NZ737199B2 - Method for producing a saccharified product - Google Patents

Abstract

Disclosed are methods for processing biomass materials that are disposed in one or more porous 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. Also disclosed is the addition of an additive such as, e.g., microorganism, a nutrient, a spore, an enzyme, an acid, a base, a gas, an antibiotic, or a pharmaceutical to the structure or carrier to convert the contained biomass into useful products. sequence during processing. Also disclosed is the addition of an additive such as, e.g., microorganism, a nutrient, a spore, an enzyme, an acid, a base, a gas, an antibiotic, or a pharmaceutical to the structure or carrier to convert the contained biomass into useful products.

Description

METHOD FOR ING A SACCHARIFIED PRODUCT by Marshall Medoff, Thomas Craig Masterman, James J. Lynch CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application Nos. 61/579,550 and 61/579,562, both filed on December 22, 201 1. The entire disclosures of the above applications are incorporated herein by reference.

FIELD OF THE INVENTION The invention pertains to ements in conducting microbiological, biological and biochemical reactions.

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

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

Lignocellulosic biomass is recalcitrant to degradation as the plant cell walls have a structure that is rigid and compact. The structure ses crystalline cellulose fibrils ed in a hemicellulose matrix, surrounded by lignin. This compact matrix is difficult to access by enzymes and other al, biochemical and biological processes. Cellulosic biomass materials (e.g., biomass material from which substantially all the lignin has been removed) can be more accessible to enzymes and other conversion processes, but even so, lly-occurring cellulosic materials often have low yields (relative to theoretical yields) when contacted with hydro lyzing enzymes. ellulosic biomass is even more recalcitrant to enzyme attack.

Furthermore, each type of lignocellulosic s has its own ic ition of cellulose, hemicellulose and lignin.

While a number of methods have been tried to t ural carbohydrates from lignocellulosic biomass, they are either are too expensive, produce too low a yield, leave undesirable chemicals in the resulting product, or simply degrade the sugars.

Monosaccharides from renewable s sources could become the basis of chemical and fuels industries by replacing, supplementing or substituting petroleum and other fossil ocks. However, techniques need to be developed that will make these monosaccharides available in large quantities and at acceptable purities and .

SUMMARY OF THE INVENTION ed herein are methods for ing a product, which s include maintaining a combination sing a liquid medium, a structure or carrier, and a reducedrecalcitrance cellulosic or lignocellulosic s disposed within the structure or carrier, under conditions that allow the passage of les out of and/or into the structure or carrier.

In another aspect, provided herein is a method comprising: providing a liquid medium, a reduced-recalcitrance cellulosic or lignocellulosic biomass, and a microorganism capable of producing an enzyme in the presence of the cellulosic or lignocellulosic biomass; wherein the reduced-recalcitrance cellulosic or lignocellulosic biomass is disposed within a first structure or carrier formed of a mesh material having a maximum opening size of less than 1 mm; the microorganism is disposed within a second structure or carrier with a pore size of below 5 microns designed to contain the rganism but to allow the enzyme to flow out of the second structure or carrier; the first structure or carrier is disposed in the second structure or carrier; both the first and second structures or carriers are disposed in the medium and can be removed or added at any time during the ; and wherein the enzyme can be manufactured and stored and then used in saccharification reactions of the same or similar s material at a later date and/or in a different location.

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 ed in a structure or carrier, and wherein the structure or carrier possesses one or more pores configured to allow the passage of molecules; 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 molecules out of and/or into the ure or carrier; and maintaining the combination under conditions that allow the additive to t the molecules to one or more products; thereby ing a product.

Additionally, provided herein are methods of producing an enzyme, where the methods include: providing a liquid medium; providing a cellulosic or lignocellulosic biomass; providing a rganism capable of producing an enzyme in the presence of the osic or lignocellulosic biomass; providing a structure or r, wherein the structure or carrier possesses one or more pores configured to allow the passage of les; disposing the cellulosic or ellulosic biomass within the structure or carrier; combining the liquid , 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.

Also provided herein is a method of ing a substance to a microorganism, where the method includes: ing a liquid medium; providing a microorganism; providing a substance; providing a structure or carrier, wherein the structure or r 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 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.

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

In any of the methods or systems provided herein, the cellulosic or lignocellulosic biomass can be disposed Within the ure or carrier, and the methods can fiarther include: disposing the additive Within a second structure or carrier; and the structure or carrier containing the cellulosic or lignocellulosic s is disposed Within the second structure or carrier.

In any of the methods or systems provided herein, the substance can be a sugar, e.g., a sugar can be disposed Within one or more structures or carriers.

In any of the s or systems provided , the product produced can be a molecule, a protein, a sugar, a filel or combinations thereof. The protein can be an enzyme.

Any of the methods or systems provided herein can further include disposing a microorganism in the structure or carrier. Alternatively, 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 rganism, an , an acid, a base or ations thereof.

In any of the methods or s provided herein, the structure or carrier can be a bag, a shell, a net, a membrane, a mesh or combinations thereof. Where the ure or carrier includes a bag, the bag can be formed of a mesh material having a maximum opening size of less than 1 mm. atively, the mesh material can have an e pore size of from about 10 mm to 1 nm. Where the structure or carrier is a bag, the bag can be made of a bioerodible polymer.

The bioerodible polymer can be selected from the group consisting of: polylactic acid, polyhydroxybutyrate, droxyalkanoate, polyhydroxybutyrate-valerate, polycaprolactone, polyhydroxybutyrate-hexanoate, polybutylene succinate, polybutyrate succinate adipate, polyesteramide, polybutylene e-co-terephthalate, mixtures thereof, and laminates thereof.

The bag can be made of a starch film.

In 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 torn open by the impellers.

In any of the methods or s provided herein, the microorganism or microorganisms can include a strain of Trichoderma reesei, e.g., a high-yielding cellulase- producing mutant of Trichoderma reesez’, e.g., the RUT-C30 strain.

In any of the methods or s provided herein, the recalcitrance of the cellulosic or lignocellulosic al can have been reduced relative to the material in its native state. Such treatment to reduce recalcitrance can be bombardment with electrons, sonication, oxidation, sis, steam explosion, chemical treatment, ical treatment, freeze grinding, or combinations of such treatments. Preferably, the recalcitrance of the cellulosic or lignocellulosic biomass has been d by exposure to an on beam.

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 fiarther include isolating a sugar from the sugar solution or suspension. The sugar isolated can be xylose.

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 papers, coated papers, filled papers, magazines, printed matter, printer paper, polycoated paper, card stock, ard, paperboard, cotton, wood, particle 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, canola straw, wheat straw, barley straw, oat straw, rice straw, jute, hemp, flax, bamboo, sisal, abaca, corn cobs, corn stover, soybean stover, corn fiber, a, hay, coconut hair, sugar processing residues, bagasse, beet pulp, agave bagasse, algae, seaweed, manure, sewage, offal, arracacha, eat, banana, barley, cassava, kudzu, oca, sago, sorghum, potato, sweet potato, taro, yams, beans, favas, lentils, 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 milling, or by wet milling. The cellulosic or lignocellulosic material can be treated to reduce its bulk y, or to increase its surface area. The osic or lignocellulosic material can have an average particle size of less than about 1 mm, or an average particle size of from about 0.25 mm to 2.5 mm.

