OA16925A - Processing of biomass materials. - Google Patents

Abstract

Provided are methods of inducing enzymes, and for treating cellulosic and lignocellulosic biomass with the enzyme.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application Nos. 61/579,550 and 61/579,562, both filed on December 22,2011. The entire disclosures of the above applications are incorporated herein by reference.

FIELD OFTHE INVENTION [0002] The invention pertains to the préparation enzymes useful in the processing of biomass materials. For example, the invention relates to producing celluiase enzymes or other enzyme types.

BACKGROUND [0003] As demand for petroleum increases, so too does interest in renewable feedstocks for manufacturing biofuels and biochemicals. The use of iïgnocellulosic biomass as a feedstock for 20 such manufacturing processes has been studied since lhe 1970s. Lignoceilulosic biomass is attractive because it is abundant, renewable, domestically produced, and does not compete with food industry uses.

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

[0005] Lignoceilulosic bîomass is récalcitrant to dégradation as the plant cell walls hâve a structure that is rigid and compact. The structure comprises crystalline cellulose fibrils embedded in a hemicellulose matrix, surrounded by lignin. This compact matrix is diffïcult to 30 access by enzymes and other chemical, biochemical and biologlcal processes. Celluiosic biomass materials (e.g., biomass material from which substantially ali the lignin has been removed) can be more accessible to enzymes and other conversion processes, but even so, naturally-occurring cellulosic materials often hâve low yîelds (relative to theoretical yields) when contacted with hydrolyzing enzymes. Lignocellulosic biomass is even more récalcitrant to enzyme attack. Furthermore, each type οΓ lignocellulosic biomass has its own spécifie composition of cellulose, hemicellulose and lignln.

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

[0007] Saccharides from renewable biomass sources could become the basis of chemical and 10 fuels industries by replacing, supplementing or substituting petroleum and other fossil feedstocks. However, techniques need to be developed that will make these monosaccharides available in large quantifies and at acceptable purifies and prices.

SUMMARY OFTHE INVENTION [0008] Provided herein are methods of inducïng the production of one or more enzymes by a microorganism, through the use of an inductant [0009] In one aspect, a method is provided that includes combining a cellulosic or lignocellulosic biomass, which has been treated to reduce its rccalcîtrance, with a microorganism, to induce the production of one or more enzyme(s) by the microorganism by 20 maintaining the microorganism-biomass combination under conditions that allow for the production of the enzyme(s) by the microorganism. In some implémentations, the enzyme(s) are then used to saccharify cellulosic or lignocellulosic biomass.

[0010] Also provided herein is a method for inducïng the production of an enzyme by a microorganism, where the method includes: providing a first cellulosic or lignocellulosic 25 biomass; treating the first biomass with a treatment method to reduce its recalcitrance, thereby producing a first treated biomass; providing a microorganism; providing a liquid medium; combining the first treated biomass, the microorganism, and the liquid medium, thereby producing a microorganism-biomass combination; and maintaining the microorganism-biomass combination under conditions allowing for the production of an enzyme by the microorganism, 30 thereby producing an inductant-enzyme combination; thereby inducing the production of the enzyme by the microorganism.

£0011J Also provided herein is a composition that includes a liquid medium, a cellulosic or lignocellulosîc biomass treated to reduce its recale itrance, a microorganism, and one or more enzymes made by the microorganism.

[0012] In any of the methods or compositions provided herein, die treatment for reducing the 5 recalcitrance of the biomass material(s) can be any of: bombardment with électrons, sonication, oxîdation, pyrolysis, steam explosion, chemical treatment, mechanîcal treatment, and freeze grinding. Preferably, the treatment method is bombardment with électrons.

[0013] The methods and compositions can also include medianically treating the first or the second cellulosic or lignocellulosîc bîomass to reduce its bulle density and/or increase its surface 10 area. The biomass material(s) can be comminuted before being combined with the microorganism and liquid medium. The comminution can be dry milling or wet miling. The bîomass material can hâve a particie size of about 30 to 1400 pm.

[0014] In any of the methods and compositions described herein, any of the cellulosic or iignocclluiosic biomasses can be: paper, paper products, paper waste, paper pulp, pïgmented 15 papers, ioaded papers, coated papers, filied papers, magazines, printed matter, printer paper, polycoated paper, card stock, cardboard, paperboard, cotton, wood, particie board, forestry wastes, sawdust, aspen wood, wood chips, grasses, switchgrass, miscanthus, cord grass, reed canary grass, grain residues, rice hulls, oat hulis, wheat chaff, barley huIIs, agricultural waste, silage, canola straw, wheat straw, barley straw, oat straw, rice straw, jute, hemp, flax, bamboo, 20 sisal, abaca, corn cobs, corn stover, soybean stover, com fiber, alfaifa, hay, coconut haïr, sugar processing residues, bagasse, beet pulp, agave bagasse, aigae, seaweed, manure, sewage, offal, agriculture! or industrial waste, arTacacha, buckwheat, banana, barley, cassava, kudzu, oca, sago, sorghum, potato, sweet potato, tara, yams, beans, favas, lentils, peas, or mixtures of any of these. Alternatively, the cellulosic or lignocellulosîc biomass can include material that was remaining 25 after a prior cellulosic or lignocellulosîc bîomass was previously converted to a product by an enzyme of a microorganism.

[0015] In these methods and compositions, the microorganism can be any of a fungus, a bacterium, or a yeast. The microorganism can actually be a population of different microorganisms. The microorganism can be a s train that produces high leveis of celluiase, 30 and/or it can be genetically engineered. The microorganism can be Trichoderma reesei, or it can be Clostridium thermocellum, for example. The microorganism can be a T. reesei strain such as RUT-NG14, PC3-7, QM9414 or RUT-C30.

[0016] In any of these methods and compositions, the cellulosic or lignoce 11 u lostc biomass can be combined with the microorganism at a time when the microorganism is In lag phase.

[0017] The methods and compositions can also include removing ail or a portion of the liquid from the mîcroorganism-inductant-enzyme combination, to produce an enzyme extract. The methods and compositions can also include concentrating one or more of the enzymes, and/or isolating one or more of the enzymes.

[0018] The methods and compositions can also include allowing saccharification of the second cellulosic or lignoceIluiosic biomass to occur, so that one or more sugars are produced.

The one or more sugars can be isolated and/or concentrated.

[0019] It should be understood that this invention is not limited to the embodiments disclosed in this Summary, and it is intended to cover modifications that are within the spirit and scope of the Invention, as defined by the claims.

BRIEF DESCRIPTION OF THE DRAWINGS [0020] The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are 20 not nccessarily to scale, emphasis instcad being placed upon illustrating embodiments of the présent invention.

[0021] FIG. I 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 (li) to cellobiose (C), which is converted to glucose (D) by cellobtase (beta25 glucosidase) (III).

[0022] ΠΟ. 2 is a flow diagram illustrating conversion of a biomass fcedstock to one or more products. Feedstock is physically pretreated (e.g., to reduce its size) (200), optionally treated to reducc its recalcitrance (210), saccharified to form a sugar solution (220), the solution is transported (230) to a manufacturing plant (e.g., by pipeline, railcar) (or if saccharification is 30 performed en route, the feedstock, enzyme and water is transported), the saccharified feedstock is bio-processed to produce a desired product (e.g., alcohol) (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 lignîn content (201) and setting or adjusting process parameters (205). Saccharifying the feedstock (220) can be modified by mixing the feedstock with medium and the enzyme (221).

[0023] FIG. 3 is a flow diagram illustrating the treatment of a first biomass (300), addition of a cellulose producing organism (310), addition of a second biomass (320), and processing the resulting sugars to make products (e.g., alcohol(s), pure sugars) (330). The first treated biomass can optionaliy be split, and a portion added as the second biomass (A).

[0024] FIG. 4 is a flow diagram illustrating the production ofenzymes. A cellula.se10 producing organism is added to growth medium (400), a treated first biomass (405) is added (A) to make a mixture (410), a second biomass ïs added (420), and the resulting sugars are processed to make products (e.g., alcohol(s), pure sugars) (430). Portions ofthe first biomass (405) can also be added (B) to the second biomass (420).

[0025] FIG. 5 shows results of protein analysis using SDS PAGE.

DETAILED DESCRIPTION [0026] Provided herein are methods of inducing the production of one or more enzymes by a microorganism, through the use of an inductant. The inductant is made from biomass (cellulosic or lignocellulosic) that has been treated to reduce its recalcitrance. The treatment method can 20 include subjecting the biomass to bombardment with électrons, sonication, oxidation, pyrolysîs, steam explosion, chemical treatment, mechanical treatment, or freeze grînding. As disclosed herein, biomass that has been treated with such a method can be combined with a microorganism in a medium (such as a liquid medium), to induce the microorganism to produce one or more enzymes.

[0027] In one aspect, the invention features a method that includes contacting an inducer comprising a lignocellulosic material with a microorganism to produce an enzyme.

[0028] Specifically, the processes described herein include saccharifying cellulosic and/or lignocellulosic materials using enzymes that hâve been produced by Trichoderma reesei fungi, as will be discussed in further detail below.

