WO2016007350A1 - Preconditioning of lignocellulosic biomass - Google Patents

Abstract

An improved process for converting lignocellulosic biomass materials into soluble sugars or fermentable products is provided, comprising a preconditioning step whereby the lignocellulosic biomass materials are preconditioned to have a pH of at least above about 5.5.

Description

PRECONDITIONING OF LIGNOCELLULOSIC BIOMASS

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/022,339, filed July 9, 2014, the contents of which are hereby incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[0002] Provided is an improved method or process of producing useful sugars and/or fermentation products from lignocellulosic biomass materials. Specifically the method or process provides for improved effectiveness and efficiency in converting such lignocellulosic biomass materials by preconditioning of such biomass materials before they are subject to enzymatic hydrolysis. The process or method of the invention substantially reduces or even eliminates the need to use certain costly preconditioning agents, and as such can confer significant cost savings to the cellulosic biorefinery.

BACKGROUND

[0003] Cellululosic and lignocellulosic plant biomass, particularly agricultural residues, forage crops and woody crops, wood, forestry wastes, sludge from paper manufacture, and municipal and industrial solid wastes, provide an abundant and renewable feedstock for the production of valuable products such as fuels and other chemicals, replacing petroleum feedstock, which is non-renewable and increasingly costly and scarce. Ethanol and other fuel alcohols have many desirable features that made them ideal petroleum substitutes. However, most of the ethanol presently in the market place is produced from food-related resources such as corn grain and sugar cane juice, which is not seen as economically feasible and sustainably practical in the long run given the rapid rise in fuel and energy demands around the globe. Widely available lignocellulosic sources of biomass are seen as a potentially inexpensive and feasible substitute feedstock, which at the same time would help avoid feedstock conflict with the prevalent food industry. [0004] Cellulose and hemicellulose are the most abundant plant materials produced by photosynthesis. Lignocellulosic biomass materials are made up of three major organic fractions, cellulose, hemicellulose and lignin. Among these major fractions, cellulose and hemicelluloses together can constitute as much as three quarters of overall biomass composition, and both of these can be degraded or converted to sugars, which can in turn be used as an energy source by numerous microorganisms {e.g., bacteria, yeast and fungi) that produce extracellular enzymes capable of hydrolysis of the polymeric substrates to monomeric sugars (Aro et al., (2001 ) J. Biol. Chem., 276: 24309-24314). Monomeric sugars can then be metabolized or fermented by ethanologen microorganisms into ethanol, by other microorganisms into chemicals, or simply used as building blocks to be converted into useful materials with chemical processes. As the limits of non-renewable resources approach, the potential of cellulose to become a major renewable energy resource is enormous (Krishna et al., (2001 ) Bioresource Tech., 77: 193-196). The effective utilization of cellulose through biological processes is one approach to overcoming the shortage of foods, feeds, and fuels (Ohmiya et al., (1997)

Biotechnol. Gen. Engineer Rev., 14: 365-414). [0005] In a typical process, the lignocellulosic biomass material is first subject to one or more pretreatements, during which the lignin becomes more or less permeabilized and the hemicelluloses disrupted to an extent such the complex carbohydrate cellulose polymers become more readily accessible to celluloytic hydrolysis enzymes. Afterwards, the pretreated lignocellulosic biomass mixture is subject to an enzymatic hydrolysis/saccharification step, whereby the enzymes solubilize the pretreated biomass, breaking it down into oligomeric saccharides and further into monosaccharides that are fermentable. In order for the entire biomass to fermentation product(s) process to be economically conducted, it is desirable that the saccharification or enzymatic hydrolysis step be as efficient and effective as possible, and the product of such a step to contain a high concentration of fermentable sugars.

[0006] It is well known that, while lignocellulosic biomass materials are renewable and abundant, their compositional recalcitrance is a key impediment to hydrolysis and production of fermentable sugars. Aside from the enzymatic process as described above, another approach to break down plant-based lignocellulosic materials is acid hydrolysis. Acid hydrolysis is inexpensive but they can cause degradation of sugars due to the high temperatures typically used with acids during the hydrolysis reaction, and the almost inevitable generation of inhibitors to various well-known ethanologens, which would severely hinders downstream processing. The use of acids also places significant burden on plant construction because all equipment and systems need to be corrosion resistant.

[0007] Enzymatic hydrolysis of lignocellulosic biomass materials is thus

comparatively more attractive in that there tends to be a higher potential of generating greater yields of fermentable sugars, and less inhibitors. But enzymatic hydrolysis has its limitations as well. Due to the complexity of the plant biomass materials, pretreatment is typically necessary to render or disrupt the cellulosic structure of the biomass and make it more accessible to the enzymes. Many enzymatic activities may need to be present in a consortium or at least applied together to the post-pretreatment lignocellulosic biomass material, delicately balanced in order to achieve effective synergism and more complete breakdown of the materials. For example, enzymatic hydrolysis of the complex lignocellulosic structure and rather recalcitrant plant cell walls involves the concerted and/or tandem actions of a number of different endo-acting and exo-acting enzymes (e.g., cellulases and hemicellulases). In addition, typically a large amount of enzymes is required in order to achieve reasonable and commercially viable rate and yields.

[0008] Most of the enzymatic hydrolysis of lignocellulosic biomass materials focus on cellulases, which are enzymes that hydrolyze cellulose (comprising beta-1 ,4- glucan or beta D-glucosidic linkages) resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. Cellulases have been traditionally divided into three major classes: endoglucanases (EC 3.2.1 .4) ("EG"),

exoglucanases or cellobiohydrolases (EC 3.2.1 .91 ) ("CBH") and beta-glucosidases ([beta]-D-glucoside glucohydrolase; EC 3.2.1 .21 ) ("BG") (Knowles et al., (1987) TIBTECH 5: 255-261 ; and Schulein, (1988) Methods Enzymol., 160: 234-243).

Endoglucanases act mainly on the amorphous parts of the cellulose fiber, whereas cellobiohydrolases are also able to degrade crystalline cellulose (Nevalainen and Penttila, (1995) Mycota, 303-319). Thus, the presence of a cellobiohydrolase in a cellulase system is required for efficient solubilization of crystalline cellulose

(Suurnakki et al., (2000) Cellulose, 7: 189-209). Beta-glucosidase acts to liberate D- glucose units from cellobiose, cello-oligosaccharides, and other glucosides (Freer, (1993) J. Biol. Chem., 268: 9337-9342). [0009] Beta-xylanases and beta-mannanases are endo-acting enzymes, beta- mannosidase, beta-glucosidase and alpha-galactosidases are exo-acting enzymes. To disrupt the hemicelulose, xylanases together with other accessory proteins (non- limiting examples of which include L-a-arabinofuranosidases, feruloyl and

acetylxylan esterases, glucuronidases, and β-xylosidases) can be applied.

[0010] Other cellulosic disrupting enzymes and proteins are increasingly gaining recognition and prominence in recent years. Thos are accessory enzymes such as mannanases (galactanases {e.g., endo- and exo-galactanases), arabinases {e.g., endo-arabinases and exo-arabinases), ligninases, amylases, glucuronidases, proteases, esterases {e.g., ferulic acid esterases, acetyl xylan esterases, coumaric acid esterases or pectin methyl esterases), lipases, other glycoside hydrolases, xyloglucanases, CIP1 , CIP2, swollenins, expansins, and cellulose disrupting proteins. For example, the cellulose disrupting proteins are cellulose binding modules. [0011] The costs of producing enzymes can be prohibitively high, therefore it is important to reduce the costs of enzyme production and/or reduce the amount of enzymes used in any given process to achieve the same level of resulting

fermentable sugars.

[0012] Other factors that may affect the efficacy and effectiveness of the hydrolysis and the yield of resulting fermentable sugars are also important. These factors typically include the operating mode and conditions of the hydrolysis reaction such that the enzymes perform their catalytic functions under as optimized conditions as possible. Prevention of enzymatic activity loss is recognized as another important consideration, therefore conditions under which the enzymes perform hydrolysis, and other conditions such enzymes are exposed to throughout the industrial processes are often carefully controlled to avoid enzymatic denaturation or deactivation, which can be irreversible.

[0013] Another important cost reduction consideration pertains to reducing the waste of enzymes and enzymatic activities, through prevention or reduction of non- productive binding and/or inactivation of such enzymes during the industrial processes. The present invention provides one such improved method or process where reduction of non-productive binding and/or inactivation of enzymes is achieved, thereby overall costs of the industrial process can be achieved through the application of such a method/process.

SUMMARY OF THE INVENTION [0014] Described is an improved and lower-cost industrial process for producing useful, soluble sugars from a lignocellulosic biomass material. The improved process leads to higher enzymatic hydrolysis efficacy and efficiency, and as such it also delivers a higher yield of fermentable sugars from a given batch of

lignocellulosic biomaterial as compared to when other previously known and practiced processes are applied. The lignocellulosic biomass material used in the process can be, and is preferably pretreated. Described is also an improved and lower-cost process for producing fermentation products from such lignocellulosic biomass materials.

[0015] In the first aspect, the invention pertains to a method or process for producing a fermentable sugar from a lignocellulosic biomass material comprising: (1 ) pretreatment of a lignocellulosic biomass material to produce a pretreated biomass substrate; (2) pre-conditioning of the pretreated biomass substrate; (3) hydrolyzing the pre-conditioned pretreated biomass substrate using an enzymatic mixture comprising at least one cellulase enzyme; and (4) a recovery step during which the soluble sugars produced in step (3) is recovered. The soluble sugars thus produced can then be further converted into a variety of useful produces including, for example, liquid fuels such as ethanol or butanol, or syrups, or biochemical building blocks or feedstock for making industrially useful materials such as polymers that may replace petrochemical derived materials. [0016] In some embodiments, the lignocellulosic biomass material is pretreated in a step comprising an acidic pretreatment and/or a steam explosion type pretreatment. The resulting pretreated lignocellulosic biomass material can have a pH of below about 3.5, or lower. For example, the resulting pretreated lignocellulosic biomass material can have a pH of about 1 to about 4, such as about 1 .5 to about 3.5, or about 2 to 3. In these embodiments, a regular preconditioning procedure is applied, which comprises a pH adjustment procedure adjusting the pretreated biomass material to a pH of at least above about 5.5, for example, at least above about 5.6, or at least about 5.7, or at least about 5.8, or at least about 5.9, or at least about 6.0, or at least about 6.1 , or at least about 6.2, or at least about 6.3, or at least about 6.4, or even at least about 6.5, prior to introducing the cellulosic hydrolysis enzymes, and the start of the enzymatic hydrolysis step. The pH of the pretreated biomass material can be pre-conditioned to be between, for example, about 5.5 to about 6.5, such as about 5.6 to about 6.4, about 5.7 to about 6.3, about 5.8 to about 6.2, or even about 5.9 to about 6.1 .

[0017] In such embodiments, the regular preconditioning step can overlap with the beginning of the enzymatic hydrolysis step by, e.g., about 2 minutes, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 1 hour, about 90 minutes, or about 2 hours. In some embodiments, the overlap of the pre-conditioning step with enzymatic hydrolysis step is for a period of between about 2 minutes to about 3 hours, for example, between about 5 minutes to about 2.5 hours, about 10 minutes to about 2.2 hours, about 30 minutes to about 2 hours, between about 1 hour to about 2 hours.

[0018] In such embodiments, the regular preconditioning step is carried out in the same reactor or vessel as the enzymatic hydrolysis step. In other embodiments, the preconditioning step is carried out in a separate reactor or vessel as the enzymatic hydrolysis step. In related embodiments, the enzyme mixture comprising at least one cellulase enzyme can be added to the preconditioning vessel or reactor when the pH of the pretreated biomass material is raised to at least about 4.0, for example, at least about 4.2, at least about 4.4, at least about 4.6, at least about 4.8, or at least about 5.0, through pre-conditioning.

[0019] In alternative embodiments, the regular preconditioning step is carried out in a separate reactor or vessel as the enzymatic hydrolysis step. Batches of pretreated and preconditioned biomass materials can be added to a saccharification reactor already containing an enzymatic hydrolysis reaction mixture comprising one or more prior batch of pretreated and/or preconditioned biomass materials and suitable hydrolytic enzymes. [0020] In some particular embodiments, no further pH adjustment is necessary from the start (i.e., the point at which the pretreated and preconditioned biomass materials come into contact with the hydrolysis enzymes) until the conclusion of the enzymatic hydrolysis step.

[0021] In some embodiments, the lignocellulosic biomass material is pretreated in a step comprising an alkaline pretreatment. The resulting pretreated biomass material can have a pH of between about 7.0 and about 1 1 .0. In these embodiments, a more involved preconditioning step is applied. The more involved preconditioning step, like the regular preconditioning step, comprises also adjusting the pretreated biomass material to a pH of at least above about 5.5, for example, at least above about 5.6, or at least about 5.7, or at least about 5.8, or at least about 5.9, or at least about 6.0, or at least about 6.1 , or at least about 6.2, or at least about 6.3, or at least about 6.4, or even at least about 6.5, prior to introducing the cellulosic hydrolysis enzymes, and the start of the enzymatic hydrolysis step. The pH of the pretreated biomass material can be preconditioned to be between, for example, about 5.5 to about 6.5, such as about 5.6 to about 6.4, about 5.7 to about 6.3, about 5.8 to about 6.2, or even about 5.9 to about 6.1 .

[0022] In such embodiments, the more involved preconditioning step can, just like the regular preconditioning step, also overlap with the beginning of the enzymatic hydrolysis step by, e.g., about 2 minutes, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 1 hour, about 90 minutes, or about 2 hours. In some embodiments, the overlap of the preconditioning step with enzymatic hydrolysis step is for a period of between about 2 minutes to about 3 hours, for example, between about 5 minutes to about 2.5 hours, about 10 minutes to about 2.2 hours, about 30 minutes to about 2 hours, between about 1 hour to about 2 hours. [0023] In such embodiments, the more involved preconditioning step can, just like the regular preconditioning step, also be carried out in the same reactor or vessel as the enzymatic hydrolysis step. In other embodiments, the more involved

preconditioning step can also be carried out in a separate reactor or vessel as the enzymatic hydrolysis step. In related embodiments, the enzyme mixture comprising at least one cellulase enzyme can be added to the preconditioning vessel or reactor when the pH of the pretreated biomass material is raised to at least about 4.0, for example, at least about 4.2, at least about 4.4, at least about 4.6, at least about 4.8, or at least about 5.0, through preconditioning. [0024] Moreover, the more involved preconditioning step, like the regular preconditioning step, can be carried out in a separate reactor or vessel as the enzymatic hydrolysis step. Batches of pretreated and preconditioned biomass materials can be added to a saccharification reactor already containing an enzymatic hydrolysis reaction mixture comprising one or more prior batch of pretreated and/or preconditioned biomass materials and suitable hydrolytic enzymes.