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

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

Cellulosic substrate (A) is converted by endocellulase (i) to cellulose (B), which is converted by exocellulase (ii) to cellobiose (C), which is ted to glucose (D) by cellobiase (beta- glucosidase) (iii). is a flow diagram illustrating conversion of a biomass feedstock to one or more products. ock is ally pretreated (e.g., to reduce its size) (200), optionally treated to reduce its recalcitrance (210), saccharified to form a sugar solution (220), the solution is orted (230) to a cturing plant (e.g., by pipeline, railcar) (or if saccharification is performed en route, the ock, enzyme and water is transported), the saccharified feedstock is bio-processed to produce a desired product (e.g., l) (240), and the product can be processed further, e.g., by distillation, to produce a final product (250). Treatment for recalcitrance can be modified by measuring lignin content (201) and setting or adjusting process parameters (205). Saccharifying the feedstock (220) can be d by mixing the feedstock with medium and the enzyme (221). is a flow diagram illustrating the treatment of a first biomass (300), addition of a cellulase producing organism (310), addition of a second s (320), and processing the resulting sugars to make products (e.g., alcohol(s), pure sugars) (330). The first treated biomass can optionally be split, and a portion added as the second biomass (A). is a flow diagram illustrating the production of s. A cellulase- producing organism is added to growth medium (400), a treated first biomass (405) is added (A) to make a mixture (410), a second s portion is added (420), and the resulting sugars are sed to make products (e.g., alcohol(s), pure sugars) (430). Portions of the first s (405) can also be added (B) to the second biomass (420).

DETAILED DESCRIPTION Provided herein are methods of conducting ical, microbiological, and biochemical reactions by using one or more structures or containers, which can have pores or other openings, or can be degradable. The structure can be a bag, net or mesh, shell (e.g., rigid or semi-rigid shell), a membrane, or combinations of these structures (e.g., one or more ures of one or more types can be disposed within a structure of the same or another type).

The structures can hold various parts or ingredients involved in ical, microbiological, and biochemical reactions. Containing the material in this manner allows parts or ingredients, 6.g. , 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 metabolite, sugar, or nutrient.

For instance, the structures can be used to provide one or more nutrients to microorganisms. The nutrients can be placed in the structure, and the ure placed in a liquid medium containing microorganisms. The nutrients are ed from the structure into the medium to be ed by the microorganisms. Alternatively, the microorganisms can be placed within the structure, and the structure placed in a liquid medium that ns the nutrients.

In a preferred ment, the structure can contain biomass which is to be acted on by microorganisms, or products of microorganisms, such as enzymes or signal molecules. For instance, the biomass can be placed in the structure, which is then placed in a liquid medium with 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 rganisms, and enzymes produced by the microorganisms can migrate into the structures and act on the biomass.

In another aspect, the invention relates to 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 ase sis, producing a cellulase complex having an activity that is tailored to the particular biomass material, which in some implementations is the same material that is to be saccharif1ed by the cellulase complex.

The invention also features a method that includes contacting a cellulosic or lignocellulosic material disposed in a structure or carrier, in a medium, with an additive to produce a product. The additive can, for example, be a rganism, 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 e, a molecule, a protein, a sugar a fuel or mixtures of any of these. The products can be produced in any order. For example, a n can be first produced followed by a sugar and finally by a filel. Optionally, the protein can be an enzyme.

The migration of substances into and out of the ure can be accomplished in a variety of ways. The structure can slowly degrade over time in the medium, the structure can be made of a porous material that releases the nutrients into the , the structure can be made of a material that is consumed by the microorganisms, the structure can be made of a material that is torn 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.

In an embodiment of the process described herein, a biomass can be ed in, on, or placed into the structure or carrier. The biomass can be treated before or after being placed into the ure or carrier. Additives, nutrients and products can also be 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.

WO 96699 Optionally, the biomass can be outside of the structure or r. For example, a microbe can be ed in, within (i.e., built into the structure or carrier), or on the structure or carrier, which is contacted with a medium containing the biomass. As another example, there may be one kind of biomass in the structure or carrier and a second kind of biomass outside the structure or r. There may be multiple biomasses inside and outside of the ure or carrier added in any combination and sequence during the process.

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 each, other made of the same material or different materials, have the same shape or different shapes, and may be used in any combination.

For example, multiple ures or carriers can be disposed within r structure or r. The various structures or rs can be of the same type, or can be of different types.

Multiple structures or carriers can be sequentially disposed, each inside r, e.g., similar to “nesting dolls.” For example, it may be convenient to have biomaterial disposed in a plurality of structures or carriers of a uniform size and , each containing the same or a similar amount 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 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.

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

As r e, one or more biomass-containing structures or carriers, and one or more microbe-containing structures or carriers can be contacted simultaneously or sequentially with a medium.

As a further example, in some processes one or more biomass-containing structures or carriers, and one or more additive-containing water-degradable structures or carriers are ted with an aqueous .

In another embodiment of the process, the structure or carrier can be removed at any point in the process and in any sequence. For example, the structure or carrier including its 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.

As another example, a biomass ed 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 ure or carrier can be added to produce more product. Optionally, the microbe can be removed before or after addition of the second biomass.

In yet another example, a s 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 uently a different microbe can be added, which causes a second product to be produced.

In a r example, a biomass can be disposed in a first structure or carrier. The first structure or carrier can be ed in a second ure 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 Sum, below about 1 um, below about 0.4 um, below 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 the first and second structures or carriers with their contents can be optionally used in another medium to produce more product.

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

The processes described herein include producing s using a microorganism in the presence of a biomass material, 6.g. a cellulosic or lignocellulosic material. Enzymes made by the processes described herein contain or manufacture various olytic enzymes (cellulases), ligninases or various small molecule biomass-destroying metabolites. These enzymes may be a x of s that act synergistically to degrade crystalline ose or the lignin portions of biomass. Examples of olytic enzymes include: endoglucanases, cellobiohydrolases, and cellobiases (beta-glucosidases).

As shown in for example, during saccharification a cellulosic substrate (A) is initially hydrolyzed by endoglucanases (i) at random locations producing oligomeric intermediates (e.g., cellulose) (B). These intermediates are then substrates for exo-splitting glucanases (ii) such as cellobiohydrolase to produce cellobiose from the ends of the cellulose polymer. Cellobiose is a water-soluble nked dimer of glucose. Finally cellobiase (iii) cleaves cellobiose (C) to yield e (D). Therefore, the endoglucanases are particularly effective in attacking the crystalline portions of ose and increasing the effectiveness of exocellulases to produce cellobiose, which then es the specificity of the cellobiose to WO 96699 produce glucose. Therefore, it is evident that depending on the nature and structure of the cellulosic substrate, the amount and type of the three different enzymes may need to be modified.

In some implementations, the enzyme is produced by a fungus, e.g., by s of the cellulolytic filamentous fungus Trichoderma reesez’. For example, high-yielding cellulase mutants of Trichoderma reesez’ may be used, e.g., RUT-NGl4, PC3-7, QM94l4 and/or Rut-C30.

Such strains are described, for example, in tive Screening Methods for the Isolation of High Yielding Cellulase Mutants of Trichoderma ’,” Montenecourt, BS. and igh, D.E., Adv. Chem. Ser. 18 1, 289-301 (1979), the full disclosure of which is incorporated herein by reference. Other cellulase-producing microorganisms may also be used.

As will be discussed fiarther 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 on to this saccharif1cation step. Such steps are described, e.g., in US. Pat. App. Pub. 2012/0100577 Al, filed r 18, 2011 and published April 26, 2012, the full disclosure of which is hereby incorporated herein by nce.

For example, referring to 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 r reduce its recalcitrance (210), then rifying the feedstock, using the enzyme complex, to form a sugar solution (220). Optionally, the method may also include transporting, e.g. truck or barge, the solution (or the feedstock, enzyme and water, if , by ne, railcar, saccharif1cation is performed en route) to a manufacturing plant (230). In some cases the saccharif1ed feedstock is r bioprocessed (e.g., fermented) to produce a desired product e.g., alcohol (240). This resulting product may in some implementations be sed further, e.g., by distillation (250), to produce a final product. One method of reducing the recalcitrance of the feedstock is by electron 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 US. Pat.

