CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the priority of U.S. Provisional Patent Application Serial Number 61/838,293, filed June 23, 2013 which is hereby incorporated by reference in its entirety.

FIELD

[0002] The present disclosure generally relates to a method for increasing the ethanol yield from the fermentation of agave by enzymatically hydro lyzing any of the fibrous materials, e.g., cellulose, the hemi-cellulose, or inulin, in agave bagasse and/or agave leaves to fermentable sugars, e.g., glucose. In some embodiments, the present disclosure is directed to a method of preparing a distilled beverage from the fermentation of fermentable sugars from combinations of the agave pina, the agave bagasse, and the agave leaves. The distilled beverage may be a tequila. In some embodiments, the present disclosure is directed to the fermentation of sugars derived from agave bagasse and/or agave leaves to produce fuel ethanol.

BACKGROUND

[0003] Tequila is an alcoholic beverage produced from the fermentation of sugars (comprising mainly fructose) obtained from Blue agave (Agave tequilana). Premium tequila requires that all of the fermentable sugars must come from the agave pina. Regular tequila allows some of the sugars (less than 50%) to be derived from other sources, such as sugar cane and molasses.

[0004] Tequila is typically produced according to a traditional method developed centuries ago, which remains substantially unchanged by modern farming and distillation developments. The blue agave that is the base ingredient for any distilled beverage called "tequila" must be grown in the state of Jalisco, Mexico and a few surrounding regions.

Typically, the first step in tequila production is the removal of the heart, or pina, in the plant's twelfth year. The pina is cut in half or chopped into smaller pieces then steamed in ovens or autoclave at about 80-90°C for many hours, typically up to about 36 hours. The cooked pina is then crushed and washed with hot water in a countercurrent extractor to recover the soluble sugars. The washed fiber (agave bagasse) is pressed to expel as much as feasible the sugar solution entrained with the fibers. The total soluble sugar recovery may be over about 95%. The combined sugar solution is then forwarded to the fermentors where yeast is added to ferment the sugars to ethanol. The wort, which contains about 6% ethanol, is distilled at least twice, or thrice, or even four times, to a 38 to 40% ethanol content, although the total ethanol content may vary from 31 to 55%. The distilled ethanol is aged in oak barrels for various length of time to achieve the final tequila product.

[0005] A new sugar extraction method was commercialized in the early 2000's where the sugar recovery from agave pina is claimed to reach above 98%. The agave pina is ground to small particles to increase the exposed surface areas. The sugar is extracted from the ground pina with hot water in a countercurrent diffuser washer. The washed fiber is pressed to improve the soluble sugar recovery. The pressate is combined with the sugar solution from the diffuser washer. The combined sugar solution is then forwarded to the cooker to hydrolyze the polysaccharides to simple sugars and to pasteurize the slurry. The slurry may be filtered to remove entrained fibers before fermentation. The fermentation and distillation steps that follow are similar to the traditional process described above.

BRIEF DESCRIPTION OF THE DISCLOSURE

[0006] In one aspect, the present disclosure is directed to a method for producing potable ethanol from agave. The method comprises combining agave bagasse, water, and an enzyme to form a hydrolysis mixture comprising a water-soluble sugar. The method also comprises separating the water-soluble sugar from the hydrolysis mixture. The method further comprises combining the water-soluble sugar with a fermentable sugar derived from an agave pina and a yeast to thereby prepare a fermentation mixture.

[0007] In another aspect, the agave bagasse is steam treated prior to combining with the enzyme. In some embodiments, the fermentation mixture is fermented to thereby prepare a wort comprising ethanol. The wort is distilled at least twice to thereby prepare a tequila. In some embodiments, the present disclosure is directed to a tequila prepared by this method.

[0008] Another aspect is a method for producing potable ethanol from agave. The method comprises combining agave bagasse with an acid to form an acid hydrolysis mixture. The method comprises steam treating the acid hydrolysis mixture to thereby prepare a steam treated mixture. The method comprises cooling the steam treated mixture to thereby form a cooled mixture having a temperature sufficient for enzyme hydrolysis. The method comprises combining the cooled mixture and an enzyme selected from the group consisting of cellulase, hemicellulase, inulinase, and combinations thereof to thereby form an enzyme hydrolysis mixture, wherein the enzyme hydrolyzes at least one of cellulose, hemicellulose, or inulin present in the agave bagasse into a water-soluble sugar. The method comprises separating the water-soluble sugar from the hydrolysis mixture. The method comprises combining the water- soluble sugar with a fermentation mixture comprising a fermentable sugar derived from an agave pina.

[0009] In some embodiments, the fermentation mixture is fermented to thereby prepare a wort comprising ethanol, and the wort is distilled at least twice to thereby prepare a tequila. In some embodiments, the present disclosure is directed to a tequila prepared by this method.

