OA16929A - Processing biomass. - Google Patents

Abstract

Provided herein are methods for processing biomass materials that are disposed in one or more structures or carriers, e.g., a bag, a shell, a net, a membrane, a mesh or any combination of these. Containing the material in this manner allows it to be readily added or removed at any point and in any sequence during processing.

Description

[0003] As demand for petroleum increases, so too does interest in renewabie fcedstocks for manufacturing biofuels and biochemicals. The use of lignocellulosic biomass as a feedstock for such manufacturing processes has been studied since the 1970s. Lignocellulosic biomass is attractive because it is obundant, renewabie, domestically produced, and does not compete with 20 food industry uses.

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

[0005] Lignocellulosic biomass is récalcitrant to dégradation as the plant cell walls hâve a structure that is rigid and compact. The structure comprises crystalline cellulose ftbrüs embedded in a hcmicclluiosc matrix, surrounded by lignin. This compact matrix is difficult to access by enzymes and other chemical, biochemical and biological processes. Cellulosic biomass materials (e.g., biomass material from which substantially ail the lignin has been removed) can be more accessible to enzymes and other conversion processes, but even so, naturel ly-occurrin g cellulosic materials often hâve low yields (relative to theoretlcal yields) when contacted with hydrolyzing enzymes. Lignocellulosic biomass is even more récalcitrant to enzyme attack. Furthermore, each type of lignocellulosic biomass has its own spécifie composition of cellulose, hcmlcellulose and lignin.

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

[0007] Monosaccharidcs from renewablc biomass sources could become the basis of chemical and fuels industries by replaclng, supplementing or substituting petroleum and other fossil feedstocks. However, techniques need to be developed that will make these monosaccharides available in large quantities and at acceptable purities and prices.

SUMMARY OFTHE INVENTION [0008] Provided herein are methods for producing a product, which methods include mnintnining a combination comprising a liquid medium, a structure or carrier, and a reducedrecalcitrance cellulosic or lignocellulosic biomass disposed within the structure or carrier, under 10 conditions that allow the passage of molécules out of and/or Into the structure or carrier.

[0010] In another aspect, provided herein is a method for producing a product, where the method Includes: providing a liquid medium; providing a cellulosic or lignocellulosic biomass, wherein the cellulosic or lignocellulosic biomass is disposed in a structure or carrier, and wherein the structure or carrier possesses one or more pores configured to allow the passage of 15 molécules; providing an additive; combining the structure or carrier and the additive in the liquid medium to make a combination; maintaining the combination under conditions that allow the passage of molécules out of and/or into tlie structure or carrier; and maintaining the combination under conditions that allow the additive to convert the molécules to one or more products; thereby producing a product.

[0011] Additionally, provided herein arc methods of producing an enzyme, where the methods include: providing a liquid medium; providing a cellulosic or lignocellulosic biomass; providing a microorganism capable of producing an enzyme in the présence of the cellulosic or lignocellulosic biomass; providing a structure or carrier, wherein the structure or carrier possesses one or more pores configured to allow the passage of molécules; disposing the cellulosic or lignocellulosic biomass within the structure or carrier; combining the liquid medium, the structure or carrier, and the microorganism to make a combination; and maintaining the combination under conditions that allow the microorganism to produce the enzyme; thereby producing an enzyme.

[0012] Also provided herein is a method of providing a substance to a microorganism, where 30 the method Includes: providing a liquid medium; providing a microorganism; providing a substance; providing a structure or carrier, wherein the structure or carrier possesses one or more pores configured to allow the passage of the substance into and out of the structure or carrier; either: by disposing the microorganism within the structure or carrier, and forming a combination by combining the liquid medium, the microorganism within the structure or carrier 35 and the substance, or by disposing the substance within the structure or carrier, and forming a combination by combining the liquid medium, the substance within the structure or carrier, and the microorganism: and maintaining the combination under conditions that allow the substance to move out of and into the structure or carrier, and to come in contact with the microorganism; thereby providing the substance to the microorganism. Such methods can also include: providing a second structure or carrier, and disposing both the microorganism and the substance each in a separate structure or carrier.

[0013} Also provided herein is a system for making a product, where the system includes: a liquid medium in a container, a microorganism capable of making a product; and a structure or carrier containing a substance, where the structure or carrier is configured to release the substance into the liquid medium.

[0014] In any of the methods or Systems provided herein, the cellulosic or lignocellulosic biomass can be disposed within the structure or carrier, and the methods can further include: disposing the additive witliin a second structure or carrier; and the structure or carrier containing the cellulosic or lignocellulosic biomass is disposed within the second structure or carrier.

[0015] ln any of the methods or Systems provided herein, the substance can be a sugar, e.g., a sugar can be disposed within onc or more structures or carriers.

[0016] ln any of the methods or Systems provided herein, the product produced can be a molécule, a protein, a sugar, a fuel or combinations thereof. The protein can be un enzyme. [0017] Any of the methods or Systems provided herein can further include disposing a microorganism in the structure or carrier. Altematively, the cellulosic or lignocellulosic material, or the additive can be disposed in the structure or carrier. The cellulosic or lignocellulosic material, the additive, or the microorganism can be disposed in a second structure or carrier. The additive can be a microorganism, an enzyme, an acid, a base or combinations thereof.

[0018] In any of the methods or Systems provided herein, the structure or carrier can be a bag, a shel I, a net, a membrane, a mesh or combinations thereof. Where the structure or carrier includes a bag, the bag can be formed of a mesh material having a maximum opening size of less than 1 mm. Altematively, the mesh material can hâve an average pore size of from about 10 mm to 1 nm. Where the structure or carrier is a bag, the bag can be mode of a biœrodible polymer. The bioerodible polymer can be selected from the group consisting of: polylactic acid, polyhydroxybutyrate, poly hydroxy alkanoatc, polyhydroxybutyrate-valerate, polycaprolactone, polyhydroxybutyrate-hexanoate, polybutylene succinate, polybutyTate succinate adipate, polycsteramidc, polybutylene adipatc-co-terephthalate, mixtures thereof, and laminates thereof. The bag can be made of a starch film.

[0019] ln any of the methods or Systems provided herein, the combination can be placed in a fermentation vessel that includes impellers, and where the combination is maintained under conditions where the bag is lom open by the impellers.

[0020] In any of the methods or Systems provided herein, the microorganism or microorganisms can include a straîn of Trichoderma reesei, e.g._t a high-yielding cellulaseproducing mutant of Trichoderma reesei, e.g., the RUT-C30 strain.

[0021] In any of the methods or Systems provided herein, the recalcitrance of the cellulosic or lignocellulosic material can hâve been reduced relative to the material in its native state. Such treatment to reduce recalcitrance can be bombardment with électrons, sonicatlon, oxi dation, pyrolysis, s team explosion, chemical treatment, mechanical treatment, freeze grinding, or combinations ofsuch treatments. Preferably, the recalcitrance of the cellulosic or lignocellulosic biomass has been reduced by exposure to an électron beam.

[0022] In any of the methods or Systems provided, the conversion can be saccharification, and the product can be a sugar solution or suspension. The methods can further include isolating a sugar from the sugar solution or suspension. The sugar isolated can be xylose.

[0023] In any of the Systems or methods provided herein. the cellulosic or lignocellulosic biomass can be: paper, paper products, paper waste, paper pulp, pigmented papers, loaded 15 papers, coated papers, filled papers, magazines, printed matter, printer paper, polycoated paper, card stock, cardboard, paperboard, cotton, wood, partide board, forestry wastes, sawdust, aspen wood, wood chips, grasses, switchgrass, 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, 20 com stover, soybean stover, com fiber, alfalfa, hay, coconut haïr, sugar processing residues, bagasse, bcct pulp, agave bagassc, algae, seaweed, manure, sewage, offal, arracacha, buckwhcat, banana, barley, cassava, kudzu, oca, sago, sorghum, potato, sweet potato, taro, yams, beans, favas, Icntils, peas, or mixtures of any of these. The cellulosic or lignocellulosic material can include com cobs. The cellulosic or lignocellulosic biomass can be comminuted, e.g., by dry 25 milling, or by wet milling. The cellulosic or lignocellulosic material can be treated to reduce its bulk density, or to increase its surface areu. The cellulosic or lignocellulosic material can hâve an average particlc size of less than about 1 mm, or an average particle size of from about 0.25 mm to 2.5 mm.

[0024] lt should be understood that this invention is not limited to the embodiments disclosed în this Summary, and it is intended to cover modifications that are within the spirit and scope ofthe invention, as defined by the claims.

BRIEF DESCRIPTION OF THE DRAWINGS [0025] The forcgoing will be apparent from the following more particular description of 35 example embodiments of the invention, as illustrated in the accompanying drawings in which like référencé characters refer to the same parts throughout the different views. The drawings are

not necessarily to scale, emphasts instead being placed upon ülustrating embodiments of the présent invention.

[0026] FIG. 1 is a diagram ülustrating the enzymatic hydrolysis of cellulose to glucose. Cellulosic substrate (A) is converted by endocellulase (i) to cellulose (B), which is converted by 5 exoceilulase (H) to cel iobiose (C), which is converted to glucose (D) by cellobiase (betaglucosidase) (iii).

[0027] FIG. 2 is a flow diagram iilustroting conversion of a biomass feedstock to one or more products. fcedstock is physicaliy pretreated (e.g., to reduce Ils size) (200), optionally treated to reduce its recalcitrance (210), saccharified to form a sugar solution (220), the solution 10 is transported (230) to a manufacturing plant (e.g., by pipeline, railcar) (or if saccharification ls performed en route, the feedstock, enzyme and water îs transported), the saccharified feedstock is bio-processed to produce a desired product (e.g., alcohol) (240), and the product cnn be processed further, e.g., by distillation, to produce a final product (250). Treatment for recaicitrance can be modified by measuring lignln content (201) and setting or adjusting process 15 parameters (205). Saccharifying the feedstock (220) can bc modified by mixing the feedstock with medium and the enzyme (221).

[0028] FIG. 3 is n flow diagram illustrating the treatment of a first biomass (300), addition of a cellulose producing organism (310), addition of a second biomass (320), and processing the resulting sugars to make products (e.g., alcohol(s), pure sugars) (330). The first treated biomass 20 can optionally be split, and a portion added os the second biomass (A).

[0029] FiG. 4 is a flow diagram illustrating the production ofenzymes. A cellulaseproducing organism is added to growth medium (400), a treated first biomass (405) is added (A) to make a mixture (410), a second biomass portion is added (420), and the resulting sugars are processed to make products (e.g., aicohol(s), pure sugars) (430). Portions of the first biomass 25 (405) can also be added (B) to the second biomass (420).

DETAILED DESCRIPTION [0030] Provided herein arc methods of conducting biological, microbiological, and biochemical réactions by using one or more structures or containers, which can hâve pores or 30 other openings, or can be degradable. The structure can bc a bag, net or mesh, shell (e.g., rigid or semî-rigid sheii), a membrane, or combinations of these structures (e.g., one or more structures of one or more types can be disposed within a structure of the same or another type). The structures can hold various parts or ingrédients involved in biological, microbiological, and biochemical reactions. Containing the material in this manner allows parts or ingrédients, e.g., 35 biomass, such as treated biomass, to be readily added or removed at any point and in any sequence during such reactions. The invention also allows simplification of purification of

products (such as e.g., sugars or other products of saccharification or fermentation), and can aid in the maintenance of the level of a métabolite, sugar, or nutrient.

[0031] For instance, the structures can be used to provide one or more nutrients lo microorganisms. The nutrients can be piaced in the structure, and the structure piaced in a liquid 5 medium containing microorganisms. The nutrients are released from the structure into the medium to be accessed by the microorganisms. Altematively, the microorganisms can be piaced within the structure, and the structure piaced in a liquid medium that contains the nutrients. [0032] In a preferred embodiment, the structure can contain biomass which is to be acted on by microorganisms, or products of microorganisms, such as enzymes or signal molécules. For instance, the biomass can be piaced in the structure, which is then piaced in a liquid medium wîth the microorganisms. Substances from the biomass are able to leach out of the structure and be accessed by the microorganisms and enzymes secreted by the microorganisms, and enzymes produced by the microorganisms can migrate into the structures and act on the biomass. [0033] In another aspect, the invention relates lo producing enzymes using a microorganism in the presence of a biomass material. The biomass material acts in the enzyme production process as an inducer for cellulase synthesis, producing a cellulase complex having an activity that is tailored lo the particular biomass material, which in some implémentations ts the same material that is to be saccharified by the cellulase complex.