App. Pub. 2010/0203495 Al by Medoff and Masterman, published August 12, 2010, the complete sure of which is incorporated herein by reference. Saccharifying the feedstock (220) can also be modified by mixing the feedstock with medium and the enzyme (221).

For example, referring to a first biomass is optionally treated (300), for example to reduce its size and/or recalcitrance, and placed into a structure or carrier. ally, 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 s medium and a cellulase ing organism (310). After an adequate time has passed for the cells to grow to a desired stage and enough enzymes have been produced, a second biomass, 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 es mixed sugars which can be fiarther processed to useful products (330). Optionally, the second structure or 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 s can be placed into a ure or carrier and ted with a medium ning the cellulase producing organism. Once some s have been produced; the enzyme containing media can be combined with the second biomass (A). Optionally, the first and second biomass may be pretreated to reduce recalcitrance. The first and second biomass can also be contained in a single ure or carrier. The structure or carrier can form a liner for a bioreactor. Multiple biomass containing structures or carrier can also be used. The aqueous media will be discussed below. In some cases, rather than adding the second biomass to the reactor, the enzyme is harvested, stored, and used in a later saccharification process.

Referring now to the cellulase-producing organism (400) can be grown in a grth medium for a time to reach a c 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 ed period of time. The enzyme containing on 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 produces mixed sugars which can be further processed to useful products (430). The first and second biomass can be portions of the same biomass or can be similar but not identical (e.g., ated and non-pretreated) material (B).

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

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 partial or almost complete l of the solvent and growth media components. For example the cells may be collected by centrifilgation and then washed with water or another solution.

In another embodiment, after enzyme is produced, the structure or carrier can be removed from the enzyme-containing medium and the enzyme can be concentrated. tration may be by any useful method including chromatography, centrifilgation, filtration, dialysis, extraction, evaporation of solvents, spray drying and tion 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.

In another entation of the method, the enzyme is produced by the selected microorganism in a liquid (6.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 ably at least 90%, at least 95% or at least 99%) of the insoluble portion of the biomass material is retained within the ure 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 s material is retained within the structure or r during enzyme production.

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 al flows out of the container during enzyme production. It is also preferred that the pore size be large enough to allow molecules such as sugars, soluble polysaccharides, proteins and ecules to pass. Preferably the pore size is large enough that large molecules such as proteins do not foul or block the pores during the course of enzyme production.

Thus, it is generally preferred that the nominal pore size or mesh size be r than most of all of the particles of the s material. In some implementations the absolute pore size is smaller than 50% (preferably smaller than 60%, 70%, 80%, 90%, 95%, 98% or 99%) of 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% ofthe particles ofthe biomass al. 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%, 97%, 98%, 99% of the particles of the biomass material.

The aqueous media used in the above described methods can contain added yeast extract, corn steep, es, amino acids, ammonium salts, phosphate salts, potassium salts, magnesium salts, calcium salts, iron salts, manganese salts, zinc salts and cobalt salts. In addition to these components, the growth media typically contains 0 to 10% glucose (e.g., 1 to % glucose) as a carbon source. The r 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 the processing to optimize the desired production of useful products.

The concentration of the biomass typically used for ng 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, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 wt %. The tration 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 %.

Any of the processes described herein may be performed as a batch, a fed-batch or a uous process. The processes are especially useful for rial scale production, e.g., having a culture medium of at least 50 liters, preferably at 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 liters. The process may be carried out aerobically or anaerobically.

Some enzymes are produced by submerged cultivation and some by e cultivation.

In any of the process described herein, the enzyme can be manufactured and stored and then used to in saccharif1cation ons at a later date and/or in a different location.

Any of the processes described herein may be conducted with agitation. In some cases, agitation may be performed using jet mixing as described in US. Pat. App. Pub. 2010/0297705 Al, filed May 18, 2010 and published on November 25, 2012, US. Pat. App.

Pub. 2012/0100572 A1, filed er 10, 2011 and published on April 26, 2012, US. Pat.

App. Pub. 2012/0091035 A1, filed November 10, 2011 and published on April 19, 2012, the full sures of which are incorporated by nce herein.

Temperatures for the growth of enzyme-producing sms are chosen to enhance organism . For example for Trichoderma reesez’ the optimal temperature is generally between 20 and 40°C (e.g., 30°C), and the temperature for enzyme production can be optimized for that part of the process. For example for Trichoderma reesez’ the optimal temperature for enzyme production is n 20 and 40°C (e.g., 27°C).

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

The structure or carrier can be made with a thermoplastic resin, for example, polyethylene, polypropylene, polystyrene, rbonate, polybutylene, a thermoplastic polyester, a polyether, a thermoplastic polyurethane, polyvinylchloride, polyvinylidene difluoride, a polyamide or any combination of these.

The structure or r can also be made of woven or non-woven fibers. Some preferred synthetic fiber or non-fiber materials are, for example, polyester, aramid, polyolefin, PTFE, enlene sulfide, polyurethane, polyimide, acrylic, nylon and any combination of these.

The structure of carrier can also be made from biodegradable and/or water e polymers, for example, aliphatic polyesters, polyhydroxyalkanoates (PHAs), poly hydroxybutyrate, polyhydroxyvalerate, polyhydroxyhexanoate, polylactic acid, polybutylene succinate, polybutylene succinate adipate, polycaprolactone, polyvinyl alcohol, polyanhydrides, starch derivatives, cellulose esters, ose acetate, nitrocellulose and any combination of these.

Other materials contemplated for the structure or carrier include, for example, metal (e. g., aluminum, ), an alloy (e.g, brass, stainless steel), a c (e.g., glass, alumina), a thermosetting polymer (6.g. , bakelite), a composite material (6.g. , fiberglass), a biopolymer and any ation of these. Any structural material, for example, as disclosed above, can be combined to provide the structure or carrier.

The structure or carrier can be made of a biodegradable, bioerodible, and/or water soluble polymer. Such a polymer can be chosen to degrade and release the material within it at 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 ed to e its ts into a culture of such organisms.

Alternatively, the structure or carrier can be configured and made from materials ed to be torn apart by the impellers of a fermentation system. The fermentation mixing cycle can be led to maintain the ure or carroer in an intact state for a period of time, and then altered to cause the structure or carrier to come in contact with the impellers.

The container or carrier can be of any suitable shape, for example, a toroid, sphere, cube, oval, , dog bone, cylindrical, hexagonal prism, cone, square based pyramid, envelope or combinations of these.

The container or structure can have a le and in some cases resealable opening such as a , VelcroTM hook and loop fastener, heat seal, clips, pressure sensitive adhesive, buttons or tie (e.g. with a string or drawstring).

The structure or container may be rigid, semi-rigid or non-rigid. A non-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 implementations, the container comprises a flexible, fabric bag.

The bag may have some rigid components such as a frame made of a metal wire or rigid r. The container or carrier can have a surface texturing, for example, grooves, corrugation, and quilting.

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

The container or carrier may be designed so as to float on top of the medium or be partially submerged therein, or it may be designed to be fully ged in the medium. For example, the bag may have hooks, loops or adhesives to allow it to attach to the wall of a bioreactor, tank or other container. It may also have weights to hold part or all of it submerged 2012/071092 in the medium, and/or buoyant parts to keep parts of it above the medium. The container or r can be designed to be free in the .

The structures or carriers can have 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 absolute or nominal. An absolute pore size rating specifies 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 ability of the permeable material to retain the majority of the ulates (e.g. 60 to 98%). Both ratings depend on process conditions such as the differential pressure, the temperature or the concentration.

In some implementations, 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, 10 um, 1um, 0.1 um, 10 nm or even less than 1 nm. In some implementations, 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.

If the structure or r is made of a polymer, the pores may be formed by hing the polymer, either uniaxially or biaxially. Such methods for formulating and stretching polymers to make films with a particular pore size are known in the art.

