CONTROL OF CONTAMINANT MICROORGANISMS IN FERMENTATION PROCESSES WITH SYNERGISTIC FORMULATIONS CONTAINING STABILIZED CHLORINE DIOXIDE AND QUATERNARY AMMONIUM COMPOUND

FIELD OF THE INVENTION

The present invention relates to synergistic combination of stabilized chlorine dioxide and quaternary ammonium compound and method of use, particularly in fermentation processes. BACKGROUND OF THE INVENTION

In the last decade, the use of ethanol as a transportation fuel has increased significantly. Ethanol production in the United States rose from approximately 6.4 billion liters in the year 2000 to over 37 billion liters in 2009. The number of ethanol plants increased from 54 in 2000 to 170 in 2009. Similar increases in production and plant construction have occurred in Latin America and Europe. In 2007, the United States Congress enacted the Energy Independence and Security Act (H.R. 6), which set the renewable fuel standard at 136 billion liters of ethanol by the year 2022. If this standard is to be met, the ethanol industry will continue to grow.

Currently both industrial ethanol (e.g., fuel) and beverage ethanol are produced on large scale from agricultural (natural) feedstocks by fermentation processes in which sugar is converted to ethanol and carbon dioxide by inoculant yeast. Many feedstocks can be used to provide the sugar for fermenting, including potentially, any starch or cellulosic material, which includes nearly all plants, as any starch or cellulose can be a precursor to sugar. Some of the common feedstocks particularly suitable for producing fuel ethanol include corn, milo, sorghum, sugar cane, sugar beets and molasses.

The feedstocks used for ethanol production are natural products therefore, a wide variety of microorganisms such as bacteria, fungi, and yeasts are likely to be naturally present in the feedstocks. Commercial fermentation process conditions are not completely sterile, hence these "contaminant microorganisms" will be present in the process. In commercial ethanol production, microorganisms of greatest concern are lactic acid-producing bacteria and acetic acid-producing bacteria. Such bacteria enter the process from several sources including raw materials, equipment, process water, air, and inoculant yeast, among others. Concentrations of such bacteria may increase in the process environment either through introduction with incoming materials (raw materials, water, air, yeast) or naturally proliferate as a result of conditions favorable to bacterial growth. The optimum atmosphere for yeast production is also extremely conducive to the growth of these bacteria. Organic acids produced by the bacteria inhibit the growth of yeasts and thus reduce ethanol production rate. The bacteria may also consume sugars and other nutrients intended for use by the yeast to produce desired products, rendering the entire process less economical.

Many fermentation processes use antibiotics as antimicrobial compositions.

Such use has become disfavored due to suspected development of antibiotic-resistant bacteria and accumulation of antibiotic residues in fermentation byproducts.

Antibiotic-resistant bacteria are a significant concern in human health.

Byproducts of ethanol production include solids that are collected after distillation of the ethanol product. Such solids include distillers dried grains with solubles (DDGS) and distiller's wet grains with solubles (DWGS). Many countries are considering regulatory actions that would limit or eliminate the use of antibiotics for ethanol production. DDGS and DWGS are sold as animal feed products.

The need for antimicrobial treatments is increasing, not only because of the growth in production volume of ethanol but also the expansion in size of ethanol production facilities. Whereas a plant producing 150-200 million liters per year (MMly) was considered a large facility just a few years ago, 380 MMly (or more) facilities are today's industry standard. In fed-batch processes, the volume of individual fermentation batches has increased significantly. To accommodate this added capacity, the flow rate of feedstock (commonly known as "mash" once it has been prepared for entry into fermentation) into a fermentation system has increased from approximately 2000-3000 liters per minute to 4500-6000 liters per minute in the largest ethanol production facilities.

WO 2007/149450 describes the use of stabilized chlorine dioxide (SCD) to prevent bacterial contamination in ethanol production. SCD is added prior to the onset of significant bacterial growth in ethanol production, as a preventive rather than as a remedial measure. The growth of contaminant bacteria prior to and during the fermentation of sugar to alcohol is thus substantially prevented, creating conditions that enhance growth of inoculant yeast and enable inoculant yeast to produce ethanol without inhibition by organic acids produced by the bacteria.

US Patent 5,611,938 discloses biocide compositions for controlling the growth of bacteria in water systems having pH of at least 7.2, such as natural water, pools, cooling water systems, and paper mills. The composition comprises a synergistic combination of chlorine dioxide and a quaternary ammonium compound, at a weight ratio of the quaternary ammonium compound to chlorine dioxide is from 1 :5 to 1 : 100, preferably 1 :5 to 1 :25. Generally a quaternary ammonium compound is used in an amount ranging from 1-25 ppm of the quaternary ammonium compound and in combination with bis(tributyltin)oxide, at a weight ratio of of the tin oxide to ammonium compound of 2: 1 to 1 :2, preferably 1 : 1.

Although some methods are known, there remains demand for improved methods for addressing contaminant microorganisms in the fermentation industry, both for carbohydrate-containing feedstocks and in fermentation processes. An improved method should preferably be antibiotic-free and not result in residues that accumulate in fermentation coproducts or give rise to antibiotic-resistant bacteria. The method should be efficacious at a wide variety of pH ranges and conditions encountered in the fermentation industry. The method should also use treatment that has a reasonable shelf life, prior to use. There is also a need to improve economics of today's larger volume fermentation processes. The present invention meets these needs.

SUMMARY OF THE INVENTION

The present invention provides a method for controlling growth of contaminant microorganisms in a fermentation process using a combination of (a) stabilized chlorine dioxide (SCD) and (b) a quaternary ammonium compound (QAC). The method comprises adding SCD and QAC to one or more steps of a fermentation process, wherein the fermentation process comprises (i) providing an inoculant, a fermentable sugar and process water; (ii) introducing separately, or in any combination, the inoculant, fermentable sugar and process water into a fermentation vessel to provide a fermentation broth; (iii) contacting the inoculant with the fermentable sugar in the fermentation vessel at a temperature at which the inoculant converts the fermentable sugar to ethanol. Optionally nutrients are added to one or more of the inoculant, fermentable sugar and process water. Nutrients may also be added directly to the fermentation vessel. The SCD and QAC may be added to the inoculant, fermentable sugar or process water prior to introducing each of these to the fermentation vessel. Alternatively the SCD and QAC may be added directly to the fermentation vessel.

The stabilized chlorine dioxide and quaternary ammonium compound, may be added separately to the process. When adding the components separately, the components may be added simultaneously or sequentially in any order. That is, components may be added at the same time (simultaneously) or one component may be added prior to adding the other component (sequentially). Alternatively, the method may comprise contacting the components (a) and (b) prior to adding to the fermentation process.

