WO2015175308A1

WO2015175308A1 - Improved enzymatic hydrolysis of biomass

Info

Publication number: WO2015175308A1
Application number: PCT/US2015/029648
Authority: WO
Inventors: Michael Bodo; Jeffrey David Cohen; Chuanbin Liu
Original assignee: Danisco Us Inc.
Priority date: 2014-05-13
Filing date: 2015-05-07
Publication date: 2015-11-19

Abstract

Provided is an improved method or process for saccharifying a lignocellulosic biomass feedstock into fementable sugars, comprising the maintenance of dissolved oxygen levels in the saccharification mixture. Provided is also an improved apparatus or reactor useful for practicing the improved method or process.

Description

IMPROVED ENZYMATIC HYDROLYSIS OF BIOMASS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority from US provisional application USSN 61 /992,738, filed 13 May 2014 and is incorporated herein by reference in entirety.

FIELD OF THE INVENTION

[0002] Provided is an improved method or process of enzymatic hydrolysis of lignocellulosic biomass materials. Specifically the process provides for improved hydrolysis efficacy and/or efficiency, and improved yields of fermentable sugars. Also provided is an apparatus or reactor that can be used to practice such a method or process.

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] Before they can be effectively degraded, however, the lignin will typically first need to be permeabilized, for example, by various pretreatment methods, and the hemicelluloses disrupted such that the complex carbohydrate cellulose polymers become more readily accessible to celluloytic hydrolysis enzymes. Afterwards, the pretreated mixture is subject to a 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 product contains a high concentration of fermentable sugars. For most types of lignocellulosic biomass, economic viability of the industrial process also would require a high biomass dry matter level prior to the saccharification step. At such high dry matter levels, efficient enzymatic hydrolysis can be a challenge.

[0006] Even with the best combination of enzymatic activities, coupled with the most effective pretreatment of the lignocellulosic biomass materials, the efficiency and efficacy of the saccharification process remain important to whether a cellulosic biorefinery operation is successful or economically viable. Accordingly, there remains a need for an economical process for saccharification of biomass which can be carried out using high dry solids weight of biomass, and giving high yields of fermentable sugars.

SUMMARY OF THE INVENTION

[0007] Described is a new and improved process for enzymatic hydrolysis of a lignocellulosic biomass material, optionally pretreated, which results in higher conversion efficacy and efficiency and production of higher yields of fermentable sugars from a given biomass material, as compared to when such a biomass material is saccharified using previously known processes.

[0008] 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. There existed in the art two main approaches to break down such plant-based lignocellulosic materials: (1 ) acid hydrolysis and (2) enzymatic 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 well-developed

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.

[0009] 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 lignocellulosic biomass material, delicately balanced in order to achieve effective synergism and more complete breakdown of the materials. Moreover typically a large amount of enzymes is required in order to achieve reasonable and commercially viable rate and yields.

[0010] 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 ai, (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). [0011] 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). 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.

[0012] 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.

[0013] 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.

[0014] Aside from the amount of enzymes and types of enzymatic activities used in a process, other factors may affect the efficacy and effectiveness of the hydrolysis and the yield of resulting fermentable sugars. 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. The factors may also include the level of dry solids in the biomass material that is subject to the hydrolysis by the enzymes. High dry solids level can present significant operational challenges to the enzymatic hydrolysis reaction due to the need to maintain suspension of the biomass such that they come into adequate contact with enzymes. Accordingly, high dry solids weight or content places high energy requirements on the biorefinery, and even with increased efforts to insure proper mixing, such high solids levels tend to render the enzymes less effective. These factors in aggregate have prompted the careful control of operational conditions such as temperature and pH in the hydrolysis step. [0015] Mixing is typically required for the hydrolysis or saccharification step, therefore the saccharification reactor typically would comprise a mixing means such as an agitator. The slurry or saccharification mixture is brought to the desirable temperature by either heating or cooling, whereas the temperature desired is set based on the temperature optima for the saccahrification enzymes to be used to hydrolyze the biomass being processed, in order to achieve the best possible saccharification raction rate. It is sometimes suitable to operate the saccharification step at a lower than optimal temperature for the enzymes, in order to accommodate other process factors. Under such circumstances, the activities of the enzymes are not damaged but the rate of hydrolysis may be reduced. Substantial deviation of temperature, especially to a level higher than the optimal temperature can lead to irreversible unfolding, denaturing or otherwise disintegration of the enzymes, thus maintenance of the temperature to below a certain threshold level is deemed one of the determinative factors of whether the process is successful.

[0016] While mixing, the slurry or saccahrification mixture is brought to the desired pH through the addition of acid or base as required, depending on the initial pH of the pretreated biomass material, which can vary depending on the pretreatment used. The specific pH that is desired is based on the pH optima of the saccharification enzymes to be used with the particular type of biomass being processed. The mixing allows a substantially uniform pH to be achieved throughout the biomass and enzyme mixture, which in turn allows optimal functioning of the enzymes.

[0017] It was surprisingly discovered that, the concentration of dissolved oxygen in the saccharification mixture, comprising a lignocellulosic biomass material and an enzyme product, can become substantially depleted during the enzymatic saccharification reaction. As a result, the saccharification performance is reduced and so are the yields of the fermentable sugars.

[0018] While not wishing to be bound by theory, it has been demonstrated that certain of the celluloytic hydrolysis enzymes may consume oxygen during the sacharification reaction. More specifically enzymes such as certain monooxygenases or glycosyl hydrolase (GH) family 61 enzymes may act upon oxygen or other oxidized substrates. There were also in the art knowledge and prior experience of oxidative inactivation and/or destabilization of enzymes or protein molecules. Accordingly at least some of loss of dissolved oxygen in the saccharification mixture may be due to enzymes.

[0019] However, it has been surprisingly found that enzymes' contribution to oxygen depletion is insignificant as compared to the depletion of oxygen by components of the pretreated biomass material. Specifically, different types of lignocellulosic biomass materials, pretreated using different methods, tend to cause different level or extent of dissolved oxygen depletion at different speed. On the other hand, it has been found that a certain minimum threshold level of dissolved oxygen, if maintained in the saccharification mixture during the saccharification step, the yields of fermentable monomeric sugars would significantly increase.

