KR101970859B1 - Methods of hydrolyzing pretreated densified biomass particulates and systems related thereto - Google Patents

Abstract

There is provided a method wherein the pretreated densified cellulose biomass particles can be hydrolyzed at a high solid loading rate compared to the solid loading rate of coarse hydrolysable cellulose biomass fibers. The resulting high concentration of sugar-containing streams can be readily converted to an entire collection of biofuels or other useful biochemicals.

Description

TECHNICAL FIELD The present invention relates to a hydrolysis method of preprocessed densified biomass particles and a system therefor. BACKGROUND OF THE INVENTION 1. Field of the Invention < RTI ID = 0.0 >

This application claims priority to U.S. Patent Application No. 13 / 458,830, filed April 27, 2012, which is a continuation-in-part of U.S. Patent Application No. 13 / 202,011 filed on August 17, 2011 , Which application is filed on August 24, 2010 and incorporated by reference in its entirety to the International Application No. PCT / US2010 / 046525 published on March 10, 2011 in WO 2011/028543, 371, the entirety of which is incorporated herein by reference. U.S. Provisional Application No. 61 / 236,403, filed August 24, 2009 under 119 (e), all of which are incorporated herein by reference in their entirety.

Declaration of Government Rights

The present invention was made with Government support under the US Department of Energy's Sungrant Research Project Grant No. DE-FG36-08-GO88073. The government has certain rights in the invention.

Current attempts to produce cellulosic ethanol are costly and involve a number of steps.

It is an object of the present invention to provide a method for hydrolyzing pretreated densified biomass particles and a system associated therewith.

summary

In one embodiment, there is provided a product comprising at least one hydrolysable densified biomass particulate consisting of a plurality of lignin-coated plant biomass fibers without binder addition, wherein at least one hydrolyzable densified biomass The fine particles have a substantially equivalent intrinsic density to the binder-containing hydrolyzable densified biomass particles and have a substantially smooth non-flakey outer surface. In one embodiment, the novel product contains a minor amount of ammonia. In one embodiment, the article of manufacture comprises at least one hydrolyzable densified biomass particulate, wherein each particulate is a hydrolyzable densified biodegradable biomass having substantially the same intrinsic density as the binder-containing hydrolyzable densified biomass particulate, Coated plant biomass fibers in an amount sufficient to form mass particulates.

In one embodiment, the at least one hydrolyzable densified biomass particulate without binder addition is characterized by increased resistance to deformation, increased hardness, increased degradation resistance, improved shelf life Or a combination thereof. In one embodiment, the novel product may be more resistant to stress, and possibly less brittle, as compared to binder-containing hydrolyzable densified biomass particles.

In one embodiment, the novel product is harder than the same mass of binder-containing hydrolyzable densified biomass particles provided, for example at least 21% harder and hardness variability less than 20%.

The novel products described herein may be of any suitable shape and size, including, for example, substantially rectangular or substantially cylindrically shaped.

In one embodiment, each of the plurality of lignin-coated plant biomass fibers in the hydrolyzable densified fine particles is completely coated 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 in the hydrolyzable densified fine particles are also coated with hemicellulose. In one embodiment, substantially all of the plurality of substantially lignin-coated plant biomass fibers in the hydrolyzable densified particulate are also coated with hemicellulose, such that the hemicellulose and lignin are surfaced as a " package " .

The use of any suitable plant biomass, including, but not limited to, corn stover, switchgrass, pine and / or prairie cord grass, A product can be produced.

In one embodiment, the novel products have improved shelf life, increased resistance to degradation, increased fluidity, and greater bulk density when compared to binder-containing hydrolyzable densified biomass particles.

In one embodiment, a container; And a fixed amount of hydrolyzed densified biomass particulate, wherein the binder is not added, wherein the amount of the hydrolyzed densified biomass particulate is equal to the amount of the binder-containing hydrolyzed densified biomass Has a bulk density greater than the bulk density of the particulates. The container may be a rigid container or a flexible bag.