[0010] In still another aspect, a method for producing ethanol comprises blending agave bagasse and agave leaves to form a blended mixture. The method comprises steam treating the blended mixture to prepare a steam treated blended mixture. The method comprises cooling the steam treated blended mixture to thereby form a cooled blended mixture having a temperature sufficient for enzyme hydrolysis. The method comprises combining the cooled blended mixture and an enzyme selected from the group consisting of cellulase, hemicellulose, inulinase, and combinations thereof to thereby form an enzyme hydrolysis mixture, wherein the enzyme hydrolyzes at least one of a cellulose, a hemicellulose, or an inulin present in the agave bagasse and the agave leaves into a water-soluble sugar. The method comprises combining the enzyme hydrolysis mixture with a fermentable sugar derived from an agave pina and with a yeast to thereby form a fermentation mixture. The method comprises fermenting the fermentation mixture to thereby prepare a wort comprising ethanol. In some embodiments, the wort is distilled at least twice to thereby prepare a tequila. In some embodiments, the present disclosure is directed to a tequila prepared by this method and in at least some embodiments to a premium tequila prepared by one or more methods of the present disclosure

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 depicts the process flow of an embodiment of a process of the present disclosure. [0012] FIG. 2 depicts the process flow of a second embodiment of a process of the present disclosure.

[0013 ] FIG. 3 depicts the process flow of a third embodiment of a process of the present disclosure.

[0014] FIG. 4 depicts the process flow of a fourth embodiment of a process of the present disclosure.

[0015] FIG. 5 depicts the process flow of a fifth embodiment of a process of the present disclosure.

[0016] FIG. 6 depicts the process flow of a sixth embodiment of a process of the present disclosure.

DETAILED DESCRIPTION

[0017] The present disclosure includes methods for increasing the ethanol yield from the fermentation of agave. Tequila is produced from the heart or pina of blue agave plant (Agave tequilana). The traditional method of tequila production produces agave bagasse, which is a fibrous by-product that may currently find use as animal feed if a feedlot is within a

commercially practicable distance. Alternatively, agave bagasse may be composted. The agave bagasse may also be burned as fuel or used in paper production. According to some

embodiments, the fibrous agave bagasse is subjected to enzyme hydrolysis of one or more of cellulose, hemi-cellulose, or inulin to thereby yield fermentable sugars that may be fermented to ethanol during a subsequent fermentation process. In some embodiments, the fermentable sugars may be combined with fermentable sugars obtained from the pina of the agave plant, fermented, and at least double distilled to thereby prepare a tequila or premium tequila, or more generally, a mezcal.

[0018] In some embodiments, fermentable sugars may be obtained from the enzymatic hydrolysis of agave leaves. In some embodiments, the present disclosure is directed to a method of preparing a distilled beverage from the fermentation of fermentable sugars from combinations of the agave pina, the agave bagasse, and the agave leaves. This distilled beverage may also be a tequila. [0019] In still other embodiments, the present disclosure is directed to the fermentation of sugars derived from agave bagasse and/or agave leaves to produce fuel ethanol.

[0020] The starting material for the process comprises feedstock derived from an agave plant. According to the regulatory requirements of Mexico, in tequila production, the feed stock, specifically, the pina, is derived from the blue agave plant (Agave tequilana), which is harvested according to the traditional techniques used in tequila production. A fermentable juice, comprising mainly fructose, is obtained from the pina. The fibrous co-product left over after the juice is extracted from the agave pina is the agave bagasse, which is a fibrous material comprising cellulose, hemi-cellulose, and inulin. In the production of other distilled beverages, specifically mezcals, other agave species may be used, such as Agave americana, Agave attenuate, Agave salmiana, Agave angustifolia, Agave cupreata, and Agave karwinskii. The method is useful for increasing the ethanol yield from any agave species useful for the production of tequila, or more generally, mezcals, by the treatment of the agave bagasse.

Accordingly, the method includes the enzymatic hydrolysis of agave bagasse to increase the fermentable sugar content. In some embodiments, the fermentable sugars may be combined with fermentable sugars obtained from the pina of the agave plant, fermented, and at least double distilled to thereby prepare a tequila, or more generally, a mezcal.

[0021] The agave plant additionally comprises spiky, fleshy leaves. The leaves contain significant fermentable sugar content, e.g., fructose and fibers that may be treated to thereby increase the fermentable sugar content. According to some embodiments, the agave leaves comprise a feedstock, optionally along with the fibrous agave bagasse, for enzymatic hydrolysis and fermentation. The combined agave bagasse and agave leaves feedstock may be used in the production of fermentable sugars that may be combined with sugars derived from the agave pina, the combination of which may be fermented and distilled to prepare a distilled beverage that may be a tequila, or more generally, a mezcal.

[0022] The method enhances the ethanol yield of tequila production through the enzymatic hydrolysis and fermentation of the co-products of the traditional process for preparing tequila. Accordingly, the agave pina is harvested and processed, i.e., steamed in an oven or autoclave at a temperature within the range of about 80-90°C for up to about 36 hours.