[0020] The present description provides an improved method or process for saccharifying a pretreated biomass at a high dry weight of biomass to produce fermentable sugars. The method or process of the invention uses a fed batch reactor system whereby the enzymatic hydrolysis step is carried out, at least partially, in a saccharification reactor that is sufficiently aerated and/or mixed such that the dissolved oxygen concentration of the saccharification mixture is maintained at above a certain threshold level.

[0021] In one aspect, the method or process comprises:

(a) a loading step comprising introducing the lignocellulosic biomass material and an enzyme composition into a reactor;

(b) a mixing step comprising stirring or agitating in the reactor such the enzyme composition and the lignocellulosic biomass material are sufficiently mixed into a substantially homogenous slurry saccharification mixture; and

(c) a saccharification step during which the saccharifciation mixture of (b) is incubated under conditions that allow the hydrolysis of the biomass material into soluble fermentable oligomer or monomer saccharifdes; wherein the level of dissolved oxygen in the saccharification mixture is maintained at above about 1 .5% of the saturation dissolved oxygen level throughout the

saccharification step.

[0022] 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. [0023] In certain embodiments, the lignocellulosic biomass material is suitably one that comprises at least about 3 wt.% lignin, for example, at least about 5 wt.%, at least about 7 wt.%, at least about 9 wt.%, at least about 10 wt.%, at least about 15 wt.% or even at least about 20 wt.% lignin, referencing the total weight of carbohydrate polymers present in the biomass material. [0024] In a certain embodiment, the enzyme composition comprises at least one cellulase. In certain other embodiments, the enzyme mixture 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 composition 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 composition 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.

[0025] In certain embodiments, the enzymes of the enzyme composition are introduced into the reactor during the loading step in two or more separate mixtures. The enzymes in these separate mixtures can be the same or different enzymes. In certain specific embodiments, the separate mixtures may comprise a different amount of a same enzyme. In alternative embodiments, the enzymes of the enzyme

composition are present in a single enzyme mixture. In a specific embodiment, the enzymes of the enzyme composition are produced by a single microorganism. The microorganism can be one that natively produce such an enzyme mixture. 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.

[0026] In certain embodiments, the mixing step comprises stirring or agitating the content of the reactor at a rate sufficient to cause effective mixing of the biomass and the enzyme composition. The mixing or agitation can be continuous or intermittent, and whether or not it is continuous may depend on the dry solids weight of the biomass material and the relative ease or difficulty in keeping the solids in suspension, the size of the reactor, and/or the available means for temperature control.

[0027] In certain embodiments, the amount of lignocellulosic biomass material present in the reactor during the mixing step is at a level of at least 10%, at least 15%, at least 17%, at least 19%, or even at least 21 % dry solids weight. In some embodiments, the dry solids weight of the lignocellulosic biomass material present in the reactor during the mixing step is at about 10% to about 40% dry solids weight, or at about 15% to about 35% dry solids weight, or at about 17% to about 30% dry solids weight, or about 19% to about 25% dry solids weight. [0028] In any of the above embodiments, the saccharification step takes place at a pH of about 3 to about 9, or at a pH of about 3.5 to about 7.5, or at a pH of about 4 to about 7, or at a pH of about 4.5 to about 6.5, or even at a pH of about 5 to about 6.

[0029] In any of the above embodiments, the saccharifcation step takes place for a period of at least 1 hour, or at least 5 hours, or at least 10 hours, or at least 15 hours, or at least 24 hours, or at least 24 hours, or even at least 72 hours. In some

embodiments, the saccharification step takes place for a period of 1 to 120 hours, or 5 to 96 hours, or 10 to 85 hours, or 15 to 80 hours, or 24 hours to 72 hours, or 48 to 65 hours.

[0030] In any of the above embodiments, the saccharification 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 saccharification 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.

[0031] In any of the above embodiments, the concentration of dissolved oxygen in the saccharification mixture is maintained at a level of above 1 .5%, preferably at a level of above 2%, more preferably at a level of above 2.5%, or above 3.0%, or above 3.5%, or above 4.0%, or above 4.5%, or above 5.0%, or above 5.5%, or above 6.0%, or above 6.5%, or above 7.0%, or above 7.5%, or even above 8.0% of the saturating dissolved oxygen concentration of such a mixture at the saccharification temperature immediately prior to the saccharification step. In some embodiments, the level of dissolved oxygen in the saccharification mixture is maintained at a level within the range of about 1 .5% to about 35%, of about 3% to about 30%, or about 5% to about 25%, or about 7.5% to about 20% of the saturating dissolved oxygen concentration of such a mixture at the saccharification temperature immediately prior to the saccharification step. In some embodiments, the dissolved oxygen level in the saccharification mixture is measured using a dissolved oxygen probe. In certain embodiments, the dissolved oxygen probe is pre-calibrated.

[0032] In another aspect, the disclosure provides an apparatus or reactor suitable for carrying out the enzymatic hydrolysis of a lignocellulosic biomass substrate, wherein the reactor comprises an off-gas condenser, an agitator, a pH probe, a temperature sensor and a dissolved oxygen probe. In some embodiments, the reactor further comprises means of adjusting such operational conditions as pH, temperature, dissolved oxygen concentrations, etc., such that the saccharification conditions can be maintained at certain preferred levels to insure a successful outcome.

[0033] In certain embodiments, the reactor is loaded with a lignocellulosic biomass material and an enzyme composition, which constitutes a sacharification mixture. In certain embodiments, the saccharification mixture has a total volume of at least 5%, at least 10%, at least 15%, or even at least 20% less than the volume of the reactor. Stated differently, the reactor, after the saccharification mixture is loaded and sufficiently mixed to become a substantially homogenous slurry, has a headspace that is at least about 5%, or at least about 10%, or at least about 15%, or at least about 20% of the total volume of the reactor. [0034] In certain embodiments, the reactor further comprises a gas inlet, which can be placed at or near the headspace of the reactor or at a position that would be

submerged when the saccharification mixture is in the reactor. In some embodiments, a sterile airflow is introduced into the reactor through the gas inlet. In particular embodiments, the sterile airflow is introduced into the reactor at a rate of about 50 to about 400 M³ per hour, for example, at a rate of about 50 M³ per hour to about 350 M³ per hour, or about 100 M³ per hour to about 300 M³ per hour, or about 150 M³ per hour to about 280 M³ per hour.