[0029] In general, the invention relates to improvements in processing biomass materials (e.g., biomass materials or biomass-derived materials) to produce intermediates and products, such as fuels and/or other products. For example, the processes may be used to produce sugars, alcohols (such as éthanol, isobutanol, or π-butanol), sugar alcohols (such as erythritol), or organic acids.

[0030] The invention also relates to the préparation of enzymes useful in the processing of 5 biomass materials. For example the invention relates to producing cellulase enzymes or other enzyme types.

[0031] A typical biomass resource contains cellulose, hemiccllulose, and lignin plus lesser amounts of proteins, extractables and minerais. The complex carbohydrates contained in the cellulose and hemicellulose fractions can be converted into sugars, e.g., fermen table sugars, by 10 saccharification, and the sugars can then be used as an end product or an Intermediate, or converted by further processing. e.g., fermentation or hydrogénation, into a variety of products, such as alcohols or organic acids. The product obtained dépends upon the method or microorganism utilized and the conditions under which the bioprocessing occurs.

[0032] In one embodiment, for instance, the invention includes a method of inducing the production of an enzyme. A cellulosic or lignocellulosic biomass is provided, treated to reduce its recalcitrance. and then combined with a microorganism in a liquid medium. The resulting microorganism-biomass combination is then maintained under conditions allowing for the growth of the organism and production of enzymes capable of degrading the biomass. The treated biomass acts as an inductant, causing the microorganism to produce enzymes. The method produces an induc tant-enzyme combination.

[0033] Without wishing to be bound by any theory, it is believed that the treatment used to reduce the recalcitrance of the biomass is important in enzyme induction. The inventors hâve found that low levels of treatment resuit in either low levels of enzyme induction, or extremely long lag times, presumably because it is di f ficult for the microorganisms to extract sugars from 25 the treated biomass material. Similarly, very high levels of treatment also cause the microorganisms to produce low levels of enzymes, possibly because relatively easy extraction of sugars from the treated biomass lessens the need for the microorganisms to produce large amounts of enzymes.

[0034] On a related matter, the recalcitrance treatment also serves to sterilize the material.

Biomass material, by its nature, contains contaminating microbes, which are often embedded deep within the material itself. Because the enzyme inductions as disclosed herein tend to be long fermentations (up to a week or more), sterilization is important It would therefore be advantagcous to treat the material as heavîly as possible to sterilize it. However, such high levels of treatment would likely be counterproductlve because high levels of treatment lessen the enzyme production by the microorganisms.

[0035] As disclosed herein. there is therefore a large benefït to be gained from carefully balancing the level of treatment to sterilize the material, yet not over-treat the material so that the microorganisms fall to produce large amounts of enzyme.

[0036] The term “inductant,” as used herein, means a ccllulosic or lignocellulosic biomass that encourages an organism to produce enzyme. An example would be biomass that has been 10 treated to reduce its recalcitrance. The treated biomass is then used as an enzyme inductant, by being combined with one or more microorganisms in a liquid medium, and then being maintained under conditions that allow the microorganism to produce one or more enzymes.

[0037] The inductant-enzyme combination can then be combined with another biomass, and used to saccharify it.

[0038] Surprisingly, it has been found that treating the biomass before inoculating it with the microorganism causes an increased amount of enzymes to be produced by the microbes. In addition, different enzymes are produced on the treated biomass, relative to the use of untreated biomass.

[0039] As described herein, the cellulosic or lignocellulosic biomass can be sourced from a 20 wide variety of materials. In one embodiment, the biomass can be lignin hulls. By “lignin hulls,” as used herein, is meant material that is remaining after a biomass has been saccharified.

[0040] In certain embodiments, the invention relates to processes for saccharifying a cellulosic or lignocellulosic material using an enzyme that has been produced by a fungus, e.g., by strains of the cel lu loi ytic filamentous fungus Trichoderma reesei. In some Implémentations, 25 high-yîelding ce) lulase mutants of Trichoderma reesei are used, e.g., RUT-NG14, PC3-7,

QM9414 and/or RUT-C30. Such strains are described, for example, in “Sélective Screening Methods for the Isolation of High Yîelding Ccllulase Mutants of Trichoderma reesei,” Montcnecourt, B.S. and Everleigh, D.EMJv. Chem. Ser. 18], 289-301 (1979). These mutants are hyperproducîng and are catabolite répression-résistant, allowing high yîelds of cellulases to 30 be achieved.

[0041] In preferred embodiments, the enzyme production is conducted in the presence of a portion ofthe lignocellulosic material to be saccharified. The lignocellulosic material can net in the enzyme production process as an inducer for cellulose synthesis, producing a cellulose complex having an activity that is tailored to the particular lignocellulosic material. In some implémentations, the recalcitrance of the lignocellulosic material is reduced prior to using it as an inducer. It is believed that this makes the cellulose within the lignocellulosic material more readily available to the fungus. Reducing the recalcitrance of the lignocellulosic material also facilitâtes saccharification.

[0042] In some cases, reducing the recalcitrance of the lignocellulosic material includes treating the lignocellulosic material with a physical treatment The physical treatment can be, for example, radiation, e.g., électron bombardaient, sonication, pyrolysis, oxidation, steam explosion, chcmical treatment, or combinations of any of these treatments. The treatments can also include any one or more of the treatments disclosed herein, applied alone or in any desired combination, and applied once or multiple times.

[0043] Enzymes and biomass-destroying organisme thaï break down biomass, such os the cellulose and/or the lignin portions of the biomass, contain or manufacture various ceilulolytic enzymes (celluloses), ligninases or various small molécule biomass-destroying métabolites. These enzymes may be a complex of enzymes thaï aci synergistically to dégradé crystalline cellulose or the lignin portions of biomass. Exemples of ceilulolytic enzymes include:

endoglucanases, cellobioliydrolases, and cellobiases (beta-glucosidases).

[0044] As shown in FIG. I, for example, during saccharification a cellulostc substrate (A) is ini tially hydrolyzed by endoglucanases (I) at random locations producing oltgomeric intermediates (e.g., cellulose) (B). These intermediates are then substrates forexo-splitling glucanases (il) such as cellobiohydrolase to produce cellobiose from the ends of the cellulose polymer. Cellobiose is a waler-soluble 1,4-linked dimer of glucose. Finally cellobiasc (iii) cleaves cellobiose (C) to yield glucose (D). Therefore, the endoglucanases are partîcularly effective in attacking the crystalline portions of cellulose and increasing the effectiveness of exocellulases to produce cellobiose, which then requires the specificity of the cellobiose to produce glucose. Therefore, it is évident that depending on the nature and structureofthe cellulosic substrate, the amount and type of the three different enzymes may need to be modified.

[0045] The enzymes produced and used in the processes described herein can be produced by a fungus, e.g., by one or more strains of the fungus Trichoderma reesei. In preferred implémentations, high-yielding cellulase mutants of Trichoderma reesei, e.g., RUT-NG14, PC37, QM9414 and/or RUT-C30, are used.

[0046] It is preferred that enzyme production be conducted in the presence of a portion of the feedstock that will be saccharifïed, thereby producing a cellulase complex that is tailored to the particular feedstock. The feedstock may be treated prior to such use to reduce its recalcitrance, e.g., using one or more of the recalcitrance-reducing processes described herein, so as to make the cellulose in the feedstock more readily available to the fungus.

[0047] In a preferred embodiment the enzyme-inducing biomass can be treated by électron bombardment. The biomass can be treated, for instance, by électron bombardment with a total dose of less than about 1 Mrad, less than about 2 Mrad,, less than about 5, about 10. about 20, about 50, about 100 or about 150 Mrad. Preferably, the enzyme-inducing biomass is treated with a total dose of about 0.1 Mrad to about 150 Mrad, about I to about 100 Mrad, preferably about 2 to about 50 Mrad, or about 5 to about 40 Mrad.

[0048] As will be discussed further below, once the enzyme has been produced, it is used to saccharify the remainïng feedstock that has not been used to produce the enzyme. The process for converting the feedstock to a desired product or intermediate generally includes other steps in addition to this saccharification step.

[0049] For example, referring to FIG. 2, a process for manufacturing an alcohol can include, for example, optionally mechanically treating a feedstock, e.g., to reduce its size (200), before and/or after this treatment, optionally treating the feedstock with another physical treatment to further rcducc its recalcitrance (210), then saccharifylng the feedstock, using the enzyme complex, to form asugar solution (220). Optionally, the method may also include transporting,

e.g., by pipeline, railcar, truck or barge, the solution (or the feedstock, enzyme and water, if saccharification is performed en route) to a manufacturing plant (230). In some cases the saccharifïed feedstock is further bioprocessed (e.g., fermented) to produce a desired product e.g., alcohoi (240). This resulting product may in some implémentations beprocessed further, e.g., by distillation (250), to produce a final product. Onc method of reducing the recalcitrance of the feedstock is by électron bombardment of the feedstock. If desired, the steps of measuring lignin content of the feedstock (201) and setting or adjusting process parameters based on this ίο measurement (205) can be performed at various stages of the process, as described in U.S. Pat. App. Pub. 2010/0203495 Al by Medoff and Masterman, published August 12,2010, the complété disclosure of which is incorporated herein by reference. Saccharifying the feedstock (220) can also be modified by mixing the feedstock with medium and the enzyme (221).