[0034] The Invention also features n method that includes contacting a celiulosic or lignoceilulosic material disposed in a structure or carrier, in a medium, with an additivc to produce a product. The additive can, for example, be a microorganism, an enzyme, an acid, a base or mixtures of any of these. The additives can be added in any order. The product can be, for example, u molécule, a protein, a sugar a fuel or mixtures of any of these. The products can be produced in any order. For example, a protein can be first produced followed by a sugar and 25 finally by a fuel. Optionally, the protein can be an enzyme.

[0035] Tlic migration of substances into and out of the structure can be accompiished in a variety of ways. The structure can slowly dégradé over time in the medium, the structure can be made of a porous material thaï releases the nutrients into the medium, the structure can be made of a material that is consumed by the microorganisms, the structure can be made of u material that is tom open by the impellers in the bottom of a fermentation vessel, or the structure can be made of a material that swells and bursts in the medium.

[0036] ln an embodiment of the process described herein, a biomass can be disposed in, on, or piaced Into the structure or carrier. The blomass can be treated before or after being piaced into the structure or carrier. Additives, nutrients and products can also bc disposed in the structure or carrier with or without the biomass. For example, a biomass with an antibiotic, a microbe, an enzyme and a sugar can be disposed in the structure, and may be combined in any amounts and in any sequence during the process.

[00371 Optionally, the biomass can be outside οΓ the structure or carrier. For example, a microbe can be disposed in, within (i.e., built into the structure or carrier), or on the structure or carrier, which is contacte d with a medium containing the biomass. As another ex ample, there may be one kind of biomass in the structure or carrier and a second kind of biomass outside the structure or carrier. There may be multiple biomasses inside and outside of the structure or carrier added in any combination and sequence during the process.

[0038] In another embodiment of the process, there may be multiple structures or carriers placed in or contacted with a medium. These can be placed in the medium in any sequence and combination during the process. The structure or carriers can be, for example, with respect to 10 each, other made of the same material or different materials, hâve the same shape or different shapes, and may be uscd in any combination.

[0039] For example, multiple structures or carriers can be disposed within another structure or carrier. The various structures or carriers can be of the same type, or can be of different types. Multiple structures or carriers can be sequentially disposed, each inside another, e.g., similar to 15 “nesting doils.” [0040] For example, it may be convenient to hâve biomaterial disposed in a plurality of structures or carriers of a uniform size and volume, each containing the same or a similar nmount of biomass. In this way, whole number amounts or units of the structure or carrier can be contacted with the medium, with the number of units used depending on the batch size in the 20 process. Such uniform volume structures or carriers may also be more convenient to store, for example, if they are designed as approximately cuboid in shape so that they can be easily stacked.

[0041] Optionally, in some implémentations, a structure or carrier containing biomnss can be contacted with a medium in combination with a structure or carrier that is designed to slowly release an additive, e.g., an enzyme, contained within the structure or carrier. For example, controlled release may be effectcd by having a controlled pore size (e.g., a pore size smaller than lOum, e.g., smaller than lum, smaller than 0. lum).

[0042] As another example, one or more biomass-contaînlng structures or carriers, and one or more microbe-containing structures or carriers can be contacted simultaneously or sequentially with a medium.

[0043] As a further example, in some processes one or more biomass-containing structures or carriers, and one or more ndditive-containing water-degradable structures or carriers are contacted with an aqucous medium.

[0044] In another embodiment of the process, the structure or carrier can be removed at any 35 point in the process and in any sequence. For example, the structure or carrier including ils contents can be removed after producing a product, and/or additional structures or carriers including their contents can be added during production of a product.

[0045] As another example, a biomass disposed in a structure or carrier is contacted with an aqueous medium, and a microbe is added to the aqueous medium, which then produces a product. Subsequently, the biomass-containing structure or carrier can be removed, and a second amount of biomass in a structure or carrier can be added to produce more product. Optionally, 5 the microbe can be removed before or after addition of the second biomass.

[0046] In yet another exemple, a biomass can be disposed in a structure or carrier and contacted with an aqueous medium containing a microbe the combination of which produces a first product. The microbe can be optionally removed (e.g., by filtration or centrifugation) or killed (e.g., by application of antibiotics, heat, or ultraviolet light) and subsequently a different 10 microbe can be added. which causes a second product to be produced.

[0047] In a further example, a biomass can be disposed in n first structure or carrier. The first structure or carrier can be disposed in a second structure or carrier containing a microbe. The two structures or carriers can be disposed in a medium. The second structure or carrier is designed to contain the microbes (e.g., has pore sizes below about 5um, below about 1 um, 15 below about 0.4 um, beiow about 0.2 um). The combination produces a product that optionally can flow out of the second structure or carrier. Once product is produced, the first and second structures and contents can be removed leaving media with product dispersed and/or dissolved within it. The combination of lhe first and second structures or carriers with their contents can be optionally used in another medium to produce more product.

[0048] The processes described herein inciude processing of biomass and biomass materials and the intermediates and products resulting from such processing. During at least a part of the processing, the biomass material can be disposed in a structure or carrier.

[0049] The processes described herein include producing enzymes using a microorganism in the presence of a biomass material, e.g., a ccllulosic or lignoceliulostc material. Enzymes tnade 25 by the processes described herein contain or manufacture various ce!lulolytic enzymes (celluloses), ligninases or various small molécule biomass-destroying métabolites. These enzymes may be a complcx of enzymes that act synergistîcally to dégrade crystalline cellulose or lhe lignin portions of biomass. Examples of ceilulolytic enzymes include: endogiucanases, cellobiohydrolascs, and cellobiases (beta-glucosidases).

[0050] As shown in FIG. I, for examplc, during saccharification a cellulosic substrate (A) is initïally hydrolyzed by endogiucanases (i) at random locations producing oligomeric intermediates (e.g., cellulose) (B). These intermediates arc then substrates for exo-splitting glucanases (li) such as cellobiohydrolase to produce cellobiose from the ends of the cellulose polymer. Cellobiose is a waler-soluble 1,4-llnked dimerof glucose. Finally cellobiase(iii) clcavcs cellobiose (C) to yield glucose (D). Therefore, the endogiucanases arc particularly effective in attacking the crystalline portions of cellulose and increasing the effectiveness of exoccllulascs to produce cellobiose, which then requires the specificily of the cellobiose to

produce glucose. Therefore, it is évident that depending on the nature and structure of the cellulosic substrate, the amount and type of the three different enzymes may need to be modified. [0051] In some implémentations, the enzyme is produced by a fungus, e.g., by strains of the cellulolytic filamentous fungus Trichodenna reesei. For example, high-yielding cellulase mutants of Trichodenna reesei may be used, e.g., RUT-NG14, PC3-7, QM9414 and/or Rut-C30. Such strains are described, for example, in Sélective Screening Methods for the Isolation of High Yielding Cellulose Mutants of Trichodenna reesei,*' Montenecourt, B.S. and Everleigh, D.E., Adv. Chem. Ser. 181,289-301 (1979), the fuit disclosure of which is incorporated herein by reference. Other cellulase-producing microorganisms may also be used.

[0052] As will be discussed further below, once the enzyme has been produced, It can be used to saccharify biomass, in some cases the same type of biomass material that has been used to produce the enzyme. The process for converting the biomass material to a desired product or intermediate generally includes other steps in addition to this saccharification step. Such steps are described, e.g., in U.S. Pat. App. Pub. 2012/0100577 Al, filedOctober 18,2011 and published April 26,2012, the full disclosure of which is hereby incorporated herein by reference. [0053] For example, rcferring to RG. 2, a process for manufacturing an alcohol can include, for example, optionally mechanically treating a feedstock, e.g., to reduce its size (200), before and/or after this treatment, optionally treating the feedstock with another physical treatment to further reduce Its recalcitrance (210), then saccharifying the feedstock, using the enzyme complex, to form a sugar solution (220). Optionally, the method may also include transporting, e.g., by pipeline, railcar, truck or barge, the solution (or the feedstock, enzyme and water, if saccharification is performed en route) to a manufacturing plant (230). In some cases the saccharified feedstock is further bioprocessed (e.g„ fermented) to producc a desired product e.g., alcohol (240). This resulting product may in some implémentations be processcd further, e.g., by distillation (250), to produce a final product. One method of reducing the recalcitrance of the feedstock is by électron bombardment of the feedstock. If desired, the steps of measuring lignin content of the feedstock (201) and setting or adjusting process parameters based on this measurement (205) can be performed at various stages of the process, as described in U.S. Pat. App. Pub. 2010/0203495 Al by Medoff and Masterman, published August 12,2010, the complété disclosure of which is incorporated herein by reference. Saccharifying the feedstock (220) can also bc modified by mixing the feedstock with medium and the enzyme (221). [0054] For example, referring to HG. 3 a first biomass is optionally treated (300), for example to reduce its size and/or recalcitrance, and placed into a structure or carrier. Optionally, the first biomass can first be placed into a first structure or carrier and then treated. The biomass containing structure or carrier is then contacted with an aqueous medium and a cellulase producing organism (310). After an adéquate time has passed for the cells to grow to a desired stage and enough enzymes hâve been produced, a second bîomass, optionally disposed in a

second structure or carrier, may be added (320). Optionally, the structure or carrier containing the first biomass can be removed prior to or at any point after addition of the second biomass. The action of the enzyme on the second and any remaining first biomass produces mixed sugars which can be further proccssed to useful products (330). Optionally, the second structure or 5 carrier containing the second biomass can be removed prior to or after the production of the useful product. The first and second biomass can be portions of the same biomass material. For example, a portion of the biomass can be placed into a structure or carrier and contacted with a medium containing the cellulase producing organism. Once some enzymes hâve been produced; the enzyme containing media can be combined with the second biomass (A). Optionally, the 10 first and second biomass may be pretreated to reduce recalcitrance. The first and second biomass can also be contained in a single structure or carrier. The structure or carrier cnn form a liner for a bioreactor. Multiple biomass containing structures or carrier can also be used. The aqueous media will be discussed below. ln some cases, rather than adding the second biomass to the reactor, the enzyme is horvested, stored, and used in a later saccharification process.

[0055] Referring now to FIG. 4, the cellulase-producing organism (400) can be grown in a growth medium for a time to reach a spécifie growth phase. For example, this growth period could extend over a period of days or even weeks. Pretreated first biomass (405) is placed in a structure or carrier and can then be contacted with the enzyme producing cells (410) so that after a time enzymes are produced. Enzyme production may also take place over an extended period 20 of time. The enzyme containing solution may then be combined with a second biomass (420).

Optionally, before addition of the second biomass or at any point after addition of the second biomass, the structure or carrier containing the first biomass can be removed. The action of the enzyme on the second and remaining first biomass produccs mixed sugars which can be further proccssed to useful products (430). Tlie first and second biomass cnn be portions of the same 25 biomass or can be similar but not identical (e.g., pretreated and non-pretreated) material (B).

Again, if desired the enzyme can be harvested and stored rather than being used immediatcly with a second biomass.

[0056] Along with the methods discussed above, the cellulose producing organism may be harvested prior to being combined with the first pretreated biomass. Harvesting may include 30 partial or almost complété removal of the solvent and growth media components. For example the ccl Is may be collected by centrifugation and then washed with water or another solution. [0057] ln another embodiment, after enzyme is produced, the structure or carrier can be removed from the enzymc-containing medium and the enzyme can be concentrated.

Concentration may be by any useful method including chromatography, centrifugation, filtration, 35 dialysis, extraction, évaporation of solvents, spray drying and adsorplion onto a solid support.

The concentrated enzyme can be stored for a time and then be used by addition to a second biomass to produce useful products.

