WO2016061339A1

WO2016061339A1 - A method for yeast modification for improved fermentation

Info

Publication number: WO2016061339A1
Application number: PCT/US2015/055721
Authority: WO
Inventors: Jayarama K. Shetty; Mark GOUTHRO
Original assignee: Danisco US Inc
Current assignee: Danisco US Inc
Priority date: 2014-10-15
Filing date: 2015-10-15
Publication date: 2016-04-21
Anticipated expiration: 2017-04-15

Abstract

Disclosed is a method for modifying yeast to enhance yield of the fermentation primary product, and decrease glycerol in fermentation co-products. The method comprises contacting yeast with a benzoic organic salt followed by yeast fermentation with a medium containing a fermentable sugar. Also, during fermentation reduced levels of glycerol are observed resulting in co-products containing reduced glycerol.

Description

A METHOD FOR YEAST MODIFICATION FOR IMPROVED

FERMENTATION

CROSS-REFERENCE TO THE PRIORITY APPLICATIONS

This application claims the benefit of U.S. Provisional Application

No. 62/064,204, filed October 15, 2014 and is incorporated herein by reference.

Described herein are methods for preparing a modified yeast cell and conducting fermentation with the modified yeast cell so as to enhance yield of the fermentation primary product, and decrease glycerol in fermentation co-products. 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.

BACKGROUND

Processes for producing yeast fermentation products, such as ethanol, butanol, etc. from starch, hydrolysed starch (liquefact or high glucose syrup), and sucrose/molasses, are well known in the art. There are several methods currently used to convert starch substrates into alcohol, which are described as follows:

1 ) 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. 2) 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. 3) 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.

4) 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.

5) 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. There is an unmet need in the marketplace to develop novel methods to reduce unproductive yeast without affecting the alcohol yield. Often during a traditional ethanol fermentation process, lactic acids and acetic acids are produced. Increased levels of lactic acids and acetic acids can result in decreased ethanol yield. Also, the presence of bacteria can alter the profile of organic acids present. 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. et al., Influence of medium buffering capacity on inhibition of Saccharomyces cerevisiae growth by acetic and lactic acids. Appl. Environ. Microbiol. 2002, 68:1616-23; Abbott, D.A. and Ingledew, W.M, Buffering capacity of whole corn mash alters concentrations of organic acids required to inhibit growth of Saccharomyces cerevisiae and ethanol production.

Biotechnol Lett. 2004, 26:1313-6; Zhao, R., Bean S.R. et al., Application of acetate buffer in pH adjustment of sorghum mash and its influence on fuel ethanol fermentation. J. Ind. Microbiol. Biotechnol. 2009, 36:75-85.

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

Saccharomyces cerevisiae on glucose and ethanol," Appl. Microbiol. Biotechnol. 1986, 24: 193-198; Warth, A.D., "Effect of benzoic acid on glycolytic metabolite levels and intracellular pH in Saccharomyces cerevisiae, " Appl. Environ. Microbiol. 1991 , 57:3415-7; Warth, A.D., "Mechanism of action of benzoic acid on

Zygosaccharomyces bailii: effects on glycolytic metabolite levels, energy production, and intracellular pH," Appl. Environ. Microbiol. 1991 , 57: 3410-4; Taherzadeh, M.J. and Niklasson, C. et al., "Acetic acid - friend or foe in anaerobic batch conversion of glucose to ethanol by Saccharomyces cerevisiae?" Chem. Eng. Sci., 1997, 52:2653- 2659; Narendranath, N.V. and Thomas, K.C. et al., "Effects of acetic acid and lactic acid on the growth of Saccharomyces cerevisiae in a minimal medium," J. Ind.

Microbiol. Biotechnol. 2001 , 26:171 -7; Narendranath, N.V. and Power, R., "Effect of yeast inoculation rate on the metabolism of contaminating lactobacilli during fermentation of corn mash," J. Ind. Microbiol. Biotechnol. 2004, 31 : 581 -4; Tara, V. et al. 2007; Zhao, R., Bean S.R. et al., "Application of acetate buffer in pH