(0050] The manufacture of the enzyme complexes wiiI now be described first, followed by a description of the method steps discussed above with reference to FIG. 2, and the materials used in the process.

[0051] A number of different conditions were tested, and the results are as foilows. In one embodiment, the enzyme induction biomass is corn cob. In this embodiment, the biomass îs 10 treated by électron bombardment with a 35 Mrad électron beam. Preferably, the biomass is comminuted to aparticle size of 10-1400 um, more preferably less than 200 um, most preferably less than 50 um. The treated biomass (in either wet or dry form) is added in a total amount of about 25 to about 133 g/L of inoculated medium, more preferably 100 g/L. The inductant biomass can be added at any point in the growth of the microorganisms up through the third day 15 after inoculation, but is preferably added 1-3 days after inoculation. The total amount of biomass to be added as an inductant can be added ail at once, or in aliquots, for instance, in two parts, or in five parts. Preferably the comcob biomass is added ail at once.

[0052] The enzyme induction biomass can be presented to the microorganisms as a solid, or as a slurry. Preferably it is added as a slurry.

ENZYME PRODUCTION [0053] Filamentous fungi, or bacteria that produce ceilulase, typically require a carbon source and an inducer for production of ceilulase. Without being bound by any theory, it is believed that the enzymes of this disclosure are particularly suîted for saccharification of the 25 substrate used for Inducing its production.

[0054] Lignocellulosic materials comprise different combinations of cellulose, hemicellulose and lignin. Cellulose is a linear poiymer of glucose forming a fairiy stiff linear structure without significant coiling. Duc to this structure and the disposition of hydroxyl groups that can hydrogen bond, cellulose contains crystalline and non-crystalline portions. The crystalline portions can also be of different types, noted as I(alpha) and I(beta) for example, depending on the location of hydrogen bonds between strands. The polymer lengths themselves can vary

II lending more variety to the form ofthe cellulose. Hemicellulose is any ofseveral heteropolymers, such as xylan, glucuronoxylan, arabinoxylans, and xyloglucan. The primary sugar monomer présent is xylose, although other monomers such as mannose, galactose, rhamnosc, arabînose and glucose are présent. Typically hemicellulose forms branched structures 5 with lower molecular weights than cellulose. Hemicellulose is therefore an amorphous material that is generally susceptible to enzymatic hydrolysis. Lignin is a complcx high molecular weight heteropolymer generally. Although al! lignins show variation in their composition, they hâve been described as an amorphous dendritic network polymer of phenyl propene units. The amounts of cellulose, hemicellulose and lignin in a spécifie biomaterial dépends on the source of 10 the biomaterial. For example wood derived biomaterial can be about 38-49% cellulose, 7-26% hemicellulose and 23-34% lignin depending on the type. Grasses typically are 33-38% cellulose, 24-32% hemicellulose and 17-22% lignin. Clearly lignocellulosic biomass constitutes a large ciass of substrates.

[0055] The diversity of biomass materials may be further increased by pretreatment, for example, by changing the crystal I inity and molecular weights ofthe polymers.

[0056] The cellulose producing organism when contacted with a biomass will rend to producc enzymes that release molécules advantageous to the organism’s growth, such as glucose. This is done through the phenomenon of enzyme induction as described above. Since there arc a variety of substrates in a particular biomaterial, there are a variety of cellulases, for exemple, the 20 endoglucanase, exoglucanase and cellobiase discussed previously. By selecting a particular lignocellulosic material as the inducerthe relative concentrations and/or activities of these enzymes can be modulated so that the resulting enzyme complex will work efficlently on the lignocellulosic material used as the înducer or a similar material. For example, a biomaterial with a higher portion of crystalline cellulose may induce a more effective or higher amount of 25 endoglucanase than a biomaterial with little crystalline cellulose.

[0057] Therefore, there may be many methods for optimal formation and use of cellulases. Some details of these processes will be described with reference to the figures.

[0058] For example, referring to FIG. 3, a first biomass Is optionally pre-treated (300), for example to reduce its recalcilrance, and is then mixed with an aqueous medium and a cellulase 30 producing organism (310). After an adéquate time has passed for the cells to grow to n desired stage and enough enzymes hâve been produced, a second biomass is added (320). The action of the enzyme on lhe second and any remaining first biomass produces a mix of sugars, which can be further processed to useful products (e.g., alcohols, pure sugars) (330). The first and second biomass can be portions of the same biomass source material. For example, a portion of the biomass can be combined with the cellulase producing organism and then another portion added 5 at a later stage (Λ) once some of the enzymes hâve been produced. Optionally, the first and second biomass may both be pretreated to reduce recalcitrance. The aqueous media will be discussed below.

[0059] Referring now to FIG. 4, the cellulase producing organism (400) can be grown in a growth medium for a time to reach a spécifie growth phase. For example, this growth period 10 could extend over a period ofdays or even weeks. Pretreated first biomass (405) can then be contacted (A) with the enzyme producing cells (410) so that after a time enzymes are produced. Enzyme production may also take place over an extended period of time. The enzyme containing solution is then combined with a second biomass (420). The action of the enzyme on the second and remaining first biomass produces mixed sugars which can be further processed to 15 useful products (430). The first and second biomass can be portions of the same biomass or could be similar but not identical (e.g., pretreated and non-pretreated) material (B).

[0060] In addition to the methods discussed above in FIG. 4, the cellulase producing organism (400) may optionally be harvested prior to being combined with the first pretreated biomass (410). Harvesting may include partial or almost complété removai of the solvent and 20 growth media components. For example the cells may be collected by centrifugation and then washed with water or another solution.

[0061] In another embodiment, after the enzyme(s) is produced (410), it can be concentrated (e.g., between steps 410 and 420 of FIG. 4). Concentration may be by any useful method including chromatography, centrifugation, filtration, dialysis, extraction, évaporation of solvents, 25 spray drying and adsorption onto a solid support. The concentrated enzyme can be stored for a time and then be used by addition of a second biomass (420) and production of useful products (430).

[0062] The aqueous media used in the above described methods can contaîn added yeast extract, com steep, peptones. amino acids, ammonium salts, phosphate salts, potassium salts, 30 magnésium salts, calcium salts, iron salts, manganèse salts, zinc salts and cobalt salts. In addition to these components, the growth media typically contains 0 to 10% glucose (e.g., I to

5% glucose) as a carbon source. Additionally the inducer media can contain, in addition to the biomass discussed previously, other inducers. For example, some known lnducers are lactose, pure cellulose and sophorose. Various components can be added and removed during the processing to optimize the desired production of useful products.

[0063] The concentration of the biomass typically used for Inducing enzyme production is greater than or equal to 0.1 wt.% and less than or equal to 50 wt.%, greater than or equal to 0.5 wt.% and less than or equal to 25 wt.%, greater than or equal to 1 wt. %, and less than or equal to 15 wt.%, and greater than or equal lo 1 wt.% and less than or equal to 10 wt. %.

[0064] Any of the processes described above may be performed as a batch, a fed-batch or a continuous process. The processes are useful for especially industrial scale production, e.g., having a culture medium ofat 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 100,000 liters or500,000 liters. The process may be carried out aerobically or anaerobically. Some enzymes are produced by submerged cultivation and some by surface cultivation.

[0065] In any of the process described above, the enzyme can be manufactured and stored and then used to saccharify at a later date and/or different location.

[0066] Any of the processes described above may be conducted with agitation. In some cases, agitation may be performed using jet mixing as described In U.S. Pat. App. Pub. 2010/0297705 Al by Medoff and Masterman, published November 25,2010, U.S. Pat. App.

Pub. 2012/0091035 Al to Medoff and Masterman, published April 19,2012, and U.S. Pat. App. Pub. 2012/0100572 AI by Medoff and Masterman, published April 26,2012, the full disclosures of which are incorporated by reference herein.

[0067] Températures for the growth of enzyme producing organisms are chosen to enhance organism growth. For example for Trichoderma reesei the optimal température is generally between 20 and 40°C (e.g., 30°C), Tlie température for enzyme production is optimized for thaï part of the process. For example for Trichoderma reesei the optimal température for enzyme production is between 20 and 40°C (e.g., 27°C).

FEEDSTOCK, BIOMASS MATERIALS, AND/OR INDUCERS [0068] The feedstock, which may also be the inducer for enzyme production, is preferably a lignocellulosic material, although the processes described herein may also be used with cellulosic materials, e.g., paper, paper products, paper pulp, cotton, and mixtures of any of these, and other types of bîomass. The processes described herein are particularly useful with lignocellulosic materials, because these processes are particularly effective in reducing lhe recalcitrance of lignocellulosic materials and allowing such materials to be processed into products and întermediates in an economically viable manner, [0069] As used herein, the term “biomass materials” includes lignocellulosic, cellulosic, starchy, and microbial materials.

[0070] Preferably the enzyme-inducing biomass materials are agricultural waste such as corn cobs, more preferably corn stover. Most preferably, the enzyme-inducing biomass material 10 comprises grasses.

