WO2016061339A1 - Procédé de modification d'une levure pour une fermentation améliorée - Google Patents

Procédé de modification d'une levure pour une fermentation améliorée Download PDF

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Publication number
WO2016061339A1
WO2016061339A1 PCT/US2015/055721 US2015055721W WO2016061339A1 WO 2016061339 A1 WO2016061339 A1 WO 2016061339A1 US 2015055721 W US2015055721 W US 2015055721W WO 2016061339 A1 WO2016061339 A1 WO 2016061339A1
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organic acid
yeast cell
fermentation
yeast
modified
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PCT/US2015/055721
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English (en)
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Jayarama K. Shetty
Mark GOUTHRO
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Danisco Us Inc.
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Publication of WO2016061339A1 publication Critical patent/WO2016061339A1/fr

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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
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • 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
    • C12N1/04—Preserving or maintaining viable microorganisms
    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • 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

  • Modification of the yeast includes contacting yeast with an organic acid or a salt of the organic acid.
  • the organic acid or the salt of the organic acid can be removed from the modified yeast by centrifugation and decantation.
  • the modified yeast can be dried for storage.
  • the yeast that are modified can be genetically-modified.
  • the modified yeast can then be used for fermentation with a medium containing a fermentable sugar.
  • the modified yeast can be resuspended to form a cream for inoculation.
  • the cream can be transferred to a fermentation medium containing a starch substrate.
  • Saccharification and fermentation may be conducted to covert the starch in the starch substrate to an end product.
  • the process can be simultaneous or conducted in series.
  • the methods described herein can lead to a reduction in glycerol levels, which can result in co-products containing reduced glycerol.
  • a wet-milling process involves fractionating corn into different components by subjecting corn to steeping processes followed by separating the components by gravity separation and washing into germ (oil), protein fiber and starch fractions. Only the starch component enters into further processing to make fermentation feed stock.
  • Dry-grind processes starts with grinding the corn kernel without separating the components of corn.
  • a dry-grind process is the most commonly used process and is generally referred to as "conventional process”.
  • the dry-grind process includes grinding the starch-containing materials and then liquefying gelatinized starch at high temperature using a thermostable alpha amylase (such as SPEZYME®RSL from DuPont Industrial Bioscience), followed by saccharification and fermentation carried out in the presence of glucoamylase (such as DISTILLASE® SSF from DuPont Industrial Bioscience) and other secondary enzymes and yeast to convert fermentable sugars into alcohol.
  • a thermostable alpha amylase such as SPEZYME®RSL from DuPont Industrial Bioscience
  • glucoamylase such as DISTILLASE® SSF from DuPont Industrial Bioscience
  • a third process that is suitable for small grains such as wheat, barley and triticale involves pretreatment steps using viscosity reducing enzymes (such as non-starch polysaccharides hydrolyzing enzymes such as cellulases, hemicellulases etc.) between 50 and 60 °C for an extended period of time followed by liquefaction, saccharification and fermentation.
  • viscosity reducing enzymes such as non-starch polysaccharides hydrolyzing enzymes such as cellulases, hemicellulases etc.
  • a "no cook process” includes grinding a starch containing material followed by saccharifying and fermenting a granular starch without gelatinization using granular starch hydrolyzing glucoamylase and acid stable alpha amylase and yeast.
  • sugar referred to as sucrose from sugar cane or beet and molasses from sugar crystallization process is also used as yeast fermentation feed stock for producing an alcohol.
  • a process involving sugar does not have any further processing, as with starch feed stocks.
  • the fermentation broth containing soluble solids, insoluble solids and alcohol is distilled to recover alcohol.
  • the fermentation efficiency of the carbon conversion into end products during yeast fermentation depends on several factors, which include pH, temperature, dry solids content, organic acid content and glycerol content.
  • Anaerobic yeast fermentation generally produces glycerol as a by-product with the production of glycerol being directly proportional to yeast growth.
  • Another by-product is the production of unproductive yeast, often referred as wild yeast. Wild yeast production is undesirable because it produces only biomass at the cost of end product.
  • Organic acids are well known for their toxicity to microorganisms and thus are used as antimicrobial food additives. There is general agreement that the toxicity of organic acid is not due to hydrogen-ion concentration alone but seems to be a function of the concentration of their un-dissociated forms See, Verduyn, C, B. Postma, W. A. Scheffers, and J. P. Van Dijken, Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 1992, 8:501 -507; Narendranath, N. V., Thomas. K. C, and Ingledew W. M., Effects of acetic acid and lactic acid on the growth of
  • Saccharomyces cerevisiae in a minimal medium Journal of Industrial Microbiology and Biotechnology. 2001 , 26/3:171 -177; Narendranath, N. V. K. C. Thomas. W. M. Ingledew, Acetic acid and lactic acid inhibition of growth of Saccharomyces cerevisiae by different mechanisms. J. Am. Soc. Brew. Chem. 2001 , 59: 187; Piper, P. and Calderon, CO. et al., Weak acid adaptation: the stress response that confers yeasts with resistance to organic acid food preservatives. Microbiology. 2001 , 147:2635-42; Thomas, K.C and Hynes, S.H.
  • the un-dissociated form of the molecule diffuses passively into the microbial cell and then dissociates inside the cell. This can lead to a massive accumulation of dissociated anions and protons within the cell, thereby acidifying the cytoplasm, disrupting homeostasis of intracellular pH, and increasing the inhibitory activity.
  • the cell tries to maintain its pH homeostasis by extruding the excess protons via the plasma H + -ATPase, which uses energy from ATP hydrolysis for its activity.