adjustment of sorghum mash and its influence on fuel ethanol fermentation," J. Ind. Microbiol. Biotechnol. 2009, 36:75-85. In the fuel ethanol industry, the presence of bacteria in fermentations is often unavoidable, and results in increasing lactic acid and acetic acid production. It has been reported that lactic acid at concentrations of 0.2-0.8% w/v (%w/v can be expressed in grams/100 ml_) and acetic acid at concentrations of 0.05-0.1 % w/v began to stress yeast growth, reduce growth rate, decrease rates of glucose consumption and ethanol production in minimal medium. See, Narendranath, N.V. and Thomas, K.C. et al., "Effects of acetic acid and lactic acid on the growth of Saccharomyces cerevisiae in a minimal medium," J. Ind. Microbiol. Biotechnol. 2001 , 26:171 -7. But, studies by M. J. Taherzadeh observed a 20% increase in ethanol yield when 3 g/L of un-dissociated acetic acid was added to the minimal medium, while the biomass and glycerol production decreased by 45 and 33%, respectively. See, Taherzadeh, M.J. and Niklasson, C. et al., "Acetic acid - friend or foe in anaerobic batch conversion of glucose to ethanol by Saccharomyces cerevisiae?" Chem. Eng. Sci. 1997, 52:2653-2659. Graves, T. and Narenranath, N.V. et al. found that the additional buffering capacity of corn mash could offer some protection to yeast against acid-induced stress. See, Graves, T. and Narenranath, N.V. et al., "Effect of pH and lactic or acetic acid on ethanol productivity by Saccharomyces cerevisiae in corn mash," J. Ind. Microbiol. Biotechnol. 2006, 33:469-74. Abbott and Ingledew demonstrated that the ethanol fermentation was elevated in the whole corn mash containing 0.025%- 0.35% w/v acetic acid and increased the final ethanol yield. See, Abbott, D.A. and Ingledew, W.M. "Buffering capacity of whole corn mash alters concentrations of organic acids required to inhibit growth of Saccharomyces cerevisiae and ethanol production," Biotechnol. Lett. 2004, 26:1313-6. Renyong Zhao noted that ethanol yields or conversion efficiencies were improved with a 5.9% relative increase in alcohol when the method of pH adjustment changed from traditional HCI to acetate buffer in sorghum mash. The biomass and glycerol was decreased by 36% and 43.6%, respectively. See, Zhao, R., Bean S.R. et al., "Application of acetate buffer in pH adjustment of sorghum mash and its influence on fuel ethanol fermentation," J. Ind. Microbiol. Biotechnol. 2009, 36:75-85; Warth, A.D., "Effect of benzoic acid on glycolytic metabolite levels and intracellular pH in Saccharomyces cerevisiae," AppI. Environ. Microbiol. 1991 , 57:3415-7; and Warth, A.D., "Mechanism of action of benzoic acid on Zygosaccharomyces bailii: effects on glycolytic metabolite levels, energy production, and intracellular pH," AppI. Environ. Microbiol. 1991 , 57:3410-4.

Chengming Zhang noted that the final ethanol yield increased 7.6% when un- dissociated propionic acid was less than 30 mM, and the ethanol fermentation was inhibited by 53.2 mM un-dissociated propionic acid. The effect of adding various acids to the medium reservoir of anaerobic glucose-limited cultures of S. cerevisiae CBS 8066 and H 1022 has been studied by Verduyn et al. See, Yeast 1990 Mar- Apr, 6:149-58. Addition of weak acids, including acetate, propionate, and butyrate, resulted in a decreased cell yield and an increased ethanol formation. Benzoic acid at concentrations up to 0.4 mM stimulated ethanol production.

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. As a food additive, 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.

SUMMARY Described herein is a method of modifying a yeast cell. 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. There can be an increase in the fermentation yield of ethanol in either simultaneous or in series saccharification and fermentation, when using modified yeast as compared to using yeast not modified with an organic acid. 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.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 illustrates a GC/FID chromatogram of transesterified yeast extract showing methyl benzoate and major lipids. DETAILED DESCRIPTION

Any starch substrate can be used with the methods described herein. A starch substrate comprises starch. The term "starch" refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and/or amylopectin with the formula (C₆H ₀O₅) _x, wherein X can be any number. In particular, 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. The term "granular starch" refers to uncooked (raw) starch, which has not been subjected to

gelatinization.

During, before or after saccharification and fermentation, various hydrolyzing enzymes may be active, including granular starch hydrolyzing enzyme and enzymes having granular starch hydrolyzing activity. The terms "granular starch hydrolyzing enzyme (GSHE)" and "enzymes having granular starch hydrolyzing activity" refer to enzymes, which have the ability to hydrolyze starch in granular form.

