WO2016044606A1

WO2016044606A1 - Simultaneous saccharification and fermentation process in the presence of benzoate

Info

Publication number: WO2016044606A1
Application number: PCT/US2015/050716
Authority: WO
Inventors: Gang Duan; Mark GOUTHRO; Jayarama K. Shetty; Bruce A. Strohm; Xin Wang; Hongxian Xu
Original assignee: Danisco Us Inc
Priority date: 2014-09-17
Filing date: 2015-09-17
Publication date: 2016-03-24

Abstract

The present invention provides fermentation methods. Benzoates have been found to improve ethanol yield and decrease glycerol production in fermentation reactions using granular starch as the feedstock.

Description

SIMULTANEOUS SACCHARIFICATION AND FERMENTATION PROCESS

IN THE PRESENCE OF BENZOATE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is related to and claims the benefit of priority from PCT Application No. PCT/CN2014/086692, filed on September 17, 2014 in the China Intellectual Property Office, the entirety of which is herein incorporated by reference.

FIELD OF THE INVENTION

The invention relates to methods for fermentation, enhancing ethanol yield from fermentation, and/or reducing glycerol. Methods provided comprise fermenting un- gelatinized starch in the presence of benzoate.

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 known in the art. There are several methods currently used to convert starch substrates into alcohol.

For example, the wet-milling process involves fractionating corn into different components by subjecting corn to steeping processes followed by separating the components by gravity separation. Only the starch component enters into further processing to make fermentation feed stock.

As another example, the dry-grind processes starts with grinding the whole corn kernel without separating the components. This is the most commonly used process, generally referred to as the "conventional process," includes grinding the starch - containing materials then liquefying starch at high temperature using a thermostable alpha amylase (e.g. SPEZYME® RSL from DuPont Industrial Bioscience), followed by simultaneous saccharification and fermentation (SSF) in the presence of glucoamylase (e.g. DISTILLASE® SSF from DuPont Industrial Bioscience) and yeast to convert fermentable sugars into alcohol. Another example is a process adapted to small grains such as wheat, barley and triticale, which requires pretreatment steps using viscosity reducing enzymes (non-starch polysaccharide hydrolyzing enzymes such as cellulases, hemicellulases etc ) between 50 and 60°C for an extended period of time followed by liquefaction and SSF.

Another example, often referred to as the "no cook process" includes grinding starch containing material followed by simultaneously saccharifying and fermenting granular starch without gelatinization using granular starch hydrolyzing glucoamylase and acid stable alpha amylase and yeast.

Aside from using starch-based substrates as a starting material, another approach for making fermentation products involves the use of "lignocellulosic material," which includes the cell walls of plants. Cell walls are composed of a heterogeneous mixture of complex polysaccharides that interact through covalent and non-covalent means. Complex poly-saccharides of higher plant cell walls include, e.g., cellulose (β-1 -4 glucan), which generally makes up 35-50% of carbon found in cell wall components. Cellulose polymers self-associate through hydrogen bonding, van der Waals interactions and hydrophobic interactions to form semi-crystalline cellulose microfibrils. These microfibrils also include non-crystalline regions, generally known as amorphous cellulose. The cellulose microfibrils are embedded in a matrix formed of hemicelluloses (including, e.g., xylans, arabinans, and mannans), pectins (e.g., galacturonans and galactans), and various other β-1 ,3 and β-1 ,4 glucans. These polymers are often substituted with, e.g., arabinose, galactose and/or xylose residues to yield highly complex arabinoxylans, arabinogalactans, galactomannans, and xyloglucans. The hemicellulose matrix is, in turn, surrounded by polyphenolic lignin. Materials derived from plants are often referred to as lignocellulosic materials, comprising cellulose and hemicellulose as well as lignin. Lignocellulosic biomass materials include seeds, grains, tubers, plant waste or byproducts of food processing or industrial processing (e.g., stalks), corn (including, e.g., cobs, stover, and the like), grasses (e.g., Indian grass, such as Sorghastrum nutans; or, switchgrass, e.g., Panicum species, such as Panicum virgatum), perennial canes, e.g., giant reeds, wood (including, e.g., wood chips, processing waste), paper, pulp, recycled paper (e.g., newspaper). Such materials can be derived from, e.g., an agricultural crop, a byproduct of a food or feed production, a lignocellulosic waste product, a plant residue, or a waste paper or waste paper product. Suitable plant residue can comprise residues of, e.g., grain, seeds, stems, leaves, hulls, husks, corncobs, corn stover, straw, grasses, canes, reeds, wood, wood chips, wood pulp and sawdust. The paper waste can be, e.g., discarded or used photocopy paper, computer printer paper, notebook paper, notepad paper, newspapers, magazines, cardboard, and paper-based packaging materials.

Fermentation efficiency or carbon conversion into end products during yeast fermentation depends on several factors, such as pH, and temperature, dry solids content, organic acid and glycerol. Anaerobic yeast fermentation generally produces glycerol as a by-product with the production of glycerol being directly proportional to yeast growth. The growth of unproductive yeast, often referred to as wild yeast, is undesirable because it produces only biomass at the cost of end product. The development of novel methods to reduce unproductive yeast without affecting the alcohol yield is one of the unmet needs in the market place.

Often times during traditional ethanol fermentation process, lactic and acetic acids are produced by contaminating bacterial species. When this happens in a fermentation, bacteria present can alter the profile of organic acids, resulting in high levels of lactic and acetic acids and consequent decreased ethanol yield.

