NZ724310B2 - Processing Biomass - Google Patents

Abstract

Disclosed is a method of saccharifying biomass, the method comprising: saccharifying recalcitrance-reduced lignocellulosic biomass with a cellulase in the presence of an isomerization agent (a strong acid) to obtain saccharified biomass, the isomerization agent reducing at least some feedback inhibition of the cellulase during saccharification, and wherein the recalcitrance-reduced lignocellulosic biomass has been pre-treated with a method of pre-treatment comprising bombardment with electrons. The method includes a way of avoiding feedback inhibition during the production of useful products. In a particular embodiment, the isomerization agent is polystyrene sulfonic acid or xylose isomerase. tion of the cellulase during saccharification, and wherein the recalcitrance-reduced lignocellulosic biomass has been pre-treated with a method of pre-treatment comprising bombardment with electrons. The method includes a way of avoiding feedback inhibition during the production of useful products. In a particular embodiment, the isomerization agent is polystyrene sulfonic acid or xylose isomerase.

Description

S FOR SACCHARIFYING BIOMASS by Marshall , Thomas Craig Masterman, Michael W. Finn CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application Nos. 61/579,552 and 61/579,559, both filed on December 22, 2011. The entire disclosures of the above applications are incorporated herein by reference.

FIELD OF THE INVENTION The invention pertains to efficiencies useful in the sing of biomass materials. For example, the invention relates to processes that circumvent negative feedback of enzymatic reactions.

BACKGROUND As demand for eum increases, so too does interest in renewable ocks for manufacturing biofuels and biochemicals. The use of lignocellulosic biomass as a feedstock for such manufacturing processes has been studied since the 1970s. Lignocellulosic biomass is attractive because it is abundant, renewable, domestically produced, and does not compete with food industry uses.

Many potential lignocellulosic feedstocks are available today, including agricultural es, woody biomass, municipal waste, ds/cakes and sea weeds, to name a few. At present these materials are either used as animal feed, biocompost als, are burned in a cogeneration facility or are landfilled.

Lignocellulosic biomass is itrant to degradation as the plant cell walls have a structure that is rigid and compact. The structure comprises crystalline ose fibrils embedded in a hemicellulose matrix, surrounded by lignin. This compact matrix is difficult to access by enzymes and other chemical, biochemical and biological processes. Cellulosic biomass materials (e.g., biomass al from which substantially all the lignin has been removed) can be more accessible to enzymes and other conversion processes, but even so, naturally-occurring cellulosic materials often have low yields (relative to theoretical yields) when contacted with yzing enzymes. ellulosic s is even more itrant to enzyme attack. Furthermore, each type of lignocellulosic biomass has its own specific composition of cellulose, hemicellulose and lignin.

While a number ofmethods have been tried to extract structural carbohydrates from lignocellulosic biomass, they are either are too expensive, produce too low a yield, leave undesirable chemicals in the resulting product, or simply degrade the sugars. ccharides from renewable biomass sources could become the basis of chemical and fuels industries by replacing, supplementing or substituting petroleum and other fossil feedstocks. However, techniques need to be developed that will make these monosaccharides available in large quantities and at acceptable purities and prices.

SUMMARY OF THE ION Provided herein are methods of increasing the efficiency of saccharification of biomass. In particular, efficiencies can be achieved by avoiding negative feedback inhibition of enzymatic reactions.

Provided herein is a method of making a product, where the method includes: saccharifying itrance-reduced lignocellulosic biomass, and adding an isomerization agent to the saccharified biomass. In some implementations, the saccharified biomass comprises a first sugar and a second sugar and the isomerization agent is used to convert the second sugar to a third sugar. The method may also e, in some cases, contacting the saccharified biomass with a microorganism to convert the first sugar and third sugar to one or more product(s).

Also provided herein is a method of making a t with a microorganism from a first sugar and a second sugar, where the rganism can convert the first sugar to the product, but cannot metabolize the second sugar, and where the method includes: providing a cellulosic or lignocellulosic biomass; saccharifying the biomass to make a saccharified biomass, wherein the saccharified s comprises a first sugar and a second sugar; providing a microorganism that is capable of converting the first sugar into a product, but wherein the microorganism cannot metabolize the second sugar; ing the microorganism and the saccharified biomass, thereby producing a microorganism-biomass combination; maintaining the microorganism-biomass combination under conditions that enable the microorganism to convert the first sugar to the product, producing a combination that comprises the product and the second sugar; converting the second sugar to a third sugar, wherein the microorganism is capable of converting the third sugar to the product; and maintaining the microorganism under conditions that enable the microorganism to convert the third sugar to the product; thereby making a t with a microorganism from the first sugar and the second sugar.

In r aspect, the invention features a method of increasing the amount of a product made by a microorganism from a saccharified s, the method comprising: providing a cellulosic or lignocellulosic biomass; saccharifying the biomass to make a rified biomass, wherein the saccharified biomass comprises a first sugar and a second sugar; providing a microorganism that is capable of converting the first sugar into a product, but wherein the microorganism cannot metabolize the second sugar; combining the microorganism and the saccharified biomass, thereby producing a microorganism-biomass combination; maintaining the microorganism-biomass combination under conditions that enable the microorganism to t the first sugar to the product, producing a combination that comprises the product and the second sugar; converting the second sugar to a third sugar, wherein the rganism is capable of converting the third sugar to the product; and maintaining the microorganism under conditions that enable the microorganism to t the third sugar to the product; thereby increasing the amount of the product made by the microorganism from the saccharified biomass. [0011B] In another aspect, the present invention provides a method of producing glucose, xylose, and fructose, the method comprising: saccharifying recalcitrance-reduced lignocellulosic biomass with one or more cellulases and an acid on a support in the presence of xylose isomerase at between 30 °C and 65 °C to produce a mixture comprising glucose, se, and xylose.

In any of the methods provided herein, the lignocellulosic biomass can be treated to reduce its recalcitrance to saccharification. The treatment method is selected from the group ting of: bombardment with electrons, sonication, ion, pyrolysis, steam explosion, chemical treatment, mechanical treatment, or freeze grinding. The ent method can be dment with electrons.

In any of the methods, the conversion of the second sugar to the third sugar can be done before maintaining the microorganism-biomass combination under ions that enable the microorganism to convert the first sugar to the product.

The sion of the second sugar to the third sugar can be done immediately after saccharification of the biomass, or it can be done during saccharification of the biomass.

In the methods provided herein, the lignocellulosic biomass can be selected from the group consisting of: wood, particle board, forestry wastes, sawdust, aspen wood, wood chips, [Text continued on page 4] grasses, switchgrass, miscanthus, cord grass, reed canary grass, grain residues, rice hulls, oat hulls, wheat chaff, barley hulls, agricultural waste, silage, canola straw, wheat straw, barley straw, oat straw, rice straw, jute, hemp, flax, bamboo, sisal, abaca, corn cobs, corn stover, soybean stover, corn fiber, alfalfa, hay, coconut hair, sugar processing residues, bagasse, beet pulp, agave bagasse, algae, seaweed, manure, sewage, offal, agricultural or rial waste, arracacha, buckwheat, banana, barley, cassava, kudzu, oca, sago, sorghum, potato, sweet potato, taro, yams, beans, favas, lentils, peas, or mixtures of any of these. The lignocellulosic biomass can be mechanically treated to reduce its bulk density and/or increase its e area. For instance, it can be comminuted, e.g., by dry g, or by wet milling. The s can be saccharified with one or more cellulases.

In the methods provided herein, the ization agent can comprise an acid, e.g., polystyrene sulfonic acid.

In the methods provided herein, the microorganism-biomass combination can be maintained at a pH of about 10 to about 14, or at a pH of about 11 to about 13. It can be maintained at a temperature of about 10°C to about 300C, or at a temperature of about 20°C. It can also be maintained at a temperature of about 60°C to about 65°C. It can be maintained at a pH of about 6.0 to about 7.5, or a pH of about 7.

In the methods, the second sugar can be glucose, and the third sugar can be fructose.

The isomerization agent can comprise an enzyme. Alternatively, the second sugar can be xylose, and the third sugar can be se. The enzyme can be xylose isomerase.

The microorganism can be yeast. The product can be alcohol. The microorganism can be z’dz’um spp., and the product can be l, butanol, butyric acid, acetic acid, or acetone. The microorganism can be acillus spp., and the t can be lactic acid.

It Should be understood that this invention is not limited to the embodiments disclosed in this Summary, and it is intended to cover modifications that are within the spirit and scope of the invention, as defined by the claims.

BRIEF DESCRIPTION OF THE DRAWINGS The foregoing will be apparent from the following more ular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the t invention. is a diagram illustrating the enzymatic ysis of cellulose to glucose.

Cellulosic substrate (A) is converted by llulase (i) to cellulose (B), which is converted by exocellulase (ii) to cellobiose (C), which is ted to glucose (D) by cellobiase (beta- glucosidase) (iii). is a flow diagram illustrating the action of ase on cellulose and cellulose derivatives. Cellulose (200) is broken down to cellobiose (210) by endoglucanases and exo- glucanases/cellobiohydrolases (205) (A), which is then broken down by beta-glucosidase (215) to glucose (220) (B). Endoglucanases and exo—glucanases/cellobiohydrolases are directly inhibited by cellobiose (210) (D) and e (E), and beta-glucosidase is inhibited by glucose (C). is a flow diagram illustrating the conversion of biomass (300) to a product (340). The feedstock (300) is combined (A) with cellulase (305) and fluid to form a e (310), which is then allowed to saccharify (B), producing sugars (320). As sed herein, an additive (325) is combined (C) with the mixture of sugars (320) to make a mixture of sugars and ve (330). The resulting sugars are then used (D) in ream processing to produce one or more products (340), such as alcohol, lactic acid, or one or more of the sugars themselves.

DETAILED DESCRIPTION ed herein are methods of increasing the efficiency of production of sugars (and/or products made from the sugars) from saccharified biomass. The methods are especially useful in cases where one or more sugars or products cause negative feedback, limiting the amount of sugars or products that can be produced.

Typically, the methods begin with saccharifying a biomass. Saccharification usually produces a mixture of sugars. The mixture includes sugars that can be converted to a useful product. However, the mixture of sugars can include sugars that cannot be metabolized by the microorganism. As these non-utilizable sugars increase in concentration, they represent a metabolic “dead-end.” rmore, some sugars may form the basis of feedback inhibition, and limit the throughput of metabolic pathways that make desired sugars or other desired products.

Disclosed herein are methods for preventing such feedback inhibition, and increasing the amount of sugars and other useful products from the saccharification of biomass.

The glucose produced during sacchariflcation can inhibit further production of glucose. In one embodiment, therefore, the invention encompasses the effective removal of glucose by converting it to fructose (which does not inhibit saccharification), thereby allowing for the production of additional glucose. Glucose can be converted to fructose by the action of enzymes (such as xylose isomerase), strong acids or chemicals (such as polystyrene sulfonic acid). Likewise, xylose, which cannot be metabolized by many microorganisms, can be converted by xylose isomerase into xylulose, which can be metabolized by many microorganisms. In addition, se often does not inhibit its own production, unlike glucose.

For ce, biomass can be saccharified to produce a mixture of sugars, including glucose and xylose. Most yeast strains can metabolize glucose, e.g., to an alcohol, but not xylose. Therefore, if the desired end product is alcohol, then increased saccharification, and increased production of glucose, followed by fermentation, will produce more alcohol, but it will also produce more . While the xylose is not harmful, it can represent a metabolic “dead end.” If the xylose is converted to xylulose, it can be fermented to alcohol, and production efficiency can be increased.

As shown in FIG. I, for example, during saccharification a cellulosic ate (A) is initially hydrolyzed by endoglucanases (i) at random locations producing oligomeric intermediates (e.g, cellulose) (B). These intermediates are then substrates for exo-splitting glucanases (ii) such as cellobiohydrolase to produce cellobiose from the ends of the cellulose polymer. Cellobiose is a soluble nked dimer of glucose. Finally cellobiase (iii) cleaves cellobiose (C) to yield glucose (D). Therefore, the endoglucanases are particularly effective in ing the crystalline portions of ose and sing the effectiveness of exocellulases to produce cellobiose, which then es the specificity of the cellobiose to produce glucose. ore, it is evident that depending on the nature and structure of the cellulosic substrate, the amount and type of the three different enzymes may need to be d.

As shown in hydrolysis of cellulose (200) to cellobiose (210) is a step process which includes initial breakdown at the liquid interface via the istic action of ucanases (EG) and exo-glucanases/cellobiohydrolases (CBH) (205) (A). This l degradation is accompanied by further liquid phase degradation, by hydrolysis of soluble intermediate products such as oligosaccharides and cellobiose that are catalytically cleaved by beta-glucosidase (BG; 215) (B) to e (220). r, cellobiose (210) directly inhibits (D) both CBH and EG (205), and glucose (220) directly inhibits (C, E) not only BG (215), but also CBH and EG (205). The invention as described herein reduces or avoids this inhibition. shows a s for manufacturing a product (340) from a feedstock (300).