The starch may or may not undergo gelatinization. The term "starch gelatinization" means solubilization of a starch molecule to form a viscous

suspension. Gelatinization may occur to various degrees at various temperatures. The term "gelatinization temperature" refers to the lowest temperature at which

gelatinization of 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.

The term "total sugar content" refers to the total sugar content present in a starch composition. The term "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.

The term "fermentation" refers to the enzymatic and anaerobic breakdown of organic substances by microorganisms to produce simpler organic compounds. While fermentation occurs under anaerobic conditions it is not intended that the term be solely limited to strict anaerobic conditions, as fermentation also occurs in the presence of oxygen.

The term "fermentation broth" refers to fermentation medium containing the end products after fermentation.

The term "fermentable sugar" refers to the sugar composition consisting of DP1 and DP2.

The term "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. The term "ds" or "DS" refers to dissolved solids and dry substance in a solution.

The term "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

Standard Analytical Methods of the Corn Refiners Association (1701 Pennsylvania Ave. NW Washington, DC 20006-5805) and may be found in their Critical Data Tables.

The term "distillers' dried grain with soluble (DDGS)" refers to the product derived by separating the liquid portion (soluble) from grain whole stillage by screening or centrifuging, then evaporating to thick syrup and drying it together with the grain solids portion.

The "distillers dry grains (DDG)" refers to the dried residual by-product of a grain fermentation process.

As used herein the term "starch" refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and amylopectin with the formula (ΰ₆Ηιο0₅)χ, wherein x can be any number.

The term "starch liquefaction" refers to a process by which starch is converted to shorter chain and less viscous dextrins.

The term "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 ).

The term "saccharification enzyme" and "glucoamylases" used

interchangeability herein refers to the amyloglucosidase class of enzymes

(EC.3.2.1 .3, glucoamylase, alpha-1 , 4-D-glucan glucohydrolase). These are exo- acting enzymes, which release glucosyl residues from the non-reducing ends of amylose and amylopectin molecules. The enzymes also hydrolyze alpha-1 , 6 and alpha-1 ,3 linkages although at a lesser rate than alpha-1 ,4 linkages.

The term "hydrolysis of starch" refers to the cleavage of glucosidic bonds with the addition of water molecules. The term "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.

The term "liquefact" refers to starch hydrolysate from a conventional high temperature liquefaction process using thermostable alpha amylase. The term "mash" refers to a mixture of a fermentable substrate in liquid used in the production of a fermented product and is used to refer to any stage of the fermentation from the initial mixing of the fermentable substrate with one or more starch hydrolyzing enzymes and fermenting organisms through the completion of the fermentation run.

The term "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.

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 term "fermentation products" includes a product produced by a

fermentation process. 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).

The term "backset/thin stillage" is the liquid portion of the stillage (soluble solids) separated from the insoluble solids of the yeast fermentation broth after distillation. 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. The terms "sugar" and "molasses" refer to sugar extracted from sugar cane or beet and molasses as a concentrate from mother liquor from sugar crystallization process.

The term "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). In dry milling, the whole grain can be milled and used in the process. In wet milling the grain is separated {e.g. the germ, protein, and fiber from the starch). In particular, 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

University Press, in particular Chapters 2 and 4. 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%).

The term "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.

Described herein is a method of modifying a yeast cell. 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.

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. In one aspect, 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. Then, 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.

Saccharification and fermentation can then be conducted for converting the starch in the starch substrate to an end product. The organic acid or the organic acid of the organic acid salt can be of the formula:

R-COO^" where R is a hydrogen or an aliphatic or aromatic group containing from 1 to 14 carbon atoms. 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. After the organic acid or the organic acid salt is added to the aqueous suspension containing the yeast cell, 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. After the organic acid or the organic acid salt is added to the aqueous suspension containing the yeast cell, 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, SUPERSTART™, FERMIOL®, RED STAR®, or Angel® alcohol yeast. Yeast can express recombinant enzymes. For example SYNERTIA® ADY (DuPont Industrial Bioscience) and TRANSFERM™ 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 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.

Any enzyme may be used in saccharification. The enzyme may be

glucoamylase. The glucoamylase may be DISTILLASE® SSF (DuPont Industrial Bioscience). The enzyme may be amyloglucosidase. The enzyme may be exo-1 ,4- alpha-D-glucosidase.

Various amounts of the 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. In 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.

In various embodiments, 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.

Another aspect relates to a dried modified yeast cell. 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-COO^" where R is a hydrogen or an aliphatic or aromatic group containing from 1 to 14 carbon atoms. 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.

