OA16617A - Processing biomass. - Google Patents

Abstract

Biomass (e.g., plant biomass, animal biomass, and municipal waste biomass) is processed to produce useful products, such as fuels. For example, systems are described that can use feedstock materials, such as cellulosic and/or lignocellulosic materials and/or starchy materials, to produce ethanol and/or butanol, e.g., by fermentation.

Description

This application daims priority to U.S. Provisional Application Serial No, 61/139,473 filed Deccmbcr 19, 2008, The complété disclosurc of this provisional application is hereby incorporated by référencé herein.

BACKGROUND

Various carbohydrates, such as ccllulosic and lignoccllulosic materials, e.g., in fibrous form, are produced, proccssed, and used in large quantities in a number of applications. Often such materials arc used once, and then discardcd as wastc, or arc simply considcred to be wastc materials, e.g., sewage, bagasse, sawdust, and stover.

Various cellulosic and lignoccllulosic materials, their uses, and applications hâve been described, for example, in U.S. Patent Nos. 7,307,108, 7,074,918,6,448,307, 6,258,876, 6,207,729, 5,973,035 and 5,952,105.

SUMMARY

Generally, this invention relates to carbohydrate-containing materials (e.g., biomass materials or biomass-derived materials, such as starchy materials and/or cellulosic or lignocelkilosic materials), methods of making and processing such materials to change their structure and/or their rccalcitrancc levd, and products made from the changed materials. For examplc, many of the methods described herein can provide cellulosic and/or lignoccllulosic materials that hâve an oxygen-rich functionality, a lower molccular weight and/or crystallinity relative to a native material. Many of the methods, such as Fenton oxidation methods, provîdc materials that can be more rcadily utilized by a variety of microorganisms (with or without cnzymatic hydrolysis) to produce useful products, such as hydrogen, alcohols (e.g., éthanol or butanol), organic acids (e.g., acetic acid), hydrocarbons, co-products (e.g., proteins) or mixtures of any of these. Many of the products obtained, such as éthanol or n-butanol, can be utilized as fuel, e.g., as an internai combustion fuel or as a fuel cell fccdstock. In addition, the products described herein can

-¼ bc utilizcd for clcctrical power génération, e.g., in a conventional stcam generating plant or in a fuel cell plant.

In one aspect, the invention features methods of changing molecular structures and/or reducing rccalcitrance in materials, such as hydrocarbon-containing materials and/or biomass materials, e.g., ccllulosic or lignocellulosic materials, such as any one or more unproccssed (e.g., eut grass), scmi-processed (e.g., comminuted grass) or processcd materials (e.g., comminuted and irradiated grass) described herein.

The methods can feature oxidativc methods of reducing rccalcitrance in cellulosic or lignocellulosic materials that cmploy Fcnton-typc chcmistry. Fcnton-typc chcmistry is discussed in Pestovsky et al., Angew. Chem., Int. Ed. 2005, 44, 6871-6874, the entire disclosurc of which is hcreby incorporated by référencé herein. The methods can also feature combinations of Fenton oxidation and any other pretreatment method described herein in any order.

Without wishing to be bound by any particular theory, it is believed that oxidation increases the number of hydrogen-bonding groups on the cellulose and/or the lîgnin, such as hydroxyl groups, aldéhyde groups, ketone groups carboxylic acid groups or anhydride groups, which can increasc its dispersability and/or its solubility.

In one aspect, the invention features methods that include contacting, in a mixture, a first ccllulosic or lignocellulosic material having a first level of recalcitrance with one or more compounds comprising one or more naturally-occurring, nonradioactive mctallic éléments, e.g., non-radioactive group 5, 6, 7, 8, 9,10 or 11 éléments, and, optionally, one or more oxidants capable of increasing an oxidation state of at least some of said éléments, to produce a second cellulosic or lignocellulosic material having a second level of rccalcitrance lower than the first level of rccalcitrance.

Other methods include combining a hydrocarbon-containing material with one or more compounds including one or more naturally-occurring, non-radioactive mctallic éléments, e.g., non-radioactive group 5, 6, 7, 8, 9, 10 or 11 éléments to providc a mixture in which the one or more compounds contact the hydrocarbon-containing material; and maintaining the contact for a period of time and under conditions sufficient to change the structure of the hydrocarbon-containing material.

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In some embodiments, the method further includes combining the first cellulosic, lignocellulosic, or hydrocarbon-containing material with one or more oxidants capable of increasing an oxidation statc of al Ieast some of the cléments. In such instances, the onc or more oxidants contact the material with the one or more compounds in the mixture. Jn some embodiments, the onc or more oxidants include ozone and/or hydrogen peroxide.

In some embodiments, the onc or more éléments are in a l +, 2+, 3+, 4+ or 5+ oxidation statc. In particular instances, the onc or more éléments arc in a 2+, 3+ or 4+ oxidation state. For exemple, iron can be In the form of iron(II), iron(III) or iron(lV).

In particular instances, the onc or more cléments include Mn, Fc, Co, Ni, Cu or Zn, preferably Fe or Co. For cxample, the Fe or Co can bc in the form of a sulfate, e.g., iron(II) or iron(lll) sulfate.

In some embodiments, the onc or more oxidants are applied to the first cellulosic or lignocellulosic material and the one or more compounds as a gas, such as by gencrating ozone in-situ by irradiating the first cellulosic or lignocellulosic material and the one or more compounds through air with a beam of particles, such as électrons or protons.

In some embodiments, the mixture further includes one or more hydroquinones, such as 2,5-dimcthoxyhydroquinone and/or one or more benzoquinones, such as 2,5dimcthoxy-l ,4-bcnzoquinone. Such compounds, which hâve similar molccular cntitics as lignin, can aid in électron transfer.

In some désirable embodiments, the one or more oxidants are clectrochemically or electromagnetically generated in-situ. For cxample, hydrogen peroxide and/or ozone can be elcctrochcmically or electromagnetically produccd within a contact or reaction vessel or outsidc the vessel and transferred into the vcsscl.

The methods may further include contacting the second cellulosic or lignocellulosic material with an enzyme and/or microorganism. Products produced by such contact can include any of thosc products described herein, such as food or fuel, e.g., éthanol, or any other products described in U.S. Provisional Application Serial No. 61/139,453, which is hereby incorporated by référence herein in its cntircty.

In another aspect, the invention features Systems that include a structure or carrier, e.g., a reaction vessel, containing a mixture including 1) any material described herein,

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such as a cellulosic or IignocelIulosic material and 2) onc or more compounds comprising one or more naturally-occurring, non-radioactive metallic cléments, e.g., non-radioactive group 5, 6, 7, 8, 9, 10 or 11 cléments. Optionally, the mixture can include 3) one or more oxidants capable of incrcasing an oxidation state of at least some of the éléments.

In another aspect, the invention featurcs compositions that include 1) any material described herein, such as a cellulosic or lignoccllulosic material and 2) one or more compounds comprising one or more naturally-occurring, non-radioactive group 5, 6,7, 8, 9, 10 or 11 éléments. Optionally, the composition can include one or more oxidants capable of incrcasing an oxidation state of at least some of the cléments.

In another aspect, the invention featurcs methods of changing molecular structures and/or reducing rccalcitrance in biomass matcrials, such as cellulosic or lignoccllulosic matcrials. The methods include combining a first lignoccllulosic material having a first level of recalcitrancc with one or more ligninases and/or one or more biomass-destroying, e.g., lignin-destroying organisms, in a manner that the onc or more ligninases and/or organisms contact the first cellulosic or lignoccllulosic material; and maintaining the contact for a period of time and under conditions sufficicnt to produce a second lignoccllulosic material having a second level of rccalcitrance lowcr than the first level of rccalcitrance. The method can further include contacting the second cellulosic or lignoccllulosic material with an enzyme and/or microorganism, e.g., to make any product described herein, e.g., food or fuel, e.g., éthanol or butanol (e.g., n-butanol) or any product described in U.S. Provisional Application Serial No. 61/139,453.

The ligninase can be, e.g., onc or more of manganèse peroxidase, lignin pcroxidasc or laccascs.

The biomass-destroying organism can be, e.g., onc or more of white rot, brown rot or soft rot. For example, the biomass-destroying organism can be a Basidiomycetcs fungus. In particular embodiments, the biomass-destroying organism is Phanerochaete chrysoporiunt or Gleophylluin trabeum.

In certain embodiments, the first material is in the form of a fibrous material that includes fibers provided by shearing a fiber source. Shearing alone can rcducc the crystallinity of a fibrous material and can work syncrgistically with any process technique that also reduces crystallinity and/or molecular weight. For example, the shearing can be

L performcd with a rotary knife cutter. In some embodiments, the fibrous material has an average length-to-diameter ratio of greater than 5/l.

The first and/or second material can bave, e.g., a BET surface arcaof greater than 0.25 m²/g and/or a porosity of greater than about 25 percent.

To further aid in the réduction of the molecular weight of the cellulose, an enzyme, e.g., a cellulolytic enzyme, or a chemical, e.g., sodium hypochloritc, an acid, a base or a swclling agent, can be utilized with any method described herein.

When a microorganism is utilized, it can be a natural microorganism or an cnginccrcd microorganism. For cxamplc, the microorganism can bc a bactcrium, e.g., a cellulolytic bactcrium, a fungus, e.g., a ycast, an enzyme, a plant or a protist, e.g., an algae, a protozoa or a fungus-like protist, e.g., a slime mold. When the organisais are compatible, mixtures may be utilized. Generally, various microorganisms can produce a number of useful products, such as a fuel, by operating on, e.g., fermenting the matcrials. For examplc, alcohols, organic acids, hydrocarbons, hydrogen, proteins or mixtures of any of these matcrials can bc produced by fermentation or other proccsscs.

Examplcs of products that may bc produced include mono- and polyfunctional C1-C6 alkyl alcohols, mono- and poly-functional carboxylîc acids, C1-C6 hydrocarbons, and combinations thereof. Spécifie examples of suitable alcohols include mcthanol, éthanol, propanol, isopropanol, butanol, ethylene glycol, propylene glycol, l,4-butanc diol, glycerin, and combinations thereof. Spécifie cxample of suitable carboxylîc acids include formic acid, acetic acid, propionic acid, bulyric acid, valeric acid, caproic acid, palmitic acid, stcaric acid, oxalic acid, malonic acid, succinic acid, glutaric acid, oleic acid, linolcic acid, glycolic acid, lactic acid, γ-hydroxybutyric acid, and combinations thereof. Examples of suitable hydrocarbons include methane, ethanc, propane, pentane, n-hexanc, and combinations thereof. Many of these products may be used as fuels.

The term “fibrous material,” as used herein, is a material that includes numerous loose, discrète and scparable fibers. For cxamplc, a fibrous material can be prepared from a bleachcd Kraft paper fiber source by shearing, e.g., with a rotary knife cutter.

The term “screen,” as used herein, means a member capable of sieving material according to size. Examples of scrcens include a perforated plate, cylindcr or the like, or a wire mesh or cloth fabric.

The term “pyrolysis,” as used herein, means to break bonds in a material by the application of heat energy. Pyrolysis can occur while the subject material is under vacuum, or immersed in a gascous material, such as ail oxidizing gas, e.g., air or oxygen, or a reducing gas, such as hydrogen,

Oxygen content is measured by elcmcntal analysis by pyrolyzing a sample in a fumacc operating at 1300 °C or above.

