BRPI1000543A2 - Method for the production of a fermentation product from a sugar hydrolyzate - Google Patents

Abstract

METHOD FOR THE PRODUCTION OF A FERMENTATION PRODUCT FROM A SUGAR HYDROLYSATE. The present invention relates to a method for producing a fermentation product of a sugar hydrolyzate. The method comprises fermenting the sugar hydrolyzate in a yeast fermentation system to produce a fermentation broth comprising a fermentation product; introduction of acid and an oxidant, such as chlorine dioxide, to the fermentation system in order to expose microbial contaminants in the fermentation system in one or more stages to chlorine dioxide and a pH less than 3.0; and recovering the fermentation product. In an example of the invention, a yeast slurry obtained from a yeast recycling step is treated with acid and the oxidizer.

Description

Report of the Invention Patent for "METHOD FOR PRODUCING A FERMENTATION PRODUCT FROM A SUGAR HYDROLISATE".

Field of the Invention

The present invention relates to a method for the production of

a fermentation product. More specifically, the present invention relates to a method for producing a fermentation product of a sugar hydrolyzate. Background of the Invention

Lignocellulosic feedstock is a term commonly used to describe plant-derived biomass comprising celluloses, hemicellulose and lignin. Much attention and effort has been applied in recent years to the production of fuels and chemicals, especially ethanol, from lignocellulosic feed stocks, such as agricultural and forest clearance, due to their low cost and wide availability. These agricultural and forest dumps are typically burned or buried; Thus using these lignocellulosic ethanol feed stocks offers an attractive alternative for availability. Yet another advantage of these feed stocks is that the lignin byproduct, which remains after the pulp production process, can be used as a fuel to energize the process instead of fossil fuels. Several studies have concluded that when total production and consumption cycle is taken into account, ethanol production from lignocellulosic feed stocks generates greenhouse gases close to zero.

The first chemical processing step for converting lignocellulosic feedstock into ethanol, or other fermentation products, involves breaking down the fibrous lignocellulosic material to release sugar monomers from feedstock for conversion into a fermentation product in a subsequent step. of fermentation.

There are several known methods for producing fermentable sugars from lignocellulosic feed stocks, one of which involves pretreatment with acid or alkali, followed by cellulose hydrolysis with cellulase and β-glucosidase enzymes. The purpose of the pretreatment is to increase the cellulose surface area and convert the fibrous feedstock into a cloudy texture with limited conversion of cellulose to glucose. Acid pretreatment typically hydrolyzes the hemicellulose component of the feedstock to produce xylose, glucose, galactose, mannose and arabinose and this is thought to improve cellulose accessibility in cellulase enzymes. Cellulase enzymes hydrolyze cellulose into cellobiose which is then hydrolyzed to glucose by β-glucosidase. Cellulose and hemicellulose hydrolysis can be achieved with a single step chemical treatment in which the lignocellulosic feedstock is contacted with a strong acid or alkali under conditions sufficient to hydrolyze both the cellulose and hemicellulose components of the feedstock. sugar monomers.

Following production of a stream comprising fermentable sugar from the lignocellulosic feedstock, a solids separation may be conducted to remove lignin, followed by fermentation of the sugars in ethanol or other fermentation products. If glucose is the predominant substrate present, fermentation is typically performed with a yeast of sp. Saccharomyces spp. which converts this sugar and other hexose sugars present in ethanol. However, glucose may also be fermented to other commercial products including lactic acid, sorbitol, acetic acid, citric acid, ascorbic acid, propanediol, butanediol, xylitol, acetone, and butanol. This conversion can be performed by a variety of organisms, including sp. Saccharomyces.

Pentose, xylose and arabinose sugars, which occur from the hemicellulose component of the feedstock during acidic pretreatment, can be fermented in ethanol. However, a vast majority of wild type Saccharomyces strains do not naturally contain all the genes required for conversion of these sugars to ethanol. Therefore, they must be introduced into the yeast to allow this conversion. Recombinant yeasts that are capable of converting xylose to ethanol are described, for example, in United States Patent Nos. 5,789,210 and 6,475,768 and EP 1,727,890.

A problem with the fermentation of sugar in ethanol or other fermentation products is that bacteria can spread rapidly, since optimal fermentation conditions are also conducive to their growth. Unwanted by-products that can be produced by bacterial contaminants during fermentation include lactic acid, acetone, propionic acid and mycotoxins. Lactic acid is a common byproduct produced by bacteria such as sp. Lactobacillus, sp. Pediococcus, sp. Leuconostoc and / or sp. Weissella (among others) during ethanol fermentations. Production of such undesirable by-products decreases the production of the desired fermentation product as the bacteria competes with yeast for fermentable sugars and converts them into undesirable by-products through the fermentation product of interest. In addition, organic acids and other byproducts may be inhibitory to yeast. Each of these factors can contribute to lower fermentation efficiency by increasing the time required to effect fermentation, increasing the amount of yeast required and / or decreasing the final yields for the desired fermentation product from fermentable sugars.

Microbial contamination is especially problematic when the yeast concentration in the fermenter is increased by yeast recycling. Yeast recycle is employed to improve the efficiency of fermentation processes that are subjected to slow glucose-related reaction kinetics such as those involving the conversion of xylose to ethanol or when it is beneficial for increasing volumetric conversion rates. Increasing the volumetric rate of conversion of fermentable sugar to ethanol can be achieved by continuously separating the yeast from the collected fermentation broth, such as by centrifugation, and then recirculating the yeast back to the fermenter. By reintroducing the yeast into the reactor in this manner, the yeast concentration in the fermenter is continuously maintained at a high level with no significant shift of sugars to cell growth and out of the desired fermentation product. However, as a result of repeated yeast recirculation, unwanted microbes such as bacteria are also recycled along with the yeast. Because bacteria tend to divide faster than yeast, this can lead to significant levels of microbial contamination.

de Oliva-Neto and Yokoya (Brazilian Journal of Microbiology, 2001, 3: 10-14) examined the effect of a variety of antimicrobial compounds on the viability of Saccharomyces eerevisiae, Lactobacillus, and Leueonostoe in cane juice fermentations to produce ethanol. This includes formulated chemicals such as zinc manganese ethylenebis (dithiocarbamate), methyl dithiocarbamate, 3-methyl-4-chlorophenol, 2-benzyl-4-chlorophenol and o-phenylphenol, 2-chloroacetamide and others, which are commonly recommended. for use in microbial control in sugar and alcohol factories. Antibiotics tested include penicillum, clindamycin and cephamandole. The results showed that current biocides used in industrial fuel alcohol fermentations reduce yeast viability, while antibiotics were effective in reducing bacterial growth without affecting yeast viability.

However, the use of antibiotics in fuel ethanol applications has its limitations as microbial contaminants are known to develop antibiotic resistance (Lushia and Heist, 2005, Ethanol Producer Magazine, Antibiotic-Resistant Bacteria in Fuel Ethanol Fermentati- ons). In addition, antibiotics can be conducted through dry distillers grain, which is a by-product of commercial ethanol plants used in animal feed, and this valuable by-product cannot be sold if antibiotics are used in the plant.

Bacterial control in alcoholic fermentation of industrial fuel may also be effected by washing with sulfuric acid from yeast cell suspensions. Commercial fuel ethanol in Brazil is produced by continuous or batch-fed fermentation of sugarcane by Saccharomyees eerevisiae and employs light-cell cell recycling (from Oliva-Neto and Yokoya, supra). The purpose of acid treatment is to destroy contaminating microorganisms that cannot withstand low pH conditions without a substantial reduction in yeast viability or fermentative capacity.

