EP2582826A2 - Production of alcohol esters and in situ product removal during alcohol fermentation - Google Patents
Production of alcohol esters and in situ product removal during alcohol fermentationInfo
- Publication number
- EP2582826A2 EP2582826A2 EP11727619.6A EP11727619A EP2582826A2 EP 2582826 A2 EP2582826 A2 EP 2582826A2 EP 11727619 A EP11727619 A EP 11727619A EP 2582826 A2 EP2582826 A2 EP 2582826A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- carboxylic acid
- butanol
- alcohol
- oil
- esters
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C29/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom not belonging to a six-membered aromatic ring
- C07C29/74—Separation; Purification; Use of additives, e.g. for stabilisation
- C07C29/76—Separation; Purification; Use of additives, e.g. for stabilisation by physical treatment
- C07C29/86—Separation; Purification; Use of additives, e.g. for stabilisation by physical treatment by liquid-liquid treatment
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/16—Butanols
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D21/00—Separation of suspended solid particles from liquids by sedimentation
- B01D21/26—Separation of sediment aided by centrifugal force or centripetal force
- B01D21/262—Separation of sediment aided by centrifugal force or centripetal force by using a centrifuge
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Definitions
- 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.
- Alcohols have a variety of applications in industry and science such as a beverage (i.e., ethanol), fuel, reagents, solvents, and antiseptics.
- a beverage i.e., ethanol
- fuel i.e., ethanol
- reagents i.e., reagents
- solvents i.e., ethanol
- antiseptics i.e., ethanol
- 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.
- alcohols such as butanol, as well as for efficient and environmentally-friendly production methods.
- Production of alcohol utilizing fernnentation by microorganisms is one such environmentally-friendly production method.
- 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.
- ISPR In situ product removal
- 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).
- 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.
- 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.
- 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.
- 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 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.
- OA oleyl alcohol
- the presence of the undissolved solids during extractive fermentation can negatively affect the efficiency of the alcohol production.
- 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.
- DDGS Dried Distillers' Grains with Solubles
- 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 containing lipids, from liquefied mash prior to use in fermentation can substantially eliminate undissolved solids as described, for example, in copending, commonly owned U.S. Provisional Application Serial No. 61/356,290, filed June 18, 2010.
- 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.
- 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.
- 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 feedstock prior to fernnentation or can be supplied to the fernnentation vessel before or contemporaneously with the supplying of the feedstock.
- 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.
- 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.
- 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.
- 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.
- 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.
- the production of butanol and the production of butyl esters occur simultaneously or sequentially.
- a feedstock in the fermentation process comprises one or more fermentable sugars.
- the feedstock in the fermentation process comprises one or more fermentable sugars derived from 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, cellulosic material, lignocellulosic material, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, and mixtures thereof.
- fermentable sugars derived from 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, cellulosic material, lignocellul
- 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.
- the carboxylic acid comprises fatty acids.
- the carboxylic acid comprises 12 to 22 carbons.
- the carboxylic acid is a mixture of carboxylic acids.
- the butyl esters of the carboxylic acid are butyl esters of fatty acids.
- the catalyst is an enzyme capable of esterifying the carboxylic acid with the butanol to form butyl esters of the carboxylic acid.
- the enzyme is an esterase, lipase, phospholipase, or lysophospholipase.
- 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
- the method further comprises obtaining an oil from the oil stream and converting at least a portion of the oil into carboxylic acid.
- 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.
- the carboxylic acid comprises fatty acids. In one embodiment, the carboxylic acid comprises 12 to 22 carbons.
- 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.
- the catalyst is an enzyme capable of esterifying the carboxylic acid with the butanol to form butyl esters of the carboxylic acid.
- the enzyme is an esterase, lipase, phospholipase, or lysophospholipase.
- the method further comprises the step of washing the solids with a solvent.
- the solvent is select from hexane, isobutanol, isohexane, ethanol, petroleum distillates such as petroleum ether, or mixtures thereof.
- the solids are processed to form an animal feed product. In one embodiment, the solids are processed to form an animal feed product.
- 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).
- the animal feed product further comprises one or more vitamins, minerals, flavoring, or coloring.
- 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.
- 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.
- 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
- the method further comprising converting at least a portion of the oil into carboxylic acid.
- 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.
- the carboxylic acid comprises fatty acids.
- the carboxylic acid comprises 12 to 22 carbons.
- the method further comprises adding oil to the fermentation vessel.
- the method further comprising adding additional carboxylic acid to the fermentation vessel.
- the oil is converted to carboxylic acid after the step of adding the additional carboxylic acid.
- 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.
- the oil comprises glycerides and the one or more catalysts hydrolyze the glycerides into fatty acids.
- the butyl esters of carboxylic acid are butyl esters of fatty acids.
- the catalyst is an enzyme capable of esterifying the carboxylic acid with the butanol to form butyl esters of the carboxylic acid.
- the enzyme is an esterase, lipase, phospholipase, or lysophospholipase.
- the method further comprises the step of washing the solids with a solvent.
- the solvent is select from hexane, isobutanol, isohexane, ethanol, petroleum distillates such as petroleum ether, or mixtures thereof.
- the solids are processed to form an animal feed product. In one embodiment, the solids are processed to form an animal feed product.
- 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).
- the animal feed product further comprises one or more vitamins, minerals, flavoring, or coloring.
- 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.
- 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.
- 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 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.
- recovering butanol from the butyl esters comprises hydrolyzing the esters into carboxylic acid and butanol.
- the butyl esters are hydrolyzed in the presence of a hydrolysis catalyst.
- 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.
- the hydrolysis catalyst comprises an enzyme capable of hydrolyzing the butyl esters to form a carboxylic acid and butanol.
- the enzyme is an esterase, lipase, phospholipase, or lysophospholipase.
- enzyme reaction conditions favor enzymatic hydrolysis over esterification.
- the enzyme reaction conditions comprise a cosolvent.
- 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.
- the cosolvent is selected from acetone, terf-butanol, 2-Me-2-butanol, 2-Me-2- pentanol, and 3-Me-3-pentanol.
- the enzyme reaction conditions comprise end-product removal.
- the end- product is isobutanol or fatty acids.
- isobutanol is removed by vacuum distillation, pervaporartion, permselective filtration, or gas sparging.
- the fatty acids are removed by precipitation, permselective filtration, or electrophorectically.
- the hydrolysis reaction occurs in a reaction vessel.
- recovering butanol from the butyl esters comprises transeste fying the butyl esters into butanol and fatty acid alkyl esters or acyl glycerides.
- the fatty acid alkyl esters comprise fatty acid methyl esters, fatty acid ethyl esters, or fatty acid propyl esters.
- 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.
- the enzyme is an enzyme capable of hydrolyzing or transesterifying the butyl esters to form butanol.
- the enzyme is an esterase, lipase, phospholipase, or lysophospholipase.
- the carboxylic acid comprises fatty acids.
- 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.
- 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.
- the distillation column is a super-atmospheric distillation column with a steam heated reboiler.
- the method further comprises the steps of recovering water and solvent from the distillation columns; and recycling the water and solvent.
- the method further comprises the steps of recovering heat from the distillation process; and recycling the heat to evaporate water.
- 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
- the method further comprises obtaining an oil from the oil stream and converting at least a portion of the oil into carboxylic acid.
- the feedstock slurry is separated by centrifugation, filtration, or decantation.
- the carboxylic acid comprises fatty acids.
- the carboxylic acid has carbon chain lengths ranging from 12 to 22 carbons.
- 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.
- the method further comprises adding additional carboxylic acid to the fermentation vessel.
- the oil is converted to carboxylic acid after the step of adding the additional carboxylic acid.
- 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.
- the oil obtained from the oil stream comprises glycerides and the one or more catalysts hydrolyze the glycerides into fatty acids.
- the butyl esters of carboxylic acid are butyl esters of fatty acids.
