NZ716083B2 - 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 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 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 ll 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 INVENTION The invention pertains to the production of products, e.g., sugar alcohols, e.g., such as erythritol.

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

Lignocellulosic biomass is recalcitrant to degradation as the plant cell walls have a structure that is rigid and t. The structure comprises lline cellulose fibrils embedded in a hemicellulose matrix, surrounded by lignin. This compact matrix is difficult to access by enzymes and other chemical, biochemical and biological ses. Cellulosic biomass materials (e.g., biomass material from which ntially all the lignin has been removed) can be more accessible to enzymes and other conversion processes, but even so, lly-occurring cellulosic materials often have low yields (relative to theoretical yields) when ted with hydrolyzing enzymes. ellulosic biomass is even more recalcitrant to enzyme attack. rmore, each type of lignocellulosic biomass has its own specific composition of ose, hemicellulose and lignin.

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

Saccharides from renewable biomass sources could become the basis of chemical and fuels industries by replacing, menting or substituting eum 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 osic or lignocellulosic biomass that contains 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 rganism-biomass combination under ions that enable the microorganism to convert at least one of the sugars to the sugar l. In some implementations, the method includes: providing a cellulosic or lignocellulosic biomass, wherein the cellulosic or lignocellulosic biomass ns one or more sugars; providing a microorganism that is capable of ting 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 ellulosic biomass. The cellulosic or lignocellulosic biomass can be saccharified. [0008A] The present invention also provides a method for making sugar ls, the method comprising: combining a slurry of cellulosic or lignocellulosic biomass that contains a first sugar with a microorganism cultured in a medium comprising phytic acid, the recalcitrance of the biomass having been reduced by bombardment with electrons, and the microorganism selected from among the species of Moniliella; and utilizing the microorganism to ferment the first sugar to a first sugar alcohol while providing aeration from 0.3 to 1.0 VVM and mixing the slurry with a jet mixer that comprises a jet-flow agitator, the aeration and the agitation speed of the jet mixer being ive, at least in part, to increase production of the first sugar alcohol.

Any of the methods provided herein can e reducing the recalcitrance of the cellulosic 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, sonication, oxidation, pyrolysis, steam explosion, chemical ent, mechanical treatment, and freeze grinding. The treatment method can be bombardment with electrons.

Any of the methods provided herein can also include mechanically treating the cellulosic or lignocellulosic biomass to reduce its bulk density and/or increase its surface area.

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

In any of the methods provided herein, the biomass can be saccharif1ed with one or more cellulases. Any of the methods can also e 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 ed to have an initial e concentration of at least 5 wt%. The saccharif1ed s 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 ed , the sugar alcohol can be glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, itol, , inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, or polyglycitol.

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., Moniliellaacetoabutans, Typhula ilis, Candida magnoliae, Ustilaginomycetes, Pseudozyma tsukubaensz's; yeast species of genera Zygosaccharomyces, Debaryomyces, Hansenula and Pichia, or fiangi of the oid genus . The microorganism can be a species of Monilz'ella, such as M. pollim's, for instance, strain CBS 461.67, or M. megachilz’ensz’s, strain CBS 567.85.

In any of the methods ed herein, the cellulosic or lignocellulosic biomass can be: paper, paper products, paper waste, paper pulp, pigmented papers, loaded papers, coated papers, filled papers, nes, printed matter, printer paper, polycoated paper, card stock, cardboard, 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, alfalfa, hay, coconut hair, sugar processing es, bagasse, beet pulp, agave bagasse, algae, seaweed, manure, sewage, offal, arracacha, buckwheat, banana, barley, 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 invention is not limited to the embodiments disclosed in this Summary, and it is intended to cover ations that are within the spirit and scope of the invention, as defined by the claims.

BRIEF PTION OF THE DRAWINGS The foregoing will be apparent from the following more ular 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 gs are not arily to scale, emphasis instead being placed upon illustrating ments of the present ion. is a diagram illustrating the enzymatic hydrolysis of cellulose to glucose.

Cellulosic substrate (A) is converted by endocellulase (i) to cellulose (B), which is converted by exocellulase (ii) to cellobiose (C), which is 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 ally pretreated (e.g., to reduce its size) (200), optionally treated to reduce its recalcitrance (210), saccharified to form a sugar solution (220), the solution is transported (230) to a cturing plant (e.g., by pipeline, railcar) (or if saccharification is performed en route, the feedstock, enzyme and water is orted), the saccharified feedstock is bio-processed to produce a desired product (e.g., alcohol) (240), and the product can be processed further, e.g., by distillation, to produce a final product (250). Treatment for recalcitrance can be modified by ing lignin content (201) and setting or adjusting process parameters (205). Saccharifying the feedstock (220) can be modified by mixing the feedstock with medium and the enzyme (221).

DETAILED DESCRIPTION This invention relates to methods of processing biomass feedstock materials (e.g., biomass materials 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 mixtures thereof.

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

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

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 recalcitrance.

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

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

In some implementations, the contacting step includes a dual stage process, comprising 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 hydrolyzed by endoglucanases (i) at random ons producing oligomeric intermediates (e.g., ose) (B). These intermediates are then substrates for exo-splitting glucanases (ii) such as cellobiohydrolase to e cellobiose from the ends of the cellulose polymer. Cellobiose is a water-soluble l,4-linked dimer of glucose. Finally cellobiase (iii) cleaves cellobiose (C) to yield glucose (D). Therefore, the endoglucanases are particularly effective in attacking the crystalline portions of cellulose and increasing the effectiveness of exocellulases to produce cellobiose, which then requires the specificity of the cellobiose to produce glucose. Therefore, it is evident that ing on the nature and structure of the cellulosic substrate, the amount and type of the three different s 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, high-yielding ase 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 disclosure of which is incorporated herein by reference. Other cellulase-producing microorganisms may also be used.