Examples of biomass fcedstock include paper, paper products, paperwaste, wood, wood wastes and residues, particle board, sawdust, agricultural waste and crop residues, sewage, silage, grasses, ricc hulls, bagassc, cotton, jute, hemp, fiax, bamboo, sisal, abaca, straw, corn cobs, corn stover, swîtchgrass, alfalfa, hay, ricc hulls, coconut hair, cotton, synthetic celluloses, seawecd, algae, municipal waste, or mixtures of these. The biomass can be or can include a natural or a synthetic material.

The terms “plant biomass” and “lignocellulosic biomass” refer to virtually any plant-derived organic matter (woody or non-woody).

For the purposes of this disclosure, carbohydrates arc materials that are composed cntirely of one or more saccharidc units or that include one or more saccharide units. Carbohydrates can bc polymeric (e.g., cqual to or greater than 10-mer, lOO-mer, 1,000mer, 10,000-mer, or 100,000-mer), oligomcric (e.g., cqual to or greater than a 4-mcr, 5mer, 6-mer, 7-mer, 8-mer, 9-mer or 10-mcr), trimcric, dimcric, or monomcric. When the carbohydrates are formed of more than a single repeat unit, each repeat unit can be the same or different. Examples of polymeric carbohydrates include cellulose, xylan, pectin, and starch, while ccllobiose and lactose arc cxamples of dimcric carbohydrates. Examplcs of monomcric carbohydrates include glucose and xylose. Carbohydrates can bc part of a supramolecular structure, e.g., covalently bonded into the structure. Examplcs of such materials include lignocellulosic materials, such as that found in wood.

A starchy material is one that is or includes significant amounts of starch or a starch dérivative, such as greater than about 5 percent by weight starch or starch dérivative. For purposes of this disclosure, a starch is a material that is or includes an amylosc, an amylopectin, or a physical and/or chemical mixture thereof, e.g., a 20:80 or 30:70 percent by weight mixture of amylosc to amylopectin. For example, rice, corn, and mixtures thereof are starchy materials. Starch dérivatives include, e.g., maltodextrin, t

acid-modified starch, basc-modificd starch, bleached starch, oxidized starch, acetylated starch, acetylated and oxidized starch, phosphatc-modïficd starch, genctically-modified starch and starch that is résistant to digestion.

Swclling agents as used herein are materials that cause a discernable swelling, e.g., a 2.5 percent increasc in volume over an unswollcn statc of cellulosic and/or lignocellulosic materials, when applicd to such materials as a solution, e.g., a water solution. Examplcs include alkalinc substances, such as sodium hydroxidc, potassium hydroxide, lithium hydroxide and ammonium hydroxides, acidifying agents, such as minerai acids (e.g., sulfuric acid, hydrochloric acid and phosphoric acid), salts, such as zinc chloridc, calcium carbonate, sodium carbonate, bcnzyltrîmethylammonium sulfate, and basic organic amines, such as cthylene diaminc.

A “sheared material,” as used herein, is a material that includes discrète ftbers in which at least about 50% of the discrète fibers, hâve a length/diamcter (L/D) ratio of at least about 5, and that has an uncompressed bulk density of less than about 0.6 g/cn?. A sheared material is thus different from a material that has been eut, choppcd or ground.

Changing a molccular structure of a biomass fccdstock, as used herein, means to change the chemical bonding arrangement or conformation of the structure. For cxamplc, the change in the molecular structure can include changing the supramolecular structure of the material, oxidation of the material, changing an average molecular weight, changing an average crystal linity, changing a surface area, changing a degrec of polymcrization, changing a porosity, changing a degrec of branching, grafting on other materials, changing a crystalline domain size, or an changing an overall domain size.

Unless otherwise defined, ail tcchnical and scicntific terms used herein hâve the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Ali publications, patent applications, patents, and other references mentioncd herein or attached hcrcto are incorporatcd by référence in theirentircty for ail that they contain.

Any biomass material, e.g., carbohydratc-containing material, e.g., cellulosic and/or lignocellulosic material described herein can bc utilized in any application or process described in any patent or patent application incorporatcd by reference herein.

Other features and advantages of the invention will bc apparent from the following detailed description, and from the daims.

DESCRIPTION OF DRAWINGS

FIG. I is block diagram illustrating conversion of a fiber source into a first and second fïbrous material.

FIG. 2 is a cross-scctional view of a rotary knife cutter.

FIG. 3 is block diagram illustrating conversion of a fiber source into a first, second and third fïbrous material.

FIG. 4 is a schcmatic cross-sectional sidc view of a reactor.

FIG. 5 shows a sequence of chemical reactions illustrating Fenton chemistry.

FIG. 6 shows a sequence of Fenton réactions illustrating conversion ofbenzène to phénol and toluene to benzaldéhyde and benzyl alcoliol.

FIG. 7 shows a réaction schcme for the préparation of a réactivé iron (IV) compound from an iron (II) compound.

FIG. 8 shows a proposed pathway for réduction of Fe (III) and production of hydrogen peroxide in the présence of 2,5-dimcthoxyhydroquinonc.

FIG. 9 is a scanning électron micrograph of a fïbrous material produced from polycoatcd paper at 25 X magnifîcation. The fïbrous material was produced on a rotary knife cutter utilizing a scrccn with I/8 inch openings.

FIG. 10 is a scanning électron micrograph of a fïbrous material produced from bleached Kraft board paper at 25 X magnifîcation. The fïbrous material was produced on a rotary knife cutter utilizing a scrccn with 1/8 inch openings.

FIG. 11 is a scanning électron micrograph of a fïbrous material produced from bleached Kraft board paper at 25 X magnifîcation. The fïbrous material was twice sheared on a rotary knife cutter utilizing a scrccn with 1/16 inch openings during each shearing.

FIG. 12 is a scanning électron micrograph of a fïbrous material produced from bleached Kraft board paper at 25 X magnifîcation. The fïbrous material was thrice sheared on a rotary knife cutter, During the first shearing, a 1/8 inch screen was used;

during the second shearing, a 1/16 inch screen was used, and dtiring the third shearing a 1/32 inch screen was used.

DETAILED DESCRIPTION

Using the methods described herein, biomass can be processed to a lower levé! of recalcitrance and converted into useful products such as fuels. Systems and proccsscs arc described below that can use as fecdstocks materials such as cellulosic and/or lignoccllulosic materials that arc rcadily availablc, but can bc difficult to proccss, for exemple, by saccharification and/or by fermentation. In some implémentations the feedstock materials arc first physically prepared for proccssing, for cxample by size réduction. The physically prepared feedstock is then pretreated using oxidation (e.g., using Fenton-typc chcmistry), and may in some cases be further treated with one or more of radiation, sonication, pyrolysis, and stcam explosion. Altcmatively, in some cases, the feedstock is first treated with one or more of radiation, sonication, pyrolysis, and stcam explosion, and then treated using oxidation, e.g., Fcnton-typc chcmistry.

Preferred oxidative methods for rcducing recalcitrance in cellulosic or lignocellulosic materials include Fcnton-type chcmistry, discussed above, in which onc or more group 5, 6, 7, 8, 9, 10 or 11 cléments, optionally along with one or more oxidants capable of increasing an oxidation statc of at least some of the éléments arc utilized.

After pretreatment, the pretreated material can be further processed, e.g., using primary proccsscs such as saccharification and/or fermentation, to produce a product.

TYPES OF BIOMASS

Generally, any biomass material that is or includes carbohydrates composcd cntirely of one or more saccharidc units or that include one or more saccharidc units can be processed by any of the methods described herein. For cxample, the biomass material can bc cellulosic or lignoccllulosic materials, or starchy materials, such as kernels of corn, grains of rice or other foods.

Fibcr sources include cellulosic fïber sources, including paper and paper products (e.g., polycoatcd paper and Kraft paper), and lignocellulosic fibcr sources, including wood, and wood-related materials, e.g., particle board. Other suitable fibcr sources include nalural fibcr sources, e.g., grasses, rice hulls, bagassc, cotton, jute, hemp, flax, bamboo, sisal, abaca, straw, corn cobs, rice huiis, coconut liair; fiber sources high in accllulosc content, e.g., cotton; and synthetic fiber sources, e.g., extruded yam (oriented yam or un-oriented yam). Natural or synthetic fiber sources can be obtained from virgin scrap textile materials, e.g., remnants or they can be post consumer wastc, e.g., rags. When paper products are used as fiber sources, they can be virgin materials, e.g., scrap virgin materials, or they can be post-consumcr wastc. Asidc from virgin raw materials, post-consumcr, industrial (e.g., offal), and processing wastc (e.g., effluent from paper processing) can also be used as fiber sources. Also, the fiber source can be obtained or derived from human (e.g., sewage), animal or plant wastes. Additional fiber sources hâve been described in U.S. Patent Nos. 6,448,307, 6,258,876,6,207,729, 5,973,035 and 5,952,105, the full disclosurcs of which arc incorporatcd by référence herein.

Starchy materials include starch itsclf, e.g., corn starch, wheat starch, potato starch or rice starch, a dérivative of starch, or a material that includes starch, such as an edible food product or a crop. For cxamplc, the starchy material can be arracacha, buckwheat, banana, barley, cassava, kudzu, oca, sago, sorghum, rcgular household potatocs, sweet potato, tara, yams, or one or more beans, such as favas, lentils or peas. Blends of any one or more starchy material is also a starchy material. In particular embodiments, the starchy material is derived from corn. Various corn starches and dérivatives are described in “Corn Starch,” Corn Refiners Association (11”' Edition, 2006), which is hcrcby incorporatcd by référence herein.

Blends of any biomass materials described herein can be utilized for making any of the products described herein, such as éthanol. For example, blends of cellulosic materials and starchy materials can be utilized for making any product described herein.

FEED PREPARATION

In some cases, methods of processing begin with a physical préparation of the feedstock, e.g., size réduction of raw feedstock materials, such as by cutting, grinding, shearing or choppïng. In some cases, loose feedstock (e.g., rccyclcd paper, starchy materials, or switchgrass) is prepared by shearing or shredding. Screcns and/or magnets can be used to remove oversized or undcsirablc objccts such as, for cxamplc, rocks or nails from the fecd stream.

Fecd préparation Systems can be confïgured to produce feed strcams with spécifie characteristics such as, for example, spécifie maximum sizes, spécifie length-to-width, or spécifie surface areas ratios. As a part of fecd préparation, the bulk density of fcedstocks can be controlled (e.g., increascd). If desired, lignin can be removed from any fcedstock that includcs lignin.

Slze Réduction

In somc embodiments, the material to be proccsscd is in the form of a fibrous material that includcs fibers provided by shearing a fiber source. For cxample, the shearing can be performed with a rotary knife cutter.

For cxample, and by reference to FIG. 1, a fiber source 210 is sheared, e.g., in a rotary knife cutter, to provide a first fibrous material 212. The first fibrous material 212 is passed through a first scrccn 214 having an average opening size of 1.59 mm or less (1/16 inch, 0.0625 inch) to providc a second fibrous material 216. If desired, fiber source can be eut prior to the shearing, e.g., with a shredder.

