Note: Descriptions are shown in the official language in which they were submitted.
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PRODUCTION OF ALCOHOL ESTERS AND IN SITU PRODUCT
REMOVAL DURING ALCOHOL FERMENTATION
[0001] This application claims the benefit of U.S. Provisional Application No.
61/356,290, filed on June 18, 2010; U.S. Provisional Application No.
61/368,451,
filed on July 28, 2010; U.S. Provisional Application No. 61/368,436, filed on
July
28, 2010; U.S. Provisional Application No. 61/368,444, filed on July 28, 2010;
U.S. Provisional Application No. 61/368,429, filed on July 28, 2010; U.S.
Provisional Application No. 61/379,546, filed on September 2, 2010; and U.S.
Provisional Application No. 61/440,034, filed on February 7, 2011; U.S. Patent
Application No. 13/160,766, filed on June 15, 2011; the entire contents of
which
are all herein incorporated by reference.
[0002] The Sequence Listing associated with this application is filed in
electronic
form via EFS-Web and hereby incorporated by reference into the specification
in
its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to the fermentative production of
alcohols
including ethanol and butanol, and all related co-products, and processes for
improving alcohol fermentation employing in situ product removal methods.
BACKGROUND OF THE INVENTION
[0004] Alcohols have a variety of applications in industry and science such as
a
beverage (i.e., ethanol), fuel, reagents, solvents, and antiseptics. For
example,
butanol is an alcohol that is an important industrial chemical and drop-in
fuel
component with a variety of applications including use as a renewable fuel
additive, as a feedstock chemical in the plastics industry, and as a food-
grade
extractant in the food and flavor industry. Accordingly, there is a high
demand for
alcohols such as butanol, as well as for efficient and environmentally-
friendly
production methods.
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[0005] Production of alcohol utilizing fermentation by microorganisms is one
such
environmentally-friendly production method. In the production of butanol, in
particular, some microorganisms that produce butanol in high yields also have
low butanol toxicity thresholds. Removal of butanol from the fermentation
vessel
as it is being produced is a means to manage these low butanol toxicity
thresholds. Thus, there is a continuing need to develop efficient methods and
systems for producing butanol in high yields despite low butanol toxicity
thresholds of the butanol-producing microorganisms in the fermentation medium.
[0006] In situ product removal (ISPR) (also referred to as extractive
fermentation)
can be used to remove butanol (or other fermentative alcohol) from the
fermentation vessel as it is produced, thereby allowing the microorganism to
produce butanol at high yields. One ISPR method for removing fermentative
alcohol that has been described in the art is liquid-liquid extraction (U.S.
Patent
Application Publication No. 2009/0305370). In general, with regard to butanol
fermentation, the fermentation medium which includes the microorganism is
contacted with an organic extractant at a time before the butanol
concentration
reaches a toxic level. The organic extractant and the fermentation medium form
a biphasic mixture. The butanol partitions into the organic extractant phase
decreasing the concentration of butanol in the aqueous phase containing the
microorganism, thereby limiting the exposure of the microorganism to the
inhibitory butanol. In order to be technically and economically viable, liquid-
liquid
extraction requires contact between the extractant and the fermentation broth
for
efficient mass transfer of the product alcohol into the extractant; phase
separation
of the extractant from the fermentation broth (during an/or after
fermentation);
efficient recovery and recycle of the extractant; and minimal decrease of the
partition coefficient of the extractant over a long-term operation.
[0007] The extractant can become contaminated over time with each recycle, for
example, by the build-up of lipids present in the biomass that is fed to the
fermentation vessel as feedstock of hydrolyzable starch. As an example, during
the conversion of glucose to butanol, a liquified corn mash loaded to a
fermentation vessel at 30 wt % dry corn solids can result in a fermentation
broth
that contains about 1.2 wt % corn oil generated by simultaneous
saccharification
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and fermentation (with saccharification of the liquified mash occurring during
fermentation by the addition of glucoamylase to produce glucose). The
dissolution of the corn oil lipids into oleyl alcohol (OA) serving as an
extractant
during ISPR can result in build-up of lipid concentration with each OA recycle
decreasing the partition coefficient for the product alcohol in OA as the
lipid
concentration in OA increases with each recycle of OA.
[0008] In addition, the presence of the undissolved solids during extractive
fermentation can negatively affect the efficiency of the alcohol production.
For
example, the presence of the undissolved solids may lower the mass transfer
coefficient inside the fermentation vessel, impede phase separation in the
fermentation vessel, result in the accumulation of corn oil from the
undissolved
solids in the extractant leading to reduced extraction efficiency over time,
increase the loss of solvent because it becomes trapped in solids and
ultimately
removed as Dried Distillers' Grains with Solubles (DDGS), slow the
disengagement of extractant drops from the fermentation broth, and/or result
in a
lower fermentation vessel volume efficiency.
[0009] Several approaches for reducing the degradation of the partition
coefficient
of the extractant used in extractive fermentation have included wet milling,
fractionation, and removal of solids. Wet milling is an expensive, multi-step
process that separates a biomass (e.g., corn) into its key components (germ,
pericarp fiber, starch, and gluten) in order to capture value from each co-
product
separately. This process gives a purified starch stream; however, it is costly
and
includes the separation of the biomass into its non-starch components which is
unnecessary for fermentative alcohol production. Fractionation removes fiber
and germ, which contains a majority of the lipids present in ground whole corn
resulting in a fractionated corn that has a higher starch (endosperm) content.
Dry
fractionation does not separate the germ from fiber and therefore, it is less
expensive than wet milling. However, fractionation does not remove the
entirety
of the fiber or germ, and does not result in total elimination of solids.
Furthermore, there is some loss of starch in fractionation. Wet milling of
corn is
more expensive than dry fractionation, but dry fractionation is more expensive
than dry grinding of unfractionated corn. Removal of solids including germ
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containing lipids, from liquefied mash prior to use in fermentation can
substantially eliminate undissolved solids as described, for example, in co-
pending, commonly owned U.S. Provisional Application Serial No. 61/356,290,
filed June 18, 2010. However, it would be advantageous if the degradation of
the
partition coefficient of the extractant caused by contamination by lipid can
be
reduced even without fractionation or removal of substantially all undissolved
solids. Converting the lipids present in a liquefied mash into an extractant
that
can be used in ISPR is another method of decreasing the amount of lipids that
are fed to the fermentation vessel as described, for example, in co-pending,
commonly owned U.S. Provisional Application Serial No. 61/368,436 and U.S.
Provisional Application Serial No. 61/368,444, both filed on July 28, 2010.
[0010] There is a continuing need for alternative extractive fermentation
methods
which do not necessitate the partitioning of the product alcohol between the
fermentation medium and the ISPR extractant as a means to reduce the toxic
effect of the product alcohol such as butanol on the microorganism, and which
can also reduce the degradation of the partition coefficient of a fermentation
product extractant.
BRIEF SUMMARY OF THE INVENTION
[0011] Conversion of alcohol such as butanol produced from a microorganism in
a fermentation medium into a substance less toxic to the microorganism can
allow increased production of alcohol such as butanol for a given fermentation
vessel volume. Alcohol esters can be formed by contacting the alcohol in a
fermentation medium with a carboxylic acid (e.g., fatty acids) and a catalyst
capable of esterifying the alcohol with the carboxylic acid. Moreover, the
carboxylic acid can serve as an ISPR extractant into which the alcohol esters
partition. The carboxylic acid can be supplied to the fermentation vessel
and/or
derived from the biomass supplying fermentable carbon feed to the fermentation
vessel. Lipids present in the feedstock can be catalytically hydrolyzed to
carboxylic acid and the same catalyst (e.g., enzymes) can esterify the
carboxylic
acid with the alcohol (e.g., butanol); lipids can also be directly
transesterified by
the catalyst to produce alcohol esters. The catalyst can be supplied to the
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feedstock prior to fermentation or can be supplied to the fermentation vessel
before or contemporaneously with the supplying of the feedstock. When the
catalyst is supplied to the fermentation vessel, alcohol esters can be
obtained by
hydrolysis of the lipids into carboxylic acid and concurrent esterification of
carboxylic acid with butanol present in the fermentation vessel; lipids can
also be
directly transesterified with butanol by the catalyst to produce alcohol
esters.
Carboxylic acid and/or native oil not derived from the feedstock can also be
fed to
the fermentation vessel, with the native oil being hydrolyzed into carboxylic
acid.
Carboxylic acid and/or native oil not derived from the feedstock can be fed
into
the fermentation vessel in an amount sufficient such that a two-phase mixture
comprising an organic phase and an aqueous phase is formed. As such, in some
embodiments, any carboxylic acid not esterified with the alcohol can serve as
the
ISPR extractant or as a part thereof. The extractant containing alcohol esters
can be separated from the fermentation medium, and the alcohol can be
recovered from the extractant. The extractant can be recycled to the
fermentation vessel. Thus, in the case of butanol production, for example, the
conversion of butanol to an ester reduces the free butanol concentration in
the
fermentation medium, shielding the microorganism from the toxic effect of
increasing butanol concentration. In addition, unfractionated grain can be
used
as feedstock without separation of lipids therein, since the lipids can be
catalytically hydrolyzed to carboxylic acid, thereby decreasing the rate of
build-up
of lipids in the ISPR extractant.
[0012] The present invention is directed to a method for producing butyl
esters
comprising contacting butanol produced in a fermentation process with at least
one carboxylic acid and at least one catalyst capable of esterifying the
carboxylic
acid with the butanol to form butyl esters of the carboxylic acid; wherein the
carboxylic acid in the fermentation process is present at a concentration
sufficient
to produce a two-phase mixture. In one embodiment, the production of butanol
and the production of butyl esters occur simultaneously or sequentially. In
one
embodiment, a feedstock in the fermentation process comprises one or more
fermentable sugars. In another embodiment, the feedstock in the fermentation
process comprises one or more fermentable sugars derived from corn grain, corn
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cobs, crop residues such as corn husks, corn stover, grasses, wheat, rye,
wheat
straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar
cane
bagasse, sorghum, sugar cane, soy, components obtained from milling of grains,
cellulosic material, lignocellulosic material, trees, branches, roots, leaves,
wood
chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure,
and mixtures thereof. In one embodiment, the method further comprises
providing a native oil and converting at least a portion of the native oil
into
carboxylic acid by contacting the oil with one or more enzymes. In one
embodiment, the carboxylic acid comprises fatty acids. In another embodiment,
the carboxylic acid comprises 12 to 22 carbons. In one embodiment, the
carboxylic acid is a mixture of carboxylic acids. In another embodiment, the
butyl
esters of the carboxylic acid are butyl esters of fatty acids. In one
embodiment,
the catalyst is an enzyme capable of esterifying the carboxylic acid with the
butanol to form butyl esters of the carboxylic acid. In another embodiment,
the
enzyme is an esterase, lipase, phospholipase, or lysophospholipase.
[0013] The present invention is also directed to a method for producing
butanol
and butyl esters from a feedstock comprising: (a) providing a feedstock; (b)
liquefying the feedstock to create a liquefied biomass comprising
oligosaccharides; (c) separating the feedstock slurry to produce a product
comprising an aqueous stream comprising oligosaccharides, an oil stream, and
solids; (d) adding the aqueous stream to a fermentation vessel containing a
fermentation broth; (e) saccharifying the oligosaccharides of the aqueous
stream;
(f) fermenting the products of the oligosaccharide saccharification present in
the
aqueous stream to produce butanol and concurrently contacting the butanol with
at least one carboxylic acid and at least one catalyst capable of esterifying
the
carboxylic acid with the butanol to form butyl esters of the carboxylic acid
wherein
the carboxylic acid is present at a concentration sufficient to produce a two-
phase
mixture; and optionally steps (e) and (f) occur concurrently. In one
embodiment,
the method further comprises obtaining an oil from the oil stream and
converting
at least a portion of the oil into carboxylic acid. In one embodiment, the
feedstock
slurry is separated by decanter bowl centrifugation, tricanter centrifugation,
disk
stack centrifugation, filtering centrifugation, decanter centrifugation,
filtration,
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vacuum filtration, beltfilter, pressure filtration, screen filtration, screen
separation,
grating, porous grating, flotation, hydroclone, filter press, screwpress,
gravity
settler, vortex separator, or combination thereof. In another embodiment, the
carboxylic acid comprises fatty acids. In one embodiment, the carboxylic acid
comprises 12 to 22 carbons. In one embodiment, the method further comprises
adding the oil to the fermentation vessel prior to the step of converting at
least a
portion of the oil into carboxylic acid. In one embodiment, the method further
comprises adding additional carboxylic acid to the fermentation vessel. In one
embodiment, the oil is converted to carboxylic acid after the step of adding
the
additional carboxylic acid. In another embodiment, the carboxylic acid is corn
oil
fatty acid, soya oil fatty acid, or a mixture of corn oil fatty acid and soya
oil fatty
acid. In one embodiment, the oil obtained from the oil stream comprises
glycerides and the one or more catalysts hydrolyze the glycerides into fatty
acids.
In another embodiment, the butyl esters of carboxylic acid are butyl esters of
fatty
acids. In one embodiment, the catalyst is an enzyme capable of esterifying the
carboxylic acid with the butanol to form butyl esters of the carboxylic acid.
In one
embodiment, the enzyme is an esterase, lipase, phospholipase, or
lysophospholipase. In one embodiment, the method further comprises the step
of washing the solids with a solvent. In one embodiment, the solvent is select
from hexane, isobutanol, isohexane, ethanol, petroleum distillates such as
petroleum ether, or mixtures thereof. In another embodiment, the solids are
processed to form an animal feed product. In one embodiment, the solids are
processed to form an animal feed product. In one embodiment, the animal feed
product comprises one or more crude protein, crude fat, triglycerides, fatty
acid,
fatty acid isobutyl ester, lysine, neutral detergent fiber (NDF), and acid
detergent
fiber (ADF). In another embodiment, the animal feed product further comprises
one or more vitamins, minerals, flavoring, or coloring. In one embodiment, the
animal feed product comprises 20-35 wt% crude protein, 1-20 wt% crude fat, 0-5
wt% triglycerides, 4-10 wt% fatty acids, and 2-6 wt% fatty acid isobutyl
esters. In
one embodiment, the step of separating the solids from the feedstock slurry
increases the efficiency of the butanol production by increasing a liquid-
liquid
mass transfer coefficient of the butanol from the fermentation broth to the
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extractant; increases the efficiency of the butanol production by increasing
an
extraction efficiency of the butanol with an extractant; increases the
efficiency of
the butanol production by increasing a rate of phase separation between the
fermentation broth and an extractant; increases the efficiency of the butanol
production by increasing recovery and recycling of an extractant; or increases
the
efficiency of the butanol production by decreasing a flow rate of an
extractant.
[0014] The present invention is also directed to a method for producing
butanol
and butyl esters from a feedstock comprising: (a) providing a feedstock; (b)
liquefying the feedstock to create a liquefied biomass comprising
oligosaccharides; (c) separating the feedstock slurry to produce a stream
comprising oligosaccharides and oil, and solids; (d) adding the stream to a
fermentation vessel containing a fermentation broth; (e) saccharifying the
oligosaccharides of the stream; (f) fermenting the products of the
oligosaccharide
saccharification present in the stream to produce butanol and concurrently
contacting the butanol with at least one carboxylic acid and at least one
catalyst
capable of esterifying the carboxylic acid with the butanol to form butyl
esters of
the carboxylic acid wherein the carboxylic acid is present at a concentration
sufficient to produce a two-phase mixture; and optionally steps (e) and (f)
occur
concurrently. In one embodiment, the method further comprising converting at
least a portion of the oil into carboxylic acid. In one embodiment, the
feedstock
slurry is separated by decanter bowl centrifugation, tricanter centrifugation,
disk
stack centrifugation, filtering centrifugation, decanter centrifugation,
filtration,
vacuum filtration, beltfilter, pressure filtration, screen filtration, screen
separation,
grating, porous grating, flotation, hydroclone, filter press, screwpress,
gravity
settler, vortex separator, or combination thereof. In another embodiment, the
carboxylic acid comprises fatty acids. In one embodiment, the carboxylic acid
comprises 12 to 22 carbons. In one embodiment, the method further comprises
adding oil to the fermentation vessel. In one embodiment, the method further
comprising adding additional carboxylic acid to the fermentation vessel. In
one
embodiment, the oil is converted to carboxylic acid after the step of adding
the
additional carboxylic acid. In one embodiment, the carboxylic acid is corn oil
fatty
acid, soya oil fatty acid, or a mixture of corn oil fatty acid and soya oil
fatty acid.
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In one embodiment, the oil comprises glycerides and the one or more catalysts
hydrolyze the glycerides into fatty acids. In one embodiment, the butyl esters
of
carboxylic acid are butyl esters of fatty acids. In one embodiment, the
catalyst is
an enzyme capable of esterifying the carboxylic acid with the butanol to form
butyl esters of the carboxylic acid. In one embodiment, the enzyme is an
esterase, lipase, phospholipase, or lysophospholipase. In one embodiment, the
method further comprises the step of washing the solids with a solvent. In one
embodiment, the solvent is select from hexane, isobutanol, isohexane, ethanol,
petroleum distillates such as petroleum ether, or mixtures thereof. In one
embodiment, the solids are processed to form an animal feed product. In one
embodiment, the solids are processed to form an animal feed product. In some
embodiments, the animal feed product comprises one or more crude protein,
crude fat, triglycerides, fatty acid, fatty acid isobutyl ester, lysine,
neutral
detergent fiber (NDF), and acid detergent fiber (ADF). In some embodiments,
the
animal feed product further comprises one or more vitamins, minerals,
flavoring,
or coloring. In some embodiments, the animal feed product comprises 20-35 wt%
crude protein, 1-20 wt% crude fat, 0-5 wt% triglycerides, 4-10 wt% fatty
acids,
and 2-6 wt% fatty acid isobutyl esters.In some embodiments, the step of
separating the solids from the feedstock slurry increases the efficiency of
the
butanol production by increasing a liquid-liquid mass transfer coefficient of
the
butanol from the fermentation broth to the extractant; increases the
efficiency of
the butanol production by increasing an extraction efficiency of the butanol
with
an extractant; increases the efficiency of the butanol production by
increasing a
rate of phase separation between the fermentation broth and an extractant;
increases the efficiency of the butanol production by increasing recovery and
recycling of an extractant; or increases the efficiency of the butanol
production by
decreasing a flow rate of an extractant.
[0015] The present invention is also directed to a method for producing
butanol
comprising (a) contacting butanol produced in a fermentation process with at
least one carboxylic acid and at least one catalyst capable of esterifying the
carboxylic acid with the butanol to form butyl esters of the carboxylic acid;
wherein the carboxylic acid in the fermentation process is present at a
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concentration sufficient to produce a two-phase mixture comprising an aqueous
phase and a butyl ester-containing organic phase; (b) separating the butyl
ester-
containing organic phase from the aqueous phase; and (c) recovering butanol
from the butyl esters. In some embodiments, recovering butanol from the butyl
esters comprises hydrolyzing the esters into carboxylic acid and butanol. In
some
embodiments, the butyl esters are hydrolyzed in the presence of a hydrolysis
catalyst. In some embodiments, the butyl esters are hydrolyzed in the presence
of water and wherein the hydrolysis catalyst comprises an acid catalyst, an
organic acid, an inorganic acid, a water soluble acid, or water insoluble
acid. In
some embodiments, the hydrolysis catalyst comprises an enzyme capable of
hydrolyzing the butyl esters to form a carboxylic acid and butanol. In some
embodiments, the enzyme is an esterase, lipase, phospholipase, or
lysophospholipase. In some embodiments, enzyme reaction conditions favor
enzymatic hydrolysis over esterification. In some embodiments, the enzyme
reaction conditions comprise a cosolvent. In some embodiments, fatty acid
butyl
esters, fatty acids, isobutanol, and water are soluble in the cosolvent, and
wherein free fatty acids do not react with the cosolvent. In some embodiments,
the cosolvent is selected from acetone, tert-butanol, 2-Me-2-butanol, 2-Me-2-
pentanol, and 3-Me-3-pentanol. In some embodiments, the enzyme reaction
conditions comprise end-product removal. In some embodiments, the end-
product is isobutanol or fatty acids. In some embodiments, isobutanol is
removed
by vacuum distillation, pervaporartion, permselective filtration, or gas
sparging.
In some embodiments, the fatty acids are removed by precipitation,
permselective filtration, or electrophorectically. In some embodiments, the
hydrolysis reaction occurs in a reaction vessel. In some embodiments,
recovering butanol from the butyl esters comprises transesterifying the butyl
esters into butanol and fatty acid alkyl esters or acyl glycerides. In some
embodiments, the fatty acid alkyl esters comprise fatty acid methyl esters,
fatty
acid ethyl esters, or fatty acid propyl esters. In some embodiments, the
method
further comprises providing a native oil and converting at least a portion of
the
native oil into carboxylic acid by contacting the oil with one or more
enzymes. In
some embodiments, the enzyme is an enzyme capable of hydrolyzing or
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transesterifying the butyl esters to form butanol. In some embodiments, the
enzyme is an esterase, lipase, phospholipase, or lysophospholipase. In some
embodiments, the carboxylic acid comprises fatty acids. In some embodiments,
the carboxylic acid has carbon chain lengths ranging from 12 to 22 carbons. In
some embodiments, at least about 10% of butanol is recovered from the butyl
esters. In some embodiments, at least about 50% of butanol is recovered from
the butyl esters. In some embodiments, at least about 90% of butanol is
recovered from the butyl esters. In some embodiments, carboxylic acid is
recovered from the butyl esters. In some embodiments, the method further
comprising the steps of removing butanol from the fermentor as extractant
stream; and adding the extractant stream to two or more distillation columns.
In
some embodiments, the distillation column is a super-atmospheric distillation
column with a steam heated reboiler. In some embodiments, the method further
comprises the steps of recovering water and solvent from the distillation
columns;
and recycling the water and solvent. In some embodiments, the method further
comprises the steps of recovering heat from the distillation process; and
recycling
the heat to evaporate water.
[0016] The present invention is also directed to method for producing butanol
from a feedstock comprising (a) providing a feedstock; (b) liquefying the
feedstock to create a feedstock slurry; (c) separating the feedstock slurry to
produce a product comprising an aqueous stream, an oil stream, and solids; (d)
adding the aqueous stream to a fermentation vessel containing a fermentation
broth; (e) saccharifying the aqueous stream; (f) fermenting the saccharified
aqueous stream to produce butanol and concurrently contacting the butanol with
at least one carboxylic acid and at least one catalyst capable of esterifying
the
carboxylic acid with the butanol to form butyl esters of the carboxylic acid
wherein
the carboxylic acid is present at a concentration sufficient to produce a two-
phase
mixture; (g) separating the butyl ester-containing organic phase from the
aqueous
phase; and (h)recovering butanol from the butyl esters; and optionally steps
(e)
and (f) occur concurrently. In some embodiments, the method further comprises
obtaining an oil from the oil stream and converting at least a portion of the
oil into
carboxylic acid. In some embodiments, the feedstock slurry is separated by
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centrifugation, filtration, or decantation. In some embodiments, the
carboxylic
acid comprises fatty acids. In some embodiments, the carboxylic acid has
carbon chain lengths ranging from 12 to 22 carbons. In some embodiments, the
method further comprises adding the oil to the fermentation vessel prior to
the
step of converting at least a portion of the oil into carboxylic acid. In some
embodiments, the method further comprises adding additional carboxylic acid to
the fermentation vessel. In some embodiments, the oil is converted to
carboxylic
acid after the step of adding the additional carboxylic acid. In some
embodiments, the carboxylic acid is corn oil fatty acid, soya oil fatty acid,
or a
mixture of corn oil fatty acid and soya oil fatty acid. In some embodiments,
the oil
obtained from the oil stream comprises glycerides and the one or more
catalysts
hydrolyze the glycerides into fatty acids. In some embodiments, the butyl
esters
of carboxylic acid are butyl esters of fatty acids. In some embodiments, the
catalyst is an enzyme capable of esterifying the carboxylic acid with the
butanol
to form butyl esters of the carboxylic acid. In some embodiments, the enzyme
is
an esterase, lipase, phospholipase, or lysophospholipase. In some
embodiments, the solids are processed to form to an animal feed product. In
some embodiments, recovering butanol from the butyl esters comprises
hydrolyzing the esters into carboxylic acid and butanol. In some embodiments,
the butyl esters are hydrolyzed in the presence of a hydrolysis catalyst. In
some
embodiments, the butyl esters are hydrolyzed in the presence of water and
wherein the hydrolysis catalyst comprises an acid catalyst, an organic acid,
an
inorganic acid, a water soluble acid, or water insoluble acid. In some
embodiments, the hydrolysis catalyst comprises an enzyme capable of
hydrolyzing the butyl esters to form a carboxylic acid and butanol. In some
embodiments, the enzyme is an esterase, lipase, phospholipase, or
lysophospholipase. In some embodiments, the hydrolysis reaction occurs in a
reaction vessel. In some embodiments, recovering butanol from the butyl esters
comprises transesterifying the butyl esters into butanol and fatty acid alkyl
esters
or acyl glycerides. In some embodiments, the fatty acid alkyl esters comprise
fatty acid methyl esters, fatty acid ethyl esters, or fatty acid propyl
esters. In
some embdodiments, the method further comprises providing a native oil and
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converting at least a portion of the native oil into carboxylic acid by
contacting the
oil with one or more enzymes. In some embdodiments,the enzyme is an enzyme
capable of hydrolyzing or transesterifying the butyl esters to form butanol.
In
some embdodiments,the enzyme is an esterase, lipase, phospholipase, or
lysophospholipase.
[0017] In other embodiments, a fermentation method may include: providing an
aqueous feedstream obtained from biomass where the aqueous feedstream
includes water, fermentable carbon source derived from the biomass, and oil;
contacting the aqueous feedstream with a catalyst whereby at least a portion
of
the oil is hydrolyzed into free fatty acids to form a catalyst-treated
feedstream
including the free fatty acids and the catalyst; contacting the catalyst-
treated
feedstream with a fermentation broth in a fermentation vessel; fermenting the
fermentable carbon source in the fermentation vessel to produce a product
alcohol; and contacting the product alcohol with the free fatty acids and the
catalyst during fermentation so as to catalyze the esterification of the free
fatty
acids and the product alcohol in the fermentation vessel to produce alcohol
esters of fatty acids. In some embodiments, the steps of contacting the
feedstream with catalyst and fermentation broth and the steps of fermenting
and
contacting the product alcohol with the free fatty acids and the catalyst may
occur
simultaneously. In some embodiments, the product alcohol is butanol and the
alcohol esters of fatty acids are butyl esters of fatty acids.
[0018] The present invention provides methods for removing alcohol from a
fermentation medium during fermentation including: providing a fermentation
medium including a microorganism that produces alcohol in the fermentation
medium; and contacting the fermentation medium during fermentation with a
carboxylic acid and a catalyst capable of esterifying the alcohol with the
carboxylic acid to form an alcohol ester. In some embodiments, the alcohol
that
is produced by the microorganism is butanol, and the alcohol ester is butyl
ester.
In some embodiments, the fermentation medium is contacted with a carboxylic
acid that is substantially insoluble in the fermentation medium and with a
catalyst
capable of esterifying the alcohol with the carboxylic acid to form an alcohol
ester.
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[0019] The present invention also provides methods for producing alcohol
esters
of fatty acids during a fermentation process including: providing a
fermentation
medium comprising alcohol, fermentable carbon source, and free fatty acids;
and
contacting the fermentation medium with one or more enzymes capable of
esterifying the free fatty acids with the alcohol whereby the free fatty acids
are
esterified with the alcohol to form alcohol esters of fatty acids. In some
embodiments, the fermentable carbon source is derived from biomass. In some
embodiments, the microorganism of the fermentation medium is a recombinant
microorganism. In some embodiments, the alcohol is butanol and the alcohol
esters of fatty acids are butyl esters of fatty acids.
[0020] In another embodiment, a method for producing a product alcohol may
include providing a biomass feedstock including water, fermentable carbon
source, and oil where the oil includes acyl glycerides; liquefying the biomass
feedstock to create a liquefied biomass comprising oligosaccharides;
contacting
the biomass feedstock or the liquefied biomass with a composition comprising
one or more enzymes capable of converting at least a portion of the acyl
glycerides into free fatty acids whereby the free fatty acids form an
extractant, the
one or more enzymes also being capable of esterifying free fatty acids with
product alcohol into alcohol esters of fatty acids; contacting the liquefied
biomass
with a saccharification enzyme capable of converting oligosaccharides into
fermentable sugar; contacting the liquefied biomass with a recombinant
microorganism capable of converting the fermentable sugar to product alcohol
whereby a fermentation product comprising product alcohol is produced;
contacting the product alcohol with the free fatty acids and the one or more
enzymes so as to catalyze the esterification of the free fatty acids and the
product
alcohol to produce alcohol esters of fatty acids; and contacting the
fermentation
product with extractant. In embodiments, the contacting with extractant
results in
the formation of a two-phase mixture including an aqueous phase and an
extractant phase and the alcohol esters of fatty acids partition into the
extractant
phase to form an ester-containing extractant phase. In some embodiments, the
product alcohol is butanol and the alcohol esters of fatty acids are butyl
esters of
fatty acids.
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[0021] In another embodiment, a method for producing a product alcohol may
include providing a biomass feedstock including water, fermentable carbon
source, and oil where the oil includes acyl glycerides; liquefying the biomass
feedstock to create a liquefied biomass comprising oligosaccharides;
contacting
the liquefied biomass with a composition comprising one or more enzymes
capable of converting at least a portion of the acyl glycerides into free
fatty acids,
the one or more enzymes also being capable of esterifying free fatty acids
with
product alcohol into alcohol esters of fatty acids; contacting the liquefied
biomass
with a saccharification enzyme capable of converting oligosaccharides into
fermentable sugar; contacting the saccharified biomass with a recombinant
microorganism capable of converting the fermentable sugar to product alcohol
during fermentation whereby a fermentation medium comprising product alcohol
is produced; contacting the fermentation medium during fermentation with a
carboxylic acid extractant, where the fermentation medium comprises one or
more enzymes capable of esterifying free fatty acids with product alcohol to
form
alcohol esters of fatty acids. In additional embodiments of this method, the
fermentation medium is contacted with a carboxylic acid that is substantially
insoluble in the fermentation medium and with a catalyst capable of
esterifying
the alcohol with the carboxylic acid to form an alcohol ester. In other
embodiments of this method, the alcohol that is produced by the microorganism
is butanol and the alcohol ester is butyl ester.
[0022] The present invention also provides a process to produce a product
alcohol from a feedstock including: liquefying starch or a fermentable carbon
source in a feedstock to create a slurry having oligosaccharides; centrifuging
the
feedstock slurry to produce a centrifuge product comprising (i) an aqueous
layer
comprising oligosaccharides, (ii) an oil layer, and (iii) solids; feeding the
aqueous
layer to a fermentation vessel containing a fermentation broth; and fermenting
the
aqueous layer to produce the product alcohol. The product alcohol is then
contacted with the carboxylic acid and the catalyst whereby the carboxylic
acid is
esterified with the product alcohol to form the alcohol esters. In some
embodiments, the oil is plant-derived oil. In other embodiments, the product
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alcohol is butanol and the alcohol esters of carboxylic acids are butyl esters
of
fatty acids.
[0023] In some embodiments, a method for producing a product alcohol includes
providing a fractionated biomass feedstock including water, starch, and/or a
fermentable carbon source, and only residual amounts of oil remaining after
fractionation of the biomass, the residual oil including acyl glycerides;
liquefying
the fractionated biomass feedstock to create a liquefied fractionated biomass
comprising oligosaccharides; contacting the liquefied fractionated biomass
with a
composition comprising one or more enzymes capable of converting at least a
portion of the residual acyl glycerides into free fatty acids, the one or more
enzymes also being capable of esterifying free fatty acids with product
alcohol to
form alcohol esters of fatty acids; contacting the liquefied fractionated
biomass
with a saccharification enzyme capable of converting oligosaccharides into
fermentable sugar; contacting the saccharified biomass with a recombinant
microorganism capable of converting the fermentable sugar to product alcohol
during fermentation whereby a fermentation medium comprising product alcohol
is produced; contacting the fermentation medium during fermentation with a
carboxylic acid extractant, where the fermentation medium comprises one or
more enzymes capable of esterifying free fatty acids with product alcohol to
form
alcohol esters of fatty acids. In additional embodiments of this method, the
fermentation medium is contacted with a carboxylic acid in the fermentation
medium and with a catalyst capable of esterifying the alcohol with the
carboxylic
acid to form an alcohol ester. In a further embodiment, the carboxylic acid
may
be substantially insoluble in the fermentation medium. In other embodiments of
this method, the alcohol that is produced by the microorganism is butanol and
the
alcohol ester is butyl ester.
[0024] The present invention also provides a composition including: a mash
formed from biomass and including water and fermentable sugar; a catalyst
capable of esterifying free fatty acids with alcohol into fatty acid alkyl
esters and
optionally capable of hydrolyzing acyl glycerides into free fatty acids;
alcohol; free
fatty acids; and fatty acid alcohol esters formed in situ from esterification
of the
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free fatty acids with the alcohol by the catalyst. In some embodiments, the
alcohol is butanol and the fatty acid alcohol esters are fatty acid butyl
esters.
[0025] The present invention also provides a fermentation broth including: a
recombinant microorganism capable of producing alcohol; a fermentable carbon
source; and fatty acid alcohol esters, wherein the fatty acid alcohol esters
are
produced during the fermentation. In some embodiments, the recombinant
microorganism is capable of producing butanol. In some embodiments, the fatty
acid alcohol esters are fatty acid butyl esters. In some embodiments, the
fermentable carbon source comprises sugar. In some embodiments, the
fermentable carbon source comprises methane, the recombinant microorganism
is capable of producing methanol, and the fatty acid alcohol esters are fatty
acid
methyl esters.
[0026] Also provided herein are recombinant yeast cells useful for production
of
product alcohols. In embodiments, the recombinant host cells disclosed herein
can be any bacteria, yeast or fungi host useful for genetic modification and
recombinant gene expression. In other embodiments, a recombinant host cell
can be a member of the genera Clostridium, Zymomonas, Escherichia,
Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas,
Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus,
Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces,
Kluyveromyces, Yarrowia, Pichia, Candida, Hansenula, Issatchenkia, or
Saccharomyces. In other embodiments, the host cell can be Saccharomyces
cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces
thermotolerans, Kluyveromyces marxianus, Candida glabrata, Candida albicans,
Pichia stipitis, Yarrowia lipolytica, E. coli, or L. plantarum. In still other
embodiments, the host cell is a yeast host cell. In some embodiments, the host
cell is a member of the genera Saccharomyces. In some embodiments, the host
cell is Kluyveromyces lactis, Candida glabrata or Schizosaccharomyces pombe.
In some embodiments, the host cell is Saccharomyces cerevisiae. S. cerevisiae
yeast are known in the art and are available from a variety of sources,
including,
but not limited to, American Type Culture Collection (Rockville, MD),
Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre,
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LeSaffre, Gert Strand AB, Ferm Solutions, North American Bioproducts, Martrex,
and Lallemand. S. cerevisiae include, but are not limited to, BY4741, CEN.PK
113-7D, Ethanol Red yeast, Gert Strand Prestige Turbo yeast, Ferm ProTM
yeast, Bio-Ferm XR yeast, Gert Strand Distillers Yeast, FerMaxTM Green yeast,
FerMaxTM Gold yeast, Thermosacc yeast, BG-1, PE-2, CAT-1, CBS7959,
CBS7960, and CBS7961.
[0027] Also provided are methods of producing isobutanol including: providing
a
recombinant host cell comprising an isobutanol biosynthetic pathway wherein at
least one of the enzyme that catalyzes the substrate to product conversion a-
ketoisovalerate to isobutyraldehyde or the enzyme that catalyzes the substrate
to
product conversion isobutyraldehyde to isobutanol are encoded by heterologous
polynucleotides integrated into the chromosome; and contacting the recombinant
host cell with a fermentable carbon source to form a fermentation broth under
conditions whereby isobutanol is produced. In some embodiments, the methods
further include: adding an extractant to form a two-phase mixture. In other
embodiments, the extractant comprises a carboxylic acid. In some embodiments,
the extractant comprises fatty acids. In other embodiments, the methods
further
include: adding an esterification enzyme capable of catalyzing the
esterification of
isobutanol with the carboxylic acid.
[0028] Also provided herein are methods including: providing a fermentation
medium comprising product alcohol, water, fermentable carbon source, and a
microorganism that produces the product alcohol; contacting the fermentation
medium during fermentation with an extractant to form a two-phase mixture
comprising an aqueous phase and an organic phase; and contacting the
fermentation medium with a carboxylic acid and an enzyme capable of
esterifying
the carboxylic acid with the product alcohol. In some embodiments, the
extractant comprises the carboxylic acid. In some embodiments, the carboxylic
acid is produced by hydrolysis of oil from a biomass feedstock. In some
embodiments, the fermentable carbon and the carboxylic acid are derived from
the same biomass feedstock source. In some embodiments, the carboxylic acid
comprises saturated, mono-unsaturated, poly-unsaturated carboxylic acids
having 12 to 22 carbons, and mixtures thereof. In some embodiments, contacting
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the fermentation medium with an extractant and a carboxylic acid and an enzyme
occur contemporaneously. In some embodiments, the microorganism is a
genetically modified microorganism (e.g., a recombinant microorganism or host
cell such as recombinant yeast cells).
[0029] Also provided herein are compositions comprising: PNY1504, PNY2205,
or a recombinant host cell; an extractant; and optionally an esterification
enzyme.
Also provided herein are compositions comprising PNY1504, PNY2205, or a
recombinant host cell and butyl ester.
[0030] Further provided herein are uses of PNY1504, PNY2205, or other
recombinant yeast cells, and compositions comprising recombinant yeast cells
for
the production of isobutanol.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0031] The accompanying drawings, which are incorporated herein and form a
part of the specification, illustrate the present invention and, together with
the
description, further serve to explain the principles of the invention and to
enable a
person skilled in the pertinent art to make and use the invention.
[0032] FIG. 1 schematically illustrates an exemplary method and system of the
present invention, in which a catalyst for alcohol esterification is supplied
to a
fermentation vessel along with carboxylic acid and/or native oil.
[0033] FIG. 2 schematically illustrates an exemplary method and system of the
present invention, in which native oil is converted into carboxylic acid using
a
catalyst, and the carboxylic acid and the catalyst are supplied to a
fermentation
vessel.
[0034] FIG. 3 schematically illustrates an exemplary method and system of the
present invention, in which a liquefied biomass is contacted with a catalyst
for
lipid hydrolysis before fermentation.
[0035] FIG. 4 schematically illustrates an exemplary method and system of the
present invention, in which a liquefied and saccharified biomass is contacted
with
a catalyst for lipid hydrolysis before fermentation.
[0036] FIG. 5 schematically illustrates an exemplary method and system of the
present invention, in which an amount of lipids and undissolved solids are
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removed from a liquefied biomass before fermentation, and in which the removed
lipids are converted into carboxylic acid using a catalyst, and the carboxylic
acid
and the catalyst are supplied to the fermentation vessel.
[0037] FIG. 6 shows the aqueous and solvent phase concentrations of isobutanol
produced by fermentation with sucrose as a carbon source. Aqueous phase titer
(Panel A) is reported in g/L and the solvent phase species (isobutanol, Panel
B
and isobutanol as FABE, Panel C. Panel D is the total isobutanol in the
solvent
phase) in weight percent.
[0038] FIG. 7 shows the effective titer of isobutanol, g/L, over time.
Effective titer
in this example was calculated as described in the text, based on the initial
volume of aqueous fermentor broth after inoculation.
[0039] FIG. 8 shows the consumption of sugars, reported in glucose
equivalents,
over time.
DETAILED DESCRIPTION OF THE INVENTION
[0040] Unless defined otherwise, 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. In case of conflict, the present
application
including the definitions will control. Also, unless otherwise required by
context,
singular terms shall include pluralities and plural terms shall include the
singular.
All publications, patents, and other references mentioned herein are
incorporated
by reference in their entireties for all purposes.
[0041] Unless otherwise specified, when the following abbreviations are used
herein, they have the following meaning:
ADH alcohol dehydrogenase
ALS acetolactate synthase
AQ aqueous fraction
BuO-COFA butyl ester(s) of corn oil fatty acid(s)
CALB Candida antarctica lipase B
COFA corn oil fatty acid(s)
DDGS Dried Distillers' Grains with Solubles
DG diglyceride(s)
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DHAD dihydroxyacid dehydratase
FOR end of run
EtOH ethanol
EtO-COFA ethyl ester(s) of corn oil fatty acid(s)
FABE fatty acid butyl ester(s)
FAEE fatty acid ethyl ester(s)
FAME fatty acid methyl ester(s)
FFA free fatty acid(s)
FOA fluoro-orotic acid
HADH horse liver alcohol dehydrogenase
IBA isobutanol
i-BuOH isobutanol
i-BuO-COFA isobutyl ester(s) of corn oil fatty acid(s)
i-BuO-oleate iso-butyl oleate
i-PrOH isopropanol
i-PrO-COFA isopropyl ester(s) of corn oil fatty acid(s)
ISPR in situ product removal
KART ketol-acid reductoisomerase
KivD ketoisovalerate decarboxylase
MAG monoacylglyceride(s)
MeBOH 2-methyl-1 -butanol
MeBO-COFA 2-methyl-1 -butyl ester(s) of corn oil fatty acid(s)
MeOH methanol
MeO-COFA methyl ester(s) of corn oil fatty acid(s)
MG monoglyceride(s)
n-BuOH n-butanol
OA oleyl alcohol
ORG organic fraction
PenOH 1-pentanol
PenO-COFA 1-pentyl ester(s) of corn oil fatty acid(s)
PrOH 1-propanol
PrO-COFA 1-propyl ester(s) of corn oil fatty acid(s)
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SOFA soya oil fatty acids
SSF simultaneous saccharification and fermentation
t-BuOH tert-butyl alcohol
TG triglyceride(s)
3M3P 3-Me-3-pentanol
[0042] In order to further define this invention, the following terms and
definitions
are herein provided.
[0043] As used herein, the terms "comprises," "comprising," "includes,"
"including," "has," "having," "contains," or "containing," or any other
variation
thereof, will be understood to imply the inclusion of a stated integer or
group of
integers but not the exclusion of any other integer or group of integers. For
example, a composition, a mixture, a process, a method, an article, or an
apparatus that comprises a list of elements is not necessarily limited to only
those
elements but can include other elements not expressly listed or inherent to
such
composition, mixture, process, method, article, or apparatus. Further, unless
expressly stated to the contrary, "or" refers to an inclusive or and not to an
exclusive or. For example, a condition A or B is satisfied by any one of the
following: A is true (or present) and B is false (or not present), A is false
(or not
present) and B is true (or present), and both A and B are true (or present).
[0044] Also, the indefinite articles "a" and "an" preceding an element or
component of the invention are intended to be nonrestrictive regarding the
number of instances, that is, occurrences of the element or component.
Therefore "a" or "an" should be read to include one or at least one, and the
singular word form of the element or component also includes the plural unless
the number is obviously meant to be singular.
[0045] The term "invention" or "present invention" as used herein is a non-
limiting
term and is not intended to refer to any single embodiment of the particular
invention but encompasses all possible embodiments as described in the
application.
[0046] As used herein, the term "about" modifying the quantity of an
ingredient or
reactant of the invention employed refers to variation in the numerical
quantity
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that can occur, for example, through typical measuring and liquid handling
procedures used for making concentrates or solutions in the real world;
through
inadvertent error in these procedures; through differences in the manufacture,
source, or purity of the ingredients employed to make the compositions or to
carry out the methods; and the like. The term "about" also encompasses
amounts that differ due to different equilibrium conditions for a composition
resulting from a particular initial mixture. Whether or not modified by the
term
"about," the claims include equivalents to the quantities. In one embodiment,
the
term "about" means within 10% of the reported numerical value, alternatively
within 5% of the reported numerical value.
[0047] "Biomass" as used herein refers to a natural product containing
hydrolyzable polysaccharides that provide fermentable sugars including any
sugars and starch derived from natural resources such as corn, cane, wheat,
cellulosic or lignocellulosic material and materials comprising cellulose,
hemicellulose, lignin, starch, oligosaccharides, disaccharides and/or
monosaccharides, and mixtures thereof. Biomass may also comprise additional
components such as protein and/or lipids. Biomass may be derived from a single
source or biomass can comprise a mixture derived from more than one source.
For example, biomass may comprise a mixture of corn cobs and corn stover, or a
mixture of grass and leaves. Biomass includes, but is not limited to,
bioenergy
crops, agricultural residues, municipal solid waste, industrial solid waste,
sludge
from paper manufacture, yard waste, waste sugars, wood and forestry waste.
Examples of biomass include, but are not limited to, corn grain, corn cobs,
crop
residues such as corn husks, corn stover, grasses, wheat, rye, wheat straw,
barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane
bagasse, sorghum, sugar cane, soy, components obtained from milling of grains,
trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes,
vegetables, fruits, flowers, animal manure, and mixtures thereof. For example,
mash, juice, molasses, or hydrolysate may be formed from biomass by any
processing known in the art for processing the biomass for purposes of
fermentation such as by milling, treating, and/or liquefying and comprises
fermentable sugar and may comprise water. For example, cellulosic and/or
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lignocellulosic biomass may be processed to obtain a hydrolysate containing
fermentable sugars by any method known to one skilled in the art. Particularly
useful is a low ammonia pretreatment as disclosed U.S. Patent Application
Publication No. 2007/0031918A1, which is herein incorporated by reference.
Enzymatic saccharification of cellulosic and/or lignocellulosic biomass
typically
makes use of an enzyme consortium for breaking down cellulose and
hemicellulose to produce a hydrolysate containing sugars including glucose,
xylose, and arabinose. (Saccharification enzymes suitable for cellulosic
and/or
lignocellulosic biomass are reviewed in Lynd, et al. (Microbiol. Mol. Biol.
Rev.
66:506-577, 2002).
[0048] Mash, juice, molasses, or hydrolysate may include feedstock 12 and
feedstock slurry 16 as described herein. An aqueous feedstream may be derived
or formed from biomass by any processing known in the art for processing the
biomass for purposes of fermentation such as by milling, treating, and/or
liquefying and comprises fermentable carbon substrate (e.g., sugar) and may
comprise water. An aqueous feedstream may include feedstock 12 and
feedstock slurry 16 as described herein.
[0049] "Biomass yield" as used herein refers to the grams of biomass produced
(i.e., cell biomass production) per gram of carbon substrate produced.
[0050] "Feedstock" as used herein means a feed in a fermentation process, the
feed containing a fermentable carbon source with or without undissolved
solids,
and where applicable, the feed containing the fermentable carbon source before
or after the fermentable carbon source has been liberated from starch or
obtained
from the break down of complex sugars by further processing such as by
liquefaction, saccharification, or other process. Feedstock includes or is
derived
from a biomass. Suitable feedstocks include, but are not limited to, rye,
wheat,
corn, corn mash, cane, cane mash, barley, cellulosic material, lignocellulosic
material, or mixtures thereof. Where reference is made to "feedstock oil," it
will
be appreciated that the term encompasses the oil produced from a given
feedstock.
[0051] "Fermentation medium" as used herein means the mixture of water,
sugars, dissolved solids, optionally microorganisms producing alcohol, product
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alcohol, and all other constituents of the material held in the fermentation
vessel
in which product alcohol is being made by the reaction of sugars to alcohol,
water, and carbon dioxide (C02) by the microorganisms present. From time to
time, as used herein the term "fermentation broth" and "fermented mixture" can
be used synonymously with "fermentation medium."
[0052] "Fermentable carbon source" or "fermentable carbon substrate" as used
herein means a carbon source capable of being metabolized by the
microorganisms disclosed herein for the production of fermentative alcohol.
Suitable fermentable carbon sources include, but are not limited to,
monosaccharides such as glucose or fructose; disaccharides such as lactose or
sucrose; oligosaccharides; polysaccharides such as starch or cellulose; C5
sugars such as xylose and arabinose; one carbon substrates including methane;
and mixtures thereof.
[0053] "Fermentable sugar" as used herein refers to one or more sugars capable
of being metabolized by the microorganisms disclosed herein for the production
of fermentative alcohol.
[0054] "Fermentation vessel" as used herein means the vessel in which the
fermentation reaction is carried out whereby product alcohol such as butanol
is
made from sugars.
[0055] "Liquefaction vessel" as used herein means the vessel in which
liquefaction is carried out. Liquefaction is the process in which
oligosaccharides
are liberated from the feedstock. In some embodiments where the feedstock is
corn, oligosaccharides are liberated from the corn starch content during
liquefaction.
[0056] "Saccharification vessel" as used herein means the vessel in which
saccharification (i.e., the break down of oligosaccharides into
monosaccharides)
is carried out. Where fermentation and saccharification occur simultaneously,
the
saccharification vessel and the fermentation vessel may be one in the same
vessel.
[0057] "Sugar" as used herein refers to oligosaccharides, disaccharides,
monosaccharides, and/or mixtures thereof. The term "saccharide" also includes
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carbohydrates including starches, dextrans, glycogens, cellulose, pentosans,
as
well as sugars.
[0058] As used herein, "saccharification enzyme" means one or more enzymes
that are capable of hydrolyzing polysaccharides and/or oligosaccharides, for
example, alpha-1,4-glucosidic bonds of glycogen, or starch. Saccharification
enzymes may include enzymes capable of hydrolyzing cellulosic or
lignocellulosic
materials as well.
[0059] "Undissolved solids" as used herein means non-fermentable portions of
feedstock, for example, germ, fiber, and gluten. For example, the non-
fermentable portions of feedstock include the portion of feedstock that
remains as
solids and can absorb liquid from the fermentation broth.
[0060] Dried Distillers' Grains with Solubles (DDGS) as used herein refers to
a
co-product or by-product from a fermentation of a feedstock or biomass (e.g.,
fermentation of grain or grain mixture that produces a product alcohol). In
some
embodiments, DDGS may also refer to an animal feed product produced from a
process of making a product alcohol (e.g., butanol, isobutanol, etc.).
[0061] "Product alcohol" as used herein refers to any alcohol that can be
produced by a microorganism in a fermentation process that utilizes biomass as
a source of fermentable carbon substrate. Product alcohols include, but are
not
limited to, C, to C8 alkyl alcohols. In some embodiments, the product alcohols
are C2 to C8 alkyl alcohols. In other embodiments, the product alcohols are C2
to
C5 alkyl alcohols. It will be appreciated that Ci to C8 alkyl alcohols
include, but
are not limited to, methanol, ethanol, propanol, butanol, and pentanol.
Likewise
C2 to C8 alkyl alcohols include, but are not limited to, ethanol, propanol,
butanol,
and pentanol. "Alcohol" is also used herein with reference to a product
alcohol.
[0062] "Butanol" as used herein refers with specificity to the butanol isomers
1-
butanol (1-BuOH), 2-butanol (2-BuOH), tert-butanol (t-BuOH), and/or isobutanol
(iBuOH or i-BuOH or I-BUGH, also known as 2-methyl -1-propanol), either
individually or as mixtures thereof. From time to time, when referring to
esters of
butanol, the terms "butyl esters" and "butanol esters" may be used
interchangeably.
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[0063] "Propanol" as used herein refers to the propanol isomers isopropanol or
1-
propanol.
[0064] "Pentanol" as used herein refers to the pentanol isomers 1-pentanol, 3-
methyl-1-butanol, 2-methyl-1-butanol, 2,2-dimethyl-1-propanol, 3-pentanol, 2-
pentanol, 3-methyl-2-butanol, or 2-methyl-2-butanol.
[0065] The term "alcohol equivalent" as used herein refers to the weight of
alcohol that would be obtained by a perfect hydrolysis of an alcohol ester and
the
subsequent recovery of the alcohol from an amount of alcohol ester.
[0066] The term "aqueous phase titer" as used herein refers to the
concentration
of a particular alcohol (e.g., butanol) in the fermentation broth.
[0067] The term "effective titer" as used herein refers to the total amount of
a
particular alcohol (e.g., butanol) produced by fermentation or alcohol
equivalent
of the alcohol ester produced by alcohol esterification per liter of
fermentation
medium. For example, the effective titer of butanol in a unit volume of a
fermentation includes: (i) the amount of butanol in the fermentation medium;
(ii)
the amount of butanol recovered from the organic extractant; (iii) the amount
of
butanol recovered from the gas phase, if gas stripping is used; and (iv) the
alcohol equivalent of the butyl ester in either the organic or aqueous phase.
[0068] The term "effective rate" as used herein is the effective titer divided
by the
fermentation time.
[0069] The term "effective yield" as used herein is the total grams of product
alcohol produced per gram of glucose consumed.
[0070] "In Situ Product Removal (ISPR)" as used herein means the selective
removal of a specific fermentation product from a biological process such as
fermentation, to control the product concentration in the biological process
as the
product is produced.
[0071] "Extractant" or "ISPR extractant" as used herein means an organic
solvent
used to extract any product alcohol such as butanol or used to extract any
product alcohol ester produced by a catalyst from a product alcohol and a
carboxylic acid or lipid. From time to time, as used herein the term "solvent"
may
be used synonymously with "extractant." For the processes described herein,
extractants are water-immiscible.
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[0072] The terms "water-immiscible" or "insoluble" refer to a chemical
component
such as an extractant or solvent, which is incapable of mixing with an aqueous
solution such as a fermentation broth, in such a manner as to form one liquid
phase.
