NZ719871B2 - Production Of Sugar And Alcohol From Biomass - Google Patents

Abstract

Discloses is a method for making a sugar alcohol comprising: combining a slurry of cellulosic or lignocellulosic biomass that contains one or more sugars with a microorganism; and utilizing jet mixing to agitate the slurry while maintaining a dissolved oxygen level of at least 10% while allowing the microorganism (e.g., species of Moniliella) to ferment a sugar to a sugar alcohol, the jet mixing providing agitation effective to increase production of the sugar alcohol, yielding at least 80 g/L of the sugar alcohol from 300 g/L of the sugar. In a particular embodiment the sugar alcohol is erythritol. microorganism (e.g., species of Moniliella) to ferment a sugar to a sugar alcohol, the jet mixing providing agitation effective to increase production of the sugar alcohol, yielding at least 80 g/L of the sugar alcohol from 300 g/L of the sugar. In a particular embodiment the sugar alcohol is erythritol.

Description

PRODUCTION OF SUGAR AND ALCOHOL FROM BIOMASS by Marshall Medoff, Thomas Craig Masterman, Jaewoong Moon, Aiichiro Yoshida CROSS REFERENCE TO RELATED APPLICATION This application claims the benefit of US. Provisional Application No. 61/579,576, filed on December 22, 2011. The entire disclosure of the above application is incorporated herein by reference.

FIELD OF THE ION The invention pertains to the production of products, e.g., sugar alcohols, e.g., such as erythritol.

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

Many potential lignocellulosic feedstocks are available today, including agricultural 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 t. The ure comprises lline cellulose fibrils embedded 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, naturally-occurring cellulosic materials often have low yields ive to theoretical yields) when contacted with hydrolyzing enzymes. Lignocellulosic biomass is even more recalcitrant to enzyme attack. Furthermore, each type of lignocellulosic biomass has its own specific ition of cellulose, hemicellulose and lignin.

While a number of methods have been tried to extract structural carbohydrates from lignocellulosic biomass, they are either are too expensive, produce too low a yield, leave rable als in the ing product, or simply e the sugars.

Saccharides from ble biomass s could become the basis of chemical and fuels ries by replacing, supplementing or substituting petroleum and other fossil feedstocks. However, techniques need to be developed that will make these monosaccharides available in large quantities and at acceptable purities and prices.

SUMMARY OF THE INVENTION A method is provided for making a sugar alcohol from a cellulosic or lignocellulosic biomass that ns one or more sugars that includes combining the cellulosic or lignocellulosic biomass with a microorganism that is capable of converting at least one of the sugars to a sugar alcohol, and maintaining the microorganism-biomass ation under conditions that enable the microorganism to t at least one of the sugars to the sugar alcohol. In some implementations, the method includes: ing a cellulosic or lignocellulosic biomass, wherein the cellulosic or lignocellulosic biomass contains one or more sugars; providing a microorganism that is capable of converting at least one of the sugars to a sugar alcohol; combining the cellulosic or lignocellulosic biomass with the microorganism, thereby producing a microorganism-biomass combination; and maintaining the microorganism-biomass combination under conditions that enable the microorganism to convert at least one of the sugars to a sugar alcohol; thereby making a sugar alcohol from a cellulosic or lignocellulosic biomass. The cellulosic or lignocellulosic biomass can be saccharified. [0008A] The present ion also provides a method for making sugar alcohols, the method comprising: combining a slurry of lignocellulosic biomass that contains a sugar solution comprising glucose and xylose derived from the biomass with a microorganism selected from the group consisting of CBS 461.67 (Moniliella pollinis) and CBS 567.85 (Moniliella megachiliensis), the recalcitrance of the or ellulosic biomass having been reduced by bombardment with electrons; and utilizing the microorganism to ferment the sugar solution to produce erythritol, while providing aeration from 0.3 to 1.0 VVM and mixing the slurry with a jet mixer.

Any of the methods provided herein can include reducing the recalcitrance of the osic or lignocellulosic biomass to saccharification prior to combining it with the microorganism. The recalcitrance can be reduced by a treatment method selected from the group consisting of: bombardment with electrons, tion, oxidation, pyrolysis, steam explosion, chemical treatment, mechanical treatment, and freeze ng. The treatment method can be bombardment with electrons.

[Text continued on page 3] Any of the methods provided herein can also include mechanically treating the cellulosic or ellulosic s to reduce its bulk density and/or increase its surface area.

For instance, the cellulosic or lignocellulosic biomass can be comminuted, for instance, it can be dry , or it can be wet .

In any of the methods provided herein, the biomass can be rif1ed with one or more cellulases. Any of the methods can also include separating one or more sugars prior to combining the cellulosic or lignocellulosic biomass with the microorganism, or the methods can include concentrating the one or more sugars prior to combining the cellulosic or lignocellulosic biomass with the microorganism. The methods can also include both concentrating and separating one or more sugars prior to combining the cellulosic or lignocellulosic biomass with the microorganism. The saccharif1ed biomass can be adjusted to have an initial glucose concentration of at least 5 wt%. The saccharif1ed biomass can also be purified, for instance, by the removal of metal ions.

Any of the methods disclosed herein can also include culturing the microorganism in a cell growth phase before combining the cellulosic or lignocellulosic biomass with the microorganism.

In any of the methods provided herein, the sugar alcohol can be glycol, glycerol, erythritol, threitol, ol, xylitol, ribitol, mannitol, sorbitol, galactitol, iditol, inositol, volemitol, t, ol, lactitol, maltotriitol, maltotetraitol, or ycitol.

The microorganism can be Monilz'ella pollim's, Monilz'ella megachz'lz'ensz’s, Yarrowz'a lz'polytl'ca, Aureobasidium 519., Trichosporonoides 519., Trigonopsz's variabilis, Trichosporon sp., ellaacetoabutans, Typhula variabilis, Candida magnoliae, Ustilaginomycetes, Pseudozyma tsukubaensz's; yeast species of genera Zygosaccharomyces, Debaryomyces, Hansenula and Pichia, or fiangi of the dematioid genus Torula. The microorganism can be a species of Monilz'ella, such as M. pollim's, for instance, strain CBS , or M. megachilz’ensz’s, strain CBS 567.85.

In any of the 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, oard, 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, buckwheat, banana, , cassava, kudzu, oca, sago, sorghum, potato, sweet potato, taro, yams, beans, favas, lentils, peas, or mixtures of any of these.

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

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

Cellulosic substrate (A) is converted by endocellulase (i) to cellulose (B), which is converted by exocellulase (ii) to cellobiose (C), which is converted to glucose (D) by cellobiase (beta- glucosidase) (iii). is a flow diagram illustrating conversion of a biomass feedstock to one or more products. Feedstock is physically 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 transported (230) to a manufacturing plant (e.g., by pipeline, railcar) (or if saccharification is performed en route, the feedstock, enzyme and water is orted), the saccharified ock is ocessed to produce a d 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 lignin content (201) and g or adjusting process ters (205). Saccharifying the feedstock (220) can be d by mixing the feedstock with medium and the enzyme (221).

DETAILED DESCRIPTION This invention s to methods of processing biomass feedstock materials (e.g., biomass als or biomass-derived materials such as cellulosic and lignocellulosic materials) to obtain sugar alcohols such as erythritol ((2R,3S)-butane-l,2,3,4-tetraol), or isomers, or es thereof.

OIIIIIII-I In some instances, the recalcitrance of the feedstock is reduced prior to saccharif1cation. In some cases, reducing the recalcitrance of the feedstock includes treating the feedstock . The treatment can, for example, be radiation, e.g., on beam radiation, sonication, pyrolysis, ion, steam explosion, chemical treatment, or combinations of any of these.

In some implementations, the method also includes mechanically treating the feedstock before and/or after reducing its recalcitrance. Mechanical treatments include, for example, cutting, milling, e.g., hammermilling, pressing, grinding, shearing and chopping.

Mechanical treatment may reduce the bulk density of the feedstock and/or increase the surface area of the feedstock. In some embodiments, after mechanical treatment the material has a bulk density of less than 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 0.025 g/cm3. Bulk density is determined using ASTM Dl895B. Under some circumstances, mechanical treatments can remove or reduce itrance.