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

For examplc, a rotary knife cutter can be used to concurrently shear the fiber source and screen the first fibrous material. Other methods of making the fibrous materials include, e.g., stone grinding, mechanical ripping or tcaring, pin grinding or air attrition milling. Refcrring to FIG. 2, a rotary knife cutter 220 includcs a hopper 222 that can be loaded with a shreddcd fiber source 224. The shrcddcd fiber source is sheared between stationary biades 230 and rotating bladcs 232 to providc a first fibrous material 240. First fibrous material 240 passes through scrccn 242, and the resulting second fibrous material 244 is capturcd in bin 250. To aid in the collection of the second fibrous material, a vacuum source 252 can be utilized to maintain the bin at a pressure below nominal atmosphcric pressure, e.g., at least 10, 25, 50 or 75 percent below nominal atmosphcric pressure.

Shearing can be advantageous for “opening up” and “stressing” the fibrous materials, making the cellulose of the materials more susceptible to chain scission and/or réduction of crystallinity. The open materials can also be more susceptible to oxidation.

The fiber source can be shcarcd in a dry state, a hydratcd statc (e.g., having up to ten percent by weight absorbed water), or in a wct state, e.g., having between about 10 percent and about 75 percent by weight water. The fiber source can even be sheared while partially or fully submerged under a liquid, such as water, éthanol, isopropanol. The fiber source can also be shcarcd under a gas (such as a stream or atmosphère of gas other than air), e.g., oxygen or nitrogen, or steam.

In some embodiments, the average opening size of the first scrcen 214 is less than 0.79 mm (0.031 inch), e.g., less than 0.51 mm (0.020 inch), 0.40 mm (0.015 inch), 0.23 mm (0.009 inch), 0,20 mm (0.008 inch), 0.18 mm (0.007 inch), 0.13 mm (0.005 inch), or even less than less than 0.10 mm (0.004 inch). The characteristics of suitable screens are described, for cxamplc, in US 2008-0206541. In some embodiments, the open area of the mesh is less than 52%, e.g., less than 41%, less than 36%, less than 31%, or less than 30%.

In some embodiments, the second fibrous is sheared and passed through the first screen, or a different sized screen. In some embodiments, the second fibrous material is passed through a second screen having an average opening size cquai to or less than that of first screen. Referring to FIG. 3, a third fibrous material 220 can be prepared from the second fibrous material 216 by shearing the second fibrous material 216 and passing the resulting material through a second screen 222 having an average opening size less than the first screen 214. In such instances, a ratio of the average length-to-diamctcr ratio of the second fibrous material to the average lcngth-to-diameter ratio of the third fibrous material can be, e.g., less than 1.5, e.g., less than 1.4, less than 1.25, or even less than 1.1.

Generally, the fibers of the fibrous materials can hâve a relatively large average lcngth-to-diameter ratio (e.g., greater than 20-to-1), even if they hâve been sheared more than once. In addition, the fibers of the fibrous materials described herein may hâve a relatively narrow length and/or length-to-diameter ratio distribution.

As used herein, average fiber widths (i.e., diameters) are thosc determined optically by randomly selecting approximately 5,000 fibers. Average fiber lengths are correctcd length-weightcd lengths. BET (Brunaucr, Emmet and Tcllcr) surface areas are multi-point surface areas, and porosities are those determined by mercury porosimetry.

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The average length-to-diameier ratio of thc second fibrous material 14 can be, e.g. greater than 8/1, e.g., grcatcrthan 10/1, greater than 15/1, grcater than 20/1, greater than 25/1, or greater than 50/1. An average Icngth of the second Elbrous material 14 can be, e.g., between about 0.5 mm and 2.5 mm, e.g., between about 0.75 mm and 1.0 mm, and an average width (i.e., diameter) of thc second fibrous material 14 can bc, e.g., between about 5 μπι and 50 pm, e.g., between about 10 pm and 30 pm.

In some embodiments, a standard déviation of thc Icngth of the second fibrous material 14 is less than 60 percent of an average Icngth of thc second fibrous material 14, e.g., less than 50 percent of thc average Icngth, less than 40 percent of thc average Icngth, less than 25 percent of the average Icngth, less than 10 percent of thc average length, less than 5 percent of the average Icngth, or even less than 1 percent of thc average length.

In some embodiments, a BET surface area of thc second fibrous material is greater than 0.1 m²/g, e.g., greater than 0.25 m²/g, greater than 0.5 m²/g, greater than 1.0 m²/g, greater than 1.5 m²/g, grcater than 1.75 m²/g, greater than 5.0 m²/g, greater than 10 m²/g, grcater than 25 m²/g, grcater than 35 m²/g, grcater than 50m²/g, grcater than 60 m²/g, greater than 75 m²/g, grcater than 100 m²/g, grcater than 150 m²/g, grcater than 200 m²/g, or even greater than 250 m²/g.

A porosity of the second fibrous material 14 can bc, e.g., grcater than 20, 25, 35, 50, 60, 70, 80, 85, 90, 92, 94, 95, 97.5 or 99 percent, or even grcater than 99.5 percent.

In some embodiments, a ratio of the average lcngth-to-diamcter ratio of the first fibrous material to the average length-to-diameter ratio of the second fibrous material is, e.g., less than 1.5, e.g., less than 1.4, less than 1.25, less than 1.1, less than 1.075, less than 1.05, less than 1.025, or even substantially cqual to 1.

In some embodiments, thc third fibrous material is passed through a third screcn to produce a fourth fibrous material. Thc fourth fibrous material can bc, e.g., passed through a fourth screcn to produce a fifth material. Similar screening processes can bc repeated as many times as desired to produce the desired fibrous material having the desired properties.

In some implémentations, thc size réduction equipment may bc portable, e.g., in the manner of the mobile processing equipment described in U.S. Provisional Patent

Application Serial 60/832,735, now Published International Application No. WO 2008/011598.

PRETREATMENT

Physically prepared fecdstock can be pretreated for use in primary production proccsscs such as saccharification and fermentation by, for example, reducing the average molecular weight and crystallinity of the fecdstock and/or increasing the surface area and/or porosity of the fecdstock. Prctreatmcnt processes include utilizing Fcntontype chcmistry, discussed above, and can further include one or more of irradiation, sonication, oxidation, pyrolysis, and stcam explosion.

Fenton Chemistry

In some embodiments, the one or more éléments used in the Fenton reaction are in a 1+, 2+, 3+, 4+ or 5+ oxidation state. In particular instances, the one or more éléments include Mn, Fc, Co, Ni, Cu or Zn, preferably Fc or Co. For cxample, the Fc or Co can be in the form of a sulfate, e.g., iron(II) or iron(lll) sulfate. In particular instances, the one or more cléments are in a 2+, 3+ or 4+ oxidation state. For example, iron can be in the form of iron(II), iron(III) or iron(IV).

Exemplary îron (II) compounds include ferrous sulfate heptahydrate, iron(II) acctylacctonatc, (+)-iron(lI) L-ascorbatc, iron(Il) bromide, iron(II) chloride, iron(II) chloride hydrate, iron(II) chloride tetrahydrate, iron(Il) ethylenediammonium sulfate tetrahydrate, iron(Il) fluoride, iron(II) gluconatc hydrate, iron(II) D-gluconate dehydrate, iron(Il) iodide, iron(H) lactate hydrate, iron(II) molybdatc, iron(ll) oxalate dehydrate, iron(ll) oxide, iron(II,III) oxide, iran(II) perchloratc hydrate, iron(II) phthalocyanine, iron(II) phthalocyanine bîs(pyridinc) complex, iron(Il) sulfate heptahydrate, iron(II) sulfate hydrate, iron(II) sulfidc, iron(II) tetrafluoroboratc hcxahydratc, iron(II) titanatc, ammonium iron(II) sulfate hexahydrate, ammonium iron(II) sulfate, cyclopentadicnyl iron(II) dicarbonyl dimer, ethylcncdiaminetetraacctic acid hydrate iron(lll) sodium sait and ferrie citrate.

Exemplary iron (III) compounds include iron(III) acctylacctonatc, iron(lll) bromide, iron(III) chloride, iron(lll) chloride hexahydrate, iron(III) chloride solution, iron(III) chloride on silica gel, iron(III) citrate, tribasic monohydratc, iron(lll) ferrocyanidc, iron(lll) fluoride, iron(Ill) fluoride trihydrate, iron(III) nitrate nonahydratc, iron(III) nitrate on silica gel, iron(III) oxalate hexahydrate, iron(III) oxide, iron(lll) pcrchlorate hydrate, iron(lll) phosphate, iron(Ill) phosphate dehydrate, îron(lll) phosphate hydrate, iron(lll) phosphate tetrahydrate, ϊιόιι(ΠΙ) phthalocyaninc chloride, iron(III) phthalocyanine-4,4',4,4'-tetrasulfonic acid, compound with oxygen hydrate monosodium sait, iron(III) pyrophosphate, iron(IIl) sulfate hydrate, iron(IIl) ptolucnesulfonate hexahydrate, iron(III) tris(2,2,6,6-tetramcthyl-3,5-heptanedionatc) and ammonium iron(IIl) citrate.

Exemplary cobalt (II) compounds include cobalt(II) acetate, cobalt(II) acetate tetrahydrate, cobalt(II) acctylacetonate hydrate, cobalt(II) benzoylacetonate, cobalt(II) bromide, cobalt(II) bromidc hydrate and cobalt(II) carbonate hydrate.

Exemplary' cobalt (111) compounds include cobalt(lll) acctylacetonate, cobalt(lll) fluoride, cobalt(lll) oxide, cobalt(IIl) sepulchrate trichloridc, hcxaminc cobalt(III) chloride, bis(cyclopentadicnyi)cobalt(lll) hexafluorophosphate and bis(cthylcyclopcntadienyl)cobalt(IIl) hexafluorophosphate.

Exemplary oxidants include pcroxîdes, such as hydrogen peroxide and bcnzoyl peroxide, pcrsulfatcs, such as ammonium persulfate, activatcd forms of oxygen, such as ozone, permanganates, such as potassium permanganate, pcrchloratcs, such as sodium pcrchlorate, and hypochlorites, such as sodium hypochlorite (houschold bleach).

Generally, Fcnton oxidation occurs in an oxidizing environment. For cxample, the oxidation can bc effectcd or aidcd by pyrolysis in an oxidizing environment, such as in air or argon cnriched in air. To aid in the oxidation, various chcmical agents, such as oxidants, acids or bases can be added to the material prior to or during oxidation. For cxample, a peroxide (e.g., benzoyl peroxide) can be added prior to oxidation.

In some cases, pH is maintained at or below about 5.5 during contact, such as between 1 and 5, between 2 and 5, between 2.5 and 5 or between about 3 and 5. The contact period may bc, for example, between 2 and 12 hours, e.g., between 4 and 10 hours or between 5 and 8 hours. In some instances, the reaction conditions arc controllcd so that the température does not exceed 300 °C, e.g., the température rcmains less than

250, 200, 150, 100 or even less than 50 °C. in some cases, the température rcmains substantially ambient, e.g., at or about 20-25 °C.