[0035] In some embodiments, the reactor has a vertical, cylindrical geometry. In some embodiments, the agitator enters the reactor from the top of the reactor, optionally but preferably in a centered location. In certain embodiments, the agitation or mixing effectuated by the agitator creases a downward-flow direction in the center of the biomass-enzyme slurry or suspension, and an upward flow near the wall of the reactor. [0036] 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, in a reactor 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

BRIEF DESCRIPTION OF THE FIGURES

[0037] FIGURE 1 : Time courses of glucose, xylose and arabinose products at varying levels of dissolved oxygen concentrations in the saccharification mixture in accordance with the small, laboratory scale experiment of Example 1 . [0038] FIGURE 2: Dissolved oxygen concentration throughout a large, industrial scale saccharification run in accordance with Example 2. This dissolved oxygen concentration profile was generated during and throughout the saccharification run.

[0039] FIGURE 3: Time courses of glucose, and xylose/arabinose products during saccharification runs. This figure depicts the comparison of the yields of such sugars in a large, industrial scale but poorly aerated reactor, with a small, laboratory scale and fully aerated reactor, as detailed in Example 2. [0040] FIGURE 4: Time course of glucose, and xylose/arabinose products during saccharification runs. This figure depicts the comparison of the yields of such sugars in a large, industrial scale, and well aerated reactor, with a small laboratory scale, fully aerated reactor, and detailed in Example 3. [0041] FIGURE 5: Dissolved oxygen levels in the saccharification mixtures of

Reactors 1 -4 of Example 4.

[0042] FIGURE 6: Time course of glucose, and xylose/arabinose products during the saccharification runs of Reactors 1 -4 of Example 4.

[0043] FIGURE 7: Dissolved oxygen levels in the saccharification mixtures of Reactors 5-8 of Example 5.

[0044] FIGURE 8: Dissolved oxygen levels in a mixture of size-reduced dilute ammonia pretreated corn stover and water over time, in accordance with Example 5.

[0045] FIGURE 9: Dissolved oxygen levels over time in a saccharification mixture prepared with Accellerase® TRIO™ and a whPCS from NREL, in accordance with Example 5.

[0046] FIGURE 10: Dissolved oxygen levels over time in a saccharification mixture prepared with Accellerase ® TRIO™ and an Avicel, in accordance with Example 5.

DETAILED DESCRIPTION I. Overview

[0047] Described is a method of improving the enzymatic saccharification of a lignocellulosic biomass material and increasing the yield of fermentable sugars from such lignocellulosic biomass materials using the same enzyme composition and dose. More specifically the improved method is one that involves the maintenance of dissolved oxygen level in the saccharification mixture and throughout the

saccharification step, in addition to carefully controlling the other conditions which are known to affect saccharification efficacy and efficiency such as pH and temperature. Provided also is a saccharification reactor comprising a dissolved oxygen probe installed in such a way as to enable the measurement and monitoring of dissolved oxygen levels in the saccharification mixture during the saccharification step of the biomass to fermentable sugar process. The reactor is optionally loaded with a volume of the saccharification mixture that is at most about 95% of its total volume so as to leave a sufficiently large head space. Additionally the reactor may further comprises a means for introducing an oxygen flow into the reactor, such that, when the dissolved oxygen level in the saccharification mixture falls below a certain threshold level, new oxygen can be added to the slurry to insure optimal saccharification efficiency and efficacy.

[0048] Specifically the improved method or process of the invention comprises:

(a) a loading step comprising introducing a lignocellulosic biomass material and an enzyme composition into a vessel; (b) a mixing step comprising stirring or agitating the content of the reactor such that the enzyme composition and the lignocellulosic biomass material are sufficiently mixed into a substantially homogenous slurry that is a saccharification mixture; and

(c) a saccharification step comprising incubating the saccharification mixture under conditions that allow the hydrolysis of the biomass material into soluble

fermentable monomeric sugars; wherein the concentration of dissolved oxygen in the saccharification mixture during the saccharification step of (c) is maintained at a level above about 1 .5%, for example, above about 1 .5%, above about 2.5%, above about 5%, above about 10%, above about 20%, or even above about 30%, of the saturating oxygen level of such a saccharification mixture at the saccharification temperature immediately prior to the saccharification step.

[0049] The lignocellulosic biomass material saccharified 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.

[0050] The lignocellulosic biomass material is also preferably pretreated before they are introduced into the reactor via the loading step. In some embodiments, the amount of lignocellulosic biomass material loaded to the reactor is such that the resulting saccharification mixture immediately before the saccharification step has a dry solids weight level of at least about 10%, for example, at least about 10%, at least about 15%, at least about 20%, or even at least about 25%.

[0051] The enzyme composition comprises at least one cellulase. In some

embodiments, the enzyme composition comprises two or more cellulases. In certain embodiments, the enzyme composition further comprises one or more hemicellulases. In further embodiments, the enzyme composition comprises one or more accessory enzymes. In certain embodiments, the enzyme composition is provided to the reactor in an amount that is sufficient to convert at least 40% of the cellulose (glucan) in the lignocellulosic biomass substrate in the saccharification mixture to glucose. In some embodiments, the enzyme composition is provided to the reactor in an amount that is sufficient to convert at least 25% of the hemicelluloses (xylan) in the lignocellulosic biomass substrate in the saccharification mixture to xylose.

[0052] In certain embodiments, the enzyme composition comprises at least one cellulase and at least one hemicellulase. The enzyme composition may be produced by a single microorganism. Or the enzyme composition may be one that is an admixture of various enzymes produced by different microorganism.