The present invention provides a method for controlling the growth of contaminant microorganisms in the reactants (defined hereinbelow) in the fermentation broth, and in the products of a fermentation process. The method also controls growth of contaminant microorganisms on surfaces of components of a fermentation system. The method consists of, consists essentially of, or comprises the step of adding stabilized chlorine dioxide and quaternary ammonium compound to a reactant or the fermentation broth, or to a surface or into a vessel of the fermentation system.

The present invention also provides a combination of components that can be used in cleaning-in-place (CIP) applications to treat surfaces of equipment used in fermentation processes. By "CIP" is meant herein that surfaces can be cleaned without the need to disassemble the equipment.

The method includes adding a a combination of components which comprise SCD and QAC in amounts effective to control the growth of contaminant

microorganisms without detrimental effect on the inoculant used in the fermentation process. The effective amount varies in accordance with how the combination of components is used, but can be determined by one skilled in the art in view of the disclosures herein. SCD is typically added in an amount to provide a concentration of SCD in the fermentation broth of 10 to 1000 ppm on a weight/weight basis, based on the total weight of the fermentation broth. QAC is also typically added in an amount to provide a concentration of SCD in the fermentation broth of 10 to 1000 ppm on a weight/weight basis, based on the total weight of the fermentation broth. The method of the present invention further provides a method to control growth of at least one contaminant microorganism in a carbohydrate feedstock wherein the step of providing a fermentable sugar comprises providing a carbohydrate feedstock and contacting the feedstock with SCD and QAC, wherein the carbohydrate content of the feedstock is at least 1% and preferably 1 to 70%, by weight, based on the total feedstock weight and wherein SCD and QAC are added in effective amounts.

Typically SCD is added in an amount of 10 ppm to 1000 ppm of SCD, based on total weight of the feedstock, and QAC is typically added in an amount of 10 ppm to 1000 ppm on a weight/weight basis of the QAC, based on the total weight of the feedstock.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for controlling the growth of a contaminant microorganism comprising, consisting essentially of, or consisting of, adding a combination of components comprising stabilized chlorine dioxide (SCD) and a quaternary ammonium compound (QAC) to one or more steps of a fermentation process. Synergistic combinations of SCD and QAC are added to the process in amounts effective to control growth of contaminant microorganisms without inhibiting the ability of inoculant yeast to convert fermentable sugars into ethanol and carbon dioxide. SCD and QAC are typically added to provide a concentration of SCD in the fermentation broth of 10 to 1000 ppm based on the total weight of the fermentation broth and a concentration of QAC in the fermentation broth of 10 to 1000 ppm based on the total weight of the fermentation broth.

The present invention further provides in the step of providing a fermentable sugar, a method to control growth of at least one contaminant microorganism in a carbohydrate feedstock comprising, consisting essentially of, or consisting of, providing a carbohydrate feedstock and adding stabilized chlorine dioxide and quaternary ammonium compound to the feedstock.

Definitions

The following terms have the definitions as provided below for use herein. Aqueous medium means the medium is substantially water, such as for example greater than 80% water, preferably greater than 90% water, more preferably greater than 95% water. The aqueous medium can be greater than 99% water. Process water is an example of an aqueous medium.

Carbohydrate feedstock means a feed used in preparation of or directly as a fermentable sugar. That is, a carbohydrate feedstock is a fermentable sugar or a composition comprising a fermentable sugar or a composition that can be converted to a fermentable sugar.

The carbohydrate feedstock may comprise up to 100% by weight of carbohydrates. Generally the carbohydrate feedstock comprises between 1% and 70% carbohydrate based on the total weight of the feedstock, preferably between 2 and 40%, as a solution or suspension in an aqueous medium. The amount and composition of the carbohydrates in the feedstock can vary depending on the intended end use. For example, corn steep liquor, which is a carbohydrate solution obtained from a wet mill process, may comprise 16.5% carbohydrates. In a wet mill process, corn is soaked or steeped and then separated into various components. The corn steep liquor is the aqueous liquid obtained after the corn has been soaked for an extended period, during which readily fermentable soluble components are extracted from the corn solids into the steep water. The starch component from the wet mill process may comprise up to 40%) by weight carbohydrates.

The carbohydrate feedstock may comprise other components generally functioning as adjuncts to the solutions and/or suspensions. For example, the carbohydrate feedstock may comprise enzymes, surfactants, dispersants, antifoaming compositions, minerals, trace elements, and combinations of two or more thereof. These components and other components that act as adjuncts are well-known to those skilled in the art.

Control as applied to growth of a microorganism herein means to reduce and/or prevent the growth of a targeted undesirable contaminant microorganism. Controlling the growth of a microorganism as used herein also includes to maintain the

microorganism population at a desired level, to reduce the population of the microorganism to a desired level (partially reduce or even reduce to undetectable limits, e.g., zero population), and/or to inhibit growth of the microorganism. An effective amount refers herein to an amount of each of the SCD and QAC that, in combination, when added to a fermentation process, is effective to control the growth of contaminant microorganisms without detrimental effect on the inoculant used in the fermentation process.

A fermentable sugar is a reactant and common nutrient for inoculants used in ethanol fermentation processes. The fermentable sugar is a sugar (e.g. glucose) or starch that is converted by action of yeast to ethanol and carbon dioxide. Equation (1) illustrates the process for glucose (CgHi 20g).

C6Hl ₂ 0 ₆ → 2 C ₂ H ₅ OH + 2 C0 ₂ (1) A fermentable sugar as used herein is a carbohydrate that is derived from essentially any plant source comprising sugar, starch and/or cellulose, generally provided in the form of a solution or suspension of the sugar, starch and/or cellulose in water. Starch and/or cellulose can be converted by processes known in the art, e.g., using enzymes, to a fermentable sugar for use in the method of this invention. The fermentable sugar can be derived from sources of starch or one or more cellulosic material such as corn, sorghum, wheat, wood chips, milo, barley, millet, sugar cane, sugar beets, molasses, whey, potatoes, wheat straw, corn stover, switch grass, algae, seaweed, and/or other biological sources. The fermentable sugar may also be derived from fruit juice (e.g., grapes, apples). The fermentable sugar may alternatively be derived from non-traditional feedstocks such as wood waste, bagasse, paper waste, and municipal solid waste. Processes are known to those skilled in the art to convert these sources to fermentable sugar. For reference, see, J. Y. Zhu, et al. Bioresource

Technology (2010) vol. 101 (13), pp. 4992-5002; and A. L. Demain, J. Ind. Microbiol. Biotechnol. (2009) vol. 36(3), pp. 319-332.