In one embodiment, the step of subjecting a given amount of biomass fibers to ammonia treatment, wherein at least a portion of the lignin contained in each fiber migrates to the outer surface of each fiber to form a certain amount of tacky (i.e. sticky) Producing mass fibers; And densifying an amount of tacky biomass fiber to produce at least one hydrolyzable densified biomass particulate, wherein the tampon is densified without the binder to which a quantity of tacky biomass fiber is added. In one embodiment, the ammonia treatment causes at least a portion of the hemicellulose contained within each fiber to migrate to the outer surface of each fiber. In one embodiment, the ammonia treatment is ammonia fiber expansion (AFEX (TM)) treatment, for example, gaseous AFEX (TM) treatment.

In one embodiment, the integrating process further comprises a hydrolysis step in which the hydrolyzable densified biomass particulates are hydrolyzed using a high, i.e., greater than 12%, solid loading. By the use of high solids loading, the per cellulose stream can be fed through the fermentation of the free sugars to a biofuel (e. G. At least about 6 to about 8% by weight of the fermentable sugar) or other useful bioproduct And become enriched enough to be converted into an entire suite. In one embodiment, the conversion comprises fermentation.

A variety of systems for producing cellulose per stream and / or converted cellulose biomass are also provided.

In one embodiment, there is provided a biofuel comprising at least one hydrolysable densified biomass particulate of a given mass, wherein the binder is not added, but consists of a plurality of lignin-coated plant biomass fibers, wherein at least one The hydrolysable densified biomass particles have a substantially uniform density and a substantially smooth non-flaky outer surface with the same mass of binder-containing hydrolyzable densified biomass particles provided. Such biofuels may be useful in biomass-burning stoves or boilers.

In one embodiment, there is provided an animal feed comprising a provided mass of at least one hydrolysable densified biomass particulate, wherein the binder is not added, but consists of a plurality of lignin-coated plant biomass fibers, wherein at least one The hydrolysable densified biomass particulate has a substantially smooth non-flaky outer surface with substantially equivalent densities to the same mass of binder-containing hydrolyzable densified biomass particulate provided, and the animal feed has a binder-containing hydrolyzable Compared with animal feed containing densified biomass particles, digestibility is improved.

In one embodiment, there is provided a solid material comprising at least one hydrolysable densified biomass particulate of a given mass, consisting of a plurality of lignin-coated plant biomass fibers, without the addition of a binder, The at least one hydrolyzable densified biomass particulate has substantially the same density as the binder-containing hydrolyzable densified biomass particulate of the same mass provided and has a substantially smooth non-flaky outer surface, For example, it is useful for fiberboard or extruded fibrous construction materials.

The resulting densified biomass particulates can be used to produce a complete collection of other biotech products using animal feed production, chemical catalysts or chemical conversion, other biochemical applications, electrogeneration applications (eg, combustion in boilers, biomass-burning stoves, etc.) For example, as a component in biofuels, solids such as fibreboards and extruded fibrous construction materials, including, but not limited to,

Figure 1 shows an image showing the AFEX (TM) preprocessed cornbore (AFEX ™ -CS), AFEX ™ Pretreatment Switchgrass (AFEX ™ -SG), AFEX ™ -CS briquette and AFEX ™ -SG briquettes according to various embodiments .
Figure 2 includes images of binder-containing non-AFEX ™ -CS briquettes and AFEX ™ -CS briquettes according to various embodiments.
3A-3E are images of three biomass samples taken at various times, including AFEX ™ -CS, AFEX ™ -CS briquettes, and immersed AFEX ™ -CS briquettes according to various embodiments.
Figure 4 is a graph showing percent conversion of glucan versus biomass at 6 hours, 24 hours, and 72 hours for the biomass samples shown in Figures 3C-3E according to various embodiments.
FIG. 5 is a graph showing the% xylen conversion versus biomass at 6 hours, 24 hours, and 72 hours for the biomass samples shown in FIGS. 3C-3E according to various embodiments.
Figure 6 is a graph showing glucose concentrations for AFEX (TM) -treated corn versus pellets produced at four different moisture ratios according to various embodiments.
Figures 7a-7h provide a visual comparison of the hydrolysis process 7a-7d using the hydrolyzable densified microparticles according to various embodiments and the conventional hydrolysis process 7e-7h using loose biomass fibers Fig.
Figure 8 is a graph showing the bulk density for AFEX (TM) -treated corn versus pellets generated with multiple sizes and moisture ratios according to various embodiments.

