiments, the methods comprise the steps of contacting at least
one carbon substrate with at least one substrate converting enzyme
to produce at least one intermediate; and contacting at least one
intermediate with an intermediate producing enzyme in a reactor
vessel, wherein at least one intermediate is substantially
completely bioconverted an end-product. In some preferred
embodiments, this bioconversion is achieved by microorganisms. By
maintaining a low concentration of the intermediate in a conversion
medium, the intermediate's catabolite repressive and/or enzymatic
inhibitive effects are altered (e.g., reduced). The present
invention also provides various levels of intermediate
concentration, substrates, intermediates and steps of converting
the intermediate to the desired end-product (e.g., ethanol).
[0030] Definitions
[0031] 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. Various references (See e.g., Singleton, et al.,
DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John
Wiley and Sons, New York [1994]; and Hale and Marham, THE HARPER
COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY [1991]) provide
general definitions of many of the terms used herein. Furthermore,
all patents and publications, including all sequences disclosed
within such patents and publications, referred to herein are
expressly incorporated by reference.
[0032] Although any methods and materials similar or equivalent to
those described herein find use in the practice of the present
invention, preferred methods and materials are described herein.
Numeric ranges are inclusive of the numbers defining the range.
Unless otherwise indicated, nucleic acids are written left to right
in 5' to 3' orientation; amino acid sequences are written left to
right in amino to carboxy orientation, respectively. It is to be
understood that this invention is not limited to the particular
methodology, protocols, and reagents described, as these may
vary.
[0033] The headings provided herein are not limitations of the
various aspects or embodiments of the invention that can be had by
reference to the specification as a whole. Furthermore, the terms
defined immediately below are more fully defined by reference to
the Specification as a whole.
[0034] As used herein, the term "carbon substrate" refers to a
material containing at least one carbon atom which can be
enzymatically converted into an intermediate for subsequent
conversion into the desired carbon end-product. Exemplary carbon
substrates include, but are not limited to biomass, starches,
dextrins and sugars.
[0035] As used herein, "biomass" refers to cellulose- and/or
starch-containing raw materials, including but not limited to wood
chips, corn stover, rice, grasses, forages, perrie-grass, potatoes,
tubers, roots, whole ground corn, cobs, grains, wheat, barley, rye,
milo, brans, cereals, sugar-containing raw materials (e.g.,
molasses, fruit materials, sugar cane or sugar beets), wood, and
plant residues. Indeed, it is not intended that the present
invention be limited to any particular material used as biomass. In
preferred embodiments of the present invention, the raw materials
are starch-containing raw materials (e.g., cobs, whole ground
corns, corns, grains, milo, and/or cereals, and mixtures thereof).
In particularly preferred embodiments, the term refers to any
starch-containing material originally obtained from any plant
source.
[0036] As used herein, "starch" refers to any starch-containing
materials. In particular, the term refers to various plant-based
materials, including but not limited to wheat, barley, potato,
sweet potato, tapioca, corn, maize, cassaya, milo, rye, and brans.
Indeed, it is not intended that the present invention be limited to
any particular type and/or source of starch. In general, the term
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.
[0037] As used herein, "cellulose" refers to any
cellulose-containing materials. In particular, the term refers to
the polymer of glucose (or "cellobiose"), with the formula
(C.sub.6H.sub.10O.sub.5).sub.x wherein "x" can be any number.
Cellulose is the chief constituent of plant cell walls and Is among
the most abundant organic substances in nature. While there is a
.beta.-glucoside linkage in cellulose, there is an
.alpha.-glucoside linkage in starch. In combination with lignin,
cellulose forms "lignocellulose."
[0038] As used herein, the term "corn solids" refers to ground
materials from corn, including but not limited to kernels, bran and
cobs.
[0039] As used herein, "intermediate" refers to a compound that
contains at least one carbon atom into which the carbon substrates
are enzymatically converted. Exemplary intermediates include, but
are not limited to pentoses, (e.g., xylose, arabinose, lyxose,
ribose, ribulose, xylulose); hexoses (e.g., glucose, allose,
altrose, mannose, gulose, idose, galactose, talose, psicose,
fructose, sorbose, and tagatose); and organic acids thereof.
[0040] As used herein, the term "enzymatic conversion" refers to
the modification of a carbon substrate to an intermediate or the
modification of an intermediate to an end-product by contacting the
substrate or intermediate with an enzyme. In some embodiments,
contact is made by directly exposing the substrate or intermediate
to the appropriate enzyme. In other embodiments, contacting
comprises exposing the substrate or intermediate to an organism
that expresses and/or excretes the enzyme, and/or metabolizes the
desired substrate and/or intermediate to the desired intermediate
and/or end-product, respectively.
[0041] As used herein, the term "starch hydrolyzing enzyme" refers
to any enzyme that is capable of converting starch to the
intermediate sugar (e.g., a hexose or pentose).
[0042] As used herein, "monosaccharide" refers to any compound
having an empirical formula of (CH.sub.2O).sub.n, wherein n is 3-7,
and preferably 5-7. In some embodiments, the term refers to "simple
sugars" that consist of a single polyhydroxy aldehyde or ketone
unit. The term encompasses, but is not limited to such compounds as
glucose, galactose, and fructose.
[0043] As used herein, "disaccharide" refers to any compound that
comprises two covalently linked monosaccharide units. The term
encompasses, but is not limited to such compounds as sucrose,
lactose and maltose.
[0044] As used herein, "oligosaccharide" refers to any compound
having 2-10 monosaccharide units joined in glycosidic linkages. In
some preferred embodiments, the term refers to short chains of
monosaccharide units joined together by covalent bonds.
[0045] As used herein, "polysaccharide" refers to any compound
having multiple monosaccharide units joined in a linear or branched
chain. In some preferred embodiments, the term refers to long
chains with hundreds or thousands of monosaccharide units. Some
polysaccharides, such as cellulose have linear chains, while others
(e.g., glycogen) have branched chains. Among the most abundant
polysaccharides are starch and cellulose, which consist of
recurring glucose units (although these compounds differ in how the
glucose units are linked).
[0046] As used herein, "culturing" refers to fermentative
bioconversion of a carbon substrate to the desired end-product
within a reactor vessel. In particularly preferred embodiments,
culturing involves the growth of microorganisms under suitable
conditions for the production of the desired end-product(s).
[0047] As used herein, the term "saccharification" refers to
converting a directly unusable polysaccharide to a useful sugar
feed-stock for bioconversion or fermentative bioconversion.
[0048] As used herein, the term "fermentation" refers to the
enzymatic and anaerobic breakdown of organic substances by
microorganisms to produce simpler organic products. In preferred
embodiments, fermentation refers to the utilization of
carbohydrates by microorganisms (e.g., bacteria) involving an
oxidation-reduction metabolic process that takes place under
anaerobic conditions and in which an organic substrate serves as
the final hydrogen acceptor (i.e., rather than oxygen). Although
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.
[0049] As used herein, the terms "substantially all consumed" and
"substantially all bioconverted" refer to the maintenance of a low
level of intermediate in a conversion medium which adversely
affects the enzymatic inhibition, oxygen transfer, yield, byproduct
minimization or catabolite repression effects of the intermediate
(e.g., a hexose), upon the ability of the intermediate converting
enzyme to convert the intermediate to the end-product or another
intermediate and/or the ability of the substrate converting enzyme
to convert the substrate to the intermediate.
[0050] As used herein, the terms "bioconversion" and "bioconverted"
refer to contacting a microorganism with the carbon substrate or
intermediate, under conditions such that the carbon substrate or
intermediate is converted to the intermediate or desired
end-product, respectively. In some embodiments, these terms are
used to describe the production of another intervening intermediate
in in vitro methods in which biocatalysts alone are used. In some
preferred embodiments, the terms encompass metabolism by
microorganisms and/or expression or secretion of enzyme(s) that
achieve the desired conversion.
[0051] As used herein, the terms "conversion media" and "conversion
medium" refer to the medium/media in which the enzymes and the
carbon substrate, intermediate and end-products are in contact with
one another. These terms include, but are not limited to
fermentation media, organic and/or aqueous media dissolving or
otherwise suspending the enzymes and the carbon substrate,
intermediate and end-products. In some embodiments, the media are
complex, while in other preferred embodiments, the media are
defined.
[0052] As used herein, the term "end-product" refers to any
carbon-source derived molecule product which is enzymatically
converted from the intermediate. In particularly preferred
embodiments, the methods of the present invention are used in order
to produce a "desired end-product" (i.e., the product that is
intended to be produced through the use of these methods). In
particularly preferred embodiments, the term refers to an alcohol,
particularly ethanol.
[0053] As used herein, "low concentration" refers to a
concentration level of a compound that is less than that would
result in the production of detrimental effects due to the presence
of the compound. In particularly preferred embodiments, the term is
used in reference to the concentration of a particular intermediate
below which the detrimental effects of catabolite suppression
and/or enzyme inhibition are observed. In some embodiments, the
term refers to the concentration level of a particular intermediate
above which triggers catabolite repression and/or enzymes
inhibition by substrate and/or products.
[0054] As used herein, the phrase "maintained at a level below
which triggers catabolite repression effects" refers to maintaining
the concentration of an intermediate to below that level which
triggers catabolite repression.
[0055] As used herein, the term "reduces catabolite repression"
means conditions under which the effects of catabolite repression
are produced. In preferred embodiments, the term refers to
conditions in which the intermediate concentration is less than
that threshold which triggers catabolite repressive effects.
[0056] As used herein, the term "reduces enzyme inhibition" means
conditions under which the inhibition of an enzyme is reduced as
compared to the inhibition of the enzyme under usual, standard
conditions. In preferred embodiments of the present invention, the
term refers to conditions in which the concentration of an
intermediate, substrate and/or product of the enzyme reaction is
less than that threshold which triggers enzyme inhibition.
[0057] As used herein, the term "substrate converting enzyme"
refers to any enzyme that converts the substrate (e.g., granular
starch) to an intermediate, (e.g., glucose). Substrate converting
enzymes include, but are not limited to alpha-amylases,
glucoamylases, pullulanases, starch hydrolyzing enzymes, and
various combinations thereof.
[0058] As used herein, the term "intermediate converting enzyme"
refers to any enzyme that converts an intermediate (e.g.,
D-glucose, D-fructose, etc.), to the desired end-product. In
preferred embodiments, this conversion is accomplished through
hydrolysis, while in other embodiments, the conversion involves the
metabolism of the intermediate to the end-product by a
microorganism. However, it is not intended that the present
invention be limited to any particular enzyme or means of
conversion. Indeed, it is intended that any appropriate enzyme will
find use in the various embodiments of the present invention.
[0059] As used herein, "yield" refers to the amount of end-product
or intermediate produced using the methods of the present
invention. In some preferred embodiments, the yield produced using
the methods of the present invention is greater than that produced
using methods known in the art. In some embodiments, the yield
refers to the volume of the end-product or intermediate, while in
other embodiments, the term is used in reference to the
concentration of the end-product or intermediate in a
composition.
[0060] As used herein, "byproduct formation" refers to the
production of products that are not desired. In some preferred
embodiments, the present invention provides methods that avoid or
reduce the production of byproducts, as compared to methods known
in the art.
[0061] As used herein, the term "enzymatic inhibition" refers to
loss of enzyme activity by either physical or biochemical effects
on the enzyme. In some embodiments, inhibition results from the
effects of the product formed by the enzyme activity, while in
other embodiments, inhibition results from the action of the
substrate or intermediate on the enzyme.
[0062] As used herein, "enzyme activity" refers to the action of an
enzyme on its substrate. In some embodiments, the enzyme activity
is quantitated using means to determine the conversion of the
substrate to the intermediate, while in other embodiments, the
conversion of the substrate to the end-product is determined, while
in still further embodiments, the conversion of the intermediate to
the end-product is determined.
[0063] As used herein, the term "enzyme unit" refers to the amount
of enzyme which converts 1 micromole of substrate per minute to the
substrate product at optimum assay conditions (unless otherwise
noted). In some embodiments, commercially available enzymes (e.g.,
SPEZYME.RTM., DISTALLASE.RTM., OPTIMAX.RTM.; Genencor
International) find use in the methods of the present
invention.