As shown in a process for manufacturing a sugar alcohol can e, for example, ally 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 rifying the feedstock, using the enzyme complex, to form a sugar solution (220). Optionally, the method may also include transporting, e.g. truck or barge, the solution (or the feedstock, enzyme and water, if , by ne, railcar, rif1cation is performed en route) to a manufacturing plant (230). In some cases the saccharif1ed feedstock is further cessed (e.g., fermented) to produce a desired product e.g., alcohol (240). This resulting product may in some implementations be processed further, e.g., by distillation (250), to produce a final product. One method of reducing the recalcitrance of the feedstock is by electron bombardment of the feedstock. If desired, the steps of measuring lignin t of the feedstock (201) and setting or adjusting process ters based on this measurement (205) can be performed at various stages of the process, as bed 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 nce. Saccharifying the feedstock (220) can also be modified by mixing the feedstock 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 r 18, 2011.

The processes described above can be partially or completely performed in a tank (e.g., a tank having a volume of at least 4000, 40,000, or 500,000 L) in a manufacturing plant, and/or can be partially or tely 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 bed in US. Pat.

App. Pub. 2010/0064746 A1, published on March 18, 2010, the entire disclosure of which is incorporated by reference 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 herein.

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

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

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

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

The optionally mechanically and/or physically treated ock can also be combined with an enzyme complex for rif1cation and after the saccharif1cation is at least lly completed, the mixture is combined with an organism that produces sugar alcohols.

The conditions for rif1cation (e.g., temperature, agitation, aeration) can be different than the conditions for fermentation. The optimum pH for fermentation is generally from about pH 4 to 6. Typical 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 concentration at the start of fermentation favors the production of sugar ls. Accordingly, the saccharified ock on can be concentrated prior to combination with the organism that es sugar alcohols to increase the glucose level of the solution. Concentration can be done by any desired technique. For example, concentration can be by heating, cooling, centrifugation, reverse s, chromatography, itation, crystallization, evaporation, adsorption and combinations thereof. Preferably concentration is done by evaporation of at least a portion of the liquids from the saccharif1ed feedstock. Concentration is preferably done to increase the glucose content 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 product from the fermentation is then isolated.

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

Purification is preferably done to se the e 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, cooling, centrifugation, reverse osmosis, chromatography, precipitation, crystallization, evaporation, adsorption or combinations of any of these.

In some implementations the fermentation is dual-stage, with a cell growth phase and a product tion 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 tation 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 growth, and high sugar levels (6.g. than 5 , greater wt.%, greater than about 10 wt.%, greater than 20 wt.%, greater than 30 wt.%, greater than 40 wt.%) in the fermentation medium favor t 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 optimize the process. For example, grth can be monitored to achieve 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 rified solution can be added to trigger the onset of product formation. ally, the process can be zed, for example, by monitoring and adjusting the pH or oxygenation level with probes and automatic g to control cell growth and product ion. Furthermore, other nutrients can be controlled and monitored to optimized the process (e.g., amino acids, vitamins, metal ions, yeast t, vegetable ts, 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: 1017-1025.

While generally a high l 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 glucose concentration can be achieved by concentrating glucose during or after saccharification as discussed above. After an initial fermentation time to allow the start of fermentation, the fermentation media is diluted with a suitable diluent so that the glucose 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 MATERIALS As used herein, the term “biomass materials” includes lignocellulosic, cellulosic, starchy, and microbial materials.

Lignocellulosic materials include, but are not limited to, wood, particle board, forestry wastes (e.g., sawdust, aspen wood, wood chips), grasses, (e.g., switchgrass, miscanthus, cord grass, reed canary grass), grain residues, (e.g., rice hulls, oat hulls, wheat chaff, barley hulls), ltural waste (e.g., silage, canola straw, wheat straw, barley straw, oat straw, rice straw, jute, hemp, flax, bamboo, sisal, abaca, corn cobs, corn , soybean stover, corn fiber, alfalfa, hay, coconut hair), sugar processing residues (e.g., bagasse, beet pulp, agave bagasse), , 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 tation of comcobs or osic or lignocellulosic materials containing significant amounts of comcobs.

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

Cellulosic als include, for e, paper, paper products, paper waste, paper pulp, pigmented papers, loaded papers, coated papers, filled papers, magazines, printed matter (e. g., books, catalogs, manuals, labels, calendars, greeting cards, res, prospectuses, newsprint), printer paper, polycoated 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. 13/396,365 (“Magazine ocks” by Medoff et al., filed February 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 materials 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, eat, banana, barley, cassava, kudzu, oca, sago, sorghum, regular household potatoes, sweet potato, taro, yams, or one or more beans, such as favas, lentils or peas. Blends of any two or more starchy materials are also starchy materials. es of starchy, osic and or lignocellulosic materials can also be used. For example, a s 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 als can be treated by any of the methods described herein.

Microbial materials include, but are not limited to, any naturally occurring or genetically modified microorganism or organism that contains or is capable of providing a source of carbohydrates (e.g., ose), for example, protists, e.g., animal protists (e.g., protozoa such as fiagellates, amoeboids, ciliates, and sporozoa) and plant protists (e.g., algae such alveolates, chlorarachniophytes, cryptomonads, euglenids, glaucophytes, haptophytes, red algae, stramenopiles, and viridaeplantae). Other examples include seaweed, plankton (e.g., macroplankton, mesoplankton, microplankton, nanoplankton, picoplankton, and femptoplankton), phytoplankton, bacteria (e.g., gram positive bacteria, gram negative bacteria, and extremophiles), yeast and/or mixtures of these. In some instances, ial 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 addition, microbial biomass can be obtained from culture systems, e.g., large scale dry and wet culture and fermentation systems.

The s material can also e offal, and similar sources of material.

In other embodiments, the biomass materials, such as cellulosic, starchy and lignocellulosic feedstock als, 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, through the iterative steps of selection and breeding to obtain desired traits in a plant.

Furthermore, the plants can have had genetic al removed, modified, silenced and/or added with respect to the wild type variety. For example, genetically modified plants can be produced by inant DNA methods, where c ations include ucing or modifying specific genes from parental varieties, or, for example, by using transgenic breeding wherein a specific gene or genes are introduced to a plant from a different species of plant and/or ia.