Organic acids are well known for their toxicity to microorganisms and 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 rather seems to be a function of the concentration of their undissociated forms (Verduyn, C, E. Postma, et al. (1990) J. Gen. Microbiol. 136(3): 405; Verduyn, C, E. Postma, et al. (1990) J. Gen. Microbiol. 136(3): 395; Verduyn, C, E. Postma, et al. (1992) Yeast 8(7): 501 -51 7; Narendranath, N. V., K. C. Thomas, et al. (2001 ) J. Am. Soc. of Brewing Chemists 59(4): 187-194; Piper, P., C. O. Calderon, et al. (2001 ) Microbiol. 147(10): 2635; Thomas, K. C, S. H. Hynes, et al. (2002). Appl. Environ. Microbiol. 68(4): 1616-1 623; Abbott, D. A. and W. M. Ingledew (2004). Biotechnol. Lett. 26(16): 1313-1316; Zhao, R., S. R. Bean, et al. (2009). J. Ind. Microbiol. Biotechnol. 36(1 ): 75-85.)

The undissociated form of the molecule diffuses passively into the microbial cell and then dissociates inside the cell. This leads 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.

A considerable amount of research has been conducted on the effect of organic acids, mainly on acetic acid and lactic acid, on ethanol fermentation (Gutierrez, U. E., A. V. K. O. Annicchino, et al. (1 991 ) Arq. Biol. Tecnol 34(235-242); Pons, M.-N., A. Rajab, et al. (1986) Appl. Microbiol. Biotechnol. (1986) 24:193-198; Warth, A. D. (1991 ) Appl. Environ. Microbiol. 57(12): 3415-3417; Taherzadeh, M. J., C. Niklasson, et al. (1 997) Chem. Eng. Sci. 52(1 5): 2653-2659; Narendranath, N. V., K. C. Thomas, et al. (2001 ) J. Am. Soc. of Brewing Chemists 59(4): 187-194; Narendranath, N. V., K. C. Thomas, et al. (2001 ) J Ind Microbiol Biotechnol 26(3): 1 71 -1 77; Narendranath, N. V. and R. Power (2004) J Ind Microbiol Biotechnol 31 (12): 581 -584; Tara, G., N. N. V, et al. (2007) Appl. Microbio. Biotechnol. 73(5): 1 190-1 196; Zhao, R., S. R. Bean, et al. (2009) J Ind Microbiol Biotechnol 36(1 ): 75-85.)

In the fuel ethanol industry, microbial contamination of fermentations with bacteria 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 and acetic acid at concentrations of 0.05-0.1 % w/v begin to stress yeast growth, reduce growth rate, decrease rates of glucose consumption and ethanol production (Narendranath, N. V., K. C. Thomas, et al. (2001 ) J. Am. Soc. of Brewing Chemists 59(4): 187-194; Narendranath, N. V., K. C. Thomas, et al. (2001 ) J. Ind. Microbiol. Biotechnol. 26(3): 171 -177). M. J. Taherzadeh observed a 20% increase in ethanol yield when 3 g/L of undissociated acetic acid is added to the minimal medium, while the biomass and glycerol production decreased by 45 and 33%, respectively (Taherzadeh, M. J., C. Niklasson, et al. (1997) Chem. Engin. Sci. 52(15): 2653-2659) , while other studies (Graves, T., N. V. Narendranath, et al. (2006) J Ind Microbiol Biotechnol 33(6): 469-474) found that the additional buffering capacity of corn mash could offer some protection to yeast against acid-induced stress. Abbot et.al (Abbott, D. A. and W. M. Ingledew (2004) Biotechnol Lett 26(1 6): 1313-1316) 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. Renyong Zhao noted that ethanol yields or conversion efficiencies were improved significantly with 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 (Zhao, R., S. R. Bean, et al. (2009). J Ind Microbiol Biotechnol 36(1 ): 75-85; Warth, A. D. (1991 ). Appl Environ Microbiol 57(12): 3415-3417). Chengming Zhang noted that the final ethanol yield increased 7.6% when undissociated propionic acid was lower than 30mM, and the ethanol fermentation was completely inhibited by 53.2mM undissociated propionic acid. The effect of addition of 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. (Verduyn, C, E. Postma, et al. (1990) J. Gen. Microbiol. 136(3): 405; Verduyn, C, E. Postma, et al. (1 990). J. Gen. Microbiol. 136(3): 395; Verduyn, C, E. Postma, et al. (1992) Yeast 8(7): 501 -517).

Addition of weak acids, including acetate, propionate and butyrate, resulted in a decreased cell yield and an increased ethanol formation. Benzoic acid at low concentrations (up to 0.4 mM) stimulated ethanol production.

Sodium benzoate has been in 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 lower, the anaerobic fermentation of glucose through phosphofructosekinase is decreased by 95% (Krebs H.A, et al. (1983) Biochem. J. 214, 657-663).

SUMMARY OF THE INVENTION

Provided herein are fermentation methods comprising performing simultaneous saccharification and fermentation of a granular starch mash in the presence of benzoate. In embodiments, the benzoate comprises sodium benzoate, potassium benzoate, or benzoic acid. In embodiments, simultaneous saccharification and fermentation of a granular starch mash are carried out in the presence of salicylic acid or acetyl salicylic acid or a combination thereof. In embodiments, the benzoate is present in an amount of 0.05-0.3, 0.05-0.2, or 0.05-0.1 grams of sodium benzoate per liter of mash. In embodiments, the benzoate is added with the fermenting organism at the beginning of fermentation, after fermentation has commenced, or both.

In some embodiments, the granular starch mash is derived from corn. In embodiments, the granular starch mash is derived from any suitable starch-bearing plant except rice and sorghum. In embodiments, the granular starch mash is produced by pretreating grain. In some embodiments, the granular starch has a particle size less than 30 mesh.