Referring to FIG. 4, reactive mixtures 2108 within a vessel 2110 can be prepared using various approachcs. For exampie, in instances in which the mixture includes one or more compounds and one or more oxidants, the first cellulosic or lignocellulosic material can be first disperscd in water or an aqueous medium, and then the one or more compounds can be added, followed by addition of the one or more oxidants. Altcmatively, the one or more oxidants can added, followed by the one or more compounds, or the one or more oxidants and the one or more compounds can be concurrcntly added separately to the dispersion (e.g., each added independently through a separate addition device 2120, 2122 to the dispersion).

In some embodiments, a total maximum concentration of the éléments in the one or more compounds mcasured in the dispersion is from about 10 μΜ to about 500 mM, e.g., between about 25 μΜ and about 250 mM or between about 100 μΜ and about 100 mM, and/or a total maximum concentration of the onc or more oxidants is from about 100 μΜ to about 1 M, e.g., between about 250 μΜ and about 500 mM, or between about 500 qm and 250 mM. In some embodiments, the mole ratio of the éléments in the onc or more compounds to the one or more oxidants is from about 1:1000 to about 1:25, such as from about 1:500 to about 1:25 or from about 1:100 to about 1:25.

In some cases, the one or more oxidants arc applied to the first cellulosic or lignocellulosic material and the one or more compounds as a gas, such as by generating ozone in-situ by irradîating the first cellulosic or lignocellulosic and the one or more compounds through air with a beam of particles, such as électrons or protons.

In other cases, the first cellulosic or lignocellulosic material is first dispersed in water or an aqueous medium that includes the one or more compounds dispersed and/or dissolved therein, and then water is removed after a soak time (e.g., loose and free water is removed by filtration), and then the onc or more oxidants arc applied to the combination as a gas, such as by generating ozone in-situ by irradîating the first cellulosic or lignocellulosic and the one or more compounds through air with a beam of particles, such as électrons (e.g., each being accelcratcd by a potential différence of between 3 McV and 10 McV).

• ·

Refcrring now to FIG. 5, in some particular embodiments, an iron (H) compound is utilîzed for the Fenton-type chemistry, such as iron (II) sulfate, and hydrogen peroxide is utilîzed as the oxidant. FIG. 5 illustrâtes that in such a system, hydrogen peroxide oxidizes the iron (11) to generate iron (111), hydroxyl radicals and hydroxide ions (équation I). The hydroxyl radicals can then rcact with the first cellulosic or lignocellulosic material, thereby oxidizing it to the second cellulosic or lignocellulosic material. The iron (III) thus produccd can be reduced back to iron (II) by the action of hydrogen peroxide and hydroperoxyl radicals (équations 2 and 3). Equation 4 illustrâtes that it is also possible for an organic radical (R) to rcducc iron (III) back to iron (II).

FIG. 6 illustrâtes that iron (II) sulfate and hydrogen peroxide in aqueous solutions and at pH below about 6 can oxidize aromatic rings to give phénols, aldéhydes and alcohols. When applied to cellulosic or lignocellulosic material, thèse Fenton-type reactions can hclp cnhance the solubility of the lignocellulosic material by functionalization of the lignin and/or cellulose or hemicellulose, and by réduction in molecular weight of the lignocellulosic material. The net effect of the Fenton-type reactions on the lignocellulosic material can bc a change in molecular structure and/or a réduction in its rccalcitrancc.

FIG. 7 shows that hydrated iron (II) compounds, such as hydrated iron (II) sulfate, can react with ozone in aqueous solutions to generate extrcmely reactive hydrated iron (IV) compounds that can react with and oxidize cellulosic and lignocellulosic materials.

In some désirable embodiments, the mixture further includes onc or more hydroquinones, such as 2,5-dimethoxyhydroquinonc (DMHQ) and/or one or more benzoquinones, such as 2,5-dimethoxy-l,4-benzoquinonc (DMBQ), which can aid in électron transfer reactions. FIG. 8 illustrâtes how iron (III) can be reduced by DMHQ to give iron (II) and DMHQ semi-quinone radical. Addition of oxygen to the scmi-quinonc then gives alpha-hydroxyperoxyl radical that éliminâtes HOO to give DMBQ. Finally, HOO oxidizes iron (II) or dismutates to generate hydrogen peroxide.

In some désirable embodiments, the one or more oxidants arc clcctrochcmically or electromagnetically generated in-situ. For cxample, hydrogen peroxide and/or ozone can be clectrochemically or electromagnetically produccd within a contact or reaction vessel.

In some implémentations, the Fenton réaction vessel may be portable, e.g., in the manner of the mobile proccssing equipment described in U.S. Provisional Patent Application Serial 60/832,735, now Publishcd International Application No. WO 2008/011598.

Radiation Treatment

Before, during or after the Fenton oxidation discussed above, one or more irradiation proccssing sequences can be used to pretreat the fcedstock. Irradiation can rcduce the molecular weight and/or crystallinity of fcedstock. In some embodiments, energy deposited in a material that rclcascs an électron from its atomic orbital is used to irradiatc the matcrials. The radiation may be provided by I) heavy charged particles, such as alpha particles or protons, 2) électrons, produced, for example, in beta dccay or électron bcam accclcrators, or 3) electromagnctic radiation, for examplc, gamma rays, x rays, or ultraviolet rays. In one approach, radiation produced by radioactive substances can bc used to irradiatc the fcedstock. In some embodiments, any combination in any order or concurrcntly of (1) through (3) may bc utilized. In another approach, electromagnctic radiation (e.g., produced using électron bcam emitters) can be used to irradiate the feedstock. The doses applicd dépend on the desired effcct and the particular fcedstock. For example, high doses of radiation can break chemical bonds within feedstock componcnts and low doses of radiation can increase chemical bonding (e.g., cross-linking) within fcedstock componcnts. In some instances when chain scission is désirable and/or polymer chain functionalization is désirable, particles heavicr than électrons, such as protons, hélium nuclci, argon ions, silicon ions, néon ions carbon ions, phoshorus ions, oxygen ions or nitrogen ions can bc utilized. When ring-opening chain scission is desired, positivcly chargcd particles can be utilized for their Lewis acid properties for enhanced ring-opening chain scission. For example, when maximum oxidation is desired, oxygen ions can bc utilized, and when maximum nitration is desired, nitrogen ions can be utilized.

Doses ln some embodiments, the irradiating (with any radiation source or a combination of sources) is performed until the material rcceivcs a dose of at least 0.25 Mrad, e.g., at least 1.0 Mrad, 2.5 Mrad, 5.0 Mrad, 10.0 Mrad, 25 Mrad, 50 Mrad, or evcn at least 100 Mrad. In some embodiments, the irradiating is performed until the material reçoives a dose of between 1.0 Mrad and 6.0 Mrad, e.g., between 1.5 Mrad and 4.0 Mrad.

In some embodiments, the irradiating is performed at a dose rate of between 5.0 and 1500.0 kilorads/hour, e.g., between 10.0 and 750.0 kilorads/hour or between 50.0 and 350.0 kilorads/hours.

In some embodiments, two or more radiation sources are used, such as two or more ionizing radiations. For example, samplcs can bc treated, in any order, with a beam of électrons, followcd by gamma radiation and UV light having wavclcngths from about 100 nm to about 280 nm.

In some embodiments, rclatively low doses of radiation can crosslink, graft, or otherwise increasc the molecular weight of a carbohydrate-containing material, such as a ccllulosic or lignocellulosic material (e.g., cellulose). For cxample, a fïbrous material that includes a first ccllulosic and/or lignocellulosic material having a first molecular weight can bc irradîated in such a manner as to provide a second ccllulosic and/or lignocellulosic material having a second molecular weight higher than the first molecular weight. For example, if gamma radiation is utilized as the radiation source, a dose of from about 1 Mrad to about 10 Mrad, about 1 Mrad to about 75 Mrad, or about 1 Mrad to about 100 Mrad can bc applied. In some implémentations, from about 1.5 Mrad to about 7.5 Mrad or from about 2.0 Mrad to about 5.0 Mrad, can be applied.

Sonication, Pyrolysis, Oxidation, and Steam Explosion

One or more sonication, pyrolysis, oxidativc processing, and/or steam explosion can be used to further pretreat the fccdstock. Such processing can rcduce the molecular weight and/or crystallinity of fecdstock and biomass, e.g., one or more carbohydrate sources, such as ccllulosic or lignocellulosic materials, or starchy materials. These processes are described în detail in U.S. Serial No. 12/429,045.

In some embodiments, biomass can bc processed by applying two or more of any of the processes described herein, such Fenton oxidation combined with any one, two or more of radiation, sonication, oxidation, pyrolysis, and steam explosion either with or without prior, intermediate, or subséquent physical fcedstock préparation. The processes can bc applied in any order or concurrcntly to the biomass. Multiple processcs can in some cases provide matcrials that can be more rcadily utilized by a variety of microorganisms because οΓ their lower molecular weight, lower crystallinity, and/or enhanced solubility. Multiple proccsscs can provide synergies and can reduce overall cnergy input rcquired in comparison to any single process,

PRIMARY PROCESSING

Primary processing of the pretreated fecdstock may include bioproccsscs such as saccharifying and/or fermenting the fccdstock, e.g., by contacting the pretreated material with an enzyme and/or microorganism. Products produced by such contact can include any of those products described herein, such as food or fuel, e.g., éthanol, or any other products described in U,S. Provisional Application Serial No. 61/139,453.

Fermentation

Gcncrally, various microorganisms can producc a number of uscful products, such as a fuel, by operating on, e.g., fermenting the pretreated biomass matcrials. For cxamplc, alcohols, organic acids, hydrocarbons, hydrogen, proteins or mixtures of any of these matcrials can be produced by fermentation or other bioproccsscs.

The microorganism can be a naturel microorganism or an cnginccrcd microorganism. For examplc, the microorganism can bc a bacterium, e.g., a cellulolytic bactcrium, a fungus, e.g., a yeast, a plant or a protist, e.g., an algae, a protozoa or a fungus-like protist, e.g., a slime mold. When the organisais arc compatible, mixtures of organisais can bc utilized.

To aid in the breakdown of the matcrials that include the cellulose, one or more enzymes, e.g., a cellulolytic enzyme can be utilized. In some embodiments, the matcrials that include the cellulose arc first treated with the enzyme, e.g., by combining the material and the enzyme in an aqueous solution. This material can then bc combined with the microorganism. In other embodiments, the matcrials that include the cellulose, the one or more enzymes and the microorganism arc combined concurrcntly, e.g., by combining in an aqueous solution.

The pretreatcd material can be treated with beat and/or a chcmical (e.g., minerai acid, base or a strong oxidizer such as sodium hypochlorite) to further facilitatc breakdown.