Another way to create c variation is through mutation breeding wherein new alleles are artificially created from endogenous genes. The artificial genes can be created by a y of ways including treating the plant or seeds with, for example, chemical mutagens (e.g., using ting 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 ques. Other methods of providing modified genes is through error prone PCR and DNA shuffling followed by ion of the desired modified DNA into the desired plant or seed. s of introducing the desired genetic variation in the seed or plant include, for example, the use of a ial carrier, biolistics, calcium phosphate precipitation, electroporation, gene splicing, gene silencing, ction, microinjection and viral carriers. Additional genetically modified als have been described in US. Application Serial No ,369 filed February 14, 2012 the full disclosure of which is incorporated herein by reference.

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

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

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

Bulk density is determined using ASTM D1895B. Briefly, the method involves filling a measuring cylinder ofknown volume with a sample and obtaining a weight of the sample. The bulk y is calculated by dividing the weight of the sample in grams by the known volume of the cylinder in cubic centimeters. If desired, low bulk density materials can be 1ed, for example, by methods described in US.

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

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

Screening of material can also be by a manual method, for example by an operator or mechanoid (e.g., a robot equipped with a color, reflectivity or other sensor) that removes unwanted material. Screening can also be by magnetic screening wherein a magnet is disposed near the conveyed material and the ic material is removed magnetically.

Optional pre-treatment processing can include heating the material. For example a portion of the conveyor can be sent through a heated zone. The heated zone can be created, for example, by IR radiation, microwaves, combustion (e.g., gas, coal, oil, biomass), resistive heating and/or inductive coils. The heat can be applied from at least one side or more than one side, can be uous or periodic and can be for only a portion of the material or all the material. For e, a portion of the ing 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 2012/071083 the material, this can also be tated, 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 conveyed.

Optionally, pre-treatment sing can include cooling the material. g 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 g fluid, for example water (6.g. with glycerol), or nitrogen (e.g. to the bottom of the conveying , , liquid en) trough. Alternatively, a cooling gas, for example, chilled nitrogen can be blown over the biomass materials or under the conveying system.

Another optional pre-treatment processing method can include adding a material to the biomass. The additional al can be added by, for example, by showering, sprinkling and or pouring the material onto the biomass as it is conveyed. als that can be added include, for example, metals, ceramics and/or ions as described in US. Pat. App. Pub. 2010/0105119 A1 (filed October 26, 2009) and US. Pat. App. Pub. 2010/0159569 A1 (filed December 16, 2009), the entire disclosures of which are incorporated herein by reference.

Optional materials that can be added include acids and bases. Other materials that can be added are oxidants (e.g., peroxides, chlorates), rs, polymerizable monomers (e.g., ning unsaturated bonds), water, catalysts, enzymes and/or organisms. Materials can be added, for example, in pure form, as a solution in a t (e.g., water or an organic solvent) and/or as a solution. In some cases the solvent is volatile and can be made to evaporate e.g., by heating and/or blowing gas as previously described. The added material may form a uniform coating on the biomass or be a neous mixture of different components (e.g., biomass and additional al). The added material can te the subsequent irradiation step by increasing the efficiency of the irradiation, damping the irradiation or changing the effect of the irradiation (e.g., from electron beams to X-rays or heat). The method may have no impact on the irradiation but may be useful for fiarther downstream processing. The added material may help in conveying the material, for example, by lowering dust levels.

Biomass can be delivered to the or by a belt conveyor, 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 conveyor 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 undesirable because these can form an explosion hazard or damage the window foils of an on gun (if such a device is used for treating the material).

The material can be leveled to form a uniform thickness between about 0.03 12 and 5 inches (e.g., between about 0.0625 and 2.000 , between about 0. 125 and 1 inches, between about 0. 125 and 0.5 inches, between about 0.3 and 0.9 inches, between about 0.2 and 0.5 inches between about 0.25 and 1.0 inches, between about 0.25 and 0.5 inches, 0.100 +/- 0.025 , 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 inches, 0.700 --/- 0.025 , 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 example the material can be conveyed at rates of at least 1 ft/min, e.g., at least 2 ft/min, at least 3 ft/min, at least 4 ft/min, at least 5 ft/min, at least 10 ft/min, at least 15 ft/min, 20, 25, 30, 35, 40, 45, 50 ft/min. The rate of conveying is related to the beam current, for example, for a 14 inch thick biomass and 100 mA, the conveyor can move at about 20 ft/min to provide a useful irradiation dosage, at 50 mA the conveyor can move at about ft/min to provide approximately the same irradiation .

After the biomass material has been ed through the ion zone, optional post-treatment processing can be done. The optional reatment processing can, for example, be a process described with respect to the pre-irradiation processing. For example, the biomass can be screened, heated, cooled, and/or combined with ves. Uniquely to post-irradiation, quenching of the radicals can occur, for example, quenching of radicals by the addition of fluids or gases(e.g., oxygen, s 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 ents can be used in on to ation to fiarther reduce the recalcitrance of the biomass material. These ses can be applied before, during and or after irradiation. 2012/071083 In some cases, the mechanical ent may include an initial preparation of the feedstock as received, e.g., size reduction of materials, such as by comminution, e.g, cutting, grinding, ng, pulverizing or chopping. For example, in some cases, loose feedstock (e.g., recycled paper, starchy materials, or grass) is prepared by shearing or shredding.

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

Alternatively, or in addition, the feedstock material can first be physically treated by one or more of the other physical treatment methods, 6.g. chemical treatment, radiation, sonication, oxidation, pyrolysis or steam explosion, and then ically treated. This sequence can be advantageous since materials treated by one or more of the other treatments, 6.g. ation 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 al 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 chemical pulping) and can partially or completely hydrolyze the material. The methods also can be used with pre-hydrolyzed material. The methods also can be used with material that has not been pre hydrolyzed The methods can be used with mixtures of yzed 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 drolyzed material, with about 80% or more non-hydrolyzed material or even with 90% or more non-hydrolyzed material.

