AU2013205681C1 - Methods for Producing and Using Densified Biomass Products Containing Pretreated Biomass Fibers - Google Patents

Abstract

Abstract A process is provided comprising subjecting a quantity of cellulosic biomass fibers to a pretreatment wherein at least a portion of lignin and/or hemicellulose contained within each fiber is moved to an outer surface of each fiber to produce a quantity of tacky cellulosic biomass fibers; and densifying the quantity of tacky cellulosic biomass fibers to produce one or more densified biomass particulates, wherein the quantity of tacky cellulosic biomass fibers is densified without adding binder.

Description

Australian Patents Act 1990 - Regulation 3.2A ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Invention Title "Methods for Producing and Using Densified Biomass Products Containing Pretreated Biomass Fibers" The following statement is a full description of this invention, including the best method of performing it known to us: H: \rr\Interwoven\NRPortbl\DCC\RR\5071784_1. DOC - 12/4/13 METHODS FOR PRODUCING AND USING DENSIFIED BIOMASS PRODUCTS CONTAINING PRETREATED BIOMASS FIBERS This application is a divisional of Australian Patent Application No. 2010289797, the 5 entire content of which is incorporated herein by reference. Statement of Government Rights This invention was made with Government support by the US Department of Energy Sungrant Research Project Award No. DE-FG36-08-GO88073. The Government 10 has certain rights in the invention. Background Primary biofuels produced today are based on corn and other grains, transesterified biodiesel from oilseed crops, such as soybeans and animal fats. Ethanol production from 15 grains is currently the mainstay of the ethanol industry. Increasingly, however, there is a demand for cellulosic based biofuels. The inventors recognize the need for economically providing pretreated densified biomass products for use in a variety of applications. Summary 20 In one embodiment, a product comprising at least one densified biomass particulate having no added binder and comprised of a plurality of lignin-coated plant biomass fibers is provided, wherein the at least one densified biomass particulate has an intrinsic density substantially equivalent to a binder-containing densified biomass particulate and has a substantially smooth, non-flakey outer surface. In one embodiment, 25 the novel product contains trace amounts of ammonia. In one embodiment, the product comprises one or more densified biomass particulates, each particulate having no added binder and an amount of lignin-coated plant biomass fiber sufficient to form a densified biomass particulate which has an intrinsic density substantially equivalent to a binder containing densified biomass particulate. 30 In one embodiment, the at least one densified biomass particulate having no added binder has an increased resistance to deformation, an increased hardness, an increased resistance to degradation, an improved shelf life, or a combination thereof, as compared with a binder-containing densified biomass particulate. In one embodiment, the novel 1 product is more able to resist stress and is likely less brittle as compared to a binder containing densified biomass particulate. In one embodiment, the novel product is harder, such as at least 21% harder, with at least 20% less variability in hardness than a binder-containing densified biomass 5 particulate of the same given mass. The novel products described herein can be any suitable shape and size, including, for example, substantially rectangular or substantially cylindrical. In one embodiment, each of the plurality of lignin-coated plant biomass fibers is 10 coated completely with lignin. In one embodiment, at least some of the plurality of lignin coated biomass fibers are also coated with hemicellulose. In one embodiment, most of the plurality of lignin-coated plant biomass fibers are also coated with hemicellulose. In one embodiment, substantially all of the plurality of lignin-coated plant biomass fibers are also coated with hemicellulose, such that the hemicelluloses and lignin appear to come to the 15 surface in a "package" rather than as separate components. Any suitable plant biomass may be used to produce the novel products described herein, including, but not limited to, corn stover, switchgrass, pine and/or prairie cord grass. In one embodiment, the novel product has an improved shelf life, increased 20 resistance to degradation, increased flowability, and greater bulk density as compared to the binder-containing densified biomass particulate. In one embodiment, a packaged product comprising a container; and a quantity of densified biomass particulates having no added binder and located within the container is provided, wherein the quantity of densified biomass particulates has a bulk density at 25 greater than a bulk density of an identical quantity of binder-containing densified biomass particulates. The container may be a rigid container or a flexible bag. In one embodiment, an integrated process comprising subjecting a quantity of biomass fibers to an ammonia treatment wherein at least a portion of lignin contained within each fiber is moved to an outer surface of each fiber to produce a quantity of tacky 30 biomass fibers; and densifying the quantity of tacky biomass fibers to produce one or more densified biomass particulates is provided, wherein the quantity of tacky biomass fibers is densified without adding binder. In one embodiment the ammonia treatment causes at least a portion of hemicellulose contained within each fiber to move to the outer surface of each 2 fiber. In one embodiment, the ammonia treatment is an ammonia fiber expansion (AFEX TM) treatment, such as a gaseous AFEX treatment. In one embodiment, a fuel comprising at least one densified biomass particulate of a given mass having no added binder and comprised of a plurality of lignin-coated plant 5 biomass fibers is provided, wherein the at least one densified biomass particulate has an intrinsic density substantially equivalent to a binder-containing densified biomass particulate of the same given mass and has a substantially smooth, non-flakey outer surface. Such a fuel may be useful in biomass-burning stoves or boilers. In one embodiment, an animal feed comprising at least one densified biomass 10 particulate of a given mass having no added binder and comprised of a plurality of lignin coated plant biomass fibers is provided, wherein the at least one densified biomass particulate has an intrinsic density substantially equivalent to a binder-containing densified biomass particulate of the same given mass and has a substantially smooth, non-flakey outer surface, wherein the animal feed has improved digestibility as compared with animal 15 feed containing binder-containing densified biomass particulates. In one embodiment, a solid material comprising at least one densified biomass particulate of a given mass having no added binder and comprised of a plurality of lignin coated plant biomass fibers is provided, wherein the at least one densified biomass particulate has an intrinsic density substantially equivalent to a binder-containing densified 20 biomass particulate of the same given mass and has a substantially smooth, non-flakey outer surface, wherein the solid material is useful in construction, such as in fiberboard or extruded fibrous building materials. The resulting pellets are useful in a variety of applications, including, but not limited to, animal feed, chemical conversion, biochemical applications, electricity 25 generating applications (e.g., for burning in a boiler, biomass-burning stove, and the like), and as a component in solid materials, such as fiberboards and extruded fibrous building materials. Brief Description of the Drawings 30 FIG. 1 comprises an image showing AFEXTM pretreated corn stover (AFEX TM CS), AFEXTM pretreated switchgrass (AFEXTM -SG), AFEXTM -CS pellets and AFEXTM SG pellets in embodiments of the present invention. 3 FIG. 2 comprises an image of a binder-containing non-AFEXT -CS pellet and an AFEXTM -CS pellet in an embodiment of the present invention. FIGS. 3A-3E are images taken at various times of three biomass samples, including AFEXTM -CS, AFEXTM -CS pellets, and soaked AFEXTM -CS pellets in embodiments of 5 the present invention. FIG. 4 is a graph show % glucan conversion versus biomass at 6 hr, 24 hr and 72 hr for the biomass samples shown in FIGS. 3C-3E in embodiments of the present invention. FIG. 5 is a graph show % xylan conversion versus biomass at 6 hr, 24 hr and 72 hr for the biomass samples shown in FIGS. 3C-3E in embodiments of the present invention. 10 Detailed Description of the Embodiments In the following detailed description of embodiments of the invention, embodiments are described in sufficient detail to enable those skilled in the art to practice them, and it is to be understood that other embodiments may be utilized and that chemical 15 and procedural changes may be made without departing from the spirit and scope of the present subject matter. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of embodiments of the present invention is defined only by the appended claims. The Detailed Description that follows begins with a definition section followed by 20 a brief overview of cellulosic biomass, a description of the embodiments, an example section and a brief conclusion. The term "biofuel" or "biomass" as used herein, refers in general to organic matter harvested or collected as a source of energy. Biofuels are originally derived from the photosynthesis process and can therefore be considered a solar energy source. A biofuel is 25 a renewable solid, liquid or gaseous fuel derived from relatively "recently" dead biological material, i.e., "biomass," as distinguished from fossil fuels, which are derived from long dead biological material and are not renewable. There are generally considered to be three types of biofuels, namely, agricultural biofuels (defined below), municipal waste biofuels (residential and light commercial garbage or refuse, with most of the recyclable materials 30 such as glass and metal removed) and forestry biofuels (e.g., trees, waste or byproduct streams from wood products, wood fiber, pulp and paper industries). Biomass can further be in the form of solid biomass, liquid biomass or gaseous biomass. 4 The term "agricultural biofuel", as used herein, refers to a biofuel derived from agricultural crops, crop residues, grain processing facility wastes (e.g., wheat/oat hulls, corn/bean fines, out-of-specification materials, etc.), livestock production facility waste (e.g., manure, carcasses, etc.), livestock processing facility waste (e.g., undesirable parts, 5 cleansing streams, contaminated materials, etc.), food processing facility waste (e.g., separated waste streams such as grease, fat, stems, shells, intermediate process residue, rinse/cleansing streams, etc.), value-added agricultural facility byproducts (e.g., distiller's wet grain (DWG) and syrup from ethanol production facilities, etc.), and the like. Examples of livestock industries include, but are not limited to, beef, pork, turkey, chicken, 10 egg and dairy facilities. Examples of agricultural crops include, but are not limited to, any type of non-woody plant (e.g., cotton), grains such as corn, wheat, soybeans, sorghum, barley, oats, rye, and the like, herbs (e.g., peanuts), short rotation herbaceous crops such as switchgrass, alfalfa, and so forth. The term "plant biomass" or "ligno-cellulosic biomass" as used herein is intended 15 to refer to virtually any plant-derived organic matter (woody or non-woody) available for energy on a sustainable basis. Plant biomass can include, but is not limited to, agricultural crop wastes and residues such as corn stover, wheat straw, rice straw, sugar cane bagasse and the like. Plant biomass further includes, but is not limited to, woody energy crops, wood wastes and residues such as trees, including fruit trees, such as fruit-bearing trees 20 (e.g., apple trees, orange trees, and the like), softwood forest thinnings, barky wastes, sawdust, paper and pulp industry waste streams, wood fiber, and the like. Additionally grass crops, such as various prairie grasses, including prairie cord grass, switchgrass, big bluestem, little bluestem, side oats grama, and the like, have potential to be produced large-scale as additional plant biomass sources. For urban areas, potential plant biomass 25 feedstock includes yard waste (e.g., grass clippings, leaves, tree clippings, brush, etc.) and vegetable processing waste. Plant biomass is known to be the most prevalent form of carbohydrate available in nature and corn stover is currently the largest source of readily available plant biomass in the United States. The term "pretreatment step" as used herein, refers to any step, i.e., treatment, 30 intended to alter native biomass so it can be more efficiently and economically converted to reactive intermediate chemical compounds such as sugars, organic acids, etc., which can then be further processed to a variety of value added products such as ethanol. Pretreatment methods can utilize acids of varying concentrations (including sulfuric acids, 5 hydrochloric acids, organic acids, etc.) and/or other components such as ammonia, ammonium, lime, and the like. Pretreatment methods can additionally or alternatively utilize hydrothermal treatments including water, heat, steam or pressurized steam. Pretreatment can occur or be deployed in various types of containers, reactors, pipes, flow 5 through cells and the like. Many pretreatment methods will cause hydrolysis of hemicellulose to pentose sugars. The term "bulk density" as used herein, refers to the mass or dry weight of a quantity of particles or particulates (granules and other "divided" solids) divided by the total volume they occupy (mass/volume). Therefore, bulk density is not an intrinsic 10 property of the particles, as it is changeable when the particles are subjected to movement from an external source. The volume measurement is a combination of the particle volume (which includes the internal pore volume of a particle) and the intra-particle void volume. Bulk density = intrinsic density (of each particle) x (1 - voids fraction). For a given intrinsic particle density, therefore, the bulk density depends only on the void fraction, 15 which is variable. The term "moisture content" as used herein, refers to percent moisture of biomass. The moisture content is calculated as grams of water per gram of wet biomass (biomass dry matter plus water) times 100%. The term "Ammonia Fiber Explosion" or "Ammonia Fiber Expansion" (hereinafter 20 "AFEXTM") pretreatment" as used herein, refers to a process for pretreating biomass with ammonia to solubilize lignin and redeposit it from in between plant cell walls to the surface of the biomass. An AFEXTM pretreatment disrupts the lignocellulosic matrix, thus modifying the structure of lignin, partially hydrolyzing hemicellulose, and increasing the accessibility of cellulose and the remaining hemicellulose to subsequent enzymatic 25 degradation. Lignin is a primary impediment to enzymatic hydrolysis of native biomass, and removal or transformation of lignin is a suspected mechanism of several of the leading TM pretreatment technologies, including AFEX . However in contrast to many other pretreatments, the lower temperatures and non-acidic conditions of the AFEXTM process reduces the conversion of lignin and sugars into lower molecular weight aromatics, 30 furfural, hydroxymethyl furfural, and organic acids that could negatively affect microbial activity. The AFEXTM process further expands and swells cellulose fibers and further breaks up amorphous hemi-cellulose in lignocellulosic biomass. These structural changes open up the plant cell wall structure enabling more efficient and complete conversion of 6 lignocellulosic biomass to value-added products while preserving the nutrient value and composition of the material. The term "condensed AFEXTM pretreatment" or "gaseous AFEXTM pretreatment" as used herein, refers to a gaseous AFEXTM pretreatment as defined herein, which uses 5 gaseous ammonia rather than liquid ammonia. By allowing hot ammonia gas to condense directly on cooler biomass, the biomass heats up quickly and the ammonia and biomass come into intimate contact. Such a pretreatment process is now more commonly referred to as a "GAP" process. The term "added binder" as used herein, refers to natural or synthetic substances or 10 energy forms added or applied to biomass to improve stability and quality of a densified biomass particulate, comprised of a plurality of biomass fibers compressed to form a single particulate product, such as a pellet. Examples of commonly added binders include, but are not limited to, heat, steam, water, air, corn starch, lignin compounds, lignite, coffee grounds, sap, pitch, polymers, salts, acids, bases, molasses, organic compounds, urea, and 15 tar. Specialty additives are also used to improve binding and other pellet properties such as color, taste, pH stability, and water resistance. A binder may be added to the pretreated biomass at any time before, during or after a pelleting process. The amount of added binder may vary depending on the substrate being densified. Typically, the amount of added binder is about one to ten pounds per ton of loose biomass. Added binder in the 20 form of added energy is typically in the form of convective or conducted heat in the range of 90 to 160 'C, although radiated heat may also be used for the same purpose. The heat can be added outright, but heated added as a result of the heat of friction developed in the pelleting equipment during operation is not "added binder." The term "particulate" as defined herein refers to solid biomass dividable into 25 separate pieces, which can range from small microscopic particles (larger than powders) up to large objects, such as bricks, or larger, such as hay bales or larger, with any suitable mass. The specific geometry and mass will depend on a variety of factors including the type of biomass used, the amount of compression used to create the particulate, the desired length of the particulate, and the particular end use. 30 The term "pellet" is used interchangeably herein with the term "briquette." More common nomenclature currently in use utilizes the term "briquette" to refer to a compressed particulate (e.g., Example 1) and the term "pellet" to refer to an extruded 7 particulate (e.g., Example 4), i.e., a compressed particulate formed with a shaping process in which material is forced through a die. The term "flowability" as used herein refers to the ability of particulates to flow out of a container using only the force of gravity. A product having increased flowability, 5 therefore, would flow out of the container at a faster rate as compared to a product having lower flowability. Nearly all forms of ligno-cellulosic biomass, i.e., plant biomass, such as monocots, comprise three primary chemical fractions: hemicellulose, cellulose, and lignin. Hemicellulose is a polymer of short, highly-branched chains of mostly five-carbon pentose 10 sugars (xylose and arabinose), and to a lesser extent six-carbon hexose sugars (galactose, glucose and mannose). Dicots, on the other hand, have a high content of pectate and/or pectin, which is a polymer of alpha-linked glucuronic acid. Pectate may be "decorated" with mannose or rhamnose sugars, also). These sugars are highly substituted with acetic acid. 15 Because of its branched structure, hemicellulose is amorphous and relatively easy to hydrolyze (breakdown or cleave) to its individual constituent sugars by enzyme or dilute acid treatment. Cellulose is a linear polymer of glucose sugars, much like starch, which is the primary substrate of corn grain in dry grain and wet mill ethanol plants. However, unlike starch, the glucose sugars of cellulose are strung together by B-glycosidic linkages 20 which allow cellulose to form closely-associated linear chains. Because of the high degree of hydrogen bonding that can occur between cellulose chains, cellulose forms a rigid crystalline structure that is highly stable and much more resistant to hydrolysis by chemical or enzymatic attack than starch or hemicellulose polymers. Lignin, which is a polymer of phenolic molecules, provides structural integrity to plants, and remains as residual material 25 after the sugars in plant biomass have been fermented to ethanol. Lignin is a by-product of alcohol production and is considered a premium quality solid fuel because of its zero sulfur content and heating value, which is near that of sub-bituminous coal. Typically, cellulose makes up 30 to 50% of residues from agricultural, municipal, and forestry sources. While cellulose is more difficult to convert to ethanol than 30 hemicellulose, it is the sugar polymers of hemicellulose which can be more readily hydrolyzed to their individual component sugars for subsequent fermentation to ethanol. Although hemicellulose sugars represent the "low-hanging" fruit for conversion to ethanol, 8 the substantially higher content of cellulose represents the greater potential for maximizing alcohol yields, such as ethanol, on a per ton basis of plant biomass. As noted above, the hemicellulose fraction of biomass contains hexose and pentose sugars, while the cellulose fraction contains glucose. In current AFEXTM pretreatment 5 operations, only limited hemicellulose conversions are obtained. It is further known that of the sugars extracted, about 30 to 35% is xylose and about 35 to 40% is glucose (most all of which is currently converted only in post-pretreatment steps). Overall conversions, as well as over-all ethanol yields, will vary depending on several factors such as biomass type, pretreatment type, and so forth. 10 Conventional methods used to convert biomass to alcohol include processes employing a concentrated acid hydrolysis pretreatment, a two-stage acid hydrolysis pretreatment as well as processes employing any known conventional pretreatment, such as hydrothermal or chemical pretreatments, followed by an enzymatic hydrolysis (i.e., enzyme-catalyzed hydrolysis) or simultaneous enzymatic hydrolysis and saccharification. 15 Such pretreatment methods can include, but are not limited to, dilute acid hydrolysis, high pressure hot water-based methods, i.e., hydrothermal treatments such as steam explosion and aqueous hot water extraction, reactor systems (e.g., batch, continuous flow, counter TM flow, flow-through, and the like), AFEX , ammonia recycled percolation (ARP), lime treatment and a pH-based treatment. 20 Several of these methods generate nearly complete hydrolysis of the hemicellulose fraction to efficiently recover high yields of the soluble pentose sugars. This also facilitates the physical removal of the surrounding hemicellulose and lignin, thus exposing the cellulose to later processing. However, most, if not all, pretreatment approaches do not significantly hydrolyze the cellulose fraction of biomass. 25 In one embodiment, an ammonia fiber expansion method (AFEX

