OA16930A - Processing biomass. - Google Patents

Abstract

Provided herein are methods of increasing the efficiency of biomass saccharification. In particular, the methods include ways of avoiding feedback inhibition during the production of useful products.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application Nos. 61/579,552 and 61/579,559, both filed on December 22, 2011. Tlie entire disclosurcs of the above applications arc incorporated herein by reference.

FIELD OFTHE INVENTION [0002] Tlie invention pcrtains to cfficiencies useful in Üic processing of biomass materials. For example, the invention relates to processes that circumvent négative feedback of enzymatic reactions.

BACKGROUND [0003] As demand for petroleum increases, so too does interesl in renewable feedstocks for manufacturing biofucls 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 wilh food industry uses.

[0004] Many potential lignocellulosic feedstocks are available today, including agricultural residues, woody biomass, municipal waste, oilseeds/cakes and sea weeds, to name a few. At présent these materials are either used as animal feed, biocompost materials, are bumed in a 25 cogénération facility or are landfillcd.

[0005] Lignocellulosic biomass is récalcitrant to dégradation as the plant cell walls hâve a structure that is rigid and compact. Tlie structure comprises crystalline cellulose fibrils embedded in a hemiccllulase matrix, surrounded by lignîn. This compact matrix is difficult to access by enzymes and other chemical, biochcmical and biological processes. Cellulosic biomass materials (e.g., biomass material from which substantially ail the lignin has been removed) can be more accessible to enzymes and other conversion processes, but even so,

nalurally-occurring cellulosic materials often hâve low yields (relative to theoretical yields) when contacted with hydrolyzing enzymes. Lignocellulosic biomass is even more récalcitrant to enzyme attack. Furthermore, each type of lignocellulosic biomass has ils own spécifie composition of cellulose, hemicellulose and lignin.

[0006] While a number of methods hâve been tried to extract structural carbohydrates from lignocellulosic biomass, lhey are either are too expensive, produce too low a yield, leave undesirable chemicals in the resulting product, or simply dégrade the sugars.

[0007J Monosaccharides from renewable biomass sources could become the basis of chemical and fuels industries by rcplacing, supplementing or substituting petroleum and other fossil feedstocks. However, techniques nced lo be developed that will make these monosaccharides available in large quantifies and at acceptable purifies and prices.

SUMMARY OFTHE INVENTION [0008] Provided herein are methods of increasing the cfficicncy of .saccharification of I5 biomass. In particular, efficicncies can be achieved by avoiding négative feedback inhibition of enzymatic réactions.

[0009] Provided herein is a method of making a product, where the method includes: saccharifying rccaicitrance-rcduccd lignocellulosic biomass, and adding an isomerization agent to the saccharified biomass. In some implémentations, the saccharified biomass comprises a first 20 sugar and a second sugar and the isomerization agent is used to convert the second sugar to a third sugar. The method may also include, in some cases, contacting the saccharified biomass with a microorganism to convert the first sugar and third sugar to one or more product(s).

[0010] Also provided herein is a method of making a product with a microorganism from a first sugar and a second sugar, where the microorganism 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 lhe 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 microorganisin-bioniass combination under conditions thaï enablc lhe 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 Lhird sugar, wherein the microorganism is capable of converting the diird 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 product with a microorganism from the first sugar and the second sugar.

[0011] In another aspect, the invention features a method of increasing the amount of a product made by a microorganism from a saccharified biomass, the method comprising: providing a celiulosic or lignoceilulosic biomass; saccharifying the biomass to make a saccharified biomass, wherein the saccharified biomass comprises a first sugar and a second 10 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 convert lhe first sugar lo the product, producing a combination that comprises 15 the product and the second sugar; converting lhe second sugar to a third sugar, wherein the microorganism is capable of converting lhe lhird sugar to the product; and maintaining the microorganism under conditions thaï enable the microorganism to convert the third sugar to the product; thereby increasing the amount of lhe product made by the microorganism from the saccharified biomass.

[0012] In any of the methods provided herein, the lignoceilulosic biomass can be treated to reduce its recalcitrance to saccharification. The treatment method is selected from the group consisting of: bombardment with électrons, sonication, oxidation, pyrolysis, steam explosion, chemical treatment, mcchanical treatment, or freeze grinding. The treatment method can be bombardment with électrons.

[0013] In any of lhe methods, lhe conversion of the second sugar to lhe third sugar can be done before maintaining the microorganism-biomass combination under conditions that enable the microorganism lo convert the first sugar to the product. The conversion of the second sugar to the third sugar can be done immedîately after saccharification of the biomass, or it can bc done during saccharification of lhe biomass.

[0014] In lhe methods provided herein, the lignoceilulosic biomass can be selected from the group consisting of: wood, particic board, forestry wastes, sawdust, aspen wood, wood chips, grasses, swtlchgrass, miscanthus, cord grass, reed canary grass, grain residues, rice hulls, oat hulls, wheat chaff, barley hulls, agricultural waste, silage, canota straw, wheat straw, barley straw, oat straw, rice straw, jute, hemp, flax, bamboo, sisal, abaca, com cobs, com stover, soybean stover, com fiber, alfalfa, hay, coconut hair, sugar processing residues, bagasse, beet pulp, agave bagasse, algae, seaweed, manure, sewage, offal, agricultural or industrial waste, arTacacha, buckwheal, banana, barley, cassava, kudzu, oca, sago, sorghum, potato, sweel potato, taro, yams, beans, favas, lentils, peas, or mixtures of any of these. The lignocellulosic biomass can be mechanically treated to rcduce ils bulk density and/or increase its surface area. For instance, it can be comminuted, e.g., by dry milling, or by wet milling. The biomass can bc saccharified with one or more celluloses, [0015] In the methods provided herein, the isomerization agent can comprise an acid, e.g., polystyrène sulfonic acid.

[0016] In the methods provided herein, the microorganism-biomass combination can be maintaincd at a pH of about 10 to about 14, or at a pH of about 11 to about 13. It can bc maintaincd at a température of about 10°C (o about 30°C, or at a température of about 20°C. It can also bc maintaincd at a teniperature of ubout 60°C to about 65°C. Il can bc maintaincd at a pH of about 6.0 to about 7.5, or a pH of about 7.

[0017] In the methods, the second sugar can be glucose, and lhe third sugar can be fructose. The isomerization agent can comprise an enzyme. Alternatively, the second sugar can bc xylose, and the third sugar can bc xylulose. The enzyme can be xylose isomerase.

[0018] The microorganism can be yeast. The product can bc alcohol. The microorganism can bc Clostridium spp., and lhe product can be éthanol, butanol, bulyric acid, acetic acid, or acetone. The microorganism can be Lactobacillux spp., and the product can be lactic acid. [0019] It should be understood thaï this invention is not limited to the embodiments disclosed in tliis Summary, and it is intended to cover modifications that are within the spiril and scope of lhe invention, as defined by the daims.

BRIEFDESCRIPTION OFTHE DRAWINGS [0020] The foregoing will be apparent from the following more particular 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 nccessariiy to scale, emphasis instead being placed upon illustrating embodiments of the présent invention.

[0021] FIG. 1 is a diagram iIIustraling the enzymatic hydrolysis of cellulose to glucose. Cellulosic subsirate (A) is converted by endocellulase (i) to cellulose (B), which is converted by exoccllulase (îi) to cellobiose (C), which is converted to glucose (D) by cellobiase (betaglucosidase) (iii).

[0022] FIG. 2 is a flow diagram ilIustraling the action of cellulase on cellulose and cellulose dérivatives. Cellulose (200) is broken down to cellobiose (210) by endoglucanases and exoglucanases/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 glucose (E), and beta-glucosidase is inhibited by glucose (C).

[0023] FIG. 3 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 fonn a mixture (310), which is then allowed to saccharify (B), producing sugars (320). As disclosed herein, an additivc (325) is combined (C) with the mixture of sugars (320) to make a mixture of sugars and addilivc (330). The resulting sugars are then used (D) in downstream processing to produce one or more products (340), such as alcohol, lactic acid, or onc or more of the sugars themselves.

DETAILED DESCRIPTION [0024] Provided herein arc melhods of increasing the efficiency of production of sugars (and/or products made from the sugars) from saccharifïed biomass. The methods arc especially useful in cases where one or more sugars or products cause négative feedback, limiting the amount of sugars or products that can be produced.

[0025] 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 inciude sugars thaï cannot bc metabolized by the microorganism. As these non-utilizablc sugars increase in concentration, they represent a metabolic “dcad-end. Furthermore, some sugars may form the basis of feedback inhibition, and limit the throughpul of metabolic pathways that make desired sugars or other desired products.

[0026] Disclosed herein are methods for preventing such feedback inhibition, and încreasing the amount of sugars and other useful products from the saccharification of biomass.

[0027] The glucose produced during saccharification can inhibit further production of glucose. In one embodiment, therefore, lhe invention encompasses the effective removal of glucose by converting it to fructose (which does not inhibit saccharification), thereby allowing for lhe production of additional glucose. Glucose can bc converted to fructose by the action of enzymes (such as xylose isomerase), strong acids or chemicals (such as polystyrène sulfonic acid). Likcwise, xylose, which cannot be metaboiized by many microorganisms, can bc converted by xylose isomerase into xylulose, which can bc metaboiized by many microorganisms. In addition, xylulose often does not inhibit ils own production, unlike glucose. [0028] For instance, biomass can bc 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 lhe desired end product is alcohol, then increased saccharification, and increased production of glucose, followed by fermentation, will produce more alcohol, but il will also producc more xylose. While lhe xylose is not harmful, it can represent a metabolic “dead end. ΙΓ lhe xylose is converted lo xylulose, it can bc fermented to alcohol, and production efficiency can be increased.

[0029] As shown in FIG. I, l'or example, during saccharification a cellulosic substratc (A) is initiully hydrolyzed by endoglucanascs (i) at random locations producing oligomeric intermédiares (e.g., cellulose) (B). These intermédiares are then substrates for exo-splitting glucanascs (ii) such as ccllobiohydrolase to produce cellobiose from the ends of the cellulose polymer. Cellobiose is a water-soluble 1,4-linked dimer of glucose. Finally cellobiasc (iii) clcavcs cellobiose (C) to yield glucose (D). Therefore, the cndoglucanases are particularly effective in attacking the crystalline portions of cellulose and incrcasing the effectiveness of exoccllulascs to producc cellobiose, which then requires the specificity of lhe cellobiose lo produce glucose' Therefore, it is évident that depending on lhe nature and structure of the cellulosic substratc, lhe amount and type of the three different enzymes may need to bc modified. [0030] As shown in FIG. 2, hydrolysis of cellulose (200) to cellobiose (210) is a multi-stcp process which includes initial breakdown at the solid-liquid interface via the synergistic action of cndoglucanases (EG) and cxo-glucanases/cellobiohydrolases (CBH) (205) (A). This initial dégradation is accompanied by further liquid phase dégradation, by hydrolysis of soluble

intermediate products such as oligosaccharides and cellobiose that are catalytically cleaved by bela-glucosidase (BG; 215) (B) to glucose (220). However, 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.

[0031] FIG. 3 shows a process for manufacturing a product (340) from a feedstock (300).

The feedstock can be pre-processed, such as by réduction of the size and recalcitrance of the feedstock. This can include, for example, optionally mechanically treating the feedstock and, before and/or aller this treatment, optionally treating the feedstock with another treatment, for examplc, particle bombardment, to further reduce its recalcitrance. 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) îs combined (C) with the mixture of sugars (320) to make a mixture of sugars and additive (330). The additive (325) increases the effeclivcness of the cellulase during saccharification, e.g., by rcducing inhibition of the cellulase by cellobiose and/or glucose. This increased effeclivcness 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 onc or more of the sugars themselves.

[0032] During saccharification, the feedstock is treated with onc or more cellulolytic enzymes, generally by combining the feedstock and the enzyme (305) in a fluid medium, e.g., an 20 aqueous solution. In some cases, the feedstock is boilcd, steeped, or cookcd in hot water prior to saccharification, as described in U.S. Pat. App. Pub. 2012/0100577 Al, filed Octobcr 18,2011 and publishcd April 26, 2012, the entire contents of which are incorporated herein by referencc. [0033] The additive can be added at the initiation of the saccharification (B), for example, with the biomass and cellulase. Altematively, llie additive can bc added after some or ail of the 25 saccharification (B) has occurrcd. H can also be added at the start of producing a product.