[0058] In another implémentation of the method, the enzyme is produced by the selected microorganism in a liquid (e.g., aqueous) medium, in the presence of the biomass material. In order to contain the biomass material within the medium the biomass material is disposed in a structure or carrier, for example a mesh bag or other porous container with openings or pores.

The pore size is such that preferably at least 80% (more preferably at least 90%, at least 95% or at least 99%) of the insoluble portion of the biomass material is retained within the structure or carrier during enzyme production. For instance, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%. 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99% ofthe insoluble portion of the biomass material is retained within the structure or carrier during enzyme production.

[0059] It is preferred that the pore size or mesh size of the container be such that substantially none of the insoluble portion of the biomass material flows out of the container during enzyme production. It is also preferred that the pore size be large enough to allow molécules such as sugars, soluble polysaccharides, proteins and biomolecules to pass. Preferably 15 the pore size is large enough that large molécules such as proteins do not foui or block the pores during the course of enzyme production.

[0060] Thus, it is generally preferred that the nominal pore size or mesh size bc smaller than most of ali of the particles of tire biomass material. In some implémentations the absolute pore size is smaller than 50% (preferably smaller than 60%, 70%, 80%, 90%, 95%, 98% or 99%) of 20 the particles of the biomass material. For instance, the absolute pore size can be smaller that

50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, or 59% of the particles of the biomass material. Preferably the absolute pore size can be smaller than 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 25 97%, 98%, 99% of the particles of the biomass material.

[0061] The aqueous media used in the above described methods can contain added yeast extract, com steep, peploncs, amino acids, ammonium salts, phosphate salis, potassium salts, magnésium salts, calcium salts, iron salts, manganèse salts, zinc salts and cobalt salts. In addition to these components, the growth media typicaliy contains 0 to 10% glucose (e.g., 1 to 30 5% glucose) as a carbon source. The inducer media can contain, in addition to the biomass discussed previously, other inducers. For example, some known inducers are lactose, pure cellulose and sophorose. Various components can be added and removed during tire processing to optimize the desired production of useful products.

[0062] The concentration of the biomass typicaliy used for inducing enzyme production is greater than 0.1 wt % (e.g., greater than or equal to 1 %) and less than or equal to 50 wt % (less than or equal to 40 wt %, less than or equal to 30 wt %, less than or equal to 20 wt %, less than or equal to 10 wt %, less than or equal to 5 wt %). For instance, the concentration of biomass

used for enzyme induction can be 0.1 wt %, 0.2,0.3,0.4,03,0.6,0.7,0.8,0.9, or 1.0 wt %. The concentration of biomass can be 2,3,4,5,6,7,8,9, or 10 wt %. The concentration of biomass can be 15,20,25,30,35,40,45, or 50 wt %.

[0063] Any of the processes described herein may be performed as a batch, a fed-batch or a continuous process. The processes are especially useful for industrial scale production, e.g., having a culture medium of at least 50 litcrs, preferably Qt least 100 liters, more preferably at least 500 liters, even more preferably at least 1,000 liters, in particular at least 5,000 liters or 50,000 liters or 500.000 Ihers. The process may be carried out aerobically or anaerobically. Some enzymes are produced by submerged cultivation and some by surface cultivation.

[0064] In any of the process described herein, the enzyme can be manufactured and stored and then used to in saccharification réactions at a later date and/or in a different location. [0065] Any of the processes described herein may be conducted with agitation. In some cases, agitation may be performed using jet mixing as described in U.S. Pat. App. Pub. 2010/0297705 Al, filed May 18,2010 and published on November 25,2012, U.S. Pat. App.

Pub. 2012/0100572 Al, filed November 10.2011 and published on April 26,2012, U.S. Pat. App. Pub. 2012/0091035 Al, filed November 10,2011 and published on April 19,2012, the full disclosures of which are incorporated by référence herein.

[0066] Températures for the growth of enzyme-produclng organisms are chosen to enhance organism growth. For example for Trichoderma reesei the optimal température is generally between 20 and 40°C (e.g,, 30°C). and the température for enzyme production can be optimized for that part of the process. For example for Trichoderma reesei the optimal température for enzyme production Is between 20 and 40°C (e.g., 27°C).

STRUCTURE OR CARRIER [0067] The structure or carrier can be, for example, a bag, net, membrane, shell or combinations of any of these.

[0068] The structure or carrier can bc made with a thermoplastie resin, for example, polyethylene, polypropylenc, polystyrène, poiycarbonate, poiybutyiene, a thermopiastic polyester, a polyether, a thermoplastic polyuréthane, polyvinylchloride, polyvinylidene difiuoride, a polyamide or any combination of these.

[0069] The structure or carrier can also be made of woven or non-woven fibers. Some preferred synthetic fiber or non-fiber materials are, for example, polyester, aramid, poiyoiefin, PTFE, polyphenlene sulfidc, polyuréthane, polyimide, acrylîc, nylon and any combination of these.

[0070] The structure of carrier can also be made from biodégradable and/or water soluble poiymers, for example, aliphatic polyesters, polyhydroxyalkanoates (PHAs), poly-3hydroxybutyrate, polyhydroxyvalerate, poiyhydroxyhexanoate, polylactic acid, poiybutyiene

succinatc, polybutylcnc succinatc adipate, polycaprolactonc, polyvinyl alcohol, polyanhydridcs, s tare h dérivatives, cellulose esters, cellulose acetate, nitrocellulose and any combination of these. [0071] Other materials contemplated for the structure or carrier include, for example, métal (e.g._t aluminum, copper), an alloy (e.g., brass. stainless steel), a ceramic (e.g., glass, alumina), a lhermosetting polymer (e.g., bakélite), a composite material (e.g., fiberglass), a biopolymer and any combination of these. Any structural material, for example, as disclosed above, can be combined to provide the structure or cairier.

[0072] The structure or carrier can be made of a biodégradable, bioerodible, and/or water soluble polymer. Such a polymer can be chosen to dégradé and release the material within it at 10 or near a designated time. The polymer can be selected so that it will serve as a carbon source or nutritive source for the microorganisms being cultured. Polyhydroxyalkanoates, for instance, are readily consumed by many composting fungi and bacteria. PHAs can be a good choice for a structure or carrier designed to release ils contents into a culture of such organisms.

[0073] Altematively, the structure or carrier can be configured and made from materials intended to be tom apart by the impellers of a fermentation system. The fermentation mixing cycle can be schedulcd to maintain the structure or carroer in an intact state for a period of time, and then altered to cause the structure or carrier to corne in contact with the impellers.

[0074] The container or carrier can be of any suitable shape, for example, a loroïd, sphère, cube, oval, cuboid, dog bone, cylindrical, hexagonal prism, cône, square based pyramid, envelopc or combinations of these.

[0075] The container or structure can hâve a scalable and in some cases rescalablc opening such as a zipper, Velcro™ hook and loop fastener, heat seal, clips, pressure sensitive adhesive, buttons or lie (e.g., with a string or drawstring).

[0076] The structure or container may be rigid, semi-rigid or non-rigid. Anon-rigid container is expected to be generally flexible in most directions. A semi-rigid container can be expected to be somewhat flexible in most directions. In some implémentations, the container comprises a flexible, fabric bag.

[0077] The bag may hâve some rigid components such as a framc made of a métal wire or rigid polymer. The container or carrier can hâve a surface texturing, for example, grooves, 30 corrugat ion, and qui 11 ing.

[0078] The container can hâve partitions, for example, it can hâve different pouches made with the same or different materials and/or there may be two or more structures or carriers nested within each other.

[0079] The container or carrier may be designed so as to float on top of the medium or be 35 partially submerged therein, or It may be designed to be fully submerged in the medium. For example, the bag may hâve hooks, loops or adhesives to allow it to attach to the wall of a bioreactor, tank or other container. Il may also hâve weighls to hold part or ail of it submerged

in the medium, and/or buoyant parts to keep parts of it above the medium. The container or carrier can be désigné d to be free in the medium.

[0080] The structures or carriers can hâve pores. With respect to pore size, it is known that permeable materials may contain a distribution of pore sizes. Typically the pore size is rated as 5 absolute or nominal. An absolute pore size rating spécifiés the pore size at which a challenge material or organism of a particular size will be retained with 100% efficiency. A nominal pore size describes the abîlity of the permeable material to retain the majority of the partlculates (e.g., 60 to 98%). Both ratings dépend on process conditions such as the diflerential pressure, the température or the concentration.

[0081] In some implémentations, the container has a nominal pore size or mesh size of less than about 10 mm, e.g., less than 1000 um, 750 um, 500 um, 250 um, 100 um, 75 um, 50 um, 25 um, lOum, lum.0.1 um, 10 nm or even less than l nm. In some implémentations, the container has a nominal pore size or mesh larger than 1 nm, e.g., larger than 10 nm, 0.1 um, 10 um, 25 um, 50 um, 75 um, 100 um, 250 um, 500 um, 750 um, 1 mm or even 10 mm.

[0082] If the structure or carrier Is made of a polymer, the pores may be formed by stretching the polymer, either uniaxially or biaxial ly. Such methods for formulating and stretching polymers to make films with a particular pore size are known in the art.

[0083] The structure or carrier may be designed to allow for the insertion of, for example, a mixing device, a monitoring device, a sampling device or combinations of any of these. The 20 design may inciude, for example a sealable opening or fittîng configured to receive such a device. The monitoring device can be, for example, a pH probe, an oxygen probe, a température probe, a chemical probe or any combinations of these. Optionally, the monitoring device can be remotely operated (e.g., by a wireless connection) and can be free or attached to the structure. The carrier or structure can bave a tagging device, for example, a tag with an identifying alphanumcrical label or identifying color.

[0084] In some Implémentations, it is preferred that the structure or carrier hâve suffirent surface area, for example, to allow good exchange between the contents of the structure or carrier and the medium or other external components, for example between the additive and the biomass material. Il can also bc advantageous to hâve a high surface area to présent a large area 30 to which a microorganism, e.g., a ccl I ul ase-producin g organism, can optional ly attach.

MEDIUM [0085] In the methods described herein, the structure or carrier is contacted or placed in a medium. The medium can be, for ex ample, a liquid, u gas, a chemical solution, a suspension, a 35 colloid, an émulsion, a non-homogenous multiphase System (e.g., a hydrophilic phase layered with a hydrophobie phase) and any combinations of these. The medium can be further manipulated during or after the process; for example, it can bc purified and reused by, for

example, by nitration, centrifugation and/or irradiation. Optionaliy, the medium can contain, for example, nutrients, particulates (e.g., inorganic or organic containing), oligomers (e.g., viscosity modifiers), carbon sources, surfactants (e.g., anti-foam agents), lipïds, fats, extracts (e.g., yeast extract, casein extracts and or vegetable extracts), meta! ions (e.g., Fe^î+, Mg^î+, Mn³⁺, Cu^î+, Na¹⁺, 5 Ca²⁺, K**), anions, nitrogen sources (e.g., amino acids, ammonia, urea), vitamins, proteins (e.g., peptones, enzymes), buffers (e.g., phosphates) added in any combination and sequence.

ADDITIVES [0086] Additives used in the processes disclosed herein can include, by way of example, a 10 microorganism, n nutricnt, a spore, an enzyme, an acid, a base, a gas, an antibiotic, a pharmaceutical and any combinations of these. The additives can be added in any sequence and combination during the process. The additives can be disposed in a structure or carrier or out of the structure or carrier in any combination or sequence.

ENZYMES [0087] In one embodiment of the process, the additive is un enzyme produced by filamentous fungi or bacteria.

[0088] Enzymes are produced by a wide variety of fungi, bacteria, yeasts, and other microorganisms, and there are many methods for optimizing the production and use of 20 cellulases.

[0089] Filamentous fungi, or bacteria that produce cellulase, typicaily require a carbon source and an induccr for production of cellulase. In prior art processes the carbon source is typicaily glucose and the induccr is typicaily pure cellulose. Apart from the cost of pure glucose and pure cellulose, the secreted enzyme produced by this method can be inferior for saccharifying biomass. Without being bound by any theory, it is believed that the rcason for this is that the enzymes produced are particularly suited for saccharification of the substrate used for inducing Its production, and thus if the inducer is cellulose the enzymes may not be well suited for degrading lignocellulosic material.