- the catalyst is an enzyme capable of esterifying the carboxylic acid with the butanol to form butyl esters of the carboxylic acid.
- the enzyme is an esterase, lipase, phospholipase, or lysophospholipase.
- the solids are processed to form to an animal feed product.
- recovering butanol from the butyl esters comprises hydrolyzing the esters into carboxylic acid and butanol.
- the butyl esters are hydrolyzed in the presence of a hydrolysis catalyst.
- 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.
- the hydrolysis catalyst comprises an enzyme capable of hydrolyzing the butyl esters to form a carboxylic acid and butanol.
- the enzyme is an esterase, lipase, phospholipase, or lysophospholipase.
- the hydrolysis reaction occurs in a reaction vessel.
- recovering butanol from the butyl esters comprises transesterifying the butyl esters into butanol and fatty acid alkyl esters or acyl glycerides.
- the fatty acid alkyl esters comprise fatty acid methyl esters, fatty acid ethyl esters, or fatty acid propyl esters.
- 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.
- the enzyme is an enzyme capable of hydrolyzing or transesterifying the butyl esters to form butanol.
- the enzyme is an esterase, lipase, phospholipase, or lysophospholipase.
- 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.
- 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.
- the product alcohol is butanol and the alcohol esters of fatty acids are butyl esters of fatty acids.
- 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.
- the alcohol that is produced by the microorganism is butanol
- the alcohol ester is butyl ester.
- 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.
- 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.
- the fermentable carbon source is derived from biomass.
- the microorganism of the fermentation medium is a recombinant microorganism.
- the alcohol is butanol and the alcohol esters of fatty acids are butyl esters of fatty acids.
- 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
- 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.
- the product alcohol is butanol and the alcohol esters of fatty acids are butyl esters of fatty acids.
- 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 ester
- 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.
- the alcohol that is produced by the microorganism is butanol and the alcohol ester is butyl ester.
- 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.
- the oil is plant-derived oil.
- the product alcohol is butanol and the alcohol esters of carboxylic acids are butyl esters of fatty acids.
- 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
- 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.
- the carboxylic acid may be substantially insoluble in the fermentation medium.
- the alcohol that is produced by the microorganism is butanol and the alcohol ester is butyl ester.
- 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 free fatty acids with the alcohol by the catalyst.
- the alcohol is butanol and the fatty acid alcohol esters are fatty acid butyl esters.
- 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.
- the recombinant microorganism is capable of producing butanol.
- the fatty acid alcohol esters are fatty acid butyl esters.
- the fermentable carbon source comprises sugar.
- the fermentable carbon source comprises methane
- the recombinant microorganism is capable of producing methanol
- the fatty acid alcohol esters are fatty acid methyl esters.
- recombinant yeast cells useful for production of product alcohols.
- the recombinant host cells disclosed herein can be any bacteria, yeast or fungi host useful for genetic modification and recombinant gene expression.
- 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.
- 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.
- the host cell is a yeast host cell.
- the host cell is a member of the genera Saccharomyces.
- the host cell is Kluyveromyces lactis, Candida glabrata or Schizosaccharomyces pombe.
- 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 1 13-7D, 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 .
- 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.
- the methods further include: adding an extractant to form a two-phase mixture.
- 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.
- 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.
- the extractant comprises the carboxylic acid.
- the carboxylic acid is produced by hydrolysis of oil from a biomass feedstock.
- the fermentable carbon and the carboxylic acid are derived from the same biomass feedstock source.
- the carboxylic acid comprises saturated, mono-unsaturated, poly-unsaturated carboxylic acids having 12 to 22 carbons, and mixtures thereof. In some embodiments, contacting the fermentation medium with an extractant and a carboxylic acid and an enzyme occur contemporaneously.
- the microorganism is a genetically modified microorganism (e.g., a recombinant microorganism or host cell such as recombinant yeast cells).
- compositions comprising: PNY1504, PNY2205, or a recombinant host cell; an extractant; and optionally an esterification enzyme.
- compositions comprising PNY1504, PNY2205, or a recombinant host cell and butyl ester.
- 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.
- 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.
- 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.
- 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.
- FIG. 5 schematically illustrates an exemplary method and system of the present invention, in which an amount of lipids and undissolved solids are 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.
- FIG. 6 shows the aqueous and solvent phase concentrations of isobutanol produced by fermentation with sucrose as a carbon source.
- 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.
- FIG. 8 shows the consumption of sugars, reported in glucose equivalents, over time.
- 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.
- 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.
- “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).
- 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.
- the term "about" modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity 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.
- the term “about” means within 10% of the reported numerical value, alternatively within 5% of the reported numerical value.
- Biomass 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.
- 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.
- 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.
- cellulosic and/or 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.
- Sacharification enzymes suitable for cellulosic and/or lignocellulosic biomass are reviewed in Lynd, et al. (Microbiol. Mol. Biol. Rev. 66:506-577, 2002).
- 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.
- Biomass yield refers to the grams of biomass produced (i.e., cell biomass production) per gram of carbon substrate produced.
- 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.
- “Fermentation medium” as used herein means the mixture of water, sugars, dissolved solids, optionally microorganisms producing alcohol, product 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 (CO 2 ) 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.”
- 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.
- “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.
- Frermentation 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.
- 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.
- 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.
- saccharide refers to oligosaccharides, disaccharides, monosaccharides, and/or mixtures thereof.
- saccharide also includes carbohydrates including starches, dextrans, glycogens, cellulose, pentosans, as well as sugars.
- 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.
- Undissolved solids as used herein means non-fermentable portions of feedstock, for example, germ, fiber, and gluten.
- the non- fermentable portions of feedstock include the portion of feedstock that remains as solids and can absorb liquid from the fermentation broth.
- Dried Distillers' Grains with Solubles 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).
- DDGS may also refer to an animal feed product produced from a process of making a product alcohol (e.g., butanol, isobutanol, etc.).
- Product alcohol 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, Ci to C 8 alkyl alcohols.
- the product alcohols are C 2 to Cs alkyl alcohols.
- the product alcohols are C 2 to C 5 alkyl alcohols.
- Ci to Cs alkyl alcohols include, but are not limited to, methanol, ethanol, propanol, butanol, and pentanol.
- C 2 to C 8 alkyl alcohols include, but are not limited to, ethanol, propanol, butanol, and pentanol.
- Alcohol is also used herein with reference to a product alcohol.
- butanol refers with specificity to the butanol isomers 1 - butanol (1 -BuOH), 2-butanol (2-BuOH), terf-butanol (f-BuOH), and/or isobutanol (iBuOH or / ' -BuOH or l-BUOH, 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. [0063] "Propanol” as used herein refers to the propanol isomers isopropanol or 1 - propanol.
- Pentanol 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.
- alcohol equivalent 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.
- aqueous phase titer refers to the concentration of a particular alcohol (e.g., butanol) in the fermentation broth.
- 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.
- 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.
- the term "effective rate” as used herein is the effective titer divided by the fermentation time.
- ISPR In situ Product Removal
- Extract 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. [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.
- aqueous phase refers to the aqueous phase of a biphasic mixture obtained by contacting a fermentation broth with a water- immiscible organic extractant.
- fermentation broth specifically refers to the aqueous phase in biphasic fermentative extraction.
- organic phase refers to the non-aqueous phase of a biphasic mixture obtained by contacting a fermentation broth with a water- immiscible organic extractant.
- 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.
- 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.
- fatty acid refers to a carboxylic acid (e.g., aliphatic monocarboxylic acid) having C to C 2 8 carbon atoms (most commonly Ci2 to C2 4 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.
- fatty alcohol refers to an alcohol having an aliphatic chain of C 4 to C 22 carbon atoms, which is either saturated or unsaturated.
- fatty aldehyde refers to an aldehyde having an aliphatic chain of C 4 to C 22 carbon atoms, which is either saturated or unsaturated.