In some embodiments, the simultaneous saccharification and fermentation is carried out in the presence of recycled backset/thin stillage. In some embodiments, the granular starch mash comprises recycled backset/thin stillage. In embodiments, the granular starch mash comprises 20-45% dry solids, 30-38% dry solids, 32-34% dry solids, or high dry solids ("high ds").

In some embodiments, the simultaneous saccharification and fermentation comprises amylase and glucoamylase. In embodiments, the simultaneous saccharfication and fermentation comprises Aspergillus kawachi alpha amylase or Aspergillus niger glucoamylase or both. In some embodiments the fermenting organism is yeast. In some embodiments, the yeast is from the genus Saccharomyces, Pichia, or Candida. In some embodiments, the fermenting organism is bacterial. In some embodiments, the bacteria is from the genus Escherichia, Zymomonas, or Klebsiella.

In embodiments, methods provided herein are carried out in at temperatures of 30-34C, 31 -33C, or 32C. In embodiments, the pH is 3.5-6, or 4-6. In embodiments, the methods of simultaneous saccharification and fermentation provided herein are carried out for 24-96 hours.

In some embodiments, the methods provided herein provide improved fermentation. In some embodiments, the improved fermentation is improved ethanol yield or reduced glycerol production or both. In some embodiments, the ethanol yield is improved as compared to the analogous fermentation process carried out in the absence of benzoate. In some embodiments, the glycerol production is reduced as compared to the analogous fermentation process carried out in the absence of benzoate.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

Figure 1 shows HPLC patterns on the comparison of ethanol and glycerol content of yeast fermentation broth from a no cook process with and without benzoic acid.

Figure 2 shows the effect of sodium benzoate and benzoic acid concentration during conversion of ground whole corn to ethanol under no cook process conditions on final alcohol yield

Figure 3 shows the effect of starting pH of the yeast fermentation on the effect of benzoic acid for a no cook process during conversion of ground whole corn to ethanol at 33% ds using 69 hours of fermentation as described in Example 2.

Figure 4 shows the effect of sodium benzoate and benzoic acid at different dry solids content of ground whole corn slurries during yeast fermentation using no cook processes at 33% ds at 69 hours of fermentation as described in Example 3. Figure 5 shows the effect of sodium benzoate concentrations on conversion of ground whole rice and ground whole sorghum to ethanol by no cook process with pretreatment step on the final alcohol yield at 69 hours of fermentation as described in Example 4.

Figure 6 shows the effect of sodium benzoate and FERMASURE® XL on yeast cell counts during yeast fermentation of whole ground corn to ethanol at 24 hours under no cook process conditions as described in Example 6 (32% ds whole ground corn,, starting pH 5.5, 32 C,0.1 g.of sodium benzoate /liter mash;200 ppm of FEMASURE® XL.) For each three sample group, the left bar indicates cell count, the middle bar indicates % viability, and the right bar indicates % bud rate.

Figure 7 shows micrographs taken during cell counts done at 48h fermentation time as described in Example 6. For the cell counts, a gram of sample is diluted 1 :20 in water, and stained with methylene blue dye. The diluted sample is placed on a hemocytometer (Bright-Line, Reichert, and Buffalo, NY). Cells are counted at 400X magnification with a light microscope for cells per 0.02 μί.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used have their ordinary meaning in the relevant scientific field. Singleton, et al., Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, New (1994), and Hale & Markham Harper Collins Dictionary of Biology, Harper Perennial, NY (1991 ) provide the ordinary meaning of many of the terms describing the invention.

As used herein, "benzoate" is used to refer generally to benzoic acid, benzoic salts (such as sodium benzoate, potassium benzoate, etc.), and combinations of benzoic acid and benzoic salts. Suitable benzoic acids include salicylic acid or acetyl salicylic acid.

As used herein, "pretreatment" refers to contacting a granular starch containing feedstock with enzymes (e.g. GC626 alpha-amylase or FERMGEN™ protease, though any enzyme can be employed; see, e.g., PCT Application Publication No. WO2009134964A2) at a suitable, preferably elevated, temperature (e.g., 65C, but any temperature from 25C to 75C can be employed) for a period of time (e.g., 60 minutes, but any period of time from 20 minutes to 6 hours (h) can be employed) before the fermentation with yeast. As used herein, "improving fermentation" refers to increasing ethanol yield, and/or decreasing glycerol production, as compared to a control fermentation process lacking benzoate. Improved fermentation may be demonstrated by increased ethanol yield, decreased glycerol production, or a combination thereof. Ethanol yield and glycerol production may be readily detemined by methods known in the art and/or described herein.

As used herein "starch" refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and/or amylopectin with the formula (C₆H₁₀O₅) 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.

"Granular starch" as used herein refers to uncooked (raw) starch, which has not been subjected to gelatinization.

The term "gelatinization" used in the context of starch means solubilization of a starch molecule to form a viscous suspension.

The term "gelatinization temperature" refers to the lowest temperature at which gelatinization of a starch substrate 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 initial starch gelatinization temperature ranges for a number of granular starches which may be used in accordance with the process herein include barley (52-59°C), wheat (58-64°C), rye (57-70°C), corn (62-72°C), high amylose corn (67-80°C), rice (68-77°C), sorghum (68- 77°C), potato (58- 68°C), tapioca (59-69°C) and sweet potato (58-72°C) (Swinkels, pg. 32-38 in STARCH CONVERSION TECHNOLOGY, Eds. Van Beynum et al., (1985) Marcel Dekker Inc. New York and The Alcohol Textbook 3.sup.rd ED. A Reference for the Beverage, Fuel and Industrial Alcohol Industries, Eds. Jacques et al., (1999) Nottingham University Press, UK). Gelatinization involves melting of crystalline areas, hydration of molecules and irreversible swelling of granules. The gelatinization temperature occurs in a range for a given grain because crystalline regions vary in size and/or degree of molecular order or crystalline perfection.