The ock can be pre-processed, such as by reduction of the size and recalcitrance of the feedstock. This can include, for example, optionally mechanically treating the feedstock and, before and/or after this treatment, optionally treating the feedstock with another treatment, for example, particle bombardment, to further reduce its itrance. The up-stream processed feedstock (300) is then combined (A) with cellulase (305) and fluid to form a mixture (310), which is then allowed to saccharify (B), producing sugars (320). As disclosed herein, an additive (325) is combined (C) with the mixture of sugars (320) to make a mixture of sugars and additive (330). The additive (325) increases the effectiveness of the cellulase during saccharification, e.g., by reducing inhibition of the cellulase by cellobiose and/or glucose. This increased effectiveness of saccharification results in increased levels of sugars, which are then used (D) in downstream processing to produce one or more products (340), such as alcohol, lactic acid, or one or more of the sugars themselves.

During rification, the ock is treated with one or more olytic enzymes, generally by combining the feedstock and the enzyme (305) in a fluid , e.g., an aqueous solution. In some cases, the feedstock is boiled, steeped, or cooked in hot water prior to saccharification, as described in US. Pat. App. Pub. 2012/0100577 A1, filed r 18, 2011 and published April 26, 2012, the entire contents of which are incorporated herein by reference.

The ve can be added at the initiation of the saccharification (B), for example, with the biomass and cellulase. Alternatively, the ve can be added after some or all of the saccharification (B) has occurred. It can also be added at the start of producing a product.

The additive can be a chemical or an enzyme. Examples of suitable additives include acids and bases. Bases can catalyze the Lobry-de-Bruyn-Alberda-van-Ekenstein transformation, as described in more detail below. Acids can catalyze the hydrolysis of iose. Boronic acids can be used to complex with the ols of glucose. Xylose isomerase (a.k.a. glucose isomerase) can be used to isomerize glucose to fructose.

The additive can be physically supported. Useful supports include but are not limited to cationic polymeric supports, anionic polymeric supports, neutral polymeric supports, metal oxide supports, metal carbonate supports, metal halide supports and/or es thereof. The support can be added to the mixed sugars or can be stationary and the mixed sugars made to pass through or over the supported additive.

The mixture containing the additive (330) can be ed to the biomass and ase stage (310) to release more sugars before being further processed. This can include returning the conditions to a state that preferably causes the saccharification of cellulose rather than conditions that favor the action of the additive. For example the pH can be optimized for saccharification in the acidic region (less than or equal to pH 7, less than or equal to pH 6, less than or equal to pH 5) and greater than or equal to pH 2 (greater than or equal to pH 3, greater than or equal to pH 4). The temperature can be optimized for the action of cellulases, e.g, to greater than or equal to 30°C (greater than or equal to 40°C, greater than or equal to 50°C, greater than or equal to 60°C) and less than or equal to 90°C (less than or equal to 80°C, less than or equal to 70°C, less than or equal to 60°C). Additional biomass, cellulase and additive can ally be added for sed production of sugars.

The sugar solution or suspension produced by saccharification can be subjected to downstream processing to obtain a desired product. For e, one or more of the sugars can be isolated, and/or the solution can be fermented. When fermentation is utilized, the fermentation product can be distilled. For example, sugars can be hydrogenated and sugar ls isolated.

Without being bound by any particular theory, it is believed that this conversion effectively removes glucose from the mix of sugars. As shown in this removal would remove the inhibition steps C and E. This increases the overall saccharification of ose in the biomass.

In many ces, the m temperature for using glucose isomerase ranges from 60 to 80°C. In the processes described , temperatures lower than the m may be preferred because of cost and because the optimum temperature for other components of the process can be different. For example cellulase activities are generally optimal between 30°C and 65°C. A ature range of about 60°C to about 65°C may therefore be preferred, particularly if the glucose isomerase and cellulase are combined and used aneously. If they are not used together, then optimal temperatures for each enzyme can be selected.

The optimum pH range for glucose isomerase activity is between pH 7 and 9. As with the selection of the temperature range, in practicing this invention a lower pH can be preferred e in some cases other components of the process may require a lower pH. For example, cellulases are active over a range ofpH of about 3 to 7. The preferred pH for the combined enzymes is therefore generally at or below pH 7. If the glucose isomerase and cellulase are not used together, then the optimal pH range for each enzyme can be selected.

Glucose isomerase can be added in any amount. For example, the concentration may be below about 500 U/g of cellulose (lower than or equal to 100 U/g cellulose, lower than or equal to 50 U/g cellulose, lower than or equal to 10 U/g cellulose, lower than or equal to 5 U/g cellulose). The concentration can be at least about 0.1 U/g cellulose to about 500 U/g ose, at least about 0.5 U/g cellulose to about 250 U/g cellulose, at least about 1 U/g cellulose to about 100 U/g cellulose, at least about 2 U/g ose to about 50 U/g cellulose.

In some cases, the addition of a glucose isomerase increases the amount of sugars produced by at least 5 % (e.g., at least 10 %, at least 15 %, at least 20 %, at least 30, 40, 50, 60, 70, 80, 90, 100 %).

Another additive that can be used in the invention is, e.g., a al that increases the ty of the saccharifying agent. The chemical can be, for example, a chemical that facilitates the Lobry-de-Bruyn-van-Alberda-van-Ekenstein transformation (also called the Lobry—de-Bruyn—van-Ekenstein transformation). This reaction forms an enol from an aldose which can then form a ketose. For example, in the pH range of 11 to 13 and at a temperature of °C, alkali will catalyze the transformation of ose into D—fructose and D-mannose.

Typically the reaction is base catalyzed, but it can also be acid catalyzed, or take place under neutral conditions. As with the use of glucose isomerase, this reaction ively removes glucose.

As r example, an acid can be used to catalyze hydrolysis of cellobiose. By using chemical means to cleave cellobiose to glucose, rather than enzymatic or microbial means, tion of these reactions by glucose does not occur.

In another example, the chemical can be one that reacts with glucose, such as a boronic acid which binds entially to cis-diols.

WO 96700 The chemical can be on a support, for example, by polystyrene ates (such as an ystTM) or polystyrene amines. The mixed sugars can be passed through the ted chemical or flow over it. For example, the chemical can be a polystyrene supported boronic acid. The glucose can be trapped as a borate by the polystyrene support and then released at a later stage, by addition of base for example.

XYLOSE ISOMERASE Xylose isomerase (ES 5) is an enzyme the catalyzes the chemical reaction back and forth between D-xylose and D-xylulose. It is also known systematically as glucose isomerase and D-xylose aldose-ketose isomerase, and belongs to a family of isomerases, specifically those olecular oxidoreductases interconverting aldoses and ketoses. Other names in common use include D—xylose isomerase, D-xylose ketoisomerase, and D—xylose ketol- isomerase. The enzyme participates in pentose and glucuronate interconversions and fructose and mannose metabolism. It is used industrially to convert glucose to fructose in the cture of ructose corn syrup. It is sometimes referred to as “glucose isomerase.” “Xylose isomerase” and “glucose isomerase” are used interchangeably herein. In vitro, glucose isomerase catalyzes the interconversion of glucose and fructose. In vivo, it catalyzes the interconversion of xylose and xylulose.

Several types of enzymes are considered xylose isomerases. The first kind is produced from Pseudomonas hydrophila. This enzyme has 160 times lower affinity to e than xylose but nonetheless is useful for increasing the amount of fructose in the presence of glucose. A second kind of enzyme is found in Escherichia intermedia. This enzyme is a phophoglucose isomerase (EC 5.3.1.9) and can ize phorylated sugar only in the ce of arsenate. A glucose isomerase (EC 5.3.16) can be isolated from Bacillus megaterz'um AI and is NAD linked and is c to glucose. Another glucose isomerase having r activity is isolated from Paracolobacterz'um aerogenoz’des. Glucose isomerases produced by heterolactic acid bacteria require xylose as an inducer and are relatively unstable at high temperature. The xylose isomerase (EC 5.3.1.5) is the most useful for commercial applications as it does not require expensive cofactors such as NAD+ or ATP and it is relatively heat stable.

The glucose isomerases are usually produced intercellularly but reports of extracellular secretion of glucose isomerases are known. The enzyme used can be isolated from many bacteria including but not limited to: Actinomyces olivocinereus, Actinomyces phaeochromogenes, Actinoplanes missouriensis, Aerobacter aerogenes,Aerobacter cloacae, Aerobacter levanicum, Arthrobacter spp., Bacillus stearothermophilus, Bacillus megabacterium, us coagulans, Bifidobacterium spp., Brevibacterium incertum, acterium pentosoaminoacidicum, Chainia spp., bacterium spp., Cortobacterium helvolum, ichiafreuna’ii, ichia intermedia, Escherichia coli, Flavobacterium arborescens, Flavobacterium devorans, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillusfermenti, Lactobacillus marmitopoeus, Lactobacillus gayonii, Lactobacillus rum, Lactobacillus lycopersici, Lactobacillus pentosus, Leuconostoc mesenteroides, Microbispora rosea, Microellobosporiaflavea, Micromonospora coerula, Mycobacterium spp., ia asteroides, Nocardia corallia, Nocardia dassonvillei, Paracolobacterium aerogenoides, Pseudonocardia spp., Pseudomonas hila, Sarcirza spp., Staphylococcus bibila, Staphylococcusflavovirerzs, Staphylococcus echinatus, Streptococcus achromogenes, Streptococcus phaeochromogenes, Streptococcusfracliae, Streptococcus roseochromogeries, ococcus olivaceus, Streptococcus californicos, Streptococcus venuceus, Streptococcus virginial, Streptomyces olivochromogenes, Streptococcus venezaelie, Streptococcus wedmorensis, Streptococcus lus, ococcus glaucescens, Streptococcus bikiniensis, Streptococcus rubiginosus, Streptococcus achinatus, ococcus cinnamonensis, ococcusfraa’iae, Streptococcus albus, Streptococcus griseus, Streptococcus hivens, Streptococcus matensis, Streptococcus murinus, Streptococcus nivens, ococcus platensis, Streptosporangium album, Streptosporarzgium oulgare, Thermopolyspora spp., Thermus spp., Xanthomonas spp. and Zymononas mobilis.

Glucose isomerase can be used free in solution or immobilized on a support. Whole cells or cell free enzymes can be immobilized. The support structure can be any insoluble material. Support structures can be cationic, anionic or neutral materials, for example diethylaminoethyl ose, metal oxides, metal chlorides, metal carbonates and polystyrenes.

Immobilization can be accomplished by any suitable means. For example immobilization can be accomplished by contacting the support and the whole cell or enzyme in a t such as water and then ng the solvent. The solvent can be removed by any suitable means, for example filtration or ation or spray drying. As another example, spray drying the whole cells or enzyme with a support can be effective.

Glucose isomerase can also be present in a living cell that produces the enzyme during the process. For example a glucose isomerase producing bacteria can be co-cultured in the process with an ethanol fermenting bacteria. atively, the glucose—isomerase-producing ia can be first contacted with the substrate, followed by inoculating with an ethanol- producing ate.

Glucose ase can also be present within or secreted from a cell also capable of a further useful transformation of sugars. For example a glucose fermenting species can be genetically modified to contain and s the gene for production of glucose isomerase.

I. TREATMENT OF BIOMASS MATERIAL A. PARTICLE BOMBARDMENT One or more treatments with energetic particle bombardment can be used to process raw ock from a wide variety of different sources to extract useful substances from the feedstock, and to provide partially degraded organic material which functions as input to further processing steps and/or sequences. Particle bombardment can reduce the molecular weight and/or crystallinity of ock. In some embodiments, energy ted in a material that releases an electron from its atomic orbital can be used to treat the materials. The bombardment may be provided by heavy charged particles (such as alpha particles or protons), electrons (produced, for example, in beta decay or electron beam accelerators), or electromagnetic radiation (for e, gamma rays, x rays, or ultraviolet rays). Alternatively, radiation ed by radioactive substances can be used to treat the feedstock. Any combination, in any order, or concurrently of these treatments may be utilized. In another approach, electromagnetic radiation (e.g., produced using electron beam emitters) can be used to treat the feedstock.

Each form of energy ionizes the s via particular interactions. Heavy charged particles primarily ionize matter via Coulomb scattering; furthermore, these interactions produce energetic electrons that may further ionize matter. Alpha particles are identical to the nucleus of a helium atom and are produced by the alpha decay of various radioactive nuclei, such as isotopes of bismuth, polonium, astatine, radon, francium, radium, several actinides, such as actinium, thorium, m, neptunium, curium, califomium, ium, and ium.

When particles are ed, they can be neutral (uncharged), vely charged or negatively charged. When charged, the charged particles can bear a single positive or negative charge, or multiple charges, e.g. , one, two, three or even four or more charges. In instances in which chain on is desired, positively charged particles may be desirable, in part, due to their acidic . When particles are utilized, the particles can have the mass of a resting electron, or greater, e.g, 500, 1000, 1500, or 2000 or more times the mass of a g electron. For example, the particles can have a mass of from about 1 atomic unit to about 150 atomic units, e.g., from about 1 atomic unit to about 50 atomic units, or from about 1 to about 25, e.g., 1, 2, 3, 4, 5, 10, 12 or 15 atomic units. Accelerators used to accelerate the particles can be electrostatic DC, electrodynamic DC, RF linear, magnetic induction linear or continuous wave. For example, cyclotron type accelerators are available from IBA (Ion Beam Accelerators, Louvain-la—Neuve, m), such as the RhodotronTM system, while DC type accelerators are available from RDI, now IBA Industrial, such as the DynamitronTM. Ions and ion accelerators are discussed in Introductory Nuclear Physics, Kenneth S. Krane, John Wiley & Sons, Inc. (1988), Krsto Prelec, FIZIKA B 6 (1997) 4, 177-206; Chu, William T., “Overview of Light-Ion Beam Therapy”, Columbus—Ohio, ICRU-IAEA Meeting, 18-20 Mar. 2006; Iwata, Y. et al., “Altemating—Phase- Focused IH—DTL for Ion l Accelerators”, Proceedings of EPAC 2006, Edinburgh, Scotland; and Leitner, C. M. et 61]., “Status of the Superconducting ECR Ion Source Venus”, Proceedings of EPAC 2000, Vienna, a.