[0053] In certain embodiments, the mixing step comprises stirring or agitating the reactor in such a way that the biomass material is sufficiently mixed and evenly exposed to the enzyme composition in the saccharification mixture, which is typically a substantially homogenous slurry.

[0054] In some embodiments, the saccharification step is carried out at a pH of about 3 to about 9, for example, about 3.5 to about 8.5, about 4 to about 8, about 4.5 to about 7.5 or even about 5 to about 6.5. In some embodiments, the pH of the saccharification mixture in the reactor is adjusted by the addition or acid or base as needed, and the pH level of the saccharification mixture is measured and continuously monitored such that, whenever the pH of the saccharification mixture deviates from the preferred range, which is pre-determined based on the pH optima of the enzymes in the enzyme composition, an acid or a base is added to bring the pH back within the desired range. [0055] In some embodiments, the sacharification 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 saccharification step is charried 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 saccharification mixture is measured and continuously monitored such that whenever the temperature of the saccharification 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.

[0056] In some embodiment, the saccharification 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 saccharification 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 saccharification 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 saccharification 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 saccharification 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.

[0057] In some embodiments, the dissolved oxygen level in the saccharification mixture is at least about 1 .5%, preferably at least about 2.5%, or about 5%, or about 7.5%, or at least about 10% of the of the saturation dissolved oxygen level in a given saccharification mixture before the saccharification. The saturation dissolved oxygen can be readily measured using a dissolved oxygen probe, immersed in a

saccharification mixture or slurry prepared via the mixing step, held at the

saccharification temperature, but immediately before the start of the saccharification step, previously calibrated at the same temperature. The level of dissolved oxygen in the saccharification mixture can be monitored continuously throughout the saccharification step, or alternatively, the level of dissolved oxygen in the saccharification mixture can be monitored at pre-determined intervals.

[0058] In certain embodiments, the desired dissolved oxygen level can be maintained by continuously directing a certain flow of oxygen or air into either the headspace of the saccharification reactor or through an aeration inlet that is submerged when the saccharification mixture is placed in the reactor. The headspace is suitably at least 5% of the volume of the reactor, to which preferably is affixed a gas inlet. Alternatively the influx of air or oxygen can be intermittent or as needed, triggered by the detection of a dissolved oxygen level below a certain threshold. The influx of air or oxygen is suitably pre-sterilized to prevent the introduction of contaminating materials such as

microorganisms and other potential contaminants to the saccharification mixture. The speed of the oxygen or air flow through the inlet is suitably adjusted in coordination with the dissolved oxygen readout of the dissolved oxygen probe immersed in the saccharification mixture during the saccharification step. [0059] Another aspect of the invention is an apparatus or reactor comprising at least a gas inlet affixed to the wall of either the headspace or at a position that is submerged when the saccharification mixture is placed in the reactor, a dissolved oxygen probe that is inserted in such a way to allow immersion of the probe in the saccharification mixture/slurry during saccharification, and monitoring and reporting out of dissolved oxygen level in the saccharification mixture continuously or intermittently. Moreover an aeration means is suitably attached to the gas inlet of the reactor, which allows for continuous air or oxygen flow into the reactor/slurry, or for intermittent air or oxygen flow into the reactor/slurry when the dissolved oxygen level in the saccharification mixture drops below a certain threshold level, as reported by the read out of the dissolved oxygen probe.

[0060] In some embodiments, the reactor further comprises a mixing means which can be suitably an agitator with an impeller or a stirrer or a mixer that can otherwise cause effective and even mixing of the saccharificaction mixture/slurry, maintain solids suspension throughout the saccharification step. The mixing means can be kept at a constant mixing speed throughout the saccharification step or it can have varying mixing speeds. [0061] In some embodiments, the reactor further comprises a temperature sensor together with a cooling and a heating means. The temperature sensor is suitably affixed to the reactor in such a way to allow measurement, monitoring and reporting out of the temperature of the saccharification mixture/slurry during the saccharification step. The cooling and/or heating means may be any suitable methodology used in industrial practices involving temperature-controlled fermentation or incubation tanks, such as, for example, running water or other fluids at certain elevated or reduced temperatures to achieve heating or cooling, respectively. The cooling or heating means of the invention is preferably triggered to turn on when the temperature readout from the temperature probe is above or below a predetermined preferred temperature range for the

saccharification step. Typically the preferred temperature range is determined based on the temperature optima of the various enzymes used in the saccharification step, but from time to time, a lower or reduced temperature may be applied to the

saccharification step as a compromise for other process conditions, requirements, and constraints.

[0062] In some embodiments, the reactor further comprises a pH sensor together with certain fluid inlet(s) or sampling outlet(s). The pH sensor can be suitably affixed to the reactor in such a way to allow measurement, monitoring and reporting out of the pH of the saccharification mixture/slurry during the saccharification step. Alternatively the pH of the saccharification can be measured by sampling the saccharification slurry through the sampling outlets. If and when the pH of the saccharification slurry deviates beyond a predetermined preferred range, an amount of acid or base can be introduced to the saccharification slurry through the fluid inlet to bring the pH of the saccharification mixture back into the preferred range. Typically the preferred pH range is determined based on the pH optima of the various enzymes used in the saccharification step.

Prior to the start of the saccharification step, the pH of the slurry is adjusted to be within the preferred pH range. The amount of acid or base required to adjust pH may vary, much dependent on the particular lignocellulosic biomass material and the particular pretreatment method applied to the material.

ii. Definitions [0063] 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.

[0064] 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.

[0065] 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 un-recited 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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).

[0073] 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).

[0074] 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.

[0075] 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.

[0076] 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. 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. [0077] 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.

[0078] The term "pretreated biomass" refers to biomass materials that have been subject to a treatment or pretreatment prior to saccharification.

[0079] In the method or process described herein, or placed in the apparatus or reactor for saccharification, 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 or saccharification. The biomass material may initially, prior to the saccharification 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. [0080] 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.

[0081] 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. [0082] 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 (C₆H ₀O₅)n, representing a polysaccharide consisting of a linear chain of β(1 ->4) linked D-glucose units.

[0083] 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. [0084] 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.