- fatty amide refers to an amide having a long, aliphatic chain of C 4 to C 22 carbon atoms, which is either saturated or unsaturated
- fatty ester refers to an ester having a long aliphatic chain of C 4 to C 22 carbon atoms, which is either saturated or unsaturated.
- Native oil refers to lipids obtained from plants (e.g., biomass) or animals.
- Plant-derived oil 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.
- 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.
- butanol biosynthetic pathway refers to an enzyme pathway to produce 1 -butanol, 2-butanol, or isobutanol.
- 1 -butanol biosynthetic pathway refers to an enzyme pathway to produce 1 -butanol from acetyl-coenzyme A (acetyl-CoA).
- 2-butanol biosynthetic pathway refers to an enzyme pathway to produce 2-butanol from pyruvate.
- isobutanol biosynthetic pathway refers to an enzyme pathway to produce isobutanol from pyruvate.
- gene refers to a nucleic acid fragment that is capable of being expressed as a specific protein, optionally including regulatory sequences 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.
- 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.
- 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.
- codon-optimized 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.
- 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).
- mRNA messenger RNA
- pDNA plasmid DNA
- a "gene” is a polynucleotide.
- a polynucleotide can contain the nucleotide sequence of the full-length cDNA sequence or a fragment thereof, including the 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).
- 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.
- a polynucleotide sequence may be referred to as "isolated,” in which it has been removed from its native environment.
- 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.
- 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).
- polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product.
- polypeptides 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 nucleic acid sequence. It may be generated in any manner including by chemical synthesis.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- feedstock 12 can be one or more components of a fractionated biomass, and in other embodiments, feedstock 12 can be a milled, unfractionated biomass.
- 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.
- feedstock 12 will often be described as constituting milled, unfractionated corn in which the undissolved solids have not been separated therefrom.
- maximizing feedstock content e.g., corn content
- 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.
- 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.
- 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.
- 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.
- a saccharification enzyme for example, glucoamylase
- a 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.
- 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.
- feedstock 12 is corn or corn kernels and therefore, feedstock slurry 16 is a corn mash slurry.
- feedstock 12 is a lignocellulosic feedstock and therefore, feedstock slurry 16 may be a lignocellulosic hydrolysate.
- feedstock 12 is sugar cane.
- 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.
- 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.
- microorganism 32 can be a bacteria such as E. coli.
- 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 5 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.
- 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.
- a 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.
- simultaneous saccharification and fermentation can occur inside fermentation vessel 30 as shown in FIG. 1 .
- an enzyme 38 such as glucoamylase, can be introduced to an inlet in fermentation vessel 30 in order to breakdown the starch or oligosaccharides to glucose capable of being metabolized by microorganism 32.
- 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.
- 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.
- catalyst 42 is capable of esterifying butanol with carboxylic acid 28 to form a butyl ester.
- 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.
- the resulting acid/oil composition includes monoglycerides and/or diglycerides from the partial hydrolysis of the acyl glycerides in the oil.
- the resulting acid/oil composition includes glycerol, a by-product of acyl glyceride hydrolysis.
- the resulting acid/oil composition includes lysophospholipids from the partial hydrolysis of phospholipids in the oil. [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.
- the remaining acyl glycerides from oil 26 are at least about 0.5 wt% of the fermentation broth composition.
- 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.
- a second catalyst (not shown) can be introduced to the fermentation vessel for hydrolysis of the acyl glycerides.
- 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).
- 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.
- 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.
- catalyst 42 and the second catalyst can be one or more enzymes, for example, lipase enzymes.
- 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.
- the source of the lipase is selected from the group consisting of Absidia blakesleena, Absidia corymbifera, Achromobacter iophagus, Alcaligenes sp., Alternaria brassiciola, Aspergillus 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 penicillat
- 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.
- Pseudomonas fluorescens Pseudomonas tragi, 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 scro
- 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 Pseudomonas fluorescens, Pseudomonas cepacia, Mucor miehei, hog pancreas, Candida cylindracea, Candida rugosa, Rhizopus niveus, Candida antarctica, Rhizopus arrhizus or Aspergillus available from SigmaAldrich.
- 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).
- LAH lipid acyl hydrolase
- the term "phospholipase” encompasses enzymes having any phospholipase activity, for example, cleaving a glycerolphosphate ester linkage (catalyzing hydrolysis of a glycerolphosphate 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 Dl or a phospholipase D2 activity; and/or a patatin activity or any combination thereof.
- PLC phospholipase C
- PDA phospholipase A
- PLB phospholipase B
- LPL lysophospholipase
- LPL lysophospholip
- phospholipase also encompasses enzymes having lysophospholipase activity, where the two substrates of this enzyme are 2- lysophosphatidylcholine and H 2 O, 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 l-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.
- phospholipases may be applied as lyophilized powder, immobilized or in aqueous solution.
- An alcohol e.g., butanol
- 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.
- oligosaccharides are additionally converted to one or more fermentable sugars
- the enzyme that converts oligosaccharides to fermentable sugars may also be inactivated by one or more of these same factors.
- 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.
- Enzymatic 8:183-192, 2000 have described the interfacial inactivation of a-chymotrypsin, ⁇ -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).
- 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.
- 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.
- 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.
- 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.
- carboxylic acid 28 is one or more of diacids.
- carboxylic acid 28 can be a mixture of two or more different fatty acids.
- 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.
- carboxylic acid 28 can be derived from native oil 26 by enzymatic hydrolysis of the oil glycerides using an enzyme as catalyst 42.
- 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.
- 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 of unsaturation (i.e., 18:1 )).
- native oil 26 is a mixture of two or more native oils such as a mixture of palm and soybean oils, for example.
- native oil 26 is a plant-derived oil.
- 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.
- oil 26 can be derived from corn, whereas feedstock 12 can be cane.
- 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.
- oil 26 is derived from the biomass used in the fermentation process.
- 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.
- ethanol 33 may be supplied to fermentation vessel 30 to be included in the fermentation broth.
- microorganism 32 may require supplementation of a 2-carbon substrate, for example, ethanol, for survival and growth.
- ethanol 33 may be supplied to fermentation vessel 30.
- the butanol production rate when fatty acid but not ethanol is in the fermentation vessel can be comparable to or greater than the butanol production rate when neither fatty acid nor ethanol is in the fermentation vessel.
- the amount of ethanol 33 supplementation is reduced compared to conventional processes.
- 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).
- 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.
- supplemental ethanol 33 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.
- 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.
- alcohol produced by microorganism 32 is esterified with carboxylic acid 28 using catalyst 42 to form alcohol esters.
- butanol produced by microorganism 32 is esterified with carboxylic acid 28 using catalyst 42 to form butyl esters.
- ISPR In situ product removal
- using catalyst to form esters in conjunction with ISPR can improve the performance of the fermentation.
- using catalyst to form esters in conjunction with ISPR 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.
- using a catalyst to form esters in conjunction with ISPR 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 1 1 -14, Table 3).
- 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%.
- 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.
- 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.
- 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.
- 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.
- SSF simultaneous saccharification and fermentation
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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 fernnentation broth, or less than about 15 g/L of fernnentation broth.
- 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.
- 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.
- the fermentation broth is contacted during fermentation with an extractant to form a two-phase mixture comprising an aqueous phase and an organic phase.
- 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.
- the extractant can be an organic extractant selected from the group consisting of saturated, mono-unsaturated, poly-unsaturated (and mixtures thereof) Ci 2 to C 2 2 fatty alcohols, Ci 2 to C 2 2 fatty acids, esters of Ci 2 to C 22 fatty acids, Ci 2 to C 22 fatty aldehydes, Ci 2 to C 22 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) C 4 to C 22 fatty alcohols, C 4 to C 28 fatty acids, esters of C 4 to C 28 fatty acids, C 4 to C 22 fatty aldehydes, and mixtures thereof.
- 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, whereby less carboxylic acid would be available for esterification with the product alcohol.
- 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, whereby less carboxylic acid would be available for esterification with the product alcohol.
- 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.