[0034] The additive can bc a chemical or an enzyme. Examplcs of suitable additives include acids and bases. Bases can calalyze the Lobry-de-Bruyn-Albcrda-van-Ekcnstein transformation, as described in more detail below. Acids can catalyze the hydrolysis of cellobiose. Boronic acids can be used to complex with the cM’-diols of glucose. Xylose isomerase (a.k.a. glucose 30 isomerase) can bc used to isomerize glucose to fructose.

[0035] The additive can be physically supported. Useful supports include but are not limited to cationic polymcric supports, anionic polymeric supports, ncutral polymeric supports, métal oxide supports, métal carbonate supports, métal halide supports and/or mixtures thereof. The support can be added to the mixed sugars or can be stationary and the mixed sugars made to pass 5 through or over the supported additive.

[0036] The mixture containing the additive (330) can be rctumcd to the bioniass and cellulose stage (310) to release more sugars before being further processed. This can include rctuming 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 lhe acidic région (less than or equal lo 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 lhan or equal to pH 3, greater than or equal to pH 4). The température can be optimized for the action of cellulases, e.g., to greater than or equal to 30°C (greater lhan or equal to 40°C, greater than or equal to 50°C, greater than or equal lo 60°C) and less lhan or equal lo 90°C (less lhan or equal to 80°C, less lhan or equal to 70°C, less thon or equal to 60°C). Additional biomass, cel lulase and additive can optionaliy be added for increased production of sugars.

[0037] The sugar solution or suspension produced by saccharification can be subjected to downstream processing to obtain a desired product. For example, one or more of the sugars can be isolated, and/or tlie solution can be fermented. When fermentation is utilized, the fermentation product can be dislilled. For example, sugars can be hydrogenated and sugar alcohols isolated.

[0038] Without being bound by any particular theory, il is believed that this conversion effectivcly removes glucose from tlie mix of sugars. As shown in FIG. 2, this removal would remove the inhibition steps C and E. This increases the overall saccharification of cellulose in 25 the biomass.

[0039] In many instances, the optimum température for using glucose isomerase ranges from 60 to 80°C. In lhe processes described herein, températures lower than the optimum may be preferred because of cost and because the optimum température for other components of the process can be different. For example cellulase activilies are generally optimal between 30°C 30 and 65°C. A température range of about 60°C to about 65°C may therefore be preferred.

particularly if the glucose isomerase and ceilulase are combined and used simultaneously. If they are not used together, then optimal températures for each enzyme can be selected. [0040] The optimum pH range for glucose isomerase activity îs between pH 7 and 9. As with lhe sélection of the température range, in praclicing this invention a lower pH can be 5 preferred because in some cases other components of the process may rcquire a lower pH. For examplc, celluloses are active over a range of pH of about 3 to 7. The preferred pH for lhe combined enzymes is therefore generally at or below pH 7. If the glucose isomerase and ceilulase are not used together, then lhe optimal pH range for each enzyme can be selected.

[0041] Glucose isomerase can be added in any amount. For example, the concentration may 10 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 cellulose, at least about 0.5 U/g cellulose to about 250 U/g cellulose, al least about 1 U/g cellulose to about 100 U/g cellulose, at least about 2 U/g cellulose to about 50 U/g cellulose.

(0042] In some cases, the addition of a glucose isomerase incrcases Lhe amount of sugars produced by at least 5 % (e.g., at least 10 %, al least 15 %, al least 20 %, at least 30, 40, 50, 60, 70, 80, 90, 100 %).

[0043] Another additive that can be used in the invention is, e.g., a chemical that increases the activity of the saccharifying agent. The chcmicai can be, for example, a chemical thaï facilitâtes the Lobry-dc-Bruyn-van-Alberdu-van-Ekenstcin transformation (also called the Lobry-dc-Bruyn-van-Ekenstein transformation). This réaction forms an cnol from an aldosc which can then form a kclosc. For examplc, in the pH range of 11 to 13 and at a température of 20°C, alkali will catalyze the transformation of D-glucosc into D-fructose and D-mannose. Typically the réaction is base catalyzcd, but it can also be acid catalyzcd, or lake place under ncutral conditions. As with the use of glucose isomerase, this reaction effectively removes glucose.

[0044] As another examplc, an acid can be used to catalyze hydrolysis of cellobiose. By using chemical means to cleavc cellobiose to glucose, rather than enzymalic or inicrobial means, inhibition of these réactions by glucose does not occur.

[0045] In another example, lhe chemical can be one that rcacts with glucose, such as a boronic acid which binds preferentially to cis-diols.

rei 16930

[0046] The chemical can bc on a support, for example, by polystyrène sulfonates (such as an Amberlysl ) or polystyrène amines. The mixed sugars can be passed through the supported chemical or flow over it. For example, the chemical can be a polystyrène supported boronic acid. The glucose can be trapped as a borate by the polystyrène support and then released at a latcr stage, by addition of base for example.

XYLOSEISOMERASE [0047] Xylose isomerase (ES 5.3.1.5) is an enzyme the catalyzes the chemical reaction back and forlh 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 intramolecular oxidoreductases interconverting aldoses and keioses. Other names in common use include D-xylose isomerase, D-xylose ketoisomerase, and D-xylose kctolisomerasc. The enzyme participâtes in pentose and glucuronate inlerconversions and fructose and mannose metabolism. It is used induslrially to convcrt glucose to fructose in the manufacture of high-fruclose corn syrup. Il is sometimes referred to as “glucose isomerase. “Xylose isomerase” and “glucose isomerase are used intcrchangcably herein. In vitro, glucose isomerase catalyzes the interconversion of glucose and fructose, in vivo, it catalyzes the interconvcrsion of xylose and xylulosc.

[0048] Several types of enzymes are considcred xylose isomerases. The first kind is produced from Pseitdomonas liydrophila. This enzyme has 160 times lower affinity to glucose than xylose but nonctlicless is useful for increasing the amount of fructose in the presence of glucose. A second kind of enzyme is found in Escherichia intennedia. This enzyme is a phophoglucose isomerase (EC 5.3.1.9) and can isomerize unphosphorylatcd sugar only in lhe presence of arscnatc. A glucose isomerase (EC 5.3.16) can be isolated from Bacillns inegaterium AI and is NAD linked and is spécifie to glucose. Another glucose isomerase having similar activity is isolated from Paracolobacterium aerogenoides. Glucose isomerases produced by heterolaclic acid bacteria require xylose as an inducer and arc reiatively unstable at high température. 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 reiatively heat stable.

[0049] The glucose isomerases are usually produced inlcrcellularly but reports of exlracellular sécrétion of glucose isomerases are known. The enzyme used can bc isolated from

many bacleria including but not limited to: Actinomyces olivocineretts, Actinomyces phaeochromogenes, Actinoplanes missouriensis, Aerobacter aerogenes Aerobacter cloacae, Aerobacter levanictun, Arthrobacter spp., Bacilhts stearodtennophihts, Bacilltts megabacterittm, Bacilhts coagulons, Biftdobacierittm spp., Brevibacterium incertum, Brevibacterium pentosoantinoacidicum, Chainia spp., Corynebacterium spp., Cortobacterium helvolam, Escherichla freandii, Escherichia intermedia, Esclterichia coli, Flavobacterium arborescens, Flavobacterium devorans, Lactobacillits brevis, Lactobacillus buchneri, Lactobacillus ferinenti, Laclobacilhts mannitopoetts, Lactobacillus gayonii, Lactobacillus plantaritm, Lactobacillus lycopersici, Lactobacilltts pentosits, Leuconostoc mesenteroides, Microbispora rosea, Microellobosporia flavea, Microntonospora coerttla, Mycobacterium spp., Nocardia astéroïdes, Nocardia corallia, Nocardia dassonvillei, Paracolobacterbmi aerogenoides, Psettdonocardia spp., Pseitdotttonas hydrophila, Sarcina spp., Staphylococctts bibila, Staphylococcusflavovirens, Staphylococcits échinants, Streptococcits achromogenes, Streptococcus phaeochromogenes, Streptococctts fracliae, Streptococcus roseochrontogenes, Streptococcus olivaceits, Streptococcits califomicos, Streptococcus venuceus, Streptococcus virginiai, Streptotnyces olivochromogenes, Streptococcus venezaelie, Streptococcus wedinorensis, Streptococcus griseoitts, Streptococcus glattcescens, Streptococcus bikiniensis, Streptococctts rttbiginosus, Streptococcits achinatus, Streptococcus cinnantonaisis, Streptococcus fradiae, Streptococctts albtts, Streptococcus grisais, Streptococcits hivens, Streptococcus matensis, Streptococctts mitrinus, Streptococcus nivens, Streptococctts platensis, Streptosporanghtm album, Streptosporangium oui gare, Thennopolyspora spp., Thennus spp., Xanfhontonas spp. and Zytnononas ntobllis.

[0050] Glucose isomerasc 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 dicthylaminoethyl cellulose, métal oxides, métal chlorides, métal carbonates and polystyrènes. Immobilîzalion can be accomplishcd by any suitable means. For examplc immobilization can be accomplislicd by contacling lhe support and the whole cell or enzyme in a solvent such as water and then removing the solvent. The solvent can be removed by any suitable means, for example filtration or évaporation or spray drying. As another cxample, spray drying the whole cells or enzyme with a support can be effective.

[0051] Glucose isomerase can also be présent 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 éthanol fermenting bacteria. Altematively, die glucose-isomerase-producing bacteria can be first contacted with die substrate, followed by inoculaling with an cthanol5 producing substrate.

[0052] Glucose isomerase can also be présent 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 express the gene for production of glucose isomerase.

I. TREATMENT OF BIOMASS MATERIAL

A. PARTICLE BOMBARDMENT [0053] One or more treatments with energetic particle bombardment can be used to process raw feedstock 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 15 processing steps and/or sequences. Particle bombardment can reduce the molecular weight and/or crystallinity of feedstock. In some embodiments, energy deposited in a material Üiat releascs an électron from its atoinic orbital can be used to treat the materials. The bombardment may be provided by heavy charged particles (sucli as alpha particles or protons), électrons (produced, for example, in beta dccay or électron beam acceleralors), or electromagnetic 20 radiation (for example, gamma rays, x rays, or ultraviolet rays). Altematively, radiation produced by radioactive substances can be used to treat the feedstock. Any combination, in any order, or concurrcntly of these treatments may bc utilized. In another approach, electromagnetic radiation (e.g., produced using électron beam cmitters) can be used to treat the feedstock.

[0054] Each form of energy ionizes the bioniass via particular interactions. Heavy charged 25 particles primarily ionize matlcr via Coulomb scattcring; furthermore, these interactions produce energetic électrons that may further ionize matlcr. Alpha particles are identical to the nucléus of a hélium atom and are produced by lhe alpha decay of various radioactive nuclei, such as isotopes of bismuth, polonium, astatinc, radon, francium, radium, scveral actinides, such as actinium, thorium, uranium, neptunium, curium, californium, américium, and plutonium.

[0055] When particles are utilized, they can be neutral (uncharged), posîtively charged or negatively charged. When charged, lhe charged particles can bear a single positive or négative

charge, or multiple charges, e.g., one, two, three or even four or more charges. In instances in which chain scission is desired, positively charged particies may be désirable, in part, due to their acidic nature, When particies are utilized, the particies can hâve the mass of a resling électron, or greater, e.g., 500, 1000, 1500, or 2000 or more times the mass of a resling électron. For examplc, the particies can hâve a mass of from about l atomic unit to about 150 atomic units, e.g., from about l atomic unit to about 50 atomic units, or from about l to about 25, e.g., I, 2, 3, 4, 5, 10, 12 or 15 atomic unies. Accelerators used to accelerate the particies can be electrostatic DC, electrodynamic DC, RF linear, magnetic induction linear or continuous wave. For examplc, cyclotron type accelerators are available from IBA (Ion Beam Accelerators, Louvain-la-Neuve, I0 Belgium), such as the Rhodotron™ system, while DC type accelerators are available from RDI, now IBA Industrial, such as the Dynamilron™. Ions and ion accelerators are discussed in Introductory Nuclear Physics, Kenneth S. Kranc, John Wiley & Sons, Inc. ( I988), 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-Phase15 Focuscd IH-DTL for Heavy-Ion Medical Accelerators, Proceedings of EPAC 2006, Edinburgh, Scotland; and Leitner, C. M. et al., “Status of the Superconducting ECR Ion Source Venus, Proceedings of EPAC 2000, Vicnna, Austria.