[0064] As used herein, the term "glucoamylase unit" (GAU) is
defined as the amount of enzyme required to produce one micromole
of glucose per minute under assay conditions of 40.degree. C. and
pH 5.0 or under the alternative assay conditions of 25.degree. and
pH 7.0.
[0065] As used herein, the term "glucose oxidase unit" (GOU) is
defined as the amount of enzyme required to oxidize one micromole
of D-glucose per minute under assay conditions of 25.degree. C. and
pH 7.0, to gluconic acid.
[0066] As used herein, the term "catalase units" (CU) is defined as
the amount of enzyme required to decompose 1 micromole of hydrogen
peroxide per minute under assay conditions of 25.degree. C. and pH
7.0.
[0067] As used herein, one AG unit (AGU) is the amount of enzyme
which splits one micromole of maltose per minute at 25.degree. C.
and pH 4.3. In some embodiments of the present invention, a
commercially available liquid form of glucoamylase (OPTIDEX.RTM.
L-400; Genencor International) with an activity of 400 GAU per ml
is used. In alternative embodiments, a commercially available
liquid form of glucoamylase (AMG NOVO 150) has an activity of 150
AGU per ml finds use.
[0068] As used herein, the terms "starch hydrolyzing unit" and "raw
starch hydrolyzing unit" (RHU) are defined as being the amount of
enzyme required to produce one gram of glucose per minute from
starch, under the assay conditions of 25.degree. C. and pH 5.0.
[0069] As used herein, "carbon end-product" means any carbon
product produced from the carbon intermediate, wherein the
substrate contains at least one carbon atom (i.e., a carbon
substrate).
[0070] As used herein, "carbon intermediate" refers to the
carbon-containing compounds that are produced during the conversion
of a carbon-containing substrate to a carbon end-product.
[0071] As used herein, "enzymatically controlled" means regulating
the amount of carbon intermediate produced from the carbon
substrate by altering the amount or activity of the enzyme used in
the reaction.
[0072] As used herein, "microorganism" refers to any organism with
cells that are typically considered to be microscopic, including
such organisms as bacteria, fungi (yeasts and molds), rickettsia,
and protozoa. It is not intended that the present invention be
limited to any particular microorganism(s) or species of
microorganism(s), as various microorganisms and microbial enzymes
are suitable for use in the present invention. It is also not
intended that the present invention be limited to wild-type
microorganisms, as microorganisms and microbial enzymes produced
using recombinant DNA technologies also find use in the present
invention.
[0073] As used herein, "microbial enzyme" refers to any enzyme that
is produced by a microorganism. As used herein, a "microbial
intermediate-converting enzyme" is an enzyme that converts an
intermediate to an end-product, while a "microbial
substrate-converting enzyme" is an enzyme that converts a substrate
to an intermediate or directly converts a substrate to an
end-product (i.e., there is not intermediate compound).
[0074] As used herein, the term "ethanologenic microorganism"
refers to a microorganism with the ability to convert a sugar or
oligosaccharide to ethanol. Ethanologenic microorganisms are known
in the art and include ethanologenic bacteria. The microorganisms
are ethanologenic by virtue of their ability to express one or more
enzymes that individually or together, convert a sugar to
ethanol.
[0075] As used herein, the terms "ethanol producer" and "ethanol
producing organism" refer to any organism or cell that is capable
of producing ethanol from a hexose or a pentose. Generally, ethanol
producing cells contain an alcohol dehydrogenase and pyruvate
decarboxylase.
[0076] As used herein, "antimicrobial" refers to any compound that
kills or inhibits the growth of microorganisms.
[0077] As used herein, the term "linked culture" refers to a
fermentation system that employs at least two cell cultures, in
which the cultures are added sequentially. In most embodiments of
linked systems, a primary culture or a set of primary cultures is
grown under optimal fermentation conditions for the production of a
desired intermediate (i.e., the intermediate is released into the
culture media to produce a "conditioned medium"). Following the
fermentation of the primary culture, the conditioned medium is then
exposed to the secondary culture(s). The secondary cultures then
convert the intermediate in the conditioned media to the desired
end-product. In some embodiments of the present invention, the
primary cultures are typically glycerol producers and the secondary
cultures are 1,3-propanediol producers.
[0078] As used herein, "mixed culture" refers to the presence of
any combination of microbial species in a culture. In some
preferred embodiments, the mixed culture is grown in a reaction
vessel under conditions such that the interaction of the individual
metabolic processes of the combined organisms results in a product
which neither individual organism is capable of producing. It is
not intended that the present invention be limited to mixed
cultures comprising a particular number of microbial species.
[0079] As used herein, "conditioned media" refers to any
fermentation media suitable for the growth of microorganisms that
has been supplemented by organic byproducts of microbial growth. In
preferred embodiments of the present invention, conditioned media
are produced during fermentation of linked cultures wherein
glycerol producing cells secrete glycerol into the fermentation
media for subsequent conversion to 1,3-propanediol.
[0080] As used herein, "oxygen uptake rate" ("OUR") refers to the
determination of the specific consumption of oxygen within the
reactor vessel. Oxygen consumption can be determined using various
on-line measurements known in the art. In one embodiment, the OUR
(mmol/(liter*hour)) is determined by the following formula:
((Airflow (standing liters per minute)/Fermentation weight (weight
of the fermentation broth in kilograms)).times.supply
O.sub.2.times.broth density.times.(a constant to correct for
airflow calibration at 21.1 C instead of standard 20.0 C)) minus
([airflow/fermentation weight].times.[offgas O.sub.2/offgas
N.sub.2].times.supply N.sub.2.times.broth
density.times.constant).
[0081] As used herein, "carbon evolution rate" ("CER") refers to
the determination of how much CO.sub.2 is produced within the
reactor vessel during fermentation. Usually, since no CO.sub.2 is
initially or subsequently provided to the reaction vessel, any
CO.sub.2 is assumed to be produced by the fermentation process
occurring within the reaction vessel. "Off-gas CO.sub.2" refers to
the amount of CO.sub.2 measured within the reactor vessel, usually
by mass spectroscopic methods known in the art.
[0082] As used herein, the term "enhanced" refers to improved
production of proteins of interest. In preferred embodiments, the
present invention provides enhanced (i.e., improved) production and
secretion of a protein of interest. In these embodiments, the
"enhanced" production is improved as compared to the normal levels
of production by the host (e.g., wild-type cells). Thus, for
heterologous proteins, basically any expression is enhanced, as the
cells normally do not produce the protein.
[0083] As used herein, the terms "isolated" and "purified" refer to
a nucleic acid or amino acid that is removed from at least one
component with which it is naturally associated.
DETAILED DESCRIPTION OF THE INVENTION
[0084] The present invention provides means for the production of
desired end-products of in vitro and/or in vivo bioconversion of
biomass-based feed stock substrates, including but not limited to
such materials as starch and cellulose. In particularly preferred
embodiments, the methods of the present invention do not require
gelatinization and/or liquefaction of the substrate. In
particularly preferred embodiments, the present invention provides
means for the production of ethanol. In some particularly preferred
embodiments, the present invention provides means for the
production of ethanol directly from granular starch, in which
altered catabolite repression is involved.
[0085] In preferred embodiments, the present invention provides
dramatic improvements in the process for directly converting a
commonly available carbon substrate (e.g., biomass and/or starch)
into an intermediate, preferably, an intermediate that is readily
convertible into a multitude of desired end-products, including
alcohols such as ethanol. In particularly preferred embodiments,
the present invention provides means for dramatically improving the
processes for directly converting granular starch into glucose, an
intermediate readily convertible into an ethanol.
[0086] In alternative embodiments, the present invention provides
means for dramatic improvements in the process for converting
starch or cellulose into glucose, which in turn is converted into
the desired end-product. By maintaining the presence of the
intermediate at a low concentration within the conversion media,
overall efficiency of the production is improved. In some
embodiments, enzymatic inhibition and/or catabolite repression,
oxygen uptake demand, and/or byproduct formation are reduced. In
additional preferred embodiments, the present invention provides
means for dramatic improvements in the non-cooking conversion of
granular starch into glucose, which in turn is converted into the
desired end-product.
[0087] In some preferred embodiments, the maintenance of minimal
intermediate concentrations is achieved by maintaining the
concentration of the intermediate at a low concentration. In one
embodiment, the concentration of the intermediate is less than or
equal to 0.25% by weight volume of the medium (e.g., 0.25% to
0.00001% by weight volume). In other embodiments, the concentration
of the intermediate is less than or equal to 0.20%, 0.10%, 0.05%,
or 0.01% by weight volume (e.g., 0.20% to 0.00001%, 0.10% to
0.00001% 0.05% to 0.00001%, 0.01% to 0.00001%, respectively).
Alternatively, the intermediate concentration is maintained at less
than or equal to a concentration of 2.0 .mu.molar in the conversion
media. In another embodiment, the concentration is maintained at
less than or equal to 1.0 .mu.molar. In still another embodiment,
the concentration of the intermediate is maintained at a
concentration of less than or equal to 0.75 .mu.molar. In any
event, maintaining a low concentration means maintaining the
concentration of the intermediate below the threshold that results
in enzyme inhibition (i.e., enzyme inhibitive effects), catabolite
repression (i.e., catabolite repressive effects).
[0088] In further embodiments, the maintenance of a minimal
concentration is achieved by maintaining the rate of conversion of
the substrate to the intermediate at less than or equal to the rate
of conversion of the intermediate to the end-product. While it is
recognized that the conversion of the substrate to the intermediate
is necessarily rate limiting for the conversion of the intermediate
to the end-product, by providing sufficient intermediate converting
enzymes for the conversion of substantially all of the intermediate
produced by the first enzymatic conversion from the carbon
substrate, substantially all of the intermediate is converted to
the end-product as fast as it is converted from the starting
substrate to minimize the presence of the intermediate in the
conversion medium. Exemplary methods of providing such excessive
intermediate conversion include providing an excess of intermediate
converting enzyme, increasing the enzyme activity of the
intermediate converting enzyme, and/or decreasing the activity of
the substrate converting enzyme to convert the intermediate to
end-product as quickly as it is converted from the substrate. As
the actual rate of conversion is contemplated to vary with the
specific end product produced, some variation in the amount and
experimentation in determining the amount are contemplated. However
guidelines for making these determinations are provided herein.
[0089] In some embodiments of the present invention, the conversion
or consumption rate of the intermediate was determined by the
calculating the amount of organism present in the mixed media,
taking into consideration the other physical parameters of the
mixed media, and multiplying that amount by the generally known
conversion rate. This provides a rate of conversion of the
intermediate, (e.g., glucose), to the end-product. In some
embodiments, this conversion of the intermediate to the desired end
product is by conversion or bioconversion of the intermediate to
the end-product by a naturally occurring organism or one mutated to
provide such bioconversion. Another embodiment of the conversion
from intermediate to end product involves the use of an enzymatic
conversion by a known enzyme to the desired end-product using known
enzymatic conversion methods. For example, in some embodiments, the
conversion of glucose to a desired end product (e.g., propanediol,
succinic acid, gluconic acid, lactic acid, amino acid,
antimicrobials, ethanol, ascorbic acid intermediates and/or
ascorbic acid) is accomplished by the addition of an amount of an
enzyme known to convert glucose to the specified end product
desired.
[0090] Once the conversion rate of the intermediate to the desired
end product is determined, the limit of the conversion of the
carbon substrate to the intermediate can be determined in the same
manner. By calculating the upper limit of the intermediate to end
product conversion, the conversion rate of the carbon substrate to
intermediate can be determined, the main consideration being that
the intermediate concentration levels in the conversion media are
maintained at a sufficiently low level to adversely effect the
normally catabolite repressive/enzymatic inhibitory effects of the
intermediate. In one embodiment, this is accomplished by
maintaining the conversion rate of the intermediate to the end
product in excess or equal to the rate of conversion of the carbon
substrate to the intermediate. Thus, the present invention provides
means for increasing the conversion rate to the end product, as
well as means for restricting the conversion of the carbon
substrate to the intermediate.