[0090] The ccllulase-producing organism’s growth rate and state is determined by particular growth conditions. When the host cell culture is introduced into the fermentation medium, containing n carbon source, the inoculated culture passes through a number of stages. Initially growth does not occur. This period is referred to as the lag phase and may be considered a period of adaptation. During the next phase referred to as the cxponential phase” the growth rate of the host cell culture gradually increases and the carbon source is consumed. After a 35 period of maximum growth the rate ceases and the culture enters statîonary phase. After a further period of time the culture enters the dcath phase and the number of viable cells déclinés.

Where in the growth phase the cellulase is expressed dépends on the cellulase and host cell. For • '6 example, the cellulose may be expressed in the exponcntial phase, in the transient phase between the exponential phase and the stotionary phase, or alternativeiy in the stationary phase and/or just before sporulation. The ccllulase may also be produced in more than one of the above mentioned phases.

[0091] When contacted with a biomass, the cellulase producing organism 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. Since there are a variety of substrates ln a particular biomaterial, there are a variety of celluloses, for example, the endoglucanase, exoglucanase and cellobiase discussed previously. By selecting a particular lignocellulosic material as the inducer the relative concentrations and/or activities of these enzymes can be modulated so that the resulting enzyme complex wiil work efficiently on the lignocellulosic material used us 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.

[0092] Since cellulose is Insoluble and imperméable to organisms, it has been suggested that when cellulose is used us an inducer, a soluble oligosaccharide(s) such us cellobiose is actually the direct inducer of cellulase. Expression at a basal level allows a small amount of ccllulase to hydrolyze cellulose to soluble oligosaccharides or to an inducer. Once the inducer enters the cell, It triggers full-scalc transcription of the cellulase gene medinted by activator proteins and activating éléments. After cellulose is degraded u large amount of glucose is liberated, which causes calabolite repression.

[0093] Lignocellulosic materials comprise different combinations of cellulose, hemicellulose and lignin. Cellulose is a linear polymer of glucose forming a fuir!y stiff linear structure without significant coiling. Due to this structure and the disposition of hydroxyl groups that can hydrogen bond, cellulose contains crystalline and non-crystalline portions. The crystalline portions can also be of different types, noted as Ralpha) and I(bcta) for example, depending on the location of hydrogen bonds between strands. The polymer lengths themselves can vary lending more variety to the form of the cellulose. Hemicellulose is any ofseverol heteropolymers, such as xylan, glucuronoxylan, orabinoxylans, and xyloglucon. The primary sugar monomer présent 1s xylose, although other monomers such ns mannose, galactose, rhamnose, arabinosc 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, they hâve been described as an amorphous dendritic network polymer of phenyl propene units. The amounts of cellulose, hemicellulose and lignin in a spécifie biomaterial 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 depcnding on the type. Grasses typically are 33-38% cellulose, 24-32% hemicellulose and 17-22% lignin. Ciearly lignocellulosic biomass constitutes a large class of substrates.

[0094] The diversity of biomass materials may be further increased by pretreatment, for example, by changing the crystailinity and molccular weights of the poiymers. The variation in the composition of the biomass may also increase due to geographical and seasonal variation, Le., where and when the material was coiiected.

[0095] One of ordinary skill in the art can optimize the production of enzymes by microorganisms by adding yeast extract, com steep, peptones, amino acids, ammonium salts, 10 phosphate salts, potassium salis, magnésium salts, calcium saits, iron salts, manganèse salts, zinc sails, cobalt salis, 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.

[0096] Température, pH and other conditions optimal for growth of microorganisms and 15 production of enzymes are generally known in the art.

BIOMASS MATERIALS [0097] As used herein, the term biomass materials” includes lignocellulosic, cellulosic, starchy, and microbial materials.

[0100] Lignocellulosic materials include, but are not limited to, wood, particle board, forestry wastes (e.g., sawdust, aspen wood, wood chips), grasses, (e.g., switchgrass, miscanthus, cord grass, reed canary grass), grain residues, (e.g., rice hulis, oat huils, wheat chaff, barley hulls), agricultural waste (e.g., silage, canola straw, wheat straw, barley straw, oat straw, rice straw, jute, hemp, (lax, bamboo, sisal, abaca, com cobs, com stover, soybean stover, com fiber, alfolfa, hay, coconut hair), sugar processing residues (e.g., bagasse, beet pulp, agave bagasse), algae, scaweed, manure, sewage, and mixtures of any of these.

[0101] In some cases, the lignocellulosic material includes comcobs. Ground or hammermiiled 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 facilitate 30 harvest and collection, in some cases the entire com plant is used, including the com stalle, com kemeis, and in some cases even the root system of the plant.

[0102] Advantagcously, no additional nutrients (other than a nitrogen source, e.g., urca or ammonia) arc required during fermentation of comcobs or cellulosic or lignocellulosic materials containing significant amounts of comcobs.

[0103] Comcobs, before and after comminution, are also easier to 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.

[0104] 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, greetingcards, brochures, prospectuses, newsprint), printer paper, polycoated paper, card stock, cardboard, paperboard, materials having 5 a high a-ccllulo$e content such as cotton, and mixtures of any of these. For example paper products as described in U.S. App. No. 13/396,365 (Magazine Feedstocks” by Medoff et ai, filed February 14,20)2), the full disclosure of which is incorporated herein by reference.

[0105] Cellulosic materials can also include lignocellulosic materials which hâve been delignified.

[0106] Starchy materials include starch itself, e.g., corn starch, wheat starch, potato starch or rice starch, a dérivative of starch, or a material that includes starch, such as an edible food product or a crop. For exampie, lhe starchy material can be arracacha, buckwheat, banana, barley, cassava, kudzu, oca, sago, sorghum, regular household potatoes, sweet potato, taro, yams, or one or more beans, such as favas, lentils or peas. Blends of any two or more starchy materials are also starchy materials. Mixtures of starchy, cellulosic and or lignocellulosic materials can also be used. For example, a biomass can be an entire plant, a part of a plant or different parts of a plant, e.g., a wheat plant, cotton plant, a corn plant, rice plant or a tree. The starchy materials can be treated by any of the methods described herein.

[0107] Microbial materials include, but are not limited to, any naturally occurring or genetically modified microorganism or organism that contains or is capable of providing a source of carbohydrates (e.g., cellulose), for example, protists, e.g., anima! protists (e.g., protozoa such as flagellâtes, amoeboids, ciliatcs, and sporozoa) and plant protists (e.g., algae such alveolates, chlorarachniophytes, cryptomonads, euglenids, glaucophytes, haptophytes, red algae, stramenopiles, and viridacplantae). Other examples include seaweed, plankton (e.g., macroplankton, mesoplankton, mîcroplankton, nanoplankton, picoplankton, and femptoplankton), phytoplankton, bacteria (e.g., gram positive bacteria, gram négative bacteria, and extremophilcs), 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. Altematively or in addition, microbial biomass can be obtained from culture

Systems, e.g., large scale dry and wet culture and fermentation Systems.

[0108] The biomass material can also include offal, and similar sources of material. [0109] In other embodiments, the biomass materials, such as cellulosic, starchy and lignocellulosic feedstock materials, can be obtained from transgenic microorganisms and plants that hâve been modified with respect to a wild type variety. Such modifications may bc, for 35 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 the wild type variety. For exampie, 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 alleles are 5 artificially created 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 nlky lating agents, epoxides, alkaloids, peroxides, formaldéhyde), irradiation (e.g., X-rays, gamma rays, neutrons, beta particles, alpha particles, protons, deuterons, UV radiation) and température shocking or other external stressîng and subséquent sélection techniques. Other 10 methods of providing modified genes is through error prone PCR and DNA shuffling followed by insertion of lhe desired modified DNA into the desired plant or sced. Methods of introducing lhe desired genetic variation in the seed or plant include, for example, the use of a bacterial carrier, biolistics, calcium phosphate précipitation, electroporation, gene splicing, gene silencing, lipofection, microinjection and viral carriers. Additional genetically modified materials hâve 15 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.

[0110] Any of the methods described herein can bc practiccd with mixtures of any biomass materials described lierein.

BIOMASS MATERIAL PREPARATION - MECHANICAL TREATMENTS [0111] The biomass can bc 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 I %). The biomass can also bc delivered in a wet state, for example as a wet solid, a sluny or a 25 suspension with al least about 10 wt% solids (e.g., al least about 20 wt.%, at least about 30 wl.

%, at least about 40 wt.%, at least about 50 wt.%, at least about 60 wt.%, al least about 70 wt%).

[0112] The processes disclosed herein can utilize low buik density materials, for example cellulosic or lignoccllulosic feedstocks that hâve been physically pretreated to hâve a bulk 30 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 Dl 895B. Briefly, lhe method involvcs filling a measuring cylinder of known volume with a sample and obtaining a weight of the sample. The bulk density is calculated by dividing the weight of the sample in grams by the known volume of the cylinder in cubic centimeters. If 35 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 hereby incorporated by référencé.

φ 20 [0113] In some cases, the pre-treatment processîng includes screening of the biomass material. Screening can be through a mesh or perforated plate with a desired opening size, for example, tess than about 6.35 mm (1/4 inch, 0.25 inch), (e.g., less than about 3.18 mm (1/8 ïnch, 0.125 inch), tess than about 1.59 mm (1/16 inch, 0.0625 Inch), ls less than about 0.79 mm (1/32 5 inch, 0.03125 inch), e.g., less than about 0.51 mm (1/50 Inch, 0.02000 ïnch), less than about 0.40 mm (1/64 inch, 0.015625 inch), less than about 0.23 mm (0.009 inch), less than about 0.20 mm (1/128 inch, 0.0078125 Inch), less than about 0.18 mm (0.007 inch), less than about 0.13 mm (0,005 Inch), or even less than about 0.10 mm (1/256 inch, 0.00390625 inch)). Inone configuration the desired biomass fails through the perforations or screen and thus biomass larger than the perforations or screen are not irradiated. These larger materials can be reprocessed, for examplc by comminuting, or they can simply be removed from processîng. In another configuration material that is larger than the perforations is irradiated and the smaller material is removed by the screening process or recycled. In this kind of a configuration, the conveyor itself (for example a part of the conveyor) can be perforated or made with a mesh. For 15 example, In one particular embodiment the biomass material may be wet and lhe perforations or mesh allow water to drain away from the biomass before irradiation.

[0114] Screening of material can also be by a manua! method, for examplc by an operator or mechanoid (e.g., a robot equipped with a color, refiectivity or other sensor) that removes unwanted material. Screening can also be by magnetïc screening wherein u magnet is disposed 20 ncar lhe convcycd material and the magnetic material is removed magnetically.

[0115] Optional pre-treatment processîng can include heating the material. For example a portion of the conveyor can be sent through a heated zone. The heated zone can be created, for example, by IR radiation, microwavcs, 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 25 side, can be continuous or periodic and can be for only a portion of the material or ail the material. For example, a portion of lhe conveying trough cnn be heated by use of a heating jackct Heating can be, for example, for the purposc of drying the material. In the case of drying the material, this can also be facilitated, with or without heating, by the movement of a gas (e.g., air, oxygen, nitrogen, He, CO₂. Argon) over and/or through lhe biomass os it is being conveyed.

[0116] Optionally, pre-treatment processîng can include cooling the material. Cooling material is described in US Pat No. 7,900,857 to Medoff, lhe disclosure of which in incorporated herein by reference. For example, cooling can be by supplying a cooling fluid, for exemple water (e.g., with glycerol), or nitrogen (e.g., liquid nitrogen) to the bottom of the conveying trough. Altematively, a cooling gas, for example, chilled nitrogen can be blown over the b iomass materi al s or under the conveying s ystem.

[0117] Another optional pre-treatment processîng method can include adding u material to the biomass. The additional material can be added by, for examplc, by showering, sprinkling

and or pouring the material onto the biomass as it is conveyed. Materials that can be added include, for example, metals, ceramics and/or ions as described in U.S. Pat. App. Pub. 2010/0105119 Al (fïled October 26,2009) and U.S. Pat App. Pub. 2010/0159569 Al (filed December 16,2009), the entire disclosurcs of which are incorporated herein by référencé.