As used herein, "no cook process" refers to a process includes grinding starch containing material followed by simultaneously saccharifying and fermenting the granular starch without gelatinization using glucoamylase capable of hydrolyzing granular starch and acid stable alpha amylase and a fermenting organism such as yeast.

A "slurry" is an aqueous mixture containing insoluble starch granules in water.

The term "dry solids" (ds) refer to dry solids dissolved in water, dry solids dispersed in water or a combination of both. Dry solids thus include granular starch, and its hydrolysis products, including glucose.

"Dry solid" content refers to the percentage of dry solids both dissolved and dispersed as a percentage by weight with respect to the water in which the dry solids are dispersed and/or dissolved. The initial dry solid content is the weight of granular starch corrected for moisture content over the weight of granular starch plus weight of water. Subsequent dry solid content can be determined from the initial content adjusted for any water added or lost and for chemical gain. Subsequent dissolved dry solid content can be measured from refractive index as indicated below.

The term "high DS" refers to aqueous starch slurry containing dry solids greater than 38% by weight of dry solids plus water.

"Dry substance starch" refers to the dry starch content of granular starch and can be determined by subtracting from the mass of granular starch any contribution of. For example, if granular starch has a water content of 20%, then 100 kg of granular starch has a dry starch content of 80 kg. Dry substance starch can be used in determining how many units of enzymes to use.

"Refractive Index Dry Substance" (RIDS) is the determination of the refractive index of a starch solution at a known DE at a controlled temperature then converting the Rl to dry substance using an appropriate relationship, such as the Critical Data Tables of the Corn Refiners Association.

The term "distillers' dried grain with solubles" (or "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 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. An "alpha-amylase (E.C. class 3.2.1 .1 )" is an enzyme that catalyze the hydrolysis of alpha-1 ,4-glucosidic linkages. These enzymes have also been described as those effecting the exo or endohydrolysis of 1 , 4-a-D-glucosidic linkages in polysaccharides containing 1 , 4-a-linked D-glucose units. Another term used to describe these enzymes is glycogenase. Exemplary enzymes include alpha-1 ,4-glucan 4-glucanohydrase glucanohydrolase.

A "glucoamylase" refers to an amyloglucosidase class of enzymes (EC.3.2.1 .3, glucoamylase, alpha-1 , 4-D-glucan glucohydrolase) are enzymes that remove successive glucose units from the non-reducing ends of starch. The enzyme can hydrolyze both linear and branched glucosidic linkages of starch, amylose and amylopectin. The enzymes also hydrolyze alpha-1 , 6 and alpha -1 , 3 linkages although at much slower rates than alpha-1 , 4 linkages.

"Pullulanase" also called debranching enzyme (E.C. 3.2.1 .41 , pullulan 6- glucanohydrolase), is capable of hydrolyzing alpha 1 -6 glucosidic linkages in an amylopectin molecule.

As used herein, the term "liquifact" refers to the starch hydrolysate from a conventional high temperature liquefaction process using thermostable alpha amylase.

As used herein, "simultaneous saccharification and fermentation," or synonymously "SSF (SSF), refers to a process in which saccharification of granular starch or hydrolysed starch (gelatinized and liquefied) mash occurs in the fermentation vessel simultaneously with the commencement of fermentation by a fermenting organism (eg. yeast) to alcohol.

The term "fermenting organism" refers to any organism, including bacterial, fungal and yeast, suitable for producing the desired fermentation end product of ethanol. Examples of fermenting organisms are known in the art and include REDSTAR™ yeast, ANGEL® yeast, and Fali yeast. Another example of a fermenting organism known in the art is Zymomonas (see, for example, U.S. Patent 8,569,458B2. Suitable fermenting organisms may be recombinant organisms.

Examples of fermenting organisms includes fungal organisms such as yeast. Preferred yeast includes strains of the genus Saccharomyces, Pichia, and Candida. Preferred bacterial fermenting organisms include strains of Escherichia, strains of Zymomonas and Klebsiella. As used herein, the term "backset/thin stillage" refers to the liquid portion of the stillage (soluble solids) separated from the insoluble solids of the yeast fermentation broth after distillation.

The term "comprising" and its cognates are used in their inclusive sense; that is, equivalent to the term "including" and its corresponding cognates.

Numeric ranges are inclusive of the numbers defining the range. Some preferred subranges are also listed, but in any case, reference to a range includes all subranges defined by integers included within a range.

Disclosed herein are fermentation methods which may provide improved ethanol yield, decreased glycerol production, or both. In simultaneous saccharification and fermentation processes, starch-based substrate is hydrolyzed while a fermenting organism converts the hydrolysate to a product. Both actions may be carried out in the same fermentation vessel, thus the vessel will contain the substrate, the fermenting organism, the enzymes for saccharification. Demonstrated herein are improved fermentation results when the simultaneous saccharification and fermentation of granular starch is carried out in the presence of benzoate (see Examples). For example, demonstrated herein is that addition of sodium or potassium benzoate results in a decrease in glycerol production and an increase in the final alcohol yield (Example 1 )- Starch-containing materials may be obtained from wheat, corn, rye, sorghum