[0091] Another method for determining whether the rate of
conversion of the intermediate to the end product is greater than
or equal to the production of the intermediate from the carbon
substrate is to measure the weight percentage of the intermediate
in the reactor vessel. The amount of the intermediate present in
the reactor vessel can be determined by various known methods,
including, but not limited to direct or indirect measurement of the
amount of intermediate present in the reactor vessel. Direct
measurement can be by periodic assays of the reactor vessel, using
assays known to identify the amount of intermediate and or
end-product in the vessel. In addition, direct measurement of the
amounts of intermediates within the reactor vessel include on-line
gas, liquid and/or high performance liquid chromatography
methodologies known in the art
[0092] Indirect measurement of the levels of intermediate or
end-products produced can be assessed by the measurement of oxygen
uptake or carbon dioxide production, using methods known in the art
(e.g., by determining the oxygen uptake rate and/or the carbon
evolution rate).
[0093] Substrates
[0094] The substrates of the present invention are carbon-based
compounds that can be converted enzymatically to intermediate
compounds. Suitable substrates include, but are not limited to
processed materials that contain constituents which can be
converted into sugars (e.g., cellulosic biomass, glycogen, starch
and various forms thereof, such as corn starch, wheat starch, corn
solids and wheat solids). During the development of the present
invention good results were obtained with corn starch and wheat
starch, although other sources, including starches from grains and
tubers (e.g., sweet potato, potato, rice and cassaya starch) also
find use with the present invention. Various starches are
commercially available. For example, corn starches are available
from Cerestar, Sigma, and Katayama Chemical Industry Co. (Japan);
wheat starches are available from Sigma; sweet potato starch is
available from Wako Pure Chemical Industry Co. (Japan); and potato
starch is available from Nakari Chemical Pharmaceutical Co.
(Japan). A particularly useful carbon substrate is corn starch. In
some embodiments of the present invention, granular starch is used
in a slurry having a percentage of starch between about 20% and
about 35%. Preferably, the starch is in a concentration between
about 10% and about 35%. In some particularly preferred
embodiments, the range for percent starch is between 30% and 32%.
In addition to granular starch, other carbon substrate sources find
use in the present invention include, but are not limited to
biomass, polysaccharides, and other carbon based materials capable
of being converted enzymatically to an intermediate.
[0095] Fermentable sugars can be obtained from a wide variety of
sources, including lignocellulosic material. Lignocellulose
material can be obtained from lignocellulosic waste products (e.g.,
plant residues and waste paper). Examples of suitable plant
residues include but are not limited to any plant material such as
stems, leaves, hulls, husks, cobs and the like, as well as corn
stover, begasses, wood, wood chips, wood pulp, and sawdust.
Examples of paper waste include but are not limited to discarded
paper of any type (e.g., photocopy paper, computer printer paper,
notebook paper, notepad paper, typewriter paper, and the like), as
well as newspapers, magazines, cardboard, and paper-based packaging
materials. The conditions for converting sugars to ethanol are
known in the art. Generally, the temperature is between about
25.degree. C. and 35.degree. C. (e.g., between 25.degree. and
35.degree., and more particularly at 30.degree. C). Useful pH
ranges for the conversion medium are provided between about 4.0 and
6.0, between 4.5 and 6.0, and between pH 5.5 and 5.8. However, it
is not intended that the present invention be limited to any
particular temperature and/or pH conditions as these conditions are
dependent upon the substrate(s), enzyme(s), intermediate(s), and/or
end-product(s) involved.
[0096] Enzymes
[0097] In some preferred embodiments of the present invention,
enzymes that are substrate-converting enzymes (i.e., enzymes that
are able to first convert the carbon substrate into the carbon
intermediate), and intermediate converting enzymes (i.e., enzymes
that are able to convert the resulting intermediate into an
intervening intermediate and/or the desired end-product) both find
use in the present invention. Enzymes that find use in some
embodiments of the present invention to convert a carbon substrate
to an intermediate include, but are not limited to alpha-amylase,
glucoamylase, starch hydrolyzing glucoamylase, and pullulanase.
Enzymes that find use in the conversion of an intermediate to an
end-product depend largely on the actual desired end-product. For
example enzymes useful for the conversion of a sugar to
1,3-propanediol include, but are not limited to enzymes produced by
E. coli and other microorganisms. For example enzymes useful for
the conversion of a sugar to lactic acid include, but are not
limited to those produced by Lactobacillus and Zymomonas. Enzymes
useful for the conversion of a sugar to ethanol include, but are
not limited to alcohol dehydrogenase and pyruvate decarboxylase.
Enzymes useful for the conversion of a sugar to ascorbic acid
intermediates include, but are not limited to glucose
dehydrogenase, gluconic acid dehydrogenase, 2,5-diketo-D-gluconate
reductase, and various other enzymes. Enzymes useful for the
conversion of a sugar to gluconic acid include, but are not limited
to glucose oxidase and catalase.
[0098] In some preferred embodiments, the alpha-amylase used in
some methods of the present invention is generally an enzyme which
effects random cleavage of alpha-(1-4) glucosidic linkages in
starch. In most embodiments, the alpha-amylase is chosen from among
the microbial enzymes having an E. C. number E. C. 3.2.1.1 and in
particular E. C. 3.2.1.1-3. In some preferred embodiments, the
alpha-amylase is a thermostable bacterial alpha-amylase. In most
particularly preferred embodiments, the alpha-amylase is obtained
or derived from Bacillus species. Indeed, during the development of
the present invention good results were obtained using the
SPEZYME.RTM. alpha-amylase obtained from Bacillus licheniformis
(Genencor). In other embodiments, black-koji amylase described in
alcoholic fermentation from starch such as corn and cassaya without
precooking (Ueda et al., J. Ferment. Technol., 50:237-242 [1980];
and Ueda et al, J. Ferment. Technol., 58:237-242 [1980]) find use
in the present invention.
[0099] As understood by those in the art, the quantity of
alpha-amylase used in the methods of the present invention will
depend on the enzymatic activity of the alpha-amylase and the rate
of conversion of the generated glucose by the end-product
converter. Generally an amount between 0.001 and 2.0 ml of a
solution of the alpha-amylase is added to 1000 gm of raw materials,
although in some embodiments, it is added in an amount between
0.005 and 1.5 ml of such a solution. In some preferred embodiments,
it is added in an amount between 0.1 and 1.0 ml of such a solution.
In further embodiments, other quantities are utilized. For example,
generally an amount between 0.01 and 1.0 kg of SPEZYME.RTM. FRED
(Genencor) is added to one metric ton of starch. In some
embodiments, the enzyme is added in an amount between 0.4 to 0.6
kg, while in other embodiments, it is added in an amount between
0.5 and 0.6 kg of SPEZYME.RTM. FRED/metric ton of starch.
[0100] In preferred embodiments of the present invention, the
glucoamylase is an enzyme which removes successive glucose units
from the non-reducing ends of starch. The enzyme can hydrolyze both
the linear and branched glucosidic linkages of starch, amylose and
amylopectin. In most embodiments, the glucoamylase used in the
methods of the present invention are microbial enzymes. In some
preferred embodiments, the glucoamylase is a thermostable fungal
glucoamylase, such as the Aspergillus glucoamylase. Indeed, during
the development of the present invention, good results were
obtained using the DISTALLASE.RTM. glucoamylase derived from
Aspergillus niger (Genencor). Glucoamylase preparations from
Aspergillus niger have also been used without the use of precooking
(See, Ueda et al, Biotechnol. Bioeng., 23:291[1981]). Three
glucoamylases have been selectively separated from Aspergillus
awamori var. kawachi for use in hydrolyzing starch (See, Hayashida,
Agr. Biol. Chem., 39:2093-2099 [1973]). Alcoholic fermentation of
sweet potato by Endomycopsis fibuligoeu glucoamylase without
cooking has also been described (Saha et al., Biotechnol. Bioeng.,
25:1181-1186 [1983]). Another enzyme that finds use in the present
invention is glucoamylase (EC 3.2.1.3), an enzyme that hydrolyzes
the alpha.-1,4-glucoside chain progressively from the non-reducing
terminal end. This enzyme also hydrolyzes the alpha-1,6-glucoside
chain. Glucoamylase is secreted from fungi of the genera
Aspergillus, Rhizopus and Mucor also find use in the methods of the
present invention. These enzymes further find use in glucose
production and quantitative determination of glycogen and starch.
Glucoamylase preparations obtained from E. fibuligera (IFO 0111)
have been used to contact sweet potato starch for alcoholic
fermentation (See, Saha et al., Biotechnol. Bioeng., 25:1181-1186
[1983]). One of this enzyme's major applications is as a
saccharifying agent in the production of ethyl alcohol from starchy
materials. However, as with the other glucoamylases described
herein, this enzyme also finds use in the methods of the present
invention.
[0101] Additional glucoamylases that find use in the methods of the
present invention include those obtained from the genera Rhizopus
and Humicola, which are characterized as having particularly high
productivity and enzymatic activity. Furthermore, in comparison
with the glucoamylase derived from other organisms, the
Rhizopus-derived glucoamylase exhibits a strong action on starch
and its enzymological and chemical properties including optimum pH
are particularly suitable for the saccharification of cereal
starch. Because of these features, the Rhizopus-derived
glucoamylase is considered to be best suited for alcohol production
using non-cooked or low-temperature cooked starch (See, U.S. Pat.
Nos. 4,514,496 and 4,092,434). It has been noted that upon the
incubation of corn starch with Rhizopus glucoamylase, was used in
conjunction with Rhizopus alpha amylase, the starch degradation by
glucoamylase was accelerated. While it is not intended that the
present invention be limited to any particular mechanism or theory,
it is believed that Rhizopus glucoamylase has a stronger
degradation activity than Aspergillus niger glucoamylase
preparations which also contain .alpha.-amylase (See, Yamamoto et
al., Denpun Kagaku, 37:129-136 [1990]). One commercial preparation
that finds use in the present invention is the glucoamylase
preparation derived from the Koji culture of a strain of Rhizopus
niveus available from Shin Nippo Chemical Co., Ltd. Another
commercial preparation that finds use in the present invention is
the commercial starch hydrolyzing composition M1 is available from
Biocon India (Bangalore, India).
[0102] As understood by those in the art, the quantity of
glucoamylase used in the methods of the present invention depends
on the enzymatic activity of the glucoamylase (e.g.,
DISTILLASE.RTM.) L-400). Generally, an amount between 0.001 and 2.0
ml of a solution of the glucoamylase is added to 450 gm of a slurry
adjusted to 20-35% dry solids, the slurry being the liquefied mash
during the saccharification and/or in the hydrolyzed starch and
sugars during the fermentation. In some embodiments, the
glucoamylase is added in an amount between 0.005 and 1.5 ml of such
a solution. In some preferred embodiments, the enzyme is added at
an amount between 0.01 and 1.0 ml of such a solution.
[0103] As indicated above, pullulanases also find use in the
methods of the present invention. These enzymes hydrolyze
alpha.-1,6-glucosidic bonds. Thus, during the saccharification of
the liquefied starch, pullulanases remove successive glucose units
from the non-reducing ends of the starch. This enzyme is capable of
hydrolyzing both the linear and branched glucosidic linkages of
starch, amylose and amylopectin.
[0104] Additional enzymes that find use in the present invention
include starch hydrolyzing (RSH) enzymes, including Humicola RSH
glucoamylase enzyme preparation (See, U.S. Pat. No. 4,618,579).
This Humicola RSH enzyme preparation exhibits maximum activity
within the pH range of 5.0 to 7.0 and particularly in the range of
5.5 to 6.0. In addition, this enzyme preparation exhibits maximum
activity in the temperature range of 50.degree. C. to 60.degree. C.
Thus, in each of the steps of the present invention in which this
enzyme is used, the enzymatic solubilization of starch is
preferably carried out within these pH and temperature ranges.