Optional materials that can be added include acids and bases. Other materials that can be added are oxidants (e.g., peroxides, chlorates), polymers, potymerizable monomers (e.g„ containing unsaturated bonds), water, catalysts, enzymes and/or organisms. Materials can be added, for example, in pure form, ns 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 to 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 efficiency 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 bc useful for further downstream processing. The added material may help in conveying the material, for example, by iowering dust levels.

[0118] Biomass can bc delivered to the conveyor by a belt conveyor, a pneumatic conveyor, a screw conveyor, a hopper, a pipe, manualiy or by a combination of these. The biomass can, for examplc, bc droppcd, poured and/or piaccd onto the conveyor by any of these methods. ln some 20 embodiments the material is delivered to the conveyor using an encloscd 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 foiIs of an électron gun (if such a device is used for treating the material). [0119] The material can be leveled to form a uniform thickness between about 0.0312 and 5 inches (e.g., between about 0.0625 and 2.000 inches, between about 0.125 and 1 înches, between about 0.125 and 0.5 inches, between about 0.3 and 0.9 inches, between about 0.2 and 05 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 inches, 0.250 +/- 0.025 inches, 0.300 +/- 0.025 inches, 0.350 +/- 0.025 inches, 0.400 +/- 0.025 inches, 0.450 +/- 0.025 inches, 0.500 +/- 0.025 inches,

0550 +/- 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 +A 0.025 inches, 0.900 +/- 0.025 inches. [0120] Generally, it is preferred to convey the material as quickly as possible through the électron beam to maximize throughput. For ex ample the material can be conveyed at rates of at least 1 ft/min, e.g., at least 2 ft/min, at least 3 ft/min, at least 4 ft/min, at least 5 ft/min, at least 10 ft/min, at least 15 ft/min, 20.25,30,35,40,45,50 ft/min. The rate of conveying is related to the beam current, for example, for a W inch thick biomass and 100 mA, the conveyor can move at

about 20 fVmin to provide a useful irradiation dosage, at 50 mA the conveyor can move at about 10 η/min to provide approximately the same irradiation dosage.

[0121] After the biomass material has been conveyed through the radiation zone, optional post-treatment processing can be done. The optional post-treatment processing can, for example, 5 be a process described with respect to the pré-irradiation processing. For example, the biomass can be screened, heated, cooled, and/or combined with additives. Uniquely to post-irradiation, quenching of the radicals can occur, for example, quenchîng of radicals by the addition of fluids or gases (e.g., oxygen, ni trous oxide, ammonia, liquids), using pressure, heat, and/or the addition of radical scavengcrs. For example, the biomass can be conveyed out of the enclosed conveyor 10 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 blomass 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.

[0122] If desired, one or more mechanical treatments can be used in addition to Irradiation to 15 further reduce the recalcitrance of the biomass material. These processes can be applied before, during and or after irradiation.

[0123] In some cases, the mechanical treatment may Include on initial préparation of the feedstock as received, e.g., size réduction of materials, such as by comminution, e.g., cutting, grinding, shearing, pulvcrizing or chopping. For example, in some cases, loose feedstock (e.g., 20 recycled paper, starchy materials, or switchgrass) is prepared by shearing or shredding.

Mechanical treatment may reduce the bulk density ofthe biomass material. increase the surface area of the biomass material and/or decrease one or more dimensions of the biomass material. [0124] Altematively, or in addition, the feedstock material can first be physically treated by one or more of the other physical treatment methods, e.g., chemical treatment, radiation, 25 sonication, oxidation, pyrolysis or sleam explosion, and then mcchanically 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 brittlc and, therefore, it may be easier to further change the structure of the material by mechanical treatment. For example, a feedstock material can be conveyed through ionizing radiation using a conveyor as described hcreîn and then 30 mcchanically treated. Chemical treatment can remove some or ali of the lignin (for example chemical pulping) and can partially or completely hydrolyze the material. The methods also can be used with pre-hydrolyzed material. The methods also can be used with material that has not been pre hydrolyzed Tlie methods can be used with mixtures of hydrolyzed and non-hydrolyzed materials, for example with about 50% or more non-hydrolyzed material, with about 60% or 35 more non- hydrolyzed material, with about 70% or more non-hydrolyzed material, with about 80% or more non-hydrolyzed material or even with 90% or more non-hydrolyzed material.

[0125] In 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 shattering the biomass materials, making the cellulose of the materials more susceptible to chain scission and/or dîsruption of crystalline structure during the physical treatment [0126] Methods of mechanically treating the biomass material include, for example, milling or grînding. Milling may be performed using. for example, a mil!, bail mi 11, colloid mil!, conical or cône mill, disk mill, edge mill, Wiley mill, grist mill or other mill. Grînding may be performed using, for example, a cutting/impact type grinder. Some exemplary grinders include ! 0 stone grinders, pin grinders, coffec grinders, and burr grinders. Grînding or milling may be provided, for example, by a rcciprocating pin or other element, as is the case in a pin mill. Other mechanical treatment methods include mechanical ripping, tearing, shearing or chopping, other methods that apply pressure to the libers, and air attrition milling. Suitable mechanical treatments further include any other technique that continues the disruption of the internai 15 structure of the material that was initiated by the previous processing steps.

[0127] Mechanical feed préparation Systems can be configured to produce streams with spécifie characteristics such as, for example, spécifie maximum sizes, spécifie length-to-width, or spécifie surface areas ratios. Physical préparation can Increase the rate of réactions, improve the movement of material on a conveyor, improve the irradiation profile of the material, improve 20 the radiation uniformity of the material, or reduce the processing time required by opening up the materials and making them more accessible to processes and/or reagents, such as reagents in a solution.

[0128] The bulk density of feedstocks can be controlled (e.g., increased). In some situations, it can bc désirable to prépare a low bulk density material, e.g., by densifying the material (e.g., 25 densification can make it easier and less costly to transport to another site) and then reverting the material to a lower bulk density state (e.g., after transport). The material can be densifled, for example from less than about 0.2 g/cc to more than about 0.9 g/cc (e.g., less than about 0.3 to more than about 0.5 g/cc, less than about 0.3 to more than about 0.9 g/cc, less than about 0.5 to more than about 0.9 g/cc, less than about 0.3 to more than about 0.8 g/cc, less than about 0.2 to 30 more than about 0.5 g/cc). For examplc, the material can be densified by the methods and equipment dîsclosed 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 the United States), the full disclosures of which are incorporated herein by référencé. Densified materials can be processed by any of the methods described herein, or any material 35 processed by any of the methods described herein can be subsequently densified.

[0129] In some embodiments, the material to be processed is in the form of a fibrous material that includes fibers provided by shearing a fiber source. For examplc, the shearing can be performed with a rotary knife cutter.

[0130] For examplc, a fiber source, e.g., that is récalcitrant or that has had its recalcitrance level redueed, 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 IJ9 mm or less (1/16 inch, 0.0625 Inch), provide a second fibrous material. If desired, the fiber source can be eut prior to the shearing, e.g., with a shredder. For example, when a paper is used as the fiber source, the paper can be first eut into strips that are, e.g., 1/4- to l/2-inch wide, using 10 a shredder, e.g., a counter-rotating screw shredder, such as those manufactured by Munson (Utica, N.Y.). As an alternative to shredding, the paper can be redueed in size by cutting to a desired size using a guillotine cutter. For example, the guillotine cutter can be used to eut the paper into sheets that are, e.g., 10 inches wide by 12 inches long.

[0131] In some embodiments, the shearing of the fiber source and the passing of the resulting 15 first fibrous material through a first screen are performed concurrently. The shearing and the passing can also be performed in a batch-typc process.

[0132] For cxample, a rotary knife cutter can be used to concurrently shear the fiber source and screen the first fibrous material. A rotary knife cutter includes a hopper that can be loadcd with a shredded fiber source prepared by shredding a fiber source. The slireddcd fiber source.

[0133] In some implémentations, the feedstock is physically treated prior to saccharification and/or fermentation. Physical treatment processes can include one or more of any of those described herein, such as mechanical treatment, chemical treatment, irradiation, sonication, oxidation, pyrolysis or stcam explosion, Treatment methods can be used ln combinations of two, three, four, or even ail of these technologies (in any order). When more than one treatment method is used, the methods can be applied at the same time or at different times. Other processes that change a molecular structure of a biomass feedstock may also be used, alone or in combination with the processcs disclosed herein.

[0134] Mechanical treatments that may be used, and the characteristîcs of the mechanically treated biomass materials, are described in further detail ln U.S. Pat. App. Pub. 2012/0100577 30 Al, filedOctober 18,2011, the full disclosureof which ishereby incorporatedhereinby reference.

TREATMENT OF BIOMASS MATERIAL - PARTICLE BOMBARDMENT [0135] One or more treatments with energetic particle bombardment can be used to process 35 raw feedstock from a wide variety of different sources to extract useful substances from the feedstock, and to provide panially degraded organic material which fonctions as input to further processing steps and/or sequences. Particle bombardment can rcduce the molecular weight

and/or crystallinity of fcedstock. In some embodiments, energy deposited in a material that releases an électron from its atomic orbital can be used to treat the materials. The bombardment may be provided by heavy charged particles (such as alpha particles or protons), électrons (produced, for example, in beta dccay or électron beam accelerators), or electromagnetic radiation (for example, gamma rays, x rays, or ultraviolet rays). Altematively, radiation produced by radioactive substances can be used lo treat the fcedstock. Any combination, in any order, or concurrcnlly of these treatments may be utilized. In another approach, electromagnetic radiation (e.g., produced using électron beam emitters) can be used to treat the feedstock. [0136] Each form of energy ionizes the biomass via particular interactions. Heavy charged particles primarily ionize matter via Coulomb scattering; furthermore, these interactions produce energetic électrons that may further ionize matter. Alpha particles are identical to the nucléus of a hélium atom and are produced by the alpha decay of various radioactive nudei, such as isotopes of bismuth, polonium, astatine, radon, francium, radium, several actinides, such as actinium, thorium, uranium, neptunium, curium, californium, américium, and plutonium.

[0137] When particles are utilized, they can be neutral (uncharged), positively charged or negatlvely charged. When charged, the 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 particles may be désirable, in part, due to their acidic nature. When particles are utilized, the particles can hâve the mass of a resting électron, or greater, e.g., 500,1000, 1500, or 2000 or more times the mass of a resting électron. For example, the particles can hâve a mass of from about 1 atomic unit to about 150 atomic units, e.g., from about I atomic unit to about 50 atomic units, or from about 1 lo about 25, e.g., 1,2,3, 4, 5, 10,12 or 15 atomic units. Accelerators used to accelerate the particles can be clectrostatic DC, electrodynamic DC, RF lincar, magnclic induction linear or continuous wave. For example, cyclotron type accelerators are available from IBA (Ion Beam Accelerators, Louvain-la-Ncuvc, Belgium), such as the Rhodolron™ system, while DC type accelerators are available from RDI, now IBA Industrial, such as the Dynamitron™. Ions and ion accelerators arc discusscd in (ntroductory Nuclcar Physics, Kenneth S. Krane, John Wiley & Sons, Inc. (1988), Krsto Prelec, FIZIKA B 6 ( 1997) 4,177-206; Chu, William T., “O vervie w of Ught-Ion Beam Therapy,

Columbus-Ohio, ICRU-IAEA Meeting, 18-20 Mar. 2006; Iwata, Y. et al., Altemating-PhascFocused (H-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, Vienna, Austria.

[0138] 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 increase chemical bonding (e.g., cross-linking) within feedstock components.

[0139] In some instances when chain scission is désirable and/or polymer chain functionalization is désirable, particles heavicr 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 particles can be 5 utilized for their Lewis acid properties for enhanced ring-opening chain scission. For example, when oxygen-containing functional groups are desired, treatment in the presence of oxygen or even treatment with oxygen ions can be performed. For example, when nitrogcn-containing functional groups are désirable, treatment in the presence of nitrogen or even treatment with nitrogen ions can be perfonned.