The structure or carrier may be designed to allow for the insertion of, for example, a mixing , a monitoring device, a sampling device or combinations of any of these. The design may include, for example a le opening or fitting configured to receive such a device. The monitoring device can be, for example, a pH probe, an oxygen probe, a temperature probe, a chemical probe or any combinations of these. ally, 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 have a tagging device, for example, a tag with an identifying alphanumerical label or identifying color.

In some implementations, it is preferred that the ure or carrier have sufficient 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. It can also be advantageous to have a high surface area to present a large area to which a microorganism, e.g., a cellulase-producing organism, can ally attach.

MEDIUM In the methods described herein, the structure or carrier is contacted or placed in a . The medium can be, for example, a liquid, a gas, a al solution, a suspension, a colloid, an emulsion, a non-homogenous multiphase system (6.g. , a hydrophilic phase layered with a hydrophobic phase) and any combinations of these. The medium can be further manipulated during or after the process; for example, it can be purified and reused by, for example, by filtration, centrifugation and/or irradiation. Optionally, the medium can contain, for example, nutrients, particulates (e.g., inorganic or organic containing), ers (e.g., viscosity modifiers), carbon sources, surfactants (e.g., anti-foam agents), lipids, fats, extracts (e.g., yeast ' ‘ 2 l 2 l 2 l 2 l l l extract, case1n extracts and or ble extracts), metal ions (e.g., Fe Mn Cu Na , Mg , , , , Ca2+ K1+), anions, n1trogen sources (e.g., am1no ac1ds, ammon1a, urea), Vitamins, prote1ns (e.g.,. . . . . . . . peptones, enzymes), buffers (e.g., phosphates) added in any combination and ce.

ADDITIVES Additives used in the ses disclosed herein can include, by way of example, a rganism, a nutrient, 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 ation 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.

In one embodiment of the s, the additive is an enzyme produced by filamentous filngi or bacteria.

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

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

The cellulase-producing organism’s growth rate and state is determined by particular grth conditions. When the host cell culture is introduced into the fermentation medium, containing a carbon source, the ated culture passes h a number of stages. Initially grth 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 “exponential phase” the growth rate of the host cell culture gradually increases and the carbon source is consumed. After a period ofmaximum growth the rate ceases and the culture enters stationary phase. After a r period of time the culture enters the death phase and the number of viable cells declines.

Where in the growth phase the cellulase is expressed depends on the cellulase and host cell. For example, the cellulase may be expressed in the exponential phase, in the transient phase between the exponential phase and the stationary phase, or alternatively in the nary phase and/or just before sporulation. The ase may also be produced in more than one of the above mentioned phases.

When contacted with a biomass, the cellulase ing organism will tend to produce s that release molecules advantageous to the sm’s growth, such as glucose.

This is done through the phenomenon of enzyme induction. Since there are a variety of substrates in a particular biomaterial, there are a variety of cellulases, for example, the endoglucanase, exoglucanase and cellobiase discussed 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 ing enzyme complex will work efficiently on the lignocellulosic material used as the inducer or a similar material. For e, 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.

Since cellulose is insoluble and impermeable to organisms, it has been suggested that when cellulose is used as an inducer, a e oligosaccharide(s) such as cellobiose is actually the direct inducer of cellulase. Expression at a basal level allows a small amount of cellulase to hydrolyze cellulose to soluble accharides or to an inducer. Once the inducer enters the cell, it triggers full-scale transcription of the ase gene mediated by activator ns and activating elements. After cellulose is degraded a large amount of glucose is liberated, which causes catabolite repression.

Lignocellulosic materials comprise different combinations of cellulose, llulose and lignin. Cellulose is a linear polymer of glucose forming a fairly stiff linear structure without significant coiling. Due to this structure and the disposition of yl groups that can hydrogen bond, cellulose contains crystalline and non-crystalline portions. The crystalline portions can also be of ent types, noted as I(alpha) and I(beta) for example, depending on the location of hydrogen bonds n strands. The polymer lengths themselves can vary lending more variety to the form of the cellulose. Hemicellulose is any of several heteropolymers, such as xylan, glucuronoxylan, arabinoxylans, and xyloglucan. The primary sugar monomer present is xylose, although other monomers such as mannose, galactose, rhamnose, arabinose and glucose are t. 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 polymer generally. Although all lignins show variation in their composition, they have been described as an amorphous tic network polymer of phenyl propene units. The amounts of cellulose, hemicellulose and lignin in a specific biomaterial s on the source of the biomaterial. For example wood derived biomaterial can be about 38-49% cellulose, 7-26% hemicellulose and 23-34% lignin depending on the type. Grasses lly are 33-38% cellulose, 24-32% hemicellulose and 17-22% lignin. Clearly lignocellulosic biomass constitutes a large class of substrates.

The diversity of biomass materials may be fiarther increased by pretreatment, for example, by changing the crystallinity and molecular weights of the polymers. The variation in the composition of the biomass may also increase due to geographical and al ion, z'.e., where and when the material was collected.

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

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

BIOMASS ALS As used herein, the term “biomass materials” includes lignocellulosic, cellulosic, starchy, and microbial materials.

Lignocellulosic als 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 hulls, oat hulls, wheat chaff, barley hulls), agricultural waste (e.g., silage, canola straw, wheat straw, barley straw, oat straw, rice straw, jute, hemp, flax, bamboo, sisal, abaca, corn cobs, corn stover, soybean stover, corn fiber, alfalfa, hay, coconut hair), sugar processing residues (e.g., bagasse, beet pulp, agave bagasse), algae, seaweed, manure, sewage, and mixtures of any of these.

In some cases, the lignocellulosic material includes comcobs. Ground or hammermilled comcobs can be spread in a layer of relatively uniform thickness for irradiation, and after irradiation are easy to se in the medium for fiarther processing. To facilitate harvest and collection, in some cases the entire corn plant is used, including the corn stalk, corn kernels, and in some cases even the root system of the plant.

Advantageously, no additional nutrients (other than a nitrogen , 6.g. urea or ammonia) are required during fermentation of s or cellulosic or lignocellulosic materials containing significant amounts of comcobs.

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

Cellulosic materials include, for example, paper, paper products, paper waste, paper pulp, pigmented papers, loaded papers, coated , filled papers, magazines, printed matter (e. g., books, catalogs, manuals, labels, calendars, greeting cards, brochures, prospectuses, newsprint), printer paper, polycoated paper, card stock, cardboard, paperboard, materials having a high lulose content such as cotton, and mixtures of any of these. For example paper products as described in US. App. No. 13/396,365 (“Magazine Feedstocks” by Medoff et al., filed February 14, 2012), the fill disclosure of which is incorporated herein by reference. osic materials can also include lignocellulosic materials which have been de- lignified.

Starchy als include starch itself, e.g., corn starch, wheat starch, potato starch or rice starch, a derivative of , or a material that includes starch, such as an edible food t or a crop. For example, the starchy material can be arracacha, buckwheat, banana, , cassava, kudzu, oca, sago, m, regular household potatoes, sweet potato, taro, yams, or one or more beans, such as favas, lentils or peas. Blends of any two or more starchy materials are also starchy materials. Mixtures of 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.

Microbial materials include, but are not limited to, any naturally occurring or genetically modified microorganism or sm that contains or is capable of providing a source of carbohydrates (e.g., cellulose), for example, protists, e.g., animal protists (e.g., protozoa such as flagellates, amoeboids, ciliates, and oa) and plant ts (e.g., algae such alveolates, chlorarachniophytes, monads, euglenids, glaucophytes, hytes, red algae, stramenopiles, and viridaeplantae). Other examples include seaweed, plankton (e.g., macroplankton, mesoplankton, microplankton, nanoplankton, ankton, and femptoplankton), phytoplankton, bacteria (e.g., gram positive bacteria, gram negative bacteria, and extremophiles), yeast and/or mixtures of these. In some instances, microbial biomass can be obtained from natural sources, e.g., the ocean, lakes, bodies of water, e.g., salt water or fresh water, or on land. atively or in addition, microbial biomass can be obtained from culture s, e.g., large scale dry and wet culture and fermentation systems.