[0071] Lignocellulosic materials include, but are not limited to, wood, partiele 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, oal hulls, wheat chaff, barley hulls), agricultural waste (e.g., silage, canola straw, wheat straw, barley straw, oal straw, rice 15 straw, jute, hemp, flax, bamboo, sisal, abaca, corn cobs, corn stover, soybean stover, corn fiber, alfalfa, hay, coconut hoir), sugar processîng residues (e.g., bagasse, bect pulp, agave bagasse), algae, seaweed, manure, sewage, and mixtures of any of these.

[0072] In some cases, the lignocellulosic material includes comcobs. Ground or hammermilled comcobs can be spread in a layer of relatively uniform thickness for irradiation, 20 and after irradiation are easy to disperse in the medium for further processîng. To facilitate harvest and collection, in some cases the entire corn plant is used, including the corn stalk, com kernels, and in some cases even the root system of the plant [0073] Advantageously, no additional nutrients (other than a nitrogen source, e.g., urea or ammonia) are required during fermentation of comcobs or cellulosic or lignocellulosic materials 25 containing signifïcant amounts of comcobs.

[0074] Comcobs, before and after comminution, are also easler to convey and disperse, and hâve a lesser tendency to form explosive mixtures in air than other cellulosic or lignocellulosic materials such as hay and grasses.

[0075] Cellulosic materials include, for example, paper, paper products, paper waste, paper 30 pulp, pigmented papers, loaded papers, coated papers, fil led papers, magazines, printed matter (e.g., books, catalogs, manuals, labels, calendars, greeting cards, brochures, prospectuscs, newsprint), printer paper, polycoated paper, card stock, cardboard, paperboard, materials having a high alpha-cellulose content such as cotton, and mixtures ofany of these. For example paper products as described in U.S. App. No. 13/396,365 (“Magazine Feedstocks by Medoff et al., filed February 14,2012), the full disclosure of which îs incorporated herein by référence.

[0076] Cellulosic materials can also Include lignocellulosic materials which hâve been delignified.

[0077] Starchy materials include starch itseif, e.g., com starch, wheat starch, potato starch or rice starch, a dérivative of starch, or a material that includes starch, such as an edible food product or a crop. For example, the starchy material can be arracacha, buckwheal, banana, barley, cassava, kudzu, oca, saga, sorghum, regular household potatoes, sweet potato, tare, yams, or one or more beans, such as favas, lentîls 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 com plant, rice plant or a tree. The starchy materials 15 can be treated by any of the methods described herein.

[0078] Microbial materials include, but are not limited to, any naturally occurring or genetically modified microorganism or organism that contains or is capable of providing a source of carbohydrates (e.g., cellulose), for example, protists, e.g., animal protists (e.g., protozoa such as flagellâtes, amoeboids, ciliates, and sporozoa) and plant protists (e.g., algae such alveolates, chlorarachniophytes, cryptomonads, cuglenids, glaucophytes, haptophytes, red algae, stramenopiles, and viridaeplantae). Other exemples include scaweed, plankton (e.g., macroplankton, mesoplankton, microplankton, nanoplankton, picoplankton, and femptoplankton), phytoplankton, bacteria (e.g., gram positive bacteria, gram négative bacteria, and extremophiles), yeast and/or mixtures of these. In some instances, microbial biomass can be obtained from naiural sources, e.g., the océan, lakes, bodies of water, e.g., sait water or fresh water, or on land. Altematively or in addition, microbial biomass can be obtained from culture Systems, e.g., large scale dry and wet culture and fermentation systems.

[0079] The bîomass material can also include ofTal, and similar sources of material.

[0080] In other embodiments, the biomass materials, such as cellulosic, starchy and lignocellulosic feedslock materials, can be obtained from transgenic microorganisms and plants that hâve been modified with respect to a wild type variety. Such modifications may bc, for example, through the itérative steps of sélection and breeding to obtain desired traits in a plant. Furthermore, the plants can hâve had gene tic material removed, modified, silenced and/or added with respect to the wild type variety. For example, genetically modified plants can be produced by recombïnant DNA methods, where genetic modifications include introducing or modifying 5 spécifie genes from parental varieties, or, for example, by using transgenic breeding wherein a spécifie gene or genes are introduced to a plant from a different species of plant and/or bacteria. Another way to create genetic variation is through mutation breeding wherein new alleles are artificially created from endogenous genes. The artificial genes can bc created by a variety of ways including treating the plant or seeds with, for example, chemical mutagens (e.g., using alkylating agents, epoxides, alkaloids, peroxides, formaldéhyde), irradiation (e.g., X-rays, gamma rays, neutrons, beta particles, alpha particles, protons, deuterons, UV radiation) and température shocking or other external stressing and subséquent sélection 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 bacterial carrier, blolistlcs, calcium phosphate précipitation, electroporation, gene splicing, gene silencing, lipofection, microinjection and viral carriers. Additional genetically modified materials hâve been described in U.S. Application Serial No 13/396,369 filed Febraary 14,2012 the full disclosure of which is incorporated herein by reference.

[0081 ] Any of the methods described herein can be practiced with mixtures of any biomass materials described herein.

BIOMASS ~ MECHANICAL PREPARATION [0082] Mechanical treatments of the feedslock may include, for cxample, cutting, milling,

e.g., hammermilling, wet milling, grînding, pressing, shearing or chopping. The initial mechanical treatment step may, in some implémentations, include reducing the size of the feedstock. In some cases, loosc feedstock (e.g., recycled paper or switchgrass) is initially prepared by cutting, shearing and/or shredding.

[0083] In addition to this size réduction, which can beperformed initially and/or laterduring 30 processing, mechanical treatment can also be advantageous for opening up, stressing, breaking or shattering the feedstock materials, making the cellulose of the materials more susceptible to chain scission and/or disruption of crystalline structure during the structural modification treatment.

[0084] Methods of mechanically treating the feedstock include, for example, milling or grinding. Milling may be performed using, for example, a hammer mi II, bail mill, colloid mil 1, 5 conlcal or cône mill, disk mill, edge mill, Wiley mill or grist mill. Grinding may be performed using, for example, a culting/impact type grinder. Spécifie examples of grinders include stone grinders, pin grinders, coffee grinders, and burr grinders. Grindingor milling may be provided, for example, by a reciprocating pin or other élément, as is the case in a pin mill. Other mechanical treatment methods include mechanical ripping or tearing, other methods that apply 10 pressure to the fibers, and air attrition milling. Suitable mechanical treatments further include any other technique that continues the disruption ofthe internai structure ofthe material that was i nitiated by the previous processing steps.

[0085] Mechanical treatments that may be used, and the characteristics of the mechanically treated feedstocks, are described in further detail in U.S. Serial No. 13/276,192, filed October 18,

2011, and published on April 26,2012 as U.S. Pat. App. Pub. 2012/0100577 AI, the full disclosure of which is hereby incorporated herein by référencé.

BIOMASS TREATMENT- ELECTRON BOMBARDMENT [0086] In some cases, the feedstock may be treated with électron bombardment to modify its 20 structure and thereby reduce its rccalcitrance. Such treatment may, for example, reduce the average molecular weight of the feedstock, change the crystalline structure of the feedstock, and/or increase the surface area and/or porosity of the feedstock.

[0087] Electron bombardment via an électron beam is generally preferred, because it provides very high throughput and because the use of a re!atively low voltage/high power 25 électron beam device éliminâtes the need for expensive concrète vault shielding, as such devices are “self-shielded” and provide a safe, efficient process. While the “self-shielded devices do include shielding (e.g., meta! plate shielding), they do not require the construction of a concrète vault, greatly reducing capital expenditure and often allowing an existing manufacturing facility to be used without expensive modification. Electron beam accelerators are available, for example, from IBA (Ion Beam Applications, Louvain-la-Neuve, Belgium), Titan Corporation (San Diego, California, USA), and NHV Corporation (Nippon High Voltage, Japan).

[0088] Electron bombardment may be performed using an électron beam device that has a nominal energy of less than 10 MeV, e.g., less than 7 MeV, less than 5 MeV, or less than 2 MeV, e.g., from about 0.5 to 1.5 MeV, from about 0.8 to 1.8 MeV, from about 0.7 to I MeV, or from about I to about 3 MeV. In some implémentations the nominal energy is about 500 to 800 keV.

[0089] The électron beam may hâve a relatively high total beam power (the combined beam power of ail accelcrating heads, or, if multiple accelerators are used, of ail accelerators and ail heads), e.g., at least 25 kW, e.g., at least 30,40,50,60,65,70, 80, 100,125, or 150 kW. In some cases, the power is even as high as 500 kW, 750 kW, or even 1000 kW or more. In some cases the électron beam has a beam power of 1200 kW or more.

[0090] This high total beam power is usually achieved by u ti lizing multiple accelerating heads. For example, the électron 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 température rise in the material, thereby preventing buming of the material, and also increases the uniformity of the dose through the thickness of the layer of material.

[0091] In some Implémentations, it is désirable to cool the material during électron bombardment. For example, the material can be cooled while it is being conveyed, for example by a screw extrader or other conveying equipment.