US2009 / 0117633 describes a process for producing corn ethanol in which combined saccharification and fermentation are conducted at pH values such as 3.5 to 4.0. The enzymes used in saccharification are amylases that are adapted for starch hydrolysis under these relatively low pH values. Low pH saccharification / fermentation is conducted with a view to reducing bacterial contaminants such as lactic acid and acetic acid bacteria. Lactic acid and acetic acid contaminating bacteria grow best at pH 5.0 and above. Thus, in the pH range 3.0 to 4.5, it is believed that ethanol fermentation will predominate because yeast will grow better than contamination bacteria.

The use of oxidants to control microbial contamination in ethanol fermentations is also known. For example, Chang et al. (Appl. Environ. Microbiol., 1997, 63: 1-6) describes the use of hydrogen sulfide and peroxide to control bacterial contamination in the fermentation of malt extract in ethanol with yeast recycle.

Chlorine dioxide is an oxidant that is known to have a bacteriocidal effect and has been used as a drinking water disinfectant and in the food and beverage industry. There are several known methods for producing chlorine dioxide, (see Alternative Disinfectants and Oxidants Guidance Manual, United States Environmental Protection Agency, April 1999, Chapter 4. Chlorine dioxide, which is incorporated herein by reference), one of the which involves reacting sodium chlorite with acid according to the following reaction:

5NaCl2 + 4H + ^ 4Cl2 + 4Na + + Na + Cr + 2H20.

Sodium chloride is often referred to as "stabilized chlorine dioxide" or "SCD".

The use of chlorine dioxide in ethanol fermentations is known as stated in WO 2007/097874, WO 2009/026706, WO 2007/149450 and Johnson and Kunz (The New Brewer, 1998, Coming Clean - A New Method of Washing Yeast Using Chloro Dioxide Vol. 15 # 5-P56). WO 2007/097874 describes a process in which chlorine dioxide is added to a fermentation tank, a fermentable carbohydrate added to a fermentation tank, or a propagation or conditioning system used to prepare the inoculum for a fermentation. WO 2009/026706 describes the use of chlorine dioxide to reduce bacterial contamination in a fermentation process employing yeast recycling and utilizing sugars from lignocellulosic feed stocks. Chlorine dioxide was used to treat a separate yeast slurry from the fermentation prior to its reintroduction to the fermenter. WO 2007/149450 describes a method for preventing the growth of bacterial contaminants in yeast fermentations to produce ethanol by the addition of stabilized chlorine dioxide. Stabilized chlorine dioxide was added prior to any significant propagation of bacteria in the system, such as for the inoculant or for fermentable sugars prior to their introduction into the fermentation system. As the pH of the solution is lowered due to the generation of organic acids produced by bacterial contaminants, activated chlorine dioxide is generated in situ from stabilized chlorine dioxide and further bacterial growth has been prevented. Johnson and Kunz (The New Brewer, 1998, Coming Clean - The New Method of Washing Yeast Using Chlorine Dioxide Vol. 15 # 5-P56) describes the use of chlorine dioxide to wash milk during brewing as an alternative to acid washing.

The effects of CIO2 concentration on bacterial cell death and yeast viability and fermentative capacity have been examined in ethanol fermentations (see copending and same applicant WO 2009/026706), but less information is available regarding the effect of other variables. on the efficiency of chlorine dioxide such as pH. However, the impact of pH on chlorine dioxide efficiency in other industrial applications has been studied. In the beverage industry, it has been reported that chlorine dioxide has a constant efficiency at a pH level between 4 and 10, with the sterilization rate being higher than at high pH. ("Chloro Dioxide in the Beverage Industrial", Petplanet Insider September 2005, 6: 46-47). Chlorine dioxide bleaching stages in pulp bleaching applications are conducted at acidic pH values, although there is still some controversy about optimal pH (Reeve, 1996, Section IV: The Technological of Chemical Pulp Bleaching) , Chapter 3: Chloro Dioxide in Delignification In Pulp Bleaching, Principles and Practice, Ed. By Dence and Reeve, Tappi Press). Foegeding et al. (1986, Journal of Food Science, 51 (1): 197-201) evaluated chlorine dioxide inactivation of Bacillus and Clostridium spores in buffered water at pH values of 4.5, 6.5 and 8.5 with phosphoric acid and it was found that C. perfuringens spores were inactivated more at pH 8.5 than at 6.5.

Summary of the Invention

The present invention provides a method for producing a fermentation product. More specifically, the present invention relates to a method for producing a fermentation product of a sugar hydrolyzate.

It is an object of the invention to provide an improved method for producing a sugar hydrolyzate fermentation product.

According to a first aspect of the invention, there is provided a method (A) for producing a sugar hydrolyzate fermentation product comprising: (i) fermenting the sugar hydrolyzate in a fermentation system to produce a fermentation broth comprising - giving a fermentation product; (ii) introducing acid and an oxidant, including, but not limited to chlorine dioxide, to said fermentation system in order to expose any microbial contaminants in said fermentation system at one or more stages to chlorine dioxide at a pH of less than 3.0; and (iii) recovery of the fermentation product.

According to a second aspect of the invention there is provided a method (B) for obtaining a sugar hydrolyzate fermentation product comprising: (i) removing suspended fiber solids from the sugar hydrolyzate to obtain a whitened sugar solution; (ii) sugar fermentation in the bleached sugar solution in a fermentation reaction using yeast to produce a fermentation broth comprising the fermentation product; (iii) separating yeast from the fermentation broth to produce a yeast slurry and a fermentation product, (iv) introducing chlorine acid and chlorine dioxide to the yeast slurry to expose any contaminants microbial and light in said chlorine dioxide yeast slurry at a pH of less than 3.0; (v) reintroducing at least a portion of the chlorine dioxide-treated yeast slurry back to the fermentation step, step (ii), to maintain the yeast concentration in the fermentation reaction; and (vi) recovery of the fermentation product.

In embodiments of any of the preceding aspects of the invention, the sugar hydrolyzate comprises at least xylose or glucose. In another embodiment, the sugar hydrolyzate comprises both xylose and glucose. Sugar hydrolysate can be obtained from a lignocellulosic feedstock. This may involve a pre-treatment step of the lignocellulosic feeding with acid or alkali.

According to another embodiment of any aspect of the invention, the acid and oxidant are introduced to said fermentation system in order to continuously expose any microbial contaminants in said fermentation system at one or more stages for the oxidant at a pH. less than 3.0.

Without limitation, the product produced by fermentation may be either ethanol or xylitol. If ethanol is the fermentation product, it can be produced by a sp. Saccharomyces that converts glucose and xylose to ethanol. If xylitol is the fermentation product, it can be produced by a sp. Candida that converts xylose to xylitol.

According to embodiments of any aspect of the invention, in the introduction step, chlorine dioxide is at a concentration of between about 0.5 and about 1500 ppm. In one example of the invention, chlorine dioxide is at a concentration of from about 100 to about 500 ppm. Preferably, microbial contaminants and yeast are exposed to chlorine dioxide at a pH of more than about 1.0, but less than 2.5. In one example of the invention, the acid is added rather than chlorine dioxide.

When yeast recycling is employed according to the second aspect of the invention, the whitened sugar solution resulting from the fiber solids removal step may comprise sugar selected from the group consisting of glucose, xylose, galactose, mannose. , arabinose, fucose and fructose. In further embodiments of this aspect of the invention, the fermentation step may be conducted in one of a series of fermentation reactors, and the chlorine dioxide-treated yeast slurry is then reintroduced back to it or a Different fermentation reactor in the series. Preferably, when the yeast slurry is treated with acid and chlorine dioxide, the temperature of the yeast slurry is between about 4 ° C and about 37 ° C.