[0025] In some embodiments, no further pH adjustment is necessary from the start (i.e., the point at which the preconditioned biomass material is exposed to the suitable hydrolysis enzyme mixture) until the conclusion of the enzymatic hydrolysis step.

[0026] In both the regular preconditioning step and the more involved

preconditioning step embodiments above, the preconditioning step further comprises a water washing step before the pH adjustment step. The water washing step can be carried out with warm or hot water, for example, with water of temperatures at least above about 40 °C, such as at least above about 45 °C, or at least above about 50 °C, or at least above about 55 °C, or even at least above about 60 °C.

Alternatively, the water washing step can be carried out with cold water, for example, with water of temperatures of about 4 °C to about 40 °C, such as about 10 °C to about 38 °C, about 15 °C to about 35 °C, about 20 °C to about 30 °C. In some embodiments, the water washing step can be carried out with rounds of warm or hot water followed by cold water, or vice versa, or in any order that is suitable. In certain specific embodiments, the pretreated biomass material can be cooled by one or more cold water-washing steps at the start of the preconditioning step, such that after each rounds of cold water washing the temperature of the pretreated biomass material is lower than prior to the water washing step.

[0027] The water washing step can be carried out by methods well known in the field. Washing can comprising exposing the pretreated biomass material to a volume of water, for example, an equal volume of water, or a half volume of water, or any fractional volume of water, or a double volume of water, optionally one or more times, for example, 2, 3, 4, 5, 6, or 7 or even 8 or more times. After each rounds of washing, the biomass and the "washate" (or waste water) can be separated or removed by extraction, centrifugation or other methods known to allow solid-liquid separation, including, for example, using a screw press, belt press, drum filter, hydrocyclone and/or filter press, using any kind of apparatus that can handle or bring about solid/liquid separation, including gravity-fed systems and/or apparatuses.

[0028] When cold water is used for washing, after a water washing step, the washed biomass material can be at least about 1 °C lower, e.g., at least about 1 °C lower, at least about 2°C lower, or at least about 5°C lower, in temperature than that of the pretreated biomass prior to the water washing step.

[0029] In certain embodiments, the washate (i.e., the used water from the water- washing step) comprises a measurable level of acetate. The level of acetate in the washates can be measured using conventional and well known analytical methods such as spectroscopy. In some embodiments, the washate comprises a measurable level of phenolic compounds. The level of phenolic compounds can be measured using conventional and known analytical methods and instruments such as GC and other spectroscopy methods.

[0030] In certain embodiments, after the water washing step, the surface of the pretreated and washed biomass substrate is more negatively charged than that of the pretreated but not-yet- washed biomass substrate. In certain embodiments, the surface of the pretreated and washed biomass substrate has a negative charge level that is at least about 1 mV in zeta potential, measured using known instruments such as, for example, the Zetasizer Nano ZS made by Malvern. For example, the surface of the pretreated and washed biomass substrate has a negative charge level that is at least about 1 mV, at least about 2 mV, at least about 3 mV, at least about 4 mV, at least about 5 mV, at least about 6 mV, at least about 7 mV, at least about 8 mV, at least about 9 mV, at least about 10 mV, at least about 1 1 mV, at least about 12 mV, at least about 13 mV, at least about 14 mV, or even at least about 15 mV, higher than the negative charge level of the same pretreated biomass substrate before it is washed.

[0031] In embodiments of the regular preconditioning step, the water washing step is followed by a pH adjustment step, during which an amount of an alkali or a chemical base is used to bring the pH of the water-washed pretreated biomass material to a pH of at least above about 5.5, for example, at least above about 5.6, or at least about 5.7, or at least about 5.8, or at least about 5.9, or at least about 6.0, or at least about 6.1 , or at least about 6.2, or at least about 6.3, or at least about 6.4, or even at least about 6.5. In certain embodiments, the pH of the water-washed pretreated biomass material is adjusted to be within a range of, for example, about 5.5 to about 6.5, such as about 5.6 to about 6.4, about 5.7 to about 6.3, about 5.8 to about 6.2, or even about 5.9 to about 6.1 . [0032] In embodiments of the more involved preconditioning step, the water washing step is followed by an acid treatment procedure, during which an amount of acid is used to bring the pH of the water-washed pretreated biomass material to a pH of about 1 .0 to about 3.5. This is followed by letting the water-washed pretreated biomass material sit at that acidic pH for a period of about 2 minutes to about 24 hours, for example, about 10 minutes to about 20 hours, about 20 minutes to about 10 hours, and so on. This is then followed by an optional water-washing procedure, which is in turn followed by a second pH adjustment step during which an amount of an alkali or chemical base is used to bring the pH of the water washed, acid treated pretreated biomass material to a pH of at least above about 5.5, for example, at least above about 5.6, or at least about 5.7, or at least about 5.8, or at least about 5.9, or at least about 6.0, or at least about 6.1 , or at least about 6.2, or at least about 6.3, or at least about 6.4, or even at least about 6.5. In certain embodiments, the pH of the water-washed, acid treated, pretreated biomass material is adjusted to be within a range of, for example, about 5.5 to about 6.5, such as about 5.6 to about 6.4, about 5.7 to about 6.3, about 5.8 to about 6.2, or even about 5.9 to about 6.1 .

[0033] In some embodiments, the amount of alkali or chemical used to adjust the water-washed pretreated biomass material to a given pH is at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or even at least about 60% less than that would be required to bring the same pretreated biomass material to the same pH if such pretreated biomass material has not been subject to a water washing step.

[0034] The enzymatic hydrolysis step of the above embodiments is carried with an enzyme mixture comprising at least a cellulase enzyme. In certain instances, the enzyme mixture comprises two or more cellulases, for example, at least one cellobiohydrolase and at least one endoglucanase. In some instances, the enzyme mixture comprises one or more beta-glucosidases. In certain instances, the enzyme mixture comprises two or more cellobiohydrolases. In further instances, the enzyme mixture also comprises one or more hemicellulases. In yet further instances, the enzyme mixture also comprises one or more accessory enzymes. In certain specific instances, the enzyme mixture comprises one or more lytic polysaccharide monooxygenases or GH61 enzymes.

[0035] The preconditioned and pretreated biomass materials prepared according to the embodiments above, loaded to a same dry solids level, when subject to hydrolysis by a given enzyme mixture, present at a given concentration or level of enzyme loading, and under the same hydrolysis conditions as well as for the same duration of time, give yield to at least about 0.5%, for example, at least about 0.5%, at least about 1 .0%, at least about 1 .5%, at least about 2.0%, at least about 2.5%, at least about 3.0%, at least about 3.5%, at least about 4.0%, at least about 4.5%, at least about 5.0%, at least about 5.5%, at least about 6.0%, at least about 6.5%, at least about 7.0%, at least about 7.5%, or even at least about 8.0% higher glucan conversion or yield of fermentable sugars, as compared to if the same pretreated biomass materials, which has not undergone the preconditioning step. [0036] In some specific embodiments, the method or process of the invention can be used to significantly reduce the amount of non-productive lignin binding blocker used in the process. Non-productive lignin binding blockers can include, for example, certain surfactants, detergents or agents that changes the surface properties of materials, or polyethylene glycol. For example, the method or process of the invention can be used to achieve the same improved enzyme hydrolysis yield as about 0.05%, for example, about 0.05%, about 0.07%, about 0.1 %, about 0.15%, about 0.2%, about 0.3%, about 0.4%, about 0.5% or even about 0.6% of PEG.

Known non-productive lignin blockers tend to be expensive commodities.

Replacement or complete displacement of their use will lead to significant cost savings in industrial and commercial scale cellulosic biorefineries.

[0037] In a second aspect, the invention pertains to a method or process for producing a fermentation product from a lignocellulosic biomass materials

comprising: (1 ) pretreatment of a lignocellulosic biomass material to produce a pretreated biomass substrate; (2) preconditioning of the pretreated biomass substrate; (3) hydrolyzing the preconditioned pretreated biomass substrate using an enzymatic mixture comprising at least one cellulase enzyme; and (4) fermenting the product of enzymatic hydrolysis step (3). Optionally the process further comprises recovery of the fermentation product. [0038] In some embodiments, the same preconditioning steps and embodiments as described in the first aspect can be used in the method or process of the second aspect for producing a fermentation product. The same enzyme mixture comprising at least one cellulase enzyme of the first aspect can also be suitable for the method or process for producing a fermentation product of the second aspect.

[0039] The preconditioned and pretreated biomass materials prepared according to the embodiments above, loaded to a same dry solids level, when subject to hydrolysis by a given enzyme mixture, present at a given concentration or level of enzyme loading, and under the same hydrolysis conditions as well as for the same duration of time, the same fermentation conditions and microorganism, give yield to at least about 0.5%, for example, at least about 0.5%, at least about 1 .0%, at least about 1 .5%, at least about 2.0%, at least about 2.5%, at least about 3.0%, at least about 3.5%, at least about 4.0%, at least about 4.5%, at least about 5.0%, at least about 5.5%, at least about 6.0%, at least about 6.5%, at least about 7.0%, at least about 7.5%, or even at least about 8.0% higher amounts of fermentation product, as compared to if the same pretreated biomass materials, which has not undergone the same preconditioning step, is subject to the same enzymes and enzyme loading, same hydrolysis conditions and durations, and same fermentation condition and microorganism. [0040] In a third aspect, the invention pertains to an improved saccharification mixture comprising a pretreated and preconditioned lignocellulosic biomass substrate and an enzymatic mixture comprising at least one cellulase enzyme, wherein the saccharification mixture, at the start of the enzymatic hydrolysis step, has a pH of at least about 5.5, for example, at least about 5.5, at least about 5.6, at least about 5.7, at least about 5.8, at least about 5.9, at least about 6.0, at least about 6.1 , at least about 6.2, at least about 6.3, at least about 6.4, or even at least about 6.5. For example, the saccharification mixture has a pH within a range of, for example, about 5.5 to about 6.5, such as about 5.6 to about 6.4, about 5.7 to about 6.3, about 5.8 to about 6.2, or even about 5.9 to about 6.1 . The saccharification mixture is then, without further adjustment of pH, incubated for a period sufficient to cause conversion of at least about 20% (e.g., at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or even at least about 60%) of the total glucan contained in the lignocellulosic cellulosic biomass material.

[0041] In certain embodiments, at the start of the enzymatic hydrolysis step, the pH of saccharification mixture is at least above about 5.5, for example, at least above about 5.6, or at least about 5.7, or at least about 5.8, or at least about 5.9, or at least about 6.0, or at least about 6.1 , or at least about 6.2, or at least about 6.3, or at least about 6.4, or even at least about 6.5. In related embodiments, at the start of the enzymatic hydrolysis step, the pH of the saccharification mixture is within a range of, for example, about 5.5 to about 6.5, such as about 5.6 to about 6.4, about 5.7 to about 6.3, about 5.8 to about 6.2, or even about 5.9 to about 6.1 .

[0042] In certain embodiments, the improved saccharification mixture further comprises one or more hemicellulases. In certain embodiments, the improved sacccharification mixture further comprises one or more accessory enzymes. In certain further embodiments, the improved saccharification mixture further comprises one or more lytic polysaccharide monooxygenases or GH61 enzymes.

[0043] In certain embodiments, the improved saccharification mixture does not contain non-productive lignin binding blocker such as surfactants or polyethylene glycol.

[0044] In a related fourth aspect, the invention pertains to a method of using the improved saccharification mixture. In some embodiments, the improved

saccharification mixture is incubated for a period that is sufficient for conversion of at least 25% of the glucan in the lignocellulosic biomass substrate. The incubation period can be as short as a few hours to as long as a few days. For example, the incubation period may be 1 hour, 2 hours, 5 hours, 10 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, 66 hours, or 72 hours or even longer. The incubation can take place at a temperature that is suitable for the enzymes in the mixture to function, for example, at a temperature of between about 20 °C and 65 °C, for example between about 25 °C and about 62 °C, between about 28 °C and about 60 °C, between about 30 °C and about 58 °C, or between about 40 °C and about 55 °C. [0045] In a fifth and sixth aspects, the invention pertains to the fermentable sugars produced using the method or process of the first aspect, and the fermentation products produced using the method or process of the second aspect.

BRIEF DESCRIPTION OF THE FIGURES

[0046] FIGURE 1 : Depicts the lack of effect of the regular preconditioning step as applied to an Avicel cellulosic substrate, as described in Example I.

[0047] FIGURE 2: Depicts the substantial improvement of enzymatic hydrolysis as reflected in increased glucan conversion as a result of applying the regular preconditioning step to a dilute acid pretreated corn stover biomass material prior to the enzymatic hydrolysis step, as described in Example 2.

[0048] FIGURE 3: Depicts the substantial improvement of enzymatic hydrolysis as reflected in increased glucan conversion as a result of applying the regular preconditioning step to a dilute acid pretreated sugarcane bagasse biomass material prior to the enzymatic hydrolysis step, as described in Example 3.

[0049] FIGURE 4: Depicts improvement of enzymatic hydrolysis as reflected in increased glucan conversion as a result of applying the regular preconditioning step to a hydrothermally pretreated switchgrass biomass material prior to the enzymatic hydrolysis step, as described in Example 4. [0050] FIGURE 5: Depicts improvement of enzymatic hydrolysis as reflected in increased glucan conversion as a result of applying the more involved

preconditioning step comprising an acid treatment step to a dilute ammonia pretreated corn stover biomass material prior to the enzymatic hydrolysis step, as described in Example 5. Example 5.

DETAILED DESCRIPTION

1. Overview

[0051] Provided are improved methods or processes for producing fermentable sugars or fermentation products from lignocellulosic biomass substrate with reduced process costs and increased yield of desirable products. Also provided are improved saccharification mixtures comprising a pretreated and preconditioned lignocellulosic biomass substrate, an enzyme mixture comprising at least one cellulase enzyme, at a pH of at least about 5.5 or higher. Provided further are methods of using such an improved saccharification mixture to produce industrially useful sugars and other fermentation products.

[0052] More specifically the improved method is one that introduces an additional preconditioning step after the pretreatment step, and prior to the enzymatic hydrolysis step. In some embodiments, when the biomass material is subject to a pretreatment method comprising an acidic pretreatment and/or a steam explosion pretreatment, a regular preconditioning step is used. During the regular

preconditioning step, a water washing procedure is applied, prior to a pH adjustment procedure, during which the pH of the pretreated biomass substrate is brought up to a level of at least above about 5.5, for example, at least above about 5.6, or at least about 5.7, or at least about 5.8, or at least about 5.9, or at least about 6.0, or at least about 6.1 , or at least about 6.2, or at least about 6.3, or at least about 6.4, or even at least about 6.5.