TM

) pretreatment is used as defined herein. See also, for example, U.S. Patent Nos. 6,106, 888 ('888), 6,176,176 ('176), 5,037,663 ('663), and 4,600,590 ('590), each of which are hereby incorporated by reference in its entirety. In one embodiment, biomass is heated to a temperature of from about 60 'C to 30 about 100 'C in the presence of concentrated ammonia. See, for example, Dale, B.E. et al., 2004, Pretreatment of corn stover using ammonia fiber expansion (AFEX TM) Applied Biochem, Biotechnol. 115: 951-963, which is incorporated herein by reference in its entirety. A rapid pressure drop then causes a physical disruption of the biomass structure, 9 exposing cellulose and hemicellulose fibers, without the extreme sugar degradation common to many pretreatments. Nearly all of the ammonia can be recovered and reused while the remaining ammonia serves as nitrogen source for microbes in fermentation. In one embodiment, 5 about one (1) to two (2) wt% of ammonia remains on the pretreated biomass. Additionally, since there is no wash stream in the process, dry matter recovery following an AFEXTM treatment is essentially quantitative. This is because AFEXTM is basically a dry to dry process. AFEXTM treated biomass is also stable for longer periods (e.g., up to at least a year) 10 than non-AFEXTM treated biomass and can be fed at very high solids loadings (such as at least about 40%) in enzymatic hydrolysis or fermentation process as compared with dilute acid or other aqueous pretreatments that cannot easily exceed 20% solids. Cellulose and hemicellulose are also well-preserved in an AFEXTM process, showing little degradation. As such, there is no need for neutralization prior to enzymatic 15 hydrolysis of AFEXTM treated biomass. Enzymatic hydrolysis of AFEXTM treated biomass also produces clean sugar streams for subsequent fermentation. Degradation products from AFEXTM treated biomass have also been identified and quantified. One such study compared AFEXTM and acid-pretreated corn stover using LC MS/GC-MS techniques. In acid-pretreated feedstock, over 40 major compounds were 20 detected, including organic acids, furans, aromatic compounds, phenolics, amides and oligosaccharides. AFEXTM pretreatment performed under mild alkaline condition produced very little acetic acid, HMF, and furfural. See, Dale, B.E. et al., 2004, supra, and Dale, B.E. et al, 2005b, Pretreatment of Switchgrass Using Ammonia Fiber Expansion (AFEX TM), Applied Biochemistry and Biotechnology. Vol. 121-124. pp. 1133 - 1142. See 25 also Dale, B.E. et al., 2005a. Optimization of the Ammonia Fiber Explosion (AFEX TM) Treatment Parameters for Enzymatic Hydrolysis of Corn Stover, Bioresource Technology. Vol. 96, pp. 2014-2018. In one embodiment, a modified AFEXTM pretreatment process is used as described in Example 1. In this method, gaseous ammonia is used, which condenses on the biomass 30 itself. In one embodiment, AFEXTM pretreatment conditions are optimized for a particular biomass type. Such conditions include, but are not limited to, ammonia loading, moisture content of biomass, temperature, and residence time. In one embodiment, corn stover is 10 subject to an AFEXTM pretreatment at a temperature of about 90 4C, ammonia: dry corn stover mass ratio of 1:1, moisture content of corn stover of 60% (dry weight basis, (dwb)), and residence time (holding at target temperature), of five (5) min. In one embodiment, switchgrass is subjected to an AFEXTM pretreatment at a temperature of about 100 4C, 5 ammonia loading of 1:1 kg of ammonia: kg of dry matter, and 80% moisture content (dwb) at five (5) min residence time. Hydrolysis results of AFEXTM treated and untreated samples show 93% vs. 16% glucan conversion, respectively. The ethanol yield of optimized AFEX treated switchgrass was measured to be about 0.2 g ethanol/g dry biomass, which is 2.5 times 10 more than that of the untreated sample. See Dale, B.E. et al., 2005b, supra. In one embodiment, approximately 98% of the theoretical glucose yield is obtained during enzymatic hydrolysis of an AFEXTM treated corn stover using 60 filter paper units (FPU) of cellulase enzyme/g of glucan (equal to 22 FPU/g of dry corn stover). Ethanol yield has been shown to increase by up to 2.2 times over that of an 15 untreated sample. In one embodiment, lower enzyme loadings of 15 and 7.5 FPU/g of glucan do not significantly affect the glucose yield, as compared with 60 FPU. In this embodiment, differences between effects at different enzyme levels decreased as the treatment temperature increased. See, for example, Dale, B.E. et al., 2004, supra: and Dale, B.E. et al., 2004, supra. 20 Optimal AFEXTM pretreatment conditions for hydrolysis and fermentation of switchgrass and corn stover are also discussed in Dale, B.E. et al., 2004, supra; Dale, B.E. et al, 2005b, supra; and Dale, B.E. et al., 2005b, supra. In one embodiment, a modified AFEXTM pretreatment with significantly reduced ammonia loadings and lower required concentrations of ammonia is used. See Elizabeth 25 (Newton) Sendich, et al., Recent process improvements for the ammonia fiber expansion (AFEXTM) process and resulting reductions in minimum ethanol selling price, 2008, Bioresource Technology 99: 8429-8435 and U.S. Patent Application Publication No. 2008/000873 to Dale, B.E. In one embodiment, steam is used as a pretreatment instead of or in addition to an 30 AFEXTM treatment. However, steam tends to reduce availability of sugars, thus reducing the overall quality of animal feed. Regardless, steam remains a viable optional embodiment for pretreatment. When biomass is being pelletized, the pellets themselves typically become hot. Additionally, water is oftentimes added to bring the biomass up to a 11 desired moisture content, such as between about 10 and 20%, such as about 12 and 18%, such as around 15% + 1%. As such, steam typically develops prior to and during an AFEXTM pretreatment anyway. Addition of additional steam may allow water to be distributed evenly through the pellet. When the pelletization process is complete, steam 5 will evaporate off and leave a pellet that is sufficiently dry, i.e., typically about five (5) to 20% moisture, although the invention is not so limited. Although a non-volatile base, such as sodium hydroxide, may also be used to move the lignin to the surface, the sodium hydroxide which remains after evaporation may negatively impact further application of the treated material, such as for animal feed and 10 other applications. Due to temperatures reaching the glass transition temperature of the oligomers within the fiber (e.g., lignin, hemicelluloses), pretreatments, such as AFEXTM (and/or steam) also transfers these oligomers (primarily lignin), and in some embodiments, an amount of hemicellulose, to the surface. Once on the surface, the lignin and hemicellulose 15 are tacky. Surprisingly, these oligomers (lignin or lignin and hemicellulose) contain sufficient tackiness to provide properties at least comparable to an added binder (as the term is defined herein). As such, the inventors have discovered there is no need to cure the pretreated biomass (e.g., with heat) prior to forming it into pellets. Additionally surprising and unexpected is the discovery that there is no need to add binder in any form 20 to produce pellets having properties at least as good as, if not better than, conventional pellets containing binder. The ability to omit the step of adding curing and/or adding binding further provides significant costs savings during production, leading to a product which is not only environmentally green but highly economical and transportable, including transportation by conventional means. 25 In one embodiment, the densification device utilizes a gear mesh system to compress biomass through a tapering channel between adjacent gear teeth. This densification device operates at temperatures less than 60 'C (See Example 2). In one embodiment energy consumption is minimized and physical and downstream processing characteristics are optimized. 30 In one embodiment, an alternative pelleting device is used to form more conventional cylindrically-shaped pellets (See Example 4). In one embodiment, an integrated biomass pretreatment and densification process is provided. In a particular embodiment, an ammonia fiber expansion (AFEX TM 12 pretreatment is used in conjunction with a compaction process to produce densified biomass particulates (hereinafter called "pellets"), in a process requiring no added binder. In one embodiment, the pellets have an inherent density at least ten (10) times that of baled biomass (which itself is about 192.2 to about 240.28 kg/m 3 (about 12 to about 15 5 lb/ft3)). Use of an integrated process as described herein eliminates the need for further pretreatment at the processing plant and further minimizes the distance that low density feedstock bales need to be transported. In one embodiment, densified pellets are transported to centralized processing 10 facilities using existing transportation and handling infrastructure used for grains. In one embodiment, AFEXTM conditions are optimized according to the type of biomass being processed to enhance binding properties and increase hydrolysis efficiency following densification and storage. It is further expected that downstream processing characteristics will be at least as 15 good as or better than non-densified biomass in terms of hydrolysis, fermentation rates, yields, and so forth. In one embodiment, AFEXTM-biomass pellets having no added binder are provided. In contrast to conventional binder-containing pellets, the novel AFEXTM pellets described herein have a substantially smooth, non-flakey outer surface, likely due to the presence of 20 lignin and, in some embodiments, hemicellulose, on the outer surface of the pellet, which essentially serve as a type of coating. As such, AFEXTM pellets are not susceptible to flaking (loss of mass) as with a conventional pellet which has no coating and contains removable flakes on its outer surface. In contrast to conventional pellets containing certain binders, such as water, which are dull in appearance, the novel AFEXTM biomass pellets 25 have a shiny appearance. In some embodiments, the presence of lignin and/or hemicellulose is not restricted to the surface only, but also is found deeper inside the microscopic pores of the biomass particle. Therefore, the AFEXTM pellet may have added benefits, such as more efficient burning/co-firing with lignite coal than a conventional pellet whose added binder is chemically restricted to the surface of the biomass particle 30 only. The AFEXTM pellets further are less bendable and therefore tend to be straighter than conventional pellets. Surprisingly, the novel AFEXTM pellets have a harder "feel" to them (and are likely less brittle) as compared with the softer feel of a conventional pellet. 