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

Methods of ically treating the s material include, for example, milling or grinding. Milling may be performed using, for e, 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 r. Some ary grinders include stone grinders, pin grinders, coffee grinders, and burr grinders. Grinding or milling may be provided, for example, by a reciprocating pin or other element, as is the case in a pin mill. Other WO 96693 mechanical treatment methods include mechanical ripping, g, ng or chopping, other s that apply re to the fibers, and air attrition milling. Suitable mechanical treatments further include any other technique that continues the disruption of the internal structure of the material that was initiated by the previous processing steps.

Mechanical feed preparation systems can be configured to produce streams with specific characteristics such as, for example, specific m sizes, specific length-to-width, or specific surface areas ratios. Physical preparation can increase the rate of ons, improve the movement of material on a conveyor, improve 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 prepare a low bulk density material, 6.g. the al (e.g., , by densifying densification can make it easier and less costly to transport to r site) and then reverting the material to a lower bulk y state (e.g., after transport). The al 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 Publication 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 incorporated herein by reference.

Densified materials can be processed by any of the methods described , 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 material that includes fibers provided by ng a fiber source. For example, the shearing can be performed with a rotary knife cutter.

For example, a fiber source, 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 shredder, such as those manufactured by Munson (Utica, N.Y.). As an alternative to shredding, the paper can be reduced in size by cutting to a desired size using a guillotine cutter. For example, 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 ng 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 es a hopper that can be loaded with a ed fiber source prepared by shredding a fiber source. The shredded fiber source.

In some implementations, the feedstock is physically treated prior to rification and/or fermentation. Physical treatment processes can include one or more of any of those bed herein, such as mechanical treatment, chemical treatment, irradiation, sonication, oxidation, pyrolysis or steam explosion. Treatment methods can be used in ations of two, three, four, or even all of these technologies (in any order). When more than one ent 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 ock may also be used, alone or in combination with the processes disclosed herein.

Mechanical treatments that may be used, and the teristics of the mechanically treated s materials, are described in fiarther detail in US. Pat. App. Pub. 2012/0100577 Al, filed October 18, 2011, the fill disclosure of which is hereby incorporated herein by reference.

TREATMENT OF BIOMASS MATERIAL -- PARTICLE BOMBARDMENT One or more treatments with energetic particle bombardment can be used to process raw ock from a wide variety of different s to extract useful substances from the feedstock, and to e partially degraded organic material which functions as input to filrther processing steps and/or sequences. Particle bombardment can reduce the molecular weight and/or crystallinity of feedstock. In some 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 ed by heavy charged particles (such as alpha particles or s), electrons (produced, for example, in beta decay or on beam accelerators), or electromagnetic radiation (for example, gamma rays, x rays, or ultraviolet rays). Alternatively, radiation produced by radioactive nces 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 ctions. Heavy charged particles primarily ionize matter via Coulomb scattering; fiarthermore, these interactions produce energetic electrons that may further ionize matter. Alpha particles are cal to the nucleus of a helium atom and are produced by the alpha decay of various ctive nuclei, such as isotopes of bismuth, polonium, astatine, radon, francium, radium, several des, such as actinium, thorium, uranium, neptunium, , califomium, americium, and plutonium.

When particles are utilized, they can be neutral (uncharged), positively charged or negatively charged. When charged, the d particles can bear a single ve or negative charge, or multiple charges, e.g., one, two, three or even four or more charges. In instances in which chain scission is desired, positively charged particles may be desirable, in part, due to their acidic nature. When particles are utilized, the particles can have the mass of a resting electron, or greater, e.g., 500, 1000, 1500, or 2000 or more times the mass of a resting electron. For example, the particles can have a mass of from about 1 atomic unit to about 150 atomic units, e.g., from about 1 atomic unit to about 50 atomic units, or from about 1 to about 25, e.g., 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 induction linear or continuous wave. For example, ron type accelerators are available from IBA (Ion Beam Accelerators, Louvain-la-Neuve, Belgium), such as the RhodotronTM , 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 Meeting, 18-20 Mar. 2006; Iwata, Y. et al., “Altemating-Phase- Focused IH-DTL for Heavy-Ion l Accelerators”, Proceedings of EPAC 2006, Edinburgh, Scotland; and Leitner, C. M. et al., “Status of the Superconducting ECR Ion Source Venus”, Proceedings of EPAC 2000, , Austria.

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

In some instances when chain scission is desirable and/or polymer chain fianctionalization is ble, particles heavier than electrons, such as s, 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 utilized for their Lewis acid ties for enhanced ring-opening chain scission. For example, when -containing fianctional groups are desired, treatment in the presence of oxygen or even treatment with oxygen ions can be performed. For example, when en-containing fianctional groups are desirable, treatment in the ce of nitrogen or even treatment with nitrogen ions can be performed.

OTHER FORMS OF ENERGY Electrons interact via Coulomb ring and bremsstrahlung radiation ed by s 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.

Electromagnetic ion interacts via three ses: photoelectric absorption, Compton scattering, and pair production. The dominating interaction is determined by the energy of the incident radiation and the atomic number of the al. The summation of interactions contributing to the absorbed radiation in cellulosic material can be expressed by the mass absorption 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 significant penetration depth into a variety of material in the sample. Sources of gamma rays include radioactive , such as isotopes of cobalt, calcium, technetium, chromium, gallium, indium, iodine, iron, krypton, samarium, selenium, , thalium, and xenon. s of x rays include electron beam collision with metal targets, such as en 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 c lamps.

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

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

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

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

Electron bombardment may be performed using an electron beam device that has a nominal energy of less than 10 MeV, e.g., less than 7 MeV, less than 5 MeV, or less than 2 MeV, agjmmdmm05mL5M&hmmamm08mL8M$meMMM07mlM&Lmfimn about 1 to 3 MeV. In some implementations the nominal 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 rators 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 al, thereby ting 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 ble 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 med at a dose rate of greater than about 0.25 Mrad per second, e.g., greater than about 0.5, 0.75, l, 1.5, 2, 5, 7, 10, l2, 15, or even greater than about 20 Mrad per second, e.g., about 0.25 to 2 Mrad per second. Higher dose rates generally require higher line speeds, to avoid thermal decomposition of the material. In one implementation, the accelerator is set for 3 MeV, 50 mAmp beam current, and the line speed is 24 feet/minute, for a sample ess 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 WO 96693 2012/071083 implementations, a total dose of 25 to 35 Mrad is preferred, applied y over a couple of seconds, e.g., at 5 Mrad/pass with each pass being applied for about one second. Applying a dose of greater than 7 to 8 Mrad/pass can in some cases cause thermal degradation of the feedstock material.