(milo), rice, millet, barley, triticale, cassava (tapioca), potato, sweet potato, sugar beets, sugarcane, and legumes such as soybean and peas. Preferred material includes 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 heterogenous genes). Any part of the plant may be used as a starch- containing material, including but not limited to, plant parts such as leaves, stems, hulls, husks, tubers, cobs, grains and the like. In some embodiments, essentially the entire plant may be used, for example, the whole ground grain or fractionated grain may be used. In some embodiments, whole grain may be used as a starch-containing material. Preferred whole grains include corn, wheat, rye, barley, sorghum and combinations thereof. In other embodiments, starch-containing material may be obtained from fractionated cereal grains including fiber, endosperm and/or germ components. Methods for fractionating plant material, such as corn and wheat, are known in the art (Alexander,A.J, 1 987 ,Corn Dry Milling: Process, Products, And Applications, Page 351 - 376, Chapter 1 1 , in Corn Chemistry and Technology, Watson, S.A.and Ramstead, P.E; editors; American Association of Cereal Chemists, lnc,3340 Pilot Knob Road, St. Paul, Minnesota, USA; Johnston et.al 2005 US Patent # 6,899,91 0). In some embodiments, starch-containing material obtained from different sources may be mixed together to obtain material used in the processes of the invention (e.g. corn and milo or corn and barley).

In some embodiments, starch-containing material may be prepared by means such as milling. Two suitable milling processes include wet milling or dry milling (grinding). In dry milling for example, the whole grain is 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 (1 999) Nottingham University Press. See, Chapters 2 and 4. In some embodiments, the milled grain which is used in the process has 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, for example, WO2004/081 193). The milled starch-containing material may be ground to a specified sieve size and combined with water resulting in an aqueous slurry (also referred to as "mash"). As shown in Example 8, methods provided herein may be employed with a range of dry solids content. The slurry will comprise between 1 5 to 55% dry solids on a weight/weight (w/w) basis. In embodiments, the slurry comprises 20 to 50%, 25 to 50%, 25 to 45%, 25 to 40%, or 20 to 35% dry solids. In some embodiments the recycled thin-stillage (backset) is used as a portion of the water for slurry make-up of 1 0 to 70% on a volume/volume (v/v) basis. In embodiments, the recycled thin-stillage (backset) is used as a portion of the water for slurry make-up of 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% V/V.

The slurry is contacted with the appropriate enzymes and a suitable fermenting organism under suitable conditions to carry out simultaneous saccharification and fermentation. Suitable conditions for fermentation are known in the art. In some embodiments, the process is carried out at temperatures of 30-34C, 31 -33C, or 32C. In some embodiments, the process is carried out at a starting pH of 3.5-6 or 4-6. As shown in Example 2, the methods provided herein may provide fermentation improvements over a range of starting pH. In some embodiments, the fermentation process proceeds for 24-96 hours. Appropriate enzymes for simultaneous saccharification and fermentation include alpha amylase and glucoamylase and mixtures thereof. An example suitable combination of alpha amylase and glucoamylase is a Stargen® brand blend of alpha-amylase and glucoamylase enzymes for hydrolyzing uncooked starch (DuPont Industrial Biosciences, Wilmington, DE). Other suitable alpha amylase and glucoamylase enzyme preparations or mixtures thereof are known in the art.

Prior to fermentation, the slurry may be contacted with enzymes suitable for pretreatment. Suitable enzymes are known in the art and include non-starch polysaccharide hydrolyzing enzymes such as cellulases, hemicellulases, and proteases. An example of a suitable protease includes FERMGEN™ protease enzyme (DuPont Industrial Biosciences, Wilmington, DE).

Benzoate is present during the methods provided herein. Benzoate may be added along with the fermenting organism (at the beginning of the fermentation process), or may be added at any time during the fermentation. Benzoate may also be added both at the beginning of the fermentation as well as during and throughout the fermentation. The addition may be a bolus or may be an addition over time. Equipped with this specification, one of skill in the art will readily be able to ascertain the appropriate protocol for adding benzoate to achieve the desired fermentation effects. Sodium benzoate, potassium benzoate, or benzoic acid may be used. Benzoate may be present in an amount of 0.05-0.3, 0.05-0.2, or 0.05-0.1 grams of sodium benzoate per liter of mash. For example, benzoate may be present in amounts such as 0.05, 0.01 , 0.1 5 and 0.20 grams of sodium or potassium benzoate per liter of mash.

Suitable measurements for fermentation performance via soluble sugar, alcohol concentration, cell counts, and byproduct production are well-known in the art. In embodiments, methods provided herein provide improved ethanol yield. Production of ethanol by fermentation is well-known in the art, as is analysis of ethanol production in fermentation. Likewise, measurement of glycerol production is known in the art. As an example, and as demonstrated herein, high pressure liquid chromatography (HPLC) is a suitable method for such measurement. Soluble sugar may be measured by assessing the amount of monosaccharides (DP1 ) and the amount of di- and higher saccharides (DP2+).

The method of practicing the present invention is further illustrated by the following non-limiting examples.

EXAMPLE 1

Effect of sodium benzoate concentration during conversion of corn to ethanol under no cook process on final alcohol yield, glycerol content and total yeast count.

An aqueous slurry of ground whole corn (33% ds) with a particle size smaller than 30 mesh was prepared and added sodium benzoate to different concentrations (0.05,0.01 , 0.15 and 0.20 grams of sodium benzoate per liter of mash). Urea was then added at 400 ppm and the pH of the slurry was adjusted to pH 4.5 using 20% sulfuric acid. Simultaneous saccharification and yeast fermentation were carried out using STARGEN® 002 (DuPont Industrial Bioscience) at 2.0 Kg. per MT ds of corn and dry ANGEL® yeast (Angel Yeast Co. Ltd; Yichang Hubei Province, China) was added at 0.10%, (w/w). The yeast fermentation was carried out by incubating the slurry at 32°C with constant stirring. The samples were taken at different time intervals and analyzed for soluble sugar, glycerol, alcohol and total yeast count. The difference between alcohol and glycerol content in the fermentation broth with and without sodium benzoate is shown in Table 1 A and Table 1 B.