13 Hardness tests (Example 4) reveal that the AFEXTM pellet is stronger initially before suddenly breaking. In contrast, a conventional pellet, such as a non-AFEXTM pellet, while maintaining strength for a longer time, is essentially more "squeezable" or "squishier" than the novel AFEXTM pellet (i.e., comparable to the softness of a "cigar"). In one 5 embodiment, an AFEXTM-CS pellet is at least 21% harder and demonstrates at least 20% less variability in hardness. In one embodiment, the novel AFEXTM pellets exhibit less deformation than conventional pellets (See, for example, Table 7). It is likely that AFEXTM pellets made from other types of biomass will demonstrate similar or better results. Lignin is generally darker than other components in plant material, so the resulting 10 material is noticeably darker in appearance than a material not substantially surrounded by lignin. In one embodiment, the AFEXTM-CS pellets have a specific gravity of up to 1.16 as compared with non-AFEXTM pellets which have a specific gravity of no more than 0.87, although the invention is not so limited. As the AFEXTM pellets appear to be less porous 15 and further demonstrate superior hardness properties as compared with conventional pellets, AFEXTM pellets are likely to show improved short and long term storage properties including, flowability, compression strength, water solubility, absorption, and overall shelf life, with reduced susceptibility to degradation due to heat, bugs, and the like. It is also expected that the AFEXTM pellets will have an improved flowability. 20 Further testing, as noted in prophetic examples will quantify the amount of improvement. The resulting pellets are useful in a variety of applications, including, but not limited to, animal feed, chemical conversion, biochemical applications, electricity generating applications (e.g., burning in a boiler), fuel for biomass-burning stoves, and as a component in solid materials, such as fiberboards and extruded fibrous building materials. 25 The ammonia pretreatment in the various AFEXTM processes described herein dissolves a certain amount of lignin and further brings a significant amount of lignin from the interior of a plant material to the outer surface or outer edges of the fiber. As a result, the material is more easily digested by animals. In one embodiment, a combination of the novel AFEXTM pellets as described herein together with suitable additives and fillers as is 30 known in the art produces a novel animal feed. A blending of the novel AFEXTM pellets with coal provides a novel feed material in power plants. 14 The logistics of harvesting, handling, transporting, and storing low bulk density feedstocks is a significant challenge to the developing bioeconomy. Assuming a yield of 70 gal/ton, biomass baled at a density 120 kg/m 3 would require over ten times the volume of material for a given volume of ethanol compared with corn grain. This lower bulk 5 density will not allow trucks to reach maximum weight capacity, further increasing the number of trucks required for feedstock supply. Biomass densification of untreated biomass through an extrusion pelleting process has been demonstrated, but at a cost that limits the ability to lower net costs for feedstock delivery. As the bioeconomy develops individual producers will need the flexibility to sell 10 their biomass into the bioenergy market as economics warrant. For example, with use of regional biomass processing centers (RBPCs) (within a 5 to 10 mile area, for example), round bales may be transported using the existing infrastructure and equipment of the trucking industry. Because the RBPCs will be scaled appropriately, trucking distances for round bales will be minimized. Moreover, the presence of multiple, distributed RBPCs will 15 minimize need for long term storage of round bales. Shorter term storage would use bale wraps and other current methods to minimize expense. With use of the novel integrated AFEXTM pretreatment/densification system described herein, densified pellets will then be more efficiently transported to centralized processing sites. The invention will be further described by reference to the following examples, 20 which are offered to further illustrate various embodiments of the present invention. It should be understood, however, that many variations and modifications may be made while remaining within the scope of the present invention. EXAMPLE 1 25 Corn stover (CS) (everything remaining after grain is harvested, typically including stalks and leaves without cobs) from a hybrid corn plant (Zea mays L.) grown at the Michigan State University (MSU) Agronomy Center Field was harvested in October 2007, and stored at room temperature in individual five (5) kg bags which were housed in a 30 gal trash bin. Switchgrass (SG) from the "Alamo" lowland variety of seed, Panicum 30 virgatum L., grown at the Thelen Field located on Farm Lane at MSU, was harvested in October, 2005, and stored in sealed Ziploc@ brand plastic bags in a freezer at four (4) 'C. The CS and SG were each subjected to an AFEXTM pretreatment comparable to the methods described in U.S. Patent Nos. '888, '176, '663, and '590 noted above, but with 15 certain modifications. Specifically, rather than applying liquid ammonia to the biomass and allowing the ammonia and biomass to react as in a conventional AFEX treatment, gaseous ammonia was used instead. By allowing hot ammonia gas to condense directly on cooler biomass, the ammonia and biomass become well-mixed. 5 The gaseous AFEXTM pretreatment was performed in the Biomass Conversion Research Laboratory at Michigan State University, East Lansing, Michigan. Unless otherwise noted, standard laboratory equipment available in conventionally stocked laboratories was used. The AFEXTM pretreatment was performed in an approved ventilation hood with protective glass sash minimum face velocity of 75 feet/minute. 10 A Parr Instruments Model 4524 bench top reactor (hereinafter "4524 reactor") was used for this testing. The reaction chamber was first placed into the heating mantle of the 4524 reactor. A J-type T-couple temperature probe was connected to a Parr Instruments Model 4843 Modular (heat) controller (hereinafter "4843 controller") on one end and to the reaction chamber on the other end by placing the temperature probe against the internal 15 wall (about half-way down) of the reaction chamber. The reaction chamber was then covered with a custom-fabricated circular stainless sheet metal piece having an approximately 12.7 cm (about five (5) in) diameter relief cut out for the temperature probe. The controller was turned on to low (with a red heater switch) and a J- type temperature (blue) controller showed a room temperature reading of about 25 'C + 5OC. 20 A (yellow) K-type thermocouple (red display) and (green) Omega brand CX105 pressure connector (having offices in Stamford, CT) (green display) from the controller were briefly connected to test the 4524 reactor cover probes. The red display showed a room temperature reading of about 25 'C + 5'C. The green display showed a one (1) atm gauge pressure reading of -0.34 to about 0.34 atm (about -5 to about 5 psig). The yellow 25 and green connecters and 4524 reactor cover were then set aside and the blue preheat temperature was turned on to preheat the 4524 reactor to a target temperature of room temperature +20 'C. The blue display was observed for about five (5) minutes to ensure that the blue temperature increased at a rate of about three (3) 'C/minute. A Sartorius MA35 moisture analyzer (Goettingen, Germany) was used to determine 30 the moisture content of each of the biomass samples. Initial moisture measurements for the samples were typically five (5) to ten (10) % total moisture wet basis (mwb). The dry weight of each sample added to the 4524 reactor was 150 g dry weight, i.e., "dry biomass." An amount of biomass was then weighed out to result in 150 g of dry biomass (as given by 16 the total moisture calculation). For example, for a biomass sample containing five (5) % total moisture (mwb), the following calculation would be made: x (g) of water in biomass = 150 g dry biomass/(1-0.05) - 150 g dry biomass). Solving for "x" results in 7.9 g of water present in the biomass. 5 A calculation was then made to determine the amount of deionized water to be added to each sample. For corn stover, the desired percentage of total water to dry biomass was 60%. For switchgrass, the desired value was 80%. These values were selected because they represent the optimal respective biomass moistures for maximum TM glucose and xylose yields from enzymatic hydrolysis after AFEX 10 Therefore, for a corn stover sample with 7.9 g of water already present, but requiring 60% (dwb) moisture content, the following calculation would be made: x (g) water to be added to biomass = (0.6 *150 g dry biomass) -7.9 g water already in biomass. Solving for "x" would result in 82.1 g of water to be added. The total weight of a 150 g (dwb) corn stover sample in this instance would be 82.1 g + 7.9 g + 150 g = 240 g. Water 15 was misted onto each biomass sample with a water bottle until the total weight (dry biomass (g) + water desired (g)) was achieved. The biomass was evenly coated with water by stirring the biomass. An empty 500 ml ammonia cylinder having a 208 g maximum fill level (Parker 500 ml spun 316 Stainless steel pressure vessel (hereinafter "Parker cylinder") with high 20 pressure Swagelok@ Series 83 two-way ball valves installed at both ends, made by Swagelok Co. (having offices in Chicago, IL) was weighed. Since eight (8) g was determined to be the approximate residual ammonia left in the cylinder after completion of this step, the total weight of the cylinder and ammonia required for AFEXTM pretreatment was determined by adding eight (8) g to the weight of the amount of ammonia needed. 25 The Parker cylinder was attached to an Airgas T M brand stock ammonia tank (with siphon tube) made by Airgas, Inc. (Radnor, PA), by opening the inlet valve on the ammonia tank, followed by opening the inlet valve on the Parker cylinder. The Parker cylinder was allowed to fill until it was cold and no more filling noise from the cylinder could be heard (elapsed time was about one (1) min). The exit valve on the ammonia tank 30 was opened about 1/4 way. After a few trials, it was determined that it took about 20 seconds to add 158 g of ammonia to the Parker cylinder. Thereafter, all valves were closed, starting with the exit valve of the Parker cylinder and finally the exit valve on the ammonia tank. The Parker cylinder was weighed to make sure the total weight was equal to the 17 expected weight. Some ammonia was released under the hood if the weight was too great. When it was not enough, the above step was repeated. The Parker cylinder, now containing ammonia, was heated by first wrapping it in BH Thermal brand Briskheat (Columbus, OH) heat tape and plugging in the BH Thermal 5 brand Briskheat (Columbus, OH) heat tape controller. Cylinder pressure started at 0-125 psig (depending on the temperature of the ammonia inside the cylinder, as it became cold during the filling step). The Parker cylinder was heated to 600 psig (40 bar), adjustable from 400 psig (27 bar) for "colder" reactions (80 'C) to 1000 psig (70 bar) for hot reactions (160 'C). The pressure increased slowly, but always at a rate less than 0.034 atm/sec (five 10 (5) psig/sec). The desired biomass was then added to the reaction chamber. The (black) temperature probe was removed from the reaction chamber and placed into the slot on the side of the heater mantle that allowed the outside surface temperature of the reaction chamber to be measured. The (blue) display temperature was adjusted (using arrow keys) 15 +20 degrees more than the original preheat to allow for the continued heating of the reaction chamber. The cover of the reaction chamber was replaced and a funnel was added. The selected biomass sample was then poured down the funnel into the reaction chamber. Once added, the (yellow) temperature probe tip was completely covered with biomass and was 20 observed to be about 2.54 cm (about one (1) in) from the ammonia input nozzle of the cover. The funnel was then removed, the cover returned on top of the 4524 reactor and brackets were tightened with bolts to seal it in place. The Parker cylinder was then attached to the reaction chamber. A Welch Model 8803 vacuum pump. (Niles, Illinois) was also attached to the reaction chamber. The 25 vacuum valve on the 4524 reactor was opened and the vacuum was turned on to pump air from the 4524 reactor for one (1) minute. The vacuum valve was closed and the vacuum was turn off. The (yellow) temperature probe and (green) pressure connector was plugged into the 4843 controller. The valve on ammonia cylinder (only) leading towards reaction chamber was opened. 