Using multiple heads as discussed above, the material can be treated in multiple passes, for example, two passes at 10 to 20 Mrad/pass, e.g., 12 to 18 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, ng the material with several relatively low doses, rather than one high dose, tends to t overheating of the material and also increases dose uniformity through the thickness of the al. In some implementations, the material is stirred or otherwise mixed during or after each pass and then smoothed into a m 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 described herein occurs on lignocellulosic material that remains dry as acquired or that has been dried, e.g., using heat and/or reduced pressure. For example, in some embodiments, the cellulosic and/or lignocellulosic material has less than about five percent by weight retained water, measured at 25°C and at fifty t relative humidity.

Electron bombardment can be applied while the cellulosic and/or lignocellulosic material is exposed to air, oxygen-enriched air, or even oxygen itself, or blanketed by an inert gas such as nitrogen, argon, or 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 maximize reactive gas formation, e.g., ozone and/or oxides of nitrogen.

In some embodiments, two or more electron sources are used, such as two or more ionizing sources. For e, samples can be treated, in any order, with a beam of ons, 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 ion sources, such as a beam of electrons, gamma radiation, and energetic UV light. The s is conveyed through the ent zone where it can be bombarded with electrons. It is generally preferred that the bed of biomass al has a relatively uniform thickness, as previously described, while being treated.

It may be advantageous to repeat the treatment to more thoroughly reduce the recalcitrance of the s 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 circular system where the biomass is conveyed multiple times through 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 biomass.

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

In some embodiments, the treatment (with any electron source or a 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 ments, the ent is performed until the material receives a dose of between 0.1-100 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 n 50.0 and 350.0 kilorads/hours. In other embodiments the treatment is performed at a dose rate of between 10 and 10000 kilorads/hr, between 100 and 1000 kilorad/hr, or between 500 and 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 , cesium, technetium, and iridium. Alternatively, an on gun can be used as an electron source via thermionic emission and accelerated through an accelerating potential. An on gun generates ons, accelerates them through a large ial (e.g., r than about 500 nd, greater than about lmillion, greater than about 2 million, greater than about 5 million, greater than about 6 million, greater than about 7 million, greater than about 8 million, greater than about 9 million, or even greater than 10 n volts) and then scans them 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 g by the electron beam. Window foil e is a cause of significant down-time due to subsequent necessary repairs and re-starting the electron gun.

Various other irradiating devices may be used in the methods disclosed herein, including field ionization sources, electrostatic ion separators, field ionization tors, thermionic emission sources, microwave discharge ion s, 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 radiation source. A beam of electrons has the advantages of high dose rates (e.g., l, 5, or even 10 Mrad per second), high throughput, less containment, and less confinement equipment. Electron beams can also have high electrical efficiency (e.g., 80%), ng for lower energy usage relative to other radiation s, which can translate into a lower cost of operation and lower greenhouse gas emissions corresponding to the smaller amount of energy used. Electron 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 rators, and pulsed accelerators.

Electrons can also be more nt at causing changes in the molecular structure of biomass materials, for example, by the mechanism of chain scission. In addition, electrons having energies of 05-10 MeV can penetrate low density materials, such as the biomass materials described herein, e.g., materials having a bulk density of less than 0.5 g/cm3, and a depth of 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, 2012/071083 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 (million electron volts), e.g., from about 0.5 MeV to about 1.5 MeV, or from about 0.7 MeV to about 1.25 MeV.

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

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

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

Tradeoffs in considering electron energies include energy costs.

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

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

USE OF TREATED BIOMASS MATERIAL Using the methods bed herein, a starting biomass material (6.g. , plant biomass, animal biomass, paper, and pal 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 described herein that can use as feedstock cellulosic and/or lignocellulosic materials that are readily available, but often can be difficult to process, e.g., pal waste streams and waste paper streams, such as streams that include newspaper, kraft paper, corrugated paper or mixtures of these.

In order to convert the feedstock to a form that can be readily processed, the glucan- or xylan-containing cellulose in the feedstock can be yzed to low molecular weight carbohydrates, such as sugars, by a saccharifying agent, e.g., an enzyme or acid, a process referred to as rif1cation. 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 ock can be hydrolyzed using an enzyme, e.g., by combining the als and the enzyme in a solvent, e.g., in an aqueous solution.

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

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

INTERMEDIATES AND PRODUCTS The processes described herein are ably used to produce l, 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 al can be converted to one or more products, such as , fuels, foods and materials. Specific examples of products include, but are not limited to, hydrogen, sugars (e.g., glucose, xylose, arabinose, mannose, galactose, fructose, disaccharides, accharides and polysaccharides), alcohols (e.g., monohydric alcohols or dihydric alcohols, such as ethanol, anol, isobutanol, sec-butanol, tert-butanol or n-butanol), hydrated or hydrous alcohols (e.g., containing greater than 10%, 20%, % or even greater than 40% water), biodiesel, organic acids, hydrocarbons (e.g., methane, ethane, propane, isobutene, e, n-hexane, biodiesel, bio-gasoline and mixtures thereof), co- products (e.g., proteins, such as cellulolytic ns (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 include carboxylic acids, salts of a carboxylic acid, a mixture of carboxylic acids and salts of carboxylic acids and esters of carboxylic acids (e.g., methyl, ethyl and n-propyl ), ketones (e.g., acetone), des (e.g., acetaldehyde), alpha and beta unsaturated acids (e.g., acrylic acid) and olef1ns (e.g., ethylene).