[0105] In some embodiments, Humicola RSH enzyme preparations
obtained from the fungal organism strain Humicola grisea var.
thermoidea find use in the methods of the present invention. In
some particularly preferred embodiments, these Humicola RSH enzymes
are selected from the group consisting of ATCC (American Type
Culture Collection) 16453, NRRL (USDA Northern Regional Research
Laboratory) 15219, NRRL 15220, NRRL 15221, NRRL 15222, NRRL 15223,
NRRL 15224, and NRRL 15225, as well as genetically altered strains
derived from these enzymes.
[0106] Additional RSH glucoamylases that find use in the methods of
the present invention include Rhizopus RSH glucoamylase enzyme
preparations. In some embodiments, the enzyme obtained from the
Koji strain of Rhizopus niveus available from Shin Nihon Chemical
Co., Ltd., Ahjyo, Japan, under the tradename "CU CONC" is used.
Another useful enzyme preparation is a commercial digestive from
Rhizopus available from Amano Pharmaceutical under the tradename
"GLUCZYME" (See, Takahashi et al., J. Biochem., 98:663-671 [1985]).
Additional enzymes include three forms of glucoamylase (EC 3.2.1.3)
of a Rhizopus sp., namely "Gluc1" (MW 74,000), "Gluc2" (MW 58,600)
and "Gluc 3" (MW 61,400). Gluc1 was found to be 22-25 times more
effective than Gluc2 or Gluc3. Thus, although Gluc2 and Gluc3 find
use in the present invention, because Gluc1 tightly binds to starch
and has an optimum pH of 4.5, Gluc1 finds particular use in the
present invention. An additional RSH glucoamylase enzyme
preparation for use in the present invention includes enzyme
preparations sold under the designation "M1," available from Biocon
India, Ltd., Bangalore, India. M1 is a multifaceted enzyme
composition or mixture, as indicated by the high performance liquid
chromatography spectra of FIG. 1 and the SDS gel of FIG. 2.
[0107] As noted above, in most embodiments, Humicola RSH
glucoamylase enzyme preparations contain glucoamylase activity as
well as a potentiating factor which solubilizes starch. The
relative proportions of potentiating factor and glucoamylase
activity in other RSH enzyme preparations may vary somewhat.
However, with RSH glucoamylase enzyme preparations that find use in
the present invention, there is usually ample potentiating factor
produced along with the glucoamylase fraction. Accordingly, the
activity of the RSH glucoamylase enzyme preparations is defined in
terms of their glucoamylase activity.
[0108] Glucoamylase activity can also be measured for purposes of
this invention in 10 D.E. units for either RSH enzyme preparation
or conventional glucoamylase. A "10 D.E. unit" is the amount of
either type of enzyme which produces 1 micromole of glucose per
minute under the assay conditions. To determine glucoamylase
activity for purposes of this invention, one-tenth ml of enzyme
preparation, diluted if necessary, containing 0.06 units to 1.1
units is added to 0.9 ml of substrate solution preheated at
50.degree. C. for 5 minutes. The substrate solution consists of 40
parts by volume 0.25M sodium acetate buffer (pH 5.5) and 50 parts
by volume 4% by weight 10 D.E. maltodextrin in water. The substrate
solution is kept at 50.degree. C. for 5 minutes before the enzyme
solution is added. After 10 minutes, the reaction is quenched by
pouring into a preheated 16 mm test tube and heating in a
100.degree. C. water bath for 6 minutes. Glucose concentration is
determined by any convenient method (e.g., glucose reagent kit No.
15-UV from Sigma Chemical Co. or with an instrument such as the
Technicon Autoanalyzer).
[0109] A particularly useful enzymatic composition includes a
mixture of glucoamylase (e.g., DISTILLASE.RTM.) and RSH (e.g., M1).
The amount of the glucoamylase useful in this combination is in the
range of 0.2 to about 1.0 GAU units of glucoamylase per gram of
granular solids. A more useful amount of glucoamylase is between
about 0.75 to 0.5 GAU per gram of solids. The range of starch
hydrolyzing enzyme (M1) present in this mixture ranges from 0.2
starch hydrolyzing units (RSHU) to about 1.0 RSHU per gram of
solids. One particularly useful mixture includes about 0.6 GAU
DISTILLASE.RTM. per gram of corn solids and 0.2 RSHU M1 per gram of
corn solids.
[0110] In addition to the use of enzymatic compositions containing
the above described enzymes, the present invention provides methods
in which a microorganism is exposed to a substrate and uses the
substrate to produce the desired end-product. Thus, in some
embodiments, contacting the substrate or intermediate with a
fungal, bacterial or other microorganism that produces the desired
end-product is used to convert the substrate or intermediate to the
desired intermediate or end-product.
[0111] In preferred embodiments of the present invention, once the
carbon source is enzymatically converted to the intermediate, it is
converted into the desired end-product by the appropriate
methodology. Conversion is accomplished via any suitable method
(e.g., enzymatic or chemical). In one preferred embodiment,
conversion is accomplished by bioconversion of the intermediate by
contacting the intermediate with a microorganism. In alternate
preferred embodiments, the respective substrate-converting enzyme
and the intermediate-converting enzyme are placed in direct contact
with the substrate and/or intermediate. In some embodiments, the
enzyme(s) are provided as isolated, purified or concentrated
preparations.
[0112] In further embodiments, the substrate and/or intermediate
are placed in direct contact with a microorganism (e.g., bacterium
or fungus) that secretes or metabolizes the respective substrate or
intermediate. Thus, the present invention provides means for the
bioconversion of a substrate to an end-product. In some
embodiments, at least one intermediate compound is produced during
this conversion process.
[0113] In some embodiments, microorganisms that are genetically
modified to express enzymes not normally produced by the wild-type
organism are utilized. In some particularly preferred embodiments,
the organisms are modified to overexpress enzymes that are normally
produced by the wild-type organism.
[0114] Indeed, commercially available alpha-amylases and
glucoamylases find use in the methods of the present invention in
economically realistic enzyme concentrations. Although commonly
used fermentation conditions do not utilize optimum temperatures,
the pH conditions for fermentation do correspond closely to the
optimum pH for commercially available saccharification enzymes
(i.e., the glucoamylases). In some embodiments of the present
invention, complete saccharification to glucose is favored by the
gradual solubilization of granular starch. Presumably, the enzyme
is always exposed to low concentrations of dextrin. In addition,
the removal of glucose throughout the fermentation maintains a low
glucose content in the fermentation medium. Thus, glucoamylase is
exposed to low concentration of glucose. In consequence, the
glucoamylase is used so effectively that economically feasible
dosage levels of glucoamylase are suitable for use in the methods
of the present invention (i.e., glucoamylase dosage of 0.05-10.0
GAU/g of starch; and preferably 0.2-2.0 GAU/g starch).
[0115] The dosages provided above for glucoamylase only approximate
the effective concentration of the enzymatic saccharification
activity in the fermentation broth, as an additional proportion of
the saccharification activity is contributed by the alpha-amylase.
Although it is not intended that the present invention be limited
to any particular mechanism or theory, it is believed that the
alpha-amylase further widens the holes bored by glucoamylase on
starch granules (See, Yamamoto et al., supra). Typically, the use
of commercially available alpha-amylases results in the production
of significant amounts of sugars, such as glucose and maltose.
[0116] Addition of the alpha-amylase from Aspergillus oryzae (e.g.,
FUNGAMYL) to wort has been suggested to the brewing industry. This
particular enzyme saccharifies dextrins to maltotriose and maltose.
Thus, although the purpose of the alpha-amylase is to liquefy the
starch, its saccharification propensity also makes the
alpha-amylase some part of the saccharifying enzyme content. It is
believed that an alpha amylase is present in the M1
composition.
[0117] It is also contemplated that addition of the alpha-amylase
from Aspergillus oryzae (e.g., CLARASE.RTM. L (Genencor
International Inc.) to wort will find use in the brewing industry.
This particular enzyme saccharifies dextrins to maltotriose and
maltose. Thus, although the purpose of the alpha-amylase is to
liquefy starch, its saccharification propensity also make the
alpha-amylase a portion of the saccharifying enzyme content.
[0118] Furthermore, some commercially available glucoamylases
contain some alpha-amylase activity. Thus, it is possible (albeit
usually not practical) to ferment particulate starch in the
presence solely of glucoamylase. However, it is not intended that
such embodiments be excluded from the present invention.
[0119] Thus, it is also contemplated that commercially available
starch hydrolyzing enzymes will find use in the present invention
as part of a enzyme mixture which includes starch hydrolyzing
enzymes, alpha amylases and glucoamylases.
[0120] In most embodiments of the methods of the present invention,
an effective amount of alpha-amylase is added to a slurry of
particulate starch. Those of skill in the art understand that in
addition to the uncertain amount of alpha-amylase activity
contributed by glucoamylase, the effective activity of the
alpha-amylase may be quite different from the unit activity values
given by the supplier. The activity of alpha-amylase is pH
dependent, and may be different at the pH range selected for the
fermentation (i.e., as compared with the test conditions employed
by the suppliers for their reported unit activity values). Thus,
some preliminary experiments are contemplated as being sometimes
necessary in order establish the most effective dosages for
alpha-amylases, including those not explicitly described herein,
but find use in the methods of the present invention.
[0121] In some most preferred embodiments, the alpha-amylase dosage
range for fungal alpha-amylases is from 0.02 GAU/g (Fungal Amylase
Units) to 2.0 FAU/g of starch, although in some particularly
preferably embodiments, the range is 0.05-0.6 FAU/g. One "FAU" is
the amount of enzyme which breaks down 5260 mg of starch per hour
under a standardized set of conditions, and corresponds to
approximately 25 SKB units (See, Cerial Chem., 16:712-723 [1939]).
In most embodiments utilizing Bacillus alpha-amylases, the range is
0.01 KNU/g to 0.6 KNU/g, preferably 0.05 to 0.15 KNU/g, the NU (or
Novo Unit) being the amount of enzyme which breaks down 5.26 mg of
starch per hour under a standardized set of conditions. One KNU
corresponds to 1000 NU.
[0122] It is contemplated that the uncertainty as to the real
activity of both the glucoamylase and the alpha-amylase in the
fermenting slurry will require some preliminary investigation into
the practice of some embodiments. Optimization considerations
include the fact that increasing the alpha-amylase dosage with a
constant glucoamylase content, increases the fermentation rate. In
addition, increasing the glucoamylase dosage with a constant
alpha-amylase content increases the fermentation rate. Holding the
dosage of enzyme constant and/or increasing the starch content in
the slurry also increase the fermentation rate. Indeed, it is
contemplated that in some embodiments, the optimum alpha-amylase
dosage well exceeds dosages heretofore recommended for liquefying
starch; the optimum glucoamylase may well exceed dosages
recommended for saccharifying syrups. However, enzyme dosage levels
should not be confused with enzyme usage. Substantial proportions
of the enzymes dosed into the starch slurry are available for
recovery from the fermentation broth for use anew to ferment
granular starch.
[0123] A further consideration arising from employment of the
enzymes at fermentation temperatures is that although the enzymes
exhibit low relative activity (e.g., activity of the alpha-amylase
from B. licheniformis at fermentation temperatures is not more than
about 25% of maximum activity), the low relative activity is
counterbalanced by the extended duration of the 48-120 hours of
fermentation, and by the extended half-life of enzymes that have
not been subjected to elevated temperatures. Indeed, it has been
determined that more than 90% of the enzyme activity remains after
72 hours of fermentation. It was also noted that the use of M1
resulted in at least 50% of the starch solids being hydrolyzed
after 72 hours, at least 90% hydrolyzed after 72 hours and in some
cases, at least 95% hydrolyzed after 72 hours.
[0124] The alpha-amylase of B. licheniformis (SPEZYME.RTM. AA or
SPEZYME.RTM. FRED enzymes; Genencor) is sufficiently stable to
withstand brief exposures to still pot temperatures. Thus, recycle
of stillage can be used as a way to recycle alpha-amylase. However,
recovery of enzyme in recycled stillage requires care, in avoiding
subjecting the fermentation broth to ethanol stripping temperatures
that deactivate the enzyme(s). For example, the alcohol may be
vacuum stripped from the fermentation broth and such stillage
recycled to recover the enzymes suitable for use in subsequent
reactions.