Conveniently, the fermentable sugar is derived from corn, using either the dry grind or wet mill process. In a dry grind process, corn is ground into meal and processed without separation into its constituent components comprising essentially fiber, starch, oil, and protein. In a wet mill process, corn is soaked or steeped in water and then mechanically separated into its constituent components. The corn starch component from the wet mill process or meal (corn flour) from the dry grind process is mixed with water and enzymes and cooked to solubilize the starch. Corn starch is a polysaccharide, that is, a polymer made of individual units of glucose. The corn starch is converted to smaller (shorter) polysaccharides, i.e., dextrins, by enzymes (a-amylase). The smaller polysaccharides are converted to glucose (monosaccharide) using the enzyme glucoamylase, thus forming the fermentable sugar.

As an alternative to corn, the fermentable sugar can be derived from molasses. Molasses can be obtained from a variety of sources including sugar cane or sugar beets, for example, as a byproduct of the process to manufacture crystalline sugar. Molasses is typically obtained as a syrup, to which other ingredients may be added in preparation for fermentation. These other ingredients include sugarcane juice, beet juice, water, and vitamins or other nutrients. Whether one or more of the other ingredients are added and the amount added will vary in a molasses-derived fermentable sugar.

As an alternative to corn, the fermentable sugar can be derived from sugar cane juice. Sugar cane juice is juice extracted with water from sugar cane. The extraction of juice from sugarcane is also accomplished by physical crushing, diffusion in water, or other methods generally well known to those skilled in the art. See, for example, H. V.

Amorim, et al, in "The Alcohol Textbook" (2009), Nottingham University Press ,

Nottingham, pp. 39-46; and EPA Food and Agricultural Industries Handbook, chapters

9.10.1.1 (sugar cane) and 0.10.1.2 (sugar beets), available at

http://www.epa.gov/ttnchiel /ap42/ch09/ (accessed December 18, 2011). Sugar cane juice can be used without further processing and added directly to a fermentation vessel as the fermentable sugar.

For purposes herein, steps used to produce carbohydrate feedstocks and fermentable sugars are steps of the fermentation process . That is, the fermentation process comprises steps to produce carbohydrate feedstocks, and steps to convert starch and/or cellulose to a fermentable sugar.

In the fermentation process, carbohydrates, including sugars and starches, is typically present in the fermentation broth at a concentration of about 5 to about 40% (weight/volume), preferably in the range of about 10 to 35% (weight/volume), based on the total volume of the fermentation broth. Fermentation broth as used herein means the contents of a fermentation vessel during the fermentation process after all reactants have been added and typically comprises fermentable sugar, inoculant, optional nutrients and process water.

Fermentation process as used herein means a process comprising contacting an inoculant, such as yeast, with a fermentable sugar, and optional nutrients, in process water to produce ethanol. A fermentation process can be batch or continuous. In addition, for purposes herein, fermentation also includes steps for preparing or pretreating one or more of the reactants, such as processes to prepare the fermentable sugar from natural sources, and yeast propagation.

The contacting step is performed at a temperature at which the inoculant converts sugar to ethanol and carbon dioxide.

When ethanol is produced via a dry grind process using corn as a feedstock, the fermentation process may comprise one or more of the steps of slurrying of the corn, liquefaction, saccharification, preparing inoculant yeast (propagation), fermenting the mash(actual step of action of inoculant on sugar to produce ethanol), separating and recovering the resultant ethanol, and optionally recycling the spent inoculant yeast. The fermentation process may comprise one or more of the steps of preparing inoculant yeast, preparing the juice by dilution, pasteurization, or other such process, fermenting, separating and recovering the ethanol, and optionally recycling the yeast. It will be understood by those skilled in the art that these process steps may vary, depending on feedstock availability and plant design and plant location.

Fermentation system as used herein comprises components (equipment), such as vessels and pipes in which and through which one or more of the reactants and products of a fermentation process resides or passes. Such equipment have surfaces on which bacteria and other contaminant (undesirable) microorganisms may be present. As part of a fermentation system is a fermentation vessel - the vessel in which the fermentation step of reacting a fermentable sugar with inoculant to produce ethanol occurs.

When ethanol is produced via a dry grind process, the fermentation system may comprise one or more vessels such as a slurry tank, liquefaction tank, saccharification tank, yeast propagation tank, and fermentation vessel. Such vessels and their use are known to those skilled in the art. See, for example, R. J. Bothast and M. A. Schlicher, Applied Microbiology and Biotechnology ( 2005), vol. 67(1), pp. 19-25. When ethanol is produced via a wet mill process, the fermentation system may comprise one or more vessels such as a steep tank, a separation tank, yeast propagation tank, and fermentation vessel. It will be understood by those skilled in the art that these process steps may vary, depending on feedstock availability and plant design and plant location.

An inoculant is any microorganism added purposely to a process in order to convert a feedstock (input) to a desired product (output). For purposes herein, an inoculant is a microorganism which is capable of converting a fermentable sugar to ethanol. Yeasts are common inoculants used in ethanol fermentation. Yeasts are microorganisms capable of living and growing in either aerobic (with oxygen) or anaerobic (lacking oxygen) environments. An inoculant may also be selected from the group consisting of fungi, and algae that are capable of converting a fermentable sugar to ethanol. For example, ethanol can be produced from cellulose using

ethanol-producing bacteria as the inoculant. When used in accordance with the method of this invention, the inoculant bacteria is not adversely affected by the stabilized chlorine dioxide and peroxide compound as are lactic acid- and acetic acid-producing bacteria.

The following discussion is directed to a process in which the inoculant is yeast. For purposes herein, an inoculant yeast is a yeast which has been deliberately selected for a particular conversion in a reaction system. For example, an inoculant yeast may be selected for production of additional yeast in yeast propagation (such as for use as baker's yeast); an inoculant yeast may be selected for metabolism of a particular nutrient for production of enzymes. In this specific application, an inoculant yeast is selected for fermentation of a fermentable sugar to produce ethanol of desired quality and in desired quantity. Inoculant yeasts are generally selected because of their ability to completely ferment available sugars, tolerance to high levels of osmotic stress, elevated temperatures, and high concentrations of ethanol. Yeast are commonly used in ethanol fermentation. Yeast are microorganisms capable of living and growing in either aerobic (with oxygen) or anaerobic (lacking oxygen) environments. Suitable inoculant yeasts for use in the process of this invention include, but are not limited to Saccharomyces cerevisiae (S. cerevisiae), Saccharomyces uvarum (S. uvarum), Schizosaccharomyces pombe (S. pombe), and Kluyveromyces sp.