  • Much research has been conducted on the effect of organic acids, mainly acetic acid and lactic acid, on ethanol fermentation. See Pons, M.N. and Rajab, A. et al., "Influence of acetate on growth kinetics and production control of
  • sodium benzoate has been mostly used as a preservative for its bacteriostatic and fungistatic effect under acidic conditions in many food formulations, such as salad dressings, carbonated drinks, jams, fruits juices, pickles, and condiments.
  • sodium benzoate has the E number E21 1 .
  • the mechanism of food preservation starts with the absorption of benzoic acid into the cells. If the intracellular pH changes to 5 or less, the anaerobic fermentation of glucose through phosphofructosekinase is decreased by 95%. See, Krebs H.A et al., "Studies on the mechanism of the antifungal action of benzoate," Biochem.J. 1983, 214:657-663.
  • Yeast are modified by contacting the yeast with organic acids and salts of such organic acids.
  • the organic acid may be benzoic acid and the salts may be sodium benzoate and potassium benzoate.
  • the modification can persist even when the organic acids and organic acid salts are removed after the contacting but before the yeast are used in fermentation.
  • the yeast can even be dried and stored after the modification and used in fermentation later.
  • a cream of modified yeast can be formed for inoculation, with the cream transferred to a fermentation medium containing a starch substrate.
  • the starch substrate may be a starch-containing material or a purified starch.
  • Saccharification and fermentation can be conducted for converting the starch in the starch substrate to an end product.
  • the modification to the yeast can result in increased fermentation yield during the conversion of starch and or sugar containing feed stocks and reduced glycerol.
  • the increase in the fermentation yield may be accomplished by first adding to yeast an organic compound containing carboxylic acid with a non-polar aliphatic or aromatic group.
  • Fig. 1 illustrates a GC/FID chromatogram of transesterified yeast extract showing methyl benzoate and major lipids.
  • starch substrate comprises starch.
  • starch refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and/or amylopectin with the formula (C 6 H 0 O 5 ) x , wherein X can be any number.
  • the term refers to any plant-based material including but not limited to grains, grasses, tubers and roots and more specifically wheat, barley, corn, rye, rice, sorghum, legumes, cassava, millet, potato, sweet potato, and tapioca.
  • the starch substrate may also comprise granular starch.
  • granular starch refers to uncooked (raw) starch, which has not been subjected to
  • various hydrolyzing enzymes may be active, including granular starch hydrolyzing enzyme and enzymes having granular starch hydrolyzing activity.
  • granular starch hydrolyzing enzyme GSHE
  • enzymes having granular starch hydrolyzing activity refer to enzymes, which have the ability to hydrolyze starch in granular form.
  • starch gelatinization means solubilization of a starch molecule to form a viscous
  • Gelatinization may occur to various degrees at various temperatures.
  • gelatinization temperature refers to the lowest temperature at which
  • a starch substrate or starch begins.
  • the exact temperature depends upon the specific starch substrate and further may depend on the particular variety of plant species from which the starch is obtained and the growth conditions.
  • the term "DE” or “dextrose equivalent” is an industry standard for measuring the concentration of total reducing sugars, calculated as D-glucose on a dry weight basis. Unhydrolyzed granular starch has a DE that is about 0 and D-glucose has a DE of 100.
  • total sugar content refers to the total sugar content present in a starch composition.
  • end product refers to any carbon-source derived product which is enzymatically converted from a fermentable substrate.
  • the end product may be an alcohol.
  • the alcohol may be ethanol.
  • fermentation refers to the enzymatic and anaerobic breakdown of organic substances by microorganisms to produce simpler organic compounds. While fermentation occurs under anaerobic conditions it is not intended that the term be solely limited to strict anaerobic conditions, as fermentation also occurs in the presence of oxygen.
  • fermentation broth refers to fermentation medium containing the end products after fermentation.
  • transferable sugar refers to the sugar composition consisting of DP1 and DP2.
  • DP refers to degree of polymerization to the number (n) of anhydroglucopyranose units in a given saccharide.
  • DP1 can include glucose, fructose, and other monosaccharides.
  • DP2 can include maltose, isomaltose, sucrose, and other disaccharides.
  • DP3 can include maltotriose and other trisaccharides.
  • DP4 + (>DP3) denotes polymers with a degree of polymerization of greater than 3, or oligosaccharides having a degree of polymerization DP4 or greater.
  • ds or "DS" refers to dissolved solids and dry substance in a solution.
  • Brix refers to a well-known hydrometer scale for measuring the sugar content of a solution at a given temperature.
  • the Brix scale measures the number of grams of sucrose present per 100 grams of aqueous sugar solution (the total solubilized solid content).
  • Brix measurements may be made by use of a hydrometer or refractometer.
  • Refractive index is a physical measurement and detection of the speed of light through air as compared to the speed through the measurement medium. The comparison of the two is the index of refraction (bending of light).
  • Refractive indices of aqueous solutions of sugars of various DE's are known and published by the Corn Refiners association (Method E-54 of
  • DDGS distalmost fine grain with soluble
  • the "distillers dry grains (DDG)” refers to the dried residual by-product of a grain fermentation process.
  • starch refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and amylopectin with the formula ( ⁇ 6 ⁇ 0 5 ) ⁇ , wherein x can be any number.
  • starch liquefaction refers to a process by which starch is converted to shorter chain and less viscous dextrins.
  • starch-liquefying enzyme refers to an enzyme that affects the hydrolysis or breakdown of granular starch.
  • exemplary starch liquefying enzymes include alpha amylases (E.C. 3.2.1 .1 ).
  • hydrolysis of starch refers to the cleavage of glucosidic bonds with the addition of water molecules.
  • contacting refers to the placing of the respective enzymes in sufficiently close proximity to the respective substrate to enable the enzymes to convert the substrate to the end product. Those skilled in the art will recognize that mixing solutions of the enzyme with the respective substrates can effect contacting.