The doses applied depend on the d effect and the particular feedstock. For example, high doses can break chemical bonds within feedstock components and low doses can increase chemical bonding (e.g., cross-linking) within feedstock components.

In some instances when chain scission is desirable and/or polymer chain filnctionalization is desirable, particles r than electrons, such as protons, helium nuclei, argon ions, silicon ions, neon ions, carbon ions, phosphorus ions, oxygen ions or nitrogen ions can be ed. When ring-opening chain scission is desired, positively charged particles can be utilized for their Lewis acid properties for enhanced ring—opening chain scission. For example, when oxygen-containing fimctional groups are desired, ent in the presence of oxygen or even treatment with oxygen ions can be performed. For example, when nitrogen-containing fimctional groups are ble, treatment in the presence of nitrogen or even treatment with nitrogen ions can be performed.

B. OTHER FORMS OF ENERGY Electrons interact via Coulomb scattering and bremsstrahlung radiation produced by changes in the velocity of electrons. Electrons may be produced by radioactive nuclei that undergo beta decay, such as isotopes of iodine, cesium, technetium, and iridium. Alternatively, an electron gun can be used as an electron source via thermionic emission.

Electromagnetic radiation interacts via three processes: lectric absorption, Compton scattering, and pair production. The dominating interaction is determined by the energy of the incident radiation and the atomic number of the material. The summation of interactions contributing to the absorbed radiation in cellulosic al can be expressed by the mass absorption coefficient.

Electromagnetic radiation is ssified as gamma rays, x rays, ultraviolet rays, infrared rays, microwaves, or radiowaves, depending on the wavelength.

For e, gamma radiation can be employed to treat the materials. Gamma radiation has the advantage of a significant penetration depth into a variety of material in the . Sources of gamma rays include radioactive nuclei, such as isotopes of cobalt, calcium, technetium, chromium, gallium, indium, , iron, krypton, samarium, selenium, sodium, thalium, and xenon.

Sources of x rays include electron beam collision with metal s, such as tungsten or molybdenum or alloys, or compact light sources, such as those ed commercially by Sources for ultraviolet radiation include deuterium or cadmium lamps.

Sources for infrared radiation include sapphire, zinc, or selenide window ceramic lamps.

Sources for aves include klystrons, Slevin type RF sources, or atom beam sources that employ hydrogen, oxygen, or nitrogen gases.

Various other devices may be used in the methods disclosed herein, including field ionization sources, electrostatic ion separators, field tion generators, thermionic emission sources, microwave discharge ion sources, recirculating or static rators, dynamic linear accelerators, van de Graaff accelerators, and folded tandem rators. Such devices are disclosed, for example, in US. Pat. No. 7,931,784 B2, the complete disclosure of which is incorporated herein by reference.

C. ELECTRON BOMBARDMENT 1. Electron Beams The feedstock may be treated with electron bombardment to modify its structure and thereby reduce its recalcitrance. Such treatment may, for example, reduce the average molecular weight of the feedstock, change the crystalline structure of the feedstock, and/or increase the surface area and/or porosity of the feedstock. on bombardment Via an electron beam is generally preferred, because it provides very high throughput and e the use of a relatively low e/high power on beam device eliminates the need for expensive concrete vault ing, as such devices are shielded” and provide a safe, efficient process. While the “self-shielded” devices do include shielding (e.g., metal plate shielding), they do not require the construction of a concrete vault, greatly reducing capital expenditure and often allowing an existing manufacturing facility to be used without expensive modification. Electron beam accelerators are available, for example, from IBA (Ion Beam Applications, Louvain—la-Neuve, Belgium), Titan Corporation (San Diego, California, USA), and NHV Corporation (Nippon High Voltage, Japan).

Electron bombardment may be performed using an electron beam device that has a l energy of less than 10 MeV, e. g., less than 7 MeV, less than 5 MeV, or less than 2 MeV, e.g., from about 0.5 to 1.5 MeV, from about 0.8 to 1.8 MeV, from about 0.7 to 1 MeV, or from about 1 to 3 MeV. In some implementations the nominal energy is about 500 to 800 keV.

The electron beam may have a relatively high total beam power (the ed beam power of all accelerating heads, or, if multiple rators are used, of all rators and all heads), e.g., at least 25 kW, e.g., at least 30, 40, 50, 60, 65, 70, 80, 100, 125, or 150 kW. In some cases, the power is even as high as 500 kW, 750 kW, or even 1000 kW or more. In some cases the electron beam has a beam power of 1200 kW or more.

This high total beam power is usually achieved by utilizing multiple accelerating heads. For example, the on beam device may include two, four, or more accelerating heads. The use ofmultiple heads, each of which has a relatively low beam power, prevents excessive temperature rise in the material, thereby preventing burning of the material, and also increases the uniformity of the dose through the thickness of the layer of material.

In some implementations, it is desirable to cool the material during electron bombardment. For example, the material can be cooled while it is being conveyed, for example by a screw extruder or other conveying equipment.

To reduce the energy required by the recalcitrance-reducing process, it is desirable to treat the material as quickly as possible. In general, it is preferred that treatment be med at a dose rate of greater than about 0.25 Mrad per second, e.g, r than about 0.5, 0.75, 1, 1.5, 2, 5, 7, 10, 12, 15, or even greater than about 20 Mrad per second, e.g., about 0.25 to 2 Mrad per second. Higher dose rates generally require higher line speeds, to avoid thermal decomposition of the al. In one implementation, the accelerator is set for 3 MeV, 50 mAmp beam current, and the line speed is 24 feet/minute, for a sample thickness of about 20 mm (6.g. , comminuted corn cob material with a bulk density of 0.5 g/cm3).

In some embodiments, electron bombardment is performed until the material receives a total dose of at least 0.5 Mrad, e.g., at least 5, 10, 20, 30 or at least 40 Mrad. In some embodiments, the treatment is performed until the material receives a dose of from about 0.5 Mrad to about 150 Mrad, about 1 Mrad to about 100 Mrad, about 2 Mrad to about 75 Mrad, 10 Mrad to about 50 Mrad, e.g., about 5 Mrad to about 50 Mrad, from about 20 Mrad to about 40 Mrad, about 10 Mrad to about 35 Mrad, or from about 25 Mrad to about 30 Mrad. In some implementations, a total dose of 25 to 35 Mrad is preferred, applied ideally over a couple of seconds, e.g. at 5 Mrad/pass with each pass being applied for about one second. ng a dose of r than 7 to 8 Mrad/pass can in some cases cause thermal degradation of the feedstock material.

Using multiple heads as discussed above, the material can be treated in multiple passes, for example, two passes at 10 to 20 Mrad/pass, e.g., 12 to 18 ass, separated by a few seconds of cool-down, or three passes of 7 to 12 Mrad/pass, e.g., 9 to 11 Mrad/pass. As discussed above, treating the material with l relatively low doses, rather than one high dose, tends to prevent overheating of the material and also increases dose uniformity h the thickness of the al. In some implementations, the material is stirred or otherwise mixed during or after each pass and then smoothed into a uniform layer again before the next pass, to further enhance treatment uniformity. -16— In some embodiments, electrons are accelerated to, for example, a speed of greater than 75 percent of the speed of light, e.g, greater than 85, 90, 95, or 99 percent of the speed of light.

In some embodiments, any processing described herein occurs on lignocellulosic material that s dry as acquired or that has been dried, e.g., using heat and/or reduced pressure. For example, in some embodiments, the cellulosic and/or lignocellulosic al has less than about five percent by weight retained water, measured at 25°C and at fifty percent relative humidity.

Electron bombardment can be applied while the cellulosic and/or lignocellulosic material is exposed to air, oxygen—enriched air, or even oxygen itself, or ted by an inert gas such as en, argon, or helium. When m oxidation is desired, an oxidizing environment is utilized, such as air or oxygen and the distance from the beam source is optimized to maximize reactive gas formation, e.g., ozone and/or oxides of nitrogen.

In some embodiments, two or more electron sources are used, such as two or more ionizing sources. For e, samples can be treated, in any order, with a beam of electrons, followed by gamma radiation and UV light having wavelengths from about 100 nm to about 280 nm. In some ments, samples are treated with three ionizing radiation sources, such as a beam of electrons, gamma radiation, and energetic UV light. The biomass is conveyed through the treatment zone where it can be bombarded with electrons. It is generally preferred that the bed of biomass material has a relatively uniform ess, as usly described, while being treated.

It may be advantageous to repeat the treatment to more thoroughly reduce the recalcitrance of the biomass and/or further modify the biomass. In particular the s parameters can be adjusted after a first (e.g., second, third, fourth or more) pass depending on the recalcitrance of the material. In some embodiments, a conveyor can be used which includes a circular system where the biomass is conveyed multiple times through the various processes described above. In some other ments multiple treatment devices (e.g., electron beam generators) are used to treat the biomass multiple (e.g., 2, 3, 4 or more) times. In yet other embodiments, a single electron beam generator may be the source of multiple beams (e.g, 2, 3, 4 or more beams) that can be used for ent of the biomass.

The effectiveness in changing the molecular/supermolecular structure and/or ng the itrance of the biomass material depends on the electron energy used and the dose applied, while exposure time depends on the power and dose.

In some embodiments, the treatment (with any electron source or a combination of sources) is performed until the material receives a dose of at least about 0.05 Mrad, e.g., at least about 0.1, 0.25, 0.5, 0.75, 1.0, 2.5, 5.0, 7.5, 10.0, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200 Mrad. In some ments, the treatment is performed until the material es a dose ofbetween 0.1-100 Mrad, 1—200, 5-200, 10-200, 5—150, 5-100, 5-50, 5—40, 10-50, -75, 15-50, 20-35 Mrad.

In some embodiments, the treatment is performed at a dose rate of between 5.0 and 1500.0 kilorads/hour, e.g., between 10.0 and 750.0 kilorads/hour or between 50.0 and 350.0 kilorads/hours. In other embodiments the treatment is performed at a dose rate of between 10 and 10000 ds/hr, between 100 and 1000 kilorad/hr, or n 500 and 1000 kilorads/hr. 2. on Sources Electrons interact via Coulomb scattering and bremsstrahlung radiation produced by changes in the velocity of electrons. Electrons may be produced by ctive nuclei that undergo beta decay, such as isotopes of iodine, cesium, technetium, and iridium. Alternatively, an electron gun can be used as an electron source via thermionic on and accelerated through an accelerating ial. An electron gun generates electrons, accelerates them through a large potential (6.g. than about 500 thousand, greater than about 1million, greater than , greater about 2 million, greater than about 5 million, greater than about 6 million, greater than about 7 million, greater than about 8 million, greater than about 9 million, or even greater than 10 million volts) and then scans them magnetically in the x-y plane, where the electrons are initially accelerated in the z ion down the tube and extracted through a foil window. Scanning the electron beam is useful for increasing the irradiation surface when irradiating materials, e.g, a biomass, that is conveyed h the scanned beam. Scanning the electron beam also distributes the thermal load homogenously on the window and helps reduce the foil window rupture due to local heating by the electron beam. Window foil rupture is a cause of significant down-time due to subsequent necessary repairs and rting the electron gun. -18— Various other irradiating devices may be used in the methods disclosed herein, including field ionization sources, electrostatic ion separators, field ionization generators, thermionic emission sources, microwave discharge ion sources, recirculating or static accelerators, dynamic linear rators, van de Graaff accelerators, and folded tandem accelerators. Such devices are sed, for example, in US. Pat. No. 7,931,784 to Medoff, the complete disclosure of which is incorporated herein by reference.

A beam of electrons can be used as the radiation source. A beam of electrons has the advantages of high dose rates (e.g, 1, 5, or even 10 Mrad per second), high throughput, less containment, and less ment equipment. Electron beams can also have high electrical efficiency (6.g. , 80%), allowing for lower energy usage relative to other ion methods, which can translate into a lower cost of ion and lower greenhouse gas emissions corresponding to the smaller amount of energy used. Electron beams can be generated, e.g., by electrostatic generators, cascade generators, transformer tors, low energy accelerators with a scanning system, low energy rators with a linear cathode, linear accelerators, and pulsed accelerators.

Electrons can also be more efficient at causing changes in the molecular structure of biomass materials, for example, by the mechanism of chain scission. In addition, ons having energies of 0.5—10 MeV can penetrate low density materials, such as the biomass materials bed herein, e.g, materials having a bulk y of less than 0.5 g/cm3, and a depth of 0.3-10 cm. Electrons as an ionizing radiation source can be useful, e. g., for relatively thin piles, layers or beds of materials, e.g., less than about 0.5 inch, e.g, less than about 0.4 inch, 0.3 inch, 0.25 inch, or less than about 0.1 inch. In some embodiments, the energy of each electron of the on beam is from about 0.3 MeV to about 2.0 MeV (million electron volts), e.g., from about 0.5 MeV to about 1.5 MeV, or from about 0.7 MeV to about 1.25 MeV.