[0053] In some embodiments, when the biomass material is subject to a

pretreatment method comprising an alkaline pretreatment, a more involved preconditioning step is used. During the more involved preconditioning step, a water washing procedure is applied, prior to an acid treatment procedure, during which the pH of the pretreated and water-washed biomass material is reduced to about 1 .0 to about 3.5. The acid treated, water-washed and pretreated biomass material is then allowed to sit at the highly acidic pH for a period of about 2 minutes to about 24 hours. Thereafter, the acid treated, water-washed, and pretreated biomass material is optionally subject to a second water-washing step, then adjusted to a higher pH using an alkali or a chemical base, to a level of at least above about 5.5, for example, at least above about 5.6, or at least about 5.7, or at least about 5.8, or at least about 5.9, or at least about 6.0, or at least about 6.1 , or at least about 6.2, or at least about 6.3, or at least about 6.4, or even at least about 6.5.

[0054] As such, at the start of the enzymatic hydrolysis step, the pH of the saccharification mixture comprising the pretreated and preconditioned lignocellulosic biomass substrate and the enzyme mixture comprising at least one cellulase enzyme is at a pH that is significantly higher than the pH optimums of the enzymes in the enzyme mixture.

2. Definitions [0055] Before the present compositions and methods are described in greater detail, it is to be understood that the present methods or apparatus are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present methods or apparatus will be limited only by the appended claims.

[0056] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the present methods or apparatus. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the present methods or apparatus, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the present methods or apparatus. [0057] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. For example, in connection with a numerical value, the term "about" refers to a range of -10% to +10% of the numerical value, unless the term is otherwise specifically defined in context. In another example, the phrase a "pH value of about 6" refers to pH values of from 5.4 to 6.6, unless the pH value is specifically defined otherwise.

[0058] The headings provided herein are not limitations of the various aspects or embodiments of the present methods or apparatus which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0059] The present document is organized into a number of sections for ease of reading; however, the reader will appreciate that statements made in one section may apply to other sections. In this manner, the headings used for different sections of the disclosure should not be construed as limiting.

[0060] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present methods or apparatus belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present methods or apparatus, representative illustrative methods and materials are now described.

[0061] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present methods or apparatus are not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0062] In accordance with this detailed description, the following abbreviations and definitions apply. Note that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an enzyme" includes a plurality of such enzymes, and reference to "the dosage" includes reference to one or more dosages and equivalents thereof known to those skilled in the art, and so forth.

[0063] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation. [0064] It is further noted that the term "consisting essentially of," as used herein refers to a composition or inventive concept wherein the component(s) or element(s) after the term is in the presence of other known component(s) or element(s) in a total amount that is less than 30% by weight or by significance of the total

composition or inventive concept and do not contribute to or interferes with the actions or activities of the component(s) or element(s).

[0065] It is further noted that the term "comprising," as used herein, means including, but not limited to, the component(s) or element(s) after the term

"comprising." The component(s) or element(s) after the term "comprising" are required or mandatory, but the composition or inventive concept comprising the component(s) or element(s) may further include other non-mandatory or optional component(s) or element(s).

[0066] It is also noted that the term "consisting of," as used herein, means including, and limited to, the component(s) after the term "consisting of." The component(s) after the term "consisting of" are therefore required or mandatory, and no other component(s) are present in the composition.

[0067] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods or apparatus described herein. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0068] The term "biomass" refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising

hemicelluloses, lignin, starch, polysaccharides, oligosaccharides, and/or

monosaccharides. For purposes of the present application, the term "biomass" is used interchangeably with the term "cellulosic biomass," "lignocellulosic biomass", "lignocellulosic biomass material" or "cellulosic biomass material" and so on.

Biomass may also comprise additional components, such as proteins and/or lipids. For purposes of the present invention, biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass may comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solids wastes. Examples of biomass include, without limitation, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetable, fruits, flowers and animal manure. In one embodiment, biomass that is useful for the invention include biomass that has a relatively high carbohydrate value, is relatively dense, and/or is relatively easy to collect, transport, store, and/or handle. In some embodiments, the biomass that is useful includes corn cobs, corn stover, sugarcane bagasse, wheat straws, and other abundantly and readily available plant-based materials.

[0069] The biomass suitably applicable to the invention includes those that comprises at least about 2.5 wt.%, for example, at least about 2.5 wt.%, at least about 5 wt.%, at least about 7.5 wt.%, or even at least about 10 wt.% of lignin among the total weight of polymeric carbohydrates.

[0070] The term "pretreated biomass" refers to biomass materials that have been subject to a treatment or pretreatment prior to enzymatic hydrolysis. [0071] In the method or process described herein, or placed in the apparatus or reactor for enzymatic hydrolysis, any pretreated biomass material may be suitably used. Biomass may be pretreated by any method known to one skilled in the art, such as with acid, base, organosolvent, oxidizing agent, or other chemicals. In addition, biomass materials may be pretreated with one or more chemicals in combination with steam, or heat, or with steam or heat alone. Suitable pretreatment may also include mechanical disruption such as by crushing, grinding or chopping, as well as application of other disrupting physical energies such as ultrasound, microwave or pressure. Certain non-pretreated biomass materials may be used with the method or process herein or in the reactor as described, but more suitable is the use of biomass materials that has been pretreated to enhance subsequent enzymatic hydrolysis. The biomass material may initially, prior to the enzymatic hydrolysis reaction/step, be in a form of high dry solids weight level (or have a dry appearance), or alternatively the biomass material may initially be in a more dilute form such as in the case of stillage. The methods or processes as described herein can be applied to biomass materials of various dry solids weight levels. For example, such dry solids levels can range from between about 1 % to about 40%, preferably between about 3% to about 30%, for example, between about 5% to about 25%, or about 5% to about 20%, or about 3% to about 18%, or about 2% to about 15%, or about 5% to about 15%.

[0072] The term "Ngnocellulosic" refers to a composition comprising both lignin and cellulose. Lignocellulosic material may further comprise hemicelluloses. The biomass materials suitably applicable to the invention herein are accordingly lignocellulosic materials.

[0073] The term "cellulosic" refers to a composition comprising cellulose. Cellulosic material may further comprise hemicelluloses. Cellulosic material may further comprise lignin and other polymeric carbohydrates.

[0074] As used herein the term "cellulose" or "cellulosic materials" refers to materials containing cellulose. Relatedly, the term "lignocellulose" or "lignocellulosic materials" refers such cellulosic materials that also contain lignin. It is known that the largest component polysaccharides constituting the cell walls of plant biomass include cellulose, hemicelluloses and pectin. Cellulose is an organic compound with the formula (C₆Hio0₅)n, representing a polysaccharide consisting of a linear chain of β(1 -»4) linked D-glucose units.

[0075] Hemicellulose" refers to any of several heteropolymers (matrix

polysaccharides) present along with cellulose in almost all plant cell walls, interconnecting the insoluble crystalline matrix of cellulose, which are further embedded or connected to lignin that help to provide for the physical integrity of the plants. Hemicellulases may be xylan, glucuronoxylan,xyloglucans, arabinoxylans, glucomannan, and mannans. When hemicellulose is broken down into sugar monomers, the monomers may include xylose, mannose, galactose, rhamnose, and arabinose, which are mostly D-pentose (C-5 sugars), and occasionally small amounts of L-sugars as well. Xylose is in most cases the most abundant sugar monomer, although in softwoods mannose can be the most abundant sugar. Not only regular sugars can be found in hemicellulose, but also their acidified form, for instance glucuronic acid and galacturonic acid can be present. [0076] Cellulose and lignocellulose are found in various plants and plant-derived materials, including stems, leaves and cobs, various parts of grains, including, for example, corn fiber, wheat hull, etc. Cellulosic materials or lignocellulosic materials can also be materials produced from plants and plant parts, such as paper and pulp. [0077] The term "saccharification" refers to the production of fermentable sugars from polysaccharides or polymeric carbohydrates, such as those contained by certain cellulosic materials or lignocellulosic materials. For the purposes of the present application, the term "saccharification" refers to enzymatic conversion from cellulosic or lignocellulosic materials to fermentation sugars. As such the term "saccharification" may be used interchangeably with the term "enzymatic hydrolysis."

[0078] The term "fermentable sugars" refers to oligosaccharides and

monosaccharides that can be metabolized or otherwise used as a carbon source by a microorganism in a fermentation process.

[0079] The term "hydrolysate" refers to the product of enzymatic hydrolysis, which contains the sugars produced in the enzymatic hydrolysis process or step, the remaining unhydrolyzed biomass, and the enzymes and breakdown products of such enzymes used for the enzymatic hydrolysis.

[0080] The term "slurry" refers to a mixture of insoluble material and a liquid.

[0081] The term "substantially homogenous slurry" refers to a slurry that is sufficiently mixed so that substantially the same composition exists throughout the slurry composition under the action of the agitation means to which it is subjected. This term is used interchangeably herein with "thoroughly mixed slurry."

[0082] The term "dry weight" or "dry solids weight" of biomass refers to the weight of the biomass having all or essentially all water removed. Dry weight is typically measured according to the American Society for Testing and Materials (ASTM) standard E1756-01 (Standard Test Method for Determination of Total Solids in Biomass) or Technical Association of the Pulp and Paper Industry, Inc. (TAPPI) Standard T412 om-01 (Moisture in Pulp, Paper and Paperboard).

[0083] The term "dry weight of biomass concentration" refers to the total amount of biomass dry weight added into a fed batch system reactor, calculated at the time of addition, as a percent of the total weight of the reacting composition in the reactor at the end of the run.

[0084] The term "suitable reaction conditions" refers to the time, temperature, pH and reactant concentrations which are described in details herein. Reaction conditions can further include parameters such as mixing or stirring by the action of an agitator system in the reactor, including without limitation to impellers. The mixing or stirring may be continuous or intermittent, with, for example, interruptions resulting from adding additional components or for temperature and pH assessment.

[0085] Enzymes have traditionally been classified by substrate specificity and reaction products. In the pre-genomic era, function was regarded as the most amenable (and perhaps most useful) basis for comparing enzymes and assays for various enzymatic activities have been well-developed for many years, resulting in the familiar EC classification scheme. Cellulases and other glycosyl hydrolases, which act upon glycosidic bonds between two carbohydrate moieties (or a

carbohydrate and non-carbohydrate moiety~as occurs in nitrophenol-glycoside derivatives) are, under this classification scheme, designated as EC 3.2.1 .-, with the final number indicating the exact type of bond cleaved. For example, according to this scheme an endo-acting cellulase (1 ,4- -endoglucanase) is designated EC 3.2.1 .4. [0086] With the advent of widespread genome sequencing projects, sequencing data have facilitated analyses and comparison of related genes and proteins.

Additionally, a growing number of enzymes capable of acting on carbohydrate moieties (i.e., carbohydrases) have been crystallized and their 3-D structures solved. Such analyses have identified discreet families of enzymes with related sequence, which contain conserved three-dimensional folds that can be predicted based on their amino acid sequence. Further, it has been shown that enzymes with the same or similar three-dimensional folds exhibit the same or similar stereospecificity of hydrolysis, even when catalyzing different reactions (Henrissat et al., FEBS Lett 1998, 425(2): 352-4; Coutinho and Henrissat, Genetics, biochemistry and ecology of cellulose degradation, 1999, T. Kimura. Tokyo, Uni Publishers Co: 15-23.).

[0087] These findings form the basis of a sequence-based classification of carbohydrase modules, which is available in the form of an internet database, the Carbohydrate-Active enZYme server (CAZy), available at http://afmb.cnrs- mrs.fr/CAZY/index.html (Carbohydrate-active enzymes: an integrated database approach. See Cantarel et al., 2009, Nucleic Acids Res. 37 (Database issue):D233- 38). [0088] CAZy defines four major classes of carbohydrases distinguishable by the type of reaction catalyzed: Glycosyl Hydrolases (GH's), Glycosyltransferases (GT's), Polysaccharide Lyases (PL's), and Carbohydrate Esterases (CE's). The enzymes of the disclosure are glycosyl hydrolases. GH's are a group of enzymes that hydrolyze the glycosidic bond between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety. A classification system for glycosyl hydrolases, grouped by sequence similarity, has led to the definition of over 85 different families. This classification is available on the CAZy web site.

[0089] The term "protein", as used herein, includes proteins, polypeptides, and peptides. [0090] The terms "protein" and "polypeptide" are used interchangeably herein.

[0091] As used herein, "cellulases" refer to all enzymes that hydrolyzes cellulose, i.e., any of its components, e.g., 1 ,4-beta-D-glycosidic linkages in cellulosic materials such as those found in various plants and plant-related or -derived materials, such as grains, seeds, cereals, etc., or plant cell walls. [0092] As typically known, "cellulase" comprises at least the enzymes classified in E.C. 3.2.1 .4 (cellulase/endocellulases or endoglucanases), E.C. 3.2.1 .91

(exocellulases), and E.C. 3.2.1 .21 (cellobiases or beta-glucosidases). Examples of endocellulases include endo-1 ,4-beta-glucanase, carboxymethyl cellulase

(CMCase), endo-1 ,4-beta-D-glucanase, beta-1 ,4-glucanase, beta-1 ,4-endoglucan hydrolase, celludextrinase, and various endoglucanases such as those produced by naturally-occurring wood-rotting fungi. Examples of exocellulases include

cellobiohydrolases, which in turn includes those that cleave the 1 ,4-beta-D-glycosidic linkages from the reducing ends of the cellulose chain and those that cleaves the same linkages from the non-reducing ends. [0093] "Cellulases" may also refer to complete enzyme systems that are useful for efficiently converting crystalline cellulose to glucose. Such complete cellulase system typically would comprise components from each of the cellobiohydrolase, endoglucanase and beta-glucosidase classifications, as it has been reported that individual isolated components are less effective in hydrolyzing crystalline cellulose (Filho et al., Can. J. Microbiol., 42:1 -5, 1996).

[0094] A synergistic relationship has been observed between cellulase components from different classifications. Endo-1 ,4-beta-glucanases (EG) and exo- cellobiohydrolases (CBH) catalyze the hydrolysis of cellulose to

cellooligosaccharides (cellobiose as a main product), while beta-glucosidases (BGL) convert the oligosaccharides to glucose. In particular, the EG-type cellulases and CBH-type cellulases synergistically interact to efficiently degrade cellulose. The beta-glucosidases serve the important role of liberating glucose from the cellooligosaccharides such as cellobiose, which is toxic to the microorganisms that are used to ferment the sugars into ethanol (e.g., yeasts) and which is also inhibitory to the activities of endoglucanases and cellobiohydrolases, thus rendering them ineffective in further hydrolyzing the crystalline cellulose. [0095] Cellulases" may further refer to complete enzyme systems that comprises not only cellulases but also certain hemicellulases, or any combination thereof.