The inoculant yeast may be introduced into the fermentation vessel as a yeast inoculum, which is an activated form of the inoculant yeast. That is, prior to introducing inoculant yeast into a fermentation vessel, a yeast inoculum is produced by contacting a yeast starter culture and a nutrient composition in a propagation tank to propagate (increase the quantity of) the yeast. The nutrient composition comprises one or more fermentable sugar, enzyme, additional nutrients, and water to grow or activate the yeast. The propagation tank is a separate vessel from the fermentation vessel and is operated at suitable conditions of temperature (e.g., 68-104°F, 20-40°C). Each inoculant will have preferred temperature for propagation. For example, a temperature of 30-32°C (86-90°F) may be particularly suitable for propagating S. cerevisiae. While it is recognized that yeast propagation can occur in the fermentation vessel during the fermentation process, activation of yeast in a propagation tank provides a highly active inoculant yeast. Thus, highly active yeast is introduced to the fermentation vessel. Such yeast propagation techniques are known to those skilled in the art. See, for example, E. Bellissimi, et al, Process Biochemistry (2005), vol. 40(6), pp. 2205-2213; and M. Knauf, et al, Sugar Industry (2006), vol. 131, pp. 753-758.

For continuous fermentation processes, there is often no separate yeast propagation tank. Yeast may or may not be recycled in a continuous process. When no recycle is available, yeasts grow and produce ethanol continuously in a stage called a primary fermentation. When recycle is available, such as is common when molasses or sugarcane juice is used as a feedstock for the fermentable sugar, yeasts are recycled by separation from the other fermentation components, (usually using centrifugation) and typically treated with acid in a separate tank (generally called a yeast recycle tank) to condition the yeast cells for a new fermentation cycle. Alternatively, the yeast may be separated from the fermentation broth, then dried and sold as a co-product. These steps are well known to those skilled in the art. See, for example, E. A. N. Fernandes, et al., Journal of Radioanalytical and Nuclear Chemistry (1998) vol. 234 (1-2), pp. 113-119; and L. C. Basso, et al, FEMS Yeast Res. (2008) vol. , pp. 1155-1163.

Relative to bacteria, yeasts may have moderate to slow fermentation rates. To compensate for their metabolic rate, large amounts of yeast may be required in large scale industrial ethanol production. Inoculant yeast is generally added to the fermentation process in an amount typically about 1 x 10^ to 1 x 10^ cells per gram of fermentation broth. It will be recognized by those skilled in the art that this amount may vary depending on the method of fermentation employed.

Mash is used herein to refer to a composition comprising a fermentable sugar.

Mash includes any mixture of mixed grain or other fermentable carbohydrates in water used in the production of ethanol at any stage from mixing of a fermentable sugar with water to prior to any cooking and saccharification through to completion of fermentation, as defined in Jacques, K.A., Lyons, T.P., Kelsall, D.R, "The Alcohol Textbook", 2003, 423-424, Nottingham University Press, UK.

Microorganisms in the context of this invention are in two categories, desirable and contaminant (undesirable) microorganisms. A desirable microorganism has the capability of consuming nutrients to convert a fermentable sugar to ethanol. Desirable microorganisms include inoculants such as the yeast, Saccharomyces cerevisiae, which is used in the fermentation of glucose into ethanol and carbon dioxide. Other desirable microorganisms are used in other biorefmery processes. Desirable microorganisms are not typically present in carbohydrate feedstocks. When desirable microorganisms are present in a carbohydrate feedstock, the number of viable cells present is typically too low to compete favorably with the other microorganisms commonly also present in the feedstock.

A contaminant microorganism is a microorganism that competes with the inoculant for and consumes the fermentable sugar and nutrients. Contaminant microorganisms produce undesirable products and/or produce alcohol at a much lower rate than would be produced by the inoculant.

Contaminant microorganisms include bacteria, fungi, wild or contaminant yeasts, and other microorganisms capable of metabolizing components of a carbohydrate feedstock to sustain the viability of the microorganism. Contaminant microorganisms contaminate carbohydrate feedstocks, consume the feedstock as a food source in support of their growth, multiply, and thus deplete the feedstock.

Contaminant microorganisms such as contaminant yeasts are often found in both industrial and beverage ethanol production, and can cause severe episodes of contamination, resulting in reduced ethanol productivity in a fermentation process. These unwanted microorganisms may be introduced into the process through the fermentable sugar (feedstock), process water, air, operators, and numerous other sources.

Contaminant microorganisms, such as bacteria, including lactic acid bacteria

(species of Lactobacillus) produce products such as acetic and lactic acids from glucose feedstocks, that not only consume the feedstock and thus prevent feedstock conversion to desired products, but also adversely affect desirable microorganisms in a biorefining process. For example, acetic and lactic acids adversely affect the rate at which Saccharomyces cerevisiae converts glucose to ethanol. Other contaminant bacteria include species of Pediococcus, Enterococcus, Acetobacter, Gluconobacter, and Clostridium.

A nutrient means an element which supports growth and/or survival of the inoculant yeast or other inoculant microorganism, for example a nutrient may be a source of carbon, nitrogen, oxygen, sulfur, phosphorus, magnesium, and a variety of trace elements such as metals. A carbon source can be an alcohol such as methanol, or a hydrocarbon such as octane. A carbon source may also be the fermentable sugar. A nitrogen source can be urea, ammonia, an ammonium salt or a nitrate. A salt such as magnesium sulfate can provide magnesium and sulfur. Phosphorus can be supplied as a sodium or potassium salt, while oxygen can be supplied by introducing air into the process. Other forms of gaseous oxygen can be used, such as pure oxygen or oxygen/nitrogen mixtures. Other elements can be added directly or may be naturally present in process components.

Preserve and preservation means treating a carbohydrate feedstock to prevent reaction or consumption of carbohydrate by contaminant microorganisms such as bacteria. Preservation provides a stable carbohydrate feedstock that does not undergo substantial change, such as would result from reaction or consumption of the carbohydrates due to microbial metabolism, over a period of time of at least one month. One measure of change is the microbial population of the preserved feedstock. When properly preserved, the carbohydrate feedstock does not undergo an increase in the microbial population in the feedstock of more than 1 logi Q CFU/ml or 1 logi Q CFU/g. Typically microbial population is expressed as logi Q CFU/ml for liquid feedstocks and as logi o CFU/g for solid/semi-solid feedstocks. The expression logi Q CFU/g can also be used for liquid feedstocks.

A second measure of change with respect to preservation is based on the concentrations of particular acids in the feedstock. Acids are known products resulting from reaction and/or consumption of carbohydrates by contaminant bacteria. For example, certain bacteria known to contaminate carbohydrate feedstocks produce lactic acid and acetic acid upon consumption of the carbohydrate. Increasing concentrations of lactic acid and acetic acid in a carbohydrate feedstock can be used to indicate whether the feedstock is properly preserved. When properly preserved, concentration of lactic acid in the carbohydrate feedstock is less than 0.60% (weight/volume) and concentration of acetic acid in the carbohydrate feedstock is less than 0.30 %

(weight/volume) .