Methods of irradiating materials are discussed in US. Pat. App. Pub. 2012/0100577 A1 filed October 18, 2011, the entire disclosure of which is herein incorporated by reference.

Electron beam irradiation devices may be procured commercially from Ion Beam Applications (Louvain-la-Neuve, m), the Titan Corporation (San Diego, rnia, USA), and NHV Corporation (Nippon High Voltage, Japan). Typical electron energies can be 0.5 MeV, 1 MeV, 2 MeV, 4.5 MeV, 7.5 MeV, or 10 MeV. Typical electron beam irradiation device power can be 1 KW, 5 KW, 10 KW, 20 KW, 50 KW, 60 KW, 70 KW, 80 KW, 90 KW, 100 KW, 125 KW, 150 KW, 175 KW, 200 KW, 250 KW, 300 KW, 350 KW, 400 KW, 450 KW, 500 KW, 600 KW, 700 KW, 800 KW, 900 KW or even 1000 KW.

Tradeoffs in considering electron beam irradiation device power specifications include cost to operate, capital costs, depreciation, and device footprint. Tradeoffs in considering exposure dose levels of on beam irradiation would be energy costs and nment, safety, and health (ESH) concerns. Typically, generators are housed in a vault, e.g., of lead or concrete, especially for production from X-rays that are generated in the process.

Tradeoffs in considering electron energies include energy costs.

The electron beam irradiation device can produce either a fixed beam or a scanning beam. A scanning beam may be advantageous with large scan sweep length and high scan , as this would effectively replace a large, fixed beam width. r, available sweep widths of 0.5 m, 1 m, 2 m or more are available. The scanning beam is preferred in most embodiments describe herein because of the larger scan width and reduced possibility of local heating and failure of the windows. 3. Electron Guns - Windows When treated with an electron gun, the biomass is irradiated as it passes under a window, which is generally a metallic foil (e.g. , titanium, titanium alloy, aluminum and/or silicon). The window is impermeable to gases, yet electrons can pass with low resistance while being impermeable to gasses. The foil windows are preferably between about 10 and 100 microns thick (e.g., a window can be 10 s thick, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 microns thick). Thin windows dissipate less energy as an electron beam passes through them (e.g, the resistive heating is less since Power = 12R) which is advantageous with respect to ating the target material (e.g., s) with as much energy as possible. Thin windows are also less mechanically strong and more likely to fail which causes sed expense and more downtime for the equipment.

The foil window can be cooled by passing air or an inert gas over the window. When using an enclosure, it is generally preferred to mount the window to the ure and to cool the window from the side e of the enclosed conveying system to avoid lofting up any particulates of the material being irradiated.

The system can include more than one , e.g., a y window and a secondary . The two windows may form the enclosure to contain the purging gases and/or the cooling gases. The secondary window may serve a function as a “sacrificial” window, to protect the primary window. The electron beam tus includes a vacuum n the electron source and the primary window, and breakage of the primary window is likely to cause biomass material to be sucked up into the electron beam apparatus, resulting in , repair costs, and equipment downtime.

The window can be polymer, ceramic, coated ceramic, composite or coated composite. The secondary window can be, for instance, a continuous sheet/roll of polymer or coated polymer, which can be advanced continuously or at intervals to e a clean or new section to serve as the secondary window.

The primary window and the secondary window can be made from the same material, or different materials. For instance, the primary window foil can be made from titanium, scandium, vanadium, chromium, nickel, zirconium, niobium, enum, ruthenium, rhodium, ium, hafnium, tantalum, tungsten, rhenium, platinum, iridium, or alloys or mixtures of any of these. The ary single-type window foil can be made from titanium, um, vanadium, chromium, nickel, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium, hafnium, tantalum, tungsten, rhenium, platinum, iridium, beryllium, aluminum, silicon, or alloys or mixtures of any of these. The primary and secondary windows can be of the same material, mixture of materials, or alloy, or different materials, mixtures of al or alloys. One or both of the windows can be laminates of the same of ent materials, mixtures of materials, or alloys.

One ofmore of the windows can have a support structure across its face. The term e-type window”, as used herein, means a window with no support structure across its face.

The term “double-type window”, as used herein, means a window with a support structure across its face, where the support structure effectively divides the surface of the window into two parts.

Such a double-type window is shown in US. Pat. No. 5,877,582 to Nishimura. Additional support ures can also be used.

The primary window foil and the secondary window foil can both be made from low Z element. Alternatively, the primary window foil can be made from a high Z element, and the secondary window foil can be made from a low Z element.

The embodiments described herein do not preclude the inclusion of additional windows, which may have a protective function, or may be included to modify the radiation exposure.

The windows can be concave, flat or convex. It is generally preferred that the window be slightly convex, in a direction away from the direction of the cooling fluid. This curvature improves the mechanical strength of the window and increases the permitted temperature levels as well as allowing a better flow path for the cooling fluid. On the side of the scanning horn the curvature tends to be s the vacuum (6g, away from the g fluid) due to the vacuum (e.g., about 10'5 to 10‘10torr, about 10‘6 to 10'9 torr, about 10'7 to 10‘8 torr).

The cooling of the window and/or concave shape of the window become especially important for high beam currents, for example at least about 100 mA electron gun currents (e.g, at least about 110 mA, at least about 120 mA, at least about 130 mA, at least about 140 mA, at least about 150 mA at least about 200 mA, at least about 500 mA, at least about 1000 mA) because ive heating is approximately related to the square of the current as sed above.

The windows can be any shape but typically are approximately rectangular with a high aspect ratio of the width to the length (where the width direction is the same as the width of the conveying system perpendicular to the conveying direction, and the length is the same as the direction of ing). The distance of the window to the conveyed material can be less than about 10 cm (e.g., less than about 5cm) and more than about 0.10m (e.g. more than about lcm, more than about 2 cm, more than about 3 cm, more than about 4 cm). It is also le to use le windows (6.g. , 3, 4, 5, 6 or more) with different and varied shapes and red in ent ways. For example a primary or secondary foil window can include one, two or more windows in the same plane or layered and can include one or more support structures. For example support structures can be a bar or a grid in the same plane and contacting the windows.

In some embodiments, the window that is mounted on the enclosed conveying system is a secondary foil window of a two foil window extraction system for a scanning on beam.

In other embodiments there is no enclosure for conveying the biomass material, e. g., the biomass is conveyed in air under the irradiation device.

A two-foil window extraction system for a scanning electron beam has two windows, a primary and a secondary . Generally the primary window is closest to the electron source, and is concave towards the top of the scanning horn due to the vacuum on that side of the window. The secondary foil window tends to be flatter but is also concave in the same direction.

This ure helps provide structural support to the window and is mechanically stronger than a flat window. atively the windows can be flat or curved in any direction. The window foils are typically at least about 10 microns thick to about 30 microns thick (e.g., about 15-40 microns, about 20-30 microns, about 5-30 microns, about 8-25 s, about 10-20 microns, about 20-25 microns thick). The distance between the front surface of the primary window foil and back surface of the secondary window foil is preferably less than 30 cm, more preferably less than 20 cm, and most preferably less than 10 cm. Sidewalls, in ation with the primary and secondary windows, can define an interior space. Electrons travel through both windows to impinge on and penetrate the material (e.g, biomass) disposed beneath. A first inlet can be included on one sidewall can be arranged to allow a cooling fluid (e.g, a liquid or a gas) to impinge on the primary window foil. The cooling fluid can run along the window and then reverse direction on meeting the far (opposite) wall and flow back generally through the center of the interior space and then out through an exhaust port and or outlet. A second inlet can be included on the sidewall and can be arranged to allow cooling fluid to impinge on the secondary window foil in a r fashion. Optionally more inlets (e.g, 2, 3, 4, 5, 6 or more) can bring cooling fluid to the primary and ary window surfaces and multiple outlets (e.g, 2, 3, 4, 5, 6 or more) can allow the cooling fluid to exit the interior space. In some embodiments one or more side walls can even be a mesh, screen or grate with many openings through which cooling gas can flow while providing ural support to the windows.

Such window systems are bed in US. ional App. No. 61/711,801, by Medoff et al., which was filed on October 10, 2012, the entire contents of which are incorporated herein by reference. A variety of rations for such a system will also be known to those of ordinary skill in the art. 4. Electron Guns - Window Spacing Although a large spacing between the windows can be advantageous, for example, for the reasons described above, the large spacing poses some disadvantages. One disadvantage of a large spacing between windows is that the electron beams will pass h a larger volume of cooling gas which can cause energy losses. For example a lMeV beam loses about 0.2 MeV/M of energy, a 5 MeV beam loses about 0.23 MeV/M and a 10 MeV beam loses about 0.26 WO 96700 MeV/M. Therefore with a 1 MeV beam of ons passing h 1 cm of air, the beam loses only 0.2% of its energy, at 10 cm of air, the beam loses 2% of its energy, at 20 cm this is 4% of its energy, while at 50 cm the energy loss is 10%. Since the electrons also have to travel from the secondary foil window to the biomass through onal air, the gap between the windows must be carefully lled. Preferably, energy losses are less that about 20% (e.g., less than %, less than 5% or even less than 1%). It is therefore advantageous to minimize the spacing between the windows to decrease energy losses. Optimal spacing (e.g., average spacing) n the s (eg, between the surface side of the electron window foil and the facing surface of the secondary window foil) for the benefit of cooling as described above and for the benefit of reducing energy loss are n about 2 and 20 cm (eg, between about 3 and 20 cm, between about 4 and 20 cm, between about 5 and 20 cm, between about 6 and 20 cm, between about 7 and 20 cm, between about 8 and 20 cm, between about 3 and 15 cm, between about 4 and 15 cm, between about 5 and 15 cm, between about 6 and 15 cm, between about 7 and 15 cm, between about 8 and 15 cm between about 3 and 10 cm, between about 4 and 10 cm, between about 5 and 10 cm, between about 6 and 10 cm, between about 7 and 10 cm, between about 8 and 10 cm).

One of ordinary skill in the art will balance the advantages and disadvantages of window spacing to suit their needs.

In some embodiments support structures for the windows can be used across the windows, although these types of structures are less preferred because of energy losses that can occur to the electron beam as it strikes these kinds of structures.

A large spacing between the windows can be advantageous because it defines a larger volume between the windows and allows for rapid flowing of a large volume g gasses for very efficient cooling. The inlets and outlets are between 1mm and 120 mm in diameter (e.g, about 2 mm, about 5 mm about 10 mm, about 20 mm, about 50 mm or even about 100 mm).

The cooling gas flow can be at between about 500-2500 CFM (e.g., about 600 to 2500 CFM, about 700—2500 CFM, about 800 to 2500 CFM, about 1000 to 2500 CFM, about 600 to 2000 CFM, about 700-2000 CFM, about 800 to 2000 CFM, about 1000 to 2000 CFM, about 600 to 1500 CFM, about 700-1500 CFM, about 800 to 1500 CFM, about 1000 to 1500 CFM). In some embodiments, about 50% ofthe gas is exchanged per about 60 seconds or less (e.g, in about 50 sec or less, in about 30 sec or less, in about 10 sec or less, in about 1 sec or less).

. Electron Guns - Cooling and Purging Gases The cooling gas in the two foil window extraction system can be a purge gas or a mixture, for example air, or a pure gas. In one ment the gas is an inert gas such as nitrogen, argon, helium and or carbon dioxide. It is preferred to use a gas rather than a liquid since energy losses to the electron beam are minimized. es ofpure gas can also be used, either pre-mixed or mixed in line prior to impinging on the windows or in the space between the windows. The cooling gas can be cooled, for example, by using a heat exchange system (e.g., a chiller) and/or by using boil off from a condensed gas (e.g., liquid nitrogen, liquid helium).

When using an enclosure, the enclosed conveyor can also be purged with an inert gas so as to maintain an atmosphere at a reduced oxygen level. Keeping oxygen levels low avoids the formation of ozone which in some ces is undesirable due to its reactive and toxic nature. For example the oxygen can be less than about 20% (e.g., less than about 10%, less than about 1%, less than about 0.1%, less than about 0.01%, or even less than about 0.001% oxygen).

Purging can be done with an inert gas including, but not limited to, nitrogen, argon, helium or carbon dioxide. This can be supplied, for example, from a boil off of a liquid source (e.g, liquid nitrogen or ), ted or separated from air in situ, or ed from tanks. The inert gas can be recirculated and any residual oxygen can be removed using a st, such as a copper catalyst bed. Alternatively, combinations of purging, recirculating and oxygen l can be done to keep the oxygen levels low.

The enclosure can also be purged with a reactive gas that can react with the biomass.

This can be done before, during or after the irradiation process. The reactive gas can be, but is not limited to, nitrous oxide, ammonia, oxygen, ozone, hydrocarbons, aromatic compounds, amides, peroxides, azides, halides, ides, phosphides, phosphines, arsines, sulfides, thiols, boranes and/or hydrides. The reactive gas can be activated in the enclosure, e.g., by irradiation (e.g, electron beam, UV irradiation, microwave irradiation, heating, IR radiation), so that it reacts with the biomass. The biomass itself can be activated, for e by irradiation.