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

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

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

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

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

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

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

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

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

Pat. No. 7,971,809 to Medoff, the full disclosure of which is hereby incorporated by reference.

In some cases, the pre-treatment processing includes ing of the biomass material. Screening can be through a mesh or perforated plate with a desired opening size, for example, less than about 6.35 mm (1/4 inch, 0.25 inch), (e.g., less than about 3.18 mm (1/8 inch, 0.125 inch), less than about 1.59 mm (1/16 inch, 0.0625 inch), is less than about 0.79 mm (1/32 inch, 0.03125 inch), e.g., less than about 0.51 mm (1/50 inch, 0.02000 inch), less than about 0.40 mm (1/64 inch, 0.015625 inch), less than about 0.23 mm (0.009 inch), less than about 0.20 mm (1/ 128 inch, 0.0078125 inch), less than about 0.18 mm (0.007 inch), less than about 0.13 mm (0.005 inch), or even less than about 0.10 mm (1/256 inch, 0.00390625 inch)). In one configuration the desired biomass falls through the perforations or screen and thus biomass larger than the perforations or screen are not irradiated. These larger materials can be re- processed, for example by comminuting, or they can simply be removed from processing. In r configuration material that is larger than the perforations is irradiated and the smaller material is removed by the ing process or recycled. In this kind of a ration, the conveyor itself (for example a part of the conveyor) can be perforated or made with a mesh. For example, in one particular embodiment the biomass material may be wet and the perforations or mesh allow water to drain away from the biomass before irradiation.

Screening of material can also be by a manual method, for example by an operator or mechanoid (e.g., a robot ed with a color, reflectivity or other sensor) that removes unwanted material. Screening can also be by magnetic ing wherein a magnet is disposed near the conveyed material and the magnetic material is removed magnetically. al pre-treatment processing can include heating the material. For example a portion of the conveyor can be sent through a heated zone. The heated zone can be created, for e, by IR radiation, microwaves, combustion (e.g., gas, coal, oil, biomass), resistive heating and/or ive coils. The heat can be applied from at least one side or more than one side, can be continuous or periodic and can be for only a portion of the material or all the material. For example, a n of the conveying trough can be heated by use of a heating jacket. Heating can be, for example, for the e of drying the material. In the case of drying the material, this can also be facilitated, with or without heating, by the nt of a gas (6.g. air, oxygen, nitrogen, He, C02, Argon) over and/or through the biomass as it is being conveyed.

Optionally, pre-treatment processing can include cooling the al. Cooling material is described in US Pat. No. 7,900,857 to , the disclosure of which in incorporated herein by reference. For example, cooling can be by supplying a cooling fluid, for e water (6.g. with glycerol), or nitrogen (e.g. to the bottom of the ing , , liquid nitrogen) trough. Alternatively, a cooling gas, for example, chilled nitrogen can be blown over the biomass als or under the conveying system.

Another optional pre-treatment processing method can include adding a material to the biomass. The additional material can be added by, for example, by showering, sprinkling and or pouring the material onto the biomass as it is conveyed. Materials that can be added include, for example, metals, ceramics and/or ions as described in US. Pat. App. Pub. 2010/01051 19 Al (filed October 26, 2009) and US. Pat. App. Pub. 2010/0159569 A1 (filed December 16, 2009), the entire disclosures of which are incorporated herein by reference.

Optional materials that can be added include acids and bases. Other materials that can be added are oxidants (e.g., peroxides, chlorates), polymers, rizable monomers (e.g., containing unsaturated bonds), water, catalysts, enzymes and/or organisms. als can be added, for example, in pure form, as a solution in a solvent (e.g., water or an organic solvent) and/or as a solution. In some cases the solvent is volatile and can be made to evaporate e.g., by g and/or g 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 te the subsequent irradiation step by increasing the efficiency of the irradiation, damping the irradiation or changing the effect of the irradiation (e.g., from electron beams to X-rays or heat). The method may have no impact on the irradiation but may be useful for r ream processing. The added material may help in conveying the material, for example, by lowering dust levels.

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

The material can be leveled to form a uniform thickness between about 0.03 12 and 5 inches (e.g., between about 0.0625 and 2.000 inches, between about 0. 125 and 1 inches, between about 0. 125 and 0.5 inches, between about 0.3 and 0.9 inches, n about 0.2 and 0.5 inches between about 0.25 and 1.0 inches, between about 0.25 and 0.5 inches, 0.100 +/- 0.025 inches, 0.150 --/- 0.025 , 0.200 --/- 0.025 inches, 0.250 --/- 0.025 , 0.300 --/- 0.025 inches, 0.350 --/- 0.025 inches, 0.400 --/- 0.025 inches, 0.450 --/- 0.025 inches, 0.500 --/- 0.025 inches, 0.550 --/- 0.025 inches, 0.600 --/- 0.025 inches, 0.700 --/- 0.025 inches, 0.750 --/- 0.025 inches, 0.800 --/- 0.025 , 0.850 --/- 0.025 inches, 0.900 --/- 0.025 inches, 0.900 --/- 0.025 inches.

Generally, it is red to convey the material as quickly as possible through the electron beam to ze throughput. For example the material can be conveyed at rates of at least 1 ft/min, e.g., at least 2 ft/min, at least 3 ft/min, at least 4 ft/min, at least 5 ft/min, at least 10 ft/min, at least 15 ft/min, 20, 25, 30, 35, 40, 45, 50 ft/min. The rate of conveying is related to the beam t, for example, for a 14 inch thick biomass and 100 mA, the conveyor can move at 2012/071092 about 20 ft/min to e a useful irradiation dosage, at 50 mA the conveyor can move at about ft/min to provide approximately the same irradiation dosage.

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

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

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

Mechanical treatment may reduce the bulk density of the biomass material, increase the surface area of the biomass al and/or se one or more dimensions of the biomass material.

Alternatively, or in addition, the feedstock material can first be physically treated by one or more of the other al treatment methods, 6.g. chemical treatment, radiation, sonication, oxidation, pyrolysis or steam explosion, and then mechanically treated. This ce can be advantageous since materials treated by one or more of the other treatments, 6.g. irradiation or pyrolysis, tend to be more brittle 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 ng radiation using a conveyor as described herein and then ically treated. Chemical treatment can remove some or all of the lignin (for example chemical pulping) and can lly or completely yze the material. The methods also can be used with pre-hydrolyzed material. The methods also can be used with material that has not been pre hydrolyzed The methods can be used with mixtures of hydrolyzed and non-hydrolyzed materials, for example with about 50% or more non-hydrolyzed material, with about 60% or more non- hydrolyzed material, with about 70% or more non-hydrolyzed material, with about 80% or more non-hydrolyzed material or even with 90% or more non-hydrolyzed material.

In addition to size reduction, which can be performed initially and/or later in processing, mechanical treatment can also be ageous for “opening up,3, sing,” breaking or shattering the biomass materials, making the cellulose of the materials more susceptible to chain scission and/or disruption of lline structure during the physical treatment.

Methods of mechanically ng the biomass material include, for example, milling or ng. g may be performed using, for example, a mill, ball mill, colloid mill, conical or cone mill, disk mill, edge mill, Wiley mill, grist mill or other mill. Grinding may be performed using, for example, a cutting/impact type grinder. Some ary grinders include stone grinders, pin grinders, coffee grinders, and burr grinders. Grinding or milling may be provided, for e, by a reciprocating 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 fibers, and air ion milling. Suitable mechanical ents further include any other technique that continues the disruption of the internal structure of the material that was initiated by the previous processing steps.