[0073] The term "aqueous phase" as used herein refers to the aqueous phase of
a biphasic mixture obtained by contacting a fermentation broth with a water-
immiscible organic extractant. In an embodiment of a process described herein
that includes fermentative extraction, the term "fermentation broth" then
specifically refers to the aqueous phase in biphasic fermentative extraction.
[0074] The term "organic phase" as used herein refers to the non-aqueous phase
of a biphasic mixture obtained by contacting a fermentation broth with a water-
immiscible organic extractant.
[0075] The term "carboxylic acid" as used herein refers to any organic
compound
with the general chemical formula -000H in which a carbon atom is bonded to
an oxygen atom by a double bond to make a carbonyl group (-C=O) and to a
hydroxyl group (-OH) by a single bond. A carboxylic acid may be in the form of
the protonated carboxylic acid, in the form of a salt of a carboxylic acid
(e.g., an
ammonium, sodium, or potassium salt), or as a mixture of protonated carboxylic
acid and salt of a carboxylic acid. The term carboxylic acid may describe a
single
chemical species (e.g., oleic acid) or a mixture of carboxylic acids as can be
produced, for example, by the hydrolysis of biomass-derived fatty acid esters
or
triglycerides, diglycerides, monoglycerides, and phospholipids.
[0076] The term "fatty acid" as used herein refers to a carboxylic acid (e.g.,
aliphatic monocarboxylic acid) having C4 to C28 carbon atoms (most commonly
C12 to C24 carbon atoms), which is either saturated or unsaturated. Fatty
acids
may also be branched or unbranched. Fatty acids may be derived from, or
contained in esterified form, in an animal or vegetable fat, oil, or wax.
Fatty acids
may occur naturally in the form of glycerides in fats and fatty oils or may be
obtained by hydrolysis of fats or by synthesis. The term fatty acid may
describe a
single chemical species or a mixture of fatty acids. In addition, the term
fatty acid
also encompasses free fatty acids.
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[0077] The term "fatty alcohol" as used herein refers to an alcohol having an
aliphatic chain of C4 to C22 carbon atoms, which is either saturated or
unsaturated.
[0078] The term "fatty aldehyde" as used herein refers to an aldehyde having
an
aliphatic chain of C4 to C22 carbon atoms, which is either saturated or
unsaturated.
[0079] The term "fatty amide" as used herein refers to an amide having a long,
aliphatic chain of C4 to C22 carbon atoms, which is either saturated or
unsaturated
[0080] The term "fatty ester" as used herein refers to an ester having a long
aliphatic chain of C4 to C22 carbon atoms, which is either saturated or
unsaturated.
[0081] "Native oil" as used herein refers to lipids obtained from plants
(e.g., biomass) or animals. "Plant-derived oil" as used herein refers to
lipids
obtain from plants in particular. From time to time, "lipids" may be used
synonymously with "oil" and "acyl glycerides." Native oils include, but are
not
limited to, tallow, corn, canola, capric/caprylic triglycerides, castor,
coconut,
cottonseed, fish, jojoba, lard, linseed, neetsfoot, oiticica, palm, peanut,
rapeseed,
rice, safflower, soya, sunflower, tung, jatropha, and vegetable oil blends.
[0082] The term "separation" as used herein is synonymous with "recovery" and
refers to removing a chemical compound from an initial mixture to obtain the
compound in greater purity or at a higher concentration than the purity or
concentration of the compound in the initial mixture.
[0083] The term "butanol biosynthetic pathway" as used herein refers to an
enzyme pathway to produce 1-butanol, 2-butanol, or isobutanol.
[0084] The term "1-butanol biosynthetic pathway" as used herein refers to an
enzyme pathway to produce 1 -butanol from acetyl-coenzyme A (acetyl-CoA).
[0085] The term "2-butanol biosynthetic pathway" as used herein refers to an
enzyme pathway to produce 2-butanol from pyruvate.
[0086] The term "isobutanol biosynthetic pathway" as used herein refers to an
enzyme pathway to produce isobutanol from pyruvate.
[0087] The term "gene" refers to a nucleic acid fragment that is capable of
being
expressed as a specific protein, optionally including regulatory sequences
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preceding (5' non-coding sequences) and following (3' non-coding sequences)
the coding sequence. "Native gene" refers to a gene as found in nature with
its
own regulatory sequences. "Chimeric gene" refers to any gene that is not a
native gene (i.e., it is modified from its native state or is from another
source)
comprising regulatory and coding sequences that are not found together in
nature. Accordingly, a chimeric gene may comprise regulatory sequences and
coding sequences that are derived from different sources or regulatory
sequences and coding sequences derived from the same source, but arranged in
a manner different than that found in nature. "Endogenous gene" refers to a
native gene in its natural location in the genome of an organism. A "foreign
gene" or "heterologous gene" refers to a gene not normally found as a native
gene in the host organism, but that is introduced into the host organism by
gene
transfer. Foreign genes can comprise native genes inserted into a non-native
organism or chimeric genes.
[0088] As used herein the term "coding region" refers to a DNA sequence that
codes for a specific amino acid sequence. "Suitable regulatory sequences"
refer
to nucleotide sequences located upstream (5' non-coding sequences), within, or
downstream (3' non-coding sequences) of a coding sequence, and which
influence the transcription, RNA processing or stability, or translation of
the
associated coding sequence. Regulatory sequences may include promoters,
translation leader sequences, introns, polyadenylation recognition sequences,
RNA processing site, effector binding site, and stem-loop structure.
[0089] The term "codon-optimized" as it refers to genes or coding regions of
nucleic acid molecules for transformation of various hosts, refers to the
alteration
of codons in the gene or coding regions of the nucleic acid molecules to
reflect
the typical codon usage of the host organism without altering the polypeptide
encoded by the DNA. Codon optimization is within the ordinary skill in the
art.
[0090] The term "polynucleotide" is intended to encompass a singular nucleic
acid
as well as plural nucleic acids, and refers to a nucleic acid molecule or
construct,
for example, messenger RNA (mRNA) or plasmid DNA (pDNA). As used herein,
a "gene" is a polynucleotide. A polynucleotide can contain the nucleotide
sequence of the full-length cDNA sequence or a fragment thereof, including the
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untranslated 5' and 3' sequences and the coding sequences. The polynucleotide
can be composed of any polyribonucleotide or polydeoxyribonucleotide, which
may be unmodified RNA or DNA or modified RNA or DNA (e.g., heterologous
DNA). For example, polynucleotides can be composed of single- and double-
stranded DNA, DNA that is a mixture of single- and double-stranded regions,
single- and double-stranded RNA, and RNA that is mixture of single- and double-
stranded regions, hybrid molecules comprising DNA and RNA that may be single-
stranded or, more typically, double-stranded or a mixture of single- and
double-
stranded regions. "Polynucleotide" embraces chemically, enzymatically, or
metabolically modified forms.
[0091] A polynucleotide sequence may be referred to as "isolated," in which it
has
been removed from its native environment. For example, a heterologous
polynucleotide encoding a polypeptide or polypeptide fragment having dihydroxy-
acid dehydratase activity contained in a vector is considered isolated for the
purposes of the present invention. Further examples of an isolated
polynucleotide include recombinant polynucleotides maintained in heterologous
host cells or purified (partially or substantially) polynucleotides in
solution.
Isolated polynucleotides or nucleic acids according to the present invention
further include such molecules produced synthetically. An isolated
polynucleotide fragment in the form of a polymer of DNA may be comprised of
one or more segments of cDNA, genomic DNA, or synthetic DNA.
[0092] As used herein, the term "polypeptide" is intended to encompass a
singular "polypeptide" as well as plural "polypeptides," and refers to a
molecule
composed of monomers (amino acids) linearly linked by amide bonds (also
known as peptide bonds). The term "polypeptide" refers to any chain or chains
of
two or more amino acids, and does not refer to a specific length of the
product.
Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid
chain," or any other term used to refer to a chain or chains of two or more
amino
acids, are included within the definition of "polypeptide," and the term
"polypeptide" may be used instead of, or interchangeably with any of these
terms.
A polypeptide may be derived from a natural biological source or produced by
recombinant technology, but is not necessarily translated from a designated
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nucleic acid sequence. It may be generated in any manner including by chemical
synthesis.
[0093] By an "isolated" polypeptide or a fragment, variant, or derivative
thereof is
intended a polypeptide that is not in its natural milieu. No particular level
of
purification is required. For example, an isolated polypeptide can be removed
from its native or natural environment. Recombinantly produced polypeptides
and proteins expressed in host cells are considered isolated for purposes of
the
invention, as are native or recombinant polypeptides which have been
separated,
fractionated, or partially or substantially purified by any suitable
technique.
[0094] As used herein, "recombinant microorganism" refers to microorganisms
such as bacteria or yeast, that are modified by use of recombinant DNA
techniques, for example, by engineering a host cell to comprise a biosynthetic
pathway such as a biosynthetic pathway to produce an alcohol such as butanol.
[0095] The present invention satisfies the need for alternative extractive
fermentation methods which do not necessitate the partitioning of the product
alcohol between the fermentation medium and the ISPR extractant as a means to
reduce the toxic effect of the product alcohol (such as butanol) on the
microorganism. It also satisfies the need to reduce the degradation of the
partition coefficient of a fermentation product ISPR extractant by providing
methods for producing alcohol such as butanol in which the product alcohol is
converted into alcohol esters which can be less toxic to the microorganism and
where there is realized a concomitant reduction in the degradation of the
partition
coefficient of a fermentation product extractant, resulting in improved
production
yields of alcohol (as a combination of free alcohol and alcohol esters that
can be
converted back to alcohol after separation from the fermentation medium).
Moreover, the present invention offers solutions to disadvantages of
alternative
alcohol product removal processes such that the methods herein can be
combined with existing processes (e.g., solids removal) to provide increased
product removal at economic and environmental advantage. As such, the
present invention provides further related advantages, as will be made
apparent
by the description of the embodiments that follow.
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[0096] The present invention provides methods for removing alcohol from a
fermentation medium by esterifying the alcohol with carboxylic acid and
extracting the resulting alcohol ester from the fermentation medium,
whereafter
the alcohol can be recovered from the alcohol ester. The acid may be added to
the fermentation medium directly as free fatty acid or may be derived from
oil.
The present invention also provides methods for removing or reducing oil from
an
alcohol fermentation process by hydrolyzing the oil derived from a feedstock
into
carboxylic acid which can be used for the esterification of alcohol and/or
serve as
an ISPR extractant or a component of the ISPR extractant for extracting the
alcohol ester.
[0097] Decreasing the amount of water present in a reaction system, or
employing a reaction system that uses only one or more non-aqueous solvents,
has typically been necessary for esterification of alcohols by carboxylic
acids
when catalyzed by enzymes such as lipases. Described herein is the surprising
finding that lipase enzymes can efficiently catalyze the esterification of a
product
alcohol with a carboxylic acid during fermentation of a fermentable carbon
source
to produce product alcohol. Also described herein is the surprising finding
that
esterification of a product alcohol with a carboxylic acid during a
fermentation can
provide improvements in the fermentation performance. For example, by
capturing the product alcohol (e.g., butanol) as produced in ester form it
effectively reduces the concentration of the product alcohol in the aqueous
phase
and thus, mitigates the toxic effects of the product alcohol on glucose
consumption and product production.
[0098] The present invention will be described with reference to the Figures.
FIG. 1 illustrates an exemplary process flow diagram for production of
fermentative alcohol such as ethanol or butanol, according to an embodiment of
the present invention. As shown, a feedstock 12 can be introduced to an inlet
in
a liquefaction vessel 10 and liquefied to produce a feedstock slurry 16.
Feedstock 12 contains hydrolysable polysaccharides that supplies a fermentable
carbon substrate (e.g., fermentable sugar such as glucose), and can be a
biomass such as, but not limited to, rye, wheat, cane or corn, or can
otherwise be
derived from a biomass. In some embodiments, feedstock 12 can be one or
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more components of a fractionated biomass, and in other embodiments,
feedstock 12 can be a milled, unfractionated biomass. In some embodiments,
feedstock 12 can be corn, such as dry milled, unfractionated corn kernels, and
the undissolved particles can include germ, fiber, and gluten. The undissolved
solids are non-fermentable portions of feedstock 12. For purposes of the
discussion herein with reference to the embodiments shown in the Figures,
feedstock 12 will often be described as constituting milled, unfractionated
corn in
which the undissolved solids have not been separated therefrom. However, it
should be understood that the exemplary methods and systems described herein
can be modified for different feedstocks whether fractionated or not, as
apparent
to one of skill in the art. Furthermore, as one skilled in the art can
appreciate,
maximizing feedstock content (e.g., corn content) can maximize sugar content
as
well as product titer. In some embodiments, feedstock 12 can be high-oleic
corn,
such that corn oil derived therefrom is a high-oleic corn oil having an oleic
acid
content of at least about 55 wt% oleic acid. In some embodiments, the oleic
acid
content in high-oleic corn oil can be up to about 65 wt%, as compared with the
oleic acid content in normal corn oil which is about 24 wt%. High-oleic oil
can
provide some advantages for use in the methods of the present invention, as
hydrolysis of the oil provides free fatty acids having a high oleic acid
content for
contacting with a fermentation broth.
[0099] The process of liquefying feedstock 12 involves hydrolysis of
polysaccharides in feedstock 12 into sugars including, for example, dextrins
and
oligosaccharides, and is a conventional process. Any known liquefying
processes as well as the corresponding liquefaction vessel, normally utilized
by
the industry can be used including, but not limited to, the acid process, the
acid-
enzyme process, or the enzyme process. Such processes can be used alone or
in combination. In some embodiments, the enzyme process can be utilized and
an appropriate enzyme 14, for example, alpha-amylase, is introduced to an
inlet
in liquefaction vessel 10. Water can also be introduced to liquefaction vessel
10.
In some embodiments, a saccharification enzyme, for example, glucoamylase,
may also be introduced to liquefaction vessel 10. In additional embodiments, a
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lipase may also be introduced to liquefaction vessel 10 to catalyze the
conversion
of one or more components of the oil to free fatty acids.
[00100] Feedstock slurry 16 produced from liquefying feedstock 12 comprises
fermentable carbon substrate (e.g., sugar), oil, and undissolved solids
derived
from the feedstock. Feedstock slurry 16 can be discharged from an outlet of
liquefaction vessel 10. In some embodiments, feedstock 12 is corn or corn
kernels and therefore, feedstock slurry 16 is a corn mash slurry. In some
embodiments, feedstock 12 is a lignocellulosic feedstock and therefore,
feedstock slurry 16 may be a lignocellulosic hydrolysate. In some embodiments,
feedstock 12 is sugar cane.
[00101] Feedstock slurry 16 is introduced into a fermentation vessel 30 along
with
a microorganism 32. Fermentation vessel 30 is configured to ferment slurry 16
to
produce alcohol. In particular, microorganism 32 metabolizes the fermentable
sugar in slurry 16 and excretes a product alcohol. Microorganism 32 is
selected
from the group of bacteria, cyanobacteria, filamentous fungi, and yeasts. In
some embodiments, microorganism 32 can be a bacteria such as E. coli. In
some embodiments, microorganism 32 can be a fermentative recombinant
microorganism. The slurry can include sugar, for example, in the form of
oligosaccharides, and water, and in some embodiments, can comprise less than
about 20 g/L of monomeric glucose, less than about 10 g/L, or less than about
g/L of monomeric glucose. Suitable methodology to determine the amount of
monomeric glucose is well known in the art. Such suitable methods known in the
art include HPLC.
[00102] In some embodiments, slurry 16 is subjected to a saccharification
process
in order to break the complex sugars (e.g., oligosaccharides) in slurry 16
into
monosaccharides that can be readily metabolized by microorganism 32. Any
known saccharification process, normally utilized by the industry can be used
including, but not limited to, the acid process, the acid-enzyme process, or
the
enzyme process. In some embodiments, simultaneous saccharification and
fermentation (SSF) can occur inside fermentation vessel 30 as shown in FIG. 1.
In some embodiments, an enzyme 38 such as glucoamylase, can be introduced
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to an inlet in fermentation vessel 30 in order to breakdown the starch or
oligosaccharides to glucose capable of being metabolized by microorganism 32.
[00103] Carboxylic acid 28 and/or native oil 26 are introduced into
fermentation
vessel 30, along with a catalyst 42. Catalyst 42 can be introduced before,
after,
or contemporaneously with enzyme 38. Thus, in some embodiments, addition of
enzyme 38 and catalyst 42 can be stepwise (e.g., catalyst 42, then enzyme 38,
or
vice versa) or substantially simultaneous (i.e., at exactly the same time as
in the
time it takes for a person or a machine to perform the addition in one stroke,
or
one enzyme/catalyst immediately following the other catalyst/enzyme as in the
time it takes for a person or a machine to perform the addition in two
strokes).
Catalyst 42 is capable of esterifying the product alcohol with carboxylic acid
28 to
form an alcohol ester. For example, in the case of butanol production,
catalyst 42
is capable of esterifying butanol with carboxylic acid 28 to form a butyl
ester.
[00104] In the instance that native oil 26 is supplied to fermentation vessel
30, at
least a portion of the acyl glycerides in oil 26 can be hydrolyzed to
carboxylic acid
28 by contacting oil 26 with catalyst 42. The resulting acid/oil composition
from
hydrolyzing oil 26 is typically at least about 17 wt% carboxylic acid 28 (as
free
fatty acids). In some embodiments, the resulting acid/oil composition from
hydrolyzing oil 26 is at least about 20 wt% carboxylic acid, at least about 25
wt%
carboxylic acid, at least about 30 wt% carboxylic acid, at least about 35 wt%
carboxylic acid, at least about 40 wt% carboxylic acid, at least about 45 wt%
carboxylic acid, at least about 50 wt% carboxylic acid, at least about 55 wt%
carboxylic acid, at least about 60 wt% carboxylic acid, at least about 65 wt%
carboxylic acid, at least about 70 wt% carboxylic acid, at least about 75 wt%
carboxylic acid, at least about 80 wt% carboxylic acid, at least about 85 wt%
carboxylic acid, at least about 90 wt% carboxylic acid, at least about 95 wt%
carboxylic acid, or at least about 99 wt% carboxylic acid. In some
embodiments,
the resulting acid/oil composition includes monoglycerides and/or diglycerides
from the partial hydrolysis of the acyl glycerides in the oil. In some
embodiments,
the resulting acid/oil composition includes glycerol, a by-product of acyl
glyceride
hydrolysis. In some additional embodiments, the resulting acid/oil composition
includes lysophospholipids from the partial hydrolysis of phospholipids in the
oil.
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[00105] In some embodiments, after hydrolysis of the acyl glycerides in oil
26, the
remaining acyl glycerides from oil 26 are from about 0 wt% to at least about
2 wt% of the fermentation broth composition. In some additional embodiments,
after hydrolysis of the acyl glycerides in oil 26, the remaining acyl
glycerides from
oil 26 are at least about 0.5 wt% of the fermentation broth composition. Thus,
in
some embodiments, the acyl glycerides from oil 26 can be catalytically
hydrolyzed to carboxylic acid 28 using catalyst 42, and catalyst 42 can also
esterify carboxylic acid 28 with the product alcohol. In some embodiments, a
second catalyst (not shown) can be introduced to the fermentation vessel for
hydrolysis of the acyl glycerides. In addition, the acyl glycerides in the oil
derived
from feedstock 12 and present in slurry 16 can also be hydrolyzed to
carboxylic
acid 28' (see, e.g., the embodiment of FIG. 3). In some embodiments, the
concentration of the carboxylic acid (such as fatty acid) in the fermentation
vessel
exceeds the solubility limit in the aqueous phase and results in the
production a
two-phase fermentation mixture comprising an organic phase and an aqueous
phase. In some embodiments, the concentration of carboxylic acids in the
fermentation broth is typically not greater than about 0.8 g/L and is limited
by the
solubility of the carboxylic acid in the broth.
[00106] In some embodiments, catalyst 42 and the second catalyst, if used, can
be
one or more enzymes, for example, lipase enzymes. In some embodiments,
catalyst 42 can be one or more enzymes, for example, hydrolase enzymes such
as lipase enzymes. Lipase enzymes used may be derived from any source
including, for example, Absidia, Achromobacter, Aeromonas, Alcaligenes,
Alternaria, Aspergillus, Achromobacter, Aureobasidium, Bacillus, Beauveria,
Brochothrix, Candida, Chromobacter, Coprinus, Fusarium, Geotricum,
Hansenula, Humicola, Hyphozyma, Lactobacillus, Metarhizium, Mucor, Nectria,
Neurospora, Paecilomyces, Penicillium, Pseudomonas, Rhizoctonia,
Rhizomucor, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Sus,
Sporobolomyces, Thermomyces, Thiarosporella, Trichoderma, Verticillium,
and/or a strain of Yarrowia. In a preferred aspect, the source of the lipase
is
selected from the group consisting of Absidia blakesleena, Absidia
corymbifera,
Achromobacter iophagus, Alcaligenes sp., Alternaria brassiciola, Aspergillus
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flavus, Aspergillus niger, Aspergillus tubingensis, Aureobasidium pullulans,
Bacillus pumilus, Bacillus strearothermophilus, Bacillus subtilis, Brochothrix
thermosohata, Candida cylindracea (Candida rugosa), Candida paralipolytica,
Candida antarctica lipase A, Candida antarctica lipase B, Candida ernobii,
Candida deformans, Chromobacter viscosum, Coprinus cinerius, Fusarium
oxysporum, Fusarium solani, Fusarium solani pisi, Fusarium roseum culmorum,
Geotricum penicillatum, Hansenula anomala, Humicola brevispora, Humicola
brevis var. thermoidea, Humicola insolens, Lactobacillus curvatus, Rhizopus
oryzae, Penicillium cyclopium, Penicillium crustosum, Penicillium expansum,
Penicillium sp. I, Penicillium sp. II, Pseudomonas aeruginosa, Pseudomonas
alcaligenes, Pseudomonas cepacia (syn. Burkholderia cepacia), Pseudomonas
fluorescens, Pseudomonas fragi, Pseudomonas maltophilia, Pseudomonas
mendocina, Pseudomonas mephitica lipolytica, Pseudomonas alcaligenes,
Pseudomonas plantari, Pseudomonas pseudoalcaligenes, Pseudomonas putida,
Pseudomonas stutzeri, and Pseudomonas wisconsinensis, Rhizoctonia solani,
Rhizomucor miehei, Rhizopus japonicus, Rhizopus microsporus, Rhizopus
nodosus, Rhodosporidium toruloides, Rhodotorula glutinis, Saccharomyces
cerevisiae, Sporobolomyces shibatanus, Sus scrofa, Thermomyces lanuginosus
(formerly Humicola lanuginose), Thiarosporella phaseolina, Trichoderma
harzianum, Trichoderma reesei, and Yarrowia lipolytica. In a further preferred
aspect, the lipase is selected from the group consisting of Thermomcyces
lanuginosus lipase, Aspergillus sp. lipase, Aspergillus niger lipase,
Aspergillus
tubingensis lipase, Candida antarctica lipase B, Pseudomonas sp. lipase,
Penicillium roqueforti lipase, Penicillium camembertii lipase, Mucor javanicus
lipase, Burkholderia cepacia lipase, Alcaligenes sp. lipase, Candida rugosa
lipase, Candida parapsilosis lipase, Candida deformans lipase, lipases A and B
from Geotrichum candidum, Neurospora crassa lipase, Nectria haematococca
lipase, Fusarium heterosporum lipase Rhizopus delemar lipase, Rhizomucor
miehei lipase, Rhizopus arrhizus lipase, and Rhizopus oryzae lipase. Suitable
commercial lipase preparations suitable as catalyst 42 include, but are not
limited
to, Lipolase 100 L, Lipex 100L, Lipoclean 2000T, Lipozyme CALB L,
Novozyme CALA L, and Palatase 20000L, available from Novozymes, or from
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Pseudomonas fluorescens, Pseudomonas cepacia, Mucor miehei, hog pancreas,
Candida cylindracea, Candida rugosa, Rhizopus niveus, Candida antarctica,
Rhizopus arrhizus orAspergillus available from SigmaAldrich.
[00107] Phospholipases are enzymes that hydrolyze the ester bonds of
phospholipids, but many phospholipases also can hydrolyze triglycerides,
diglycerides, and monoglycerides (lipid acyl hydrolase (LAH) activity). As
used
herein, the term "phospholipase" encompasses enzymes having any
phospholipase activity, for example, cleaving a glycerol phosphate ester
linkage
(catalyzing hydrolysis of a glycerol phosphate ester linkage), for example, in
an
oil, such as a crude oil or a vegetable oil. The phospholipase activity of the
invention can generate a water extractable phosphorylated base and a
diglyceride. The phospholipase activity can comprise a phospholipase C (PLC)
activity; a PI-PLC activity, a phospholipase A (PLA) activity such as a
phospholipase Al or phospholipase A2 activity; a phospholipase B (PLB)
activity
such as a phospholipase B1 or phospholipase B2 activity, including
lysophospholipase (LPL) activity and/or lysophospholipase- transacylase (LPT
A)
activity; a phospholipase D (PLD) activity such as a phospholipase DI or a
phospholipase D2 activity; and/or a patatin activity or any combination
thereof.
[00108] The term "phospholipase" also encompasses enzymes having
lysophospholipase activity, where the two substrates of this enzyme are 2-
lysophosphatidylcholine and H2O, and where its two products are
glycerophosphocholine and carboxylate. Phospholipase Al (PLA1) enzymes
remove the 1-position fatty acid to produce free fatty acid and 1-lyso-2-
acylphospholipid. Phospholipase A2 (PLA2) enzymes remove the 2-position fatty
acid to produce free fatty acid and I-acyl-2-lysophospholipid. PLA1 and PLA2
enzymes can be intra- or extra-cellular, membrane-bound or soluble.
Phospholipase C (PLC) enzymes remove the phosphate moiety to produce 1 ,2
diacylglycerol and a phosphate ester. Phospholipase D (PLD) enzymes produce
1 ,2-diacylglycerophosphate and base group. A phospholipase useful in the
present invention may be obtained from a variety of biological sources, for
example, but not limited to, filamentous fungal species within the genus
Fusarium, such as a strain of F. culmorum, F. heterosporum, F. solani, or F.
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oxysporum; or a filamentous fungal species within the genus Aspergillus, such
as
a strain of Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,
Aspergillus niger or Aspergillus oryzae. Also useful in the present invention
are
Thermomyces lanuginosus phospholipase variants such as the commercial
product Lecitase Ultra (Novozymes A'S, Denmark). One or more
phospholipases may be applied as lyophilized powder, immobilized or in aqueous
solution.
[00109] An alcohol (e.g., butanol) that is produced by fermentation of one or
more
fermentable sugars may be converted to a carboxylic acid ester by an enzyme-
catalyzed reaction where the carboxylic acid is esterified with the alcohol.
Enzymes such as lipase, phospholipase, and lysophospholipase may catalyzed
this reaction; however, these enzymes may be inactivated due to one or more
factors including, but not limited to, hydrodynamic shear or inactivation at
gas-
liquid and liquid-liquid interfaces. In fermentations where oligosaccharides
are
additionally converted to one or more fermentable sugars, the enzyme that
converts oligosaccharides to fermentable sugars (e.g., glucoamylase) may also
be inactivated by one or more of these same factors.
[00110] Inactivation of enzymes at a gas-liquid interface (e.g., may occur at
the
interface of bubbles with the fermentation broth) that results from aeration
of the
fermentation broth and/or is produced by the evolution of gaseous carbon
dioxide
in the broth during fermentation of one or more fermentable sugars, is well-
known
in the art. Inactivation of Hen egg white lysozyme and Thermomyces
lanuginosus lipase produced in Aspergillus oryzae (Novozymes Lipolase ) was
observed at the gas-liquid interface in three different reactor
configurations:
bubble column, stirred vessel with baffles (with no aeration by gas sparging),
and
falling film (Ghadge, et al., Chem. Eng. Sci. 58:5125-5134, 2003). The
mechanism of inactivation of Thermomyces lanuginosus lipase (produced in
Aspergillus oryzae; Novozymes Lipolase 100L ) at the gas-liquid interface in a
baffled stirred-tank reactor (with no aeration by gas sparging) has been
reported
(Patil, et al., AIChE J. 46:1280-1283, 2000).
[00111] Stahmann, et al. (Eur. J. Biochem. 244:220-225, 1997) have reported
that
Ashbya gossypii lipase was inactivated within minutes in stirred gas/water,
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trioleoylglycerol/water or oleic acid/water mixtures, due to interfacial
inactivation
at either a gas/liquid or liquid/liquid interface. Elias, et al. (Adv.
Biochem.
Engineering/Biotechnology 59:47-71, 1998) have reported that: (i) some enzymes
are inactivated by hydrodynamic shear even in the absence of a gas-liquid
interface; (ii) for enzymes that are inactivated by hydrodynamic shear, the
rate of
inactivation increases in the presence of gas-liquid interface; (iii) some
enzymes
are not inactivated in the absence of gas-liquid interface regardless of the
applied hydrodynamic shear; and (iv) for enzymes that require a gas-liquid
interface for inactivation, the rate of inactivation increases with an
increase in
hydrodynamic shear. Ross, et al. (J. Mol. Catal. B: Enzymatic 8:183-192, 2000)
have described the interfacial inactivation of a-chymotrypsin, R-chymotrypsin,
papain, and pig liver esterase in a variety of aqueous/organic solvent
mixtures by
passing solvent droplets up through an aqueous enzyme solution in a bubble
column apparatus. The kinetics and mechanism of shear inactivation of Candida
cylindracea lipase in a stirred tank reactor has also been reported, where the
mechanism of inactivation was found to be due to a shear-induced gas-liquid
interface effect (Lee, et al., Biotechnol. Bioeng. 33:183-190, 1989).
[00112] Under the fermentation conditions employed in some methods described
herein, hydrodynamic shear and gas-liquid and liquid-liquid interfaces are
each
present over the course of the fermentation, and capable of causing enzyme
inactivation. The potential effect of each of these factors on the stability
and
activity of one or more of the enzymes (e.g., glucoamylase, lipase,
phospholipase, and lysophospholipase) present in the two-phase mixture (e.g.,
fermentation broth and carboxylic acid) during fermentation under the
conditions
described herein could not have been anticipated based on the prior art.
Although each of these factors could have resulted in the inactivation of one
or
more enzymes in the fermentation mixture, sufficient enzyme activity to
catalyze
esterification of the product alcohol by carboxylic acid to produce carboxylic
acid
esters was maintained over the course of the fermentation. In reactions where
glucoamylase was also present in the two-phase fermentation mixture of
fermentation broth and carboxylic acid, sufficient enzyme activity (i.e., to
convert
oligosaccharide to fermentable sugars) was also maintained.
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[00113] Carboxylic acid 28 can be any carboxylic acid capable of esterifying
with a
product alcohol such as butanol or ethanol, to produce an alcohol ester of the
carboxylic acid. For example, in some embodiments, carboxylic acid 28 can be
free fatty acid, and in some embodiments, the carboxylic acid or free fatty
acid
has 4 to 28 carbons, 4 to 22 carbons in other embodiments, 8 to 22 carbons in
other embodiments, 10 to 28 carbons in other embodiments, 7 to 22 carbons in
other embodiments, 12 to 22 carbons in other embodiments, 4 to 18 carbons in
other embodiments, 12 to 22 carbons in other embodiments, and 12 to 18
carbons in still other embodiments. In some embodiments, carboxylic acid 28 is
one or more of the following fatty acids: azaleic, capric, caprylic, castor,
coconut
(i.e., as a naturally-occurring combination of fatty acids including lauric,
myrisitic,
palmitic, caprylic, capric, stearic, caproic, arachidic, oleic, and linoleic,
for
example), isostearic, lauric, linseed, myristic, oleic, palm oil, palmitic,
palm kernel,
pelargonic, ricinoleic, sebacic, soya, stearic acid, tall oil, tallow, and #12
hydroxy
stearic. In some embodiments, carboxylic acid 28 is one or more of diacids.
[00114] In some embodiments, carboxylic acid 28 can be a mixture of two or
more
different fatty acids. In some embodiments, carboxylic acid 28 comprises free
fatty acid derived from hydrolysis of acyl glycerides by any method known in
the
art including chemical or enzymatic hydrolysis. In some embodiments as noted
above, carboxylic acid 28 can be derived from native oil 26 by enzymatic
hydrolysis of the oil glycerides using an enzyme as catalyst 42. In some
embodiments, the fatty acids or mixtures thereof comprise unsaturated fatty
acids. The presence of unsaturated fatty acids decreases the melting point,
providing advantages for handling. Of the unsaturated fatty acids, those which
are monounsaturated, that is, possessing a single carbon-carbon double bond,
may provide advantages with respect to melting point without sacrificing
suitable
thermal and oxidative stability for process considerations.
[00115] In some embodiments, native oil 26 can be tallow, corn, canola,
capric/caprylic triglycerides, castor, coconut, cottonseed, fish, jojoba,
lard,
linseed, neetsfoot, oiticica, palm, peanut, rapeseed, rice, safflower, soya,
sunflower, tung, jatropha, pumpkin, grape seed, and vegetable oil blends (or
oils
that can be purified into higher concentrations of different chain length and
levels
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of unsaturation (i.e., 18:1)). In some embodiments, native oil 26 is a mixture
of
two or more native oils such as a mixture of palm and soybean oils, for
example.
In some embodiments, native oil 26 is a plant-derived oil. In some
embodiments,
the plant-derived oil can be, though not necessarily, derived from biomass
that
can be used in a fermentation process. The biomass can be the same or
different source from which feedstock 12 is obtained. Thus, for example, in
some
embodiments, oil 26 can be derived from corn, whereas feedstock 12 can be
cane. For example, in some embodiments, oil 26 can be derived from corn, and
the biomass source of feedstock 12 is also corn. Any possible combination of
different biomass sources for oil 26 versus feedstock 12 can be used, as
should
be apparent to one of skill in the art. In some embodiments, oil 26 is derived
from
the biomass used in the fermentation process. Thus, in some embodiments, as
later described with reference to FIG. 3, oil 26 is derived directly from
feedstock
12 as oil 26'. For example, when feedstock 12 is corn, then oil 26' is the
feedstock's constituent corn oil.
[00116] Optionally, ethanol 33 may be supplied to fermentation vessel 30 to be
included in the fermentation broth. In some embodiments, when a recombinant
microorganism having a butanol biosynthetic pathway and/or reduced or
eliminated expression of pyruvate decarboxylase is used as microorganism 32,
microorganism 32 may require supplementation of a 2-carbon substrate, for
example, ethanol, for survival and growth. Thus, in some embodiments, ethanol
33 may be supplied to fermentation vessel 30.
[00117] However, it has been surprisingly found that methods of the present
invention, in which carboxylic acid such as fatty acid, is present in the
fermentation vessel, can allow reduction of the amount of ethanol 33 typically
supplied for a given recombinant microorganism without detriment to the
vitality
of the recombinant microorganism. Further, in some embodiments of the
methods of the present invention, the alcohol (e.g., butanol) production rate
without ethanol supplementation can be comparable with the production rate
that
can be realized when ethanol 33 is supplemented. As further demonstrated by
the comparative examples presented in the Examples 1-14 below, the butanol
production rate when fatty acid but not ethanol is in the fermentation vessel
can
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be comparable to or greater than the butanol production rate when neither
fatty
acid nor ethanol is in the fermentation vessel. Thus, in some embodiments, the
amount of ethanol 33 supplementation is reduced compared to conventional
processes. For example, a typical amount of ethanol added to a fermentation
vessel for microorganisms requiring supplementation of a 2-carbon substrate is
about 5 g/L anhydrous ethanol (i.e., 5 g anhydrous ethanol per liter of
fermentation medium). In some embodiments, the fermentation is not
supplemented with any ethanol 33. In the latter case, the stream of ethanol 33
is
entirely omitted from the fermentation vessel. Thus, in some embodiments of
the
present invention, it is possible to reduce or eliminate the cost associated
with
supplemental ethanol 33, as well as the inconvenience associated with storing
vats of ethanol 33 and supplying it to the fermentation vessel during butanol
fermentation or other alcohol fermentation that employs a microorganism that
may require supplementation of a 2-carbon substrate to survive and grow.
[00118] Moreover, regardless of ethanol supplementation, in some embodiments,
the methods of the present invention can provide a higher rate of glucose
uptake
by microorganism 32 by virtue of the presence of fatty acids during the
fermentation. The fatty acids can be introduced into fermentation vessel 30 as
carboxylic acid 28, hydrolyzed from supplied oil 26, and/or derived from
hydrolysis of constituent biomass oil of slurry 16. Methods for producing a
product alcohol from a fermentation process in which free fatty acids are
produced at a step in the process and are contacted with microorganism
cultures
in a fermentation vessel for improving microorganism growth rate and glucose
consumption are described in co-pending, commonly owned U.S. Provisional
Application Serial No. 61/368,451, filed on July 28, 2010, which is
incorporated
herein in its entirety by reference thereto.
[00119] In fermentation vessel 30, alcohol produced by microorganism 32 is
esterified with carboxylic acid 28 using catalyst 42 to form alcohol esters.
For
example, in the case of butanol production, butanol produced by microorganism
32 is esterified with carboxylic acid 28 using catalyst 42 to form butyl
esters. In
situ product removal (ISPR) can be utilized to remove the alcohol esters from
the
fermentation broth. As demonstrated herein, using catalyst to form esters in
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conjunction with ISPR can improve the performance of the fermentation. In some
embodiments, using catalyst to form esters in conjunction with ISPR (such as,
for
example, liquid-liquid extraction) can increase the effective titer by at
least about
10%, at least about 20%, at least about 30%, at least about 40%, at least
about
50%, at least about 60%, at least about 70%, at least about 80%, at least
about
90%, or at least about 100% as compared to the effective titer in an analogous
fermentation using ISPR without a catalyst forming esters. Similarly, in some
embodiments, using a catalyst to form esters in conjunction with ISPR (such
as,
for example, liquid-liquid extraction) can increase the effective rate by at
least
about 10%, at least about 20%, at least about 30%, at least about 40%, at
least
about 50%, at least about 60%, at least about 70%, at least about 80%, at
least
about 90%, or at least about 100% as compared to the effective rate in an
analogous fermentation using ISPR without a catalyst forming esters (see,
e.g.,
Examples 9 and 11-14, Table 3). In some embodiments, the effective yield is
increased by at least about 10%, at least about 20%, at least about 30%, at
least
about 40%, or at least about 50%. In some embodiments, the resulting
fermentation broth after alcohol esterification can comprise free (i.e.,
unesterified)
alcohol and in some embodiments, the concentration of free alcohol in the
fermentation broth after alcohol esterification is not greater than 1, 3, 6,
10, 15,
20, 25, 30 25, 40, 45, 50, 55, or 60 g/L when the product alcohol is butanol,
or
when the product alcohol is ethanol, the concentration of free alcohol in the
fermentation broth after alcohol esterification is not greater than 15, 20,
25, 30
25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 g/L. In some
embodiments, the ratio of alcohol ester to alcohol in the fermentation vessel
may
be about 1:1. In some embodiments, at least about 20%, at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least about 70%,
at
least about 80%, or at least about 90% of the effective titer of alcohol is
converted to alcohol ester.
[00120] In some embodiments, a grain load on water at a sufficient
concentration
to achieve a final effective titer of at least about 50 g/L, at least about 75
g/L, or at
least about 100 g/L may be used in a grain mash fermentation comprising a
microorganism capable of producing an alcohol such as butanol. In other
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embodiments, the grain mash fermentation may use simultaneous
saccharification and fermentation (SSF), and the concentration of glucose may
remain relatively low, for example, at least about 75 g/L glucose in the
fermentation broth phase over the course of the fermentation.
[00121] In some embodiments, fatty acids may be added to the fermentor in an
amount that is less than about 70% of the volume of the fermentor, less than
about 50% of the volume of the fermentor, or less than about 30% of the volume
of the fermentor. The amount of fatty acid added to the fermentor may be a
means to maintain the aqueous phase titer of butanol during fermentation. In
other embodiments, the aqueous phase titer of butanol may be maintained at a
level less than about 35 g/L of fermentation broth, less than about 25 g/L of
fermentation broth, or less than about 20 g/L of fermentation broth. In other
embodiments, the amount of active esterification enzyme in the fermentation
broth may be less than about 100 ppm, less than about 50 ppm, or less than
about 10 ppm active enzyme. In some embodiments, the cell mass employed in
a fermentation broth may be less than about 50 g dcw/L, less than about 20 g
dcw/L, or less than about 10 g dcw/L. In other embodiments, the fermentation
process may run at least about 30 hours to at least about 100 hours, at least
about 40 hours to at least about 80 hours, or at least about 50 hours to at
least
about 70 hours.
[00122] In some embodiments, a brix on water at a sufficient concentration to
achieve a final effective titer of at least about 30 g of butanol per liter of
fermentation broth phase, at least about 45 g of butanol per liter of
fermentation
broth phase, or at least about 60 g of butanol per liter of fermentation broth
phase
may be used in a sugarcane fermentation comprising a microorganism capable of
producing butanol. In some embodiments, fatty acids may be added to the
fermentor in an amount that is less than about 70% of the volume of the
fermentor, less than about 50% of the volume of the fermentor, or less than
about
30% of the volume of the fermentor. The amount of fatty acid added to the
fermentor may be a means to maintain the aqueous phase titer of butanol during
fermentation. In other embodiments, the aqueous phase titer of butanol may be
maintained at a level less than about 35 g/L of fermentation broth, less than
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about 25 g/L of fermentation broth, or less than about 15 g/L of fermentation
broth. In other embodiments, the amount of active esterification enzyme in the
fermentation broth may be less than about 200 ppm, less than about 100 ppm, or
less than about 20 ppm active enzyme. In some embodiments, the cell mass
employed in a fermentation broth may initially be at least about 100 g of cell
per
liter of broth in the initial charge occupying at least about 30% of the
fermentor
volume. After 3-7 hours of fermentation, the cell mass may be diluted to at
least
about 25 g of cell per liter of fermentation broth by the addition of a
sugarcane
feed. The cells may continue to grow to at least about 30 g of cell per liter
of
fermentation broth over the 8 to 15 hours of total fermentation time.
[00123] In some embodiments, the fermentation broth is contacted during
fermentation with an extractant to form a two-phase mixture comprising an
aqueous phase and an organic phase. In such embodiments, ISPR including
liquid-liquid extraction may be conveniently carried out. Liquid-liquid
extraction
can be performed according to the processes described in U.S. Patent
Application Publication No. 2009/0305370, the disclosure of which is hereby
incorporated in its entirety. U.S. Patent Application Publication No.
2009/0305370 describes methods for producing and recovering butanol from a
fermentation broth using liquid-liquid extraction, the methods comprising the
step
of contacting the fermentation broth with a water-immiscible extractant to
form a
two-phase mixture comprising an aqueous phase and an organic phase.
Typically, the extractant can be an organic extractant selected from the group
consisting of saturated, mono-unsaturated, poly-unsaturated (and mixtures
thereof) C12 to C22 fatty alcohols, C12 to C22 fatty acids, esters of C12 to
C22 fatty
acids, C12 to C22 fatty aldehydes, C12 to C22 fatty amides, and mixtures
thereof.
The extractant may also be an organic extractant selected from the group
consisting of saturated, mono-unsaturated, poly-unsaturated (and mixtures
thereof) C4 to C22 fatty alcohols, C4 to C28 fatty acids, esters of C4 to C28
fatty
acids, C4 to C22 fatty aldehydes, and mixtures thereof. For use with the
processes described herein, the extractant(s) for ISPR are typically non-
alcohol
extractants, so as to avoid consuming carboxylic acid 28 in fermentation
vessel
30 by catalytic esterification of carboxylic acid 28 with an alcohol
extractant,
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whereby less carboxylic acid would be available for esterification with the
product
alcohol. For example, if oleyl alcohol is used as an ISPR extractant, then
oleyl
alcohol esters of the carboxylic acid can be produced in fermentation vessel
due
to the presence of active catalyst 42, as further demonstrated in the Example
24
below.
[00124] With reference to the embodiment of FIG. 1, the carboxylic acid 28 can
also serve as an ISPR extractant 28 or a component thereof. As earlier noted,
carboxylic acid 28 can be supplied, and/or formed in situ in the case when
native
oil 26 is supplied to fermentation vessel 30, and/or formed in situ in the
case
when feedstock 16 includes oil that can be hydrolyzed. In some embodiments,
ISPR extractant 28 includes free fatty acids. In some embodiments, ISPR
extractant 28 includes corn oil fatty acids (COFA). In some embodiments, oil
26
is corn oil, whereby ISPR extractant 28 is COFA. ISPR extractant (carboxylic
acid) 28 contacts the fermentation broth and forms a two-phase mixture
comprising an aqueous phase 34 and an organic phase. The product alcohol
ester formed in the fermentation vessel preferentially partitions into the
organic
phase to form an ester-containing organic phase 36. That is, the product
alcohol
esters are produced at a concentration in excess of the equilibrium
concentration
of alcohol ester present in the aqueous phase 34 and therefore, preferentially
partition into the organic phase. Any free product alcohol in the fermentation
broth also preferentially partitions into the ester-containing organic phase.
The
biphasic mixture can be removed from fermentation vessel 30 as stream 39 and
introduced into a vessel 35, in which the ester-containing organic phase 36 is
separated from aqueous phase 34. Separation of biphasic mixture 39 into ester-
containing organic phase 36 and aqueous phase 34 can be achieved using any
methods known in the art, including but not limited to, siphoning, aspiration,
decantation, centrifugation, using a gravity settler, membrane-assisted phase
splitting, hydroclyclone, and the like. All or part of aqueous phase 34 can be
recycled into fermentation vessel 30 as fermentation medium (as shown), or
otherwise discarded and replaced with fresh medium, or treated for the removal
of any remaining product alcohol and then recycled to fermentation vessel 30.
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[00125] With reference to FIG. 1, ester-containing organic phase 36 is
introduced
into vessel 50 in which the alcohol esters are reacted with one or more
substances 52 to recover product alcohol 54. Product alcohol 54 can be
recovered using any method known in the art for obtaining an alcohol from an
alcohol ester. For example, in some embodiments, the product alcohol can be
recovered from the alcohol ester by hydrolysis with base followed by
acidification.
In other embodiments the product alcohol esters can be hydrolyzed by water in
the presence of a hydrolysis catalyst as substance 52. For example, in some
embodiments, hydrolysis of the product alcohol esters to alcohol and
carboxylic
acid 28 (e.g., fatty acid when carboxylic acid 28 is a fatty acid) can be
achieved
using a lipase, a water soluble acid, an inorganic acid, an organic acid, or a
solid
acid catalyst as substance 52. For example, sulfuric acid can be used as an
inorganic acid catalyst for alcohol ester hydrolysis. Some suitable hydrolysis
catalysts are lipase enzymes; esterase enzymes; strong inorganic acids such as
sulfuric acid, hydrochloric acid, or phosphoric acid; strong organic acids
such as
toluenesulfonic acid or naphthalenesulfonic acid; or solid acid catalysts such
as
Amberlyst sulfonated polystyrene resins, or zeolites. In some embodiments,
hydrolysis of the alcohol esters can be achieved using steam as substance 52,
by
increasing temperature, and/or by application of pressure. In some
embodiments, hydrolysis of the alcohol esters can be carried out in a column,
for
example, a reactive distillation column. Examples 45 to 54 and 56 to 58
demonstrate several methods to recover the product alcohol from an alcohol
ester. In some embodiments, by-products 56 are obtained from recovering
product alcohol 54. By-products 56 do not include carboxylic acid 28 that can
be
recovered from hydrolysis of the alcohol esters.
[00126] In some embodiments, hydrolysis of the alcohol esters of fatty acids
present in the ester-containing organic phase 36 into the product alcohol and
free
fatty acids occurs at a fatty acid to water ratio from about 10:1 to about
1:10 or in
other embodiments, at a fatty acid to water ratio from about 100:1 to about
1:100.
In some embodiments, the alcohol esters of fatty acids are hydrolyzed with
water
at a temperature less than about 100 C. In some embodiments, the hydrolysis
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occurs at a temperature greater than 100 C, greater than 150 C, greater than
200 C, or greater than 250 C.
[00127] For example, in some embodiments, the alcohol esters can be
transesterified to produce product alcohol 54 and in some embodiments, a
second alcohol ester 56, for example, fatty acid alkyl esters, can also be
produced as by-product 56. To achieve such transesterification, the alcohol
esters can be contacted with catalysts capable of transesterifying the alcohol
esters to release butanol. In some embodiments, the alcohol esters can be
transesterified using glycerol to produce product alcohol 54 and acyl
glycerides
as by-product 56. The acyl glycerides produced may comprise mono- and
diacylglycerides. Some suitable catalysts for transesterification reactions
are, for
example, lipase enzymes, alkoxide salts particularly of the second alcohol,
alkyl
titanates, soluble inorganic acids such as sulfuric acid and phosphoric acid,
soluble organic acids such as toluenesulfonic acid and naphthalenesulfonic
acid,
and solid acids such as Amberlyst sulfonated polystyrene resins, or zeolites.
Suitable lipases for transesterifications or hydrolysis include, but are not
limited
to, lipases derived from Burkholderia cepacia, Thermomyces lanuginosa, or
Candida antarctica. In some embodiments, the lipases are immobilized on a
soluble or insoluble support using methods well-known to those skilled in the
art
(see, e.g., Immobilization of Enzymes and Cells; Gordon F. Bickerstaff,
Editor;
Humana Press, Totowa, NJ, USA, 1997). The immobilization of enzymes may be
performed using a variety of techniques including 1) binding of the enzyme to
a
porous or non-porous carrier support, via covalent support, physical
adsorption,
electrostatic binding, or affinity binding; 2) crosslinking with bifunctional
or
multifunctional reagents; 3) entrapment in gel matrices, polymers, emulsions,
or
some form of membrane; and 4) a combination of any of these methods. In other
embodiments, the lipases may not be immobilized. In some embodiments, the
lipases are soluble. Fatty acid alkyl esters 56 can include fatty acid methyl
esters, for example. Other fatty acid alkyl esters 56 can include C2 to C12
linear,
branched, and cyclic alcohol esters, for example. Product alcohol 54 can then
be
separated from the reaction mixture including by-products 56 using any
separation means known in the art such as distillation, for example. Other
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suitable separation mechanisms can include extraction and membrane
separation, for example.
[00128] In some embodiments, at least about 5%, at least about 10%, at least
about 20%, at least about 30%, at least about 40%, at least about 50%, at
least
about 60%, at least about 70%, at least about 80%, at least about 90%, or at
least about 99% of the product alcohol is recovered from the alcohol esters.
[00129] ISPR extractant (carboxylic acid) 28 can be separated from the alcohol
esters before reaction of the alcohol esters for recovery of product alcohol
54.
Alternatively, ISPR extractant 28 can be separated from the product alcohol
and
any by-products after the reaction of the alcohol esters. The resulting
recovered
lean extractant 27 can then be recycled back into fermentation vessel 30,
usually
in combination with fresh make-up extractant 28 (which can be derived from oil
26, if supplied) for further production and/or extraction of alcohol esters.
Alternatively, fresh extractant 28 (or oil 26) can be continuously added to
the
fermentation vessel to replace the extractant removed in biphasic mixture
stream
39.
[00130] In some embodiments, catalyst 42 can be recovered from biphasic
mixture
39 and reused at a step in the fermentation process such as in the
fermentation
itself or in recovery of the product alcohol.
[00131] In some embodiments, one or more additional ISPR extractants 29 (see
FIG. 2) can be introduced into fermentation vessel 30 to form a two-phase
mixture comprising an aqueous phase and an organic phase. In such
embodiments, ISPR extractant 29 can be an exogenous organic extractant such
as oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl
alcohol,
stearyl alcohol, 1-undecanol, oleic acid, lauric acid, myristic acid, stearic
acid,
methyl myristate, methyl oleate, undecanal, lauric aldehyde, 20-
methylundecanal,
and mixtures thereof. However, for the reasons noted above, ISPR extractant 29
is preferably not an alcohol. Rather, ISPR extractant 29 is preferably a
carboxylic
acid (e.g. free fatty acids). In some embodiments, ISPR extractant 29 is COFA.
In some embodiments, ISPR extractant 29 is linseed oil fatty acid, soybean oil
fatty acid, jatropha oil fatty acid, or fatty acids derived from palm oil,
castor oil,
olive oil, coconut oil, peanut oil, or any seed oil. In some embodiments, ISPR
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extractant 29 can be a fatty acid extractant selected from the group
consisting of
fatty acids, fatty alcohols, fatty amides, fatty esters (particularly those
comprising
1 to 8 carbon atoms in the alcohol portion, e.g., fatty acid methyl esters and
lower
alcohol esters of fatty acids), fatty acid glycol esters, hydroxylated
triglycerides,
and mixtures thereof, obtained from chemical conversion of native oil such as
biomass lipids as described, for example, in co-pending, commonly owned U.S.
Provisional Application Serial No. 61/368,436, filed on July 28, 2010, herein
incorporated by reference. In some embodiments, ISPR extractant 29 is free
fatty acids obtained by chemical hydrolysis of biomass lipids. In some
embodiments, ISPR extractant 29 can be free fatty acids produced from
enzymatic hydrolysis of native oil such as biomass lipids as described, for
example, in co-pending, commonly owned U.S. Provisional Application Serial No.
61/368,444, filed on July 28, 2010, herein incorporated by reference.
[00132] In situ product removal can be carried out in a batch mode or a
continuous
mode in fermentation vessel 30. In a continuous mode of in situ product
removal,
product is continually removed from the vessel (or reactor). In a batchwise
mode
of in situ product removal, a volume of organic extractant is added to the
fermentation vessel and the extractant is not removed during the process. For
in
situ product removal, the organic extractant can contact the fermentation
medium
at the start of the fermentation forming a biphasic fermentation medium.