In the following detailed description, implementations are described in sufficient detail to enable those skilled in the art to practice them, and that other implementations may be used and that changes in the chemical and procedural aspects may be made without departing from the purpose and scope of the subject matter of the present invention It will be understood. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the embodiments is defined only by the appended claims.

As used herein, the term " biomass " refers generally to organic matter obtained or collected from renewable biomass as an energy source. The renewable biomass may comprise plant material, animal material and / or biologically produced material. The term " biomass " is not to be construed as including non-renewable fossil fuels.

The term " plant biomass " or " lignocellulosic biomass (LCB) ", as used herein, refers to cellulose and / or lignocellulosic biomass as its major carbohydrate (woody or non- woody) Lt; / RTI > is intended to refer to virtually any plant-derived organism containing hemicellulose or hemicellulose. The plant biomass may include, but is not limited to, crop waste and residues such as corn stover, straw, rice straw, sugar cane bagasse, and the like. The plant biomass may be selected from the group consisting of woody energy crops, wood waste and residues such as wood, such as fruit trees such as fruit trees (eg apple trees, orange trees, etc.), coniferous trees, Wastes, sawdust, paper and pulp industrial waste streams, wood fibers, and the like. In addition, herbaceous crops such as a variety of preygrass, such as praligy cordgrass, switchgrass, big bluestem, little bluestem, side oats grama, As a biomass source of plant biomass. In urban areas, potential plant biomass feedstock includes yard waste (eg, shredded grass, leaves, cut trees, dumplings, etc.) and vegetable processing waste. Plant biomass is known to be the most common form of carbohydrates available in nature, and corn is the largest source of readily available plant biomass in the United States. When used without modifiers, the term " biomass " is intended to refer to an LCB.

The term " biofuel " as used herein refers to any biologically and / or chemically generated renewable solid, liquid or gaseous fuel, for example originating from biomass. Most biofuels originate from the original biological processes, for example photosynthesis processes, and can therefore be considered as solar or chemical energy sources. Other biofuels, such as natural polymers (e.g., chitin or certain sources of microbial cellulose), are not synthesized during photosynthesis, but nevertheless can be considered biofuels because they are biodegradable. Generally, there are three types of biofuels derived from biomass that are synthesized during photosynthesis: agricultural biofuels (defined below), municipal waste biofuels (most recyclable materials, such as glass and metals, (Eg, residential and small commercial garbage or waste) and forest biofuels (eg, wood or wood product waste or by-product streams, wood fibers, pulp and paper industries). Biofuels produced from biomass that are not synthesized during photosynthesis include, but are not limited to, those derived from chitin, a chemical variant form of cellulose known as N-acetylglucosamine polymer. Chitin is an important component of the waste generated by the aquaculture industry because the wastes contain shells of seafood.

As used herein, the term " agricultural biofuel " includes crops, lignocellulose crop residues, grain processing facility wastes (e.g., wheat / oat husks, corn / (E. G., A separate waste stream, e. G., A waste stream, e. G., A waste stream) (For example, distiller's wet grain (DWG) and syrup from an ethanol production facility, etc.), and the like (for example, oil, fat, stems, husks, intermediate process residues, rinsing / Biofuels derived from biofuels. Examples of livestock industries include, but are not limited to, beef, pork, turkey, chicken, laying and dairy facilities. Examples of crops include, but are not limited to, any type of non-ligneous plants (e.g. cotton), grains such as corn, wheat, soybeans, sorghum, barley, oats, rye, Short-term cultivated herbaceous crops, such as, for example, switchgrass, alpapa, and the like.