In one aspect, the invention features a method that includes contacting a sugar, produced by saccharifying a cellulosic or lignocellulosic feedstock with a microorganism to produce a product, such as a sugar alcohol e.g., itol. Other products e, for e, citric acid, lysine and glutamic acid.

In some implementations, the microorganism includes Monilz'ella pollim's, Yarrowz'a lz'polytl'ca, Aureobasidium 519., sporonoides 519., Trigonopsz's variabilis, Trichosporon sp., Moniliellaacetoabutans, a variabilis, Candida magnoliae, Ustilaginomycetes, Pseudozyma tsukubaensz's; yeast species of genera Zygosaccharomyces, Debaryomyces, Hansenula and Pichia; and fungi of the dematioid genus .

In some implementations, the contacting step includes a dual stage process, sing a cell growth step and a fermentation step. Optionally, the fermentation is performed using a glucose solution having an initial glucose concentration of at least 5 wt.% at the start of the fermentation. Furthermore, the glucose solution can be diluted after fermentation has begun.

As shown in for example, during saccharif1cation a cellulosic substrate (A) is initially yzed by endoglucanases (i) at random locations producing oligomeric intermediates (e.g., cellulose) (B). These ediates are then substrates for exo-splitting glucanases (ii) such as cellobiohydrolase to produce cellobiose from the ends of the cellulose r. Cellobiose is a water-soluble l,4-linked dimer of glucose. y cellobiase (iii) s cellobiose (C) to yield glucose (D). Therefore, the endoglucanases are particularly effective in attacking the lline portions of cellulose and sing the effectiveness of exocellulases to produce cellobiose, which then requires the city of the cellobiose to produce e. 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 strains of the cellulolytic filamentous fungus Trichoderma reesez’. For example, ielding 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 "Selective Screening Methods for the Isolation of High Yielding Cellulase Mutants of Trichoderma reesez’," Montenecourt, BS. and Everleigh, D.E., Adv. Chem. Ser. 18 1, 289-301 (1979), the full sure of which is incorporated herein by reference. Other ase-producing microorganisms may also be used.

As shown in a process for manufacturing a sugar 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 filrther reduce its recalcitrance (210), then saccharifying the feedstock, using the enzyme complex, to form a sugar solution (220). Optionally, the method may also include transporting, e.g. truck or barge, the solution (or the feedstock, enzyme and water, if , by pipeline, railcar, rif1cation is performed en route) to a cturing plant (230). In some cases the saccharif1ed feedstock is further bioprocessed (e.g., fermented) to produce a desired product e.g., alcohol (240). This resulting t may in some implementations be processed r, e.g., by distillation (250), to produce a final product. One method of ng the recalcitrance of the feedstock is by electron bombardment of the feedstock. If d, 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 disclosure of which is incorporated herein by reference. Saccharifying the feedstock (220) can also be modified by mixing the ock with medium and the enzyme (221).

In some cases, the feedstock is boiled, steeped, or cooked in hot water prior to saccharification, as described in US. Serial No. 13/276,192, filed October 18, 2011.

The processes described above can be lly 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 rail car, tanker truck, or in a supertanker or the hold of a ship. Mobile fermenters can be utilized, as described in US. Pat.

App. Pub. 064746 A1, published on March 18, 2010, the entire sure of which is incorporated by nce herein.

It is generally preferred that the tank and/or fermenter contents be mixed during all or part of the process, e.g., using jet mixing as described in US. Pat. App. Pub. 2010/0297705 A1, filed May 18, 2010 and published on November 25, 2012, US. Pat. App. Pub. 2012/0100572 A1, filed November 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 filll disclosures of which are incorporated by reference .

The addition of additives such as e.g., surfactants or nutrients, can e the rate of saccharification. Examples of surfactants include non-ionic surfactants, such as a Tween® 20 or Tween® 80 polyethylene glycol surfactants, ionic tants, or amphoteric surfactants.

One or more useful products may be produced. For example glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, ol, galactitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, and polyglycitol can be produced by fermentation. In addition, butyric acid, gluconic acid and citric acid also can be produced.

WO 96693 In some embodiments, polyols can be made by fermentation, including monomeric polyols such as glycerin, pentaerythritol, ethylene glycol, and sucrose. These can be built up into polymeric polyols such as polyether polyols.

In some embodiments, the optionally mechanically and/or physically treated feedstock can be combined with an enzyme complex for saccharif1cation and is also combined with an organism that ferments at least a part of the released sugars to a sugar alcohol. The sugar alcohol is then ed from other products and non-fermented material such as solids, un- fermentable sugars and cellular debris.

The optionally mechanically and/or physically treated feedstock can also be ed with an enzyme complex for saccharif1cation and after the saccharif1cation is at least partially completed, the mixture is combined with an organism that es sugar alcohols.

The conditions for saccharif1cation (e.g., temperature, agitation, aeration) can be different than the conditions for tation. The optimum pH for fermentation is generally from about pH 4 to 6. l fermentation times are about 24 to 120 hours with temperatures in the range of °C to 40°C, e.g., 25°C to 30°C. Fermentation is typically done with aeration using a sparging tube and an air and/or oxygen supply to maintain the dissolved oxygen level above about 10% ( e.g., above about 20%). The saccharification and fermentation can be in the same or different reactor/vessel. The sugar alcohol is then isolated. As discussed above, the fermentation can be performed during a transportation process.

Generally, a high initial sugar tration at the start of fermentation favors the production of sugar alcohols. Accordingly, the saccharified feedstock solution can be concentrated prior to combination with the organism that produces sugar alcohols to se the glucose level of the solution. Concentration can be done by any desired technique. For example, concentration can be by heating, cooling, centrifugation, reverse osmosis, tography, precipitation, crystallization, evaporation, adsorption and combinations thereof. Preferably concentration is done by ation of at least a portion of the liquids from the saccharif1ed feedstock. Concentration is preferably done to increase the glucose t to greater than about wt%, e.g., greater than 10 wt.%, greater than 15 wt.%, greater than 20 wt.%, greater than 30 wt.%, greater than 40 wt.% or even greater than 50 wt.%. The t from the fermentation is then isolated.

The saccharif1ed feedstock can also be purified before or after concentration.

Purification is preferably done to increase the glucose content to greater than about 50 wt.% of all components other than water (e.g., greater than about 60wt.%, greater than about 70 wt.%, greater than about 80 wt.% than about 90 wt.% and even greater than about 99wt.%). , greater Purification can be done by any desired technique, for e, by heating, g, centrifugation, reverse osmosis, chromatography, precipitation, crystallization, evaporation, adsorption or combinations of any of these.

In some implementations the fermentation is tage, with a cell growth phase and a product production phase. In the growth phase, conditions are selected to optimize cell growth, while in the production phase conditions are selected to optimize production of the desired fermentation products. Generally, low sugar levels (e.g., between 0.1 and 10 wt.% ,between 0.2 and 5 wt.%) in the growth medium favor cell , and high sugar levels (6.g. than 5 , greater wt.%, r than about 10 wt.%, greater than 20 wt.%, greater than 30 wt.%, greater than 40 wt.%) in the fermentation medium favor product production. Other conditions can be optionally modified in each stage, for example, temperature, agitation, sugar levels, nutrients and/or pH.

Monitoring of conditions in each stage can be done to ze the process. For example, grth can be monitored to e an optimum density, e.g., about 50 g/L (e.g., greater than 60 g/L, greater than 70 g/L or greater than about 75 g/L), and a concentrated saccharified solution can be added to trigger the onset of t formation. Optionally, the process can be optimized, for example, by monitoring and adjusting the pH or oxygenation level with probes and automatic feeding to control cell growth and product formation. Furthermore, other nutrients can be controlled and monitored to optimized the process (e.g., amino acids, vitamins, metal ions, yeast extract, vegetable extracts, peptones, carbon sources and proteins).

Dual-stage fermentations are described in Biotechnologicalproduction oferythritol and its applications, Hee-Jung Moon et al., Appl. Microbiol. Biotechnol. (2010) 86: 025.