After the organic acid or the organic acid salt is added to the aqueous suspension containing the yeast cell, 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.

After the organic acid or the organic acid salt is added to the aqueous suspension containing the yeast cell, 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,

SUPERSTART™, FERMIOL®, RED STAR®, and Angel® alcohol yeast. Yeast can express recombinant enzymes. For example SYNERTIA® ADY (DuPont Industrial Bioscience) and TRANSFERM™ 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

conducted simultaneously.

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.

Additional Variations

1 . A method of modifying a yeast cell comprising 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 the modified yeast cell;

centrifuging and decanting to remove a supernatant comprising the organic acid or the organic acid salt from the modified yeast cell;

wherein the 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.

2. The method of variation 1 , wherein the organic acid or the organic acid of the organic acid salt is of the formula:

R-COO^" where R is a hydrogen or an aliphatic or aromatic group containing from 1 to

14 carbon atoms.

3. The method of variation 2, wherein R is selected from the group consisting of methyl, ethyl, n-or isopropyl, n-or iso-butyl, phenyl and substituted phenyl.

4. The method of variation 2, wherein R is an aromatic ring with a carboxylic group.

5. The method of any of variations 1 -4, wherein the organic acid of the organic acid salt is benzoic acid, phenyl acetic acid or phenyl propionic acid. The method of any of variations 1 -5, wherein the organic acid salt is sodium benzoate. The method of any of variations 1 -6, wherein the organic acid salt is potassium benzoate. The method of any of variations 1 -7, wherein the yeast cell is selected from the group consisting of FALI, SUPERSTART™, FERMIOL®, RED STAR®, and Angel® alcohol yeast. The method of any of variations 1 -8, wherein the modified yeast cell is dried for storage after the centrifuging and decanting step. The method of any of variations 1 -9, further comprising

resuspending the modified yeast cell with de-ionized water,

centrifuging and decanting a second supernatant from the modified yeast cell. The method of any of variations 1 -10, further comprising

resuspending the modified yeast cell to form a cream for inoculation; transferring the cream to a fermentation medium containing a starch substrate; and

conducting saccharification and fermentation for converting the starch in the starch substrate to an end product. The method of variation 1 1 , wherein the saccharification and fermentation are conducted in a cold cook process.

13. A method of modifying a genetically-modified yeast cell comprising preparing an aqueous suspension containing a genetically-modified 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 genetically-modified yeast cell to form the improved 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;

wherein the 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. The method of any of variation 13, wherein the organic acid or the organic acid of the organic acid salt is of the formula:

R-COO^" where R is a hydrogen or an aliphatic or aromatic group containing from 1 to 14 carbon atoms. The method of variation 14, wherein R is selected from the group consisting of methyl, ethyl, n-or isopropyl, n-or iso-butyl, phenyl and substituted phenyl. The method of variation 14, wherein R is an aromatic ring with a carboxylic group. The method of any of variations 13-16, wherein the organic acid of the organic acid salt is benzoic acid, phenyl acetic acid or phenyl propionic acid. The method of any of variations 13-17, wherein the organic acid salt is sodium benzoate. The method of any of variations 13-18, wherein the organic acid salt is potassium benzoate. The method of any of variations 13-19, wherein the yeast cell is selected from the group consisting of FALI, SUPERSTART™, FERMIOL®, RED STAR®, and Angel® alcohol yeast. The method of any of variations 13-20, wherein the modified yeast cell is dried for storage after the centrifuging and decanting step. The method of any of variations 13-21 , further comprising after the

centrifuging and decanting step:

resuspending the modified yeast cell with de-ionized water,

centrifuging and decanting a second supernatant from the modified yeast cell. The method of any of variations 13-22, further comprising after the

centrifuging and decanting step:

conducting saccharification and fermentation for converting the starch in the starch substrate to an end product. The method of variation 23, wherein the saccharification and fermentation are conducted in a cold cook process. A dried modified yeast cell prepared by a method comprising

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 to remove a supernatant comprising the organic acid or the organic acid salt from the modified yeast cell; and

drying the modified yeast cell. 26. The dried modified yeast cell of variation 25, wherein the organic acid or the organic acid of the organic acid salt is of the formula:

R-COO^" where R is a hydrogen or an aliphatic or aromatic group containing from 1 to 14 carbon atoms.

27. The dried modified yeast cell of variation 26, wherein R is selected from the group consisting of methyl, ethyl, n-or isopropyl, n-or iso-butyl, phenyl, and substituted phenyl.