Preferably the biomass is activated by the electron beam, to produce radicals which then react with the ted or unactivated reactive gas, e. g., by radical coupling or quenching.

Purging gases supplied to an enclosed conveyor can also be cooled, for example below about 25°C, below about 0°C, below about -40°C, below about -80°C, below about - 120°C. For example, the gas can be boiled off from a compressed gas such as liquid nitrogen or sublimed from solid carbon dioxide. As an alternative example, the gas can be cooled by a chiller or part of or the entire conveyor can be cooled. 6. Electron Guns - Beam Stops In some embodiments the systems and methods include a beam stop (e.g., a shutter).

For example, the beam stop can be used to quickly stop or reduce the irradiation of material without ng down the electron beam device. Alternatively the beam stop can be used while powering up the electron beam, e.g., the beam stop can stop the on beam until a beam current of a desired level is achieved. The beam stop can be placed between the primary foil window and secondary foil window. For example the beam stop can be mounted so that it is movable, that is, so that it can be moved into and out of the beam path. Even partial coverage of the beam can be used, for example, to control the dose of irradiation. The beam stop can be mounted to the floor, to a conveyor for the s, to a wall, to the radiation device (e.g, at the scan horn), or to any structural support. Preferably the beam stop is fixed in relation to the scan horn so that the beam can be effectively controlled by the beam stop. The beam stop can orate a hinge, a rail, wheels, slots, or other means allowing for its operation in moving into and out of the beam. The beam stop can be made of any material that will stop at least 5% of the electrons, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even about 100% ofthe ons.

The beam stop can be made of a metal including, but not limited to, stainless steel, lead, iron, molybdenum, silver, gold, titanium, aluminum, tin, or alloys of these, or laminates (layered materials) made with such metals (e.g., coated c, metal-coated polymer, metal-coated composite, multilayered metal materials).

The beam stop can be cooled, for example, with a g fluid such as an aqueous solution or a gas. The beam stop can be partially or completely hollow, for example with cavities. Interior spaces of the beam stop can be used for cooling fluids and gases. The beam stop can be of any shape, ing flat, curved, round, oval, square, rectangular, beveled and wedged shapes.

The beam stop can have perforations so as to allow some electrons through, thus lling (e.g., reducing) the levels of radiation across the whole area of the window, or in -26— specific regions of the window. The beam stop can be a mesh formed, for example, from fibers or wires. Multiple beam stops can be used, together or independently, to control the irradiation.

The beam stop can be remotely controlled, e.g, by radio signal or hard wired to a motor for moving the beam into or out of on.

D. TREATMENT OF BIOMASS MATERIAL —- SONICATION, PYROLYSIS, OXIDATION, STEAM EXPLOSION If desired, one or more sonication, pyrolysis, oxidative, or steam explosion processes can be used in addition to or instead of other treatments to further reduce the recalcitrance of the biomass material. These processes can be applied before, during and or after another treatment or treatments. These processes are described in detail in US. Pat. No. 7,932,065 to Medoff, the full disclosure of which is incorporated herein by reference. 11. BIOMASS MATERIALS As used herein, the term “biomass materials” includes lignocellulosic, cellulosic, starchy, and microbial materials.

Lignocellulosic materials include, but are not limited to, wood, le board, ry wastes (e.g., sawdust, aspen wood, wood chips), grasses, (e.g., switchgrass, miscanthus, cord grass, reed canary grass), grain residues, (e.g., rice hulls, oat hulls, wheat chaff, barley hulls), agricultural waste (e.g., silage, canola straw, wheat straw, barley straw, oat straw, rice straw, jute, hemp, flax, bamboo, sisal, abaca, corn cobs, corn stover, soybean stover, corn fiber, alfalfa, hay, coconut hair), sugar processing residues (e.g., bagasse, beet pulp, agave e), , algae, seaweed, manure, sewage, and mixtures of any of these.

In some cases, the lignocellulosic al includes corncobs. Ground or hammermilled corncobs can be spread in a layer of relatively uniform thickness for irradiation, and after irradiation are easy to disperse in the medium for further processing. To facilitate harvest and collection, in some cases the entire corn plant is used, including the corn stalk, corn kernels, and in some cases even the root system of the plant. ageously, for ethanol production, no additional nts (other than a nitrogen source, e.g., urea or a) are required during fermentation of corncobs or osic or ellulosic als containing significant s of corncobs. Other products may require addition of trace metals, vitamins, or buffering capacity, but these adjustment are well within the knowledge of those of ry skill in the art.

Comcobs, before and after comminution, are also easier to convey and disperse, and have a lesser tendency to form explosive mixtures in air than other cellulosic or lignocellulosic materials such as hay and grasses.

Cellulosic materials include, for example, paper, paper products, paper waste, paper pulp, ted papers, loaded papers, coated papers, filled papers, magazines, printed matter (e.g, books, catalogs, s, labels, calendars, ng cards, brochures, ctuses, newsprint), printer paper, polycoated paper, card stock, cardboard, paperboard, materials having a high oz—cellulose content such as cotton, and mixtures of any of these. For example paper products as described in US. App. No. 13/396,365 (“Magazine ocks” by Medoff et al., filed February 14, 2012), the full disclosure ofwhich is incorporated herein by reference.

Cellulosic materials can also e lignocellulosic materials which have been de- ied.

Starchy materials include starch itself, e. g., corn starch, wheat starch, potato starch or rice starch, a derivative of starch, or a material that includes starch, such as an edible food t or a crop. For example, the starchy material can be arracacha, buckwheat, banana, , cassava, kudzu, oca, sago, m, regular household potatoes, sweet potato, taro, yams, or one or more beans, such as favas, lentils or peas. Blends of any two or more starchy materials are also starchy materials. Mixtures of y, cellulosic and or lignocellulosic materials can also be used. For example, a biomass can be an entire plant, a part of a plant or ent parts of a plant, e.g., a wheat plant, cotton plant, a corn plant, rice plant or a tree. The starchy materials can be treated by any of the methods described herein.

Microbial materials include, but are not limited to, any naturally occurring or cally modified microorganism or organism that contains or is e of providing a source of carbohydrates (e.g, cellulose), for example, protists, e.g., animal protists (e.g, protozoa such as flagellates, amoeboids, ciliates, and sporozoa) and plant protists (e.g., algae such alveolates, chlorarachniophytes, cryptomonads, euglenids, glaucophytes, haptophytes, red algae, nopiles, and viridaeplantae). Other examples include seaweed, plankton (e.g., macroplankton, mesoplankton, microplankton, nanoplankton, picoplankton, and femptoplankton), phytoplankton, bacteria (e.g., gram positive bacteria, gram negative bacteria, -28— and extremophiles), yeast and/or mixtures of these. In some ces, ial biomass can be obtained from natural sources, e.g., the ocean, lakes, bodies of water, e.g., salt water or fresh water, or on land. Alternatively or in addition, microbial biomass can be obtained from e systems, e.g, large scale dry and wet culture and fermentation systems.

The biomass material can also include offal, and similar sources of material.

In other embodiments, the biomass materials, such as cellulosic, y and lignocellulosic feedstock materials, can be obtained from transgenic microorganisms and plants that have been modified with respect to a wild type variety. Such modifications may be, for example, through the iterative steps of selection and breeding to obtain desired traits in a plant.

Furthermore, the plants can have had genetic material removed, modified, silenced and/or added with respect to the wild type variety. For example, cally modified plants can be produced by recombinant DNA methods, where genetic ations include introducing or modifying specific genes from al varieties, or, for example, by using transgenic breeding wherein a specific gene or genes are introduced to a plant from a different species of plant and/or bacteria.

Another way to create genetic variation is through mutation ng n new alleles are artificially created from nous genes. The artificial genes can be created by a variety of ways including treating the plant or seeds with, for example, chemical mutagens (e.g., using alkylating agents, epoxides, alkaloids, peroxides, formaldehyde), irradiation (e.g., X-rays, gamma rays, neutrons, beta particles, alpha particles, protons, deuterons, UV radiation) and temperature shocking or other external stressing and subsequent selection techniques. Other methods of providing modified genes is through error prone PCR and DNA shuffling followed by insertion of the desired modified DNA into the desired plant or seed. s of introducing the desired genetic variation in the seed or plant include, for example, the use of a bacterial carrier, biolistics, calcium phosphate precipitation, oporation, gene splicing, gene silencing, ction, microinjection and viral carriers. Additional genetically d materials have been described in US. Application Serial No 13/396,369 filed February 14, 2012 the full disclosure of which is incorporated herein by nce.

Any of the methods described herein can be practiced with mixtures of any biomass materials described herein.

WO 96700 III. BIOMASS MATERIAL PREPARATION -- MECHANICAL TREATMENTS The biomass can be in a dry form, for example with less than about 35% moisture content (e.g, less than about 20 %, less than about 15 %, less than about 10 % less than about 5 %, less than about 4%, less than about 3 %, less than about 2 % or even less than about 1 %).

The s can also be delivered in a wet state, for example as a wet solid, a slurry or a sion with at least about 10 wt% solids (e.g., at least about 20 wt%, at least about 30 wt. %, at least about 40 wt%, at least about 50 wt%, at least about 60 wt.%, at least about 70 wt%).

The processes disclosed herein can utilize low bulk density materials, for example cellulosic or lignocellulosic feedstocks that have been physically ated to have a bulk density ofless than about 0.75 g/cm3, e.g., less than about 0.7, 0.65, 0.60, 0.50, 0.35, 0.25, 0.20, 0.15, 0.10, 0.05 or less, e.g., less than about 0.025 g/cm3.

Bulk density is determined using ASTM D1895B. Briefly, the method involves filling a measuring er of known volume with a sample and obtaining a weight of the sample. The bulk density is calculated by dividing the weight of the sample in grams by the known volume of the cylinder in cubic centimeters. If desired, low bulk density materials can be densified, for example, by methods described in US.

Pat. No. 809 to Medoff, the full sure of which is hereby incorporated by reference.

In some cases, the eatment processing includes screening of the biomass material. Screening can be through a mesh or perforated plate with a desired opening size, for example, less than about 6.35 mm (1/4 inch, 0.25 inch), (e.g., less than about 3.18 mm (1/8 inch, 0.125 inch), less than about 1.59 mm (1/16 inch, 0.0625 inch), is less than about 0.79 mm (1/32 inch, 5 inch), e.g., less than about 0.51 mm (1/50 inch, 0.02000 inch), less than about 0.40 mm (1/64 inch, 0.015625 inch), less than about 0.23 mm (0.009 inch), less than about 0.20 mm (1/ 128 inch, 0.0078125 inch), less than about 0.18 mm (0.007 inch), less than about 0.13 mm (0.005 inch), or even less than about 0.10 mm (1/256 inch, 0.00390625 inch)). In one configuration the desired biomass falls through the perforations or screen and thus biomass larger than the perforations or screen are not irradiated. These larger materials can be re- processed, for example by comminuting, or they can simply be removed from processing. In another configuration material that is larger than the perforations is irradiated and the smaller material is removed by the screening process or recycled. In this kind of a configuration, the conveyor itself (for example a part of the conveyor) can be perforated or made with a mesh. For example, in one particular embodiment the biomass material may be wet and the perforations or mesh allow water to drain away from the biomass before irradiation.

Screening of material can also be by a manual method, for e by an operator or oid (e.g., a robot equipped with a color, reflectivity or other sensor) that removes unwanted material. Screening can also be by magnetic screening wherein a magnet is disposed near the conveyed material and the ic material is removed magnetically.

Optional pre-treatment processing can include heating the material. For example a portion of the conveyor can be sent through a heated zone. The heated zone can be created, for example, by IR radiation, aves, combustion (e.g., gas, coal, oil, biomass), resistive g and/or inductive coils. The heat can be applied from at least one side or more than one side, can be continuous or periodic and can be for only a portion of the material or all the material. For example, a n of the ing trough can be heated by use of a heating jacket. Heating can be, for example, for the purpose of drying the material. In the case of drying the material, this can also be facilitated, with or without heating, by the movement of a gas (e.g., air, oxygen, en, He, C02, Argon) over and/or through the biomass as it is being conveyed.

Optionally, pre—treatment processing can include cooling the material. Cooling material is described in US Pat. No. 7,900,857 to Medoff, the disclosure of which in incorporated herein by reference. For e, cooling can be by supplying a cooling fluid, for example water (e.g. with glycerol), or nitrogen (e.g. to the bottom of the ing , , liquid nitrogen) trough. Alternatively, a g gas, for example, chilled nitrogen can be blown over the biomass materials or under the conveying system.

Another optional pre-treatment processing method can include adding a material to the biomass. The additional material can be added by, for example, by showering, sprinkling and or pouring the material onto the biomass as it is ed. Materials that can be added include, for example, metals, ceramics and/or ions as described in US. Pat. App. Pub. 2010/0105119 A1 (filed October 26, 2009) and US. Pat. App. Pub. 2010/0159569 A1 (filed December 16, 2009), the entire disclosures of which are incorporated herein by reference.

Optional materials that can be added e acids and bases. Other materials that can be added are oxidants (e.g. monomers (e.g, containing , des, chlorates), polymers, polymerizable unsaturated bonds), water, catalysts, enzymes and/or organisms. Materials can be added, for example, in pure form, as a solution in a solvent (e.g, water or an c solvent) and/or as a solution. In some cases the solvent is volatile and can be made to evaporate e.g. , by heating and/or g gas as previously bed. The added material may form a uniform coating on the biomass or be a homogeneous mixture of different components (e.g., s and additional material). The added material can modulate the subsequent irradiation step by increasing the efficiency of the irradiation, damping the irradiation or changing the effect of the irradiation (e.g, from electron beams to X-rays or heat). The method may have no impact on the irradiation but may be useful for further downstream processing. The added material may help in conveying the al, for example, by lowering dust levels.