  • liquefact refers to starch hydrolysate from a conventional high temperature liquefaction process using thermostable alpha amylase.
  • ash refers to a mixture of a fermentable substrate in liquid used in the production of a fermented product and is used to refer to any stage of the fermentation from the initial mixing of the fermentable substrate with one or more starch hydrolyzing enzymes and fermenting organisms through the completion of the fermentation run.
  • saccharification and fermentation refers to a process in which saccharification of a hydrolysed starch (gelatinized and liquefied) mash occurs in the fermentor using glucoamylase with the commencement of fermentation by yeast to alcohol. It can occur simultaneously (i.e., simultaneously saccharification and fermentation or "SSF"), or it can occur in series. If conducted in series, it can be conducted with one or more hours separating the initiation of saccharification from the initiation of fermentation.
  • SSF simultaneous saccharification and fermentation
  • the term "fermenting organism” refers to any organism, including bacterial, fungal and yeast, suitable for producing a desired fermentation end products like alcohol (e.g., ethanol, methanol and butanol), amino acids, organic acids (e.g., lactic acid, citric acid, succinic acid), monosodium glutamate, 1 ,3-propane diol, etc.
  • the fermenting organism can be any fungal organism.
  • the fungal organism may be yeast.
  • the yeast can be of any strain.
  • the yeast strain can be of the genera Saccharomyces, Pichia, Candida, etc.
  • the fermenting organism can be bacterial.
  • the bacteria can be of any strain.
  • the bacterial fermenting organisms may be strains of Escherichia, strains of Zymomonas, and strains of Klebsiella. Any strain may be used that is capable of producing alcohol, e.g., ethanol, methanol, butanol, etc.
  • the fermentation products include alcohol (e.g., ethanol, methanol, and butanol), and are also contemplated to include organic acids (e.g., citric acid, lactic acid, succinic acid, and gluconic acid) and amino acids (e.g., glutamic acid, tryptophan, threonine, and methionine).
  • alcohol e.g., ethanol, methanol, and butanol
  • organic acids e.g., citric acid, lactic acid, succinic acid, and gluconic acid
  • amino acids e.g., glutamic acid, tryptophan, threonine, and methionine
  • starch-containing materials can include any starch-containing material.
  • the starch-containing material may be obtained from wheat, corn, rye, sorghum (milo), rice, millet, barley, triticale, cassava (tapioca), potato, sweet potato, sugar beets, sugarcane, and legumes.
  • the legumes can be soybeans or peas.
  • the material may be corn, barley, wheat, rice, milo, and combinations thereof.
  • Plant material may include hybrid varieties and genetically modified varieties ⁇ e.g., transgenic corn, barley, or soybeans comprising heterogeneous genes).
  • any part of the plant may be used as a starch-containing material, including but not limited to, leaves, stems, hulls, husks, tubers, cobs, grains, roots, and the like.
  • the whole plant or nearly the whole plant may be used.
  • the whole ground grain or fractionated grain may be used.
  • the whole grain may be used as a starch-containing material.
  • Whole grains may be corn, wheat, rye, barley, sorghum, and combinations thereof.
  • the starch- containing material may be obtained from fractionated cereal grains including fiber, endosperm, and/or germ components.
  • sugar and "molasses” refer to sugar extracted from sugar cane or beet and molasses as a concentrate from mother liquor from sugar crystallization process.
  • milling starch-containing material refers to some embodiments where the starch-containing material may be prepared by milling.
  • Two general milling processes include wet milling or dry milling (grinding).
  • dry milling the whole grain can be milled and used in the process.
  • wet milling the grain is separated ⁇ e.g. the germ, protein, and fiber from the starch).
  • means of milling whole cereal grains are well known and include the use of hammer mills and roller mills.
  • Methods of milling are well known in the art and reference is made to THE ALCOHOL TEXTBOOK: A REFERENCE FOR THE BEVERAGE, FUEL AND INDUSTRIAL ALCOHOL INDUSTRIES 3 rd ED. K.A. Jacques et al., Eds., and (1999) Nottingham
  • the milled grain which is used in the process may have a particle size such that more than 50% of the material will pass through a sieve with a 500 micron opening and in some embodiments more than 70% of the material will pass through a sieve with a 500 micron opening (see, WO2004/081 193).
  • a "slurry of starch-containing material” refers to milled starch-containing materials ground to a specified sieve size and combined with water resulting in aqueous slurry.
  • the slurry may comprise between 1 5 to 55% ds w/w (e.g., 20 to 50%, 25 to 50%, 25 to 45%, 25 to 40%, and 20 to 35% ds).
  • the recycled thin- stillage (backset) may be used as a portion of the water for slurry make-up of 1 0 to 70% v/v ⁇ e.g., 1 0 to 60%, 1 0 to 50%, 1 0 to 40%, 1 0 to 30%, 1 0 to 20%, 20 to 60%, 20 to 50%, 20 to 40%, and also 20 to 30%).
  • no cook process is also used alternatively as GSHE process (granular starch hydrolyzing enzyme process) and refers to a process of using granular starch (un-gelatinized starch) containing feed stock in yeast fermentation at the pH and temperature conditions of yeast fermentation without subjecting the starch to gelatinization process.
  • Yeast are modified by contacting the yeast with organic acids and salts of such organic acids.
  • the organic acid may be benzoic acid and the salts may be sodium benzoate and potassium benzoate.
  • the modification can persist even when the organic acids and organic acid salts are removed after the contacting but before the yeast are used in fermentation.
  • the yeast can even be dried and stored after the modification and later used in fermentation.
  • a cream of modified yeast can be formed for inoculation, with the cream transferred to a fermentation medium containing a starch substrate.
  • the starch substrate may be a starch-containing material or a purified starch.
  • Saccharification and fermentation can be conducted for converting the starch in the starch substrate to an end product.