OTHER FORMS OF ENERGY [0140] Electrons Internet via Coulomb scattering and bremsstrahlung radiation produced by changes in the velocity of électrons. Electrons may be produced by radioactive nuclei that undergo beta decay, such as isotopes of lodine, césium, technetium, and iridium. Altematively, 15 an électron gun can be used as an électron source via thermionic émission.

[0141] Electromagnetic radiation interacts via three processes: photoelectric absorption, Compton scattering, and pair production. The dominating interaction is determined by the energy of the incident radiation and the atomic number of the material. The summation of interactions contributing to the absorbed radiation in cellulosic material can be expressed by the 20 mass absorption coefficient.

[0142] Electromagnetic radiation is subclassifîed as gamma rays, x rays, ultraviolet rays, infrared rays, microwavcs, or radiowaves, depending on the wavelength.

[0143] For example, gamma radiation can bc employed to treat the materials. Gamma radiation has the advantage of a significant pénétration depth into a variety of material in the 25 samplc. Sources of gamma rays include radioactive nudei, such as isotopes of cobalt, calcium, technetium, chromium, gallium, indium, lodine, iron, krypton, samarium, sélénium, sodium, thalium, and xénon.

[0144] Sources of x rays include électron beam collision with métal targets, such as tungsten or molybdenum or alloys, or compact light sources, such as those produced commercially by 30 Lyncean.

[0145] Sources for ultraviolet radiation include deuterium or cadmium Jamps.

[0146] Sources for infrared radiation include sapphîre, zinc, or selenide window ceramic lamps.

[0147] Sources for microwaves include klystrons, Slevin type RF sources, or atom beam 35 sources that employ hydrogen, oxygen, or nitrogen gases.

[0148] Various other devices may bc used in the methods disclosed herein, including field ionization sources, elcctrostatic ion separators, field ionization generators, thermionic émission sources, microwave discharge ion sources, recirculating or static accelerators, dynamic linear accelerators, van de Graaff accelerators, and folded tandem accelerators. Such devices are disclosed, for example, in U.S. Pat. No. 7,931,784 B2, the complété disclosure of which is incorporated herein by reference.

TREATMENT OF BIOMASS MATERIAL - ELECTRON BOMBARDMENT (0149] The feedstock may be treated with électron bombardment to modify lis structure and thereby reduce its recalcitrance. Such treatment may, for example, reduce the average molecular weight of the feedstock, change the crystalline structure of the feedstock, and/or increase the surface area and/or porosity of the feedstock.

[0150] Electron bombardment via an électron beam is generaily preferred, because it provides very high throughput and because the use of a relatively low voltage/high power électron beam device éliminâtes the need for expensive concrète vaull shielding, as such devices are “self-shiclded” and provide a safe, efficient process. While the “self-shielded devices do include shielding (e.g., métal plate shielding), they do not require the construction of a concrète vaull, greally reducing capital expenditure and often allowing an existing manufacturing facility to be used without expensive modification. Electron beam nccelerators are available, for example, from IBA (Ion Beam Applications, Louvain-la-Neuve, Belgium), Titan Corporation (San Diego, California, USA), and NHV Corporation (Nippon High Voltage, Japan).

[0151] Electron bombardment may be performed using an électron beam device that has a nominal energy of less than 10 MeV, e.g., less than 7 MeV, less than 5 MeV, or less than 2 MeV, e.g., from about 0.5 to 1.5 MeV, from about 0.8 to 1.8 MeV, from about 0.7 to 1 MeV, or from about I to 3 MeV. In some implémentations the nominal energy Is about 500 to 800 keV.

[0152] The électron beam may hâve a relatively high total beam power (the combined beam power of ail accelerating heads, or, if multiple accelerators are used, of ail accelerators and ail heads), e.g., al least 25 kW, e.g., at least 30,40,50,60,65,70, 80,100,125, or 150 kW. In some cases, the power is even as high as 500 kW, 750 kW, or even 1000 kW or more. In some cases the électron beam has a beam power of 1200 kW or more.

[0153] This high total beam power is usualiy achieved by utilizing multiple accelerating heads. For example, the électron beam device may include two, Γοιιτ, or more accelerating heads. The use of multiple heads, each of which has a relatively low beam power, prevents excessive température rise in the material, thereby preventing buming of the material, and also increases the uniformily of the dose through the thickness of the layer of material.

[0154] In some implémentations, it is désirable to cool the material during électron bombardment. For example, the material can be cooled while it is being conveyed, for example by a screw extrader or other convcying equipment.

[0155] To reducc the energy required by the recalcitrance-reducmg process, it is désirable to treat the material as quickly as possible. In general, it is preferred that treatment be performed at a dose rate of greater than about 025 Mrad per second, e.g., greater than about 0.5,0.75,1,13, 2,5,7,10,12,15, or even gTeater than about 20 Mrad per second, e.g., about 0.25 to 2 Mrad per second. Higher dose rates generally requirc higher line speeds, to avoid thermal décomposition of the material. In one implémentation, the accelerator is set for 3 MeV, 50 mAmp beam current, and the line speed is 24 feel/minute, for a sample thîckness of aboul 20 mm (e.g., comminuted corn cob material with a bulk density of 0.5 g/cm³).

[0156] In some embodiments, électron bombardment is performed until the material receives a total dose of al least 0.5 Mrad, e.g., al leasi 5,10,20,30 or at least 40 Mrad. In some embodiments, the treatment is performed until the material receives a dose of from about 0.5 Mrad to aboul 150 Mrad, about I Mrad to aboul 100 Mrad, aboul 2 Mrad to about 75 Mrad, 10 Mrad lo aboul 50 Mrad. e.g., aboul 5 Mrad to about 50 Mrad, from aboul 20 Mrad to about 40 Mrad, about 10 Mrad lo aboul 35 Mrad. or from aboul 25 Mrad to about 30 Mrad. In some implémentations, a total dose of 25 to 35 Mrad is preferred, applied ideally over a couple of seconds, e.g., at 5 Mrad/pass with each pass being applied for aboul one second. Applying a dose of greater than 7 to 8 Mrad/pass can in some cases cause thermal dégradation of the feedstock material.

[0157] Using multiple heads as discussed above, the material can be treated in multiple passes, for examplc, two passes at 10 to 20 Mrad/pass, e.g., 12 to 18 Mrad/pass, separated by a fewsecondsofcool-down,orthreepassesof7to 12Mrad/pass,e.g.,9to II Mrad/pass. As discussed above, treating the material with several relatively low doses, rather than one high dose, tends to prevent ovcrheatïng of the material and also incrcases dose uniformity through the thickness ofthematerial. In some implémentations, the materialisstirredorotherwise mixed during or after each pass and then smoothed into a uniform layer again before the next pass, to further enhance treatment uniformity.

[0158] In some embodiments, électrons are accelcrated lo, 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.

[0159] 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 redueed pressure. For examplc, in some embodiments, the cellulosic and/or lignocellulosic material has less than about five percent by weight retained water, measured at 25°C and at fifly percent relative humidity.

[0160] Electron bombardment can be applied while the cellulosic and/or lignocellulosic material is exposed to air, oxygen-enriched air, or even oxygen ilself, or blanketed b y 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 optimizcd to maximize réactivé gas formation, e.g., ozone and/or oxides of nitrogen.

[0161] In some embodiments, two or more électron sources are used, such as two or more ionizing sources. For example, samples can be treated, in any order, with a beam of électrons, 5 followed 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 beam of électrons, gamma radiation, and energctic UV light. The biomass is conveyed through llie treatment zone where it can be bombarded with électrons. It is generally preferred that the bed of biomass material has a relatively uniform thickness, as previously described, while being 10 treated.

[0162] It may be advantageous to repeat the treatment to more thoroughly reduce the recaicitrance of the biomass and/or further modîfy the biomass. In particular the process parameters can bc adjusted after a first (e.g., second, third, fourth or more) pass depending on the recaicitrance of the material. In some embodiments, a conveyor can be used which includes a 15 circular system where the biomass is conveyed multiple tintes through the various processes described above. In some other embodiments multiple treatment devices (e.g., électron beam generators) are used to Ireat the biomass multiple (e.g., 2,3,4 or more) times. In yet other embodiments, a single électron beam gcncrator may be the source of multiple beams (e.g., 2,3,4 or more beams) that can be used for treatment of the biomass.

[0163] The effectivcness in changing the molecular/supermolecular structure and/or rcducîng the recaicitrance of the biomass biomass dépends on the électron energy used and the dose applied, while exposure time dépends on the power and dose.

[0164] In some embodiments, the treatment (with any électron source or u combination of sources) is performed until tire material reçoives a dose of at least about 0.05 Mrad, e.g., at least 25 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 receïves 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.

[0165] In some embodiments, the treatment is performed at a dose rate of between 5.0 and 30 1500.0 kilorads/liour, e.g., between 10.0 and 750.0 kilorads/hour or between 50.0 and 350.0 kilorads/hours. In other embodiments the treatment is performed at a dose rate of between 10 and 10000 kilorads/hr, between 100 and 1000 kilorad/hr, or between 500 and lOOOkilorads/hr.

ELECTRON SOURCES [0166] Electrons interact via Coulomb scattering and brcmsstrahlung radiation produced by changes in the vclocity of électrons. Electrons may be produced by radioactive nuclei that undergo beta decay, such as isotopes of iodine, césium, technetium, and iridium. Alternatively,

an électron gun can be used as an électron source via thermionic émission and accelerated through an accelerating potential. An électron gun generates électrons, accélérâtes them through a large potential (e.g., greater than about 500 thousand, greater than about 1 million, 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 are initially accelerated in the z direction down the tube and extracted through a foil window. Scanning the électron beam is useful for Increasing the irradiation surface when inadiating materials, e.g., u biomass, that is conveyed through the scanned beam. Scanning the électron beam also distributes the thermal load homogenously on the window and helps reduce the foil window rupture due to local heating by the électron beam. Window foil rupture is a cause of significant down-time due to subséquent necessary repairs and re-starting the électron gun.

[0167] Various other irradiating devices may be used in the methods disclosed herein, including field ionization sources, electrostatic ion separators, field lonization generators, thermionic émission sources, microwave discharge ion sources, recirculating or static accclcrators, dynamic linear accelerators, van de Graaff accelerators, and folded tandem accelcrators. 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.

[0168] A beam of électrons can be used as the radiation source. A beam of électrons has the 20 advantages of high dose rates (e.g., 1,5, or even 10 Mrad per second), high throughput, less containment, and less confinement equipment. Electron bcams can also hâve high electrical efficicncy (e.g., 80%), allowing for lower energy usage relative lo other radiation methods, which can translate into a lower cost of operation and lower greenhouse gas émissions corresponding to the smaller amount ofenergy used. Electron bcams can be generated, e.g., by 25 electrostatic generators, cascade generators, transformer generators, low energy accelerators with a scanning System, low energy accelerators with a linear cathode, linear accelerators, and pulsed accelerators.

[0169] Electrons can also be more efficient at causing changes in the molecular structure of biomass materials, for example, by the mechanism of chain scission. In addition, électrons 30 having energies of 0.5-10 MeV can penetrate low density materials, such as the 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 be useful, e.g., for relatively thin piles, layers or beds of materials, e.g., less than about 0.5 inch, e.g., less than about 0.4 inch, 0.3 inch, 0.25 inch, or less than about 0.1 inch. In some embodiments, the energy of each 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 1.5 MeV, or from about 0.7 MeV to about 1.25 MeV.

Melhods of irradiating materials are discussed ΐη U.S. Pat. App. Pub. 2012/0100577 Al, filed October 18,2011, the entire disclosure of which is herein incorporated by reference.

[0170] Electron beam irradiation devices may be procured commercially from Ion Beam Applications (Louvain-la-Neuve, Belgium), the Titan Corporation (San Diego, California, USA), 5 and NHV Corporation (Nippon High Voltage, Japon). Typical électron energies can be 05

MeV, l MeV, 2 MeV, 45 MeV, 75 MeV, or 10 MeV. Typical électron beam irradiation device power can be 1 KW, 5 KW, 10 KW. 20 KW, 50 KW, 60 KW, 70 KW, 80 KW, 90 KW, 100 KW, 125 KW, 150 KW, 175 KW, 200 KW, 250 KW, 300 KW, 350 KW, 400 KW, 450 KW, 500 KW, 600 KW, 700 KW, 800 KW, 900 KW or even 1000 KW.