[0056] The doses applied dépend on the desired effect and the particular feedstock. For example, high doses can break chemical bonds within feedstock components and low doses can 20 increase chemical bonding (e.g., cross-linking) within feedstock components.

[0057] In some instances when chain scission is désirable and/or polymer chain functionalization is désirable, particies heavier than électrons, such as protons, hélium nuclei, argon ions, silicon ions, néon ions, carbon ions, phosphores ions, oxygen ions or nitrogen ions can be utilized. When ring-opening chain scission is desired, positively charged particies can be 25 utilized for their Lewis acid properties for enhanced ring-opening chain scission. For example, wlicn oxygen-containing functional groups are desired, treatment in the presence of oxygen or even treatment with oxygen ions can be performed. For cxample, when nitrogen-containing functional groups arc désirable, treatment in tlie presence of nitrogen or even treatment with nitrogen ions can be performed.

irrrÆA irr*¹ ·η^·,~π£

B. OTHER FORMS OF ENERGY [0058] Electrons internet via Coulomb scatlering and bremsstrahlung radiation produced by changes in the velocily of électrons. Electrons may be produced by radioactive nuclei that undergo beta decay, such as isotopes of iodïne, césium, technetium, and iridium. Altematively, an électron gun can be used as an électron source via thermionic émission.

[0059] Electromagnetic radiation interacts via three processes: photoclectric 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 lhe absorbed radiation in cellulosic material can be expressed by the mass absorption coefficient.

[0060] Electromagnetic radiation is subclassified as gamma rays, x rays, ultraviolet rays, infrared rays, microwavcs, or radiowaves, depending on lhe wavelength.

[0061] For example, gamma radiation can be employed to treat the materials. Gamma radiation lias the advanlage of a significanl pénétration depth into a variety of material in the sample. Sources of gamma rays include radioactive nuclei, such as isotopes of cobalt, calcium, technetium, chromium, gallium, indium, iodine, iron, krypton, samarium, sélénium, sodium, thulium, and xenon.

[0062] Sources of x rays include électron beam collision with métal targets, such as lungsten or molybdenum or alloys, or compact liglil sources, such as those produced commercially by Lynccan.

[0063] Sources for ultraviolet radiation include deuterium or cadmium lamps.

[0064] Sources for infrared radiation include sapphire, zinc, or selenide window ccramic lamps. | [0065] Sources for microwaves include klystrons, Slcvin type RF sources, or atom beam sources that cinploy hydrogen, oxygen, or nitrogen gascs.

[0066] Various other devices may be used in the methods disciosed herein, including field ionization sources, clcctrostatic ion separators, field ionization generators, thermionic émission sources, microwave discharge ion sources, recirculating or static accelerators, dynamic linear accelerators, van de Graaff accelerators, and foldcd tandem accelerators. Such devices are disciosed, for example, in U.S. Pat. No. 7,931,784 B2, the complète disclosure of which is incorporated herein by reference.

C. ELECTRON BOMBARDMENT

l. Electron Beams [0067] The feedstock may be treated with électron bombardment to modify ils structure and thereby reduce ils recalcitrance. Such treatment may, for example, reduce lhe average molecular weight of tlie feedstock, change the crystalline structure of the feedstock, and/or increase the surface area and/or porosily of the feedstock.

[0068] Electron bombardment via an électron beam is generally preferred, because it provides very high throughput and because lhe use of a relatively low vohage/high power électron beam device éliminâtes the need for expensive concrète vault shielding, as such devices are “self-shielded and provide a safe, efficient process. While lhe self-shielded devices do include shielding (e.g., métal plaie shielding), they do not rcquire the construction of a concrète vault, grcatly reducing capital expenditure and often allowing an existîng manufacturing facility to be used without expensive modification. Electron beam accelerators are available, for example, from IBA (Ion Beam Applications, Louvain-la-Neuve, Bclgium), Titan Corporation (San Diego, California, USA), and NHV Corporation (Nippon High Voltage, Japan).

[0069] Electron bombardment may be performed using an électron beam device that has a nominal energy of less than 10 McV, e.g., less than 7 McV, less than 5 MeV, or less than 2 McV, e.g., from about 0.5 to l .5 MeV, from about 0.8 to l .8 MeV, from about 0.7 to l MeV, or from about l to 3 MeV. In some implémentations the nominal energy is about 500 to 800 keV. [0070] The électron beam may hâve a relatively high total beam power (the combined beam power ol'all accelcrating heads, or, if multiple accelerators are used, of ail accelerators and ail heads), e.g., at least 25 kW, e.g., at least 30,40,50,60,65, 70, 80, I00, 125, or 150 kW. ln some cases, the power is even as high as 500 kW, 750 kW, or even 1000 kW or more. In some cases the électron beam has a beam power of 1200 kW or more.

[0071] This high total beam power is usually achieved by utilizing multiple accelcrating heads. For example, lhe électron beam device may include two, four, or more accelcrating heads. The use of multiple heads, each of which has a relatively low beam power, prevenls excessive température rise in the material, thereby preventing buming of the material, and also increases the uniformity of lhe dose through tlie thickncss of lhe layer of material.

[0072] In some implémentations, it is désirable to cool the material during électron bombardment. For example, lhe material can be cooled while it is being conveyed, for example by a screw extrader or other conveying equipment.

[0073] To rcduce the energy required by the recalcitrance-reducing process, il is désirable to treat the material as quîckly as possible. In general, it is preferred thaï treatment be performed at a dose rate of greater than about 0,25 Mrad per second, e.g., greater than about 0.5,0.75, l, 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 décomposition of the material. In one implémentation, lhe accelerator is set for 3 MeV, 50 mAmp beam

I0 current, and the line speed is 24 feet/minute, for a sample thickness of about 20 mm (e.g., comminutcd com cob material with a bulk density of 0.5 g/cm³).

[0074] In some embodiments, électron bombardment is performed unlîl die material receives a total dose of at least 0.5 Mrad, e.g., al least 5, 10, 20, 30 or at least 40 Mrad. In some embodiments, the treatment is performed unlil lhe material receives a dose of from about 0.5

I5 Mrad to about 150 Mrad, about l Mrad to about 100 Mrad, about 2 Mrad lo about 75 Mrad, 10 Mrad to about 50 Mrad, e.g., about 5 Mrad to about 50 Mrad, from about 20 Mrad lo about 40 Mrad, about I0 Mrad lo about 35 Mrad, or from about 25 Mrad to about 30 Mrad. In some implémentations, a total dose of 25 lo 35 Mrad is preferred, applied ideally over a couple of seconds, e.g., al 5 Mrad/pass with each pass being applied for about one second. Applying a dose of greater than 7 to S Mrad/pass can in some cases cause thermal dégradation of the feedstock material.

[0075] 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 lo 18 Mrad/pass, separated by a few seconds of cool-down, or three passes of 7 lo 12 Mrad/pass, e.g., 9 to 11 Mrad/pass. As discussed above, treating the material widi scvcral relatively low doses, rather than one high dose, tends lo prevent overheating of lhe material and also increases dose uniformity through lhe thickness of the material. In some implémentations, the material is stirred or otherwise mixed during or after each pass and then smoodicd into a uniform layer again before the next pass, to further enhancc treatment uniformity.

[0076] In some embodiments, électrons 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.

[0077] In some embodiments, any processing described herein occurs on lignocellulosic material that remains 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 material has less than about five percent by weight retained water, measured at 25°C and al fïfty percent relative humidity.

[0078] Electron bombardment can be applied while the cellulosic and/or lignocellulosic material is exposed to air, oxygen-enriched air, or even oxygen itself, or blanketed by an inert gas such as nitrogen, argon, or hélium. When maximum oxidation is desired, an oxidizing environment is utilized, such as air or oxygen and the distance from the beam source is optimized to maximize réactivé gas formation, e.g., ozone and/or oxides of nitrogen.

[0079] ln some embodiments, two or more électron sources are used, such as two or more |5 ionizing sources. For example, samples can be treated, in any order, with a beam of électrons, followcd by gamma radiation and UV light having wavelengths from about 100 nm to about 280 nm. In some embodiments, samples arc treated with three ionizing radiation sources, such as a ι

beam of électrons, gamma radiation, and energetic UV light. The biomuss is conveyed through the treatment zone where it can be bombarded with électrons. Il is generally preferred that the 20 bed of biomass material has a relatively uniform thickncss, as previously described, while being treated. ¹ [0080] Il may be advantageous to repeat the treatment to more thoroughly rcducc the recalcitrance of the biomass and/or further modify lhe biomass. In particular Ote process parameters can bc adjusted afler a first (e.g., second, third, fourth or more) pass depending on the 25 recalcitrance of the material. ln some embodiments, a conveyor can be used which includes a circular system where lhe biomass is conveyed multiple times through the various processes described above. ln some other embodiments multiple treatment devices (e.g., électron beam generators) are used to treal the biomass multiple (e.g., 2, 3,4 or more) times. In yet other embodiments, a single électron beam generator may be lhe source of multiple bean» (e.g., 2, 3, 4 30 or more bcams) thaï can be used for treatment of lhe biomass.

(0081] The effectiveness in changing the molecular/supermolecular structure and/or reducing die rccalcitrance of the biomass material dépends on the électron energy used and the dose applied, while exposure lime dépends on the power and dose.

[0082] In some embodiments, the treatment (with any électron 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 embodiments, the treatment is performed until the material receives a dose of between 0,1-100 Mrad, 1-200, 5-200, 10-200,5-150, 5-100,5-50, 5-40, 10-50, 10-75, 15-50, 20-35 Mrad.

[0083] ln 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. ln other embodiments the treatment is performed at a dose rate of between 10 and 10000 kilorads/hr, between 100 and 1000 kilorad/hr, or between 500 and 1000 kilorads/hr.

2. Electron Sources [0084] Electrons interact via Coulomb scattering and brcmsstrahlung radiation produced by changes in the velocity of électrons. Electrons may be produced by radioactive nuclei that undergo beta dccay, such as isotopes of iodinc, césium, technetium, and iridium. Alternatively, an électron gun can bc used as an électron source via lhermionic émission and aceelerated through un acccleraling potential. An électron gun generales électrons, accélérâtes them through a large potential (e.g., greater than about 500 thousand, greater than about lmillion, greater than 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 électrons arc initially aceelerated in die z direction down the tube and extracted üirougli a foil window. Scanning the électron beam is useful for increasing the irradiation surface when irradiating materials, e.g., a biomass, that is conveycd through the scanned beam. Scanning the électron beam also distribuas the thermal load honiogenously on die window and hclps reduce the foil window rupture duc to local heatîng by lhe électron beam. Window foil rupture is a cause of significant down-time duc to subséquent necessary repairs and re-starting die électron gun.

[0085] Various other irradiating devices may be used in the methods disclosed herein, including field ionization sources, elcclrostalic ion separators, field ionization generators, thermionic émission sources, microwavc discharge ion sources, recirculating or slalic acceleralors, dynamic linear accelerators, van de Graaff accclcraiors, and folded tandem accclcraiors. Such devices are disclosed, for example, in U.S. Pat. No. 7,931,784 to Medoff, the complété disclosure of which is incorporated herein by reference.

[0086] A beam of électrons can be uscd as tlie radiation source. A beam of électrons has the advantages of high dose rates (e.g., I, 5, or even 10 Mrad per second), high throughput, less contaminent, and less confinement equipmenl. Electron beams can also hâve high electrical efficiency (e.g., 80%), allowing for lower energy usage relative to other radiation methods, which can translate into a lower cost of operation and lower greenhouse gas émissions corresponding to the smaller amount of energy used. Electron beams can be gcneraled, e.g., by electrostatic generators, cascade generators, transformer generators, low energy accelerators with a scanning system, low energy accclcraiors with a linear cathode, linear accelerators, and pulsed 15 accelerators.

[0087] Electrons can also be more efficient at causing changes in the molecular structure of biomass materials, for examplc, by lhe mechanism of chain scission. In addition, électrons having énergies of 0.5-10 McV can penetratc low density materials, such as lhe biomass materials described herein, e.g., materials having a bulk density of less than 0.5 g/cm³, and a depth of 0.3-10 cm. Electrons as an ionizing radiation source can bc uscful, e.g., for relatively thin piles, layers or beds of malcriais, e.g., less lhan 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 électron of the électron beam is from about 0.3 MeV to about 2.0 MeV (million électron volts), e.g., from about 0.5 MeV to about i.5 McV, or from about 0.7 MeV to about 1.25 McV.