Biomass can be red to the conveyor by a belt conveyor, a pneumatic conveyor, a screw conveyor, a hopper, a pipe, manually or by a ation of these. The biomass can, for example, be dropped, poured and/or placed onto the conveyor by any of these methods. In some embodiments the material is delivered to the conveyor using an enclosed material distribution system to help maintain a low oxygen atmosphere and/or control dust and fines. Lofted or air suspended biomass fines and dust are undesirable e these can form an explosion hazard or damage the window foils of an electron gun (if such a device is used for treating the material).

The material can be leveled to form a uniform thickness between about 0.0312 and 5 inches (e.g., between about 0.0625 and 2.000 inches, between about 0.125 and 1 , n about 0.125 and 0.5 inches, between about 0.3 and 0.9 inches, n about 0.2 and 0.5 inches n about 0.25 and 1.0 inches, between about 0.25 and 0.5 inches, 0.100 +/— 0.025 inches, 0.150 --/— 0.025 inches, 0.200 +/- 0.025 inches, 0.250 +/- 0.025 inches, 0.300 --/— 0.025 inches, 0.350 --/— 0.025 inches, 0.400 +/- 0.025 inches, 0.450 +/- 0.025 inches, 0.500 --/— 0.025 inches, 0.550 --/— 0.025 inches, 0.600 +/- 0.025 inches, 0.700 --/- 0.025 inches, 0.750 --/— 0.025 inches, 0.800 --/— 0.025 inches, 0.850 +/- 0.025 inches, 0.900 --/- 0.025 , 0.900 --/— 0.025 inches.

Generally, it is preferred to convey the material as quickly as possible through the on beam to maximize hput. For example the material can be conveyed at rates of at least 1 ft/min, e.g., at least 2 ft/min, at least 3 ft/min, at least 4 ft/min, at least 5 ft/min, at least 10 ft/min, at least 15 ft/min, 20, 25, 30, 35, 40, 45, 50 ft/min. The rate of conveying is related to the beam current, for example, for a 14 inch thick biomass and 100 mA, the conveyor can move at about 20 ft/min to provide a useful irradiation dosage, at 50 mA the conveyor can move at about ft/min to provide approximately the same irradiation dosage.

After the biomass material has been conveyed through the radiation zone, optional post-treatment processing can be done. The optional post-treatment sing can, for example, be a process described with t to the pre-irradiation processing. For example, the biomass can be screened, heated, , and/or combined with additives. Uniquely to post-irradiation, quenching of the ls can occur, for example, quenching of radicals by the addition of fluids or gases (e.g., oxygen, nitrous oxide, a, liquids), using pressure, heat, and/or the addition of radical scavengers. For example, the s can be conveyed out of the enclosed conveyor and exposed to a gas (e.g., oxygen) where it is quenched, forming caboxylated groups. In one embodiment the s is exposed during irradiation to the reactive gas or fluid. Quenching of biomass that has been irradiated is described in US. Pat. No. 8,083,906 to Medoff, the entire disclosure of which is incorporate herein by reference.

If desired, one or more mechanical treatments can be used in on to irradiation to flirther reduce the recalcitrance of the biomass material. These processes can be d before, during and or after irradiation.

In some cases, the mechanical treatment may include an l preparation of the feedstock as received, e.g., size reduction of materials, such as by comminution, e.g., cutting, grinding, shearing, pulverizing or chopping. For example, in some cases, loose feedstock (e.g., recycled paper, starchy materials, or switchgrass) is prepared by shearing or shredding.

Mechanical treatment may reduce the bulk density of the biomass material, increase the surface area of the biomass material and/or decrease one or more dimensions of the biomass material. atively, or in addition, the feedstock material can first be physically treated by one or more of the other physical treatment methods, e.g, al treatment, radiation, sonication, oxidation, pyrolysis or steam explosion, and then mechanically treated. This sequence can be advantageous since materials d by one or more of the other treatments, e.g. irradiation or pyrolysis, tend to be more brittle and, therefore, it may be easier to further change the structure of the material by ical treatment. For example, a feedstock material can be conveyed through ionizing radiation using a conveyor as described herein and then ically treated. Chemical ent can remove some or all of the lignin (for example chemical pulping) and can partially or completely hydrolyze the material. The methods also can be used with pre-hydrolyzed material. The methods also can be used with material that has not been pre hydrolyzed The methods can be used with mixtures of hydrolyzed and non-hydrolyzed materials, for example with about 50% or more non-hydrolyzed material, with about 60% or more non- hydrolyzed material, with about 70% or more non-hydrolyzed material, with about 80% or more non-hydrolyzed material or even with 90% or more non-hydrolyzed material.

In addition to size reduction, which can be performed initially and/or later in processing, mechanical ent can also be ageous for “opening up, ,7 ‘6stressing,” breaking or shattering the biomass materials, making the cellulose of the materials more susceptible to chain scission and/or disruption of lline structure during the al treatment.

Methods of ically ng the biomass material e, for e, milling or grinding. Milling may be performed using, for example, a hammer mill, ball mill, colloid mill, conical or cone mill, disk mill, edge mill, Wiley mill, grist mill or other mill. Grinding may be performed using, for example, a cutting/impact type grinder. Some exemplary grinders include stone grinders, pin grinders, coffee grinders, and burr grinders. Grinding or milling may be provided, for example, by a ocating pin or other t, as is the case in a pin mill.

Other mechanical ent methods include mechanical ripping or tearing, other methods that apply pressure to the fibers, and air attrition milling. Suitable mechanical treatments further include any other technique that continues the disruption of the internal ure of the material that was initiated by the previous processing steps.

Mechanical feed preparation systems can be configured to produce streams with specific characteristics such as, for example, specific maximum sizes, specific length-to-width, or specific surface areas ratios. Physical preparation can increase the rate of reactions, improve the movement of material on a conveyor, e the irradiation profile of the material, improve the radiation uniformity of the material, or reduce the processing time required by opening up the materials and making them more accessible to processes and/or reagents, such as reagents in a solution.

The bulk density of feedstocks can be controlled (e.g., increased). In some situations, it can be desirable to prepare a low bulk y material, e.g. the material (e.g, , by densifying cation can make it easier and less costly to transport to another site) and then reverting the material to a lower bulk density state (e.g. after transport). The material can be densified, for example from less than about 0.2 g/cc to more than about 0.9 g/cc (e.g, less than about 0.3 to more than about 0.5 g/cc, less than about 0.3 to more than about 0.9 g/cc, less than about 0.5 to more than about 0.9 g/cc, less than about 0.3 to more than about 0.8 g/cc, less than about 0.2 to more than about 0.5 g/cc). For example, the al can be densified by the methods and equipment disclosed in US. Pat. No. 7,932,065 to Medoff and International Publication No. WO 2008/073186 (which was filed October 26, 2007, was hed in English, and which designated the United States), the full disclosures of which are incorporated herein by reference.

Densified als can be processed by any of the methods described herein, or any material processed by any of the methods described herein can be subsequently densified.

In some embodiments, the al to be processed is in the form of a fibrous material that includes fibers provided by shearing a fiber . For example, the shearing can be performed with a rotary knife cutter.

For example, a fiber source, e. g., that is recalcitrant or that has had its recalcitrance level reduced, can be sheared, e.g., in a rotary knife cutter, to provide a first fibrous al.

The first fibrous material is passed through a first screen, e.g. an average opening size of , having 1.59 mm or less (1/16 inch, 0.0625 inch), provide a second fibrous material. If desired, the fiber source can be cut prior to the shearing, e.g, with a shredder. For example, when a paper is used as the fiber source, the paper can be first cut into strips that are, e.g. 1/4- to 1/2-inch wide, using a shredder, e.g. a counter-rotating screw shredder, such as those manufactured by Munson , NY.) As an alternative to shredding, the paper can be reduced in size by cutting to a d size using a guillotine cutter. For example, the guillotine cutter can be used to cut the paper into sheets that are, e.g., 10 inches wide by 12 inches long.

In some embodiments, the shearing of the fiber source and the passing of the resulting first fibrous material through a first screen are performed concurrently. The shearing and the passing can also be performed in a batch—type s.

For example, a rotary knife cutter can be used to concurrently shear the fiber source and screen the first fibrous material. A rotary knife cutter includes a hopper that can be loaded with a shredded fiber source prepared by shredding a fiber source. The ed fiber source.

In some implementations, the feedstock is physically treated prior to saccharification and/or fermentation. Physical treatment processes can include one or more of any of those described herein, such as mechanical treatment, chemical treatment, irradiation, sonication, ion, sis or steam explosion. Treatment methods can be used in combinations of two, three, four, or even all of these technologies (in any order). When more than one treatment method is used, the methods can be applied at the same time or at different times. Other processes that change a molecular structure of a biomass feedstock may also be used, alone or in combination with the processes disclosed herein.

Mechanical treatments that may be used, and the characteristics of the ically treated s materials, are described in further detail in US. Pat. App. Pub. 100577 A1, filed October 18, 2011, the full disclosure of which is hereby incorporated herein by reference.

IV. USE OF TREATED BIOMASS MATERIAL Using the methods described herein, a starting biomass material (6.g. , plant biomass, animal biomass, paper, and municipal waste biomass) can be used as feedstock to produce useful intermediates and products such as organic acids, salts of organic acids, anhydrides, esters of organic acids and fuels, e.g., fuels for internal combustion engines or feedstocks for fuel cells.

Systems and processes are bed herein that can use as ock cellulosic and/or ellulosic materials that are readily available, but often can be difficult to process, e.g., municipal waste streams and waste paper streams, such as streams that include newspaper, kraft paper, ated paper or mixtures of these.

In order to convert the feedstock to a form that can be readily processed, the glucan- or containing cellulose in the feedstock can be hydrolyzed to low molecular weight carbohydrates, such as sugars, by a saccharifying agent, e.g., an enzyme or acid, a process referred to as saccharification. The low molecular weight carbohydrates can then be used, for example, in an existing manufacturing plant, such as a single cell protein plant, an enzyme manufacturing plant, or a fuel plant, e.g., an ethanol manufacturing facility.

The feedstock can be hydrolyzed using an enzyme, e.g., by combining the materials and the enzyme in a solvent, e.g., in an aqueous solution.

Alternatively, the enzymes can be supplied by organisms that break down biomass, such as the cellulose and/or the lignin portions of the biomass, contain or manufacture various olytic enzymes (cellulases), ligninases or various small molecule s-degrading metabolites. These s may be a x of enzymes that act synergistically to e crystalline cellulose or the lignin portions of biomass. es of cellulolytic enzymes include: endoglucanases, cellobiohydrolases, and cellobiases (beta-glucosidases). -36— During saccharification a cellulosic substrate can be initially hydrolyzed by endoglucanases at random locations producing oligomeric intermediates. These intermediates are then substrates for exo—splitting glucanases such as cellobiohydrolase to produce cellobiose from the ends of the cellulose polymer. Cellobiose is a water-soluble 1,4-linked dimer of e. Finally, iase cleaves cellobiose to yield glucose. The efficiency (e.g., time to hydrolyze and/or completeness of hydrolysis) of this process depends on the itrance of the cellulosic material.

V. EDIATES AND TS Using the processes described herein, the biomass al can be converted to one or more products, such as energy, fuels, foods and materials. c examples of products include, but are not limited to, hydrogen, sugars (e.g., glucose, xylose, arabinose, mannose, ose, fructose, disaccharides, accharides and polysaccharides), alcohols (e.g., monohydric alcohols or ic alcohols, such as ethanol, n—propanol, isobutanol, sec-butanol, tert—butanol or n-butanol), hydrated or hydrous alcohols (e.g, containing greater than 10%, 20%, % or even greater than 40% water), biodiesel, organic acids, hydrocarbons (e.g, methane, ethane, propane, isobutene, pentane, n-hexane, biodiesel, bio-gasoline and mixtures thereof), co- products (e.g., proteins, such as cellulolytic proteins (enzymes) or single cell proteins), and mixtures of any of these in any combination or relative concentration, and optionally in combination with any additives (e.g., fuel additives). Other examples include carboxylic acids, salts of a carboxylic acid, a mixture of carboxylic acids and salts of carboxylic acids and esters of carboxylic acids (e.g., methyl, ethyl and n-propyl esters), ketones (e.g., acetone), aldehydes (e.g., acetaldehyde), alpha and beta unsaturated acids (e.g., acrylic acid) and olefins (e.g., ethylene).

Other ls and alcohol derivatives e propanol, propylene glycol, 1,4-butanediol, 1,3- propanediol, sugar alcohols and polyols (e.g., glycol, glycerol, erythritol, threitol, ol, l, ribitol, mannitol, sorbitol, itol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, and polyglycitol and other polyols), and methyl or ethyl esters of any of these alcohols. Other products include methyl acrylate, methylmethacrylate, lactic acid, citric acid, formic acid, acetic acid, propionic acid, butyric acid, succinic acid, valeric acid, caproic acid, 3-hydroxypropionic acid, palmitic acid, stearic acid, oxalic acid, malonic acid, glutaric acid, oleic acid, linoleic acid, glycolic acid, gamma-hydroxybutyric acid, and mixtures f, salts of any of these acids, mixtures of any of the acids and their respective salts.