  • the modification to the yeast can result in increased fermentation yield during the conversion of starch and or sugar containing feed stocks and reduced glycerol. There can be an increase in the fermentation yield of ethanol.
  • the increase in the fermentation yield may be accomplished by first adding to yeast an organic compound containing carboxylic acid with a non-polar aliphatic or aromatic group. Because the benefits of the modification persist after the removal of carboxylic acid from the yeast culture, there is no need to include carboxylic acid in the fermentation media, resulting in substantial cost savings.
  • a modified yeast cell is prepared.
  • An aqueous suspension containing a yeast cell is prepared.
  • An organic acid or an organic acid salt is added to the aqueous suspension. The organic acid or the organic acid salt is allowed to enter the yeast cell to form the modified yeast cell.
  • centrifuging and decanting are performed to remove a supernatant comprising the organic acid or the organic acid salt from the modified yeast cell.
  • a further resuspension of the modified yeast cell in deionized water followed by centrifugation and decanting can be performed.
  • the modified yeast cell may be dried and stored.
  • the modified yeast cell may be suitable for performing fermentation with an increased amount of one or more fermentation products produced from fermentation with the modified yeast cell as compared to fermentation with a yeast cell that is not modified with an organic acid or an organic acid salt.
  • the modified yeast cell can be resuspended to form a cream for inoculation.
  • the cream can then be transferred to a fermentation medium containing a starch substrate.
  • the starch substrate may be a starch-containing material.
  • the organic acid or the organic acid of the organic acid salt can be of the formula:
  • R can be methyl, ethyl, n-or isopropyl, n-or iso-butyl, phenyl or substituted phenyl.
  • R can also be aromatic ring with carboxylic group.
  • the carboxylic group can be benzoic, phenyl acetic, or phenyl propionic acids.
  • the carboxylate ion containing organic acid can be introduced to the aqueous medium of yeast in the form of carboxylic acid or its' salts, e.g. a sodium, potassium or calcium salt of the corresponding carboxylic acid.
  • the concentration of the organic acid or the organic acid salt can be from 100 ppm to 5,000 ppm, from 100 ppm to 500 ppm, from 500 ppm to 1000 ppm, from 1000 ppm to 5000 ppm, from 100 ppm to 1000 ppm, or from 500 ppm to 5000 ppm.
  • the organic acid or the organic acid salt may be added to the aqueous suspension containing the yeast cell at a temperature from 20 to 35 °C, 21 to 34 °C, 22 to 33 °C, 23 to 32°C, 24 to 31 °C, 25 to 30 0, 26 to 31 °C, 27 to 32°C, 28 to 33 °C, 29 to 34 °C, 30 to 35 °C, at 32 °C or at room temperature.
  • the pH can be adjusted to 5.0, about 5, from 4.5 to 5.5, from 4.6 to 5.4, from 4.7 to 5.3, from 4.8 to 5.2, from 4.9 to 5.3, or from 5.0 to 5.5.
  • the suspension may be shaken for a time.
  • the time can be 1 hour, about 1 hour, from 30 minutes to 2 hours, or from 1 hour to 3 hours.
  • the shaking can be conducted at a temperature from 20 to 35 °C, 21 to 34 °C, 22 to 33 °C, 23 to 32°C, 24 to 31 °C, 25 to 30 0, 26 to 31 °C, 27 to 32°C, 28 to 33 °C, 29 to 34 °C, 30 to 35 °C, at 32 °C or at room temperature.
  • the organic acid can be benzoic acid.
  • the organic acid salt can be sodium benzoate or potassium benzoate.
  • the yeast cell can be a S. cerivisiae cell.
  • the S. cerivisiae cell can be FALI, SUPERSTARTTM, FERMIOL®, RED STAR®, or Angel® alcohol yeast.
  • Yeast can express recombinant enzymes. For example SYNERTIA® ADY (DuPont Industrial Bioscience) and TRANSFERMTM from Lallimond, Inc. are genetically modified (GMO) yeast that produces glucoamylase.
  • the centrifuging can be performed at from 4500 to 6000 rcf (relative centrifugal force), 4600 to 5900 rcf, 4700 to 5800 rcf, 4800 to 5700 rcf, 4900 to 5600 rcf, 5000 to 5500 rcf, 5100 to 5400 rcf, 5200 to 5500 rcf, or at about 5300 rcf.
  • the centrifuging can be performed at 10°C, about 10 °C, from 7 °C to 15*0, from 8°C to 13°C, or from 9°C to 12°C.
  • the modified yeast cell can be dried for storage after the centrifuging and decanting step and prior to the resuspending step.
  • the modified yeast cell can be pelleted and then resuspended one or more times in de-ionized water so as to wash the cream yeast.
  • the amount of deionized water can be less than, equal to, or greater than the amount of the supernatant decantated after the centrifugation step.
  • the starch substrate may be dry grind corn.
  • the starch substrate may in the form of a liquefact.
  • the liquefact may be from corn and may be collected from dry grind corn.
  • the pH of the liquefact can be adjusted to 4.8, about 4.8, from 4.5 to 5.0, from 4.6 to 4.9, or from 4.7 to 5.0. Any suitable acid may be used to adjust the pH.
  • the acid used to adjust the pH may be sulfuric acid.
  • a nitrogen supplement may be added to the liquefact.
  • the amount of nitrogen supplement may be urea.
  • the amount of nitrogen supplement can be from 0.04 to 0.07% w/w, from 0.05 to 0.07% w/w, from 0.06 to 0.08% w/w, about 0.06% w/w, or 0.06% w/w.
  • the amount of nitrogen supplement can be added to have a final concentration of nitrogen supplement of from 400 to 700 ppm, from 500 to 700 ppm, from 600 to 800 ppm, about 600 ppm, or 600 ppm.