While generally a high initial concentration of glucose at the start of the fermentation favors erythritol production, if this high concentration is maintained too long it may be detrimental to the organism. A high initial e concentration can be ed by concentrating glucose during or after saccharification as discussed above. After an initial tation time to allow the start of fermentation, the fermentation media is diluted with a suitable diluent so that the e level is brought below about 60 wt.% (e.g., below about 50 wt.%, below about 40 wt.%).

The diluent can be water or water with additional components such as amino acids, vitamins, metal ions, yeast extract, vegetable ts, peptones, carbon sources and proteins.

BIOMASS ALS As used herein, the term ss materials" includes lignocellulosic, cellulosic, starchy, and microbial materials.

Lignocellulosic materials include, but are not limited to, wood, particle board, ry wastes (e.g., sawdust, aspen wood, wood chips), grasses, (e.g., switchgrass, miscanthus, cord grass, reed canary grass), grain residues, (e.g., rice hulls, oat hulls, wheat chaff, barley hulls), agricultural waste (e.g., silage, canola straw, wheat straw, barley straw, oat straw, rice straw, jute, hemp, flax, bamboo, sisal, abaca, corn cobs, corn stover, soybean stover, corn fiber, alfalfa, hay, coconut hair), sugar processing residues (e.g., bagasse, beet pulp, agave e), , algae, seaweed, , 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 disperse 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 source, 6.g. urea or ammonia) are required during fermentation of comcobs or cellulosic or ellulosic materials containing significant amounts of comcobs.

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

Cellulosic materials include, for example, paper, paper products, paper waste, paper pulp, pigmented papers, loaded , coated papers, filled papers, magazines, printed matter (e. g., books, catalogs, manuals, labels, calendars, greeting cards, brochures, prospectuses, newsprint), r paper, ated paper, card stock, cardboard, paperboard, materials having a high oc-cellulose content such as cotton, and mixtures of any of these. For example paper products as described in US. App. No. ,365 ("Magazine Feedstocks" by Medoff et al., filed ry 14, 2012), the fill disclosure of which is incorporated herein by reference.

Cellulosic 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 starch, or a material that includes starch, such as an edible food product or a crop. For example, the starchy material can be arracacha, buckwheat, , barley, cassava, kudzu, oca, sago, sorghum, regular household potatoes, sweet potato, taro, yams, or one or more beans, such as favas, lentils or peas. Blends of any two or more starchy materials are also starchy materials. Mixtures of starchy, cellulosic and or lignocellulosic materials can also be used. For e, 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 s described herein.

Microbial als e, 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 ates, amoeboids, ciliates, and sporozoa) and plant protists (e.g., algae such alveolates, chlorarachniophytes, monads, euglenids, glaucophytes, haptophytes, red algae, nopiles, and viridaeplantae). Other examples include seaweed, plankton (e.g., macroplankton, mesoplankton, microplankton, nanoplankton, picoplankton, and femptoplankton), phytoplankton, bacteria (e.g., gram positive bacteria, gram ve bacteria, and extremophiles), yeast and/or mixtures of these. In some ces, 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. Alternatively or in on, microbial biomass can be obtained from culture systems, e.g., large scale dry and wet culture and tation systems.

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

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

Furthermore, the plants can have had genetic material removed, modified, silenced and/or added with respect to the wild type variety. For example, genetically modified plants can be produced by recombinant DNA methods, where genetic modifications include introducing or modifying WO 96693 specific genes from parental varieties, or, for e, by using transgenic breeding wherein a specific gene or genes are introduced to a plant from a different s 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 be d by a variety of ways including ng the plant or seeds with, for example, chemical mutagens (e.g., using alkylating agents, epoxides, alkaloids, peroxides, formaldehyde), irradiation (e.g., X-rays, gamma rays, neutrons, beta particles, alpha particles, protons, deuterons, UV radiation) and temperature shocking or other external stressing and subsequent selection techniques. Other methods of providing modified genes is through error prone PCR and DNA shuffling followed by insertion of the desired modified DNA into the d plant or seed. Methods of introducing the desired genetic variation in the seed or plant include, for example, the use of a bacterial carrier, biolistics, calcium phosphate precipitation, electroporation, gene splicing, gene silencing, lipofection, microinjection and viral carriers. Additional genetically d als 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 bed herein can be practiced with mixtures of any s materials described herein.

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

The biomass can also be red 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 y materials, for example cellulosic or lignocellulosic feedstocks that have been physically pretreated 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 ing cylinder ofknown volume 2012/071083 with a sample and obtaining a weight of the sample. The bulk density is calculated by ng 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 densif1ed, for e, by methods described in US.

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

In some cases, the pre-treatment processing includes screening of the biomass al. 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 als can be re- processed, for example by comminuting, or they can simply be removed from processing. In another configuration material that is larger than the perforations is irradiated and the smaller material is removed by the screening process or recycled. In this kind of a configuration, the conveyor itself (for example a part of the conveyor) can be perforated or made with a mesh. For e, 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 , for example by an operator or mechanoid (e.g., a robot ed with a color, reflectivity or other sensor) that removes unwanted material. ing can also be by magnetic screening wherein a magnet is disposed near the conveyed material and the magnetic material is removed magnetically.

Optional eatment processing can include heating the material. For example a n of the conveyor can be sent through a heated zone. The heated zone can be created, for example, by IR radiation, microwaves, combustion (e.g., gas, coal, oil, biomass), ive heating and/or inductive coils. The heat can be applied from at least one side or more than one side, can be continuous or periodic and can be for only a portion of the material or all the material. For example, a portion of the conveying trough can be heated by use of a heating jacket. Heating can be, for example, for the purpose of drying the material. In the case of drying the material, this can also be facilitated, with or without heating, by the movement of a gas (6.g. air, oxygen, nitrogen, He, C02, Argon) over and/or through the biomass as it is being ed. ally, pre-treatment processing can include cooling the material. Cooling material is described in US Pat. No. 7,900,857 to Medoff, the disclosure of which in incorporated herein by reference. For example, cooling can be by supplying a cooling fluid, for example water (6.g. with glycerol), or nitrogen (e.g. to the bottom of the conveying , , liquid nitrogen) trough. Alternatively, a cooling gas, for example, chilled en can be blown over the biomass materials or under the conveying .

Another optional pre-treatment processing method can include adding a al 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. 2010/0105119 A1 (filed r 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 als that can be added include acids and bases. Other materials that can be added are oxidants (e.g., peroxides, chlorates), polymers, polymerizable monomers (e.g., containing unsaturated bonds), water, sts, enzymes and/or organisms. Materials can be added, for example, in pure form, as a solution in a solvent (e.g., water or an organic t) and/or as a solution. In some cases the solvent is volatile and can be made to evaporate e.g., by heating and/or blowing gas as previously described. The added material may form a uniform coating on the biomass or be a homogeneous mixture of different components (e.g., biomass and additional material). The added al can modulate the subsequent irradiation step by sing the efficiency of the irradiation, damping the irradiation or ng 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 fiarther downstream processing. The added material may help in conveying the material, for example, by lowering dust levels.

Biomass can be red to the conveyor by a belt or, a pneumatic conveyor, a screw conveyor, a hopper, 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 or using an enclosed material distribution system to help maintain a low oxygen atmosphere and/or control dust and fines. Lofted or air ded biomass fines and dust are rable 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 ess between about 0.03 12 and 5 inches (e.g., between about 0.0625 and 2.000 inches, n about 0. 125 and 1 inches, between about 0. 125 and 0.5 inches, between about 0.3 and 0.9 inches, between about 0.2 and 0.5 inches between about 0.25 and 1.0 inches, between about 0.25 and 0.5 , 0.100 +/- 0.025 inches, 0.l50 --/- 0.025 inches, 0.200 --/- 0.025 inches, 0.250 --/- 0.025 inches, 0.300 --/- 0.025 inches, 0.350 --/- 0.025 inches, 0.400 --/- 0.025 inches, 0.450 --/- 0.025 inches, 0.500 --/- 0.025 inches, 0.550 --/- 0.025 inches, 0.600 --/- 0.025 , 0.700 --/- 0.025 inches, 0.750 --/- 0.025 inches, 0.800 --/- 0.025 inches, 0.850 --/- 0.025 inches, 0.900 --/- 0.025 inches, 0.900 --/- 0.025 inches.