[0125] However, as earlier described, some RSHs (e.g., the enzyme
obtained from Rhizopus) are available that convert starch to
glucose at non-cooking temperatures (e.g., 25 to 35.degree. C.),
reducing the need for exposing the enzymatic composition to still
pot temperatures. This reduces the energy costs of converting the
carbon substrate to the desired end-product, thereby reducing the
overall costs of manufacturing. Thus, these enzymes find particular
use in the methods of the present invention.
[0126] In preferred embodiments of the present invention, once the
carbon source is enzymatically converted to the intermediate, it is
converted into the desired end-product by the appropriate
methodology. Conversion is accomplished via any suitable method
(e.g., enzymatic or chemical). In one preferred embodiment,
conversion is accomplished by bioconversion of the intermediate by
contacting the intermediate with a microorganism. In alternate
preferred embodiments, the respective substrate-converting enzyme
and the intermediate-converting enzyme are placed in direct contact
with the substrate and/or intermediate. In some embodiments, the
enzyme(s) are provided as isolated, purified or concentrated
preparations.
[0127] The desired end-product can be any product that may be
produced by the enzymatic conversion of the substrate to the
end-product. In some preferred embodiments, the substrate is first
converted to at least one intermediate and then converted from the
intermediate to an end-product. For example, hexoses can be
bioconverted into numerous products, such as ascorbic acid
intermediates, ethanol, 1,3-propanediol, and gluconic acid.
Ascorbic acid intermediates include but are not limited to
2,5-diketogluconate, 2 KLG (2-keto-L-gluconate), and 5-KDG
(5-keto-D-gluconate). Gluconate can be converted from glucose by
contacting glucose with glucose dehydrogenase (GDH). In addition,
gluconate itself can be converted to 2-KDG (2-keto-D-gluconate) by
contacting gluconate with GDH. Furthermore, 2-KDG can be converted
to 2,5-DKG by contacting 2-KDG with 2-KDGH. Gluconate can also be
converted to 2-KDG by contacting gluconate with 2KR. Glucose can
also be converted to 1,3-propanediol by contacting glucose with E.
coli. In addition, glucose can be converted to succinic acid by
contacting glucose with E. coli.
[0128] Additional embodiments, as described herein are also
provided by the present invention. In one particularly preferred
embodiment of the present invention, the end-product is ethanol. In
some embodiments in which glucose is an intermediate, it is
converted to ethanol by contacting glucose with an ethanologenic
microorganism. In contacting the intermediate with an intermediate
converting enzyme, it is contemplated that isolated and/purified
enzymes are placed into contact with the intermediate. In yet
another embodiment, the intermediate is contacted with
bioconverting agents such as bacteria, fungi or other organism that
takes in the intermediate and produces the desired end-product. In
some embodiments, the organism is wild-type, while in other
embodiments it is mutated.
[0129] Preferred examples of ethanologenic microorganisms include
ethanologenic bacteria expressing alcohol dehydrogenase and
pyruvate decarboxylase, such as can be obtained with or from
Zymomonas mobilis (See e.g., U.S. Pat. Nos. 5,028,539, 5,000,000,
5,424,202, 5,487,989, 5,482,846, 5,554,520, 5,514,583, and
copending applications having U.S. Ser. No. 08/363,868 filed on
Dec. 27, 1994, U.S. Ser. No. 08/475,925 filed on Jun. 7, 1995, and
U.S. Ser. No. 08/218,914 filed on Mar. 28, 1994, the teachings of
all of which are hereby incorporated by reference, in their
entirety).
[0130] In additional embodiments, the ethanologenic microorganism
expresses xylose reductase and xylitol dehydrogenase, enzymes that
convert xylose to xylulose. In further embodiments, xylose
isomerase is used to convert xylose to xylulose. In additional
embodiments, the ethanologenic microorganism also expresses
xylulokinase, an enzyme that catalyzes the conversion of xylulose
to xylulose-5-phosphate. Additional enzymes involved in the
completion of the pathway include transaldolase and transketolase.
These enzymes can be obtained or derived from Escherichia coli,
Klebsielia oxytoca and Erwinia species (See e.g., U.S. Pat. No. No.
5,514,583).
[0131] In some particularly preferred embodiments, a microorganism
capable of fermenting both pentoses and hexoses to ethanol are
utilized. For example in some embodiments, a recombinant organism
which inherently possesses one set of enzymes and which is
genetically engineered to contain a complementing set of enzymes is
used (See e.g., U.S. Pat. Nos. 5,000,000, 5,028,539, 5,424,202,
5,482,846, 5,514,583, and WO 95/13362). In some embodiments,
particularly preferred microorganisms include Klebsiella oxytoca P2
and E. coli KO11.
[0132] In some embodiments, supplements are added to the nutrient
medium (i.e., the culture medium), including, but not limited to
vitamins, macronutrients, and micronutrients. Vitamins include, but
are not limited to choline chloride, nicotinic acid, thiamine HCl,
cyanocobalamin, p-aminobenzoic acid, biotin, calcium pantothenate,
folic acid, pyridoxine.HCl, and riboflavin. Macronutrients include,
but are not limited to (NH.sub.4).sub.2SO.sub.4, K.sub.2HPO.sub.4,
NaCl, and MgSO.sub.4. 7H.sub.2O. Micronutrients include, but are
not limited to FeCl.sub.36H.sub.2O, ZnCl.sub.2.4H.sub.2O,
CoCl.sub.2.6H.sub.2O, molybdic acid (tech), CuCl.sub.3.2H.sub.2O,
CaCl.sub.2.2H.sub.2O, and H.sub.3BO.sub.3.
[0133] Media and Carbon Substrates
[0134] The conversion media in the present invention must contain
suitable carbon substrates. Suitable carbon substrates include, but
are not limited to biomass, monosaccharides (e.g., glucose and
fructose), disaccharides (e.g., lactose and sucrose),
oligosaccharides, polysaccharides (e.g., starch and cellulose), as
well as mixtures thereof, and unpurified mixtures from renewable
feedstocks such as cheese whey permeate, cornsteep liquor, sugar
beet molasses, and barley malt. In additional embodiments, the
carbon substrate comprises one-carbon substrates such as carbon
dioxide, or methanol for which metabolic conversion into key
biochemical intermediates has been demonstrated.
[0135] Although it is contemplated that all of the above mentioned
carbon substrates and mixtures thereof will find use in the methods
of the present invention, preferred carbon substrates include
monosaccharides, disaccharides, oligosaccharides, polysaccharides,
and one-carbon substrates. In more particularly preferred
embodiments, the carbon substrates are selected from the group
consisting of glucose, fructose, sucrose and single carbon
substrates such as methanol and carbon dioxide. In a most
particularly preferred embodiment, the substrate is glucose.
[0136] As known in the art, in addition to an appropriate carbon
source, fermentation media must contain suitable nitrogen
source(s), minerals, salts, cofactors, buffers and other components
suitable for the growth of the cultures and promotion of the
enzymatic pathway necessary for the production of the desired
end-product (e.g., glycerol). In some embodiments, (II) salts
and/or vitamin B.sub.12 or precursors thereof find use in the
present invention.
[0137] Culture Conditions
[0138] Typically, cells are grown at approximately 30.degree. C. in
appropriate media. Preferred growth media utilized in the present
invention include common commercially prepared media such as Luria
Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast Malt
Extract (YM) broth. However, other defined or synthetic growth
media may also be used, as appropriate. Appropriate culture
conditions are well-known to those in the art.
[0139] In some embodiments, agents known to modulate catabolite
repression directly or indirectly (e.g., cyclic adenosine
2':3'-monophosphate or cyclic adenosine 2':5'-monophosphate), are
incorporated into the reaction media. Similarly, the use of agents
known to modulate enzymatic activities (e.g., sulphites,
bisulphites and alkalis) that lead to enhancement of glycerol
production also find use in conjunction with or as an alternative
to genetic manipulations.
[0140] Suitable pH ranges for fermentation are between pH 5.0 to pH
9.0; while the range of pH 6.0 to pH 8.0 is particularly preferred
for the initial conditions of the reaction system. Furthermore,
reactions may be performed under aerobic, microaerophilic, or
anaerobic conditions, as suited for the organism utilized.
[0141] Batch and Continuous Fermentations
[0142] In some preferred embodiments, the present process uses a
batch method of fermentation. A classical batch fermentation is a
closed system, wherein the composition of the media is set at the
beginning of the fermentation and is not subject to artificial
alterations during the fermentation. Thus, at the beginning of the
fermentation the medium is inoculated with the desired organism(s).
In this method, fermentation is permitted to occur without the
addition of any components to the system. Typically, a batch
fermentation qualifies as a "batch" with respect to the addition of
the carbon source and attempts are often made at controlling
factors such as pH and oxygen concentration. The metabolite and
biomass compositions of the batch system change constantly up to
the time the fermentation is stopped. Within batch cultures, cells
moderate through a static lag phase to a high growth log phase and
finally to a stationary phase where growth rate is diminished or
halted. If untreated, cells in the stationary phase eventually die.
In general, cells in log phase are responsible for the bulk of
production of end product or intermediate.
[0143] A variation on the standard batch system is the "fed-batch
fermentation" system, which also finds use with the present
invention. In this variation of a typical batch system, the
substrate is added in increments as the fermentation progresses.
Fed-batch systems are useful when catabolite repression is apt to
inhibit the metabolism of the cells and where it is desirable to
have limited amounts of substrate in the media. Measurement of the
actual substrate concentration in fed-batch systems is difficult
and is therefore estimated on the basis of the changes of
measurable factors such as pH, dissolved oxygen and the partial
pressure of waste gases such as CO.sub.2. Batch and fed-batch
fermentations are common and well known in the art.
[0144] It is also contemplated that the methods of the present
invention are adaptable to continuous fermentation methods.
Continuous fermentation is an open system where a defined
fermentation media is added continuously to a bioreactor and an
equal amount of conditioned media is removed simultaneously for
processing. Continuous fermentation generally maintains the
cultures at a constant high density where cells are primarily in
log phase growth.
[0145] Continuous fermentation allows for the modulation of one
factor or any number of factors that affect cell growth and/or end
product concentration. For example, in one embodiment, a limiting
nutrient such as the carbon source or nitrogen level is maintained
at a fixed rate an all other parameters are allowed to moderate. In
other systems, a number of factors affecting growth can be altered
continuously while the cell concentration, measured by media
turbidity, is kept constant. Continuous systems strive to maintain
steady state growth conditions. Thus, cell loss due to media being
drawn off must be balanced against the cell growth rate in the
fermentation. Methods of modulating nutrients and growth factors
for continuous fermentation processes as well as techniques for
maximizing the rate of product formation are well known in the art
of industrial microbiology.
[0146] In some embodiments, the present invention is practiced
using batch processes, while in other embodiments, fed-batch or
continuous processes, as well as any other suitable m0de of
fermentation are used. Additionally, in some embodiments, cells are
immobilized on a substrate as whole-cell catalysts and are
subjected to fermentation conditions for the appropriate
end-product production.
[0147] Identification and Purification of the End-Product
[0148] Methods for the purification of the end-product from
fermentation media are known in the art. For example, propanediols
can be obtained from cell media by subjecting the reaction mixture
to extraction with an organic solvent, distillation and column
chromatography (See e.g., U.S. Pat. No. 5,356,812). A particularly
good organic solvent for this process is cyclohexane (See, U.S.
Pat. No. 5,008,473).
[0149] In some embodiments, the end-product is identified directly
by submitting the media to high pressure liquid chromatography
(HPLC) analysis. One method of the present invention involves
analysis of fermentation media on an analytical ion exchange column
using a mobile phase of 0.01 N sulfuric acid in an isocratic
fashion.
[0150] Identification and Purification of the Enzymes
[0151] The enzyme levels in the media can be measured by enzyme
assays. For example in the manufacture of 1,3-propanediol, the
levels of expression of the proteins G3PDH and G3P phosphatase are
measured by enzyme assays. The G3PDH activity assay relies on the
spectral properties of the cosubstrate, NADH, in the DHAP
conversion to G-3-P. NADH has intrinsic UV/vis absorption and its
consumption can be monitored spectrophotometrically at 340 nm. G3P
phosphatase activity can be measured by any method of measuring the
inorganic phosphate liberated in the reaction. The most commonly
used detection method used the visible spectroscopic determination
of a blue-colored phosphomolybdate ammonium complex.