It will be further appreciated by those skilled in the art that other measures of change may be used. For example, detection of the presence of undesired compounds may indicate change, such as a product from metabolism of the carbohydrate feedstock. Detection methods may include spectrophotometry, chromatography, and other methods known to those skilled in the art. Still other measures may include physical changes to the carbohydrate feedstock such as specific gravity or viscosity.

Quaternary ammonium compounds (QAC) have the general formula R ₄ N X ^" and are a type of cationic organic nitrogen compound, where X is any halogen. R is selected from the group consisting of saturated and unsaturated alkyl, aryl, alkylaryl, phenyl, allyl, and alkylphenyl groups which may be connected together in any combination. QAC are salts having quaternary ammonium cations and halogen anions, wherein the cations remain permanently charged independent of the pH of the solution. R may either be equivalent or correspond to two to four distinctly different moieties. R may be any type of hydrocarbon: saturated, unsaturated, aromatic, aliphatic, branched chain, or normal chain with 1-24 carbons. R may also be an alkylamidoalkylene, hydroxyalkylene, or alkoxyalkylene group having 1-8 carbon atoms, wherein the hydrocarbon or alkylene chain may be interrupted by one or more heteroatoms selected from the group consisting of nitrogen , sulfur, and phosphorus. R may also contain additional functionality and heteroatoms. The nitrogen atom, covalently bonded to four organic groups, bears a positive charge that is balanced by the anion "X". Heterocyclics, in which the nitrogen is bonded to two carbon atoms by single bonds and to one carbon by a double bond, are also considered quaternary ammonium

compounds. Examples of QAC include (1) methylpyridinium iodide, (2)

benzyldimethyloctadecylammonium chloride, and (3) di(hydrogenated

tallow)alkyldimethylammonium chloride, where the alkyl groups, R, can be the same or different and have 14 to 18 carbon atoms, which are illustrated below.

H ₃ CI

(1) (2) (3)

Quaternary ammonium compounds also include sources of protonizable nitrogen. Various sources of quaternary ammonium compounds or protonizable nitrogen can be used in this invention, including cholines, lecithins, betaines, and amine oxides.

Quaternary ammonium compounds useful in the present invention may be selected from the group consisting of alkyl dimethyl benzylammonium chloride; dioctyl-dimethylammonium chloride; didecyl dimethyl ammonium chloride;

diallyl-dimethylammonium chloride; di-n-decyl-dimethylammonium chloride;

cetylpyridinium chloride; benzethonium chloride; and mixtures thereof.

Reactant means inoculant, fermentable sugar, optional nutrients as well as any other optional components in a fermentation broth. For purposes herein, reactant also includes process water.

Stabilized chlorine dioxide otherwise referred to herein as SCD means one or more chlorine dioxide-containing oxy-chlorine complexes, one or more

chlorite-containing compounds, one or more other entities capable of forming chlorine dioxide when exposed to acid, and combinations thereof. Thus, stabilized chlorine dioxide comprises at least one of a chlorine dioxide-containing oxy-chlorine complex, a chlorite-containing compound, or an entity capable of forming chlorine dioxide in a liquid medium when exposed to acid. SCD are defined herein are commercially available products. A preferred chlorine dioxide-containing oxy-chlorine complex is selected from the group consisting of a complex of chlorine dioxide with carbonate, a complex of chlorine dioxide with bicarbonate and mixtures thereof. Examples of

chlorite-containing compounds include metal chlorites, and in particular alkali metal and alkaline earth metal chlorites. A specific example of a chlorite-containing compound that is useful as SCD (a chlorine dioxide precursor) is sodium chlorite, which can be used as technical grade sodium chlorite.

SCD is preferably used in the form of an aqueous solution, such as that of an alkali metal or alkaline earth metal chlorite, typically sodium chlorite (NaClC^). Sodium chlorite in solution is generally stable at pH above 7, but releases the active chlorine dioxide (CIO2), when the pH is lowered below neutral (pH 7). The rate of activation of SCD, that is, the rate at which the active CIO2 is released from the stable form, increases as pH decreases.

The exact chemical composition of many of SCD compositions, and in particular, chlorine dioxide-containing oxy-chlorine complexes, is not completely understood. The manufacture or production of certain chlorine dioxide precursors is described by Gordon, U.S. Patent 3,585,147 and Lovely, U.S. Patent 3,591,515.

Specific examples of commercially available and useful stabilized chlorine dioxide include, for example, ANTHIUM DIOXCIDE and FERMASURE, available from E. I. du Pont de Nemours and Company, Wilmington DE; OXINE and PUROGENE, available from Bio-Cide International, Inc., Norman, OK.

SCD may be provided as a solution of the one or more chlorine

dioxide-containing oxy-chlorine complexes, one or more chlorite-containing compounds, or one or more other entities capable of forming chlorine dioxide when exposed to acid, and combinations thereof. The solution provides SCD in a liquid medium at a predetermined concentration of "available chlorine dioxide". The concentration of "available chlorine dioxide" is based on complete (100%) release of chlorine dioxide from the one or more chlorine dioxide-containing oxy-chlorine complexes and/or complete (100%) conversion of the one or more chlorite-containing compounds to chlorine dioxide and/or complete (100%) formation of chlorine dioxide from the one or more other entities capable of forming chlorine dioxide. Preferably, the SCD solution has sufficient SCD to have an available chlorine dioxide concentration in the range of about 0.002% to about 40%> by weight, preferably, in the range of about 2% to about 25% by weight, more preferably in the range of about 5% to about 15% by weight, based on the total weight of the SCD solution.

SCD may be provided as a solid material, such as a composition comprising an alkali or alkaline earth metal chlorite solid, inert ingredients, and optionally dry activator such as a dry acid.

SCD may also be provided as a mixture (or slurry) comprising a saturated solution of alkali or alkaline earth metal chlorite and additional solid alkali or alkaline earth metal chlorite. Such slurries provide a liquid SCD with a higher active ingredient level than available in solution form.

The invention is hereinafter described in terms of SCD as stabilized alkali metal chlorite, more specifically sodium chlorite (NaC102). Typically sodium chlorite is used as an aqueous solution comprising 5 - 22% by weight, based on weight of the solution. Hereinafter SCD concentrations are described in terms of the concentration of chlorine dioxide available when the chlorite is stoichiometrically converted to chlorine dioxide, "available CIO2". The content of potential chlorine dioxide in 1 g of sodium chlorite is 0.597 g. Sodium chlorite solutions comprising 5 - 22% by weight of sodium chlorite thus contain 2.98 - 13.13% available chlorine dioxide. The generation of CIO2 is illustrated by the following equation (2):

5 NaC102 + 4H+ -> 4 CIO2 (g) + 2 H ₂ 0 + CI ^" + 5 Na+ (2) wherein one mole of NaC102 provide 0.8 mole of CIO2 (5:4 ratio). (See for example, Ulllmann's Encyclopedia of Industrial Chemistry, Wiley Online Library,

http://onlinelibrarv.wilev.com/doi/10.1002/14356007.a06 483.pub2/pdf, accessed September 30, 2010.)