28. The dried modified yeast cell of variation 26, wherein R is an aromatic ring with a carboxylic group.

29. The dried modified yeast cell of any of variations 25-28, wherein the organic acid of the organic acid salt is benzoic acid, phenyl acetic acid, or phenyl propionic acid.

30. The dried modified yeast cell of any of variations 25-29, wherein the organic acid salt is sodium benzoate or potassium benzoate.

31 . The dried modified yeast cell of any of variations 25-30, wherein the yeast cell is selected from the group consisting of FALI, SUPERSTART™, FERMIOL®, RED STAR®, and Angel® alcohol yeast. 32. A method of conducting fermentation comprising

resuspending a dried modified yeast cell prepared according to a method of any of variations 25-31 to form a cream for inoculation;

transferring the cream to a fermentation medium containing a starch substrate; and

conducting saccharification and fermentation for converting the starch in the starch substrate to an end product The following examples serve to illustrate the above aspects and embodiments without limiting them in any way.

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.

As an alternative to shearing, a trans-esterification procedure for

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.

Yeast samples from control and benzoate-enhanced processes were directly derivatized with acetyl chloride after adding a C15:0 triglyceride internal standard in toluene. The reaction was then quenched by adding 1 M NaCI and hexane was added. The resulting methyl benzoate was extracted into the hexane/toluene layer and subjected to GC/FID analysis. The resulting chromatogram is shown in Figure 1 . Sample preparation: A 50 mg sample was weighed. If the sample was liquid, it was placed on a nitrogen evaporator with water bath set at 30-35 °C and

evaporated until only a dry residue remained. 100 μΙ_ of the internal standard solution was pipetted into the 13 X100 mm glass culture tube containing the sample. 0.5 ml_ of 5% acetyl chloride in methanol solution was added. The tubes were capped tightly and placed in a heat block for 60 minutes at 80 °C. The tubes were then cooled for 5 minutes. When cooled, 400 μΙ_ heptane and 500 μΙ_ 1 M NaCI in water solution were added to each tube. The tubes were vortexed for 6 seconds, paused, and vortexed for an additional 6 seconds to fully mix the sample. If the layers could not be separated without centrifuging, centrifuging was performed to separate the layers. About 200 μΙ_ of the top (organic) layer was carefully removed with a Pasteur pipette and placed into a GC vial with insert. The sample was then ready to be analyzed by GC/FID.

GC Conditions: Column Omegawax 320 (Sigma Aldrich catalog no 24136). Initial flow 2.6 ml/min with average velocity 54 cm/sec. Oven Initial temp 160°C with equilibration time 0.5 min. Step to 5.0 ml/min at 200 °C, ramp to 10.0 ml/min at 250 °C over 10.0 min. Run time 23.0 min. Inlet split flow (split liner); temperature 260 °C, pressure: 33.4 psi, split flow 77.0 ml/min, total flow 82.6 ml/min, and gas saver: On, 20 ml/min after 2 min. Detector: FID; temperature 260 °C, H₂ flow 30 ml/min, air flow 400 ml/min, makeup flow: 25.0 ml/min, and makeup gas helium. See Fig. 1 .

Example 2: Determination of Carbohydrate Composition by High Pressure Liquid Chromatography (HPLC)

The 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,

Monosaccharides), maintained at 65 °C fitted with a refractive index (Rl) detector

(ERC-7515A, Rl Detector from The Anspec Companyjnc). Dilute sulfuric acid (0.01 N) was used as the mobile phase at a flow rate of 0.6 ml per minute. Twenty microliters (μΙ_) of solution were injected onto the column. The column separates based on the molecular weight of the saccharides. 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 μιτι 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.

Sodium Benzoate ppm

Treatment Supernatant Cell Paste

100 ppm 74 128

500 ppm 479 628

1000 ppm 979 1389

5000 ppm 3808 2640 Example 4: Modification of yeast used to inoculate simultaneous

saccharification and fermentation.

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.

• Dry yeast: The pellet was extruded out as a 2 mm tube and allowed to air dry.

After drying 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.

Soluble Sugar

Yeast DP2+ DP1 Glycerol Ethanol

%w/v %w/v %w/v %v/v

Un-treated yeast 0.57 0.000 1 .38 1 1 .1

20,000 ppm treated yeast 0.56 0.004 1 .27 1 1 .4

20,000 ppm treated and washed yeast 0.56 0.000 1 .29 1 1 .3

20,000 ppm treated and dried yeast 0.58 0.003 1 .25 1 1 .3

The results in 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.