- the carboxylic acid 28 can also serve as an ISPR extractant 28 or a component thereof.
- 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.
- ISPR extractant 28 includes free fatty acids.
- ISPR extractant 28 includes corn oil fatty acids (COFA).
- 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.
- 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. [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.
- the product alcohol can be recovered from the alcohol ester by hydrolysis with base followed by acidification.
- the product alcohol esters can be hydrolyzed by water in the presence of a hydrolysis catalyst as substance 52.
- hydrolysis of the product alcohol esters to alcohol and carboxylic acid 28 e.g., fatty acid when carboxylic acid 28 is a fatty acid
- hydrolysis of the product alcohol esters to alcohol and carboxylic acid 28 can be achieved using a lipase, a water soluble acid, an inorganic acid, an organic acid, or a solid acid catalyst as substance 52.
- sulfuric acid can be used as an inorganic acid catalyst for alcohol ester hydrolysis.
- 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.
- hydrolysis of the alcohol esters can be achieved using steam as substance 52, by increasing temperature, and/or by application of pressure.
- 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.
- 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.
- 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.
- the alcohol esters of fatty acids are hydrolyzed with water at a temperature less than about 100°C.
- the hydrolysis occurs at a temperature greater than 100°C, greater than 150°C, greater than 200°C, or greater than 250°C.
- 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.
- the alcohol esters can be contacted with catalysts capable of transesterifying the alcohol esters to release butanol.
- 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.
- 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.
- 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.
- 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 suitable separation mechanisms can include extraction and membrane separation, for example.
- 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.
- 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.
- 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.
- ISPR extractant 29 can be introduced into fermentation vessel 30 to form a two-phase mixture comprising an aqueous phase and an organic phase.
- 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.
- 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
- 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.
- 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, 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.
- ISPR extractant 29 is free fatty acids obtained by chemical hydrolysis of biomass lipids.
- 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.
- In situ product removal can be carried out in a batch mode or a continuous mode in fermentation vessel 30.
- product is continually removed from the vessel (or reactor).
- 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.
- the organic extractant can contact the fermentation medium at the start of the fermentation forming a biphasic fermentation medium.
- 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.
- 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 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- aqueous phase stream 34 can include microorganism 32 such as yeast.
- Microorganism 32 can be easily separated 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.
- 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).
- 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 s/ft/ 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.
- catalyst 42 can be added to feedstock slurry 16 comprising oil 26' derived from the biomass from which feedstock 12 was formed.
- catalyst 42 is capable of hydrolyzing the glycerides in oil 26' to free fatty acids 28'.
- feedstock slurry 16 comprising oil 26' derived from the biomass from which feedstock 12 was formed.
- catalyst 42 is capable of hydrolyzing the glycerides in oil 26' to free fatty acids 28'.
- feedstock slurry 16 After introduction of catalyst 42 to feedstock slurry 16, at least a portion of the glycerides in oil 26' are hydrolyzed, resulting in a feedstock slurry 18 having free fatty acids 28' and catalyst 42.
- oil 26' is the feedstock's constituent corn oil and the free fatty acids 28' are corn oil fatty acids (COFA).
- COFA corn oil fatty acids
- Feedstock slurry 18 is introduced to fermentation vessel 30 along with alcohol-producing microorganism 32 to be included in a fermentation medium.
- 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 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 .
- butanol- producing microorganism 32 is introduced in fermentation vessel 30 along with feedstock slurry 18.
- Catalyst 42 in fermentation vessel 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.
- 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.
- ISPR extractant 29 can be carboxylic acid 28 described with reference to the embodiments of FIGs. 1 and 2.
- 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.
- oil 26 is corn oil, whereby ISPR extractant 29 is COFA.
- 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 reference to the embodiments of FIGs. 1 and 2.
- ISPR extractant 29 can be free fatty acids obtained by chemical or enzymatic hydrolysis of biomass lipids.
- the biomass lipids for producing extractant 29 can be from a same or different biomass source from which feedstock 12 is obtained.
- 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.
- 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.
- 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.
- 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.
- 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 COFA have a high partition coefficient for dissolution into the COFA phase 36 during liquid-liquid extraction ISPR.
- 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.
- 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.
- 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.
- slurry 62 can be introduced to fermentation vessel 30, with catalyst 42 being added directly to the fermentation vessel 30.
- 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 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.
- 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.
- undissolved solids can be removed from feedstock slurry 16 prior to introduction into fermentation vessel 30.
- 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.
- 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.
- 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.
- 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.
- separator 20 can also be configured to remove some or substantially all of oil 26' present in feedstock slurry 16.
- 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.
- 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').
- 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').
- 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 .
- 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.
- 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.
- wet cake 24 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 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.
- DDGS Dried Distillers' Grains with Solubles
- 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.
- 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.
- oil 26' may be separated from DDGS using any suitable known process including, for example, a solvent extraction process.
- DDGS are loaded into an extraction vessel and washed with a solvent such as hexane to remove oil 26'.
- solvents include, for example, isobutanol, isohexane, ethanol, petroleum distillates such as petroleum ether, or mixtures thereof.
- DDGS may be treated to remove any residual solvent.
- DDGS may be heated to vaporize any residual solvent using any method known in the art.
- 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.
- the resulting oil 26' and solvent mixture may be collected for separation of oil 26' from the solvent.
- the oil 267solvent mixture may be processed by evaporation whereby the solvent is evaporated and may be collected and recycled.
- the recovered oil may be converted to an ISPR extractant 29 for subsequent use in the same or different alcohol fermentation process.
- fatty acid esters e.g., fatty acid isobutyl esters
- 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.
- 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.
- 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.
- the miscella may be discharged from the extractor or separator through a device that separates or scrubs the fines from the miscella.
- the miscella may be subjected to a distillation 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.
- the solvent evaporates, it may be collected, for example, in a condenser, and recycled for 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.
- fatty acid esters e.g., fatty acid isobutyl esters
- fatty acids e.g., fatty acids, and triglycerides.
- 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.
- 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.
- 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.
- the trays carrying the solids may include openings that allow the solids to pass from one tray to the next.
- the solids may be conveyed to, optionally, a mixer where the solids are mixed with other byproducts before being conveyed into a dryer.
- a mixer where the solids are mixed with other byproducts before being conveyed into a dryer.
- An example of solids extraction is described in Example 63.
- the solids are fed to a desolventizer where the solids are contacted by steam.
- 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.
- 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.
- the overhead and bottom products may be recycled to the fermentation process.
- 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 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.
- 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.
- 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.
- 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.
- by-products may be derived from the mash used in the fermentation process.
- 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 byproduct 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.
- 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.
- 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.
- 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.
- these solids may be of particular value in a high protein, low fat feed.
- 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.
- 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.
- 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.
- 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.
- fat may be added to this composition, for example, from another by-product stream if a higher fat, low fiber animal feed is desired.
- this higher fat, low fiber animal feed may be used for swine or poultry.
- a non-aqueous composition of Condensed Distillers Solubles 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.
- oil from the fermentation process may be recovered by evaporation.
- This non-aqueous composition may comprise fatty 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.
- this stream may be used as feedstock for biodiesel production.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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 detergent fiber (ADF).
- 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 detergent fiber (ADF).
- 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 1 1 -23 wt% NDF, and at least about 5-1 1 wt% ADF.
- 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.
- 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.
- 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.
- the various streams may be combined in such a way as to customize a feed product for a specific animal species.
- 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.
- milled grain e.g., corn processed by hammer mill
- one or more enzymes are 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.
- 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.
- the washed filter cake may be reslurried with beer at a number of stages during the production process.
- 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.
- the filtrate produced as a result of the initial mix step may be further processed as described herein.
- 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.
- 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.
- a microorganism that is suitable for alcohol production
- 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 a scrubber in order to reduce air emissions (e.g., butanol air emissions) and to increase product yield.
- 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.
- 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.
- 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).
- 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 part to reslurry the filter cake described above. The split also provides thin stillage which may be pumped to evaporators for further processing.