Other ls and alcohol derivatives include propanol, propylene glycol, 1,4-butanediol, 1,3- propanediol, sugar alcohols and polyols (e.g., glycol, glycerol, itol, threitol, arabitol, xylitol, l, mannitol, sorbitol, galactitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, and polyglycitol and other polyols), and methyl or ethyl esters of any of these alcohols. Other products include methyl te, methylmethacrylate, lactic acid, citric acid, formic acid, acetic acid, propionic acid, butyric acid, succinic acid, valeric acid, caproic acid, 3-hydroxypropionic acid, palmitic acid, stearic acid, oxalic acid, malonic acid, glutaric acid, oleic acid, ic 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 products 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, blended or co-dissolved, or may simply be packaged or sold together.

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

The ses 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 s. As another example, electricity generated from burning by-product streams can be used to power electron beam generators used in pretreatment.

The by-products used to generate steam and electricity are derived from a number of sources throughout the s. For example, anaerobic digestion of ater 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 ethanol or n-butanol, can be utilized as a filel for powering cars, trucks, tractors, ships or , e.g., as an al combustion filel or as a fuel cell feedstock. Many of the products ed can also be utilized to power aircraft, such as planes, e.g., having jet engines or helicopters. In addition, the products described herein can be utilized for electrical power generation, e.g., in a conventional steam generating plant or in a fuel cell plant. 2012/071083 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, distillation, centrifugation, tography, precipitation, crystallization, evaporation, adsorption and combinations thereof. In some cases, the fermentation product is also ized, e.g., by heat or irradiation.

SACCHARIFICATION To obtain a se solution from the reduced-relacitrance feedstock, the treated biomass materials can be rified, generally by combining the material and a cellulase enzyme in a fluid , e.g., an aqueous solution. In some cases, the material is boiled, steeped, or cooked in hot water prior to saccharif1cation, as described in US. Pat. App. Pub. 2012/0100577 Al by Medoff and man, published on April 26, 2012, the entire ts of which are incorporated herein.

The saccharif1cation process can be lly 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 anker or the hold of a ship. The time required for complete saccharif1cation will depend on the process conditions and the biomass material and enzyme used. If saccharification is performed in a manufacturing plant under controlled conditions, the cellulose may be substantially entirely converted to sugar, e.g., glucose in about 12-96 hours. If saccharif1cation is performed partially or completely in transit, saccharif1cation may take longer.

It is generally 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 ated the United States, the filll disclosure of which is incorporated by reference herein.

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

It is lly 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 d, e.g., by ation, to increase the concentration of the sugar solution. This reduces the volume to be shipped, and also inhibits microbial growth in the solution.

Alternatively, sugar solutions of lower concentrations may be used, in which case it may be desirable to add an antimicrobial additive, e.g., a broad spectrum antibiotic, in a low concentration, e.g., 50 to 150 ppm. Other suitable antibiotics include amphotericin B, ampicillin, chloramphenicol, oxacin, gentamicin, hygromycin B, kanamycin, neomycin, llin, cin, streptomycin. Antibiotics will inhibit growth of microorganisms during transport and storage, and can be used at appropriate concentrations, e.g., between 15 and 1000 ppm by weight, e.g., between 25 and 500 ppm, or between 50 and 150 ppm. If desired, an antibiotic can be included even if the sugar concentration is relatively high. Alternatively, other ves with anti-microbial of preservative properties 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 lled, 6.g. by controlling how much saccharification takes place. For example, concentration can be increased by adding more s 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 sed above.

Solubility can also be increased by increasing the ature of the solution. For example, the solution can be maintained at a temperature of 40-50°C, 60-80°C, or even higher.

By adding glucose isomerase to the ts 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 ose (lower than or equal to 100 U/g cellulose, lower than or equal to 50 U/g cellulose, lower than or equal to 10 U/g cellulose, lower than or equal to 5 U/g cellulose).

The concentration is at least about 0.1 U/g cellulose (at least about 0.5 U/g cellulose, at least about 1U/g cellulose, 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 concentration of sugars in the solution can also be enhanced by limiting the amount of water added to the feedstock with the enzyme, 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 on. For example, the solution can be maintained at a temperature of 40-50°C, 60-80°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, Thielavz'a, Acremom’um, ChrySOSporz'um and Trichoderma, especially those produced by a strain ed from the s 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 uS, Fusarium oxySporum, Mycelz'ophthora thermophila, Merlpz'luS eus, Thielavz'a terrestriS, Acremonium Sp. (including, but not limited to, A. perSl'cz'num, A. nium, A. brachypem'um, A. dichromosporum, A. obclavatum, A. pinkertonz'ae, A. roseogriseum, A. incoloratum, and A. furatum). Preferred s include Humicola insolenS DSM 1800, Fusarz’um oxySporum DSM 2672, Mycelz'ophthora phila CBS 117.65, Cephalosporium Sp. RYM-202, Acremonium Sp. CBS 478.94, Acremonium Sp. CBS 265.95, nium perSicz'num CBS 169.65, nium acremonium AHU 9519, Cephalosporium Sp. CBS 535.71, Acremonium brachypenium CBS 866.73, Acremonium dichromosporum CBS 683.73, Acremonium obclavatum CBS 311.74, Acremonium pinkertonz'ae CBS 157.70, Acremonium roseogriseum CBS 134.56, nium incoloratum 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. , 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 rganisms that can be used to saccharify biomass material and produce sugars can also be used to ferment and convert those sugars to useful products.

SUGARS In the processes described 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 re tography), centrifilgation, extraction, any other isolation method known in the art, and combinations thereof.

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

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

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

The optimum pH for fermentations is about pH 4 to 7. For example, the optimum pH for yeast is from about pH 4 to 5, while the optimum pH for Zymomonas is from about pH 5 to 6. Typical fermentation times are about 24 to 168 hours (e.g., 24 to 96 hrs) with temperatures in the range of 20°C to 40°C (e.g., 26°C to 40°C), however thermophilic microorganisms prefer higher temperatures. 2012/071083 In some embodiments, e.g., when bic organisms are used, at least a portion of the fermentation is ted in the absence 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 ed 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 e sugar and carbohydrates in high concentrations. The sugars and carbohydrates can be isolated via any means known in the art. These ediate fermentation products can be used in preparation of food for human or animal consumption.