[0085] 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.

[0086] 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. [0087] The term "hydrolysate" refers to the product of saccharification, which contains the sugars produced in the saccharification process or step, the remaining unhydrolyzed biomass, and the enzymes and breakdown products of such enzymes used for saccharification.

[0088] The term "slurry" refers to a mixture of insoluble material and a liquid.

[0089] 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."

[0090] 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).

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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 ai, FEBS Lett. (1998) 425(2): 352-4;

Coutinho and Henrissat, G ENETICS, BIOCHEMISTRY AND ECOLOGY OF CELLULOSE

DEGRADATION, 1999, T. Kimura. Tokyo, Uni Publishers Co: 15-23.).

[0095] 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 ai, (2009) Nucleic Acids Res. 37 (Database issue):D233- 38).

[0096] 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.

[0097] The term "protein", as used herein, includes proteins, polypeptides, and peptides.

[0098] The terms "protein" and "polypeptide" are used interchangeably herein.

[0099] 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.

[00100] 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.

[00101] "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 ai, (1996) Can. J. Microbiol., 42:1 -5).

[00102] 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. [00103] Cellulases" may further refer to complete enzyme systems that comprises not only cellulases but also certain hemicellulases, or any combination thereof. [00104] 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.

[00105] 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, Pure & App. Chem, (1987) 59:257-268.

[00106] 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 II (CBH II) 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 II 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.

[00107] 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 . [00108] 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.

[00109] 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.

[00110] 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.

[00111] 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.

[00112] The term "saccharification enzyme" refers to an enzyme that can catalyze conversion of a component of biomass to fermentable sugars. [00113] The term "microorganism" as used herein refers to any bacterium, yeast, or fungal species. [00114] 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.

[00115] 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.

[00116] 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.

[00117] 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.

[00118] 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.

[00119] 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.

[00120] 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.

Mi. Saccharification of Lignocellulosic Biomass

[00121 ] 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. The invention further provides an improved apparatus or reactor wherein the improved method or process as above can be carried out.

[00122] 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. [00123] 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 saccharification step. Removal of such inhibitors can be costly and cumbersome.

[00124] 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 saccharification 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 lignicellulosic biomass to fuel operation.

iv. Pretreatment

[00125] 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. [00126] 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.

[00127] 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. [00128] 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

[00129] 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. [00130] 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.

[00131] 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 .

[00132] 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.

[00133] Further pretreatment methods can involve the use of hydrogen peroxide H₂0₂. See Gould, 1984, Biotech, and Bioengr. 26:46-52.

[00134] 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., Appl. Biochem.and Biotech. (1999) 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. [00135] 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 .

v. Enzymes [00136] Saccharification enzymes, which also may be referred to as a saccharification enzyme consortium, are used to hydrolyze the biomass 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). [00137] A saccharification enzyme consortium 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 s (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. 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. [00138] Saccharification enzymes may 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

[00139] In addition, saccharification enzymes may be produced biologically, including using recombinant microorganisms. New saccharification enzymes may be developed, which may be used in the present process.

[00140] 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 saccharification of a given pretreatment product under the selected conditions. Preferably, saccharification is performed at or near the pH and temperature optima for the saccharification 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.

[00141] As saccharification 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.

[00142] As additional biomass is added, the pH and temperature are controlled within the preferred ranges while mixing and the saccharification reaction continues. The thorough mixing of the slurry allows control of pH in a narrow range as more biomass is added and acid or base is added to make pH adjustments. The tight pH control may help to improve saccharification enzyme function. The thorough mixing of the slurry allows better control of the temperature of the reactor contents in a narrow range as more biomass is added, which also improves saccharification enzyme function.

[00143] Sources or means of heating or cooling that may be used are well known to one skilled in the art, and may include a jacket on the reactor, internal coils in the reactor, or a heat exchanger through which the reactor contents is pumped. The tight temperature control enhances saccharification by allowing the saccharification to run at the highest temperature possible without overshooting the reactor temperature and thermally inactivating the enzymes. [00144] Additional portions of a saccharification enzyme consortium may optionally be added following one or more new biomass loadings. Each added portion of a

saccharification enzyme consortium may include the same enzymes as in the initially added saccharification enzyme consortium, or it may include a different enzyme mixture. For example, the first added saccharification enzyme consortium may include only or primarily cellulases, while a later added saccharification enzyme consortium may include only or primarily hemicellulases. Any saccharification enzyme consortium loading regime may be used, as determined to be best at saccharifying the specific biomass in the reactor. One skilled in the art can readily determine a useful

saccharification enzyme consortium loading regime, such as is described in the examples herein. vi. Saccharification process conditions

[00145] Liquefaction of biomass results from further saccharification, thereby again reducing biomass slurry viscosity, allowing 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 saccharification. 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.

[00146] 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 saccharification 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 saccharification, prior to addition of pretreated biomass to an existing saccharifying slurry, and/or during saccharification of a slurry.

[00147] Alternatively to providing a fully saccharified hydrolysate product, the saccharification 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).

vi. Fermentation to Produce Cellulosic Fuels or Chemicals

[00148] 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.

[00149] 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 [00150] 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. [00151] 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, Microbiol. Rev. (1986) 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. 5,192,673. 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. 6,358,717. Published international patent applications WO 2007/041269 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 US Patent No.

8,206,970 and published US patent application 20070292927, 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.

[00152] Genetically modified strains of E. coli have also been used as biocatalysts for ethanol production (Underwood et al., Appl. Environ. Microbiol. (2002) 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 US Patent Nos. 7,741 ,1 19 and 7,741 ,084, 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.

[00153] 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. 6,013,494, 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 10 of co- owned US Patent No. 7,781 ,191 , which is herein incorporated by reference.

[00154] Lactic acid has been produced in fermentations by recombinant strains of E. Coli (Zhou et al., Appl. Environ. Microbiol. (2003) 69:399-407), natural strains of Bacillus (US20050250192), and Rhizopus oryzae (Tay and Yang, Biotechnol.