  • the enzyme may be any enzyme used in saccharification.
  • the enzyme may be any enzyme used in saccharification.
  • the enzyme may be any enzyme used in saccharification.
  • the enzyme may be any enzyme used in saccharification.
  • the enzyme may be any enzyme used in saccharification.
  • the enzyme may be any enzyme used in saccharification.
  • the enzyme may be any enzyme used in saccharification.
  • the enzyme may be any enzyme used in saccharification.
  • the enzyme may be any enzyme used in saccharification.
  • the enzyme may be any enzyme used in saccharification.
  • the glucoamylase may be DISTILLASE® SSF (DuPont Industrial Bioscience).
  • the enzyme may be amyloglucosidase.
  • the enzyme may be exo-1 ,4- alpha-D-glucosidase.
  • yeast suspension cream may be transferred to a fermentation medium containing a starch substrate, including 0.5% v/w of the cream, about 0.5% v/w of the cream, from 0.4% to 0.6% v/w of the cream, from 0.45% to 0.55% v/w of the cream and from 0.5% to 0.6% v/w of the cream. Saccharification and fermentation can be conducted at various temperatures, from 30 to 34 °C, from 30 to 32 °C, from 31 to 33 °C, from 32 to 34 °C, at about 32 °C or at 32 °C.
  • the saccharification and fermentation can be conducted under agitation at various rpm, from 100 to 200 rpm, from 120 to 180 rpm, at about 150 rpm, or at 150 rpm.
  • the end product can be ethanol.
  • the end products can also be ethanol and glycerol.
  • the ratio of ethanol %v/v to glycerol %w/v can range from 8.75 to 12.79.
  • S The amount of ethanol in %v/v can be increased by at least 1 %, 1 .5%, 2.0%, or 2.5%, as compared to the amount of ethanol in %v/v obtained from a process conducted in a similar manner in which the yeast are not modified with an organic acid or an organic acid salt.
  • the saccharification and fermentation can be conducted in a cold cook process.
  • a cold cook saccharification process the temperature is kept below the temperature of starch gelatinization so that saccharification may occur directly from raw native insoluble starch to soluble glucose.
  • the end products can be ethanol and glycerol.
  • the ratio of ethanol %v/v to glycerol %w/v can be from 38-40 in a cold cook process.
  • the starting organism can be a fermenting yeast.
  • the yeast can be any strain of Saccharomyces cerevisiae, and can be genetically modified.
  • the yeast can be treated prior to or before fermentation with organic acids or salts of organic acids.
  • the organic acid can be one with aromatic rings.
  • the organic acid can be benzoic acid.
  • the organic acid salts can be salts of organic acids with aromatic rings.
  • the organic acids salts can be salts of benzoic acid.
  • the salts of benzoic acid can be sodium benzoate or potassium benzoate.
  • the starch substrate can be plant material.
  • the starch substrate can be obtained from one or more of wheat, corn, rye, sorghum (milo), rice, millet, barley, triticale, cassava (tapioca), potato, sweet potato, sugar beets, sugarcane, and legumes.
  • the legumes can be soybeans or peas.
  • the plant material may include hybrid varieties and genetically modified varieties. Any part of the plant may be used as a starch-containing material.
  • the parts of the plant used can be one or more of leaves, stems, hulls, husks, tubers, cobs, grains and the like. The whole plant or nearly the whole plant may be used.
  • the whole ground grain or fractionated grain may be part of the starch substrate.
  • the whole grain may be may be part of the starch substrate.
  • the starch substrate may be obtained from fractionated cereal grains including fiber, endosperm, and/or germ components.
  • the starch substrate can be any plant material containing starch.
  • the plant material may be from corn, rice, Milo, barley or wheat.
  • the plant material can be from a portion of the whole plant.
  • the plant material may be a mixture from two or more of corn, Milo, barley, rice, or wheat.
  • methods for fractionating plant material that are known in the art (Alexander, A.J. 1987, “Corn Dry Milling: Process, Products, And Applications", pp. 351 -376, Chapter 1 1 , in CORN CHEMISTRY AND TECHNOLOGY, Watson, S.A. and Ramstead, P.E; editors; American Association of Cereal Chemists, Inc., 3340 Pilot Knob Road, St. Paul, Minnesota, USA; Johnston et ai, 2005 US Patent # 6,899,910) can be used.
  • the plant material may be corn or wheat.
  • the starch-containing material obtained from different sources may be mixed together to obtain material used in the processes described herein (e.g., (a) corn and Milo or (b) corn and barley).
  • the fermentation producing alcohol may be produced by cultivating suitable ethanologenic yeast in a suitable fermentation medium containing starch and or hydrolyzed starch substrate with yeast nutrients; the fermentation converts said starch substrate into alcohol.
  • the yeast may be Red Star® S. cerevisiae.
  • the fermentation process may be carried out at 32 °C, at pH 3.5 to 6.0, for a period of 24 to 96 hours.
  • the yeast cell may be genetically-modified.
  • the modified yeast cell is made by preparing an aqueous suspension containing a yeast cell, adding an organic acid or an organic acid salt to the aqueous suspension and allowing the organic acid or the organic acid salt to enter the yeast cell to form a modified yeast cell. Centrifuging and decanting are performed to remove a supernatant comprising the organic acid or the organic acid salt from the modified yeast cell.
  • the modified yeast cell can be resuspended in deionized water followed by centrifugation and decanting.
  • the modified yeast cell is dried.
  • the organic acid or the organic acid of the organic acid salt can be of the formula:
  • R can be methyl, ethyl, n-or isopropyl, n-or iso-butyl, phenyl or substituted phenyl.
  • R can also be aromatic ring with carboxylic group.
  • the carboxylic group can be benzoic, phenyl acetic, or phenyl propionic acids.