Optionally, the agave pina may be chopped in half, or even into smaller pieces, prior to steaming in the oven or autoclave. The cooked pina is then crushed and washed with hot water in a countercurrent extractor to recover the soluble sugars. This traditional technique is suitable for extracting about 95% of the fermentable sugars in the agave pina. According to a recently developed technique, the agave pina is ground to small particles to increase the exposed surface areas. The sugar is extracted from the ground pina with hot water in a countercurrent diffuser washer. The washed fiber is pressed to improve the soluble sugar recovery. The pressate is combined with the sugar solution from the diffuser washer. The combined sugar solution is then forwarded to the cooker to hydrolyze the polysaccharides to simple sugars and to pasteurize the slurry. The washed fiber (agave bagasse) is pressed to expel as much as feasible the sugar solution entrained with the fibers. The slurry may be filtered to remove entrained fibers before fermentation. This technique may extract up to 98% of the fermentable sugars from the agave pina. The resulting co-product of the extraction technique is a fibrous co-product called the agave bagasse. Agave bagasse is the residual fiber remaining after cooked agave heads are shredded, milled and the sugars water-extracted. The bagasse is primarily the rind and fibrovascular bundles dispersed throughout the interior of the agave head. It represents about 40% of the total weight of the milled agave on a wet weight basis. Bagasse is composed of fiber and pith. The fiber is thick walled and long (10-12 cm). The fiber comprises cellulose, hemi- cellulose, inulin, and lignin.

[0023] According to some embodiments, agave bagasse from the existing tequila operation is subjected to enzyme hydrolysis to thereby increase the yield of fermentable sugars obtainable from the agave pina. See FIG. 1, which depicts a modified process for the production of tequila. As shown in FIG. 1 , agave pina is slow cooked with steam at a temperature between about 80 to 90°C for a duration up to about 36 hours, according to the traditional tequila production process. Thereafter, the steamed agave pina is then crushed, washed (generally via countercurrent), and pressed in order to extract about 95% of the fermentable sugar (mostly fructose) from the pina. According to some embodiments, the agave bagasse is contacted with water to thereby form a slurry. The agave bagasse is suitably slurried with sterilized process water.

[0024] Optionally, the slurried agave bagasse may be pre-treated and conditioned prior to combining the bagasse with enzyme. See FIG. 2. Pre-treatment and conditioning may comprise grinding and/or disc refining to decrease the particle sizes of the agave bagasse. It should be noted, that the particle size of the agave bagasse may be reduced using other processes and equipment suitable for the reduction in particles size of fibrous materials such as, for example, slicing, hammer milling, jet milling, ball milling etc. and the processed material may be classified to separate the particle size into one or more particle size distributions which may then be further processed or recycled without departing from the scope of the present disclosure. Pre-treatment and conditioning may comprise at least one or both of acidic hydrolysis and steam treatment. In some embodiments, the total solids content of the bagasse entering the

pretreatment reactors range from about 30% total solids to about 60% total solids, or about 40% total solids to about 55% total solids.

[0025] Optionally, the process may be integrated with newly developed processes for grinding and diffuser washing the agave pina. See, for example, FIGS. 3 and 4, which depict processes according to the present disclosure in which the agave pina is processed to extract about 98% of the fermentable sugars from the pina.

[0026] In an optional acid-pretreatment first step, the agave bagasse and water mixture is subjected to acid hydrolysis under generally mild conditions of pH and temperature. The pH of the slurry may be adjusted to between about 3.5 and about 6.5, such as between about 4.0 and about 6.5, such as between about 4.5 and about 6.0, such as about 5.2. For acidic pH adjustment, if necessary, sulfuric acid, hydrochloric acid, phosphoric acid, nitric acid, acetic acid, lactic acid, citric acid, tartaric acid, and the like, may be used. Suitable acids include sulfuric acid, hydrochloric acid, phosphoric acid, and nitric acid. The acid loading inside the pretreatment reactor ranges from about 0.002 g acid/g dry bagasse to about 0.02 g acid/g dry bagasse. Acid addition to bagasse is accomplished in a spray impregnation system or a soaking impregnation system prior to steam pretreatment. If necessary, for alkaline pH adjustment, ammonia, sodium hydroxide and potassium hydroxide, are suitable. The slurry may be agitated by conventional means during acid hydrolysis, such as by paddle stirring, stir plate, vortex, or shaker. Acid hydrolysis may occur under these mild conditions for a duration of at least about five minutes, such as between about five minutes and about 120 minutes.