Methods of irradiating materials arc discusscd in U.S. Pat. App. Pub. 2012/0100577 Al, filed Oclobcr 18, 2011, the enlire disclosure of which is herein incorporated by reference. [0088] Electron beam irradiation devices may be procurcd commercially from Ion Beam Applications (Louvain-la-Ncuve, Belgium), the Titan Corporation (San Diego, California, USA), and NHV Corporation (Nippon High Voltage, Japan). Typical électron energies can be 0.5

McV, I McV, 2 MeV, 4.5 MeV, 7.5 MeV, or 10 MeV. Typical électron beam irradiation device power can bc I KW, 5 KW, 10 KW, 20 KW, 50 KW, 60 KW, 70 KW, 80 KW, 90 KW, 100 KW.

I25 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.

[0089] Tradeoffs in considering électron beam irradiation device power spécifications include cost to operate, capital costs, dépréciation, and device foolprint. Tradeoffs in considering exposurc dose levels of électron beam irradiation would be energy costs and environment, safety, and health (ESH) concems. Typically, generalors are housed in a vault, e.g., of lead or concrète, especially for production from X-rays that are generated in the process. Tradeoffs in considering électron energies include energy costs.

[0090] The électron 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 speeds, as this wouid effectively replace a large, fixed beam width. Further, available sweep widlhs of 0.5 m, I m, 2 m or more are available. The scanning beam is preferred in most embodiments describe herein because of lhe larger scan width and reduced possibility of local heating and failure of the Windows.

3. Electron Guns - Windows [0091] When treated with an électron gun, lhe 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 imperméable to gascs, yet électrons can pass with low résistance while 20 being imperméable to gasscs. The foil Windows are preferably between about 10 and 100 microns thick (e.g., a window can be 10 microns 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). Tliin Windows dissipate less energy as an électron beam passes through them (e.g., the résistive heating is less since Power = 25 I²R) which is advantageous with respect to irradiating the target material (e.g., biomass) with as much energy as possible. Thin Windows are also less mechanicaily strong and more likely to fail which causes increased expense and more downtime for the equipment.

[0092] 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 die enclosure and to cool the 30 window from the side outside of the encloscd conveying system to avoid lofting up any particulatcs of the material being irradiated.

2I [0093] The system can include more than one window, e.g., a primary window and a secondary window, 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 électron beam apparatus includes a vacuum between lhe électron source and lhe primary window, and breakage ofthe primary window is likely to cause biomass material to be sucked up into the électron beam apparatus, resulting in damage, repair costs, and equipment downlime.

[0094] The window can be polymer, ceramic, coated ceramic, composite or coated composite. The secondary window can be, for instance, a continuous sheet/roil of polymer or coated polymer, which can be advanced continuously or at intervals to provide a clean or new section to serve as lhe secondary window.

[0095] The primary window and lhe 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, molybdenum, ruthénium, rhodium, palladium, hafnium, tantalum, tungsten, rhénium, platinum, iridium, or alloys or mixtures of any of these. The secondary single-type window foil can be made from titanium, scandium, vanadium, chromium, nickel, zirconium, niobium, molybdenum, ruthénium, rhodium, palladium, hafnium, tantalum, tungsten, rhénium, platinum, iridium, béryllium, aluminum, silicon, or alloys or mixtures of any of these. The primary and secondary Windows can be of lhe same material, mixture of materials, or alloy, or different materials, mixtures of material or alloys. One or both ofthe Windows can be laminates of the same of different materials, mixtures of materials, or alloys.

[0096] One of more of die Windows can hâve a support structure across its face. The term “single-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 lhe surface of lhe window into two parts. Such a double-type window is shown in U.S. Pat. No. 5,877,582 to Nishimura. Additional support structures can also be used.

[0097] The primary window foil and the secondary window foil can both be made from low Z élément. Alternatively, lhe primary window foil can be made from a high Z element, and the secondary window foil can be made from a low Z element.

[0098] The embodiments described herein do not precludc lhe inclusion of additional Windows, which may hâve a protective function, or may be included to modify the radiation exposure, [0099] The Windows can be concave, fiat or convex. It is generally preferred that the window be slighlly convex, in a direction away from the direction of the cooling iluid. This curvature improves the mechanical strength of the window and increases the permitled température levels as well as allowing a better flow path for lhe cooling fluid. On the side of the scanning hom tlie curvature tends to be towards the vacuum (e.g., away from tlie cooling fluid) due to the vacuum (e.g., aboul 10⁵ to 10'¹⁰ torr, about 10'⁶ to 10‘⁹ torr, about l0‘⁷ to 10‘⁸ torr). [0100] 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 électron gun currents (e.g., al least aboul 110 mA, at least about 120 mA, al least about 130 mA, at least aboul 140 mA, at least about 150 mA at least about 200 mA, at least about 500 mA, al least about 1000 mA) because résistive heating is approximately related lo lhe square of the current as discussed above. The Windows can be any shape but typicaily are approximately rectangular with a high aspect ratio of tlie width to tlie length (where lhe width direction is the same as the width of the conveying system perpcndicular to lhe conveying direction, and tlie length is the same as tlie direction of conveying). The distance of the window to tlie convcycd material can be less than about 10 cm (e.g., less lhan about 5cm) and more than about 0.1cm (e.g., more than about 1cm, more than about 2 cm, more than about 3 cm, more than about 4 cm). It is also possible to use multiple Windows (e.g., 3,4, 5, 6 or more) with different and varied shapes and configured in different ways. For example a primary or secondary foil window can include one, two or more Windows in die same plane or layered and can include one or more support structures. For example support structures can be a bar or a grid in tlie same plane and contacting the Windows.

[0101] 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 électron beam. In odier embodiments there is no enclosure for conveying the biomass material, e.g., lhe biomass is conveycd in air under the irradiation device.

[0102] A two-foil window extraction system for a scanning électron beam has two Windows, a primary and a secondary window. Generally the primary window is closest to the électron source, and is concave towards the top of the scanning hom duc to tlie vacuum on thaï side of the

window. The secondary foil window tends to be flatter but is also concave in the same direction. Tlîis curvature helps provide structural support to the window and is mechanically sironger than a fiat window. Altematîvely lhe Windows can be fiat or curved in any direction. The window foils are typically at least about 10 microns thick to aboul 30 microns thick (e.g., about 15-40 microns, about 20-30 microns, about 5-30 microns, aboul 8-25 microns, about 10-20 microns, about 20-25 microns thick). The distance between lhe front surface of die primary window foil and back surface of die secondary window foil is preferably less than 30 cm, more preferably less than 20 cm, and most preferably less than 10 cm. Sidewalis, in combination with the primary and secondary Windows, can define an interior space. Electrons travel through both

Windows lo impinge on and penetratc die material (e.g., biomass) disposed beneath. A first inlet can be included on one sidewali can be arranged lo 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 lhen reverse direclion on meeting the far (opposite) wall and flow back generally through lhe center of lhe interior space and then oui through an exhaust port and or outlet. A second inlet can be included on die sidewali and can be arranged to allow cooling fluid to impinge on the secondary window foil in a similar fashion. Optionally more inlcls (e.g., 2, 3,4, 5,6 or more) can bring cooling fluid to lhe primary and secondary window surfaces and multiple oullets (e.g., 2,3, 4, 5, 6 or more) can allow die cooling fluid lo exit lhe interior space. In some embodimenls one or more side walls can even be a mesh, screen or grate wiüi many openings through which cooling gas can flow while providing structural support lo the Windows.

[0103] Such window Systems arc described in U.S. Provisional App. No. 61/711,801, by Mcdoff et al., which was fded on October 10,2012, the entire contents of which are incorporated herein by reference. A variety of configurations for such a system will also be known to those of ordinary skill in lhe art.

4. Electron Guns - Window Spacing [0104] Although a large spacing between the Windows can be advantageous, for example, for lhe reasons described above, the large spacing poses some disadvantages. One disadvantage of a large spacing between Windows is that the électron beams will pass through a larger volume of cooling gas which can cause energy losses. For example a 1 MeV beam loses aboul 0.2 Me V/M of energy, a 5 McV beam loses about 0.23 MeV/M and a 10 MeV beam loses about 0.26

MeV/M. Therefore wilh a l McV beam of électrons passing through l cm of air, the beam loses only 0.2% of its energy, at 10 cm of air, lhe beam loses 2% of its energy, at 20 cm this is 4% of ils energy, while at 50 cm lhe energy loss is 10%. Since the électrons also hâve to travel from the secondary foil window to the biomass through additional air, the gap between the Windows must bc carcfully controlled. Preferably, energy losses are less that about 20% (e.g., less than 10%, less lhan 5% or even less than I %). It is therefore advantageous to minimize lhe spacing between Lhe Windows to decrease energy losses. Optimal spacing (e.g., average spacing) between the Windows (e.g., between the surface side ofthe électron 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 between about 2 and 20 cm (e.g., between aboul 3 and 20 cm, between about 4 and 20 cm, between aboul 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 aboul 8 and 15 cm between about 3 and 10 cm, between about 4 and 10 cm, between about 5 and 10 cm, between aboul 6 and 10 cm, between about 7 and 10 cm, between about 8 and 10 cm).

[01051 Onc of ordinary skill in the art will balance lhe advantages and disadvantages of window spacing to suit their needs.

[0106] In some embodiments support structures for the Windows can be used across Lhe

Windows, although these types of structures are less preferred because of energy losses that can occur to the électron beam as it strikes these kinds of structures.

[0107] A large spacing between the Windows can bc advantageous because it defines a larger volume between die Windows and allows for rapid ilowing of a large volume cooling gasses for very efficient cooling. The inlets and outlets arc between 1mm and 120 mm in diameter (e.g., about 2 ntm, about 5 mm about 10 mm, about 20 mm, aboul 50 mm or even about 100 mm).

The cooling gas flow can be al 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 lo 2000 CFM, about 700-2000 CFM, about 800 lo 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% of the gas is cxchangcd 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).

5. Electron Guns - Cooling and Purging Gases [0108] 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 embodiment the gas is an inert gas such as nitrogen, argon, hélium and or carbon dioxide. Il is preferred to use a gas rather than a liquid since encrgy fosses to the électron beam are minimized. Mixtures of pure 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 condenscd gas (e.g., liquid nitrogen, liquid hélium).

[0109] When using an enclosure, the enclosed conveyor can also be purged with an inert gas so as to mainlain an atmosphère al a reduced oxygen level. Keeping oxygen levels low avoids the formation of ozone which in some instances is undesirable due to its reactive and loxic nature. For examplc Lhe oxygen can be less titan about 20% (e.g., less than about 10%, less than about 1%, less titan 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, hélium or carbon dioxide. This can be supplicd, for examplc, front a boil off of a liquid source (e.g., liquid nitrogen or hélium), generated or separated froin air in situ, or supplied from tanks. The inert gas can be recirculated and any residual oxygen can be removed using a catalyst, such as a copper catalyst bed. Alternatively, combinations of purging, recirculating and oxygen rentoval can be done to keep lhe oxygen levels low.

[0110] The enclosure can also be purged with a reactive gas that can react with the biomass. This can be done beforc, during or after lhe irradiation process. The réactivé gas can be, but is not limited to, nitrous oxide, ammonia, oxygen, ozone, hydrocarbons, aromatic compounds, amides, peroxides, azides, halides, oxyhalides, phosphides, phosphines, arsines, sulfîdes, thiols, borancs and/or hydrides. The réactivé gas can be activated in the enclosure, e.g., by irradiation (e.g., électron beam, UV irradiation, microwavc irradiation, heating, IR radiation), so that il rcacts with lhe biomass. The biomass itself can be activated, for example by irradiation. Preferably the biomass is activated by the électron beam, to produce radicals which then react with lhe activated or unactivated reactive gas, e.g., by radical coupling or quenching.

[0111] Purging gascs supplicd 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 16930

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 [0112] In some embodiments the Systems and methods include a beam stop (e.g., a shuttcr). For example, the beam stop can bc used to quickly stop or reduce the irradiation of material without powering down the électron beam device. Altematively the beam stop can be used while powering up the électron beam, e.g., the beam stop can stop the électron 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 il can be moved into and out of the beam path. Even partial coverage of the beam can be used, for examplc, to control the dose of irradiation. The beam stop can be mounted to tlie floor, to a conveyor for tlie biomass, 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 hom so that the beam can be effectively controllcd by the beam stop. The beam stop can incorporate a hinge, a rail, whccls, 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 tire électrons, 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% of the électrons.