The concentration of microbial contaminants in the yeast slurry may be reduced to at least 100 times lower than that of yeast. In another embodiment, the concentration of microbial contaminants in the yeast slurry is reduced to below about 103 CFU / mL.

In yet another embodiment, the concentration of yeast cells in the yeast slurry is from about 10 g / l to about 300 g / l, or from about 20 g / l to about 200 g / l (weight dry cell).

The present invention overcomes difficulties in the prior art in conjunction with the efficient conversion of feedstock into ethanol or other fermentation products due to the presence of microbial contaminants. In particular, the invention is based on the discovery that the combined effect of an oxidant and low pH may result in significant improvements in reducing microbial contaminants in fermentation systems. Advantageously, the process of the present invention does not result in any substantial reduction in yeast viability or fermentative capacity. Therefore, the yield of the desired fermentation product and the purity of the fermentation product can be significantly improved compared to conventional systems. In addition, due to improved efficiency at lower pH, lower levels of an oxidant may be required for non-operated processes at pH values below 3.0. Advantageously, this can reduce the chemical demand and thereby the cost of the process.

Brief Description of the Drawings

These and other features of the invention will become more apparent from the following description in which reference is made to the accompanying drawings, in which:

FIGURE 1 shows a process flow diagram illustrating yeast recycling during acid addition fermentation followed by chlorine dioxide to a yeast slurry (also referred to herein as "yeast cream") obtained after separating yeast from the fermenter according to one embodiment of the invention.

FIGURE 2 is a bar graph showing bacterial counts (CFU / mL) of a contaminated yeast cream after treatment with a mass dose of 0, 200 and 500 ppm chlorine dioxide or at pH 5 (full bars), or preceded by by titration with sulfuric acid to pH 2 (open bars). FIGURE 3 is a bar graph showing bacterial counts (CFU / mL) of a contaminated yeast cream after treatment with no chlorine acid or dioxide (control), titration to pH 2 with mineral acid with no chlorine dioxide treatment. (pH control) and titration to pH 2, followed by treatment with a mass dose of 50, 100, 150 and 200 ppm chlorine dioxide.

FIGURE 4 is a bar graph showing yeast cell counts (CFU / mL) of a contaminated yeast cream after treatment with 0, 200 and 500 ppm chlorine dioxide or at pH 5 (full bars), or preceded by by titration with sulfuric acid to pH 2 (open bars). FIGURE 5 is a bar graph showing ethanol fermentation yields of glucose and xylose and xylose entry rates. Fermentation of lignocellulosic hydrolyzate was performed with contaminated yeast cream after treatment with 0, 200 and 500 ppm chlorine dioxide preceded by titration with ρH 2 sulfuric acid. FIGURE 6 is a bar graph showing bacterial counts (CFU / mL) of a yeast cream obtained from an industrial fermentation before and after acid titration at a pH of 2,0 and before and after combined treatment of chlorine dioxide and acid to reach a pH of 2,0. Full bars and open bars indicate bacterial counts before treatment and after treatment, respectively.

Detailed Description

The present invention relates to a method for producing a fermentation product from a lignocellulosic feedstock.

The following description is of one embodiment by way of example only and without limitation to the combination of features necessary to effect the invention in effect.

The sugar hydrolyzate for the process may be derived from sugar and starch groups including, but not limited to, wheat, corn, sugar beets and sugarcane. Methods for producing fermentable sugar containing sugars hydrolyzate from such feed stocks are well known.

The feedstock for the process of the present invention may also be a lignocellulosic material, which includes any type of plant biomass such as, but not limited to, non-woody plant biomass, cultivated groups such as, but not limited to, grams, for example, but not limited to C4 grams, such as switch grass, rope grass, rye grass, miscanthus, reed grass, or a combination thereof, sugar processing wastes, for example, but not limited to, bagasse, such as sugarcane bagasse, beet pulp, or a combination thereof, agricultural waste, for example, but not limited to, soybean fodder, maize fodder , rice straw, rice husks, barley straw, sugar cane straw, corncobs, wheat straw, canola straw, oat straw, oat husks, corn fiber, or a combination forest biomass, for example, but not limited to, fiber recycled wood pulp, sawdust, hardwood, for example, aspen wood, or a combination thereof. In addition, the lignocellulosic feedstock may comprise cellulosic dump material or forest dump materials such as, but not limited to, newsprint, cardboard and the like. The lignocellulosic feedstock may comprise one kind of fiber or, alternatively, the lignocellulosic feedstock may comprise a mixture of fibers originating from different lignocellulosic feedstock.

Lignocellulosic feed stocks comprise in a cellulose in an amount greater than about 20%, more preferably greater than about 30%, more preferably greater than about 40% (w / w). For example, the lignocellulosic material may comprise from about 20% to about 50% (w / w) of cellulose, or any amount in between. The lignocellulosic feedstock also comprises lignin in an amount greater than about 10%, more typically in an amount greater than about 15% (w / w). The lignocellulosic feedstock may also comprise small amounts of sucrose, fructose and starch. Additionally, the food supply may contain pectin.

The present invention may be practiced with a pre-treated lignocellulosic feed material. Pretreatment methods are intended to deliver a sufficient combination of mechanical and chemical action to disrupt the fiber structure and increase the surface area of feedstock to make it accessible to hydrolytic enzymes such as cellulases. Mechanical action typically includes the use of pressure, grinding, grinding, stirring, tearing, compression / expansion, and mechanical action includes the use of heat (often steam), acid or alkali, and solvents.

Pretreatment is preferably a chemical treatment involving the addition of acid or alkali. This includes any acid or alkali that is suitable for disrupting the fiber structure of the lignocellulosic feedstock and increasing the accessibility of the lignocellulosic feedstock to be hydrolyzed in subsequent enzymatic hydrolysis. Nonlimiting examples of acid and alkali suitable for such a proposal include sulfuric acid, nitric acid, hydrochloric acid, sulfurous acid, phosphoric acid, ammonia, ammonium hydroxide, sodium hydroxide, potassium hydroxide, lime and magnesium hydroxide.

Pretreatment with acid hydrolyzes hemicellulose, or a portion thereof, that is present in the lignocellulosic feedstock for monomeric sugars including, but not limited to, xylose, arabinose, mannose, and / or galactose, and Organic acids such as acetic acid, galacturonic acid and glucuronic acid. Sucrose, fructose and starch may also be present in sugar hydrolyzate. Preferably, the acid pretreatment is carried out so that almost complete hydrolysis of hemicellulose and a small amount of cellulose to glucose conversion occurs. Cellulose is hydrolyzed to glucose in a subsequent step using cellulase enzymes. Typically a dilute acid, at a concentration of about 0.02% (w / v) to about 2% (w / v), or any amount in between (measured as the weight percent pure acid in total weight dry stock plus aqueous solution) is used for pretreatment. Preferably, the acid pretreatment is carried out at a temperature of from about 180 ° C to about 250 ° C, or any temperature between them, for a time from about 60 seconds to about 600 seconds, or any other temperature. between them at a pH of about 0.8 to about 2.0, or any pH between them.

One method of performing acid pretreatment of the feedstock is steam blast using the process conditions described in U.S. Patent No. 4,461,648 (which is incorporated herein by reference). Another method of pretreatment feedstock slurry involves continuous pretreatment, meaning that the lignocellulosic feedstock is pumped through a reactor continuously. Continuous acid pretreatment is familiar to those skilled in the art, see, for example, U.S. Patent 5,536,325, WO 2006/128304 and U.S. Patent No. 4,237,226 (which are incorporated herein by reference). Other techniques which are known in the art and which may be used as required include, but are not limited to, those described in U.S. Patent No. 4,556,430 (Converse et al; which is incorporated herein by reference).