  • the carboxylate ion containing organic acid can be introduced to the aqueous medium of yeast in the form of carboxylic acid or its' salts, e.g. a sodium, potassium or calcium salt of the corresponding carboxylic acid.
  • the concentration of the organic acid or the organic acid salt can be from 100 ppm to 5,000 ppm, from 100 ppm to 500 ppm, from 500 ppm to 1000 ppm, from 1000 ppm to 5000 ppm, from 100 ppm to 1000 ppm, or from 500 ppm to 5000 ppm.
  • the organic acid or the organic acid salt may be added to the aqueous suspension containing the yeast cell at a temperature from 20 to 35 °C, 21 to 34 °C, 22 to 33 °C, 23 to 32°C, 24 to 31 °C, 25 to 30 0, 26 to 31 °C, 27 to 32°C, 28 to 33 °C, 29 to 34 °C, 30 to 35 °C, at 32 °C or at room temperature.
  • the pH can be adjusted to 5.0, about 5, from 4.5 to 5.5, from 4.6 to 5.4, from 4.7 to 5.3, from 4.8 to 5.2, from 4.9 to 5.3, or from 5.0 to 5.5.
  • the suspension may be shaken for a time.
  • the time can be 1 hour, about 1 hour, from 30 minutes to 2 hours, or from 1 hour to 3 hours.
  • the shaking can be conducted at a temperature from 20 to 35 °C, 21 to 34 °C, 22 to 33 °C, 23 to 32°C, 24 to 31 °C, 25 to 30*0, 26 to 31 °C, 27 to 32°C, 28 to 33 °C, 29 to 34 °C, 30 to 35 °C, at 32 °C or at room temperature.
  • the organic acid salt can be sodium benzoate.
  • the organic acid salt can be potassium benzoate.
  • the organic acid salt can be potassium sorbate.
  • the yeast cell can be selected from the group consisting of FALI,
  • SUPERSTARTTM, FERMIOL®, RED STAR®, and Angel® alcohol yeast can express recombinant enzymes.
  • SYNERTIA® ADY DuPont Industrial Bioscience
  • TRANSFERMTM from Lallimond, Inc. are genetically modified (GMO) yeast that produces glucoamylase.
  • the modified yeast cell can be pelleted and then resuspended one or more times in de-ionized water so as to wash the cream yeast.
  • the amount of deionized water can be less than, equal to, or greater than the amount of the supernatant decanted after the centrifugation step.
  • Another aspect relates to a method of conducting fermentation.
  • a dried modified yeast cell is resuspended to form a cream for inoculation.
  • the cream is transferred to a fermentation medium containing a starch substrate. Saccharification and fermentation are conducted for converting the starch in the starch substrate to an end product.
  • the saccharification and fermentation may or may not be
  • the starch substrate can be plant material.
  • the starch substrate can be obtained from one or more of wheat, corn, rye, sorghum (milo), rice, millet, barley, triticale, cassava (tapioca), potato, sweet potato, sugar beets, sugarcane, and legumes.
  • the legumes can be soybeans or peas.
  • the plant material may include hybrid varieties and genetically modified varieties. Any part of the plant may be used as a starch-containing material.
  • the parts of the plant used can be one or more of leaves, stems, hulls, husks, tubers, cobs, grains and the like.
  • the whole plant or nearly the whole plant may be used.
  • the whole ground grain or fractionated grain may be part of the starch substrate.
  • the whole grain may be may be part of the starch substrate.
  • the starch substrate may be obtained from fractionated cereal grains including fiber, endosperm, and/or germ components.
  • the plant material may be from corn, rice, Milo, barley or wheat.
  • the plant material can be from a portion of the whole plant.
  • the plant material may be a mixture from two or more of corn, Milo, barley, rice, or wheat.
  • the starch substrate can be dry grind corn.
  • the end product can be ethanol.
  • the end product can be ethanol.
  • the end products can also be ethanol and glycerol.
  • the ratio of ethanol (%v/v) to glycerol (%w/v) can range from 8.75 to 12.79.
  • the process can be a cold cook process.
  • the end products may be ethanol and glycerol.
  • the ratio of ethanol (%v/v) to glycerol (%w/v) may be from 38-40 in the cold cook process.
  • the yeast can be modified and used for fermentation as follows. An aqueous suspension containing yeast cells is made. Benzoic acid, sodium benzoate, or potassium benzoate can be added and allowed to enter the cells. Centrifugation and decanting can be performed to remove the benzoate solution. Yeast cells are resuspended as a cream for inoculation. The cream yeast is transferred to the fermentation medium containing dry grind corn. Saccharification and fermentation are conducted to produce ethanol from dry grind corn. The saccharification and fermentation may or may not be conducted simultaneously. The fermentation may or may not be conducted as a cold cook process.
  • the yeast can be modified and used for fermentation as follows. Benzoic acid, sodium benzoate, or potassium benzoate can be added and allowed to enter the cells. Centrifugation and decanting are performed to remove the benzoate solution. The modified yeast cells are dried for storage. The modified yeast cells are then resuspended as a cream for inoculation. The cream yeast is transferred to the fermentation medium containing dry grind corn. Saccharification and fermentation are conducted to produce ethanol from dry grind corn. The saccharification and fermentation may or may not be conducted simultaneously. The fermentation may or may not be conducted as a cold cook process.
  • the yeast can also be modified and used for fermentation as follows.
  • An aqueous suspension containing yeast cells is made. Potassium benzoate is added and allowed to enter the cells. Centrifugation and decanting are performed to remove the benzoate solution.
  • Yeast cells are resuspended as a cream for inoculation. The cream yeast is transferred to the fermentation medium containing dry grind corn. Saccharification and fermentation are conducted to produce ethanol from dry grind corn. The saccharification and fermentation may or may not be conducted simultaneously. The fermentation may or may not be conducted as a cold cook process.