The term " pretreatment step " as used herein refers to any step, i.e., treatment, that is intended to alter the inherent biomass so that it can be converted more efficiently and economically into a reactive intermediate compound, e.g., a sugar, an organic acid, , The reactive intermediate compound can then be processed into various end products, such as ethanol, iso-butanol, long-chain alkanes, and the like. Pretreatment reduces the crystallinity of the polymer matrix, reduces the lignin from interfering with biomass conversion, hydrolyzes a portion of the structural carbohydrates, thereby increasing their degradability by enzymes and degrading the biomass to useful products Can be accelerated. The pretreatment method can use various concentrations of acid (including sulfuric acid, hydrochloric acid, organic acid and the like) and / or an alkali such as ammonia, ammonium hydroxide, sodium hydroxide, lime and the like. The pretreatment method may additionally or alternatively employ hydrothermal treatment including water, heat, steam or pressurized steam. Pretreatment can occur or be used in various types of containers, reactors, pipes, flows through cells, and the like. Most pretreatment methods will result in partial or complete solubilization and / or destabilization of lignin, and / or hydrolysis of hemicellulose per pentose.

As used herein, the term " moisture content " refers to the percentage moisture of the biomass. The water content is calculated as the liquid per gram of wet biomass (biomass dry material + liquid), for example, 100 grams of water multiplied by grams. Thus, when used herein without the clue condition, the% moisture content refers to the total weight basis.

As used herein, the term " AFEX (TM) " pretreatment refers to the process of pretreating biomass with ammonia, solubilizing lignin from plant cell walls, and re-depositing it on the surface of the biomass. AFEX ™ pretreatment destroys the lignocellulose matrix, thereby modifying the structure of the lignin, partially hydrolyzing the hemicellulose, and increasing the accessibility of the cellulose and residual hemicellulose to subsequent degradation by the enzyme. Lignin is a major barrier to enzymatic hydrolysis of native biomass, and removal, rearrangement or alteration of lignin is an anticipatory mechanism for some of the leading pretreatment technologies, including AFEX ™.

However, in contrast to many other pretreatments, the lower temperature and non-acidic conditions of the AFEX (TM) process can be attributed to the fact that lignin and / or sugars can be used as furfural, hydroxymethyl furfural, and organic acids that can negatively affect microbial activity Do not switch. The process further inflates and swells the cellulosic fibers and further destroys the amorphous hemicellulose in the lignocellulosic biomass. These structural changes can open the plant cell wall structure and convert the lignocellulosic biomass into a more efficient and complete value-added product while preserving the nutritive value and composition of the material. See, for example, U.S. Patent No. 6,106,888; 6,176,176; 5,037,663; And 4,600,590, all of which are incorporated herein by reference in their entirety as if fully set forth herein.

As used herein, the term "condensation AFEX ™ pretreatment" refers to AFEX ™ pretreatment as defined herein using gaseous ammonia instead of liquid ammonia. By allowing the hot ammonia gas to condense directly on the cooler biomass, the biomass is heated quickly and the ammonia and biomass are intimately contacted.

The term " additive binder " as used herein refers to natural and / or synthetic materials and / or energy forms added to or applied to biomass fibers that have been pretreated in an amount sufficient to enhance the stability of the densified biomass particulates. Examples of binders that are typically added include extrinsic heat, steam, water, corn starch, lignin compounds, lignite, coffee grounds, sap, pitch, polymers, salts, acids, bases, molasses, organic compounds, urea, But are not limited to these. In addition, special additives are used to improve bonding and other properties such as color, taste, pH stability and water resistance.

The added binder of the added energy type is typically in the form of apparently added heat, i.e. exogenous heat, e.g. convection heat or conduction heat, but radiant heat can also be used for the same purpose. The intentional addition of exogenous heat is in contrast to the intrinsic heat that occurs as a result of processing the material, such as frictional heat that occurs during operation in a densification machine. Thus, the heat inherent in the pretreatment and / or densification of the biomass is not considered herein as " added binder ". The added binder may be added to the pretreated biomass at any time before, during, or after the densification process. The amount of binder added may vary depending on the substrate being densified.

The term " particulate " or " biomass particulate " as used herein refers to densified (i.e., rigid) biomass formed from a plurality of spongy biomass fibers that are compressed to form a single particulate product that can be divided into individual pieces. The microparticles may be hydrolyzable or non-hydrolysable and may range in size from pellets and briquettes or large objects, such as bricks or more, for example, from a size smaller than a hay bale, And may have any suitable mass. The specific geometry and mass of the particulate will depend on various factors including the type of biomass used, the amount of pressure used to produce the particulate, the length of the particulate desired, the particular end use, and the like.

The term " briquettes " as used herein refers to compressed particulates.