After pretreatment, the lignocellulosic feedstock may be separated to obtain a solids stream comprising the pretreated feedstock and an aqueous stream comprising soluble components. This can be accomplished by washing the pretreated feedstock with an aqueous solution to produce the wash stream, and a solids stream comprising the pretreated feedstock. Alternatively, the pretreated feedstock is subjected to solid-liquid separation using known methods such as centrifugation, microfiltration, frame and plate filtration, cross flow filtration, pressure filtration, vacuum filtration, and the like. . When an acidic pretreatment is employed, the aqueous phase comprises sugars produced by hemicellulose hydrolysis, as well as the acid added during the pretreatment and any organic acids released during the pretreatment. This stream may subsequently be processed to remove mineral acid and organic acid, and then optionally fed back to the solids stream comprising the pretreated feed stock. The aqueous stream obtained from the acid pretreated feedstock may also undergo fermentation to ferment the sugars. For example, xylose present in this stream may be fermented to alcohols, including ethanol and butanol; sugar acids including xylonic acid and arabic acid; sugar alcohols including xylitol, arbitol, erythritol, galactitol and mannitol; organic acids including citric acid, malic acid, succinic acid, pyruvic acid, acetic acid, itaconic acid and lactic acid; ketones including acetone; and amino acids, including glutamic acid.

Pretreated lignocellulosic feedstock is typically fluidized in an aqueous solution such as process water, fresh water, steam condensate or process recycle streams. The concentration of pretreated lignocellulosic feedstock in the slurry depends on particle size, water retention, pump capacity and other properties of the feedstock. Typically, the concentration is between about 3% and 30% (w / w), or any amount between fiber solids (also known as suspended or undissolved solids), or between about 10% and about 20% (w / w) fiber solids, or any amount in between. The concentration of fiber solids may be higher if dehydration of the feedstock slurry is effected prior to pretreatment, for example as set forth in PCT / CA2009 / 001191 (incorporated herein by reference). The aqueous slurry preferably has a solids concentration which enables it to be pumped. As is well known in the art, the concentration of suspended or undissolved solids may be determined by filtration of a sample of the slurry using microfiber glass filter paper, washing the filter mass with water, and drying the mass overnight at 105 ° C. It is preferred that fiber solids comprise at least about 20% to about 70% cellulose by weight, or any weight percent thereof. For example, fiber solids may comprise 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% by weight of cellulose.

The pH of the pretreated feedstock is typically adjusted so that it is within a range that is optimal for the cellulase enzymes used. Generally, the pH of the pretreated feedstock is adjusted to be within a range of about 3.0 to about 7.0, or any pH in between. For example, the pH may be within a range of from about 4.0 to about 6.0, or any pH between them, between about 4.5 and about 5.5, or any pH between them, or about 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, or any pH in between.

The temperature of the pretreated feedstock is adjusted so that it is within the optimal range for cellulase enzyme activity. Generally, a temperature of about 45 ° C to about 55 ° C, or any temperature in between, is suitable for many cellulase enzymes, for example a temperature of 45, 46, 48, 49, 50, 51, 52, 53, 54, 55 ° C, or any temperature between them.

Cellulase enzymes and β-glucosidase enzyme are added to the pretreated feedstock before, during, or after adjusting the temperature and pH of the aqueous slurry after pretreatment. Preferably, the cellulase enzymes and the β-glucosidase enzyme are added to the pretreated lignocellulosic feedstock after adjusting the temperature and pH of the slurry.

By the term "cellulase enzymes" or "cellulases," is meant a mixture of enzymes that hydrolyze cellulose. The mixture may include cellobiohydrolases (CBH), endoglucanases (EG) and β-glucosidase. In a non-limiting example, a cellulase mixture may include CBH, EG and β-glucosidase enzymes. The CBH enzyme mainly hydrolyzes cellulose polymer chains from their ends to release cellobiose, and the EG enzyme mainly hydrolyzes cellulose polymer in the middle of the chain. If the pretreated feedstock comprises xylan, it is especially advantageous if enzyme hydrolysis is also performed in the presence of one or more xylanase enzymes. Examples of xylanase enzymes that may be used for this purpose include xylanase 1, 2 (Xyn1 and Xyn2) and β-xylosidase, which are typically present in cellulase mixtures.

The conversion of cellobiose to glucose is effected by the enzyme β-glucosidase. By the term "β-glucosidase" is meant any enzyme that hydrolyzes the glucose dimer, cellobiose, into glucose. The activity of the enzyme β-glucosidase is defined by its activity by the Enzyme Commission as EC 3.2.1.21.

The process of the present invention may be carried out with any type of cellulase enzymes suitable for glucose hydrolysis, regardless of their source. Non-limiting examples of cellulases that may be used in the practice of the invention include those obtained from fungi of the genus Aspergillus, Humicola, Chrysosporium, Myceliopthora, Penicillium, Neurospora and Trichoderma, and bacteria of the genus Bacillus and Thermobifida.

The cellulase enzyme dosage is chosen to convert the cellulose from the pretreated feedstock into glucose. For example, an appropriate cellulase dosage may be from about 0.1 to about 40.0 Paper Filter Units (FPU or IU) per gram of cellulose, or any amount in between, for example 0.1. 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 14.0, 16.0, 18.0, 20.0, 22 , 0, 24.0, 26.0, 28.0, 30.0, 32.0, 34.0, 36.0, 38.0, 40.0 FPU (or UI) per gram of cellulose, or any amount between these.

Enzymatic hydrolysis with cellulase enzymes yields a solution comprising glucose, unconverted cellulose, and lignin. Other components that may be present in the hydrolyzate slurry include xylose, arabinose, mannose and galactose, acetic acid, glucuronic acid, and galacturonic acid, as well as silica, insoluble salts and other compounds.

Although the production of a sugar hydrolyzate by pretreatment followed by cellulose hydrolysis from the cellulase enzyme pretreated feedstock is described, it should be understood that the aqueous sugar stream may occur from an acid or alkaline treatment. cali to perform a complete hydrolysis of hemicellulose and cellulose components from the feedstock to their respective monomer constituents. Hydrolysis may be performed in two stages (see U.S. Patent No. 5,536,325, which is incorporated herein by reference), or may be performed in a single stage.

According to the invention, a sugar hydrolyzate is fermented by one or more yeast to produce a fermentation broth comprising the fermentation product. Sugar hydrolysate may occur at various stages in the processing of feedstock. As described above, a separated hemicellulose hydrolyzate from a solid stream comprising the pretreated feedstock may be sent for fermentation. Such sugar hydrolyzate will typically comprise xylose, glucose, arabinose, mannose and galactose. After separation of hemicellulose hydrosylate from solids, cellulose in the pretreated feedstock may be subjected to enzymatic hydrolysis to produce glucose and the resulting glucose stream may then be sent for fermentation. Alternatively, a pretreated feedstock stream comprising cellulose as well as monomeric sugars resulting from hemicellulose hydrolysis is subjected to enzymatic hydrolysis with cellulase enzymes. This produces a sugar hydrolyzate comprising sugars released from hemicellulose during pretreatment, as well as glucose resulting from enzymatic hydrolysis of cellulose. In a further embodiment, a hemicellulose hydrolyzate is separated from the pretreated feedstock and then added to the glucose comprising stream obtained from enzymatic cellulose hydrolysis, thereby producing a stream comprising both glucose. and monomeric sugars derived from hemicellulose, which, in turn, is sent for fermentation. In yet another embodiment of the invention, the sugar hydrolyzate sent for fermentation is obtained by complete acid or alkali hydrolysis wherein both the cellulose and hemicellulose components of the feedstock are hydrolyzed to their monomeric constituents.