  • the yeast can also be modified and used for fermentation as follows. An aqueous suspension containing yeast cells is made. Sodium benzoate is added and allowed to enter the cells.
  • Centrifugation and decanting are performed to remove the benzoate solution.
  • Yeast cells are resuspended as a cream for inoculation.
  • the cream yeast is transferred to the fermentation medium containing dry grind corn. Saccharification and fermentation are conducted to produce ethanol from dry grind corn.
  • the saccharification and fermentation may or may not be conducted simultaneously.
  • the fermentation may or may not be conducted as a cold cook process.
  • a method of modifying a yeast cell comprising preparing an aqueous suspension containing a yeast cell;
  • modified yeast cell is suitable for performing fermentation and wherein an increased amount of one or more fermentation products is produced from fermentation with the modified yeast cell as compared to fermentation with a yeast cell that is not modified with an organic acid or an organic acid salt.
  • R is selected from the group consisting of methyl, ethyl, n-or isopropyl, n-or iso-butyl, phenyl and substituted phenyl.
  • a method of modifying a genetically-modified yeast cell comprising preparing an aqueous suspension containing a genetically-modified yeast cell;
  • centrifuging and decanting to remove a supernatant comprising the organic acid or the organic acid salt from the improved genetically-modified yeast cell
  • modified yeast cell is suitable for performing fermentation and wherein an increased amount of one or more fermentation products is produced from fermentation with the modified yeast cell as compared to fermentation with a yeast cell that is not modified with an organic acid or an organic acid salt.
  • organic acid or the organic acid of the organic acid salt is of the formula:
  • the method of any of variations 13-21 further comprising after the
  • a dried modified yeast cell prepared by a method comprising
  • a method of conducting fermentation comprising
  • Example 1 Determination of Sodium Benzoate in Dried Spent Yeast by Direct Trans-esterification and Gas Chromatography
  • Yeast fermentation to produce ethanol biofuel is a well-known process that is routinely done on large commercial scales. Any yield improvements, even less than 1 %, can impart a large cost savings.
  • the concern of using sodium benzoate to improve ethanol yield was that sodium benzoate cannot be present at levels greater than 0.1 % in the spent, dried yeast that is sold as animal feed.
  • Analytical methods are helpful to determine sodium benzoate in yeast samples from fermentation processes. Determination of intracellular components of yeast from fermentation experiments is very challenging, due to the challenge in quantitatively extracting the analytes.
  • the yeast cell wall is very resistant to shearing, which often makes conventional extraction methods, which include bead milling and homogenization, ineffective.
  • lipids in yeast determination of lipids in yeast is performed where the esterification reagent (5% acetyl chloride in methanol) is directly added to the dry, intact yeast, and incubated at 80 °C for one hour.
  • the action of the HCI and methanol not only trans-esterifies glycerides and free fatty acids to their methyl ester forms, but they also appear to dissolve the cell wall and release the contents into solution. This method was tested for benzoate determination in the distillers dry solids yeast, from yeast fermentation solids derived, as described below.
  • Example 2 Determination of Carbohydrate Composition by High Pressure Liquid Chromatography (HPLC)
  • composition of the reaction products of oligosaccharides was measured by high pressure liquid chromatographic method (Beckman System Gold 32 Karat Fullerton, California, USA) equipped with a HPLC column (Rezex 8 u8% H,
  • Example 3 Preparation of modified yeast cells and determination of benzoic acid content in the yeast cells.
  • Suspensions of 20% w/v ADY, at pH 6.0 were prepared with different concentrations of sodium benzoate (100 ppm, 500 ppm, 1000 ppm, and 5,000 ppm) in 0.1 M 2-(N-morpholino)ethanesulfonic (MES) acid buffer.
  • the yeast suspensions were incubated at room temperature for 10 minutes. The suspensions were centrifuged to separate the cells (cell paste) and supernatant. Supernatant was filtered through 0.45 ⁇ nylon filter. The paste was washed three times to remove any free sodium benzoate from yeast cake. The cake was then dried at 60 °C, for 24 hours.
  • the sodium benzoate content in the dry yeast and cell free supernatant from incubated slurry was determined (Table 1 ).
  • Table 1 Sodium benzoate content in the liquid and yeast after incubation at different concentration of sodium benzoate.
  • Yeast was treated with sodium benzoate. Mixtures of 20% w/w dry yeast, 20,000 ppm sodium benzoate, and de-ionized water were prepared. Mixtures were shaken to suspend the yeast Ethanol Red® Fermentis and dissolve the sodium benzoate. After the salts were dissolved, the pH was adjusted to pH 5.0 and the suspension was placed in a shaker incubator, at 32°C with 150 rpm shaking, for 1 h. Following incubation the suspension was centrifuged at 531 1 rcf (relative centrifugal force) for 5 minutes at 10°C. The supernatant was discarded, and the yeast pellet was used in three different ways.
  • Cream yeast the pellet was re-suspended with water equal to the discarded supernatant.
  • Washed cream yeast the pellet was re-suspended with de-ionized water equal to the discarded supernatant. The suspension was re-centrifuged and the supernatant discarded. The pellet was re-suspended as before.
  • the dried yeast was broken into pellets.
  • the dried yeast was suspended in water at 20% w/w in de-ionized water.
  • Fermentations used corn liquefact collected from a commercial dry grind corn to ethanol plant (29% dry solids). Liquefact was pH adjusted to 4.8 with 10 N sulfuric acid. Urea was added as a nitrogen supplement at 0.06% w/w. The gluco-amylase DISTILLASE® SSF (DuPont Industrial Bioscience) was used for saccharification at 0.325 GAU/g ds. Fermentations were inoculated with 0.5% v/w of yeast suspension, and placed in an incubator at 32 °C with 150 rpm agitation. Samples were taken at intervals and analyzed for soluble sugar, glycerol, and alcohol. End of fermentation results are shown in Table 2. Table 2: Three forms of modified yeast fermentations. End of fermentation soluble sugars, glycerol and ethanol from ethanol fermentations using modified yeast for inoculation.