[0113] The beam stop can bc made of a métal 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., mctal-coatcd ceramic, metal-coatcd polymer, melal-coated composite, multilayered métal materials).

[0114] The beam stop can be cooled, for example, with a cooling fluid such as an aqueous solution or a gas. The beam stop can be partially or completely hollow, for example with cavilies. Interior spaces of the beam stop can be used for cooling fluids and gases. The beam stop can be of any shape, including fiat, curved, round, oval, square, rectangular, beveled and wcdged shapes.

[0115] The beam stop can hâve perforations so as to allow some électrons through, thus controlling (e.g., reducing) tlie levels of radiation across the whole area of the window, or in

spécifie régions 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 position.

D. TREATMENT OF BIOMASS MATERIAL ~ SONICATION, PYROLYSIS, OXIDATION, STEAM EXPLOSION [0116] If desired, one or more sonication, pyrolysis, oxidative, or steam explosion processes can be used in addition to or inslead of other treatments lo further reduce the recalcitrance of the 10 biomass material. These processes can be applied before, during and or after another treatment or treatments. These processes are described in detail in U.S. Pat. No. 7,932,065 to Medoff, lhe full disclosure of which is incorporated herein by référencé.

II. BIOMASS MATERIALS [0117] As used herein, lhe term “biomass materials includes lignoceilulosic, celiulosic, starchy, and microbial materials.

[0118] Lignoceilulosic materials include, but are not limited to, wood, particle board, foreslry wastes (e.g., sawdust, aspen wood, wood chips), grasses, (e.g., switchgrass, miscanthus, cord grass, rced canary grass), grain residues, (e.g., rice hulls, oat hulls, wheat chaff, barley hulls), agricullural waste (e.g., silage, canola straw, wheat straw, barley straw, oat straw, rice straw, jute, hemp, flax, bamboo, sisal, abaca, com cobs, com stover, soybean stover, com fiber, alfalfa, hay, coconul hair), sugar processing residues (e.g., bagasse, beet pulp, agave bagasse),, algac, scawced, manure, sewage, and mixtures of any of these.

[0119] ln some cases, lhe lignoceilulosic material includes comcobs. Ground or hainmermillcd comcobs 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 facilitaie harvest and collection, in some cases lhe entire com plant is used, including lhe com stalk, com kernels, and in some cases even lhe root system of the plant.

[0120] Advantageously, for éthanol production, no additional nutrients (other than a nitrogen 30 source, e.g., urea or ammonia) are required during fermentation of comcobs or celiulosic or lignoceilulosic materials containing significant amounts of comcobs. Other products may

require addition of trace metals, vilamins, or buffering capacity, but these adjustment are well within the knowledge of those of ordinary skill in lhe art.

[0121] Comcobs, before and after comminution, are also easier (o convey and disperse, and hâve a lesser tendency to form explosive mixtures in air than other cellulosic or lignocellulosic materials such as hay and grasses.

[0122] Cellulosic materials include, for example, paper, paper products, paper waste, paper pulp, pigmented papers, loaded papers, coated papers, filled papers, magazines, printed matter (e.g., books, catalogs, manuals, labels, calendars, greeting cards, brochures, prospectuses, newsprint), printer paper, polycoated paper, card stock, cardboard, paperboard, materials having 10 a high ot-cellulose content such as colton, and mixtures of any of these. For example paper products as described in U.S. App. No. 13/396,365 (“Magazine Feedstocks by Mcdoff et al., filed February 14, 2012), the full disclosure of which is incorporated herein by référencé.

[0123] Cellulosic materials can also include lignocellulosic materials which hâve been delignified.

[0124] Starchy materials include starch itsclf, e.g., com starch, wheat starch, potato starch or rice starch, a dérivative of starch, or a material tliat includes starch, such as an cdiblc food product or a crop. For example, tire starchy material can be arracacha, buckwheat, banana, barley, cassava, kudzu, oca, sago, sorghum, regular household potalocs, sweet potato, laro, yams, or one or more beans, such as favas, lcntils or peas. Blonds of any two or more starchy materials arc also starchy materials. Mixtures of starchy, cellulosic and or lignocellulosic materials can also be used. For example, a biomass can be an entirc plant, a part of a plant or different parts of a plant, e.g., a wheat plant, cotton plant, a com plant, rice plant or a tree. The starchy materials can be treated by any of the methods described herein, [0125] Microbiai materials include, but are not limited to, any naturally occurring or genetically modified microorganism or organism thaï contains or is capable of providing a source of carbohydrates (e.g., cellulose), for example, protists, e.g., animal protists (e.g., protozoa such as flagellâtes, amoeboids, cil talcs, and sporozoa) and plant protists (e.g., algae such alvcolales, chiorarachniophytcs, cryptomonads, cuglenids, glaucophytes, haptophytes, red aigac, stramenopiles, and viridaeplantae). Other examples include seaweed, planklon (e.g., macroplanklon, mesoplankton, microplankton, nanoplankton, picoplankton, and femptoplanklon), phytoplanklon, bacteria (e.g., gram positive bacteria, gram negalive bacteria,

and extrcmophiles), yeast and/or mixtures of these. In some instances, microbial biomass can be obtained from natural sources, e.g., the océan, lakes, bodies of water, e.g., sait water or fresh water, or on land. Alternativeiy or in addition, microbial biomass can be obtained from culture Systems, e.g., large scale dry and wet culture and fermentation Systems.

[0126] Tlie biomass material can also include offal, and similar sources of material.

[0127] In ollier embodiments, the biomass materials, such as cellulosic, starchy and lignocellulosic feedstock materials, can be obtained from transgenic microorganisms and plants that bave been modified with respect to a wild type variety. Such modifications may be, for example, through the itérative steps of sélection and breeding to obtain desired traits in a plant.

Furthermore, the plants can hâve had genetic material removed, modified, silenced and/or added with respect to lhe wild type variety. For example, genetically modified plants can be produced by recombinant DNA methods, where genetic modifications include introducing or modifying spécifie genes from parental varieties, or, for example, by using transgenic breeding wherein a spécifie 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 breeding wherein new allèles are artificially creatcd from endogenous 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 alkylaling agents, epoxîdes, alkaloids, peroxides, formaldéhyde), irradiation (e.g., X-rays, gamma rays, neutrons, bêla particles, alpha particles, protons, deulerons, UV radiation) and température shocking or other extemal stressing and subséquent sélection techniques. Other methods of providing modified genes is through error prône PCR and DNA shuffling followed by insertion ofthe desired modified DNA into lhe desired plant or seed. Methods of introducing lhe desired genetic variation in lhe seed or plant include, for example, tlie use of a bacterial carrier, biolistics, calcium phosphate précipitation, cicciroporation, gene splicing, gene silencing, lipofeelion, microinjection and viral carriers. Additional genetically modified materials hâve been described in U.S. Application Serial No 13/396,369 filed February 14, 2012 the full disclosure of which is incorporated herein by référencé.

[0128] Any of the methods described herein can be practiced with mixtures of any biomass materials described herein.

III. BIOMASS MATERIAL PREPARATION - MECHANICAL TREATMENTS [0129] The bîomass 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 bîomass can also be delivercd in a wet state, for example as a wet solid, a slurry or a suspension 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.%).

[0130] The processes disclosed herein can ulilize low bulk density materials, for example cellulosic or lignocellulosîc feedstocks that hâve been physîcally pretreated to hâve a bulk density of less 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, lhe method involves filling a measuring cylinder of known volume with a sample and oblaining a weight of Lhe sample. The bulk density is calculated by dividîng lhe weight of the sample in grams by lhe known volume of the cylinder in cubic centimeters. If desired, low bulk density materials can be densified, for example, by methods described in US. Pal. No. 7,971,809 to Medoff, the full disclosure of which is hcreby incorporated by reference. [0131] In some cases, Üic pre-treatment processing includes screening of the biomass material. Screening can be through a mesh or perforaled plate with a desired opening size, for example, less than about 6.35 mm (1/4 inch, 0.25 inch), (e.g., less lhan aboul 3.18 mm (1/8 inch, 0.125 inch), less than about 1.59 mm (1/16 inch, 0.0625 inch), is less lhan about 0.79 mm (1/32 inch, 0.03125 inch), e.g., less than aboul 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 lhan about 0.13 mm (0.005 inch), or even less lhan aboul 0.10 mm (1/256 inch, 0.00390625 inch)). In one configuration lhe desired biomass l’alls through the perforations or screen and thus biomass larger than the perforations or screen are not irradiated. These larger materials can bc rcprocessed, for example by comminuting, or they can simply bc removed from processing. In another configuration material that is larger than the perforations is irradiated and lhe smaller material is removed by lhe screening process or rccycled. 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 β

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.

[0132] Screening of material can also be by a manual method, for example by an operator or mechanoid (e.g., a robot equipped wilh a color, reflectivily or other sensor) that removes unwanled material. Screening can also be by magnetic screening wherein a magnet is disposed near the conveyed material and lhe magnetic material is removed magnetically.

[0133] Optîonal pre-lreaiment processing can include heating lhe material. For example a portion of the conveyor can be sent through a heated zone. The heated zone can be created, l'or example, by IR radiation, microwaves, combustion (e.g., gas, coal, oil, biomass), résistive heating 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 ail tlie material. For example, a portion of the conveying trough can be heated by use of a heating jacket. Heating can be, for example, for lhe purposc of drying Lhe material. ln 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, nitrogen, He, CO₂, Argon) over and/or through lhe biomass as it is being conveyed. [0134] Optionally, pre-treatment processing can include cooling lhe material. Cooling material is described in US Pal. No. 7,900,857 to Medoff, the disclosure of which in incorporated herein by reference. For example, cooling can be by supplying a cooling fluid, for example water (e.g., wilh glycerol), or nitrogen (e.g., liquid nitrogen) to the bottom ofthe conveying trough. Altematively, a cooling gas, for example, chillcd nitrogen can be blown over the biomass materials or under the conveying system.

[0135] Another optîonal pre-treatment processing method can include adding a material to the biomass. The additional material can be added by, for example, by showering, sprinkJing and or pouring the material onto lhe biomass as it is conveyed. Materials that can be added include, for example, metals, ccramics and/or ions as described in U.S. Pat. App. Pub. 2OJO/O1O5119 Al (filed October 26,2009) and U.S. Pat. App. Pub. 2010/0159569 Al (filed December 16, 2009), lhe entire disclosures of which are incorporated herein by reference. Optîonal materials that can be added include acids and bases. Other materials Üiat can be added are oxidants (e.g., peroxides, chlorates), polymers, polymerizable monomers (t-.g., containing 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 organic solvent) and/or as a

solution. In some cases the solvent is volatile and can be made lo evaporate e.g., by heating and/or blowing gas as previously described. The added material may form a uniform coating on the biomass or be a homogeneous mixture of different components (e.g., biomass and additional material). The added material can modulate the subséquent irradiation step by increasing the effïciency of the irradiation, damping the irradiation or changing the effect of the irradiation (e.g., from électron beams to X-rays or heat). The method may hâve no impact on the irradiation but may be useful for further downstream processing. The added material may help in conveying the material, for example, by lowering dust levels.

[0136] Biomass can be delivered to the conveyor by a belt conveyor, a pneumalic conveyor, a screw conveyor, a hopper, a pipe, manually or by a combination of these, The biomass can, for examplc, 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 atmosphère and/or control dust and fines. Lofted or air suspended biomass fines and dust are undesirable because these can form an explosion hazard or damage the window foils of an électron gun (if such a device is used for treating the material). [0137] The material can be levcled 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 inches, between about 0.125 and 0.5 inches, between about 0.3 and 0.9 inches, between about 0.2 and 0.5 inches between 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 incites, 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 inches, 0.900 +/- 0.025 inches.

[0138] Generally, it is preferred to convey the material as quickly as possible through the électron beam to maximize throughput. For examplc the material can be conveyed at rates of at least I ft/min. e.g., at least 2 ft/min, at least 3 ft/min, al 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 tlie beam current, for example, for a W incli tliick 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 10 ft/min to provide approximately the same irradiation dosage.