The term " pellets " as used herein refers to extruded particulates, i.e., compressed particulates formed in a molding process to force the material through the die.

As used herein, the term " fluidity " refers to the ability of a particulate to flow out of a container using only gravity. Accordingly, the product with increased fluidity will flow out of the container at a higher rate than the product with lower fluidity.

The term " transport properties " as used herein refers to one or more properties of the particulate associated with storage, handling and transport, including stability, shelf life, fluidity, high bulk density, high true density, compressibility, durability, , Springback, permeability, unconfined yield strength, and the like.

As used herein, the term " solid loading " refers to the weight percent of solids in the hydrolysis mixture, including solids, liquids, and hydrolysis additives (e.g., enzymes). The solids may be coarse cellulosic fibers or densified cellulose microparticles.

Cellulose biofuel generation from lignocellulosic biomass is highly resilient due to both environmental and social sustainability benefits. However, the technology is not yet fully commercialized. One problem that hinders the production of cellulosic biofuels using this platform is the hydrolysis of certain components in the lignocellulosic biomass.

Almost all forms of lignocellulosic biomass, i.e., plant biomass, e.g., monocotyledonous plants, comprise three major chemical fractions: hemicellulose, cellulose and lignin. Lignin, a polymer of phenolic molecules, provides structural integrity to plants and is difficult to hydrolyse. Thus, after the sugar in the biomass is fermented with a biosynthetic, for example, alcohol, the lignin remains a residual material (i.e., a recalcitrant lignin matrix).

Cellulose and hemicellulose in plant cell walls are present in a complex structure within the degradable lignin matrix. Hemicellulose is a short, highly branched polymer of mostly 5-carbon pentoses (xylose and arabinose) and less 6-carbon hexose sugars (galactose, glucose and mannose). Hemicellulose is amorphous because of its branching structure and is relatively easy to hydrolyse into its individual constituents by enzymatic or refluxing treatment. Cellulose is very similar to starch with linear / branched polymers composed of wet milled ethanol plants and α (1 → 4) linked D-glucose, the main substrate of corn grains in dry grains, 1 → 4) linked D-glucose. However, unlike starch, the glucose sugars of cellulose are bound together by a [beta] -glucosid bond that allows the cellulose to form tightly-associated linear chains. Because of the high degree of hydrogen bonding that can occur between the cellulose chains, cellulose is very stable and forms a hard crystalline structure that is much more resistant to hydrolysis by chemical or enzymatic attack than starch or hemicellulose polymers. Although the hemicellulose sugars represent easily achievable targets for conversion to biofuels, substantially higher cellulosic content has the ability to maximize biofuel yield based on tonnes of plant biomass Indicating a larger one.

Thus, using a pretreatment process, it modifies and opens the cell wall matrix, hydrolyzes hemicellulose, and reduces crystallinity. Pretreatment destroys the lignocellulosic biomass, for example, the refractory portion of cellulose and lignin, thereby improving its degradability. After pretreatment, a large amount of biomass is easily degradable, while a significant amount is retained. Ultimately, the pretreatment process makes cellulose more accessible for conversion of carbohydrate polymers to fermentable sugars (during subsequent hydrolysis processes) (Balan et al. 2008; Sierra et al. 2008; Sun and Cheng 2002). For example, ammonia fiber expansion (AFEX ™) can open cell walls in agricultural residues, greatly reducing degradation products compared to acid pretreatment (Chundawat et al., 2010) It is a viable option.

Other pretreatment methods include, for example, ammonia recycle leaching (ARP), high concentration acid hydrolysis pretreatment, dilute acid hydrolysis, two-step acid hydrolysis pretreatment, high pressure hot water based method, i.e., hydrothermal treatment, Explosion and hydrothermal hydrolysis by enzymatic hydrothermal hot water extraction, reactor systems (e.g., batch, continuous flow, reflux, perfusion, etc.), lime treatment and pH-based treatment, hydrothermal or chemical pretreatment, Catalytic hydrolysis) or hydrolysis and glycosylation by simultaneous enzymes. As mentioned above, some methods produce nearly complete hydrolysis of the hemicellulose fraction for efficient recovery per high yield of soluble pentos. The recovery of these sugars also facilitates physical removal of the surrounding hemicellulose and lignin, thereby exposing the cellulose to further processing.