Equations providing stoichiometry for generation of CIO2 from other chlorite-containing compounds or from one or more chlorine dioxide-containing oxy-chlorine complexes, or from one or more other entities capable of forming chlorine dioxide when exposed to acid are known to those skilled in the art.

Method for Growth Control

The method of this invention for controlling growth of one or more contaminant microorganisms comprises, consists essentially of or consists of adding a combination of the components (a) stabilized chlorine dioxide (SCD) and (b) quaternary ammonium compound (QAC), to one or more steps of a fermentation process.

The components SCD and QAC may be added separately to the fermentation process. When added separately, the SCD and QAC may be added simultaneously (at the same time) or sequentially (at different times) in any order to one or more different process steps.

The components SCD and QAC may be added separately or in combination to any of the reactants such as the inoculant, fermentable sugar or process water prior to introducing the reactant and component into the fermentation vessel.

Alternatively, the method may comprise contacting components SCD and QAC prior to adding to the fermentation process. Preferably components SCD and QAC are contacted prior to adding and are thus added as a single composition to the fermentation process.

The fermentation process may be batch or continuous. In a batch fermentation, preferably the components SCD and QAC are added as a single composition (contacted prior to adding) or separately to the fermentation vessel prior to adding a fermentable sugar, more preferably prior to adding any of the reactants. In a continuous fermentation process, components SCD and QAC may be added as a single composition in a yeast propagation step or in the contacting step to the fermentation vessel. If more than one fermentation vessel is used in series, preferably the components are added to the first or primary fermentation vessel in the series.

Preferably SCD and QAC are added to the process prior to the production of significant amount of ethanol, or prior to the production of significant amounts of organic acid associated with growth of contaminant bacteria. By "significant amounts of ethanol", it is meant that the amount of ethanol in the fermentation broth is not more than 10 % by weight, based on the total volume of the fermentation broth. By

"significant amounts of organic acids", it is meant that the amount of organic acids in the fermentation broth is not more than 0.3% by weight, based on the total volume of the fermentation broth. Preferably SCD and QAC are added early in the fermentation process, such as during the propagation of inoculant yeast, before or just after inoculant yeast has been introduced into the fermentation vessel. In another embodiment, SCD and QAC are added to the fermentation process prior to the introduction of inoculant into the fermentation vessel. In this embodiment, SCD and QAC may be added to the fermentable sugar or process water or both, prior to introducing either of these reactants to the fermentation vessel and the combined SCD/QAC/fermentable sugar and/or SCD/QAC/process water is introduced to the fermentation vessel prior to introducing the inoculant to the fermentation vessel. For example SCD and QAPC may be added to one or more steps of providing the fermentable sugar, such as to one or more steps of a wet mill or dry grind process.

When the fermentation process includes a sugarcane- or sugar beet- or molasses-based fermentable sugar, it is typical to have fermentation vessels in series, having a first, second or even third or more fermentation vessel as a series of vessels where the fermentation broth is successively transferred into each fermentation vessel as part of the fermentation cycle. In this embodiment, SCD and QAC are preferably added to the first fermentation vessel.

Alternatively, in a fermentation process where sugarcane or sugar beet or molasses is used as the fermentable sugar, SCD and QAC are added to the fermentable sugar prior to contacting the fermentable sugar with inoculant. SCD and QAC may be added during the storage of the fermentable sugar. Such step of adding SCD and QAC during storage is contemplated herein as a step of the process for providing the fermentable sugar, prior to introducing the fermentable sugar into the fermentation vessel.

SCD and QAC may also be added to an empty fermentation vessel, prior to adding the reactants.

In another alternative, for a batch process in which sugarcane or molasses is the fermentable sugar, and in which the inoculant is recycled at the completion of each fermentation cycle, the SCD and QAC may be added to a yeast treatment stage (yeast propagation) prior to the beginning of a subsequent batch in a fermentation process. That is, the SCD and QAC are added separately, simultaneously, or as a combined composition, into the yeast recycle stream, which is commonly referred to as yeast cream. In one embodiment, SCD and QAC are added to the process water. Such a step of adding SCD and QAC to process water is contemplated herein as a step of the process for providing the process water, prior to introducing process water into the fermentation vessel. Process water includes any water that is introduced into the fermentation process from either external sources or recycled from other parts of the fermentation process.

In a molasses-based fermentation process, process water includes water used to dilute incoming molasses prior to fermentation, water added as part of the yeast preparation process, or water recycled from preceding fermentation steps, including yeast recycle stream.

In a dry grind fermentation process, process water includes water added into the steep tank, slurry tank, yeast propagation, or fermentation vessels. The process water may also be a recycle stream in a dry grind batch fermentation process, wherein the recycle stream is a stream produced after fermentation is complete, and is commonly referred to as backset or process condensate. These stages of the dry grind

fermentation process are well known to those skilled in the art. See, for example, R. J. Bothast and M. A. Schlicher, Applied Microbiology and Biotechnology as cited hereinabove.

The fermentation process may further comprise or alternatively comprise following the contacting step, adding SCD and QAC to the product of the contacting step which product comprises ethanol, separating ethanol from the product.

Alternatively, the fermentation process may further comprise, following the contacting step, separating the ethanol from the remaining product, wherein the remaining product is fed, for example, into a beer-well, or a thin stillage tank and adding the SCD and QAC to the beer-well or thin stillage tank.

Stabilized chlorine dioxide and the quaternary ammonium compound are added to the fermentation process in an amount to provide an effective amount to control growth of one or more contaminant microorganisms. Typically SCD is added in an amount to provide a concentration of the stabilized chlorine dioxide of 10 to 1000 ppm based on the total weight of the fermentation broth. Typically QAC is also added in an amount to provide a concentration of QAC of 10 to 1000 ppm based on the total weight of the fermentation broth. Preferably, SCD and QAC are added to the fermentation process to provide a concentration of SCD of 50 to 500 ppm, based on the total weight of the fermentation broth and a concentration of QAC of 50 to 500 ppm, based on the total weight of the fermentation broth. More preferably, SCD and QAC are added to provide a concentration of SCD of 50 to 200 ppm, based on the total weight of the fermentation broth and a concentration of QAC of 50 to 200 ppm, based on the total weight of the fermentation broth. Most preferably SCD and QAC are added to provide a concentration of SCD of 50 to 100 ppm, based on the total weight of the fermentation broth and a concentration of QAC of 50 to 100 ppm, based on the total weight of the fermentation broth.