During fermentation, sugars released from cellulolytic hydrolysis or saccharification are fermented to, e.g., éthanol, by a fermenting microorganism such as ycast. Suitable fermenting microorganisms hâve the ability to convcrt carbohydrates, such as glucose, xylose, arabinose, mannose, galactose, oligosaccharides or polysaccharides into fermentation products. Fermenting microorganisms include strains of the genus Sacchromyces spp. e.g., Sacchromyces cerevisiae (bukcr’s ycast), Saccharomyces distaticus, Saccharomyces uvarunv, the genus Kluyveromyces, e.g., spccies Kluyveromyces marxianus, Kluyveromyces fragilis', the genus Candida, e.g., Candida pseudotropicalis, and Candida brassicat, the genus Clavispora, e.g., spccies Clavispora lusitaniae and Clavispora opuntiae the genus Pachysolen, e.g., spccies Pachysolen tannophilus, the genus Bretannomyces, e.g., spccies Bretannomyces clausenii (Philippidis, G. P., 1996, Cellulose bioconversion technology, in Handbook on Biocthanol: Production and Utilization, Wyman, C.E., cd., Taylor & Francis, Washington, DC, 179-212).

Commercially available yeasts include, for cxamplc, Rcd StaiO/Lcsaffre Ethanol Rcd (available from Red Star/Lcsaffre, USA) FALI® (available from Fleischmann’s Yeast, a division of Bums Philip Food Inc., USA), SUPERSTART® (available from Alltcch), GERT STRAND® (available from Gcrt Strand AB, Swcdcn) and FERMOL® (available from DSM Specialties).

Bactcria that can ferment bimoss to éthanol and other products include, e.g., Zymomonas mobilis and Clostridium thermocellum (Philippidis, 1996, supra). Leschine et al. (International Journal ofSyslematic and Evolutionary Microbiology 2002,52, 1155-1160) isolated an anacrobic, mcsophilic, cellulolytic bactcrium from forest soil, Clostridium phytofermentans sp. nov., which converts cellulose to éthanol.

Fermentation of biomass to éthanol and other products may be carricd out using certain types of thermophilic or gcnctically cnginccrcd microorganisms, such Thcrmoanaerobactcr spccies, including T. malhranii, and yeast spccies such as Pichia specîes. An example of a strain of T. mathranii is A3M4 described in Sonnc-Hansen et al. (Applied Microbiology and Biotechnology 1993, 38, 537-541 ) or Ahring et al. (Arch. Microbiol. 1997,168, 114-119).

Yeast and Zymomonas bacterïa can be used for fermentation or conversion. 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 arc about 24 to 96 hours with températures in the range of 26 °C to 40 °C, however thcrmophilic microorganisms prefer higher températures.

Enzymes and biomass-destroying organisms that break down biomass, such as the cellulose and/or the lignin portions of the biomass, to lowcr molecular weight ofthe carbohydrate-containing matcrials contain or make various ccllulolytic enzymes (cellulascs), ligninases or various small molécule biomass-destroying métabolites. These enzymes may be a complex of enzymes that act syncrgistically to dégrade crystalline cellulose or the lignin portions of biomass. Examples of ccllulolytic enzymes include: cndoglucanases, ccllobiohydrolascs, and ccllobiascs (β-glucosidascs). A cellulosic substrate is initially hydrolyzed by cndoglucanases at random locations producing oligomcric intermediates. These intermediates arc then substrates for cxo-splitting glucanascs such as ccllobiohydrolase to produce cellobiosc from the ends of the cellulose polymcr, Cellobiose is a water-solublc 1,4-linkcd dimer of glucose. Finally cellobiase cleaves cellobiosc to yield glucose.

A cellulase is capable of degrading biomass and may be of fungal or bacterial origin. Suitable enzymes include cellulascs from the gênera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, Chrysosporium and Trichoderma, and include spccies of Humicola, Coprinus, Thielavia, Fusarium, Myceliophthora, Acremonium, Cephalosporium, Scytalidium, Pénicillium or Aspergilltis (see, e.g., EP 458162), especially those produccd by a strain selected from the spccics Humicola insolens (rcclassifîcd as Scytalidium thermophilum, sce, e.g., U.S. Patent No. 4,435,307), Coprinus cinereus, Fusarium oxysporum, Myceliophthora thermophila, Meripilus giganteus, Thielavia terrestris, Acremonium sp., Acremonium persicinum, Acremonium acremonium, Acremonium brachypenium, Acremonium dichromosporum, Acremonium obclavatum, Acremonium pinkertoniae, Acremonium roseogriseum, Acremonium incoloratum, and Acremonium furatum-, preferably from the spccies Humicola insolens • k

DSM 1800, Fusarium oxysportmi DSM 2672, Myceliaphthora thermophila CBS 117.65, Cephalosporium sp. RYM-202, Acremonium sp. CBS 478.94, Acremonium sp. CBS 265.95, Acremoniumpersicinum CBS 169.65, Acremonium acremonium AHU 9519, Cephalosporium sp. CBS 535.71, Acremonïum brachypenium CBS 866.73, Acremonium dichromosporum CBS 683.73, Acremonium obclavatum CBS 311.74, Acremonium pinkertoniae CBS 157.70, Acremonium roseogriseum CBS J 34.56, Acremonium incoloratum CBS 146.62, and Acremonium furatum CBS 299.70H. Ccllulolytic enzymes may also bc obtained from Chrysosporium, preferably a strain of Chrysosporium lucknowense. Additionally, Trichoderma (particularly Trichoderma viride, Trichoderma reesei, and Trichoderma koningiï), alkalophilic Bacillus (see, for cxample, U.S. Patent No. 3,844,890 and EP 458162), and Streptomyces (sec, e.g., EP 458162) may bc used.

Anacrobic ccllulolytic bacteria hâve also bcen isolated from soil, e.g., a novei ccllulolytic specics of Clostiridium, Clostridium phylofermentans sp. nov. (sec Leschine et. al, International Journal ofSystematic and Evolutionary Microbiology (2002), 52, 1155-1160).

Cellulolytic enzymes using recombinant tcchnology can also be used (sec, e.g., WO 2007/071818 and WO 2006/110891).

The cellulolytic enzymes used can be produced by fermentation of the abovenoted microbial strains on a nutrient medium containing suitable carbon and nitrogen sources and inorganic salts, using procedures known in the art (sec, e.g., Bennett, J.W. and LaSure, L. (cds.), More Gene Manipulations in Fungi, Academie Press, CA 1991 ). Suitable media arc available from commercial supplicrs or may be prepared according to publishcd compositions (e.g., in catalogues of the Amcrican Type Culture Collection). Température ranges and other conditions suitable for growth and cellulase production are known in the art (see, e.g., Bailcy, J.E., and Ollis, D.F., Biochemical Engineering Fundamentals, McGraw-Hill Book Company, NY, 1986).

Treatment of cellulose with cellulase is usually carricd out at températures between 30 °C and 65 °C. Cellulases arc active over a range of pH of about 3 to 7. A saccharification step may last up to 120 hours. The cellulase enzyme dosage achicvcs a sufficiently high level of cellulose conversion. For cxample, an appropriate cellulase dosage is typieally between 5.0 and 50 Filter Paper Units (FPU or 1U) per gram of cellulose. The FPU is a standard mcasurcmcnt and is defincd and mcasurcd according to Ghose (l 987, Pure and Appl. Chem. 59:257-268).

Mobile fermentors can be utilized, as described in U.S. Provisional Patent Application Serial 60/832,735, now Published International Application No. WO 5 2008/011598.

PRODUCTS / CO-PRODUCTS

Using such primary proccsscs and/or post-proccssing, the treated biomass can be converted to one or more products, for cxample alcohols, e.g., methanol, éthanol, propanol, isopropanol, butanol, e.g., n-, sec- or t-butanol, ethylene glycol, propylene glycol, 1,4-butane diol, glycerin or mixtures of these alcohols; organic acids, such as formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic, palmitic acid, stcaric acid, oxalic acid, malonic acid, succinic acid, glutaric acid, oleic acid, linolcic acid, glycolic acid, lactic acid, γ-hydroxybutyric acid or mixtures of these acids; food products; animal feed; pharmaccuticals; or nutriccuticals. Co-products that may be produced include lignin residue.

EXAMPLES

The following Examplcs arc intended to illustratc, and do not limit the teachingsof this disclosure.

Example 1 - Préparation Of Fibrous Material From Polycoated Papar

A 1500 pound skid of vîrgin, half-gallon juice cartons made of un-printed polycoated white Kraft board having a bulk density of 20 lb/fi^J was obtained from International Paper. Each carton was foldcd fiat, and then fed into a 3 hp Flinch Baugh shredder at a rate of approximately 15 to 20 pounds per hour. The shredder was equipped 25 with two 12 inch rotary blades, two fixed blades and a 0.30 inch discharge scrcen. The gap between the rotary and fixed blades was adjusted to 0.10 inch. The output from the shredder resembled confetti having a width of between 0.1 inch and 0.5 inch, a length of between 0.25 inch and 1 inch and a thickness équivalent to that of the starting material (about 0.075 inch).

The confctti-like material was fed to a Munson rotary knife cutter, Model SC30. Model SC30 is equipped with four rotary blades, four fïxcd blades, and a discharge screen having l/8 inch openings. The gap between the rotary and fixed blades was set to approximatcly 0.020 inch. The rotary knife cutter sheared the confctti-like pièces across the knife-edges, tcaring the pièces apart and releasîng a fïbrous material at a rate of about one pound per hour. The fïbrous material had a BET surface area of 0.9748 m²/g+/0.0167 m²/g, a porosity of 89.0437 percent and a bulk density (@0.53 psia) of 0.1260 g/mL. An average length of the fïbers was 1.141 mm and an average width of the fïbers was 0.027 mm, giving an average L/D of 42: l. A scanning électron micrograph ofthe fîbrous material is shown in FIG. 9 at 25 X magnîfïcation.

Example 2 - Préparation Of Flbrous Material From Bleached Kraft Board

A 1500 pound skid of virgin bleached white Kraft board having a bulk density of 30 lb/ft’ was obtained from International Paper. The material was folded fiat, and then fed into a 3 hp Flînch Baugh shredder at a rate of approximately 15 to 20 pounds per hour. The shredder was equipped with two 12 inch rotary blades, two fixed blades and a 0.30 inch discharge screen. The gap between the rotary and fixed blades was adjusted to 0.10 inch. The output from the shredder rescmbled confetti having a width of between 0.1 inch and 0.5 inch, a length of between 0.25 inch and 1 inch and a thickness équivalent to that of the starting material (about 0.075 inch). The confetti-likc material was fed to a Munson rotary knife cutter, Model SC30. The discharge screen had 1/8 inch openings. The gap between the rotary and fixed blades was set to approximatcly 0.020 inch. The rotary knife cutter sheared the confctti-like pièces, rclcasing a fïbrous material at a rate of about one pound per hour. The fïbrous material had a BET surface area of 1.1316 m²/g +/- 0.0103 m²/g, a porosity of 88.3285 percent and a bulk density (@0.53 psia) of 0.1497 g/mL. An average length of the fïbers was 1.063 mm and an average width of the fïbers was 0.0245 mm, giving an average L/D of 43:1. A scanning électron micrographs ofthe fïbrous material is shown in FIG. 10 at 25 X magnifïcation.