Alternatively, the organic extractant can contact the fermentation medium
after
the microorganism has achieved a desired amount of growth which can be
determined by measuring the optical density of the culture. Further, the
organic
extractant can contact the fermentation medium at a time at which the product
alcohol level in the fermentation medium reaches a preselected level. In the
case
of butanol production according to some embodiments of the present invention,
at a time before the butanol concentration reaches a toxic level, the
carboxylic
acid extractant can contact the fermentation medium to esterify the butanol
with
the carboxylic acid to produce butyl esters and in some embodiments, produce a
two-phase mixture comprising an aqueous phase and an organic phase
comprising the butyl esters. Consequently, the concentration of butanol is
reduced in the fermentation vessel and as a result, minimizes the toxic
effects of
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butanol on the microorganism. The ester-containing organic phase can then be
removed from the fermentation vessel (and separated from the fermentation
broth which constitutes the aqueous phase) after a desired effective titer of
the
butyl esters is achieved. For example, in some embodiments, the ester-
containing organic phase can be separated from the fermentation broth after
the
effective titer of butyl esters is greater than about 10 g/kg of fermentation
broth.
In other embodiments, the ester-containing organic phase can be separated from
the fermentation medium after the effective titer of butyl esters is greater
than
about 230 g/kg fermentation broth, greater than about 300 g/kg fermentation
broth, greater than about 400 g/kg fermentation broth, greater than about 500
g/kg fermentation broth, or greater than about 600 g/kg fermentation broth. In
another embodiment, the ester-containing organic phase can be separated from
the fermentation medium after the % conversion of COFA is at least about 10%,
at least about 25%, at least about 50%, at least about 75%, or at least about
100%. In some embodiments, the ester-containing organic phase is separated
from the aqueous phase after fermentation of the available fermentable sugar
in
the fermentation vessel is substantially complete.
[00133] In the example embodiment depicted in FIG. 1, the alcohol ester is
extracted from the fermentation broth in situ, with the separation of the
biphasic
mixture 39 occurring in a separate vessel 35. In some embodiments, separation
of the biphasic mixture can occur in the fermentation vessel, as shown in the
example embodiments of later described FIGs. 3 and 4 in which the ester-
containing organic phase stream 36 exits directly from fermentation vessel 30.
Aqueous phase stream 34 can also exit directly from fermentation vessel 30, be
treated for the removal of any remaining alcohol ester or product alcohol, and
recycled, or discarded and replaced with fresh fermentation medium. The
extraction of the alcohol ester and the product alcohol by the organic
extractant
can be done with or without the removal of microorganism 32 from the
fermentation broth. Microorganism 32 can be removed from the fermentation
broth by means known in the art including, but not limited to, filtration or
centrifugation. For example, aqueous phase stream 34 can include
microorganism 32 such as yeast. Microorganism 32 can be easily separated
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from the aqueous phase stream, for example, in a centrifuge (not shown).
Microorganism 32 can then be recycled to fermentation vessel 30 which over
time can increase the production rate of alcohol production, thereby resulting
in
an increase in the efficiency of the alcohol production.
[00134] In some embodiments, the system and processes of FIG. 1 can be
modified such that simultaneous saccharification and fermentation in
fermentation vessel 30 is replaced with a separate saccharification vessel 60
prior to fermentation vessel 30, as should be apparent to one of skill in the
art
(see, e.g., the embodiment of FIG. 4).
[00135] In still other embodiments, as shown, for example, in the example
embodiment of FIG. 2, native oil 26 (instead of being supplied directly to
fermentation vessel 30) is supplied to a vessel 40 to which catalyst 42 is
also
supplied, whereby at least a portion of the acyl glycerides in oil 26 are
hydrolyzed
to form carboxylic acid 28. A product stream from vessel 40 containing
carboxylic acid 28 and catalyst 42 are then introduced into fermentation
vessel
30. Carboxylic acid 28 and catalyst 42 contact the product alcohol produced in
the fermentation medium whereby alcohol esters of the product alcohol are
formed in situ from catalyzed esterification of the carboxylic acid with the
product
alcohol, in a same manner as described above with reference to FIG. 1.
Carboxylic acid 28 can also serve as an ISPR extractant and in some
embodiments, sufficient carboxylic acid 28 and/or one or more additional ISPR
extractants 29 can be introduced into fermentation vessel 30 to form a two-
phase
mixture comprising an aqueous phase and an organic phase, with the alcohol
ester partitioning into the organic phase. The remaining process operations of
the embodiment of FIG. 2 are identical to FIG. 1 and therefore, will not be
described in detail again.
[00136] In some embodiments of the present invention, as shown, for example,
in
the embodiment of FIG. 3, catalyst 42 can be added to feedstock slurry 16
comprising oil 26' derived from the biomass from which feedstock 12 was
formed.
In the embodiment shown, catalyst 42 is capable of hydrolyzing the glycerides
in
oil 26' to free fatty acids 28'. Thus, after introduction of catalyst 42 to
feedstock
slurry 16, at least a portion of the glycerides in oil 26' are hydrolyzed,
resulting in
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a feedstock slurry 18 having free fatty acids 28' and catalyst 42. For
example,
when feedstock 12 is corn, then oil 26' is the feedstock's constituent corn
oil and
the free fatty acids 28' are corn oil fatty acids (COFA).
[00137] Feedstock slurry 18 is introduced to fermentation vessel 30 along with
alcohol-producing microorganism 32 to be included in a fermentation medium. In
some embodiments, an enzyme 38 such as glucoamylase, can also be
introduced into fermentation vessel for simultaneous saccharification of
sugars in
slurry 18 and fermentation of alcohol inside fermentation vessel 30. The
presence of catalyst 42 in fermentation vessel (introduced via slurry 18)
catalyzes
the esterification of the alcohol with the free fatty acids 28' (introduced
via slurry
18) to form fatty acid alcohol esters in situ, in a same manner as described
above
with reference to FIG. 1. In some embodiments, for butanol production, butanol-
producing microorganism 32 is introduced in fermentation vessel 30 along with
feedstock slurry 18. Catalyst 42 in fermentation vessel (introduced via slurry
18)
catalyzes the esterification of the butanol with the free fatty acids 28'
(introduced
via slurry 18) to form fatty acid butyl esters (FABE) in situ. Free fatty
acids 28'
can also serve as an ISPR extractant. For example, when free fatty acids 28'
are
COFA, then alcohol esters of COFA are formed in situ, and COFA serves as an
ISPR extractant or a portion thereof.
[00138] In some embodiments, one or more additional ISPR extractants 29 can be
introduced into fermentation vessel 30 for preferentially partitioning the
alcohol
ester (and any free alcohol) from the aqueous phase. In some embodiments,
ISPR extractant 29 can be carboxylic acid 28 described with reference to the
embodiments of FIGs. 1 and 2. In some embodiments, ISPR extractant 29 is
introduced in fermentation vessel 30 as oil 26 which is then hydrolyzed into
fatty
acids by catalyst 42 so as to become ISPR extractant 29. In some embodiments,
oil 26 is corn oil, whereby ISPR extractant 29 is COFA. In some embodiments,
ISPR extractant 29 can be a fatty acid extractant selected from the group
consisting of fatty acids, fatty alcohols, fatty amides, fatty esters
(particularly
those comprising 1 to 8 carbon atoms in the alcohol portion, e.g., fatty acid
methyl esters and lower alcohol esters of fatty acids), fatty acid glycol
esters,
hydroxylated triglycerides, and mixtures thereof, as described above with
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reference to the embodiments of FIGs. 1 and 2. In still other embodiments,
ISPR
extractant 29 can be free fatty acids obtained by chemical or enzymatic
hydrolysis of biomass lipids. In such embodiments, the biomass lipids for
producing extractant 29 can be from a same or different biomass source from
which feedstock 12 is obtained. For example, in some embodiments, the
biomass lipids for producing extractant 29 can be derived from soya, whereas
the
biomass source of feedstock 12 is corn. Any possible combination of different
biomass sources for extractant 29 versus feedstock 12 can be used, as should
be apparent to one of skill in the art. The remaining process operations of
the
embodiment of FIG. 2 are identical to FIG. 1 and therefore, will not be
described
in detail again.
[00139] As a non-limiting prophetic example, with reference to the embodiment
of
FIG. 3, an aqueous suspension of ground whole corn (as feedstock 12) which
can nominally contain ca. 4 wt% corn oil, can be treated with amylase (as
liquefaction enzyme 14) at ca. 85 C to 1200C for 30 minutes to 2 hours, and
the
resulting liquefied mash 16 cooled to between 65 C and 30 C and treated with
0.1 ppm to 10 ppm (in some embodiments, 0.5 ppm to 1.0 ppm) of lipase (as
catalyst 42) at pH 4.5 to 7.5 (in some embodiments, between pH 5.5 and 6.5)
for
sufficient time to produce from at least 30% to as high as at least 99%
conversion
of the available fatty acid content in lipids to free fatty acids. The
liquefied and
lipase-treated mash 18 can be cooled to ca. 30 C (e.g., using a heat-
exchanger)
and loaded to fermentation vessel 30 at ca. 25% to 30 wt% dry corn solids.
Saccharification of the liquefied mash 18 during fermentation by the addition
of
glucoamylase (as saccharification enzyme 38) can result in the production of
glucose. The resulting fermentation broth can contain significantly less than
the
amount of corn oil (e.g., about 1.2 wt% corn oil) that can be present in a
broth
using a liquefied mash that has not been treated with lipase 42. In
particular, the
lipase treatment 42 can result in the conversion of corn oil lipids 26'
(triglycerides
(TG)) into COFA 28' (and some diglycerides (DG) or monoglycerides (MG)),
decreasing the rate of build-up of lipids 26' in the COFA ISPR extraction
solvent
28' or 29. The lipase treatment 42 can also result in the conversion of
butanol
produced during fermentation to butyl esters of COFA, where the butyl esters
of
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COFA have a high partition coefficient for dissolution into the COFA phase 36
during liquid-liquid extraction ISPR. At the end of fermentation, the COFA
phase
36 containing butyl esters of COFA can be separated from the fermentation
broth
(at vessel 30/35), and the butanol 54 can be recovered (at vessel 50) from
this
organic mixture 36 using one of several methods including, but not limited to,
hydrolysis of the ester using, for example, a lipase 52, a solid acid catalyst
52, or
steam 52, to produce butanol 54 and COFA 27.
[00140] In still other embodiments, as shown, for example, in the embodiment
of
FIG. 4, the system and processes of FIG. 3 can be modified such that
simultaneous saccharification and fermentation (SSF) in fermentation vessel 30
is replaced with a separate saccharification vessel 60 prior to fermentation
vessel
30. FIG. 4 is substantially identical to FIG. 3 except for the inclusion of a
separate saccharification vessel 60 receiving enzyme 38, with catalyst 42
being
introduced to a liquefied, saccharified feedstock stream 62. Feedstock slurry
16
is introduced into saccharification vessel 60 along with enzyme 38 such as
glucoamylase, whereby sugars in the form of oligosaccharides in slurry 16 can
be
broken down into monosaccharides. A liquefied, saccharified feedstock stream
62 exits saccharification vessel 60 to which catalyst 42 is introduced.
Feedstock
stream 62 includes monosaccharides, and oil 26' and undissolved solids derived
from the feedstock. Oil 26' is hydrolyzed by the introduction of catalyst 42,
resulting in a liquefied, saccharified feedstock slurry 64 having free fatty
acids 28'
and catalyst 42.
[00141] Alternatively, in some embodiments, catalyst 42 can be added along
with
saccharification enzyme 38 to simultaneously produce glucose and hydrolyze oil
lipids 26' to free fatty acids 28', in a like manner as the introduction of
catalyst 42
with enzyme 38 to the fermentation vessel 30 for SSF in the embodiment of FIG.
1. The addition of enzyme 38 and catalyst 42 can be stepwise (e.g., catalyst
42,
then enzyme 38, or vice versa), or simultaneous. However, in contrast with the
embodiment of FIG. 1 in which the addition of catalyst 42 into fermentation
vessel
30 during SSF also substantially simultaneously converts the product alcohol
to
the alcohol esters, alcohol esters are not formed until slurry 64 containing
catalyst
42 is introduced to fermentation vessel 30. Alternatively, in some
embodiments,
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slurry 62 can be introduced to fermentation vessel 30, with catalyst 42 being
added directly to the fermentation vessel 30.
[00142] In the embodiment of FIG. 4, slurry 64 is introduced to fermentation
vessel
30 along with alcohol-producing microorganism 32 which metabolizes the
monosaccharides to produce product alcohol. The presence of catalyst 42 in
fermentation vessel (introduced via slurry 64) catalyzes the esterification of
the
alcohol with the free fatty acids 28' (introduced via slurry 62) to form fatty
acid
alcohol esters in situ, in the same manner as described above with reference
to
FIG. 1. Free fatty acids 28' can also serve as an ISPR extractant for
preferentially partitioning the alcohol ester (and any free alcohol) from the
aqueous phase. In some embodiments, one or more additional ISPR extractants
29 can also be introduced into fermentation vessel 30 as described above with
reference to FIG. 3. The remaining process operations of the embodiment of
FIG. 4 are identical to FIG. 3 and therefore, will not be described in detail
again.
[00143] In some embodiments, including any of the earlier described
embodiments with respect to FIGs. 1-4, undissolved solids can be removed from
feedstock slurry 16 prior to introduction into fermentation vessel 30. For
example, as shown in the embodiment of FIG. 5, feedstock slurry 16 is
introduced into an inlet of a separator 20 which is configured to discharge
the
undissolved solids as a solid phase or wet cake 24. For example, in some
embodiments, separator 20 can include a filter press, vacuum filtration,
mechanical pressure filtration, or a centrifuge (e.g., decanter centrifuge)
for
separating the undissolved solids from feedstock slurry 16. In some
embodiments, any any conventional centrifuge utilized in the industry,
including,
for example, a decanter bowl centrifuge, tricanter centrifuge, disk stack
centrifuge, filtering centrifuge, or decanter centrifuge may be used to
separate the
undissolved solids. In some embodiments, removal of the undissolved solids
from feedstock slurry 16 can be accomplished by filtration, vacuum filtration,
beltfilter, pressure filtration, filtration using a screen, screen separation,
grates or
grating, porous grating, flotation, hydroclone, filter press, screwpress,
gravity
settler, vortex separator, or any method that may be used to separate solids
from
liquids. Optionally, in some embodiments, separator 20 can also be configured
to
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remove some or substantially all of oil 26' present in feedstock slurry 16. In
such
embodiments, separator 20 can be any suitable separator known in the art for
removing oil from an aqueous feedstream including, but not limited to,
siphoning,
decantation, centrifugation, using a gravity settler, membrane-assisted phase
splitting, and the like. The remaining feedstock including sugar and water is
discharged as an aqueous stream 22 to fermentation vessel 30.
[00144] For example, in some embodiments, separator 20 includes a tricanter
centrifuge 20 that agitates or spins feedstock slurry 16 to produce a
centrifuge
product comprising an aqueous layer containing sugar and water (i.e., stream
22), a solids layer containing the undissolved solids (i.e., wet cake 24), and
an oil
layer (i.e., oil stream 26'). In such a case, catalyst 42 can be contacted
with the
removed oil 26' to produce a stream of free fatty acid 28' and catalyst 42.
The
stream of free fatty acid 28' and catalyst 42 can then be introduced into
fermentation vessel 30 to contact with the fermentation medium, whereby
catalytic esterification of product alcohol in the fermentation medium into
fatty
acid alcohol esters can be achieved in situ, in a same manner as described
above with reference to FIG. 1.
[00145] Free fatty acids 28' can also serve as an ISPR extractant 28', and one
or
more additional ISPR extractants 29 can also be introduced into fermentation
vessel 30. Thus, feedstock oil 26' can be catalytically hydrolyzed to
carboxylic
acid, thereby decreasing the amount of lipids present in an ISPR extractant
while
also producing an ISPR extractant. The ester-containing organic phase 36 can
be separated from the aqueous phase 34 of the biphasic mixture 39 at vessel
35,
and the product alcohol can be recovered from the alcohol esters in vessel 50
(see FIG. 1). The remaining process operations of the embodiment of FIG. 5 are
identical to FIG. 3 and therefore, will not be described in detail again.
[00146] When wet cake 24 is removed via centrifuge 20, in some embodiments, a
portion of the oil from feedstock 12, such as corn oil when the feedstock is
corn,
remains in wet cake 24. Wet cake 24 can be washed with additional water in the
centrifuge once aqueous solution 22 has been discharged from the centrifuge
20.
Washing wet cake 24 will recover the sugar (e.g., oligosaccharides) present in
the wet cake and the recovered sugar and water can be recycled to the
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liquefaction vessel 10. After washing, wet cake 24 may be combined with
solubles and then dried to form Dried Distillers' Grains with Solubles (DDGS)
through any suitable known process. The formation of the DDGS from wet cake
24 formed in centrifuge 20 has several benefits. Since the undissolved solids
do
not go to the fermentation vessel, the DDGS does not have trapped extractant
and/or product alcohol such as butanol, it is not subjected to the conditions
of the
fermentation vessel, and it does not contact the microorganisms present in the
fermentation vessel. All these benefits make it easier to process and sell
DDGS,
for example, as animal feed. In some embodiments, oil 26' is not discharged
separately from wet cake 24, but rather oil 26' is included as part of wet
cake 24
and is ultimately present in the DDGS. In such instances, the oil can be
separated from the DDGS and converted to an ISPR extractant 29 for
subsequent use in the same or different alcohol fermentation process. Methods
and systems for removing undissolved solids from feedstock slurry 16 via
centrifugation are described in detail in co-pending, commonly owned U.S.
Provisional Application Serial No. 61/356,290, filed June 18, 2010, which is
incorporated herein in its entirety by reference thereto.
[00147] As described above, oil 26' may be separated from DDGS using any
suitable known process including, for example, a solvent extraction process.
In
one embodiment of the invention, DDGS are loaded into an extraction vessel and
washed with a solvent such as hexane to remove oil 26'. Other solvents that
may
be utilized include, for example, isobutanol, isohexane, ethanol, petroleum
distillates such as petroleum ether, or mixtures thereof. After oil 26'
extraction,
DDGS may be treated to remove any residual solvent. For example, DDGS may
be heated to vaporize any residual solvent using any method known in the art.
Following solvent removal, DDGS may be subjected to a drying process to
remove any residual water. The processed DDGS may be used as a feed
supplement for animals such as poultry, livestock, and domestic pets.
[00148] After extraction from DDGS, the resulting oil 26' and solvent mixture
may
be collected for separation of oil 26' from the solvent. In one embodiment,
the oil
26'/solvent mixture may be processed by evaporation whereby the solvent is
evaporated and may be collected and recycled. The recovered oil may be
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converted to an ISPR extractant 29 for subsequent use in the same or different
alcohol fermentation process.
[00149] In addition to the recovery of solids, it may be desired to recover
other by-
products of the fermentation process. In one embodiment, fatty acid esters
(e.g.,
fatty acid isobutyl esters) may be recovered, for example, to increase the
yield of
carbohydrate to product alcohol (e.g., butanol). This may be accomplished, for
example, by using a solvent to extract fatty acid isobutyl esters from, for
example,
the by-product formed by combining and mixing several by-product streams and
drying the product of the combining and mixing steps. Such a solvent-based
extraction system for recovering corn oil triglyceride from DDGS is described
in
U.S. Patent Application Publication No. 2010/0092603, the teachings of which
are incorporated by reference herein.
[00150] In one embodiment of solvent extraction of fatty acid esters, solids
may be
separated from whole stillage ("separated solids") since that stream would
contain the largest portion, by far, of fatty acid esters in uncombined
byproduct
streams. These separated solids may then be fed into an extractor and washed
with solvent. In one embodiment, the separated solids are turned at least once
in
order to ensure that all sides of the separated solids are washed with
solvent.
After washing, the resulting mixture of lipid and solvent, known as miscella,
is
collected for separation of the extracted lipid from the solvent. For example,
the
resulting mixture of lipid and solvent may be deposited to a separator for
further
processing. During the extraction process, as the solvent washes over the
separated solids, the solvent not only brings lipid into solution, but it
collects fine,
solid particles. These "fines" are generally undesirable impurities in the
miscella
and in one embodiment, the miscella may be discharged from the extractor or
separator through a device that separates or scrubs the fines from the
miscella.
[00151] In order to separate the lipid and the solvent contained in the
miscella, the
miscella may be subjected to a distillation step. In this step, the miscella
can, for
example, be processed through an evaporator which heats the miscella to a
temperature that is high enough to cause vaporization of the solvent, but is
not
sufficiently high to adversely affect or vaporize the extracted lipid. As the
solvent
evaporates, it may be collected, for example, in a condenser, and recycled for
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future use. Separation of the solvent from the miscella results in a stock of
crude
lipid which may be further processed to separate water, fatty acid esters
(e.g.,
fatty acid isobutyl esters), fatty acids, and triglycerides.
[00152] After extraction of the lipids, the solids may be conveyed out of the
extractor and subjected to a stripping process that removes residual solvent.
Recovery of residual solvent is important to process economics. In one
embodiment, the wet solids can be conveyed in a vapor tight environment to
preserve and collect solvent that transiently evaporates from the wet solids
as it
is conveyed into the desolventizer. As the solids enter the desolventizer,
they
may be heated to vaporize and remove the residual solvent. In order to heat
the
solids, the desolventizer may include a mechanism for distributing the solids
over
one or more trays, and the solids may be heated directly, such as through
direct
contact with heated air or steam, or indirectly, such as by heating the tray
carrying the meal. In order to facilitate transfer of the solids from one tray
to
another, the trays carrying the solids may include openings that allow the
solids
to pass from one tray to the next. From the desolventizer, the solids may be
conveyed to, optionally, a mixer where the solids are mixed with other by-
products before being conveyed into a dryer. An example of solids extraction
is
described in Example 63. In this example, the solids are fed to a
desolventizer
where the solids are contacted by steam. In one embodiment, the flows of steam
and solids in the desolventizer may be countercurrent. The solids may then
exit
the desolventizer and may be fed to a dryer or optionally a mixer where
various
by-products may be mixed. Vapor exiting the desolventizer may be condensed
and optionally mixed with miscella and then fed to a decanter. The water-rich
phase exiting the decanter may be fed to a distillation column where hexane is
removed from the water-rich stream. In one embodiment, the hexane-depleted
water rich stream exits the bottom of the distillation column and may be
recycled
back to the fermentation process, for example, it may be used to slurry the
ground corn solids. In another embodiment, the overhead and bottom products
may be recycled to the fermentation process. For example, the lipid-rich
bottoms
may be added to the feed of a hydrolyzer. The overheads may be, for example,
condensed and fed to a decanter. The hexane rich stream exiting this decanter
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can optionally be used as part of the solvent feed to the extractor. The water-
rich
phase exiting this decanter may be fed to the column that strips hexane out of
water. As one skilled in the art can appreciate, the methods of the present
invention may be modified in a variety of ways to optimize the fermentation
process for the production of a product alcohol such as butanol.
[00153] In another embodiment of solvent extraction of fatty acid esters,
solids
may be separated from beer and solvent discharged from fermentation before
they are introduced into a preflash column as a heterogeneous mixture. A wet
cake of these solids can be formed using a separation device such as a screen
filter or a centrifuge. A screened cake of solids can be displacement washed
using hydrous isobutanol to remove fatty acid esters that were retained in the
wet
solids. Alternatively, a centrifuged cake of solids can be re-pulped in
hydrous
isobutanol and separated again to effect the removal of fatty acid esters that
were
retained in the wet solids. An example of this embodiment of solids extraction
is
described in Example 63.
[00154] In a further embodiment, by-products (or co-products) may be derived
from the mash used in the fermentation process. For example, corn oil may be
separated from mash and this corn oil may contain triglycerides, free fatty
acids,
diglycerides, monoglycerides, and phospholipids (see, e.g., Example 66). The
corn oil may optionally be added to other by-products (or co-products) at
different
rates and thus, for example, creating the ability to vary the amount of
triglyceride
in the resulting byproduct. In this manner, the fat content of the resulting
by-
product could be controlled, for example, to yield a lower fat, high protein
animal
feed that would better suit the needs of dairy cows compared to a high fat
product.
[00155] In one embodiment, crude corn oil separated from mash may be further
processed into edible oil for consumer use, or it could also be used as a
component of animal feed because its high triglyceride content would make it
an
excellent source of metabolizable energy. In another embodiment, it could also
be used as feedstock for biodiesel or renewable diesel.
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[00156] In one embodiment, extractant by-product may be used, all or in part,
as a
component of an animal feed by-product or it can be used as feedstock for
biodiesel or renewable diesel.
[00157] In a further embodiment, solids may be separated from mash and may
comprise triglycerides and free fatty acids. These solids (or stream) may be
used
as an animal feed, either recovered as discharge from centrifugation or after
drying. The solids (or wet cake) may be particularly suited as feed for
ruminants
(e.g., dairy cows) because of its high content of available lysine and by-pass
or
rumen undegradable protein. For example, these solids may be of particular
value in a high protein, low fat feed. In another embodiment, these solids may
be
used as a base, that is, other by-products such as syrup may be added to the
solids to form a product that may be used as an animal feed. In another
embodiment, different amounts of other by-products may be added to the solids
to tailor the properties of the resulting product to meet the needs of a
certain
animal species.
[00158] The composition of solids separated from whole stillage as described
in
Example 62 may include, for example, crude protein, fatty acid, and fatty acid
isobutyl esters. In one embodiment, this composition (or by-product) may be
used, wet or dry, as an animal feed where, for example, a high protein (e.g.,
high
lysine), low fat, and high fiber content is desired. In another embodiment,
fat may
be added to this composition, for example, from another by-product stream if a
higher fat, low fiber animal feed is desired. In one embodiment, this higher
fat,
low fiber animal feed may be used for swine or poultry. In a further
embodiment,
a non-aqueous composition of Condensed Distillers Solubles (CDS) (see, e.g.,
Example 66) may include, for example, protein, fatty acids, and fatty acid
isobutyl
esters as well as other dissolved and suspended solids such as salts and
carbohydrates. This CDS composition may be used, for example, as animal
feed, either wet or dry, where a high protein, low fat, high mineral salt feed
component is desired. In one embodiment, this composition may be used as a
component of a dairy cow ration.
[00159] In another embodiment, oil from the fermentation process may be
recovered by evaporation. This non-aqueous composition may comprise fatty
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acid isobutyl esters and fatty acids (see, e.g., Example 66) and this
composition
(or stream) may be fed to a hydrolyser to recover isobutanol and fatty acids.
In a
further embodiment, this stream may be used as feedstock for biodiesel
production.
[00160] The various streams generated by the production of an alcohol (e.g.,
butanol) via a fermentation process may be combined in many ways to generate
a number of co-products. For example, if crude corn from mash is used to
generate fatty acids to be utilized as extractant and lipid is extracted by
evaporators for other purposes, then the remaining streams may be combined
and processed to create a co-product composition comprising crude protein,
crude fat, triglycerides, fatty acid, and fatty acid isobutyl ester. In one
embodiment, this composition may comprise at least about 20-35 wt% crude
protein, at least about 1-20 wt% crude fat, at least about 0-5 wt%
triglycerides, at
least about 4-10 wt% fatty acid, and at least about 2-6 wt% fatty acid
isobutyl
ester. In one particular embodiment, the co-product composition may comprise
about 25 wt% crude protein, about 10 wt% crude fat, about 0.5 wt%
triglycerides,
about 6 wt% fatty acid, and about 4 wt% fatty acid isobutyl ester.
[00161] In another embodiment, the lipid is extracted by evaporators and the
fatty
acids are used for other purposes and about 50 wt% of the crude corn from mash
and the remaining streams are combined and processed, the resulting co-product
composition may comprise crude protein, crude fat, triglycerides, fatty acid,
and
fatty acid isobutyl ester. In one embodiment, this composition may comprise at
least about 25-31 wt% crude protein, at least about 6-10 wt% crude fat, at
least
about 4-8 wt% triglycerides, at least about 0-2 wt% fatty acid, and at least
about
1-3 wt% fatty acid isobutyl ester. In one particular embodiment, the co-
product
composition may comprise about 28 wt% crude protein, about 8 wt% crude fat,
about 6 wt% triglycerides, about 0.7 wt% fatty acid, and about 1 wt% fatty
acid
isobutyl ester.
[00162] In another embodiment, the solids separated from whole stillage and 50
wt% of the corn oil extracted from mash are combined and the resulting co-
product composition may comprise crude protein, crude fat, triglycerides,
fatty
acid, fatty acid isobutyl ester, lysine, neutral detergent fiber (NDF), and
acid
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detergent fiber (ADF). In one embodiment, this composition may comprise at
least about 26-34 wt% crude protein, at least about 15-25 wt% crude fat, at
least
about 12-20 wt% triglycerides, at least about 1-2 wt% fatty acid, at least
about 2-
4 wt% fatty acid isobutyl ester, at least about 1-2 wt% lysine, at least about
11-23
wt% NDF, and at least about 5-11 wt% ADF. In one particular embodiment, the
co-product composition may comprise about 29 wt% crude protein, about 21 wt%
crude fat, about 16 wt% triglycerides, about 1 wt% fatty acid, about 3 wt%
fatty
acid isobutyl ester, about 1 wt% lysine, about 17 wt% NDF, and about 8 wt%
ADF. The high fat, triglyceride, and lysine content and the lower fiber
content of
this co-product composition may be desirable as feed for swine and poultry.
[00163] As described above, the various streams generated by the production of
an alcohol (e.g., butanol) via a fermentation process may be combined in many
ways to generate a co-product composition comprising crude protein, crude fat,
triglycerides, fatty acid, and fatty acid isobutyl ester. For example, a
composition
comprising at least about 6% crude fat and at least about 28% crude protein
may
be utilized as an animal feed product for dairy animals. A composition
comprising at least about 6% crude fat and at least about 26% crude protein
may
be utilized as an animal feed product for feedlot cattle whereas a composition
comprising at least about 1 % crude fat and at least about 27% crude protein
may
be utilized as an animal feed product for wintering cattle. A composition
comprising at least about 13% crude fat and at least about 27% crude protein
may be utilized as an animal feed product for poultry. A composition
comprising
at least about 18% crude fat and at least about 22% crude protein may be
utilized
as an animal feed product for monogastric animals. Thus, the various streams
may be combined in such a way as to customize a feed product for a specific
animal species.
[00164] In one embodiment, one or more streams generated by the production of
an alcohol (e.g., butanol) via a fermentation process may be combined in many
ways to generate a composition comprising at least about 90% COFA which may
be used as fuel source such as biodiesel.
[00165] As an example of one embodiment of the methods of the invention,
milled
grain (e.g., corn processed by hammer mill) and one or more enzymes are
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combined to generate a slurried grain. This slurried grain is cooked,
liquified, and
optionally flashed with flash vapor resulting in a cooked mash. The cooked
mash
is then filtered to remove suspended solids, generating a wet cake and a
filtrate.
The filtration may be accomplished by several methods such as centrifugation,
screening, or vacuum filtration and this filtration step may remove at least
about
80% to at least about 99% of the suspended solids from the mash.
[00166] The wet cake is reslurried with water and refiltered to remove
additional
starch, generating a washed filter cake. The reslurry process may be repeated
a
number of times, for example, one to five times. The water used to reslurry
the
wet cake may be recycled water generated during the fermentation process. The
filtrate produced by the reslurry/refiltration process may be returned to the
initial
mix step to form a slurry with the milled grain. The filtrate may be heated or
cooled prior to the mix step.
[00167] The washed filter cake may be reslurried with beer at a number of
stages
during the production process. For example, the washed filter cake may be
reslurried with beer after the fermentor, before the preflash column, or at
the
feedpoint to the distillers grain dryer. The washed filter cake may be dried
separately from other by-products or may be used directly as wet cake for
generation of DDGS or an animal feed product.
[00168] The filtrate produced as a result of the initial mix step may be
further
processed as described herein. For example, the filtrate may be heated with
steam or process to process heat exchange. A saccharification enzyme may be
added to the filtrate and the dissolved starch of the filtrate may be
partially or
completely saccharified. The saccharified filtrate may be cooled by a number
of
means such as process to process exchange, exchange with cooling water, or
exchange with chilled water.
[00169] The cooled filtrate may then be added to a fermentor as well as a
microorganism that is suitable for alcohol production, for example, a
recombinant
yeast capable of producing butanol. In addition, ammonia and recycle streams
may also be added to the fermentor. This process may include at least one
fermentor, at least two fermentors, at least three fermentors, or at least
four
fermentors. Carbon dioxide generated during the fermentation may be vented to
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a scrubber in order to reduce air emissions (e.g., butanol air emissions) and
to
increase product yield.
[00170] Solvent may be added to the fermentor via a recycled loop or may be
added directly into the fermentor. The solvent may be one or more organic
compounds which have the ability to dissolve or react with the alcohol (e.g.,
butanol) and may have limited solubility in water. The solvent may be taken
from
the fermentor continually as a single liquid phase or as a two liquid phase
material, or the solvent may be withdrawn batchwise as a single or two liquid
phase material.
[00171] Beer may be degassed. The beer may be heated before degassing, for
example, by process to process exchange with hot mash or process to process
exchange with preflash column overheads. Vapors may be vented to a
condenser and then, to a scrubber. Degassed beer may be heated further, for
example, by process to process heat exchange with other streams in the
distillation area.
[00172] Preheated beer and solvent may enter a preflash column which may be
retrofit from a beer column of a conventional dry grind fuel ethanol plant.
This
column may be operated at sub-atmospheric pressure, driven by water vapor
taken from an evaporator train or from the mash cook step. The overheads of
the
preflash column may be condensed by heat exchange with some combination of
cooling water and process to process heat exchange including heat exchange
with the preflash column feed. The liquid condensate may be directed to an
alcohol/ water decanter (e.g., butanol/water decanter).
[00173] The preflash column bottoms may be advanced to a solvent decanter.
The preflash column bottoms may be substantially stripped of free alcohol
(e.g.,
butanol). The decanter may be a still well, a centrifuge, or a hydroclone.
Water
is substantially separated from the solvent phase in this decanter, generating
a
water phase. The water phase including suspended and dissolved solids may be
centrifuged to produce a wet cake and thin stillage. The wet cake may be
combined with other streams and dried to produce DDGS, it may be dried and
sold separate from other streams which produce DDGS, or it may be sold as a
wet cake. The water phase may be split to provide a backset which is used in
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part to reslurry the filter cake described above. The split also provides thin
stillage which may be pumped to evaporators for further processing.
[00174] The organic phase produced in the solvent decanter may be an ester of
an
alcohol (e.g., butanol). The solvent may be hydrolyzed to regenerate reactive
solvent and to recover additional alcohol (e.g., butanol). Alternatively, the
organic
phase may be filtered and sold as a product. Hydrolysis may be thermally
driven,
homogeneously catalyzed, or heterogeneously catalyzed. Hydrolysis may also
occur by enzymatic reaction. The heat input to this process may be a fired
heater, hot oil, electrical heat input, or high pressure steam. Water added to
drive the hydrolysis may be from a recycled water stream, fresh water, or
steam.
[00175] Cooled hydrolyzed solvent may be pumped into a sub-atmospheric solvent
column where it may be substantially stripped of alcohol (e.g., butanol) with
steam. This steam may be water vapor from evaporators, it may be steam from
the flash step of the mash process, or it may be steam from a boiler (see,
e.g.,
U.S. Patent Application Publication No. 2009/0171129, incorporated herein by
reference). A rectifier column from a conventional dry grind ethanol plant may
be
suitable as a solvent column. The rectifier column may be modified to serve as
a
solvent column. The bottoms of the solvent column may be cooled, for example,
by cooling water or process to process heat exchange. The cooled bottoms may
be decanted to remove residual water and this water may be recycled to other
steps with the process or recycled to the mash step.
[00176] The solvent column overheads may be cooled by exchange with cooling
water or by process to process heat exchange, and the condensate may be
directed to a vented alcohol/ water decanter (e.g., butanol/water decanter)
which
may be shared with the preflash column overheads. Other mixed water and
alcohol (e.g., butanol) streams may be added to this decanter including the
scrubber bottoms and condensate from the degas step. The vent which
comprises carbon dioxide, may be directed to a water scrubber. The aqueous
layer of this decanter may also be fed to the solvent column or may be
stripped of
alcohol (e.g., butanol) in a small dedicated distillation column. The aqueous
layer
may be preheated by process to process exchange with the preflash column
overheads, solvent column overheads, or solvent column bottoms. This
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dedicated column may be modified from the side stripper of a conventional dry
grind fuel ethanol process.
[00177] The organic layer of the alcohol/water decanter (e.g., butanol/water
decanter) may be pumped to an alcohol (e.g., butanol) column. This column may
be a super-atmospheric column and may be driven by steam condensation within
a reboiler. The feed to the column may be heated by process to process heat
exchange in order to reduce the energy demand to operate the column. This
process to process heat exchanger may include a partial condenser of the
preflash column, a partial condenser of a solvent column, the product of the
hydrolyzer, water vapor from the evaporators, or the butanol column bottoms.
The condensate of the alcohol (e.g., butanol) column vapor may be cooled and
may be returned to the alcohol/water decanter (e.g., butanol/water decanter).
The alcohol (e.g., butanol) column bottoms may be cooled by process to process
heat exchange including exchange with the alcohol (e.g., butanol) column feed
and may be further cooled with cooling water, filtered, and are sold as
product
alcohol (e.g., butanol).
[00178] Thin stillage generated from the preflash column bottoms as described
above may be directed to a multiple effect evaporator. This evaporator may
have
two, three, or more stages. The evaporator may have a configuration of four
bodies by two effects similar to the conventional design of a fuel ethanol
plant, it
may have three bodies by three effects, or it may have other configurations.
Thin
stillage may enter at any of the effects. At least one of the first effect
bodies may
be heated with vapor from the super-atmospheric alcohol (e.g., butanol)
column.
The vapor may be taken from the lowest pressure effect to provide heat in the
form of water vapor to the sub-atmospheric preflash column and solvent column.
Syrup from the evaporators may be added to the distiller's grain dryer.
[00179] Carbon dioxide emissions from the fermentor, degasser, alcohol/water
decanter (e.g., butanol/water decanter) and other sources may be directed to a
water scrubber. The water supplied to the top of this scrubber may be fresh
makeup water or may be recycled water. The recycled water may be treated
(e.g., biologically digested) to remove volatile organic compounds and may be
chilled. Scrubber bottoms may be sent to the alcohol/water decanter (e.g.,
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butanol/water decanter), to the solvent column, or may be used with other
recycled water to reslurry the wet cake described above. Condensate from the
evaporators may be treated with anaerobic biological digestion or other
processes to purify the water before recycling to reslurry the filter cakes.
[00180] If corn is used as the source of the milled grain, corn oil may be
separated
from the process streams at any of several points. For example, a centrifuge
may be operated to produce a corn oil stream following filtration of the
cooked
mash or the preflash column water phase centrifuge may be operated to produce
a corn oil stream. Intermediate concentration syrup for final syrup may be
centrifuged to produce a corn oil stream.
[00181] In another example of an embodiment of the methods of the invention,
the
material discharged from the fermentor may be processed in a separation system
that involves devices such as a centrifuge, settler, hydrocyclone, etc., and
combinations thereof to effect the recovery of live yeast in a concentrated
form
that can be recycled for reuse in a subsequent fermentation batch either
directly
or after some re-conditioning. This separation system may also produce an
organic stream that comprises fatty esters (e.g. isobutyl fatty esters) and an
alcohol (e.g., isobutanol) produced from the fermentation and an aqueous
stream
containing only trace levels of immiscible organics. This aqueous stream may
be
used either before or after it is stripped of the alcohol (e.g., isobutanol)
content to
re-pulp and pump the low starch solids that was separated and washed from
liquefied mash. This has the advantage of avoiding what might otherwise be a
long belt-driven conveying system to transfer these solids from the
liquefaction
area to the grain drying and syrup blend area. Furthermore, this whole
stillage
that results after the alcohol (e.g., isobutanol) has been stripped will need
to be
separated into thin stillage and wet cake fractions either using existing or
new
separation devices and this thin stillage will form in part the backset that
returns
to combine with cook water for preparing a new batch of fermentable mash.
Another advantage of this embodiment is that any residual dissolved starch
that
was retained in the moisture of the solids separated from the liquefied mash
would in part be captured and recovered through this backset. Alternatively,
the
yeast contained in the solids stream may be considered nonviable and may be
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redispersed in the aqueous stream and this combined stream distilled of any
alcohol (e.g., butanol) content remaining from fermentation. Non viable
organisms may further be separated for use as a nutrient in the propagation
process.
[00182] In another embodiment, the multi-phase material may leave the bottom
of
the pre-flash column and may be processed in a separation system as described
above. The concentrated solids may be redispersed in the aqueous stream and
this combined stream may be used to re-pulp and pump the low starch solids
that
were separated and washed from liquefied mash.
[00183] The process described above as well as other processes described
herein
may be demonstrated using computational modeling such as Aspen modeling
(see, e.g., U.S. Patent No. 7,666,282). For example, the commercial modeling
software Aspen Plus (Aspen Technology, Inc., Burlington, MA) may be use in
conjunction with physical property databases such as DIPPR, available from
American Institute of Chemical Engineers, Inc. (New York, NY) to develop an
Aspen model for an integrated butanol fermentation, purification, and water
management process. This process modeling can perform many fundamental
engineering calculations, for example, mass and energy balances, vapor/liquid
equilibrium, and reaction rate computations. In order to generate an Aspen
model, information input may include, for example, experimental data, water
content and composition of feedstock, temperature for mash cooking and
flashing, saccharification conditions (e.g., enzyme feed, starch conversion,
temperature, pressure), fermentation conditions (e.g., microorganism feed,
glucose conversion, temperature, pressure), degassing conditions, solvent
columns, preflash columns, condensers, evaporators, centrifuges, etc.
[00184] The present invention provides systems and methods for producing a
fermentative product such as a product alcohol, through fermentation as well
as
increasing biomass processing productivity and cost effectiveness. In some
embodiments, the product alcohol is butanol. A feedstock can be liquefied to
create a feedstock slurry, wherein the feedstock slurry includes soluble sugar
and
undissolved solids. If the feedstock slurry is fed directly to the fermentor,
the
undissolved solids may interfere with efficient removal and recovery of a
product
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alcohol such as butanol from the fermentor. In particular, when liquid-liquid
extraction is utilized to extract butanol from the fermentation broth, the
presence
of the undissolved particulates may cause system inefficiencies including, but
not
limited to, decreasing the mass transfer rate of the butanol to the extractant
by
interfering with the contact between the extractant and the fermentation
broth;
creating an emulsion in the fermentor and thereby interfering with good phase
separation of the extractant and the fermentation broth; reducing the
efficiency of
recovering and recycling the extractant because at least a portion of the
extractant and butanol becomes "trapped" in the solids which are ultimately
removed as DDGS; a lower fermentor volume efficiency because there are solids
taking up volume in the fermentor and because there is a slower disengagement
of the extractant from the fermentation broth; and shortening the life cycle
of the
extractant by contamination with corn oil. All of these effects result in
higher
capital and operating costs. In addition, the extractant "trapped" in the DDGS
may detract from DDGS value and qualification for sale as animal feed. Thus,
in
order to avoid and/or minimize these problems, at least a portion of the
undissolved particles (or solids) are removed from the feedstock slurry prior
to
the addition of sugar present in the feedstock slurry to the fermentor.
Extraction
activity and the efficiency of the butanol production are increased when
extraction
is performed on a fermentation broth containing an aqueous solution wherein
undissolved particles have been removed relative to extraction performed on a
fermentation broth containing an aqueous solution wherein undissolved
particles
have not been removed.
[00185] Extractive fermentation without the presence of the undissolved solids
can
lead to higher mass transfer rate of the product alcohol from the fermentation
broth to the extractant, better phase separation of the extractant from the
fermentation inside or external to the fermentor, and lower hold up of the
extractant as a result of higher extractant droplet rise velocities. Also, for
example, the extractant droplets held up in the fermentation broth during
fermentation will disengage from the fermentation broth faster and more
completely, thereby resulting in less free extractant in the fermentation
broth and
can decrease the amount of extractant lost in the process. In addition, for
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example, the microorganism can be recycled and additional equipment in the
downstream processing can be eliminated, such as for example, a beer column
and/or some or all of the whole stillage centrifuges. Further, for example,
the
possibility of extractant being lost in the DDGS is removed. Also, for
example,
the ability to recycle the microorganism can increase the overall rate of
product
alcohol production, lower the overall titer requirement, and/or lower the
aqueous
titer requirement, thereby leading to a healthier microorganism and a higher
production rate. In addition, for example, it can be possible to eliminate an
agitator in the fermentor to reduce capital costs; to increase the fermentor
productivity since the volume is used more efficiently because the extractant
hold
up is minimized and the undissolved solids are not present; and/or to use
continuous fermentation or smaller fermentors in a greenfield plant.
[00186] Examples of increased extraction efficiency can include, for example,
a
stabilized partition coefficient, enhanced (e.g., quicker or more complete)
phase
separation, enhanced liquid-liquid mass transfer coefficient, operation at a
lower
titer, increased process stream recyclability, increased fermentation volume
efficiency, increased feedstock (e.g., corn) load feeding, increased butanol
titer
tolerance of the microorganism (e.g., a recombinant microorganism), water
recycling, reduction in energy, increased recycling of extractant, and/or
recycling
of the microorganism.
[00187] For example, the volume of the fermentor taken up by solids will be
decreased. Thus, the effective volume of the fermentor available for the
fermentation can be increased. In some embodiments, the volume of the
fermentor available for the fermentation is increased by at least about 10%.
[00188] For example, there can be a stabilization in partition coefficient.
Because
the corn oil in the fermentor can be reduced by removing the solids from the
feedstock slurry prior to fermentation, the extractant is exposed to less corn
oil
which combines with the extractant and may lower the partition coefficient if
present in sufficient amount. Therefore, reduction of the corn oil introduced
into
the fermentor results in a more stable partition coefficient of the extractant
phase
in the fermentor. In some embodiments, the partition coefficient is decreased
by
less than about 10% over 10 fermentation cycles.
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[00189] For example, there can be an increase in the extraction efficiency of
the
butanol with extractant because there will be a higher mass transfer rate
(e.g., in
the form of a higher mass transfer coefficient) of the product alcohol from
the
fermentation broth to the extractant, thereby resulting in an increased
efficiency
of product alcohol production. In some embodiments, the mass transfer
coefficient is increased at least 2-fold (see Examples 4 and 5).
[00190] In addition, there can be an increase in phase separation between the
fermentation broth and the extractant that reduces the likelihood of the
formation
of an emulsion, thereby resulting in an increased efficiency of product
alcohol
production. For example, the phase separation can occur more quickly or can be
more complete. In some embodiments, a phase separation may occur where
previously no appreciable phase separation was observed in 24 hours. In some
embodiments, the phase separation occurs at least about 2x as quickly, at
least
about 5x as quickly, or at least about 10x as quickly as compared to the phase
separation where solids have not been removed (see Examples 6 and 7).
[00191] Further, there can be an increase in the recovery and recycling of the
extractant. The extractant will not be "trapped" in the solids which may
ultimately
be removed as DDGS, thereby resulting in an increased efficiency of product
alcohol production (see Examples 8 and 9). Also, there will be less dilution
of the
extractant with corn oil, and there may be less degradation of the extractant
(see
Example 10).
[00192] Also, the flow rate of the extractant can be reduced which will lower
operating costs, thereby resulting in an increased efficiency of product
alcohol
production.
[00193] Further still, hold up of the extractant will be decreased as a result
of
extractant droplets rising at a higher velocity, thereby resulting in an
increased
efficiency of product alcohol production. Reducing the amount of undissolved
solids in the fermentor will also result in an increased efficiency of product
alcohol
production.
[00194] In addition, an agitator can be removed from the fermentor because it
is no
longer needed to suspend the undissolved solids, thereby reducing capital
costs
and energy, and increasing the efficiency of the product alcohol production.
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[00195] Figures 1-5 provide various non-limiting embodiments of methods and
systems involving fermentation processes in which alcohol esters are produced
in
situ, extracted from the fermentation medium, and reacted to recover product
alcohol. FIGs. 1-5 also provide various non-limiting embodiments of methods
and systems of using carboxylic acid that can be esterified with product
alcohol
and can contemporaneously serve as an ISPR extractant. FIGs. 1-5 also provide
various non-limiting embodiments of methods and systems of converting lipids
in
a feedstock to carboxylic acid that can be esterified with product alcohol and
can
contemporaneously serve as an ISPR extractant.
[00196] In some embodiments, including any of the aforementioned embodiments
described with reference to FIGs. 1-5, the fermentation broth in fermentation
vessel 30 includes at least one recombinant microorganism 32 which is
genetically modified (that is, genetically engineered) to produce butanol via
a
biosynthetic pathway from at least one fermentable carbon source. In
particular,
recombinant microorganisms can be grown in a fermentation broth which
contains suitable carbon substrates. Additional carbon substrates may include,
but are not limited to, monosaccharides such as fructose and galactose;
oligosaccharides such as lactose maltose, or sucrose; polysaccharides such as
starch or cellulose; or mixtures thereof and unpurified mixtures from
renewable
feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet
molasses, and barley malt. Other carbon substrates may include ethanol,
lactate, succinate, or glycerol.
[00197] Additionally, the carbon substrate may also be one-carbon substrates
such
as carbon dioxide or methanol for which metabolic conversion into key
biochemical intermediates has been demonstrated. In addition to one and two
carbon substrates, methylotrophic organisms are also known to utilize a number
of other carbon containing compounds such as methylamine, glucosamine, and a
variety of amino acids for metabolic activity. For example, methylotrophic
yeasts
are known to utilize the carbon from methylamine to form trehalose or glycerol
(Bellion, et al., Microb. Growth C1 Compd., [Int. Symp.], 7th (1993), 415-32,
Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover,
UK).
Similarly, various species of Candida will metabolize alanine or oleic acid
(Sulter
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et al., Arch. Microbiol. 153:485-489, 1990). Hence, it is contemplated that
the
source of carbon utilized in the present invention may encompass a wide
variety
of carbon containing substrates and will only be limited by the choice of
organism.
[00198] Although it is contemplated that all of the above mentioned carbon
substrates and mixtures thereof are suitable, in some embodiments, the carbon
substrates are glucose, fructose, and sucrose, or mixtures of these with C5
sugars such as xylose and/or arabinose for yeasts cells modified to use C5
sugars. Sucrose may be derived from renewable sugar sources such as sugar
cane, sugar beets, cassava, sweet sorghum, and mixtures thereof. Glucose and
dextrose may be derived from renewable grain sources through saccharification
of starch-based feedstocks including grains such as corn, wheat, rye, barley,
oats, and mixtures thereof. In addition, fermentable sugars may be derived
from
renewable cellulosic or lignocellulosic biomass through processes of
pretreatment and saccharification, as described, for example, U.S. Patent
Application Publication No. 2007/0031918 Al, which is herein incorporated by
reference. In addition to an appropriate carbon source (from aqueous stream
22), fermentation broth must contain suitable minerals, salts, cofactors,
buffers,
and other components, known to those skilled in the art, suitable for the
growth of
the cultures and promotion of an enzymatic pathway for production of a product
alcohol.
[00199] From the above discussion and the Examples, one skilled in the art can
ascertain essential characteristics of the present invention and can make
various
changes and modifications of the invention to adapt to various uses and
conditions without departing from the present invention. For example, in some
embodiments, alcohol esterification and extraction according to the present
invention can be employed pre-fermentation, that is, during seed culturing of
microorganisms 32 prior to fermentation in fermentation vessel 30. Typically,
microorganisms 32 such as yeast can be grown from a seed culture to a desired
cell concentration before being harvested and inoculated into fermentation
vessel
30, as known in the art.
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[00200] The carbon source feedstock is an important cost factor in
microorganism
production such as yeast production and consequently, the biomass yield on
sugar is an important optimization criterion. Because the ATP yield from the
alcoholic fermentation is much lower than that from the respiratory sugar
dissimilation, occurrence of alcoholic fermentation negatively affects the
biomass
yield and is sought to be avoided during the yeast production (i.e., seed
culturing). Nonetheless, the culturing of microorganisms in a seed culture
medium can produce an amount of fermentation product including alcohol. For
example, in S. cerevisiae yeast, the alcoholic fermentation and respiration
occur
simultaneously whenever the specific growth rate (p) and/or the sugar
concentration in aerobic cultures exceed a critical value (see, e.g., van
Hoek, et
al., Biotechnol. Bioeng. 68:517-523, 2000). In order to achieve high biomass
yield, the yeast growth is typically controlled, for example, by respiratory
conditions using fed-batch fermentation technology for seed culturing. For
example, sugar is fed at a low rate resulting in a low sugar concentration in
the
culture and a low rate of sugar uptake such that sugar metabolism can be
substantially respiratory. Under these conditions, high biomass yields can be
obtained and accumulation of toxic products can be minimized. In practice, in
large scale fed-batch industrial processes, the cells can be exposed to
concentration gradients due to an inefficient mixing (see, e.g., Enfors, et
al., J.
Biotechnol. 85:175-185, 2001). Production and reassimilation of fermentation
by-
products can be one of the reasons for reduction of biomass yield per glucose
in
large scale bioreactors compared to laboratory scale.
[00201] However, at these conditions, when culturing butanol-producing yeast,
for
example, the fermentation product including butanol cannot be reassimilated
and
may accumulate in the culture medium which can be toxic to the microorganisms
at high concentration. If product accumulation exceeds critical cell growth
inhibitory concentrations (e.g., cell growth is lower than the growth that may
be
limited by the feed), then a loss of fed-batch control may occur. According to
the
present invention, using alcohol esterification and extraction to remove
butanol
from the culture medium can allow the fed-batch fermentation to proceed
despite
the problems with inefficient mixing and butanol toxicity.