In a preferred embodiment, the sugar hydrolyzate sent for fermentation is substantially free of undissolved solids such as lignin and other non-hydrolysed components. This is particularly advantageous in embodiments of the invention by employing a subsequent separation step and recycling yeast from the fermentation broth, as it is desirable to avoid any significant recycling of undissolved solids together with the yeast. Separation can be effected by known techniques including centrifugation, microfiltration, frame plate filtration, cross flow filtration, pressure filtration, vacuum filtration, and the like.

Any of a number of known yeasts may be used to convert sugar in sugar hydrolyzate to ethanol or other fermentation products. These include, but are not limited to, yeast of the genus Saccharomyces, Hansenula, Pichia, Kluyveromyces and Candida. In addition, commercially available yeast may be used, including, but not limited to, Turbo Yeast, Red® Safdistil® Ethanol, Thermosac®, Fermiol®, Fermivin® or Superstart®. Yeast can be genetically engineered to ferment both hexose and pentose sugars in ethanol. Alternatively, the yeast may be a strain that has been made capable of fermenting xylose by one or more non-recombinant methods, such as adaptive evolution or random mutagenesis and selection.

For example, fermentation may be performed with recombinant Saccharomyces yeast. The recombinant yeast strain may be a strain that has been capable of fermenting xylose by recombinantly incorporating (a) genes encoding xylose reductase (XR) and xylitol dehydrenase (XDH) (see, for example, US Patent Nos. 5,789,210, 5,866,382, 6,582,944 and 7,527,927 and EP 450 530) and / or (b) gene (s) encoding one or more xylose isomerase (XI) (see, for example). U.S. Patent Nos. 6,475,768 and 7,622,284). In addition the modified yeast strain may also over express an endogenous or heterologous gene encoding xylulokinase.

Yeast other than Saccharomyces eerevisiae can ferment hexose and pentose sugars in ethanol. This includes, but is not limited to, yeast of the genus Hansenula, Piehia, Kluyveromyces and Candida. WO 2008/130603 describes strains of Hansenula polymorpha with increased xylose ethanol production. In addition, mutants of Piehia stipitis and Candida shehatae were isolated by the method described in U.S. Patent No. 5,126,266.

In another example of the invention, xylose is converted to a sugar alcohol. Sugar alcohol may be selected from xylitol, arbitrol, erythritol, mannitol and galactitol. Preferably, the sugar alcohol is xylitol. In one embodiment of the invention, xylose is fermented in xylitol by yeast. Yeasts that are capable of converting xylose to xylitol include Candida, Piehia, Paehysolen, Hansenula, Debaryomyees, Kluyveromyees, Saccharomyees eerevisiae and Schizosaceharomyees strains. According to one embodiment of the invention, the yeast strain is Candida, preferably C. tropiealis.

Fermentation may be performed at or near the optimum temperature and pH of the fermentation microorganism. The temperature range for fermentation may be from about 10 ° C to about 70 ° C, although the temperature may be higher if the yeast is naturally or genetically modified to be thermostable. For example, the temperature may be from about 10 ° C to about 55 ° C, or any temperature in between, or about 15 ° C to about 45 ° C, or any temperature in between. The pH of the fermentation may be from about 3 to about 6, or any pH between these, for example a pH of 3.0, 3.5, 4.0, 4.5, 5.0, 5 , 5, 6.0, or any pH in between. The dose of the fermentation microorganism will depend on other factors, such as the activity of the fermentation microorganism, the desired fermentation time, the reactor volume and other parameters. It will be appreciated that these parameters may be adjusted as desired by one of ordinary skill in the art to achieve optimal fermentation conditions.

Sugar hydrolyzate may also be supplemented with additional nutrients required for growth and fermentation performance of the fermentation microorganism. For example, yeast extract, specific amino acids, phosphate, nitrogen sources, salts, trace elements and vitamins can be added to sugar hydrolyzate to support growth and optimize microorganism productivity. (See also Verduyn et al., 1992, Yeast 8 (7): 501-170, Jorgensen, 2009, Appl Bio-chem Biotechnol, 153: 44-57 and Zhao et al., 2009, Journal of Biotechnology, 139 : 55-60, which are each incorporated herein by reference). Fermentation is typically conducted under anaerobic conditions, although aerobic or microaerobic fermentations are also included within the scope of the invention.

The term "fermentation system" includes any arrangement of one or more fermentation reactors for sugar fermentation to a yeast fermentation product. In embodiments of the invention, this includes systems employing yeast recycling. The fermentation system may also comprise yeast conditioning tanks. In a typical commercial scale operation, fermentation is conducted in a system using multiple reactors, such as 2 to 6, or any number between them. Fermentation reactors may be arranged in series or parallel. Fermentation may be conducted in batch, continuous or batch-fed modes, with or without agitation. In one embodiment of the invention, the fermentation reactor (s) are stirred with mixing.

Microbial contaminants are exposed to oxidant at a pH that is less than 3.0 by the addition of acid. The pH may be greater than about 1 but less than 3.0. This includes all sub-values between these, for example a pH of 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 or 2.9, or any pH between them, is included within the scope of the invention. Examples of pH ranges that may be employed include about 1.0 to 3.0, 1.0 to 2.9, 1.0 to 2.8, 1.0 to 2.75, 1.0 to 2, 5, 1.0 to 2.4, 1.0 to 2.3, 1.0 to 2.2, 1.0 to 2.1, or 1.0 to 2.0,

In one embodiment of the invention, the acid is a mineral acid such as sulfuric acid, hydrochloric acid, sulfuric acid, phosphoric acid, or nitric acid.

Any fermentation system stream containing microbial contaminants may be exposed to the oxidant at a pH of less than 3.0. Typically each stream will also comprise yeast, along with microbial contaminants, although the oxidant and acid may be introduced into a sugar hydrolyzate prior to the addition of yeast.

When chlorine dioxide is the oxidant, the acid may be added prior to the addition of chlorine dioxide or a mixture of chlorine dioxide and acid may be introduced to the fermentation system. Chlorine dioxide reacts rapidly and thus is typically not advantageous for adding this oxidant prior to the addition of acid.

In embodiments of the invention employing yeast recycle, the acid may first be added to the yeast slurry that is separated from the fermentation broth, followed by the addition of the oxidant. Alternatively, a mixture of acid and oxidant is added to the lightweight slurry prior to oxidant treatment.

An oxidant suitable for use in the invention reduces bacterial contaminants to a level whereby they do not reduce the productivity or yield of the fermentation product. The oxidant should not have any significant effect on yeast viability or fermentative capacity. In addition, the oxidant selected for use in the invention has maximum efficiency in reducing bacterial contaminants to a pH of less than 3.0, or less than 2.5. A suitable oxidant may be selected by those skilled in the art by routine experimentation. Without being limited, the oxidant may be chlorine dioxide or ozone.

Preferably, oxidant treatment reduces the concentration of microbial contaminants (in colony forming units per mL of culture or CFU / mL) to about 100 times less than the yeast concentration (in colony forming units per mL of culture). culture or CFU / mL). More preferably, oxidant treatment reduces the concentration of microbial contaminants to about 103 CFU / mL or less. For example, oxidant treatment may reduce the concentration of microbial contaminants from about 1010 to about 103 CFU / mL. Bacterial colonies are enumerated using standard plaque counting methods. Methods are known to selectively place bacterial colonies and inhibit yeast growth. An example of such a method involves preparing serial dilutions and placing cyclohexamide agar plates, which selectively kill yeast but not bacteria (see, for example, Example 1.1).