- 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).
- 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.
- 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. 2009/0171 129, 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.
- 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.
- a vented alcohol/ water decanter e.g., butanol/water decanter
- 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 dedicated column may be modified
- the organic layer of the alcohol/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).
- 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.
- the super-atmospheric alcohol e.g., butanol
- 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., 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.
- corn oil may be separated from the process streams at any of several points.
- 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.
- 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.
- the alcohol e.g., isobutanol
- 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.
- 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.
- the yeast contained in the solids stream may be considered nonviable and may be 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.
- 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.
- 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.
- saccharification conditions e.g., enzyme feed, starch conversion, temperature, pressure
- fermentation conditions e.g., microorganism feed, glucose conversion, temperature, pressure
- degassing conditions e.g., solvent columns, preflash columns, condensers, evaporators, centrifuges, etc.
- 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.
- 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 alcohol such as butanol from the fermentor.
- 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.
- the extractant "trapped" in the DDGS may detract from DDGS value and qualification for sale as animal feed.
- 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.
- 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.
- 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.
- 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.
- the possibility of extractant being lost in the DDGS is removed.
- 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.
- 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.
- a stabilized partition coefficient e.g., 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
- the volume of the fermentor taken up by solids will be decreased.
- the effective volume of the fermentor available for the fermentation can be increased.
- the volume of the fermentor available for the fermentation is increased by at least about 10%.
- partition coefficient 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.
- the mass transfer coefficient is increased at least 2-fold (see Examples 4 and 5).
- phase separation 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.
- the phase separation can occur more quickly or can be more complete.
- a phase separation may occur where previously no appreciable phase separation was observed in 24 hours.
- 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).
- the flow rate of the extractant can be reduced which will lower operating costs, thereby resulting in an increased efficiency of product alcohol production.
- FIGs. 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.
- 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.
- 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.
- 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.
- 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.
- 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).
- Candida will metabolize alanine or oleic acid (Suiter et al., Arch. Microbiol. 153:485-489, 1990).
- 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.
- 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.
- 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 A1 , which is herein incorporated by reference.
- fermentation broth 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.
- 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.
- 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.
- 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.
- the culturing of microorganisms in a seed culture medium can produce an amount of fermentation product including alcohol.
- the alcoholic fermentation and respiration occur simultaneously whenever the specific growth rate ( ⁇ ) 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).
- the yeast growth is typically controlled, for example, by respiratory conditions using fed-batch fermentation technology for seed culturing.
- 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.
- high biomass yields can be obtained and accumulation of toxic products can be minimized.
- 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 byproducts can be one of the reasons for reduction of biomass yield per glucose in large scale bioreactors compared to laboratory scale.
- 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.
- critical cell growth inhibitory concentrations e.g., cell growth is lower than the growth that may be limited by the feed
- 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.
- 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.
- 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.
- 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.
- 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.
- 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).
- 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.
- 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.
- 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, varnishes, resins, gums, dyes, fats, waxes, resins, shellac, rubbers, and alkaloids.
- the present invention provides alternative methods to produce alcohols including butanol, which can support the high demand for these industrial chemicals.
- Alcohol-producing microorganisms are known in the art.
- fermentative oxidation of methane by methanotrophic bacteria 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.PK1 13-7D (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).
- 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.
- 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 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.
- recombinant microorganisms can be selected from the group consisting of Escherichia coli, Lactobacillus plantarum, Kluyveromyces lactis, Kluyveromyces marxianus, and Saccharomyces cerevisiae.
- the recombinant microorganism is yeast.
- 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.
- 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 1 13-7D, 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 .
- microorganisms comprise a butanol biosynthetic pathway.
- at least one, at least two, at least three, or at least four polypeptides catalyzing substrate to product conversions of a pathway are encoded by heterologous polynucleotides in the microorganism.
- all polypeptides catalyzing substrate to product conversions of a pathway are encoded by heterologous polynucleotides in the microorganism.
- 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.
- Suitable biosynthetic pathways for production of butanol are known in the art, and certain suitable pathways are described herein.
- 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.
- 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 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%.
- 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.
- This biosynthetic pathway comprises the following substrate to product conversions:
- butyryl-CoA to butyraldehyde which may be catalyzed, for example, by butyraldehyde dehydrogenase;
- 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.
- the recombinant host cell comprises a heterologous gene for each substrate to product conversion of a 1 -butanol biosynthetic pathway.
- 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 A1 and 2007/0292927A1 , and in PCT Application Publication No. WO 2007/130521 , all of which are incorporated herein by reference.
- One 2- butanol biosynthetic pathway comprises the following substrate to product conversions:
- 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.
- the recombinant host cell comprises a heterologous gene for each substrate to product conversion of a 2-butanol biosynthetic pathway.
- 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 A1 and PCT Application Publication No. WO 2007/050671 , incorporated herein by reference.
- One isobutanol biosynthetic pathway comprises the following substrate to product conversions:
- 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/).
- 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.
- both an enzyme catalyzing step c) is encoded by a heterologous polynucleotide integrated into the chromosome of the microorganism
- enzyme catalyzing step e) is encoded by a heterologous polynucleotide integrated into the chromosome of the microorganism.
- 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.
- 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 KARI, as well as plasmids comprising either or both chimeric genes.
- Suitable polynucleotides include the coding region of the ilvD gene from Streptococcus mutans (nt position 3313-4849 of SEQ ID NO: 2), the coding 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.
- 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.
- 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.
- 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.
- the recombinant host cell comprises a heterologous gene for each substrate to product conversion of an isobutanol biosynthetic pathway.
- 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.
- strain BP1064 was derived from CEN.PK 1 13-7D (CBS 8340;
- Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre, 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 (BP1083, PNY1504).
- 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.
- the scarless deletion procedure was adapted from Akada, et al., (Yeast 23:399-405, 2006).
- 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 1 13-7D 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.
- 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.
- 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).
- 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 1 13-7D 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 1 13-7D Aura3::kanMX.
- 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 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).
- Competent cells of CEN.PK 1 13-7D 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 1 13-7D Aura3::kanMX Ahis3::URA3.
- the KanMX marker was removed by transforming CEN.PK 1 13-7D 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 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 1 13-7D Aura3::loxP Ahis3 and designated as BP857.
- 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).
- 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 1 13-7D 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 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).
- Competent cells of CEN.PK 1 13-7D Aura3::loxP 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 1 13-7D Aura3::loxP Ahis3 Apdc6::URA3.
- CEN.PK 1 13-7D Aura3::loxP 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 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 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.
- 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 1 13-7D 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 NO: 38) and oBP521 (SEQ ID NO: 45).
- the PCR product was purified with a PCR Purification kit (Qiagen, Valencia, CA).
- Competent cells of CEN.PK 1 13-7D Aura3::loxP Ahis3 Apdc6 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 pdd knockout ilvDSm integration were screened for by PCR with primers oBP51 1 (SEQ ID NO: 46) and oBP512 (SEQ ID NO: 47) using genomic DNA prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, CA).
- CEN.PK 1 13-7D Aura3::loxP Ahis3 Apdc6 Apdd ::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 oBP51 1 (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 1 13-7D Aura3::loxP Ahis3 Apdc6 Apdd ::ilvDSm and designated as BP907.
- 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.
- 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.
- the sequences from upstream and downstream of 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.
- 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 1 13-7D genomic DNA was amplified with primers oBP438 (SEQ ID NO: 12) containing BamHI, AscI, Pmel, and Fsel restriction sites, and oBP439 (SEQ ID NO: 13) containing Xbal, Pad, 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 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: 1 1 ).
- 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 AscI 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 AscI 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 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).
- Competent cells of CEN.PK 1 13-7D Aura3::loxP Ahis3 Apdc6 Apdc1 ::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 1 13-7D Aura3::loxP Ahis3 Apdc6 Apdd ::ilvDSm Apdc5::sadB-URA3.