Bioeng. (2002) 80:1 -12). Recombinant strains of E. coli 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., Biotechnol. Prog. (2002) 18:201 -21 1 ). Acetic acid has been made by fermentation using recombinant Clostridia (Cheryan et al., Adv. Appl. Microbiol. (1997) 43:1 -33), and newly identified yeast strains (Freer, World J. Microbiol. Biotechnol. (2002) 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 recombinant E. coli \n Lin et al., Metab. Eng. (2005) 7:1 16-127). Pyruvic acid has been produced by mutant Torulopsis glabrata yeast (Li et al., Appl. Microbiol.

Technol. (2001 ) 55:680-685) and by mutant E. coli (Yokota et al., Biosci. Biotech. Biochem. (1994) 58:2164-2167). Recombinant strains of E. coli have been used as biocatalysts for production of para-hydroxycinnamic acid (US20030170834) and quinic acid (U.S. Patent No. 7,642,083).

[00155] A mutant of Propionibacterium acidipropionici has been used in fermentation to produce propionic acid (Suwannakham and Yang, Biotechnol. Bioeng. (2005) 91 :325- 337), and butyric acid has been made by Clostridium tyrobutyricum (Wu and Yang, Biotechnol. Bioeng. (2003) 82:93-102). Propionate and propanol have been made by fermentation from threonine by Clostridium sp. strain 17cr1 (Janssen, Arch. Microbiol. (2004) 182:482-486). A yeast-like Aureobasidium pullulans has been used to make gluconic acid (Anantassiadis et al., Biotechnol. Bioeng. (2005) 91 :494-501 ), by a mutant of Aspergillis niger (Singh et al., Indian J. Exp. Biol. (2001 ) 39:1 136-43). 5-keto- D-gluconic acid was made by a mutant of Gluconobacter oxydans (Elfari et al., Appl Microbiol. Biotech. (2005) 66:668-674), itaconic acid was produced by mutants of Aspergillus terreus (Reddy and Singh, Bioresour. Technol. (2002) 85:69-71 ), citric acid was produced by a mutant Aspergillus niger strain (Ikram-UI-Haq et al., Bioresour. Technol. (2005) 96:645-648), and xylitol was produced by Candida guilliermondii FTI 20037 (Mussatto and Roberto, J. Appl. Microbiol. (2003) 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.,

Biomacromolecules, (2001 ) 2:45-57). L-2,3-butanediol was made by recombinant E. coli (Ui et al., Lett. Appl. Microbiol. (2004) 39:533-537). [00156] Production of amino acids by fermentation has been accomplished using auxotrophic strains and amino acid analog-resistant strains of Corynebacterium, 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 (1976) 50 (1 ) R79-R87, 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, J. Biosci Bioeng. (2000) 89:87-79. Methionine was produced by a mutant strain of Corynebacterium lilium (Kumar et al, Bioresour. Technol. (2005) 96: 287-294).

[00157] 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). [00158] 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. (1998) 49:639-648, Groot et al., (1992) Process. Biochem. 27:61 -75, 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

[00159] 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 : Dissolved Oxygen (DO) Levels in Saccharification Mixtures at

Laboratory Scale Substantially Affect Yield of Fermentable Sugars.

[00160] Five (5) Biostat B water-jacketed glass fermenter systems (Sartorius) each of 1 .6 L in size were used. Each fermenter was also equipped with an off-gas condenser, a headspace gas inlet, an agitator with a single marine impeller, a pH probe, a temperature sensor and an amperometric dissolved oxygen probe calibrated in air- saturated water at 47 °C.

[00161] Into each of the fermenters, a dilute ammonia pretreated corn stover biomass material was added together with Accellerase® TRIO™ in a fed-batch mode for about 2 hours, starting with an initial 10% dry solids weight mixture. Ultimately in each of the fermenters, the following components were added to make a 25% dry matter weight saccharification mixture: 266.27 g moist dilute ammonia pretreated corn stover prepared in accordance with the method described by Schell et al. DILUTE-SULFURIC

ACID PRETREATMENT OF CORN STOVER IN PILOT-SCALE REACTOR - INVESTIGATION OF YIELDS,

KINETICS, AND ENZYMATIC DIGESTIBILITIES OF SOLIDS. Appl Biochem Biotechnol (2003), 105-108:69-85, which together constitutes 157.5 dry matter weight, 31 1 .93 g water, 39.2 g of 1 N sulfuric acid, 31 .5 mg Lactrol® (an antibiotic) PhiBroChem), and 12.60 g enzyme solution of Accellerase® TRIO™.

[00162] Mixing started at a speed sufficient to maintain the suspension of the solids in a substantially homogenous slurry. Some amounts of a 1 N Sulfuric acid was added to adjust the pH of the saccharification mixture to 5.3. Warm water was coursed through the jacket of the fermenters to raise the temperatures in the saccharification mixture to about 47 °C. [00163] Ten (10) hours after the addition of enzymes different gas streams were directed to the headspace gas inlets of the fermenters. The gases were ambient air, nitrogen, and blends of the ambient air and nitrogen in various proportions as described below. The gas proportions and gas flow rates were adjusted to achieve a set of time- weighted average dissolved oxygen concentrations in the saccharification mixtures of 0.3%, 13%, 49%, 78% and 92% of the saturation dissolved oxygen level, respectively. The dissolved oxygen concentrations were maintained throughout the remainder of the saccharification step until it is terminated at 70.1 hours.

[00164] Hydrolysate samples were taken at intervals during the saccharification step above. Each of the hydrolysate samples was immediately separately into solids and clarified liquids. The clarified liquids were each diluted 10x with water and its content was analyzed by high performance liquid chromatography (Agilent HPLC, with a Bio- Rad Aminex HPX-87H column, using 0.01 N sulfuric acid as eluent, at 55oC column temperature, and using a refractive index detector (Agilent), precalibrated with glucose, xylose and arabinose standard solutions.

[00165] The fermentable sugar levels in the hydrolysates produced at different dissolved oxygen levels were determined and plotted as shown in FIGURE 1 .