30 The AFEXTM reaction was started by opening the 4524 reactor valve connected to the Parker cylinder. When the pressure between the Parker ammonia cylinder and the reaction chamber was equalized, the valves between the ammonia cylinder and the reaction chamber were closed (i.e., after about one (1) min). The heat tape on the Parker cylinder 18 was also turned off. The 4843 controller heater was left on a low setting at 20 'C above the original temperature used at pre-heat. After about one (1) minute the peak (red) display temperature and (green) pressure were recorded. When the (red) display temperature did not get > 100 'C within 1 minute, it meant the feedstock is not touching the temperature 5 probe. The temperature and pressure were recorded approximately every five (5) minutes thereafter. Starting approximately five (5) minutes before the expansion step noted below, the vacuum was detached from the 4524 reaction chamber cover. The ammonia cylinder pipe was removed from the reaction chamber cover. The reaction chamber was rotated so that 10 the 4524 pressure release valve was facing toward the back of the fume ventilation hood. The ventilation hood sash was adjusted for maximum face velocity (75 feet/minute recommended). Expansion step: Ear protection was worn. The ammonia pressure in the 4524 was released by opening the pressure release valve quickly. The reaction chamber cover was removed. The biomass was removed and placed 15 in a tray and left under the ventilation hood to allow ammonia vapor to volatilize. The AFEXTM biomass was allowed to air-dry over-night. The Parker cylinder was weighed to determine residual grams of ammonia applied to the biomass and the weight was recorded. The remaining ammonia (approximately 8 g) was released from the Parker cylinder inside of ventilation hood. 20 FIG. 2 comprises an image of a binder-containing non-AFEXTM-CS pellet and an AFEXTM-CS. EXAMPLE 2 Starting Materials and Sample Preparation 25 Corn stover (CS) obtained from the same source as in Example 1 was used. Two samples, two (2) kg each, of each type of biomass were then subjected to the AFEXTM pretreatment according to the method described in Example 1. After pretreatment, samples were densified using a pelleting device (Federal Machine Co. d/b/a ComPAKco, LLC, Fargo, ND) to produce AFEX TMCOrn stover (AFEXTM -CS) pellets and AFEXTM 30 switchgrass (AFEXTM -SG) pellets. FIG. 1 shows an image of the four resulting products, which include seven (7) g of AFEXTM -CS 102, 12 g of AFEXTM -SG 104, a 22 g AFEXTM -CS 106 pellet and a 23 g AFEXTM -SG pellet 108). The AFEXTM -CS and AFEXTM SG pellets, 106 and 108, 19 respectively, had a substantially rectangular shape. Both pellets 106 and 108 were about 2.54 cm (about one (1) in) wide, about 1.27 (0.5 in) depth and about 10.16 to about 12.7 cm (about four (4) to about five (5) in) in length. (Pellet length is dependent on the particular setting use on the ComPAKco machine). 5 This image illustrates that just seven (7) to 12 grams of unpelleted biomass, such as AFEXTM -CS 102 and AFEXTM -SG 104, occupies more space than a 22 or 23 g pellet, such as AFEXTM -CS pellet 106 and AFEXTM -SG pellet 108. In this instance, the unpelleted biomass (102 and 104) occupies about 570 to about 980% more space than the pelleted biomass (106 and 108). FIG. 2 comprises an image of a binder-containing non 10 AFEX-CS pellet and an AFEX-CS pellet. Testing Performed Several additional samples were prepared in the manner described above and subjected to preliminary physical tests, such as Angle of Repose (0), according to the 15 method described in Carr, R. L. Jr. 1965. Evaluating flow properties of solids. Chemical Engineering 72(3): 163-168. Thermal Conductivity (W/m 0 C) was determined with a thermal properties meter (KD2, Decagon Devices, Pullman, WA) that utilized the line heat source probe technique described in Baghe-Khandan, M., S. Y Choi, and M.R. Okos. 1981, Improved line heat 20 source thermal conductivity probe, J. of Food Science 46(5):1430-1432. Water activity was measured using a calibrated water activity meter (AW Sprint TH 500, Novasina, Talstrasse, Switzerland). Bulk density (kg/m 3 ), true density (kg/m 3 ) and porosity were determined using a multivolume pycnometer (Micromeritics model 1305, Norcross, GA) as described in 25 Sahin, S. and S. G. Sumnu. 2006, Physical properties of foods, New York, NY: Springer Science Media, LLC. Moisture Content (% db) was determined by ASAE standard method S352.1 using ISOTEMP laboratory scale (model no: 838F, Fisher Scientific, Pittsburg, PA) as described in ASAE Standards. 5 1" ed.2004. S352.1: Moisture measurement -- Grain and seeds, St. 30 Joseph, Mich.: ASABE. Color properties (L*, a*, b*) were measured using a spectrocolorimeter (LabScan XE, Hunter Associates Laboratory, Reston, VA). 20 Roundness and sphericity were determined using an Olympus SZH10 stereo microscope with a DP digital camera, followed by image analysis of the particles by Image Pro Plus@ software. Water Solubility Index (%) and Water Absorption Index (-) were calculated using 5 the method described in Anderson, R. A., H. F. Conway, V. F. Pfeifer, and E. L. Griffin. 1969, Gelatinization of corn grits by roll and extrusion cooking, Cereal Science Today 14 (1): 4. Results are shown in Table 1 below 21 Table 1: Physical properties of AFEXTM -CS and SG vs. AFEXTM -CS and AFEXTM -SG Pellets* Biomass AoR TC aw BD Porosity TD MC Round Sphericity WAI WSI type (0) (W/m (-) (kg/m 3 ) (kg/m 3 ) (% dwb) Color -nes ( ( C) L* a* b* (-) AFEXm 57.4a 0.035b 0.575c 547.2a 0.487 918a 13.9b 21.7 2.21c 6.47b 0.56a 0.64a 6.30b 6.74a switchgrass b pellets AFEXm 56a 0.055a 0.787a b 0.640a c 29.3a 17.8 2.20c 5.94b 0.635a 0.52c 6.17b 6.14a switchgrass c biomass AFEXm 60.6a 0.04ab 0.451b 549.2a 0.376b 722b 7.41c 21.5 3.14b 6.70b 0.45b 0.6b 7.14ab 4.36a Corn stover b pellets AFEXm 54.4a 0.045ab 0.672b b 0.657a c 16.7b 24.2 3.69a 8.81a 0.56a 0.61ab 8.03a 5.63a corn stover a biomass Similar letters for a given property is not significantly different at =0.05 n=2 for all the properties analyzed AoR - Angle of Repose (0); TC - Thermal Conductivity (W/m 0 C); aw - Water activity (-); BD - Bulk density (kg/m 3 ); TD - True Density (kg/M 3 ); MC - Moisture Content (% db); L* - Brightness or luminosity; a* - redness or greenness; b* - yellowness or blueness; WAI - Water Absorption Index (-); WSI - Water Solubility Index (%) Conclusions The AFEXTM-CS pellets (e.g., 106) and AFEXTM -SG pellets (e.g., 108), had a relatively smooth surface and held together well during handling. The AFEXTM pellets of both the corn stover and switchgrass possess lower porosity, water adsorption index, water activity, and moisture content as compared to the non-pelleted AFEXTM samples. Such properties are an indication of improved storability for the pelleted biomass. Lower porosity, higher bulk density and higher true density of the pellets are also indicative of reduced shipping costs. The pellets exhibited other desirable properties as shown in Table 1. In particular, the pellets demonstrated a high angle of repose. A pellet's angle of repose is defined as the angle between the horizontal and the plane of contact between two pellets when the upper pellet is just about to slide over the lower. This is also known as angle of friction. Therefore, particles have an expected value of 45 degrees. Both the corn stover pellets and switchgrass pellets tested herein exhibited higher than expected angles of repose of 57.4 and 60.6, respectively, as shown in Table 1. These values are likely related to the pellets' substantially rectangular geometry. EXAMPLE 3 The purpose of this experiment was to compare hydrolysis properties of AFEXTM -CS pellets as compared with AFEXTM -CS biomass (i.e., unpelleted). Starting Materials Corn stover (CS) obtained from the same source as described in Example 1 was used. An AFEXTM pretreatment was performed on the CS in the same manner as described in Example 1. Pellets were made according to the method described in Example 2. Tested samples included 1.7 g of AFEXTM -CS biomass, a 1.6 g AFEXTM -CS pellet, and a 2.2 g AFEXTM -CS soaked in 100 ml amount of de-ionized water at 25 'C for five (5) minutes before hydrolysis to produce a soaked AFEXTM -CS pellet. Procedure After being placed in a 500 ml beaker, an enzymatic hydrolysis was performed on each sample according to a standard laboratory protocol at one (1)% solids loading. See, for 23 example, Shishir P.S. Chundawat, Balan Venkatesh, Bruce E. Dale, 2005, Effect of particle size based separation of milled corn stover on AFEXTM pretreatment and enzymatic digestibility, Biotechnology and Bioengineering, Vol. 96, Issue 2, pp 219-23 1. Fifteen Filter Paper Units (FPU) of an enzyme, specifically Spezyme* CP (Genencor@, a Danisco Division, having offices in Rochester, NY whole cellulose, was added. The samples were incubated at 50 'C in a New Brunswick incubator Innova 44, (Edison, NJ) while being shaken at 150 RPM within the incubator. Observations and samples were taken at 6 hrs, 24 hrs and 72 hrs incubation time. Results A visual inspection of the resulting hydrolysates indicates that each of the three samples completely dissolved immediately upon water addition (FIG. 3B). Therefore, it is apparent that all three samples hydrolyzed to substantially the same extent in substantially the same amount of time. Approximately two (2) ml samples were taken from the incubator were filtered and run through a Shimadzu high pressure liquid chromatographer (HPLC) Model LC-2010HT w/ELSD-LT to determine glucan and xylan conversions. FIGS. 3A-3E are images taken at various times of three biomass samples, including AFEX-CS, AFEX-CS pellets, and soaked AFEX-CS pellets. FIGS. 4A and 4B are comparative hydrolysis graphs showing glucan conversions of the samples shown in FIGS. 3A-3E. As can be seen, the glucan conversions remain substantially the same across each sample. Table 2 shows percent of glucan converted to glucose at various times in each of the samples. 24 Table 2. Percent of Glucan converted to Glucose % glucan % glucan % glucan conversion conversion conversion (to glucose) (to glucose) (to glucose) Biomass type 6h 24h 72h