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

The bulk density of feedstocks can be controlled (e.g., increased). In some situations, it can be desirable to prepare a low bulk density material, 6.g. the material (e.g., , by densifying densif1cation 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 densif1ed, for example from less than about 0.2 g/cc to more than about 0.9 g/cc (e.g., less than about 0.3 to more than about 0.5 g/cc, less than about 0.3 to more than about 0.9 g/cc, less than about 0.5 to more than about 0.9 g/cc, less than about 0.3 to more than about 0.8 g/cc, less than about 0.2 to more than about 0.5 g/cc). For example, the material can be 1ed by the methods and equipment disclosed in US. Pat. No. 7,932,065 to Medoff and International Publication No. WO 2008/073186 (which was filed October 26, 2007, was published in English, and which designated the United ), the filll disclosures of which are incorporated herein by reference.

Densifled materials can be processed by any of the methods described herein, or any material processed by any of the methods described herein can be subsequently densif1ed.

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 example, the ng can be performed with a rotary knife .

For example, a fiber source, e.g., that is recalcitrant or that has had its recalcitrance level reduced, can be d, 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 e opening size of 1.59 mm or less (1/16 inch, 0.0625 inch), e a second fibrous material. If desired, the fiber source can be cut prior to the shearing, e.g., with a shredder. For example, when a paper is used as the fiber source, the paper can be first cut into strips that are, e.g. 1/4- to ch wide, using a shredder, e.g., a counter-rotating screw shredder, such as those ctured by Munson (Utica, N.Y.). As an alternative to shredding, the paper can be d in size by cutting to a desired size using a guillotine cutter. For example, the guillotine cutter can be used to cut the paper into sheets that are, e.g, 10 inches wide by 12 inches long.

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

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

In some implementations, the feedstock is physically treated prior to saccharification and/or fermentation. Physical treatment processes can include one or more of any of those bed herein, such as mechanical treatment, chemical treatment, irradiation, sonication, oxidation, pyrolysis or steam explosion. Treatment methods can be used in ations of two, three, four, or even all of these technologies (in any order). When more than one treatment method is used, the methods can be applied at the same time or at different times. Other processes that change a molecular ure of a biomass feedstock may also be used, alone or in combination with the processes disclosed herein. ical treatments that may be used, and the characteristics of the mechanically treated biomass materials, are described in fiarther detail in US. Pat. App. Pub. 2012/0100577 A1, filed October 18, 2011, the fill disclosure of which is hereby incorporated herein by nce.

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

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

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

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

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

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

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

Electromagnetic ion is subclassif1ed as gamma rays, x rays, ultraviolet rays, infrared rays, microwaves, or radiowaves, depending on the wavelength.

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

Sources of x rays include electron beam collision with metal targets, such as tungsten or molybdenum or alloys, or compact light sources, such as those produced commercially by Lyncean.

Sources for ultraviolet radiation include deuterium or m lamps.

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

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

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

TREATMENT OF BIOMASS MATERIAL -- ELECTRON BOMBARDMENT The feedstock may be treated with electron bombardment to modify its structure and y reduce its recalcitrance. Such treatment may, for example, reduce the e molecular weight of the feedstock, change the crystalline structure of the feedstock, and/or increase the surface area and/or porosity of the feedstock.

Electron bombardment via an electron beam is generally preferred, e it es very high throughput and because the use of a relatively low voltage/high power electron beam device eliminates the need for expensive te vault shielding, as such s are “self-shielded” and provide a safe, efficient process. While the “self-shielded” devices do include shielding (e.g. metal plate shielding), they do not e the construction of a concrete vault, greatly reducing l expenditure and often allowing an existing manufacturing facility to be used without expensive modification. Electron beam accelerators are available, for e, from IBA (Ion Beam Applications, Louvain-la-Neuve, Belgium), Titan Corporation (San Diego, California, USA), and NHV Corporation (Nippon High Voltage, Japan).

Electron bombardment may be performed using an electron beam device that has a 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 1 to 3 MeV. In some implementations the nominal energy is about 500 to 800 keV.

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

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

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

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

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

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

In some embodiments, electrons are rated to, for example, a speed of r than 75 percent of the speed of light, e.g., greater than 85, 90, 95, or 99 percent of the speed of light.

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

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

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

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

The iveness in changing the molecular/supermolecular structure and/or reducing the recalcitrance of the biomass biomass depends on the electron energy used and the dose applied, while exposure time depends on the power and dose.

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

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

ELECTRON SOURCES Electrons interact via Coulomb scattering and bremsstrahlung radiation produced by changes in the velocity of electrons. Electrons may be produced by radioactive nuclei that undergo beta decay, such as isotopes of iodine, cesium, technetium, and iridium. Alternatively, an electron gun can be used as an electron source via thermionic on and accelerated h an accelerating potential. An electron gun generates electrons, accelerates them through a large potential (e.g., greater than about 500 thousand, greater than about lmillion, greater than about 2 million, greater than about 5 million, greater than about 6 million, greater than about 7 million, greater than about 8 million, greater than about 9 million, or even greater than 10 n volts) and then scans them magnetically in the x-y plane, where the electrons are initially accelerated in the z direction down the tube and extracted through a foil window. Scanning the on beam is useful for increasing the irradiation surface when irradiating materials, e.g., a biomass, that is conveyed through the scanned beam. Scanning the on beam also distributes the thermal load homogenously on the window and helps reduce the foil window rupture due to local heating by the electron beam. Window foil rupture is a cause of significant ime due to subsequent necessary repairs and re-starting the electron gun.

Various other irradiating devices may be used in the methods disclosed herein, including field ionization sources, electrostatic ion separators, field ionization generators, onic emission 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 US. Pat. No. 7,931,784 to Medoff, the te disclosure of which is incorporated herein by reference.

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

Electrons can also be more efficient at causing s in the molecular structure of biomass materials, for example, by the ism of chain scission. In addition, electrons having energies of 0.5-10 MeV can ate low density materials, such as the biomass materials described herein, e.g., als having a bulk density of less than 0.5 g/cm3, and a depth of 03-10 cm. Electrons as an ng radiation source can be useful, e.g., for relatively thin piles, layers or beds of materials, e.g., less than about 0.5 inch, e.g., less than about 0.4 inch, 0.3 inch, 0.25 inch, or less than about 0.1 inch. In some embodiments, the energy of each electron of the on beam is from about 0.3 MeV to about 2.0 MeV (million electron volts), e.g., from about 0.5 MeV to about 1.5 MeV, or from about 0.7 MeV to about 1.25 MeV.

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

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

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

Tradeoffs in ering electron energies include energy costs.

The electron beam irradiation device can produce either a fixed beam or a scanning beam. A scanning beam may be advantageous with large scan sweep length and high scan speeds, as this would effectively replace a large, fixed beam width. Further, ble sweep widths of 0.5 m, 1 m, 2 m or more are available. The scanning beam is preferred in most embodiments describe herein e of the larger scan width and reduced possibility of local heating and e of the windows.

TREATMENT OF BIOMASS AL -- SONICATION, PYROLYSIS, OXIDATION, STEAM EXPLOSION If desired, one or more sonication, sis, oxidative, or steam explosion processes can be used in addition to or instead of other treatments to further reduce the recalcitrance of the biomass material. These processes can be applied before, during and or after another treatment or treatments. These processes are bed in detail in US. Pat. No. 7,932,065 to Medoff, the filll disclosure of which is incorporated herein by nce.

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

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

In order to convert the feedstock to a form that can be readily processed, the glucan- or 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 saccharif1cation. The low molecular weight carbohydrates can then be used, for e, in an existing manufacturing plant, such as a single cell protein plant, an enzyme manufacturing plant, or a fuel plant, 6.g. , an ethanol manufacturing facility.