[0092] To reduce the energy required by the recalcitrance-reducing process, it is désirable to treat the material as quickly as possible. In general, it is preferred that treatment be performed at 20 a dose rate of greater than about 0.25 Mrad per second, e.g., greater than about 0.5,0.75,1, IJ,

2,5,7,10,12,15, or even greater than about 20 Mrad per second, e.g., about 0.25 to 2 Mrad per second. Higher dose rates generally require higher line speeds, to avoid thermal décomposition of the material. ln one implémentation, the accelerator is set for 3 MeV, 50 mAmp beam current, and the line speed is 24 feet/mlnute, fora sample thickness of about 20 mm (e.g., comminuted corn cob material with a bulk density of 0.5 g/cm³).

[0093] In some embodiments, électron bombardment is performed until the material receivcs a total dose of at least 0.5 Mrad, e.g., at least 5, 10,20,30 or at least 40 Mrad. In some embodiments, the treatment is performed until the material receives a dose of from about 0.5 Mrad to about 150 Mrad, about 1 Mrad to about 100 Mrad, about 2 Mrad to about 75 Mrad, 10

Mrad to about 50 Mrad, e.g., about 5 Mrad to about 50 Mrad, from about 20 Mrad to about 40 Mrad, about 10 Mrad to about 35 Mrad, or from about 25 Mrad to about 30 Mrad. In some implémentations, a total dose of 25 to 35 Mrad is preferred, applied ideally over a couple of seconds, e.g., al 5 Mrad/pass with each pass being applied for about one second. Applying a dose of greater than 7 to 8 Mrad/pass can in some cases cause thermal dégradation of the feedstock material.

[0100] 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 Mrad/pass, 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 ovcrheatîng of the material and also increases dose uniformity through the thickness of the material. In some implémentations, the material is stirred or otherwise mixed during or after each pass and then smoothed into a uniform layer again before the next pass. to further enhance treatment uniformity.

[0101] In some embodiments. électrons are accelerated to, for cxample, a speed of greater than 75 percent of the speed of light, e.g., greater than 85,90,95, or 99 percent of the speed of light.

[0102] In some embodiments, any processing described herein occurs on lignocellulosic material that remains dry as acquired or that has been dried, e.g., using heat and/or redueed pressure. For example, in some embodiments, the cellulosic and/or lignocellulosic material has less than about fivc percent by weight retained water, measured at 25°C and at fifty percent relative humidity.

[0103] Electron bombardment can be applied while the cellulosic and/or lignocellulosic material îs exposed to air, oxygen-enrichcd air, or even oxygen itself, or blanketed by an inert gas such as nitrogen, argon, or hélium. When maximum oxidation is desired, an oxidizing environment is utilized, such as air or oxygen and the distance from the beam source is optimized to maximize réactivé gas formation, e.g., ozone and/or oxides of nitrogen.

BIOMASS TREATMENT - SONICATION, PYROLYSIS, OXIDATION, STEAM EXPLOSION [0104] lf desired, one or more sonîcation, pyrolysis, oxidative, or steam explosion processes can be used in addition to or instead of électron bombardment to reduce the recalcitrance of the

feedstocL These processes are described in detail in U.S. Pat. No. 7,932,065 to Medoff, the full disclosure of which is incorporated herein by reference.

USE OF TREATED BIOMASS MATERIAL [0105] The biomass material (e.g., plant biomass, animal biomass, paper, and municipal waste biomass) can be used as feedstock to produce useful intermediates and products such ns organic acids, salts of organic acids, anhydrides, esters of organic acids and fuels, e.g., fuels for internai combustion engînes or feedstocks for fuel cells. Systems and processes are described herein that can use as feedstock cellulosic and/or lignoceilulosic 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 inciude newspaper, kraft paper, corrugated paper or mixtures of these.

[0106] In order to convert the feedstock to a form that can be readily processed, the glucanor xylan-containing cellulose in the feedstock can be hydrolyzed to low molecular weight carbohydrates, such as sugars, by a saccharifying agent, e.g., an enzyme or acid, a process referred to as saccharification. The low molecular weight carbohydrates can then be used, for example, in an exîstîng manufacturing plant, such as a single cetl protein plant, an enzyme manufacturing plant, or a fuel plant, e.g., an éthanol manufacturing facility. · [0107] The feedstock can be hydrolyzed using an enzyme, e.g., by combîning the materials 20 and the enzyme in a solvent, e.g., in an aqueous solution. The enzymes can be made/induced according to the methods described herein.

[0108] Specifïcally, the enzymes can be supplied by organisms that are capable of breaking down biomass (such as the cellulose and/or the lignin portions ofthe biomass), or thaï contain or manufacture various cellulolytîc enzymes (cellulases), ligninases or various small molécule biomass-degrading métabolites. Thèse enzymes may be a complex of enzymes that act synergisticaliy to dégradé crystalline cellulose or the lignin portions of biomass. Examples of cellulolytic enzymes inciude: cndoglucanascs, cellobiohydrolases, and cellobiascs (betaglucosîdases).

[0109] During saccharification a cellulosic substrate can be initially hydrolyzed by endoglucanases at random locations producing oligomcric intermediates. These intermediates are then substrates for exo-spiitting glucanases such as cellobiohydrolase to produce cellobiose from the ends ofthe cellulose polymer. Cellobiose is a watcr-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 dépends on the recalcitrance of the cellulosic material.

[0110]

INTERMEDIATES AND PRODUCTS [OUI] Using the processes described herein, the biomass material can be converted to one or more products. such as energy, fuels, foods and materials. Spécifie examples of products include, but are not limited to, hydrogen, sugars (e.g., glucose, xylose, arabinose, mannose, galactose, fructose, disaccharides, oligosaccharides and polysaccharides), alcohols (e.g., monohydric alcohols ordihydric alcohols, such as éthanol, n-propano|, Isobutanol, jec-butanol, tert-butanol or n-butanol), hydrated or hydrous alcohols (e.g„ containing greater than 10%, 20%, 30% or even greater than 40% water), biodiesel, organic acids, hydrocarbons (e.g„ methane, ethane, propane, isobutene, pentane, n-hexane, biodiesel, bio-gasoline and mixtures thereof), coproduc ts (e.g., proteins, such as cellulolytic proteins (enzymes) or single cell proteins), and mixtures of any of these in any combination or relative concentration, and optionally in combination with any additives (e.g., fuel additives). Other examples include carboxylic acids, salts of a carboxylic acid, a mixture of carboxylic acids and salts of carboxylic acids and esters of carboxylic acids (e.g„ methyl, ethyl and n-propyl esters), ketones (e.g., acetone), aldéhydes (e.g., acetaldehyde), alpha and beta unsaturated acids (e.g., acrylic acid) and olefîns (e.g., ethylene). Other alcohols and alcohol dérivatives include propanol, propylene glycol, 1,4-butanedîol, 1,3propanediol, sugar alcohols and polyols (e.g., glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, iditol, inositol, volemitol, isomalt, maltitoi, lactitol, maltotriitol, maltotetnütol, and polyglycitol and other polyols), and methyl or ethyl esters of any ofthese alcohols. Other products include methyl acrylate, methylmethacrylate, lactic acid, citric acid, formic acid, acetic acid, propionic acid, butyric acid, succinic acid, valeric acid, caproic acid, 3-hydroxypropionic acid, palmîtic acid, stearic acid, oxalic acid, malonic acid, glutaric acid, oleic acid, linoleic acid, glycolïc acid, gamma- hydroxybutyric acid, and mixtures thereof, salts of any of these acids, mixtures of any ofthe acids and their respective salts.

[0112] Any combination of the above products with each other, and/or of the above products with other products, which other products may be made by the processes described herein or otherwise, may be packaged together and sold as products. The products may be combined, e.g., mîxed, blended or co-dissolved, or may simply be packaged or sold together.

[0113] 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 neutralize one or more potentially undeslrable contaminants that could be présent in the product(s). Such sanitation can be done with électron bombardment, for example, be at a dosage of less than about 20 Mrad, e.g., from about 0.1 to 15 Mrad, from about 0.5 to 7 Mrad, or from about I to 3 Mrad.

[0114] 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-gencration) or sold on the open market. For example, steam generated from buming by-product streams can be used in a distillation process. As another example, electricity generated from buming by-product streams can be used to power électron beam généra tors used in pretreatment [0115] The by-products used to generate steam and electricity are derived from a number of sources throughout the process. For example, anaérobie digestion of wastewater can produce a biogas high in methane and a small amount of waste biomass (sludge). As another example, post-saccharification and/or post-distillate solids (e.g., unconverted lignin, cellulose, and hemicellulose remainîng from the pretreatment and primary processes) can be used, e.g., bumed, as a fuel. ¹ [0116] Many of the products obtained, such as éthanol or n-butanol, can be utilized as a fuel for powering cars, trucks, tractors, ships or trains, e.g., as an internai combustion fuel or as a fuel cell feedstock. Many of the products obtained can also be utilized to power aircraft, such as planes, e.g., having jet engines or helicopters. In addition, the products described herein can be utilized for electrical power génération, e.g., in a conventional steam generating plant or in a fuel cell plant.