[0139] After the biomass material has been conveyed through the radiation zone, optional post-ire aiment proccssing can bc done. The optional post-treatment processing can, for example, be a process described with respect lo the pre-irradialion processing. For example, the biomass can bc screened, heated, cooled, and/or combined with additives. Uniquely to post-irradiation, quenching of the radicals can occur, for examplc, quenching of radicals by the addition of fluids or gases (e.g., oxygen, nitrous oxide, ammonia, liquids), using pressure, heat, and/or the addition of radical scavengcrs. For examplc, the biomass 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 biomass is exposed during irradiation to the reactive gas or fluid. Quenching of biomass that has been irradiated is described in U.S. Pat. No, 8,083,906 to Medoff, the entire disclosure of which is incorporate herein by reference.

[0140] If desired, one or more mechanical treatments can be used in addition to irradiation to further reduce tlie recalcitrance of the biomass material. These processes can be applied before, during and or after irradiation.

[0141] In some cases, the mechanical treatment may include an initial préparation of the feedstock as received, e.g., size réduction 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 mutcrials, or switchgrass) is prepared by shearing or shredding. Mechanical treatment may rcduce 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. [0142] Alternative!y, or in addition, the feedstock material can first be physically treated by one or more of tbe other physical treatment methods, e.g., chemical treatment, radiation, sonication, oxidation, pyrolysis or steam explosion, and then mechanically treated. This sequence can be advantageous since materials treated by one or more of the other treatments, e.g., irradiation or pyrolysis, tend to be more britlle and, therefore, it may be casier lo further change the structure of the material by mechanical treatment. For example, a feedstock material can bc conveyed through ionizing radiation using a conveyor as described herein and then mechanically treated. Chemical treatment can remove some or ail of the Hgnin (for example chemical pulping) and can partially or complctely 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 hydrolyzcd and non-hydrolyzed

materials, for example with about 50% or more non-hydrolyzed material, with about 60% or more non- hydrolyzed material, will) about 70% or more non-hydrolyzed material, with about 80% or more non-hydrolyzed material or even with 90% or more non-hydrolyzed material. [0143] ln addition to size réduction, which can be performed initially and/or later in processing, mechanical treatment can also be advantageous for opening up, “stressing, breaking or shattcring the biomass materials, making lhe cellulose of the materials more susceptible to chain scission and/or disruption of crystalline structure during the physical Irealnient.

[0144] Methods of mechanically treating the biomass material include, for example, milling or grînding. Milling may be performed using, for exemple, a hammer mill, bail mill, colloid mill, conical or cône mill, disk mill, edge mill, Wiley mill, grist mill or Ollier mill. Grînding may be performed using, for example, a cutting/impact type grinder. Some exemplary grinders include stone grinders, pin grinders, coffee grinders, and burr grinders. Grînding or milling may be provided, for example, by a reciprocating pin or other element, as is the case in a pin mill.

Ollier mechanical treatment methods include mechanical ripping or tearing, other methods thaï apply pressure to the fibers, and air attrition milling. Suitable mechanical treatments further include any other technique thaï continues the disruption of lhe internai structure of lhe material that was initiated by the previous processing steps.

[0145] Mechanical feed préparation Systems can be configured to produce streams with spécifie characleristics such as, for example, spécifie maximum sizes, spécifie length-to-width, or spécifie surface areas ratios. Physical préparation can increase the rate of reactions, improve the movement of material on a conveyor, improve lhe irradiation profile of the material, improve lhe radiation uniformily of the material, or reduce the processing time required by opening up lhe materials and making them more accessible to processes and/or reagents, such as reagents in a 25 solution.

[0146] The bulk density of feedstocks can be controlled (e.g., increased). In some situations, it can be désirable to prépare a low bulk density material, e.g., by dcnsifying the material (e.g., densification can make il casier and less costly to transport lo 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 i

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 material can be densified by the methods and equipment disclosed in U.S. Pat. No. 7,932,065 to Medoff and International Publication No. WO 2008/073186 (which was filed October 26, 2007, was published in English, and which designated die United States), the full disclosures of which are incorporated herein by reference. Densified materials can bc proccsscd by any of the methods described herein, or any material processed by any of the mediods described herein can be subsequently densified.

[0147] In some embodiments, the material to be processed is in the form of a fi brous material that includes ftbers provided by shearing a liber source. For examplc, die shearing can be performed with a rotary knife cutter.

[0148] For example, a fiber source, e.g., that is récalcitrant or that has had its recalcitrance level reduced, can be sheared, e.g., in a rotary knife cutter, to provide a first fibrous material. The first fibrous material is passed through a first screen, e.g., having an average opening size of 1,59 mm or less (1/16 inch, 0.0625 inch), provide a second fibrous material. If desired, die fiber source can be eut prior to die shearing, e.g., with a shredder. For example, when a paper is used as the fiber source, the paper can be first cul into strips that arc, e.g., 1/4- to 1/2-inch wide, using a shredder, e.g., a counter-rolaling screw shredder, such as those manufactured by Munson (Utica, N.Y.). As an alternative to shredding, the paper can be reduced in size by cutting to a desired size using a guillotine cutter. For example, die guillotine cutter can be used to eut the paper into shccts that arc, e.g., 10 inchcs wide by 12 inclics long.

[0149] In some embodiments, the shearing of the fiber source and the passing of the resulting first fibrous material through a first screen arc performed concurrcntly. The shearing and die passing can also bc performed in a batch-typc process.

[0150] For examplc, a rotary knife cutter can be used to concurrcntly shear die fiber source and screen the first fibrous material. A rotary knife cutter includes a hopper diat can be loaded with a shredded fiber source prepared by shredding a fiber source. The shreddcd fiber source.

[0151] In some implémentations, the fcedstock 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 mcchanical treatment, chemical treatment, irradiation, sonication, oxidation, pyrolysis or steam explosion. Treatment mcdiods can be used in combinations of two, three, four, or even ail of these technologies (in any order). When more dian one treatment

I 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.

[0152] Mechanîcal treatments thaï may be used, and the characteristics ofthe mechanically treated biomass materials, are described in further detail in U.S. Pat. App. Pub. 2012/0100577 Al, filed Octobcr 18, 2011, the full disclosure of which is hereby incorporated herein by reference.

IV. USE OF TREATED BIOMASS MATERIAL [0153] Using the methods described herein, a starting biomass material (e.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 internai combustion engines or feedstocks for fuel cells. Systems and processes are described herein that can use as feedstock cellulosic and/or lignoceilulosic materials thaï are readily availabie, but often can be difficult to process, e.g., municipal waste streams and waste paper streams, such as streams that inciude newspaper, kraft paper, corrugated paper or mixtures of these.

[0154] In order to convert the feedstock to a form that can be readily proccssed, the glucanor xylan-containing cellulose in the feedstock can bc hydrolyzed to low molecular weight carbohydrates, such as sugars, by a saccharil'ying 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 exislîng manufacturing plant, such as a single cell protein plant, an enzyme manufacturing plant, or a fuel plant, e.g., an éthanol manufacturing facility.

[0155] The feedstock can bc hydrolyzed using an enzyme, e.g., by combining the materials and the enzyme in a solvent, e.g., in an aqueous solution.

[0156] Alternatively, the enzymes can be supplicd by organisms thaï break down biomass, such as the cellulose and/or the lignin portions ofthe biomass, contain or manufacture various cellulolytic enzymes (celluloses), ligninases or various small molécule biomass-degrading métabolites. These enzymes may be a complex of enzymes thaï act syncrgistically to dégradé crystalline cellulose or the lignin portions of biomass. Examplcs of cellulolytic enzymes inciude: endoglucanases, ccllobiohydrolascs, and ccllobiases (bctu-glucosidases).

-î-,ι [0157] During saccharification a cellulosic substrate can be initially hydrolyzed by endoglucanases at random locations producing oligomeric intermediates. These intermediates are then substrates for cxo-splitting glucanases such as cellobiohydrolase to produce cellobiose from the ends of the cellulose polymer. Cellobiose is a water-soluble 1,4-linkcd dimer of glucose. Finally, cellobiase cleaves cellobiose to yield glucose. The efficiency (e.g., time to hydrolyze and/or completeness of hydrolysis) of this process dépends on lhe recalcitrance of lhe cellulosic material.

V. INTERMEDIATES AND PRODUCTS [0158] Using the processes described herein, the biomass material can be converted to one or more products. such as energy, fuels, foods and materials. Spécifie examples of products include, but are not limited to, hydrogen, sugars (e.g., glucose, xylose, arabinose, mannose, galactose, fructose, disaccharidcs, oligosaccharides and polysaccharides), alcohols (e.g., monohydric alcohols or dihydric alcohols, such as éthanol, n-propanol, isobulanol, .vec-butanol, tcrt-butanol or n-butanol), hydrated or hydrous alcohols (e.g., containing greater than 10%, 20%, 30% or even greater than 40% water), biodiesel, organic acids, hydrocarbons (e.g., methane, ethane, propane, isobulcne, pentane, n-hexane, biodiesel, bio-gasoiinc and mixtures thereof), coproduis (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((,',g., methyl, ethyl and n-propyl esters), ketoncs (e.g., acétone), aldéhydes (e.g., acetaldéhyde), alpha and beta unsaturated acids (e.g., acrylic acid) and olcfins (e.g., ethylene). Ollier alcohols and alcohol dérivatives include propanol, propylene glycol, 1,4-butancdiol, 1,3propancdiol, sugar alcohols and polyols (e.g., glycol, glycerol, erythritol, threitol, arabitol, xylilo], ribitol, mannitol, sorbitol, galactitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, mallotriitol, maltotetraîtol, and polyglycitol and other polyols), and methyl or ethyl esters of any of these alcohols. Ollier products include methyl acrylate, mcthylmethacrylate, lactic acid, citric acid, formic acid, accLic acid, propionic acid, butyric acid, succinic acid, valeric acid, caproic acid, 3-hydroxypropionic acid, palmitic acid, stcaric acid, oxalîc acid, malonic acid, glularic

acid, oleic acid, linolcic acid, glycolic acid, gamma-hydroxybutyric acid, and mixtures thereof, salts of any of these acids, mixtures of any of the acids and their respective salts.

[0159] Any combination of lhe above products with each ollier, and/or of the above products with other products, which other products may bc made by lhe 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.

[0160] Any of the products or combinations of products described herein may be sanitized or sterilized prior to selling the products, e.g., after purification or isolation or even after packaging, to nculralizc one or more potenlially undesirable contaminants thaï could be présent in the product(s). Such sanilation can be donc with électron 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 I to 3 Mrad.

[0161] The processes described herein can produce various by-product streams useful for generaling steam and electricity to be uscd in other parts of lhe plant (co-gcncration) or sold on 15 lhe open market. For example, steam generated from buming by-product streams can be used in a distillation process. As another example, electricity generated from buming by-product streams can be used to power électron beam generators used in pretreatment.

[0162] The by-producls uscd to generate steam and electricity are derived from a number of sources throughout tlie process. For examplc, anaérobie digestion of wastewater can produce a 20 biogas high in méthane and a small amount of waste biomass (sludge). As another example, post-saccharification and/or posl-distillatc solids (e.g., uneonverted lignin, cellulose, and hemicellulosc remaining from the pretreatment and primary processes) can be used, e.g., bumed, as a fuel.

[0163] Many of lhe products obtained, such as éthanol or n-butanol, can bc utilized as a fuel 25 for powering cars, trucks, trac lors, ships or trains, e.g., as an internai combustion fuel or as a fuel cell feedstock. Many of lhe products obtained can also bc 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 génération, e.g., in a conventional steam generating plant or in a fuel cell plant.

[0164] Olher tntcniiediates and products, including food and pharmaceutical products, aie described in U.S. Pat. App. Pub. 2010/0124583 Al, published May 20,2010, to Medoff, the full disclosure of which is hereby incorporated by référencé herein.

VI. PRODUCTION OF ENZYMES BY MICROORGANISMS [0165] Fîlamentous fungi, or bacteria that produce cellulase, typically require a carbon source and an inducer for production of cellulase.