[0096] A number of commercial cellulase compositions are available and suitable for use in the methods/processes and/or with the reactors described herein, including, for example, products of Genencor, Danisco US Inc., such as

ACCELLERASE® 1000 and ACCELLERASE® 1500, ACCELLERASE® BG,

ACCELLERASE® DUET, and ACCELLERASE® TRIO™; products of Novozymes, such as its Celluclast, Novozyme 188, Cellic CTec2, Cellic CTec3; products of AB Enzymes, such as its Flashzyme; products of Codexis, such as its CodeXyme® cellulase products; products of Dyadic, such as its CMax® products. Certain of the commercial compositions as listed above also contains hemicellulases. For example, about 1 /5 to 1 /4 of the total proteins of ACCELLERASE® DUET are hemicellulases, and about 1 /3 of the proteins in ACCELLERASE® TRIO™ are hemicellulases. CMax®, certain of CodeXyme® products, as well as Cellic Ctec3 all contain certain amounts of hemicellulases. [0097] The term "endoglucanase" as used herein refers to an enzyme of

classification E.C. 3.2.1 .4, which catalyzes the hydrolysis of 1 ,4-beta-D-glycosidic linkages that are found in cellulosic materials. Methods of measuring endoglucanase activities are known, including, for example, the one measuring the hydrolysis of carboxymethyl cellulose (CMC) as described by Ghose, 1 987, Pure & App. Chem, 59:257-268.

[0098] The term "cellobiohydrolase" refers to an enzyme with cellobiohydrolase activity or capable of catalyzing the hydrolysis of a particular glocosidic linkage in cellulose. Specifically, the cellobiohydrolase (CBH) activity may be CBH class I (CBH I) or CBH class I I (CBH I I) activity or a combination of both CBH I and CBH II. Suitably the cellobiohydrolase may hydrolyse (1→4)^-D-glucosidic linkages in cellulose and cellotetraose, releasing cellobiose from the non-reducing ends of the chains. Another term for cellobiohydrolase activity may be exo-cellobiohydrolase activity or cellulose 1 ,4 β-cellobiosidase activity. The cellobiohydrolase I I activity can be classified under E.C. classification EC. 3.2.1 .91 . The cellobiohydrolase I activity can be classified under E.C. classification EC. 3.2.1 .176.

[0099] The term "beta-glucosidase" as used herein refers to an enzyme having beta-glucosidase activity or one that is capable of catalyzing the hydrolysis of terminal non-reducing β-D-glucosyl residues and release of monomer β-D-glucose from cellobiose. β-glucosidase activity can be classified under E.C. classification E.C. 3.2.1 .21 .

[00100] The term "hemicellulase" as used herein refers to a group of enzymes capable of catalyzing the hydrolysis of a hemicellulosic materials. The term

"hemicellulases" as used herein refer to three major types of enzymes: beta- xylosidases, L-cc-arabinofuranosidases, and xylanases. Those enzymes include, for example, arabinases, arabinofuranosidases, certain acetylmannan esterases, acetylxylan esterases, ferulyoyl esterases, mannanases, mannosidases, xylanases, and xylosidases, etc. Hemicellulases can be from many different glycosyl hydrolase families, including, without limitation, beta-xylosidases of GH3; beta-xylosidases of GH39; L-a-arabinofuranosidase (EC 3.2.1 .55), β-xylosidase (EC 3.2.1 .37), endo- arabinanase (EC 3.2.1 .99), and/or galactan 1 ,3^-galactosidase (EC 3.2.1 .145) of GH43; and L-a-arabinofuranosidase (EC 3.2.1 .55) of GH51 , as well as the xylanases of GH10 and GH1 1 , and the beta-xylosidases of GH30, for example.

[00101 ] The term "xylanase" refers to a 1 ,4-beta-D-xylan xylohydrolase of E.C.

3.2.1 .8, which catalyzes the hydrolysis of 1 ,4-beta-D-xylosidic linkages in xylan. Xylanase activities can be measured, for example, by the PHBAH assay as described by Lever, 1972, A new reaction for colorimetric determination of carbohydrates, Anal. Biochem. 47:273-279.

[00102] β-xylosidase activity may hydrolyse successive xylose residues from the non-reducing termini of (1→3)^-D-xylans, e.g. the β-xylosidase may be a 1 ,3 β-D- xylosidase. 1 ,3 β-D-xylosidases may be classified under E.C. classification E.C. 3.2.1 .72 or may catalyse the hydrolysis of (1→4)^-D-xylans, to remove successive D-xylose residues form the non-reducing termini, e.g. the β-xylosidase may be a 1 ,4 β-xylosidase. 1 ,4 β-xylosidases may be classified under E.C. classification E.C. 3.2.1 .37.

[00103] L-alpha arabinofuranosidases may hydrolyse (1→6)^-D-galactosidic linkages in arabinogalactan proteins and (1→3):(1→6)^-galactans to yield galactose and (1→6)^-galactobiose. L-alpha-arabinofuranosidases may be classified under E.C. classification E.C. 3.2.1 .164. [00104] The term "saccharification enzyme" refers to an enzyme that can catalyze conversion of a component of biomass to fermentable sugars.

[00105] The term "microorganism" as used herein refers to any bacterium, yeast, or fungal species.

[00106] As used herein the term "ethanologen" and "ethanologenic microorganism" are used interchangeably to refer to a microorganism with the ability to convert a sugar or oligosaccharide to ethanol. The ethanologenic microorganism are ethanologenic by virtue of their ability to express one or more enzymes that individually or collectively convert soluble sugars to ethanol.

[00107] Such an ethanolgen can also be referred as an "ethanol producing microorganism" which is an organism or cell that is capable of producing ethanol from a hexose or a pentose. Generally ethanol producing cells would contain at least one alcohol dehydrognase and a pyruvate decarboxylase. Examples of ethanol producing microorganisms include fungal microorganisms such as yeast, such as, for example, the species and strains of Saccharomyces, e.g., S. cerevisiae. [00108] The term "heterologous" with reference to a polynucleotide or

polypepide/protein refers to a polynucleotide or polypeptide/protein, or an enzyme that does not naturally occur in a host cell. In some embodiments, the protein is a commercially important industrial protein. It is intended that the term encompass proteins that are encoded by naturally occurring genes, mutated genes, and/or synthetic genes. [00109] The term "endogenous" as used herein with reference to a polynucleotide or polypeptide/protein refers to a polynucleotide or polypeptide/protein that occurs naturally in the host cell.

[00110] The term "fermentation" as used herein refers to the enzymatic and/or anaerobic breakdown of organic substances by microorganisms to produce simpler organic compounds. Although fermentation occurs under anaerobic conditions, the term "fermentation" as used herein is not intended to be limited to strict anaerobic conditions, because fermentation also can occur in the presence of oxygen at various levels. Accordingly, in the context of the present invention, fermentation encompasses at least some fermentative conversion of a soluble cellulosic fermentable sugar into an end product.

[00111] The term "contacting" as used herein refers to placing of the enzyme(s) in a reactor, vessel or the like, such that the enzymes can come into sufficiently close proximity to the substrate so as to enable the enzymes to convert the substrate to the end product. The skilled persons in the art would recognize that mixing an enzyme (e.g., in a solution form) with one or more substrates, whether in a relatively pure or crude form, constitutes contacting.

[00112] The term "yield" with reference to the ethanol production refers to the production of a compound, e.g., ethanol, from a certain amount of a starting material, e.g., a lignocellulosic based biomass feedstock. The term "yield" is also suitably used herein with reference to the production of fermentable sugars, and in that context, it refers to the amount of fermentable sugars produced from a given lignocellulosic biomass materials. "Yield" may be expressed as the product formed over a particular amount of time from the starting material.

3. Conversion of Lignocellulosic Biomass Material into Fermentable Sugars and/or Fermentation Products [00113] The invention provides an improved method or process for enzymatic hydrolysis or saccharfication of a lignocellulosic biomass material. The improved method or process when applied can lead to an increased yield of fermentable sugars from a given biomass material where the same amount or concentration of enzymes are used under the same conditions.

[00114] There is estimated hundreds of millions of tonnes of lignocellulosic biomass materials available in the United States each year, which can be converted into fermentable sugars, and then further into cellulosic fuels or chemicals, (see, e.g., Stanford University Global Climate & Energy Report, 2005, An Assessment of Biomass Feedstock and Conversion Research Opportunities). Among the many different available lignocellulosic biomass materials available for conversion, corn stover is the most abundant agricultural residue produced in the United States each year, making it a highly suitable feedstock for fermentable sugars, cellulosic fuels, and chemicals production. Just like the other lignocellulosic biomass feedstock, however, corn stover composition can vary with climate conditions, harvest seasons, location, or the plant variety, which all affects the content of cellulose, hemicellulose, lignin and other components. Many of these components can confound the efforts to convert these materials, some by being recalcitrant, while other negatively affect conversion by being inhibitory to certain biological processes. [00115] There are two main approaches to convert the complex plant polymeric carbohydrates into simple, fermentable sugars, such as glucose and xylose. The first approach is acid hydrolysis. It is a relatively inexpensive and simple process, but the involvement of acids, typically also accompanied by heating, makes the process and the equipment used to carry out the process challenging. For example, the equipment and connectors must be made of materials that are corrosion resistant in an acidic, humid and heated environment for sustained periods of time. The used acids and other process wastes are hazardous and must also be handled with substantial care. On the other hand, the conversion can be unsatisfactory in that the resulting sugars can be further degraded under high temperature. High concentration of inhibitors can also form, including, for example, furfural, which are inhibitors to fermenting organisms or ethanologens involved in downstream processing of the sugars produced by the enzymatic hydrolysis step. Removal of such inhibitors can be costly and cumbersome. [00116] The second known approach is enzymatic hydrolysis. Such processes are typically carried out in mild, physiological conditions, having the potential of achieving high yields of fermentable sugars that are not subsequently degraded. Handling of the materials used in the enzymatic hydrolysis step as well as the waste, unrelated residual biomass, is also much less cumbersome. On the other hand, the costs of producing enzymes, which are required in high quantities in order to sustain cellulosic biorefineries, and in consortiums of many types of enzymatic activities, can be prohibitively high for an economically viable lignocellulosic biomass to fuel operation. [00117] The lignocellulosic biomass material hydrolyzed using such an improved method or process of the invention is suitably one that comprises at least 3 wt.% lignin, for example, at least about 3 wt.%, at least about 5 wt.%, at least about 10 wt.%, at least about 15 wt.%, or even at least about 20 wt.%, referencing the total weight of polymeric carbohydrates in the biomass material. As such, substantially purified or "clean" cellulose model biomass materials such as Avicel would be unsuitable for the purpose of the invention herein.

[00118] The lignocellulosic biomass material is also preferably pretreated before they are placed into a preconditioning procedure.

4. Pretreatments

[00119] One way of making enzymatic hydrolysis of lignocellulosic biomass more effective and efficient is to pretreat the biomass feedstock, in order to render or disrupt the lignin tightly wound around the lignocellulosic structure and make the cellulose and hemicellulose part of the biomass more readily accessible to the enzymes. Prior to saccharification, a biomass material is preferably subject to one or more pretreatment step(s) in order to render xylan, hemicellulose, cellulose and/or lignin material more accessible or susceptable to enzymes and thus more amenable to hydrolysis by the enzyme(s) and/or enzyme blends/compositions of the disclosure.

[00120] Pretreatment may include chemical, physical, and biological pretreatment. For example, physical pretreatment techniques can include without limitation various types of milling, crushing, steaming/steam explosion, irradiation and

hydrothermolysis. Chemical pretreatment techniques can include without limitation dilute acid, alkaline, organic solvent, ammonia, sulfur dioxide, carbon dioxide, and pH-controlled hydrothermolysis. Biological pretreatment techniques can include without limitation applying lignin-solubilizing microorganisms. The pretreatment can occur from several minutes to several hours, such as from about 1 hour to about 120.

[00121] In some aspects, any of the methods or processes provided herein may further comprise pretreating the biomass material, such as pretreating the biomass with acid or base. The acid or base may be ammonia, sodium hydroxide, or phosphoric acid. The method may further comprise pretreating the biomass material with ammonia. The pretreatment may be steam explosion, pulping, grinding, acid hydrolysis, or combinations thereof.

[00122] In one embodiment, the pretreatment may be by elevated temperature and the addition of either of dilute acid, concentrated acid or dilute alkali solution. The pretreatment solution can added for a time sufficient to at least partially hydrolyze the hemicellulose components and then neutralized

[00123] In some embodiments, the pretreatment entails subjecting biomass material to a catalyst comprising a dilute solution of a strong acid and a metal salt in a reactor. The biomass material can, e.g., be a raw material or a dried material. This pretreatment can lower the activation energy, or the temperature, of cellulose hydrolysis, ultimately allowing higher yields of fermentable sugars. See, e.g., U.S. Patent Nos. 6,660,506; 6,423,145.

[00124] Another example of a pretreatment method entails hydrolyzing biomass by subjecting the biomass material to a first hydrolysis step in an aqueous medium at a temperature and a pressure chosen to effectuate primarily depolymerization of hemicellulose without achieving significant depolymerization of cellulose into glucose. This step yields a slurry in which the liquid aqueous phase contains dissolved monosaccharides resulting from depolymerization of hemicellulose, and a solid phase containing cellulose and lignin. The slurry is then subject to a second hydrolysis step under conditions that allow a major portion of the cellulose to be depolymerized, yielding a liquid aqueous phase containing dissolved/soluble depolymerization products of cellulose. See, e.g., U.S. Patent No. 5,536,325. [00125] A further example of method involves processing a biomass material by one or more stages of dilute acid hydrolysis using about 0.4% to about 2% of a strong acid; followed by treating the unreacted solid lignocellulosic component of the acid hydrolyzed material with alkaline delignification. See, e.g., U.S. Patent No.

6,409,841 .

[00126] Another example of pretreatment method comprises prehydrolyzing biomass {e.g., lignocellulosic materials) in a prehydrolysis reactor; adding an acidic liquid to the solid lignocellulosic material to make a mixture; heating the mixture to reaction temperature; maintaining reaction temperature for a period of time sufficient to fractionate the lingo-cellulosic material into a solubilized portion containing at least about 20% of the lignin from the lignocellulosic material, and a solid fraction containing cellulose; separating the solubilized portion from the solid fraction, and removing the solubilized portion while at or near reaction temperature; and

recovering the solubilized portion. The cellulose in the solid fraction is rendered more amenable to enzymatic digestion. See, e.g., U.S. Patent 5,705,369.

[00127] Further pretreatment methods can involve the use of hydrogen peroxide H₂0₂. See Gould, 1984, Biotech, and Bioengr. 26:46-52.