[0152] Thus, although there are various superficial resemblances
between the methods known in the art and the methods of the present
invention, the present invention provides more comprehensive
objectives that are reflected in a great number of detail features
believed to be unique to practice of this invention, including
notably enzyme recycling, biomass and starch recycling.
[0153] Recovery
[0154] Overall, recovery of enzymes in recycled stillage requires
care, in order to avoid subjecting the conversion media to
temperatures that deactivate the enzymes. In one example, for the
recovery of ethanol, the alcohol is vacuum stripped from the
fermentation broth and the stillage is recycled, in order to
recover the enzymes. In embodiment, enzymes are recovered through
the use of ultrafiltration or an electrodialysis device and
recycled.
[0155] Process Considerations
[0156] As indicated above, fermentation of granular starch slurry
has completely different characteristics than fermentation of a
syrup. Generally, a concentration of about 20% solids in solution
is considered the maximum sugar content in a fermentation medium,
as higher concentrations create difficulties at the onset and at
the end of fermentation. However, no similar limits exist in the
fermentation of a starch slurry. The concentration of starch in the
slurry may vary from 10-35%, with no discernable consequences at
the onset of fermentation. Increasing starch concentration (e.g.,
at constant enzyme dosages) speeds up the bioconversion rate, or
conversely, allows for lowering the enzyme dosages required to
achieve a given bioconversion rate. In any event fermenting until
the broth has 7-10% alcohol, as is prevalent in the fermentation
arts, is still possible. The excess (i.e., residual) granular
starch may be recovered, along with substantial amounts of enzymes
and subjected to renewed fermentation. Thus, control over starch
concentration is a major process parameter for practice of this
invention.
[0157] In one preferred embodiment, means for bioconversion and
fermentation of a granular starch slurry having 10-35% starch by
weight are provided. In some preferred embodiments, fermentation of
a 10-35% starch slurry with E. coli results in the production of
residual starch when fermentation has proceeded to the intended
organic acid or 1,3-propane diol content levels. However, this
reaction is dependent on the microorganism and bioprocessing
conditions used and, therefore, recycling of the enzymes on the
starch particles occurs when the residual starch is again
fermented. However, even when a 25-35% starch slurry is fermented,
in preferred embodiments, the fermentation is halted before
complete disappearance of the granular starch, for fermentation
anew. Thus, recycling of starch is a facile way to recover enzymes
for reuse.
[0158] In an alternative embodiment, means for fermentation of a
granular starch slurry of 25-25% by weight are provided. Fermenting
a 25-35% starch slurry with common baker's yeast will invariably
result in residual starch when fermentation has proceeded to the
intended alcohol content levels (e.g., 7-10%), dependent on the
microorganism used and the recycling of the enzymes on the starch
particles occurs when the residual starch is again fermented.
However, it is not intended that the present invention be limited
to this range, as other weight percentages will find use in the
present invention, depending upon the substrate and/or enzyme
system utilized in the methods. For example, in some embodiments, a
granular starch slurry of 10-35% by weight is preferred. A
particularly useful microorganism is one that is resistant to the
alcohol produced by the process.
[0159] In one preferred embodiment of the present invention, the
(granular) starch and microorganisms are removed together (e.g., by
centrifugation or filtration). This removed starch and
microorganisms are mixed with fresh granular starch and additional
aliquot(s) of enzyme(s) as needed, to produce a fermentation charge
for another fermentation run.
[0160] In another embodiment, bioconversion and fermentation of a
corn-stover slurry having 10-35% cellulosics by weight is provided.
In one embodiment, fermenting a 10-35% cellulosic slurry with E.
coli results in residual cellulosic when fermentation has proceeded
to the intended organic acid or 1,3-propane diol content levels.
This reaction is dependent upon the microorganism and bioprocessing
conditions used. As above, recycling of the enzymes on the
cellulosics occurs when the residual corn-stover is again
fermented. However, even when a 25-35% cellulosics slurry is
fermented, in some preferred embodiments, the fermentation is
halted before the complete disappearance of the stover, for
fermentation anew. Thus, recycling stover is a facile way to
recover enzymes for reuse.
[0161] In yet another preferred embodiment, the granular starch or
corn stover and microorganisms are removed together (e.g., by
centrifugation or filtration). This mixture of removed granular
starch or corn stover and microorganisms is mixed with fresh
granular starch or corn stover and additional aliquot(s) of
enzyme(s) as needed, to produce a fermentation charge for another
fermentation run.
[0162] As recognized by those of skill in the art, engineering
trade-offs are contemplated in arriving at optimum process details;
these trade-offs are contemplated to vary, depending upon each
particular situation. Nonetheless, the methods provided herein
provide the necessary teachings to make such trade-offs to obtain
optimum processes. For example, to achieve the most rapid
fermentation reasonable, high starch or cellulosic content, and
high enzymes dosage are indicated. But, the consequential rapid
fermentation tails off into generation of a level of nutrients in
the fermentation broth, when then dictates recovery of the
nutrients, or, alternatively that fermentation be halted at a
relatively low end-product (e.g., alcohol) content. However, in
situations where relatively low fermentation rates are acceptable,
then (with high starch content slurries) enzyme dosage is
relatively low and nutrient losses are held to levels heretofore
accepted by the fermentation arts. In cases where maximum yield of
end-product (e.g., alcohol) is a principal objective, then low
starch content slurries, moderate alpha-amylase dosage, and high
glucoamylase dosage find use in the present invention. However, it
is not intended that the present invention be limited to any
particular method design.
[0163] As indicated herein, the present invention saves
considerable thermal energy. However, just as the starting
substrate (e.g., starch) is never subjected to the thermal
conditions used for liquefactions, the substrate is not thermally
sterilized. Thus, it is contemplated that is some embodiments, the
starting substrate (e.g., granular starch) adds contaminating
microorganisms to the fermentation medium. Thus, in some
embodiments, it is advantageous to seed the fermentation medium
with a large number of the product-producing microorganisms that
are associated with recycled substrate (e.g., starch). By greatly
outnumbering the contaminants, the recycled microorganisms
overwhelm any contaminating microorganisms, thereby dominating the
fermentation, resulting in the production of the desired
end-product. Thus, in some embodiments, the method involves seeding
the fermentation medium with the great number of the ethanol
producing microorganism that are likely to accompany the recycled
granular starch. Through their great numbers, the recycled
microorganisms overwhelm any contaminating microorganisms, thereby
dominating the fermentation, as is, of course, desired.
[0164] In some embodiments, the quantities of yeast initially
charged into the fermentation vat may be in accord with prior art
practices for ethanol fermentation, and can vary widely since the
yeast cells will multiply during the course of the fermentation.
Recycling of yeast cells is not necessary, although may be
performed. In some embodiments, the yeast is removed from the
residual starch particles prior to recycling of the residual
starch. However, it is noted once again that practice of the
present invention does not necessarily require a thermal treatment
of the starch (i.e., thermal conditions that would heat sterilize
the starch). Thus, as with bacteria, it is advisable in some
embodiments of the present invention to charge relatively large
proportions of yeast cells into the fermentation in order to help
overcome the likelihood of (inadvertent) contamination. In
addition, in some embodiments, antimicrobials are added to the
fermentation medium to suppress growth of contaminating
microorganisms. In further embodiments, cold sterilization
techniques are utilized with the materials involved in the
methods.
[0165] In most preferred embodiments, the practice of the present
invention controls the fermentation rate by releasing metabolizable
sugars to the microorganisms (e.g., yeast) at a controlled rate and
maintaining the concentration of the intermediate (e.g., glucose)
at a level that does not trigger enzyme inhibition or catabolite
repression. This approach is very different from what was done
prior to the development of the present invention. Indeed, the
prior art suggests treating solid starch with enzymes prior to
fermentation and/or including enzymes in the fermentation medium to
conserve energy and/or to improve fermentation efficiency. However,
these teachings do not alter the character of the fermentation so
as to avoid the adverse effects of catabolite repression and/or
enzymatic inhibition. The present invention also provide means to
counter the adverse effects of producing undesired by-products from
glucose. The present invention also provides means to conserve
energy, particularly in comparison with prior art methods involving
high temperature starch liquefaction. Indeed, the present invention
provides means to conserve more thermal energy than other methods.
The present invention provides methods that operate with high
fermentation efficiency, in part because product losses due to
starch retrogradation, incomplete saccharification, and incomplete
fermentation of fermentables are reduced. The ability to tailor the
fermentation rate through control of starch concentration and
enzyme content and proportions includes the capability of creating
a fermentation broth product with minimal carbohydrate content.
[0166] As indicated above, in some embodiments, the quantities of
microorganisms and enzymes initially charged into the fermentation
vat or bioreactor are in accord with prior art practices for the
fermentation or bioconversion of various products. These quantities
will vary, as the microbial cells multiply during the course of the
fermentation whereas enzymes used for bioconversion will have a
limited half-life. Although in some embodiments, recycling of
microorganisms is utilized, in many embodiments, it is not required
for the practice of the present invention. In contrast, in
particularly preferred embodiments, it is desirable to recycle
enzymes (although it is not intended that the present invention be
limited to methods which require the recycling of enzymes).
[0167] Thus, in some embodiments, the microbes are removed from the
residual starch or biomass particles prior to recycling of the
residual starch or biomass. However, it is again noted that
practice of the present invention does not necessarily require
thermal treatment of the starting substrate (e.g., starch). Thus,
in some embodiments, the starting substrate is heat-sterilized,
while in other embodiments, it is not. Therefore, in some
embodiments, the fermentation/bioconversion is conducted in the
presence of a relatively large proportion of microorganisms, in
order to overcome the effects of any contamination. In alternative
embodiments, antimicrobials are added to the fermentation medium to
suppress growth of contaminating microorganisms. In additional
embodiments, cold sterilization techniques, UV radiation,
65.degree. C. pasteurization are used to sterilize the starting
(e.g., substrate) materials. However, biomass poses no problem
regarding sterilization of fermentation vats or bioreactors.
[0168] Use of starch as the starting material does not only address
the above shortcomings of currently used methods, but has three
additional significant benefits in terms of the raw material cost
of corn starch vs. D-glucose, reduction of substrate and/or product
based inhibition of enzymes employed in the bioconversion, and a
concomitant significant reduction in the requirement of high enzyme
dosages.
[0169] Various other examples and modifications of the description
and Examples are apparent to a person skilled in the art after
reading the disclosure without departing from the spirit and scope
of the invention; it is intended that all such examples or
modifications be included within the scope of the appended claims.
All publications and patents referenced herein are hereby
incorporated by reference in their entirety.
[0170] Experimental
[0171] 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.
[0172] In the experimental disclosure which follows, the following
abbreviations apply: wt % (weight percent); .degree. C. (degrees
Centigrade); rpm (revolutions per minute); H.sub.2O (water);
dH.sub.2O (deionized water); (HCl (hydrochloric acid); aa (amino
acid); bp (base pair); kb (kilobase pair); kD (kilodaltons); gm
(grams); .mu.g (micrograms); mg (milligrams); ng (nanograms); .mu.l
(microliters); ml (milliliters); mm (millimeters); nm (nanometers);
.mu.m (micrometer); M (molar); mM (millimolar); .mu.M (micromolar);
U (units); V (volts); MW (molecular weight); psi (pounds per square
inch); sec (seconds); min(s) (minute/minutes); hr(s) (hour/hours);
Q.S. and q.s. (quantity sufficient); OD (optical density);
OD.sub.280 (optical density at 280 nm); OD.sub.600 (optical density
at 600 nm); PAGE (polyacrylamide gel electrophoresis); PBS
(phosphate buffered saline [150 mM NaCl, 10 mM sodium phosphate
buffer, pH 7.2]); Cerestar (Cerestar, a Cargill, inc., company,
Minneapolis, Minn.); SDS (sodium dodecyl sulfate); Tris
(tris(hydroxymethyl)aminomethane); w/v (weight to volume); v/v
(volume to volume); ATCC (American Type Culture Collection,
Rockville, Md.); Difco (Difco Laboratories, Detroit, Mich.); GIBCO
BRL or Gibco BRL (Life Technologies, Inc., Gaithersburg, Md.);
Genencor (Genencor International, Inc., Palo Alto, Calif.); Shin
Nihon (Shin Nihon, Japan); BioRad (BioRad Laboratories, Hercules,
Calif.); and LeSaffre (LeSaffre Yeast Corporation, Milwaukee,
Wis.).