[0027] In an optional pre-treatment step, the agave bagasse and water mixture may be steam treated. The agave bagasse and water mixture may be steam treated prior to, after, or simultaneous with acidic pre-treatment. Steam pretreatment reactors may be batch or continuous. In some aspects, steam treating may occur at a temperature of at least about 100°C, such as from about 100°C to about 200°C, from about 120°C to about 160°C, from about 140°C to about 160°C, or from about 100°C to about 110°C. Steam treating may occur for a duration of at least about 1 minute, such as between about 1 minute and about 30 minutes, such as from about 5 minutes to about 20 minutes, such as about 10 minutes. [0028] The optional acid hydrolysis and steam treatment are effective to disrupt and separate the fibrous strands in the agave bagasse, thereby making the fiber more available to the enzymes in a subsequent enzymatic hydrolysis. The acidic conditions may also hydrolyze at least a portion of the celluloses, hemicelluloses, inulins, and lignins into lower molecular weight polymers. Pretreatment of bagasse enhances the enzyme accessibility to cellulose, and sterilizes the fiber to minimize the risk of bacterial contamination in the subsequent enzymatic hydrolysis step. In addition, the acidic and high temperature conditions in the steam treatment may break down lignin-hemicellulose complexes and may hydrolyze the hemicellulose, thereby producing soluble oligomers and monomers of the pentose sugars xylose and arabinose, and other sugars.

[0029] After one or more pre-treatment steps (i.e., grinding, disc refining, acid hydrolysis, steam treating), the agave bagasse and water mixture is pH adjusted to a pH suitable for enzymatic hydrolysis. Additionally, the agave bagasse and water mixture is cooled to a temperature suitable for enzymatic hydrolysis. In some embodiments, a chilled dilute aqua ammonia is blended with the pretreated bagasse to achieve a slurry having between about 10% total solids and about 30% total solids content, such as between about 20% total solids and about 25% total solids, such as between about 15% to about 25% total solids content. The pH of the pretreated bagasse is adjusted to between about 4.5 and about 6.0, such as between about 5.0 and about 5.5, such as about 5.2. In some embodiments, the temperature may be between about 35°C and about 60°C, such as between about 50°C to about 55°C, such as about 50°C. For steam pretreatment, the pretreated bagasse slurry is held for a period of time from 5 minutes to about 30 minutes to ensure the pH is stabilized before enzyme is added.

[0030] Enzymes are then blended into the slurry using a dynamic mixer (e.g., pug mill mixer, paddle mixer, mixing screw conveyor, dynamic in-line mixer, or homogenizer) to thereby form an enzyme hydrolysis mixture. The enzymes may include cellulases, hemicellulases, and inulases. Enzymatic hydrolysis may be batch, fed-batch or continuous. The hydrolysis time may range from about 24 hours to about 72 hours.

[0031] According to a method of the present disclosure, the agave bagasse, which has been optionally acid hydrolyzed and/or steam treated, is combined with a source of enzymes comprising at least one cellulase to form a hydrolysis mixture. Cellulases are a class of enzymes produced chiefly by fungi, bacteria, and protozoans that catalyze the cellulolysis (hydrolysis) of cellulose into glucose, cellobiose, cellotriose, cellotetrose, cellopentose, cellohexose, and longer chain cellodextrins. Combinations of the three basic types of cellulases may be employed. For example, endo-cellulases may be added to randomly hydro lyze internal p-l,4,-D-glucosidic linkages in order to disrupt the crystalline structure of cellulose and expose individual cellulose chains. Exo-cellulases may be added to cleave off two units (cellobiose), three units

(cellotriose), or four units (cellotetrose) from the exposed chains, while β-glucosidase may be added to hydrolyze these compounds into glucose, which is available for fermentation.

Examples of suitable cellulases include Cellic ^® CTec2, Cellic ^® CTec3, CELLUCLAST ^® , CELLUZYME ^® , CEREFLO ^® and ULTRAFLO ^® (available from Novozymes A/S),

LAMINEX ^® , SPEZYME ^® CP (Genencor Int.), and ROHAMENT ^® 7069 W (Rohm GmbH), and GC-220 (Genencor International). The cellulase enzymes are added in amounts effective from about 0.001% to about 2.0% wt. of solids, from about 0.025% to about 1.0% wt. of solids, or from about 0.01% to about 1.0% wt. of solids. Cellulase loading may suitably vary with coarse fraction cellulose content, but typical loading may be expressed as about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45 or about 50, and ranges thereof, such as from about 5 to about 50, from about 10 to about 50, from about 20 to about 50, from about 10 to about 50, from about 10 to about 40, from about 10 to about 30, from about 20 to about 50 or from about 20 to about 40 filter paper units (FPU) per gram of cellulose. Alternatively expressed, cellulase loading is about 0.3, 0.6, 1.0 or 2 FPU per gram of solids in the agave bagasse slurry.

[0032] Cellulase enzymes may be combined with the agave bagasse by any means known in the art to achieve a substantially homogeneous admixture, including agitated mixing tanks, in line mixers, pug mill mixers, paddle mixers, ribbon mixers, or in liquefaction reactors such as reactors having at least one mixing section and at least one plug flow section. Cellulase is suitably added to the coarse fraction at pH of from about 4.5 to about 6.0 or from about 5.0 to about 5.5 and a temperature of from about 35°C to about 70°C, from about 45°C to about 65°C, or from about 50°C to about 55°C.