[0117] Other intermediates and products, including food and pharmaceutical products, are described in U.S. Pat. App. Pub. 2010/0124583 Al, pubiished May 20,2010, to Medoff, the full 30 disclosure of which is hereby incorporated by reference herein.

SACCHARIFICATION [0118] The rcduced-recalcitrance feedstock is treated with the enzymes discussed above, generally by combining the material and the enzyme in a fluid medium, e.g., an aqueous solution. In some cases, the feedstock is boiled, steeped, or cooked in hot water prior to saccharification, as described in U.S. Pat. App. Pub. 2012/0100577 Al by Medoff and Masterman, published on April 26,2012, the entire contents of which are incorporated herein. [0119] The saccharification process can be partially or completely performed in a tank (e.g., a tank having a volume of at least 4000,40,000, or 500,000 L) in a manufacturing plant, and/or can be partially or completely performed in transit, e.g., in a rai! car, tanker truck, or in a supertanker or the hold of a ship. The time required for complété saccharification will dépend on the process conditions and the biomass material and enzyme used. If saccharification is performed in a manufacturing plant under controlled conditions, the cellulose may be substantially cntirely converted to sugar, e.g., glucose in about 12-96 hours. If saccharification is performed partially or completely in transit, saccharification may takc longer.

[0120] It is generally preferred that the tank contents be mixed during saccharification, e.g., usïngjet mixing as described in International App. No. PCT/US2010/035331, filed May 18, 2010, which was published in English as WO 2010/135380 and designated the United States, the full disclosure of which is incorporated by reference herein.

[0121] The addition of surfactants can enhance the rate of saccharification. Examples of surfactants include non-ionîc surfactants, such as a Tween® 20 or Tween® 80 polyethylene glycol surfactants, ionic surfactants, or amphoteric surfactants.

[0122] It is generally preferred that the concentration of the sugar solution resulting from saccharification be relatively high, e.g., greater than 40%, or greater than 50,60,70,80,90 or even greater than 95% by weight. Water may be removed, e.g., by évaporation, to increase the concentration of the sugar solution. This reduces the volume to be shipped, and also inhibits microbial growth in the solution.

[0123] Altcrnatively, sugar solutions of lower concentrations may be used, in which case it may be désirable to add an antimicrobial additivc, e.g., a broad spectrum antibiotic, in a low concentration, e.g., 50 to 150 ppm. Other suitabie antibiotics include amphotericin B, ampicillin, chloramphenicol, ciprofïoxacin, gentamicin, hygromycin B, kanamycin, neomycin, penicillin, puromycin, streptomycîn. Antibiotics will inhibit growth of microorganisms during transport and storage, and can be used at appropriate concentrations, e.g., between 15 and 1000 ppm by weight, e.g., between 25 and 500 ppm, or between 50 and 150 ppm. If desired, an antibiotic can be included even if the sugar concentration is relatively high. Altematively, other additives with anti-microbial ofpreservativc properties may be used. Preferably the antimicrobial additive(s) are food-grade.

[0124] A relatively high concentration solution can be obtained by Iimiting the amount of water added to the biomass material with the enzyme. The concentration can be controlled, e.g., by controlling how much saccharification takes place. For example, concentration can be increased by adding more biomass material to the solution. In order to keep the sugar that is being produced in solution, a surfactant can be added, e.g., one of those discussed above. Solubility can also be increased by increasing the température of the solution. For example, the solution can be maintaincd at a température of40-50°C, 60-80°C, or even higher.

SACCHARIFYING AGENTS [0125] Suitable cellulolytic enzymes include celluloses from specics in the généra Bacillus,

Coprinus, Myceliophthara, Cephalosporium, Scytalidium, Pénicillium, Aspergillus, Psetidomonas, Humicola, Fusarium, Thlelavla.Acremoniunt, Chrysasporium and Trichoderma, especially those produced by a slrain selected from the species Aspergillus (see, e.g., EP Pub.

No. 0 458 162), Humicola Insolens (reclussified as Scytalidium thermophilum, see, e.g., U.S. Pat.

No. 4,435,307), Coprinus cinereus, Fusarium oxysporum, Myceliophthora thermophila, Meripilus giganteus, Thlelavia terrestris, Acremonium sp. (including. but noi limited to, A. persicinttm, A. acremonium, ri. brachypenlttm, ri. dichromospontm, ri. abclavatum, A pinkertoniae, ri. roseogrisetim, ri. incoloratutn, and A. furattim). Preferred strains include Humicola insolens DSM 1800, Fusarium oxysporum DSM 2672, Myceliophthara thermophila

CBS 117.65, Cephalosporium sp. RYM-202, Acremonium sp. CBS 478.94, Acremonium sp. CBS 265.95, Acremonium persiclntim CBS 169.65, Acremonium acremonium AHU 9519, Cephalosporium sp. CBS 535.H, Acremonium brachypenittm CBS 866.73, Acremonium dichrontosporum CBS 683.73, Acremonium abclavatum CBS 311.74, Acremonium pinkertoniae CBS \51.19, Acremonium roseogrisettm CBS 134.56, Acremonium Incoloratum CBS 146.62, and Acremonium furattim CBS 299.70H. Cellulolytic enzymes may also be obtained from

Chrysosporium, preferably a strain of Chrysosporitint lucknowense. Additional strains that can be used include, but are not limited to, Trichoderma (particularly T. viride, T. reesei, and T.

koningiï), alkalophilie Bacillus (see, for example, U.S. Pat No. 3,844,890 and EP Pub. No. 0 458

162), and Streptomyces (see, e.g., EP Pub. No. 0 458 162).

[0126] 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 [0127] In the processes described herein, for example after saccharification, sugars (e.g., glucose and xylose) can be isolated. For example sugars can be isolated by précipitation, crystallization, chromatography (e.g., simulated moving bed chromatography, high pressure chromatography), centrifugation, extraction, any other isolation method known in the art, and combinations thereof.

HYDROGENATION AND OTHER CHEMICAL TRANSFORMATIONS [0128] The processes described herein can include hydrogénation. For example glucose and xylose can be hydrogenated to sorbitol and xylito! respectively. Hydrogénation can be accomplished by use of a catalyst (e.g., Pt/gamma-AIjOj, Ru/C, Raney Nickel, or other catalysts know in the art) in combination with H₂ under high pressure (e.g., 10 to ! 2000 psi). Other types of chemical transformation of the products from the processcs described herein can be used, for example production of organic sugar derived products such (e.g., furfura! and furfüral-derived products). Chemical transformations of sugar derived products are described in U.S. Prov. App. No. 61/667,481, filed July 3,2012, the disclosure of which is incorporated herein by reference in its entirety.

FERMENTATION [0129] The sugars produced by saccharification can be isolated as a final product, or can be fermented to produce other products, e.g., alcohols, sugar alcohols, such as erythritol, or organic acids, e.g., lactic, glutamic or ci trie acids or amino acids.

[0130] Yeast and Zymomonas bacteria, for example, can be used for fermentation or conversion ofsugar(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 Îs from about pH 5 to 6. Typical fermentation limes are about 24 to ! 68 hours (e.g., 24 to 96 hrs) with températures in the range of 20°C to 40®C (e.g., 26°C to 40°C), however thermophilic microorganisms prefer higher températures. [0131] In some embodiments, e.g., when anaérobie organisms are used, at least a portion of the fermentation is conducted in the absence of oxygen, e.g., under a blanket of an inert gas such as Ni, Ar, He, CO2 or mixtures thereof. Additionally, the mixture may hâve a constant purge of an inert gas flowing through the tank during part of or al I of the fermentation. In some cases, anaérobie condition, can be achieved or maintained by carbon dioxide production during the fermentation and no additional inert gas is needed.

[0132] In some embodiments, ail or a portion of the fermentation process can be interrupted before the low molecular weight sugar is completely converted to a product (e.g., éthanol). The intermediate fermentation products include sugar and carbohydrates in high concentrations. The sugars and carbohydrates can be isolated via any means known in the art. These intermediate fermentation products can be used in préparation of food for human or animai consumption.

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

[0133] Jet mixing may be used during fermentation, and in some cases saccharification and fermentation are performed in the same tank.

[0134] Nutrients for the microorganisms may be added during saccharification and/or fermentation, for example the food-based nu trient packages described in U.S. Pat. App. Pub.

2012/0052536, filed July 15,2011, the complété disclosure of which is incorporated herein by reference.

[0135] “Fermentation” includes the methods and products that are disclosed in U.S. Prov. App. No. 61/579,559, filed December 22,2012, and U.S. Prov. App. No. 61/579,576, filed

December 22,2012, the contents of both of which are incorporated by reference herein in their entirety, [0136] Mobile fermentera can be utilized, as described in International App. No. PCT/US2007/074028 (which was filed July 20,2007, was published in English as WO 2008/011598 and designated the United States), the contents of which is incorporated herein in 30 its entirety. Similarfy, the saccharification equipment can be mobile. Further, saccharification and/or fermentation may be performed tn part or entirety during transit.