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[00202] Thus, according to some embodiments, the seed culture medium can be
contacted with catalyst 42 and carboxylic acid 28 leading to the production of
alcohol esters by esterification of the product alcohol and ultimately, an
improved
biomass yield per glucose in large scale bioreactors. Furthermore, the
concentration of product alcohol in the culture medium can be controlled by
alcohol esterification and thus, minimizing or avoiding the deleterious
effects of
the product alcohol on the microorganisms. In some embodiments, alcohol
esters can be extracted from the seed culture medium and the alcohol recovered
from the alcohol esters in the same manner as described above with respect to
extraction of alcohol esters from fermentation vessel 30 and recovery of
product
alcohol 54. In some embodiments, alcohol esterification according to the
present
invention can be employed to esterify the product alcohol in both the seed
culture
medium and the fermentation medium. In such embodiments, a higher yield of
product alcohol can be achieved for the fermentation process as a whole by
recovering not only alcohol esters (and free product alcohol) from the
fermentation medium, but also recovering alcohol esters produced during the
seed culturing (e.g., recovering alcohol esters and/or product alcohol from a
propagation tank). In some embodiments, alcohol esterification according to
the
present invention can be employed pre-fermentation for removal of alcohol from
the seed culture medium, while conventional ISPR of product alcohol can be
employed for removal of product alcohol during fermentation in fermentation
vessel 30.
[00203] Thus, it should be apparent that alcohol esterification and extraction
according to the present invention can be employed at various stages in an
alcohol fermentation process without departing from the present invention.
[00204] The alcohol products produced by the methods of the present invention
have a number of applications, for example, as reagents, solvents, and fuel.
Butanol produced by the claimed methods may be used directly as a fuel (e.g.,
biofuel), a fuel additive, an alcohol used for the production of esters that
can be
used as diesel or biodiesel fuel, a feedstock chemical in the plastics
industry, an
ingredient in formulated products such as cosmetics, and a chemical
intermediate. Butanol may also be used as a solvent for paints, coatings,
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varnishes, resins, gums, dyes, fats, waxes, resins, shellac, rubbers, and
alkaloids. Thus, the present invention provides alternative methods to produce
alcohols including butanol, which can support the high demand for these
industrial chemicals.
Recombinant microorganisms and butanol biosynthetic pathways
[0128] While not wishing to be bound by theory, it is believed that the
processes
described herein are useful in conjunction with any alcohol producing
microorganism, particularly recombinant microorganisms which produce alcohol
at titers above their tolerance levels.
[0129] Alcohol-producing microorganisms are known in the art. For example,
fermentative oxidation of methane by methanotrophic bacteria (e.g.,
Methylosinus
tfichosoorium) produces methanol, and contacting methanol (a Ci alkyl alcohol)
with a carboxylic acid and a catalyst capable of esterifying the carboxylic
acid
with methanol forms a methanol ester of the carboxylic acid. The yeast strain
CEN.PK113-7D (CBS 8340, the Centraal Buro voor Schimmelculture; van Dijken,
et al., Enzyme Microb. Techno. 26:706-714, 2000) can produce ethanol, and
contacting ethanol with a carboxylic acid and a catalyst capable of
esterifying the
carboxylic acid with the ethanol forms ethyl ester (see, e.g., Example 36).
[0130] Recombinant microorganisms which produce alcohol are also known in
the art (e.g., Ohta, et al., Appl. Environ. Microbiol. 57:893-900, 1991;
Underwood,
et al., Appl. Environ. Microbiol. 68:1071-1081, 2002; Shen and Liao, Metab.
Eng.
10:312-320, 2008; Hahnai, et al., Appl. Environ. Microbiol. 73:7814-7818,
2007;
U.S. Patent No. 5,514,583; U.S. Patent No. 5,712,133; PCT Application
Publication No. WO 1995/028476; Feldmann, et al., Appl. Microbiol. Biotechnol.
38: 354-361, 1992; Zhang, et al., Science 267:240-243, 1995; U.S. Patent
Application Publication No. 2007/0031918 Al; U.S. Patent No. 7,223,575; U.S.
Patent No. 7,741,119; U.S. Patent Application Publication No. 2009/0203099 Al;
U.S. Patent Application Publication No. 2009/0246846 Al; and PCT Application
Publication No. WO 2010/075241, which are herein incorporated by reference).
[0131] Suitable recombinant microorganisms capable of producing butanol are
known in the art, and certain suitable microorganisms capable of producing
butanol are described herein. Recombinant microorganisms to produce butanol
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via a biosynthetic pathway can include a member of the genera Clostridium,
Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella,
Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes,
Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium,
Schizosaccharomyces, Kluyveromyces, Yarrowia, Pichia, Candida, Hansenula,
Issatchenkia, or Saccharomyces. In one embodiment, recombinant
microorganisms can be selected from the group consisting of Escherichia coli,
Lactobacillus plantarum, Kluyveromyces lactis, Kluyveromyces marxianus, and
Saccharomyces cerevisiae. In one embodiment, the recombinant microorganism
is yeast. In one embodiment, the recombinant microorganism is crabtree-
positive
yeast selected from Saccharomyces, Zygosaccharomyces,
Schizosaccharomyces, Dekkera, Torulopsis, Brettanomyces, and some species
of Candida. Species of crabtree-positive yeast include, but are not limited
to,
Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces
pombe, Saccharomyces bayanus, Saccharomyces mikitae, Saccharomyces
paradoxus, Zygosaccharomyces rouxii, and Candida glabrata.
[0132] In some embodiments, the host cell is Saccharomyces cerevisiae. S.
cerevisiae yeast are known in the art and are available from a variety of
sources
including, but not limited to, American Type Culture Collection (Rockville,
MD),
Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre,
LeSaffre, Gert Strand AB, Ferm Solutions, North American Bioproducts, Martrex,
and Lallemand. S. cerevisiae include, but are not limited to, BY4741, CEN.PK
113-7D, Ethanol Red yeast, Ferm ProTM yeast, Bio-Ferm XR yeast, Gert
Strand Prestige Batch Turbo alcohol yeast, Gert Strand Pot Distillers yeast,
Gert
Strand Distillers Turbo yeast, FerMaxTM Green yeast, FerMaxTM Gold yeast,
Thermosacc yeast, BG-1, PE-2, CAT-1, CBS7959, CBS7960, and CBS7961.
[0133] The production of butanol utilizing fermentation with a microorganism,
as
well as microorganisms which produce butanol, is disclosed, for example, in
U.S.
Patent Application Publication No. 2009/0305370, herein incorporated by
reference. In some embodiments, microorganisms comprise a butanol
biosynthetic pathway. In some embodiments, at least one, at least two, at
least
three, or at least four polypeptides catalyzing substrate to product
conversions of
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a pathway are encoded by heterologous polynucleotides in the microorganism.
In some embodiments, all polypeptides catalyzing substrate to product
conversions of a pathway are encoded by heterologous polynucleotides in the
microorganism. In some embodiments, the microorganism comprises a reduction
or elimination of pyruvate decarboxylase activity. Microorganisms
substantially
free of pyruvate decarboxylase activity are described in US Application
Publication No. 2009/0305363, herein incorporated by reference.
Microorganisms substantially free of an enzyme having NAD-dependent glycerol-
3-phosphate dehydrogenase activity such as GPD2 are also described therein.
[0134] Suitable biosynthetic pathways for production of butanol are known in
the
art, and certain suitable pathways are described herein. In some embodiments,
the butanol biosynthetic pathway comprises at least one gene that is
heterologous to the host cell. In some embodiments, the butanol biosynthetic
pathway comprises more than one gene that is heterologous to the host cell. In
some embodiments, the butanol biosynthetic pathway comprises heterologous
genes encoding polypeptides corresponding to every step of a biosynthetic
pathway.
[0135] Certain suitable proteins having the ability to catalyze indicated
substrate
to product conversions are described herein and other suitable proteins are
provided in the art. For example, U.S. Patent Application Publication Nos.
2008/0261230, 2009/0163376, and 2010/0197519, incorporated herein by
reference, describe acetohydroxy acid isomeroreductases; U.S. Patent
Application Publication No. 2010/0081154, incorporated by reference, describes
dihydroxyacid dehydratases; an alcohol dehydrogenase is described in U.S.
Patent Application Publication No. 2009/0269823, incorporated herein by
reference.
[0136] It is well understood by one skilled in the art that many levels of
sequence
identity are useful in identifying polypeptides from other species, wherein
such
polypeptides have the same or similar function or activity and are suitable
for use
in the recombinant microorganisms described herein. Useful examples of
percent identities include, but are not limited to, 75%, 80%, 85%, 90%, or
95%, or
any integer percentage from 75% to 100% may be useful in describing the
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present invention such as 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99%.
1 -Butanol Biosynthetic Pathway
[0137] A biosynthetic pathway for the production of 1-butanol as well as
suitable
polypeptides and polynucleotides encoding such polypeptides that may be used
is described by Donaldson, et al., in U.S. Patent Application Publication No.
2008/0182308 Al, incorporated herein by reference. This biosynthetic pathway
comprises the following substrate to product conversions:
[0138] a) acetyl-CoA to acetoacetyl-CoA, which may be catalyzed, for example,
by acetyl-CoA acetyltransferase;
[0139] b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA, which may be catalyzed, for
example, by 3-hydroxybutyryl-CoA dehydrogenase;
[0140] c) 3-hydroxybutyryl-CoA to crotonyl-CoA, which may be catalyzed, for
example, by crotonase;
[0141] d) crotonyl-CoA to butyryl-CoA, which may be catalyzed, for example, by
butyryl-CoA dehydrogenase;
[0142] e) butyryl-CoA to butyraldehyde, which may be catalyzed, for example,
by
butyraldehyde dehydrogenase; and
[0143] f) butyraldehyde to 1-butanol, which may be catalyzed, for example, by
1-
butanol dehydrogenase
[0144] In some embodiments, the 1-butanol biosynthetic pathway comprises at
least one gene, at least two genes, at least three genes, at least four genes,
or at
least five genes that is/are heterologous to the yeast cell. In some
embodiments,
the recombinant host cell comprises a heterologous gene for each substrate to
product conversion of a 1 -butanol biosynthetic pathway.
2-Butanol Biosynthetic Pathway
[0145] Biosynthetic pathways for the production of 2-butanol as well as
suitable
polypeptides and polynucleotides encoding such polypeptides that may be used
are described by Donaldson, et al., in U.S. Patent Application Publication
Nos.
2007/0259410 Al and 2007/0292927A1, and in PCT Application Publication No.
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WO 2007/130521, all of which are incorporated herein by reference. One 2-
butanol biosynthetic pathway comprises the following substrate to product
conversions:
[0146] a) pyruvate to alpha-acetolactate, which may be catalyzed, for example,
by acetolactate synthase;
[0147] b) alpha-acetolactate to acetoin, which may be catalyzed, for example,
by
acetolactate decarboxylase;
[0148] c) acetoin to 2,3-butanediol, which may be catalyzed, for example, by
butanediol dehydrogenase;
[0149] d) 2,3-butanediol to 2-butanone, which may be catalyzed, for example,
by
butanediol dehydratase; and
[0150] e) 2-butanone to 2-butanol, which may be catalyzed, for example, by 2-
butanol dehydrogenase.
[0151] In some embodiments, the 2-butanol biosynthetic pathway comprises at
least one gene, at least two genes, at least three genes, or at least four
genes
that is/are heterologous to the yeast cell. In some embodiments, the
recombinant
host cell comprises a heterologous gene for each substrate to product
conversion
of a 2-butanol biosynthetic pathway.
Isobutanol Biosynthetic Pathway
[0152] Biosynthetic pathways for the production of isobutanol as well as
suitable
polypeptides and polynucleotides encoding such polypeptides that may be used
are described in U.S. Patent Application Publication No. 2007/0092957 Al and
PCT Application Publication No. WO 2007/050671, incorporated herein by
reference. One isobutanol biosynthetic pathway comprises the following
substrate to product conversions:
[0153] a) pyruvate to acetolactate, which may be catalyzed, for example, by
acetolactate synthase;
[0154] b) acetolactate to 2,3-dihydroxyisovalerate, which may be catalyzed,
for
example, by acetohydroxy acid reductoisomerase;
[0155] c) 2,3-dihydroxyisovalerate to a-ketoisovalerate, which may be
catalyzed,
for example, by acetohydroxy acid dehydratase;
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[0156] d) a-ketoisovalerate to isobutyraldehyde, which may be catalyzed, for
example, by a branched-chain keto acid decarboxylase; and
[0157] e) isobutyraldehyde to isobutanol, which may be catalyzed, for example,
by a branched-chain alcohol dehydrogenase.
[0158] Suitable polypeptide sequences that encode enzymes which catalyze the
substrate to product conversions of the isobutanol biosynthetic pathway as
well
as E.C. numbers corresponding to suitable enzymes for the indicated pathway
steps include, but are not limited to, those in Tables AA and BB. Suitable
enzymes associated with the given E.C. numbers will be readily available to
those of skill in the art, for example, through the BRENDA database
(http://www.brenda-enzymes.org/).
Table AA: Example polypeptides
Pathway step Enzyme SEQ ID
NO:
a) pyruvate to acetolactate Bacillus subtilis alsS (acetolactate 144
synthase, "ALS")
b) acetolactate to 2,3- Lactococcus lactis ilvC (ketol-acid 145
dihydroxyisovalerate reductoisomerase, "KART")
c) 2,3-dihydroxyisovalerate to a- Streptococcus mutans ilvD 146
ketoisovalerate (dihydroxyacid dehydratase, "DHAD")
d) a-ketoisovalerate to Lactococcus lactis kivD (branched-chain 147
isobutyraldehyde a-keto acid decarboxylase), codon
optimized
e) isobutyraldehyde to isobutanol horse liver alcohol dehydrogenase 148
("AD H")
e) isobutyraldehyde to isobutanol Achromobacterxylosoxidans sadB 149
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Table BB: E.C. numbers
Pathway step E.C. Number:
a) pyruvate to acetolactate 2.2.1.6
b) acetolactate to 2,3-dihydroxyisovalerate 1.1.1.86
c) 2,3-dihydroxyisovalerate to a-ketoisovalerate 4.2.1.9
d) a-ketoisovalerate to isobutyraldehyde 4.1.1.72 or 4.1.1.1
e) isobutyraldehyde to isobutanol 1.1.1.265, 1.1.1.1 or 1.1.1.2
[0159] Provided herein are recombinant microorganisms comprising an
isobutanol biosynthetic pathway comprising steps a)-e) (above) wherein at
least
one of the enzymes selected from the group of the enzyme catalyzing step c)
and
the enzyme catalyzing step e) is encoded by a heterologous polynucleotide
integrated into the chromosome of the microorganism. In some embodiments,
both an enzyme catalyzing step c) is encoded by a heterologous polynucleotide
integrated into the chromosome of the microorganism, and enzyme catalyzing
step e) is encoded by a heterologous polynucleotide integrated into the
chromosome of the microorganism.
[0160] Provided herein are polynucleotides suitable for recombinant
microorganisms comprising a butanol biosynthetic pathway such as an isobutanol
biosynthetic pathway. Such polynucleotides include the coding region of the
alsS
gene from Bacillus subtilis (nt position 457-2172 of SEQ ID NO: 1) and the
ilvC
gene from Lactococcus lactis (nt 3634-4656 of SEQ ID NO: 1) as well as
plasmids comprising either or both. Also suitable is a chimeric gene having
the
coding region of the alsS gene from Bacillus subtilis (nt position 457-2172 of
SEQ
ID NO: 1) expressed from the yeast CUP1 promoter (nt 2-449 of SEQ ID NO: 1)
and followed by the CYC1 terminator (nt 2181-2430 of SEQ ID NO: 1) for
expression of ALS, and a chimeric gene having the coding region of the ilvC
gene
from Lactococcus lactis (nt 3634-4656 of SEQ ID NO: 1) expressed from the
yeast ILV5 promoter (2433-3626 of SEQ ID NO: 1) and followed by the ILV5
terminator (nt 4682-5304 of SEQ ID NO: 1) for expression of KART, as well as
plasmids comprising either or both chimeric genes.
[0161] Suitable polynucleotides include the coding region of the ilvD gene
from
Streptococcus mutans (nt position 3313-4849 of SEQ ID NO: 2), the coding
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region of codon optimized horse liver alcohol dehydrogenase (nt 6286-7413 of
SEQ ID NO: 2), the coding region of the codon-optimized kivD gene from
Lactococcus lactis (nt 9249-10895 of SEQ ID NO: 2) as well as plasmids
comprising any or all or any combination thereof. Also suitable is a chimeric
gene having the coding region of the ilvD gene from Streptococcus mutans (nt
position 3313-4849 of SEQ ID NO: 2) expressed from the S. cerevisiae FBA1
promoter (nt 2109 - 3105 of SEQ ID NO: 2) followed by the FBA1 terminator (nt
4858 - 5857 of SEQ ID NO: 2) for expression of DHAD; a chimeric gene having
the coding region of codon optimized horse liver alcohol dehydrogenase (nt
6286-7413 of SEQ ID NO: 2) expressed from the S. cerevisiae GPM1 promoter
(nt 7425-8181 of SEQ ID NO: 2) followed by the ADH1 terminator (nt 5962-6277
of SEQ ID NO: 2) for expression of ADH; and a chimeric gene having the coding
region of the codon-optimized kivD gene from Lactococcus lactis (nt 9249-10895
of SEQ ID NO: 2) expressed from the TDH3 promoter (nt 10896-11918 of SEQ
ID NO: 2) followed by the TDH3 terminator (nt 8237-9235 of SEQ ID NO: 2) for
expression of KivD as well as plasmids containing any, all, or any combination
of
such chimeric genes. In addition, suitable polynucleotides include those
having
at least about 75% identity to the coding regions and chimeric genes
specified, as
well as plasmids comprising such polynucleotides.
[0162] In some embodiments, the isobutanol biosynthetic pathway comprises at
least one gene, at least two genes, at least three genes, or at least four
genes
that is/are heterologous to the yeast cell. In some embodiments, the
recombinant
host cell comprises a heterologous gene for each substrate to product
conversion
of an isobutanol biosynthetic pathway.
[0163] Suitable strains include those described in certain applications cited
and
incorporated by reference herein as well as in U.S. Provisional Application
Serial
No. 61/380,563, filed on September 7, 2010. Construction of certain suitable
strains including those used in the Examples, is provided herein.
Construction of Saccharomyces cerevisiae strain BP1083 ("NGCI-070";
PNY1504)
[0164] The strain BP1064 was derived from CEN.PK 113-7D (CBS 8340;
Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre,
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Netherlands) and contains deletions of the following genes: URA3, HIS3, PDC1,
PDC5, PDC6, and GPD2. BP1064 was transformed with plasmids pYZ090 (SEQ
ID NO: 1, described in U.S. Provisional Application Serial No. 61/246,844) and
pLH468 (SEQ ID NO: 2) to create strain NGCI-070 (BP1 083, PNY1 504).
[0165] Deletions, which completely removed the entire coding sequence, were
created by homologous recombination with PCR fragments containing regions of
homology upstream and downstream of the target gene and either a G418
resistance marker or URA3 gene for selection of transformants. The G418
resistance marker, flanked by loxP sites, was removed using Cre recombinase.
The URA3 gene was removed by homologous recombination to create a scarless
deletion or if flanked by loxP sites, was removed using Cre recombinase.
[0166] The scarless deletion procedure was adapted from Akada, et al., (Yeast
23:399-405, 2006). In general, the PCR cassette for each scarless deletion was
made by combining four fragments, A-B-U-C, by overlapping PCR. The PCR
cassette contained a selectable/counter-selectable marker, URA3 (Fragment U),
consisting of the native CEN.PK 113-7D URA3 gene, along with the promoter
(250 bp upstream of the URA3 gene) and terminator (150 bp downstream of the
URA3 gene). Fragments A and C, each 500 bp long, corresponded to the 500 bp
immediately upstream of the target gene (Fragment A) and the 3' 500 bp of the
target gene (Fragment C). Fragments A and C were used for integration of the
cassette into the chromosome by homologous recombination. Fragment B (500
bp long) corresponded to the 500 bp immediately downstream of the target gene
and was used for excision of the URA3 marker and Fragment C from the
chromosome by homologous recombination, as a direct repeat of the sequence
corresponding to Fragment B was created upon integration of the cassette into
the chromosome. Using the PCR product ABUC cassette, the URA3 marker was
first integrated into and then excised from the chromosome by homologous
recombination. The initial integration deleted the gene, excluding the 3' 500
bp.
Upon excision, the 3' 500 bp region of the gene was also deleted. For
integration
of genes using this method, the gene to be integrated was included in the PCR
cassette between fragments A and B.
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URA3 Deletion
[0167] To delete the endogenous URA3 coding region, a ura3::loxP-kanMX-loxP
cassette was PCR-amplified from pLA54 template DNA (SEQ ID NO: 3). pLA54
contains the K. lactis TEF1 promoter and kanMX marker, and is flanked by loxP
sites to allow recombination with Cre recombinase and removal of the marker.
PCR was done using Phusion DNA polymerase (New England BioLabs Inc.,
Ipswich, MA) and primers BK505 and BK506 (SEQ ID NOs: 4 and 5). The URA3
portion of each primer was derived from the 5' region upstream of the URA3
promoter and 3' region downstream of the coding region such that integration
of
the loxP-kanMX-loxP marker resulted in replacement of the URA3 coding region.
The PCR product was transformed into CEN.PK 113-7D using standard genetic
techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY, pp. 201-202) and transformants were selected on
YPD containing G418 (100 pg/mL) at 30 C. Transformants were screened to
verify correct integration by PCR using primers LA468 and LA492 (SEQ ID NOs:
6 and 7) and designated CEN.PK 113-7D Aura3::kanMX.
HIS3 Deletion
[0168] The four fragments for the PCR cassette for the scarless HIS3 deletion
were amplified using Phusion High Fidelity PCR Master Mix (New England
BioLabs Inc., Ipswich, MA) and CEN.PK 113-7D genomic DNA as template,
prepared with a Gentra Puregene Yeast/Bact, kit (Qiagen, Valencia, CA).
HIS3 Fragment A was amplified with primer oBP452 (SEQ ID NO: 14) and primer
oBP453 (SEQ ID NO: 15) containing a 5' tail with homology to the 5' end of
HIS3
Fragment B. HIS3 Fragment B was amplified with primer oBP454 (SEQ ID NO:
16) containing a 5' tail with homology to the 3' end of HIS3 Fragment A, and
primer oBP455 (SEQ ID NO: 17) containing a 5' tail with homology to the 5' end
of HIS3 Fragment U. HIS3 Fragment U was amplified with primer oBP456 (SEQ
ID NO: 18) containing a 5' tail with homology to the 3' end of HIS3 Fragment
B,
and primer oBP457 (SEQ ID NO: 19) containing a 5' tail with homology to the 5'
end of HIS3 Fragment C. HIS3 Fragment C was amplified with primer oBP458
(SEQ ID NO: 20) containing a 5' tail with homology to the 3' end of HIS3
Fragment U, and primer oBP459 (SEQ ID NO: 21). PCR products were purified
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with a PCR Purification kit (Qiagen, Valencia, CA). HIS3 Fragment AB was
created by overlapping PCR by mixing HIS3 Fragment A and HIS3 Fragment B
and amplifying with primers oBP452 (SEQ ID NO: 14) and oBP455 (SEQ ID NO:
17). HIS3 Fragment UC was created by overlapping PCR by mixing HIS3
Fragment U and HIS3 Fragment C and amplifying with primers oBP456 (SEQ ID
NO: 18) and oBP459 (SEQ ID NO: 21). The resulting PCR products were
purified on an agarose gel followed by a Gel Extraction kit (Qiagen, Valencia,
CA). The HIS3 ABUC cassette was created by overlapping PCR by mixing HIS3
Fragment AB and HIS3 Fragment UC and amplifying with primers oBP452 (SEQ
ID NO: 14) and oBP459 (SEQ ID NO: 21). The PCR product was purified with a
PCR Purification kit (Qiagen, Valencia, CA).
[0169] Competent cells of CEN.PK 113-7D Aura3::kanMX were made and
transformed with the HIS3 ABUC PCR cassette using a Frozen-EZ Yeast
Transformation IITM kit (Zymo Research Corporation, Irvine, CA).
Transformation
mixtures were plated on synthetic complete media lacking uracil supplemented
with 2% glucose at 30 C. Transformants with a his3 knockout were screened for
by PCR with primers oBP460 (SEQ ID NO: 22) and oBP461 (SEQ ID NO: 23)
using genomic DNA prepared with a Gentra Puregene Yeast/Bact. kit
(Qiagen, Valencia, CA). A correct transformant was selected as strain CEN.PK
113-7D Aura3::kanMX Ahis3::URA3.
KanMX Marker Removal from the Aura3 Site and URA3 Marker Removal from
the Ahis3 Site
[0170] The KanMX marker was removed by transforming CEN.PK 113-7D
Aura3::kanMX Ahis3::URA3 with pRS423::PGAL1-cre (SEQ ID NO: 66,
described in U.S. Provisional Application No. 61/290,639) using a Frozen-EZ
Yeast Transformation IITM kit (Zymo Research Corporation, Irvine, CA) and
plating on synthetic complete medium lacking histidine and uracil supplemented
with 2% glucose at 30 C. Transformants were grown in YP supplemented with
1% galactose at 30 C for -6 hours to induce the Cre recombinase and KanMX
marker excision and plated onto YPD (2% glucose) plates at 30 C for recovery.
An isolate was grown overnight in YPD and plated on synthetic complete medium
containing 5-fluoro-orotic acid (5-FOA, 0.1%) at 30 C to select for isolates
that
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lost the URA3 marker. 5-FOA resistant isolates were grown in and plated on
YPD for removal of the pRS423::PGAL1-cre plasmid. Isolates were checked for
loss of the KanMX marker, URA3 marker, and pRS423::PGAL1-cre plasmid by
assaying growth on YPD+G418 plates, synthetic complete medium lacking uracil
plates, and synthetic complete medium lacking histidine plates. A correct
isolate
that was sensitive to G418 and auxotrophic for uracil and histidine was
selected
as strain CEN.PK 113-7D Aura3::loxP Ahis3 and designated as BP857. The
deletions and marker removal were confirmed by PCR and sequencing with
primers oBP450 (SEQ ID NO: 24) and oBP451 (SEQ ID NO: 25) for Aura3 and
primers oBP460 (SEQ ID NO: 22) and oBP461 (SEQ ID NO: 23) for Ahis3 using
genomic DNA prepared with a Gentra Puregene Yeast/Bact. kit (Qiagen,
Valencia, CA).
PDC6 Deletion
[0171] The four fragments for the PCR cassette for the scarless PDC6 deletion
were amplified using Phusion High Fidelity PCR Master Mix (New England
BioLabs Inc., Ipswich, MA) and CEN.PK 113-7D genomic DNA as template,
prepared with a Gentra Puregene Yeast/Bact. kit (Qiagen, Valencia, CA).
PDC6 Fragment A was amplified with primer oBP440 (SEQ ID NO: 26) and
primer oBP441 (SEQ ID NO: 27) containing a 5' tail with homology to the 5' end
of PDC6 Fragment B. PDC6 Fragment B was amplified with primer oBP442 (SEQ
ID NO: 28), containing a 5' tail with homology to the 3' end of PDC6 Fragment
A,
and primer oBP443 (SEQ ID NO: 29) containing a 5' tail with homology to the 5'
end of PDC6 Fragment U. PDC6 Fragment U was amplified with primer oBP444
(SEQ ID NO: 30) containing a 5' tail with homology to the 3' end of PDC6
Fragment B, and primer oBP445 (SEQ ID NO: 31) containing a 5' tail with
homology to the 5' end of PDC6 Fragment C. PDC6 Fragment C was amplified
with primer oBP446 (SEQ ID NO: 32) containing a 5' tail with homology to the
3'
end of PDC6 Fragment U, and primer oBP447 (SEQ ID NO: 33). PCR products
were purified with a PCR Purification kit (Qiagen, Valencia, CA). PDC6
Fragment
AB was created by overlapping PCR by mixing PDC6 Fragment A and PDC6
Fragment B and amplifying with primers oBP440 (SEQ ID NO: 26) and oBP443
(SEQ ID NO: 29). PDC6 Fragment UC was created by overlapping PCR by
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mixing PDC6 Fragment U and PDC6 Fragment C and amplifying with primers
oBP444 (SEQ ID NO: 30) and oBP447 (SEQ ID NO: 33). The resulting PCR
products were purified on an agarose gel followed by a Gel Extraction kit
(Qiagen, Valencia, CA). The PDC6 ABUC cassette was created by overlapping
PCR by mixing PDC6 Fragment AB and PDC6 Fragment UC and amplifying with
primers oBP440 (SEQ ID NO: 26) and oBP447 (SEQ ID NO: 33). The PCR
product was purified with a PCR Purification kit (Qiagen, Valencia, CA).
[0172] Competent cells of CEN.PK 113-7D Aura3::IoxP Ahis3 were made and
transformed with the PDC6 ABUC PCR cassette using a Frozen-EZ Yeast
Transformation IITM kit (Zymo Research Corporation, Irvine, CA).
Transformation
mixtures were plated on synthetic complete media lacking uracil supplemented
with 2% glucose at 30 C. Transformants with a pdc6 knockout were screened for
by PCR with primers oBP448 (SEQ ID NO: 34) and oBP449 (SEQ ID NO: 35)
using genomic DNA prepared with a Gentra Puregene Yeast/Bact. kit
(Qiagen, Valencia, CA). A correct transformant was selected as strain CEN.PK
113-7D Aura3::IoxP Ahis3 Apdc6::URA3.
[0173] CEN.PK 113-7D Aura3::IoxP Ahis3 Apdc6::URA3 was grown overnight in
YPD and plated on synthetic complete medium containing 5-fluoro-orotic acid
(0.1 %) at 30 C to select for isolates that lost the URA3 marker. The deletion
and
marker removal were confirmed by PCR and sequencing with primers oBP448
(SEQ ID NO: 34) and oBP449 (SEQ ID NO: 35) using genomic DNA prepared
with a Gentra Puregene Yeast/Bact. kit (Qiagen, Valencia, CA). The absence
of the PDC6 gene from the isolate was demonstrated by a negative PCR result
using primers specific for the coding sequence of PDC6, oBP554 (SEQ ID NO:
36) and oBP555 (SEQ ID NO: 37). The correct isolate was selected as strain
CEN.PK 113-7D Aura3::IoxP Ahis3 Apdc6 and designated as BP891.
PDC1 Deletion ilvDSm Integration
[0174] The PDC1 gene was deleted and replaced with the ilvD coding region from
Streptococcus mutans ATCC No. 700610. The A fragment followed by the ilvD
coding region from Streptococcus mutans for the PCR cassette for the PDC1
deletion-ilvDSm integration was amplified using Phusion High Fidelity PCR
Master Mix (New England BioLabs Inc., Ipswich, MA) and NYLA83 genomic DNA
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as template, prepared with a Gentra Puregene Yeast/Bact. kit (Qiagen,
Valencia, CA). NYLA83 is a strain (construction described in U.S. Patent
Application Publication No. 2011/0124060, incorporated herein by reference in
its
entirety) which carries the PDC1 deletion-ilvDSm integration described in U.S.
Patent Application Publication No. 2009/0305363, herein incorporated by
reference in its entirety). PDC1 Fragment A-ilvDSm (SEQ ID NO: 141) was
amplified with primer oBP513 (SEQ ID NO: 38) and primer oBP515 (SEQ ID NO:
39) containing a 5' tail with homology to the 5' end of PDC1 Fragment B. The
B,
U, and C fragments for the PCR cassette for the PDC1 deletion-ilvDSm
integration were amplified using Phusion High Fidelity PCR Master Mix (New
England BioLabs Inc., Ipswich, MA) and CEN.PK 113-7D genomic DNA as
template, prepared with a Gentra Puregene Yeast/Bact. kit (Qiagen, Valencia,
CA). PDC1 Fragment B was amplified with primer oBP516 (SEQ ID NO: 40)
containing a 5' tail with homology to the 3' end of PDC1 Fragment A-ilvDSm,
and
primer oBP517 (SEQ ID NO: 41) containing a 5' tail with homology to the 5' end
of PDC1 Fragment U. PDC1 Fragment U was amplified with primer oBP518
(SEQ ID NO: 42) containing a 5' tail with homology to the 3' end of PDC1
Fragment B, and primer oBP519 (SEQ ID NO: 43) containing a 5' tail with
homology to the 5' end of PDC1 Fragment C. PDC1 Fragment C was amplified
with primer oBP520 (SEQ ID NO: 44), containing a 5' tail with homology to the
3'
end of PDC1 Fragment U, and primer oBP521 (SEQ ID NO: 45). PCR products
were purified with a PCR Purification kit (Qiagen, Valencia, CA. PDC1 Fragment
A-ilvDSm-B was created by overlapping PCR by mixing PDC1 Fragment A-
ilvDSm and PDC1 Fragment B and amplifying with primers oBP513 (SEQ ID NO:
38) and oBP517 (SEQ ID NO: 41). PDC1 Fragment UC was created by
overlapping PCR by mixing PDC1 Fragment U and PDC1 Fragment C and
amplifying with primers oBP518 (SEQ ID NO: 42) and oBP521 (SEQ ID NO: 45).
The resulting PCR products were purified on an agarose gel followed by a Gel
Extraction kit (Qiagen, Valencia, CA). The PDC1 A-ilvDSm-BUC cassette (SEQ
ID NO: 142) was created by overlapping PCR by mixing PDC1 Fragment A-
ilvDSm-B and PDC1 Fragment UC and amplifying with primers oBP513 (SEQ ID
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NO: 38) and oBP521 (SEQ ID NO: 45). The PCR product was purified with a
PCR Purification kit (Qiagen, Valencia, CA).
[0175] Competent cells of CEN.PK 113-7D Eura3::IoxP Ehis3 Epdc6 were made
and transformed with the PDC1 A-ilvDSm-BUC PCR cassette using a Frozen-EZ
Yeast Transformation IITM kit (Zymo Research Corporation, Irvine, CA).
Transformation mixtures were plated on synthetic complete media lacking uracil
supplemented with 2% glucose at 30 C. Transformants with a pdcl knockout
ilvDSm integration were screened for by PCR with primers oBP511 (SEQ ID NO:
46) and oBP512 (SEQ ID NO: 47) using genomic DNA prepared with a Gentra
Puregene Yeast/Bact. kit (Qiagen, Valencia, CA). The absence of the PDC1
gene from the isolate was demonstrated by a negative PCR result using primers
specific for the coding sequence of PDC1, oBP550 (SEQ ID NO: 48) and oBP551
(SEQ ID NO: 49). A correct transformant was selected as strain CEN.PK 113-7D
Eura3::IoxP Ehis3 Epdc6 Epdcl ::ilvDSm-URA3.
[0176] CEN.PK 113-7D Eura3::IoxP Ehis3 Epdc6 Epdcl::ilvDSm-URA3 was
grown overnight in YPD and plated on synthetic complete medium containing 5-
fluoro-orotic acid (0.1 %) at 30 C to select for isolates that lost the URA3
marker.
The deletion of PDC1, integration of ilvDSm, and marker removal were confirmed
by PCR and sequencing with primers oBP511 (SEQ ID NO: 46) and oBP512
(SEQ ID NO: 47) using genomic DNA prepared with a Gentra Puregene
Yeast/Bact. kit (Qiagen, Valencia, CA). The correct isolate was selected as
strain
CEN.PK 113-7D Eura3::IoxP Ehis3 Epdc6 Epdcl::ilvDSm and designated as
BP907.
PDC5 Deletion sadB Integration
[0177] The PDC5 gene was deleted and replaced with the sadB coding region
from Achromobacter xylosoxidans. A segment of the PCR cassette for the PDC5
deletion-sadB integration was first cloned into plasmid pUC19-URA3MCS.
[0178] pUC19-URA3MCS is pUC19 based and contains the sequence of the
URA3 gene from Saccharomyces cerevisiae situated within a multiple cloning
site
(MCS). pUC19 contains the pMB1 replicon and a gene coding for beta-
lactamase for replication and selection in Escherichia coli. In addition to
the
coding sequence for URA3, the sequences from upstream and downstream of
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this gene were included for expression of the URA3 gene in yeast. The vector
can be used for cloning purposes and can be used as a yeast integration
vector.
[0179] The DNA encompassing the URA3 coding region along with 250 bp
upstream and 150 bp downstream of the URA3 coding region from
Saccharomyces cerevisiae CEN.PK 113-7D genomic DNA was amplified with
primers oBP438 (SEQ ID NO: 12) containing BamHI, Ascl, Pmel, and Fsel
restriction sites, and oBP439 (SEQ ID NO: 13) containing Xbal, Pacl, and Notl
restriction sites, using Phusion High Fidelity PCR Master Mix (New England
BioLabs Inc., Ipswich, MA). Genomic DNA was prepared using a Gentra
Puregene Yeast/Bact. kit (Qiagen, Valencia, CA). The PCR product and
pUC19 (SEQ ID NO: 150) were ligated with T4 DNA ligase after digestion with
BamHI and Xbal to create vector pUC19-URA3MCS. The vector was confirmed
by PCR and sequencing with primers oBP264 (SEQ ID NO: 10) and oBP265
(SEQ ID NO: 11).
[0180] The coding sequence of sadB and PDC5 Fragment B were cloned into
pUC19-URA3MCS to create the sadB-BU portion of the PDC5 A-sadB-BUC PCR
cassette. The coding sequence of sadB was amplified using pLH468-sadB (SEQ
ID NO: 67) as template with primer oBP530 (SEQ ID NO: 50) containing an Ascl
restriction site, and primer oBP531 (SEQ ID NO: 51) containing a 5' tail with
homology to the 5' end of PDC5 Fragment B. PDC5 Fragment B was amplified
with primer oBP532 (SEQ ID NO: 52) containing a 5' tail with homology to the
3'
end of sadB, and primer oBP533 (SEQ ID NO: 53) containing a Pmel restriction
site. PCR products were purified with a PCR Purification kit (Qiagen,
Valencia,
CA). sadB-PDC5 Fragment B was created by overlapping PCR by mixing the
sadB and PDC5 Fragment B PCR products and amplifying with primers oBP530
(SEQ ID NO: 50) and oBP533 (SEQ ID NO: 53). The resulting PCR product was
digested with Ascl and Pmel and ligated with T4 DNA ligase into the
corresponding sites of pUC19-URA3MCS after digestion with the appropriate
enzymes. The resulting plasmid was used as a template for amplification of
sadB-Fragment B-Fragment U using primers oBP536 (SEQ ID NO: 54) and
oBP546 (SEQ ID NO: 55) containing a 5' tail with homology to the 5' end of
PDC5 Fragment C. PDC5 Fragment C was amplified with primer oBP547 (SEQ
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ID NO: 56) containing a 5' tail with homology to the 3' end of PDC5 sadB-
Fragment B-Fragment U, and primer oBP539 (SEQ ID NO: 57). PCR products
were purified with a PCR Purification kit (Qiagen, Valencia, CA). PDC5 sadB-
Fragment B-Fragment U-Fragment C was created by overlapping PCR by mixing
PDC5 sadB-Fragment B-Fragment U and PDC5 Fragment C and amplifying with
primers oBP536 (SEQ ID NO: 54) and oBP539 (SEQ ID NO: 57). The resulting
PCR product was purified on an agarose gel followed by a Gel Extraction kit
(Qiagen, Valencia, CA). The PDC5 A-sadB-BUC cassette (SEQ ID NO: 143)
was created by amplifying PDC5 sadB-Fragment B-Fragment U-Fragment C with
primers oBP542 (SEQ ID NO: 58) containing a 5' tail with homology to the 50
nucleotides immediately upstream of the native PDC5 coding sequence, and
oBP539 (SEQ ID NO: 57). The PCR product was purified with a PCR Purification
kit (Qiagen, Valencia, CA).
[0181] Competent cells of CEN.PK 113-7D Aura3::IoxP Ahis3 Apdc6
Apdcl::ilvDSm were made and transformed with the PDC5 A-sadB-BUC PCR
cassette using a Frozen-EZ Yeast Transformation IITM kit (Zymo Research
Corporation, Irvine, CA). Transformation mixtures were plated on synthetic
complete media lacking uracil supplemented with 1% ethanol (no glucose) at
30 C. Transformants with a pdc5 knockout sadB integration were screened for
by PCR with primers oBP540 (SEQ ID NO: 59) and oBP541 (SEQ ID NO: 60)
using genomic DNA prepared with a Gentra Puregene Yeast/Bact. kit
(Qiagen, Valencia, CA). The absence of the PDC5 gene from the isolate was
demonstrated by a negative PCR result using primers specific for the coding
sequence of PDC5, oBP552 (SEQ ID NO: 61) and oBP553 (SEQ ID NO: 62). A
correct transformant was selected as strain CEN.PK 113-7D Aura3::IoxP Ahis3
Apdc6 Apdcl ::ilvDSm Apdc5::sadB-URA3.
[0182] CEN.PK 113-7D Aura3::IoxP Ahis3 Apdc6 Apdcl::ilvDSm Apdc5::sadB-
URA3 was grown overnight in YPE (1% ethanol) and plated on synthetic
complete medium supplemented with ethanol (no glucose) and containing 5-
fluoro-orotic acid (0.1 %) at 30 C to select for isolates that lost the URA3
marker.
The deletion of PDC5, integration of sadB, and marker removal were confirmed
by PCR with primers oBP540 (SEQ ID NO: 59) and oBP541 (SEQ ID NO: 60)
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using genomic DNA prepared with a Gentra Puregene Yeast/Bact. kit
(Qiagen, Valencia, CA). The correct isolate was selected as strain CEN.PK 113-
7D Aura3::loxP Ahis3 Apdc6 Apdcl::ilvDSm Apdc5::sadB and designated as
BP913.
GPD2 Deletion
[0183] To delete the endogenous GPD2 coding region, a gpd2::loxP-URA3-loxP
cassette (SEQ ID NO: 151) was PCR-amplified using loxP-URA3-loxP (SEQ ID
NO: 68) as template DNA. loxP-URA3-loxP contains the URA3 marker from
(ATCC No. 77107) flanked by IoxP recombinase sites. PCR was done using
Phusion DNA polymerase (New England BioLabs Inc., Ipswich, MA) and
primers LA512 and LA513 (SEQ ID NOs: 8 and 9). The GPD2 portion of each
primer was derived from the 5' region upstream of the GPD2 coding region and
3'
region downstream of the coding region such that integration of the IoxP-URA3-
IoxP marker resulted in replacement of the GPD2 coding region. The PCR
product was transformed into BP913 and transformants were selected on
synthetic complete media lacking uracil supplemented with 1% ethanol (no
glucose). Transformants were screened to verify correct integration by PCR
using primers oBP582 and AA270 (SEQ ID NOs: 63 and 64).
[0184] The URA3 marker was recycled by transformation with pRS423::PGAL1-
cre (SEQ ID NO: 66) and plating on synthetic complete media lacking histidine
supplemented with 1 % ethanol at 30 C. Transformants were streaked on
synthetic complete medium supplemented with 1% ethanol and containing 5-
fluoro-orotic acid (0.1 %) and incubated at 30 C to select for isolates that
lost the
URA3 marker. 5-FOA resistant isolates were grown in YPE (1% ethanol) for
removal of the pRS423::PGAL1-cre plasmid. The deletion and marker removal
were confirmed by PCR with primers oBP582 (SEQ ID NO: 63) and oBP591
(SEQ ID NO: 65). The correct isolate was selected as strain CEN.PK 113-7D
Aura3::IoxP Ahis3 Apdc6 Apdcl::ilvDSm Apdc5::sadB Agpd2::loxP and
designated as PNY1503 (BP1064).
[0185] BP1064 was transformed with plasmids pYZ090 (SEQ ID NO: 1) and
pLH468 (SEQ ID NO: 2) to create strain NGCI-070 (BP1 083; PNY1 504).
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Construction of Saccharomyces cerevisiae strain PNY2205
[0186] The strain, PNY2205, was derived from PNY1503 (BP1064) which is
described above.
[0187] Deletions, which generally removed the entire coding sequence, were
created by homologous recombination with PCR fragments containing regions of
homology upstream and downstream of the target gene and the URA3 gene for
selection of transformants. The URA3 gene was removed by homologous
recombination to create a scarless deletion. Gene integrations were generated
in
a similar manner.
[0188] The scarless deletion procedure was adapted from Akada et al., (Yeast,
23:399, 2006). In general, the PCR cassette for each scarless deletion was
made by combining four fragments, A-B-U-C, by overlapping PCR. In some
instances, the individual fragments were first cloned into a plasmid prior to
the
entire cassette being amplified by PCR for the deletion/integration procedure.
The PCR cassette contained a selectable/counter-selectable marker, URA3
(Fragment U), consisting of the native CEN.PK 113-7D URA3 gene, along with
the promoter (250 bp upstream of the URA3 gene) and terminator (150 bp
downstream of the URA3 gene) regions. Fragments A and C, each generally 500
bp long, corresponded to the 500 bp immediately upstream of the target gene
(Fragment A) and the 3' 500 bp of the target gene (Fragment C). Fragments A
and C were used for integration of the cassette into the chromosome by
homologous recombination. Fragment B (500 bp long) corresponded to the 500
bp immediately downstream of the target gene and was used for excision of the
URA3 marker and Fragment C from the chromosome by homologous
recombination, as a direct repeat of the sequence corresponding to Fragment B
was created upon integration of the cassette into the chromosome.
[0189] Using the PCR product ABUC cassette, the URA3 marker was first
integrated into and then excised from the chromosome by homologous
recombination. The initial integration deleted the gene, excluding the 3' 500
bp.
Upon excision, the 3' 500 bp region of the gene was also deleted. For
integration
of genes using this method, the gene to be integrated was included in the PCR
cassette between fragments A and B.
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FRA2 Deletion
[0190] The FRA2 deletion was designed to delete 250 nucleotides from the 3'
end
of the coding sequence, leaving the first 113 nucleotides of the FRA2 coding
sequence intact. An in-frame stop codon was present 7 nucleotides downstream
of the deletion. The four fragments for the PCR cassette for the scarless FRA2
deletion were amplified using Phusion High Fidelity PCR Master Mix (New
England BioLabs Inc., Ipswich, MA) and CEN.PK 113-7D genomic DNA as
template, prepared with a Gentra Puregene Yeast/Bact. kit (Qiagen, Valencia,
CA). FRA2 Fragment A was amplified with primer oBP594 (SEQ ID NO: 152)
and primer oBP595 (SEQ ID NO: 153), containing a 5' tail with homology to the
5'
end of FRA2 Fragment B. FRA2 Fragment B was amplified with primer oBP596
(SEQ ID NO: 154), containing a 5' tail with homology to the 3' end of FRA2
Fragment A, and primer oBP597 (SEQ ID NO: 155), containing a 5' tail with
homology to the 5' end of FRA2 Fragment U. FRA2 Fragment U was amplified
with primer oBP598 (SEQ ID NO: 156), containing a 5' tail with homology to the
3'
end of FRA2 Fragment B, and primer oBP599 (SEQ ID NO: 157), containing a 5'
tail with homology to the 5' end of FRA2 Fragment C. FRA2 Fragment C was
amplified with primer oBP600 (SEQ ID NO: 158), containing a 5' tail with
homology to the 3' end of FRA2 Fragment U, and primer oBP601 (SEQ ID NO:
159). PCR products were purified with a PCR Purification kit (Qiagen,
Valencia,
CA). FRA2 Fragment AB was created by overlapping PCR by mixing FRA2
Fragment A and FRA2 Fragment B and amplifying with primers oBP594 (SEQ ID
NO: 152) and oBP597 (SEQ ID NO: 155). FRA2 Fragment UC was created by
overlapping PCR by mixing FRA2 Fragment U and FRA2 Fragment C and
amplifying with primers oBP598 (SEQ ID NO: 156) and oBP601 (SEQ ID NO:
159). The resulting PCR products were purified on an agarose gel followed by a
Gel Extraction kit (Qiagen, Valencia, CA). The FRA2 ABUC cassette was
created by overlapping PCR by mixing FRA2 Fragment AB and FRA2 Fragment
UC and amplifying with primers oBP594 (SEQ ID NO: 152) and oBP601 (SEQ ID
NO: 159). The PCR product was purified with a PCR Purification kit (Qiagen,
Valencia, CA).
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[0191] Competent cells of PNY1503 were made and transformed with the FRA2
ABUC PCR cassette using a Frozen-EZ Yeast Transformation IITM kit (Zymo
Research Corporation, Irvine, CA). Transformation mixtures were plated on
synthetic complete media lacking uracil supplemented with 1% ethanol at 30 C.
Transformants with a fra2 knockout were screened for by PCR with primers
oBP602 (SEQ ID NO: 160) and oBP603 (SEQ ID NO: 161) using genomic DNA
prepared with a Gentra Puregene Yeast/Bact. kit (Qiagen, Valencia, CA). A
correct transformant was grown in YPE (yeast extract, peptone, 1 % ethanol)
and
plated on synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at
30 C to select for isolates that lost the URA3 marker. The deletion and marker
removal were confirmed by PCR with primers oBP602 (SEQ ID NO: 160) and
oBP603 (SEQ ID NO: 161) using genomic DNA prepared with a Gentra
Puregene Yeast/Bact. kit (Qiagen, Valencia, CA). The absence of the FRA2
gene from the isolate was demonstrated by a negative PCR result using primers
specific for the deleted coding sequence of FRA2, oBP605 (SEQ ID NO: 162)
and oBP606 (SEQ ID NO: 163). The correct isolate was selected as strain
CEN.PK 113-7D MATa ura3&::IoxP his3E pdc6E pdcl&::P[PDC1]-
DHADIiIvD_Sm-PDC1t pdc5&::P[PDC5]-ADHIsadB_Ax-PDC5t gpd2&::IoxP fra2E
and designated as PNY1 505 (BP1 135).
ADH1 Deletion and kivD LI(y) Integration
[0192] The ADH1 gene was deleted and replaced with the kivD coding region
from Lactococcus lactis codon optimized for expression in Saccharomyces
cerevisiae. The scarless cassette for the ADH1 deletion-kivD LI(y) integration
was first cloned into plasmid pUC19-URA3MCS, as described in U.S. Provisional
Application Serial No. 61/356,379, filed June 18, 2010, incorporated herein by
reference. The vector is pUC19 based and contains the sequence of the URA3
gene from Saccharomyces cerevisiae CEN.PK 113-7D situated within a multiple
cloning site (MCS). pUC19 contains the pMB1 replicon and a gene coding for
beta-lactamase for replication and selection in Escherichia coli. In addition
to the
coding sequence for URA3, the sequences from upstream (250 bp) and
downstream (150 bp) of this gene are present for expression of the URA3 gene
in
yeast.
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[0193] The kivD coding region from Lactococcus lactis codon optimized for
expression in Saccharomyces cerevisiae was amplified using pLH468 (U.S.
Provisional Application Serial No. 61/246,709, filed September 29, 2009) as
template with primer oBP562 (SEQ ID NO: 164), containing a Pmel restriction
site, and primer oBP563 (SEQ ID NO: 165), containing a 5' tail with homology
to
the 5' end of ADH1 Fragment B. ADH1 Fragment B was amplified from genomic
DNA prepared as above with primer oBP564 (SEQ ID NO: 166), containing a 5'
tail with homology to the 3' end of kivD LI(y), and primer oBP565 (SEQ ID NO:
167), containing a Fsel restriction site. PCR products were purified with a
PCR
Purification kit (Qiagen, Valencia, CA). kivD_Ll(y)-ADH1 Fragment B was
created by overlapping PCR by mixing the kivD_Ll(y) and ADH1 Fragment B
PCR products and amplifying with primers oBP562 (SEQ ID NO: 164) and
oBP565 (SEQ ID NO: 167). The resulting PCR product was digested with Pmel
and Fsel and ligated with T4 DNA ligase into the corresponding sites of pUC19-
URA3MCS after digestion with the appropriate enzymes. ADH1 Fragment A was
amplified from genomic DNA with primer oBP505 (SEQ ID NO: 168) containing a
Sacl restriction site, and primer oBP506 (SEQ ID NO: 169), containing an Ascl
restriction site. The ADH1 Fragment A PCR product was digested with Sacl and
Ascl and ligated with T4 DNA ligase into the corresponding sites of the
plasmid
containing kivD_Ll(y)-ADH1 Fragment B. ADH1 Fragment C was amplified from
genomic DNA with primer oBP507 (SEQ ID NO: 170), containing a Pacl
restriction site, and primer oBP508 (SEQ ID NO: 171), containing a Sall
restriction site. The ADH1 Fragment C PCR product was digested with Pacl and
Sall and ligated with T4 DNA ligase into the corresponding sites of the
plasmid
containing ADH1 Fragment A-kivD_Ll(y)-ADH1 Fragment B. The hybrid
promoter UAS(PGK1)-PFBA1 was amplified from vector pRS316-UAS(PGK1)-
PFBA1-GUS (SEQ ID NO: 172) with primer oBP674 (SEQ ID NO: 173), containing
an Ascl restriction site, and primer oBP675 (SEQ ID NO: 174), containing a
Pmel
restriction site. The UAS(PGK1)-PFBA1 PCR product was digested with Ascl and
Pmel and ligated with T4 DNA ligase into the corresponding sites of the
plasmid
containing kivD_Ll(y)-ADH1 Fragments ABC. The entire integration cassette was
amplified from the resulting plasmid with primers oBP505 (SEQ ID NO: 168) and
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oBP508 (SEQ ID NO: 171) and purified with a PCR Purification kit (Qiagen,
Valencia, CA).