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

Alternatively, the enzymes can be ed by organisms that break down biomass, such as the cellulose and/or the lignin portions of the biomass, contain or manufacture various cellulolytic enzymes (cellulases), ligninases or various small molecule biomass-degrading metabolites. These s may be a complex of enzymes that act synergistically to degrade crystalline ose or the lignin portions of biomass. Examples of olytic enzymes include: endoglucanases, cellobiohydrolases, and cellobiases (beta-glucosidases).

During saccharif1cation a cellulosic substrate can be lly hydrolyzed by endoglucanases at random ons producing oligomeric intermediates. These intermediates are then substrates for litting glucanases such as iohydrolase to produce cellobiose from the ends of the cellulose polymer. iose is a soluble 1,4-linked dimer of glucose. Finally, cellobiase cleaves cellobiose to yield glucose. The efficiency (e.g., time to hydrolyze and/or completeness of hydrolysis) of this process depends on the recalcitrance of the cellulosic al.

INTERMEDIATES AND PRODUCTS Using the processes described herein, the biomass al can be converted to one or more products, such as energy, fuels, foods and als. Specific examples of ts include, but are not limited to, hydrogen, sugars (e.g., e, xylose, arabinose, mannose, galactose, fructose, disaccharides, oligosaccharides and polysaccharides), alcohols (e.g., monohydric alcohols or dihydric alcohols, such as ethanol, n-propanol, isobutanol, sec-butanol, tert-butanol or n-butanol), hydrated or hydrous alcohols (e.g., containing greater than 10%, 20%, % or even greater than 40% water), biodiesel, organic acids, hydrocarbons (e.g, methane, ethane, propane, isobutene, pentane, n-hexane, biodiesel, bio-gasoline and mixtures thereof), co- products (e.g., proteins, such as cellulolytic proteins (enzymes) or single cell ns), and mixtures of any of these in any combination or relative concentration, and optionally in ation with any additives (e.g, fuel additives). Other examples include carboxylic acids, salts of a carboxylic acid, a mixture of carboxylic acids and salts of carboxylic acids and esters of carboxylic acids (e.g., methyl, ethyl and n-propyl esters), ketones (e.g., acetone), aldehydes (e.g., acetaldehyde), alpha and beta unsaturated acids (e.g., acrylic acid) and olef1ns (e.g., ethylene).

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

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 ise, may be packaged together and sold as products. The products may be combined, e.g., mixed, blended or co-dissolved, or may simply be packaged or sold together.

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

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

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

Many of the products ed, such as ethanol or n-butanol, can be ed as a filel for powering cars, trucks, tractors, ships or trains, e.g., as an internal tion filel or as a fuel cell feedstock. Many of the products obtained can also be utilized to power aircraft, such as planes, e.g., having jet engines or helicopters. In addition, the products described herein can be utilized for electrical power generation, e.g., in a conventional steam ting plant or in a fuel cell plant.

Other intermediates and products, including food and pharmaceutical products, are described in US. Pat. App. Pub. 2010/0124583 A1, published May 20, 2010, to Medoff, the fill disclosure of which is hereby incorporated by reference herein.

SACCHARIFICATION The d s materials can be rified, generally by combining the material and a cellulase enzyme in a fluid medium, e.g., an aqueous on. In some cases, the material is boiled, steeped, or cooked in hot water prior to saccharification, as described in US.

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

The saccharif1cation process can be partially or completely performed in a tank (6.g. a tank having a volume of at least 4000, 40,000, or 500,000 L) in a manufacturing plant, and/or can be partially or completely med in transit, e.g., in a rail car, tanker truck, or in a supertanker or the hold of a ship. The time required for complete saccharif1cation will depend on the process conditions and the biomass material and enzyme used. If saccharification is performed in a manufacturing plant under controlled conditions, the cellulose may be substantially ly converted to sugar, e.g., glucose in about 12-96 hours. If saccharif1cation is performed partially or completely in transit, saccharif1cation may take .

It is generally preferred that the tank contents be mixed during saccharif1cation, e.g., using jet mixing as described in International App. No. 2010/03533 l , filed May 18, 2010, which was published in English as WC 2010/135380 and designated the United States, the filll disclosure of which is incorporated by reference herein.

The addition of surfactants can enhance the rate of saccharif1cation. Examples of surfactants include nic surfactants, such as a Tween® 20 or Tween® 80 polyethylene glycol surfactants, ionic surfactants, or eric surfactants.

It is generally preferred that the concentration of the sugar on resulting from saccharif1cation be vely high, e.g., greater than 40%, or greater than 50, 60, 70, 80, 90 or even greater than 95% by weight. Water may be removed, e.g., by evaporation, to increase the concentration of the sugar solution. This reduces the volume to be shipped, and also inhibits microbial growth in the solution.

Alternatively, sugar solutions of lower concentrations may be used, in which case it may be desirable to add an antimicrobial additive, e.g., a broad spectrum antibiotic, in a low concentration, e.g., 50 to 150 ppm. Other suitable antibiotics e amphotericin B, ampicillin, chloramphenicol, ciprofloxacin, gentamicin, hygromycin B, kanamycin, neomycin, penicillin, puromycin, streptomycin. Antibiotics will inhibit growth of microorganisms during transport WO 96699 and storage, and can be used at appropriate concentrations, e.g., between 15 and 1000 ppm by weight, e.g., between 25 and 500 ppm, or between 50 and 150 ppm. If desired, an antibiotic can be included even if the sugar concentration is relatively high. Alternatively, other additives with anti-microbial of preservative properties may be used. Preferably the antimicrobial additive(s) are rade.

A relatively high concentration solution can be ed by limiting the amount of water added to the biomass material with the enzyme. The concentration can be controlled, 6.g. by controlling how much rif1cation 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 ature of the solution. For example, the solution can be maintained at a temperature of 40-50°C, 60-80°C, or even .

SACCHARIFYING AGENTS Suitable olytic enzymes include cellulases from species in the genera Bacillus, CaprinuS, Myceliophthora, Cephalosporz'um, Scytalz'dz'um, Penicillium, ASpergz'lluS, monas, Humicola, Fusarium, Thielavz'a, Acremonium, ChrySOSporz'um and Trichoderma, ally those produced by a strain selected from the species ASpergz'lluS (see, e.g., EP Pub.

No. 0 458 162), Humicola insolenS (reclassified as z'clz'um thermophilum, see, e.g., US. Pat.

No. 4,435,307), CaprinuS cinereuS, Fusarium oxySporum, Myceliophthora thermophila, Merlpl'luS giganteus, Thielavz'a triS, Acremonium Sp. ding, but not limited to, A. perSl'cz'num, A. acremonium, A. brachypem'um, A. dichromosporum, A. obclavatum, A. pinkertonz'ae, A. roseogriseum, A. incoloratum, and A. furatum). Preferred strains include Humicola insolenS DSM 1800, Fusarium oxySporum DSM 2672, Myceliophthora phila CBS 117.65, osporz'um Sp. 2, Acremonium Sp. CBS 478.94, Acremonium Sp.

CBS 265.95, Acremonium persicinum CBS 169.65, Acremonium acremonium AHU 9519, Cephalosporz'um Sp. CBS , Acremonium brachypem'um CBS 866.73, Acremonium dichromosporum CBS 683.73, Acremonium obclavatum CBS 311.74, Acremonium pinkertoniae CBS 157.70, Acremonium roseogriseum CBS 134.56, Acremonium incoloratum CBS 146.62, and Acremom’umfuratum CBS 299.70H. Cellulolytic enzymes may also be obtained from ChrySOSporz'um, preferably a strain of ChrySOSporz'um lucknowense. Additional strains that can be used e, but are not limited to, Trichoderma (particularly T. viride, T. reesez’, and T. koningii), alkalophilic Bacillus (see, for example, US. Pat. No. 3,844,890 and EP Pub. No. 0 458 162), and Streptomyces (see, e.g., EP Pub. No. 0 458 162).