Soluble Sugar

Yeast DP2+ DP1 Glycerol Ethanol

%w/v %w/v %w/v %v/v

Un-treated yeast 0.96 0.00 1 .55 15.6

Potassium Benzoate treated yeast 0.91 0.02 1 .37 16.0

The use of a potassium benzoate salt for yeast treatment resulted in a decrease in glycerol and an increase in ethanol. The replacement of sodium with potassium shows that alternate salts of benzoate will function in the same manner.

Example 6: Modification of a genetically modified yeast used to inoculate simultaneous saccharification and fermentation.

SYNERTIA® ADY (DuPont Industrial Bioscience), a genetically modified

(GMO) yeast that produces Glucoamylase, was treated with sodium benzoate.

Mixtures of 20% w/w dry yeast, 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 pellet re-suspended.

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.

Soluble Sugar

Yeast DP2+ DP1 Glycerol Ethanol

%w/v %w/v %w/v %v/v

Un-treated yeast 1 .04 0.00 1 .59 15.27

Sodium Benzoate treated yeast 0.92 0.02 1 .24 15.86

The results in 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. Urea was added for a concentration of 600 ppm. Slurry pH was adjusted to 4.5 with 10N sulfuric acid. Saccharification was accomplished with STARGEN® 002 dosed at 1 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 5.

Table 5: Cold Cook end of fermentation soluble sugars, glycerol and ethanol from fermentations with untreated yeast and benzoate treated yeast.

Yeast DP4+ Glycerol Ethanol

%W/V %W/V %V/V

Un-treated yeast 0.39 0.32 1 1 .89

40,000 ppm treated yeast 0.39 0.32 12.40

The results in Table 5 show an increase in ethanol for modified yeast.

Claims

1 . A method of preparing a modified yeast cell comprising preparing an aqueous suspension containing a yeast cell;

2. The method of claim 1 , wherein the organic acid or the organic acid of the organic acid salt is of the formula:

3. The method of claim 2, wherein R is selected from the group consisting of methyl, ethyl, n-or isopropyl, n-or iso-butyl, phenyl, and substituted phenyl.

4. The method of claim 2, wherein R is an aromatic ring with a carboxylic

group.

5. The method of claim 1 , wherein the organic acid of the organic acid salt is benzoic acid, phenyl acetic acid, or phenyl propionic acid.

6. The method of claim 1 , wherein the organic acid salt is sodium benzoate or potassium benzoate.

7. The method of claim 1 , wherein the yeast cell is selected from the group consisting of FALI, SUPERSTART™, FERMIOL®, RED STAR®, and Angel® alcohol yeast.

8. The method of claim 1 , wherein the modified yeast cell is dried for storage after the centrifuging and decanting step and prior to the resuspending step.

9. The method of claim 1 , further comprising

resuspending the modified yeast cell with de-ionized water,

centrifuging and decanting a second supernatant from the modified yeast cell.

10. The method of claim 1 , further comprising

conducting saccharification and fermentation for converting the starch in the starch substrate to an end product.

1 1 . The method of claim 1 , wherein the saccharification and fermentation are conducted in a cold cook process.

12. The method of claim 1 , wherein the yeast cell is genetically-modified and wherein the modified yeast cell is genetically-modified.

13. A dried modified yeast cell prepared by a method comprising preparing an aqueous suspension containing a yeast cell;

drying the modified yeast cell.

14. The dried modified yeast cell of claim 13, wherein the organic acid or the organic acid of the organic acid salt is of the formula:

15. The dried modified yeast cell of claim 14, wherein R is selected from the group consisting of methyl, ethyl, n-or isopropyl, n-or iso-butyl, phenyl, and substituted phenyl.

16. The dried modified yeast cell of claim 14, wherein R is an aromatic ring with a carboxylic group.

17. The dried modified yeast cell of claim 13, wherein the organic acid of the organic acid salt is benzoic acid, phenyl acetic acid, or phenyl propionic acid.

18. The dried modified yeast cell of claim 13, wherein the organic acid salt is sodium benzoate or potassium benzoate.

19. The dried modified yeast cell of claim 13, wherein the yeast cell is selected from the group consisting of FALI, SUPERSTART™, FERMIOL®, RED STAR®, and Angel® alcohol yeast.

20. A method of conducting fermentation comprising resuspending a dried modified yeast cell prepared according to the method of claim 13 to form a cream for inoculation;