[0194] Competent cells of PNY1505 were made and transformed with the ADH1-
kivD_LI(y) PCR cassette constructed above using a Frozen-EZ Yeast
Transformation IITM kit (Zymo Research Corporation, Irvine, CA).
Transformation
mixtures were plated on synthetic complete media lacking uracil supplemented
with 1 % ethanol at 30 C. Transformants were grown in YPE (1 % ethanol) and
plated on synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at
30 C to select for isolates that lost the URA3 marker. The deletion of ADH1
and
integration of kivD LI(y) were confirmed by PCR with external primers oBP495
(SEQ ID NO: 175) and oBP496 (SEQ ID NO: 176) and with kivD LI(y) specific
primer oBP562 (SEQ ID NO: 164) and external primer oBP496 (SEQ ID NO: 176)
using genomic DNA prepared with a Gentra Puregene Yeast/Bact. kit
(Qiagen, Valencia, CA). The correct isolate was selected as strain CEN.PK 113-
7D MATa ura3&::IoxP his3A pdc6A pdcl&::P[PDC1]-DHADIiIvD_Sm-
PDCltpdc5&::P[PDC5]-ADHIsadB_Ax-PDC5t gpd2&::IoxP fra2A
adhl A::UAS(PGK1)P[FBA1 ]-kivD_LI(y)-ADH1 t and designated as PNY1507
(BP1201). PNY1507 was transformed with isobutanol pathway plasmids pYZ090
(SEQ ID NO: 1) and pBP915 (described below).
Construction of the pRS316-UAS(PGK1)-FBA1 p-GUS vector
[0195] To clone a cassette UAS(PGK1)-FBA1 p (SEQ ID NO: 177, first a 602bp
FBA1 promoter (FBA1 p) was PCR-amplified from genomic DNA of CEN.PK with
primers T-FBA1(Sall) (SEQ ID NO: 178) and B-FBA1(Spel) (SEQ ID NO: 179),
and cloned into Sall and Spel sites on the plasmid pWS358-PGK1 p-GUS (SEQ
ID NO: 180) after the PGK1p promoter was removed with a Sall/Spel digest of
the plasmid, yielding pWS358-FBA1 p-GUS. The pWS358-PGK1 p-GUS plasmid
was generated by inserting a PGK1p and beta-glucuronidase gene (GUS) DNA
fragments into multiple cloning site of pWS358, which was derived from pRS423
vector (Christianson, et al., Gene 110:119-122, 1992). Secondly, the resulting
pWS358-FBA1 p-GUS plasmid was digested with Sall and Sacl, a DNA fragment
containing a FBA1p promoter, GUS gene, and FBAt terminator gel-purified, and
cloned into Sall/Sacl sites on pRS316 to create pRS316-FBA1 p-GUS. Thirdly, a
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118 bp DNA fragment containing an upstream activation sequence (UAS) located
between positions -519 and -402 upstream of the 3-phosphoglycerate kinase
(PGK1) open reading frame, namely UAS(PGK1), was PCR-amplified from
genomic DNA of CEN.PK with primers T-U/PGK1(Kpnl) (SEQ ID NO: 181) and
B-U/PGK1(Sall) (SEQ ID NO: 182). The PCR product was digested with KpnI
and Sall and cloned into Kpnl/SalI sites on pRS316-FBA1p-GUS to create
pRS316-UAS(PGK1)-FBA1 p-GUS.
Construction of integration vector PUC19-kan::pdcl ::FBA-alsS::TRX1
[0196] The FBA-alsS-CYCt cassette was constructed by moving the 1.7 kb
BbvCI/PacI fragment from pRS426::GPD::alsS::CYC (U.S. Patent Application
Publication No. 2007/0092957) to pRS426::FBA::ILV5::CYC (U.S. Patent
Application Publication No. 2007/0092957, previously digested with BbvCI/PacI
to release the ILV5 gene). Ligation reactions were transformed into E. coli
TOP10 cells and transformants were screened by PCR using primers N98SegF1
(SEQ ID NO: 183) and N99SeqR2 (SEQ ID NO: 184). The FBA-alsS-CYCt
cassette was isolated from the vector using BglII and Notl for cloning into
pUC19-
URA3::ilvD-TRX1 (as described in U.S. Provisional Application Serial No.
61/356,379, filed June 18, 2010, incorporated herein by reference, clone "B")
at
the AfIll site (Klenow fragment was used to make ends compatible for
ligation).
Transformants containing the aIsS cassette in both orientations in the vector
were
obtained and confirmed by PCR using primers N98SeqF4 (SEQ ID NO: 185) and
N1111 (SEQ ID NO: 186) for configuration "A" and N98SeqF4 (SEQ ID NO: 185)
and N1110 (SEQ ID NO: 187) for configuration "B". A geneticin selectable
version of the "A" configuration vector was then made by removing the URA3
gene (1.2 kb Notl/Nael fragment) and adding a geneticin cassette previously
described (SEQ ID NO: 655 of U.S. Provisional Application Serial No.
61/356,379, filed June 18, 2010, incorporated herein by reference). Klenow
fragment was used to make all ends compatible for ligation, and transformants
were screened by PCR to select a clone with the geneticin resistance gene in
the
same orientation as the previous URA3 marker using primers BK468 (SEQ ID
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NO: 188) and N160SegF5 (SEQ ID NO: 189). The resulting clone was called
pUC1 9-kan::pdcl::FBA-alsS::TRX1 (clone A)(SEQ ID NO: 190).
[0197] The pUC19-kan::pdcl::FBA-alsS integration vector described above was
linearized with Pmel and transformed into PNY1507 (described above). Pmel
cuts the vector within the cloned pdcl-TRX1 intergenic region and thus, leads
to
targeted integration at that location (Rothstein, Methods Enzymol. 194:281-
301,
1991). Transformants were selected on YPE plus 50 pg/ml G418. Patched
transformants were screened by PCR for the integration event using primers
N160SegF5 (SEQ ID NO: 189) and oBP512 (SEQ ID NO: 47). Two
transformants were tested indirectly for acetolactate synthase function by
evaluating the strains ability to make isobutanol. To do this, additional
isobutanol
pathway genes were supplied on E. co/i-yeast shuttle vectors (pYZ090AalsS and
pBP915, described below). One clone, strain MATa ura3&::loxP his3E pdc6E
pdcl A::P[PDC1 ]-DHADIilvD_Sm-PDC1 t-pUC19-loxP-kanMX-loxP-P[FBA1 ]-
ALSIaIsS_Bs-CYC1t pdcs&::P[PDC5]-ADHIsadB_Ax-PDC5t gpd2&::loxP fra2E
adhl&::UAS(PGK1)P [FBA1]-kivD_LI(y)-ADH1t was designated as PNY2204.
PNY2205 is PNY2204 transformed with pYZ090AalsS and pBP915 plasmids.
Isobutanol pathway plasmids (pYZ090AalsS and pBP915)
[0198] pYZ090 (SEQ ID NO: 1) was digested with Spel and Notl to remove most
of the CUP1 promoter and all of the alsS coding sequence and CYC terminator.
The vector was then self-ligated after treatment with Klenow fragment and
transformed into E. coli Stbl3 cells, selecting for ampicillin resistance.
Removal
of the DNA region was confirmed for two independent clones by DNA sequencing
across the ligation junction by PCR using primer N191 (SEQ ID NO: 191). The
resulting plasmid was named pYZ090AalsS (SEQ ID NO: 192).
[0199] pBP915 was constructed from pLH468 (SEQ ID NO: 2; U.S. Provisional
Application Serial No. 61/246,709, filed September 29, 2009) by deleting the
kivD
gene and 957 base pairs of the TDH3 promoter upstream of kivD. pLH468 was
digested with Swal and the large fragment (12896 bp) was purified on an
agarose
gel followed by a Gel Extraction kit (Qiagen, Valencia, CA). The isolated
fragment of DNA was self-ligated with T4 DNA ligase and used to transform
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electrocompetent TOP10 Escherichia coli (Invitrogen, Carlsbad, CA). Plasmids
from transformants were isolated and checked for the proper deletion by
restriction analysis with the Swal restriction enzyme. Isolates were also
sequenced across the deletion site with primers oBP556 (SEQ ID NO: 193) and
oBP561 (SEQ ID NO: 194). A clone with the proper deletion was designated
pBP915 (pLH468EkivD)(SEQ ID NO: 195).
Construction of Strains NYLA74, NYLA83, and NYLA84
[0200] Insertion-inactivation of endogenous PDC1 and PDC6 genes of S.
cerevisiae. PDC1, PDC5, and PDC6 genes encode the three major isozymes of
pyruvate decarboxylase is described as follows:
Construction of r)RS425::GPM-sadB
[0201] A DNA fragment encoding a butanol dehydrogenase (SEQ ID NO: 70)
from Achromobacter xylosoxidans (disclosed in U.S. Patent Application
Publication No. 2009/0269823) was cloned. The coding region of this gene
called sadB for secondary alcohol dehydrogenase (SEQ ID NO: 69) was
amplified using standard conditions from A. xylosoxidans genomic DNA,
prepared using a Gentra Puregene kit (Qiagen, Valencia, CA) following the
recommended protocol for gram negative organisms using forward and reverse
primers N473 and N469 (SEQ ID NOs: 74 and 75), respectively. The PCR
product was TOPO -Blunt cloned into pCR 4 BLUNT (InvitrogenTM, Carlsbad,
CA) to produce pCR4Blunt::sadB, which was transformed into E. coli Mach-1
cells. Plasmid was subsequently isolated from four clones, and the sequence
verified.
[0202] The sadB coding region was PCR amplified from pCR4Blunt::sadB. PCR
primers contained additional 5' sequences that would overlap with the yeast
GPM1 promoter and the ADH1 terminator (N583 and N584, provided as SEQ ID
NOs: 76 and 77). The PCR product was then cloned using "gap repair"
methodology in Saccharomyces cerevisiae (Ma, et al., Gene 58:201-216, 1987)
as follows. The yeast-E. coli shuttle vector pRS425::GPM::kivD::ADH which
contains the GPM1 promoter (SEQ ID NO: 72), kivD coding region from
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Lactococcus lactis (SEQ ID NO: 71), and ADH1 terminator (SEQ ID NO: 73)
(described in U.S. Patent Application Publication No. 2007/0092957 Al, Example
17) was digested with BbvCI and Pacl restriction enzymes to release the kivD
coding region. Approximately 1 g of the remaining vector fragment was
transformed into S. cerevisiae strain BY4741 along with 1 g of sadB PCR
product. Transformants were selected on synthetic complete medium lacking
leucine. The proper recombination event, generating pRS425::GPM-sadB, was
confirmed by PCR using primers N142 and N459 (SEQ ID NOs: 108 and 109).
Construction of pdc6:: PGPM1-sadB integration cassette and PDC6 deletion:
[0203] A pdc6::PGPM1-sadB-ADH1t-URA3r integration cassette was made by
joining the GPM-sadB-ADHt segment (SEQ ID NO: 79) from pRS425::GPM-sadB
(SEQ ID NO: 78) to the URA3r gene from pUC19-URA3r. pUC19-URA3r (SEQ
ID NO: 80) contains the URA3 marker from pRS426 (ATCC No. 77107) flanked
by 75 bp homologous repeat sequences to allow homologous recombination in
vivo and removal of the URA3 marker. The two DNA segments were joined by
SOE PCR (as described by Horton, et al., Gene 77:61-68, 1989) using as
template pRS425::GPM-sadB and pUC19-URA3r plasmid DNAs, with Phusion
DNA polymerase (New England BioLabs Inc., Ipswich, MA) and primers 114117-
11A through 114117A-11 D (SEQ ID NOs: 81, 82, 83, and 84), and 114117-13A
and 114117-13B (SEQ ID NOs: 85 and 86).
[0204] The outer primers for the SOE PCR (114117-13A and 114117-13B)
contained 5' and 3' -50 bp regions homologous to regions upstream and
downstream of the PDC6 promoter and terminator, respectively. The completed
cassette PCR fragment was transformed into BY4700 (ATCC No. 200866) and
transformants were maintained on synthetic complete media lacking uracil and
supplemented with 2% glucose at 30 C using standard genetic techniques
(Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY, pp. 201-202). Transformants were screened by PCR using
primers 112590-34G and 112590-34H (SEQ ID NOs: 87 and 88), and 112590-
34F and 11 2590-49E (SEQ ID NOs: 89 and 90) to verify integration at the PDC6
locus with deletion of the PDC6 coding region. The URA3r marker was recycled
by plating on synthetic complete media supplemented with 2% glucose and 5-
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FOA at 30 C following standard protocols. Marker removal was confirmed by
patching colonies from the 5-FOA plates onto SD-URA media to verify the
absence of growth. The resulting identified strain has the genotype: BY4700
pdc6::PGPM1-sadB-ADH1 t.
Construction of pdcl :: PPDC1-ilvD integration cassette and PDC1 deletion:
[0205] A pdcl:: PPDC1-ilvD-FBA1t-URA3r integration cassette was made by
joining the ilvD-FBA1t segment (SEQ ID NO: 91) from pLH468 (SEQ ID NO: 2) to
the URA3r gene from pUC19-URA3r by SOE PCR (as described by Horton, et
al., Gene 77:61-68, 1989) using as template pLH468 and pUC19-URA3r plasmid
DNAs, with Phusion DNA polymerase (New England BioLabs Inc., Ipswich, MA)
and primers 114117-27A through 114117-27D (SEQ ID NOs: 111, 112, 113, and
114).
[0206] The outer primers for the SOE PCR (114117-27A and 114117-27D)
contained 5' and 3' -50 bp regions homologous to regions downstream of the
PDC1 promoter and downstream of the PDC1 coding sequence. The completed
cassette PCR fragment was transformed into BY4700 pdc6::PGPM1-sadB-
ADH1t and transformants were maintained on synthetic complete media lacking
uracil and supplemented with 2% glucose at 30 C using standard genetic
techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY, pp. 201-202). Transformants were screened by
PCR using primers 114117-36D and 135 (SEQ ID NOs: 92 and 93), and primers
112590-49E and 112590-30F (SEQ ID NOs: 90 and 94) to verify integration at
the PDC1 locus with deletion of the PDC1 coding sequence. The URA3r marker
was recycled by plating on synthetic complete media supplemented with 2%
glucose and 5-FOA at 30 C following standard protocols. Marker removal was
confirmed by patching colonies from the 5-FOA plates onto SD-URA media to
verify the absence of growth. The resulting identified strain "NYLA67" has the
genotype: BY4700 pdc6:: PGPM1-sadB-ADH1t pdcl:: PPDC1-iIvD-FBA1t.
HIS3 deletion
[0207] To delete the endogenous HIS3 coding region, a his3::URA3r2 cassette
was PCR-amplified from URA3r2 template DNA (SEQ ID NO: 95). URA3r2
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contains the URA3 marker from pRS426 (ATCC No. 77107) flanked by 500 bp
homologous repeat sequences to allow homologous recombination in vivo and
removal of the URA3 marker. PCR was done using Phusion DNA polymerase
(New England BioLabs Inc., Ipswich, MA) and primers 114117-45A and 114117-
45B (SEQ ID NOs: 96 and 97) which generated a -2.3 kb PCR product. The
HIS3 portion of each primer was derived from the 5' region upstream of the
HIS3
promoter and 3' region downstream of the coding region such that integration
of
the URA3r2 marker results in replacement of the HIS3 coding region. The PCR
product was transformed into NYLA67 using standard genetic techniques
(Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY, pp. 201-202) and transformants were selected on synthetic
complete media lacking uracil and supplemented with 2% glucose at 30 C.
Transformants were screened to verify correct integration by replica plating
of
transformants onto synthetic complete media lacking histidine and supplemented
with 2% glucose at 30 C. The URA3r marker was recycled by plating on
synthetic complete media supplemented with 2% glucose and 5-FOA at 30 C
following standard protocols. Marker removal was confirmed by patching
colonies from the 5-FOA plates onto SD-URA media to verify the absence of
growth. The resulting identified strain, called NYLA73, has the genotype:
BY4700 pdc6:: PGPM1-sadB-ADH1 t pdcl:: PPDC1-iIvD-FBA1 t Ahis3.
Construction of pdc5::kanMX integration cassette and PDC5 deletion:
[0208] A pdc5::kanMX4 cassette was PCR-amplified from strain YLR134W
chromosomal DNA (ATCC No. 4034091) using Phusion DNA polymerase (New
England BioLabs Inc., Ipswich, MA) and primers PDC5::KanMXF and
PDC5::KanMXR (SEQ ID NOs: 98 and 99) which generated a -2.2 kb PCR
product. The PDC5 portion of each primer was derived from the 5' region
upstream of the PDC5 promoter and 3' region downstream of the coding region
such that integration of the kanMX4 marker results in replacement of the PDC5
coding region. The PCR product was transformed into NYLA73 using standard
genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY, pp. 201-202) and transformants were
selected on YP media supplemented with 1% ethanol and geneticin (200 g/mL)
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at 30 C. Transformants were screened by PCR to verify correct integration at
the
PDC locus with replacement of the PDC5 coding region using primers PDC5kofor
and N175 (SEQ ID NOs: 100 and 101). The identified correct transformants have
the genotype: BY4700 pdc6:: PGPM1-sadB-ADH1t pdcl:: PPDC1-iIvD-FBA1t
Ahis3 pdc5::kanMX4. The strain was named NYLA74.
[0209] Plasmid vectors pRS423::CUP1-alsS+FBA-budA and pRS426::FBA-
budC+GPM-sadB were transformed into NYLA74 to create a butanediol
producing strain (NGCI-047).
[0210] Plasmid vectors pLH475-Z4B8 (SEQ ID NO: 140) and pLH468 were
transformed into NYLA74 to create an isobutanol producing strain (NGCI-049).
Deletion of HXK2 (hexokinase II):
[0211] A hxk2::URA3r cassette was PCR-amplified from URA3r2 template
(described above) using Phusion DNA polymerase (New England BioLabs Inc.,
Ipswich, MA) and primers 384 and 385 (SEQ ID NOs: 102 and 103) which
generated a -2.3 kb PCR product. The HXK2 portion of each primer was derived
from the 5' region upstream of the HXK2 promoter and 3' region downstream of
the coding region such that integration of the URA3r2 marker results in
replacement of the HXK2 coding region. The PCR product was transformed into
NYLA73 using standard genetic techniques (Methods in Yeast Genetics, 2005,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202) and
transformants were selected on synthetic complete media lacking uracil and
supplemented with 2% glucose at 30 C. Transformants were screened by PCR
to verify correct integration at the HXK2 locus with replacement of the HXK2
coding region using primers N869 and N871 (SEQ ID NOs: 104 and 105). The
URA3r2 marker was recycled by plating on synthetic complete media
supplemented with 2% glucose and 5-FOA at 30 C following standard protocols.
Marker removal was confirmed by patching colonies from the 5-FOA plates onto
SD-URA media to verify the absence of growth, and by PCR to verify correct
marker removal using primers N946 and N947 (SEQ ID NOs: 106 and 107). The
resulting identified strain named NYLA83 has the genotype: BY4700 pdc6::
PGPM1-sadB-ADH1 t pdcl:: PPDC1-iIvD-FBA1 t Ahis3 Ahxk2.
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Construction of pdc5::kanMX integration cassette and PDC5 deletion:
[0212] A pdc5::kanMX4 cassette was PCR-amplified as described above. The
PCR fragment was transformed into NYLA83, and transformants were selected
and screened as described above. The identified correct transformants named
NYLA84 have the genotype: BY4700 pdc6:: PGPM1-sadB-ADH1t pdcl:: PPDC1-
ilvD-FBA1t Ahis3 Ahxk2 pdc5::kanMX4.
[0213] Plasmid vectors pLH468 and pLH532 were simultaneously transformed
into strain NYLA84 (BY4700 pdc6::PGPM1-sadB-ADH1t pdcl::PPDC1-ilvD-
FBA1t Ahis3 Ahxk2 pdc5::kanMX4) using standard genetic techniques (Methods
in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY) and the resulting "butanologen NYLA84" was maintained on
synthetic complete media lacking histidine and uracil, and supplemented with
1%
ethanol at 30 C.
Expression Vector pLH468
[0214] The pLH468 plasmid (SEQ ID NO: 2) was constructed for expression of
DHAD, KivD, and HADH in yeast and is described in U.S. Patent Application
Publication No. 2009/0305363, herein incorporated by reference. pLH486 was
constructed to contain: a chimeric gene having the coding region of the ilvD
gene
from Streptococcus mutans (nt position 3313-4849) expressed from the S.
cerevisiae FBA1 promoter (nt 2109 - 3105) followed by the FBA1 terminator (nt
4858 - 5857) for expression of DHAD; a chimeric gene having the coding region
of codon optimized horse liver alcohol dehydrogenase (nt 6286-7413) expressed
from the S. cerevisiae GPM1 promoter (nt 7425-8181) followed by the ADH1
terminator (nt 5962-6277) for expression of ADH; and a chimeric gene having
the
coding region of the codon-optimized kivD gene from Lactococcus lactis (nt
9249-
10895) expressed from the TDH3 promoter (nt 10896-11918) followed by the
TDH3 terminator (nt 8237-9235) for expression of KivD.
[0215] Coding regions for Lactococcus lactis ketoisovalerate decarboxylase
(KivD) and horse liver alcohol dehydrogenase (HADH) were synthesized by
DNA2.0, Inc. (Menlo Park, CA) based on codons that were optimized for
expression in Saccharomyces cerevisiae (SEQ ID NO: 71 and 118, respectively)
and provided in plasmids pKivDy-DNA2.0 and pHadhy-DNA2Ø The encoded
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proteins are SEQ ID NOs: 117 and 119, respectively. Individual expression
vectors for KivD and HADH were constructed. To assemble pLH467
(pRS426::PTDH3-kivDy-TDH3t), vector pNY8 (SEQ ID NO: 121; also named
pRS426.GPD-ald-GPDt, described in U.S. Patent Application Publication No.
2008/0182308, Example 17, which is herein incorporated by reference) was
digested with Ascl and Sfil enzymes, thus excising the GPD promoter and the
ald
coding region. A TDH3 promoter fragment (SEQ ID NO: 122) from pNY8 was
PCR amplified to add an Ascl site at the 5' end and an Spel site at the 3'
end,
using 5' primer OT1068 and 3' primer OT1067 (SEQ ID NOs: 123 and 124). The
Ascl/Sfil digested pNY8 vector fragment was ligated with the TDH3 promoter
PCR product digested with Ascl and Spel, and the Spel-Sfil fragment containing
the codon optimized kivD coding region isolated from the vector pKivD-DNA2Ø
The triple ligation generated vector pLH467 (pRS426::PTDH3-kivDy-TDH3t).
pLH467 was verified by restriction mapping and sequencing.
[0216] pLH435 (pRS425::PGPM1-Hadhy-ADH1t) was derived from vector
pRS425::GPM-sadB (SEQ ID NO: 78) which is described in U.S. Provisional
Application Serial No. 61/058,970, Example 3, which is herein incorporated by
reference. pRS425::GPM-sadB is the pRS425 vector (ATCC No. 77106) with a
chimeric gene containing the GPM1 promoter (SEQ ID NO: 72), coding region
from a butanol dehydrogenase of Achromobacter xylosoxidans (sad B; DNA SEQ
ID NO: 69; protein SEQ ID NO: 70: disclosed in U.S. Patent Application
Publication No. 2009/0269823), and ADH1 terminator (SEQ ID NO: 73).
pRS425::GPMp-sadB contains Bbvl and Pacl sites at the 5' and 3' ends of the
sadB coding region, respectively. A Nhel site was added at the 5' end of the
sadB coding region by site-directed mutagenesis using primers OT1074 and
OT1075 (SEQ ID NOs: 126 and 127) to generate vector pRS425-GPMp-sadB-
Nhel, which was verified by sequencing. pRS425::PGPM1-sadB-Nhel was
digested with Nhel and Pacl to drop out the sadB coding region, and ligated
with
the Nhel-Pacl fragment containing the codon optimized HADH coding region from
vector pHadhy-DNA2.0 to create pLH435.
[0217] To combine KivD and HADH expression cassettes in a single vector, yeast
vector pRS411 (ATCC No. 87474) was digested with Sacl and Noti, and ligated
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with the Sacl-Sall fragment from pLH467 that contains the PTDH3-kivDy-TDH3t
cassette together with the Sall-Notl fragment from pLH435 that contains the
PGPM1-Hadhy-ADH1t cassette in a triple ligation reaction. This yielded the
vector pRS411::PTDH3-kivDy-PGPM1-Hadhy (pLH441) which was verified by
restriction mapping.
[0218] In order to generate a co-expression vector for all three genes in the
lower
isobutanol pathway: ilvD, kivDy, and Hadhy, pRS423 FBA ilvD(Strep) (SEQ ID
NO: 128) which is described in U.S. Patent Application Publication No.
2010/0081154 as the source of the IIvD gene, was used. This shuttle vector
contains an F1 origin of replication (nt 1423 to 1879) for maintenance in E.
coli
and a 2 micron origin (nt 8082 to 9426) for replication in yeast. The vector
has an
FBA1 promoter (nt 2111 to 3108; SEQ ID NO: 120) and FBA terminator (nt 4861
to 5860; SEQ ID NO: 129). In addition, it carries the His marker (nt 504 to
1163)
for selection in yeast and ampicillin resistance marker (nt 7092 to 7949) for
selection in E. coli. The ilvD coding region (nt 3116 to 4828; SEQ ID NO: 115;
protein SEQ ID NO: 116) from Streptococcus mutans UA159 (ATCC No. 700610)
is between the FBA promoter and FBA terminator forming a chimeric gene for
expression. In addition, there is a lumio tag fused to the ilvD coding region
(nt
4829-4849).
[0219] The first step was to linearize pRS423 FBA ilvD(Strep) (also called
pRS423-FBA(Spel)-IIvD(Streptococcus mutans)-Lumio) with Sacl and Sacll (with
Sacll site blunt ended using T4 DNA polymerase), to give a vector with total
length of 9,482 bp. The second step was to isolate the kivDy-hADHy cassette
from pLH441 with Sacl and KpnI (with KpnI site blunt ended using T4 DNA
polymerase), which gives a 6,063 bp fragment. This fragment was ligated with
the 9,482 bp vector fragment from pRS423-FBA(Spel)-IIvD(Streptococcus
mutans)-Lumio. This generated vector pLH468 (pRS423::PFBA1-ilvD(Strep)
Lumio-FBA1 t-PTDH3-kivDy-TDH3t-PGPM1-had hy-ADH1 t) which was confirmed
by restriction mapping and sequencing.
pLH532 construction
[0220] The pLH532 plasmid (SEQ ID NO: 130) was constructed for expression of
ALS and KART in yeast. pLH532 is a pHR81 vector (ATCC No. 87541)
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containing the following chimeric genes: 1) the CUP1 promoter (SEQ ID NO:
139), acetolactate synthase coding region from Bacillus subtilis (AIsS; SEQ ID
NO: 137; protein SEQ ID NO: 138) and CYC1 terminator2 (SEQ ID NO: 133); 2)
an ILV5 promoter (SEQ ID NO: 134), Pf5.IIvC coding region (SEQ ID NO: 132)
and ILV5 terminator (SEQ ID NO: 135); and 3) the FBA1 promoter (SEQ ID NO:
136), S. cerevisiae KART coding region (ILV5; SEQ ID NO: 131); and CYC1
terminator.
[0221] The Pf5.IIvC coding region is a sequence encoding KART derived from
Pseudomonas fluorescens that was described in U.S. Patent Application
Publication No. 2009/0163376, which is herein incorporated by reference.
[0222] The Pf5.IIvC coding region was synthesized by DNA2.0, Inc. (Menlo Park,
CA; SEQ ID NO: 132) based on codons that were optimized for expression in
Saccharomyces cerevisiae.
pYZ090 construction
[0223] pYZ090 (SEQ ID NO: 1) is based on the pHR81 (ATCC No. 87541)
backbone and was constructed to contain a chimeric gene having the coding
region of the alsS gene from Bacillus subtilis (nt position 457-2172)
expressed
from the yeast CUP1 promoter (nt 2-449) and followed by the CYC1 terminator
(nt 2181-2430) for expression of ALS, and a chimeric gene having the coding
region of the ilvC gene from Lactococcus lactis (nt 3634-4656) expressed from
the yeast ILV5 promoter (2433-3626) and followed by the ILV5 terminator (nt
4682-5304) for expression of KART.
pYZ067 construction
[0224] pYZ067 was constructed to contain the following chimeric genes: 1) the
coding region of the ilvD gene from S. mutans UA159 (nt position 2260-3971)
expressed from the yeast FBA1 promoter (nt 1161-2250) followed by the FBA
terminator (nt 4005-4317) for expression of dihydroxy acid dehydratase (DHAD),
2) the coding region for horse liver ADH (nt 4680-5807) expressed from the
yeast
GPM promoter (nt 5819-6575) followed by the ADH1 terminator (nt 4356-4671)
for expression of alcohol dehydrogenase, and 3) the coding region of the KivD
gene from Lacrococcus lactis (nt 7175-8821) expressed from the yeast TDH3
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promoter (nt 8830-9493) followed by the TDH3 terminator (nt 5682-7161) for
expression of ketoisovalerate decarboxylase.
pRS423::CUP1-aIsS+FBA-budA and pRS426::FBA-budC+GPM-sadB and
pLH475-Z4B8 construction
[0225] Construction of pRS423::CUP1-alsS+FBA-budA and pRS426::FBA-
budC+GPM-sadB and pLH475-Z4B8 is described in U.S. Patent Application
Publication No. 2009/0305363, incorporated herein by reference.
Construction of Saccharomyces cerevisiae Strain PNY2242
[0226] Strain PNY2242 was constructed in several steps from PNY1507
(described above). First, a chimeric gene comprised of the FBA1 promoter, the
alsS coding region, and the CYC1 terminator was integrated into Chromosome
XII, upstream of the TRX1 gene. The sequence of the modified locus is provided
as SEQ ID NO: 196. Next, two copies of a gene encoding horse liver alcohol
dehydrogenase were integrated into Chromosomes VII and XVI. On
Chromosome VII, a chimeric gene comprised of the PDC1 promoter, the hADH
coding region, and the ADH1 terminator were placed into the fra20 locus (the
original deletion of FRA2 is described above). The sequence of the modified
locus is provided as SEQ ID NO: 197. On Chromosome XVI, a chimeric gene
comprised of the PDC5 promoter, the hADH coding region, and the ADH1
terminator were integrated in the region formerly occupied by the long term
repeat element YPRCdeltal 5. The sequence of the modified locus is provided as
SEQ ID NO: 198. Then the native genes YMR226c and ALD6 were deleted.
Elimination of YMR226c was a scarless deletion of only the coding region. The
sequence of the modified locus is provided as SEQ ID NO: 199. The ALD6
coding region plus 700 bp of upstream sequence were deleted using CRE-lox
mediated marker removal (methodology described above), so the resulting locus
contains one loxP site. The sequence of the modified locus is provided as SEQ
ID NO: 200. Finally, plasmids were introduced into the strain for expression
of
KART (pLH702, SEQ ID NO: 201) and DHAD (pYZ067DkivDDhADH, SEQ ID NO:
202), resulting in strain PNY2242.
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[0227] Where the recombinant microorganism produces isobutanol, under certain
embodiments, microorganisms show higher specific productivity. Further, the
volumetric rate was improved by about 50%.
[0228] While not wishing to be bound by theory, it is believed that the
methods
described herein provide extractive fermentation methods with improved
production yields of product alcohol. As discussed above, alcohol production
utilizing fermentation by microorganisms may be inefficient due to the alcohol
toxicity thresholds of the microorganism. In some embodiments, the methods
herein provide a means to convert the product alcohol into a substance less
toxic
to the microorganism. For example, the product alcohol may be contacted with
carboxylic acid in the presence of a catalyst which esterifies the alcohol
with the
carboxylic acid and thereby, produces alcohol esters which are less toxic to
the
microorganism. In addition, the generation of alcohol esters from the product
alcohol results in a lower concentration of the product alcohol in the
fermentation
medium. The reduced concentration of product alcohol minimizes the toxic
effects of the product alcohol on the microorganism and thus, leads to
improved
production yields of product alcohol.
[0229] Carboxylic acid may serve as an extractant, and alcohol esters can
partition into the extractant. However, the partition coefficient of the
extractant
may be degraded by lipid contamination. To reduce the degradation of the
partition coefficient of the extractant, lipids present in the fermentation
medium
may be converted to extractant and consequently, minimize lipid contamination.
In some embodiments, the methods herein provide a means to convert the lipids
present in the feedstock or biomass into an extractant by catalytically
hydrolyzing
the lipids to carboxylic acid. The carboxylic acid produced by this hydrolysis
may
serve as an extractant or esterified with the product alcohol to form alcohol
esters. Thus, the methods described herein provide a means to preserve the
partition coefficient of the extractant (e.g., lipid hydrolysis) as well as
minimize the
toxic effects of the product alcohol (e.g., esterification of the product
alcohol.
[0230] Carboxylic acid may be supplied to the fermentation vessel or derived
by
hydrolysis from lipids (e.g., biomass) supplied to the fermentation vessel.
The
amount of carboxylic acid should be sufficient to form a two-phase mixture
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comprising an organic phase and an aqueous phase. That is, carboxylic acid
(i.e., extractant) in an appropriate concentration contacts the fermentation
broth
and forms the two-phase mixture. The alcohol esters formed in the fermentation
broth will preferentially partition into the organic phase because these
esters are
formed at a concentration in excess of the equilibrium concentration of the
aqueous phase. The alcohol ester-containing organic phase may be separated
from the fermentation broth, the product alcohol may be recovered from organic
phase, and the extractant may be recycled to the fermentation vessel.
[0231] Further, while various embodiments of the present invention have been
described above, it should be understood that they have been presented by way
of example only, and not limitation. It will be apparent to persons skilled in
the
relevant art that various changes in form and detail can be made therein
without
departing from the spirit and scope of the invention. Thus, the breadth and
scope
of the present invention should not be limited by any of the above-described
exemplary embodiments, but should be defined only in accordance with the
claims and their equivalents.
[0232] All publications, patents, and patent applications mentioned in this
specification are indicative of the level of skill of those skilled in the art
to which
this invention pertains, and are herein incorporated by reference to the same
extent as if each individual publication, patent, or patent application was
specifically and individually indicated to be incorporated by reference.
EXAMPLES
[0233] The following nonlimiting examples will further illustrate the
invention. It
should be understood that, while the following examples involve corn as
feedstock and COFA as carboxylic acid, other biomass sources can be used for
feedstock and acids other than COFA can serve as carboxylic acid, without
departing from the present invention. Moreover, while the following examples
involve butanol and butyl ester production, other alcohols including ethanol,
and
alcohol esters can be produced without departing from the present invention.
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[0234] As used herein, the meaning of abbreviations used was as follows: "g"
means gram(s), "kg" means kilogram(s), "L" means liter(s), "mL" means
milliliter(s), "pL" means microliter(s), "mL/L" means milliliter(s) per liter,
"mL/min"
means milliliter(s) per min, "DI" means deionized, "uM" means micrometer(s),
"nm" means nanometer(s), "w/v" means weight/volume, "OD" means optical
density, "OD600" means optical density at a wavelength of 600 nM, "dcw" means
dry cell weight, "rpm" means revolutions per minute, " C" means degree(s)
Celsius, " C/min" means degrees Celsius per minute, "slpm" means standard
liter(s) per minute, "ppm" means part per million, "pdc" means pyruvate
decarboxylase enzyme followed by the enzyme number.
GENERAL METHODS
Seed Flask Growth
[0235] A Saccharomyces cerevisiae strain that was engineered to produce
isobutanol from a carbohydrate source, with pdcl deleted, pdc5 deleted, and
pdc6 deleted, was grown to 0.55-1.1 g/L dcw (OD600 1.3-2.6 - Thermo Helios a
Thermo Fisher Scientific Inc., Waltham, Massachusetts) in seed flasks from a
frozen culture. The culture was grown at 23-26 C in an incubator rotating at
300 rpm. The frozen culture was previously stored at - 80 C. The composition
of the first seed flask medium was:
3.0-5.0 g/L dextrose
3.0-3.5 g/L ethanol, anhydrous
3.7 g/L ForMediumTM Synthetic Complete Amino Acid (Kaiser) Drop-Out:
without HIS, without URA (Reference # DSCK162CK)
6.7 g/L Difco Yeast Nitrogen Base without amino acids (No. 291920).
[0236] Eight to twelve milliliters from the first seed flask culture was
transferred to
a 2 L flask and grown at 30 C in an incubator rotating at 300 rpm. The second
seed flask has 220 mL of the following medium:
30.0 g/L dextrose
5.0 g/L ethanol, anhydrous
3.7 g/L ForMediumTM Synthetic Complete Amino Acid (Kaiser) Drop-Out:
without HIS, without URA (Reference # DSCK162CK)
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6.7 g/L Difco Yeast Nitrogen Base without amino acids (No. 291920)
0.2 M MES Buffer titrated to pH 5.5-6Ø
[0237] The culture was grown to 0.55-1.1 g/L dcw (OD600 1.3-2.6). An addition
of
30 mL of a solution containing 200 g/L peptone and 100 g/L yeast extract was
added at this cell concentration. Then, an addition of 250-300 mL of 0.2 uM
filter
sterilized HD OCENOL 90/95 oleyl alcohol (Cognis, Monheim, DE) was added
to the flask. The culture continues to grow to > 4 g/L dcw (OD600 > 10) before
being harvested and added to the fermentation.
Fermentation Preparation
Initial Fermentation Vessel Preparation
[0238] A glass jacked, 2 L fermentation vessel (Sartorius AG, Goettingen,
Germany) was charged with house water to 66% of the liquefaction weight. A pH
probe (Hamilton Easyferm Plus K8, part number: 238627, Hamilton Bonaduz AG,
Bonaduz, Switzerland) was calibrated through the Sartorius DCU-3 Control
Tower Calibration menu. The zero was calibrated at pH=7. The span was
calibrated at pH=4. The probe was then placed into the fermentation vessel
through the stainless steel head plate. A dissolved oxygen probe (p02 probe)
was also placed into the fermentation vessel through the head plate. Tubing
used for delivering nutrients, seed culture, extracting solvent, and base were
attached to the head plate and the ends were foiled. The entire fermentation
vessel was placed into a Steris (Steris Corporation, Mentor, Ohio) autoclave
and
sterilized in a liquid cycle for 30 minutes.
[0239] The fermentation vessel was removed from the autoclave and placed on a
load cell. The jacket water supply and return line was connected to the house
water and clean drain, respectively. The condenser cooling water in and water
out lines were connected to a 6-L recirculating temperature bath running at 7
C.
The vent line that transfers the gas from the fermentation vessel was
connected
to a transfer line that was connected to a Thermo mass spectrometer (Prima dB,
Thermo Fisher Scientific Inc., Waltham, Massachusetts). The sparger line was
connected to the gas supply line. The tubing for adding nutrients, extract
solvent,
seed culture, and base was plumbed through pumps or clamped closed.
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[0240] The fermentation vessel temperature was controlled at 55 C with a
thermocouple and house water circulation loop. Wet corn kernels (#2 yellow
dent) were ground using a hammer mill with a 1.0 mm screen, and the resulting
ground whole corn kernels were then added to the fermentation vessel at a
charge that was 29-30% (dry corn solids weight) of the liquefaction reaction
mass.
Lipase Treatment Pre-Liquefaction
[0241] A lipase enzyme stock solution was added to the fermentation vessel to
a
final lipase concentration of 10 ppm. The fermentation vessel was held at 55
C,
300 rpm, and 0.3 slpm N2 overlay for > 6 hrs. After the lipase treatment was
complete, liquefaction was performed as described below (Liquefaction).
Liquefaction
[0242] An alpha-amylase was added to the fermentation vessel per its
specification sheet while the fermentation vessel was mixing at 300-1200 rpm,
with sterile, house N2 being added at 0.3 slpm through the sparger. The
temperature set-point was changed from 55 C to 85 C. When the temperature
was > 80 C, the liquefaction cook time was started and the liquefaction cycle
was
held at > 80 C for 90-120 minutes. The fermentation vessel temperature set-
point was set to the fermentation temperature of 30 C after the liquefaction
cycle
was complete. N2 was redirected from the sparger to the head space to prevent
foaming without the addition of a chemical antifoaming agent.
Lipase Treatment Post-Liquefaction
[0243] The fermentation vessel temperature was set to 55 C instead of 30 C
after
the liquefaction cycle was complete (Liquefaction). The pH was manually
controlled at pH=5.8 by making bolus additions of acid or base when needed. A
lipase enzyme stock solution was added to the fermentation vessel to a final
lipase concentration of 10 ppm. The fermentation vessel was held at 55 C,
300 rpm, and 0.3 slpm N2 overlay for > 6 hrs. After the Lipase Treatment was
complete, the fermentation vessel temperature was set to 30 C.
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Lipase Heat Inactivation Treatment (Heat Kill Treatment Method)
[0244] The fermentation vessel temperature was held at > 80 C for > 15
minutes
to inactivate the lipase. After the Heat Inactivation Treatment was complete,
the
fermentation vessel temperature was set to 30 C.
Nutrient Addition Prior to Inoculation
[0245] Ethanol (7 mL/L, post-inoculation volume, 200 proof, anhydrous) was
added to the fermentation vessel just prior to inoculation. Thiamine was added
to
a final concentration of 20 mg/L and 100 mg/L nicotinic acid was also added
just
prior to inoculation.
Oleyl Alcohol or Corn Oil Fatty Acids Addition Prior to Inoculation
[0246] Added 1 L/L (post-inoculation volume) of oleyl alcohol or corn oil
fatty
acids immediately after inoculation.
Fermentation Operation
Fermentation Vessel Inoculation
[0247] The fermentation vessels p02 probe was calibrated to zero while N2 was
being added to the fermentation vessel. The fermentation vessels p02 probe was
calibrated to its span with sterile air sparging at 300 rpm. The fermentation
vessel was inoculated after the second seed flask with > 4 g/L dcw. The shake
flask was removed from the incubator/shaker for 5 minutes allowing a phase
separation of the oleyl alcohol phase and the aqueous phase. The aqueous
phase (110 mL) was transferred to a sterile, inoculation bottle. The inoculum
was
pumped into the fermentation vessel through a peristaltic pump.
Fermentation Vessel Operating Conditions
[0248] The fermentation vessel was operated at 30 C for the entire growth and
production stages. The pH was allowed to drop from a pH between 5.7-5.9 to a
control set-point of 5.2 without adding any acid. The pH was controlled for
the
remainder of the growth and production stage at a pH=5.2 with ammonium
hydroxide. Sterile air was added to the fermentation vessel, through the
sparger,
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at 0.3 slpm for the remainder of the growth and production stages. The p02 was
set to be controlled at 3.0% by the Sartorius DCU-3 Control Box PID control
loop,
using stir control only, with the stirrer minimum being set to 300 rpm and the
maximum being set to 2000 rpm. The glucose was supplied through
simultaneous saccharification and fermentation of the liquified corn mash by
adding a a-amylase (glucoamylase). The glucose was kept excess (1-50 g/L)
for as long as starch was available for saccharification.
Analytical
Gas Analysis
[0249] Process air was analyzed on a Thermo Prima (Thermo Fisher Scientific
Inc., Waltham, Massachusetts) mass spectrometer. This was the same process
air that was sterilized and then added to each fermentation vessel. Each
fermentation vessel's off-gas was analyzed on the same mass spectrometer.
This Thermo Prima dB has a calibration check run every Monday morning at
6:00 am. The calibration check was scheduled through the Gas Works v1.0
(Thermo Fisher Scientific Inc., Waltham, Massachusetts) software associated
with the mass spectrometer. The gas calibrated for were:
GAS Calibration Concentration mole % Cal Frequency
Nitrogen 78 % weekly
Oxygen 21 % weekly
Isobutanol 0.2 % yearly
Argon 1 % weekly
Carbon Dioxide 0.03 % weekly
[0250] Carbon dioxide was checked at 5% and 15% during calibration cycle with
other known bottled gases. Oxygen was checked at 15% with other known
bottled gases. Based on the analysis of the off-gas of each fermentation
vessel,
the amount of isobutanol stripped, oxygen consumed, and carbon dioxide
respired into the off-gas was measured by using the mass spectrometer's mole
fraction analysis and gas flow rates (mass flow controller) into the
fermentation
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vessel. Calculate the gassing rate per hour and then integrating that rate
over
the course of the fermentation.
Cell Mass Measurement
[0251] A 0.08% Trypan Blue solution was prepared from a 1:5 dilution of 0.4%
Trypan Blue in NaCl (VWR BDH8721-0) with 1X PBS. A 1.0 mL sample was
pulled from a fermentation vessel and placed in a 1.5 mL Eppendorf centrifuge
tube and centrifuged in an Eppendorf, 5415C at 14,000 rpm for 5 minutes. After
centrifugation, the top solvent layer was removed with an m200 Variable
Channel
BioHit pipette with 20-200 pL BioHit pipette tips. Care was made not to remove
the layer between the solvent and aqueous layers. Once the solvent layer was
removed, the sample was re-suspended using a Vortex-Genie set at 2700 rpm.
[0252] A series of dilutions was required to prepare the ideal concentration
for
hemacytometer counts. If the OD was 10, a 1:20 dilution would be performed to
achieve 0.5 OD which would give the ideal amount of cells to be counted per
square, 20-30. In order to reduce inaccuracy in the dilution due to corn
solids,
multiple dilutions with cut 100-1000 pL BioHit pipette tips were required.
Approximately, 1 cm was cut off the tips to increase the opening which
prevented
the tip from clogging. For a 1:20 final dilution, an initial 1:1 dilution of
fermentation sample and 0.9% NaCl solution was prepared. Then, a 1:1 dilution
of the previous solution (i.e., the initial 1:1 dilution) and 0.9% NaCl
solution (the
second dilution) was generated followed by a 1:5 dilution of the second
dilution
and Trypan Blue Solution. Samples were vortexed between each dilution and cut
tips were rinsed into the 0.9% NaCl and Trypan Blue solutions.
[0253] The cover slip was carefully placed on top of the hemacytometer
(Hausser
Scientific Bright-Line 1492). An aliquot (10 pL) was drawn of the final Trypan
Blue dilution with an m20 Variable Channel BioHit pipette with 2-20 pL BioHit
pipette tips and injected into the hemacytometer. The hemacytometer was
placed on the Zeis Axioskop 40 microscope at 40x magnification. The center
quadrant was broken into 25 squares and the four corner and center squares in
both chambers were then counted and recorded. After both chambers were
counted, the average was taken and multiplied by the dilution factor (20),
then by
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25 for the number for squares in the quadrant in the hemacytometer, and then
divided by 0.0001 mL which is the volume of the quadrant that was counted. The
sum of this calculation is the number cells per mL.
LC Analysis of Fermentation Products in the Aqueous Phase
[0254] Samples were refrigerated until ready for processing. Samples were
removed from refrigeration and allowed to reach room temperature (about one
hour). Approximately 300 pL of sample was transferred with a m1000 Variable
Channel BioHit pipette with 100-1000 pL BioHit pipette tips into a 0.2 um
centrifuge filter (Nanosep MF modified nylon centrifuge filter), then
centrifuged
using a Eppendorf, 5415C for five minutes at 14,000 rpm. Approximately 200 pL
of filtered sample was transferred into a 1.8 auto sampler vial with a 250 pL
glass
vial insert with polymer feet. A screw cap with PTFE septa was used to cap the
vial before vortexing the sample with a Vortex-Genie set at 2700 rpm.
[0255] Sample was then run on Agilent 1200 series LC equipped with binary,
isocratic pumps, vacuum degasser, heated column compartment, sampler
cooling system, UV DAD detector and RI detector. The column used was an
Aminex HPX-87H, 300 X 7.8 with a Bio-Rad Cation H refill, 30X4.6 guard column.
Column temperature was 40 C, with a mobile phase of 0.01 N sulfuric acid at a
flow rate of 0.6 mL/min for 40 minutes. Results are shown in Table 1.
Table 1: Retention times of fermentation products in aqueous phase
HPLC 302/310 FW RID Retention Range of UV
Normalized to 10 pL Time, min Standards, g/L Retention
injections Time, min
citric acid 192.12 8.025 0.3-17 7.616
glucose 180.16 8.83 0.5-71
pyruvic acid (Na) 110.04 9.388 0.1-5.2 8.5
A-Kiv (Na) 138.1 9.91 0.07-5.0 8.55
2,3-dihydroxyisovaleric 156.1 10.972 0.2-8.8 10.529
acid (Na)
succinic acid 118.09 11.561 0.3-16 11.216
lactic acid (Li) 96.01 12.343 0.3-17 11.948
glycerol 92.09 12.974 0.8-39
formic acid 46.03 13.686 0.2-13 13.232
acetate (Na) 82.03 14.914 0.5-16 14.563
meso-butanediol 90.12 17.583 0.1-19
+/- -2,3-butanediol 90.12 18.4 0.2-19
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isobutyric acid 88.11 19.685 0.1-8.0 19.277
ethanol 46.07 21.401 0.5-34
isobut raldeh de 72.11 27.64 0.01-0.11
isobutanol 74.12 32.276 0.2-15
3-OH-2-butanone (acetoin) 88.11 0.1-11 17.151
GC Analysis of Fermentation Products in the Solvent Phase
[0256] Samples were refrigerated until ready for processing. Samples were
removed from refrigeration and allowed to reach room temperature (about one
hour). Approximately 150 pL of sample was transferred using a m1000 Variable
Channel BioHit pipette with 100-1000 pL BioHit pipette tips into a 1.8 auto
sampler vial with a 250 pL glass vial insert with polymer feet. A screw cap
with
PTFE septa was used to cap the vial.
[0257] Sample was then run on Agilent 7890A GC with a 7683B injector and a
G2614A auto sampler. The column was a HP-InnoWax column (30 m x 0.32 mm
ID, 0.25 pm film). The carrier gas was helium at a flow rate of 1.5 mL/min
measured at 45 C with constant head pressure; injector split was 1:50 at 225
C;
oven temperature was 45 C for 1.5 minutes, 45 C to 160 C at 10 C/min for
0 minutes, then 230 C at 35 C/min for 14 minutes for a run time of 29 minutes.
Flame ionization detection was used at 260 C with 40 mL/min helium makeup
gas. Results are shown in Table 2.
Table 2: Retention times of fermentation products in solvent phase
GC 302/310 FW Solvent Range of Standards,
Normalized to 10 lJL Retention g/L
injections Time, min
isobut raldeh de 72.11 2.75 0.7-10.4
ethanol 46.07 3.62 0.5-34
isobutanol 74.12 5.53 0.2-16
3-OH-2-butanone (acetoin) 88.11 8,29 0.1-11
(+/-)-2,3-butanediol 90,12 10.94 0.1-19
isobut ric acid 88.11 11.907 0.1-7.9
meso-butanediol 90.12 11.26 0.1-6.5
glycerol 92.09 16.99 0.8-9
[0258] Samples analyzed for fatty acid butyl esters were run on Agilent 6890
GC
with a 7683B injector and a G2614A auto sampler. The column was a HP-DB-
FFAP column (15 meters x 0.53 mm ID (Megabore), 1-micron film thickness
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column (30 m x 0.32 mm ID, 0.25 pm film). The carrier gas was helium at a flow
rate of 3.7 mL/min measured at 45 C with constant head pressure; injector
split
was 1:50 at 225 C; oven temperature was 100 C for 2.0 minutes, 100 C to 250 C
at 10 C/min, then 250 C for 9 minutes for a run time of 26 minutes. Flame
ionization detection was used at 300 C with 40 mL/min helium makeup gas. The
following GC standards (Nu-Chek Prep; Elysian, MN) were used to confirm the
identity of fatty acid isobutyl ester products: iso-butyl palmitate, iso-butyl
stearate,
iso-butyl oleate, iso-butyl linoleate, iso-butyl linolenate, iso-butyl
arachidate.
[0259] Examples 1-14 describe various fermentation conditions that may be used
for the claimed methods. As an example, some fermentations were subjected to
Lipase Treatment pre-liquefaction and others were subjected to Lipase
Treatment
post-liquefaction. In other examples, the fermentation was subjected to Heat
inactivation Treatment. Following fermentation, the effective isobutanol titer
(Eff
Iso Titer) was measured, that is, the total grams of isobutanol produced per
liter
aqueous volume. Results are shown in Table 3.
Example 1 - (control)
[0260] Experiment identifier 2010YO14 included: Seed Flask Growth method,
Initial Fermentation Vessel Preparation method, Liquefaction method, Nutrient
Addition Prior to Inoculation method, Fermentation Vessel Inoculation method,
Fermentation Vessel Operating Conditions method, and all of the Analytical
methods. Oleyl alcohol was added in a single batch between 0.1- 1.0 hr after
inoculation. The butanologen was NGCI-070.
Example 2
[0261] Experiment identifier 2010YO15 included: Seed Flask Growth method,
Initial Fermentation Vessel Preparation method, Liquefaction method, Lipase
Treatment Post-Liquefaction method, Nutrient Addition Prior to Inoculation
method, Fermentation Vessel Inoculation method, Fermentation Vessel
Operating Conditions method, and all of the Analytical methods. Oleyl alcohol
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was added in a single batch between 0.1- 1.0 hr after inoculation. The
butanologen was NGCI-070.