[00128] Pretreatment can also comprise contacting a biomass material with stoichiometric amounts of sodium hydroxide and ammonium hydroxide at a very low concentration. See Teixeira et al., 1999, Appl. Biochem.and Biotech. 77-79:19-34. Pretreatment can also comprise contacting a lignocellulose with a chemical {e.g., a base, such as sodium carbonate or potassium hydroxide) at a pH of about 9 to about 14 at moderate temperature, pressure, and pH. See PCT Publication

WO2004/081 185. [00129] Ammonia may be used in a pretreatment method. Such a pretreatment method comprises subjecting a biomass material to low ammonia concentration under conditions of high solids. See, e.g., U.S. Patent Publication 20070031918, PCT publication WO 061 10901 .

[00130] The improved methods or processes carry certain distinction in the specifics of the preconditioning step. For example, when the lignocellulosic biomass material is pretreated using a method comprising an acidic pretreatment and/or a steam explosion pretreatment, a regular preconditioning step is suitable, wherein a water- washing procedure is followed by a pH adjustment procedure to bring the pH of the pretreated and water-washed biomass material to a level of at least above about 5.5, before an enzyme mixture comprising at least one cellulase is added to make a saccharification mixture. On the other hand, when the lignocellulosic biomass material is pretreated using a method comprising an alkaline pretreatment, a more involved preconditioning step is suitable, wherein a water-washing procedure is followed by first an acid treatment procedure involving the adjustment of pH down to very acidic level of about 2.0 to about 3.5, and an incubation period of about 10 minutes to about 24 hours. The pretreated, water-washed, and acid treated biomass substrate is then brought to a pH of at least above about 5.5, before an enzyme mixture comprising at least one cellulase is added to make a saccharification mixture.

5. Enzyme Mixture [00131] Lignocellulosic hydrolysis enzymes, which also may be referred to as a saccharification enzymes or consortiums, are used to hydrolyze lignocellulosic biomass materials, releasing oligosaccharides and/or monosaccharides in a hydrolysate. Saccharification enzymes are reviewed in Lynd, L. R., et al. (Microbiol. Mol. Biol. Rev. (2002) 66:506-577). [00132] A suitable lignocellulosic hydrolysis enzyme mixture comprising at least one cellulase for the present invention comprises one or more enzymes selected primarily, but not exclusively, from the group "glycosidases" which hydrolyze the ether linkages of di-, oligo-, and polysaccharides and are found in the enzyme classification EC 3.2.1 .x (Enzyme Nomenclature 1992, Academic Press, San Diego, Calif, with Supplement 1 (1993), Supplement 2 (1994), Supplement 3 (1995,

Supplement 4 (1997) and Supplement 5 [in Eur. J. Biochem. (1994) 223:1 -5, Eur. J. Biochem. (1995) 232:1 -6, Eur. J. Biochem. (1996) 237:1 -5, Eur. J. Biochem. (1997) 250:1 -6, and Eur. J. Biochem. (1999) 264:610-650, respectively]) of the general group "hydrolases" (EC 3.). Glycosidases useful in the present method can be categorized by the biomass component that they hydrolyze. Glycosidases useful for the present method include cellulose-hydrolyzing glycosidases (for example, cellulases, endoglucanases, exoglucanases, cellobiohydrolases, β-glucosidases), hemicellulose-hydrolyzing glycosidases, called hemicellulases, (for example, xylanases, endoxylanases, exoxylanases, β-xylosidases, arabinoxylanases, mannases, galactases, pectinases, glucuronidases), and starch-hydrolyzing glycosidases (for example, amylases, a-amylases, β-amylases, glucoamylases, a- glucosidases, isoamylases). In addition, it may be useful to add other activities to the saccharification enzyme consortium such as peptidases (EC 3.4.x.y), lipases (EC 3.1 .1 .x and 3.1 .4.x), ligninases (EC 1 .1 1 .1 .x), and feruloyl esterases (EC 3.1 .1 .73) to help release polysaccharides from other components of the biomass.

[00133] It is well known in the art that microorganisms that produce polysaccharide- hydrolyzing enzymes often exhibit an activity, such as cellulose degradation, that is catalyzed by several enzymes or a group of enzymes having different substrate specificities. Thus, a "cellulase" from a microorganism may comprise a group of enzymes, all of which may contribute to the cellulose-degrading activity. Commercial or non-commercial enzyme preparations, such as cellulase, may comprise numerous enzymes depending on the purification scheme utilized to obtain the enzyme. Thus, the saccharification enzymes used in the present method comprise at least one "cellulase", and this activity may be catalyzed by more than one enzyme. Optionally, the saccharification enzymes used in the present method may comprise at least one hemicellulase, generally depending on the type of pretreated biomass used in the present process. For example, hemicellulase is typically not needed when

saccharifying biomass pretreated with acid and is typically included when

saccharifying biomass pretreated under neutral or basic conditions.

[00134] Saccharification enzymes may be preparations that can be obtained commercially, such as Spezyme® CP cellulase (Genencor International, Rochester, N.Y.) and Multifect® xylanase (Genencor). Other commercial cellulase compositions are available and suitable for use in the methods/processes and/or with the reactors described herein, including, for example, products of Genencor, Danisco US Inc., such as ACCELLERASE® 1000 and ACCELLERASE® 1500, ACCELLERASE® BG, ACCELLERASE® DUET, and ACCELLERASE® TRIO™; products of Novozymes, such as its Celluclast, Novozyme 188, Cellic CTec2, Cellic CTec3; products of AB Enzymes, such as its Flashzyme; products of Codexis, such as its CodeXyme® cellulase products; products of Dyadic, such as its CMax® products. Certain of the commercial compositions as listed above also contains hemicellulases. For example, about 1 /5 to 1 /4 of the total proteins of ACCELLERASE® DUET are hemicellulases, and about 1 /3 of the proteins in ACCELLERASE® TRIO™ are hemicellulases. CMax®, certain of CodeXyme® products, as well as Cellic Ctec3 all contain certain amounts of hemicellulases. [00135] In addition, lignocellulosic hydrolysis enzymes may be produced biologically, including using recombinant microorganisms. New lignocellulosic hydrolysis enzymes may be developed, which may be used in the present process.

[00136] One skilled in the art will know how to determine the effective amounts of enzymes to use in the present process and how to adjust conditions for optimal enzyme activity. One skilled in the art will also know how to optimize the classes of enzyme activities required to obtain optimal hydrolysis of a given pretreatment product under the selected conditions. Conventional wisdom is that enzymatic hydrolysis is best performed at or near the pH and temperature optima for the lignocellulosic hydrolysis enzymes being used. The pH optimum can range from about 3 to about 9, but is more typically between about 4.5 and about 7. The temperature optimum can range between about 20 °C to about 80 °C, and is more typically between about 25 °C and about 60 °C.

[00137] Typically the saccharification pH is brought to and subsequently buffered to be within a narrow pH optima of the enzyme mixture. The mixing allows a

substantially uniform pH to be achieved throughout the biomass and enzyme mixture, which in turn are thought to allow optimal functioning of the enzymes. In some previously known processes, the pH control of the enzymatic hydrolysis step is typically managed by thorough mixing of the slurry comprising some pretreated biomass material that is dropped or otherwise introduced into the saccharification vessel or tank wherein the hydrolysis enzymes are already loaded. Thorough mixing is carried out continuously throughout saccharification, and optionally as more pretreated biomass materials are added to the slurry in batches.

[00138] In some embodiments, the enzymatic hydrolysis step is carried at a temperature of at least about 25 °C, for example, at least about 25 °C, about 30 °C, about 35 °C, at least about 40 °C, at least about 45 °C, or at least about 50 °C. In some embodiments, the enzymatic hydrolysis step is carried out at a temperature within the range of about 25 °C to about 65 °C, for example, about 25 °C to about 62 °C, about 30 °C to about 60 °C, about 35 °C to about 58 °C, or about 40 °C to about 55 °C. In certain embodiments, the temperature of the enzymatic hydrolysis mixture is measured and continuously monitored such that whenever the temperature of the enzymatic hydrolysis mixture deviates from the preferred range, which is

predetermined based on the temperature optima of the enzymes in the enzyme composition, the cooling or heating means attached to the reactor is engaged to adjust the temperature back to within the desired range.

[00139] In some embodiment, the enzymatic hydrolysis step is conducted for a period of at least 1 hour, for example, about 2 hours, about 5 hours, about 6 hours, about 12 hours, about 24 hours, about 30 hours, about 36 hours or longer. In certain embodiments, the enzymatic hydrolysis step is conducted for a period within the range of 1 hour and 120 hours, for example, about 2 hours to about 1 10 hours, about 5 hours to about 100 hours, about 10 hours to about 96 hours, about 12 hours to about 90 hours, about 12 hours to about 84 hours, about 24 hours to about 80 hours, about 30 hours to about 72 hours, or even about 36 hours to about 68 hours. In some embodiments, the enzymatic hydrolysis step is conducted for a period sufficient to convert or enzymatically hydrolyze at least 30% of the glucan in the lignocellulosic biomass material into glucose. In certain embodiments, the enzymatic hydrolysis is conducted for a period sufficient to convert or enzymatically hydrolyze at least 20% of the xylan in the lignocellulosic biomass material to xylose. In certain further embodiments, the enzymatic hydrolysis step is conducted for a period sufficient to convert at least 30% of the glucan and at least 20% of the xylan in the lignocellulosic biomass material into fermentable monomeric sugars.

[00140] The processes or methods of the present invention uses preconditioned pretreated lignocellulosic biomass materials, adjusted to a pH of at least above about 5.5. When such preconditioned and pretreated lignocellulosic biomaterials are mixed with the hydrolysis enzyme mixture comprising at least one cellulase, the pH at the start of the enzymatic hydrolysis reaction is at least above about 5.5, for example, at least above about 5.6, at least above about 5.7, at least above about 5.8, at least above about 5.9, at least above about 6.0, at least above about 6.1 , at least above about 6.2, at least above about 6.3, at least above about 6.4, or even at least above about 6.5. Each further batch of the preconditioned and pretreated biomass materials are adjusted to a pH of at least above about 5.5. This, in combination with the fact that the enzyme mixture comprising at least one cellulase to be used for hydrolysis is typically buffered, some reduced level of mixing to create a relatively homogenous slurry is sufficient to insure the pH at the start of hydrolysis step to be at least above about 5.5, which is significantly above the pH optimums of the enzymes in the enzyme mixture.

[00141] As enzymatic hydrolysis of lignocellulosic biomass material proceeds, soluble sugars are produced from the cellulose and/or hemicellulose in the biomass, thereby liquefying non-soluble components of the biomass slurry. The biomass in the slurry becomes partially hydrolyzed. The slurry becomes less viscous, allowing additional biomass to be added to the slurry while maintaining the mixability of the slurry with the agitator in the reactor, even with a fairly rudimentary and inexpensive agitator system. The additional portion of biomass adds more solids and thus increases the percent of total solids loaded in the saccharifying slurry.

6. Other process conditions affecting the efficiency and efficacy of conversion

[00142] Liquefaction of biomass results starts at partial enzymatic hydrolysis, and becomes more prominent with further enzymatic hydrolysis, allowing reduction of biomass slurry viscosity and addition of more biomass while retaining mixability. Thus additional biomass may be added following a fed batch system, while maintaining stirring by the agitator. The additional biomass feedings may be semi- continuous, allowing periods of liquefaction between additions. Alternatively, the biomass feeding may be continuous, at a rate that is slow enough to balance the continuous liquefaction occurring during the enzymatic hydrolysis step. In either case, mixability of the slurry is monitored and biomass addition is controlled to maintain thorough mixing as determined by the agitator system overcoming the yield stress of the slurry.

[00143] In addition to pretreatment size reduction of the lignocellulosic biomass material, the particle size of the non-soluble biomass can be repeatedly further reduced during the enzymatic hydrolysis step. For example, particle size reduction can be achieved by multiple applications of mechanical force for this purpose. A mechanical particle size reduction mechanism may be, for example, a blender, grinder, shearer, chopper, sheer disperser, disperser, or roto-stat. Particle size reduction may also be imposed by other non-mechanical methods, such as ultrasonic methods. The particle size may be reduced prior to initial production of a slurry for enzymatic hydrolysis, prior to addition of pretreated biomass to an existing saccharifying slurry, and/or during hydrolysis of a slurry. [00144] Alternatively to providing a fully saccharified hydrolysate product, the enzymatic hydrolysis reaction may be run until the final percent solids target is met and then the saccharifying biomass may be transferred to a fermentation process, where saccharification continues along with fermentation (called SSF: simultaneous saccharification and fermentation.) [00145] Even with carefully controlled saccharification process conditions, other factors can substantially affect the efficacy and/or efficiency of the enzymes. For example, unless lignin is completely stripped from the pretreated lignocellulosic material, a costly and very involved process, the presence of lignin can cause significant non-specific and/or non-productive binding of the enzymes to the pretreated biomass material, resulting in low enzymatic hydrolysis efficacy and efficiency. While the enzymes also typically would have some level of nonproductive binding to other parts of the pretreated biomass, the non-specific and non-productive binding to lignin is most prominent. See, e.g., Zhu et al., Prog.

Energy Combust. Sci. 2012, 38:583-589; and Lynd et al., Nat. Biotechnol. 2008, 26:169-172. Therefore high lignin content in the biomass material can be directly correlated to high enzyme costs and low hydrolysis efficiency, giving rise to processes that are economically wasteful not only in terms of the amount of enzymes used but also in terms of the pretreated biomass materials.

[00146] Some passive approaches have been undertaken, although to varying degrees of effectiveness. For example, washing the pretreated solid materials to remove free lignin (i.e., lignin separated from the lignocellulosic biomass through chemical pretreatment). See, e.g., Nagel et al., Biotechnol. Prog. 2002, 18:734-738. Washing, however, consumes a significant amount of water, in the order of 10m³ water per ton of lignocellulse, which causes environmental concerns both in terms of use and waste of this resource. See, Liu et al, Bioresour. Technol. 2010, 101 :9120- 9127. Other strategies have been applied to reduce the non-productive and nonspecific binding of enzymes to the pretreated biomass materials. Surfactants, metal compounds, or certain polymers have been employed. See, e.g., Tu et al., Recycling Cellulases During the Hydrolysis of Steam Exploded and Ethanol

Pretreated Logepole Pine, Biotechnol. Prog. (2007) 23:1 130; and Tu. et al.,

Evaluating the Distribution of Cellulases and the Recycling of Free Cellulases During the Hydrolysis of Lignocellulosic Substrates. Biotechnol. Prog. (2007), 23:398; Liu et al., J. Agric. Food Chem. 2010, 58:7233-38; Ooshima et al., Biotechnol. Bioeng., 1990, 36:446-52; Zheng et al., Appl. Biochem. Biotechnol. 2008, 146:231 -248;

Eriksson et al., Enzyme Microb. Technol. 2002, 31 :353-64. However such surfactants, metal compounds, or polymers are costly commodities and required in such high amounts in order to be effective, adding substantially to the costs of the process.