Example 3 - Préparation Of Twlca Sheared Fibrous Material From Bleached Kraft Board

A 1500 pound skid of virgin bleached white Kraft board having a bulk density of 30 lb/ft³ was obtained from International Paper, The material was folded fiat, and then

« · fcd into a 3 hp Flinch Baugh shredder at a rate of approximately I5 to 20 pounds per hour. The shredder was cquipped with two 12 inch rotary blades, two fixed bladcs and a 0.30 inch discharge screen. The gap between the rotary and fixed blades was adjuslcd to 0.10 inch. The output from the shredder resemblcd confetti (as above). The confetti-like material was fed to a Munson rotary knife cutter, Modcl SC30. The discharge screen had 1/16 inch openings. The gap between the rotary and fixed blades was set to approximately 0.020 inch. The rotary knife cutter the confetti-like pièces, releasing a fibrous material at a rate of about one pound per hour. The material resulting from the first shearing was fcd back into the same setup described above and sheared again, The resulting fibrous material had a BET surface area of 1.4408 m²/g +/- 0.0156 m²/g, a porosity of 90.8998 percent and a bulk density (@0.53 psia) of 0.1298 g/mL. An average length of the fibers was 0.891 mm and an average width ofthe fibers was 0.026 mm, giving an average L/D of 34:1. A scanning électron micrograph of the fibrous material is shown in FIG. 11 at 25 X magnification.

Example 4 - Préparation Of Thrlce Sheared Fibrous Material From Bleached Kraft Board

A 1500 pound skid of virgin bleached white Kraft board having a bulk density of 30 lb/ft³ was obtained from International Papcr. The material was foldcd fiat, and then fcd into a 3 hp Flinch Baugh shredder at a rate of approximately 15 to 20 pounds per hour. The shredder was equipped with two 12 inch rotary bladcs, two fixed blades and a

0.30 inch discharge screen. The gap between the rotary and fixed bladcs was adjusted to

0.10 inch. The output from the shredder rcsembled confetti (as above). The confetti-like material was fed to a Munson rotary knife cutter, Modcl SC30. The discharge screen had 1/8 inch openings. The gap between the rotary and fixed bladcs was set to approximately 0.020 inch. The rotary knife cutter sheared the confetti-like pièces across the knife25 edges. The material resulting from the first shearing was fcd back into the same setup and the screen was replaced with a 1/16 inch screen. This material was sheared. The material resulting from the second shearing was fed back into the same setup and the screen was replaced with a 1/32 inch screen. This material was sheared. The resulting fibrous material had a BET surface area of 1.6897 m²/g +/- 0.0155 m²/g, a porosity of

87.7163 percent and a bulk density (@0.53 psia) of 0.1448 g/mL. An average length of the fibers was 0.824 mm and an average width of the fibers was 0.0262 mm, giving an average L/D of 32; I. A scanning clectron micrograph of the fibrous material is shown in FIG. 12 at 25 X magnification.

Example 5 - Methods of Determining Molecular Weight of Cellulosic and Lignocellulosic Materials by Gel Perméation Chromatography

Cellulosic and lignocellulosic materials for analysis were treated according to Example 4. Sample materials presented in the following tables include Kraft paper (P), wheat straw (WS), alfalfa (A), and switchgrass (SG). The number “132” of the Sample ID refers to the particle size of the material after shearing through a I/32 inch screcn. The number after the dash refers to the dosage of radiation (MRad) and “US” refers to ultrasonic treatment. For cxample, a sample ID “P132-10” refers to Kraft paper that has been sheared to a particle size of 132 mesh and has been irradiated with 10 MRad.

Table 1. Peak Average Molecular Weight of Irradiated Kraft Paper

Sample Source	Sample II)	Dosage1 (MRad)	Ultrasound1	Average MW ± Std Dev.
Kraft Peper	P132	0	No	32853+10006
	P132-1O	10	u	61398 ±2468**
	P132-100	100	*4	8444 ±580
	P132-181	181		6668 ± 77
	P132-US	0	Ycs	3095 ±1013

**Low doses of radiation appcar to incrcase the molecular weight of sonie materials 'Dosage Rate = lMRad/hour ³Trcatnient for 30 minutes with 20kHz ultrasound using a 1000W liorn under re-circu!ating conditions with the material dispersed in water.

Table 2. Peak Average Molecular Weight of Irradiated Materials

Sample ID	Peak #	Dosage1 (MRad)	Ultrasound1	Average MW ± Std Dev.
WS132	1	0	No	1407411 ±175191
	2	41	44	39145 + 3425
	3	44	44	2886 +177
WS132-I0*	1	10		26040 ± 3240
WS132-100*	1	100	41	23620 ±453
AI32	1	0	<4	1604886± 151701
	2	44	<4	37525 ±3751
	3		44	2853 ± 490
Λ132-10*	1	10		50853 ±1665
	2	44		2461±17
Λ132-100*	1	100		38291 ±2235
	2	<4		2487+ 15
SGI 32	1	0	Cf	1557360 + 83693
	2	«4		42594 + 4414

SG 132-10*	3 1	10	LL <«	3268 ±249 60888 ±9131
SG132-100*	1	1Ü0	H	22345 ± 3797
SG132-10-US	1	10	Ycs	86086 ±43518
	2	1«		2247 ± 468
SG132-100-US	1	100	4L	4696± 1465

*Pcaks coalescc after treal ment **Low doses of radiation appcar to increase the molecular weight of somc materials ‘Dosage Ratc= lMRad/hour treatment for 30 minutes witli 20kIIz ultrasound using a 100DW hom under rc-circulating conditions wilh the material disperscd in water.

Gel Perméation Chromatography (GPC) is used to détermine the molecular weight distribution of polymers. During GPC analysis, a solution of the polymer sample is passcd through a column packcd with a porous gel trapping small molécules. The sample is separated based on molecular size with larger molécules cluting sooner than smaller molécules. The rétention time of each component is most often dctccted by rcfractivc index (RI), evaporative light scattering (ELS), or ultraviolet (UV) and compared to a calibration curve. The resulting data is then used to calculatc the molecular weight distribution for the sample.

A distribution of molecular weights rather than a unique molecular weight is used to characterize synthetic polymers. To charactcrize this distribution, statistical averages arc utilized. The most common of these averages are the “number average molecular weight” (M„) and the “weight average molecular weight” (M_w).

M_n is similar to the standard arithmetic mean associated with a group of numbers. When applied to polymers, M_n refers to the average molecular weight of the molécules in the polymer. M_n is calculatcd afïbrding the same amount of significancc to cach molécule rcgardless of its individual molecular weight. The average M_n is calculated by the following formula where Nj is the number of molécules with a molar mass equal to M_;.

N,M_t

M_w is another statistical descriptor of the molecular weight distribution that places a greater emphasis on largcr molécules than smallcr molécules in the distribution. The formula below shows the statistical calculation of the weight average molecular weight.

The polydispersity index or PI is defined as the ratio of M_w/M_n. The largcr the PI, the broader or more disperse the distribution. The lowcst value that a PI can be is l. This represents a monodisperse sample; that is, a polymer with ail of the molécules in the distribution being the same molecular weight.

The peak molecular weight value (Mp) is another descriptor defined as the mode of the molecular weight distribution. It signifies the molecular weight that is most abundant in the distribution. This value also gives insight to the molecular weight distribution.

Most GPC mcasuremcnts are made relative to a different polymer standard. The accuracy of the results dépends on how closcly the characteristics of the polymer being analyzcd match thosc of the standard used. The cxpcctcd error in rcproducibility between different sériés of déterminations, calibrated separately, is ca. 5-10% and is characteristic to the limited précision of GPC déterminations. Therefore, GPC results are most uscful when a comparison between the molecular weight distribution of different samples is made during the same sériés of déterminations.

The lignocellulosic samples required sample préparation prior to GPC analysis. First, a saturated solution (8.4% by weight) of lithium chloride (LiCl) was prepared in dimcthyl acctamidc (DMAc). Approximately 100 mg of the sample was added to approximately 10 g of a freshly prepared saturated LiCI/DMAc solution, and the mixture was heated to approximately 150°C-170°C with stirring for 1 hour. The resulting solutions were generally light- to dark-yellow in color. The température of the solutions were decreased to approximately IOO°C and heated for an additional 2 hours. The température of the solutions were then decreased to approximately 50°C and the sample solution was heated for approximately 48 to 60 hours. Of note, samplcs irradiated at 100 MRad were more easily solubilized as compared to their untreated countcrpart. Additionally, the sheared samplcs (denoted by the number 132) had slightly lower average molccular weights as compared with uncut samples.

The resulting sample solutions were diluted l : l using DMAc as solvent and were filtered through a 0.45 pm PTFE filter. The filtered sample solutions were then analyzed by GPC. The peak average molccular weight (Mp) of the samplcs, as determined by Gel Perméation Chromatography (GPC), arc summarized in Tables l and 2.Each sample was prepared in duplicate and each préparation of the sample was analyzed in duplicate (two injections) for a total of four injections per sample. The EasiCal® polystyrène standards PS l A and PS l B were used to gcncratc a calibration curve for the molccular weight scale from about 580 to 7,500,00 Daltons. Table 3 récites the GPC analysis conditions.

Table 3. GPC Analysis Conditions

Instrument:	Watcrs Alliance GPC 2000 Plgel ΙΟμ Mixed-B
Columns (3):	S/N’s; 10M-MB-148-83,10M-MB-148-84, 10M-MBI74-129
Mobile Phase (solvent):	0.5% LiCl in DMAc (1.0 mL/min.)
Column/Detector Température:	70 °C
Injecter Température:	70 °C
Sample Loop Size:	323.5 pL

Example 6 - Poroslmotrv Analysis of Irradiated Materials

Mercury porc size and pore volume analysis (Table 4) is based on forcing mercury (a non-wetting liquid) into a porous structure under tightly controllcd pressures. Since mercury does not wet most substances and will not spontaneously penctratc pores by capillary action, it must be foreed into the voids of the sample by applying extcmal pressure. The pressure rcquired to fill the voids is inversely proporlional to the size of the porcs. Only a small amount of force or pressure is rcquired to fill large voids, whereas much greater pressure is rcquired to fill voids of very small pores.