Example 3
[0262] Experiment identifier 2010YO16 included: Seed Flask Growth method,
Initial Fermentation Vessel Preparation method, Liquefaction method, Lipase
Treatment Post-Liquefaction method, Nutrient Addition Prior to Inoculation
method with the exception of the exclusion of ethanol, Fermentation Vessel
Inoculation method, Fermentation Vessel Operating Conditions method, and all
of
the Analytical methods. Oleyl alcohol was added in a single batch between 0.1-
1.0 hr after inoculation. The butanologen was NGCI-070.
Example 4
[0263] Experiment identifier 2010YO17 included: Seed Flask Growth method,
Initial Fermentation Vessel Preparation method, Liquefaction method, Heat Kill
Treatment method Post-Liquefaction, Nutrient Addition Prior to Inoculation
method with the exception of the exclusion of ethanol, Fermentation Vessel
Inoculation method, Fermentation Vessel Operating Conditions method, and all
of
the Analytical methods. Oleyl alcohol was added in a single batch between 0.1-
1.0 hr after inoculation. The butanologen was NGCI-070.
Example 5
[0264] Experiment identifier 2010YO18 included: Seed Flask Growth method,
Initial Fermentation Vessel Preparation method, Liquefaction method, Lipase
Treatment Post-Liquefaction method with the exception of only adding 7.2 ppm
lipase after liquefaction, Heat Kill Treatment method Post-Liquefaction,
Nutrient
Addition Prior to Inoculation method, Fermentation Vessel Inoculation method,
Fermentation Vessel Operating Conditions method, and all of the Analytical
methods. Oleyl alcohol was added in a single batch between 0.1- 1.0 hr after
inoculation. The butanologen was NGCI-070.
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Example 6 - (control)
[0265] Experiment identifier 2010YO19 included: Seed Flask Growth method,
Initial Fermentation Vessel Preparation method, Liquefaction method, Heat Kill
Treatment method Post-Liquefaction, Nutrient Addition Prior to Inoculation
method, Fermentation Vessel Inoculation method, Fermentation Vessel
Operating Conditions method, and all of the Analytical methods. Oleyl alcohol
was added in a single batch between 0.1- 1.0 hr after inoculation. The
butanologen was NGCI-070.
Example 7 - (control)
[0266] Experiment identifier 2010YO21 included: Seed Flask Growth method,
Initial Fermentation Vessel Preparation method, Lipase Treatment Pre-
Liquefaction method, Liquefaction method, the Heat Kill Treatment during
liquefaction, Nutrient Addition Prior to Inoculation method, Fermentation
Vessel
Inoculation method, Fermentation Vessel Operating Conditions method, and all
of
the Analytical methods. Oleyl alcohol was added in a single batch between 0.1-
1.0 hr after inoculation. The butanologen was NGCI-070.
Example 8
[0267] Experiment identifier 2010YO22 included: Seed Flask Growth method,
Initial Fermentation Vessel Preparation method, Liquefaction method, Nutrient
Addition Prior to Inoculation method, Fermentation Vessel Inoculation method,
Fermentation Vessel Operating Conditions method, and all of the Analytical
methods. Oleyl alcohol was added in a single batch between 0.1- 1.0 hr after
inoculation. The butanologen was NGCI-070.
Example 9
[0268] Experiment identifier 2010YO23 included: Seed Flask Growth method,
Initial Fermentation Vessel Preparation method, Liquefaction method, Lipase
Treatment Post-Liquefaction method, no Heat Kill Treatment, Nutrient Addition
Prior to Inoculation method, Fermentation Vessel Inoculation method,
Fermentation Vessel Operating Conditions method, and all of the Analytical
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methods. Corn oil fatty acids made from crude corn oil was added in a single
batch between 0.1- 1.0 hr after inoculation. The butanologen was NGCI-070.
Example 10
[0269] Experiment identifier 2010YO24 included: Seed Flask Growth method,
Initial Fermentation Vessel Preparation method, Lipase Treatment Pre-
Liquefaction method, Liquefaction method, Heat Kill Treatment during
liquefaction, the Nutrient Addition Prior to Inoculation method with the
exception
of there being no addition of ethanol, Fermentation Vessel Inoculation method,
Fermentation Vessel Operating Conditions method, and all of the Analytical
methods. Oleyl alcohol was added in a single batch between 0.1- 1.0 hr after
inoculation. The butanologen was NGCI-070.
Example 11
[0270] Experiment identifier 2010YO29 included: Seed Flask Growth method,
Initial Fermentation Vessel Preparation method, Lipase Treatment Pre-
Liquefaction method, Liquefaction method, Heat Kill Treatment during
liquefaction, Nutrient Addition Prior to Inoculation method, Fermentation
Vessel
Inoculation method, Fermentation Vessel Operating Conditions method, and all
of
the Analytical methods. Corn oil fatty acids made from crude corn oil was
added
in a single batch between 0.1-1.0 hr after inoculation. The butanologen was
NGCI-070.
Example 12
[0271] Experiment identifier 2010YO30 included: Seed Flask Growth method,
Initial Fermentation Vessel Preparation method, Lipase Treatment Pre-
Liquefaction method, Liquefaction method, Heat Kill Treatment during
liquefaction, Nutrient Addition Prior to Inoculation method with the exception
of
there being no addition of ethanol, Fermentation Vessel Inoculation method,
Fermentation Vessel Operating Conditions method, and all of the Analytical
methods. Corn oil fatty acids made from crude corn oil was added in a single
batch between 0.1- 1.0 hr after inoculation. The butanologen was NGCI-070.
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Example 13 - (control)
[0272] Experiment identifier 2010YO31 included: Seed Flask Growth method,
Initial Fermentation Vessel Preparation method, Liquefaction method, Lipase
Treatment Post Liquefaction method, no Heat Kill Treatment, Nutrient Addition
Prior to Inoculation method with the exception of there being no addition of
ethanol, Fermentation Vessel Inoculation method, Fermentation Vessel
Operating Conditions method, and all of the Analytical methods. Corn oil fatty
acids made from crude corn oil was added in a single batch between 0.1- 1.0 hr
after inoculation. The butanologen was NGCI-070.
Example 14
[0273] Experiment identifier 2010YO32 included: Seed Flask Growth method,
Initial Fermentation Vessel Preparation method, Liquefaction method, Lipase
Treatment Post-Liquefaction method, no Heat Kill Treatment, Nutrient Addition
Prior to Inoculation method, Fermentation Vessel Inoculation method,
Fermentation Vessel Operating Conditions method, and all of the Analytical
methods. Corn oil fatty acids made from crude corn oil was added in a single
batch between 0.1- 1.0 hr after inoculation. The butanologen was NGCI-070.
Table 3: Fermentation conditions for Examples 1-14
Example Experimental Lipase Max cell Ethanol Solvent Heat Kill EffIso max Eff
# Identifier Count x g/L Lipase Titer Iso rate
10' /L* /L/h
1 2010YO14 none 27.2 5 Oleyl none 56.0 0.79
alcohol
2 2010YO15 10 ppm 31.5 5 Oleyl none 52.4 0.74
alcohol
3 2010YO16 10 ppm 6.7 0 Oleyl none 25.9 0.36
alcohol
4 2010YO17 none 7.9 0 Oleyl post - 17.2 0.25
alcohol liquefaction
2010YO18 7.2 ppm 16.2 5 Oleyl post - 45.8 0.66
alcohol liquefaction
6 2010YO19 none 17.5 5 Oleyl post - 48.1 0.69
alcohol liquefaction
7 2010YO21 10 ppm 21.2 5 Oleyl during 46.8 0.82
alcohol liquefaction
8 2010YO22 none 9 5 Oleyl during 56.2 0.87
alcohol liquefaction
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9 2010YO23 10 ppm 12.8 5 Corn Oil none 60.3 1.3
Fatty
Acids
2010YO24 10 ppm 25.3 0 Oleyl during 19.8 0.33
alcohol liquefaction
11 2010YO29 10 ppm 21.2 5 Corn Oil during 28.36 0.52
Fatty liquefaction
Acids
12 2010YO30 10 ppm 9 0 Corn Oil during 12.71 0.24
Fatty liquefaction
Acids
13 2010YO31 10 ppm 12.8 0 Corn Oil none 18.86 0.35
Fatty
Acids
14 2010YO32 10 ppm 25.3 5 Corn Oil none 53.36 0.92
Fatty
Acids
The "Eff Iso Titer g/L" = total grams of isobutanol produced per liter aqueous
volume
[0274] Examples 15 and 16 represent a comparison of fermentation and
isobutanol production in the presence and absence of post-liquefaction lipase
treatment. Results are shown in Tables 4 and 5.
Example 15
[0275] Experiment identifier 2010YO26 included: Seed Flask Growth method,
Initial Fermentation vessel Preparation method, the Liquefaction method, the
Lipase Treatment Post-Liquefaction method, the Nutrient Addition Prior to
Inoculation method, Fermentation vessel Inoculation method, Fermentation
vessel Operating Conditions method, and all of the Analytical methods. Corn
oil
fatty acids made from crude corn oil was added in a single batch between 0.1-
1.0 hr after inoculation. The corn oil fatty acids extracting solvent was
added in
equal volume to the broth volume. The butanologen was PNY2205. Between 46
hrs and 61 hrs fermentation time, the addition of 274 g of a 50% w/w sterile,
glucose solution was made because the glucose made from corn mash had been
all but depleted.
Example 16
[0276] Experiment identifier 2010YO27 included: Seed Flask Growth method,
Initial Fermentation vessel Preparation method, the Liquefaction method, the
Nutrient Addition Prior to Inoculation method, Fermentation vessel Inoculation
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method, Fermentation vessel Operating Conditions method, and all of the
Analytical methods. HD OCENOL 90/95 (oleyl alcohol, CAS No. 143-28-2,
Cognis, Monheim, DE) was added in a single batch between 0.1- 1.0 hr after
inoculation. The oleyl alcohol extracting solvent was added in equal volume to
the broth volume. The butanologen was PNY2205.
Table 4
Experimental ID Lipase Extracting Glucose Biomass
Addition Solvent Consumed produced
(cfu/mL)
2010YO26 Yes corn oil fatty acids 326.3 34.2 x 10
2010YO27 No oleyl alcohol 234.9 33.0 x 10
Table 5
Exp. ID Lipase Maximum Effective Maximum effective Yield
Addition residual i-BuOH i-BuOH production rate g i-BuOH/
i-BuOH in titer g/L/hr g glucose
aqueous (g/L) (aqueous volume)
/L
2010YO26 Yes 4.7 72.2 1.41 0.26
2010YO26 No 10.0 55.4 1.19 0.25
[0277] Examples 17 to 22 represent a comparison of the effect of fresh
extractant
versus recycled extractant on fermentation and isobutanol production. Results
are shown in Table 6. For these examples, 2 L and 10 L fermentations were
prepared as described below.
L Pre-Seed Flask Growth
[0278] A Saccharomyces cerevisiae strain (strain PNY2242 described above) that
was engineered to produce isobutanol from a carbohydrate source, with pdcl
deleted, pdc5 deleted, and pdc6, deleted was grown to 0.6-0.7 g/L dcw (OD600
1.5-2.5 - Thermo Helios a Thermo Fisher Scientific Inc., Waltham,
Massachusetts) in seed flasks (10 mL synthetic medium in a 125 mL, vented
flask) from a frozen culture. The culture was grown at 29-31 C in an
incubator
rotating at 260 rpm. The frozen culture was previously stored at -80 C. The
composition synthetic seed flask medium was:
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10.0 g/L dextrose
3.5 mL/L ethanol, anhydrous
3.7 g/L ForMediumTM Synthetic Complete Amino Acid (Kaiser) Drop-Out:
without HIS, without URA (Reference No. DSCK162CK)
6.7 g/L Difco Yeast Nitrogen Base without amino acids (No. 291920)
1% Ergersterol in 1:1::Tween 80:Ethanol
[0279] Two milliliters from the first seed flask culture was transferred to 25
mL in
a 250 mL, vented flask and grown at 29-31 C in an incubator rotating at 260
rpm.
The second seed flask uses the same synthetic medium as used above.
[0280] The culture was grown to 0.6-0.7 g/L dcw (OD600 1.0-3.0). Then, 8 mL of
this second flask culture was added to three flasks (2 L, vented, baffled
flasks)
with 200 mL of synthetic medium. The culture was grown in an incubator at 29-
31 C for 18-24 hrs. The three seed flasks use the same synthetic medium as
used in the first two seed flasks. These three flasks (600 mL of flask broth)
are
used to inoculate the propagation tank at a final aqueous volume of 6 L.
L Propagation Tank Liquefaction
[0281] A 10 L, B. Braun BioStat C fermentor was prepared for use. An inline pH
probe was placed in the fermentor. The zero was calibrated at pH=7. The span
was calibrated at pH=4. The probe was then placed into the fermentation
vessel,
through a side port. A dissolved oxygen probe (P02 probe) was also placed into
the fermentor through a side port. Tubing used for delivering nutrients, seed
culture, extracting solvent, and base were attached to the head plate and the
ends were foiled. The valve for harvesting and sampling were sterilized with
low
pressure steam and a steam trap at > 121 C for > 20 minutes.
[0282] The fermentation vessel temperature was controlled at 30 C with a
thermocouple and house water circulation loop. Wet corn kernels (#2 yellow
dent) were ground using a hammer mill with a 1.0 mm screen, and the resulting
ground whole corn kernels were then added to the fermentation vessel at a
charge that was 10-20% (dry corn solids weight) of the liquefaction reaction
mass. Difco Yeast Extract was added to the fermentor at 0.5% w/w of the total
batch weight.
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[0283] An alpha-amylase was added to the fermentation vessel per its
specification sheet while the fermentation vessel was mixing at 300-1500 rpm,
with sterile, house N2 being added at 1-2 slpm through the sparger. The
temperature set-point was changed from 55 C to 95 C in 5 C step changes with
a 5-15 minute hold at each step to ensure good mixing. When the temperature
was > 90 C, the liquefaction cook time was started and the liquefaction cycle
was
held at > 90 C for 60 minutes. The fermentation vessel temperature set-point
was set to the fermentation temperature of 30 C after the liquefaction cycle
was
complete. N2 was redirected from the sparger to the head space to prevent
foaming without the addition of a chemical antifoaming agent.
L Propagation Tank Operation
[0284] The fermentation vessels p02 probe was calibrated to zero while N2 was
being added to the fermentation vessel. The fermentation vessels p02 probe was
calibrated to its span with sterile air sparging at 400 rpm. The fermentation
vessel was inoculated from the final stage of the Pre-Seed Flask Growth step.
The three shake flasks were removed from the incubator/shaker and added to a
sterile vessel. The content of the sterile vessel was added to 5.3-5.5 L of
the
liquefied mash that was made during the Propagation Tank Liquefaction method.
[0285] The fermentation temperature was controlled between 29-31 C. The
agitation speed was fixed at 400 rpm. Air was sparged for the entire
fermentation
at 2.0 slpm. The pH was controlled at 5.4-5.5 by using NH4OH and the PID
control loop for the fermentor. There was 0.3-0.5 bar of back pressure set on
the
fermentor, controlled by a PID loop that controlled a back pressure control
valve.
[0286] At 16-20 hrs after inoculation, a glucoamylase (1.8 mL of Distillase L-
400, Genencor, Palo Alto, CA) was added to start simultaneous saccharification
and fermentation, releasing glucose from the dissolved starch. Also, 5.5 L of
HD
OCENOL 90/95 (oleyl alcohol, Cognis, Monheim, DE) was added to the
fermentor. At 34-36 hrs, the agitator speed was reduced to 100 rpm. After
10 minutes, the agitator was turned off and the airflow to the fermentor was
changed from sparge mode to overlay mode.
10 L Production Tank Liquefaction
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[0287] A 10 L Production Tank Liquefaction was performed as described above.
The fermentation vessel temperature was controlled at 30 C with a thermocouple
and house water circulation loop. Wet corn kernels (#2 yellow dent) were
ground
using a hammer mill with a 1.0 mm screen, and the resulting ground whole corn
kernels were then added to the fermentation vessel at a charge that was 25-35%
(dry corn solids weight) of the liquefaction reaction mass. A 75 mL addition
of a
100X Vitamin Solution (2 g/L thiamine and 10 g/L nicotinic acid) was made to
the
fermentor. An alpha-amylase was added to the fermentation vessel was added
as described above. Also, an addition of 6-7 mL/L anhydrous ethanol was
made to the fermentor after the fermentor was returned to 30 C
L Production Tank Operation
[0288] The fermentation vessels p02 probe was calibrated to zero while N2 was
being added to the fermentation vessel. The fermentation vessels p02 probe was
calibrated to its span with sterile air sparging at 400 rpm. The fermentation
vessel was inoculated from Propagation Tank. An aseptic transfer was made
from the Propagation Tank after 36 hrs of growth time in the propagation tank
and the fermentation agitation was turned off for > 10 minutes. This allowed
for
significant separation of the oleyl alcohol and the aqueous phase. The aseptic
transfer was made from the harvest valve on the Propagation Tank, which is
located at the bottom of this fermentor. Approximately 10% v/v was added to
the
Production Tank, based on the tanks final non-solvent volume after transfer.
[0289] The fermentation temperature was controlled between 29-31 C. The
agitation speed was fixed at 400 rpm. Air was sparged for the entire
fermentation
at 2.0 slpm. The pH was controlled at 5.2-5.3 by using NH4OH and the PID
control loop for the fermentor. There was 0.3-0.5 bar of back pressure set on
the
fermentor, controlled by a PID loop that controlled a back pressure control
valve.
[0290] Just prior to inoculation, 25-35% v/v Cognis Emery 610 SOYA Fatty Acid
was aseptically added to the fermentor. The fermentor was inoculated with 10%
v/v fermentation broth after the completion of the 10 L Propagation Tank
Operation method. Just after inoculation, a glucoamylase (Distillase L-400)
was
added to release glucose from the starch. Additional glucoamylase additions
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were made when needed to maintain the glucose excess. Just after inoculation,
a lipase (Novozymes Lipolase 100L) was added to the fermentor at 4-15 ppm.
2 L Pre-Seed Flask Growth
[0291] A 2 L pre-seed flask growth was prepared using a Saccharomyces
cerevisiae strain (strain PNY2242 described above) and was grown to 0.6-0.7
g/L
dcw (OD600 1.5-2.5 - Thermo Helios a Thermo Fisher Scientific Inc., Waltham,
Massachusetts) in seed flasks (10 mL synthetic medium in a 125 mL, vented
flask) from a frozen culture. The culture was grown at 29-31 C in an
incubator
rotating at 260 rpm. The frozen culture was previously stored at -80 C. The
composition synthetic seed flask medium was:
10.0 g/L dextrose
3.5 mL/L ethanol, anhydrous
3.7 g/L ForMedium Synthetic Complete Amino Acid (Kaiser) Drop-Out:
without HIS, without URA (Reference No. DSCK162CK)
6.7 g/L Difco Yeast Nitrogen Base without amino acids (No. 291920)
1 % Ergersterol in 1:1::Tween 80: Ethanol.
[0292] Two milliliters from the first seed flask culture was transferred to 25
mL in
a 250 mL, vented flask and grown at 29-31 C in an incubator rotating at 260
rpm.
The second seed flask uses the same synthetic medium as used above.
[0293] The culture was grown to 0.6-0.7 g/L dcw (OD600 1.0-3.0). Then 4 mL of
this second flask culture was added to 100 mL of corn mash centrate in a 2 L
flask. The culture was grown in an incubator at 29-31 C for 18-24 hrs. Then
500
mL of HD OCENOL 90/95 (oleyl alcohol, Cognis, Monheim, DE) was added to
the flask. The flask was allowed to grow for 6-8 hrs. Then 2 mL of a 1.2 g
Distillase L-400, (Genencor, Palo Alto, CA) in 80 mL of deionized water was
added to the centrate to release glucose from the dissolved starch in the
centrate. The culture continued to grow for 18-24 hrs. The final biomass
concentration was 6-12 g/L dcw.
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[0294] The corn mash centrate was made by liquefying corn in the following
recipe:
1150 g tap water
340.5 g 1 mm screened ground corn
13.5 g yeast extract (Difco No. 9102333, low dusting)
27 g peptone
4.1 g urea
40.5 mg nicotinic acid
40.5 mg thiamine.
[0295] Then the material was centrifuged for 30 minutes in a Sorval RC5C
centrifuge. The supernatant was separated from the solids pellet. The
supernatant was heated in a Steris autoclave for a 5 minute liquid cycle and
is
defined as centrate.
2 L Fermentation Preparation
2 L Initial Fermentation Vessel Preparation
[0296] A 2 L Initial Fermentation Vessel Preparation was prepared as described
above. The fermentation vessel temperature was controlled at 55 C with a
thermocouple and house water circulation loop. Wet corn kernels (#2 yellow
dent) were ground using a hammer mill with a 1.0 mm screen, and the resulting
ground whole corn kernels were then added to the fermentation vessel at a
charge that was 25-30% (dry corn solids weight) of the liquefaction reaction
mass. In addition, Liquefaction was conducted as described above. An alpha-
amylase was added to the fermentation vessel per its specification sheet while
the fermentation vessel was mixing at 300-1200 rpm, with sterile, house N2
being
added at 0.3 slpm through the sparger.
2 L Additions Prior to Inoculation
[0297] The following nutrients were added to the fermentation vessel prior to
inoculation, after liquefaction, on a post-inoculation volume basis:
30 mg/L of nicotinic acid
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30 mg/L of thiamine
1 mL/L of a 1 % ergersterol w/v solution in 1:1:Tween 80:Ethanol
6.3 mL/L ethanol
2 g/L urea.
2 L Fermentation Vessel Inoculation
[0298] The fermentation vessels p02 probe was calibrated to zero while N2 was
being added to the fermentation vessel. The fermentation vessels p02 probe was
calibrated to its span with sterile air sparging at 300 rpm. The fermentation
vessel was inoculated from the final stage of the Pre-Seed Flask Growth step.
The shake flask was removed from the incubator/shaker and centrifuged for
30 minutes. The liquid (oleyl alcohol and aqueous supernatant) was discarded
and the cell pellet was re-suspended in the Pre-Seed Flask Growth medium
(synthetic medium). The 100 mL of the aqueous phase was transferred to a
sterile inoculation bottle. The inoculum was pumped into the fermentation
vessel
through a peristaltic pump.
2 L Lipase Addition After Inoculation
[0299] A Lipolase solution (100 L stock solution) was prepared to an
enzyme concentration of 1.2-1.4 mg/mL. The solution was added to the
fermentation after inoculating the fermentor to the desired part per million
concentration based on the non-solvent volume. The addition time occurred < 1
hr after inoculating the fermentor.
2 L Soy Bean Oil Fatty Acid Addition
[0300] To the fermentation vessel was added 0.1-0.5 L/L (post-inoculation
volume) of either virgin Cognis Emery 610 SOYA Fatty Acid or recycled Cognis
Emery 610 SOYA Fatty Acids that contains 0-30 weight percent fatty acid butyl
ester.
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2 L Fermentation Vessel Operating Conditions
[0301] The fermentation vessel was operated at 300C for the entire growth and
production stages. The pH was allowed to drop from a pH between 5.7-5.9 to a
control set-point of 5.25 without adding any acid. The pH was controlled for
the
remainder of the growth and production stage at a pH =5.2 with ammonium
hydroxide. Sterile air was added to the fermentation vessel, through the
sparger,
at 0.2-0.3 slpm for the remainder of the growth and production stages. The p02
was not controlled. The agitator was set to a fixed rpm at 300 rpm. The stir
shaft
had two Rushton impellers below the aqueous level and one pitched blade
impeller above the aqueous level. The glucose was supplied through
simultaneous saccharification and fermentation of the liquified corn mash by
adding a glucoamylase. The glucose was kept excess (1-50 g/L) for as long as
starch was available for saccharification.
[0302] A 5-20 mL sample was pulled from a fermentation vessel and placed in a
centrifuge tube and centrifuged for cell mass measurement using the procedure
described above. In addition, Analytical methods such as gas analyses as well
as LC analyses of fermentation products in the aqueous phase and GC analyses
of fermentation products in the solvent phase were conducted as described
above.
[0303] The fermentation conditions for Examples 17 to 22 are provided below
and
a summary of the results (virgin soy bean oil fatty acids and recycled soy
bean
fatty acids with fatty acid butyl esters) are shown in Table 6.
Example 17
[0304] Experimental Identifier GLNOR1050 included: 10 L Pre-Seed Flask
Growth, 10 L Propagation Tank Liquefaction, 10 L Propagation Tank Operation,
L Production Tank Liquefaction, 10 L Production Tank Operation with 10 ppm
Lipolase 100L (Genencor) added to the fermentor, extractant: Virgin Cognis
Emery 610 SOYA Fatty Acid (virgin soy bean oil fatty acid). The liquid
solvent
and non-solvent material was separated in a Sorval RC-12 centrifuge, and all
Analytical methods.
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Example 18
[0305] Experimental Identifier GLNOR1051 included: 10 L Pre-Seed Flask
Growth, 10 L Propagation Tank Liquefaction, 10 L Propagation Tank Operation,
L Production Tank Liquefaction, 10 L Production Tank Operation with 4 ppm
Lipolase 100L (Genencor) added to the fermentor, extractant: Virgin Cognis
Emery 610 SOYA Fatty Acid (virgin soy bean oil fatty acid). The liquid
solvent
and non-solvent material was separated in a Sorval RC-12 centrifuge, and all
Analytical methods.
Example 19
[0306] Identifier 2011YO29 included: 2 L Pre-Seed Flask Growth, 2 L
Fermentation Preparation, 2 L Liquefaction, 2 L Additions Prior to
Inoculation, 2 L
Fermentation Vessel Inoculation, 2 L Lipase Addition After Inoculation at a
final
concentration of 10 ppm, 2 L Recycled Soy Bean Oil Fatty Acid Addition
(Recycled Cognis Emery 610 SOYA Fatty Acid and fatty acid butyl ester from
Example 56A - 50% v/v solvent load), 2 L Fermentation Vessel Operating
Conditions, and all Analytical methods.
Example 20
[0307] Identifier 2011YO30 included: 2 L Pre-Seed Flask Growth, 2 L
Fermentation Preparation, 2 L Liquefaction, 2 L Additions Prior to
Inoculation, 2 L
Fermentation Vessel Inoculation, 2 L Lipase Addition After Inoculation at a
final
concentration of 10 ppm, added 0.4 L/L (post-inoculation volume) Virgin Cognis
Emery 610 SOYA Fatty Acids that included 20-30% fatty acid butyl esters, 2 L
Fermentation Vessel Operating Conditions, and all Analytical methods.
Example 21
[0308] Identifier 2011YO31 included: 2 L Pre-Seed Flask Growth, 2 L
Fermentation Preparation, 2 L Liquefaction, 2 L Additions Prior to
Inoculation, 2 L
Fermentation Vessel Inoculation, 2 L Lipase Addition After Inoculation at a
final
concentration of 10 ppm, 2 L Recycled Soy Bean Oil Fatty Acid Addition
(Recycled Cognis Emery 610 SOYA Fatty Acid and fatty acid butyl ester from
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Example 56B - 10% v/v solvent load), 2 L Fermentation Vessel Operating
Conditions, and all Analytical methods.
Example 22
[0309] Identifier 2011YO32 included: 2 L Pre-Seed Flask Growth, 2 L
Fermentation Preparation, 2 L Liquefaction, 2 L Additions Prior to
Inoculation, 2 L
Fermentation Vessel Inoculation, 2 L Lipase Addition After Inoculation at a
final
concentration of 10 ppm, added 0.4 L/L (post-inoculation volume) Virgin Cognis
Emery 610 SOYA Fatty Acids, 2 L Fermentation Vessel Operating Conditions,
and all Analytical methods.
Table 6
Exp. ID Lipase Max Cell Solvent Max i- FOR Extractant
ppm Count x Loading BuOH (aq) Volumetric
107 Vol % /L Rate /L/hr
GLNOR 10 21.3 28% 6.9 0.97 Virgin Soy Bean Oil
1050 Fatty Acid
GLNOR 4 20.9 28% 8.9 0.83 Virgin Soy Bean Oil
1051 Fatty Acid
2011YO29 10 20.9 50% 8.2 0.85 Recycled Soy Bean
Oil Fatty Acid and
Fatty Acid Butyl
Ester
2011YO30 10 24.4 40% 9.8 0.88 Virgin Soy Bean Oil
Fatty Acid and
Fatty Acid Butyl
Ester
2011YO31 10 9.8 10% 12.7 0.42 Recycled Soy Bean
Oil Fatty Acid and
Fatty Acid Butyl
Ester
2011YO32 10 26.5 40% 6.8 0.94 Virgin Soy Bean Oil
Fatty Acid
Example 23
[0310] The following example describes the production of isobutanol by
fermentation using sucrose as a fermentable carbon source.
Generation of Biomass
[0311] Inoculum: A seed medium was prepared to initiate the growth of the
isobutanologen. The composition of the seed medium was as follows:
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ammonium sulfate, 5 g/L; potassium phosphate monobasic, 3 g/L; magnesium
sulfate heptahydrate, 0.5 g/L; ethanol, 3.2 g/L; yeast extract (BBL), 5 g/L;
glucose, 10 g/L; MES buffer, 150 mmol/L; biotin, 50 pg/L; and a trace element
solution, 1 mL/L, which contains in 1 L water, 15 g EDTA, 4.5 g zinc sulfate
heptahydrate, 0.8 g manganese chloride dehydrate, 0.3 g cobalt chloride
hexahydrate, 0.3 g copper sulfate pentahydrate, 0.4 g disodium molybdenum
dehydrate, 4.5 g calcium chloride dihydrate, 3 g iron sulfate heptahydrate, 1
g
boric acid, 0.1 g potassium iodide. The pH was adjusted to 5.5, and then the
medium filter sterilized through an 0.22 p sterile filter apparatus.
Preparation of the 10 L Fermentor for Biomass Production
[0312] A single vial of the isobutanologen PNY2205 was aseptically transferred
to
15 mL seed medium in a 125 mL vented flask for over night growth at 30 C and
260 rpm shaking. The culture was aseptically transferred to 500 mL of the same
medium in a 2 L baffled, vented flask for over night growth at 30 C and 260
rpm
shaking, and transferred to a prepared 10 L Sartorius C fermentor (Sartorius
AG,
Goettingen, Germany) when the culture reached OD600 7.
[0313] A 10 L Sartorius C fermentor was prepared with 6 L initial volume of
growth medium. The growth medium composition and preparation was as
follows: prior to sterilization, ammonium sulfate, 1 g/L; potassium phosphate
monobasic, 5 g/L; magnesium sulfate, heptahydrate, 2 g/L; yeast extract
(AmberexTM 695), 2 g/L; Antifoam Sigma 204, 0.5 mL/L; biotin, 100 pg/L; and
1 mL/L trace element solution (prepared in 1 L water: 15 g EDTA, 4.5 g zinc
sulfate heptahydrate, 0.8 g manganese chloride dehydrate, 0.3 g cobalt
chloride
hexahydrate, 0.3 g copper sulfate pentahydrate, 0.4 g disodium molybdenum
dehydrate, 4.5 g calcium chloride dihydrate, 3 g iron sulfate heptahydrate, 1
g
boric acid, 0.1 g potassium iodide). After steam sterilization at 121 C in
place,
the vessel was cooled, and 60 g of the feed medium was added. The feed
medium was prepared as follows: sucrose, 50% solution, 2.97 L; biotin, 1.4 mg;
34 mL of the trace mineral solution; titrated to pH 7.5 with 5N sodium
hydroxide
and steam sterilized; post sterilization and cooling, 130 mL ethanol and 320
mL
of a 20% (w/v) filter sterilized solution of yeast extract (AmberexTM 695) was
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added. The initial sugar concentration in the 10 L fermentor was thus 3.7 g/L
sucrose, 0.8 g/L glucose, and 0.8 g/L fructose.
[0314] The fermentation was controlled at pH 5.5 (with ammonium hydroxide
addition), 30 C, airflow at 2.0 standard liters per minute, dissolved oxygen
at 30%
by agitation control, and 0.5 barg back pressure. After inoculation, the sugar
was
consumed until the residual measurement of glucose was less than 0.1 g/L, and
then the feed program began; this occurred at 11 hours elapsed fermentation
time. The program was established to maintain sucrose limitation until OD600
of
20 (approximately 8 g/L dry cell weight) was achieved, with a programmed
growth rate of 0.1/hr. The actual measured growth rate in this experiment was
0.18/hr. The targeted OD600 was reached after 20 hours fermentation time.
[0315] Once the target was achieved, the culture was harvested aseptically,
and
centrifuged in a Sorvall RC12BP centrifuge. The resulting pellet was
resuspended to a final volume of 300 mL with isobutanol production medium,
described below. This culture was used as the inoculum for the isobutanol
production fermentors.
Isobutanol Production
[0316] Preparation of production fermentors: Two one liter glass Applikon
(Applikon, Inc, Holland) fermentors associated with a Sartorius BioStat B Plus
Twin control unit (Sartorius AG, Goettingen, Germany) were used for the
isobutanol production. The fermentors were prepared with 1 L deionized water,
and sterilized by autoclaving at 121 C for 30 minutes. Once the fermentors
cooled, the water was aseptically removed, and the volume of filter sterilized
production medium, as indicated in Table 7, was added. The production medium
composition was as follows: yeast nitrogen base without amino acids (Difco),
6.7 g/L; Yeast Synthetic Drop-out Medium Supplements without histidine,
leucine,
tryptophan, and uracil (Sigma), 2.70 g/L; tryptophan, 1.6 mg/L; leucine, 8
mg/L;
ethanol, 2.8 g/L; Antifoam Sigma 204, 0.2 mL/L; sucrose, 25 g/L. Just before
inoculation, filter sterilized lipase solution was as indicated in Table 7.
The lipase
solution was prepared by dilution of Lipolase L100 (Sigma) in 10 mM potassium
phosphate buffer, pH 7, to a final concentration of 1.25 mg protein/mL. The
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solution was prepared and stored for one day at 5 C before addition to the
fermentors.
Table 7
Fermentor Fermentation Broth SOFA lipase
mL mL m /L
A 440 320 0
B 440 320 10
C 520 240 10
D 520 240 25
[0317] The fermentors were controlled at pH 5.2 (by addition of 20% potassium
hydroxide), 30 C, airflow at 0.2 standard liters per minute, and dissolved
oxygen
at 3% by agitation control.
[0318] The fermentors were each inoculated with 40 mL of the concentrated
biomass, to initial OD600 20-25 (approximately 8-10 g/L dry cell weight). An
addition 4 mL of a filter sterilized vitamin solution (thiamine-HCI, 1 mg/mL;
nicotinic acid, 1 mg/mL, in water) was added at inoculation, as was the volume
of
filter sterilized Soya Oil Fatty Acids (SOFA) indicated in Table 7. Samples (5-
ml-) were drawn every 2-3 hours, and assayed for glucose and sucrose by a
YSI Select Biochemistry Analyzer (YSI, Inc., Yellow Springs, Ohio). As sucrose
was consumed, a feed of 50% sucrose (w/w) was added to maintain a
concentration of 5-30 g/L. The aqueous and organic phases of the samples were
separated and analyzed by the HPLC method described above via an Agilent
1100 HPLC. For analysis of organic acids and alcohols, a Shodex Sugar
SH1011 column was used with 0.01 N sulfuric acid mobile phase. For analysis of
sucrose, glucose, and fructose, a BioRad Aminex HPX-87N column with 0.01 M
Na2HPO4 (pH 8) mobile phase was used.
[0319] Each of the fermentors with lipase added had lower concentrations of
isobutanol in the aqueous phase and free isobutanol in the solvent phase. The
aqueous and solvent phase concentrations of isobutanol are shown in FIG. 6.
Addition of more lipase at the same solvent loading also resulted in lower
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aqueous titers of isobutanol and lower free isobutanol in the solvent, and
more
isobutanol as FABE.
[0320] The cultivations which included lipase resulted in a higher effective
titer of
isobutanol than the control fermentor without lipase. FIG. 7 shows the
effective
titer of isobutanol. In this example, the effective titer was calculated based
on the
initial measured weight of broth in the fermentor after inoculation and the
initial
measured weight of solvent charged to the fermentor. The solvent density was
assumed to be 0.88 g/mL and the aqueous broth density 1.00 g/mL throughout
the fermentation. Addition of more lipase at the lower solvent loading did
result in
higher effective titers of isobutanol (D vs C), but not as much as increasing
the
relative volume of solvent (C vs B).
[0321] Sugar consumed, calculated in glucose equivalents, was higher in
fermentors with lipase added, shown in FIG. 8. Glucose equivalents consumed is
calculated from the measured sugars fed and remaining in the fermentor, with
each mole of sucrose counted as two moles of glucose and each mole of fructose
counted as one mole of glucose, then converted to grams via the molecular
weight of glucose. The concentration of glucose equivalents consumed is also
calculated on the basis of the initial volume of fermentation broth after
inoculation.
Example 24
Lipase Treatment of Liquefied Corn Mash for Simultaneous Saccharification and
Fermentation with In-situ Product Removal Using Oleyl Alcohol
[0322] Samples of broth and oleyl alcohol taken from fermentations run as
described above in Examples 1, 2, and 3 were analyzed for wt% lipid
(derivatized
as fatty acid methyl esters, FAME) and for wt% free fatty acid (FFA,
derivatized
as fatty acid methyl esters, FAME) according to the method described by E. G.
Bligh and W. J. Dyer (Canadian Journal of Biochemistry and Physiology, 37:911-
17, 1959, hereafter Reference 1). The liquefied corn mash that was prepared
for
each of the three fermentations was also analyzed for wt% lipid and for wt%
FFA
after treatment with Lipolase 100 L (Novozymes) (10 ppm of Lipolase total
soluble protein (BCA protein analysis, Sigma Aldrich)) per kg of liquefaction
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reaction mass containing 30 wt% ground corn kernels). No lipase was added to
the liquefied corn mash in Example 1 (control), and the fermentations
described
in Examples 2 and 3 containing liquefied corn mash treated with lipase (no
heat
inactivation of lipase) were identical except that no ethanol was added to the
fermentation described in Example 3.
[0323] The % FFA in lipase-treated liquefied corn mash prepared for
fermentations run as described in Examples 2 and 3 was 88% and 89%,
respectively, compared to 31 % without lipase treatment (Example 1). At 70 h
(end of run (EOR)), the concentration of FFA in the OA phase of fermentations
run as described in Examples 2 and 3 (containing active lipase) was 14% and
20%, respectively, and the corresponding increase in lipids (measured as corn
oil
fatty acid methyl ester derivatives) was determined by GC/MS to be due to the
lipase-catalyzed esterification of COFA by OA, where COFA was first produced
by lipase-catalyzed hydrolysis of corn oil in the liquefied corn mash; the
production of oleyl palmitate, oleyl stearate, and oleyl oleate was confirmed
by
GC/MS, and a fourth ester was tentatively identified as oleyl linoleate.
Results for
FFA and lipid analysis are shown in Table 8.
Table 8: Lipid and free fatty acid content of fermentations containing
oleyl alcohol as ISPR extractant and active lipase
fermentation lipase time (h), lipids FFA lipids FFA lipids + % FFA
sample (wt%) (wt%) (g) (g) FFA(g)
Example 1 none liq. mash 0.61 0.28 5.3 2.4 7.7 31
Example 1 none 0.8 h, broth 0.49 0.22 5.5 2.5 8.0 31
Example 1 none 31 h, broth 0.19 0.03 2.1 0.3 2.4 13
Example 1 none 31 h, OA 0.36 0.21 3.4 2.0 5.3 37
Example 1 none 70 h, broth 0.15 0.03 1.7 0.3 2.0 15
Example 1 none 70 h, OA 0.57 0.25 5.3 2.3 7.7 31
Example 2 10 ppm liq. mash 0.13 0.97 1.1 8.5 9.6 88
Example 2 10 ppm 0.8 h, broth 0.15 0.62 1.7 7.0 8.7 81
Example 2 10 ppm 31 h, broth 0.16 0.05 1.8 0.5 2.3 23
Example 2 10 ppm 31 h, OA 0.37 0.23 3.5 2.2 5.7 38
Example 2 10 ppm 70 h, broth 0.17 0.02 1.9 0.3 2.2 13
Example 2 10 ppm 70 h, OA 0.60 0.10 5.7 1.0 6.7 14
Example 3 10 ppm liq. mash 0.12 0.97 1.0 8.5 9.5 89
Example 3 10 ppm 0.8 h, broth 0.32 0.40 3.6 4.5 8.1 56
Example 3 10 ppm 31 h, broth 0.17 0.05 1.9 0.6 2.5 24
Example 3 10 ppm 31 h, OA 0.38 0.22 3.6 2.1 5.7 37
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Example 3 10 ppm 70 h, broth 0.15 0.02 1.7 0.2 1.9 13
Example 3 10 ppm 70 h, OA 0.46 0.12 4.4 1.1 5.6 20
Example 25
Heat Inactivation of Lipase in Lipase-treated Liquefied Corn Mash to Limit
Production of Oleyl Alcohol Esters of Corn Oil Free Fatty Acids
[0324] Tap water (918.4 g) was added to a jacketed 2-L resin kettle, then
474.6 g
wet weight (417.6 g dry weight) of ground whole corn kernels (1.0 mm screen on
hammer mill) was added with stirring. The mixture was heated to 55 C with
stirring at 300 rpm, and the pH adjusted to 5.8 with 2 N sulfuric acid. To the
mixture was added 14.0 g of an aqueous solution containing 0.672 g of
Spezyme -FRED L (Genencor , Palo Alto, CA), and the temperature of the
mixture increased to 85 C with stirring at 600 rpm and pH 5.8. After 120
minutes
at 85 C, the mixture was cooled to 50 C and 45.0 mL aliquots of the resulting
liquefied corn mash were transferred to 50-mL polypropylene centrifuge tubes
and stored frozen at -80 C.
[0325] In a first reaction, 50 g of liquefied corn mash prepared as described
above was mixed with 10 ppm Lipolase 100 L (Novozymes) for 6 h at 55 C and
with no inactivation of lipase at 85 C for 1 h, the mixture was cooled to 30
C. In a
second reaction, 50 g of liquefied corn mash was mixed with 10 ppm Lipolase
for 6 h at 55 C, then heated to 85 C for 1 h (lipase inactivation), then
cooled to
30 C. In a third reaction, 50 g of liquefied corn mash without added lipase
was
mixed for 6 h at 55 C, and with no heating at 85 C for 1 h, the mixture was
cooled to 30 C, 38 g of oleyl alcohol was added, and the resulting mixture
stirred
for 73 h at 30 C. In a fourth reaction, 50 g of liquefied corn mash without
added
lipase was mixed for 6 h at 55 C, then heated to 85 C for 1 h, then cooled to
30 C. Each of the four reaction mixtures was sampled at 6 h, then 38 g of
oleyl
alcohol added, and the resulting mixtures stirred at 30 C and sampled at 25 h
and 73 h. Samples (both liquefied mash and oleyl alcohol (OA)) were analyzed
for wt% lipid (derivatized as fatty acid methyl esters, FAME) and for wt% free
fatty
acid (FFA, derivatized as fatty acid methyl esters, FAME) according to the
method described by Reference 1.
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[0326] The % FFA in the OA phase of the second reaction run with heat
inactivation of lipase prior to OA addition was 99% at 25 h and 95% at 73 h,
compared to only 40% FFA and 21% FFA at 25 h and 73 h, respectively, when
the lipase in lipase-treated liquefied corn mash was not heat inactivated
(first
reaction). No significant change in % FFA was observed in the two control
reactions without added lipase. Results are shown in Table 9.
Table 9: Lipid and free fatty acid content of a mixture of liquefied corn mash
and oleyl alcohol in the presence or absence of active or heat-inactivated
lipase
reaction time (h), lipids FFA lipids FFA lipid+FFA % FFA
conditions sample (wt%) (wt%) (mg) (mg) (mg)
ppm active lipase, 6 h, liq. mash 0.08 0.71 41 345 386 89
no 85 C heat treatment 25 h, liq. mash 0.22 0.06 105 27 132 20
25 h, OA 0.58 0.39 212 143 355 40
73 h, liq. mash 0.25 0.05 121 22 143 18
73 h, OA 0.91 0.24 333 88 420 21
10 ppm inactive lipase, 6 h, liq. mash 0.06 0.45 28 224 252 89
85 C heat treatment 25 h, liq. mash 0.10 0.11 49 54 103 53
25 h, OA 0.02 0.96 8 366 374 99
73 h, liq. mash 0.24 0.15 117 72 189 62
73 h, OA 0.06 1.11 23 424 447 95
no lipase, 6 h, liq. mash 0.80 0.40 401 199 599 33
no 85 C heat treatment 25 h, liq. mash 0.30 0.05 147 25 173 15
25 h, OA 0.55 0.36 212 139 351 40
73 h, liq. mash 0.23 0.05 117 26 143 23
73 h, OA 0.79 0.42 305 162 467 34
no lipase, 6 h, liq. mash 0.74 0.36 370 183 553 33
85 C heat treatment 25 h, liq. mash 0.31 0.05 156 27 183 15
25 h, OA 0.60 0.35 233 136 369 37
73 h, liq. mash 0.20 0.05 99 23 121 23
73 h, OA 0.84 0.41 326 159 486 33
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Example 26
Heat Inactivation of Lipase in Lipase-treated Liquefied Corn Mash for
Simultaneous Saccharification and Fermentation with In-situ Product Removal
Using Oleyl Alcohol
[0327] Three fermentations were run as described above in Examples 4, 5, and
6.
No lipase was added to the liquefied corn mash in Examples 4 and 6 prior to
fermentation, and the Lipase Treatment of the liquefied corn mash in the
fermentation described in Example 5 (using 7.2 ppm of Lipolase total soluble
protein) was followed immediately by Heat Inactivation Treatment (to
completely
inactivate the lipase), and subsequently followed by Nutrient Addition Prior
to
Inoculation and fermentation. The % FFA in liquefied corn mash prepared
without lipase treatment for fermentations run as described in Examples 4 and
6
was 31 % and 34%, respectively, compared to 89% with lipase treatment
(Example 5). Over the course of the fermentations listed in Table 10, the
concentration of FFA in the OA phase did not decrease in any of the three
fermentations, including that containing heat-inactivated lipase. The % FFA in
the OA phase of the fermentation run according to Example 5 (with heat
inactivation of lipase prior to fermentation) was 95% at 70 h (end of run
(EOR)),
compared to only 33% FFA for the remaining two fermentations (Examples 4 and
6) where liquefied corn mash was not treated with lipase. Results are shown in
Table 10.
Table 10: Lipid and free fatty acid content of fermentations containing oleyl
alcohol
as ISPR extractant and heat-inactivated lipase (after lipase treatment of
liquefied
mash)
fermentation lipase time (h), lipids FFA lipids FFA lipid + % FFA
sample (wt%) (wt%) (g) (g) FFA (g)
Example 4 none liquefied mash 0.65 0.30 7.2 3.3 10.4 31
Example 4 none 0.2 h, broth 0.56 0.28 6.6 3.3 9.9 33
Example 4 none 4.3 h, broth 0.28 0.09 3.3 1.0 4.4 24
Example 4 none 4.3 h, OA 0.45 0.27 4.0 2.4 6.4 37
Example 4 none 30 h, broth 0.17 0.05 2.0 0.6 2.7 24
Example 4 none 30 h, OA 0.63 0.29 5.7 2.6 8.3 32
Example 4 none 53 h, broth 0.13 0.04 1.5 0.5 2.0 23
Example 4 none 53 h, OA 0.67 0.32 6.0 2.9 8.9 32
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Example 4 none 70 h, broth 0.13 0.04 1.5 0.4 1.9 23
Example 4 none 70 h, OA 0.64 0.31 5.8 2.8 8.5 33
Example 5 7.2 ppm liquefied mash 0.11 0.89 1.3 9.9 11.2 89
Example 5 7.2 ppm 0.2 h, broth 0.25 0.83 2.9 9.8 12.8 77
Example 5 7.2 ppm 4.3 h, broth 0.14 0.17 1.6 2.1 3.7 56
Example 5 7.2 ppm 4.3 h, OA 0.02 0.84 0.2 7.9 8.1 97
Example 5 7.2 ppm 30 h, broth 0.08 0.18 1.0 2.1 3.1 68
Example 5 7.2 ppm 30 h, OA 0.04 0.92 0.3 8.6 8.9 96
Example 5 7.2 ppm 53 h, broth 0.07 0.11 0.9 1.3 2.2 61
Example 5 7.2 ppm 53 h, OA 0.08 0.95 0.7 8.9 9.6 93
Example 5 7.2 ppm 70 h, broth 0.08 0.10 0.9 1.2 2.1 55
Example 5 7.2 ppm 70 h, OA 0.05 0.94 0.4 8.8 9.2 95
Example 6 none liquefied mash 0.66 0.34 7.3 3.8 11.1 34
Example 6 none 0.2 h, broth 0.63 0.34 7.6 4.0 11.6 34
Example 6 none 4.3 h, broth 0.33 0.10 3.9 1.2 5.1 23
Example 6 none 4.3 h, OA 0.45 0.27 4.0 2.4 6.4 38
Example 6 none 30 h, broth 0.17 0.06 2.1 0.8 2.8 26
Example 6 none 30 h, OA 0.69 0.33 6.2 3.0 9.1 32
Example 6 none 53 h, broth 0.14 0.05 1.6 0.5 2.2 25
Example 6 none 53 h, OA 0.72 0.35 6.4 3.1 9.5 33
Example 6 none 70 h, broth 0.15 0.05 1.8 0.6 2.4 25
Example 6 none 70 h, OA 0.70 0.34 6.2 3.0 9.2 33
Example 27
Lipase Treatment of Ground Whole Corn Kernels prior to Liquefaction
[0328] Tap water (1377.6 g) was added into each of two jacketed 2-L resin
kettles, then 711.9 g wet weight (625.8 g dry weight) of ground whole corn
kernels (1.0 mm screen on hammer mill) was added to each kettle with stirring.
Each mixture was heated to 55 C with stirring at 300 rpm, and the pH adjusted
to
5.8 with 2 N sulfuric acid. To each mixture was added 21.0 g of an aqueous
solution containing 1.008 g of Spezyme -FRED L (Genencor , Palo Alto, CA).
To one mixture was then added 10.5 mL of aqueous solution of Lipolase 100L
Solution (21 mg total soluble protein, 10 ppm lipase final concentration) and
to
the second mixture was added 1.05 mL of aqueous solution of Lipolase 100L
Solution (2.1 mg total soluble protein, 1.0 ppm lipase final concentration).
Samples were withdrawn from each reaction mixture at 1 h, 2 h, 4 h and 6 h at
55 C, then the temperature of the mixture was increased to 85 C with stirring
at
600 rpm and pH 5.8, and a sample was taken when the mixture first reached
85 C. After 120 minutes at 85 C, a sample was taken and the mixtures were
cooled to 50 C and final samples of the resulting liquefied corn mash were
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transferred to 50-mL polypropylene centrifuge tubes; all samples were stored
frozen at -800C.
[0329] In two separate reactions, a 50 g sample of the 10 ppm lipase-treated
liquefied corn mash or a 55 g sample of the 1.0 ppm lipase-treated liquefied
corn
mash prepared as described above was mixed with oleyl alcohol (OA) (38 g) at
300C for 20 h, then the liquefied mash and OA in each reaction mixture were
separated by centrifugation and each phase analyzed for wt% lipid (derivatized
as fatty acid methyl esters, FAME) and for wt% free fatty acid (FFA,
derivatized
as fatty acid methyl esters, FAME) according to the method described by
Reference 1. The % FFA in the OA phase of the liquefied mash/OA mixture
prepared using heat inactivation of 10 ppm lipase during liquefaction was 98%
at
20 h, compared to only 62% FFA in the OA phase of the liquefied mash/OA
mixture prepared using heat inactivation of 1.0 ppm lipase during
liquefaction.
Results are shown in Table 11.
Table 11: Lipid and free fatty acid content of a mixture of liquefied corn
mash and
oleyl alcohol, using lipase treatment of ground corn suspension prior to
liquefaction
(heat inactivation of lipase during liquefaction)
reaction time (h), sample lipids FFA lipids FFA lipid+FFA % FFA
conditions (wt%) (wt%) (mg) (mg) (mg)
ppm lipase 1 h, pre-liquefaction 0.226 0.627 112 311 424 74
at 55 C prior to 2 h, pre-liquefaction 0.199 0.650 99 323 422 77
liquefaction at 4 h, pre-liquefaction 0.151 0.673 75 334 410 82
85 C, mix with 6 h, pre-liquefaction 0.101 0.700 50 348 398 87
OA for 20 h 0 h, 85 C, liq. mash 0.129 0.764 64 380 444 86
2 h, 85 C, liq. mash 0.129 0.751 64 373 437 85
h, 30 C, liq. mash 0.074 0.068 37 34 71 48
20 h, 30 C, OA 0.015 1.035 5.7 394 400 98
1.0 ppm lipase 1 h, pre-liquefaction 0.408 0.480 226 266 492 54
at 55 C prior to 2 h, pre-liquefaction 0.401 0.424 222 235 457 51
liquefaction at 4 h, pre-liquefaction 0.299 0.433 165 240 405 58
85 C, mix with 6 h, pre-liquefaction 0.346 0.453 192 251 442 57
OA for 20 h 0 h, 85 C, liq. mash 0.421 0.407 233 225 458 49
2 h, 85 C, liq. mash 0.424 0.429 235 237 472 50
20 h, 30 C, liq. mash 0.219 0.054 121 30 151 20
20 h, 30 C, OA 0.344 0.573 140 233 373 62
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Example 28
Lipase Screening for Treatment of Ground Whole Corn Kernels prior to
Liquefaction
[0330] Seven reaction mixtures containing tap water (67.9 g) and ground whole
corn kernels (35.1 g wet wt., ground with 1.0 mm screen using a hammer mill)
at
pH 5.8 were stirred at 55 C in stoppered flasks. A 3-mL sample (t = 0 h) was
removed from each flask and the sample immediately frozen on dry ice, then ca.