Although the cellulose is more available for conversion to its constituent sugars during hydrolysis after the pretreatment, in order for the fermentation to take place downstream, the sugar concentration obtained may be adjusted to an appropriate level (e. G., At least about 6% In embodiments, at least about 7% or about 8% or more, up to about 9% or more, such as up to about 18% or more, including any range therein, should be present. Some attempts to increase the per-stream concentration include using a smaller amount of pretreated biomass to produce a more dilute per cellulose stream and then concentrating this stream to achieve higher sugar levels. However, the concentration of the sugar stream in this manner is expensive.

In addition, since the pretreated coarse biomass fibers rapidly absorb the liquid, a larger amount of coarse biomass fibers, i.e., 12% (e.g., pre-treated coarse grains per kilogram of total weight of biomass, The use of solid loading of biomass above 120 grams of biomass fiber produces products that may be difficult to mix and / or that are not efficiently hydrolyzed. Attempts to overcome this problem include batching each successive load by adding a small amount of pretreated virgin biomass fiber, while adding to the hydrolysis tank only after liquefaction of previously added biomass fibers has been achieved . Even though the batch process includes only two or three batches, an extended initial liquefaction period results because a continuous liquefaction step is required.

Other options to overcome this problem include the use of reactors and impellers, which are now considered to be " specialized " due to the size of the impeller relative to the inner diameter of the reactor. This reactor has an impeller with a diameter that is substantially the same as the inner diameter of the reactor, i. E., The impeller size to reactor diameter ratio is greater than about 3: 4. Examples include, but are not limited to, horizontal paddle mixers, horizontal ribbon mixers, vertical spiral ribbons, anchor-type impellers, and the like. However, these reactors tend to be more expensive than those with smaller impellers. Also, because of their weight, they are not always appropriate for large vessels (over 500,000 liters).

Various embodiments provide methods of pretreatment and densification of spongy biomass fibers to produce hydrolyzable pretreated densified biomass particulates (hereinafter " hydrolyzable particulates "). In contrast to conventional densification processes, the embodiments described herein do not require a binder that is added to improve the transport properties or stability of the resulting hydrolyzable microparticles. Rather, as discussed herein, the present inventors have surprisingly and unexpectedly found that highly stable, high-quality, hydrolysable particulates are " without binder added " during the densification step, without the addition of a binder, Can be produced without added binder during the pretreatment step or at any time after densification.

These particulates improve the hydrolysis efficiency in terms of time and / or yield and ultimately have been found to cause the conversion to occur downstream. These improvements are partly due to the fact that the hydrolyzable microparticles described herein unexpectedly allow higher solids loading during hydrolysis compared to virgin biomass fibers comprising pretreated virgin biomass fibers. A visual comparison of one embodiment of the novel hydrolysis process described herein using hydrolysable densified microparticles and a conventional hydrolysis process using coarse biomass fibers is shown in the schematic diagram of Figures 7a-7h. Figures 7a-7h are further described in Example 8 because this visual representation correlates with the test performed in Example 8. < RTI ID = 0.0 > Not only can the sugar stream be obtained at a concentration high enough to provide efficient conversion, but also downstream bio-products can now be produced more efficiently and cost-effectively.

In one embodiment, the hydrolyzable microparticles have a high solids loading (i.e., greater than 12%, up to about 15% or more, such as up to about 35% of any combination of hydrolyzable microparticles, Hydrolyzable < / RTI > particulate content of the hydrolyzable microparticles). Using high solids loading of the hydrolyzable microparticles, the per cellulose stream is allowed to concentrate sufficiently for conversion, e. G. Fermentation.

Any suitable pretreatment method may be used. In one embodiment, the ammonia fiber expansion method (AFEX (TM)) pre-treatment is used.

In one embodiment, the coarse biomass fibers are heated to a temperature of from about 60 ° C to about 100 ° C in the presence of high concentration ammonia. For example, Dale, BE et al., 2004, Pretreatment of corn stover using ammonia fiber expansion (AFEXTM) , Applied Biochem, Biotechnol. 115: 951-963, which is incorporated herein by reference in its entirety. Subsequently, rapid pressure drop causes physical breakdown of the biomass structure, exposing cellulose and hemicellulose fibers without severe sugar degradation typical of many pretreatments.