[0171] Tradeoffs in considering électron beam irradiation device power spécifications inciude cost to operate, capital costs, dépréciation, and device footprint. Tradeoffs ln considering exposure dose levels of électron beam irradiation would be energy costs and environment, safety, and health (ESH) conccms. Typically, generators are houscd 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 inciude energy costs.

[0172] 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 would cffectively replace a large, fixed beam width. Further, availabie sweep widths of 05 m, l m, 2 m or more are availabie. The scanning beam is preferred in most 20 embodiments describe herein because of the larger scan width and reduced possibility of local heatîng and failure of the Windows.

TREATMENTOF BIOMASS MATERIAL - SONICAT1ON, PYROLYSIS, OXIDATION, STEAM EXPLOSION [0173] If desired. one or more sonication, pyrolysis, oxidative, or steam explosion processes can be used in addition to or instead of other treatments to further reduce the recalcitrancc of the biomass material. These processes can be applied before, during and or after another treatment or treatments. These processes are described in detail in U.S. PaL No. 7,932,065 to Medoff, the full disclosure of which is incorporated herein by reference.

USE OF TREATED BIOMASS MATERIAL [0174] Using the melhods described herein, a starting biomass material (e.g., plant biomass, animal biomass, paper, and municipal waste biomuss) can be used as feedstock to produce useful intermediates and products such as organic acids, salis of organic acids, anhydrides, esters of 35 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 that are readily availabie, but often can be d ifficult to process, e.g.,

municipal waste streams and waste paper streams, such as streams that include newspaper, kraft paper, comigated paper or mixtures of these.

[0175] In order to convert the feedstock to a form that can be readily processed, the glucanor xylan-containing cellulose in the feedstock can be hydrolyzed to low molecular weight carbohydrates, such as sugars, by a saccharifying agent, e.g., an enzyme or acid, a process referred to as saccharification. The low molecular weight carbohydrates can then be used, for example, in on existing manufacturing plant, such as a single cell protein plant, an enzyme manufacturing plant, or a fuel plant, e.g., an éthanol manufacturing facility.

[0176] The feedstock can be hydrolyzed using on enzyme, e.g., by combining the materials 10 and the enzyme in a solvent, e.g., in an aqueous solution.

[0177] Altematively, the enzymes can be supplicd by organisms that break down biomass, such as the cellulose and/or the lignin portions of the biomass, contain or manufacture various cellulolytïc enzymes (cellulases), ligninases or various smatl molécule bîomass-degrading métabolites. Thèse enzymes may be a complex of enzymes that act synergistically to dégradé 15 crystalline cellulose or the lignin portions of biomass. Examples of cellulolytïc enzymes include: endoglucanases, cellobiohydrolascs, and cellobiases (bcta-glucosidascs).

[0178] During saccharification a cellulosic substrale can be Initially hydrolyzed by endoglucanases at random locations producing oligomeric intermediates. These intermediates aie then substrates for exo-splitting glucanases such as ccllobiohydrolase to producc cellobiose 20 from the ends of the cellulose polymer. Cellobiose is a water-soluble 1,4-linked dimer of glucose. Final! y, cellobiose cleaves cellobiose to yield glucose. The efficiency (e.g., time to hydrolyze and/or completeness of hydrolysis) of this process dépends on the recalcitrance of the cellulosic material.

INTERMEDIATES AND PRODUCTS [0179] Using the processes described herein, the biomass material can be convertcd 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., 30 monohydric alcohols or dihydric alcohols, such os éthanol, n-propanol, isobutanol, sec-butnnol, tert-butanol or n-butanol), hydrated or hydrous alcohols (e.g., containing greater than 10%, 20%, 30% or even greater than 40% water), biodicsel, organic acids, hydrocarbons (e.g., methane, ethane, propane, isobutene, pentane, n-hexane, biodiesel, bio-gasoline and mixtures theteof), coproducts (e.g., proteins, such as cellulolytïc proteins (enzymes) or single cell proteins), and 35 mixtures of any of these in any combination or relative concentration, and optionally in combination with any additives (e.g., fuel additives). Other examples include carboxylic acids, salts of a carboxylic acid, a mixture of carboxylic acids and salts of carboxylic acids and esters of carboxylic acids (e.g., methyl, ethyl and n-propyl esters), ketones (e.g., acetone), aldéhydes (e.g., acétaldéhyde), alpha and bêla unsaturated acids (e.g., acrylic acid) and olefins (e.g., ethylene). Other alcohols and alcohol dérivatives include propanol, propylene glycol, 1,4-butanediol, 1,3propanedîol, sugar alcohols and polyols (e.g., glycol, glycerol, erythritol, threitol, arabîtol, xylîtol, ribitol, mannitol, sorbitol, galactitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, and polygiycitoi and other polyols), and methyl or ethyl esters of any of these alcohols. Other products include methyl acryiate, methylmethacryiate, lactic acid, citric acid, formic acid, acetîc acid, propionic acid, bulyric acid, succinic acid, valeric acid, caproic acid, 3-hydroxypropionic acid, palmitic acid, stearic acid, oxalic acid, malonic acid, gluturic acid, oleic acid, iïnoieic acid, giycoüc acid, gamma-hydroxybutyric acid, and mixtures thereof, salts of any of these acids, mixtures of any of the acids and their respective salts.

[0180] Any combination of the above products with each other, and/or of the above products with other products, which other products may be made by the processes described herein or otherwise, may be packaged together and sold as products. The products may be combined, e.g., mixed, blendcd or co-dissolvcd, or may simply be packaged or sold together.

[0181] Any of the products or combinations of products described herein may be sanitized or sterilized prior to seliing the products, e.g., after purification or isolation or even after packaging, to ncutralize one or more potcntially undesirable contaminants that could be présent in the product(s). Such sanitation 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 t to 3 Mrad.

[0182] The processes described herein can produce various by-producl streams useful for generating steam and elcctricity to be used in other parts of the plant (co-generatîon) or sold on the open market. For example, steam generated from buming by-producl streams can be used in a distillation process. As another example, elcctricity generated from buming by-product streams can be used to power électron bcam generators used in pretreatment.

[0183] The by-products used to generate steam and elcctricity are derived from a number of sources throughout the process. For example, anaérobie digestion of wastewater can produce a biogas high in methane and a small amount ofwaste biomass (sludge). As another example, post-saccharification and/or pos t-distil late solids (e.g., unconverted lignin, cellulose, and hemicellulose remaining from the pretreatment and primary processes) can be used, e.g., bumed, as u fuel.

[0184] Many of the products obtained, such as éthanol or n-butanol, can be utilized as a fuel for powering cars, trucks, tractors, ships or trains, e.g., as an internai combustion fuel or as a fuel cell feedstock. Many of the products obtained can also be utilized to power aircraft, such as planes, e.g., having jet engines or helicopters. ln addition, the products described herein can be utilized for electrical power génération, e.g., ιη a conventional steam generating plant or in a fuel cell plant [0185] Other intermediates and products, including food and pharmaceutical products, are described in U.S. Pat. App. Pub. 2010/0124583 Al, published May 20,2010, to Medoff, the full disclosure of which is heieby incorporated by référencé herein.

SACCHARIFICATION [0186] The treated biomass materials can be sacchorifîed, generally by combining the material and a cellulase enzyme in a fluid medium, e.g., on aqueous solution, ln some cases, lhe material is boiled, steeped, or cooked in hot 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.

[0187] The saccharification process can be partially or completely performed in a tank (e.g., a tank having a volume of ai least 4000,40,000, or 500,000 L) in a manufacturing plant, and/or can be partially or completeiy performed in transit, e.g., in a rail car, tanker truck, or in n supertanker or the hold of a ship. The time required for complété saccharification will dépend on tlie 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 entirety converted to sugar, e.g., glucose in about 12-96 hours. 1Γ saccharification is performed partially or completely in transit, saccharification may take longer.

[0188] It is generally preferred that the tank contents bc 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 référencé herein.

[0189] The addition of surfactants can enhance the rate of saccharification. Examples of surfactants include non-ionic surfactants, such as a Twecn® 20 or Twcen® 80 polyethylene glycol surfactants, ionic surfactants, or amphoteric surfactants.

[0190] It is generally preferred that the concentration of the sugar solution resulting from saccharification be relativeiy 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 to be shipped, and also inhibits microbial growth in the solution.

[0191] Aitcmativciy, sugar solutions of lower concentrations may bc used, in which case it may be désirable to add an antimicrobiol additivc, e.g., a broad spectrum antibiotic, in a low concentration, e.g., 50 to 150 ppm. Other suitable ontibiotics include omphotericin B, ampiciliin, chloromphenicol, ciprofloxacin, gentumicin, hygromycin B, kanamycin, neomycin, peniciliin, puromycin, streptomycin. Antibiotics will inhibit growth of microorganisms during transport

T

T

and storagc, and can be used at appropriate concentrations, e.g., between 15 and 1000 ppm by weight, e.g., between 25 and 500 ppm, or between 50 and 150 ppm. If desired, an antibiotic can be included even if the sugar concentration ls relalively high. Altematively, other additives with anti-microbial of preservative properties may be used. Preferably the anlimicrobial additîvefs) 5 are food-grade.

[0192] A relatively high concentration solution can be obtained by Hmiting the amount of water uddcd to the biomass material with the enzyme. The concentration can be controlled, e.g., by controüing how much saccharification takes place. For example, concentration can be increased by adding more biomass material to the solution. In order to keep the sugar that is being produced in solution, a surfactant can be added, e.g., one of those discussed above.

Solubility can also be increased by increasing the température of the solution. For exampic, the solution can be maintained at a température of 40-50°C, 6O-8O°C, or even higher.

SACCHARIFYING AGENTS [0193] Suitable cellulolytic enzymes include celluloses from species in the gênera Bacillus,

Coprinus, Myceliophthora, Ceplialosporium, Scytalidium, Pénicillium, Aspergillus, Pseudomonas, Humicola, Fusarimn, Thielavla, Acremonium, Chrysosporium and Trichoderma, especially those produced by a straîn selected from the species Aspergillus (see, e.g.. EP Pub. No. 0 458 162), Humicola Insolens (reclassified as Scytalidium tliermophilum, see, e.g.. U.S. Pat.

No. 4,435,307), Coprinus cinereus, Fusarimn oxysportun, Myceliophthora tltcrmopltila, Meripüus giganteus, Thielavla terrestris, Acremonium sp. (including, but not limited to, A. persicinum, A. acremonium, A. brachypenium, A dichromosporum, A. obclavatwn, A plnkertoniae, A roseogriseum,A. incoloratum, and A. furatum). Preferred strains include Humicola Insolens DSM 1800, Fusarium oxysportun DSM 2672, Myceliophthora thermophiia

CBS 117.65, Ceplialosporium sp. RYM-202, Acremonium sp. CBS 478.94, Acremonium sp. CBS 265.95, Acremonium persicinum CBS 169.65, Acre/noniurn acremonium AHU 9519, Ceplialosporium sp. CBS 535.71. Acremonium brachypenium CBS 866.73, Acremonium dichromosporum CBS 683.73, Acremonium obclavanmt CBS 311.74, Acremonium pinkerfoniae CBS 157.70, Acremonium roseogriseum CBS 134.56. Acremonium incoloratum CBS 146.62, and Acremonium furatum CBS 299.70H. Cellulolytic enzymes may also be obtained from

Chrysosporium, preferably a strain of Cltrysosporium lucknowense. Additional strains that can be used include, but are not limited to, Trichoderma (particularly T. vtride, T. reesel, and 7*. koningii), alkalophïlic Bacillus (sec, for example, U.S. PaL No. 3,844,890 and EP Pub. No. 0 458 162), and Streptomyces (see, e.g., EP Pub. No. 0 458 162).

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

SUGARS [0195] 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., simulated moving bed chromatography, high pressure chromatography), centrifugation, extraction, any other isolation method known in the art, and combinations thereof.