FERMENTATION AGENTS [0137] The microorganism(s) used in fermentation can be naturally-occum'ng microorganisms and/or en gineered microorganisms. For example, the microorganism can be a bacterium (including, but not limited to, e.g., a cel lulolytic bacterium), a fungus, (including, but not limited to. e.g., a yeast), a plant, a protist, e.g., a protozoa or a fungus-like protest (including, but not limited to, e.g„ a slime mold), or an alga. When the organisms are compatible, mixtures of organisms can be utîlized.

[0138] Suitable fermenting microorganisms hâve the abilîty to convert carbohydrates, such us glucose, fructose, xylose, arabinose, mannose, galactose, oligosaccharides or polysaccharides into fermentation products. Fermenting microorganisms include strains of the genus Saccharomyces spp. (including, but not limited to, S. cerevislae (baker’s yeast), S. distaticus, S. uvarum), the genus Kluyveromyces, (including, but not limited to, K. marxianus, K.fragilis), the genus Candida (including, but not limited to, C. pseudatropicalis, and C. brassicae), Piclda sdpitis (a relative of Candida shehatae), the genus Ciavispara (including, but not limited to, C. lusitaniae and C. opuntiae), the genus Pachysalen (including, but not limited to, P. tannophihts), the genus Bretannamyces (including, but not limited to, e.g., B. clausenii (Philippidis, G. P„ 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and UtilizatÎon, Wyman, C.E., ed., Taylor & Francis, Washington, DC, 179-212)). Other suitable microorganisms include, for example, Zytnamanas mobllis, Clostridium spp. (including, but not limited to, C. thermocellum (Philippidis, 1996, supra), C. saccharobutylacetanlcum, C. saccharobutylicum, C. Puniceum, C. beijemckii, and C. acetobutyiicum), Moniliella pollinis, Maniliella megachiliensls, Lactobacillas spp. Yarrawia lipalytica, Aureobasidtum sp., Trichasparonaldes sp., Trigonapsis variabilis, Trichosparan sp., Maniliellaacetaabiitans sp.,

Typluda variabilis, Candida rnagnoliae, Usiilaginamycetes sp., Pseudozyma tsukubaensis, yeast species of généra Zygosacckaromyces, Debaryomyces, Hansenula and Piclda, and fungi of the dematioid genus Tarula.

[0139] For instance. Clostridium spp. can be used to produce éthanol, butanol, butyric acid, acetic acid, and acétone. Lactobacillas spp., can be used to produce lactice acid.

[0140] Many such microbial strains are publicly available, either commercially or through depositories such as the ATCC (American Type Culture Cbllectron, Manassas, Virginia, USA), the NRRL (Agricultural Research Sevlce Culture Collection, Peoria, Illinois, USA), or the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). to name a few.

[0141] Commercially available yeasts include, for example, Red StartB/Lesaffrc Ethanol Red (available from Red Star/Lesaffre, USA), FALI® (available from Fleischmann’s Yeast, a division of Bums Philip Food Inc., USA), SUPERSTART® (available from Alltech, now Lalemand), GERT STRAND® (available from Gert Strand AB, Sweden) and FERMOL® (available from DSM Specialties).

[0142] 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 [0143] After fermentation, the resulting fluids can be distilled using, for example, a “beer column” to separate éthanol and other alcohols from the majority of water and residual solids.

The vapor exiting the beer column can be, e.g., 35% by weight éthanol and can be fed to a rectification column. A mixture of nearly azeotropic (92.5%) éthanol and water from the rectification column can be purified to pure (99.5%) éthanol using vapor-phase molecular sieves. The beer column bottoms can be sent to the first effect of a three-effect evaporator. The rectification column reflux condenser can provide heat for this first effect After the first effect, 20 solids can be separated using a centrifuge and dried in a rotary dryer. A portion (25%) of the centrifuge effluent can be recycled to fermentation and the rest sent to the second and third evaporator effects. Most of the evaporator condensate can be retumed to the process as fairly clean condensate with a small portion split off to waste water treatment to prevent build-up of low-boiling compounds.

[0144] Other than in the examples herein, or unless otherwise expressly specified, ail of the numerical ranges, amounts, values and percentages, such as those for amounts of materials, elemental contents, times and températures of reaction, ratios of amounts, and others, in the following portion of the spécification and attached claims may be read as if prefaced by the word 30 “about even though the term about may not expressly appear with the value, amount, or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following spécification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the présent invention. At the very least, and not as an attempt to limit the application of the doctrine of équivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0145] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the spécifie examples are reported as precisely as possible. Any numerical value, however, inherently contains errer necessarily resulting from the standard déviation found in its underlying respective 10 testing measurements. Furthermore. when numerical ranges are set forth herein, these ranges are inclusive of the recited range end points (Le., end points may be used). When percentages by weight are used herein, the numerical values reported are relative to the total weight.

[0146] Also, It should be understood that any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, a range of “I to 10 is intended to 15 include all sub-ranges between (and including) the recited minimum value of I and the recited maximum value of 10, that is, having a minimum value equal to or greater than I and a maximum value of equal to or less than 10. The terms one,” a, or an as used herein are intended to include “at least one” or “one or more,” unless otherwise indicated.

EXAMPLES [0147] Materials & Methods [0148] The following procedures and materials were used in the following examples.

[0149] Cell banking: The following Trichiderma reesei strains were banked: ATCC 66589, PC3-7; ATCC 56765, RUT-C30; ATCC 56767, NG-14; ATCC 26921, QM 9414.

[0150] Each cell was rehydrated and propagated in potato dextrose (PD) media at 25°C.

[0151] For production of master cell banks, each strain was rehydrated ovemight in 0.5ml stérile water. To propagate cells. 40 ul of rehydrated cells were used to înoculate potato dextrose agar (PDA) solid medium. Rehydrated cells were also inoculated into 50 ml of PD liquid medium and incubated at 25°C and 200 rpm. After 2 weeks culture in PDA media, spores were resuspended in stérile NaCl (9g/L), 20% glycérol solution, and stored in -80°C freezer for use as a cell bank.

[0152] Protein measurement and cellulase assay: Protein concentration was measured by the Bradford method using bovine sérum albumin as a standard.

[0153] filter paper assay (FPU), cellobase activity and CMC activity was carried out using the IUPAC method (T.K. Chose, PureAppl. Chem. 59:257-68,1987).

[0154] The reaction product (glucose) was analyzed on a YSI7100 Multiparameter

Bioanalytical System (YSI Life Sciences, Yellow Springs, Ohio, USA) or HPLC. [0155] Media: The media included corn steep (2 g/L), ammonium sulfate ( 1.4 g/L), potassium hydroxide (0.8 g/L), Phosphoric acid (85%, 4mL/L), phthalic acid dipotassium sait (5 g/L), magnésium sulfate heptahydrate (0.3g/L), calcium chloride (0.3g/L), ferrous sulfate heptahydrate (5 mg/L), manganèse sulfate mono hydrate (1.6 mg/L), zinc sulfate heptahydrate (5 mg/L) and cobalt chloride hexahydrate (2mg/L). The media is described in Herpoel-Gïmbert et al., Biotechnology for Biofuels, 2008, 1:18.

[0156] Bio-reactor: The freezer stock from the cell banking was used to make the seed culture using the media described above, with 2.5% additional glucose. The seed culture was typically made in a flask using an incubator set at 30°C and 200rpm for 72hrs. Seed culture broth (50mL) was used as an inoculum in the 1L starting medium in a 3L fermenter. In growth phase, 35 g/Lof lactose was added to the medium. The culture conditions were as follows: 27°C, pH 4.8 (with 6M ammonia), air flow 0.5 WM, stirring 500 rpm, and dissolved oxygen (DO) was maintained above 40 % oxygen saturation. In the induction phase, lhe desired inducer 20 (discussed below) was added. During fermentation, Antiform 204 (Sigma) was injected into the culture when the foam reachcd the fermentor head.

[0157] ShakeJlask: In addition to the media described above, for the flask culture. Tris buffer (12.1 g/L), maleic acid (11.06g/L) and sodium hydroxide (2.08g/L) were added. A starter culture was prepared in the media with added glucose. After 3 days of growth, the cell mass was 25 harvested by centrifugation. The cell mass was rc-suspended ln 50 ml of media with the desired inducer. The flasks were placed in a shaker incubator set at an agitation speed of 200 rpm and température of 30“C.

[0158] Example I. Cellulase Performance Test on Paper, Treated Corn Cob and Untreated Com Cob [0159] Various inducers (treated biomass (TBM), untreated biomass (UBM), paper (P) and carboxylmethylcellulose (CMC, Aldrich)) were used to produce enzymes. The biomass (TBM 5 and UBM) was milled com cobcoilected between mesh sizes of 15 and40. Treatmentofthe biomass (UBM) to produce the TBM involved électron bombardment with an électron beam to a total dose of 35 Mrad. The paper was shredded and screened to hâve a nominal particle size smaller than 0.16 inch. The inducer experiments were conducted using shake flasks and PC3-7 and RUT-C30 strains. After 3 days of the growth culture, the harvested cell mass was added to a 10 sériés of shake flasks each containing 50 m! of medium and 1 wt. % of one of the inducers.