AFEX

M CS 44.3 61.7 71.4 AFEXTM CS-Pellet 48.3 65.9 73.7 Soaked AFEXM CS Pellet 47.5 64.0 71.3 Table 3 shows the percentage of total glucose produced between samplings. Table 3. Percentage of total glucose produced between samplings % total % total % total glucose glucose glucose Biomass type 6h 24h 72h AETM AFEX"' CS 44.3 17.4 9.7

AFEX

M

CS

Pellet 48.4 17.5 7.8 Soaked AFEXI' M CS-Pellet 47.5 16.5 7.3 Table 4 shows percentage of total xylan converted to xylose and total xylan in each sample before hydrolysis. Table 4. Percentage of total xylan converted to xylose Biomass % xylan % xylan % xylan Total Type conversion conversion conversion Xylan (g) (to xylose) (to xylose) (to xylose) 6h 24h 72h AFEXTM CS 16.5 29.7 37.9 0.42 AFEXTM CS-Pellet 24.1 39.6 48.0 0.38 Soaked AFEXTM CS-Pellet 11.8 19.3 23.4 0.72 25 Table 5 shows the percentage of total xylose produced between samplings. Table 5. Percentage of total xylose produced between samplings % total % total % total xylose xylose xylose Biomass type 6h 24h 72h AFEXTM CS 16.5 13.2 8.1

AFEX'

T M

CS

Pellet 24.1 15.5 8.4 Soaked AFEXTM CS-Pellet 11.8 7.5 4.0 Conclusion The substantially instantaneous hydrolyzing (e.g., wetting and dispersion) in the AFEXTM -CS pellet demonstrates that pelleting of corn stover biomass does not affect hydrolysis. It is likely that other AFEXTM pellets made from other biomass materials will behave in a similar manner. Indeed, as FIG. 3B shows, most of the biomass in each pellet is TM converted to sugar within six (hrs), which compares favorably with the unpelleted AFEX CS biomass sample. Additionally, both pellets (AFEXTM -CS pellet and the soaked AFEXTM CS pellet) hydrolyzed to nearly the same extent as the unpelleted sample. This determination was made by observing the lack of solids remaining after 72 hours (FIG. 3E). Since the three samples had virtually the same conversions, the test was concluded at 72 hours. These results are confirmed in FIGS. 4A and 4B. EXAMPLE 4 This test was performed to determine the comparative hardness between AFEXTM-CS pellets and non AFEXTM-CS pellets. Starting Materials CS obtained from the same source as described in Example 1 was used in this testing. Some of the CS was subjected to the AFEXTM pretreatment as described in Example 1. No additional treatment was performed on the AFEXTM treated biomass prior to pelleting, 26 including no added binder and no artificial drying (any evaporation occurring in open air at room temperature is considered to be negligible during the course of the testing procedure). The remaining portion underwent a different (non-AFEX