Additionally or alternatively, the ediate 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.

Nutrients for the microorganisms may be added during saccharif1cation and/or fermentation, for example the food-based nutrient packages described in US. Pat. App. Pub. 2012/0052536, filed July 15, 2011, the te sure of which is incorporated herein by reference.

“Fermentation” includes the methods and ts that are disclosed in US. Prov.

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

Mobile fermenters can be utilized, as described in International App. No. (which was filed July 20, 2007, was published 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, rification and/or fermentation may be performed in part or entirely during transit.

FERMENTATION AGENTS Although 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 l(s). Other microorganisms are discussed 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 sms are compatible, mixtures of organisms can be utilized.

Suitable fermenting microorganisms have the ability to convert carbohydrates, such as glucose, fructose, xylose, arabinose, mannose, galactose, oligosaccharides or polysaccharides into fermentation products. Fermenting microorganisms include strains 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. marxianas, K. fragilis), the genus Candida (including, but not limited to, C. pseudotropicalis, and C. brassicae), Pichia stipitis (a relative of a shehatae), the genus pora (including, but not limited to, C. niae and C. opuntiae), the genus Pachysolen (including, but not limited to, P. hilus), the genus Bretannomyces (including, but not limited to, e.g., B. clausenii (Philippidis, G. P., 1996, Cellulose bioconversion technology, in Handbook on anol: Production and Utilization, Wyman, C.E., ed., Taylor & Francis, Washington, DC, 179-212)). Other suitable microorganisms include, for example, Zymomonas mobilis, Clostridiam spp. (including, but not limited to, C. thermocellum (Philippidis, 1996, supra), C.saccharobutylacetonicum, C. saccharobutylicum, C. Puniceum, C. nckii, and C. utylicum), Moniliella pollinis, Moniliella megachiliensis, Lactobacillus spp. ia lipolytica, Aureobasidium 519., sporonoides 519., Trigonopsis variabilis, Trichosporon sp., Moniliellaacetoabutans sp., Typhula variabilis, Candida magnoliae, Ustilaginomycetes sp.,Pseudozyma aensis,yeast species of genera Zygosaccharomyces, omyces, Hansenula and Pichia,and fungi of the oid genus Torala.

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

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

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

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

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

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 ) l and water from the rectification column can be purified to pure (99.5%) l using vapor-phase molecular sieves.

The beer column bottoms can be sent to the first effect of a 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 fuge 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 portion split off to waste water treatment to prevent build-up of low-boiling compounds.

Other than in the examples herein, or unless ise expressly specified, all of the numerical ranges, amounts, values and percentages, such as those for amounts of materials, elemental contents, times and temperatures of reaction, ratios of amounts, and others, in the ing portion of the specification and attached claims may be read as if prefaced by the word 2012/071083 “about” even though the term “about” may not expressly appear with the value, amount, or range. Accordingly, unless indicated to the contrary, the cal parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding 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 precisely as le. Any numerical value, however, inherently contains error necessarily resulting from the standard deviation found in its underlying respective g measurements. rmore, when numerical ranges are set forth herein, these ranges are inclusive of the recited range end points , end points may be used). When percentages by weight are used herein, the numerical values ed are relative to the total weight.

Also, it should be understood that any numerical range d 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 including) the recited m value of l and the recited maximum value of 10, that is, having a minimum value equal to or greater than 1 and a maximum value of equal to or less than 10. The terms “one,a) :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: Monilz'ella cells stored at -80°C were used to inoculate propagation medium (20 g/L malt t, 1 g/L peptone, 20 g/L glucose), and incubated at 30°C and agitation of 200 rpm for 72 hours. The culture was then erred to a bioreactor (either 3L, 20L, or 400L) for itol 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.

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 purified by cation exchange n PK228, Mitsubishi al Corporation) and anion exchange (Diaion JA300, Mitsubishi Chemical ation).

Example 2. Determination of e 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. um for each consisted of 72-hour cultured seed culture, added at 5% of the volume in the bioreactor. on was adjusted 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 itol 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), a (108 g/L), Acros (93 g/L), Boston (96 g/L), Sunrise (8 g/L), US Biochem (88 g/L), Sigma (76 g/L), and BD (90-120 g/L). Granulated Fisherm Bacto Tech, Fluka, Thermo Remel, Teknova, Acros, Boston, US Biochem, 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 Performance Materials).

Table 1a. Medium Components Tested for Erythritol Production Medium Range Working Range* Optimal Range ent 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 2012/071083 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 Yeast extract 12 different 9 different brands Fluka YE ) 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 Nitrogen source 5 different Urea; Sodium nitrate; Ammonium Potassium nitrate sources nitrate; Ammonium sulfate; Potassium nitrate Potassium e 0.5-5 g/L 0.5-5 g/L 1 g/L (amount) * ng Range” was determined as conditions that produced greater than 80 g/L erythritol from 300 g/L glucose.

Table lb. Culture Conditions Tested for Erythritol 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 0 rpm 400-650 rpm 650 rpm 20L bioreactor) Aeration (VVM) 0.3-1 VVM 0.3-1 VVM 0.6 VVM Culture 30-40°C 30-370C 35°C Temperature Turbulence (dip ithout dip With dip tube with dip tube tube in 400L tube bioreactor) 2012/071083 * ng 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 Bioreactor.

Moniliella pollim's (strain CBS 461.67; Centraalbureau voor Schimmelcultures, Utrecht, 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 culture period to 3 to 4 days, while it took 10 to 12 days for erythritol production without phytic acid (Table la). Each ent c acid, yeast extract, sodium phosphate monobasic, calcium de, glucose, cupric sulfate, zinc chloride, potassium nitrate) was tested for obtaining optimal concentration (Table la). Physical conditions including ion, aeration, 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 e, 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 l 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 e 4. Bioreactor Culture of Moniliella in a 20L Bioreactor.