[0160] The induction experiment was allowed to proceed for 11 days. The culture supematant was then harvested by centrifugation at 14,500 rpm for 5 minutes, and stored at4°C. [0161] Protein concentration of culture supematant: Using the cell culture grown in the shake flasks and derived from PC3-7, protein concentrations after 11 days were 1.39,1.18, 1.06 15 and 0.26 mg/mL for TBM, UBM, P and CMC respectively. For RUT-C30, the protein concentrations were 1.26, 1.26,1.00 and 0.26 mg/mL for TBM, P, UBM and CMC respectively.

[0162] Cellulase activity: The cellulase activities were assessed and are listed in the table below.

[0163] Table 1. Cellulase activity for different inducers and cell strains.

Inducer	Cell type	FPU (U/mL)	Cellobiase aciivity (U/mL)	FPU (U/mg)	Cellobiase activity (U/mg)
TBM	PC-3-7	0.57	0.47	1.04	0.86
UBM	PC-3-7	0.45	0.39	1.08	0.93
P	PC-3-7	0.57	0.39	0.96	0.66
CMC	PC-3-7	0.06	0.11	0.55	0.99
TBM	RUT-C30	1.02	0.53	1.97	1.03
UBM	RUT-C30	0.72	0.42	1.76	1.03
P	RUT-C30	0.71	0.40	1.31	0.74
CMC	RUT-C30	0.24	0.18	1.77	1.31

[0164] These results show that treated biomass serves to induce enzyme production at a greater rate than untreated biomass.

[0165] Example 2. Enzyme Production ln Different Concentrations ofTBM Inducer [0166] This Example was done using bioreactors. Cell strain RUT-C30 was propagated in the media with 2J % glucose. After 3 days of growth the culture was centrifuged and the cells was re-suspended in 50 ml of media with ), 3,5,7 and 9 wt.% TBM. The protein concentrations 5 and activilies after 11 days of incubation at 27°C and 200 rpm are shown ln the table below.

[0167] Table 2. Amountsof Protein and Enzyme Made With Differing Amounts of Inducer

Inducer amount (wt.%)	Protein (g/L)	FPU (U/mL)	CMC (U/mL)
1	0.7	1.4	1.3
2	1.4	3.1	1.7
5	3.4	6.2	2.6
7	2.9	2.5	1.5
9	1.5	0.6	1.0

[0168] These results show that higher levels of enzymes were produced when the treated biomass (TBM) was added at a rate of 5 %.

[0169] Saccharification of biomass with enzymes: Saccharification of biomass (TBM) using enzymes produced by addition of 2,5 and 7 wt.% treated biomass inducer (TBM) versus a commercial enzyme (Duet™ Accellerase, Genencor) was conducted. The biomass, lOwt. %

TBM, was combined with either 0.25 ml/g of enzyme culture broth or commercial enzyme. The saccharification was carried out at 50°C and 200 rpm in a shaking incubator. After 24 hours the amountofgenerated glucose was measured by YSL The amount ofglucose produced per L of solution and mg of protein is shown in the table below.

[0170] Table 3. Amount of Glucose Produced From Varying Levels of Inducer

Enzyme produced from	Glucose (g/L)	Glucose (g/mg)
2% TBM inducer	4.04	2.31
5% TBM inducer	4.06	1.08
7% TBM inducer	3.02	0.83
Commercial enzyme	14.4	0.50

[0171] Example 3. SDS-PAGE of Enzyme Produced With Treated Biomass [0172] A bioreactor culture was prepared using the method described above except that the mixing was done at 50 rpm rather than 500 rpm. The protein assay showed that 3.4 g/L protein was produced.

[0173] The analysis of the protein using SDS PAGE is shown in FIG. 5. Lane 1 and 5 are molecular weight markers, Lane 2 is a 30 uL load of the protein, Lane 3 is a 40 uL load of the protein, Lane 4 is Duet™ Accelerase enzyme complex (Genencor).

[0174] Example 4. Range ofConditions Tested

Induction	Parameters Tested	Range Tested	Working Range	Best Range
Corn Cob	Particle Size	1400-<5 mm	|400-<5mm	<50mm
	Amount Added	25-133g/L	25-133g/L	tOOg/L
	Timing of Addition	Day 0-3	1-3	Day 1-3
	Frequency of Addition	1,2, and 5	1.2, and 5	1
	Présentation	wet or dry	wet or dry	wet or dry
	Treatment Levels	35	35	35
Lactose	Timing of Addition	Day 3	Day 3	Day 3
	Amount Added	4.7-40g/L/d	4.7-l8.7g/L/d	18.7g/L/d
		continuous	continuous	continuous
	Frequency of Addition	feed	feed	feed

[0175] Any patent, publication, or other disclosure material, in whole or in part, that is said to be incorporated by reference herein is incorporated herein only to the extent that the incorporated material does not conflict with existing définitions, statements, or other disclosure material set forth in this disclosure. As such, and to the extent necessary, the disclosure as 15 explicitly set forth herein supersedes any conflicting material incorporated heretn by reference.

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

[0176] While this invention has been particularly shown and described with référencés to preferred embodiments thereof, it will be understood by those skilled 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 daims.

combining a cellulosic or lignocellulosic biomass, which has been treated to reducc its recalcitrance, with a microorganism, and inducing the production of one or more enzymes by the microorganism by maintaining the microorganîsm-bïomass combination under conditions that allow for the production of the enzyme(s) by the microorganism.

Claims (21)

1. 2. The method of claim I further comprising combining a second cellulosic or lignocellulosic biomass with the miccroorganism-biomass combination.
2. 3. The method of claim 2 wherein the second biomass is lignocellulosic.
3. 4. The method of claim 1,2 or 3, where the biomass has been treated to reduce its recalcitrance by a treatment method selected from the group consisting of; bombardment with électrons, sonication, oxidation, pyrolysis, steam explosion, chemical treatment, mechanical treatment, freeze grinding.
4. 5. The method of daim 4, wherein the treatment method is bombardment with eiectrons.
5. 6. The method of any of the above daims, further comprising mechanically treating the first cellulosic or lignocellulosic biomass to reduce its buik de nsi ty and/or increase its surface

   25 area.
6. 7. The method of any of the above daims, wherein the first cellulosic or lignocellulosic biomass is comminuted before being combined with the microorganism.

   30
7. 8. The method of claim 7, wherein the comminution comprises dry milling.
8. 9. The method of claim 7, wherein the comminution comprises wet miiiing.
9. 10, The method of any of the above daims, wherein the cellulosic or lignocellulosic biomass

   35 has a partie le size of about 30 to 1400 μπι.
10. 11. The method of any ofthe above ciaims wherein the biomass is a lignocellulosic biomass selected from the group consisting of wood, grasses, and agricultural residues.
11. 12. The method of any one of the above ciaims, wherein the cellulosic or lignocellulosic biomass is selected from the group consisting of: paper, paper products, paper waste, paper pulp, pigmented papers, loaded papers, coated papers, fil lcd papers, magazines, printed matter, printer paper, polycoated paper, card stock, cardboard, paperboard, cotton, wood, partide board, forestry wastes, sawdust, aspen wood, wood chips, grasses, switchgrass, miscanthus, cord grass, reed canary grass, grain residues, rice hulls, oat hulls, wheat chaff, barley hulls, agricultural waste, silage, canota straw, wheat straw, barley straw, oat straw, rice straw, jute, hemp, flax, bamboo, sisal, abaca, corn cobs, corn stover, soybean stover, corn fiber, alfalfa, hay, coconut hair, sugar processing residues, bagasse, beet pulp, agave bagasse, algae, seaweed, manure, sewage, offal, agricultural or industrial waste, arracacha, buckwheat, banana, barley, cassava, kudzu, oca, sago, sorghum, potato, sweet potato, tare, yams, beans, favas, lentils, peas, and mixtures of any of these.
12. 13. The method of any one of the above daims, wherein the cellulosic or lignocellulosic biomass comprises a residue of a saccharification or fermentation process.
13. 14. The method of any one of the above ciaims, wherein the microorganism is selected from the group consisting of a fungus, a bacterium, and a yeast.
14. 15. The method of any one ofthe above ciaims, wherein the microorganism Is a s train that produces high levels of ccllulase.
15. 16. The method of any one of the above ciaims, wherein the microorganism is genetically engineered.
16. 17. The method of daim 14 wherein the microorganism is a fungus.
17. 18. The method of any one of the above daims, wherein the microorganism is selected from the group consisting of Trichoderma reesei, and Clostridium thermocellum.
18. 19. The method of claim 18, wherein the T. reesei strain is selected from lhe group consisting of: RUT-NG 14, PC3-7, QM9414 and/or RUT-C30, φ ³⁷
19. 20. The method of any one of the above daims, wherein the cellulosic or lignocellulosîc bîomass is combined with the microorganism when the microorganism is in a lag phase.
20. 21. The method of any one of the above daims, further comprising saccharifying the

    5 cellulosic or lignocellulosîc bîomass, and/or additional cellulosic or lignocellulosîc bîomass, using the enzyme(s).
21. 22. A composition comprising: a liquid medium, a cellulosic or lignocellulosîc bîomass treated to reduce its recalcitrance, a microorganism, and one or more enzymes made by

    10 the microorganism.