- CEN.PK 1 13-7D Aura3::loxP Ahis3 Apdc6 Apdd ::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) using genomic DNA prepared with a Gentra® Puregene® Yeast/Bact.
- 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 loxP 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 loxP-URA3- loxP 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).
- 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 1 13-7D Aura3::loxP Ahis3 Apdc6 Apdd ::ilvDSm Apdc5::sadB Agpd2::loxP and designated as PNY1503 (BP1064).
- BP1064 was transformed with plasmids pYZ090 (SEQ ID NO: 1 ) and pLH468 (SEQ ID NO: 2) to create strain NGCI-070 (BP1083; PNY1504). Construction of Saccharomyces cerevisiae strain PNY2205
- the strain, PNY2205 was derived from PNY1503 (BP1064) which is described above.
- 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.
- the scarless deletion procedure was adapted from Akada et al., (Yeast, 23:399, 2006).
- the PCR cassette for each scarless deletion was made by combining four fragments, A-B-U-C, by overlapping PCR.
- 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 1 13-7D 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.
- 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.
- the 3' 500 bp region of the gene was also deleted.
- the gene to be integrated was included in the PCR cassette between fragments A and B.
- the FRA2 deletion was designed to delete 250 nucleotides from the 3' end of the coding sequence, leaving the first 1 13 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 1 13-7D 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 0BP6OO (SEQ ID NO: 158), containing a 5' tail with homology to the 3' end of FRA2 Fragment U, and primer 0BP6OI (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 0BP6OI (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 0BP6OI (SEQ ID NO: 159).
- the PCR product was purified with a PCR Purification kit (Qiagen, Valencia, CA).
- 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.
- 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 0BP6O6 (SEQ ID NO: 163).
- the correct isolate was selected as strain CEN.PK 1 13-7D MATa ura3A::loxP his3A pdc6A pdc1A::P[PDC1 ]- DHAD
- 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 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 1 13-7D 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.
- the sequences from upstream (250 bp) and downstream (150 bp) of this gene are present for expression of the URA3 gene in yeast.
- the kivD coding region from Lactococcus lactis codon optimized for expression in Saccharomyces cerevisiae was amplified using pLH468 (U.S. Provisional Application Serial No.
- 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_LI(y)-ADH1 Fragment B was created by overlapping PCR by mixing the kivDJJ(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_LI(y)-ADH1 Fragment B.
- ADH1 Fragment C was amplified from genomic DNA with primer oBP507 (SEQ ID NO: 170), containing a Pad restriction site, and primer oBP508 (SEQ ID NO: 171 ), containing a Sail restriction site.
- the ADH1 Fragment C PCR product was digested with Pad and Sail and ligated with T4 DNA ligase into the corresponding sites of the plasmid containing ADH1 Fragment A-kivD_LI(y)-ADH1 Fragment B.
- the hybrid promoter UAS(PGK1 )-P FB AI was amplified from vector pRS316-UAS(PGK1 )- PFBAI -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 )-P F BAI PCR product was digested with Ascl and Pmel and ligated with T4 DNA ligase into the corresponding sites of the plasmid containing kivD_LI(y)-ADH1 Fragments ABC.
- the entire integration cassette was amplified from the resulting plasmid with primers oBP505 (SEQ ID NO: 168) and C-BP508 (SEQ ID NO: 171 ) and purified with a PCR Purification kit (Qiagen, Valencia, CA).
- 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.
- kivD_LI(y) 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 1 13- 7D MATa ura3A::loxP his3A pdc6A pdc1A::P[PDC1 ]-DHAD
- the pWS358-PGK1 p-GUS plasmid was generated by inserting a PGK1 p and beta-glucuronidase gene (GUS) DNA fragments into multiple cloning site of pWS358, which was derived from pRS423 vector (Christianson, et al., Gene 1 10:1 19-122, 1992).
- GUS beta-glucuronidase gene
- the resulting pWS358-FBA1 p-GUS plasmid was digested with Sail and Sacl, a DNA fragment containing a FBA1 p promoter, GUS gene, and FBAt terminator gel-purified, and cloned into Sall/Sacl sites on pRS316 to create pRS316-FBA1 p-GUS.
- the PCR product was digested with Kpnl and Sail and cloned into Kpnl/Sall sites on pRS316-FBA1 p-GUS to create pRS316-UAS(PGK1 )-FBA1 p-GUS.
- the FBA-alsS-CYCt cassette was constructed by moving the 1 .7 kb BbvCI/Pacl 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/Pacl to release the ILV5 gene). Ligation reactions were transformed into E.
- coli TOP10 cells and transformants were screened by PCR using primers N98SeqF1 (SEQ ID NO: 183) and N99SeqR2 (SEQ ID NO: 184).
- the FBA-alsS-CYCt cassette was isolated from the vector using Bglll 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 Aflll site (Klenow fragment was used to make ends compatible for ligation).
- 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 NO: 188) and N160SeqF5 (SEQ ID NO: 189).
- the resulting clone was called pUC19-kan::pdc1 ::FBA-alsS::TRX1 (clone A)(SEQ ID NO: 190).
- Isobutanol pathway plasmids (pYZ090AalsS and pBP915)
- pYZ090 (SEQ ID NO: 1 ) was digested with Spel and Not ⁇ 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).
- 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 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 (pl_H468AkivD)(SEQ ID NO: 195).
- 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.
- 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 The yeast-E.
- coli shuttle vector pRS425::GPM::kivD::ADH which contains the GPM1 promoter (SEQ ID NO: 72), kivD coding region from Lactococcus lactis (SEQ ID NO: 71 ), and ADH1 terminator (SEQ ID NO: 73) (described in U.S. Patent Application Publication No. 2007/0092957 A1 , Example 17) was digested with BbvCI and Pad 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).
- 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 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 1 141 17- 1 1A through 1 141 17A-1 1 D (SEQ ID NOs: 81 , 82, 83, and 84), and 1 141 17-13A and 1 141 17-13B (SEQ ID NOs: 85 and 86).
- the outer primers for the SOE PCR (1 141 17-13A and 1 141 17-13B) 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 1 12590-34G and 1 12590-34H (SEQ ID NOs: 87 and 88), and 1 12590- 34F and 1 12590-49E (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- 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.
- a pdd :: PPDC1 -ilvD-FBA1 t-URA3r integration cassette was made by joining the ilvD-FBA11 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 1 141 17-27A through 1 141 17-27D (SEQ ID NOs: 1 1 1 1 , 1 12, 1 13, and 1 14).
- the outer primers for the SOE PCR (1 141 17-27A and 1 141 17-27D) 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 1 141 17-36D and 135 (SEQ ID NOs: 92 and 93), and primers 1 12590-49E and 1 12590-30F (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:: PGPM 1 -sad B-ADH 11 pdd :: PPDC1 -ilvD-FBA1t.
- URA3r2 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 1 141 17-45A and 1 141 17- 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 pdd :: PPDC1 -ilvD-FBA1 t Ahis3.
- 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) at 30°C. Transform ants 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 pdd :: PPDC1 -ilvD-FBA1 t Ahis3 pdc5::kanMX4.
- the strain was named NYLA74.
- 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).
- Plasmid vectors pLH475-Z4B8 (SEQ ID NO: 140) and pLH468 were transformed into NYLA74 to create an isobutanol producing strain (NGCI-049).
- HXK2 hexokinase II
- 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.
- 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:: PGPM 1 -sad B-ADH 11 pdd :: PPDC1 - ilvD-FBA1 t Ahis3 Ahxk2 pdc5::kanMX4.
- Plasmid vectors pLH468 and pLH532 were simultaneously transformed into strain NYLA84 (BY4700 pdc6::PGPM1 -sadB-ADH1 t pdd ::PPDC1 -ilvD- FBA1 t 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.
- 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.
- 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.
- 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 1 18, respectively) and provided in plasmids pKivDy-DNA2.0 and pHadhy-DNA2.0.