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

SUGARS In the processes described , for example after saccharif1cation, sugars (e.g, glucose and xylose) can be isolated. For example sugars can be isolated by precipitation, crystallization, chromatography (6.g. , simulated moving bed chromatography, high pressure chromatography), centrifilgation, extraction, any other isolation method known in the art, and combinations thereof.

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

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

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

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

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

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

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

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

“Fermentation” includes the s and products that are disclosed in US. Prov.

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

Mobile fermenters can be utilized, as described in ational App. No. (which was filed July 20, 2007, was published in h as WO 2008/01 1598 and ated the United States), the contents of which is incorporated herein in its entirety. Similarly, the saccharification equipment can be mobile. r, saccharification and/or fermentation may be med in part or entirely during transit.

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

Suitable fermenting microorganisms have the ability to convert carbohydrates, such as glucose, fructose, xylose, arabinose, mannose, galactose, oligosaccharides or polysaccharides into fermentation products. Fermenting microorganisms e strains of the genus Saccharomyces spp. (including, but not d to, S. cerevisiae (baker’s yeast), S. z'cas, S. avaram), the genus Klayveromyces, ding, but not limited to, K. marxz’anas, K. fragilis), the genus a (including, but not limited to, C. pseudotropz'calz’s, and C. brassz'cae), Pichia stz’pz’tz’s (a relative of Candida shehatae), the genus Clavz'spora ding, but not limited to, C. lasitam'ae and C. opantz'ae), the genus olen (including, but not limited to, P. tannophz'las), the genus Bretannomyces (including, but not limited to, e.g., B. clausem'z' (Philippidis, G. P., 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization, Wyman, C.E., ed., Taylor & Francis, Washington, DC, 2)). Other suitable microorganisms include, for example, Zymomonas mobilis, Clostrz'dz'am spp. (including, but not limited to, C. thermocellam ppidis, 1996, supra), C. saccharobatylacetom’cam, C. saccharobatylicam, C. Paniceam, C. beijemckl’z’, and C. acetobatylicam), Moniliella pollinis, Moniliella megachl'liensz's, Lactobacz'llas spp. Yarrowz'a lipolytl'ca, Aareobasidz'am 519., Trichosporonoides 519., Trigonopsz's variabilis, Trichosporon sp., Moniliellaacetoabatans sp., Typhala variabilis, Candida magnoliae, Ustz'laginomycetes sp., zyma tsakabaensz's, yeast species of genera Zygosaccharomyces, Debaryomyces, Hansenala and Pichia, and fiJngi of the dematioid genus Torala.

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

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

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

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

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

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

The beer column bottoms can be sent to the first effect of a 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 n (25%) of the centrifuge effluent can be recycled to tation and the rest sent to the second and third evaporator effects. Most of the evaporator condensate can be returned to the process as fairly clean condensate with a small portion split off to waste water treatment to prevent build-up of iling compounds.

Other than in the examples herein, or unless otherwise expressly specified, all of the cal , amounts, values and percentages, such as those for amounts of materials, tal contents, times and temperatures of reaction, ratios of amounts, and others, in the following portion of the specification and attached claims may be read as if prefaced by the word “about” even though the term “about” may not expressly appear with the value, , or range. Accordingly, unless indicated to the ry, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents 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 ques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the ion are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently ns error necessarily resulting from the standard deviation found in its underlying respective testing ements. Furthermore, when numerical ranges are set forth herein, these ranges are inclusive of the recited range end points (2'.e., end points may be used). When percentages by weight are used herein, the numerical values reported are relative to the total weight.

Also, it should be understood that any numerical range recited herein is intended to include all sub-ranges subsumed n. For example, a range of “l to 10” is ed to include all sub-ranges between (and including) the recited minimum value of l and the d maximum value of 10, that is, having a minimum value equal to or r than 1 and a maximum value of equal to or less than 10. The terms “one,a) :4 a) a or “an” as used herein are intended to include “at least one” or “one or more,” unless otherwise indicated.

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

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

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those d in 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 .

Throughout the specification and claims, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the ion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Claims (17)

CLAIMS What is claimed is:

1. A method comprising: providing a liquid medium, a reduced-recalcitrance cellulosic or lignocellulosic biomass, and a microorganism capable of producing an enzyme in the ce of the cellulosic or lignocellulosic biomass; wherein the reduced-recalcitrance osic or lignocellulosic biomass is disposed within a first structure or carrier formed of a mesh material having a maximum opening size of less than 1 mm; the microorganism is disposed within a second ure or carrier with a pore size of below 5 microns designed to contain the microorganism but to allow the enzyme to flow out of the second structure or carrier; the first structure or carrier is disposed in the second structure or r; both the first and second structures or carriers are disposed in the medium and can be removed or added at any time during the method; and wherein the enzyme can be manufactured and stored and then used in saccharification reactions of the same or similar s material at a later date and/or in a different location.

2. The method of claim 1, wherein the first structure or carrier is porous.

3. The method of claim 1, wherein the first structure or carrier and the second structure or carrier with their contents can be used in another medium to produce more enzymes.

4. The method of any one of claims 1-3, wherein the first ure or carrier is made of a bio-erodible polymer.

5. The method of claim 4, wherein the bio-erodible polymer is selected from the group consisting of: ctic acid, polyhydroxybutyrate, droxyalkanoate, polyhydroxybutyrate- valerate, polycaprolactone, polyhydroxybutyrate-hexanoate, polybutylene succinate, polybutyrate succinate adipate, polyesteramide, polybutylene adipate-co-terephthalate, mixtures thereof, and laminates thereof.

6. The method of claim 4, wherein the first structure or carrier is made of a starch film.

7. The method of any one of claims 1-6, further comprising utilizing further processing to tear or rupture the structure or carrier.

8. The method of any one of claims 1-7, n the microorganism comprises a strain of Trichoderma reesei.

9. The method of claim 8, wherein the strain is a high-yielding cellulaseproducing mutant of Trichoderma reesei.

10. The method of claim 9, wherein the strain comprises RUT-C30.

11. The method of any one of claims 1-10, n the recalcitrance of the cellulosic or lignocellulosic biomass has been reduced by exposure to an electron beam.

12. The method of any one of claims 1-11, wherein the cellulosic or ellulosic biomass is selected from the group consisting of: paper, cotton, wood, forestry wastes, grasses, grain residues, agricultural waste, sugar sing residues, algae, seaweed, industrial waste, arracacha, buckwheat, banana, barley, cassava, kudzu, sago, sorghum, potato, sweet potato, taro, yams, beans, and mixtures of any of these.

13. The method of any one of claims 1-11, wherein the cellulosic or lignocellulosic s is a paper product.

14. The method of any one of claims 1-11, wherein the cellulosic or lignocellulosic biomass is a printed matter.

15. The method of any one of claims 1-11, wherein the cellulosic or ellulosic biomass is selected from the group consisting of: paper waste, paper pulp, pigmented papers, loaded papers, coated papers, filled papers, magazines, r paper, ated paper, card stock, cardboard, oard, particle board, sawdust, aspen wood, wood chips, switchgrass, miscanthus, cord grass, reed canary grass, rice hulls, oat hulls, wheat chaff, barley hulls, silage, canola straw, wheat straw, barley straw, oat straw, rice straw, jute, hemp, flax, bamboo, sisal, abaca, corn cobs, corn stover, n stover, corn fiber, alfalfa, hay, coconut hair, bagasse, beet pulp, agave bagasse, manure, offal, oca, favas, lentils, peas, and mixtures of any of these.

16. The method of any one of claims 1-15, wherein the cellulosic or lignocellulosic material has an e particle size of less than 1 mm.

17. The method of any one of claims 1-16, wherein the cellulosic or lignocellulosic material has an average particle size of 0.25 mm to 2.5 mm.