TM

) procedure, which included adding approximately five (5) to ten (10) g of water per 100 g of CS to bring the moisture content of the biomass to 15% wet basis (wb) prior to pelleting. Lodgepole pine biomass from the Driftmier Engineering Laboratory at the University of Georgia (Athens, GA) also underwent a similar non-AFEXTM procedure, and because the biomass moisture was measured to be greater than 15%, it was put in a dryer until it was at 12 15% moisture. Ten (10) AFEXTM -CS pellets and ten (10) non-AFEXTM -CS pellets were formed with a Yankee Pellet Machine Model 400 (Yankee Pellet Mill, Effingham, NH), a centrifugal die mill which produces pellets currently considered the industry standard. Ten (10) non-AFEXTM pine pellets were pelletized using a California Pellet Machine, Model CL (CPM, Crawfordsville, IN). Pellets produced on both these machines have a substantially cylindrical shape and are about six (6) mm in diameter. Length can be varied as desired, but is generally more uniform than the device used above in Example 2. For purposes of testing, the pellets were about one (1) inch. Procedure The pellets were tested for hardness using a 12T Carver Laboratory Hydraulic Press/Hardness testing apparatus with 400 PSI gauge (Carver, Wabash, IN). Specifically, this test measured the amount of force needed to crush each pellet beyond its yield strength. The determination of "yield strength" was made through trained observation and "feel." Specifically, pressure was applied to each pellet until the tester observed and felt the pellet "give." Multiple pellets were tested and an average hardness, i.e., pressure required to cause pellets to yield (Table 6), and average deformation (Table 7) were determined. 27 Results Comparative hardness results are shown below in Table 6: Table 6. Comparative Pellet Hardness for AFEXTM and non-AFEXTM pellets non-AFEXTM AFEXTM pellets Non-AFEXTM pellets (psi) (psi) Pine pellet 140 120 125 130 120 125 70 100 75 100 140 90 90 140 90 70 110 110 120 130 130 70 130 75 90 120 80 Measurements of the final diameter of each pellet after it "gave" were also made. These measurements are shown in Table 7. (Note that the data is randomized as compared with Table 6). Table 7. Comparative Pellet Deformation for AFEXTM and non-AFEXTM Pellets (initial diameter: 6 mm) non-AFEX M Non-AFEXI' M CS pellets AFEXTM pellets Pine pellet (mm) CS (mm) (mm) 5.26 4.66 5.08 4.67 5.28 5.07 4.96 5.28 5.13 4.84 4.98 5.1 5.2 4.73 5.28 5.08 5.18 4.59 4.76 5 4.75 28 4.15 5.12 4.61 5.39 5.36 4.98 The untreated, binder-added corn stover pellets average yield point was 98 psi +25 psi. The AFEXTM, no binder added corn stover pellets average yield point was 119 psi +20 psi, and the non-AFEXTM binder-added pine pellet average yield point was 98 psi +23 psi. All cylindrical pellets had a beginning diameter of 6.00 mm. The untreated, binder added corn stover pellets average deformation at yield was 1.06 mm +0.36 mm. The AFEXTM no binder added corn stover pellets average deformation at yield was 0.95 mm +0.24 mm, and the non-AFEXTM , binder-added pine pellet average deformation at yield was 1.06 mm +0.23 mm. Conclusion The AFEXTM pellets showed greater durability as compared to non-AFEXTM pellets. AFEXTM pellet quality is also more consistent than the non-AFEXTM pellets. As such, it is expected that any given AFEXTM pellet is less likely to be deformed or disfigured (not a cylindrical shape) as compared with a non-AFEXTM pellet. EXAMPLE 5 This test was performed to determine the bulk density of AFEXTM -CS pellets as compared to non-AFEXTM CS pellets. AFEXTM -CS pellets and non-AFEXTM CS produced according to the method described in Example 4 (about six (6) mm in diameter and about one (1) inch in length) were added to a 500 ml beaker and weighed. The non-AFEXTM-CS pellets had a bulk density of about 36 lb/ft3 (553 g/L), while the AFEXTM-CS pellets had a bulk density of about bout 38 lb/ft 3 (578 g/L). As this preliminary test indicates, the AFEXTM -CS pellets showed a higher bulk density than the non-AFEXTM CS pellets. This is likely due to their smooth non-flaky outer surface (which also is expected to improve their flowability), as compared to the rough flaky outer surface of the non-AFEXTM pellets. It is expected that a test performed on a larger scale would demonstrate an even greater difference in bulk density. Likely, the edge effects caused by the small size of the container were a significant factor in this preliminary testing. 29 It is also possible that pellets which are longer than the one (1) inch pellets may weigh each other down to create a higher mass at a higher density. Alternatively, shorter pellets may pack better. Additional testing (including in larger containers) will be performed to optimize pellet size, and therefore, overall bulk density, for a given application. EXAMPLE 6 In this testing, various properties of untreated corn stover pellets were compared with TM AFEX -treated corn stover pellets. Starting Materials Corn stover (CS) obtained from the same source as in Example 1 was used. An AFEXTM pretreatment was performed on the CS in the same manner as described in Example 1. Pellets were made according to the method described in Example 2. Procedure Standard procedures were followed to obtain the results shown in Tables 8 and 9. Specifically, Moisture Total: ASTM E871; Ash Content: ASTM D 1102; Sulfur Content: ASTM D4239; Gross Caloric Value at Constant Volume: ASTM E71 1; Chlorine Content: ASTM D6721; Bulk Density: ASTM E873; Fines (Particles less than 0.32 cm (0.125 in): Twin Peaks Test CH-P-06; Durability Index: Kansas State Method; Sample above 3.8 cm (1.5 in): Twin Peaks Test CH-P-06; Maximum Length: Twin Peaks Test CH-P-06; Diameter, Range: Twin Peaks Test CH-P-05. The tumbling method used to arrive at the durability indices noted herein is known as the "Kansas State Method." Results The results are shown below in Tables 8 and 9: Table 8. Corn Stover Pellets, Untreated METHOD UNITS MOISTURE AS FREE RECEIVED Moisture Total ASTM E871 wt% 12.08 Ash ASTM D1102 wt% 4.13 3.63 Sulfur ASTM D4239 wt% 0.095 0.084 30 Gross Cal. Value ASTM E711 Btu/lb 8017 7048 at Const. (Btu/kg) (17,638) (15,506) Chlorine ASTM D6721 mg/kg 4218 3709 Bulk Density ASTM E873 lbs/ft 3 44.08 (kg/m 3 ) (706) Fines < 0.125 in (< TPT CH-P-06 wt% 0.57 0.32 cm) Durability Index Kansas State PDI 97.9 Sample >1.5 in TPT CH-P-06 wt% 4 (3.8 cm) Maximum Length TPT CH-P-06 in (cm) 1.6 (4.1) (Single Pellet) Diameter, Range TPT CH-P-05 in (cm) 0.235-0.241 (0.597-0.612) Diameter, Average TPT CH-P-05 in (cm) 0.239 (0.607) Bag Weight lbs (kg) 3.5(1.6) Table 9. Corn Stover Pellets, AFEXTM METHOD UNITS MOISTURE AS FREE RECEIVED Moisture Total ASTM E871 wt% 7.39 Ash ASTM D1102 wt% 4.03 3.73 Sulfur ASTM D4239 wt% 0.087 0.08 Gross Cal. Value at ASTM E711 Btu/lb 7977 7388 Const. (Btu/kg) (17,550) (16,254) Chlorine ASTM D6721 mg/kg 3484 3226 Bulk Density ASTM E873 lbs/ft 3 47.15 (kg/m 3 ) (765) Fines < 0.125 in TPT CH-P-06 wt% 0.2 (<0.32 cm) Durability Index Kansas State PDI 97.9 Sample >1.5 in (3.8 TPT CH-P-06 wt% 3.9 cm) Maximum Length TPT CH-P-06 in (cm) 1.85(4.7) (Single Pellet) Diameter, Range TPT CH-P-05 in (cm) 0.232-0.242 (0.589 0.615) Bag Weight lbs (kg) 3.5 (1.6) 31 Conclusion As the results in Tables 8 and 9 show, the AFEXTM pellet has an increased gross caloric value, i.e., an AFEXTM pellet burns about 4.8% more efficiently due to the presence of less moisture in the AFEXTM pellet as compared with an untreated pellet. Specifically, the caloric increase, non-AFEXTM to AFEXTM was calculated as follows: 7388 Btu/lb - 7048 Btu/lb = 340 Btu/lb (or 748 Btu/kg); therefore % increase, non AFEXTM to AFEXTM is (340 Btu/lb)/(7048 Btu/lb) * 100% = 4.8%. Additionally, bulk density increased by an average of seven (7)% and there is an approximately 65% reduction in the amount of fines (i.e., broken pieces having a diameter less than 0.125 cm) in an AFEXTM pellet bag weighing about 3.5 lb (1.6 kg) as compared with a pellet bag of untreated corn stover having approximately the same weight. Additionally, although the "durability indices" between AFEXTM and non-AFEXTM pellets are substantially the same in this testing, the method of testing durability was a simple tumbling experiment ("Kansas State Method"), as compared with the destructive testing described in the above examples. As such, insufficient energy is provided to create the separation required to be able to properly distinguish between the pellets. Regardless, a high durability indice shows that the AFEXTM pellets are suitable for use in the pellet industry. EXAMPLE 7 (PROPHETIC) Samples of biomass, such as switchgrass and prairie cord grass will be collected at various maturities, and corn stover will be collected following grain harvest. Biomass composition will be determined at harvest, during storage in round bales, after initial AFEXTM processing and densification, and after storage of densified pellets. AFEXTM pretreatment will be statistically optimized for hydrolysis and binding properties based on parameters of time, temperature, biomass moisture, and ammonia to biomass ratio. AFEXTM conditions providing at least 90% of glucan conversion and 80% xylan conversion will be used to prepare materials for densification. Densification will be performed using any suitable method, including the methods used in Examples 2 and 3. The resulting pellets will be subjected to various environmental conditions to simulate long-term storage, and then evaluated for flowability, compression strength, water solubility, water absorption, etc. Downstream processing characteristics will be evaluated using a 32 standardized set of hydrolysis and fermentation conditions, including separate hydrolysis and fermentation (SHF) vs. simultaneous saccharification and fermentation (SSF). In one embodiment a comparison of these properties will be made between freshly prepared pellets (i.e., within about one (1) month), stored pellets and non-densified biomass. EXAMPLE 8 (PROPHETIC) AFEXTM pretreatment of prairie cord grass will be statistically optimized for time, temperature, biomass moisture, and ammonia to biomass ratio. A fairly broad range of AFEXTM conditions gives similar hydrolysis results, giving us confidence that there are sets of pretreatment conditions that also enhance binding properties. AFEXTM conditions providing at least 90% of glucan conversion and 80% xylan conversion will be identified and used to prepare materials for densification. We will characterize these pretreated materials for surface properties using various methods developed in our lab (ESCA, Prussian blue staining, SEM), and will correlate those properties with the pellet density and durability. EXAMPLE 9 (PROPHETIC) Ten (10) kilograms each of corn stover and switchgrass will be subjected to AFEXTM pretreatment. These materials will preferably be chopped and milled into 5 different particle sizes, ranging from 850 microns to 2.5 cm prior to receipt by the supplier. A ten (10) kg sample of this species will be used to do a statistically-optimized AFEXTM study. The optimal AFEXTM pretreatment conditions identified providing at least 90% of glucan conversion to glucose and 80% xylan conversion to xylose will be identified. These conditions will be used to prepare a 10 kg batch of AFEXTM prairie cord grass, at the varied particle sizes. It is estimated that each kilogram of AFEXTM biomass should produce approximately forty (40) of the approximately 2.54 x 1.59 x 10.16 cm (about one (1) x 0.625 x four (4) in) "single-stroke" pellets. Therefore, for each biomass species, 80 AFEXTM -treated pellets of each of the five biomass particle size should be obtained, for a total of 400 pellets per feedstock to be tested for durability and suitability. These AFEXTM pretreated materials will also be evaluated for their surface properties using various methods (e.g., ESCA, Prussian blue staining, SEM), and correlate those properties with the pellet density. 33 EXAMPLE 10 (PROPHETIC) Operating variables will be investigated to optimize operating conditions for converting pretreated biomass into densified pellets. These variables include AFEXTM pretreatment conditions, moisture content, particle size, die temperature versus bond strength, rate of compaction versus quality of output, energy usage, existing surface chemistry and variations, compaction ratios and resultant density, and compacted package size and shape. Attrition and wear of mechanical components will also be assessed. EXAMPLE 11 (PROPHETIC) Biomass pretreated using any known AFEXTM procedure or according to the procedure in Example 1 or with any other appropriate modification of an AFEXTM procedure will be densified using any suitable method, including the methods described in Examples 2 and 3. The densified biomass will then be subjected to various environmental conditions, including temperature (25 to 40'C), relative humidity (60 to 90%), consolidation stress (0 to 120 kPa), and storage time (0 to 6 mo). Following storage, physical characteristics will be evaluated as described below: TM Flowability may be evaluated with a simple test in which a number of AFEX pellets are placed in a container, such as the bed of a truck and tipped to about 45 degrees. A comparison with conventional pellets may be made by noting the time it takes for the pellets to flow out of the container. Flowability will also be evaluated using Carr Indices. See ASTM D6393. 1999, Standard test method for bulk solids characterization by Carr indices, ASTM Standards, W. Conshohocken. PA. Flowability is comprehensively defined as the ability of a material to flow un-abruptly under a given environmental condition. The flowability measurement is most often done by Carr Indices, by calculating the total flowability index and total floodability index. Carr, R. L. Jr. 1965, Evaluating flow properties of solids, Chemical Engineering 72(3): 163-168. A higher value to total flowability index and lower value to total floodability index will yield an ideal material with low or no flow problems. Another way to quantify flowability is by measuring the Jenike Shear Stress properties. See Jenike, A. W. 1964, Storage and flow 34 of Bulletin No. 123, Utah Engineering station, Bulletin of University of Utah. Jenike's method will also be used to determine particle cohesion, yield locus, angle of internal friction, yield strength, and flow function, and particle size distribution. See ASTM D6128. 2000, Standard Test Method for Shear Testing of Bulk Solids Using the Jenike Shear Cell, ASTM Standards, W. Conshohocken. PA, and ASAE S 19.3. 