[0033] For highly viscous agave bagasse mixtures, mixing can be done in two stages. In a first stage, cellulase can be admixed with viscous material in a mixer particularly suited for the processing of highly viscous materials, for instance, a pug mill mixer, a paddle mixer (single or double shaft), or a ribbon mixer (single or double shaft). High viscosity mixers are particularly suited to the process of the present disclosure because thorough mixing of cellulase with the agave bagasse enables a rapid viscosity reduction in the hydrolysis step that follows. The high viscosity mixer may optionally have a jacket to receive cooling or heating medium in order to maintain the temperature of the agave bagasse during cellulase addition. In some aspects, cellulase addition can be done through one or more addition points, for example, multiple spray nozzles, positioned near the inlet. In a second stage, the mixture may be processed in a mix tank or fiber liquefaction bioreactor. In some aspects, the mixture may be processed in a fiber liquefaction bioreactor to further reduce the viscosity prior to transfer to a cellulose hydrolysis reactor. The fiber liquefaction bioreactor may be of either a continuous mixing design or a design having at least one continuous mixing section and at least one plug flow section. Optionally, two or more fiber liquefaction bioreactors may be operated in series. In some particular aspects, the fiber liquefaction bioreactor comprises alternating mixing zones and near plug flow zones and the coarse fraction-cellulase admixture either flows downward through the tower by gravity or is moved upward by pumping. The mixture is typically processed in a fiber liquefaction bioreactor until the mixture viscosity is less than about 8,000 cP, less than about 7,000 cP, less than about 6,000 cP, less than about 5,000 cP or less than about 4,000 cP, whereafter it is transferred to a cellulose hydrolysis reactor.

[0034] The cellulose hydrolysis reactor is typically an agitated vessel designed to hold the mixture at a temperature suitable for cellulose hydrolysis by cellulase wherein the volume is sufficient to provide a hold time to allow for a significant yield of cellulose-derived C6 sugars including, for instance, glucose, dextrose, fructose and levulose. To maintain the temperature inside the hydrolysis reactor, the vessel wall is thermally insulated and/or equipped with heating jacket where heating fluid is circulated through. Glucose yields, based on total cellulose content of the coarse fraction, of at least about 30%, about 40%, about 50%, about 60%, about 70% or at least about 80%, and ranges thereof, such as from about 30% to about 80%, from about 40% to about 80% from about 30% to about 70% or from about 40% to about 70% are suitable. Total cellulose hydrolysis cycle times of 8 hours, 12 hours, 24 hours, 30 hours, 36 hours, 42 hours or 48 hours, or 72 hours and ranges thereof, such as from 8 to 72 hours, or from 8 hours to 48 hours, are within the scope of the present disclosure.

[0035] Optionally, other enzymes such as at least one hemicellulase (e.g., a xylanase), an a-amylase, a β-amylase, a glucoamylase, an arabinoxylanase, a pullanase and/or a protease can be added to the agave bagasse to generate additional C6 sugars and/or C5 sugars. The optional enzymes may be admixed with the agave bagasse slurry at any point of hydrolysis including with the cellulase during high viscosity admixing, at one or more locations in the fiber liquefaction bioreactor and/or in the cellulose hydrolysis reactor. [0036] Hemicellulases may be added to further hydro lyze the various types of hemicelluloses and to further breakdown the hemicellulose fibers to soluble C5 sugars including xylose and arabinose. A hemicellulase, as used herein, refers to a polypeptide that can catalyze hydrolysis of hemicellulose into small polysaccharides such as oligosaccharides, or

monosaccharides including xylose and arabinose. Hemicellulases can be placed into three general categories: the endo-acting enzymes (e.g., endo-l,4- -D-xylanases) that hydrolyze internal bonds within the polysaccharide (xylan) chain; the exo-acting enzymes (e.g., 1,4-β-Ο- xylosidases) that act processively from either the nonreducing end of polysaccharide chain and liberate D-xylose residues; and accessory enzymes. Hemicellulases include, for example, the following: endoxylanases, β-xylosidases, a-L-arabinofuranosidases, a-D-glucuronidases, feruloyl esterases, coumarolyl esterases, a galactosidases, β-galactosidases, β-mannanases, and β-mannosidases. Of the accessory enzymes, an a-L-arabinofuranosidase catalyzes the hydrolysis of terminal non-reducing a-L-arabinofuranoside residues in a-L-arabinosides. An a- glucuronidase catalyzes the hydrolysis of an a-D-glucuronoside to D-glucuronate and an alcohol. An acetylxylanesterase catalyzes the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha-napthyl acetate, and p-nitrophenyl acetate. An a- galactosidase catalyzes the hydrolysis of terminal, non-reducing a-D-galactose residues in a-D- galactosides, including galactose oligosaccharides, galactomannans, galactans and

arabinogalactans. A β-galactosidase catalyzes the hydrolysis of terminal non-reducing β-D- galactose residues in β-D-galactosides. Such a polypeptide may also be capable of hydrolyzing a-L-arabinosides. A 6-mannanase catalyzes the random hydrolysis of l,4^-D-mannosidic linkages in mannans, galactomannans and glucomannans. A β-mannosidase catalyzes the hydrolysis of terminal, non-reducing β D mannose residues in β-D-mannosides. In some aspects, the hemicellulase is an exo-acting hemicellulase, such as an exo-acting hemicellulase which has the ability to hydrolyze hemicellulose under acidic conditions of below pH 7. A xylanase may be obtained from any suitable source, including fungal and bacterial organisms, such as