Generally, it is preferred to convey the material as quickly as possible through the electron beam to maximize throughput. For e 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 about 20 ft/min to e a useful irradiation dosage, at 50 mA the conveyor can move at about ft/min to e approximately the same irradiation dosage.

After the biomass material has been conveyed through the radiation zone, optional post-treatment processing can be done. The optional post-treatment processing can, for example, be a process described with respect to the pre-irradiation processing. For example, the biomass can be ed, heated, , and/or combined with additives. Uniquely to post-irradiation, quenching of the radicals can occur, for example, ing of radicals by the addition of fluids or e.g., oxygen, nitrous oxide, ammonia, liquids), using pressure, heat, and/or the addition of radical scavengers. For example, the biomass can be conveyed out of the enclosed conveyor and exposed to a gas (e.g., oxygen) where it is quenched, forming caboxylated groups. In one embodiment the biomass is exposed during irradiation to the reactive gas or fluid. Quenching of biomass that has been irradiated is described in 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 materials, such as by comminution, e.g, cutting, grinding, shearing, pulverizing or ng. 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 s material, se the surface area of the biomass material and/or decrease one or more dimensions of the s material.

Alternatively, or in addition, the ock material can first be physically treated by one or more of the other physical treatment methods, 6.g. chemical treatment, ion, tion, ion, pyrolysis or steam explosion, and then mechanically treated. This sequence can be advantageous since als 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 ionizing radiation using a conveyor as described herein and then mechanically treated. Chemical treatment can remove some or all of the lignin (for example al pulping) and can partially or completely hydrolyze the material. The methods also can be used with pre-hydrolyzed material. The methods also can be used with material that has not been pre hydrolyzed The methods can be used with mixtures of hydrolyzed and non-hydrolyzed materials, for example with about 50% or more non-hydrolyzed material, with about 60% or more non- hydrolyzed material, with about 70% or more non-hydrolyzed material, with about 80% or more non-hydrolyzed material or even with 90% or more non-hydrolyzed al.

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

Methods of mechanically treating the biomass material include, for example, milling or grinding. Milling 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 exemplary grinders include stone grinders, pin grinders, coffee rs, and burr rs. Grinding or milling may be provided, for example, 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 attrition milling. Suitable mechanical treatments further include any other technique that continues the disruption of the internal ure of the material that was ted by the previous processing steps.

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

The bulk density of feedstocks can be controlled (e.g., increased). In some situations, it can be desirable to e a low bulk density material, 6.g. the material (e.g., , by densifying densification can make it easier and less costly to transport to another site) and then reverting the material to a lower bulk density state (e.g., after transport). The material can be densified, for example from less than about 0.2 g/cc to more than about 0.9 g/cc (e.g., less than about 0.3 to more than about 0.5 g/cc, less than about 0.3 to more than about 0.9 g/cc, less than about 0.5 to more than about 0.9 g/cc, less than about 0.3 to more than about 0.8 g/cc, less than about 0.2 to more than about 0.5 g/cc). For example, the material can be densified by the methods and equipment disclosed in US. Pat. No. 7,932,065 to Medoff and International ation No. WO 2008/073186 (which was filed October 26, 2007, was published in English, and which designated the United States), the filll disclosures of which are orated herein by reference.

Densified 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 densified.

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

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

The first fibrous material is passed through a first screen, e.g., having an average opening size of 1.59 mm or less (1/16 inch, 0.0625 inch), provide a second fibrous material. If desired, 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., l/4- to l/2-inch wide, using a shredder, e.g., a counter-rotating screw er, such as those manufactured 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 described herein, such as ical treatment, chemical treatment, irradiation, sonication, ion, pyrolysis or steam explosion. Treatment methods can be used in combinations of two, three, four, or even all of these technologies (in any order). When more than one treatment method is used, the methods can be applied at the same time or at different times. Other processes that change a molecular structure of a biomass feedstock may also be used, alone or in combination with the processes disclosed herein.

Mechanical ents that may be used, and the characteristics of the mechanically d biomass materials, are described in fiarther detail in US. Pat. App. Pub. 2012/0100577 Al, filed r 18, 2011, the fill disclosure of which is hereby incorporated herein by reference.

ENT 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 organic material which functions as input to filrther processing steps and/or sequences. le bombardment can reduce the molecular weight and/or llinity of feedstock. In some embodiments, energy deposited in a material that releases an electron from its atomic orbital can be used to treat the materials. The bombardment may be provided by heavy charged particles (such as alpha particles or protons), ons (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 combination, in any order, or concurrently of these treatments may be utilized. In another approach, electromagnetic radiation (e.g., produced using electron beam emitters) can be used to treat the feedstock.

Each form of energy ionizes the biomass via particular interactions. Heavy charged particles primarily ionize matter via Coulomb ring; fiarthermore, these ctions produce energetic ons 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 isotopes of bismuth, polonium, astatine, radon, francium, radium, several actinides, such as actinium, thorium, m, ium, curium, califomium, americium, and plutonium.

When particles are utilized, they can be neutral (uncharged), positively charged or negatively charged. When charged, the charged particles can bear a single positive or negative charge, or multiple charges, e.g., one, two, three or even four or more charges. In instances in which chain scission is desired, positively d 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., l, 2, 3, 4, 5, 10, 12 or 15 atomic units. Accelerators used to accelerate the particles can be electrostatic DC, electrodynamic DC, RF linear, magnetic ion linear or continuous wave. For example, ron type accelerators are available from IBA (Ion Beam Accelerators, Louvain-la-Neuve, Belgium), such as the RhodotronTM system, while DC type accelerators are available from RDI, now IBA Industrial, such as the DynamitronTM. Ions and ion accelerators are discussed in Introductory Nuclear Physics, Kenneth S. Krane, John Wiley & Sons, Inc. (1988), Krsto Prelec, FIZIKA B 6 (1997) 4, 177-206; Chu, William T., "Overview of Light-Ion Beam Therapy", Columbus-Ohio, ICRU-IAEA g, 18-20 Mar. 2006; Iwata, Y. et al., "Altemating-Phase- Focused IH-DTL for Ion l Accelerators", dings of EPAC 2006, Edinburgh, Scotland; and Leitner, C. M. et al., "Status of the Superconducting ECR Ion Source Venus", Proceedings of EPAC 2000, Vienna, Austria.

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

In some instances when chain scission is desirable and/or polymer chain fianctionalization is desirable, les heavier than ons, such as protons, helium nuclei, argon ions, silicon ions, neon ions, carbon ions, phosphorus ions, oxygen ions or nitrogen ions can be utilized. 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 performed. 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 iodine, , technetium, and iridium. Alternatively, an electron gun can be used as an electron source via thermionic emission.

Electromagnetic ion interacts via three ses: photoelectric absorption, Compton scattering, and pair production. The dominating interaction is ined by the energy of the incident radiation and the atomic number of the material. The summation of interactions contributing to the ed radiation in cellulosic material can be expressed by the mass tion coefficient.

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

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

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

Sources for ultraviolet radiation include deuterium or cadmium lamps.

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

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

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

TREATMENT OF BIOMASS MATERIAL -- ELECTRON BOMBARDMENT The feedstock may be d with electron bombardment to modify its structure and thereby 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 red, because it provides very high throughput and because the use of a relatively low voltage/high power electron beam device eliminates the need for ive te vault shielding, as such devices are "self-shielded" and provide a safe, efficient s. 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 capital expenditure and often allowing an existing manufacturing facility to be used without expensive modification. on beam accelerators are ble, for example, from IBA (Ion Beam Applications, Louvain-la-Neuve, Belgium), Titan Corporation (San Diego, California, USA), and NHV Corporation (Nippon High Voltage, Japan).

Electron bombardment may be performed using an electron beam device that has a l energy of less than 10 MeV, e.g., less than 7 MeV, less than 5 MeV, or less than 2 MeV, agjmmdmm05mL5M&hmmamm08mL8M$meMMM07mlM&Lmfimn about 1 to 3 MeV. In some implementations the l energy is about 500 to 800 keV.

The electron 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 increases the uniformity of the dose through the thickness of the layer of material.