0.5 mL of 10 mM sodium phosphate buffer (pH 7.0) containing 1 mg total soluble
protein (10 ppm final concentration in reaction mixture) of one of the
following
lipases (Novozymes) were added to one of each flask: Lipolase 100 L, Lipex
100L, Lipoclean 2000T, Lipozyme CALB L, Novozyme CALA L, and
Palatase 20000L; no lipase was added to the seventh flask. The resulting
mixtures were stirred at 55 C in stoppered flasks, and 3-mL samples were
withdrawn from each reaction mixture at 1 h, 2 h, 4 h and 6 h and immediately
frozen in dry ice until analyzed for wt% lipid (derivatized as fatty acid
methyl
esters, FAME) and for wt% free fatty acid (FFA, derivatized as fatty acid
methyl
esters, FAME) according to the method described by Reference 1, and the
percent free fatty acid content was calculated relative to the total combined
concentrations of lipid and free fatty acid was determined for each sample.
Results are shown in Table 12.
Table 12: Percent free fatty acid content (% FFA) of a mixture of ground
whole corn kernels using lipase treatment at 55 C prior to liquefaction
% FFA
time Oh 1 h 2h 4h 6h
Lipolase 100L 33 56 74 76 79
Lipex 100L 34 66 81 83 83
Lipoclean 2000T 38 55 73 69 65
Lipozyme CALB L 39 38 37 43 41
Novozyme CALA L 37 40 44 44 45
Palatase 20000L 37 49 59 62 66
no enzyme 38 33 37 41 42
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Example 29
Lipase treatment of Ground Whole Corn Kernels prior to Simultaneous
Saccharification and Fermentation with In-situ Product Removal Using Oleyl
Alcohol
[0331] Three fermentations were run as described above in Examples 7, 8, and
10. For fermentations run as described in Examples 7 and 10, lipase (10 ppm of
Lipolase total soluble protein) was added to the suspension of ground corn
and
heated at 55 C for 6 h prior to Liquefaction to produce a liquefied corn mash
containing heat-inactivated lipase. No lipase was added to the suspension of
ground corn used to prepare liquefied corn mash for the fermentation described
in Example 8, but the suspension was subjected to the same heating step at
55 C prior to liquefaction. The % FFA in lipase-treated liquefied corn mash
prepared for fermentations run as described in Examples 7 and 10 was 83% and
86%, respectively, compare to 41% without lipase treatment (Example 8). Over
the course of the fermentations, the concentration of FFA did not decrease in
any
of the fermentations, including that containing heat-inactivated lipase. The %
FFA in the OA phase of the fermentation run according to Examples 7 and 10
(with heat inactivation of lipase prior to fermentation) were each 97% at 70 h
(end
of run (EOR)), compared to only 49% FFA for the fermentation run according to
Example 8 where ground whole corn kernels had not been treated with lipase
prior to liquefaction. Results are shown in Table 13.
Table 13: Lipid and free fatty acid content of fermentations containing oleyl
alcohol
as ISPR extractant and heat-inactivated lipase (lipase treatment of ground
corn
suspension prior to liquefaction)
fermentation lipase time (h), sample lipids FFA lipids FFA lipid + % FFA
(wt%) (wt%) (g) (g) FFA(g)
Example 7 10 ppm pre-lipase/pre-liq. 0.65 0.22 7.1 2.4 9.4 25
Example 7 10 ppm post-lipase/pre-liq. 0.22 0.65 2.4 7.0 9.5 74
Example 7 10 ppm liquefied mash 0.17 0.79 1.8 8.5 10.3 83
Example 7 10 ppm 0.3 h, broth 0.16 0.79 1.8 8.9 10.7 83
Example 7 10 ppm 4.8 h, broth 0.14 0.31 1.6 3.5 5.1 69
Example 7 10 ppm 4.8 h, OA 0.04 0.68 0.3 5.4 5.6 95
Example 7 10 ppm 29 h, broth 0.10 0.12 1.2 1.3 2.5 53
Example 7 10 ppm 29 h, OA 0.03 1.05 0.2 8.2 8.4 98
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Example 7 10 ppm 53 h, broth
Example 7 10 ppm 53 h, OA 0.07 1.14 0.5 9.0 9.5 95
Example 7 10 ppm 70 h, broth 0.11 0.07 1.2 0.8 2.0 39
Example 7 10 ppm 70 h, OA 0.03 1.10 0.2 8.7 8.9 97
Example 8 none pre-lipase/pre-liq. 0.62 0.23 6.7 2.5 9.2 27
Example 8 none post-lipase/pre-liq. 0.57 0.26 6.2 2.8 9.0 31
Example 8 none liquefied mash 0.52 0.36 5.6 4.0 9.6 41
Example 8 none 0.3 h, broth 0.50 0.33 5.7 3.8 9.4 40
Example 8 none 4.8 h, broth 0.47 0.14 5.3 1.6 6.9 24
Example 8 none 4.8 h, OA 0.12 0.32 1.0 2.9 3.9 73
Example 8 none 29 h, broth 0.30 0.05 3.4 0.6 4.0 16
Example 8 none 29 h, OA 0.31 0.46 2.7 4.1 6.9 60
Example 8 none 53 h, broth
Example 8 none 53 h, OA 0.47 0.50 4.2 4.4 8.6 51
Example 8 none 70 h, broth 0.22 0.04 2.5 0.5 3.0 17
Example 8 none 70 h, OA 0.40 0.39 3.6 3.5 7.0 49
Example 10 10 ppm pre-lipase/pre-liq. 0.67 0.23 7.4 2.5 9.9 25
Example 10 10 ppm post-lipase/pre-liq. 0.19 0.69 2.1 7.6 9.7 78
Example 10 10 ppm liquefied mash 0.14 0.85 1.6 9.4 11.0 86
Example 10 10 ppm 0.3 h, broth 0.13 0.82 1.5 9.4 10.9 86
Example 10 10 ppm 4.8 h, broth 0.11 0.29 1.3 3.3 4.6 72
Example 10 10 ppm 4.8 h, OA 0.04 0.60 0.3 5.2 5.6 94
Example 10 10 ppm 29 h, broth 0.09 0.14 1.0 1.6 2.6 61
Example 10 10 ppm 29 h, OA 0.01 0.96 0.1 8.4 8.5 99
Example 10 10 ppm 53 h, broth
Example 10 10 ppm 53 h, OA 0.02 0.95 0.2 8.3 8.4 98
Example 10 10 ppm 70 h, broth 0.09 0.08 1.1 0.9 1.9 45
Example 10 10 ppm 70 h, OA 0.03 0.99 0.3 8.7 9.0 97
Example 30
Lipase Treatment of Ground Whole Corn Kernels or Liquefied Corn Mash for
Simultaneous Saccharification and Fermentation with In-situ Product Removal
Using Corn Oil Fatty Acids (COFA)
[0332] Five fermentations were run as described above in Examples 9, 11, 12,
13, and 14. For the fermentations run as described in Examples 9, 13, and 14,
lipase (10 ppm of Lipolase total soluble protein) was added after
Liquefaction
and there was no heat-inactivation of lipase. Fermentations run as described
in
Examples 9 and 14 had 5 g/L of ethanol added prior to inoculation, whereas the
fermentation run as described in Example 13 had no added ethanol. The
fermentations run as described in Examples 11 and 12 employed the addition of
ppm Lipolase total soluble protein to the suspension of ground corn prior to
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liquefaction, resulting in heat inactivation of lipase during liquefaction.
The
fermentation run as described in Example 11 had 5 g/L of ethanol added prior
to
inoculation, whereas the fermentation run as described in Example 12 had no
added ethanol. The final total grams of isobutanol (i-BuOH) present in the
COFA
phase of the fermentations containing active lipase was significantly greater
than
the final total grams of i-BuOH (including i-BuOH present as FABE) present in
the
COFA phase of the fermentations containing inactive lipase. The final total
grams of isobutanol (i-BuOH) present in the fermentation broths (aqueous
phase)
containing active lipase were only slightly less than the final total grams of
i-
BuOH present in the fermentation broths containing inactive lipase, such that
the
overall production of i-BuOH (as a combination of free i-BuOH and isobutyl
esters
of COFA (FABE)) was significantly greater in the presence of active lipase
when
compared to that obtained in the presence of heat-inactivated lipase. Results
are
shown in Tables 14 and 15.
Table 14: Dependence of the production of free isobutanol (i-BuOH) and
isobutyl
esters of COFA (FABE) in fermentations containing corn oil fatty acids (COFA)
as
ISPR extractant in presence (Examples 9, 13, and 14) or absence (Examples 11
and
12) of active lipase (COFA phase analysis)
fermentation g i-BuOH / g FABE/ g i-BuOH from FABE/ total g i-BuOH /
fermentation time (h) kg COFA kg COFA kg COFA kg COFA
Example 9 4.5 2.4 0.0 0 2.4
Example 9 28.8 5.4 70.9 16.5 22.0
Example 9 52.4 8.9 199.0 46.4 55.3
Example 9 69.3 4.9 230.9 53.9 69.3
Example 11 6.6 2.3 0.0 0.0 2.3
Example 11 53.5 25.1 2.9 0.6 25.7
Example 11 71.1 24.4 6.3 1.4 25.8
Example 12 6.6 2.3 0.0 0.0 2.3
Example 12 53.5 12.8 1.6 0.4 13.2
Example 12 71.1 12.8 3.0 0.7 13.5
Example 13 6.6 2.3 0.0 0.0 2.3
Example 13 53.5 4.9 72.1 16.0 20.9
Example 13 71.1 4.6 91.4 20.3 24.9
Example 14 6.6 2.1 0.0 0.0 2.1
Example 14 53.5 9.8 197.2 43.8 53.6
Example 14 71.1 4.9 244.5 54.3 59.2
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Table 15: Dependence of the production of free isobutanol (i-BuOH) and
isobutyl
esters of COFA (FABE) in fermentations containing corn oil fatty acids (COFA)
as
ISPR extractant on presence (Examples 9, 13 and 14) or absence (Examples 11
and
12) of active lipase (fermentation broth analysis)
fermentation g i-BuOH / g FABE/ g i-BuOH from FABE/ total g i-BuOH /
sample time (h) kg broth kg broth kg broth kg broth
Example 9 4.5 0.0 0.0 0 0
Example 9 28.8 0.0 12.6 2.9 2.9
Example 9 52.4 0.0 30.3 7.1 7.1
Example 9 69.3 0.0 24.7 5.8 5.8
Example 11 6.6 0.0 0.0 0 0.0
Example 11 53.5 9.8 0.0 0 9.8
Example 11 71.1 9.5 0.0 0 9.5
Example 12 6.6 0.0 0.0 0 0
Example 12 53.5 3.8 0.0 0.0 3.8
Example 12 71.1 5.1 0.0 0.0 5.1
Example 13 6.6 0.0 0.0 0 0
Example 13 53.5 2.1 3.0 0.7 2.8
Example 13 71.1 2.1 7.4 1.6 3.7
Example 14 6.6 0.0 0.0 0 0.0
Example 14 53.5 2.9 22.4 5.0 7.9
Example 14 71.1 3.3 19.3 4.3 7.6
Example 31
Production of iso-butyl COFA esters by phospholipase-catalyzed reaction of iso-
butanol and corn oil fatty acids (COFA)
[0333] Reaction mixtures containing aqueous 2-(N-morpholino)ethanesulfonic
acid buffer (0.20 M, pH 5.3), isobutanol (2-methyl-1-propanol), phospholipase
(Phospholipase A; SigmaAldrich, L3295-250) and corn oil fatty acids prepared
from corn oil were stirred at 300C (Table 16), and samples were withdrawn from
each reaction mixture at predetermined times, immediately centrifuged, and the
aqueous and organic layers separated and analyzed for isobutanol (i-BuOH) and
isobutyl esters of corn oil fatty acids (i-BuO-COFA) (Table 17).
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Table 16: Reaction conditions for conversion of isobutanol (i-BuOH) to
isobutyl esters of corn oil fatty acids (i-BuO-COFA)
MES buffer i-BuOH COFA lipase
reaction (0.2 M) (g) (g) (g) (ppm)
1 46.1 3.6 14.7 10
2 46.1 3.6 14.7 3
3 46.1 3.6 14.7 0
Table 17: Weights of isobutanol (i-BuOH) and isobutyl esters of corn oil fatty
acids (i-BuO-COFA) present in the aqueous fraction (AQ) and organic fraction
(ORG) for reactions described in Table 16.
free i-BuOH from
total i- total i-BuOH i-BuOH i-BuO-COFA i-BuO-
BuOH COFA
reaction time (h) (g) (AQ) (g) (ORG) (g) (ORG) (g) (ORG) (g) (ORG)
1 0.1 1.29 2.39 2.39 0.00 0.00
1 2 1.24 2.44 2.38 0.06 0.26
1 20 1.25 2.43 2.22 0.21 0.96
1 24 1.26 2.42 2.19 0.23 1.03
1 44 1.27 2.41 2.13 0.28 1.28
1 48 1.22 2.46 2.15 0.31 1.41
2 0.1 1.27 2.34 2.34 0.00 0.00
2 2 1.25 2.35 2.33 0.02 0.08
2 20 1.24 2.37 2.30 0.07 0.30
2 24 1.22 2.38 2.31 0.07 0.32
2 44 1.33 2.28 2.18 0.10 0.44
2 48 1.23 2.38 2.27 0.11 0.48
3 0.1 1.27 2.33 2.33 0.00 0.00
3 2 1.26 2.34 2.34 0.00 0.00
3 20 1.22 2.38 2.37 0.01 0.07
3 24 1.25 2.35 2.33 0.02 0.08
3 44 1.24 2.36 2.32 0.04 0.18
3 48 1.24 2.36 2.32 0.04 0.18
Example 32
Dependence of Isobutyl-COFA Ester Concentration on Aqueous/COFA Ratio in
Lipase-catalyzed Reactions
[0334] Reaction mixtures containing aqueous 2-(N-morpholino)ethanesulfonic
acid buffer (0.20 M, pH 5.2), isobutanol (2-methyl -l-propanol), lipase
(Lipolase
100 L; Novozymes) and corn oil fatty acids prepared from corn oil (Table 18)
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were stirred at 30 C, and samples were withdrawn from each reaction mixture at
predetermined times, immediately centrifuged, and the aqueous and organic
layers separated and analyzed for isobutanol (i-BuOH) and isobutyl esters of
corn
oil fatty acids (i-BuO-COFA) (Table 19).
Table 18: Reaction conditions for conversion of isobutanol (i-BuOH) to
isobutyl
esters of corn oil fatty acids (i-BuO-COFA)
MES buffer i-BuOH COFA lipase
reaction # (0.2 M) (g) (g) (g) (ppm)
1 45.96 3.6 43.4 10
2 45.96 3.6 21.7 10
3 45.96 3.6 10.85 10
4 45.96 3.6 43.4 4
45.96 3.6 43.4 0
Table 19: Weights of isobutanol (i-BuOH) and isobutyl esters of corn oil fatty
acids (i-
BuO-COFA) present in the aqueous fraction (AQ) and organic fraction (ORG) for
reactions described in Table 18
free i-BuOH from
total i- total i-BuOH i-BuOH i-BuO-COFA i-BuO-
BuOH COFA
reaction time (h) (g) (AQ) (g) (ORG) (g) (ORG) (g) (ORG) (g) (ORG)
1 0.1 0.77 2.83 2.77 0.05 0.24
1 1 0.76 2.84 2.58 0.25 1.13
1 2 0.74 2.86 2.41 0.44 2.00
1 4 0.66 2.94 2.05 0.89 4.03
1 6 0.63 2.97 1.43 1.54 6.93
1 21.5 0.28 3.32 0.34 2.98 13.4
1 25.5 0.23 3.37 0.29 3.08 13.8
2 0.1 1.17 2.43 2.36 0.07 0.30
2 1 1.09 2.51 2.26 0.24 1.10
2 2 1.07 2.53 2.19 0.34 1.52
2 4 1.03 2.57 1.99 0.59 2.64
2 6 1.00 2.60 1.70 0.90 4.04
2 21.5 0.75 2.85 0.58 2.27 10.2
2 25.5 0.59 3.01 0.49 2.52 11.4
3 0.1 1.56 2.04 1.98 0.06 0.27
3 1 1.55 2.05 1.77 0.28 1.24
3 2 1.49 2.11 1.65 0.46 2.08
3 4 1.45 2.15 1.28 0.87 3.92
3 6 1.33 2.27 0.96 1.31 5.92
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3 21.5 1.12 2.48 0.26 2.22 10.0
3 25.5 0.88 2.72 0.26 2.46 11.1
4 0.1 0.84 2.76 2.75 0.02 0.07
4 1 0.78 2.82 2.73 0.09 0.40
4 2 0.83 2.77 2.59 0.17 0.79
4 4 0.78 2.82 2.44 0.38 1.71
4 6 0.78 2.82 2.10 0.72 3.25
4 21.5 0.58 3.02 1.12 1.90 8.57
4 25.5 0.51 3.09 0.97 2.11 9.51
0.1 0.90 2.70 2.70 0.00 0.00
5 1 0.90 2.70 2.70 0.00 0.00
5 2 0.92 2.68 2.68 0.00 0.00
5 4 0.89 2.71 2.70 0.00 0.02
5 6 0.92 2.68 2.62 0.06 0.29
5 21.5 0.90 2.70 2.62 0.08 0.37
5 25.5 0.89 2.71 2.62 0.09 0.41
Example 33
Dependence of Butyl-COFA Ester Concentration on Esterification Alcohol in
Lipase-catalyzed Reactions
[0335] Reaction mixtures containing aqueous 2-(N-morpholino)ethanesulfonic
acid buffer (0.20 M, pH 5.2), isobutanol (2-methyl-1 -propanol) or n-butanol,
lipase
(Lipolase 100 L; Novozymes) and corn oil fatty acids prepared from corn oil
(Table 20) were stirred at 30 C, and samples were withdrawn from each reaction
mixture at predetermined times, immediately centrifuged, and the aqueous and
organic layers separated and analyzed for isobutanol (i-BuOH) or n-butanol (n-
BuOH) and isobutyl- or butyl esters of corn oil fatty acids (BuO-COFA) (Table
21).
Table 20: Reaction conditions for conversion of isobutanol (i-BuOH)
or n-butanol (n-BuOH) to butyl esters of corn oil fatty acids (BuO-COFA)
MES buffer butanol COFA lipase
Reaction butanol (0.2 M) (g) (g) (g) (ppm)
6 iso-butanol 45.96 3.6 13.5 10
7 n-butanol 45.96 3.6 13.5 10
8 iso-butanol 45.96 3.6 13.5 0
9 isobutanol 45.96 3.6 13.5 4
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Table 21: Weights of isobutanol (i-BuOH) or n-butanol (n-BuOH) and butyl
esters of
corn oil fatty acids (BuO-COFA) present in the aqueous fraction (AQ) and
organic
fraction (ORG) for reactions described in Table 20
i-BuOH from i- i-BuO-
total i-BuOH total i-BuOH i-BuOH BuO-COFA COFA
reaction time (h) (g) (AQ) (g) (ORG) (g) (ORG) (g) (ORG) (g) (ORG)
6 0.1 1.46 2.14 2.11 0.04 0.16
6 2 1.41 2.19 1.63 0.56 2.51
6 4 1.27 2.33 1.31 1.02 4.58
6 21 0.66 2.94 0.29 2.65 12.0
6 25 0.60 3.00 0.26 2.73 12.3
6 46 0.54 3.06 0.22 2.83 12.8
n-BuOH from n- n-BuO-
total n-BuOH total n-BuOH n-BuOH BuO-COFA COFA
(g) (AQ) (g) (ORG) (g) (ORG) (g) (ORG) (g) (ORG)
7 0.1 1.31 2.29 2.26 0.03 0.11
7 2 1.26 2.34 1.89 0.45 2.03
7 4 1.20 2.40 1.66 0.74 3.35
7 21 0.81 2.79 0.50 2.29 10.3
7 25 0.77 2.83 0.40 2.43 11.0
7 46 0.50 3.10 0.23 2.87 12.9
i-BuOH from i- i-BuO-
total i-BuOH total i-BuOH i-BuOH BuO-COFA COFA
(g) (AQ) (g) (ORG) (g) (ORG) (g) (ORG) (g) (ORG)
8 0.1 1.62 1.98 1.98 0.00 0.01
8 2 1.56 2.04 2.04 0.00 0.00
8 4 1.59 2.01 2.01 0.00 0.00
8 21 1.59 2.01 2.00 0.01 0.04
8 25 1.55 2.05 2.04 0.01 0.04
8 46 1.45 2.15 2.12 0.02 0.11
i-BuOH from i- i-BuO-
total i-BuOH total i-BuOH i-BuOH BuO-COFA COFA
(g) (AQ) (g) (ORG) (g) (ORG) (g) (ORG) (g) (ORG)
9 0.1 1.57 2.03 2.02 0.01 0.04
9 2 1.54 2.06 1.86 0.19 0.86
9 4 1.44 2.16 1.79 0.36 1.64
9 21 1.14 2.46 0.95 1.51 6.82
9 25 1.10 2.50 0.83 1.67 7.50
9 46 0.78 2.82 0.44 2.37 10.7
EXAMPLE 34
Production of Iso-butyl Oleate by Lipase-catalyzed Reaction of
Isobutanol and Oleic Acid
[0336] Reaction mixtures containing aqueous 2-(N-morpholino)ethanesulfonic
acid buffer (0.20 M, pH 5.2), isobutanol (2-methyl-1-propanol), lipase (0 ppm
or
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ppm Lipolase 100 L; Novozymes) and oleic acid (Alfa Aesar) (Table 22)
were stirred at 30 C, and samples were withdrawn from each reaction mixture at
predetermined times, immediately centrifuged, and the aqueous and organic
layers separated and analyzed for isobutanol (i-BuOH) and iso-butyl oleate (i-
BuO-oleate) (Table 23).
Table 22: Reaction conditions for conversion of isobutanol (i-BuOH) to iso-
butyl oleate (i-BuO-oleate)
MES buffer i-BuOH oleic acid lipase
reaction # (0.2 M) (g) (g) (g) (ppm)
10 46.11 3.64 14.62 10
11 46.10 3.59 14.40 0
Table 23: Weights of isobutanol (i-BuOH) and iso-butyl oleate (i-BuO-COFA)
present in the aqueous fraction (AQ) and organic fraction (ORG) for reactions
described in Table 22
i-BuOH from i- i-BuO-
total i- total i-BuOH i-BuOH BuO-oleate oleate
BuOH
reaction time (h) (g) (AQ) (g) (ORG) (g) (ORG) (g) (ORG) (g) (ORG)
10 0.1 1.37 2.28 2.24 0.04 0.18
10 2 1.30 2.34 1.95 0.40 1.81
10 4 1.28 2.37 1.82 0.55 2.53
10 6 1.22 2.42 1.71 0.72 3.27
10 23 0.92 2.72 0.71 2.01 9.20
10 27 0.89 2.75 0.65 2.11 9.62
10 47 0.81 2.84 0.55 2.29 10.5
10 51 0.82 2.83 0.54 2.29 10.5
11 0.1 1.44 2.16 2.16 0.00 0.00
11 2 1.45 2.15 2.15 0.00 0.00
11 4 1.44 2.16 2.16 0.00 0.00
11 6 1.43 2.16 2.16 0.00 0.00
11 23 1.49 2.10 2.10 0.01 0.02
11 27 1.46 2.14 2.13 0.01 0.04
11 47 1.48 2.12 2.09 0.02 0.10
11 51 1.52 2.07 2.05 0.02 0.11
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Example 35
Comparison of Production of Iso-butyl Oleate by Lipase-catalyzed Reactions
of Isobutanol and Oleic Acid
[0337] Reaction mixtures containing aqueous 2-(N-morpholino)ethanesulfonic
acid buffer (MES, 0.20 M, pH 5.2), isobutanol (2-methyl-1-propanol), oleic
acid
(Alfa Aesar), and lipase (10 ppm) from Lipolase 100L, Lipex 100L, Lipozyme
CALB L, Novozyme CALA L, Palatase from Novozymes, or lipase (10 ppm)
from Pseudomonas fluorescens, Pseudomonas cepacia, Mucor miehei, hog
pancreas, Candida cylindracea, Rhizopus niveus, Candida antarctica, Rhizopus
arrhizus or Aspergillus from SigmaAldrich (Table 24), were stirred at 30 C,
and
samples were withdrawn from each reaction mixture at predetermined times,
immediately centrifuged, and the aqueous and organic layers separated and
analyzed for isobutanol (i-BuOH) and iso-butyl oleate (i-BuO-oleate) (Table
25).
Table 24: Reaction conditions for conversion of isobutanol (i-BuOH) to iso-
butyl oleate (i-BuO-oleate)
MES buffer i-BuOH oleic acid lipase
( 0.2 M) (g) (g) (g) (ppm)
46.105 3.601 13.72 10
Table 25: Weights of isobutanol (i-BuOH) and iso-butyl oleate (i-BuO-oleate)
present in the aqueous fraction (AQ) and organic fraction (ORG) for reactions
described in Table 24
total i- total i- i-BuOH from i-BuO-
BuOH BuOH i-BuOH i-BuO-oleate oleate
lipase time (h) (g) (AQ) (g) (ORG) (g) (ORG) (g) (ORG) (g) (ORG)
Lipolase 100L 23 0.92 2.72 0.71 2.01 9.20
Lipex 100L 23 0.65 2.95 0.30 2.65 12.09
Lipozyme CALB L 23 1.01 2.59 0.82 1.77 8.08
Novozyme CALA L 23 1.39 2.22 2.16 0.06 0.27
Palatase 23 1.27 2.33 1.43 0.91 4.14
Pseudomonas fluorescens 23 1.38 2.22 1.97 0.25 1.14
Pseudomonas cepacia 23 1.39 2.21 1.95 0.26 1.20
Mucor miehei 23 1.29 2.31 1.57 0.75 3.42
hog pancreas 23 1.40 2.20 2.19 0.01 0.04
Candida cylindracea 23 1.15 2.45 1.08 1.37 6.25
Rhizopus niveus 23 1.39 2.21 2.19 0.02 0.11
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Candida antarctica 23 1.37 2.24 2.08 0.15 0.69
Rhizopus arrhizus 23 1.01 2.59 0.81 1.78 8.12
Aspergillus 23 1.36 2.24 2.06 0.18 0.82
no lipase 23 1.49 2.10 2.10 0.01 0.02
Example 36
Production of Ethyl-COFA Ester by Lipase-catalyzed Reaction of Ethanol and
Corn Oil Fatty Acids (COFA)
[0338] Reaction mixtures containing aqueous 2-(N-morpholino)ethanesulfonic
acid buffer (0.20 M, pH 5.5), ethanol, lipase (Lipolase 100 L or Lipozyme
CALB L; Novozymes) and corn oil fatty acids prepared from corn oil (Table 26)
were stirred at 30 C, and samples were withdrawn while stirring from each
reaction mixture at predetermined times, immediately centrifuged, and the
aqueous and organic layers separated and analyzed for ethanol and ethyl esters
of corn oil fatty acids (EtO-COFA) (Table 27).
Table 26: Reaction conditions for conversion of ethanol (EtOH) to ethyl esters
of corn oil fatty acids (EtO-COFA)
MES buffer ethanol COFA lipase
Reaction (0.2 M) (g) (g) (g) lipase (ppm)
12 46.11 3.60 14.48 Lipolase 100L 10
13 46.10 3.60 14.47 Lipozyme CALB L 10
14 46.11 3.61 14.47 no lipase 0
Table 27: Weights of ethanol (EtOH) and ethyl esters of corn oil fatty acids
(EtO-
COFA) present in the aqueous fraction (AQ) and organic fraction (ORG) for
reactions
described in Table 26
EtOH from ETO-
total EtOH total EtOH EtOH EtO-COFA COFA
reaction time (h) (g) (AQ) (g) (ORG) (g) (ORG) (g) (ORG) (g) (ORG)
12 0 2.94 0.655 0.634 0.021 0.01
12 2 3.09 0.504 0.105 0.398 0.81
12 20 2.74 0.854 0.030 0.824 4.46
12 24 2.43 1.167 0.032 1.135 5.25
12 44 2.37 1.230 0.022 1.208 7.28
12 48 2.24 1.360 0.022 1.338 7.63
13 0 2.94 0.659 0.635 0.024 0.01
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13 2 2.83 0.773 0.074 0.699 1.88
13 20 2.10 1.501 0.000 1.501 9.72
13 24 2.07 1.532 0.000 1.532 10.14
13 44 1.94 1.673 0.014 1.659 10.93
13 48 1.72 1.882 0.016 1.865 11.05
14 0 2.96 0.646 0.624 0.023 0.01
14 2 2.93 0.679 0.661 0.018 0.01
14 20 2.75 0.857 0.779 0.079 0.02
14 24 2.87 0.738 0.662 0.075 0.03
14 44 2.79 0.813 0.688 0.126 0.04
14 48 2.82 0.785 0.671 0.114 0.05
Example 37
Production of Ethyl-COFA Ester by Lipase-catalyzed Reaction of Ethanol and
Corn Oil Fatty Acids (COFA) during Fermentation of Yeast
The wild-type yeast strain CEN.PK1 13-7D was propagated overnight in medium
containing yeast nitrogen base without amino acids (6.7 g/L), dextrose (25
g/L),
and MES buffer (0.1 M at pH 5.5). The overnight culture was diluted into fresh
medium such that the resulting optical density at 600 nm was 0.1. The diluted
culture was aliquoted, 25 mL per flask, into six 250 mL sealed-cap shake
flasks.
Four of the cultures were supplemented with either of two lipase enzyme stock
solutions (2 mg protein/mL 10 mM phosphate buffer (pH 7.0) of Lipozyme CALB
L or Lipolase 100L) to a final lipase concentration of 10 ppm in the media.
Corn
oil fatty acids (COFA) were added at a 1:1 volume ratio to the aqueous culture
in
three of the flasks (no enzyme, CALB L, or Lipolase 100L). One flask had no
supplements. The cultures were grown in a temperature-controlled shaking
incubator at 30 C and a shaking speed of 250 rpm for 23 hours. Samples for
cell
mass determination were allowed to phase separate in 15 mL conical bottom
tubes. The sample's optical density at 600 nm was measured at a 20-fold
dilution
in saline. Samples (5 mL aqueous or 10 mL culture/COFA emulsion) for
chromatographic analysis were immediately centrifuged for 5 minutes at 4000
rpm in a TX-400 swinging bucket rotor in 15 mL conical bottom tubes. For
aqueous samples, a 0.22 m spin filter was used prior to analysis. Aqueous
samples were analyzed on a Shodex SH1011 column with a SH-G guard column
using 0.01 M sulfuric acid mobile phase at 50 C and a flow rate of 0.5 mL per
minute. Detection of sugars and alcohols was by Refractive Index and 210 nm
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absorption, and quantitation was performed using standard curves. Samples
were taken of the aqueous culture (no added COFA) or culture/COFA emulsion,
and analyzed as described in previous Examples for ethyl esters of COFA.
Results are shown in Tables 28 and 29.
Table 28: Weights of ethanol (EtOH), glucose and fermentation byproducts
present in
the aqueous media (AQ) from 23 h fermentations
glucose glycerol acetate acetoin EtOH
(g/L) (g/L) (g/L) (g/L) (g/L)
Media 0 0.62 1.01 0.08 9.98
media + CALB L 0 0.72 0.94 0.06 9.94
media + Lipolase 100L 0 0.61 0.99 0.05 9.87
media + COFA 0 0.68 0.32 0.15 7.73
media + COFA + CALB L 0 0.74 0.09 0.11 3.92
media + COFA + Lipolase 100L 0 0.63 0.23 0.18 7.19
Table 29: Weights of ethanol (EtOH) and ethyl esters of corn oil fatty acids
(EtO-
COFA) present in the aqueous fraction (AQ) and the organic fraction (ORG) for
23 h
fermentations
EtOH from ETO-COFA
EtOH EtOH EtO-COFA
Reaction (g/L) (AQ) (g/L) (ORG) (g/L) (ORG) (g/L) (ORG)
media + COFA 6.7 0 0.18 1.2
media + COFA + CALB L 3.4 0 4.52 30.0
media + COFA + Lipolase 100L 6.1 0 0.72 4.8
Example 38
Production of Methyl-COFA Ester by Lipase-catalyzed Reaction of Methanol and
Corn Oil Fatty Acids (COFA)
[0339] Reaction mixtures containing aqueous 2-(N-morpholino)ethanesulfonic
acid buffer (0.20 M, pH 5.5), methanol, lipase (Lipolase 100 L (Novozymes),
Lipozyme CALB L (Novozymes), Rhizopus arrhizus lipase (SigmaAldrich), and
Candida cylindracea lipase (SigmaAldrich) and corn oil fatty acids prepared
from
corn oil (Table 30) were stirred at 30 C, and samples were withdrawn while
stirring from each reaction mixture at predetermined times, immediately
centrifuged, and the aqueous and organic layers separated and analyzed for
ethanol and ethyl esters of corn oil fatty acids (EtO-COFA) (Table 31).
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Table 30: Reaction conditions for conversion of methanol (MeOH) to methyl
esters of corn oil fatty acids (MeO-COFA)
MES buffer methanol COFA lipase
Reaction (0.2 M) (g) (g) (g) lipase (ppm)
15 46.11 3.60 14.51 Lipolase 100L 10
16 46.10 3.59 14.49 Lipozyme CALB L 10
17 46.11 3.60 14.49 R. arrhizus 10
18 46.10 3.60 14.48 C. cylindracea 10
19 46.10 3.60 14.51 no lipase 10
Table 31: Weights of methanol (MeOH) and methyl esters of corn oil fatty acids
(MeO-COFA) present in the aqueous fraction (AQ) and organic fraction (ORG) for
reactions described in Table 30
MeOH from MeO-
total MeOH total MeOH MeOH MeO-COFA COFA
reaction time (h) (g) (AQ) (g) (ORG) (g) (ORG) (g) (ORG) (g) (ORG)
15 0 3.33 0.26 0.05 0.01 0.02
15 2 3.09 0.50 0.05 0.13 0.16
15 4 3.09 0.51 0.04 0.33 0.73
15 20 2.81 0.79 0.04 0.70 3.03
15 24 2.72 0.87 0.04 0.79 3.47
15 44 2.53 1.06 0.03 1.00 4.97
15 48 2.48 1.12 0.03 1.05 5.18
16 0 3.07 0.53 0.04 0.02 0.02
16 2 3.01 0.59 0.04 0.20 0.22
16 4 2.92 0.67 0.03 0.56 1.32
16 20 2.54 1.06 0.03 0.99 5.25
16 24 2.43 1.16 0.03 1.09 5.90
16 44 2.28 1.32 0.02 1.27 7.63
16 48 2.22 1.37 0.03 1.32 7.89
17 0 3.09 0.52 0.04 0.02 0.02
17 2 3.05 0.56 0.06 0.05 0.06
17 4 2.98 0.63 0.04 0.25 0.24
17 20 3.03 0.57 0.04 0.32 0.49
17 24 2.98 0.63 0.04 0.35 0.52
17 44 2.99 0.62 0.04 0.38 0.62
17 48 2.94 0.67 0.04 0.40 0.61
18 0 3.17 0.43 0.05 0.02 0.02
18 2 3.12 0.49 0.04 0.02 0.02
18 4 2.96 0.64 0.00 0.64 1.24
18 20 2.64 0.96 0.03 0.89 3.97
18 24 2.58 1.03 0.03 0.95 4.49
18 44 2.37 1.23 0.03 1.18 6.40
18 48 2.30 1.30 0.03 1.25 6.71
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19 0 3.08 0.52 0.04 0.03 0.02
19 2 3.08 0.52 0.04 0.02 0.02
19 4 3.04 0.56 0.04 0.03 0.02
19 20 3.08 0.53 0.04 0.03 0.03
19 24 3.04 0.56 0.05 0.03 0.04
19 44 3.01 0.59 0.04 0.06 0.04
19 48 2.95 0.65 0.05 0.06 0.04
Example 39
Production of 1-Propyl-COFA Ester by Lipase-catalyzed Reaction of 1-Propanol
and Corn Oil Fatty Acids (COFA)
[0340] Reaction mixtures containing aqueous 2-(N-morpholino)ethanesulfonic
acid buffer (0.20 M, pH 5.5), 1-propanol, lipase (Lipolase 100 L (Novozymes),
Lipozyme CALB L (Novozymes), Rhizopus arrhizus lipase (SigmaAldrich), and
Candida cylindracea lipase (SigmaAldrich) and corn oil fatty acids prepared
from
corn oil (Table 32) were stirred at 30 C, and samples were withdrawn while
stirring from each reaction mixture at predetermined times, immediately
centrifuged, and the aqueous and organic layers separated and analyzed for 1-
propanol and 1 -propyl esters of corn oil fatty acids (PrO-COFA) (Table 33).
Table 32: Reaction conditions for conversion of 1-propanol (PrOH) to 1-propyl
esters of corn oil fatty acids (PrO-COFA)
MES buffer 1-propanol COFA lipase
Reaction (0.2 M) (g) (g) (g) lipase (ppm)
20 46.11 3.60 14.47 Lipolase 100L 10
21 46.12 3.60 14.48 Lipozyme CALB L 10
22 46.10 3.60 14.48 R. arrhizus 10
23 46.13 3.62 14.49 C. cylindracea 10
24 46.13 3.60 14.48 no lipase 0
Table 33: Weights of 1 -propanol (PrOH) and 1 -propyl esters of corn oil fatty
acids
(PrO-COFA) present in the aqueous fraction (AQ) and organic fraction (ORG) for
reactions described in Table 32
PrOH from PrO-
total PrOH total PrOH PrOH PrO-COFA COFA
reaction time (h) (g) (AQ) (g) (ORG) (g) (ORG) (g) (ORG) (g) (ORG)
20 0 2.54 1.05 0.80 0.00 0.02
20 2 2.39 1.20 0.70 0.11 0.44
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20 4 2.00 1.60 0.61 0.55 1.88
20 20 1.65 1.95 0.31 1.50 6.96
20 24 1.51 2.08 0.28 1.69 7.97
20 44 1.13 2.46 0.16 2.23 11.09
20 48 1.09 2.51 0.15 2.29 11.27
21 0 2.44 1.16 0.79 0.00 0.02
21 2 2.38 1.22 0.65 0.13 0.49
21 4 2.07 1.53 0.52 0.73 2.94
21 20 1.16 2.43 0.17 2.18 10.80
21 24 1.08 2.51 0.16 2.28 11.26
21 44 1.00 2.60 0.13 2.40 11.86
21 48 0.98 2.62 0.13 2.42 11.91
22 0 2.49 1.11 0.80 0.00 0.02
22 2 2.42 1.18 0.76 0.10 0.38
22 4 2.23 1.37 0.71 0.29 1.08
22 20 2.09 1.51 0.56 0.71 2.96
22 24 2.06 1.54 0.54 0.77 3.17
22 44 1.87 1.73 0.47 0.58 1.75
22 48 1.88 1.73 0.46 0.60 1.82
23 0 2.49 1.13 0.80 0.00 0.02
23 2 2.45 1.17 0.77 0.07 0.29
23 4 2.35 1.27 0.71 0.21 0.82
23 20 2.00 1.61 0.50 0.89 3.74
23 24 1.93 1.68 0.49 0.99 4.23
23 44 1.57 2.04 0.33 1.56 6.83
23 48 1.49 2.13 0.31 1.67 7.33
24 0 2.49 1.11 0.81 0.00 0.02
24 2 2.47 1.13 0.81 0.00 0.02
24 4 2.38 1.21 0.78 0.01 0.03
24 20 2.46 1.14 0.79 0.01 0.05
24 24 2.42 1.17 0.79 0.01 0.05
24 44 2.41 1.19 0.76 0.02 0.09
24 48 2.32 1.28 0.77 0.03 0.10
Example 40
Production of 1-Pentyl-COFA Ester by Lipase-catalyzed Reaction of 1-Pentanol
and Corn Oil Fatty Acids (COFA)
[0341] Reaction mixtures containing aqueous 2-(N-morpholino)ethanesulfonic
acid buffer (0.20 M, pH 5.5), 1-pentanol, lipase (Lipolase 100 L (Novozymes),
Lipozyme CALB L (Novozymes), Rhizopus arrhizus lipase (SigmaAldrich), and
Candida cylindracea lipase (SigmaAldrich) and corn oil fatty acids prepared
from
corn oil (Table 34) were stirred at 30 C, and samples were withdrawn while
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stirring from each reaction mixture at predetermined times, immediately
centrifuged, and the aqueous and organic layers separated and analyzed for 1-
pentanol and 1 -pentyl esters of corn oil fatty acids (PenO-COFA) (Table 35).
Table 34: Reaction conditions for conversion of 1-pentanol (PenOH) to 1-
pentyl esters of corn oil fatty acids (PenO-COFA)
MES buffer 1-pentanol COFA lipase
Reaction (0.2 M) (g) (g) (g) lipase (ppm)
25 46.11 3.60 14.47 Lipolase 100L 10
26 46.12 3.60 14.48 Lipozyme CALB L 10
27 46.10 3.60 14.48 R. arrhizus 10
28 46.13 3.62 14.49 C. cylindracea 10
29 46.13 3.60 14.48 no lipase 0
Table 35: Weights of 1 -pentanol (PenOH) and 1 -pentyl esters of corn oil
fatty acids
(PenO-COFA) present in the aqueous fraction (AQ) and organic fraction (ORG)
for
reactions described in Table 34
PenOH from PenO-
total PenOH total PenOH PenOH PenO-COFA COFA
reaction time (h) (g) (AQ) (g) (ORG) (g) (ORG) (g) (ORG) (g) (ORG)
25 0 0.364 3.238 3.091 0.002 0.006
25 2 0.339 3.264 2.745 0.446 1.760
25 4 0.373 3.229 2.761 0.557 2.196
25 20 0.336 3.266 1.833 1.002 3.953
25 24 0.325 3.277 1.575 1.257 4.960
25 44 0.226 3.377 0.921 2.383 9.400
25 48 0.206 3.396 0.723 2.524 9.957
26 0 0.364 3.243 3.105 0.002 0.006
26 2 0.317 3.290 2.462 0.512 2.019
26 4 0.320 3.287 2.287 0.652 2.574
26 20 0.130 3.477 0.387 3.007 11.860
26 24 0.094 3.513 0.215 3.251 12.823
26 44 0.075 3.532 0.165 3.312 13.067
26 48 0.081 3.526 0.165 3.326 13.120
27 0 0.384 3.216 3.102 0.002 0.006
27 2 0.356 3.244 2.957 0.437 1.725
27 4 0.333 3.267 2.912 0.388 1.532
27 20 0.363 3.237 2.664 0.433 1.707
27 24 0.367 3.233 2.597 0.665 2.623
27 44 0.366 3.234 2.473 0.549 2.166
27 48 0.347 3.253 2.473 0.559 2.205
28 0 0.369 3.244 3.086 0.002 0.006
28 2 0.329 3.284 2.523 0.435 1.717
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28 4 0.332 3.281 2.496 0.493 1.944
28 20 0.304 3.309 1.575 1.321 5.209
28 24 0.270 3.343 1.292 1.868 7.367
28 44 0.186 3.427 0.596 2.722 10.735
28 48 0.162 3.451 0.509 2.846 11.224
29 0 0.375 3.239 3.102 0.001 0.006
29 2 0.366 3.248 3.117 0.009 0.034
29 4 0.377 3.237 3.099 0.023 0.089
29 20 0.380 3.234 3.092 0.032 0.125
29 24 0.379 3.235 3.058 0.039 0.154
29 44 0.374 3.240 3.013 0.053 0.209
29 48 0.373 3.241 2.950 0.059 0.233
Example 41
Production of 2-Methyl-1-butyl-COFA Ester by Lipase-catalyzed Reaction of 2-
Methyl-1 -butanol and Corn Oil Fatty Acids (COFA)
[0342] Reaction mixtures containing aqueous 2-(N-morpholino)ethanesulfonic
acid buffer (0.20 M, pH 5.5), 2-methyl -l-butanol, lipase (Lipolase 100 L
(Novozymes), Lipozyme CALB L (Novozymes), Rhizopus arrhizus lipase
(SigmaAldrich), and Candida cylindracea lipase (SigmaAldrich) and corn oil
fatty
acids prepared from corn oil (Table 36) were stirred at 30 C, and samples were
withdrawn while stirring from each reaction mixture at predetermined times,
immediately centrifuged, and the aqueous and organic layers separated and
analyzed for 2-methyl-1 -butanol and 2-methyl-1 -butyl esters of corn oil
fatty acids
(MeBO-COFA) (Table 37).
Table 36: Reaction conditions for conversion of 2-methyl-1-butanol (MeBOH)
to 2-methyl-1 -butyl esters of corn oil fatty acids (MeBO-COFA)
MES buffer 2-methyl- COFA lipase
1-butanol
Reaction (0.2 M) (g) (g) (g) lipase (ppm)
30 46.27 3.60 14.48 Lipolase 100L 10
31 46.14 3.60 14.48 Lipozyme CALB L 10
32 46.12 3.60 14.47 R. arrhizus 10
33 46.11 3.49 14.47 C. cylindracea 10
34 46.18 3.60 14.47 no lipase 0
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Table 37: Weights of 2-methyl-1-butanol (MeBOH) and 2-methyl-1-butyl esters of
corn oil fatty acids (MeBO-COFA) present in the aqueous fraction (AQ) and
organic
fraction (ORG) for reactions described in Table 36
MeBOH from MeBO-
total MeBOH total MeBOH MeBOH MeBO-COFA COFA
reaction time (h) (g) (AQ) (g) (ORG) (g) (ORG) (g) (ORG) (g) (ORG)
30 0 0.000 3.603 3.103 0.002 0.008
30 2 0.009 3.593 2.919 0.630 2.484
30 4 0.058 3.545 2.766 0.673 2.653
30 20 0.005 3.598 2.041 1.331 5.250
30 24 0.029 3.574 1.967 1.418 5.594
30 44 0.017 3.585 1.218 2.174 8.577
30 48 0.008 3.595 1.099 2.085 8.224
31 0 0.000 3.595 3.129 0.003 0.010
31 2 0.003 3.592 2.665 0.692 2.730
31 4 0.012 3.583 2.510 0.839 3.308
31 20 0.001 3.594 1.408 1.932 7.622
31 24 0.005 3.590 1.293 2.082 8.214
31 44 0.006 3.589 0.970 2.437 9.612
31 48 0.007 3.588 0.918 2.495 9.840
32 0 0.000 3.597 3.100 0.003 0.011
32 2 0.017 3.580 2.855 0.588 2.321
32 4 0.000 3.597 2.783 0.675 2.664
32 20 0.000 3.597 2.392 1.027 4.051
32 24 0.000 3.597 2.337 1.081 4.266
32 44 0.001 3.596 2.209 1.191 4.697
32 48 0.000 3.597 2.174 1.216 4.798
33 0 0.000 3.597 3.093 0.002 0.008
33 2 0.001 3.596 1.756 1.398 5.514
33 4 0.003 3.594 2.116 1.026 4.046
33 20 0.027 3.570 0.607 2.865 11.302
33 24 0.000 3.597 0.429 3.097 12.216
33 44 0.007 3.590 0.205 3.345 13.194
33 48 0.003 3.594 0.202 3.353 13.228
34 0 0.000 3.485 3.014 0.003 0.011
34 2 0.000 3.485 2.991 0.021 0.083
34 4 0.000 3.485 3.020 0.012 0.046
34 20 0.000 3.485 2.970 0.029 0.115
34 24 0.002 3.483 2.949 0.037 0.148
34 44 0.000 3.485 2.912 0.047 0.185
34 48 0.000 3.485 2.909 0.051 0.200
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Example 42
Production of Isopropyl-COFA Ester by Lipase-catalyzed Reaction of Isopropanol
and Corn Oil Fatty Acids (COFA)
[0343] Reaction mixtures containing aqueous 2-(N-morpholino)ethanesulfonic
acid buffer (0.20 M, pH 5.5), isopropanol (2-propanol), lipase (Lipolase 100
L
(Novozymes), Lipozyme CALB L (Novozymes), Rhizopus arrhizus lipase
(SigmaAldrich), and Candida cylindracea lipase (SigmaAldrich) and corn oil
fatty
acids prepared from corn oil (Table 38) were stirred at 30 C, and samples were
withdrawn while stirring from each reaction mixture at predetermined times,
immediately centrifuged, and the aqueous and organic layers separated and
analyzed for isopropanol and isopropyl esters of corn oil fatty acids (i-PrO-
COFA)
(Table 39).
Table 38: Reaction conditions for conversion of isopropanol (i-PrOH) to
isopropyl esters of corn oil fatty acids (i-PrO-COFA)
MES buffer isopropanol COFA lipase
Reaction (0.2 M) (g) (g) (g) lipase (ppm)
35 46.14 3.60 14.48 Lipozyme CALB L 10
36 46.11 3.49 14.47 C. cylindracea 10
37 46.18 3.60 14.47 no lipase 0
Table 39: Weights of isopropanol (i-PrOH) and isopropyl esters of corn oil
fatty
acids (i-PrO-COFA) present in the organic fraction (ORG) for reactions
described in
Table 38
i-PrOH from i-PrO-
i-PRO-COFA COFA
reaction time (h) (g) (ORG) (g) (ORG)
35 0 0.001 0.00
35 2 0.013 0.07
35 4 0.038 0.20
35 20 0.132 0.71
35 24 0.177 0.94
35 44 0.291 1.55
35 48 0.301 1.61
36 0 0.001 0.01
36 2 0.051 0.27
36 4 0.163 0.87
36 20 0.532 2.84
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36 24 0.652 3.48
36 44 0.916 4.89
36 48 0.959 5.12
37 0 0.001 0.01
37 2 0.001 0.01
37 4 0.003 0.02
37 20 0.009 0.05
37 24 0.011 0.06
37 44 0.016 0.09
37 48 0.023 0.12
Example 43
Comparison of Partition Coefficients for Isobutanol between Water and
Extractant
[0344] Aqueous solutions of isobutanol (30 g/L) were mixed with corn oil fatty
acids (COFA), or oleic acid or corn oil triglycerides, and their measured
partition
coefficients reported in the table relative to the measured partition
coefficient for
oleyl alcohol. Results are shown in Table 40.
Table 40: Relative partition coefficients for isobutanol (30 g/L) between
water
and extractant
isobutanol partition coefficient,
extractant relative to oleyl alcohol
oleyl alcohol 100%
corn oil fatty acids 91 %
corn oil fatty acid isobutyl esters 43 %
corn oil triglycerides 10%
Example 44
Production of Corn Oil Fatty Acids
[0345] A five-liter (5L) round bottom flask equipped with a mechanical
stirrer,
thermocouple, heating mantle, condenser and nitrogen tee was charged with
750 g of crude corn oil (non-food grade, recovered from an ethanol
fermentation
facility), 2112 g of water and 285 g of 50% sodium hydroxide solution. Mixture
was heated to 90 C and held for two hours, during which time it became a
single
thick, emulsion-like single phase. At the end of this time, TLC shows no
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remaining corn oil in the mixture. The mixture was then cooled to 74 C and 900
g
of 25% sulfuric acid was added to acidify the mixture. It was then cooled to
50 C
and the aqueous layer was drained. The oil layer was washed twice with 1500
mL of 40 C water and then once with 1 liter of saturated brine. It was dried
over
magnesium sulfate and filtered through Celite. Yield was 610 g of clear red
oil.
Titration for free fatty acids via AOCS method Ca 5a-40 shows a fatty acid
content of 95% expressed as oleic acid. A sample was silanized by reacting 104
mg with 100 uL of N-methyl-N-(trimethylsilyl)trifluoroacetamide in 1 mL of dry
pyridine. Gas chromatography-mass spectrometry (GCMS) analysis of the
silanized product shows the presence of the TMS derivatives of the 16:0, 18:2,
18:1, 18:0, and 20:0 acids.
Example 45
Chemical Synthesis of FABE
[0346] A 3L flask was equipped with a mechanical stirrer, thermocouple,
nitrogen
inlet, heating mantle and a condenser. The flask was charged with COFA (595 g)
(prepared as in Example 44), isobutanol (595 g), and sulfuric acid (12 g). The
mixture was refluxed for 1.5 hours at which time the condenser was removed and
replaced with a still head. Distillate was collected over three hours with an
initial
head temperature of 90 C and a final head temperature of 105 C. The mixture
was then cooled to room temperature and 500 mL of DI water was added. The
layers were separated and the organic layer was washed five times with 500 mL
of DI water. It was then washed once with 500 mL of a 10% calcium chloride
solution followed by six washings with 500 mL of DI water. The oil was then
dried
over magnesium sulfate and filtered through a bed of Celite yielding 601 g of
a
clear red oil. GC analysis shows the presence of 0.36 wt% of isobutanol.
GC/MS analysis shows the presence of isobutyl palmitate, isobutyl stearate,
isobutyl oleate, isobutyl linoleate, and isobutyl linolenate.
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Example 46
Recovery of Butanol using an Inorganic Acid Catalyst
[0347] A 1 liter round bottom flask with magnetic stirring and a 12" column
packed
with Rasching rings topped with a still head and nitrogen inlet was used. The
flask was charged with 254 g FABE synthesized as in Example 45, 255 g COFA,
100 mL water, and 5 g sulfuric acid, and heated to a pot temperature of 93 C.