  • Un-treated yeast 0.57 0.000 1 .38 1 1 .1
  • Table 2 show a decrease in glycerol production and an increase in ethanol for modified yeast without a change in soluble sugars.
  • the increase in ethanol and decrease in glycerol was seen after a wash step removing any residual benzoate in the liquid phase.
  • the modification of the yeast is maintained after drying the yeast.
  • Example 5 Sodium benzoate was replaced with potassium benzoate for yeast modification to show an independence from the cation.
  • Yeast was treated with potassium benzoate.
  • Mixtures of 20% w/w dry yeast Bio-Ferm®XP Lallemand Biofuels & Distilled Spirits, 40,000 ppm potassium benzoate, and de-ionized water were prepared. Mixtures were shaken to suspend the yeast and dissolve the sodium benzoate. After the salts were dissolved the suspension was placed in a shaker incubator, at 32°C with 150 rpm shaking, for 1 h. Following incubation the suspension was centrifuged at 531 1 rcf for 5 minutes at 10°C. The supernatant was discarded, and the yeast re-suspended.
  • Fermentations used corn liquefact collected from a commercial dry grind ethanol plant (32% solids). Liquefact was pH adjusted to 4.8 with 10 N sulfuric acid. Urea was added as a nitrogen supplement at 0.06% w/w. The gluco-amylase
  • DISTILLASE® SSF was used for saccharification at 0.325 GAU/g ds. Fermentations were inoculated with 0.5% v/w of yeast suspension, and placed in an incubator at 32 °C with 150 rpm agitation. Samples were taken at intervals and analyzed for soluble sugar, glycerol, and alcohol. End of fermentation results are shown in Table 3.
  • Table 3 Potassium benzoate modified yeast end of fermentation soluble sugars, glycerol and ethanol, compared with untreated yeast and used for inoculation.
  • Example 6 Modification of a genetically modified yeast used to inoculate simultaneous saccharification and fermentation.
  • Fermentations used corn liquefact collected from a commercial dry grind ethanol plant (32% solids). Liquefact was pH adjusted to 4.8 with 10 N sulfuric acid. Urea was added as a nitrogen supplement at 0.06% w/w. The gluco-amylase
  • SYNERTIA® LC was used for saccharification at 0.16 GAU/g ds. Fermentations were inoculated with 0.5% v/w of yeast suspension, and placed in an incubator at 32 °C with 150 rpm agitation. Samples were taken at intervals and analyzed for soluble sugar, glycerol, and alcohol. End of fermentation results are shown in Table 4.
  • Table 4 GMO yeast, benzoate modified, end of fermentation soluble sugars, glycerol, and ethanol, compared with untreated yeast and used for inoculation.
  • Table 4 show a decrease in glycerol production and an increase in ethanol for modified yeast with a small reduction in soluble sugars.
  • the GMO yeast functioned well with fermentations completing with less soluble sugars and increased ethanol.
  • Example 7 Modification of yeast for inoculation of fermentations using the no cook process which converts whole ground corn to ethanol.
  • Yeast was treated with sodium benzoate as follows. Mixtures of 20% w/w dry yeast Ethanol Red® Fermentis, 40,000 ppm sodium benzoate, and de-ionized water were prepared. Mixtures were shaken to suspend the yeast and dissolve the sodium benzoate. After the salts were dissolved, the suspension was placed in a shaker incubator, at 32°C with 150 rpm shaking, for 1 hour. Following incubation, the suspension was centrifuged at 531 1 rcf for 5 minutes at 10°C. The supernatant was discarded and the yeast re-suspended. Whole corn was ground with the AIC M-101 on grind setting 10. An aqueous slurry of ground corn was prepared of 30% ds.
  • Table 5 Cold Cook end of fermentation soluble sugars, glycerol and ethanol from fermentations with untreated yeast and benzoate treated yeast.

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Abstract

Cette invention concerne un procédé permettant de modifier une levure pour améliorer le rendement d'un produit de fermentation primaire, et réduire le glycérol dans les co-produits de fermentation. Le procédé comprend la mise en contact de la levure avec un sel organique benzoïque suivie par la fermentation de la levure dans un milieu contenant un sucre fermentable. Pendant la fermentation, des niveau réduits de glycérol sont en outre observés ayant pour résultat des co-produits contenant une quantité réduite de glycérol.
PCT/US2015/055721 2014-10-15 2015-10-15 Procédé de modification d'une levure pour une fermentation améliorée WO2016061339A1 (fr)

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* Cited by examiner, † Cited by third party
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WO2016140966A1 (fr) * 2015-03-04 2016-09-09 Danisco Us Inc Procédé pour la production de bio-alcool
CN114231570A (zh) * 2022-01-08 2022-03-25 张超龙 一种以白酒酒糟为原料的生物活性发酵滤液制备工艺

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WO2004081193A2 (fr) 2003-03-10 2004-09-23 Broin And Associates, Inc. Procede de production d'ethanol a partir d'amidon brut
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016140966A1 (fr) * 2015-03-04 2016-09-09 Danisco Us Inc Procédé pour la production de bio-alcool
CN114231570A (zh) * 2022-01-08 2022-03-25 张超龙 一种以白酒酒糟为原料的生物活性发酵滤液制备工艺
CN114231570B (zh) * 2022-01-08 2024-03-26 张超龙 一种以白酒酒糟为原料的生物活性发酵滤液制备工艺

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