Table 4. Pore Size and Volume Distribution by Mercury Porosimetry

Sample II)	Total Intrusion Volume (mL/g)	Total Porc Area (m’/g)	Médian Pore Dlamcter (Volume) Qun)	Médian Pore Dlamcter (Area) (/nu)	Average Pore Dlamcter (4V/A) (jim)	Rulk Density @ 0.50 psia (g/mL)	Apparent (skelctal) Density (g/mL)	Porosity (%)
P132	6.0594	1.228	36.2250	13,7278	19.7415	0.1448	1.1785	87.7163
P132-10	5.5436	1.211	46.3463	4.5646	18.3106	0,1614	1.5355	89.4875
P132-100	5.3985	0.998	34.5235	18.2005	21.6422	0.1612	1.2413	87.0151
P132-181	3,2866	0.868	25.3448	12.2410	15.1509	0.2497	1.3916	82.0577
P132-IJS	60005	14.787	98.3459	0.0055	1.6231	0.1404	0.8894	84.2199
Λ132	20037	11.759	64.6308	0.0113	0.6816	0.3683	1.4058	73.7990
A132-10	1.9514	10.326	53.2706	0,0105	0.7560	0.3768	1.4231	73.5241
A132-10«	1.9394	10205	43.8966	0.0118	0.7602	0.3760	1.3889	72.9264
SG132	2.5267	8.265	57.6958	0.0141	1.2229	0.3119	1.4708	78.7961
SG132-10	2.1414	8.643	26.4666	0,0103	0.9910	0.3457	1.3315	74.0340
SG132-100	2.5142	10.766	32.7118	0.0098	0.9342	0.3077	1.3590	77.3593
SG132-I0-US	4.4043	1.722	71.5734	1.1016	10.2319	0.1930	1.2883	85.0169
SG132-10D-US	4.9665	7.358	24.8462	0.0089	2.6998	0.1695	1.0731	84.2010
WS132	2.9920	5.447	76.3675	0.0516	2.1971	0.2773	1.6279	82.9664
WS132-I0	3.1138	2.901	57.4727	0.3630	4.2940	0.2763	1.9808	86.0484
WS132-I00	3.2077	3.114	52.3284	0.2876	4.1199	0.2599	1.5611	83.3538

The AutoPore® 9520 can attaîn a maximum pressure of 414 MPa or 60,000 psia. Thcre are four low pressure stations for sample préparation and collection of macroporc data from 0.2 psia to 50 psia. Thcre are two high pressure chambcrs which collccts data from 25 psia to 60,000 psia. Thc sample is placed in a bowl-likc apparatus callcd a penctromcter, which is bondcd to a glass capillary stem with a métal coating. As mercury invades the voids in and around the sample, it moves down the capillary stem. The loss of mercury from the capillary stem results in a change in the clectrical capacitance. Thc change in capacitance during the experiment is converted to volume of mercury by knowing thc stem volume of the penctromcter in use. A varicty of pcnctrometers with different bowl (sample) sizes and capillaries are available to accommodate most sample sizes and configurations. Table 5 below defïnes some of the key parameters calculatcd for each sample.

Table 5. Définition of Parameters

Parameter	Description
	The total volume of mercury inlrudcd during an cxpcrimcnl. This
Total Intrusion Volume:	can include intcrstitial filling between small particles, porosity of sample, and compression volume of samplc.
Total Porc Area:	The total intrusion volume convcrled lo an area assuming cylindrical shaped pores.
Médian Porc Diameter (volume):	Tlie size at lhe 50* percentile on the cumulative volume graph.
Médian Porc Diameter (area): Avcrngc Pore Diameter:	The size at the 50Λ percentile on the cumulative arca graph. The total pore volume dividcd by the total pore area (4V/A).
Bulk Dcnsity:	The ntass of the samplc dividcd by the bulk volume. Bulk volume is determined at lhe filling pressure, typically 0.5 psia.
Apparent Dcnsity:	The mass of samplc dividcd hy the volume of sample measured al highesl pressure, lypîcally 60,000 psia.
Porosity:	(Bulk Dcnsity/ Apparent Dcnsity) x 100%

Example 7 Particle Size Analysis of Irradlated Materials

The technique of particle sizing by static light scattering is bascd on Mic theory (which also encompasses Fraunhofer theory). Mie theory predicts the intensity vs. angle rclationship as a function of the size for spherical scattering particles provided that other system variables are known and hcld constant. These variables are the wavelength of incident light and the relative rcfructivc index of the samplc material. Application of Mic 10 theory provides the detailed particle size information. Table 6 summarizes particle size using médian diameter, mean diameter, and modal diameter as parameters.

Table 6. Particle Size by Laser Light Scattering (Dry Samplc Dispersion)

Sample ID	Médian Diameter (pm)	Mean Diameter (μπι)	Modal Diameter (jim)
A132	380.695	418.778	442.258
A132-10	321.742	366,231	410.156
A132-10Ü	301.786	348.633	444.169
SGI 32	369.400	411.790	455.508
SG132-II)	278.793	325.497	426.717
SG132-100	242.757	298.686	390.097
WS132	407.335	445.618	467.978
WS132-10	194.237	226.604	297.941
WS132-10(1	201.975	236.037	307,304

Particle size was determined by Laser Light Scattcring (Dry Sample Dispersion) using a Malvern Mastersizcr 2000 using the following conditions:

Fced Rote:

35%

Disperser Pressure: 4 Bar

Oplical Model: (2.610, l.OOOi), 1.000

An appropriate amount of sample was introduccd onto a vibratory tray. The feed rate and air pressure were adjusted to ensure that the particles were properly dispersed. The key component is selecting an air pressure that will break up agglomérations, but docs not compromise the sample integrity. The amount of sample needed varies depending on the size of the particles. In general, samplcs with fine particles rcquirc less material than samples with coarse particles.

Exampla 8 - Surface Area Analysis of Irradiated Materials

Surface area of each sample was analyzcd using a Micromcritics® ASAP 2420 Acceleratcd Surface Area and Porosimctry System. The samples were prepared by first degassing for 16 hours at 40 °C. Ncxt, free space (both warm and cold) with hélium is calculated and then the sample tube is cvacuated again to remove the hélium. Data collection begins aller this second évacuation and consists of defining target pressures which controls how much gas is doscd onto the sample. At each target pressure, the quantity of gas adsorbed and the actual pressure arc determined and rccorded. The pressure inside the sample tube is measured with a pressure transducer. Additional doses of gas will continue until the target pressure is achicvcd and allowcd to cquilibratc. The quantity of gas adsorbed is determined by summing multiple doses onto the sample. The pressure and quantity dcfinc a gas adsorption isothcrm and are used to calculatc a number of parameters, including BET surface area (Table 7).

Table 7. Summary of Surface Area by Gas Adsorptîon

Sample ID	Single point surface area (m’/g)	BET Surface Area (m’/g)
P132	@P/Po= 0.250387771	1.5253	1.6897
P132-10	@P/Po= 0.239496722	1.0212	1.2782
P132-100	@ P/Po= 0.240538100	1.0338	1.2622
P132-181	@ P/Po= 0.239166091	0.5102	0.6448
P132-US	@P/Po= 0.217359072	1.0983	1.6793
Λ132	@ P/Po= 0.240040610	0.5400	0.7614
A132-10	@P/Po= 0.211218936	0.5383	0.7212
Λ132-Ι0Ι)	@ P/Po= 0.238791097	0.4258	0.5538
SG132	@ P/Po= 0.237989353	0,6359	O.83SO
SG132-10	@ P/Po= 0.238576905	0.6794	0.8689
SG132-100	@ P/Po= 0.24196036]	0.5518	0.7034
SG 132-10-US	@P/Po= 0.225692889	0.5693	0.7510
SG132-I00-US	@ P/Po= 0.225935246	1.0983	1.4963
WSI32	@ P/Po= 0.237823664	0.6582	0.8663
WS132-10	@ P/Po= 0.238612476	0.6191	0.7912
WS132-100	(ô) P/Po- 0.238398832	0.6255	0.8143

The BET model for isotherms is a widcly used theory for calculating the spécifie surface area. The analysis involves determining the monolayer capacîty of the sample surface by calculating the amount requtred to covcr the entire surface with a single denscly packcd layer of krypton, The monolayer capacity is multiplîcd by the cross scctional area of a molécule of probe gas to détermine the total surface area. Spécifie surface area is the surface area of the sample aliquot divided by the mass of the sample.

Example 9 · Fiber Length Détermination of Irradiated Matériels

Fiber length distribution testing was performed in triplicate on the samplcs submitted using the Tcchpap MorFi LB01 system, The average length and width arc reported in Table 8.

Table 8. Summary of Llgnocellulosic Fiber Length and Width Data

Sa ni pic ID	Arithmetic Average (mm)	Average Length Weighted in Length (mm)	Statîstically Correeted Average Length Weighted in Length (mm)	Width (micrometers) (pm)
P132-10	0.484	0.615	0.773	24.7
P132-100	0.369	0.423	0.496	23.8
P132-181	0.312	0.342	0.392	24.4
A132-10	0.382	0.423	0.650	43.2
A132-100	0.362	0.435	0.592	29.9

SG132-10	0.328	0.363	0.521	44.0
SG 132-100	0,325	0.351	0.466	43.8
WS132-10	0,353	0.381	0.565	44.7
WS132-100	0,354	0.371	0.536	45.4

OTHER EMBODIMENTS

A number of embodiments of the invention hâve been described. Ncvertheless, it will bc understood that various modifications may be made without departing from the spirit and scope of the invention.

Lignases antl Biomass Destroving Enzymes

For cxamplc, some methods utilize one or more ligninascs and/or biomassdestroying enzymes, instead of or in addition to Fenton chcmistry, to reducc rccalcitrance in cellulosic or lignocellulosic materials. In such methods, a first ccllulosic or lignocellulosic material having a first level of recalcitrancc is provided and combined with one or more ligninases and/or one or more biomass-destroying, e.g., lignindestroying organisms, so as to contact the first ccllulosic or lignocellulosic material. The contact is maintaincd for a period of time, such as between 2 and 24 hours, e.g., between 6 and 12 hours, and under conditions sufficient, e.g., below a pH of about 6, such as between pH 3 and 5.5, to produce a second lignocellulosic material having a second level of rccalcitrance lowcr than the first level of rccalcitrance. After réduction of the recalcitrancc, the second cellulosic or lignocellulosic material can be contacted with one or more enzymes and/or microorganisms, e.g., to makc any product described herein, e.g., food or fuel, e.g., éthanol or butanol (e.g., n-butanol) or any product described in any application incorporated by référencé herein.

The ligninasc can bc, e.g., one or more of manganèse peroxidasc, lignin peroxidasc or laccases.

In particular implémentations, the biomass-destroying organism can bc, e.g., one or more of whitc rot, brown rot or soft rot. For cxamplc, the biomass-destroying organism can bc a Basidiomycctcs fungus. In particular embodiments, the biomassdestroying organism Phanerochaete chiysoporium or Gleophyiluin trabeum.

Ligninascs, biomass-destroying organisms and small molécule métabolites are described in Kirk et al., Enzyme Microb. Technol. 1986, vol. 8, 27-32, Kirk et al., Enzymes for Pulp and Papcr Processing, Chapter l (Rôles for Microbial Enzymes in Pulp and Paper Processing and Kirk et al., The Chemistry of Solid Wood, Chapter 12 (Biological Décomposition of Solid Wood (pp. 455-487).

Hydrocarbon-Containlng Materials ln some embodiments, the methods and Systems disclosed herein can be used to process hydrocarbon-containîng materials such as tar or oil sands, oil shalc, crude oil (e.g., heavy crude oil and/or light crude oil), bitumen, coal, petroleum gases (e.g., méthane, ethane, propane, butane, isobutanc), liqucfied natural and/or synthetic gas, asphalt, and other natural materials that include various types of hydrocarbons. For example, a processing facility for hydrocarbon-containîng materials receives a supply of raw material. The raw material can bc delivercd directly from a mine, e.g., by convcyor bclt and/or rail car system, and in certain embodiments, the processing facility can bc constructed in relativcly close proximity to, or even atop, the mine. In some embodiments, the raw material can bc transported to the processing facility via railway freight car or another motorized transport system, and/or pumped to the processing facility via pipeline.

When the raw material enters the processing facility, the raw material can be broken down mcchanically and/or chcmically to yield starting material. As an cxample, the raw material can include material derived from oil sands and containing crude bitumen. Bitumen can then bc processed into onc or more hydrocarbon products using the methods disclosed herein. In some embodiments, the oil sands material can be extracted from surface mines such as open pit mines. In certain embodiments, subsurface oil sands material can be extracted using a bot water flotation process that removes oil from sand particles, and then adding naphtha to allow pumpingof the oil to the processing facility.