2003, Method of determining and expressing fineness of feed materials by sieving, ASAE Standards. St Joseph, MI: ASABE. Additionally, glucan, xylan, galactan, arabinan, mannan, lignin, ash and fiber levels will be evaluated to determine their effect on storage and flowability behavior. Furthermore, several other physical properties will be measured as indicators of poor flowability (i.e., particle size, particle shape, thermal properties, moisture properties, and color). See Selig, M, et al., 2008, Enzymatic saccharification of lignocellulosic biomass, Technical report NREL/TP-510-42629; Sluiter, A, B. Hames, R. Ruiz, C.Scarlata, J. Sluiter, and D. Templeton, 2008a, Determination of ash in biomass, Technical report NREL/TP-510-42622; Sluiter, A, B. Hames, R. Ruiz, C.Scarlata, J. Sluiter, D. Templeton, and D. Crocker. 2008b, Determination of structural carbohydrates and lignin in biomass, Technical report NREL/TP 510-42618. Rheological material properties that affect the ability of biomass to be handled pre- and post-densification will be established. Such properties include, but are not limited to, bulk density, true density, compressibility, relaxation, springback, permeability, unconfined yield strength, and frictional qualities. These properties are a function of the feedstock particle size and distribution, shape factor, moisture condition, and consolidation pressure and time. Since commercial rheological testers are typically designed for use with small grains and fine powders; and consequently, do not accommodate particulate that is greater than inch in diameter, we will develop new measurement systems for characterizing larger feedstock particles. Systems include compaction and shear cells that can be scaled for various material sizes, integrated with commercial load frames, and operated over a range of consolidation pressures. Data will be analyzed to determine conditions which lead to improved (or optimized) flowability, using formal statistical methods such as general linear models, regression, response surface analysis, multivariate analysis, and other techniques as appropriate. See Myers, H. R. 1986, Classical and modem regression applications, 2 edition. Duxbury 35 publications, CA. USA. Draper, N. R., and Smith, H. 1998, Applied Regression Analysis, New York, NY: John Wiley and Sons, Inc. EXAMPLE 12 (PROPHETIC) The impact on downstream processing, such as feedstock pretreatment, densification, and storage on bioconversion efficiency from the integrated AFEXTM /densification process will be evaluated. Tests will be carried out using a standardized set of conditions for both enzymatic hydrolysis and microbial fermentation. At least three types of biomass will be evaluated, namely corn stover, switchgrass, and TM prairie cord grass. For each of these feedstocks, samples of raw ground biomass, AFEX treated biomass, and AFEXTM -treated and densified biomass (before and after storage) will be collected. Thus, 3 x 4 = 12 total biomass sample types will be evaluated. Conversion trials will be conducted in 500 ml Erlenmeyer flasks containing 48 dry g biomass and 152 ml of 2 M phosphate buffer (pH 4.3). The 24% solids loading rate was selected to simulate industrial conditions. Preliminary trials have shown that AFEXTM /densified pellets solubilize rapidly upon immersion in water, therefore grinding will not be necessary (See Example 2). At this point the pH should be 4.7-4.9, and will be adjusted to that level if necessary. To prevent bacterial contamination, 12 ml of a 10 mg/ml tetracycline stock solution will be added. Beyond comparing the three types of feedstocks and four pretreatment combinations, conversion methods, enzyme sources, and enzyme dosage will also be compared as outlined below. Therefore, the nominal enzyme dosage of 15 FPU Spezyme@ CP and 64 pNPGU Novozyme@188 per g glucan will be compared, with a more challenging dosage of one third those rates. A similar pair of enzyme dosages will be calculated for the extremophile enzyme source. Separate hydrolysis and fermentation (SHF) will be evaluated. For saccharification, flasks will be incubated for 48 h at 50 'C and 250 rpm in an orbital shaker. Samples will be removed at 0, 2, 4, 6, 8, 18, 24, 30, 36, and 48 hr. Flasks will then be cooled to 30 'C and inoculated with 2 ml of a 12-18 h culture of a recombinant strain of Saccharomyces cerevisiae which possesses pentose-fermenting capabilities grown in a medium containing two (2) g/l glucose and two (2) g/l yeast extract. Flasks will be incubated for an additional 96 h at 30 'C and 150 rpm in an orbital shaker. Samples will be removed at 0, 3, 6, 9, 18, 24, 36, 48, 60, 72, 84, and 96 hr during fermentation. 36 Simultaneous saccharification and fermentation (SSF) will also be performed to evaluate conversion. The main difference will be that flasks will be dosed with enzyme and immediately inoculated with yeast as noted above, then incubated for 144 hr at 30'C. Samples will be removed at 0, 2, 4, 6, 8, 18, 24, 36, 48, 60, 72, 96, 120, and 144 hr. Enzyme and biomass loadings and other conditions will be identical to those listed above. Additionally, both SHF and SSF, replicating the conditions listed above, except for the enzyme source. Crude enzyme preparations from lignocellulose degrading extremophiles isolated from the Homestake Mine in Lead, SD, now known as the Deep Underground Science and Engineering Laboratory (DUSEL), will be used. See Bang, S., et al, 2008. Biofuels group NSF DUSEL project [abstract]. Homestake DUSEL Spring Workshop. Talk 10. p. 2. These enzymes will be added in appropriate amounts to achieve comparable enzyme dosages to those above. Samples will be analyzed for carbohydrates, organic acids, and ethanol via HPLC using a Biorad@ HPX 87H column and refractive index detector. As noted previously, the AFEXTM process does not produce inhibitors such as furfural and hydroxymethyl furfural, and thus it will not be necessary to measure these. During fermentation, yeast and/or bacterial populations will be measured by hemocytometer or plate counts using potato dextrose agar. Three replications of each saccharification/fermentation will be performed for each treatment. Parameters to be calculated will include rates and yields for both saccharification and fermentation. Results will be averaged and statistically analyzed. EXAMPLE 13 (PROPHETIC) Techno-economic models will be developed for the AFEXTM pretreatment and densification processes of the pretreatment and densification facility. These models will be constructed using material and energy balance data collected upon completion of the aforementioned objectives and the general principles of engineering design. Both capital and operating costs will be estimated by the techno-economic model for each process. The feedstock cost will be assigned a value reflective of an informed estimate of its delivered cost. Once the costs are modeled, optimization methods, such as linear programming, will be used to minimize overall costs and determine an optimum capacity for the pretreatment and densification center facilities. The minimum feasible scale will be determined to assess the efficacy of the process for distributed adoption. 37 A preliminary model of the AFEXTM process will be constructed to interface with the subsequent densification process. Specifically, an AFEXTM pretreatment will be modeled as either a batch or a continuous process, depending upon the origin of the collected data. The capital and operating costs associated with feeding the AFEXTM reactor will be included in the model. AFEXTM reactors will be sized to achieve the desired capacity of pretreated biomass. Heat will be generated in the reactor as ammonia dissolves in the water present in the biomass. Additional heat will be provided by saturated steam at moderate to high pressures, either by direct injection or by indirect contact. Ammonia will be recovered by steam stripping the pretreated biomass using distillation. The bottom stage of the column will produce pretreated biomass that is relatively low in ammonia concentration. The pretreated biomass may be dried in a rotary dryer prior to compaction in the densification process. Both the ammonia-rich distillate and the volatilized gas from the rotary dryer will be combined and re-pressurized for recycle to the AFEXTM reactor. The amount of ammonia recycle is expected to comprise in excess of 95% of the ammonia needed for pretreatment. The costs of biomass, fresh ammonia feed, steam, and electricity will reflect industry values at the time the techno-economic model is constructed. Capital costs will be based upon the cost of fabrication using materials of construction that are compatible with the ammonia-biomass mixtures. All cost inputs will be adjustable to enable a subsequent sensitivity analysis. This analysis will determine the variables which are likely to result in marked increases in the cost of pretreated biomass. Subsequently the external costs for the pretreatment and densification (P&D) facility, including transportation, storage, and material handling will be determined. The overall cost to-benefit ratios for the proposed P&D systems will then be compared to a centralized pretreatment and processing alternative without densification in order to quantify system advantages. It is anticipated that some components, such as transportation costs and material loss, may favor the proposed distributed processing system due to reduced feedstock transportation distance. The additional processing required by the proposed distributed P&D system may increase operating and processing costs at that location, but replace similar processing costs at the centralized processing facility. Additionally, the uniformity and densification of the raw material may yield significant advantages for large-scale material handling, storage and production. Quantifying these potential advantages will be a key outcome of the proposed project. 38 These studies will optimize the AFEXTM process for both pretreatment and subsequent densification; develop and optimize the densification process for pellet formation; determine physical characteristics of pellets before and after storage; evaluate hydrolysis and fermentation of fresh and stored pellets; and conduct an in-depth economic and energy analysis of the process. Techno-economic models will be developed for the AFEXTM and densification processes using data collected above. Delivered feedstock costs will be based on informed estimates. Optimization methods (e.g., linear programming) will be used to minimize overall costs and determine an optimum and minimum capacity for the pretreatment & densification facility. The analysis will then compare the regional biomass processing center (RBPC) versus traditional systems without combined pretreatment and densification. Costs associated with pretreatment and densification of biomass in RBPC will be studied, including optimal and minimal scale of RBPCs; sensitivity analysis to elucidate the variables with greatest impact capital and operating costs; a comparison of decentralized and centralized systems; and a rationale to assist in facility location relative to main biorefinery. An economic model will be developed to provide decision-making capability to those adopting decentralized pretreatment and densification technology. See, for example Flowchart 1 below: 39 Flowchart 1. Comparison of Distributed and Centralized processing models Distributed Processing Model Centralized Processing Model Feedstock Bales Feedstock Bales Transport to Regional Biomass Transport bales to Centralized Processing Center (5-10 miles)* Processing Facility (50 miles)** Hammermill ] Hammermill AFEXTM processing and AFEXTM Processing densification Simultaneous Transport of PELLETS to Saccharification & Centralized Processing Facility, Fermentation Commutate, Solubilize *8 to 16 km; **80.5 km The logistics of harvesting, handling, transporting, and storing low bulk density feedstocks is a significant challenge to the bioeconomy. These issues are especially critical for herbaceous feedstocks, which may have low per-acre productivities. For example, biomass that yields 70 gallons of ethanol per ton, baled at a density of 120 Kg/n3 would require over 10 times the volume of material for a given volume of ethanol, compared with corn grain. Therefore, biomass densification at distributed locations (to minimize transport of feedstock bales) is critically needed, but conventional extrusion pelleting of untreated biomass has proven too costly. Novel densified biomass products and methods for making and using same are described herein. In one embodiment, an AFEXTM pretreatment is used to produce a tacky biomass which, surprisingly, is easily convertible to a solid briquette or pellet without the use of additional binder. The AFEXTM pellets are also surprisingly at least as dense and demonstrate superior hardness properties as compared with conventional pellets containing added binders. 40 In one embodiment, pellets comprising more than one type of biomass material (e.g., corn stover, grasses, wood, and the like) are provided. In this way, a commodity pelleted biomass product having relatively uniform properties which may be more easily adopted into the biomass processing industry, can be provided. Such properties may include, but are not limited to, BTU content, sugar content, and so forth. Any suitable type of densification process may be used to produce products having a variety of sizes and shapes. In one embodiment, the densification process device uses a gear mesh system to compress biomass through a tapering channel between adjacent gear teeth, forming high density pellets. In one embodiment, the system operates at lower temperature, pressure, and energy requirements than comparable pelleting systems. In one embodiment, the AFEXTM pellets "hold up" better, i.e., are more resistant to physical forces, than non-AFEXTM pellets during shipping, handling and/or storing. In one embodiment, the resulting products have an increased flowability as compared with conventional biomass solids, which allow for automated loading and unloading of transport vehicles and storage systems, as well as transport through the processing facility. All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention. Although specific embodiments have been illustrated and described herein, it will be appreciated by those of ordinary skill in the art that any procedure that is calculated to achieve the same purpose may be substituted for the specific embodiments shown. For example, although the process has been discussed using particular types of plant biomass, any type of plant biomass or other types of biomass or biofuels, such as agricultural biofuels, for example, may be used. This application is intended to cover any adaptations or variations of the present subject matter. Therefore, it is manifestly intended that embodiments of this invention be limited only by the claims and the equivalents thereof. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. 41 The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. 42