The oxidant may be introduced into the fermentation system comprising yeast and microbial contaminants at a concentration of about 0.5 ppm and about 1500 ppm, or any concentration between them. In a continuous system, the oxidant would typically be added to one or more streams containing microbial contaminants. In another embodiment of the invention, the oxidant including but not limited to chlorine dioxide may be added at a concentration of from about 60 to about 1000 ppm or from about 75 to about 1000 ppm, including from about from 75 ppm to about 500 ppm, or from about 80 to about 1000 ppm, or from about 90 to about 1000 ppm, or from about 100 to about 500 ppm, or any concentration in between. For example, the oxidant, including but not limited to chlorine dioxide, may be added at a concentration of 0.5, 5, 10, 20, 30, 40, 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500 ppm, or any concentration between them.

Chlorine dioxide can be generated using known methods, for example by reacting chlorine gas with water and then adding sodium chlorite, or by reacting sodium hypochlorite with an acid. and adding sodium chlorite. In one example of the invention, stabilized chlorine dioxide (SCD) is acidified and the chlorine dioxide then produced can be introduced into fermentation such as acidified yeast slurry if yeast recycle is used. Alternatively, upon acid addition, SCD may be added directly to the fermentation. In the latter example, chlorine dioxide will be generated in situ. (See, for example, Kim et al., 2008, Food Microbiology, 25: 964-969 and WO 2007/149450).

The oxidant treatment is preferably conducted at a temperature of from about 4 ° C to about 40 ° C, or any temperature between them, for example 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 ° C, or any temperature between them. The duration of treatment can be from 5 seconds to about 60 min, or any time between them, for example 5, 15, 20, 25, 30, 35, 40, 45 or 50 seconds, or 10, 20, 30, 40, 50, 60 minutes or any time in between. As would be appreciated by those skilled in the art, reactions involving chlorine dioxide can be rapidly or even instantaneously. Consequently, when this oxidant is used, shorter residence times may be used or the residence time may be eliminated. However, the practice of this invention is not limited by any particular choice of residence time during oxidant treatment.

In those embodiments employing yeast recycling, the concentration of cells in the yeast slurry (also referred to herein as "yeast cream") that is exposed to the oxidant and acid is typically from about 50 g / l to about 300 g / l (dry cell weight). For example, the cell concentration in the yeast slurry may be 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 180, 200, 220, 240, 260, 280 or 300 g / l (dry cell weight). More preferably, the concentration of cells in the yeast slurry is from about 50 g / l to about 300 g / l, or from about 150 g / l to about 250 g / l, or about 175 g / L at about 225 g / l (dry cell weight).

Fermentation can be conducted so that the yeast is separated from the fermentation and sent back to the fermentation reaction (also referred to herein as "yeast recycling"). This involves removing fermentation broth from the fermentation reactor and separating the yeast from its solution by known separation techniques to produce a fluid yeast paste. Examples of suitable separation techniques include, but are not limited to, centrifugation, microfiltration, plate and frame filtration, cross flow filtration, pressure filtration, settling, vacuum filtration, and the like. In one example of the invention, the recycle is continuous, meaning that yeast is continuously recycled through the fermentation system. In an alternative embodiment, the yeast recycle is operated in batch mode.

After treatment of the acidified yeast slurry with the oxidant, the oxidant-treated yeast slurry is reintroduced back to the fermentation reaction. Yeast bleed may be used after yeast separation from fermentation and prior to exposure to oxidant and low pH. Preferably, from about 10% to about 99%, or any amount thereof, of the total yeast cells in the yeast slurry are treated with the oxidant and acid and then recycled. More preferably, between 80% and 95% of the yeast cells are treated and recycled, and more preferably at least 90% of the yeast cells are treated and recycled.

It should be understood that the practice of the invention is not limited by the number of yeast cell recycle cycles. Yeast recycling can be repeated at least once, or between 5 and 70 times, or even more times than this. Without wishing to be limited in any way, the yeast container may be repeated 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 times. It should also be understood that oxidant treatment need not be performed with every recycle. The treatment frequency can be adjusted by a person skilled in the art as desired to optimize performance and minimize bacterial numbers.

A fermentation may employ multiple fermentation reactors. In such embodiments, yeast is removed from a reactor in the system, treated with chlorine acid and dioxide or another suitable oxidant and then reintroduced back to one or more of the fermentation reactors. The acidified oxidant treated yeast can be fed back to the same reactor in the series or a different reactor. By recirculating the yeast in this manner, its concentration is maintained and conditioned in lignocellulosic hydrolysate which increases the volumetric reaction rate and also maximizes the yield for the desired product by minimizing carbon drift and other nutrients in bacterial cell production.

Referring now to Figure 1, a yeast recycle fermentation system is shown. Figure 1 is included as an example of how the present invention may be practiced and is not significant to be limiting in any way. That is, the invention may be practiced with or without yeast recycling.

An aqueous sugar hydrolyzate 6 obtained from pretreatment of the lignocellulosic feedstock is fed to a first fermentation reactor 8. The sugar stream is previously treated to remove insoluble lignin and other suspended solids. Sugar stream 6 is combined with yeast from a conditioning tank 14 of line 16 or line 38 containing recycled yeast. The conditioning tank 14, in turn, is fed with an air-containing stream and a sugar portion of stream 6. A fermented solution comprising ethanol is withdrawn from reactor 8 through line 18 and fed to a unit of Separation 22, typically a centrifuge, which separates the yeast from the fermented solution. Separate ethanol-containing beer is sent for distillation to obtain an ethanol-enriched solution. A portion of the yeast slurry on line 26 is bled and after bleeding, the remainder of the yeast is acidified then washed with an aqueous chlorine dioxide solution at a pH of less than 3 and subsequently fed through line 26. to a holding tank 30 where they are kept under appropriate conditions. The acid chlorine dioxide-treated yeast is then fed along with line 34, which branches off at line 38, which in turn introduces a portion of the yeast back into fermentor 8 to convert xylose to ethanol. The remainder of the yeast can be sent through line 34 to the cell growth conditioning tank 14 and subsequent to this, the yeast is sent to the second fermenter 42, and the cycle is repeated once again. This cycle can then be repeated with the three fermenters 46.

Although three fermenters are depicted in Figure 1, it will be appreciated by those skilled in the art that the number of fermenters may be varied as required. In addition, although fermenters are shown in parallel, they can instead be arranged in series. Furthermore, it is contemplated that the holding tank 30 may be excluded, in which case the yeast is subsequently kept, for example, in the fermenter 8. In yet another variation, all yeast of line 34 is sent from returns to fermenter 8 through line 38 without a portion being diverted for conditioning. Alternatively, the whole lye in line 34 is sent to the conditioning tank 14 and subsequently to the fermenter 2.

When ethanol is the product of fermentation, it is recovered by distillation. Separate fermentation broth or beer sent for distillation is a dilute alcohol solution that is substantially solids-free, including unconverted cellulose, although it may contain components added during fermentation to support the growth of microorganisms, as well as small amounts of yeast that may remain after separation 16. The beer is preferably degassed to remove carbon dioxide and then pumped through one or more distillation columns to separate the alcohol from the other components in the beer. The column (s) in the distillation unit are preferably operated in a continuous mode, although it should be understood that batch processes are also involved by the present invention. In addition, the column (s) may be operated at any pressure and heat suitable for the distillation process to be added at one or more points or by direct or indirect current injection. through heat exchangers. The distillation unit may contain one or more separate beer and rectifying columns, in which case dilute beer is sent to the beer column where it is partially concentrated. From each beer column, steam goes to a rectifying column for further purification. Alternatively, a distillation column is employed which comprises an integral enrichment or rectifying section. Remaining water may be removed from the vapor by a molecular sieve resin, by adsorption, or other methods known to those skilled in the art. The steam may then be condensed and denatured.