[00147] Recently there have been some publications suggesting that an elevated pH, at a level higher than the optimal pH for certain well known fungal enzymes and products, could help improve the efficiency of enzymatic hydrolysis. See, e.g., Lou et al., ChemSusChem (2013), 6:919-927. It was reported that improved enzymatic hydrolysis was observed when certain lignocellulosic biomass substrates produced from a softwood and a hardwood pretreated by different processes (i.e., dilute acid, alkaline, and SPORL) were hydrolyzed by Novozymes Cellic CTec2 product at a pH of 5.5, as compared to when the enzymatic hydrolysis reactions were carried out at a pH of 4.8. This is in contrast with the fact that, when a pure cellulose substrate was used (i.e., a Whatman paper substrate), the hydrolysis efficacy and efficiency is higher at the enzyme product's optimal pH of 4.8 over that was achieved at pH 5.5. The observed higher hydrolysis efficacy at a pH that is not the pH optimum of the enzyme product has been attributed to the loss of enzyme activity being more than adequately compensated by the reduction of non-productive binding of these enzymes . It can be envisioned, however, that at a pH that deviates further from the pH optimum of the enzyme mixture, the loss of enzyme activity will no longer be able to be counterbalanced by the improved enzymatic hydrolysis resulting from less nonspecific and non-productive binding to lignin. Adjusting the pH of a commercial- scale volume of pretreated biomass materials, especially when such biomass material has previously been subject to acidic pretreatment therefore having a starting pH of lower than about 3 or lower, requires the use of expensive base (as well as acid for adjustments), and as such can add significantly to the cost of the overall process. [00148] Preconditioning of pretreated biomass materials to a pH of at least above about 5.5, for example, at least above about 5.6, at least above about 5.8, at least above about 6.0, or even at least above about 6.2, can require a large amount of strong chemical base or alkali. In the case of a commercial scale or even

demonstration scale cellulosic biorefinery, such quantities of alkali required would add significant cost to the process, even counterbalance any improvement achieved from reduced non-productive lignin binding as a result of the preconditioning of the pretreated biomass material.

[00149] It was surprisingly discovered that, in the pre-conditioning step, if a water washing step is introduced prior to the pH adjustment step, a drastically reduced amount of alkaline solution or base (for example, reduction by at least about 20%, or about 30%, or about 40%, or even about 50% of alkali) can be used to adjust the pH to a desired saccharification starting pH. The water washing step can be more easily leveraged as a cooling step for the pretreated biomass, because cold water, even in large volumes, is much less expensive than a cold alkaline solution. Subsequent to the water washing step, the pH of the pre-treated and water-washed biomass can be adjusted with a small volume of an alkaline solution, bringing the pH of the

saccharification mixture to above about pH 6.5, for example to above about pH 6.5, or above about pH 6.4, or above about pH 6.3, or above about pH 6.2, or above about pH 6.1 , or above about pH 6.0, or above about pH 5.9, or above about pH 5.8, or above about pH 5.7, or above about pH 5.6, or at least above about pH 5.5.

[00150] While not wishing to be bound by theory, it has been demonstrated the water used in the water-washing step prior to the pH adjustment effectively removes or substantially reduces the level of buffering components (e.g., acetate, etc) in the pre-treated biomass materials. The pH of the resulting water-washed biomass materials is therefore much more readily adjusted up to a desired enzymatic hydrolysis pH, and a greatly reduced amount of alkaline would be required, simply because the alkaline does not need to first overcome or neutralize the buffering components. It is further demonstrated that increasing the pH of the saccharification mixture at the start of enzymatic hydrolysis step to be as high as about pH 6.5, for example to as high as about pH 6.5, or about pH 6.4, or about pH 6.3, or about pH 6.2, or about pH 6.1 , or about pH 6.0, or about pH 5.9, or about pH 5.8, or about pH 5.7, or about pH 5.6, or about pH 5.5, substantially changes the surface of the lignin, reducing hyrophibic interactions between enzymes and lignin, and/or electrostabic interactions between enzymes and lignin. Furthermore it has also been observed that the washates of the water-washing procedure in the preconditioning step contains various phenolic compounds that are known inhibitors of enzymes. The water-washing procedure thus not only removes buffering components from the biomass materials, change their surface charge or property, but also helps to reduce the amount of enzyme inhibitors in the biomass materials.

[00151] It is believed that at pH as high as about 6.5, the enzymes applied has somewhat reduced functionality (or hydrolysis performance) as compared to the functionality at the optima pH, which is about 4.5 to about 5.2, for fungal-derived celluloytic enzymes, but that small loss of functionality is compensated by the substantially reduced non-specific and/or non-productive binding of the enzymes to the lignin, such that the overall conversion is improved.

[00152] It is further noted that, even at a higher starting saccharification pH, of as high as about pH 6.5, for example to as high as about pH 6.5, or about pH 6.4, or about pH 6.3, or about pH 6.2, or about pH 6.1 , or about pH 6.0, or about pH 5.9, or about pH 5.8, or about pH 5.7, or about pH 5.6, or about pH 5.5, once the enzymatic hydrolysis reaction begins, the pH of the saccharifciation mixture comprising the pretreated biomass material and the celluloytic hydrolysis enzymes will begin and continue to drop slowly until at about 20 to 24 hours after the start of hydrolysis reaction, the pH drops to about 5.0. Typically there would be no need to increase the pH back up to the starting hydrolysis pH, and mere maintenance of the saccharification mixture at pH about 5.0, which does not require the use of substantial amounts of alkali or acid, would be sufficient to achieve a high yield of fermentable sugars.

[00153] The present description accordingly provides an improved method or process for converting a pretreated biomass material into fermentable sugars. The method or process of the invention comprises a biomass preconditioning procedure which begins with a water washing step after the lignocellulosic biomass is subject to a pretreatment, followed by a pH adjustment step, prior to contacting the

preconditioned biomass with hydrolytic enzymes. In addition, the present description provides a saccharification mixture composition, comprising a preconditioned biomass material at a pH of at least about 5.5 and a cellulosic hydrolysis enzyme consortium.

[00154] The method or process described herein can result in a substantial and measurable reduction in process cost of enzymatic conversion of lignocellulosic biomass materials. For example, the method results in a cost saving of at least about 0.01 %, for example, at least about 0.01 %, at least about 0.02%, at least about 0.05%, at least about 0.1 % or even at least about 0.2%, as compared to a process without the water washing step prior to the pH adjusting step, starting from the same pretreated lignocellulosic biomass materials, with enzymatic hydrolysis at a pH of at least about 5.5, for the same duration and at the same temperature and other enzymatic hydrolysis conditions, reaching the same level of glucan and/or xylan conversion.

[00155] The method or process described herein can confer substantial and measurable reduction in process cost of enzymatic conversion of lignocellulosic biomass material for the preconditioning step helps to prevent or reduce non-specific and/or non-productive binding of cellulosic hydrolysis enzymes to lignin. For example, the method can confer at least about 0.01 %, such as at least about 0.01 %, at least about 0.02%, at least about 0.05%, at least about 0.10%, at least about 0.25%, at least about 0.5%, at least about 1 .0%, at least about 2%, or at least about 5% or even higher cost savings due to the substantial reduction of non-productive lignin binding by the cellulosic hydrolysis enzymes.

[00156] The method or process can result in a substantial and measurable improvement in enzymatic hydrolysis efficacy and/or efficiency. For example, the method can result in a yield of at least about 0.5%, at least about 1 .0%, at least about 1 .5%, at least about 2.0%, or even at least about 2.5% more of fermentable sugars, as compared to when the same pretreated lignocellulosic biomass material is subject to a preconditioning procedure without the water washing step, but with enzyme hydrolysis carried out at a pH of about 4.0 to about 5.3. In some

embodiments, the method results in a yield of at least about 1 .0%, e.g., at least about 1 .0%, at least about 1 .5%, at least about 2.0%, at least about 2.5%, or even at least about 5% or about 10% increased glucan conversion or glucose yield from the same pretreated lignocellulosic biomass, which has been subject to a

preconditioning procedure without the water washing step, and with the enzyme hydrolysis being carried out at a pH of about 4.0 to about 5.3. In some embodiments, the method results in a yield of at least about 0.5%, e.g., at least about 0.5%, at least about 1 .0%, at least about 1 .5%, at least about 2.0%, or even at least about 2.5% or about 5% increased xylan conversion or xylose yield from the same pretreated lignocelllulosic biomass, which has been subject to a

preconditioning procedure without the water washing step, and with the enzyme hydrolysis being carried out at a pH of about 4.0 to about 5.3.

[00157] In some embodiments, the method or process of the invention can reduce the amount of lignin blocker used to reduce the non-specific and/or non-productive binding of the enzymes of the cellulosic hydrolysis enzyme consortium to the lignin components of the pretreated lignocellulosic biomass materials. Such lignin blocker reagents can suitably be, for example, surfactants, detergents, metal compounds and polymers such as polyethylene glycols. In certain specific embodiments, the application of the present method or process can reduce the amount of lignin blockers added to the saccharification mixture by at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or even completely remove the need to use such lignin blockers altogether and still achieve the same efficacy and/or efficiency of enzymatic hydrolysis, as compared to a process carried out with the water washing step, and/or with the pH of the saccharification mixture to be at about 4.5 to about 5.3 at the start of the enzymatic hydrolysis step. [00158] In a certain embodiment, the lignocellulosic biomass material has been subject to one or more pretreatment or size reduction steps. In some cases, such pretreatment or size reduction steps are selected from one or a combination of one or more of (1 ) a mechanical pretreatment, (2) an acidic pretreatment, (3) a steam and/or heating and/or pressure- based pretreatment, (4) a cryopretreatment, (5) an alkaline pretreatment, and/or (6) an enzymatic pretreatment.

[00159] In certain embodiments, the water washing step comprising washing with a cold water, for example, one that is only slightly above freezing, at about 5°C, at about 10°C, at about 15°C, or at about 20°C. The water used can be any suitable water, including recycled process water or sea water. Some antimicrobial

procedures may be necessary to prevent microbial contamination of the resulting sugars and biological reagents and/or organisms used during the hydrolysis and fermentation subprocesses, but those procedures, if applicable or required, would not differ substantially from known antimicrobial procedures already applied to these industrial processes.

[00160] The water washing step can be carried out by methods well known in the field. Washing can comprising exposing the pretreated biomass material to a volume of water, for example, an equal volume of water, or a half volume of water, or any fractional volume of water, or a double volume of water, optionally one or more times, for example, 2, 3, 4, 5, 6, or 7 or even 8 or more times. After each rounds of washing, the biomass and the "washate" (or waste water) can be separated by extraction. After a water washing step, the washed biomass material can be at least about 2°C lower, e.g., at least about 2°C lower, at least about 5°C lower, or at least about 10°C lower, in temperature than that of the pretreated biomass prior to the water washing step. The washate comprises a measurable level of acetate.

[00161] In some embodiments, the pH adjustment step adjusts the pH of the pretreated biomass material to at least about 5.5, for example, at least about 5.7, at least about 5.9, at least about 6.0, at least about 6.1 , at least about 6.2, at least about 6.3, at least about 6.4, or at least about 6.5.

[00162] In some embodiments, the pH adjustment is performed using one or more pH adjusting agents. Suitable pH adjusting agents include, for example, an acid, a buffer, a base, or a material that can react with another material that is present in a mixture of solids, liquids, or slurries of solids and liquids to change or alter the pH of the overall mixture. One or more of such pH adjusting agents can be used as appropriate, for example, an acid can be used with a buffer, a base can be used with a buffer, an acid can be used with another acid, a base can be used with another base, a few bases can be used together, and a few bases and a buffer can be used together, etc. When more than one of such pH adjusting agents are used with each other, the pH adjusting agents can be added together or one after another.

[00163] In a certain embodiment, the enzyme mixture comprises at least one cellulase. In certain other embodiments, the enzyme mixture suitably comprises two or more cellulases. In certain embodiments, the enzyme mixture further comprises one or more hemicellulases. In certain embodiments, the enzyme mixture further comprises one or more accessory enzymes. In some embodiments, the enzyme mixture comprises a number of enzymes in amounts sufficient to cause hydrolysis of at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or even at least 60% or more of the cellulose (glucan) in the biomass substrate. In some embodiments, the enzyme mixture comprises a number of enzymes in amounts sufficient to cause hydrolysis of at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or even at least 60% or more of the hemicelluloses (xylan) in the biomass substrate.

[00164] In certain embodiments, the enzymes of the enzyme mixture are put into contact with the preconditioned biomass material, mixed, thereby creating a saccharification slurry or mixture having a pH of at least about 5.5, for example, at least about 5.5, at least about 5.7, at least about 5.9, at least about 6.0, at least about 6.1 , at least about 6.2, at least about 6.3, at least about 6.4, or at least about 6.5, at the start of the enzymatic hydrolysis step.

[00165] The enzymes that form the enzyme mixture can be from different or separate enzyme mixtures, comprising the same or different enzymes in each mixture. In certain specific embodiments, the separate mixtures may comprise a different amount of a same enzyme. In alternative embodiments, the enzymes of the enzyme mixture are present in a single enzyme mixture. In a specific embodiment, the enzymes of the enzyme mixture are produced by a single microorganism. The microorganism can be one that natively produce such an enzyme mixture. Further alternatively the microorganism can be genetically engineered to produce such an enzyme mixture, wherein certain of the enzymes of the enzyme mixture are heterologous to the microorganism, or are expressed at different than native levels as compared to the levels of these enzymes that would be produced by the native microorganism.

[00166] In some embodiments, the saccharification mixture or slurry is agitated or otherwise sufficiently mixed to achieve a substantially uniform mixture, which is then incubated under suitable enzymatic hydrolysis conditions for a sufficient time period to convert the biomass material into fermentable sugars. [00167] In certain embodiments, the amount of preconditioned lignocellulosic biomass in the saccharification mixture or slurry is at a level of at least about 1 %, at least about 2%, at least about 3%, at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 17%, at least about 19%, or even at least about 21 % dry solids weight. In some embodiments, the dry solids weight of the lignocellulosic biomass material present in the saccharification mixture or slurry is at about 1 % to about 40% dry solids weight, or at about 3% to about 35% dry solids weight, or at about 5% to about 30% dry solids weight, or about 7% to about 25% dry solids weight. [00168] In any of the above embodiments, the enzymatic hydrolysis step takes place at a temperature of at least about 20°C, or at least about 25°C, or at least about 30°C, or at least about 35°C, or at least about 40°C, or at least about 45°C, or at least about 50°C, or at least about 55°C, or at least about 60°C. In some

embodiments, the enzymatic hydrolysis step takes place at a temperature within the range of 20°C to 65°C, or the range of 25°C to 60°C, or the range of 30°C to 58°C, or the range of 35°C to 55°C.