[0166] Lignocellulosic materials comprise 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 Lhe 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 I(beta) for example, depending on the location of hydrogen bonds between strands. The polymer lengths themselves can vary lending more variety to lhe form of lhe cellulose. Hemicellulose is any of several heteropolymers, such as xylan, glucuronoxylan, arabinoxylans, and xyloglucan. The primary sugar monomer présent is xylose, although other monomers such as mannose, galactose, rhamnose, arabinose and glucose are présent. Typically hemicellulose forms branched structures with lower molecular weights than cellulose. Hemicellulose is therefore an amorphous material that is generally susceptible to enzymatic hydrolysis. Lignin is a complex high molecular weight heteropolymer generally. Although ail lignins show variation in their composition, theyhave been described as an amorphous dendritic network polymer of phenyl propene units. The amounls ol'cellulose, hemicellulose and lignin in a spécifie biomatcrial dépends on the source of the biomaterial. For example wood derived biomaterial can be about 38-49% cellulose, 7-26% hemicellulose and 23-34% lignin depending on the type. Grasses typically are 33-38% cellulose, 24-32% hemicellulose and 17-22% lignin. Clearly lignocellulosic biomass conslitutes a large class of substrates.

[0167] The diversity of biomass materials may be further increased by pretrcatmenl, for example, by changing the crystallinity and molecular weights of the polymers.

[0168] The cellulase producing organism when contacted with a biomass will tend to produce enzymes that release molécules advantageous to the organism’s growth, such as glucose. This is donc 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 cndoglucanase, cxoglucanase and cellobiase discussed previously. By selecting a particular lignocellulosic material as the induccr lhe relative concentrations and/or activities of these enzymes can be modulated so that the resulting enzyme complex will work effîcicntly on the 5 lignocellulosic material used as the inducer or a similar material. For example, a biomaterial with a higher portion of crystalline cellulose may induce a more effective or higher amount of endoglucanase than a biomaterial with little crystalline cellulose.

[0169] One of ordinary skill in the art can optimize the production of enzymes by microorganisms by adding yeast extract, corn steep, peptones, amino acids, ammonium salts, 10 phosphate salts, potassium salts, magnésium salts, calcium salis, iron salts, manganèse salts, zinc salis, 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.

[0170] Température, pH and other conditions optimal for growth of microorganisms and 15 production of enzymes are generally known in the art.

VII. SACCHARIFICATION [0171] The treated biomass materials can be saccharified, generally by combining lhe material and a cdlulase enzyme in a fluid medium, e.g., an aqueous solution. In some cases, the 20 material is boilcd, steeped, or cooked in hol water prior to saccharification, as described in U.S.

Pat. App. Pub. 2012/0100577 Al by Medoff and Masterman, published on April 26,2012, the entire contents of which are incorporated herein.

[0172] The saccharification process can be partially or completely 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 25 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 lime required for complété saccharification will dépend on the process conditions and the biomass material and enzyme used. If saccharification is performed in a manufacturing plant under controlled conditions, the cellulose may be substantially entircly converted lo sugar, e.g., glucose in about 12-96 hours. If saccharification is 30 performed partially or completely in transit, saccharification may take longer.

4I [0173] Il is generally preferTed lhal lhe tank contents be mixed during saccharification, e.g., using jet mixing as described in International App. No. PCT/US2010/035331, filed May 18, 2010, which was published in English as WO 2010/135380 and designated the United States, the full disclosure of which is incorporated by reference herein.

[0174] Tlie addition of surfactants can enhance the rate of saccharification. Examples of surfactants include non-ionic surfactants, such as a Twcen® 20 or Twcen® 80 polyethylene glycol surfactants, ionic surfactants, or amphoteric surfactants.

[0175] lt is generally preferred lliat the concentration of tlie sugar solution resulting from saccharification be relativcly 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 évaporation, to increase the concentration of the sugar solution. This reduces the volume lo be shipped, and also inhibits microbial growtb in Lhe solution.

[0176] Altematively, sugar solutions of lower concentrations may be used, in which case it may bc désirable lo add an antimicrobial additive, e.g., a broad spectrum antibiotic, in a low concentration, e.g., 50 to 150 ppm. Other suitable antibiotics include amphotericin B, ampicillin, chloramphcnicol, ciprofloxacin, gcnlamicin, hygromycin B, kanamycin, ncomycîn, penicillin, puromycin, strcptomycin. Anlibiotics will inhibit growth of microorganisms during transport and storage, and can be used al 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 antibiolic can be includcd even if the sugar concentration is relativcly high. Altematively, other additives with anti-microbial of preservative properties may be used. Preferably the antimicrobial additive(s) are food-grade.

[0177] A relativcly high concentration solution can be obtained by limiting the amount of water added to the biomass material with the enzyme. The concentration can be controlled, e.g., by conlrolling how mucli saccharification takes place. For example, concentration can be increased by adding more biomass material to lhe solution. In order to keep lhe sugar that is being produced in solution, a surfactant can be added, e.g., one of those discussed above. Solubilily can also be increased by increasing (lie température of the solution. For example, tlie solution can be mainlaincd at a température of 40-50°C, 60-80°C, or even higher.

VIII. SACCHARIFYING AGENTS [0178] Suitable cellulolytic enzymes include ccllulases from species in the généra Bacillus,

Coprinus, Myceliophthora, Cephalosporium, Scytalidium, Pénicillium, Aspergillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremoniuin, Chrysosporium and Trichodenna, especially chose produced by a strain selected from the species Aspergillus (see, e.g., EP Pub. No. 0 458 162), Humicola insolens (reclassified as Scytalidium thennophilum, see, e.g., U.S. Pat. No. 4,435,307), Coprinus cinereus, Fusarium oxysporum, Myceliophthora thermophila, Meripilus giganteus, Thielavia terrestris, Acremoniuin sp. (including, but not limited to, A. persicinum, A. acremoniuin, A. hrachypenium, A. dichromosporum, A. obclavatum, A.

pinkerfoniae, A. roseogriseum, A. încolorafum, and A. furatum). Preferred strains include Humicola insolens DSM 1800, Fusarium oxysporum DSM 2672, Myceliophthora thermophila CBS 117.65, Cephalosportum sp. RYM-202, Acremoniuin sp. CBS 478.94, Acremoniuin sp. CBS 265.95, Acremoniuin persicinum CBS 169.65, Acremoniuin acremoniuin AHU 9519, Cephalosporium sp. CBS 535.71, Acremoniuin hrachypenium CBS 866.73, Acremonittm dichromosporum CBS 683.73, Acremoniuin obclavatum CBS 311.74, Acremoniuin pinkerfoniae CBS \5Ί.Ί0, Acremoniitm roseogriseum CBS 134.56, Acremoniuin incoloratum CBS 146.62, and Acremoniuin fitramm CBS 299.70H. Celluloiytic enzymes may also be obtained from Chrysosporium, preferably a strain of Chrysosporium lucknowense. Additional strains that can bc used include, but arc not limited lo, Trichodenna (particularly T. viride, T. reesei, and T.

koningii), alkalophilic Bacillus (sec, for example, U.S. Pat. No. 3,844,890 and EP Pub. No. 0 458 162), and Streptomyces (see, e.g., EP Pub. No. 0 458 162).

[0179] Many microorganisms that can be used to saccharify biomass material and producc sugars can also be used to ferment and convcrt Chose sugars to useful products.

IX. SUGARS [0180] In the processes described herein, for example after saccharification, sugars (e.g., glucose and xylose) can be isolated. For example sugars can be isolated by précipitation, crystallization, chromatography (e.g., simulatcd moving bed chromatography, high pressure chromatography), centrifugation, extraction, any other isolation method known in tlie art, and 30 combinations thereof.

X. HYDROGENATION AND OTHER CHEMICAL TRANSFORMATIONS [0181] The processes described herein can include hydrogénation. For example glucose and xylose can be hydrogcnated lo sorbitol and xylitol respectlvely. Hydrogénation can be accomplished by use of a catalyst (e.g., Pt/gamma-AhCh, Ru/C, Raney Nickel, or other catalysts know in the art) in combination with H₂ under high pressure (e.g., 10 to 12000 psi). Other types of chemical transformation of the products from the processes 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 arc described in US Prov. App.

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

XI. FERMENTATION [0182] Yeast and Zymomonas bacteria, for example, can be used for fermentation or conversion of sugar(s) lo alcohol(s). Other microorganisms are discussed below. The optimum pH for fermentations is about pH 4 to 7. For example, the optimum pH for yeast is from about pH 4 lo 5, while the optimum pH for Zymomonas is from about pH 5 lo 6. Typical fermentation limes are about 24 to 168 hours (t’.g., 24 to 96 hrs) with températures in the range of 20°C to 40°C (e.g., 26°C to 40°C), however thermophilic microorganisms prefer higher températures.

[0183] In some embodiments, e.g., when anaérobie organisms are used, at least a portion of the femientation is conducted in the absence of oxygen, e.g., under a blanket of an inert gas such as N_2t Ar, Hc, CO₂ or mixtures thereof. Additionally, the mixture may hâve a constant purge of an inert gas ilowing through the tank during part of or ail of the fermentation. In some cases, anaérobie condition, can be achieved or maintaincd by carbon dioxide production during the i'ermentation and no additional inert gas is needed.

[0184] In some embodiments, ail or a portion of the fermentation process can be interrupted beforc the low molecular weight sugar is complctcly converted to a product (e.g., elhanol). 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 intermcdiate i fermentation products can be used in préparation of food for human or animal consumption.

i

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

[0185] Jet mixing may be used during fermentation, and in some cases saccharification and fermentation are performed in the same tank.

[0186] Nutrients for die microorganisms may be added during saccharification and/or fermentation, for example the food-based nutrient packages described in U.S. Pat. App. Pub. 2012/0052536, filed July 15, 2011, lhe complété disclosure of which is incorporated herein by reference.

[0187] “Fermentation” includes lhe methods and products that are disclosed in U.S. Prov. App. No. 61/579,559, filed December 22, 2012, and U.S. Prov. App. No. 61/579,576, filed Dccembcr 22,2012, the contents of both of which are incorporated by reference herein in their entirety.

[0188] Mobile fermenlers can be utilized, as described in International App. No. PCT/US2007/074028 (which was filed July 20, 2007, was published in English as WO 2008/011598 and designated the United Siales), lhe contents of which is incorporated herein in its entirety. Similarly, the saccharification equipment can be mobile. Further, saccharification and/or fermentation may be performed in part or enlirely during transit.

Xll, FERMENTATION AGENTS [0189] The microorganism(s) used in fermentation can be natural ly-occurring microorganisms and/or cngîneercd microorganisms. For example, the microorganism can be a bactcrium (including, but not limited lo, e.g., a cellulolytic bacterium), a fungus, (including, but not limited to, e.g., a yeast), a plant, a protist, e.g., a protozoa or a fungus-like protest (including, but not limited lo, e.g., a slime mold), or an alga. When the organisms are compatible, mixtures of organisms can bc utilized.

[0190] Suitable fcmienting microorganisms hâve the ability to convert carbohydrates, such as glucose, fructose, xylose, arabinosc, mannose, galactose, oligosaccharides or polysaccharides into fermentation products. Fermenting microorganisms include strains of the genus Saccliaromyces spp. (including, but not limited to, S. cerevisiae (bakcr’s yeast), S. distalicus, S. uvarutri), the genus Kluyveromyces, (including, but not limited to, K. marxianus, K.fragilis), the genus Candida (Including, but not limited to, C. pseudoiropicalis, and C. brassicae), Picltia stipitis (a relative of Candida shehafae), the genus Clavispora (including, but not limited to, C. hisiianiae and C. opitntiae), lhe genus Pachysolen (including, but not limited to, P. taimophilits),

the genus Bretannomyces (including, but not limited to, e.g., B. clausenii (Philippidis, G. P., 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization, Wyman, C.E., ed„ Taylor & Francis, Washington, DC, I79-212)). Other suitable microorganisms include, for example, Zymomonas mobilis. Clostridium spp. (including, but not limited to, C. fhermocellmn (Philippidis, 1996, supra), C.saccharobutylacetonicum, C. saccharobittylicum, C. Punicettm, C. beijeritckii, and C. acetobutylicum), Moniliella pollinis, Moniliella megachiliensis, Lactobacillus spp. Yarrowia lipolytica, Aureobasidium sp., Trichosporonoides sp., Trigonopsis variabilis, Trichosporon sp., Moniliellaacetoabutans sp., Typlutla variabilis, Candida magnoliae, Ustilaginomycetes sp.,Pseudozyma tsukubaensis,yeast species of généra Zygosaccbaromyces, Deharyomyces, Hansenula and Pichia,and fungi of the dcmalioid genus Tonda.