Tables 1 A and 1 B

Effect of benzoate salts, i.e. sodium (1 A) and potassium (1 B) during conversion of ground whole corn to ethanol under no cook process conditions on final soluble sugar, alcohol yield, glycerol content and total yeast count at 69 hours.

1 B

The results in Table 1 A and 1 B show that the addition of either sodium or potassium benzoate results in a significant decrease in glycerol production which corresponds to a decrease in the total yeast count. A concomitant increase in the final alcohol yield is obtained.

EXAMPLE 2

Effect of starting pH of the fermentation on the effect of benzoic acid under no cook process during conversion of ground whole corn to ethanol at 33 % ds corn solids.

Aqueous slurries of ground whole corn (33% ds) with a particle size smaller than 30 mesh were prepared with sodium benzoate added at increasing concentrations (0.05, 0.10 and 0.20 grams of benzoic acid per liter of mash). Urea was then added at 400 ppm and the pH of the slurry was adjusted to pH 5.5, 5.0, 4.5, 4.0 and 3.5 using 20% sulfuric acid. Simultaneous saccharification and yeast fermentation was carried out using STARGEN® 002 at 2.0 Kg. per MT ds corn and dry Angel yeast (Angel Yeast Co.Ltd; Yichang Hubei Province, China) was added at 0.10% w/v . The yeast fermentation was carried by incubating the slurry at 32°C with constant stirring. Samples were taken at the end of the fermentation, 69 hours and analyzed for soluble sugar; alcohol and total yeast count (Figure 3, Table 2).

Table 2

Effect of starting pH of the fermentation on the effect of benzoic acid under no cook process during conversion of whole ground corn to ethanol at 33 % ds starting

PH Sod. Benzoate Soluble Sugar % yeast g/liter mash g/liter ethanol %,Glycerol count/mL

DP2

DP1 and + v/v w/v x10 ⁸

5.50 0.00 0.97 4.19 15.81 0.85 3.15

0.05 0.29 4.16 16.06 0.65 2.25

0. 10 0.84 4.26 16.16 0.58 1 . 53

0. 20 5.29 4.00 15.59 0.57 1 . 13

5.00 0.00 1.30 4.55 15.28 0.83 2. 73

0.05 0.21 4.56 16.01 0.62 1 . 88

0. 10 0.99 4.74 16.42 0.53 1 . 50

0. 20 4.98 4.66 15.93 0.52 1 . 13

4. 50 0.00 0.16 4.67 15.27 0.72 2. 88

0.05 0.29 4.38 15.29 0.54 1 . 78

0. 10 0.60 4.56 15.69 0.47 1 . 03

0. 20 7.39 4.60 14.89 0.44 1 . 10

4.00 0.00 0.99 5.59 15.39 0.78 3. 03

0.05 1.06 5.42 15.26 0.46 1 . 70

0. 10 1.07 5.58 15.49 0.41 1 .05

0. 20 4.13 5.77 14.81 0.32 0. 80

3. 50 0.00 0.76 5.89 14. 60 0.74 3. 18

0.05 0.33 5.86 14.85 0.49 1 .55

0.10 1.31 6.01 14.53 0.35 0.85

0. 20 6.22 6.20 14.06 0.29 0.75

The data in Table 2 (Figure 3) showed that initial pH in the range of 4-5.5 with sodium benzoate addition of 0.05 and 0.10 g/L of mash resulted in increased ethanol over the control with decreasing glycerol and yeast counts. Increasing sodium benzoate additions to 0.2 g/L resulted in decreased ethanol over the control. At initial pH of 3.5, ethanol production is decreased in the control, with little benefit shown by the addition of sodium benzoate.

EXAMPLE 3

Effect of sodium benzoate and benzoic acid at different dry solids content of whole ground corn during yeast fermentation under no cook process.

The test was conducted with corn meal smaller than 30 mesh. The fermentation was carried out in 500 ml Erienmeyer flasks containing 200 grams mash, over the DS range of 28% to 38%. Sodium benzoate or benzoic acid was added at 0.1 gram per liter of mash and then adjusted pH to 4.5. Simultaneous saccharification and yeast fermentation was carried out using STARGEN® 002 (DuPont Industrial Bioscience) at 2.0 kg. per MT ds of corn and dry ANGEL® yeast (Angel Yeast Co. Ltd; Yichang Hubei Province, China) was added at 0.1 % w/v. The yeast fermentation was carried by incubating the slurry at 32°C with constant stirring. The samples were taken during different intervals of time and analyzed for soluble sugar, alcohol and total yeast count at 69 hours are shown (Figure 4,Table 3).

Table 3

EXAMPLE 4

Effect of sodium benzoate and benzoic acid during conversion of whole ground rice and whole ground sorghum to ethanol under no cook process on ethanol, glycerol and total yeast count. In this experiment, whole ground rice and whole ground sorghum with a particle size less than 30 mesh were used as feed stock in yeast fermentation under no cook process. The fermentation was carried out in 500 ml Erlenmeyer flasks containing 200 grams of mash containing different levels of benzoic acid and sodium benzoate. Urea was added at 600 ppm and the pH was adjusted to pH 4.5 using 20% sulfuric acid. Simultaneous saccharification and yeast fermentation was carried out using STARGEN® 002 at 2.0 kg. per MT ds of corn and dry Angel yeast ( Angel Yeast CO. Ltd; Yichang Hubei Province, China) was added at 0.1 % w/v. The yeast fermentation was carried by incubating the slurry at 32°C with constant stirring. The samples were taken during different intervals of time and analyzed for soluble sugar; alcohol and total viable yeast count (Figure 5, Table 4 A and 4B).