Claims (30)

1. A process comprising: subjecting a quantity of plant biomass fibers to a pretreatment to cause at least a portion of lignin and/or hemicellulose contained within each fiber to move to an outer surface of said fiber, wherein a quantity of pretreated tacky plant biomass fibers is produced; and densifying the quantity of pretreated tacky plant biomass fibers to produce one or more densified biomass particulates, wherein said biomass fibers are densified without using added binder.

2. The process of claim I wherein each of said densified biomass particulates has a substantially smooth, non-flakey outer surface.

3. The process of claim I or claim 2, wherein the pretreatment is a sodium hydroxide pretreatment or an ammonia pretreatment selected from a liquid ammonia fiber expansion (AFEX) pretreatment or a gaseous AFEX pretreatment.

4. The process of any one of claims I to 3, further comprising adding water and/or steam prior to and/or during the pretreatment.

5. The process of any one of claims I to 4, wherein said biomass fibers comprise reduced lignin and/or hemicellulose levels in their interiors and increased lignin and/or hemicellulose levels on their surfaces as compared with the lignin and/or hemicellulose levels of said fibers prior to the pretreatment, wherein said surface lignin and/or hemicellulose levels provide a degree of tackiness thereon.

6. The process of any one of claims I to 4, wherein said biomass fibers are coated with lignin on their surfaces and at least some of said biomass fibers are also coated with hemicellulose. 43

7. The process of any one of claims 1 to 6, wherein said densified biomass particulates have a substantially rectangular shape or a substantially cylindrical shape.

8. The process of any one of claims I to 7, wherein said biomass fibers are corn stover fibers, switchgrass fibers, prairie cord grass fibers, or combinations thereof.

9. The process of any one of claims I to 8, comprising a plurality of densified biomass particulates of a given mass have a higher bulk density than baled or loose plant biomass of the same given mass.

10. The process of claim 1 wherein the pretreatment and densifying are performed in a regional biomass process center.

11. The process of any one of claims I to 10, wherein said biomass fibers contain a trace amount of base from the pretreatment.

12. The process of claim I1, wherein the trace amount of base comprises about 1% to about 2% ammonia.

13. A product produced according to the process of claim 2.

14. A process comprising: subjecting a quantity of plant biomass fibers to a pretreatment to cause at least a portion of lignin and/or hemicellulose contained within each fiber to move to an outer surface of said fiber to produce a quantity of pretreated tacky plant biomass fibers, wherein the pretreatment is an ammonia pretreatment that generates reduced levels of lignin and/or hemicellulose in an interior of said fiber and increased lignin and/or hemicellulose on its surface as compared with pretreatment levels of lignin and/or hemicellulose, wherein said surface lignin and/or hemicellulose has a degree of tackiness; and densifying the quantity of pretreated tacky plant biomass fibers to produce one or more densified biomass particulates, wherein the quantity of said biomass fibers is densified without using added binder. 44

15. The process of claim 14, wherein the ammonia pretreatment is a liquid ammonia fiber expansion (AFEX) pretreatment or a gaseous AFEX pretreatment.

16. The process of claim 14 or claim 15, wherein the ammonia pretreatment comprises heating said biomass fibers to a temperature between about 60 and about 100 *C and said densification comprises performing compression at a temperature less than 60 *C.

17. The process of any one of claims 14 tol6 wherein said densification is performed without a curing step.

18. The process of any one of claims 14 to 17, wherein said densification comprises increasing pressure at a rate of less than 0.034 atm/sec.

19. The process of any one of claims 14 to 18, wherein said densified biomass particulates have an increased gross caloric value as compared with densified biomass particulates containing untreated plant biomass fibers.

20. The process of any one of claims 14 to 19, wherein said densified biomass particulate burns about 4.8% more efficiently as compared with a densified biomass particulate containing untreated plant biomass fibers.

21. The process of any one of claims 14 to 20, wherein a given mass of a plurality of densified biomass particulates has a reduced amount of fines as compared with a plurality of densified biomass particulates of the same given mass containing untreated plant biomass fibers.

22. The process of any one of claims 14 to 21, wherein said densification comprises forming a plurality of densified biomass particulates of a given mass having a higher bulk density than baled or loose plant biomass of the same given mass. 45

23. The process of any one of claims 14 to 22, wherein said densified biomass particulates have a bulk density of between about 547.2 kg/m 3 and about 765 kg/m 3 .

24. The process of any one of claims 14 to 23, wherein said biomass fibers comprise an agricultural crop waste, a woody energy crop, a wood waste and/or a grass crop.

25. The process of any one of claims 14 to 24, wherein said densification comprises compressing said densified biomass particulates through a tapering channel.

26. The process of any one of claims 14 to 25, further comprising adding water and/or steam prior to and/or during the ammonia pretreatment step.

27. The process of any one of claims 14 to 26, wherein the pretreatment and densifying steps are performed as an integrated process at a single location.

28. A product made according to the process of any one of claims 14 to 27.

29. A process comprising using one or more densified biomass particulates in a bioproduct production facility to produce a product, wherein said densified biomass particulates are each comprised of a plurality of pretreated lignin-coated plant biomass fibers, wherein the pretreatment causes at least a portion of lignin and/or hemicellulose contained within each fiber to move to an outer surface of the fiber, and each fiber contains a trace amount of base from the pretreatment, and wherein said densified biomass particulates have a substantially smooth, non-flakey outer surface and contain no added binder.

30. The process according to any one of claims I to 12, 14 to 27 or 29, or the product of claim 13 or claim 28, substantially as hereinbefore described with reference to the figures and/or examples. 46