[00166] It was observed that glucose release was significantly lower when the saccharification mixture had a 0.3% dissolved oxygen concentration. When the dissolved oxygen levels in the saccharification mixtures increased to above 13%, the glucose release kinetics were significantly higher than that observed at 0.3% dissolved oxygen concentration, but indistinguishable from each other.

[00167] A slight reduction of xylose release was also observed when the

saccharification mixture had a low 0.3% dissolved oxygen concentration. At DO levels above 13%, xylose release was faster but the levels of xylose release were

indistinguishable from each other when the level of DO reaches 13% and above.

[00168] No significant difference in arabinose release was observed at the different saccharification mixture DO levels tested.

Example 2: Effects of Depleted Dissolved Oxygen Concentration in

Saccharification Mixtures [00169] Millimeter-sized particles of corn stover were thermo-chemically pretreated using water and ammonia in a steam and air environment. The pretreated corn stover was enzymatically saccharified in a reactor equipped with process controls including controls and monitoring of temperature, pH, agitation intensity, head pressure and sterile air-flow rate through the vessel's head space. The rector was equipped also with a dissolved oxygen probe.

[00170] For this example, the reactor used had a vertical, cylindrical geometry with the shaft of the agitation means entering the vessel from the top of the vessel at a centered location on the head plate of the reactor. During the mixing step, the agitation produced a downward flow direction in the center of the biomass solids suspension formed with the enzyme solution, and an upward flow near the walls of the vessel.

[00171] The reactor was further equipped with an external, pumped, re-circulation loop containing a heat exchanger for temperature control.

[00172] The reactor had a total volume of about 90 M³. The batch volume used for this example was about 65 M³ after the addition of all components, including water, pretreated corn stover, acid and enzymes.

[00173] The process operated in a fed-batch mode wherein the pretreated corn stover was gradually added to the water heel while enzymatic saccharification was on-going. After all components were added to the reactor, at about 18 hours of batch

saccharification process, a sample of the suspension was retrieved and transferred to an Erlenmeyer flask, then placed on a temperature-controlled, laboratory shaker table to enable the testing of saccharification performance in parallel at both a large, industrial scale, and a small, benchtop laboratory scale.

[00174] The saccharification temperature was maintained at 47 °C in both the large, industrial scale saccharification reaction, as well as the small, laboratory scale saccharification reaction. The sterile air flow was maintained in a range of 50 to 100 M³/hour at standard pressure and temperature (STP) 0 °C and 1 atm pressure

[00175] After all pretreated solids were added to the reactor, the dissolved oxygen concentration was measured. The dissolved oxygen concentration was monitored throughout the batch and presented in FIGURE 2, and it was observed that the dissolved oxygen concentration substantially decreased from a near saturation level of about 7,000 ppb to about 0 ppb. [00176] The resulting fermentable sugar concentrations were measured from the hydrolysates of both the large scale and the small scale saccharification reactors, using liquid chromatography. The results are depicted in FIGURE 3. As saccharification progressed the glucose yield in the laboratory scale reactor exceeds the glucose yield in the large, industrial scale reactor.

Example 3: Maintenance of Dissolved Oxygen Concentrations in Saccharification Mixtures

[00177] The same experimental conditions of Example 2 (above) were used for this experiment under Example 3, with the exception that the sterile air flow rates into the large, industrial scale reactor and the small, laboratory scale reactor were maintained at 250 M³/hour at STP. The dissolved oxygen concentrations were measured throughout the saccharification step, and it was determined that at this sterile air flow, the dissolved oxygen concentration in the saccharification mixture never dropped below about 4,800 ppb.

[00178] The yields of fermentable sugars were measured from the resulting

hydrolysates from the large, industrial scale reactor and the small, laboratory scale reactor, using liquid chromatography. The results are reported in FIGURE 4, which indicated no significant decrease of glucose, xylose or arabinose yields from the saccharification reactions carried out at the small laboratory scale.

Example 4: Optimization of Saccharification Reactor Design Parameters for High Dissolved Oxygen Concentrations in the Saccharification Mixture

[00179] In this experiment, a mini bioreactor system was designed to accommodate the requirement for good mixing, small but representative working volumes, good

temperature, pH, and dissolved oxygen level controls. To satisfy these purposes, certain box shaped glass bioreactors having working volumes of about 60 ml_ to 250 ml_ were designed, equipped with overhead mixing, standard probes for precise measurements and controls of temperatures, pH, and dissolved oxygen levels. A controlled gas flow system was also attached allowing complete mass flow-controlled mixing of individual gases including, for example, ambient air (optionally sterilized), 0₂, C0₂, and nitrogen. The inlet of these gases can be placed at either the head space or submerged. The reactor was also designed to have off-gas analysis capabilities measuring 0₂ and C0₂.

[00180] For the first 24 hours, a previously size-reduced dilute ammonia pretreated corn stover substrate, was mixed with certain amounts of Accellerase® TRIO™, in a fed batch mode, to generate a well-liquefied saccharification mixture. Two hundred and fifty (250) mg of such a mixture was then transferred into each of the reactors of this experiment, giving about 100 ml_ of headspace in such a reactor. Reactor 1 was sealed from external environment. Reactors 2-4 each had constant flow of air through their respective headspaces, at the speed of 6 L/hour, 3 L/h, or 12 L/h.

[00181] During the saccharification step, dissolved oxygen levels of each of these reactors were monitored, as shown in FIGURE 5, and it was observed that in Reactor 1 , the dissolved oxygen concentration in the saccharification mixture/slurry dropped to below about 4% of the saturation level in about 31 hours. In Reactors 2-4, a high, near saturation dissolved oxygen level was observed throughout the process aside from the initial 2 hours of saccharification (when the system was potentially still in flux before gaining equilibrium).

[00182] During saccharification, and at various intervals, samples were taken out of the saccharification mixture. Measurements of soluble fermentable sugars were carried out and the results are in FIGURE 6.

Example 5: What causes the depletion of dissolved oxygen?

[00183] In this experiment, the same box reactors as described in Example 4 (above) were used to determine if it was certain enzymatic oxygenase components or the pretreated biomass that caused the depletion of dissolved oxygen in the

saccharification mixture.