- the encoded proteins are SEQ ID NOs: 1 17 and 1 19, respectively.
- Individual expression vectors for KivD and HADH were constructed.
- 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
- Ascl and Sfil enzymes were digested with Ascl and Sfil enzymes, thus excising the GPD promoter and the aid 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-DNA2.0.
- the triple ligation generated vector pLH467 (pRS426::PTDH3-kivDy-TDH3t). pLH467 was verified by restriction mapping and sequencing.
- pLH435 (pRS425::PGPM1 -Hadhy-ADH1 t) 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.
- pRS425::GPMp-sadB contains Bbvl and Pad 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 Pad 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.
- yeast vector pRS41 1 (ATCC No. 87474) was digested with Sad and Notl, and ligated
- pRS423 FBA ilvD(Strep) (SEQ ID NO: 128) which is described in U.S. Patent Application Publication No. 2010/0081 154 as the source of the IlvD 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 21 1 1 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 1 163) for selection in yeast and ampicillin resistance marker (nt 7092 to 7949) for selection in E. coli.
- the ilvD coding region (nt 31 16 to 4828; SEQ ID NO: 1 15; protein SEQ ID NO: 1 16) 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).
- the first step was to linearize pRS423 FBA ilvD(Strep) (also called pRS423-FBA(Spel)-llvD(Sfrepfococcus mufans)-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 Kpnl (with Kpnl 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)-llvD(Sfrepfococcus mutans)-Lum o.
- This generated vector pLH468 (pRS423::PFBA1 -ilvD(Strep) Lumio-FBA1 t-PTDH3-kivDy-TDH3t-PGPM1 -hadhy-ADH1 t) which was confirmed by restriction mapping and sequencing.
- the pLH532 plasmid (SEQ ID NO: 130) was constructed for expression of ALS and KARI in yeast.
- pLH532 is a pHR81 vector (ATCC No. 87541 ) containing the following chimeric genes: 1 ) the CUP1 promoter (SEQ ID NO: 139), acetolactate synthase coding region from Bacillus subtilis (AlsS; SEQ ID NO: 137; protein SEQ ID NO: 138) and CYC1 terminator2 (SEQ ID NO: 133); 2) an ILV5 promoter (SEQ ID NO: 134), PfS.llvC coding region (SEQ ID NO: 132) and ILV5 terminator (SEQ ID NO: 135); and 3) the FBA1 promoter (SEQ ID NO: 136), S. cerevisiae KARI coding region (ILV5; SEQ ID NO: 131 ); and CYC1 terminator.
- the Pf5.llvC coding region is a sequence encoding KARI derived from Pseudomonas fluorescens that was described in U.S. Patent Application Publication No. 2009/0163376, which is herein incorporated by reference.
- the Pf5.llvC 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
- 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 KARI.
- 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 1 161 -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 promoter (nt 8830-9493) followed by the TDH3 terminator (nt 5682-7161 ) for expression of ketoisovalerate decarboxylase.
- pRS
- 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 fra2A locus (the original deletion of FRA2 is described above).
- the sequence of the modified locus is provided as SEQ ID NO: 197.
- 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 YPRCdelta15.
- the sequence of the modified locus is provided as SEQ ID NO: 198.
- 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.
- plasmids were introduced into the strain for expression of KARI (pLH702, SEQ ID NO: 201 ) and DHAD (pYZ067DkivDDhADH, SEQ ID NO: 202), resulting in strain PNY2242.
- the methods described herein provide extractive fermentation methods with improved production yields of product alcohol.
- alcohol production utilizing fermentation by microorganisms may be inefficient due to the alcohol toxicity thresholds of the microorganism.
- the methods herein provide a means to convert the product alcohol into a substance less toxic to the microorganism.
- 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.
- 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.
- Carboxylic acid may serve as an extractant, and alcohol esters can partition into the extractant.
- the partition coefficient of the extractant may be degraded by lipid contamination.
- lipids present in the fermentation medium may be converted to extractant and consequently, minimize lipid contamination.
- 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.
- 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.
- 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 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.
- carboxylic acid i.e., extractant
- 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.
- the culture was grown to 0.55-1 .1 g/L dew (OD 6 oo 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 dew (OD 6 oo > 10) before being harvested and added to the fermentation.
- a glass jacked, 2 L fermentation vessel (Sartorius AG, Goettingen, Germany) was charged with house water to 66% of the liquefaction weight.
- the probe was then placed into the fermentation vessel through the stainless steel head plate.
- a dissolved oxygen probe (pO 2 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.
- 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.
- 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.
- 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 N 2 overlay for > 6 hrs. After the lipase treatment was complete, liquefaction was performed as described below (Liquefaction).
- 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 N 2 being added at 0.3 slpm through the sparger.
- the temperature set-point was changed from 55°C to 85°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.
- N 2 was redirected from the sparger to the head space to prevent foaming without the addition of a chemical antifoaming agent.
- the fermentation vessel temperature was set to 55°C instead of 30°C after the liquefaction cycle was complete (Liquefaction).
- 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 N 2 overlay for > 6 hrs. After the Lipase Treatment was complete, the fermentation vessel temperature was set to 30°C.
- Lipase Heat Inactivation Treatment Heat Kill Treatment Method
- 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.
- 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.
- the fermentation vessels pO 2 probe was calibrated to zero while N 2 was being added to the fermentation vessel.
- the fermentation vessels pO 2 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 dew.
- 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 (1 10 mL) was transferred to a sterile, inoculation bottle.
- the inoculum was pumped into the fermentation vessel through a peristaltic pump.
- 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.
- Sterile air was added to the fermentation vessel, through the sparger, at 0.3 slpm for the remainder of the growth and production stages.
- the pO 2 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.
- 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:
- 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.
- 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 vessel. Calculate the gassing rate per hour and then integrating that rate over the course of the fermentation.
- a 0.08% Trypan Blue solution was prepared from a 1 :5 dilution of 0.4% Trypan Blue in NaCI (VWR BDH8721 -0) with 1 X 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 ⁇ _ 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.
- Samples were refrigerated until ready for processing. Samples were removed from refrigeration and allowed to reach room temperature (about one hour). Approximately 300 ⁇ _ of sample was transferred with a m1000 Variable Channel BioHit pipette with 100-1000 ⁇ _ BioHit pipette tips into a 0.2 urn centrifuge filter (Nanosep® MF modified nylon centrifuge filter), then centrifuged using a Eppendorf, 5415C for five minutes at 14,000 rpm. Approximately 200 ⁇ _ of filtered sample was transferred into a 1 .8 auto sampler vial with a 250 ⁇ _ 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.
- Samples were refrigerated until ready for processing. Samples were removed from refrigeration and allowed to reach room temperature (about one hour). Approximately 150 ⁇ _ of sample was transferred using a m1000 Variable Channel BioHit pipette with 100-1000 ⁇ _ BioHit pipette tips into a 1 .8 auto sampler vial with a 250 ⁇ _ glass vial insert with polymer feet. A screw cap with PTFE septa was used to cap the vial.
- 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.
- 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.
- Experiment identifier 2010Y014 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.
- Experiment identifier 2010Y015 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 was added in a single batch between 0.1 - 1 .0 hr after inoculation.
- the butanologen was NGCI-070.
- Experiment identifier 2010Y016 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.
- Experiment identifier 2010Y017 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.
- Experiment identifier 2010Y018 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.
- Experiment identifier 2010Y019 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.
- Experiment identifier 2010Y021 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.
- Experiment identifier 2010Y022 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.
- Experiment identifier 2010Y023 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.
- Experiment identifier 2010Y024 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.
- Experiment identifier 2010Y029 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.
- Experiment identifier 2010Y030 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. Example 13 - (control)
- Experiment identifier 2010Y031 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.
- Experiment identifier 2010Y032 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.
- 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.
- Experiment identifier 2010Y026 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.
- 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.