The present invention will be further illustrated in the following examples. However, it is to be understood that these examples are for illustrative purposes only, and should not be used to limit the scope of the present invention in any way.

EXAMPLES

Example 1: Acidified yeast cream chlorine dioxide treatments: antibacterial efficiency and dose requirements Example 1.1: Antibacterial synergistic effect of chlorine dioxide and pH.

Pretreated dilute acid wheat straw was produced on an industrial scale as set forth in US Patent No. 4,461,648 (incorporated herein by reference) and then hydrolyzed with cellulase and β-glucosidase enzymes to produce a sugar-derived hydrolysate. of the hemicellulose and cellulose components of the feedstock. This hydrolyzate was fermented in an industrial fermentation to produce ethanol with a yeast strain of Saccharomyces cerevisiae placed in co-pending United States Serial No. 61 / 291,011 (incorporated herein by reference). Light recycling was employed during fermentation. A yeast cream (also referred to herein as a "yeast slurry") separated from the fermentation broth and contaminated with proliferating bacterial fermentation was treated at 20 ° C with acid alone, chlorine dioxide alone or a combination of both. . In the acid treated condition, the pH was lowered from 5.0 to 2.0 with sulfuric acid. Under the condition treated with chlorine dioxide, a concentrated stock of chlorine dioxide in water (10,000 ppm) was prepared by passing a mixture of chlorine and nitrogen gas through sodium chlorite columns. The resulting CIO2 gas was sprayed into 4 L of cold deionized water until the desired concentration was reached. This chlorine dioxide solution was used to treat yeast cream.

In the combination treatment, the yeast cream was first lowered to pH 2.0 with sulfuric acid for 5 minutes and subsequently at the indicated mass dose of chlorine dioxide for 5 minutes. In cases where the pH was lowered, the yeast cream was titrated back to 5.0 with sodium hydroxide after 10 minutes and prior to placement for bacterial numeration. The duration of acid exposure was limited to 10 minutes to match the exposure duration for the other treatments.

Bacterial colonies were enumerated via standard plaque counting methods. In order to selectively place bacterial colonies and inhibit yeast growth, typical soy agar plates (15 g / l pancreatic casein digestion, 5 g / l enzymatic digestion of soy flour, 5 g / l sodium chloride and 15 g / l agar) with 100 mg / l cycloheximide were used. The samples were serially diluted in sterile saline (0.9% NaCl w / v) prior to placement.

Figure 2 illustrates the synergistic effect of chlorine dioxide and low pH. Error bars represent the standard deviation of the mean of duplicate experiments, each placed in triplicate. These data demonstrate that lowering pH alone or chlorine dioxide alone is not as effective in reducing bacterial population compared to when lowering pH first and then followed by a low pH chlorine dioxide treatment. Increasing the chlorine dioxide dose has a negligible effect on bacterial populations at pH 5.0 (compare bars filled at 0, 200 and 500 ppm chlorine dioxide). Similarly, lowering chlorine dioxide-free pH does not significantly reduce the bacterial population (compare closed and open bars at 0 ppm chlorine dioxide). When combined with lowered pH, 200 ppm and 500 ppm chlorine dioxide treatments reduced the bacterial population by 102 CFU / mL and 103 CFU / mL, respectively (see bars open at 200 ppm and 500 ppm). Example 1.2: Chlorine dioxide dose effect on bacterial contamination of a yeast fermentation culture.

Contaminated yeast cream obtained from the fermentation of Example 1.1 was titrated to pH 2.0 with sulfuric acid, treated with various chlorine dioxide concentrations (0, 50, 100, 150 and 200 ppm) and placed to determine the bacterial reduction to pH 2.0 as outlined above, with the exception that the treatment temperature was 30 ° C, preferably 20 ° C. Bacterial populations were enumerated as previously described (Example 1.1). Bacterial populations were also enumerated in a control sample that was not subjected to chlorine dioxide or pH titration.

Figure 3 illustrates the results. Error bars represent the standard deviation of the medium of duplicate experiments, each placed in triplicate. As shown in the figure, a 100 ppm chlorine dioxide dose at pH 2 and 30 ° C reduced contamination in a yeast cream 100 times. Yeast cream treated with 200 ppm chlorine dioxide at 30 ° C decreased bacterial counts by more than 105, which is a reduction greater than 500 ppm treatment at room temperature (100 times reduction). see Figure 2). These results demonstrate that increasing the treatment temperature by 10 ° C further increases the reactivity of CIO 2, which allows for increased efficiency at decreased dosage. Notably, at 200 ppm, the 103 CFU / mL dilution plates had no bacterial growth, indicating that the maximum bacterial reduction was greater than 105 CFU / mL at 200 ppm. Addition of acid alone (pH control) did not reduce the bacterial population.

Example 2: Low pH and antibacterial chlorine dioxide treatments on yeast culture viability and fermentation performance.

A sample of the yeast cream of Example 1.1 was placed for yeast growth after the treatments outlined therein. That is, contaminated yeast creams were treated at 20 ° C with sulfuric acid alone at pH 2 or in combination with chlorine dioxide at 0, 200 and 500 ppm. Selective yeast growth was enabled by serial dilution of the cream in sterile saline (0.9% NaCl w / v) and plating YM (10 g / L glucose, 5 g / L peptone, 3 g / L L yeast extract, 3 g / L malt extract, 20 g / L agar) containing 34 mg / L chloramphenicol to inhibit bacterial growth.

The results are shown in Figure 4. Error bars represent the standard deviation of the mean of duplicate experiments, each placed in triplicate. The figure clearly indicates that the viability of the fermentation yeast culture is unaffected by neither acid treatment nor the combination of acid and chlorine dioxide. As can be seen, increasing the chlorine dioxide dose has a negligible effect on fermentation yeast populations regardless of the treatment pH.

Yeast fermentation performance was assessed by inoculating the contaminated control yeast, acid treated yeast and chlorine dioxide-treated yeast creams (treated as placed in Example 1.1) in 400 mL hydrolysate. lignocellulosic. Lignocellulosic hydrolyzate was produced as set forth in Example 1.1. Prior to inoculation, the medium was sprayed with pure CO2 for two minutes to ensure anaerobicity. Cells were allowed to simmer at 30 ° C, 150 rpm until sugar exhaustion. CO2 production was monitored for the course of fermentation and samples were taken for dry cell weight and HPLC analysis.

Samples were analyzed for cell mass using dry cell weight (Rice etal. (1980) Am. Soc. Brew. Chem. J. 38: 142-45, which is incorporated herein by reference). For fermentability analysis, samples were taken from bioreactors using a 10 mL syringe. From each sample, 2 mL volumes were centrifuged and the supernatant was decanted and filtered through a 0.2 μηι syringe filter. Each supernatant sample was diluted with 5 mM sulfuric acid. All dilutions were analyzed for glucose, xylose, xylitol, glycerol, and ethanol content in the Agilent 1100 Series Refractive Index Detector HPLC1 while acetic and lactic acid were analyzed concurrently using an Agilent 1200 Series Variable Wavelength Detector HPLC. The column used for separation was the Varian Metacarb 87H Organic Acid column, maintained at 50 ° C with 5 mM sulfuric acid mobile phase at a flow rate of 0.6 mL / min. The unit was equipped with the 1100 Series Autosampler and Pump System and controlled with Chemstation software.