Head temperature was equilibrated at 89.7 C. The first cut was collected with
a
reflux ratio that maintained the head temperature between 89 and 94 C.
[0348] The reaction was cooled and sat at room temperature for three days. GC
analysis of the pot shows a total of 1 g of isobutanol in the pot. The
distillation
was restarted and three more cuts, each of 25 mL, were collected. One hundred
(100) mL of water was added to the pot after collecting cut #2. Four cuts were
collected and analyzed with the results shown in Table 41.
[0349] GC analysis was done using a Hewlett Packard 6890 GC using a 30m
FFAP column. Samples were dissolved in isopropanol and 1-pentanol was
added as an internal standard. Standard curves were made for isobutanol,
isobutyl palmitate, isobutyl stearate, isobutyl oleate, isobutyl linoleate,
isobutyl
linolenate, isobutyl arachidate, palmitic acid, stearic acid, oleic acid,
linoleic acid
and linolenic acid. FABE content is reported as the sum of the butyl esters
and
COFA content as the sum of the fatty acids.
Table 41: Composition analysis of cuts collected
i-BuOH mL wt of i-
m /ml BuOH
Cut 1 60 43 2.6
Cut 2 41 23.4 1
Cut 3 29 24.2 0.7
Cut 4 30 27 0.8
Total 117.6 5.1
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Example 47
Recovery of Butanol using an Organic Acid Catalyst
[0350] A 1 liter 3 neck round bottom flask equipped with magnetic stirrer,
thermocouple, addition funnel and still head was used. The flask charged with
100 g FABE synthesized as in Example 45, 100 g COFA, 5 g p-toluenesulfonic
acid, and 25 mL water. Isobutanol analysis of initial pot shows 1.1 g of
isobutanol
present (contaminant in FABE). The pot was heated to 125 C. When the pot
reached 116 C head temperature was 96 C, and 125 mL water was added over
2.5 hours. Six cuts were collected over the time that the water was added and
they were analyzed by GC as in Example 46. Results are provided in Table 42.
Table 42: Composition analysis of cuts collected
cut pot head mL of mg/ml mL g i-
temp temp water i-BuOH BuOH
added in cut in cut
#1 116 96 25 53 13 0.7
#2 117 98 47 52 26 1.4
#3 117 99 70 37 24 0.9
#4 117 99 95 30 22 0.7
#5 117 99 125 23 31 0.7
#6 117 99 39 41 1.6
Total 5.9
[0351] Butanol analysis of the remaining still pot shows 0.9 g of free
isobutanol
present. The initial COFA:FABE mixture analyzed was 45 wt% FABE. The final
pot analyzed was 32 wt% FABE.
Example 48
Hydrolysis of FABE with Water at High Temperature
[0352] A 1 liter autoclave was charged with FABE synthesized as in Example 45,
300 mL and 300 mL water. It was sealed and purged with nitrogen. Stirring was
started and it was then heated to 250 C over 45 minutes and samples were
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removed every hour after reaching temperature. The samples were analyzed by
GC as in Example 46. The oil phase samples showed the compositions as a
function of time shown in Table 43.
Table 43: Composition of organic phase of samples
Time wt% i- wt% FABE wt%
BuOH COFA
0 0 97 2
1 3 76 18
2 6 50 41
3 7 36 45
4 7 34 48
7 35 51
Example 49
Hydrolysis of FABE with Dilute Acid at High Temperature
[0353] A 1 liter autoclave was charged with 450 g of a 75/25 mixture of FABE
synthesized as in Example 45 and COFA and with 150 g of 2% sulfuric acid. It
was sealed and purged with nitrogen. Stirring was started and it was then
heated
to 225 C over 45 minutes and samples were removed every hour after reaching
temperature. The samples were analyzed by GC as in Example 46. The oil
phase samples showed the compositions as a function of time shown in
Table 44.
Table 44: Composition analysis of cuts collected
Time (h) wt% i- wt% wt%
BuOH FABE COFA
0 2.2 61.8 34.1
1 3.8 47.8 42.0
2 5.2 38.4 48.2
3 5.3 38.4 53.5
4 5.1 33.4 48.2
5 5.1 31.9 43.3
6 5.5 35.2 51.7
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Example 50
Hydrolysis of FABE with Sulfuric Acid in Solvent at 100 C
[0354] A solution of 5 g FABE synthesized as in Example 45, 5 g of 25%
sulfuric
acid, and 60 g of diethyleneglycol dimethyl ether was prepared. Ten (10) g of
the
solution was added to each of five vials which were then sealed. All of the
vials
were heated to 100 C and one vial was removed from the heater and analyzed
every hour. The resulting compositions were determined by GC (as described in
Example 46) and are reported in Table 45.
Table 45: Composition analysis of cuts collected
Time (h) wt% i- wt% wt%
BuOH FABE COFA
0 0.11 5.74 0
1 1.1 1.72 1.23
2 1.32 0.96 1.77
3 1.35 0.76 1.8
4 1.38 0.7 1.81
1.37 0.72 1.82
1.37 0.84 2.12
Example 51
Hydrolysis of FABE by Reactive Distillation
[0355] A 12 liter flask was equipped with an insulated 2"x30" column topped
with
a feed inlet and a still head. The column was randomly packed with one liter
of
Pro-pak (316 SS 0.16") still packing and 500 g of Amberlyst 36 solid acid
catalyst (Dow). The flask was charged with 6 liters of water and brought to a
boil.
The heat was controlled to have a water distillation rate of about 1.8 mL/min.
FABE synthesized as per the method described in Example 45 was added to the
top of the column at a rate of 2 g/min. The feed was continued for a total of
60 minutes. The distillation was continued for another 30 minutes. A total of
194 g of distillate was collected which contained 2.1 g of isobutanol. Based
on
the amount of FABE fed this represents a 9% conversion of FABE to butanol.
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Example 52
Hydrolysis of FABE by Counter Current Steam
[0356] The apparatus as described in example 50 was modified by the addition
of
heat tape wrapped around the still column. The temperature in the upper half
of
the column was adjusted to 115 C and the temperature in the lower half of the
column was adjusted to 104 C. The pot was brought to a boil and the pot heat
was adjusted until water was distilling at a rate of 1.5-2 mL/min. FABE (346
g)
synthesized as per the method described in Example 45 was fed to the top of
the
packed column over a period of three hours while the distillation continued.
After
the feed period the distillation was continued for another 90 minutes. A total
of
486 g of distillate was collected that contained 30.1 g of isobutanol. This
represents a conversion of FABE to isobutanol of 39%.
Example 53
Hydrolysis Catalyzed by a Water Insoluble Organic Acid
[0357] A one liter 3n round bottom flask equipped with an oil bath, mechanical
stirrer, nitrogen inlet, subsurface water inlet, and a still head was charged
with
150 g of FABE, 50 g water, and 5 g dodecylbenzene sulfonic acid. An oil bath
was heated to 95-100 C and a slow nitrogen sweep started. Distillate cuts were
collected every half hour for a total of five hours. After three hours, water
was fed
to the still pot at a rate of 15 mL/hr. Distillate cuts were analyzed for
isobutanol
content by the GC method described in Example 46 and the results are shown in
Table 46. Approximately 44% of the isobutanol contained in the FABE was
collected over five hours.
Table 46
Sample Cumulative i-BuOH collected
1 0.80
2 1.47
3 2.46
4 3.99
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5.71
6 7.75
7 9.16
8 10.33
9 13.76
14.37
Example 54
Hydrolysis Catalyzed by Solid Acid Catalyst
[0358] A one liter 3n round bottom flask equipped with an oil bath, mechanical
stirrer, subsurface nitrogen inlet, subsurface water inlet, and a still head
was
charged with 150 g of FABE and 50 g of dry Amberlyst 15 solid acid catalyst.
The flask was heated to 110 C with the oil bath and water was added via a
syringe pump at a rate of 15 mL/hr. Distillation fractions were collected
every half
hour for a total of five hours. The fractions were analyzed for isobutanol
content
by the GC method described in Example 46 and the results are shown in Table
47. Approximately 44% of the theorectical amount of isobutanol contained in
the
FABE was collected over five hours.
Table 47
Sample Cumulative i-BuOH collected
1 0.3
2 0.9
3 2.3
4 3.4
5 4.4
6 5.5
7 6.3
8 6.9
9 7.4
10 8.0
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Example 55
Hydrolysis Catalyzed by Water Soluble Organic Acid Catalyst
[0359] A one liter flask with mechanical stirrer, subsurface nitrogen inlet,
subsurface water inlet, and a still head was charged with 200 g of FABE and 10
g
of p-toluenesulfonic acid. The flask was stirred and heated to 110 C with an
oil
bath at which time water was added at a rate of 20 mL/hr via a syringe pump.
Still fractions were collected every half hour for a total of three hours. The
fractions were analyzed for isobutanol content by the GC method described in
Example 46 and the results are shown in Table 48. Approximately 30% of the
theorectical amount of isobutanol contained in the FABE was collected over
five
hours.
Table 48
Fraction Cumulative amount of
isobutanol
1 1.0
2 4.0
3 7.2
4 9.5
11.8
6 13.4
Example 56
Hydrolysis of Solvent Phases from Fermentation
A. Solvent Phase 1
[0360] The solvent phase from the fermentation shown in Example 17 was
analyzed by the GC method shown in Example 46 and the results are shown in
Table 49. The analysis shows primarily FABE and fatty acids with a small
amount of material with a retention time consistent with FAEE. Analysis of
just
the butyl esters and acids shows a ratio of 62% FABE and 39% fatty acids.
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[0361] The solvent phase (1.25 liters, 1090 g) and 1.25 liters of water were
charged to a one gallon autoclave. The autoclave was sealed and heated to
2500C and held at temperature for four hours. The autoclave was then cooled
and opened, giving an emulsion. The mixture was filtered through a bed of
Celite
and the layers were separated. The organic layer was washed three times with
one liter of water. The sample was then heated to 50 C and purged with
nitrogen
for six hours. GC analysis shows no i-BuOH and a ratio of 33% FABE and 67%
fatty acids. An amber oil (993.9 g) was obtained. A detailed compositional
analysis of the original solvent phase from fermentation Example 17 and the
post-hydrolysis solvent phase is shown in Table 50.
B. Solvent Phase 2
[0362] The solvent phase from the fermentation shown in Example 18 was
analyzed by the GC method shown in Example 46 and the results are shown in
Table 49. The analysis shows primarily FABE and fatty acids with a small
amount of material with a retention time consistent with FAEE. Analysis of
just
the butyl esters and acids shows a ratio of 45% FABE and 55% fatty acids.
[0363] The solvent(1.25 liters, 1100 g) and 1.25 liters of water charged to a
one
gallon autoclave. The autoclave was sealed and heated to 250 C and held at
temperature for four hours. The autoclave was then cooled and opened, giving
an emulsion. The mixture was filtered through a bed of Celite and the layers
were separated. The organic layer was washed three times with one liter of
water. The sample was then heated to 50 C and purged with nitrogen for six
hours. GC analysis shows no i-BuOH and a ratio of 28% FABE and 72% fatty
acids. An amber oil (720.5 g) was obtained.
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Table 49
Pre-hydrolysis Post-hydrolysis
Sample FABE (%) Fatty Acid FABE (%) Fatty Acid
(%) (%)
Solvent 62 39 33 67
Phase 1
Solvent 45 55 28 72
Phase 2
Table 50
Solvent Phase 1 Post Hydrolysis
(wt%) Composition (wt%)
Isobutyl palmitate 7.28 4.24
Isobutyl stearate 3.41 2.25
Isobutyl oleate 13.92 7.69
Isobutyl linoleate 33.09 17.73
Isobutyl linolenate 2.78
Palmitic acid 3.79 7.3
Stearic acid 2.58 4.07
Oleic acid 9.14 16.61
Linoleic acid 19.06 33.54
Linolenic acid 2.3 2.65
Example 57
Recovery of Product Alcohol - Hydrolysis using a Lipase Catalyst
[0364] FABE was synthesized from corn oil fatty acid as per the method
described in Example 44. Novozyme 435 (Novo 435, Candida antarctica lipase
B, immobilized on an acrylic resin) was purchased from Sigma Aldrich (St.
Louis,
Mo). Candida antarctica Lipase B was purchased from Novozymes (Franklinton,
NC). t-BuOH, acetone, ethanol, methanol, and glycerol were all purchased from
Sigma Aldrich (St. Louis, Mo). For gas chromatography (GC) analysis, the gas
chromatograph used was Hewlett Packard 5890 Series II GC chromatogram and
methyl pentadecanoate was used as an internal standard.
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A. Atmospheric pressure, 40 C
[0365] To a mixture of 2 mL FABE and 5 mL water was added 40 mg Novozyme
435, and the reaction mixture was placed in a 20 mL vial and incubated at 40 C
in a rotary shaker (300 rpm). The reaction mixture was analyzed using GC
during 24 h of the reaction, to generate the following % conversion profile
given in
Table 51:
Table 51: % Conversion Profile for Example 57A
Reaction time (h) % FABE conversion
0 0
1 6.7
1.5 10.5
2 15.1
4 17.0
6 17.4
8 17.7
24 18.2
B. Atmospheric Pressure, 40 C, 65 C and 80 C, no organic solvent
[0366] Together with part A of this example, these data show how equilibrium
changes with temperature
[0367] To a mixture of 1 mL FABE and 2 mL water was added 20 mg Novozyme
435 and the reaction mixture was rotated at 40 C for 45 h in a 6 mL septum-
capped vial. The reaction mixture was analyzed using GC to reveal 18.2%
conversion of FABE at equilibrium.
[0368] To a mixture of 1 g FABE and 2 mL water was added 20 mg Novozyme
435, and the reaction mixture was rotated at 65 C for 42 h in a 6 mL septum-
capped vial. The reaction mixture was analyzed using GC to reveal 19.8%
conversion of FABE at equilibrium.
[0369] To a mixture of 1 g FABE and 2 mL water was added 20 mg Novozyme
435, and the reaction mixture was rotated at 80 C for 42 h in a 6 mL septum-
capped vial. The reaction mixture was analyzed using GC to reveal 21.4%
conversion of FABE at equilibrium.
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C. Example showing the effect of organic solvent (t-BuOH) on the equilibrium
[0370] To three reaction mixtures containing 0.25 mL FABE, 0.75 mL t-BuOH,
and 0.1-0.3 mL water was added 20 mg Novozyme 435, and the mixtures, in
6 mL septum-capped vials, were left rotating at 40 C overnight, at which point
they had reached equilibrium. The reaction mixtures were analyzed using GC
after 24 h of the reaction, to generate 77-82% FABE conversions given in
Table 52. Replacing t-BuOH with 3-Me-3-pentanol under similar reaction
conditions gave FABE hydrolysis yields of 70-80%.
Table 52: % Conversion Profile for Example 57C
Reaction mixture Novozyme 435 loading %FABE conversion
0.75 mL t-BuOH, 0.25 mL 20 mg 77%
FABE, 0.1 g H2O
0.75 mL t-BuOH, 0.25 mL 20 mg 81%
FABE, 0.2 gH2O
0.75 mL t-BuOH, 0.25 mL 20 mg 82%
FABE, 0.3 g H2O
D. Acetone as solvent
[0371] To three reaction mixtures containing 0.25 mL FABE, 0.75 mL acetone,
and 0.1-0.3 mL water was added 20 mg Novozyme 435, and the mixtures, in
6 mL septum-capped vials, were left rotating at 40 C overnight, at which point
they had reached equilibrium. The reaction mixtures were analyzed using GC
after 24 h of the reaction, to show 71-78% FABE conversions given in Table 53.
Table 53: % Conversion Profile for Example 57D
Reaction mixture Novozyme 435 loading %FABE conversion
0.75 mL acetone, 0.25 mL 20 mg 71%
FABE, 0.1 g H2O
0.75 mL acetone, 0.25 mL 20 mg 74%
FABE, 0.2 g H2O
0.75 mL acetone, 0.25 mL 20 mg 78%
FABE, 0.3 g H2O
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E. Example showing the effect of removing i-BuOH during hydrolysis on FABE
conversion - nitrogen purge at atmospheric pressure
[0372] A 25 mL round bottom flask was charged with 2 mL FABE, 5 mL water,
and 40 mg Novozyme 435. The reaction mixture was heated to 95 C, and the i-
BuOH that was forming in the reaction was removed by bubbling nitrogen through
the reaction mixture. Samples were taken from the mixture during the reaction,
and the organic phase was analyzed using GC. Conversion of 94% was
achieved after 6 h as shown in Table 54:
Table 54: FABE Conversion Profile for Example 57E
Reaction time (h) Mole % COFA in COFA + FABE
0 1
1 62
2 76
3 86
4 90
6 94
F. Example showing the effect of removing i-BuOH during hydrolysis on the
conversion-
vacuum distillation
[0373] A 25 mL round bottom flask was charged with 3 mL FABE, 7.5 mL of
water, and 60 mg Novozyme 435. The flask was attached to a vacuum
distillation apparatus, and the pressure was set to 91 mm Hg. The reaction
mixture was then heated to 74 C, and the i-BuOH that was forming in the
reaction
was distilled off. Samples were taken from the mixture during the reaction,
and
the organic phase was analyzed using GC. Conversion of 91 % was achieved
after 1 Oh as shown in Table 55.
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Table 55: % Conversion Profile for Example 57F
Reaction time (h) %FABE conversion
0 0
1 16
2 37
3 57
72
7 83
91
G. Example showing the effect of removing i-BuOH during hydrolysis on the
conversion-vacuum distillation example-varying FABE/COFA starting ratio: 23%
FABE:77% COFA v/v
[0374] A 25 mL round bottom flask was charged with 0.69 mL FABE, 2.31 mL
COFA, 7.5 mL water, and 60 mg Novozyme 435. The flask was attached to a
vacuum distillation apparatus, and the pressure was set to 91 mm Hg. The
reaction mixture was then heated to 74 C, and the i-BuOH that was forming in
the
reaction was distilled off. Samples were taken from the mixture during the
reaction, and the organic phase was analyzed using GC. Conversion of 98%
was achieved after 10 h as shown in Table 56.
Table 56: FABE Conversion Profile for Example 57G
Reaction time (h) Mole % COFA in COFA + FABE
0 77
1 83
2 86
3 90
4 92
6 93
7 96
10 98
H. Example showing the effect of removing i-BuOH during hydrolysis on the
conversion-vacuum distillation example-varying FABE/COFA starting ratio:
70%FABE:30%COFA v/v
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[0375] A 25 mL round bottom flask was charged with 2.1 mL FABE, 0.9 mL
COFA, 7.5 mL water, and 60 mg Novozyme 435. The flask was attached to a
vacuum distillation apparatus, and the pressure was set to 91 mm Hg. The
reaction mixture was then heated to 74 C, and the i-BuOH that was forming in
the
reaction was distilled off. Samples were taken from the mixture during the
reaction, and the organic phase was analyzed using GC. Conversion of 96%
was achieved after 10 h as shown in Table 57:
Table 57: FABE Conversion Profile for Example 57H
Reaction time (h) Mole % COFA in COFA + FABE
0 30
1 52
2 64
84
7 89
96
1. Example showing the free Cal B enzyme in FABE hydrolysis under vacuum
distillation
conditions
[0376] Two round bottom flasks were charged with 3 mL (2.7 g) FABE and
7.5 mL H2O each. To one mixture was added 5.9 mg Candida antarctica Lipase
B, and to the other was added 0.59 mg enzyme. The reaction flasks were
separately connected to the distillation apparatus and exposed to pressure of
91 mm Hg. The reaction mixtures were heated to 65-68 C. Samples were taken
from the reaction mixtures over a ten-hour period, and analyzed using gas
chromatography. The final FABE conversions were 96 and 78%, respectively.
The experiments show that reducing the amount of enzyme concentration by a
factor of ten reduces the rate and conversion by 3x and 18%, respectively. The
results are shown in Table 58.
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Table 58: FABE Conversion Profile for Example 571
Reaction time (h) Mole % COFA in COFA + Mole % COFA in COFA +
FABE with 5.9 mg CALB/2.7 g FABE with 0.59 mg
FABE CALB/2.7 FABE
0 0 0
1 44 21
2 62 31
6 89 61
96 78
Example 58
Recovery of Product Alcohol - Transesterification
[0377] FABE was synthesized from corn oil fatty acid as per the method
described in Example 44; Novozyme 435 (Candida antarctica lipase B,
immobilized on an acrylic resin) was purchased from Sigma Aldrich (St. Louis,
Mo). Candida antarctica Lipase B was purchased from Novozymes (Franklinton,
NC). t-BuOH, acetone, ethanol, methanol, and glycerol were all purchased from
Sigma Aldrich (St. Louis, Mo). For GC analysis, the gas chromatograph used
was Hewlett Packard 5890 Series II GC chromatogram and methyl
pentadecanoate was used as an internal standard.
A. Testing Iipases - FABE to FAME
[0378] Reagents used were t-BuOH (Aldrich); MeOH (Aldrich); Novozyme 435
(Aldrich); PS30 (Burkholderia cepacia, Amano Enzymes, Inc, Elgin, IL);
Lipolase 100T (Thermomyces lanuginosa, immobilized on silica, Novozymes,
Franklinton, NC); Lipolase 100L (Thermomyces lanuginosa, Novozymes,
Franklinton, NC); Lipozyme TLIM (immobilized Thermomyces lanuginosa,
Novozymes, Franklinton, NC); Lipoclean 2000T (immobilized mixture of lipases;
Novozymes, Franklinton, NC); NZL-103-LYO (Lipase from Rhizomucor miehi,
Novozymes, Franklinton, NC.
[0379] To a 6 mL vial was added 500 mg FABE (1.48 mmol), 400 pL t-BuOH,
60 pL MeOH (1.48 mmol), 3 pL water, and 2.5 mg lipase (see Table 57). The
resulting mixture was placed in an incubator/shaker, and left at 40 C
overnight.
GC analysis of the reaction mixture revealed the conversions from 9-56%.
Results are shown in Table 59.
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Table 59: Equilibrium concentrations [mg/mL] and % conversion of
FABE-*FAME using different lipases
Lipase FABE COFA i-BuOH % conversion
(mg/mL) (mg/mL) (mg/mL)
PS30 471 2.48 11.2 9.73%
NOVOZYME 435 224 16.8 63.7 56.30%
LTLIM 266 15.9 67.7 53.50%
L100T 455 1.8 8.9 8.80%
2000T 452 1.8 9.1 8.31%
NZL-103-LYO 429 3.94 15.3 13.90%
B. FABE to FAME transformation - Optimizing the amount of methanol
[0380] To a 6 mL vial was added 500 mg FABE (1.48 mmol), 400 pL t-BuOH, 60-
240 pL MeOH (1.48-5.92 mmol), 3 pL water, and 2.5 mg Novozyme 435. The
resulting mixture was placed in an incubator/shaker, and left at 400C
overnight.
GC analysis of the reaction mixture revealed the conversions from 53-73% as
shown in Table 60.
Table 60: % Conversion Profile for Example 58B
MeOH FABE COFA i-BuOH % conversion
e g's m /mL m /mL m /mL
1.2 232 2.4 66.9 56.64
1.4 168 2.4 70.4 65.50
1.6 167 0 72.1 66.17
1.8 184 0 80.3 66.41
2.0 131 0 72.4 71.46
2.2 134 1.2 81 73.25
2.4 136 0 80 72.71
2.6 161 0 78 68.70
2.8 168 0 73 66.31
3.0 155 0 74 68.38
3.2 180 0 73 64.75
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3.4 161 0 78 68.70
3.6 192 0 66 60.89
3.8 236 0 63 54.74
4.0 243 0 61 53.21
C. Optimizing the amount of enzyme - FABE to FAME
[0381] To a 6 mL vial was added 500 mg FABE (1.48 mmol), 400 pL t-BuOH,
132 pL MeOH (3.26 mmol), and 5-25 mg Novozyme 435. The resulting mixture
was placed in an incubator/shaker, and left at 40 C overnight. GC analysis of
the
reaction mixture revealed the conversions from 76-79% as shown in Table 61.
Table 61: % Conversion Profile for Example 58C
Novozyme 435 (mg) FABE COFA i-BuOH % conversion
(mg/mL) (mg/mL) (mg/mL)
mg 129.4 0 88 75.49
7.5 mg 119.3 0 89.2 77.21
mg 110.9 0 89 78.43
12.5 mg 109.2 0 84.9 77.89
mg 112.4 0 86.1 77.63
17.5 mg 108.4 0 87.4 78.51
mg 115.6 0 91.8 78.25
22.5 mg 108.6 0 87.7 78.53
mg 111.6 0 89 78.32
D. Minimizing the amount of solvent (t-BuOH) - FABE to FAME
[0382] To a 6 mL vial was added 500 mg FABE (1.48 mmol), 0-300 pL t-BuOH,
132 pL MeOH (3.26 mmol), and 10 mg Novozyme 435. The resulting mixture
was placed in an incubator/shaker, and left at 40 C overnight. GC analysis of
the
reaction mixture revealed the conversions from 30-81 % as shown in Table 62.
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Table 62: % Conversion Profile for Example 58D
Amount FABE COFA i-BuOH % conversion FAME
(mg/mL) (mg/mL) (mg/mL) (mg/mL)
300 pL t-BuOH 94.3 0 83 79.95 295
200 pL t-BuOH 94.1 0 83.2 80.02 295
100 pL t-BuOH 91.7 0 84.2 80.62 300
50 pL t-BuOH 92.9 0 81 79.80 290.8
<50 pL t-BuOH 137 0 70.5 69.98 249.4
no t-BuOH 322.7 1.39 30.2 29.77 98.1
E. Minimization of the amount of solvent (3-Me-3-pentanol) - FABE to FAME
[0383] To a 6 mL vial was added 500 mg FABE (1.48 mmol), 0-300 pL 3-Me-3-
pentanol, 132 pL MeOH (3.26 mmol), and 10 mg Novozyme 435. The resulting
mixture was placed in an incubator/shaker, and left at 40 C overnight. GC
analysis of the reaction mixture revealed the conversions from 30-78% as shown
in Table 63.
Table 63: % Conversion Profile for Example 58E
Amount FABE COFA i-BuOH % Conversion FAME
(mg/mL) (mg/mL) (mg/mL) (mg/mL)
0.3 mL 3M3P 115.6 3.75 81.4 76.13 296
0.2 mL 3M3P 106.3 3.13 80 77.32 294
0.1 mL 3M3P 104.8 2.36 80.8 77.74 296
0.05 mL 3M3P 102.9 2.3 79.6 77.80 295
soak 3M3P 102.5 2.4 79.9 77.93 296
no 3M3P 322.7 1.39 30.2 29.77 98.1
F. Conversion of FABE to FAME without solvent
[0384] To a mixture of FABE (500 mg, 1.48 mmol) and methanol (0.13 pL,
3.25 mmol) was added 40 mg Novozyme 435, and the reaction mixture was
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stirred at 40 C overnight. The mixture was then filtered and analyzed by GC to
reveal 76% conversion.
G. Enzyme Recycle - FABE to FAME
[0385] To a 6 mL vial was added 500 mg FABE (1.48 mmol), 400 pL t-BuOH,
132 pL MeOH (3.26 mmol), and 10 mg Novozyme 435. The resulting mixture
was placed in an incubator/shaker, and left at 40 C overnight. After that
time, the
reaction mixture was filtered and analyzed for conversion using GC, and the
filter
cake containing the immobilized enzyme, was used for another conversion of
FABE to FAME. The process was repeated ten times (Table 64). The
experiment shows that it is possible to recycle the enzyme up to ten times
without
the loss in conversion in the overnight reaction.
Table 64: % Conversion Profile for Enzyme Recycle in
Example 58G Concentrations are in mg/mL
FABE to FABE COFA i-BuOH % Conversion FAME
FAME
1st 115.8 5.29 82.1 76.26 298.1
2nd 124 5.36 82.7 75.13 293.2
3rd 112 4.82 85 77.47 303.1
4th 111 9.06 85.1 77.64 306.3
5th 99.4 5.1 82.1 78.91 284.5
6th 98.2 6 81.2 78.93 283.8
7th 115.6 6.8 78.9 75.56 262.7
8th 114.8 6.5 77.6 75.38 257.2
9th 99 5.7 78 78.12 241
10th 109 8.6 73.7 75.39 226.7
H. Conversion of FABE to FAEE
[0386] To 6 mL septum-capped vials were added 0.8 mL FABE (2.08 mmol) and
0.2 mL EtOH (3.43 mmol), forming a single phase. No enzyme or 20 mg
Novozyme 435 was added to the vials. The vials were then incubated at 25 C
and 40 C in an incubator shaker (300 rpm) for 17 h after which the solution
was
analyzed by gas chromatography, giving the contents and percent conversion of
FABE to FAEE shown in Table 65.
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Table 65
Sample FABE i-BuOH COFA % Conversion
(mg/mL) (mg/mL) (mg/mL)
EtOH/FABE + 93.2 38.5 0 41.6
enzyme, me, 25 C
EtOH/FABE, no 159.6 3.5 0.75 0
enz me, 25 C
EtOH/FABE + 90 35.7 0 43.3
enz me, 40 C
EtOH/FABE, no 158.7 3.1 0.75 0
enzyme, 40 C
Example 59
Glycerolysis of FABE
[0387] To a septum-capped 6 mL vial was added 0.75 mL t-BuOH, 0.25 mL
FABE, 0.1 mL (0.126 g) glycerol + 4 pL H2O, enzyme 20 mg each, forming a
single phase. The reactions were incubated with various lipases at 40 C on a
rotary shaker at 300 rpm. After 20 h, the samples were analyzed by gas
chromatography, giving the contents shown in Table 66. A comparison of the
COFA and FABE contents indicates that the products are i-BuOH and a mixture
of COFA and acyl glycerol (-64% acyl glycerol/36% COFA on molar basis).
Table 66: Percent conversion of FABE to a mixture of COFA and acyl glycerol
Lipase FABE COFA i-BuOH Percent
(mg/mL) (mg/mL) (mg/mL) conversion of
FABE
Amano PS-30 powder 73 43 32 64%
IM-20, powder 162.4 10.2 10.9 19%
Lipolase 100T 190 0 4 -0%
immobilized
Novozyme 435 73 39.5 30.8 64%
immobilized
Lipozyme TL IM 101.7 29 26.1 49%
immobilized
Lipoclean 2000T 200.9 0 3.9 -0%
immobilized
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A. Dependence on water and glycerol/FABE
[0388] To 6 mL septum capped vials were added 0.75 mL t-BuOH, 0.25 mL
FABE, 0.1 or 0.2 g glycerol + 20 mg each of either Amano PS-30 or Novozyme
435, forming a single phase. No water was added. The reactions were
incubated at 40 C on a rotary shaker at 300 rpm. After 20 h, the samples were
analyzed by gas chromatography, giving the contents shown in Table 67. A
comparison of the COFA and FABE contents indicates that the products are i-
BuOH and primarily acyl glycerol (mostly monoglyceride). Relative to the
previous example, the percent of product in the form of acyl glycerol
increases
with the absence of added water and with the increase in glycerol/FABE (-91 %
acyl glycerol/9% COFA on molar basis with 1.6 glycerol/FABE, mole/mole and
-95% acyl glycerol/5% COFA on molar basis with 3.2 glycerol/FABE, mole/mole).
The absence of added water eliminates the enzyme activity of Amano PS-30.
The Novozyme 435 which has water in the acrylic resin (-3% w/w) to which it is
immobilized is, however, still active.
Table 67: Percent conversion of FABE to acyl glyceride
Lipase Glycerol FABE COFA i-BuOH Percent
(mg/mL) (mg/mL) (mg/m L) conversion
of FABE
Amano PS-30 0.1 g 209 1.2 4.4 -0%
powder
Novozyme 0.1 g 84.2 10.7 28.6 58%
435,
immobilized
Amano PS-30 0.2 g 182 0.74 3.8 -0%
powder
Novozyme 0.2 g 61.5 7.1 30.6 -69%
435
immobilized
B. Dependence on enzyme concentration
[0389] To 6 mL septum capped vials was added 0.75 mL t-BuOH, 0.25 mL FABE,
0.2 g glycerol (glycerol/FABE 3.2/1, mole/mole) + 2 or 20 mg of Novozyme 435
(Novo 435). No water was added. The reactions were incubated at 40 C on a
rotary shaker at 300 rpm and the reaction was followed as a function of time
by
gas chromatography. The yields are indicated in Table 68. Approximately 97%
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of the FABE that reacted was converted to acyl glycerol (mostly monoglyceride)
on a mole basis.
Table 68
Time (h) % FABE conversion % FABE conversion
2 mg Novo 435 20 mg Novo 435
0 0 0
0.167 13.2
0.333 38
0.5 3.37 42.9
1 10.8 59.1
2 21.9 65.5
4 31.6 67.1
6.5 32.1
7 45.7 67.1
24 64 67.3
[0390] The rate of the reaction is linear with enzyme concentration, with t112
for 2
and 20 mg of Novozyme 435 of 208 and 20 minutes, respectively. The reaction,
however, reaches nearly the same yield of FABE conversion after 24 h.
[0391] These last reactions were repeated with 0.75 mL of 3-methyl-3-pentanol
replacing 0.75 mL of t-BuOH. The extent of FABE hydrolysis obtained after 24 h
was the same for both solvents. The advantage of 3-methyl-3-pentanol is that
with a boiling point of 122 C, the i-BuOH can be distilled off first in pure
form (b.p.
108 C). The 3-methyl-3-pentanol can then be distilled off and recycled for the
hydrolysis reaction, leaving in the retentate acyl glycerol, COFA, and
glycerol to
be recycled to the fermentation tank for reuse in the generation of FABE.
Tertiary
alcohols act as a solvent alone and have the advantage of not reacting with
the
fatty acid to form fatty acid alkyl esters in the presence of CALB.
C. Glycerolysis of FABE (FABE to COFA + acyl glycerol) in the absence of
organic
cosolvent - dependence on glycerol concentration
[0392] One gram (1 g) of FABE was mixed with 2 mL of 50, 70, 90, and 100%
(w/w) glycerol and placed in a 6 mL septum-sealed vial in the presence of 20
mg
Lipobond (Sprin Technologies, Trieste, Italy). The vial was tumbled end-over-
end
for 24 h at 62 C. With increasing glycerol concentration in the aqueous phase,
the percent of the product in the form of acyl glycerol increases (Table 69,
mostly
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monoglyceride). The extent of FABE conversion, however, does not show a
dependence on the glycerol concentration.
Table 69
Condition % of FABE % of COFA in % of acyl glycerol in
(% glycerol in conversion product product
aqueous phase) (mole basis) (mole basis)
50 18 100 0
70 17 84 16
90 17 34 66
100 17 3 97
Example 60
Conversion of COFA to FAEE and monoacyl glycerol
[0393] The following examples show that COFA can be esterified with EtOH or
with glycerol at high yield under mild conditions using immobilized enzyme.
[0394] Novozyme 435 (Candida antarctica lipase B, immobilized on an acrylic
resin) was purchased from Sigma Aldrich (St. Louis, Mo). Acetone, t-BuOH,
ethanol, methanol, and glycerol were all purchased from Sigma Aldrich (St.
Louis,
Mo). For GC analysis, the gas chromatograph used was Hewlett Packard 5890
Series II GC chromatogram and methyl pentadecanoate was used as an internal
standard.
A. Conversion of COFA to FAEE + i-BuOH using ethanol
[0395] Corn oil fatty acid (COFA, 0.25 g) was dissolved in 2.0 mL EtOH forming
a
single phase. Twenty mg of Candida antarctica lipase B (CALB) immobilized on
acrylic resin (Novozyme 435) was added (contains 1.7 mg of enzyme) and the
suspension was incubated for 24 h on a rotary shaker (300 rpm) at 40 C in a
6 mL glass vial sealed with a septum cap. The reaction went practically to
completion with 98% of the COFA converted to FAEE (fatty acid ethyl ester).
The
GC analysis after 24 h showed 98% conversion of COFA to fatty acid ethyl ester
as shown in Table 70.
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Table 70: % Conversion Profile for Example 60A
Reaction mix Novozyme 435 % Conversion of Phases
bp of EtOH = 78.1 C loading COFA to FAEE
2.0 mL EtOH + 0.25 g COFA 20 mg
Moles EtOH/moles COFA = 38.7 (contains 98 1 throughout
1.7 mg CALB)
B. Conversion of COFA to monoacylglycerides (MAG) + i-BuOH using glycerol
[0396] Corn oil fatty acid (COFA, 0.25 g) plus 0.325 g of glycerol were
dissolved
in 2.0 mL acetone. There was a large upper phase in which most of the
components were dissolved and a small residual glycerol-containing phase.
Twenty mg of Candida antarctica lipase B (CALB) immobilized on acrylic resin
(Novozyme 435) was added (contains 1.7 mg of enzyme) and the suspension
was incubated for 24 h on a rotary shaker (300 rpm) at 40 C in a 6 mL glass
vial
sealed with a septum cap. GC of the upper phase indicated that 87% of the
COFA had been converted to acyl glyceride (expected to be mostly mono-
acylglyceride). Results are shown in Table 71.
Table 71: % Conversion Profile for Example 60B
Reaction mix Novozyme % Conversion of Phases
435 loading COFA to acyl
bp of acetone = 56 C glyceride
most) MAG
2.0 mL acetone + 0.25 g COFA + 20 mg Minor glycerol
0.325 g glycerol (contains 87 phase throughout
Moles glycerol/moles COFA = 4 1.7 mg at 40 C
CALB)
Example 61
Conversion of COFA to FAME
[0397] The following examples show that COFA can be esterified with MeOH, with
EtOH, and with glycerol at high yield under mild conditions using immobilized
lipase.
A. Conversion of COFA to FAME without solvent
[0398] To a 6 mL vial was added 500 mg COFA (1.48 mmol), 132 pL of MeOH
(3.26 mmol), and 10 mg Novozyme 435. The resulting mixture was placed in an
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incubator/shaker, and left at 40 C overnight. GC analysis of the reaction
mixture
revealed 95% conversion.
B. Time course measurement of COFA to FAME reaction
[0399] To a 6 mL vial was added 500 mg COFA (1.48 mmol), 132 pL of MeOH
(3.26 mmol), and 10 mg Novozyme 435. The samples were incubated at 40 C in
an incubator/shaker and time points were taken during the reaction, and
analyzed
using GC. Results are shown in Table 72.
Table 72
COFA FAME Conversion
Time [mg/mL] [mg/mL] [%]
0 min 377 0 0%
min 242.2 135 35%
min 173.6 231.8 56%
min 122.8 274.3 68%
1 hr 59.2 373.2 86%
2 hr 18.2 389.9 95%
3 hr 16.8 406.2 96%
4 hr 15.6 404.4 96%
7 hr 16 411.2 96%
C. Adding more MeOH to the COFA-FAME reaction
[0400] To a 6 mL vial was added 500 mg COFA (1.48 mmol), 180, 240, 300, or
1320 pL of MeOH (4.44, 5.92, 7.41, and 14.82 mmol), and 10 mg Novozyme 435.
The resulting mixture was placed in an incubator/shaker, and left at 40 C
overnight. GC analysis of the reaction mixture revealed 96-97% conversion.
The results are shown in Table 73.
Table 73
MeOH COFA FAME
[eq] [mg/mL] [mg/mL] % Conversion
3 13.26 377 96.44%
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4 13.15 398.9 96.65%
12.12 391.8 96.85%
12.65 399.5 96.78%
Example 62
[0401] This example illustrated the removal of solids from stillage and
extraction
by desolventizer to recover fatty acids, esters, and triglycerides from the
solids.
During fermentation, solids are separated from whole stillage and fed to a
desolventizer where they are contacted with 1.1 tons/hr of steam. The flow
rates
for the whole stillage wet cake (extractor feed), solvent, the extractor
miscella,
and extractor discharge solids are as shown in Table 74. Table values are
short
tons/hr.
Table 74
Solids from Solvent Miscella Extractor
whole stillage discharge
solids
Fatty acids 0.099 0 0.0982 0.001
Undissolved solids 17.857 0 0.0009 17.856
Fatty acid butyl esters 2.866 0 2.837 0.0287
Hexane 0 11.02 10.467 0.555
Tri I ceride 0.992 0 0.982 0.0099
Water 29.762 0 29.464 0.297
[0402] Solids exiting the desolventizer are fed to a dryer. The vapor exiting
the
desolventizer contains 0.55 tons/hr of hexane and 1.102 tons/hr of water. This
stream is condensed and fed to a decanter. The water-rich phase exiting the
decanter contains about 360 ppm of hexane. This stream is fed to a
distillation
column where the hexane is removed from the water-rich stream. The hexane
enriched stream exiting the top of the distillation column is condensed and
fed to
the decanter. The organic-rich stream exiting the decanter is fed to a
distillation
column. Steam (11.02 tons/hr) is fed to the bottom of the distillation column.
The
composition of the overhead and bottom products for this column are shown in
Table 75. Table values are tons/hr.
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Table 75
Bottoms Overheads
Fatty acids 0.0981 0
Fatty acid butyl esters 2.8232 0
Hexane 0.0011 11.12
Tri I ceride 0.9812 0
Water 0 11.02
Example 63
Solids Extraction
Preparation of hydrous isobutanol
[0403] Into a 100 mL volumetric flask, 65 g of anhydrous reagent grade
isobutanol (sourced from Aldrich) was combined with 10 g of distilled water
and
shaken until a clear colorless homogeneous phase resulted. Another 10 g of
distilled water was added to the volumetric flask and shaken again resulting
in
two persistent clear colorless liquid layers. The top layer is considered to
be
hydrous isobutanol containing typically 20 wt% moisture and the bottom layer
is
predominantly water with typically 8 wt% dissolved alcohol.
Extraction using screen filtration and displacement wash
[0404] A fermentation was completed using recycled fatty acid (Example 19). A
185 g portion representative of the resulting heterogeneous mixture was
removed
and passed through a 80 MESH screen dish supported and sealed within a
Nalgene plastic filter funnel over 5 minutes using slight vacuum (-20 in H2O)
on
the underside. The filtrate partitioned into 90.5 g of a reddish brown oil
phase
and 50.9 g of a hazy aqueous phase containing dispersed fines but no settling
particulates. A wet cake remained on the screen dish. A sample of 1.5 g of
this
unwashed wet cake was removed and air dried. Hydrous isobutanol (23 g) was
drawn from the top layer inside the volumetric flask and passed through the
wet
cake over 5 minutes while mild vacuum on the underside of the screen dish was
maintained until no more liquid droplets were collected. The total filtrate
mass of
18 g consisted of a small amount of an immiscible bottom hazy aqueous layer
and a yellow clear hydrous isobutanol layer. The wet cake was removed from the
screen dish and a total mass of 38.4 g was recovered. A sample of 1.5 g of
this
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washed wet cake was removed and air dried. The dried sample of unwashed
solids was analyzed and found to contain 53.35 wt% total fat on a triglyceride
basis and the dried sample of washed solids was analyzed and found to contain
15.9 wt% total fat on a triglyceride basis.
Extraction using centrifugation and reslurry wash
[0405] A fermentation was completed using recycled fatty acid (Example 19). A
225 g portion representative of the resulting heterogeneous mixture was
removed
and centrifuged using a Beckman Coulter Allegra 64R machine at 10,000 rpm for
minutes. A clear reddish brown oil phase amounting to 67.2 g was decanted
off. The remaining material was centrifuged again and 95.1 g of a cloudy
aqueous centrate was decanted off. A 1.5 g sample of the wet solids was
removed and air dried and 56.5 g were recovered and transferred to a 400 mL
beaker. Hydrous isobutanol (20 g) drawn from the top layer inside the
volumetric
flask was added to the beaker to repulp the wet solids and stirring was
carried out
for 5 minutes. Another 32 g of hydrous isobutanol along with 32 g of the
centrate
were added and the solids were agitated in aqueous suspension beneath a
quiescent organic layer for 5 minutes. The mixture was then centrifuged at
10,000 rpm for 10 minutes to decant off a clear yellow hydrous isobutanol
layer
and centrifuged again in order to isolate and dry a 1.5 g sample of washed wet
solids. The dried sample of unwashed wet solids were analyzed and found to
contain 21.6 wt% total fat on a triglyceride basis and the dried sample of
washed
wet solids were analyzed and found to contain 4.04 wt% total fat on a
triglyceride
basis.
Example 64
Removal of Corn Oil by Removing Undissolved Solids
[0406] Approximately 1000 g of liquefied corn mash was prepared in a 1 L
glass,
jacketed resin kettle. The kettle was set up with mechanical agitation,
temperature control, and pH control. The following protocol was used: mixed
ground corn with tap water (26 wt% corn on a dry basis), heated the slurry to
55 C while agitating, adjusted pH to 5.8 with either NaOH or H2SO4, added
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alpha-amylase (0.02 wt% on a dry corn basis), continued heating to 85 C,
adjusted pH to 5.8, held at 85 C for 2 hrs while maintaining pH at 5.8, cool
to
25 C. The corn used was whole kernel yellow corn from Pioneer (3335). It was
ground in a hammer-mill using a 1 mm screen. The moisture content of the
ground corn was measured to be about 11.7 wt%, and the starch content of the
ground corn was measured to be about 71.4 wt% on a dry corn basis. The
alpha-amylase enzyme was Liquozyme SC DS from Novozymes (Franklinton,
NC). The total amounts of the ingredients used were: 294.5 g of ground corn
(11.7% moisture), 705.5 g of tap water, and 0.059 g of Liquozyme SC DS. H2O
(4.3 g) was added to dilute the enzyme, and a total of 2.3 g of 20% NaOH
solution was added to control pH. About 952 g of mash was recovered. Note
that there were losses due to mash sticking on walls of kettle and CF bottles.
[0407] The liquefied corn mash was centrifuged at 5000 rpm (7260 g's) for 30
minutes at 40 C to remove the undissolved solids from the aqueous solution of
oligosaccharides. Removing the solids by centrifugation also resulted in the
removal of free corn oil as a separate organic liquid layer on top of the
aqueous
phase. Approximately 1.5 g of corn oil was recovered from the organic layer
floating on top of the aqueous phase. It was determined by hexane extraction
that the ground corn used to produce the liquefied mash contained about 3.5
wt%
corn oil on a dry corn basis. This corresponds to about 9 g of corn oil fed to
the
liquefaction process with the ground corn.
[0408] Approximately 1 g of corn oil was recovered from the organic layer
floating
on top of the aqueous phase. About 617 g of liquefied starch solution was
recovered leaving about 334 g of wet cake. The wet cake contained most of the
undissolved solids that were in the liquefied mash. The liquefied starch
solution
contained about 0.2 wt% undissolved solids. The wet cake contained about
21 wt% undissolved solids. The wet cake was washed with 1000 g of tap water
to remove the oligosaccharides still in the cake. This was done by mixing the
cake with the water to form a slurry. The slurry was then centrifuged under
the
same conditions used to centrifuge the original mash in order to recover the
washed solids. Removing the washed solids by centrifugation also resulted in
the removal of some additional free corn oil as a separate organic liquid
layer on
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top of the aqueous phase. Corn oil was recovered from the organic layer
floating
on top of the aqueous phase.
[0409] The wet solids were washed two more times using a 1000 g of tap water
each time to remove essentially all of the liquefied starch. The final washed
solids were dried in a vacuum oven overnight at 80 C and about 20 inches Hg
vacuum. The amount of corn oil remaining in the dry solids, presumably still
in
the germ, was determined by hexane extraction. It was measured that a 3.60 g
sample of relatively dry solids (about 2 wt% moisture) contained 0.22 g of
corn
oil. This result corresponds to 0.0624 g corn oil/g dry solids. This was for
washed solids which means there are no residual oligosaccharides in the wet
solids. After centrifuging the liquefied corn mash to separate the layer of
free
corn oil and the aqueous solution of oligosaccharides from the wet cake, it
was
determined that about 334 g of wet cake containing about 21 wt% undissolved
solids remained. This corresponds to the wet cake comprising about 70.1 g of
undissolved solids. At 0.0624 g corn oil/g dry solids, the solids in the wet
cake
should contain about 4.4 g of corn oil.
Example 65
Lipid Analysis
[0410] Lipid analysis was conducted by conversion of the various fatty acid-
containing compound classes to fatty acid methyl esters ("FAMEs") by
transesterification. Glycerides and phospholipids were transesterified using
sodium methoxide in methanol. Glycerides, phospholipids, and free fatty acids
were transesterified using acetyl chloride in methanol. The resulting FAMEs
were analyzed by gas chromatography using an Agilent 7890 GC fitted with a 30-
m X 0.25 mm (i.d.) OMEGAWAXTM (Supelco, SigmaAldrich, St. Louis, MO)
column after dilution in toluene/hexane (2:3) . The oven temperature was
increased from 160 C to 200 C at 5 C/min then 200 C to 250 C (hold for 10
min) at 10 C/min. FAME peaks recorded via GC analysis were identified by their
retention times, when compared to that of known methyl esters (MEs), and
quantitated by comparing the FAME peak areas with that of the internal
standard
(C15:0 triglyceride, taken through the transesterification procedure with the
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sample) of known amount. Thus, the approximate amount (mg) of any fatty acid
FAME ("mg FAME") is calculated according to the formula: (area of the FAME
peak for the specified fatty acid/ area of the 15:0 FAME peak) * (mg of the
internal standard C15:0 FAME). The FAME result can then be corrected to mg
of the corresponding fatty acid by dividing by the appropriate molecular
weight
conversion factor of 1.052. All internal and reference standards are obtained
from Nu-Chek Prep, Inc.
[0411] The fatty acid results obtained for samples transesterified using
sodium
methoxide in methanol are converted to the corresponding triglyceride levels
by
multiplying the molecular weight conversion factor of 1.045. Triglycerides
generally account for approximately 80 to 90% of the glycerides in the samples
studies for this example, with the remainder being diglycerides. Monoglyceride
and phospholipid contents are generally negligible. The total fatty acid
results
obtained for a sample transesterified using acetyl chloride in methanol are
corrected for glyceride content by subtracting the fatty acids determined for
the
same sample using the sodium methoxide procedure. The result is the free fatty
acid content of the sample.
[0412] The distribution of the glyceride content (monoglycerides,
diglycerides,
triglycerides, and phospholipids) is determined using thin layer
chromatography.
A solution of the oil dissolved in 6:1 chloroform/methanol is spotted near the
bottom of a glass plate precoated with silica gel. The spot is then
chromatographed up the plate using a 70:30:1 hexane/diethyl ether/acetic acid
solvent system. Separated spots corresponding to monoglycerides, diglycerides,
triglycerides, and phospholipids are then detected by staining the plate with
iodine vapor. The spots are then scraped off the plate, transesterified using
the
acetyl chloride in methanol procedure, and analyzed by gas chromatography.
The ratios of the totaled peak areas for each spot to the totaled peak areas
for all
the spots are the distribution of the various glycerides.
Example 66
[0413] This example illustrates the recovery of by-products from mash. Corn
oil
was separated from mash under the conditions described in Example 64 with the
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exception that a tricanter centrifuge (Flottweg Z23-4, bowl diameter 230 mm,
length to diameter ratio 4:1) was used with these conditions:
= Bowl Speed: 5000 rpm
= Differential Speed: 10 rpm
= Feed Rate: 3 gpm
= Phase Separator Disk: 138 mm
= Impeller Setting 144 mm.
[0414] The corn oil separate had 81 % triglycerides, 6% free fatty acids, 4%
diglyceride, and 5% total of phospholipids and monoglycerides as determined by
the methods described in Example 65 and thin layer chromatography.
[0415] The solids separated from mash under the conditions described above
had a moisture content of 58% as determined by weight loss upon drying and
had 1.2% triglycerides and 0.27% free fatty acids as determined by the method
described in Example 65.
[0416] The composition of solids separated from whole stillage, oil extracted
between evaporator stages, by-product extractant and Condensed Distillers
Solubles (CDS) in Table 78 were calculated assuming the composition of whole
stillage shown in Table 76 and the assumptions in Table 77 (separation at
tricanter centrifuge. The values of Table 75 were obtained from an Aspen Plus
model (Aspen Technology, Inc., Burlington, MA). This model assumes that corn
oil is not extracted from mash. It is estimated that the protein content on a
dry
basis of cells, dissolved solids, and suspended solids is approximately 50%,
22%, and 35.5%, respectively. The composition of by-product extractant is
estimated to be 70.7% fatty acid and 29.3% fatty acid isobutyl ester on a dry
basis.
Table 76
Component Mass %
Water 57.386%
Cells 0.502%
Fatty acids 6.737%
Isobut l esters of fatty acids 30.817%
Tri I ceride 0.035%
Suspended solids 0.416%
Dissolved solids 4.107%
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Table 77
Hydrolyzer Thin Solids
feed stillage
Organics 99.175% 0.75% 0.08%
Water and dissolved solids 1 % 96% 3%
Suspended solids and cells 1 % 2% 97%
Table 78
Stream C. protein tri I ceride FFA FABE
Whole stillage wet cake 40% trace 0.5% 2.2%
Oil at evaporator 0% 0.08% 16.1% 73.8%
CDS 22% trace% 0.37% 1.71%
[0417] While various embodiments of the present invention have been described
above, it should be understood that they have been presented by way of example
only, and not limitation. It will be apparent to persons skilled in the
relevant art
that various changes in form and detail can be made therein without departing
from the spirit and scope of the invention. Thus, the breadth and scope of the
present invention should not be limited by any of the above-described
exemplary
embodiments, but should be defined only in accordance with the following
claims
and their equivalents.
[0418] All publications, patents and patent applications mentioned in this
specification are indicative of the level of skill of those skilled in the art
to which
this invention pertains, and are herein incorporated by reference to the same
extent as if each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by reference.
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