Almost all of the ammonia can be recovered and reused, while residual ammonia is used as a nitrogen source for microorganisms in fermentation. In one embodiment, about one (1) to two (2) wt% of the ammonia remains in the pretreated biomass.

Also, since the wash stream is not present in the process, the dry matter recovery after AFEX ™ treatment is essentially quantitative. This is because AFEX ™ is basically a drying-to-drying process.

In addition, AFEX (TM) -treated biomass is stable for a longer period of time (e.g., up to at least one year) than non-AFXTM-treated biomass, and can be readily diluted with dilute or other aqueous pretreatment (E. G., At least about 40%) in a hydrolysis or fermentation process by enzymes.

In addition, cellulose and hemicellulose are well preserved in the AFEX (TM) process and show little degradation. Thus, neutralization is not required prior to hydrolysis by AFEX ™ - treated biomass enzymes. In addition, enzymatic hydrolysis of AFEX (TM) -treated biomass produces a clean sugar stream for subsequent fermentation.

In addition, the degradation products from AFEX ™ -treated biomass were identified and quantified. One such study compared AFEX ™ and acid-pretreated corn bands using LC-MS / GC-MS techniques. In acid-pretreatment feedstocks, over 40 major compounds were detected, including organic acids, furans, aromatics, phenols, amides and oligosaccharides. Very little acetic acid, HMF and furfural were produced by AFEX (TM) pretreatment performed under moderate alkaline conditions. Supra [Dale, BE et al., 2004] and Reference [Dale, BE et al, 2005b , Pretreatment of Switchgrass Using Ammonia Fiber Expansion (AFEX ™), Applied Biochemistry and Biotechnology. Vol. 121-124. pp. 1133-1142. See also Dale, BE et al., 2005a. Optimization of the Ammonia Fiber Explosion (AFEXTM) Treatment Parameters for Enzymatic Hydrolysis of Corn Stover , Bioresource Technology . Vol. 96, pp. 2014-2018].

In one embodiment, a modified AFEX (TM) pretreatment process, i.e., gas AFEX (TM) pretreatment, is used as described in Example 1. In this way, gaseous ammonia is used, which condenses on the biomass itself.

In one embodiment, the AFEX (TM) pretreatment conditions are optimized for a particular biomass type. These conditions include, but are not limited to, ammonia loading, water content of biomass, temperature and residence time. In one embodiment, the cornstarch is subjected to AFEX (TM) pretreatment with a temperature of about 90 DEG C, a ammonia: dry corn to mass ratio of 1: 1, a water content of 37.5% cornice and a residence time of 5 . In one embodiment, the switchgrass has an ammonia loading of 1: 1 ammonia (kg): dry matter (kg) and a water content (based on total weight) of 45% at a temperature of about 100 deg. With AFEX ™ pre-treatment.

The hydrolysis results of AFEX ™ -treated and untreated samples show 93% versus 16% glucan conversion, respectively. The ethanol yield of the optimized AFEX ™ -treated switchgrass was measured to be about 0.2 g (ethanol) / g (dry biomass), 2.5 times greater than the ethanol yield of the untreated sample. See Dale, BE et al., 2005b.

In one embodiment, approximately 98% of the theoretical glucose yield is obtained from AFEX (TM) -treated corn using a filter paper unit of 60 cellulase / g (glucan) equivalent to 22 FPU / g (dry corn versus) Lt; / RTI > enzymatic hydrolysis.

The ethanol yield is 2.2 times higher than that of untreated samples. In one embodiment, lower enzyme loading of 15 and 7.5 FPU / g (glucan) has no significant effect on glucose yield compared to 60 FPU. In this embodiment, the difference between the effects at different enzyme levels decreases as the treatment temperature increases. For example, the literature [Dale, BE et al, 2004 .]; And refer to the literature [Dale, BE et al., 2004].

In addition, the above document optimum ™ AFEX pre-treatment conditions for the switch grass and corn cause hydrolysis and fermentation [. Dale, BE et al, 2004]; Supra [Dale, BE et al, 2005b ]; And it is discussed in the literature [Dale, BE et al., 2005b].