[0173] In the following Examples, additional various media and
buffers known to those in the art were used, including the
following:
EXAMPLE 1
Fermentation of Non-Cooked Corn Mash
[0174] In this Example, experiments conducted to compare starch
hydrolyzing enzyme activity with a glucoamylase on uncooked starch
are described.
[0175] Fermentation experiments were carried out in 250 ml flasks
that were incubated in a 30.degree. C. shaker water bath. For this
experiment 112 gm of 32.1% ground corn slurry containing 0.5% dry
corn steep was placed in 250 ml flasks. The pH of the slurry was
about 5.7, which required no further adjustment. The desired
enzymes (DISTILLASE.RTM. or Sumizyme CU; Shin Nihon) were added,
along with 0.37 gm of Red Star active dry yeast (LeSaffre) to start
the saccharification and fermentation. During the fermentation a
sample of the beer was centrifuged and 0.5 ml of supernatant was
added to a test tube containing 0.05 ml of 1.1 N H.sub.2SO.sub.4
containing 5% glutaraldehyde to terminate both the fermentation and
enzyme action. The sample was then diluted with 5.0 ml water and
then subjected to HPLC analysis on Bio Rad HPX-87H column. The
results are shown in Table 1 below.
1TABLE 1 Fermentation of Uncooked Ground Corn Comparing Sumizyme CU
With Distillase Fermentation at 30.degree. C. With enzyme dosage as
GAU per gm of corn. % W/V % W/V % W/V % W/V % W/V % V/V Flask
Enzyme Hours % DP > 2 DP-2 DP-1 Lactic Glycerol Ethanol 1 0.20
GAU/g CU 24 0.27 0 0.02 0.12 0.66 8.91 48 0.29 0 0 0.06 0.71 13.68
72 0.30 0 0 0.02 0.59 15.07 2 0.20 GAU/g Dist 24 0.25 0 0 0.13 0.28
3.05 48 0.20 0 0 0.37 0.26 4.59 72 0.19 0 0 0.49 0.24 6.10 3 0.40
GAU/g Dist 24 0.20 0 0 0.09 0.34 4.59 48 0.21 0 0 0.12 0.38 7.24 72
0.17 0 0 0.11 0.38 9.45 4 0.75 GAU/g Dist 24 0.22 0 0 0.08 0.40
5.51 48 0.20 0 0 0.06 0.44 9.54 72 0.21 0 0 0.03 0.45 12.13 5 1.00
GAU/g Dist 24 0.24 0 0 0.11 0.49 6.38 48 0.22 0 0 0.13 0.41 10.64
72 0.18 0 0 0.07 0.61 13.29 CU = Sumizyme CU from Shin Nihon Dist =
Distillase L-400
[0176] As indicated in the Table 1, little if any detectable
glucose is found in the beer, which indicated as the starch is
being hydrolyzed it quickly was converted to ethanol by the yeast.
FIG. 1 provides a graph showing the ethanol content of the various
tests.
[0177] there results could show that a starch hydrolyzing enzyme
could convert the uncooked starch much more efficiently than
DISTILLASE.RTM.. The rate of fermentation seems more related to the
RSU activity. The 0.2 GAU/gm level of CU corresponds to 0.590
RHU/gm, while the 1.0 GAU/gm level of DISTILLASE.RTM. corresponds
to only 0.108 RHU/gm. The RHU/GAU ratio for DISTILLASE.RTM. is 0.54
whereas the RHU/GAU ratio for CU is 2.98, which shows an enzyme
with a high RHU/GAU ratio can better hydrolyze uncooked starch.
EXAMPLE 2
Fermentation of Ground Corn Slurry
[0178] In these experiments, the same procedure was used for this
experiment as in Example 1, except that 35.9% ground corn slurry
was used (instead of corn mash), and prior to starting the
fermentation the slurry was placed in a 65.degree. C. water for one
hour as a pasteurization step. No observed gelatinization of the
slurry was observed. The enzymes tested were Sumizyme CU (Example
1), a Rhizopus glucoamylase preparation (M1) from Biocon assayed at
178 GAU/gm and 277 RHU/gm, and DISTILLASE.RTM. L-400 (Dist.) at 361
GAU/gm and 196 RHU/gm. Table 2 provides the conditions used for
this study, and also summarizes the results.
2TABLE 2 Fermentation Uncooked Starch With Separate And Enzyme
Combinations % W/V % W/V % W/V % W/V % W/V % V/V Sample Enzyme
Level Enzme Level Hours DP > 2 DP-2 DP-1 Lactic Glycerol Ethanol
1 M1 .20 GAU/g 24 0.40 0.04 0.02 0.17 0.67 8.66 1 48 0.35 0.02 0.00
0.10 0.74 12.28 1 72 0.36 0.01 0.00 0.04 0.76 14.02 2 M1 .50 GAU/g
24 0.42 0.03 0.01 0.13 0.80 11.91 2 48 0.41 0.04 0.00 0.04 0.82
15.24 2 72 0.54 0.03 0.00 0.02 0.84 15.23 3 M1 .75 GAU/g 24 0.42
0.03 0.01 0.12 0.86 12.43 3 48 0.53 0.02 0.01 0.06 0.91 15.30 3 72
0.55 0.03 0.01 0.03 0.94 15.43 4 Cu .20 GAU/g 24 0.34 0.03 0.10
0.14 0.92 10.59 4 48 0.35 0.07 0.05 0.09 1.03 14.96 4 72 0.40 0.04
0.04 0.04 1.04 15.63 5 Cu .50 GAU/g 24 0.37 0.13 0.80 0.13 0.96
12.20 5 48 0.45 0.24 1.15 0.08 1.05 14.96 5 72 0.45 0.25 1.46 0.07
1.08 14.96 6 Cu .75 GAU/g 24 0.43 0.16 1.15 0.13 0.97 12.69 6 48
0.51 0.30 2.19 0.08 1.05 14.90 6 72 0.51 0.33 2.67 0.07 1.08 14.83
7 M1 .20 GAU/g Dist .2 GAU/g 24 0.41 0.04 0.02 0.14 0.71 9.19 7 48
0.35 0.01 0.00 0.07 0.75 13.06 7 72 0.40 0.02 0.00 0.02 0.78 15.20
8 M1 .20 GAU/g Dist .6 GAU/g 24 0.33 0.04 0.03 0.15 0.77 9.56 8 48
0.39 0.02 0.00 0.09 0.84 13.56 8 72 0.38 0.03 0.00 0.04 0.86 15.02
9 M1 .20 GAU/g Dist 2.0 GAU/g 24 0.30 0.03 0.03 0.13 0.82 10.46 9
48 0.33 0.02 0.01 0.08 0.89 14.66 9 72 0.38 0.03 0.01 0.03 0.90
15.74
[0179] The ethanol results from the fermentations with M1 and CU
are plotted in FIGS. 2A and 2B. At the 0.2 GAU/gm level for M1 the
rate and yield of ethanol is less than the 0.5 and 0.75 levels
indicating the 0.2 level is enzyme limiting. The 0.5 and 0.75
levels of M1 seem to give very similar results indicating that
enzyme is no longer limiting. The results from CU similarly shows
that the 0.2 enzyme level is somewhat limiting the fermentation,
but is faster than 0.2 GAU/gm for M1 results. This indicates that
the RHU activity is a better parameter that indicates the
hydrolysis of uncooked starch. CU has about twice the RHU activity
per GAU as does M1, and CU is seen to hydrolyze the uncooked starch
faster at similar GAU levels. At the 0.5 and 0.75 GAU/gm dosage
excess glucose is observed particularly at the higher enzyme level.
Actually it appears that starch hydrolyzing rate is faster than the
fermentation rate. These results also show that at around 15%
ethanol, the ethanol seems to become toxic to the yeast since the
fermentations appeared to stop.
[0180] The graph provided at FIG. 2, Panel C shows the ethanol
results from the fermentations where M1 was added to
DISTILLASE.RTM.. As the results show, adding DISTILLASE.RTM. to a
low level of M1, 0.2 GAU/gm, both the rate and yield of ethanol
increased improving the performance of M1. These results show that
by adding a glucoamylase preparation with a GSH ratio greater than
1.5 to DISTILLASE.RTM., which has only a GSH ratio less than 0.6,
can hydrolyze uncooked starch so that ethanol can be made by a
process that eliminates the cooking step.
EXAMPLE 3
Comparison of Cooked and Uncooked Corn Mash
[0181] In these experiments, fermentations were conducted similar
to that described in Example 1, except 83 gm of 28.9% ground corn
slurry were placed in 250 ml bottles containing a magnetic bar. The
bottles were placed on a submersion magnetic stirrer in a
30.degree. C. water bath so that the mash was gently mixed during
the fermentation. Combinations of DISTILLASE.RTM.) and M1 were
tested as shown in Table 3. These fermentation were started with
0.27 gm of dry yeast. After the fermentation, the beer was dried in
a 65.degree. C. forced air oven to obtain what was considered the
DDGS (Distillers Dry Grains plus the Solubles). In this manner a
quantitative estimate of the DDGS was obtained, and the starch
contend of the DDGS was obtained by a starch analysis technique.
The HPLC profiles of the fermentations are also shown in Table
3.
3 TABLE 3 HPLC Profile M1 Dist. % W/V % W/V % W/V % W/V % W/V % V/V
Trial GAU/g GAU/g Hours DP > 2 DP-2 DP-1 Lactic Glycerol Ethanol
1 0.10 0.00 24 0.23 0 0 0.08 0.28 3.40 1 48 0.26 0 0 0.46 0.56 5.66
1 72 0.27 0 0 0.62 0.33 6.95 2 0.10 0.20 24 0.24 0 0 0.11 0.41 5.70
2 48 0.27 0 0 0.09 0.46 9.21 2 72 0.32 0 0 0.04 0.48 11.44 3 0.10
0.40 24 0.19 0 0 0.14 0.52 6.87 3 48 0.26 0 0 0.12 0.61 10.87 3 72
0.27 0 0 0.05 0.63 12.98 4 0.10 0.60 24 0.22 0 0 0.15 0.59 7.79 4
48 0.26 0 0 0.12 0.67 11.93 4 72 0.29 0 0 0.05 0.69 13.63 5 0.10
1.00 24 0.18 0 0.01 0.15 0.69 9.07 5 48 0.23 0 0 0.10 0.76 12.74 5
72 0.29 0 0 0.05 0.79 14.14 6 0.20 0.00 24 0.22 0 0 0.12 0.42 5.64
6 48 0.27 0 0 0.08 0.43 8.86 6 72 0.32 0 0 0.03 0.46 11.33 7 0.20
0.20 24 0.23 0 0 0.15 0.54 7.29 7 48 0.25 0 0 0.13 0.53 11.15 7 72
0.22 0 0 0.07 0.66 13.09 8 0.20 0.40 24 0.21 0 0 0.15 0.62 8.46 8
48 0.25 0 0 0.13 0.70 12.38 8 72 0.27 0 0 0.06 0.53 13.65 9 0.20
0.60 24 0.25 0 0 0.14 0.63 9.43 9 48 0.21 0 0 0.08 0.72 13.15 9 72
0.29 0 0 0.03 0.73 14.40 10 0.20 1.00 24 0.24 0 0.02 0.14 0.75
10.32 10 48 0.25 0 0 0.08 0.78 14.12 10 72 0.31 0 0.01 0.04 0.80
14.31 11 0.40 0.00 24 0.26 0 0 0.16 0.56 8.00 11 48 0.31 0 0 0.10
0.64 12.04 11 72 0.27 0 0 0.04 0.67 13.77 12 0.40 0.20 24 0.22 0 0
0.14 0.62 9.24 12 48 0.29 0 0 0.09 0.69 13.55 12 72 0.28 0 0 0.03
0.70 14.01 13 0.40 0.40 24 0.25 0 0 0.15 0.69 10.15 13 48 0.29 0 0
0.09 0.75 13.64 13 72 0.33 0 0 0.05 0.77 14.40 14 0.40 0.60 24 0.24
0 0.02 0.14 0.73 10.89 14 48 0.31 0 0 0.09 0.79 13.84 14 72 0.34 0
0 0.04 0.78 14.19 15 0.40 1.00 24 0.26 0 0.02 0.13 0.76 11.35 15 48
0.32 0 0 0.08 0.82 14.30 15 72 0.29 0 0 0.05 0.83 14.54
[0182] At each level of M1 tested the addition of DISTILLASE.RTM.
improved the fermentation rate and yield of ethanol, as shown in
FIG. 3, Panels A, B and C.