Any ation of the above products with each other, and/or of the above products with other products, which other products may be made by the processes described herein or otherwise, may be packaged together and sold as products. The products may be combined, e.g., mixed, blended or co-dissolved, or may simply be packaged or sold together.

Any of the products or combinations of ts described herein may be sanitized or sterilized prior to selling the products, e.g., after purification or isolation or even after packaging, to lize one or more potentially undesirable contaminants that could be present in the product(s). Such sanitation can be done with on bombardment, for example, be at a dosage of less than about 20 Mrad, e. g., from about 0.1 to 15 Mrad, from about 0.5 to 7 Mrad, or from about 1 to 3 Mrad.

The processes described herein can produce various by-product streams useful for generating steam and electricity to be used in other parts of the plant (co-generation) or sold on the open . For example, steam generated from burning by-product streams can be used in a lation process. As another example, electricity generated from burning by—product s can be used to power electron beam generators used in pretreatment.

The by-products used to generate steam and electricity are derived from a number of sources throughout the s. For example, anaerobic digestion ofwastewater can produce a biogas high in methane and a small amount of waste biomass (sludge). As r e, post—saccharification and/or post—distillate solids (e.g., unconverted lignin, cellulose, and hemicellulose remaining from the pretreatment and primary processes) can be used, e.g., burned, as a fuel.

Many of the products obtained, such as ethanol or n-butanol, can be utilized as a filel for powering cars, trucks, tractors, ships or trains, e.g., as an internal combustion fuel or as a fuel cell feedstock. Many of the products obtained can also be utilized to power aircraft, such as planes, e. g., having jet engines or helicopters. In addition, the products described herein can be utilized for electrical power generation, e.g., in a conventional steam generating plant or in a fuel cell plant. -38— Other intermediates and products, including food and pharmaceutical products, are described in US. Pat. App. Pub. 2010/0124583 A1, published May 20, 2010, to Medoff, the full disclosure of which is hereby incorporated by reference herein.

VI. PRODUCTION OF S BY MICROORGANISMS Filamentous fungi, or bacteria that produce cellulase, typically require a carbon source and an inducer for production of cellulase.

Lignocellulosic materials se different combinations of cellulose, hemicellulose and lignin. Cellulose is a linear polymer of glucose forming a fairly stiff linear structure without significant coiling. Due to this structure and the disposition of hydroxyl groups that can hydrogen bond, cellulose contains crystalline and non-crystalline portions. The crystalline portions can also be of different types, noted as I(alpha) and ) for example, depending on the location of hydrogen bonds between strands. The polymer lengths themselves can vary lending more variety to the form of the cellulose. Hemicellulose is any of several heteropolymers, such as xylan, glucuronoxylan, arabinoxylans, and ucan. The primary sugar monomer present is xylose, although other monomers such as mannose, galactose, rhamnose, arabinose and glucose are present. Typically hemicellulose forms branched structures with lower molecular weights than cellulose. Hemicellulose is therefore an amorphous al that is lly susceptible to enzymatic hydrolysis. Lignin is a complex high molecular weight heteropolymer generally. Although all lignins show variation in their composition, they have been described as an amorphous dendritic network r of phenyl propene units. The amounts of ose, hemicellulose and lignin in a specific biomaterial s on the source of the erial. For example wood derived biomaterial can be about 38-49% ose, 7—26% hemicellulose and 23-34% lignin ing on the type. Grasses typically are 33-38% cellulose, 24-32% hemicellulose and 17-22% lignin. Clearly ellulosic biomass constitutes a large class of substrates.

The diversity of biomass materials may be further increased by pretreatment, for example, by changing the crystallinity and molecular s of the rs.

The cellulase producing organism when contacted with a biomass will tend to produce enzymes that release molecules advantageous to the organism’s , such as glucose.

This is done through the phenomenon of enzyme induction as described above. Since there are a variety of substrates in a particular biomaterial, there are a variety of cellulases, for example, the endoglucanase, exoglucanase and cellobiase discussed usly. By selecting a particular lignocellulosic material as the r the relative concentrations and/or activities of these enzymes can be modulated so that the resulting enzyme complex will work ntly on the lignocellulosic material used as the inducer or a similar material. For example, a erial with a higher portion of crystalline cellulose may induce a more effective or higher amount of endoglucanase than a biomaterial with little crystalline cellulose.

One of ordinary skill in the art can optimize the production of enzymes by microorganisms by adding yeast extract, corn steep, peptones, amino acids, um salts, phosphate salts, potassium salts, magnesium salts, calcium salts, iron salts, manganese salts, zinc salts, cobalt salts, or other additives and/or nutrients and/or carbon sources. Various components can be added and removed during the processing to optimize the desired production of useful products.

Temperature, pH and other conditions optimal for grth of microorganisms and production of s are generally known in the art.

VII. SACCHARIFICATION The treated biomass materials can be saccharified, generally by combining the material and a cellulase enzyme in a fluid medium, 6.g. an aqueous solution. In some cases, the material is boiled, steeped, or cooked in hot water prior to saccharification, as described in US.

Pat. App. Pub. 2012/0100577 A1 by Medoff and Masterman, hed on April 26, 2012, the entire contents of which are orated .

The rification process can be partially or tely performed in a tank (e.g., a tank having a volume of at least 4000, 40,000, or 500,000 L) in a manufacturing plant, and/or can be partially or completely performed in transit, e.g, in a rail car, tanker truck, or in a supertanker or the hold of a ship. The time required for complete saccharification will depend on the process conditions and the biomass material and enzyme used. If saccharification is performed in a manufacturing plant under lled conditions, the cellulose may be substantially entirely converted to sugar, e.g., glucose in about 12-96 hours. If saccharification is performed partially or completely in transit, rification may take longer.

It is generally preferred that the tank contents be mixed during rification, e.g., using jet mixing as described in International App. No. PCT/U82010/035331, filed May 18, 2010, which was published in English as W0 2010/135380 and designated the United States, the full disclosure of which is incorporated by reference herein.

The addition of surfactants can enhance the rate of saccharification. Examples of surfactants include non-ionic surfactants, such as a Tween® 20 or Tween® 80 hylene glycol surfactants, ionic tants, or amphoteric surfactants.

It is generally preferred that the concentration of the sugar solution ing from saccharification be relatively high, e.g., greater than 40%, or greater than 50, 60, 70, 80, 90 or even greater than 95% by weight. Water may be removed, e.g, by evaporation, to increase the concentration of the sugar solution. This reduces the volume to be shipped, and also inhibits microbial growth in the solution.

Alternatively, sugar solutions of lower concentrations may be used, in which case it may be desirable to add an antimicrobial additive, e.g. , a broad spectrum antibiotic, in a low concentration, e.g., 50 to 150 ppm. Other le otics include ericin B, ampicillin, chloramphenicol, ciprofloxacin, gentamicin, hygromycin B, kanamycin, neomycin, penicillin, cin, streptomycin. Antibiotics will t growth of microorganisms during transport and storage, and can be used at appropriate concentrations, e.g, between 15 and 1000 ppm by weight, e.g, between 25 and 500 ppm, or between 50 and 150 ppm. If desired, an antibiotic can be included even if the sugar concentration is relatively high. Alternatively, other additives with anti-microbial of preservative properties may be used. Preferably the antimicrobial ve(s) are food-grade.

A relatively high concentration solution can be ed by limiting the amount of water added to the biomass material with the enzyme. The concentration can be controlled, e.g. by controlling how much saccharification takes place. For example, concentration can be increased by adding more biomass material to the solution. In order to keep the sugar that is being produced in solution, a surfactant can be added, e.g., one of those sed above.

Solubility can also be increased by increasing the temperature of the solution. For example, the solution can be maintained at a temperature of 40-50°C, 60-80°C, or even higher.

VIII. SACCHARIFYING AGENTS Suitable cellulolytic enzymes include cellulases from species in the genera Bacillus, Caprinus, Mycelz'ophthora, Cephalosporz'um, Scytalz'dz'um, Penicillium, Aspergz'llus, Pseudomonas, Humicola, Fusarium, Thielavz'a, Acremonium, ChrySOSporz'um and Trichoderma, especially those ed by a strain selected from the species ASpergz'lluS (see, e.g., EP Pub.

No. 0 458 162), Humicola nS (reclassified as lz'dz'um thermophilum, see, e.g., US. Pat.

No. 4,435,307), CaprinuS cinereuS, Fusarium oxySporum, Myceliophthora phila, Merz'pz'luS eus, Thielavz'a terrestriS, Acremom’um Sp. (including, but not limited to, A. perSl'cz'num, A. acremonium, A. brachypenium, A. dichromosporum, A. atum, A. pinkertoniae, A. roseogriseum, A. incoloratum, and A. furatum). Preferred strains include Humicola insolenS DSM 1800, Fusarz'um oxysporum DSM 2672, Mycell'ophthora thermophila CBS 117.65, Cephalosporz'um Sp. RYM-202, Acremonium Sp. CBS 478.94, Acremonium Sp.

CBS 265.95, Acremonium persicinum CBS 169.65, Acremonium acremonium AHU 9519, osporl'um Sp. CBS 535.71, Acremonium brachypenium CBS 866.73, Acremonium dichromospomm CBS 683.73, Acremonium obclavatum CBS , Acremom’um pinkertonz'ae CBS 157.70, Acremonium r0Seogrz'Seum CBS 134.56, Acremonium incoloratum CBS 146.62, and Acremoniumfuratum CBS 299.70H. Cellulolytic enzymes may also be obtained from ChrySOSporium, preferably a strain of ChrySOSporl'um lucknowense. Additional strains that can be used include, but are not limited to, Trichoderma (particularly T. viride, T. ', and T. koningii), alkalophilic BacilluS (see, for example, US. Pat. No. 3,844,890 and EP Pub. No. 0 458 162), and Streptomyces (see, e.g, EP Pub. No. 0 458 162).

Many microorganisms that can be used to saccharify biomass al and produce sugars can also be used to ferment and t those sugars to useful products.

IX. SUGARS In the processes described herein, for example after rification, sugars (e.g, e and xylose) can be ed. For example sugars can be isolated by precipitation, crystallization, chromatography (e.g, ted moving bed chromatography, high pressure chromatography), centrifugation, extraction, any other isolation method known in the art, and combinations thereof.

WO 96700 X. HYDROGENATION AND OTHER CHEMICAL TRANSFORMATIONS The processes bed herein can include hydrogenation. For example glucose and xylose can be hydrogenated to sorbitol and xylitol respectively. Hydrogenation can be accomplished by use of a catalyst (e.g., Pt/gamma-A1203, Ru/C, Raney , or other catalysts know in the art) in combination with H2 under high pressure (e.g., 10 to 12000 psi). Other types of al transformation of the products from the ses described herein can be used, for example production of organic sugar derived products such (e.g., furfural and furfural-derived products). Chemical transformations of sugar derived products are described in US Prov. App.

No. 61/667,481, filed July 3, 2012, the disclosure of which is incorporated herein by reference in its ty.

XI. FERMENTATION Yeast and nas bacteria, for example, can be used for fermentation or conversion of sugar(s) to l(s). Other microorganisms are discussed below. The optimum pH for ferrnentations is about pH 4 to 7. For example, the optimum pH for yeast is from about pH 4 to 5, while the optimum pH for Zymomonas is from about pH 5 to 6. Typical fermentation times are about 24 to 168 hours (e.g., 24 to 96 hrs) with temperatures in the range of 20°C to 40°C (e.g., 26°C to 40°C), however thermophilic microorganisms prefer higher temperatures.

In some embodiments, e.g., when anaerobic sms are used, at least a portion of the fermentation is conducted in the absence of oxygen, e. g., under a blanket of an inert gas such as N2, Ar, He, CO2 or mixtures thereof. Additionally, the mixture may have a constant purge of an inert gas flowing through the tank during part of or all of the tation. In some cases, bic condition, can be achieved or maintained by carbon dioxide production during the fermentation and no additional inert gas is needed.

In some embodiments, all or a portion of the fermentation s can be interrupted before the low molecular weight sugar is completely converted to a product (e.g., ethanol). The intermediate fermentation products include sugar and carbohydrates in high concentrations. The sugars and carbohydrates can be isolated Via any means known in the art. These intermediate fermentation products can be used in preparation of food for human or animal consumption.

Additionally or alternatively, the intermediate fermentation products can be ground to a fine particle size in a stainless-steel laboratory mill to produce a flour-like substance.

Jet mixing may be used during fermentation, and in some cases riflcation and fermentation are performed in the same tank.

Nutrients for the microorganisms may be added during saccharification and/or fermentation, for example the food-based nutrient es described in US. Pat. App. Pub. 2012/0052536, filed July 15, 2011, the complete disclosure of which is incorporated herein by nce.

“Fermentation” includes the methods and ts that are disclosed in US. Prov.

App. No. ,559, filed December 22, 2012, and US. Prov. App. No. 61/579,576, filed December 22, 2012, the contents of both of which are incorporated by reference herein in their entirety.