Table 4

Effect of sodium benzoate (4A) and benzoic acid (4B) on conversion of whole ground rice and whole ground sorghum to ethanol under no cook process

4A: Sodium benzoate

The results in Tables 4A and 4B indicated that while the addition of sodium benzoate or benzoic acid reduced the production of glycerol and reduced total viable yeast counts, the improvement in ethanol was there but marginal. EXAMPLE 5

Effect of sodium benzoate and benzoic acid during yeast fermentation of pretreated ground whole corn to ethanol

The effect of sodium benzoate and benzoic acid during yeast fermentation of pretreated ground whole corn to ethanol was studied. In this experiment, ground whole corn with a particle size less than 30 mesh was used as feed stock in yeast fermentation under no cook with pretreatment process. The fermentation was carried out in 500 ml Erlenmeyer flasks containing 200 grams of mash containing different level of benzoic acid or sodium benzoate. The pH was adjusted to 4.5 using 20% sulfuric acid. To the aqueous slurry containing whole ground corn 15Kg GC 626 /MT corn (DuPont Industrial Biosciences) and 1 Kg FERMGEN® /MT of corn (DuPont Industrial Biosciences) were added and incubated at 65°C for 60 min with constant stirring. After pretreatment, the slurry was cooled to 32°C and then 400ppm urea, 2.0Kg STARGEN® 002 /MT corn (DuPont Industrial Biosciences) and 0.1 % w/v dry Angel® yeast (Angel Yeast Co. Ltd; Yichang Hubei Province, China) were added to start the fermentation. Samples were taken at the end of the fermentation, 69 hours and analyzed for soluble sugar; alcohol and total yeast count (Table 5 A and 5B). Table 5

Effect of sodium benzoate (9A) and benzoic acid (9B) on soluble sugar; alcohol and total yeast count during conversion of ground whole corn to ethanol under no cook with pretreatment process.

5A: Sodium benzoate

5B: Benzoic acid

The data in Table 5A and 5B indicated that either sodium benzoate or benzoic acid when used in a process that includes a pretreatment step will reduce glycerol and yeast count while improving ethanol content.

EXAMPLE 6

Sodium benzoate and FERMASURE® fermentation additive in yeast fermentation of ground whole corn to ethanol under no cook process.

Aqueous slurry of ground corn was prepared with 32% dry solids in Erlenmeyer flasks. The pH of the slurry was adjusted to 5.5 with 10 N sulfuric acid. Urea was added for a concentration of 600 ppm. STARGEN® 002™(DuPont Industrial

Biosciences) was added at 3 kg/MT ds, and FERMGEN™ (DuPont Industrial

Biosciences) was added at 0.1 kg/MT ds.

1 ) Control

2) Sodium benzoate added at 0.15 g/L

3) FERMASURE® added at 200 ppm.

4) Sodium benzoate added at 0.15 g/L, after 30 min FERMASURE® added at 200 ppm.

5) FERMASURE® added at 200 ppm, after 30 min Sodium benzoate added at 0.15 g/L.

6) Sodium benzoate added at 0.15 g/L, and FERMASURE® added at 200 ppm concurrently.

An aqueous 1 0% w/v solution of yeast (ETHANOL RED® yeast, Fermentis, Marq-en-Barceul, Franc ) was hydrated for 10 minutes. Yeast slurry was added to the ground corn slurry at 500 μΙ/I OOg, 1 hour after addition of STARGEN™ 002. Flasks were capped with a vented stopper and placed in a forced air shaker/incubator at 32°C with 250 rpm orbital shaking. Samples were taken at end of fermentation, 72 hours for HPLC analysis. Sugars, organic acids and ethanol levels were measured with a PHENOMENEX REZEX™ organic acid column (Figure 6 Table 6).

Table 6

Effect of sodium benzoate and FERMASURE® during conversion of ground whole corn to ethanol under no cook process conditions, i.e. 32% ds whole ground corn, pH

5.5,32°C.

Sugars and organic acids showed little variation. Glycerol was reduced and ethanol was increased in the presence of sodium benzoate. The effect of the addition of FERMASURE® stayed neutral on the effect of sodium benzoate. Sodium benzoate was shown to provide a positive benefit to ethanol production. Final ethanol levels were significantly higher when sodium benzoate was present. Differences between the timing of addition were not significant. FERMASURE® XL™ did not show an interaction with sodium benzoate. Fermentations with benzoate and FERMASURE® XL™ were not lower than those without FERMASURE ® XL. Glycerol levels were lower with benzoate, no interaction was seen with FERMASURE® XL™ and benzoate. Cell count results were impacted by both FERMASURE® XL and benzoate (Figure 7) FERMASURE® XL by itself had a dramatic impact on yeast, cell counts were higher, budding lower and cell size much smaller (Figure 7). Benzoate had a smaller impact on yeast increasing bud count and reducing cell count. Cell size was not noticeably different. EXAMPLE 7

Effect of benzoate on fermentation

Two 14 Liter pilot plant scale yeast fermentations , i.e. control ( #1 ) and the test with sodium benzoate added at 0.15 g/L mash (# 2) were carried out using whole ground corn as feed stock under no cook process conditions. Aqueous slurry of ground corn was prepared with 28% dry solids in a 14L Biostat V, Sartorius, Goettingen, Germany. The pH of the slurry was adjusted to 5.5 with 10 N sulfuric acid. Urea was added for a concentration of 600 ppm. STARGEN® 002 (DuPont Industrial Bioscience) was added at 3 kg/MT ds. ETHANOL RED® yeast (Fermentis, Marq-en-Barceul, France) was added (dry) at 0.1 % w/w concentration.