In some implementations, it is desirable to cool the material during on 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 y 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., r than about 0.5, 0.75, l, 1.5, 2, 5, 7, 10, l2, 15, or even greater than about 20 Mrad per second, e.g., about 0.25 to 2 Mrad per . Higher dose rates generally require higher line speeds, to avoid thermal osition 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, on bombardment is performed until the material receives a total dose of at least 0.5 Mrad, e.g., at least 5, 10, 20, 30 or at least 40 Mrad. In some embodiments, the treatment is performed until the material receives a dose of from about 0.5 Mrad to about 150 Mrad, about 1 Mrad to about 100 Mrad, about 2 Mrad to about 75 Mrad, 10 Mrad to about 50 Mrad, e.g., about 5 Mrad to about 50 Mrad, from about 20 Mrad to about 40 Mrad, about 10 Mrad to about 35 Mrad, or from about 25 Mrad to about 30 Mrad. In some entations, 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 ass can in some cases cause thermal ation of the feedstock material.

Using multiple heads as discussed above, the material can be treated in multiple , 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 overheating of the material and also increases dose uniformity through the thickness of the material. In some implementations, the al 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 accelerated to, for example, a speed of greater than 75 percent of the speed of light, e.g., greater than 85, 90, 95, or 99 percent of the speed of light.

In some embodiments, any processing bed herein occurs on lignocellulosic material that remains dry as acquired or that has been dried, e.g., using heat and/or reduced re. 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 percent relative humidity.

Electron dment can be applied while the cellulosic and/or lignocellulosic material is d to air, oxygen-enriched air, or even oxygen itself, 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 distance from the beam source is optimized to ze reactive gas formation, e.g., ozone and/or oxides of nitrogen.

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

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

The effectiveness in changing the molecular/supermolecular structure and/or reducing the recalcitrance of the s depends on the on energy used and the dose d, while exposure time depends on the power and dose.

In some embodiments, the treatment (with any electron source or a combination of sources) is performed until the material receives a dose of at least about 0.05 Mrad, e.g., at least about 0.1, 0.25, 0.5, 0.75, 1.0, 2.5, 5.0, 7.5, 10.0, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100,125, 150, 175, or 200 Mrad. In some embodiments, the treatment is performed until the material receives a dose of between 0 Mrad, 1-200, 5-200, , 5-150, 5-100, 5-50, 5-40, 10-50, -75, 15-50, 20-35 Mrad.

In some embodiments, the treatment is performed at a dose rate of between 5.0 and 1500.0 kilorads/hour, e.g., between 10.0 and 750.0 kilorads/hour or between 50.0 and 350.0 ds/hours. In other embodiments the treatment is performed at a dose rate of between 10 and 10000 kilorads/hr, between 100 and 1000 kilorad/hr, or between 500 and 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 emission and accelerated through an accelerating ial. An electron gun generates electrons, rates 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 n, greater than about 6 million, greater than about 7 million, greater than about 8 million, greater than about 9 million, or even greater than 10 million volts) and then scans them ically 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 electron beam is useful for increasing the irradiation surface when irradiating materials, e.g., a biomass, that is conveyed through the scanned beam. Scanning the electron beam also distributes the thermal load homogenously on the window and helps reduce the foil window rupture due to local heating by the electron beam. Window foil rupture is a cause of significant down-time due to uent 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, thermionic emission s, 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 complete disclosure of which is incorporated herein by reference.

A beam of electrons can be used as the ion 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 nment, and less confinement equipment. Electron beams can also have high electrical efficiency (e.g., 80%), allowing for lower energy usage ve to other radiation methods, which can ate into a lower cost of operation and lower greenhouse gas emissions corresponding to the smaller amount of energy used. on beams can be ted, e.g., by electrostatic generators, cascade generators, transformer generators, low energy accelerators with a scanning system, low energy accelerators with a linear cathode, linear accelerators, and pulsed rators. ons can also be more efficient at causing changes in the molecular structure of biomass materials, for example, by the mechanism of chain scission. In on, ons having energies of 05-10 MeV can penetrate low density als, such as the biomass materials described herein, e.g., materials having a bulk density of less than 0.5 g/cm3, and a depth of 03-10 cm. Electrons as an ionizing radiation source can be useful, e.g., for relatively thin piles, layers or beds of materials, e.g., less than about 0.5 inch, e.g., less than about 0.4 inch, 0.3 inch, 0.25 inch, or less than about 0.1 inch. In some embodiments, the energy of each electron of the electron beam is from about 0.3 MeV to about 2.0 MeV on 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 r 18, 2011, the entire sure of which is herein orated by reference.

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

Tradeoffs in considering electron beam irradiation device power specifications include cost to operate, capital costs, depreciation, and device footprint. Tradeoffs in considering exposure dose levels of 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, available sweep widths of 0.5 m, 1 m, 2 m or more are ble. The ng beam is red in most embodiments describe herein because of the larger scan width and reduced possibility of local heating and failure of the windows.

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

USE OF TREATED BIOMASS AL 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 fuel cells.

Systems and processes are bed herein that can use as feedstock cellulosic and/or lignocellulosic materials that are y 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 ock 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 example, 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 , e.g., by ing 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 s cellulolytic enzymes (cellulases), ligninases or various small le biomass-degrading metabolites. These s may be a complex of enzymes that act synergistically to e crystalline cellulose or the lignin portions of biomass. Examples of cellulolytic enzymes include: ucanases, cellobiohydrolases, and cellobiases (beta-glucosidases).

During saccharif1cation a cellulosic substrate can be initially hydrolyzed by endoglucanases at random locations producing oligomeric ediates. These intermediates are then substrates for exo-splitting glucanases such as cellobiohydrolase to produce iose from the ends of the cellulose polymer. Cellobiose is a water-soluble l,4-linked dimer of glucose. Finally, cellobiase cleaves cellobiose to yield glucose. The efficiency (e.g., time to 2012/071083 yze and/or completeness of hydrolysis) of this process depends on the recalcitrance of the cellulosic material.

INTERMEDIATES AND PRODUCTS The processes described herein are preferably used to produce butanol, e.g., isobutanol or n-butanol, and derivatives. However, the processes may be used to produce other products, co-products and intermediates, for example, the products described in US. Pat. App.

Pub. 2012/0100577 Al, filed October 18, 2011 and published April 26, 2012, the full disclosure of which is incorporated herein by reference.

Using the processes described herein, the biomass material can be converted to one or more products, such as energy, fuels, foods and materials. Specific examples of products e, but are not limited to, hydrogen, sugars (e.g., e, , arabinose, mannose, galactose, fructose, harides, 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., ning greater than 10%, 20%, % or even greater than 40% water), biodiesel, c acids, hydrocarbons (e.g., methane, ethane, propane, isobutene, pentane, n-hexane, biodiesel, bio-gasoline and mixtures thereof), co- products (e.g., proteins, such as cellulolytic proteins (enzymes) or single cell proteins), and mixtures of any of these in any combination or relative concentration, and ally in combination with any additives (e.g., fuel additives). Other examples e carboxylic acids, salts of a carboxylic acid, a mixture of ylic 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., dehyde), alpha and beta unsaturated acids (e.g., acrylic acid) and olef1ns (e.g., ethylene).

Other alcohols and alcohol derivatives include propanol, propylene glycol, 1,4-butanediol, 1,3- propanediol, sugar alcohols and polyols (e.g., glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, iditol, inositol, volemitol, isomalt, maltitol, ol, maltotriitol, etraitol, and polyglycitol and other polyols), and methyl or ethyl esters of any of these alcohols. Other products include methyl acrylate, methylmethacrylate, lactic acid, citric acid, formic acid, acetic acid, propionic acid, butyric acid, ic acid, valeric acid, caproic acid, 3-hydroxypropionic acid, palmitic acid, stearic acid, oxalic acid, malonic acid, glutaric acid, oleic acid, linoleic acid, glycolic acid, gamma-hydroxybutyric acid, and mixtures thereof, salts of any of these acids, mixtures 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 ts may be made by the processes described herein or otherwise, may be ed together and sold as products. The products may be combined, e.g., mixed, d or co-dissolved, or may simply be packaged or sold er.

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 undesirable contaminants that could be t 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 market. For example, steam generated from burning by-product streams can be used in a distillation process. As another e, electricity generated from g 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 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 remaining from the pretreatment and primary processes) can be used, e.g., burned, as a fuel.