Aspergillus, Disporotrichum, Penicillium, Neurospora, Fusarium, Trichoderma, Humicola, Thermomyces, and Bacillus. Commercially available preparations comprising xylanase include SHEARZYME®, BIOFEED WHEAT®, BIO-FEED Plus®L, ULTRAFLO®, VISCOZYME®, PENTOPAN MONO®BG, and PULPZYME®HC (Novozymes A/S), and LAMI EX® and SPEZYME®CP (Genencor Int.) An example of a hemicellulase suitable for use in the present disclosure includes VISCOZYME ^® (available from Novozymes A/S, Denmark). Hemicellulase loadings vary with slurry hemicellulose content and is generally about 5, 10, 20 or 30, and ranges thereof, mg enzyme protein per gram of hemicellulose in the agave bagasse slurry. The C5 sugar content of the agave bagasse slurry hydro lyzate represents a yield of at least about 25%, about 30%, about 35%, about 40%, about 45% or at least about 50% based on the hemicellulose content of the agave bagasse.

[0037] Also useful are multienzyme complexes containing multiple carbohydrases, such as Viscozyme® L, available from Novozyme Corporation, which contains arabanase, cellulase, β-glycanase, hemicellulase, and xylanase.

[0038] At the end of the hydrolysis period, the slurry is dewatered in a solid-liquid separation device (e.g., centrifuge, filter press, belt press, screw press, roller press). Dewatering may be accompanied by filtration to remove solids, e.g., fibers. The solid materials may be suitable for the same uses as untreated agave bagasse, e.g., animal feed, paper production, etc. Sterilized process water may be used to rinse the cake to recover sugar entrained with the residual solids. The filtrate, which contains fermentable sugars, is forwarded to the agave juice fermenters.

[0039] In a fermentation step, the filtrate comprising fermentable sugars may be re- combined with the fermentable sugar stream derived from the traditional treatments of the agave pina and the combined stream is inoculated with a yeast useful for the preparation of a wort, or mosto, in the preparation of tequila, or more generally, a mezcal. The wort generally has a low alcohol content of about 6%. This wort is then distilled once to produce what is called

"ordinario," and then a second time to produce clear "silver tequila." After at least a double distillation, the ethanol content may range from about 31% to about 55%, with ethanol contents in the range of 38% to 40% being more suitable for a tequila. A few producers distill the product a third time, or even a fourth time. The residual fibrous solids may be useful as an animal feed component, in the production of paper, or may be burned to provide heat for an industrial process. From there the tequila is either bottled as "silver tequila" or bianco, or it is pumped into wooden barrels to age, where it develops a mellower flavor and amber color.

[0040] The application of the term "tequila" to a distilled beverage derived from blue agave is regulated by Mexican laws. In fact, Mexico has been granted the international right to the word "tequila." In order to be a tequila, therefore, the distilled beverage must conform with Mexican regulations. Mexican regulations recognize two types of tequila. In order to be called a "premium tequila," the source of fermentable sugars must come entirely from the Blue agave pina. Using current production technology, these sugars are primarily inulin (or fructan). It is not clear at this time whether glucose obtained from hydrolysis of the pina fiber (or agave bagasse) qualifies as substrate for premium tequila. A "regular tequila" must comprise greater than 50% of the fermentable sugar must come from blue agave pina, the remainder of the fermentable sugars can come from other sources. Typically, such other sources include sugar cane, molasses, or the like. In some embodiments, the method of the present disclosure is directed to the production of a tequila in which greater than 50% of the sugars are derived from the blue agave pina, while the remainder of the sugar is derived from the agave bagasse and/or agave leaves. The agave bagasse may be treated according to the process steps described above in order to increase the fermentable sugar content. The agave leaves contain a high content of extractable inulin. A tequila prepared by the fermentation of sugars derived from the blue agave pina (greater than 50%) with the remainder derived from the agave bagasse and/or agave leaves is of higher quality than a regular tequila in which the fermentable sugars are derived from the blue agave pina (greater than 50%) and the remainder from sugar cane and/or molasses.