Xylose entry rates and glucose and xylose ethanol yields for the fermentation of lignocellulosic hydrolysates are shown in Figure 5. Using equivalent cell concentrations, the xylose consumption rate in lignocellulosic hydrolysate exhibits a relative decrease with both. acid treatment and chlorine dioxide-acid treatment combination. It follows that the rate of xylose consumption would be higher in untreated control as contamination microbes consume xylose. This is further supported by the observed change in xylose ethanol yield. Decreased rates indicate that bacteria are no longer consuming xylose and allow much of this sugar to be converted to ethanol by yeast fermentation, thereby increasing yield. It may also be observed that the decrease in rates coupled with the increase in yields is greater with the combination of acid chlorine dioxide treatment than with acid treatment alone.

Example 3: Antibacterial effect of chlorine dioxide and pH in industrial scale operations.

Hydrolysate from a pretreated dilute acid lignocellulosic feedstock was produced as set forth in U.S. Patent No. 4,461,648 (incorporated herein by reference). The hydrolyzate was fermented to produce ethanol with the yeast strain placed in the co-pending United States Serial No. 61 / 291,011 (incorporated herein by reference). A volume of 100,000 L of total fermentation broth was separated into a beer fraction and a yeast cream upon completion of fermentation. The yeast cream was concentrated to approximately 170 g dry cell weight / L. Prior to recycling at the next batch of fermentation, the bacterially contaminated yeast cream was treated at 20 ° C with acid alone (batch 1) or treated with acid in combination with chlorine dioxide (batch 2). During acid treatment for each of the two batches, the pH of the concentrated yeast cream was lowered to 2.0 using inline addition of 93% (w / v) sulfuric acid. Chlorine dioxide was generated in concentrated form at a nominal concentration of 2700 ppm using a commercially available system from Pureline, Palatine, IL. In the combination treatment, the acidity of the yeast cream was first lowered to pH 2.0 (as per Example 1.1) and subsequently treated at a mass dose of 200 ppm chlorine dioxide with a residence time of approximately 45 ° C. seconds After treatment, the yeast cream was fed back into the fermentation.

Bacterial colonies were enumerated by standard plaque counting methods. In order to selectively place bacterial colonies and inhibit yeast growth, tryptic soy agar plates (15 g / l pancreatic casein digestion, 5 g / l enzymatic digestion of soybean flour, 5 g / l sodium chloride and 15 g / l agar) with 100 mg / l cycloheximide were used. Samples were serially diluted in sterile saline (NaCl w / v 0.9%) prior to placement.

Figure 6 illustrates the efficiency of combined acid treatment and use of low pH chlorine dioxide. Lowering pH by the addition of sulfuric acid reduced bacterial contamination counts 100-fold, while acid treatment in combination with chlorine dioxide addition was able to decrease bacterial combination counts by 104 CFU / mL.

The present invention has been described with respect to one or more embodiments. However, it will be apparent to those skilled in the art that a number of variations and modifications may be made without departing from the scope of the invention as defined in the claims.

Claims (25)

A method for producing a sugar hydrolyzate fermentation product comprising: (i) fermenting the sugar hydrolyzate in a yeast fermentation system to produce a fermentation broth comprising a fermentation product; (ii) introducing chlorine acid and chlorine dioxide to said fermentation system to expose any microbial contaminants in said one or more stage fermentation system to chlorine dioxide at a pH of less than 3.0; and (iii) recovery of the fermentation product. A method for obtaining a sugar hydrolyzate fermentation product comprising: (i) removing suspended fiber solids from the sugar hydrolyzate to obtain a whitened sugar solution; (ii) sugar fermentation in the bleached sugar solution in a fermentation reaction using yeast to produce a fermentation broth comprising the fermentation product; (iii) separating the yeast from the fermentation broth to produce a yeast slurry and a fermentation product; (iv) introducing chlorine acid and chlorine dioxide to the yeast slurry to expose any microbial contaminants and yeast to said chlorine dioxide yeast slurry at a pH of less than 3.0; (v) reintroducing at least a portion of the chlorine dioxide-treated Yeast slurry back to the fermentation step to maintain the yeast concentration in the fermentation reaction; and (vi) recovery of the fermentation product. A method according to claim 2, wherein the fermentation step (step ii) is conducted in one of a series of fermentation reactors and in which in the reintroduction step (step v) the treated yeast slurry Chlorine dioxide is reintroduced back to the same or a different fermentation reactor in the series. A method according to claim 2, wherein in the removal step (step i), the whitened sugar solution comprises a sugar selected from the group consisting of glucose, xylose galactose, mamose, arabinose, fucose and fructose. A method according to claim 1 or 2, wherein the sugar hydrolyzate comprises at least xylose or glucose. The method according to claim 5, wherein the sugar hydrolyzate comprises xylose and glucose. A method according to claim 6, wherein in the fermentation step the fermentation product is ethanol and in which the yeast is a sp. Saccharomyces that converts glucose and xylose to ethanol. A method according to claim 5, wherein the sugar hydrolyzate comprises xylose. A method according to claim 8, wherein in the fermentation step the yeast is a sp. Candida that converts xylose to xylitol. A method according to any one of claims 1 to 9 wherein in the introduction step the chlorine dioxide is added at a concentration of from about 0.5 to about 1500 ppm. A method according to claim 10, wherein in the introduction step the chlorine dioxide is added at a concentration of between about 75 and about 500 ppm. A method according to any one of claims 1 to 11, wherein the sugar hydrolyzate is derived from a lignocellulosic feedstock. The method according to claim 2, wherein in the introduction step, the concentration of microbial contaminants in the yeast slurry is reduced to at least 100 times lower than that of yeast. A method according to claim 2, wherein in the introduction step the concentration of microbial contaminants in the yeast slurry is reduced below about 103 CFU / mL. The method according to claim 2, wherein in the introduction step, the concentration of yeast cells in the yeast slurry is from about 10 g / l to about 300 g / l cell weight. dry. The method of claim 15, wherein at the introduction step, the concentration of yeast cells in the yeast slurry is from about 20 g / l to about 200 g / l cell weight. dry. A method according to claim 2, wherein in the introduction step the temperature of the yeast slurry is between about 4 ° C and about 37 ° C. A method according to claim 12, wherein the sugar hydrolyzate is obtained by pretreatment of the lignocellulosic feedstock with acid or alkali to produce a pretreated feedstock. The method of claim 18, wherein the pretreatment is with a mineral acid. A method according to any one of claims 1- -19, wherein during said exposure, the pH is greater than about 1, but less than 2.5. A method according to any one of claims 1-20, wherein during said exposure, the acid is added before to the chlorine dioxide. A method according to claim 1, wherein in the introduction step the yeast in said fermentation system is exposed to chlorine dioxide at a pH of less than 3.0. A method for producing a sugar hydrolyzate fermentation product comprising: (i) fermenting the sugar hydrolyzate in a yeast fermentation system to produce a fermentation broth comprising a fermentation product; (ii) introducing acid and an oxidant into said fermentation system to expose any microbial contaminants in said fermentation system in one or more stages to the oxidant at a pH lower than 3.0; and (iii) recovery of the fermentation product. A method for producing a sugar hydrolyzate fermentation product comprising: (i) fermenting the sugar hydrolyzate in a yeast fermentation system to produce a fermentation broth comprising a fermentation product; (ii) continuously introducing acid and an oxidant into said fermentation system to continuously expose microbial contaminants in said fermentation system in one or more stages to the oxidizer at a pH of less than 3.0; and (iii) recovery of the fermentation product. A method according to claim 23 or 24, wherein at the introduction step the yeast in said fermentation system is exposed to chlorine dioxide at a pH of less than 3.0.