Many of the products obtained, such as l or nol, can be utilized as a filel for powering cars, trucks, tractors, ships or trains, e.g., as an al combustion 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 on, the products described herein can be utilized for electrical power generation, e.g., in a conventional steam generating plant or in a fuel cell plant.

Other intermediates and products, including food and pharmaceutical products, are described in US. Pat. App. Pub. 2010/01245 83 Al, published May 20, 2010, to Medoff, the full disclosure of which is hereby incorporated by reference herein.

POST-PROCESSING The process for purification of products may include using ion-exchange resins, activated charcoal, filtration, lation, centrifugation, chromatography, precipitation, crystallization, evaporation, adsorption and combinations f. In some cases, the fermentation product is also sterilized, e.g., by heat or irradiation.

SACCHARIFICATION To obtain a fructose solution from the reduced-relacitrance feedstock, the treated biomass materials can be saccharified, generally by combining the al and a ase enzyme in a fluid medium, e.g., an aqueous solution. In some cases, the al is boiled, steeped, or cooked in hot water prior to saccharif1cation, as described in US. Pat. App. Pub. 2012/0100577 Al by Medoff and Masterman, published on April 26, 2012, the entire contents of which are incorporated 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 performed in transit, e.g., in a rail car, tanker truck, or in a supertanker or the hold of a ship. The time ed 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 ose may be substantially entirely converted to sugar, e.g., e in about 12-96 hours. If saccharif1cation is performed partially or completely in t, saccharif1cation may take longer.

It is lly preferred that the tank contents be mixed during saccharif1cation, e.g., using jet mixing as described in International App. No. PCT/USZOlO/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 tants include non-ionic surfactants, such as a Tween® 20 or Tween® 80 polyethylene glycol tants, ionic surfactants, or amphoteric tants.

It is generally preferred that the concentration of the sugar solution resulting from saccharif1cation 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 ation, to increase the tration of the sugar solution. This reduces the volume to be shipped, and also inhibits ial growth in the solution.

Alternatively, sugar ons 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 include amphotericin B, ampicillin, chloramphenicol, ciprofloxacin, gentamicin, hygromycin B, kanamycin, neomycin, penicillin, puromycin, streptomycin. Antibiotics will inhibit growth of microorganisms during transport and storage, and can be used at appropriate trations, e.g., between 15 and 1000 ppm by weight, e.g., between 25 and 500 ppm, or n 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 vative ties may be used. Preferably the antimicrobial additive(s) are food-grade.

A relatively high concentration solution can be obtained by limiting the amount of water added to the biomass material with the enzyme. The concentration can be controlled, 6.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 temperature of the on. For example, the solution can be maintained at a temperature of 40-50°C, 60-80°C, or even higher.

By adding glucose isomerase to the contents of the tank, a high concentration of fructose can be obtained without saccharification being inhibited by the sugars in the tank.

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

The tration is at least about 0.1 U/g cellulose (at least about 0.5 U/g cellulose, at least about 1U/g ose, at least about 2 U/g cellulose, at least about 3 U/g cellulose).

The addition of glucose isomerase increases the amount of sugars produced by at least 5 % (at least 10 %, at least to 15 %, at least 20 %).

The tration of sugars in the solution can also be enhanced by limiting the amount of water added to the feedstock with the , and/or the concentration can be increased by adding more feedstock to the solution during saccharification. 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 temperature of the solution. For example, the solution can be maintained at a temperature of 40-50°C, C, or even higher.

SACCHARIFYING AGENTS Suitable cellulolytic enzymes include cellulases. Cellulases can be obtained, for example, from species in the genera Bacillus, CaprinuS, Mycelz'ophthora, Cephalosporz'um, Scytalz'dium, Penicillium, ASpergz'lluS, Pseudomonas, la, Fusarz'um, vz'a, Acremom’um, 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), la insolenS (reclassified as Scytalidz'um thermophilum, see, e.g., US. Pat. No. 4,435,307), CaprinuS cinereuS, Fusarium oxySporum, Mycelz'ophthora thermophila, Merlpz'luS giganteus, Thielavz'a terrestriS, nium Sp. (including, 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 nS DSM 1800, Fusarz’um rum DSM 2672, Mycelz'ophthora thermophila CBS 117.65, Cephalosporium Sp. RYM-202, Acremonium Sp. CBS 478.94, Acremonium Sp. CBS 265.95, Acremonium perSicz'num CBS 169.65, Acremonium acremonium AHU 9519, Cephalosporium Sp. CBS 535.71, Acremonium brachypenium CBS 866.73, Acremonium dichromosporum CBS 683.73, Acremonium obclavatum CBS 311.74, nium pinkertonz'ae CBS 157.70, Acremonium roseogriseum CBS 134.56, Acremonium ratum CBS 146.62, and Acremoniumfuratum CBS 299.70H.

Cellulolytic enzymes may also be obtained from Chrysasporz’um, preferably a strain of Chrysasporz’um lucknowense. Additional strains that can be used include, 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 s material and produce sugars can also be used to ferment and convert those sugars to useful products.

SUGARS 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 precipitation, crystallization, chromatography (6.g. , simulated moving bed chromatography, high pressure chromatography), filgation, extraction, any other isolation method known in the art, and combinations thereof.

HYDROGENATION AND OTHER CHEMICAL TRANSFORMATIONS The processes described herein can include hydrogenation. For example glucose and xylose can be hydrogenated to ol and xylitol respectively. 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 ormation of the products from the processes described herein can be used, for example production of organic sugar derived ts such (e.g., furfural and al-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.

FERMENTATION Preferably, Clostrz'dz'um spp. are used to convert sugars (e.g., se) to butanol.

The optimum pH for tations is about pH 4 to 7. For example, the optimum pH for yeast is from about pH 4 to 5, while the optimum pH for Zymomonas is from about pH 5 to 6. Typical fermentation times are about 24 to 168 hours (e.g., 24 to 96 hrs) with temperatures in the range of 20°C to 40°C (e.g., 26°C to 40°C), however thermophilic rganisms prefer higher temperatures.

In some embodiments, e.g., when anaerobic organisms are used, at least a n of the fermentation is conducted in the e of oxygen, e.g., under a blanket of an inert gas such as N2, Ar, He, C02 or mixtures thereof. Additionally, the mixture may have a constant purge of an inert gas flowing through the tank during part of or all of the fermentation. In some cases, anaerobic condition, can be achieved or maintained by carbon dioxide production during the fermentation and no additional inert gas is needed.

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

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

Jet mixing may be used during fermentation, and in some cases saccharif1cation and fermentation are performed in the same tank. nts for the microorganisms may be added during saccharif1cation and/or fermentation, for example the food-based nutrient packages bed 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 ts that are disclosed in US. Prov.

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

Mobile fermenters can be utilized, as described in International App. No. (which was filed July 20, 2007, was hed in English as WC 2008/01 1598 and designated the United States), the contents of which is incorporated herein in its entirety. Similarly, the saccharif1cation equipment can be mobile. Further, saccharification and/or fermentation may be performed in part or entirely during transit. 2012/071083 FERMENTATION AGENTS gh Clostridiam is preferred, other microorganisms can be used. For instance, yeast and Zymomonas bacteria can be used for fermentation or conversion of sugar(s) to other alcohol(s). Other microorganisms are sed below. They can be naturally-occurring microorganisms and/or engineered microorganisms. For example, the microorganism can be a bacterium (including, but not limited to, e.g., a cellulolytic bacterium), a fiangus, (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 ible, mixtures of organisms can be ed.