[0041] FIG. 5 depicts a flow diagram for an embodiment directed to the production of tequila prepared by the fermentation of sugars derived from the blue agave pina (greater than 50%) with the remainder derived from the agave bagasse and/or agave leaves. In some embodiments, the tequila is prepared by the fermentation of sugars in which greater than 50% of the fermentable sugars are derived from the blue agave pina, which is prepared for fermentation according to traditional tequila production techniques. In some embodiments, greater than 50.1%, or greater than 50.2%, or greater than 50.5%, or greater than 51%, or greater than 52%, or greater than 53%, or greater than 54%, or 55%, or greater than 56%, or greater than 57%, or greater than 58%, or greater than 59%, or greater than 60%, or greater than 65%, or greater than 70%, or greater than 75% of the fermentable sugars are derived from the blue agave pina, which is prepared for fermentation according to traditional tequila production techniques. Accordingly, the portion of fermentable sugars derived from the agave bagasse and/or agave leaves is less than 50%), or less than 49.9%, or less than 49.8%, or less than 49.5%, or less than 49%, or less than 48%, or less than 47%, or less than 46%, or less than 45%, or less than 44%, or less than 43%, or less than 42%, or less than 41%, or less than 40%, or less than 35%, or less than 30%. The feedstock derived from the agave bagasse and/or agave leaves may comprise chopped Blue agave leaves and agave bagasse in the proportion ranging from 100% to 0% leaves. In one embodiment, the agave leaves are chopped using suitable equipment such as forage chopper, hammer mill, shredder, and the like. The particle size of chopped leaves ranges from about 5 mm to about 20 mm. Agave bagasse is blended with the chopped leaves in a mixer (e.g., pug mill mixer, paddle mixer, ribbon blender, mixing screw conveyor).

[0042] Dilute acid may be added in the form of fine mist via spray nozzles. The pH of the mixture of agave bagasse and/or agave leaves may be adjusted to between about 3.5 and about 6.5, such as between about 4.0 and about 6.5, such as between about 4.5 and about 6.0, such as about 5.2. For acidic pH adjustment, if necessary, sulfuric acid, hydrochloric acid, phosphoric acid, nitric acid, acetic acid, lactic acid, citric acid, tartaric acid, and the like, may be used. Suitable acids include sulfuric acid, hydrochloric acid, phosphoric acid, and nitric acid. The acid loading inside the pretreatment reactor ranges from about 0.002 g acid/g dry materials to about 0.02 g acid/g materials. Acid addition to mixture may be accomplished in a spray impregnation system or a soaking impregnation system prior to steam pretreatment. If necessary, for alkaline pH adjustment, ammonia, sodium hydroxide and potassium hydroxide, are suitable. The slurry may be agitated by conventional means during acid hydrolysis, such as by paddle stirring, stir plate, vortex, or shaker. Acid hydrolysis may occur under these mild conditions for a duration of at least about five minutes, such as between about five minutes and about 120 minutes.

[0043] The feedstock is then pretreated in direct steam injection pretreatment reactors (batch or continuous). The agave bagasse and/or agave leaves and water mixture may be steam treated prior to, after, or simultaneous with acidic pre-treatment. Steam pretreatment reactors may be batch or continuous. In some aspects of the present disclosure, steam treating may occur at a temperature of at least about 100°C, such as from about 100°C to about 200°C, from about 120°C to about 160°C, from about 140°C to about 160°C, or from about 100°C to about 110°C. Steam treating may occur for a duration of at least about 1 minute, such as between about 1 minute and about 30 minutes, such as from about 5 minutes to about 20 minutes, such as about 10 minutes.

[0044] The optional acid hydrolysis and steam treatment are effective to disrupt and separate the fibrous strands in the agave bagasse and/or agave leaves, thereby making the fiber more available to the enzymes in a subsequent enzymatic hydrolysis. The acidic conditions may also hydrolyze at least a portion of the celluloses, hemicelluloses, inulins, and lignins into lower molecular weight polymers. Pretreatment of bagasse and/or leaves enhances the enzyme accessibility to cellulose, and sterilizes the fiber to minimize the risk of bacterial contamination in the subsequent enzymatic hydrolysis step. In addition, the acidic and high temperature conditions in the steam treatment may break down lignin-hemicellulose complexes and may hydro lyze the hemicellulose, thereby producing soluble oligomers and monomers of the pentose sugars xylose and arabinose, and other sugars.

[0045] After one or more pre-treatment steps (i.e., grinding, disc refining, acid hydrolysis, and/or steam treating), the agave bagasse and/or agave leaves and water mixture is pH adjusted to a pH suitable for enzymatic hydrolysis. Additionally, the agave bagasse and/or agave leaves and water mixture is cooled to a temperature suitable for enzymatic hydrolysis. In some embodiments, a chilled dilute aqua ammonia is blended with the pretreated bagasse and/or leaves to achieved a slurry ranging having between about 10% total solids and about 30% total solids content, such as between about 20% total solids and about 25% total solids, such as between about 15% to about 25% total solids content. The pH of the pretreated bagasse and/or leaves is adjusted to between about 4.5 and about 6.0, such as between about 5.0 and about 5.5, such as about 5.2. In some embodiments, the temperature may be between about 35°C and about 60°C, such as between about 50°C to about 55°C, such as about 50°C. For steam pretreatment, the pretreated bagasse and/or leaves slurry is held for a period of time from 5 minutes to about 30 minutes to ensure the pH is stabilized before enzyme is added.