[0191] For instance. Clostridium spp. can be used to produce éthanol, butanol, butyric acid, acelic acid, and acelone. Lactobacillus spp., can be used to produce lactice acid.

[0192] 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 Research Sevice Culture Collection, Peoria, Illinois, USA), or the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany), to name a fcw.

[0193] Commercially available ycasls include, for example, Red Slar®/Lcsaffre Ethanol Red (available Iront Red Slar/Lesaffrc, USA), FALI® (available from Fleischmann’s Yeast, a division of Bums Philip Food Inc., USA), SUPERSTART® (available from Alltcch, now Lalemand), GERT STRAND® (available from Gert Strand AB, Swcden) and FERMOL® (available from DSM Spécialités).

[0194] Many microorganisms that can be used to saccharify biomass material and produce sugars can also be used to ferment and convert those sugars to useful products.

XIII. DISTILLATION [0195] After fermentation, the resulting fluids can be distilled using, for example, a “becr column to separate clhanol and other alcohols from the majority of water and residual solids.

Tlie vapor exiting the bcer column can bc, e.g., 35% by weight éthanol and can be fed to a rectification column. A mixture of nearly azcotropic (92.5%) éthanol and water from tlie rectification column can be purified to pure (99.5%) éthanol using vapor-pha.se molecular sieves.

The beer column bottoms can be sent to the first efTect of a three-effect evaporator. The rectification column reflux condenser can provide heat for this first effect. Afler the first effect, solids can be separated using a centrifuge and dried in a rotary dryer. A portion (25%) of the 5 centrifuge effluent can be rccycled to fermentation and lhe rest sent to the second and ihird evaporator effects. Most of lhe evaporator condensate can be retumed 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.

I0 EXAMPLES [0196] Example I. Effect of Exogenous Fructose on Saccharification [0197] This example lests whether or not exogenous fructose inhibits saccharification enzymes.

[0198] Three 225mL Erlenmeyer flasks were prepared, each wilh 10g of treated corn cob biomass (mesh size between 15 and 40, and irradiated to 35 Mrad with an électron beam) lOOmL of water and 2.5mL of Duet Accelerase™ (Danisco). To the first, second, and third flask were added, respectively; Og, 5g and 10g of fructose. The flasks were covered with aluminum foil and sel in an incubator shaker at 50°C and 200rpm for four days. The amount of xylosc and glucose was monitored by HPLC. The results of lhe saccharification are shown in the table below.

[0199] Table I. Saccharification under varying levels of exogenous fructose.

Sample	Glucose yield (g/L)	Xylosc yield (g/L)	% Glucose
0g added fructose	17.9	13.8	100.0
5g added fructose	16.7	12.3	93.5
10g added fructose	18.1	12,6	101.3

[0200] Unlike glucose (a known inhibitor of ccllobiase), 5% or 10% added fructose does not inhibit the saccharification of corncob.

[0201] Example 2. Effect of Xylose Isomerase on Saccharification [0202] Glucose is a known inhibitor of cellobiase. This example tests if tlie conversion of glucose lo the isomer fructose by xylose isomerase can increase saccharification.

[0203] Four 225mL Erlenmeyer flasks were prepared, each with 10g of treated corn cob biomass and lOOmL of water. The biomass was treated as described in Example 1. To the first, second, and third fiask was added 2.5mL of Duet Accelerase™ (Danisco). To lhe second, third, and fourth flasks were added, respectively: 1 g, 0.1g and 0.1g of glucose isomerase (Sweelzyme™, Aldrich). The flasks were covered with aluminum foil and set in an incubalor shaker al 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.

[0204] Table 2. Effectiveness of cellulase with added xylose isomerase.

Sample	Glucose yield (g/L)	Xylose yield (g/L)	% Glucose	%Xylose
2.5mL Duet	22.6	16.9	100.1	100.0
2.5mL Ducl+ 1 g GI	28.3	20.6	125.2	122.3
2.5mL Duel + 0.1 g GI	24.6	18.5	109.0	109.4
0.1g GI	1.6	Not detcclcd	6.9	Not detected

[0205] The addition of glucose isomerase was observed to increase the effectiveness of tlie ccllulase enzyme, with fiask 2 producing about 25% more sugars than fiask 1.

[0206] Example 3. Use of a Strong Acid to Cleave Cellobiose [0207] This example tests the use of a strong acid to cleave cellobiose to glucose, to increase saccharification yield. The strong acid used was Amberlyst-15™, a polystyrène sulfonic acid. This is a strongly acidic sulfonic acid macrorelicular polymeric resin thaï is based on crosslinked styrene divinylbenzenc copolymcrs. Published studies indicate that Amberlyst-15 can cleave tlie dimer cellobiose lo glucose.

[0208] Three 225mL Erlenmeyer flasks were prepared, each with 10g of treated corn cob biomass, lOOmLof waler and 2.5mL Duet Accelerase™, The biomass was treated as described in Example 1. ln the second fiask lg of glucose isomerase (Sweetzyme™, Aldrich) was added;

and in the third Ig of glucose isomerase and 0.Ig of polystyrène sulfonic acid (Amberiyst-l5™, DOW) was udded.

[0209] The flasks were covered wiLh 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 5 results of the saccharification are shown in the table below.

[0210] Table 3. Effect of an Acid on Saccharification.

Sample	Glucose yield (g/L)	Xylose yield (g/L)	% Glucose	% Xylose	% Amcrlyst-15 improved with GI
Duet alone	21.1	16.1	100	100	NA
Duet + GI	26.5	19.2	125	119	NA
Duet + GI +	27.9	20.5	131	127	14
Amberlyst

[0211] The results show an iinprovement in the saccharification with Üie addition of glucose 10 isomerase. The experiment also shows an improvement in the saccharification with the addition of polystyrène sulfonic acid.

[0212] Examplc 4. Removal of Cellobiasc [0213] This example examines saccharification where cellobiasc has been removed, while the endo- and exo-cellulases hâve been retained.

[0214] Chromatofocusing was used to separate the enzymes. Duet Accelerase™ (Danisco) was injeclcd onto a Mono P column using an AKTA system. The endo-and exo-cellulases bound to tlie column, while the cellobiasc passed through and was removed. The exo- and endocelluloses were then eluted from tlie column by shilling the pH to 4.0. Tlie resulting fractions were combined and immediately applied to a saccharification réaction.

[0215] Table 4. Accumulation of Cellobiose and Sugars in the Absence of Cellobiase. Sample Cellobiose Glucose Xylose Xylitol Lactose

AKTA 1.057 purified Duet

4.361

5.826

0.556

Duel 0.398	16.999	14.830	0.726
Comcob (no	0.673		0.550
enzymes)
Spun/Filtered	17.695	15.053	0.770
Duel

[0216] The expected resull was thaï without cellobiase, there would be an accumulation of cellobiose. Although the yield was low, lhe table below shows that a détectable amount of cellobiose was indeed generated.

[0217] Other than in the examples herein, or unless otherwise expressly specified, ail of the numerical ranges, amounts, values and percentages, such as those for amounts of materials, elemental contents, times and températures of réaction, ratios of amounts, and olhers, in the following portion of lhe spécification and attached ciaims may be read as if prefaced by lhe word “about even though lhe term “about” may not expressly appear with the value, amount, or range. Accordingly, unless indicated to the contrary, the numerical parameters sel forth in the following spécification and attached ciaims are approximations thaï may vary depending upon the desired properties sought lo be obtained by the présent invention. Al lhe very least, and not as an atlempl lo limît lhe application of lhe doctrine of équivalents to the scope of the ciaims, each numerical parameter should at least bc construed in light of Lhe number of reported significant digits and by applying ordinary rounding techniques.

[0218] Notwithstanding that the numerical ranges and parameters setling forth the broad scope ofthe invention arc approximations, the numerical values set forth in the spécifie examplcs are reported as prccisely as possible. Any numerical value, however, inherently contains error necessarily resulting from the standard déviation found in its underlying respective testing measurements. Furthermore, when numerical ranges are set forth herein, these ranges are inclusive of the recited range end points (i.e., end points may be used). When percentages by weight arc used herein, lhe numerical values reported are relative to Lhe total weight.

[0219] Also, it should bc understood thaï any numerical range reciled herein is intended to inciude ail sub-ranges subsumed lherein. For example, a range of “l to 10” is intended to inciude ail sub-ranges between (and including) the recited minimum value of 1 and lhe recited

Φ 50 maximum value of 10, that is, having a minimum value equal to or greater than l and a maximum value of equal to or less than 10. The terms “one,” “a, or an as used herein are intended to include at least one or “one or more, unless otherwise indicatcd, [0220] 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 conflict with existing définitions, 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 confiicting material incorporated herein by reference. Any material, or portion thereof, thaï is said to be incorporated by reference herein, but which conflicts with existing définitions, statements, 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.

[0221] While this invention lias been particularly shown and described with références to preferred embodiments thereof, it will bc understood by those skilled in the art that various changes in form and details may be made therein without departing from llte scope of the invention cncompassed by the appendcd daims.

Claims (20)

1. Wlial is claimed is:

   5 l, A method of making a product, lhe method comprising: saccharifying recalcilrance-reduced lignocellulosic biomass, and adding an isomerization agent to the saccharified biomass.
2. 2. The method of claim 1 wherein the saccharified biomass comprises a first sugar and a

   10 second sugar and lhe isomerization agent is used to convert the second sugar to a third sugar.
3. 3. The method of claim 2 further comprising contacting lhe saccharified biomass with a microorganism to convert lhe first sugar and third sugar to one or more product(s).
4. 4. The method of claim 3, where the recalcilrance-reduced biomass lias been pre-treated with a treatment method selected from lhe group consisting of: bombardment with électrons, sonication, oxidation, pyrolysis, steam explosion, chemical treatment, mechanical treatment, freeze grinding.
5. 5. The method of claim 4, wherein the treatment method is bombardment with électrons.
6. 6. The method of claim 2, wherein the conversion of the second sugar to lhe third sugar is donc before maintaining lhe microorganism-biomass combination under conditions that

   25 enable lhe microorganism to convert the first sugar to lhe product.
7. 7. The method of claim 2, wherein lhe conversion of the second sugar to the third sugar is doue immediately after saccharification of the biomass.

   30
8. 8. The meiliod of claim 2, wherein the conversion of Lhe second sugar to Lhe third sugar is donc during saccharification of the biomass.
9. 9. The method of any one of (lie above claims, wherein Üie lignoceliulosic biomass is selected from lhe group consisting of: wood, particle board, forestry wastes, sawdust, aspen wood, wood chips, grasses, switchgrass, miscanthus, cord grass, reed canary grass,

   5 grain residues, rice hulls, oat hulls, wheal 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, com fiber, alfalfa, hay, coconut hair, sugar processing residues, bagasse, bcct pulp, agave bagasse, algae, seaweed, nianure, sewage, offal, agricultural or industrial waste, arracacha, buckwheal, banana, barley,
10. 10 cassava, kudzu, oca, sago, sorghum, potato, sweet potato, taro, yams, beans, favas, lentils, peas, and mixtures of any of these.

    10. The method of any one of the above claims, wherein the isomerization agent comprises an acid.
11. 11. The method of claim 10, wherein the acid is polystyrène sulfonic acid.
12. 12. The method of any one of the above claims, wherein the isomerization agent comprises an enzyme immobilized on a support.
13. 13. The method of claim 12, wherein lhe enzyme is xylose isomerase.
14. 14. The method of any one of claims 3-13, wherein the microorganism-saccharified biomass combination is maintained at a pH of about 6.0 to about 7.5.
15. 15. The method of any one of claims 2-14, wherein the second sugar is glucose, and lhe third sugar is fructose.
16. 16. The method of any one of claims 2-14, wherein the second sugar is xylose, and Üic third

    30 sugar is xylulose.
17. 17. The method of any one of daims 3-16, wherein lhe microorganism is yeast.
18. 18. The method of any one of daims 3-16, wherein the microorganism is Clostridium spp.

    5
19. 19. The method of any one of daims 3-18, wherein lhe product is selected from lhe group consisting of: éthanol, butanol, bulyric acid, acelic acid, and acetone.
20. 20. The method of any one of daims 3-16, wherein the microorganism is Lactobacillus spp.

    10 21. The method of daim 20, wherein lhe product is lactic acid.

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    FIG

    SUBSTITUTE SHEET (RULE 26)