Preservation method

As an embodiment of the present invention for controlling growth of contaminant microorganisms in a fermentation process, SCD and QAC may be added as a step in providing the fermentable sugar. More particularly, as defined herein, the fermentation process comprises process steps to produce and store carbohydrate feedstock wherein the carbohydrate feedstock comprises a fermentable sugar or is used to prepare a fermentable sugar. Thus, the step of providing a fermentable sugar may comprise contacting a carbohydrate feedstock with SCD and QAC. Thus, the method of this invention is also useful to prevent degradation in storage of a carbohydrate feedstock.

The carbohydrate content of the feedstock is at least 1% and preferably 1 to

70%, by weight, based on the total feedstock weight. The stabilized chlorine dioxide and quaternary ammonium compound are added in effective amounts. Typically SCD is added in an amount of 10 ppm to 1000 ppm of SCD, based on total weight of the feedstock, and QAC is added in an amount of 10 ppm to 1000 ppm of QAC, based on the total weight of the feedstock, wherein all amounts herein expressed as ppm are on a weight/weight basis. Preferably, SCD is added in an amount of 50 ppm to 500 ppm, more preferably 100 ppm to 500 ppm, and most preferably 100 ppm to 200 ppm of SCD, based on the total weight of the feedstock. Preferably, QAC is added in an amount of 50 ppm to 500 ppm, more preferably 100 ppm to 500 ppm, and most preferably 100 ppm to 200 ppm of QAC, based on total weight of the feedstock.

In this method, SCD and QAC are contacted with a carbohydrate feedstock in an effective amount to protect the carbohydrate from the growth of undesirable microorganisms and thus to prevent deterioration of the feedstock. Deterioration of the feedstock can be determined by the populations of contaminant microorganisms present, or the concentration of microbial metabolites, such as organic acids, that generally indicate unintended and undesirable microbial activity in the feedstock. Microorganisms are thus substantially prevented from proliferating in the stored or transported feedstock following the addition of SCD and QAC.

The present invention may be used to control contaminant microorganisms during storage and transport, preserving the carbohydrate feedstock.

Surprisingly, the carbohydrate feedstock treated according to this invention remains stable for at least one month. By "stable", it is meant herein the addition of SCD and QAC preserves the carbohydrate feedstock, where "preserve" is defined hereinabove as preventing reaction of or consumption of carbohydrate by contaminant microorganisms. A stable carbohydrate feedstock does not undergo an increase in the microbial population in the feedstock of more than 1 logi Q CFU/ml or 1 logi Q CFU/g. CFU, an abbreviation for colony forming unit, is a measure of microbial population in the feedstock. CFU is used to determine the number of viable microbial cells in a sample per unit volume or per unit mass, or the degree of microbial contamination in samples. A second measure of change is pH of the preserved feedstock. The pH of a properly preserved feedstock should not change by more than 0.5 pH units. However, as previously stated, pH change may not be sufficient under all circumstances to monitor preservation of a carbohydrate feedstock.

The carbohydrate feedstock is defined hereinabove.

In the present invention, the combination of SCD and QAC is used as a preservative for carbohydrate feedstocks to impede contaminant microorganism activity and subsequent deterioration of the carbohydrate feedstock as a step in providing a fermentable sugar. Contaminant microorganisms include bacteria as disclosed in WO 2007/149450 and contaminant yeast as disclosed in U.S. Patent Application Serial No. 12/467,728, filed May 18, 2009. The combination inhibits growth of certain bacteria that cause undesired decomposition of carbohydrates such as simple sugars to deleterious acids and also selectively to reduce the activity of contaminant yeasts. EXAMPLES

Example 1

In this example, stabilized chlorine dioxide (SCD) and a quaternary ammonium chloride (QAC) mixture were used in a checkerboard assay to determine whether their interaction was synergistic against Lactobacillus brevis. The checkerboard assay method is used to analyze interactions between compounds by mixing them in increasing concentrations to determine whether the compounds act synergistically, additively or antagonistically. (See, M. Najjar, D. Kashtanov, M. Chikindas, Letters in Applied Microbiology, vol. 45 (2007) 13-18.) L. brevis was cultured overnight in MRS broth (deMan Rogosa and Sharp broth, Difco Laboratories, Inc., Sparks, MD) at 32°C. The culture was then diluted and resuspended in fresh MRS broth adjusted to pH 5.5. The number of bacteria in the resuspended culture was determined using standard plate counts. Samples were diluted (1 : 10) in sterile phosphate -buffered saline

(Sigma- Aldrich Co., St. Louis, MO) and plated onto the surface of MRS plates. The resuspended culture (150 μΐ) was then used to fill the wells of a 96 well plate (Becton, Dickinson and Co., Franklin Lakes, NJ). SCD (FermaSure® fermentation additive, E. I. du Pont de Nemours and Co., Wilmington DE) and a quaternary ammonium compound (BTC 1010, a solution of didecyl dimethyl ammonium chloride, DDAC, Stepan Company, Northfield, IL) were then added at the concentrations shown in the

"checkerboard" plate diagram below. The plate was covered and incubated for 28 hours at 32°C. Following incubation, the plate was removed and the optical density of each well was determined using a plate reader at 630 nm (Power Wave, BioTek Instruments Inc., Winooski, VT). Based on the optical density, each well was scored to determine whether there was no inhibition of growth (NI), partial inhibition (PI), or complete inhibition (CI). Results are provided in Table 1.

To determine whether any synergistic activity occurred between SCD and the quaternary ammonium compound, the fractional inhibitory concentration (FIC) was determined using the formula: FIC= [SCD]/MICSCD + [DDAC]/MIC _D DAC where [SCD] and [DDAC] are the concentrations of SCD and BTC 1010 that result in partial inhibition and MICSC _D and MIC _DD AC are the minimum inhibitor concentration of SCD and DDAC, respectively. Synergy between the two antimicrobials is determined when the resulting FIC is <1.0.

Table 1. Checkerboard assay to determine response of Lactobacillus brevis to treatment with SCD and a mixture of

didecyl dimethyl ammonium chlorides (DDAC) in MRS broth at pH 5.5.