[0046] Enzymes are then blended into the slurry using a dynamic mixer (e.g., pug mill mixer, paddle mixer, mixing screw conveyor, dynamic in-line mixer, or homogenizer) to thereby form an enzyme hydrolysis mixture. Enzymes may include cellulases, hemicellulases, and inulases. Enzymatic hydrolysis can be batch, fed-batch or continuous. The hydrolysis time may range from about 8 to about 72 hours, or from about 24 hours to about 72 hours. The enzymes suitable for the enzymatic hydrolysis of the agave bagasse and/or agave leaves comprise any of those enzymes previously described herein.

[0047] At the end of the hydrolysis period, the slurry is dewatered in a solid-liquid separation device (e.g., centrifuge, filter press, belt press, screw press, roller press). Dewatering may be accompanied by filtration to remove solids, e.g., fibers. The solid materials may be suitable for the same uses as untreated agave bagasse, e.g., animal feed, paper production, etc. Sterilized process water may be used to rinse the cake to recover sugar entrained with the residual solids. The filtrate, which contains fermentable sugars, is forwarded to the agave juice fermenters. [0048] In a fermentation step, the filtrate comprising fermentable sugars derived from the agave bagasse and/or agave leaves may be combined with the fermentable sugar stream derived from the traditional treatments of the agave pina and the combined stream is inoculated with a yeast useful for the preparation of a wort, or mosto, in the preparation of tequila, or more generally, a mezcal. The wort generally has a low alcohol content of about 6%. This wort is then distilled once to produce what is called "ordinario," and then a second time to produce clear "silver tequila." After at least a double distillation, the ethanol content may range from about 31% to about 55%, with ethanol contents in the range of 38% to 40% being more suitable for a tequila. A few producers distill the product a third time, or even a fourth time. The residual fibrous solids may be useful as an animal feed component, in the production of paper, or may be burned to provide heat for an industrial process. As steam pretreatment of agave leaves and bagasse may produce a small amount of furfural and HMF, a purifying system is integrated into the distillation system to remove these contaminants. Suitable purifiers include adsorbers (liquid and/or vapor phase) using appropriate adsorbents (such as copper, activated charcoal, oxidizing agents, resins that are design to remove the contaminants). From there the tequila is either bottled as "silver tequila" or bianco, or it is pumped into wooden barrels to age, where it develops a mellower flavor and amber color. The tequila prepared from the fermentation of sugars derived from the agave pina (greater than 50%) and the balance of the fermentable sugars derived from the agave bagasse and/or agave leaves is higher quality than the tequilas available on the market in which the fermentable sugars (less than 49%) are derived from cane sugar or molasses.

[0049] In some embodiments, the method of the present disclosure is directed to the production of fuel ethanol from waste agave bagasse, leaves and other available biomass residues. In this regard, FIG. 6 depicts a simplified block flow diagram for the production of fuel ethanol from agave bagasse and leaves.

[0050] According to embodiments wherein fermentable sugars derived from the agave bagasse and/or agave leaves are used in the production of biofuel ethanol, the feedstock preparation, pretreatment and enzyme hydrolysis steps are identical to the corresponding steps described above in connection with the production of a tequila. In embodiments wherein the final production is biofuel ethanol, the fermentable sugars derived from the agave bagasse and/or agave leaves are not combined with fermentable sugars derived from the agave pina, since these fermentable sugars are a high value product suitable for use in the production of tequila. Additionally, the ethanol distillation step does not require a purification system to remove contaminants, e.g., furfural, that are not suitable for use of the ethanol product as alcohol beverage.

[0051 ] After the fermentation is complete, the wort is fed to a reboiler where ethanol and volatile impurities are separated by vaporization in a distillation column leaving reboiler bottoms containing dissolved solids. Generally, conventional distillation apparatuses known in the art are suitable for use in accordance with the present disclosure. Conventional apparatuses are described, for instance, in Distillation Technology, GEA Wiegand GmbH, publication P06E 022009 (2013) and Bioethanol Technology, GEA Wiegand GmbH, publication P I IE (2013), the entire contents of which are incorporated herein by reference for all relevant purposes.

Examples of suitable distillation columns include columns having dual flow and cross flow trays, such as dual flow sieve trays or cross-flow valve trays. In some aspects, cross flow valve trays are used because of the higher turn down ratio and higher they provide. Suitable valve trays include, for example, NORPRO PRO VALVE trays. Ethanol is condensed and purified in the distillation column. The liquid ethanol exits the top of the distillation column at about 95% purity from where it passes through a molecular sieve dehydration column which removes at least about 75%, about 80%, about 85%, about 90%, about 95% or even at least about 99% of the remaining residual water. The residual fibrous solids may be useful as an animal feed component, in the production of paper, or may be burned to provide heat for an industrial process.

[0052] As various changes could be made in the above compositions and processes without departing from the scope of the disclosure, it is intended that all matter contained in the above description be interpreted as illustrative and not in a limiting sense.

[0053] When introducing elements of the present disclosure or the embodiment(s) thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.