HYDROGENATION AND OTHER CHEMICAL TRANSFORMATIONS [0196] The processes described herein can include hydrogénation. For example glucose and 10 xylose can be hydrogenated to sorbitol and xylitol respectively. Hydrogénation can be accomplished by use of a catalyst (e.g., Pt/gamma-AhOj, 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 15 products). Cbemical transformations of sugar derived products are described in US Prov. App.

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

FERMENTATION [0197] Yeast and Zymomonas bacteria, for example, can be used for fermentation or conversion of sugar(s) to alcohol(s). Other microorganisms arc 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 to 5, while the optimum pH for Zymomonas is from about pH 5 to 6. Typical fermentation limes are about 24 to 168 hours (e.g., 24 to 96 hrs) wilh températures in the range of 20°C to

40°C (e.g., 26°C to 40°C), however lhermophilic microorganisms prefer higher températures.

[0198] In some embodiments, e.g., when anaérobie organisms are used, at least a portion of lhe fermentation is conducted in lhe absence of oxygen, e.g., under a blank et of on inert gas such as N₂, Ar, He, CO2 or mixtures thereof. Additionaily, the mixture may hâve a constant purge of an inert gas fiowing through the tank during part of or oil of lhe fermentation. In some cases, anaérobie condition, can be aebieved or maintained by carbon dioxide production during the fermentation and no additional inert gas is needed.

[0199] In some embodiments, ail or a portion of the fermentation process can be interrupted before the low molecular weight sugar is completely converted to a product (e.g., éthanol). The intermediate fermentation products include sugar and carbohydrates in high concentrations. The 35 sugars and carbohydrates can be isolated via any means known in the art. These intermediate fermentation products can be used in préparation of food for human or animal consumption.

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

[0200] Jet mixing may be used during fermentation, und in some cases saccharification und fermentation are performed in the same tank.

[0201] Nutrients for the microorganisms may be added during saccharification und/or fermentation, for example the food-based nutrient packages described in U.S. Pat. App. Pub. 2012/0052536, filed July 15,2011, the complété disclosure of which is incorporated herein by référence.

[0202] “Fermentation” includes the methods und products that are disclosed in U.S. Prov. App. No. 61/579,559, filed Decembcr 22,2012, and U.S. Prov. App. No. 61 /579,576, filed December 22,2012, the contents of both of which are incorporated by référencé herein in their entirety.

[0203] Mobile fermenters 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 und designated the United States), the contents of which is incorporated herein in its entirety. Similarly, the saccharification equipment cun be mobile. Further, saccharification und/or fermentation may bc performed in part or cntirely during transit.

FERMENTATION AGENTS [0204] The microorgunism(s) used in fermentation cun be naturally-occurring microorganisms und/or engineered microorganisms. For example, the microorganism cun be a bacterium (including, but not limited to, e.g., a ce! luloly tic bacterium), a fungus, (including. but not limited to, e.g., a yeast), a plant, a protist, e.g., a protozoa or a fungus-like prolest (including, but not limited to, e.g., u slime mold), or an aigu. When the organisms arc compatible, mixtures of organisms can be utilized.

[0205] Suitable fermenting microorganisms hâve the ability to convert carbohydrates, such as glucose, fructose, xylose, arabinose, mannose, galactose, oligosaccharides or polysaccharides into fermentation products. Fermenting microorganisms include strains of the genus Saccharomyces spp. (including, but not limited to, 5. cerevisiae (baker’s yeast), S. distaticiis, S. itvamin), the genus Kluyveroinyces, (including, but not limited to, K. tnarxianus, K. fragiiis), the genus Candida (including, but not limited to, C. pseadotropicalis, und C. brassicae), Pichia sdpilis (a relative of Candida shehatae'), the genus Clavispora (including, but not limited to, C. lusitaniae und C. opuntiae), the genus Pachysolen (including, but not limited to, P. tannophilus), the genus Bretannomyces (including, but not limited to, e.g., B. clausenii (Philippidis, G. P., 1996, Cellulose bioconversion technology, in Handbook on Bioethunok Production und Utilization, Wyman, C.E., ed„ Tuylor & Francis, Washington, DC, 179-212)). Other suitable microorganisms include, for example, Zymomonas inobilis, Clostridium spp. (including, but not

T «twr

limited to, C. tkennoceiktm (Philippidis, 1996, supra), C. saccltarobutylacetonlcum, C. saccharobutylicum, C, Puniceum, C. beijemckti, and C. acetobutylicum), MonUiella poliinis, Moniliellatnegacluliensis, Lactobaciltus spp. Yarrowla lipolytica,Aureobasidiuin sp., Trichosporonoides sp., Trigonopsis variabilis, Trlchosporon sp., Moniliellaacetoabitians sp„ 5 Typhula variabilis, Candida magnoliae, Ustilaginomycetes sp., Pseudozyma tsukubaensis, yeast species of généra Zygosaccltaromyces, Debaryomyces, Hansenula and Piclda, and fungî of the dematioid genus Torula.

[0206] For instance, Clostridium spp. can be used to produce éthanol, butanol, butyric acid, acetic acid, and acétone. Lactobaciltus spp., can be used to produce factice acid.

[0207] 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 Rescarch Sevice Culture Collection, Peoria, Illinois, USA), or the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturcn GmbH, Braunschweig, Germany), to name a few.

[0208] Commercially available yeasts include, for example, Red Star®/Lesaffre Ethanol Red (available from Red Star/Lesaffrc, USA), FALI® (available from Fleischmann’s Yeast, a division of Bums Philip Food Inc., USA). SUPERSTART® (available from Alltech, now Lalemand), GERT STRAND® (available from Gert Strand AB, Sweden) and FERMOL® (available from DSM Specialties).

[0209] Many microorganisms that can bc used to saccharify biomass material and produce sugars can also be used to ferment and convert those sugars to useful products.

DISTILLATION [0210] After fermentation, the resulting fluids can bc distilled using, for example, a “bcer column to separate éthanol and other alcohols from the majority of water and residual solids. The vapor exiting the bcer column can bc, e.g., 35% by weight éthanol and can bc fed to a rectification column. A mixture of nearly azeotropic (92.5%) éthanol and water from the rectification column can bc purified to pure (99.5%) éthanol using vapor-phase molecular sieves. The bcer column bottoms can bc sent to the first effect of a thrce-effect evaporator. The rectification column reflux condenser can provide heat for this first effect. After the first effect, solids can be separated using a centrifuge and dried in a rotary dryer. A portion (25%) of the centrifuge effluent can bc recycled to fermentation and the rest sent to the second and third evaporator effects. Most of the evaporator condcnsate can be retumed to the process as fairly dean condensatc with a small portion split off to waste water treatment to prevent build-up of 35 low-boiling compounds.

[0211] Other than in the examples herein, or unless otherwise expressly specifïed. oil of the numerical ranges, amounts, values and percentages, such as those for amounts of materials, elemental contents, limes and températures of reaction, ratios of amounts, and others, in the following portion of the spécification and attached claims may be read as if prefaced by the word “about” even though the term “about may not expressly appear with the value, amount, or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following spécification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the présent invention. At the very least, and not as an attempt to limit the application of the doctrine of équivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0212] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the spécifie examplcs are reported as precisely as possible. Any numerical value, bowever. inherently contains error nccessarily resulting from the standard déviation found in ils underlying respective testing mcasurements. 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, the numerical values reported are relative to the total weight.

[0213] Also, il should be understood that any numerical range recited herein is intended to include ail sub-ranges subsumed therein. For examplc, a range ofl to 10 is intended to include ail sub-ranges between (and including) the recited minimum value of 1 and the recited maximum value of 10, that is, having a minimum value equal to or greater than 1 and a maximum value of equal to or less than 10. The terms “one, “a, or “an as used herein arc intended to include “at least one” or “one or more, unless otherwise indicated.

[0214] Any patent, publication, or other disclosure material, in wbole or in part, that is said to be incorporated by reference herein is Incorporated herein only to the extent that tire incorporated material does not conflict with existing définitions, statements, or other disclosure material set forth in this disclosure. As such, and to the extent neccssary, the disclosure as explicltly set forth herein supersedes any conflicting material incorporated herein by reference. Any material. or portion thereof, that 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 anses between that incorporated material and the existing disclosure material.

[0215] While this invention has been particularly shown and described with référencés to preferred embodiments thereof. it will be understood by those skilled ln the art that various

changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims (18)

1. What is claimed is:

   5 I. A method comprising: maintaining a combination comprising a liquid medium, a structure or carrier, and a reduced-recalcitrance cellulosic or lignocellulosic biomass disposed within the structure or carrier, under conditions that allow the passage of molécules oui of and/or into the structure or carrier.
2. 2. The method of claim 1, wherein the structure or carrier is porous.
3. 3. The method of claim l, further comprising disposing an additive in the structure or carrier.
4. 4. The method of claim 3 further comprising maintaining the combination under conditions that allow the additive to convert the molécules to one or more products.
5. 5. Tlic method of any one of the above daims, wherein the additive is selected from the

   20 group consisting of a microorganism, an enzyme, an acid, a base, a chemical solution, a nutrient, a minerai, and combinations thereof.
6. 6. The method of claim 5 wherein the additive comprises a microorganism.

   25
7. 7. The method of any one of claims 4-6, wherein the product is selected from the group consisting of: a molécule, a protein, a sugar, a fuel and combinations thereof.
8. 8. The method of daim 7, wherein the proiein comprises an enzyme.

   30
9. 9. The method of any one of the above claims, wherein the structure or carrier is selected from the group consisting of a bag, a shcll, a net, a membrane, a mesh and combinations thereof.
10. 10. The method of daim 2, wherein the structure or carrier comprises a bag, and is formed of 35 a mesh material having a maximum opening size of less than 1 mm.
11. 11. The method of claim 8, where the structure or carrier is a bag, and the bag ls made of a bioerodible polymer, e.g., a polymer selected from the group consisting of: polylactïc acid, polyhydroxybutyrate, polyhydroxyalkanoatc, polyhydroxybutyrate-valerate, polycaprolactone, polyhydroxybutyrate-hexanoate, polybutylene succinate, polybutyrate 5 succinate adipate, polyesteramide, polybutylene adipatc-co-terephthalate, mixtures thereof, and lamlnates thereof.

    i 2. The method of claim 11, wherein the bag is made of a starch film.

    10 i 3. The method of any one of the above claims, further comprising utilizing further processing to tear or rupture the structure or carrier.
12. 14. The method of claim 6, wherein the mîcroorganism comprises a strain of Trichoderma reesei.
13. 15. The method of claim 14, wherein the strain ls a high-yielding cellulase-producing mutant of Trichodenna reesei.
14. 16. The method of claim 15, wherein the strain comprises RUT-C30.
15. 17. The method of any one of the above claims, wherein the recalcitrance of the cellulosic or lignocellulosic blomass has been reduced by exposure to an électron beam.
16. 18. The method of any one of claims 4-17, wherein the conversion comprises

    25 saccharification.
17. 19. The method of any of the above claims, wherein the cellulosic or lignocellulosic biomass is selected from the group consisting of: paper, paper products, paper waste, paper pulp, pigmented papers, ioaded papers, coated papers, filled papers, magazines, printed matter,

    30 printer paper, polycoated paper, card stock, cardboard, paperboard, cotton. wood, parlicle board, forcstry wastes, sawdust, aspen wood, wood chips, grasses, swilchgrass, miscanthus, cord grass, rced canary grass, grain residues, rice hulls, oat hulls, wheat chaff, barley hulls, agricultural waste, silage, canola straw, wheat straw, barley straw, oat straw, rice straw, jute, hemp, flax, bamboo, sisal, abaca, com cobs, com stover, soybean 35 stover, com fiber, alfalfa, hay, coconut hoir, sugar processing residues, bagasse, beet pulp, agave bagasse, aigae, seaweed, manure, sewage, offal, agricultural or industrial

    I waste, arracacha, buckwheat, banana, barley, cassava. kudzu, oca, sago, sorghum, potato, sweet potato, tara, yams, beans, favas, lentils, peas, and mixtures of any of these.
18. 20. The method of any of the above daims, wherein the celiulosic or lignoceilulosic material 5 has an average particic size of less than about l mm. e.g.. about 025 mm to 2.5 mm.