Bitumen processing gcnerally includes two stages. In a first stage, relativcly large bitumen hydrocarbons are crackcd into smaller molécules using coking, hydrocracking, or a combination of the two techniques. In the coking process, carbon is removed from

bitumen hydrocarbon molécules at high températures (e.g., 400° C or more), leading to cracking of the molécules. In hydrocracking, hydrogen is added to bitumen molécules, which are then crackcd over a catalyst system (e.g., platinum).

In a second stage, the crackcd bitumen molécules arc hydrotreated. In general, hydrotreating includcs heating the crackcd bitumen molécules in a hydrogen atmosphère to remove mctals, nitrogen (e.g., as ammonia), and sulfur (e.g., as clcmcntal sulfur).

The overall bitumen processing procedure typically produccs approximately one barrel of synthetic crude oîl for every 2.5 tons of oil sand material processed. Morcovcr, an energy équivalent of approximately one barrel of oil is used to producc three barrcls of synthetic crude oil from oil sand-derived bitumen sources.

As another cxamplc, oil shale typically includes fine-graincd sedimentary rock that includcs significant amounts of kerogen (a mixture of various organic compounds in solid form). By heating oil shale, a vapor is liberated which can bc purified to yield a hydrocarbon-rich shale oil and a combustible hydrocarbon shale gas. Typically, the oil shale is heated to between 250° C and 550° C in the absence of oxygen to libcratc the vapor.

The effïciency and cost-effectivencss with which usablc hydrocarbon products can bc extracted from oil sands material, oil shale, crude oil, and other oil-bascd raw materials can bc improved by applying the methods disclosed herein. In addition, a variety of different hydrocarbon products (including various hydrocarbon fractions that are présent in the raw material, and other types of hydrocarbons that arc formed during processing) can be extracted from the raw materials.

In certain embodiments, in addition to Fenten oxidation, other methods can also bc used to process raw and/or intermediate hydrocarbon-containing materials. For cxample, électron bcams or ion beams can bc used to process the materials. For cxamplc, ion beams that include one or more different types of ions (e.g., protons, carbon ions, oxygen ions, hydridc ions) can be used to process raw materials. The ion bcams can include positive ions and/or négative ions, in doses that vary from l Mrad to 2500 Mrad or more, e.g., 50, 100, 250, 350, 500, 1000, 1500, 2000, or 2500 MRad, oreven higher lcvels.

Other additional processing methods can be used, including oxidation, pyrolysis, and sontcation. In general, process parametcrs for each of these techniques when treating hydrocarbon-based raw and/or inlcrmcdiate materials can bc the same as those disclosed above in connection with biomass materials. Various combinations of these techniques can also bc used to proccss raw or intermediate materials.

Generally, the various techniques can bc used in any order, and any number of times, to treat raw and/or intermediate materials. For example, to proccss bitumen from oil sands, one or more of the techniques disclosed herein can be used prior to any mcchanical breakdown steps, following one or more mcchanical breakdown steps, prior to cracking, after cracking and/or prior to hydrotreatment, and after hydrotreatment. As another example, to proccss oil shale, one or more of the techniques disclosed herein can be used prior to either or both of the vaporization and purification steps discussed above. Products derived from the hydrocarbon-based raw materials can bc treated again with any combination of techniques prior to transporting the products out of the processing facility (e.g., either via motorized transport, or via pipeline).

The techniques disclosed herein can bc applied to proccss raw and/or intermediate material in dry form, in a solution or slurry, or in gaseous form (e.g,, to process hydrocarbon vapors at elevated température). The solubility of raw or intermediate products in solutions and slurries can bc controlled through sélective addition of one or more agents such as acids, bases, oxidizing agents, rcducing agents, and salts. In general, the methods disclosed herein can bc used to initiatc and/or sustain the reaction of raw and/or intermediate hydrocarbon-containing materials, extraction of intermediate materials from raw materials (e.g., extraction of hydrocarbon components from other solid or liquid components), distribution of raw and/or intermediate materials, and séparation of intermediate materials from raw materials (e.g., séparation of hydrocarboncontaining components from other solid matrix components to increase the concentration and/or purity and/or homogeneity of the hydrocarbon components).

In addition, microorganisms can be used for processing raw or intermediate materials, either prior to or following the use of the methods described herein. Suitable microorganisms include various types of bacteria, yeasts, and mixtures thereof, as disclosed previously. The processing facility can be equipped to remove harmful byproducts that resuit from the processing of raw or intermediate materials, including gaseous products that are harmful to human operators, and chcmical byproducts that arc harmful to humans and/or various microorganisms.

In some embodiments, the use of onc or more of the techniques disclosed herein results in a molecular weight réduction of onc or more components of the raw or intermediate material that is proccssed. As a resuit, various lower weight hydrocarbon substances can be produced from onc or more higher weight hydrocarbon substances, in certain embodiments, the use of one or more of the techniques disclosed herein results in an incrcasc in molecular weight of onc or more components of the raw or intermediate material that is proccssed. For cxamplc, the various techniques disclosed herein can induce bond-formation between molécules of the components, leading to the formation of incrcased quantities of certain products, and even to new, higher molecular weight products. In addition to hydrocarbon products, various other compounds can be extracted from the raw materials, including nitrogen bascd compounds (e.g., ammonia), sulfurbased compounds, and silicates and other silicon-based compounds. In certain embodiments, one or more products extracted from the raw materials can be combustcd to gencrate process heat for heating water, raw or intermediate materials, generating electrical power, or for other applications.

Processing oil sand materials (including bitumen) using one or more of the techniques disclosed herein can lead to more efficient cracking and/or hydrotreatment of the bitumen. As another example, processing oil shalc can lead to more efficient extraction of various products, including shale oil and/or shale gas, from the oil shale. In certain embodiments, steps such as cracking or vaporization may not even be neccssary if the techniques disclosed herein arc first used to treat the raw material. Further, in some embodiments, by treating raw and/or intermediate materials, the products can bc made more soluble in certain solvents, in préparation for subséquent processing steps in solution (e.g., steam blasting, sonication). Improving the solubility of the products can improve the efficiency of subséquent solution-based treatment steps. By improving the effîciency of other processing steps (e.g., cracking and/or hydrotreatment of bitumen, vaporization of oil shalc), the ovcrall energy consumed in processing the raw materials

Z can be reduced, making extraction and processing of the raw materials economically feasible.

Accordingly, other embodiments are within lhe scopc of the following daims.

Claims (22)

1. A method of reducing recalcitrance in ccllulosic or lignocellulosic materials, the method comprising:

contacting, in a mixture, a first ccllulosic or lignocellulosic material having a first level of recalcitrance with one or more compounds comprising one or more natuiallyoccurring, non-radioactive group 5,6, 7, 8, 9, 10 or 11 cléments, to produce a second ccllulosic or lignocellulosic material having a second level of recalcitrance lower than the first level of recalcitrance.

2. The method of claim 1, wherein the one or more cléments arc in a 1+, 2+, 3+, 4+ or 5+ oxidation state.

3. The method of claim 1 or claim 2, wherein the one or more éléments comprise Mn, Fc, Co, Ni, Cu or Zn.

4. The method of any one of the above claims, wherein the onc or more cléments comprise Fe in the 2+, 3+ or 4+ oxidation state.

5. The method of any one of the above claims, wherein the mixture further comprises one or more oxidants capable of incrcasing an oxidation state of at least some of said éléments.

6. The method of any onc of the above claims in which the oxidant comprises ozone and/or hydrogen pcroxidc.

7. The method of any one of the above claims further comprising maintaining pH at or below about 5.5 during contact, such as between 1 and 5, between 2 and 5, between 2.5 and 5 or between about 3 and 5.

8. The method of any one daims 5-7 further comprising dispersing the first cellulosic or lignocellulosic material in water or an aqueous medium, and then adding first the one or more compounds and then the one or more oxidants.

9. The method of any one of daims 5-7 further comprising dispersing the first cellulosic or lignocellulosic material in water or an aqueous medium, and then adding first the onc or more oxidants and then the one or more compounds,

10. The method of any onc of the above daims, wherein a total maximum concentration of the éléments in the onc or more compounds mcasured in the dispersion is from about ΙΟ μΜ to about 500 mM, e.g., between about 25 μΜ and about 250 mM or between about 100 μΜ and about 100 mM.

11. The method of any onc of daims 5-9, wherein a total maximum concentration of the onc or more oxidants is from about 100 μΜ to about l M, e.g., between about 250 μΜ and about 500 mM, or between about 500 μπι and 250 mM.

12. The method of any one of daims 5-9 in which the one or more oxidants are applied to the first cellulosic or lignocellulosic material and the onc or more compounds as a gas, such as by gcncrating ozone in-situ by irradiating the first cellulosic or lignocellulosic and the onc or more compounds through air with a beam of particles, such as électrons or protons.

13. The method of any one of the above daims wherein the mixture includes onc or more compounds and onc or more oxidants, and wherein a mole ratio of the clement(s) in the onc or more compounds to the onc or more oxidants is from about l : 1000 to about l :25, such as from about l :500 to about l :25 or from about l : 100 to about l :25.

14. The method of any one of the above daims, wherein the mixture further includes one or more hydroquinoncs, such as 2,5-dimethoxyhydroquinone and/or one or more benzoquinones, such as 2,5-dimethoxy-l,4-benzoquinonc.

15. The method of any one of the above daims, wherein the onc or more oxîdants are clectrochemically or dectromagnetically generatcd in-silu.

16. A composition comprising l) a cellulosic or lignoccllulosic material, 2) one or more compounds comprising onc or more naturally-occurring, non-radioactive group 5, 6, 7, 8, 9, 10 or 11 éléments, and, optionally, 3) onc or more oxidants capable of increasing an oxidation state of at least some of said cléments.

17. A method of rcducing recalcitrance in cellulosic or lignocellulosic materials, the method comprising:

contacting a first lignocellulosic material having a first level of recalcitrance with onc or more ligninases and/or one or more biomass-destroying, e.g., lignin-destroying organisms, to produce a second lignocellulosic material having a second level of recalcitrance lowcr than the first level of recalcitrance and contacting the second lignoccllulosic material with an enzyme and/or microorganism.

18. The method of claim 17, wherein the ligninases arc selected from the group consisting of manganèse peroxidascs, lignin peroxidases, laccascs and mixtures thereof.

19. The method of claim 17, wherein the biomass-destroying organisms are selected from the group consisting of white rot, brown rot, soft rot and mixtures thereof.

20. The method of claim l, wherein the method further comprises contacting the second cellulosic or lignoccllulosic material with an enzyme and/or microorganism.

21. The method of claim l, wherein the method further comprises saccharifying the reduced recalcitrance material and then fermenting the saccharified material.

J

22. A method of rcducing recalcitrancc in cellulosic or lignocellulosic materials, the method comprising:

contacting, in a mixture, a first cellulosic or lignocellulosic material having a first level of recalcitrance with one or more compounds comprising one or more naturallyoccurring, non-radioactive metallic éléments, to producc a second cellulosic or lignocellulosic material having a second level of recalcitrancc lower than the first level of recalcitrancc.