[00169] In another aspect, the disclosure provides a saccharification mixture that comprises a pretreated and preconditioned lignocellulosic biomass material at a pH of at least about 5.5, and a cellulosic hydrolysis enzyme consortium. [00170] In certain embodiments, the saccharification mixture comprises a pretreated and preconditioned lignocellulosic biomass material at a pH of at least about 5.6, at least about 5.8, at least about 6.0, at least about 6.1 , at least about 6.2, at least about 6.3, at least about 6.4, or at least about 6.5, and a cellulosic hydrolysis enzyme consortium. Typically as the enzymatic hydrolysis reaction starts and progresses, the pH of the saccharification mixture would drop, to about 5.5, or about 5.4, or about 5.3, or about 5.2, or about 5.1 , or about 5.0, or about 4.9, or about 4.8, or even about 4.7 or lower. However suitably the pH decrease during

saccharification will reach a lower plateau, which is at a pH of about 5.0, or about 4.9, or about 4.8, or about 4.7, or even about 4.6. The initial saccharification mixture is suitably sufficiently buffered such that the pH does not continuously decrease to a level that is significantly below about 5.0. [00171] In certain embodiments, the saccharification mixture is suitably a slurry that is at least about 2%, at least about 3%, at least about 5%, at least about 8%, at least about 10%, at least about 12%, at least about 15%, at least about 18%, or at least about 20%, or even at least about 22%, or at least about 25% or higher in dry solids weight.

[00172] In some embodiments, the saccharification mixture composition is suitably a slurry comprising a cellulosic hydrolysis enzyme at a total protein concentration or dose of about 100 milligram (mg) or less of enzymes per gram (g) of cellulose plus hemicelluloses (glucan plus xylan) in the pretreated and preconditioned biomass material. For example, the total protein concentration or enzyme dose of cellulosic hydrolysis enzyme consortium in the saccharification mixture is about 90 mg/g of glucan plus xylan or less, about 80 mg/g of glucan plus xylan or less, about 75 mg/g of glucan plus xylan or less, about 70 mg/g glucan plus xylan or less, about 65 mg/g of glucan plus xylan or less, about 60 mg/g glucan plus xylan or less, about 50 mg/g of glucan plus xylan or less, about 45 mg/g of glucan plus xylan or less, about 40 mg/g of glucan plus xylan or less, about 35 mg/g of glucan plus xylan or less, about 30 mg/g of glucan plus xylan or less, about 25 mg/g of gllucan plus xylan or less, about 20 mg/g of glucan plus xylan or less, or even about 15 mg/g of glucan plus xylan or less, wherein the glucan plus xylan levels are determined by the amount of total cellulose and hemicelluloses in the pretreated and preconditioned biomass material. Persons skilled in the art will readily know how to determine the total glucan and xylan levels of a given lignocellulosic biomass material.

[00173] In some embodiments, the saccharification mixture composition further comprises a lignin blocker, at a level of about 0.001 % to about 2%, for example, between about 0.001 % to about 1 .75%, between about 0.02% to about 1 .5%, or between about 0.05% to about 1 .0%.

[00174] In another aspect, the invention pertains to the improved and higher levels of fermentable sugars in the product resulting from practicing the method of the first aspect, or by incubating or using an saccharification mixture composition of the second aspect. The thus-produced fermentable sugars can then be used for the production of high value chemicals, fuels and/or other useful products. 7. Producing Fermentation Products

[00175] Fermentable sugars produced in the present process may be fermented by suitable microorganisms that either naturally or through genetic manipulation are able to produce substantial quantities of desired target chemicals. Target chemicals that may be produced by fermentation include, for example, acids, alcohols, alkanes, alkenes, aromatics, aldehydes, ketones, biopolymers, proteins, peptides, amino acids, vitamins, antibiotics, and pharmaceuticals. Alcohols include, but are not limited to methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol, propanediol, butanediol, glycerol, erythritol, xylitol, and sorbitol. Acids may include acetic acid, lactic acid, propionic acid, 3-hydroxypropionic acid, butyric acid, gluconic acid, itaconic acid, citric acid, succinic acid and levulinic acid. Amino acids may include glutamic acid, aspartic acid, methionine, lysine, glycine, arginine, threonine, phenylalanine and tyrosine. Additional target chemicals include methane, ethylene, acetone and industrial enzymes. [00176] The fermentation of sugars to target chemicals may be carried out by one or more appropriate biocatalysts in single or multistep fermentations. Biocatalysts may be microorganisms selected from bacteria, filamentous fungi and yeast. Biocatalysts may be wild type microorganisms or recombinant microorganisms, and may include Escherichia, Zymomonas, Saccharomyces, Candida, Pichia, Streptomyces, Bacillus, Lactobacillus, and Clostridiuma. Typically, biocatalysts may be

recombinant Escherichia coli, Zymomonas mobilis, Bacillus stearothermophilus, Saccharomyces cerevisiae, Clostridia thermocellum, Thermoanaerobacterium saccharolyticum, and Pichia stipitis

[00177] Many biocatalysts used in fermentation to produce target chemicals have been described and others may be discovered, produced through mutation, or engineered through recombinant means. Any biocatalyst that uses fermentable sugars produced in the present method may be used to make the target chemical(s) that it is known to produce by fermentation.

[00178] Particularly of interest are biocatalysts that produce biofuels including ethanol and butanol. For example, fermentation of carbohydrates to acetone, butanol, and ethanol (ABE fermentation) by solventogenic Clostridia is well known (Jones and Woods (1986) Microbiol. Rev. 50:484-524). A fermentation process for producing high levels of butanol, also producing acetone and ethanol, using a mutant strain of Clostridium acetobutylicum is described in U.S. Pat. No. 5, 1 92,673. The use of a mutant strain of Clostridium beijerinckii to produce high levels of butanol, also producing acetone and ethanol, is described in U.S. Pat. No. 6,358,71 7. Co-owned and co-pending patent applications WO 2007/041 269 and WO 2007/050671 , which are herein incorporated by reference, disclose the production of 1 -butanol and isobutanol, respectively, in genetically engineered microbial hosts. Co-owned and co-pending U.S. patent applications No. 1 1 /741 ,892 and No. 1 1 /741 ,91 6, which are herein incorporated by reference, disclose the production of 2-butanol in genetically engineered microbial hosts. Isobutanol, 1 -butanol or 2-butanol may be produced from fermentation of hydrolysate produced using the present process by a microbial host following the disclosed methods.

[00179] Genetically modified strains of E. coli have also been used as biocatalysts for ethanol production (Underwood et al., (2002) Appl. Environ. Microbiol. 68:6263- 6272). A genetically modified strain of Zymomonas mobilis that has improved production of ethanol is described in US 2003/0162271 A1 . A further engineered ethanol-producing strain of Zymomonas mobilis and its use for ethanol production are described in co-owned and co-pending U.S. patent applications 60/847,81 3 and 60/847,856, respectively, which are herein incorporated by reference. Ethanol may be produced from fermentation of hydrolysate produced using the present process by Zymomonas mobilis following the disclosed methods. In Example 1 3 herein, the present process is used for saccharification of pretreated corn cob biomass to fermentable sugars, followed by fermentation of the sugars for the production of ethanol using Z. mobilis as the biocatalyst. [00180] The present process may also be used in the production of 1 ,3-propanediol from biomass. Recombinant strains of E. coli have been used as biocatalysts in fermentation to produce 1 ,3 propanediol (U.S. Pat. No. 6,01 3,494, U.S. Pat. No. 6,514,733). Hydrolysate produced by saccharification using the present process may be fermented by E. Coli to produce 1 ,3-propanediol as described in Example 1 0 of co-owned and co-pending U.S. application Ser. No. 1 1 /403,087, which is herein incorporated by reference.

[00181 ] Lactic acid has been produced in fermentations by recombinant strains of E. Coli (Zhou et al., (2003) Appl. Environ. Microbiol. 69:399-407), natural strains of Bacillus (US20050250192), and Rhizopus oryzae (Tay and Yang (2002)

Biotechnol. Bioeng. 80:1 -12). Recombinant strains of E. coll have been used as biocatalysts in fermentation to produce 1 ,3 propanediol (U.S. Pat. No. 6,013,494, U.S. Pat. No. 6,514,733), and adipic acid (Niu et al., (2002) Biotechnol. Prog.

18:201 -21 1 ). Acetic acid has been made by fermentation using recombinant

Clostridia (Cheryan et al., (1997) Adv. Appl. Microbiol. 43:1 -33), and newly identified yeast strains (Freer (2002) World J. Microbiol. Biotechnol. 18:271 -275). Production of succinic acid by recombinant E. coli and other bacteria is disclosed in U.S. Pat. No. 6,159,738, and by mutant recombinantE. coli in Lin et al., (2005) Metab. Eng. 7:1 16-127). Pyruvic acid has been produced by mutant Torulopsis glabrata yeast (Li et al., (2001 ) Appl. Microbiol. Technol. 55:680-685) and by mutant E. coli (Yokota et al., (1994) Biosci. Biotech. Biochem. 58:2164-2167). Recombinant strains ofE.

coli have been used as biocatalysts for production of para-hydroxycinnamic acid (US20030170834) and quinic acid (US20060003429). [00182] A mutant of Propionibacterium acidipropionici has been used in fermentation to produce propionic acid (Suwannakham and Yang (2005) Biotechnol. Bioeng. 91 :325-337), and butyric acid has been made by Clostridium tyrobutyricum (Wu and Yang (2003) Biotechnol. Bioeng. 82:93-102). Propionate and propanol have been made by fermentation from threonine by Clostridium sp. strain 17cr1 (Janssen (2004) Arch. Microbiol. 182:482-486). A yeast-like Aureobasidium pullulans has been used to make gluconic acid (Anantassiadis et al., (2005) Biotechnol. Bioeng. 91 :494- 501 ), by a mutant of Aspergillis niger (Singh et al., (2001 ) Indian J. Exp. Biol.

39:1 136-43). 5-keto-D-gluconic acid was made by a mutant of Gluconobacter oxydans (Elfari et al., (2005) Appl Microbiol. Biotech. 66:668-674), itaconic acid was produced by mutants of Aspergillus terreus (Reddy and Singh (2002) Bioresour. Technol. 85:69-71 ), citric acid was produced by a mutant Aspergillus niger strain (Ikram-UI-Haq et al., (2005) Bioresour. Technol. 96:645-648), and xylitol was produced by Candida guilliermondii FT I 20037 (Mussatto and Roberto (2003) J. Appl. Microbiol. 95:331 -337). 4-hydroxyvalerate-containing biopolyesters, also containing significant amounts of 3-hydroxybutyric acid 3-hydroxyvaleric acid, were produced by recombinant Pseudomonas putida and Ralstonia eutropha (Gorenflo et al., (2001 ) Biomacromolecules 2:45-57). L-2,3-butanediol was made by

recombinant E. coli (Ui et al., (2004) Lett. Appl. Microbiol. 39:533-537). [00183] Production of amino acids by fermentation has been accomplished using auxotrophic strains and amino acid analog-resistant strains ofCorynebacterium, Brevibacterium, and Serratia. For example, production of histidine using a strain resistant to a histidine analog is described in Japanese Patent Publication No.

56008596 and using a recombinant strain is described in EP 136359. Production of tryptophan using a strain resistant to a tryptophan analog is described in Japanese Patent Publication Nos. 47004505 and 51019037. Production of isoleucine using a strain resistant to an isoleucine analog is described in Japanese Patent Publication Nos. 47038995, 51006237, 54032070. Production of phenylalanine using a strain resistant to a phenylalanine analog is described in Japanese Patent Publication No. 56010035. Production of tyrosine using a strain requiring phenylalanine for growth, resistant to tyrosine (Agr. Chem. Soc. Japan 50 (1 ) R79-R87 (1976), or a

recombinant strain (EP263515, EP332234), and production of arginine using a strain resistant to an L-arginine analog (Agr. Biol. Chem. (1972) 36:1675-1684, Japanese Patent Publication Nos. 54037235 and 57150381 ) have been described.

Phenylalanine was also produced by fermentation in Eschericia coli strains ATCC 31882, 31883, and 31884. Production of glutamic acid in a recombinant coryneform bacterium is described in U.S. Pat. No. 6,962,805. Production of threonine by a mutant strain of E. coli \s described in Okamoto and Ikeda (2000) J. Biosci Bioeng. 89:87-79. Methionine was produced by a mutant strain of Corynebacterium lilium (Kumar et al, (2005) Bioresour. Technol. 96: 287-294).

[00184] Useful peptides, enzymes, and other proteins have also been made by biocatalysts (for example, in U.S. Pat. No. 6,861 ,237, U.S. Pat. No. 6,777,207, U.S. Pat. No. 6,228,630). [00185] Target chemicals produced in fermentation by biocatalysts may be recovered using various methods known in the art. Products may be separated from other fermentation components by centrifugation, filtration, microfiltration, and nanofiltration. Products may be extracted by ion exchange, solvent extraction, or electrodialysis. Flocculating agents may be used to aid in product separation. As a specific example, bioproduced 1 -butanol may be isolated from the fermentation medium using methods known in the art for ABE fermentations (see for example, Durre, Appl. Microbiol. Biotechnol. 49:639-648 (1998), Groot et al., Process.

Biochem. 27:61 -75 (1992), and references therein). For example, solids may be removed from the fermentation medium by centrifugation, filtration, decantation, or the like. Then, the 1 -butanol may be isolated from the fermentation medium using methods such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, or pervaporation. Purification of 1 ,3-propanediol from fermentation media may be accomplished, for example, by subjecting the reaction mixture to extraction with an organic solvent, distillation, and column chromatography (U.S. Pat. No. 5,356,812). A particularly good organic solvent for this process is cyclohexane (U.S. Pat. No. 5,008,473). Amino acids may be collected from fermentation medium by methods such as ion-exchange resin adsorption and/or crystallization.

EXAMPLES

[00186] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present methods or apparatus, and are not intended to limit the scope of what the inventors regard as their inventive methods or apparatus nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.

Example 1 : Preconditioning of Avicel

[00187] A synthetic Avicel (Sigma-Aldrich, Cat#1 1365) cellulosic substrate was divided into three parts. Each part was subject to a water-washing procedure using water of 50 °C and at a volume of 5 x of that of the substrate, prior to the pH adjustment procedure, during which the pH of the first sample was adjusted to 5.10, the pH of the second sample was adjusted to 5.50, and the pH of the third sample was adjusted to 5.85. Each of the preconditioned avicel samples was then mixed with a 0.13 mL/gram of avicel glucan of Accellerase® TRIO™, at a dry solids level of 10%. The enzymatic hydrolysis step was carried out at a temperature of 50°C, and for a duration of up to 72 hours. [00188] The levels of glucan conversion were measured from each of the Avicel samples at time points 24 hours, 48 hours and 72 hours. As indicated in FIGURE 1 , no improvement of glucan conversion is seen as a result of preconditioning the biomass substrate to a higher than pH 5.5, prior to enzymatic hydrolysis. [00189] As expected, a drop in glucan conversion was observed for pH 5.5 and pH 5.85, which were caused by the loss of enzymatic performance at such high pHs, outside the optimal range where Accellerase® TRIO™ is known to function in enzymatic hydrolysis.