[00184] The same size-reduced and pretreated corn stover biomass as described above was used. Two (2) enzyme mixtures were prepared, each comprising the same levels of Trichoderma reesei CBH1 , CBH2, EG1 , EG2, beta-glucosidase 1 , xylanases, plus a beta-xylosidase and an arabinofuranosidase from Fusarium vertiticillioides, wherein the first enzyme mixture also comprises a certain amount of a T. reesei GH61 enzyme, but the second enzyme mixture does not. Enzyme mixtures 1 and 2 were used to dose the saccharification mixtures, and the each of the reactors was loaded with 250 g of saccharification mixture leaving about 100 ml_ headspace.

[00185] Reactor 5 was dosed with Enzyme 1 , and was sealed. [00186] Reactor 6 was dosed with Enzyme 2, and was also sealed.

[00187] Reactors 7 was dosed with Enzyme 2, but with addition of 1 mg of T. reesei GH61 enzyme; the reactor was also sealed.

[00188] Reactor 8 was dosed with Enzyme 2, which had been previously heat inactivated; and the reactor was also sealed. [00189] The levels of dissolved oxygen concentrations in those reactors were monitored and shown in FIGURE 7. It was apparent that the T. reesei GH61 enzyme contributes only to a limited extent to the depletion of dissolved oxygen in the

saccharification mixtures.

[00190] In order to determine whether the pretreated biomass substrates caused the depletion of dissolved oxygen, Accellerase® TRIO™ was used to saccharify a dilute acid pretreated corn stover biomass obtained from NREL (Schell DJ et al., DILUTE-

SULFURIC ACID PRETREATMENT OF CORN STOVER IN PILOT-SCALE REACTOR - INVESTIGATION

OF YIELDS, KINETICS, AND ENZYMATIC DIGESTIBILITIES OF SOLI DS. Appl Biochem Biotechnol (2003) 105-108:69-85), as well as an Avicel (Sigma-Aldrich) in a reactor that is sealed just like Reactor 6 above. In another reactor, the dilute ammonia pretreated corn stover biomass was mixed with water (without Accellerase ® TRIO™). It was observed that, even without enzymes, the dilute ammonia pretreated corn stover biomass consumes and depletes the dissolved oxygen. See, FIGURE 8. It was observed that the whPCS substrate depletes oxygen, similarly to what was observed above in the experiment involving dilute ammonia pretreated corn stover. See, FIGURE 9. Avicel, on the other hand, does not deplete dissolved oxygen in the saccharification mixture. See, FIGURE 10.

[00191 ] 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.

[00192] 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 . A method of hydrolyzing a lignocellulosic biomass material comprising:

(b) a mixing step comprising stirring or agitating the content of the reactor such that the enzyme composition and the lignocellulosic biomass material are sufficiently mixed into a substantially homogenous slurry that is a saccharification mixture; and

(c) a saccharification step comprising incubating the saccharification mixture under conditions that allow the hydrolysis of biomass materials into soluble fermentable sugars; wherein the level of dissolved oxygen in the saccharification mixture is maintained at a level of above 1 .5% of the saturation dissolved oxygen level during the saccharification step.

2. The method of claim 1 wherein the lignocellulosic biomass material has been pretreated prior to the loading step.

3. The method of claim 2, wherein the pretreatment step is one selected from: (a) a mechanical pretreatment; (b) an acidic pretreatment; (c) an alkaline

pretreatment; (d) a steam, heat, pressure related pretreatment; (e) a

cryopretreatment; or (f) a combination of two or more of (a) to (e).

4. The method of any one of claims 1 -3, wherein the enzyme composition

comprises a cellulase.

5. The method of any one of claims 1 -4, wherein the enzyme composition

comprises two or more cellulases.

6. The method of any one of claims 1 -5, wherein the enzyme composition further comprises a hemicellulase.

7. The method of any one of claims 1 -6, wherein the enzyme composition further comprises an accessory enzyme.

8. The method of any one of claims 1 -7, wherein the lignocellulosic biomass material comprises at least 3 wt.% of lignin, referencing the total polymeric carbohydrates in the lignocellulosic biomass material.

9. The method of any one of claims 1 -8, wherein the dry weight of lignocellulosic biomass material in the saccharification mixture is at least 10%.

10. The method of any one of claims 1 -9, wherein the dry weight of the

lignocellulosic biomass material in the saccharification mixture is at least 15%.

1 1 . The method of any one of claims 1 -10, wherein the saccharification step is

carried out at a pH of about 3 to about 9.

12. The method of claim 1 1 , wherein the sacchaffication step is carried out at a pH of about 4 to about 8.

13. The method of any one of claims 1 -12, wherein the sacchafication step is carried out at a temperature of about 25°C to about 65°C.

14. The method of claim 13, wherein the saccharification step is carried out at a

temperature of about 35°C to about 60°C.

15. The method of any one of claims 1 -14, wherein the sacchafification is carried out for a period of 2 hours to about 120 hours.

16. The method of claim 15, wherein the saccharification step is carried out for a period of 24 hours to about 96 hours.

17. The method of any one of claims 1 -16, wherein the dissolved oxygen level in the sacchafication mixture is at least about 1 .5% of the saturation dissolved oxygen level.

18. The method of claim 17, wherein the dissolved oxygen level in the

saccharification mixture is at least about 5% of the saturation dissolved oxygen level.

19. The method of claim 18, wherein the dissolved oxygen level in the

saccharification mixture is at least about 10% of the saturation dissolved oxygen level.

20. An apparatus useful for carrying out a lignocellulosic biomass enzymatic

hydrolysis process comprising a gas inlet, and a dissolved oxygen probe.

21 . The apparatus of claim 20, whereby an aeration means is attached to the gas inlet.

22. The apparatus of claim 20 or 21 , further comprising a mixing means selected from an agitator, a stirrer, or a mixer.

23. The apparatus of any one of claims 20-22, further comprising a temperature sensor and a cooling and/or heating means.

24. The apparatus of any one of claims 20-23, further comprising a pH sensor and a fluid inlet.