Mobile fermenters can be utilized, as described in International App. No. (which was filed July 20, 2007, was hed in English as WO 2008/011598 and designated the United States), the contents of which is incorporated herein in its entirety. Similarly, the saccharification equipment can be mobile. Further, saccharification and/or fermentation may be med in part or entirely during transit.

XII. FERMENTATION AGENTS The microorganism(s) used in fermentation can be naturally-occurring microorganisms and/or engineered microorganisms. For example, the microorganism can be a bacterium (including, but not limited to, e.g., a cellulolytic ium), a fungus, (including, but not d to, e.g, a yeast), a plant, a protist, e.g. a protozoa or a fungus—like protest (including, but not d to, e.g., a slime mold), or an alga. When the sms are compatible, mixtures of organisms can be utilized.

Suitable fermenting microorganisms have the ability to convert ydrates, such as glucose, fructose, xylose, arabinose, mannose, galactose, oligosaccharides or polysaccharides into fermentation products. ting microorganisms include strains of the genus Saccharomyces spp. (including, but not limited to, S. cerevisiae (baker’s yeast), S. distatz'cas, S. avaram), the genus Klayveromyces, (including, but not limited to, K. marxianas, K. fragilis), the genus Candida (including, but not limited to, C. pseudotropz'calz’s, and C. brassz'cae), Pichia stz'pitz's (a relative of Candida she/zatae), the genus Clavz'spora (including, but not limited to, C. lasitanz'ae and C. opantz'ae), the genus Pachysolen (including, but not limited to, P. tannophz'lus), the genus Bretannomyces (including, but not limited to, e. g., B. clausem'z' ppidis, G. P., 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization, Wyman, C.E., ed., Taylor & Francis, Washington, DC, 179-212)). Other suitable microorganisms include, for example, Zymomonas mobilis, z'dz'am spp. (including, but not d to, C. cellam (Philippidis, 1996, supra), C.saccharobatylacetonicam, C. saccharobatylz'cam, C. Paniceam, C. beg'jernckz'z', and C. acetobalylz'cum), Mom'lz'ella pollim's, Mom'lz'ella megachz'll'ensz's, Lactobacz'llas spp. Yarrowia lipolytica, Aureobasidl'am 519., Trichosporonoz’des sp., Trigonopsz's variabilis, Trichosporon sp., Monilz'ellaacetoabatans Sp.

Typhala ilis, Candida magnoliae, Ustilaginomycetes sp.,Pseadozyma tsakabaensis,yeast s of genera Zygosaccharomyces, Debaryomyces, Hansenala and Pichia,and fungi of the dematioid genus .

For instance, Clostrz'dz'um spp. can be used to produce ethanol, l, butyric acid, acetic acid, and acetone. Lactobacz'llas spp., can be used to produce lactice acid.

Many such microbial strains are publicly available, either commercially or through depositories such as the ATCC (American Type Culture Collection, Manassas, Virginia, USA), the NRRL (Agricultural ch Sevice Culture Collection, Peoria, is, USA), or the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany), to name a few.

Commercially available yeasts e, for example, Red Lesaffre Ethanol Red (available from Red esaffre, USA), FALI® (available from Fleischmann’s Yeast, a division of Burns Philip Food Inc., USA), SUPERSTART® (available from Alltech, now Lalemand), GERT STRAND® (available from Gert Strand AB, Sweden) and FERMOL® (available from DSM Specialties).

Many microorganisms that can be used to rify biomass material and produce sugars can also be used to ferment and convert those sugars to useful products.

XIII. DISTILLATION After fermentation, the resulting fluids can be distilled using, for example, a “beer column” to separate ethanol and other alcohols from the majority of water and residual solids.

The vapor exiting the beer column can be, e.g., 35% by weight ethanol and can be fed to a rectification column. A mixture of nearly azeotropic (92.5%) ethanol and water from the rectification column can be purified to pure (99.5%) ethanol using phase molecular sieves.

The beer column bottoms can be sent to the first effect of a three-effect evaporator. The rectification column reflux condenser can e heat for this first effect. After the first effect, solids can be separated using a centrifuge and dried in a rotary dryer. A portion (25%) of the centrifuge effluent can be ed to fermentation and the rest sent to the second and third evaporator s. Most of the evaporator condensate can be returned to the process as fairly clean condensate with a small portion split off to waste water treatment to prevent build-up of low-boiling compounds.

EXAMPLES Example 1. Effect of Exogenous Fructose on Saccharification This example tests whether or not exogenous fructose inhibits saccharification enzymes.

Three 225mL Erlenmeyer flasks were prepared, each with 10g of treated corn cob s (mesh size between 15 and 40, and ated to 35 Mrad with an electron beam) 100mL of water and 2.5mL of Duet AcceleraseTM (Danisco). To the first, , and third flask were added, respectively: 0g, 5g and 10g of fructose. The flasks were covered with aluminum foil and set in an incubator shaker at 50°C and 200rpm for four days. The amount of xylose and glucose was monitored by HPLC. The results of the saccharification are shown in the table below.

Table 1. Saccharification under varying levels of exogenous fructose. 5g added fructose 16.7 12.3 93.5 10g added fructose 18.1 12.6 101.3 Unlike e (a known inhibitor of cellobiase), 5% or 10% added fructose does not inhibit the saccharification of corncob. -46— 2012/071093 Example 2. Effect of Xylose Isomerase on Saccharification Glucose is a known inhibitor of cellobiase. This example tests if the conversion of glucose to the isomer fructose by xylose isomerase can increase saccharification.

Four 225mL Erlenmeyer flasks were prepared, each with 10g of d corn cob biomass and lOOmL of water. The biomass was treated as described in e 1. To the first, second, and third flask was added 2.5mL of Duet AcceleraseTM (Danisco). To the second, third, and fourth flasks were added, respectively: 1g, 0.1g and 0.1g of glucose isomerase zymeTM, Aldrich). The flasks were covered with aluminum foil and set in an incubator shaker at 50°C and 200rpm for four days. The amount of xylose and glucose was monitored by HPLC. The results ofthe saccharification are shown in the table below.

Table 2. Effectiveness of cellulase with added xylose isomerase. 2.5mL Duet -- 1g G] 28.3 20.6 125.2 122.3 2.5mL Duet -- 0.1g G] 24.6 18.5 109.0 109.4 0.1 g G1 1.6 Not detected 6.9 Not detected The addition of glucose isomerase was observed to increase the effectiveness of the ase enzyme, with flask 2 producing about 25% more sugars than flask 1.

Example 3. Use of a Strong Acid to Cleave Cellobiose This example tests the use of a strong acid to cleave cellobiose to e, to se saccharification yield. The strong acid used was Amberlyst-ISTM, a polystyrene sulfonic acid.

This is a strongly acidic sulfonic acid macroreticular polymeric resin that is based on crosslinked styrene divinylbenzene copolymers. Published studies indicate that Amberlyst-15 can cleave the dimer cellobiose to glucose.

Three 225mL Erlenmeyer flasks were prepared, each with 10g of d corn cob biomass, IOOmL of water and 2.5mL Duet AcceleraseTM. The biomass was treated as described in e 1. In the second flask 1g of glucose isomerase (SweetzymeTM, Aldrich) was added; and in the third 1 g of glucose isomerase and 0.1 g of polystyrene sulfonic acid (Amberlyst—15m, DOW) was added.

The flasks were covered with um foil and set in an incubator shaker at 50°C and 200rpm for four days. The amount of xylose and glucose was red by HPLC. The results of the saccharification are shown in the table below.

Table 3. Effect of an Acid on Saccharification. (g/L) (g/L) improved with G1 Duet alone 21.1 16.1 100 100 NA Duet + GI 26.5 19.2 125 119 NA Duet + G1 + 27.9 20.5 131 127 14 Amberlyst The results show an ement in the saccharification with the addition of glucose isomerase. The experiment also shows an improvement in the saccharification with the on of yrene sulfonic acid.

Example 4. Removal of Cellobiase This example examines saccharification where iase has been removed, while the endo- and exo-cellulases have been retained.

Chromatofocusing was used to separate the enzymes. Duet AcceleraseTM (Danisco) was injected onto a Mono P column using an AKTA system. The endo-and exo—cellulases bound to the column, while the cellobiase passed through and was removed. The exo- and endo- cellulases were then eluted from the column by shifting the pH to 4.0. The resulting fractions were combined and immediately applied to a saccharification reaction.

Table 4. Accumulation of Cellobiose and Sugars in the Absence of Cellobiase.

Sample Cellobiose Glucose Xylose Xylitol Lactose AKTA 1.057 4.361 5.826 0.556 purified Duet -48— Duet 0.398 16.999 14.830 0.726 Comcob (no 0.673 0.550 enzymes) Spun/Filtered 17.695 15.053 0.770 1.052 Duet The expected result was that without iase, there would be an accumulation of cellobiose. Although the yield was low, the table below shows that a detectable amount of cellobiose was indeed generated.

Other than in the examples herein, or unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages, such as those for s of materials, elemental contents, times and temperatures of reaction, ratios of amounts, and , in the following portion of the specification and attached claims may be read as if prefaced by the word “about” even though the term “about” may not expressly appear with the value, amount, or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the t invention. At the very least, and not as an attempt to limit the ation of the doctrine of equivalents to the scope of the claims, each numerical ter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. hstanding that the numerical ranges and ters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains error necessarily resulting from the standard deviation found in its underlying respective g measurements. Furthermore, when numerical ranges are set forth herein, these ranges are inclusive of the d range end points (i.e., end points may be used). When percentages by weight are used , the numerical values reported are relative to the total weight.

Also, it should be understood that any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, a range of “l to 10” is intended to include all sub-ranges between (and including) the recited minimum value of l and the recited maximum value of 10, that is, having a minimum value equal to or greater than 1 and a maximum value of equal to or less than 10. The terms “one,n u a) a or “an” as used herein are intended to include “at least one” or “one or more,” unless ise indicated.

Any patent, publication, or other disclosure material, in whole or in part, that is said to be incorporated by reference herein is incorporated herein only to the extent that the incorporated material does not t with ng definitions, statements, or other disclosure material set forth in this disclosure. As such, and to the extent necessary, the disclosure as explicitly set forth herein supersedes any conflicting material incorporated herein by reference.

Any material, or portion f, that is said to be incorporated by reference herein, but which conflicts with existing definitions, ents, or other disclosure material set forth herein will only be incorporated to the extent that no conflict arises between that incorporated material and the existing disclosure material.

While this invention has been particularly shown and described with references to preferred ments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without ing from the scope of the invention encompassed by the appended claims.

Claims (15)

CLAIMS What is claimed is:

1. A method of producing glucose, , and fructose, the method comprising: saccharifying recalcitrance-reduced ellulosic biomass with one or more ases and an acid on a support in the presence of xylose isomerase at between 30 °C and 65 °C to produce a mixture comprising glucose, fructose, and xylose.

2. The method of claim 1, wherein the one or more cellulases is an endoglucanase, an exo-splitting glucanase, a cellobiase, or a combination thereof.

3. The method of claim 1, wherein the xylose isomerase is produced from Pseudomonas hydrophila, Escherichia, intermedia, Bacillus megaterium, Paracolobacterium aerogenoides, or combination thereof.

4. The method of any one of claims 1-3, wherein the temperature is in the range of 60 to 65 degrees Celsius.

5. The method of any one of claims 1-4, wherein the pH is 7 or below.

6. The method of any one of claims 1-5, wherein said rifying to produce the mixture takes place under conditions of a pH in the range of 3 to 7.

7. The method of any one of claims 1-5, wherein the saccharifying to produce the mixture takes place under conditions of a pH in the range of 7 to 9.

8. The method of any one of claims 1-7, wherein the concentration of xylose isomerase is in the range of 0.1 to 500 U/g cellulose.

9. The method of any one of claims 1-8, wherein the itrance-reduced biomass has been eated with a treatment method selected from the group ting of: bombardments with electrons, sonication, oxidation, pyrolysis, steam explosion, chemical treatment, mechanical treatment, and freeze grinding.

10. The method of any one of claims 1-9, further comprising inoculating the glucose, , and fructose with an sm that metabolizes the glucose, but not the xylose, to form a metabolic product.

11. The method of claim 10, wherein the metabolic product is ethanol, butanol, butyric acid, acetic acid, acetone, or a combination thereof.

12. The method of any one of claims 1-11, wherein the lignocellulosic biomass is selected from the group ting of: wood, particle board, ry wastes, sawdust, aspen wood, wood chips, grasses, switchgrass, miscanthus, cord grass, reed canary grass, grain es, rice hulls, oat hulls, wheat chaff, barley hulls, agricultural waste, silage, canola straw, wheat straw, barley straw, oat straw, rice straw, jute, hemp, flax, bamboo, sisal, abaca, corn cobs, corn stover, soybean stover, corn fiber, alfalfa, hay, coconut hair, sugar processing residues, bagasse, beet pulp, agave e, algae, seaweed, manure, sewage, offal, agricultural or industrial waste, arracacha, eat, banana, barley, cassava, kudzu, oca, sago, sorghum, , sweet potato, taro, yams, beans, favas, lentils, peas, and mixtures of any of these.

13. The method of any one of claims 1-12, wherein the acid on the support is a strong acid.

14. The method of claim 13, wherein the acid on the support is polystyrene sulfonic acid.

15. The method of claim 14, wherein the acid on the support is a sulfonic acid macroreticular polymeric resin that is based on crosslinked styrene divinylbenzene copolymers.