Suitable fermenting microorganisms have the ability to convert carbohydrates, such as glucose, fructose, xylose, arabinose, mannose, ose, oligosaccharides or polysaccharides into fermentation products. Fermenting microorganisms include s of the genus Saccharomyces spp. (including, but not limited to, S. cerevisiae (baker’s yeast), S. distaticas, S. uvaram), the genus Klayveromyces, (including, but not limited to, K. nas, K. fragilis), the genus Candida (including, but not limited to, C. pseudotropicalis, and C. brassicae), Pichia is (a relative of Candida shehatae), the genus Clavispora (including, but not limited to, C. lasitaniae and C. opuntiae), the genus Pachysolen (including, but not limited to, P. tannophilus), the genus Bretannomyces (including, but not limited to, e.g., B. clausenii (Philippidis, G. P., 1996, Cellulose bioconversion technology, in ok on Bioethanol: Production and Utilization, Wyman, C.E., ed., Taylor & Francis, Washington, DC, 179-212)). Other suitable microorganisms include, for e, Zymomonas mobilis, Clostridiam spp. (including, but not limited to, C. thermocellum (Philippidis, 1996, supra), C.saccharobutylacetonicum, C. saccharobutylicum, C. Puniceum, C. beijernckii, and C. utylicum), Moniliella pollinis, Moniliella megachiliensis, Lactobacillus spp. Yarrowia lipolytica, Aureobasidium 519., Trichosporonoides 519., Trigonopsis variabilis, Trichosporon sp., Moniliellaacetoabutans sp., Typhula variabilis, Candida magnoliae, Ustilaginomycetes eudozyma tsukubaensis,yeast species of genera Zygosaccharomyces, Debaryomyces, Hansenula and ,and fungi of the dematioid genus Torala.

For instance, idiam spp. can be used to produce ethanol, butanol, c acid, acetic acid, and acetone. Lactobacillas spp., can be used to produce lactic acid.

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

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

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

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

The vapor exiting the beer column can be, e. g., 35% by weight ethanol and can be fed to a rectification column. A mixture of nearly azeotropic (92.5%) ethanol and water from the rectification column can be purified to pure ) ethanol 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, 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 returned to the process as fairly clean condensate with a small n split off to waste water treatment to prevent build-up of low-boiling compounds.

Other than in the examples herein, or unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages, such as those for s of materials, 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, amount, 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 d properties sought to be obtained by the present invention. At the very least, and not as an t to limit the application of the doctrine of equivalents to the scope of the claims, each cal parameter should at least be ued in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and ters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as ely as possible. Any numerical value, however, inherently contains error necessarily resulting from the rd deviation found in its underlying respective testing measurements. Furthermore, when numerical ranges are set forth herein, these ranges are inclusive of the recited range end points (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 cal range recited herein is intended to include all sub-ranges subsumed therein. For example, a range of "l to 10" is intended to include all sub-ranges between (and ing) the recited minimum value of l and the d maximum value of 10, that is, having a minimum value equal to or greater than 1 and a maximum value of equal to or less than 10. The terms "one,a) :4 a) a or "an" as used herein are intended to include "at least one" or "one or more," unless otherwise indicated.

EXAMPLES Example 1. Materials & Methods Preparation of Seed Cultures: 'ella cells stored at -80°C were used to inoculate propagation medium (20 g/L malt extract, 1 g/L peptone, 20 g/L glucose), and incubated at 30°C and agitation of 200 rpm for 72 hours. The e was then transferred to a bioreactor (either 3L, 20L, or 400L) for erythritol production.

Main Culture: The erythritol production medium consists of 10 g/L yeast extract, 1 g/L phytic acid, 1 g/L potassium nitrate, 100 g/L calcium chloride, 10 mg/L cupric sulfate, 50 mg/L zinc chloride and either 300 g/L glucose (reagent grade from Sigma) or purified saccharif1ed comcob prepared in-house.

WO 96693 The corn cob was treated with 35 Mrad from an electron beam, and saccharified with cellulase prepared in-house. The saccharif1ed corn cob was then d by cation exchange (Diaion PK228, Mitsubishi Chemical Corporation) and anion exchange (Diaion JA300, Mitsubishi Chemical Corporation).

Example 2. Determination of Culture Conditions The bioreactor culture consisted of 1.5 L in a 3 L vessel, 10 L in a 20 L vessel, or 250 L in a 400L vessel. Inoculum for each consisted of 72-hour ed seed culture, added at 5% of the volume in the bioreactor. Aeration was ed to 0.3 to 1 VVM, the agitation was 300 - 1000 rpm, and the temperature was 35°C. Antifoam 204 was added continuously at a rate of 1.5 ml/L/day.

Twelve different yeast extracts were tested for their effect on erythritol production.

The results were: Granulated Fisher (105 g/L erythritol production), Thermo Oxoid (30 g/L), Bacto Tech (94 g/L), Fluka (108 g/L), Thermo Remel (111 g/L), Teknova (108 g/L), Acros (93 g/L), Boston (96 g/L), Sunrise (8 g/L), US m (88 g/L), Sigma (76 g/L), and BD 0 g/L). ated Fisherm Bacto Tech, Fluka, Thermo Remel, Teknova, Acros, Boston, US m, and BD were carried over for additional testing.

Twelve different antifoam agents were tested. These were: Antifoam A, B, C, O-30, SE-15, Y-30, Silicone Antifoam, Antifoam 204 (all from Sigma Chemical Company, St, Louis, Missouri, USA), Antifoam AF (from Fisher), KFO 880, KFO 770, and Foam Blast 779 (from Emerald mance Materials).

Table 1a. Medium Components Tested for Erythritol Production Medium Range Working Range* Optimal Range component Tested Phytic acid with phytic 3-4 days to reach max. prod. with phytic acid (culture period) acid Phytic acid without 10-12 days to reach max. prod. (culture period) phytic acid Phytic acid 0.3 — 9 g/L 0.3 — 1.0 g/L 0.3 — 1.0 g/L (amount) Sodium phosphate 2-12 g/L 2-12 g/L (3-4 days to reach max. prod. lower yield than monobasic phytic acid (culture period) Calcium chloride 10-300 10-150 mg/L 100 mg/L (amount) mg/L Glucose 150-600 g/L 200—400 g/L 300 g/L (amount) Cupric sulfate 2-250 mg/L 2-250 mg/L 10 mg/L (amount) Yeast extract 5—20 g/L 9—13 g/L 10 g/L (amount) Yeast extract 12 different 9 different brands Fluka YE (brand) brands Zinc chloride 25 - l 00 25-100 mg/L 50 mg/L (amount) mg/L Antifoam agent 12 different KFO 880; Antifoam 204 (brand) agents Antifoam 204 en source 5 ent Urea; Sodium nitrate; Ammonium Potassium nitrate sources e; um sulfate; Potassium nitrate Potassium nitrate 0.5-5 g/L 0.5-5 g/L 1 g/L * "Working Range" was determined as conditions that produced greater than 80 g/L erythritol from 300 g/L glucose.

Table lb. Culture Conditions Tested for itol Production Condition Tested Range Tested Working Range* Optimum Range Agitation (speed in 450-1000 rpm 600-1000 rpm 800 rpm 3L bioreactor) Agitation (speed in 300-650 rpm 400-650 rpm 650 rpm 20L bioreactor) Aeration (VVM) 0.3-1 VVM 0.3-1 VVM 0.6 VVM e 30-40°C 30-370C 35°C Temperature Turbulence (dip With/without dip With dip tube with dip tube tube in 400L tube bioreactor) * "Working Range" was determined as conditions that produced greater than 80 g/L erythritol from 300 g/L glucose.

Example 3. Bioreactor Culture of Moniliella in a 3L ctor.

Moniliella 's (strain CBS 461.67; albureau voor Schimmelcultures, t, The Netherlands) was cultured in production medium in the 3L bioreactor (1.5L culture volume) with various medium components conditions (Table 1a). Phytic acid shortened e period to 3 to 4 days, while it took 10 to 12 days for itol tion without phytic acid (Table la). Each ent (phytic acid, yeast extract, sodium phosphate monobasic, m chloride, glucose, cupric sulfate, zinc chloride, potassium nitrate) was tested for obtaining optimal concentration (Table la). Physical conditions including agitation, on, temperature were also tested (Table lb). Typical erythritol production was 80 to 120 g/L of erythritol from 300 g/L of glucose.

The table below shows erythritol production in a 3L bioreactor culture ofMoniliella strain CBS 461.67 with optimal concentrations of media components (300 g/L glucose, 10 g/L yeast extract, 1 g/L phytic acid, 1 g/L potassium nitrate).