Example 2: Preconditioning of Dilute Acid Pretreated Corn Stover (PCS)

Substantially Improves Conversion

[00190] A dilute sulfuric acid pretreated corn stover substrate, prepared in

accordance with the method described in Schell, D.J et al., App. Biochem.

Biotechnol. 2003, 105 (1-3):69-85, was obtained from the National Renewable Energy Laboratory (NREL). Fifteen (15) grams of this pretreated biomass was split into three equal parts.

[00191] The first part was subject to a 5x volume water wash, five times, then about 0.1 15 ml_ of 0.125 N NaOH was used to adjusted to a pH of about 6. The second part was also subject to a 5x volume water wash, five times, then about 0.09 ml_ of 0.125 N NaOH was used to adjust to a pH of about 5.65. The third part was also subject to a 5x volume water wash, five times, then about 0.035 ml_ of 0.125 N NaOH was used to adjust to a pH of about 5.15.

[00192] A 5% dry solids of each of the PCS samples was then mixed with 0.13 mL/gram of glucan in PCS of Accellerase® TRIO™ to make 3 saccharification mixtures. The first saccharification mixture was measured to have pH of about 6.02. The second saccharification mixture was measured to have a pH of about 5.65. The third saccharification mixture was measured to have a pH of about 5.15.

[00193] The enzymatic hydrolysis reactions were carried out at a temperature of 50°C. The reactions were taken to 72 hours each, with glucan conversion

measured for each at 24 hours, 48 hours and 72 hours. [00194] As can be seen from FIGURE 2, preconditioning of the dilute acid pretreated corn stover biomass by water washing followed by pH adjustment to at least above about 5.5 or higher significantly improves the efficiency and effectiveness of enzymatic hydrolysis of the biomass substrate under the same hydrolysis conditions, using the same enzymes at the same doses.

Example 3: Preconditioning of Dilute Acid Pretreated Sugarcane Bagasse Substantially Improves Conversion

[00195] A dilute sulfuric acid pretreated sugarcane bagasse substrate, prepared in accordance with the method described in Schell, D.J et al., App. Biochem.

Biotechnol. 2003, 105 (1-3):69-85, was obtained from the National Renewable Energy Laboratory (NREL).

[00196] Fifteen (15) grams of this pretreated biomass material was split into three equal parts. The first part was subject to a 10x volume water wash, one time, then about 0.230 mL of 0.125 N NaOH was used to adjusted it to a pH of about 5.89. The second part was also subject to a 10x volume water wash, one time, followed by adjustment of pH to about 5.47 using about 0.170 mL of 0.125 N NaOH. The third part was likewise subject to a 10x volume water wash, once, followed by adjustment of pH to about 5.15, using about 0.120 mL of 0.125 N NaOH. [00197] A 5% dry solids of each of the dilute acid pretreated bagasse samples was then mixed with 0.13 mL/gram of glucan in bagasse of Accellerase® TRIO™ to make 3 saccharification mixtures. The first saccharification mixture was measured to have pH of about 5.89. The second saccharification mixture was measured to have a pH of about 5.47. The third saccharification mixture was measured to have a pH of about 5.15.

[00198] The enzymatic hydrolysis reactions were carried out at a temperature of 50°C. The reactions were taken to 72 hours each, with glucan conversion measured for each at 24 hours, 48 hours and 72 hours.

[00199] As can be seen from FIGURE 3, preconditioning of the dilute acid pretreated sugarcane bagasse biomass by water washing followed by pH adjustment to at least above about 5.5 or higher significantly improves the efficiency and effectiveness of enzymatic hydrolysis of the biomass substrate under the same hydrolysis conditions, using the same enzymes at the same doses.

Example 4: Preconditioning of Hydrothermally Pretreated Switchqrass

Improves Conversion

[00200] A hydrothermally pretreated switchgrass substrate, prepared in accordance with the method described in Shi, J et al., Bioresource Technol. 2011, 102

(10) :5952-5961, was used for this set of experiments.

[00201] A 15 g of this pretreated biomass material was split into three equal parts. The first part was subject to a 10x volume water wash, 1 time, then about 0.130 ml_ of 0.125 N NaOH was used to adjusted to a pH of about 5.75. The second part was also subject to a 10x volume water wash, one time, followed by adjustment of pH to about 5.45 using about 0.095 ml_ of 0.125 N NaOH. The third part was likewise subject to a 10x volume water wash, one time, followed by adjustment to pH 5.10, using about 0.055 ml_ of 0.125 N NaOH.

[00202] A 5% dry solids of each of the hydrothermally pretreated switchgrass samples was then mixed with 0.13 mL/gram of glucan in the pretreated switchgrass of Accellerase® TRIO™ to make 3 saccharification mixtures. The first

saccharification mixture was measured to have pH of about 5.75. The second saccharification mixture was measured to have a pH of about 5.45. The third saccharification mixture was measured too have a pH of about 5.10.

[00203] The enzymatic hydrolysis reactions were carried out at a temperature of 50°C. The reactions were taken to 72 hours each, with glucan conversion measured for each at 24 hours, 48 hours and 72 hours. [00204] As can be seen from FIGURE 4, preconditioning of the hydrothermally pretreated biomass by water washing followed by pH adjustment to at least above about 5.5 or higher significantly improves the efficiency and effectiveness of enzymatic hydrolysis of the biomass substrate under the same hydrolysis conditions, using the same enzymes at the same doses. Example 5: Preconditioning of Acid Treated Water Washed Dilute Ammonia Pretreated Corn Stover also Substantially Improves Conversion.

[00205] A dilute ammonia pretreated corn stover substrate, prepared in accordance with the method described in PCT Publication WO061 1 0901 was used for this experiment.

[00206] Fifteen (1 5) grams of the dilute ammonia pretreated corn stover biomass was split into three equal parts. The first part was subject to a 5x volume water wash, six times, then treated with a 72% sulfuric acid, and incubated at about pH 2.0, for about 12 hours at 50°C. This was followed by a second round of 5x volume wash, twice. Then the material was adjusted to pH of about 6.00 using about 0.1 10 ml_ of 0.1 25 N NaOH. The second part was also subject to a 5x volume water wash, six times, then treated with a 72% sulfuric acid, and incubated at about 2.0 for about 1 2 hours at 50°C. This material was then adjusted to about pH 5.60 using about 0.095 ml_ of 0.1 25 N NaOH. The third part was likewise subject to the exact same water washing, acid treatment, followed by water washing, then adjusted to pH about 5.1 0 using about 0.080 mL of 0.125 N NaOH.

[00207] A 5% dry solids of each of the dilute ammonia pretreated corn stover samples was then mixed with 0.1 3 mL/gram of glucan in the dilute ammonia pretreated corn stover of Accellerase® TRIO™ to make 3 saccharification mixtures. The first saccharification mixture was measured to have pH of about 6.00. The second saccharification mixture was measured to have a pH of about 5.60. The third saccharification mixture was measured to have a pH of about 5.1 0.

[00208] The enzymatic hydrolysis reactions were carried out at a temperature of 50°C. The reactions were taken to 72 hours each, with glucan conversion

measured for each at 24 hours, 48 hours and 72 hours.

[00209] As can be seen from FIGURE 5, preconditioning of the dilute ammonia pretreated corn stover biomass by water washing followed by acid treatment, followed by water washing, then pH adjustment to at least above about 5.5 or higher significantly improves the efficiency and effectiveness of enzymatic hydrolysis of the biomass substrate under the same hydrolysis conditions, using the same enzymes at the same doses. [00210] In a separate experiment, three samples of the dilute ammonia pretreated corn stover were taken and each were subject to 5x volume water washing, 5 times, then adjusted using an alkali or a base to the final pH of about 5.95, 5.60 and 5.1 3, respectively. The same enzyme dosing and dry solids levels were used to hydrolyze these non-acid treated but washed and pH adjusted biomass samples. No improvement of enzymatic hydrolysis was observed.

Example 6: Preconditioning Step Comprising a Water Washing Step Followed by pH Adjustment Step significantly Reduces the Alkaline Use to Reach a Desired Preconditioning pH

[00211 ] In this experiment, the amount of NaOH used to adjust the pH of an acid pretreated corn stover sample, at 5% dry solids and 1 5% dry solids was compared when no water-washing step was included in the preconditioning step to a pH of about 5.9. The enzymatic hydrolysis step was then carried out at a starting pH of about 5.9.

[00212] We observed that adding a water-washing step prior to the pH adjustment step, saves at least about 80% in the amount of NaOH required to adjust the pH of the pretreated biomass to 5.9 as compared to when no water-washing procedure was used but NaOH was used to adjust the biomass to the same pH. The resulting glucan conversion levels of these differently preconditioned saccharification mixtures are similar.

Example 7: Determination of components of washates

[00213] Compositions of the washates (used or waste water batches collected from the water washing step) were determined using pyrolysis-GC/MS, by applying the washates of the preconditioning step of the PCS substrate of Example 2, using an instrument that is the combination of two parts, with the Pyrolysis Analytical instrument made by Frontier Lab, and the GC/MS made by Shimadzu. Acetate was found to be an important component in the washate. So was a number of phenolic compounds. [00214] Although the foregoing method/process and/or apparatus has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings herein that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[00215] Accordingly, the preceding merely illustrates the principles of the present invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the present invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the present compositions and methods and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the present invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present compositions and methods, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

WHAT IS CLAIMED IS:

1 . An improved process for producing a fermentable sugar from a

lignocellulosic biomass material comprising the following:

(a) a pretreatment step during which the lignocellulosic biomass material is subject to a pretreatment to produce a pretreated biomass substrate;

(b) a preconditioning step during which the pretreated biomass substrate of (a) is preconditioned;

(c) an enzymatic hydrolysis step during which the preconditioned and

pretreated biomass material is hydrolyzed with an enzyme mixture comprising at least one cellulase enzyme; and

(d) a recovery step during which the soluble sugars produced in step (c) are recovered.

2. The process of claim 1 , further comprising producing a fermentation

product from the soluble sugars recovered under (d).

3. The process of claim 1 or 2, wherein the lignocellulosic biomass material is pretreated with one or more of (1 ) a mechanical pretreatment; (2) an acidic pretreatment; (3) a steam and/or heating and/or pressure-based pretreatment; (4) a cryopretreatment; (5) an alkaline pretreatment, and/or an enzymatic pretreatment.

4. The process of any one of claims 1 -3, wherein the preconditioning step comprises a pH adjustment procedure which adjusts the pretreated biomass material to a pH of at least above about 5.5.

5. The process of claim 4, wherein the pH adjustment procedure adjusts the pretreated biomass material to a pH of at least about 5.7.

6. The process of claim 5, wherein the pH adjustment procedure adjusts the pretreated biomass material to a pH of at least about 5.9.

7. The process of claim 6, wherein the pH adjustment procedure adjusts the pretreated biomass material to a pH of at least about 6.1 .

8. The process of any one of claims 1 -7, wherein the preconditioning step further comprises a water washing procedure that takes place prior to the pH adjustment procedure.

9. The process of claim 8, wherein the water washing procedure is carried out using warm water of a temperature about 30°C or above.

10. The process of claim 9, wherein the water washing procedure is carried out using cold water of a temperature about 29°C or below.

1 1 . The process of any one of claims 1 -10, wherein the water washing

procedure is carried out with a volume of water that is about half of that of the pretreated biomass material to about 8x or more fold of that of the pretreated biomass material.

12. The process of claim 1 1 , wherein the water washing procedure can be repeated 2 to 10 times or more.

13. The process of any one of claims 1 -12, wherein the water washing

procedure reduces the surface negative charge of the pretreated biomass material by at least about 1 mV in zeta potential.

14. The process of claim 13, wherein the water washing procedure reduces the surface negative charge of the pretreated biomass material by at least about 5 mV in zeta potential.

15. The process of any one of claims 1 -14, wherein the enzyme mixture

comprises at least two cellulases.

16. The process of claim 15, wherein the enzyme mixture comprises at least one cellobiohydrolase and at least one endoglucanases.

17. The process of claim 15 or 16, wherein the enzyme mixture further

comprises one or more beta-glucosidases.

18. The process of any one of claims 15-17, wherein the enzyme mixture further comprises one or more hemicellulases.

19. The process of any one of claims 15-18, wherein the enzyme mixture further comprises one or more accessory enzymes.

20. The process of any one of claims 15-18, wherein the enzyme mixture further comprises a GH61 family enzyme.

21 . A process for producing a fermentation product from a lignocellulosic

biomass material comprising the following:

(a) a pretreatment step during which the lignocellulosic biomass material is pretreated to produce a pretreated biomass substrate;

(b) a preconditioning step during which the pretreated biomass substrate of (a) is preconditioned;

(c) an enzymatic hydrolysis step during which the preconditioned

pretreated biomass substrate is hydrolyzed using an enzyme mixture comprising at least one cellulase enzyme; and

(d) a fermentation step during which the product of step (c) is fermented to produce a fermentation product.

22. A saccharification mixture comprising a pretreated and preconditioned lignocellulosic biomass substrate and an enzyme mixture comprising at least one cellulase enzyme, wherein the saccharification mixture has a pH within the range of about 5.5 to about 6.5, which saccharification mixture is directly incubated for enzymatic hydrolysis without adjustment of pH for at least 24 hours.

23. The saccharification mixture of claim 22, further comprises one or more hemicellulases.

24. The saccharification mixture of claim 22 or 23, further comprising one or more accessory enzymes.

25. The saccharification mixture of any one of claims 22-24, further comprising one or more GH61 family enzymes.

26. The saccharification mixture of any one of claims 22-25, which does not contain polyethylene glycol or surfactants.

27. The saccharification mixture of any one of claims 22-26, which contains less than about 0.05% of polyethylene.

28. The sacccharification mixture of any one of claims 22-26, which contains less than about 0.05% of surfactants.

29. A method of using the sacchafication mixture of any one of claims 22-28, comprising incubation of the saccharification mixture for about 1 hour to about 120 hours, whereby soluble sugars are produced from the lignocellulosic biomass materials.

30. The method of claim 29, wherein the incubation of the saccharifiation mixture takes place at a temperature of between about 20°C to about 70°C.

31 . A fermentable sugars produced by applying the process of any one of claims 1 -20, and 29-30.

32. A fermentation product produced by applying the process of claim 21 .