Fermentations were carried out at 32°C with moderate agitation, which prevented settling. Slurry pH was measured online with a submerged pH probe.

Weight loss was measured to determine ethanol production as carbon dioxide off gassing. Samples were taken at end of fermentation for HPLC analysis. pH, soluble sugars, organic acids and ethanol levels were measured with a PHENOMENEX

REZEX™ organic acid column (Table 7 ).

Table 7

Composition of soluble sugars, glycerol, organic acid, pH and ethanol content during yeast fermentation of whole ground corn with and without sodium benzoate

Sugars show little variation after 22 h and are similar for both conditions. Lactic acid and glycerol levels were lower in the presence of sodium benzoate. Ethanol levels were higher by HPLC and mass balance in the presence of sodium benzoate. Fermentation pH did not drop as low with the addition of sodium benzoate. Cell counts were done using fermentation samples at 24 hr and 48 hr under 400X magnifications with a hemocytometer. (Table 8).

Table 8

Yeast Cell counts at 24hr and at 48hr.

Cell counts were lower and budding rate higher in the presence on sodium benzoate at 24 and 48h. Viability was similar in both conditions at 24 h, but at 48 h viability was higher with sodium benzoate (Table 8). Cell size was larger with the addition of sodium benzoate (Figure 7).

The fermentation broth from both trials were distilled to remove alcohol and then dried at 80C. The dried samples were analyzed for total protein, residual sugars, fiber, fat and glycerol content and summarized in Table 9.

Table 9

Composition of dried fermentation broths: 1 ) Control, 2) with sodium benzoate

Liu reported that a commercial DDGS generally contains glycerol content of about 7.8 % (Liu, K. 201 1 , J. Agric. Food Chem. 59, 1508-1526). The data in Table 9 showed a significant reduction in glycerol content between the two fermentation broths. Addition of sodium benzoate during yeast fermentation resulted in approximately 30% reduction in glycerol formation.

EXAMPLE 8 Effect of salicylic acid and acetyl salicylic acid (aspirin)

The effect of salicylic acid and acetyl salicylic acid during yeast fermentation of whole corn to ethanol was studied. In this experiment, ground whole corn with a particle size less than 30 mesh was used as feed stock in yeast fermentation under no cook process. The fermentation was carried out in 500 ml Erlenmeyer flasks containing 200 grams of mash containing different level of salicylic acid (Sino-pharm chemical regent Co., Ltd., China) or acetyl salicylic acid (Sigma-Aldrich Co. LLC). The pH was adjusted to 4.5 using 20% sulphuric acid, and then added 400ppm urea, 20Kg STARGEN® 002 per-MT corn (DuPont Industrial Bioscience) and 0.1 % w/v dry ANGEL® yeast (Angel Yeast Co.Ltd;Yichang Hubei Province, China). The slurry was held in 32°C using a water bath. Samples were taken at the end of the fermentation, 69 hours and analyzed for soluble sugar; alcohol and total yeast count (Table 10 A and 10B). Table 10 A and 10B

Effect of salicylic acid (10A) and acetyl salicylic acid (aspirin) (10B) during conversion of ground whole corn to ethanol under no cook process on soluble sugar; alcohol and total yeast count.

10A: Salicylic acid

The data in Table 1 0A and 10B indicated that either salicylic acid or acetyl salicylic acid when used in a process will reduce the glycerol and yeast count while improving the ethanol content.

Claims

What is claimed is:

1 . A fermentation method comprising

performing simultaneous saccharification and fermentation of a granular starch mash in the presence of benzoate.

2. The method of claim 1 wherein the simultaneous saccharification and

fermentation is carried out in the presence of recycled backset/thin stillage.

3. The method of any of the preceding claims wherein the granular starch mash comprises 20-45% dry solids.

4. The method of any of the preceding claims wherein the simultaneous

saccharification and fermentation comprises Aspergillus kawachi alpha amylase and Aspergillus niger glucoamylase.

5. The method of any of the preceding claims wherein the fermenting organism is yeast.

6. The method of any of the preceding claims wherein the simultaneous

saccharification and fermentation is carried out at a temperatures of 30-34C.

7. The method of any of the preceding claims wherein the pH is 3.5-6.

The method of any of the preceding claims wherein the simultaneous

saccharification and fermentation is carried out for 24-96 hours.

8. The method of any of the preceding claims wherein the benzoate comprises sodium benzoate, potassium benzoate, or benzoic acid.

9. The method of any of the preceding claims wherein the benzoate is present in an amount of 0.05-0.3 grams of sodium benzoate per liter of mash.

10. The method of any of the preceding claims wherein the granular starch mash is derived from corn.

1 1 . The method of any of the preceding claims wherein the granular starch mash is derived from any suitable starch-bearing plant except rice and sorghum.

12. The method of any of the preceding claims wherein the preparation of granular starch mash comprises pretreatment.

13. The method of any of the preceding claims wherein the ethanol yield is improved as compared to the analogous fermentation process carried out in the absence of benzoate.

14. The method of any of the preceding claims wherein the glycerol production is reduced as compared to the analogous fermentation process carried out in the absence of benzoate.

15. The method of any of the preceding claims wherein the granular starch of the granular starch slurry has a particle size less than 30 mesh.

16. The method of any of the preceding claims wherein the benzoate is added with the fermenting organism at the beginning of fermentation.