- Experiment identifier 2010Y027 included: Seed Flask Growth method, Initial Fermentation vessel Preparation method, the Liquefaction method, the Nutrient Addition Prior to Inoculation method, Fermentation vessel Inoculation 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
- the oleyl alcohol extracting solvent was added in equal volume to the broth volume.
- the butanologen was PNY2205.
- 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.
- a Saccharomyces cerevisiae strain (strain PNY2242 described above) that was engineered to produce isobutanol from a carbohydrate source, with pdd deleted, pdc5 deleted, and pdc6, deleted was grown to 0.6-0.7 g/L dew (OD 6 oo 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
- the culture was grown to 0.6-0.7 g/L dew (OD 6 oo 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.
- a 10 L, B. Braun BioStat C fermentor was prepared for use.
- the probe was then placed into the fermentation vessel, through a side port.
- a dissolved oxygen probe (pO 2 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.
- the fermentation vessel temperature was controlled at 30°C with a thermocouple and house water circulation loop.
- Wet corn kernels #2 yellow dent
- Difco Yeast Extract was added to the fermentor at 0.5% w/w of the total batch weight.
- 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 N 2 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.
- 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.
- N 2 was redirected from the sparger to the head space to prevent foaming without the addition of a chemical antifoaming agent.
- the fermentation vessels pO 2 probe was calibrated to zero while N 2 was being added to the fermentation vessel.
- the fermentation vessels pO 2 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.
- 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 NH OH 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.
- 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.
- 5.5 L of HD OCENOL® 90/95 was added to the fermentor.
- 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 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.
- an addition of 6-7 mL/L anhydrous ethanol was made to the fermentor after the fermentor was returned to 30°C
- the fermentation vessels pO 2 probe was calibrated to zero while N 2 was being added to the fermentation vessel.
- the fermentation vessels pO 2 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.
- 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 NH OH 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.
- 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 dew (OD 6 oo 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:
- the culture was grown to 0.6-0.7 g/L dew (OD 6 oo 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.
- HD OCENOL® 90/95 oleyl alcohol, Cognis, Monheim, DE
- the corn mash centrate was made by liquefying corn in the following recipe:
- yeast extract (Difco No. 9102333, low dusting)
- 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.
- 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 N 2 being added at 0.3 slpm through the sparger.
- the fermentation vessels pO 2 probe was calibrated to zero while N 2 was being added to the fermentation vessel.
- the fermentation vessels pO 2 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.
- 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.
- the fernnentation 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 5.25 without adding any acid.
- 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 pO 2 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.
- 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.
- 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.
- Experimental Identifier GLNOR1050 included: 10 L Pre-Seed Flask Growth, 10 L Propagation Tank Liquefaction, 10 L Propagation Tank Operation, 10 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.
- Experimental Identifier GLNOR1051 included: 10 L Pre-Seed Flask Growth, 10 L Propagation Tank Liquefaction, 10 L Propagation Tank Operation, 10 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.
- Identifier 201 1Y029 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.
- Identifier 201 1Y030 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.
- Identifier 201 1Y031 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 56B - 10% v/v solvent load), 2 L Fermentation Vessel Operating Conditions, and all Analytical methods.
- Identifier 201 1Y032 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.
- Inoculum A seed medium was prepared to initiate the growth of the isobutanologen.
- the composition of the seed medium was as follows: 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 g/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
- 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 OD 6 oo 7.
- 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
- 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 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.
- 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.
- 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 1 1 hours elapsed fermentation time.
- the program was established to maintain sucrose limitation until OD 6 oo 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 OD 6 oo was reached after 20 hours fermentation time.
- 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.
- 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.
- 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 solution was prepared and stored for one day at 5°C before addition to the fermentors.
- 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.
- the fermentors were each inoculated with 40 mL of the concentrated biomass, to initial OD 6 oo 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- 10 mL) were drawn every 2-3 hours, and assayed for glucose and sucrose by a YSI Select Biochemistry Analyzer (YSI, Inc., Yellow Springs, Ohio).
- sucrose 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 1 100 HPLC.
- a Shodex® Sugar SH101 1 column was used with 0.01 N sulfuric acid mobile phase.
- a BioRad Aminex® HPX-87N column with 0.01 M Na 2 HPO 4 (pH 8) mobile phase was used.
- 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 aqueous titers of isobutanol and lower free isobutanol in the solvent, and more isobutanol as FABE.
- FIG. 7 shows the effective titer of isobutanol.
- 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).
- 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 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.
- 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
- Example 1 none 31 h, broth 0.19 0.03 2.1 0.3 2.4 13
- Example 1 none 70 h, broth 0.15 0.03 1.7 0.3 2.0 15
- Example 2 10 ppm 0.8 h, broth 0.15 0.62 1 .7 7.0 8.7 81
- 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 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
- 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
- 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.
- 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 .
- 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)
- 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) (9) FFA (g)
- Example 4 none 30 h, broth 0.17 0.05 2.0 0.6 2.7 24
- Example 4 none 53 h, OA 0.67 0.32 6.0 2.9 8.9 32
- Example 4 none 70 h, broth 0.13 0.04 1.5 0.4 1 .9 23
- Example 5 7.2 ppm 30 h, broth 0.08 0.18 1.0 2.1 3.1 68
- Example 5 7.2 ppm 53 h, broth 0.07 0.1 1 0.9 1.3 2.2 61
- Example 5 7.2 ppm 70 h, broth 0.08 0.10 0.9 1.2 2.1 55
- Example 6 none 30 h, broth 0.17 0.06 2.1 0.8 2.8 26
- Example 6 none 53 h, broth 0.14 0.05 1.6 0.5 2.2 25
- Example 6 none 70 h, broth 0.15 0.05 1.8 0.6 2.4 25
- 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 1 1 .
- Table 1 1 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)
- 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
- 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
- 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 0.1 1 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 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 29 h, broth 0.30 0.05 3.4 0.6 4.0 16
- Example 8 none 70 h, broth 0.22 0.04 2.5 0.5 3.0 17
- Example 10 10 ppm liquefied mash 0.14 0.85 1 .6 9.4 1 1.0 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 0.1 1 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 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 1 1 The fermentation run as described in Example 1 1 had 5 g/L of ethanol added prior to inoculation, whereas the fermentation run as described in Example 12 had no added ethanol.
- Table 14 Dependence of the production of free isobutanol (/-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 1 1 and 12) of active lipase (COFA phase analysis) fermentation g /-BuOH / g FABE/ g /-BuOH from FABE/ total g /-BuOH / fermentation time (h) kg COFA kg COFA kg COFA kg COFA kg COFA kg COFA kg COFA kg COFA
- Example 1 1 6.6 2.3 0.0 0.0 2.3
- Example 1 1 53.5 25.1 2.9 0.6 25.7
- Example 1 1 71.1 24.4 6.3 1 .4 25.8
- Example 12 71.1 12.8 3.0 0.7 13.5
- Example 14 71.1 4.9 244.5 54.3 59.2
- Table 15 Dependence of the production of free isobutanol (/-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 1 1 and 12) of active lipase (fermentation broth analysis) fermentation g /-BuOH / g FABE/ g /-BuOH from FABE/ total g /-BuOH / sample time (h) kg broth kg broth kg broth kg broth kg broth kg broth kg broth kg broth kg broth kg broth kg broth kg broth
- Example 1 1 6.6 0.0 0.0 0 0.0
- Example 1 1 53.5 9.8 0.0 0 9.8
- Example 1 1 71.1 9.5 0.0 0 9.5
- 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 53.5 2.1 3.0 0.7 2.8
- Table 17 Weights of isobutanol (/-BuOH) and isobutyl esters of corn oil fatty acids (/-BuO-COFA) present in the aqueous fraction (AQ) and organic fraction (ORG) for reactions described in Table 16.
- AQ aqueous fraction
- ORG organic fraction
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US13/160,766 US9012190B2 (en) | 2011-06-15 | 2011-06-15 | Use of thiamine and nicotine adenine dinucleotide for butanol production |
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