Table 2. Production of Erythritol and Other Products From 300 g/L Glucose Day Glycerol Erythritol Ribitol Ethanol 0 0 0 0 0 l 7.13 3.66 0 5.39 2 33.50 35.69 3.51 9.68 3 33.77 92.13 4.79 2.86 4 16.89 88.51 4.92 0.45 Example 4. Bioreactor Culture of Moniliella in a 20L Bioreactor.

Agitation speed was found to greatly affect erythritol production. Erythritol was produced in a 10L culture volume in a 20L bioreactor at three different speeds (300 rpm, 400 rpm, 650 rpm), at 1 VVM and 35°C, in medium composed of yeast extract (10 g/L), KN03 (1 g/L), phytic acid (1 g/L), CuSO4 (2 mg/L). The 400 rpm and 650 rpm cultures also included three impellers. The 650 rpm culture was aerated at 0.6 VVM, rather than 1 VVM.

WO 96693 The bioreactor culture with 300 rpm of ion speed resulted in much lower erythritol production than the same culture at 650 rpm. Ethanol production, on the other hand, was decreased by increasing agitation speed.

Table 3. Effect of Agitation Speed on Erythritol Production.

Day Glycerol Erythritol Ribitol Ethanol Glucose 300 rpm 0 4.09 3.35 0 2.63 > 50 1 10.80 5.95 3.06 15.15 > 50 2 18.48 19.39 0 24.44 > 50 3 24.24 48.09 0 32.37 70.74 4 25.27 59.51 0 25.15 0 23.36 64.09 3.60 8.48 0 6 21.59 63.70 3.66 2.32 7 19.35 59.69 3.65 1.50 400 rpm 0 0 0 0 0 300 1.3 7.09 4.21 0 21.16 >150 3 16.07 80.01 3.41 22.43 48.70 4 9.56 92.08 3.88 11.04 0 4.3 7.16 94.70 3.94 4.57 0 4.08 86.30 3.68 1.31 0 650 rpm 0 0 0 0 0 300 2 18.01 89.13 4.13 6.57 112.57 3 30.72 145.67 6.86 1.61 4.31 4 16.02 129.69 6.59 1.39 0 12.65 147.54 6.87 0 Example 5. Bioreactor Culture of Moniliella in a 400L Bioreactor.

It was found that the oxygen transfer rate was a key factor in erythritol production in the 400L bioreactor. Two dip tubes were used to increase the turbulence, an air sparger was installed in the bottom of the vessel, and the aspect ratio was increased. The results (in g/L) are shown in the table below.

Table 4. Production of itol and Other Products in a 400L Bioreactor Day Glycerol itol Ribitol Ethanol 0 0 0 0 0 1 6.1 9.2 1.5 15.3 2 10.0 60.3 1.7 19.3 3 11.8 75.3 0 27.7 e 6. Purification of Saccharification Product Corn cob was saccharified and the resulting sugar mixture purified by ion exchange.

Cation exchange and anion exchange were used to remove the metal ents listed in the table below.

Table 5. Metal elements in ppm in solution of saccharified corn cob containing 100 g/L glucose, before and after ion exchange.

Element Before ion After cation After cation and exchange exchange anion exchange Mn 9 0 0 Zn 9 0 0 Si 71 70 0 Fe 14 0 0 P 668 704 0 K 495 1 20 0 Mg 418 0 0 Na 10099 0 0 Ca 342 0 0 S 2048 2372 37 The purified saccharif1ed corn cob solution was then used for erythritol production by two different Mailiella strains, CBS 461.67 (Monillz'ela pollim's) and CBS 567.85 (Molim'ella megachz'lz'ensz’s). Flask cultures were used, and the media components ed 10 g/L yeast t, 1 g/L potassium nitrate, 0.3 g/L phytic acid, 2 mg/L of cupric sulfate as well as purif1ed saccharif1ed corncob. Glucose was consumed in 2 days and little xylose was consumed.

Table 6. Erythritol production by two different strains from purified saccharifled corn cob containing 160 g/ glucose and 140 g/L xylose.

Day Glycerol Erythritol Ribitol Ethanol Fructose Strain CBS 461.67 0 6.85 4.54 0 0.36 9.78 2 9.22 31.20 0 22.35 0 3 7.30 33.46 0 19.80 0 Strain CBS 567.85 0 0 4.54 0 0.21 10.30 2 9.72 29.36 0 22.52 0 3 7.82 45.99 0 19.47 0 itol production yield was 21% in CBS 461.67 and 28 % in CBS 567.85. This yield is comparable to the erythritol production with reagent grade glucose (30 to 40% yield).

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 def1nitions, statements, or other disclosure material set forth in this disclosure. As such, and to the extent necessary, the sure as itly set forth herein supersedes any conflicting material orated herein by reference.

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

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

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 inclusion of a stated integer or group of integers but not the ion of any other integer or group of integers.

Claims (24)

CLAIMS What is claimed is:

1. A method for making sugar alcohols, the method comprising: combining a slurry of ellulosic biomass that contains a sugar solution comprising glucose and xylose derived from the s with a microorganism selected from the group consisting of CBS 461.67 (Moniliella pollinis) and CBS 567.85 (Moniliella megachiliensis), the recalcitrance of the or ellulosic biomass having been reduced by bombardment with electrons; and utilizing the microorganism to t the sugar solution to produce erythritol, while providing aeration from 0.3 to 1.0 VVM and mixing the slurry with a jet mixer.

2. The method of claim 1, further comprising saccharifying the lignocellulosic biomass.

3. The method of claim 1 or claim 2, further comprising adjusting the concentration of the glucose to at least 5 wt. %.

4. The method of claim 2, wherein the biomass is saccharified with one or more ases.

5. The method any one of claims 1-4, wherein the lignocellulosic s is selected from the group consisting of: paper, paper products, paper waste, paper pulp, pigmented papers, loaded papers, coated papers, filled papers, magazines, printed , printer paper, polycoated paper, card stock, cardboard, 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, n stover, corn fiber, a, 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, taro, yams, beans, favas, s, peas, and mixtures of any of these.

6. The method of any one of claims 1-5, further comprising mechanically treating the lignocellulosic biomass to reduce its bulk density and/or increase its surface area.

7. The method of any one of claims 1-6, further sing comminuting the lignocellulosic biomass.

8. The method of claim 7, wherein the comminution is dry milling.

9. The method of claim 7, wherein the comminution is wet milling.

10. The method of any one of claims 1-9, further comprising culturing the microorganism in a cell growth phase before combining the lignocellulosic biomass with the microorganism.

11. The method of any one of claims 1-10, n the bombardment with electrons is provided at a dose of at least 5 Mrad.

12. The method of any one of claims 1-11, wherein the bombardment with electrons is provided at a dose of between 5 Mrad to 50 Mrad.

13. The method of any one of claims 1-12, wherein the bombardment with electrons is provided at a dose of between 20 Mrad to 40 Mrad.

14. The method of any one of claims 1-13, wherein the bombardment with electrons is provided at a dose rate of greater than 0.25 Mrad/sec.

15. The method of any one of claims 1-14, wherein the bombardment with electrons is ed at a dose rate of between 0.25 to 2 Mrad/sec.

16. The method of any one of claims 1-14, wherein the dment with electrons is provided at a dose rate of greater than 2 Mrad/sec.

17. The method of any one of claims 1-16, wherein the jet mixer further comprises an er, the impeller rotating at a rate between 400 to 650 tions per minute while mixing.

18. The method of claim 1, wherein the yield of erythritol is able to a yield of erythritrol using reagent grade glucose instead of the slurry.

19. The method of claim 18, wherein the yield of erthritol using the slurry is 21% to 28%.

20. The method of claim 18, wherein the yield of erthritol using reagent grade glucose is 30% to 40%.

21. The method of claim 1, wherein the yield of erthyritol is 70% to 93% of the yield of erythritol using reagent grade glucose d of the slurry.

22. The method of claim 1, wherein the xylose concentration in the sugar solution is r than 10 wt%.

23. The method of claim 1, wherein the xylose concentration in the sugar solution is greater than 20 wt%.

24. The method of claim 1, wherein the yield of erthyritol is 70% to 93% of the yield of erythritol using reagent grade glucose instead of the slurry and the xylose concentration in the sugar solution is greater than 20 wt%. WO 96693 SUBSTITUTE SHEET (RULE 26)