Agitation speed was found to greatly affect erythritol production. Erythritol was ed 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. 2012/071083 The bioreactor culture with 300 rpm of agitation 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 itol 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 itol 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 s (in g/L) are shown in the table below.

Table 4. Production of Erythritol and Other Products in a 400L Bioreactor Day Glycerol Erythritol 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 Example 6. Purification of Saccharification Product Corn cob was saccharified and the resulting sugar mixture purified by ion exchange.

Cation exchange and anion ge were used to remove the metal components 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 included 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. itol production by two ent strains from purified saccharifled corn cob ning 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 Erythritol 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 nce 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 disclosure as explicitly set forth herein supersedes any conflicting al incorporated herein by reference.

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

While this invention has been particularly shown and described with nces to preferred ments 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 tood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Claims (38)

CLAIMS What is claimed is:

1. A method for making sugar alcohols, the method comprising: combining a slurry of cellulosic or lignocellulosic biomass that contains a first sugar with a microorganism cultured in a medium sing phytic acid, the recalcitrance of the biomass having been reduced by bombardment with electrons, and the microorganism selected from among the species of ella; and utilizing the microorganism to ferment the first sugar to a first sugar alcohol while providing aeration from 0.3 to 1.0 VVM and mixing the slurry with a jet mixer that comprises a jet-flow agitator, the aeration and the agitation speed of the jet mixer being effective, at least in part, to increase production of the first sugar l.

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

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

4. The method of claim 2 or claim 3, further sing purifying the saccharified biomass.

5. The method of claim 4, wherein the purification comprises the removal of metal ions.

6. The method ing to any one of claims 2-5, n the biomass is saccharified with one or more cellulases.

7. The method according to any one of claims 1-6, wherein the first sugar alcohol is selected from the group consisting of: , glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, etraitol, and polyglycitol.

8. The method of claim 7, wherein the sugar alcohol comprises erythritol.

9. The method of any one of claims 1-8, wherein the microorganism is selected from the group consisting of Moniliella pollinis, Moniliella megachiliensis, and Moniliellaacetoabutans.

10. The method of claim 9, wherein the microorganism is M. pollinis.

11. The method of claim 10, wherein the microorganism is M. pollinis strain CBS 461.67.

12. The method of claim 9, wherein the microorganism is M. megachiliensis.

13. The method of claim 12, wherein the microorganism is M. megachiliensis strain CBS 567.85.

14. The method ing to any one of claims 1-13, wherein the cellulosic or lignocellulosic biomass is selected from the group consisting of: paper, paper products, paper waste, paper pulp, pigmented papers, loaded papers, coated papers, filled papers, magazines, printed matter, printer paper, polycoated paper, card stock, cardboard, paperboard, cotton, wood, particle board, forestry , sawdust, aspen wood, wood chips, grasses, grass, thus, cord grass, reed canary grass, grain residues, rice hulls, oat hulls, wheat chaff, barley hulls, agricultural waste, , 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, 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, lentils, peas, and mixtures of any of these.

15. The method according to any one of claims 1-14, wherein the biomass material is a ellulosic biomass.

16. The method according to any one of claims 1-15, further comprising mechanically treating the cellulosic or lignocellulosic biomass to reduce its bulk density and/or increase its surface area.

17. The method according to any one of claims 1-16, further comprising comminuting the cellulosic or lignocellulosic biomass.

18. The method of claim 17, wherein the comminuting is dry milling.

19. The method of claim 17, wherein the uting is wet milling.

20. The method ing to any one of claims 1-19, further comprising separating the first sugar prior to ing the cellulosic or lignocellulosic biomass with the microorganism.

21. The method according to any one of claims 1-20, further comprising trating the first sugar prior to combining the cellulosic or lignocellulosic biomass with the microorganism.

22. The method according to any one of claims 1-21, further comprising culturing the microorganism in a cell growth phase before combining the cellulosic or lignocellulosic biomass with the microorganism.

23. The method of any one of claims 1-22, wherein the lignocellulosic biomass further ns a second sugar, and the method further comprises converting the second sugar to a second sugar alcohol.

24. The method of claim 23, wherein the second sugar is converted to xylitol.

25. The method of claim 23 or 24, wherein the act of converting the second sugar is ted under pressure and utilizes a catalyst.

26. The method ing to any one of claims 23-25, wherein converting the second sugar comprises fermenting the second sugar ing the microorganism.

27. The method of claim 26, wherein the second sugar is converted to erythritol.

28. The method according to any one of claims 23-27, wherein the first sugar is isolated from the second sugar prior to converting the second sugar to the second sugar l.

29. The method according to any one of claims 1-28, n the bombardment with electrons is provided at a dose of at least 5 Mrad.

30. The method according to any one of claims 1-29, wherein the bombardment with electrons is provided at a dose of n 5 Mrad to 50 Mrad.

31. The method according to any one of claims 1-30, wherein the bombardment with ons is provided at a dose of between 20 Mrad to 40 Mrad.

32. The method according to any one of claims 1-31, wherein the bombardment with electrons is provided at a dose rate of greater than 0.25 Mrad/sec.

33. The method according to any one of claims 1-32, wherein the bombardment with electrons is provided at a dose rate of between 0.25 to 2 Mrad/sec.

34. The method according to any one of claims 1-32, wherein the bombardment with electrons is provided at a dose rate of greater than 2 Mrad/sec.

35. The method according to any one of claims 1-34, wherein the jet mixer further ses an impeller, the impeller ng at a rate between 400 to 650 revolutions per minute while mixing.

36. The method according to any one of claims 1-35, wherein the jet mixer further comprises a shaft, and the method further comprising aerating the slurry through a bore in the shaft.

37. The method of claim 36, wherein the bore provides aeration at 0.6 VVM.

38. The method according to any one of claims 1-37, wherein 300 g/L of the first sugar yields at least 80 g/L of the first sugar alcohol, such yield being at least partly attributable to agitation from the jet mixer that comprises a jet-flow agitator. WO 96693 SUBSTITUTE SHEET (RULE 26)