SCD

0 5 12.5 15 25 37.5 50 62.5 75 ppm ppm ppm ppm ppm ppm ppm ppm ppm

0 NI NI NI NI NI PI PI PI CI ppm

2 PI PI PI PI PI PI PI PI ppm

4 PI PI PI PI PI PI CI CI ppm

DDAC

5 PI PI PI PI PI CI

ppm

6 PI PI CI CI CI

ppm

7 CI CI

ppm

For the checkerboard assay above, the FIC is determined to be 0.61 , indicating a synergistic effect for the interaction between SCD and the DDAC quaternary ammonium compound against Lactobacillus brevis. Thus, Example 1 demonstrates that the combination of DDAC quaternary ammonium compound and SCD will enable a reduction in the dose of SCD required to inactivate bacteria in a fermentation process. Example 2

In this example, SCD (FermaSure® fermentation additive, E. I. du Pont de Nemours and Co., Wilmington DE) and a second quaternary ammonium compound (BTC 2125, a mixture of n-alkyl dimethyl benzyl ammonium chlorides and n-alkyl dimethyl ethylbenzyl ammonium chlorides, ADBAC/ADEBAC, Stepan Company, Northfield, IL) were used in a checkerboard assay to determine whether their interaction was synergistic against Lactobacillus brevis. L. brevis was cultured as above, resuspended in MRS at pH 5.5, and then added (150 μΐ) the wells of a 96 well plate (Becton Dickinson, Franklin Lakes, NT). SCD (FermaSure®, DuPont,

Wilmington DE) and ADBAC/ADEBAC (BTC 2125) were then added at the concentrations shown in the "checkerboard" plate diagram below. The plate was covered and incubated for 28 hours at 32°C. Following incubation, the plate was removed and the optical density of each well was determined using a plate reader at 630 nm. Based on the optical density, each well was scored to determine whether there was no inhibition (NT), partial inhibition (PI), or complete inhibition (CI). Results are provided in Table 2.

Table 2. Checkerboard assay to determine response of Lactobacillus brevis to treatment with SCD and ADBAC/ADEBAC in MRS broth at pH 5.5

SCD

0 5 12.5 15 25 37.5 50 62.5 75 ppm ppm ppm ppm ppm ppm ppm ppm ppm

0 NI NI NI NI PI PI PI PI CI ppm

2 PI PI PI PI PI PI PI PI ppm

4 PI PI PI PI PI PI PI CI

ADBAC / ppm

ADEBAC 5 PI PI PI PI PI PI CI

ppm

6 PI PI PI PI PI CI

ppm

7 CI CI CI CI CI

ppm

To determine whether any synergistic activity occurred between SCD and the

ADBAC/ADEBAC quaternary ammonium compound, the fractional inhibitory concentration (FIC) was determined as above using the formula: FIC= [SCD]/MICSCD + [ACBAC/ADEBAC]/MIC _A DBAC/ADEBAC where [SCD] and [ACBAC/ADEBAC] are the concentrations of SCD and BTC 2125 that result in partial inhibition and MICSC _D and MICA _DB AC/A _DEB AC are the minimum inhibitor concentration of SCD and ACBAC/ADEBAC, respectively. Synergy between the two antimicrobials is determined when the resulting FIC is <1.0.

For the checkerboard assay above, the FIC is determined to be 0.61 , indicating a synergistic effect for the interaction between SCD and ADBAC/ADEBAC against Lactobacillus brevis. Thus, Example 2 demonstrates that the combination of the quaternary ammonium compounds and SCD will enable a reduction in the dose of SCD required to inactivate bacteria in a fermentation process.

Example 3

In this example, samples of recycle streams comprising primarily inoculant yeast were collected from a molasses-based ethanol plant in Brazil. The samples were used to complete bench-scale fermentations in which the samples were treated with a combination of SCD (FermaSure® fermentation additive, E. I. du Pont de Nemours and Co., Wilmington DE) with didecyl dimethyl ammonium chloride (BTC 1010, a solution of didecyl dimethyl ammonium chloride, DDAC, same as in Example 1), as quaternary compound. The treated samples were used to carry out successive 8-hour long fermentations, using recovered yeast at the end of each cycle to begin the next run. Viable bacteria were enumerated over 5 fermentation cycles. Results are provided in Table 3 where ND means the combination was not tested.

Table 3. Efficacy of SCD and DDAC combination in yeast recycle stream from a molasses-based fermentation

Log Viable Bacteria

DDAC Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 0 ppm 0 ppm 7.77 7.86 8.01 9.01 ND

75 ppm 0 ppm < 5 < 5 6.31 6.52 6.69

45 ppm 60 ppm < 5 < 5 < 5 6.39 6.39

0 ppm 60 ppm 7.79 7.97 ND ND ND

As shown in Table 3, the combination of DDAC plus SCD showed superior efficacy compared to either of the treatments alone. When 45 ppm SCD and 60 ppm of the quaternary ammonium compound were used to treat the recycle stream, the number of viable bacteria remained lower than all the other treatments through 5 cycles of fermentation. As further shown in Table 3, the quaternary ammonium compound by itself did not result in any significant inactivation of bacteria.

Example 4

In this example, a molasses-based fermentation medium was prepared and inoculated with both yeast and lactic acid bacteria. The medium comprised various components as follows:

Molasses 17.65%

Urea 0.5%

Potassium phosphate 0.2%>

Yeast extract 0.1%

Water 81.55%

Following inoculation, the mixed culture was grown overnight in the molasses medium, after which a recycle stream was prepared by centrifuging the broth at 3000 rpm for 5 minutes. The resulting pellet was separated from the supernatant and reconstituted using sterile deionized water, then adjusted to pH 3.0 using sulfuric acid. 5 ml portions of the reconstituted pellet were then treated as follows:

1. Untreated

2. QAC only (60 ppm)

3. SCD (45 ppm) 4. SCD/QAC (45 ppm/ 60 ppm)

The SCD was FermaSure® fermentation additive, E. I. du Pont de Nemours and Co., Wilmington DE. The quaternary ammonium compound (QAC) was the same DDAC (BTC 1010, Stepan Company, Northfield, IL) as used in Example 1.

Samples were stored in a shaking incubator at 32°C for 2 hours following which

0.1ml aliquots were serially diluted and plated on selective media to enumerate surviving yeast and bacteria cells. After 2hours of incubation, the samples were reconstituted with molasses medium (up to 25 ml) and reincubated at 32°C for 24 hours to simulate a second fermentation cycle. Following this cycle, the SCD and SCD/QAC treated samples were centrifuged as before, and the number of viable yeast and bacterial cells in the resulting pellet was determined. Results are provided in Table 4.

Table 4: Viable bacteria and yeast cells in a molasses-based recycle stream treated with

SCD, QAC, or combinations of SCD and QAC

Treatment Viable Cells* (2 hrs) Viable cells

(24 hrs)

Yeast Bacteria Bacteria

Untreated 8.2 8.2

QAC only 8.8 8.1

SCD only 9.0 6.5 7.98

SCD/QAC 8.8 6.2 6.38

^Numbers of viable cells are represented as log colony forming units (logCFU) per milliliter.

As shown in Table 4 above, the number of viable bacteria was reduced by treatment using SCD or combinations of SCD and QAC. However, the number of viable bacteria was lower after 24 hours in the sample treated with the SCD/QAC combination compared to SCD alone. Treatment with QAC alone did not result in any reduction in viable bacterial cells.