[0183] The starch analyses of the DDGS are shown in Table 4. From
these analyses and the amount of DDGS, an estimate was then made of
the amount of starch that remained unconverted in the fermenter. As
indicated by these results, the addition of DISTILLASE.RTM.) to M1
helps improve the hydrolysis of uncooked starch.
4 TABLE 4 M1 Dist. % V/V DDGS % Unused Trial GAU/g GAU/g Ethanol gm
DS % Starch Starch 1 0.10 0.00 6.95 13.93 52.37 46.75 2 0.10 0.20
11.44 8.88 26.84 15.29 3 0.10 0.40 12.98 7.29 15.60 7.29 4 0.10
0.60 13.63 6.74 9.53 4.12 5 0.10 1.00 14.14 6.68 5.60 2.40 6 0.20
0.00 11.33 9.51 33.64 20.51 7 0.20 0.20 13.09 7.53 16.84 8.13 8
0.20 0.40 13.65 6.72 8.88 3.83 9 0.20 0.60 14.40 6.37 3.75 1.53 10
0.20 1.00 14.31 6.36 2.50 1.02 11 0.40 0.00 13.77 7.00 9.43 4.23 12
0.40 0.20 14.01 6.78 8.57 3.73 13 0.40 0.40 14.40 6.38 3.12 1.28 14
0.40 0.60 14.19 6.44 2.02 0.83 15 0.40 1.00 14.54 6.41 1.61
0.66
[0184] Thus, the results obtained in these Examples indicate that
adding a glucoamylase preparation with a GSH ratio greater than 1.5
to a glucoamylase with GSH ratio less than 0.6 can hydrolyze
uncooked starch such that ethanol fermentations can be carried out
on mashes that are not cooked. These results demonstrated the
percent composition of a high GSH ratio glucoamylase to a low GSH
ratio as low as 9% is very effective in hydrolyzing uncooked
starch.
EXAMPLE 4
Influence of Stillage on the Fermentation of Cooked and Non-Cooked
Mash
[0185] This Example describes experiments designed to evaluate the
fermentation of liquefied corn mash containing various levels of
stillage compared to the fermentation of non-cooked corn mash
containing various levels of stillage. The enzymes used in the
fermentations were different. FERMENZYME.RTM. was used for
fermenting the liquefied mash, which is a preparation that is
similar to what is used commercially. For the non-cooked mash
fermentation, a combination of DISTILLASE.RTM. and the RSH enzyme
M1 were used.
[0186] The experiment was set up so that the corn solids would be
constant while the solids from the stillage would vary. This meant
that the total solids in the fermenters increased as the stillage
increased. Thin stillage was obtained from a local dry mill ethanol
plant. The thin stillage was concentrated in a vacuum rotary
evaporator to 44% solids. It was necessary to concentrate the thin
stillage, so that the total solids in the fermenters would be
manageable. The mash composition for each fermenter is shown in
Table 5.
5TABLE 5 Fermenter Mash Composition 31% Liquefied Corn at pH 5.0,
.25 gm yeast,150 gm Total .4 GAU/gm Fermenzyme Liquefact Stillage
Syrup Water Mash No gm gm gm % DS 1 130 0 20.0 31.1 2 130 2 18.0
31.7 3 130 5 15.0 32.5 4 130 10 10.0 34.0 5 130 15 5.0 35.5 6 130
20 0.0 36.9 31% Noncooked Corn <30 mesh at pH 5.0, .25 gm Yeast,
150 gm Total .6 GAU/gm Distillase + .19 RHU/gmM1 + GC106 equivalent
Corn Stillage Syrup Water Mash No gm gm gm % DS 7 52.4 0 97.1 30.9
8 52.4 2 95.1 31.5 9 52.4 5 92.1 32.4 10 52.4 10 87.1 33.9 11 52.4
15 82.1 35.3 12 52.4 20 77.1 36.8 13 52.4 25 72.1 38.3 14 52.4 30
67.1 39.7
[0187] The enzyme used for the liquefied corn mash was 0.4 GAU/gm
of corn liquefact of FERMENZYME.RTM.). FERMENZYME.RTM., a special
blend of DISTILLASE.RTM. and a fungal protease for fermenting corn
mash, is commercially available from Genencor.RTM.. For the
non-cooked fermentations, a combination of DISTILLASE.RTM. and M1
were used along with the equivalent amount of protease that was in
the FERMENZYME.RTM.) used in the liquefied corn mash test runs. As
indicated in Table 5, the solids in the fermenters varied (mash %
DS). The results of the fermentations are given in Table 6,
below.
6TABLE 6 HPLC Profile During Fermentation Stillage % W/V % W/V %
W/V % W/V % W/V % V/V Ferm Mash gm Enzyme Hours DP > 2 DP-2 DP-1
Lactic Glycerol Ethanol 1 Liq 0 Fermenzyme 24 4.93 3.63 1.66 0.69
0.60 8.74 1 48 1.50 0.56 2.17 0.88 0.83 14.08 1 72 0.71 0.56 0.17
0.84 0.85 16.28 2 Liq 2 Fermenzyme 24 5.34 3.94 1.72 0.82 0.79 9.33
2 48 1.77 0.61 4.06 0.99 1.03 12.85 2 72 0.90 0.61 3.14 0.99 1.04
14.04 3 Liq 5 Fermenzyme 24 5.75 4.40 1.80 0.98 1.07 9.35 3 48 2.07
0.65 4.66 1.13 1.27 11.92 3 72 1.14 0.68 3.26 1.16 1.35 13.54 4 Liq
10 Fermenzyme 24 5.74 4.31 1.55 1.15 1.28 9.41 4 48 2.28 0.72 5.21
1.36 1.56 11.80 4 72 1.35 0.75 4.73 1.36 1.59 12.88 5 Liq 15
Fermenzyme 24 6.26 4.65 1.64 1.47 1.56 9.66 5 48 2.56 0.87 5.50
1.59 1.82 11.56 5 72 1.57 0.84 4.71 1.58 1.83 12.58 6 Liq 20
Fermenzyme 24 6.16 1.67 1.67 1.64 1.81 9.30 6 48 2.75 0.91 5.79
1.77 2.05 11.57 6 72 1.83 1.00 6.48 1.82 2.00 11.63 7 g. corn 0
Distillase + M1 + GC106 24 0.34 0 0.01 0.14 0.67 11.86 7 48 0.36 0
0 0.17 0.78 15.65 7 72 0.39 0 0 0.18 0.86 17.48 8 g. corn 2
Distillase + M1 + GC107 24 0.35 0 0.02 0.23 0.77 11.96 8 48 0.40 0
0 0.25 0.88 16.09 8 72 0.38 0 0 0.19 0.95 17.56 9 g. corn 5
Distillase + M1 + GC108 24 0.40 0 0.02 0.38 0.88 12.07 9 48 0.42 0
0 0.38 0.99 15.99 9 72 0.43 0 0 0.31 1.07 18.03 10 g. corn 10
Distillase + M1 + GC109 24 0.54 0 0.03 0.65 1.02 11.31 10 48 0.53 0
0 0.66 1.18 15.95 10 72 0.52 0 0 0.65 1.24 17.69 11 g. corn 15
Distillase + M1 + GC110 24 0.70 0 0 0.83 1.18 10.22 11 48 0.66 0
0.10 0.87 1.40 15.53 11 72 0.62 0 0 0.83 1.42 17.14 12 g. corn 20
Distillase + M1 + GC111 24 0.82 0 0 1.15 1.47 10.04 12 48 0.78 0 0
1.09 1.52 14.32 12 72 0.78 0 0 1.04 1.70 17.24 13 g. corn 25
Distillase + M1 + GC112 24 1.04 0 0 1.48 1.78 7.71 13 48 1.01 0 0
1.40 1.77 11.40 13 72 0.99 0 0 1.42 1.97 14.72 14 g. corn 30
Distillase + M1 + GC113 24 1.17 0 0 1.74 2.02 7.68 14 48 1.18 0 0
1.70 2.06 11.61 14 72 1.12 0 0 1.65 2.18 14.58
[0188] In commercial practice, a certain amount of the stillage is
recycled for the yeast nutrient content and to help the water
balance in the plant. Thus, fermentation systems that are less
influenced by stillage is very desirable in industrial fermentation
plants. The results of these experiments show that stillage effects
the fermentation of liquefied mash more than in non-cooked
mash.
[0189] FIG. 4 shows the response of ethanol to the amount of
stillage added in both types of mashes. In both cases, increasing
the stillage solids reduced the ethanol level. But as FIG. 4 shows,
the non-cooked mash is much less sensitive to stillage than the
cooked mash.
[0190] FIG. 5 shows the glucose profile after 72 hour of
fermentation and the results are very striking. As the stillage
solids increased in cooked mash more glucose was left unfermented,
while in the non-cooked mash, essentially non-detectable levels of
glucose were observed at all levels of stillage. This observation
is very significant because as the starch was hydrolyzed it was
immediately to ethanol by the yeast. This level of glucose build-up
is very unusual. This observation is also important particularly
with respect to the yeast, in that even though the glucose level is
extremely low, the yeast remain very active in fermenting. In
contrast, in the cooked mash, even when glucose was in ample
supply, the yeast could not ferment the glucose.
[0191] FIG. 6 is a plot of the disaccharides after 72 hours of
fermentation with respect to stillage added. The disaccharide
levels for the non-cooked mash were found to be essentially
non-detectable throughout the range of stillage added, but in the
cooked mash as the stillage level increased the disaccharides
increased.
[0192] As indicated in FIG. 7, the higher sugars (i.e.,
oligosaccharides greater than disaccharides) provided a somewhat a
similar picture, as the level of higher sugars for the cooked mash
were higher with respect to stillage added than for the non-cooked
mash.
[0193] FIG. 8 shows the lactic acid level after 72 hours of
fermentation. As indicated, the levels are higher for the cooked
mash than the non-cooked mash. One consideration with lactic acid
is that it is a measure of contamination. Although it is not
intended that the present invention be limited to any particular
theory or mechanism, it is possible that since both the glucose and
disaccharide levels are always very low in the non-cooked mash,
contaminating microorganisms have very little substrate to
utilize.
[0194] FIG. 9 provides a summary of the glycerol levels after 72
hour of fermentation. As indicated, again the levels are lower at
the respective stillage addition levels with the non-cooked mash
than with the cooked mash. A contributing factor for glycerol
formation during fermentation with yeast is the stress the yeast is
under. Generally, the more stress the yeast is under, the more
glycerol that will be formed. The results in FIG. 9 would indicate
that at similar stillage levels, the yeast in the non-cooked mash
are under less stress. But even at higher stillage levels that
could be run with cooked mash, higher levels of glycerol were
formed. Even with the higher glycerol level in the non-cooked mash,
the yeast produced more ethanol. Thus, it appears that yeast seem
to ferment more efficiently in the non-cooked mash than in the
cooked mash.
[0195] Various other examples and modifications of the foregoing
description and examples will be apparent to a person skilled in
the art after reading the disclosure without departing from the
spirit and scope of the invention, and it is intended that all such
examples or modifications be included within the scope of the
appended claims. All publications and patents referenced herein are
hereby incorporated by reference in their entirety.
* * * * *