In one embodiment, a modified AFEX (TM) pretreatment is used wherein the ammonia loading is significantly reduced and the required ammonia concentration is lower. Document [Elizabeth (Newton) Sendich, et al, Recent process improvements for the ammonia fiber expansion (AFEX ™) process and resulting reductions in minimum ethanol selling price, 2008, Bioresource Technology 99:. 8429-8435] and US Patent Application Publication No. 2008/000873 (Dale, BE).

In one embodiment, the steam is used as a pretreatment instead of or in addition to the AFEX (TM) treatment. However, steam tends to reduce the availability of sugar, thereby lowering the overall quality of the animal feed. Nevertheless, steam remains an optional implementation that is viable for pretreatment.

When the biomass fibers are densified, the fibers themselves typically become hot as they are formed into hydrolyzable particulates. Such intrinsic heat may include frictional heat that occurs during the extrusion or compression process, as is known in the art. As defined herein, such heat is not considered to be " added binder ".

Although the binder to be added is not used during the densification process described herein, the binder to which it is added may be added to or applied to the virulent biomass fiber prior to densification, in one embodiment. The addition of a liquid such as water during the pretreatment can raise the water content of the hydrolyzable microparticles from about 10 to about 50%.

Steam may be used in the reaction vessel prior to pretreatment, e. G., Before and / or during AFEX (TM) pretreatment. The addition of steam to coarse biomass fibers during pretreatment can make the water more evenly distributed throughout the hydrolyzable microparticles during hydrolysis. In one embodiment, the added binder is applied or added to the hydrolyzable microparticles (i.e., after densification), but this step can increase processing costs.

When the densification process is complete, the steam is evaporated in the hydrolyzed particulate so that the product remains sufficiently dry, i.e., typically from about 5 to about 20% moisture, but the embodiment is not so limited.

The minimum amount of various substrates and energy sources referred to in the definition of " binder to which " is added to improve the transport properties and / or stability of the biomass particulates during the pre-treatment and / or densification process and / Quot; binder " as defined herein, and therefore does not function as an " additive binder " as defined herein. However, this addition can increase the processing cost.

In addition, although non-volatile bases, such as sodium hydroxide, can be used to transfer lignin to the surface, the sodium hydroxide remaining after evaporation can be used for further applications of the treated material, such as animal feed and other applications Can be negatively affected.

Pre-treatment, e.g., AFEX (TM) and / or steam) is also preferred because of the temperature at which the glass transition temperature of the oligomers in the fiber (e.g., lignin, hemicellulose) A certain amount of hemicellulose is delivered to the surface. Once transferred onto the surface, lignin and hemicellulose become sticky. Surprisingly, these oligomers (lignin or lignin and hemicellulose) contain sufficient tackiness to provide at least similar properties to those of the hydrolyzable microparticles densified with the added binder (the term is defined herein). In various embodiments, the added binder is not used at any point in the process, including before, during, or after densification.

Thus, the present inventors have applied the pre-treated biomass to the pre-treated biomass prior to forming the pretreated biomass into hydrolyzable fine particles, which may also be referred to as " binder " (typically through & (E. G., Using extraneous heat). ≪ / RTI > In addition, surprisingly and unexpectedly, to produce hydrolyzable fine particles having at least as good transport properties, if not better than conventional hydrolyzable fine particles comprising the added binder, any form of " &Quot; (and, in various embodiments, no need to apply or add " binder " prior to or after densification) has been found. The ability to omit the steps of adding and / or applying a binder that is added during the process, especially during densification at any time, additionally provides significant cost savings during production so that the product is not only environmentally friendly but also very economical, Including those transportable by the < / RTI >

In one embodiment, the densifier uses a gear mesh system to compress the biomass through a tapering channel between adjacent gear teeth. Such a densification apparatus operates at a temperature of less than 60 캜 (see Example 2). Such a densifier can be used to produce briquettes, as the term is defined herein. In one embodiment, energy consumption is minimized, and physical and downstream machining characteristics are optimized.

In one embodiment, the densifier is an extrusion device which is capable of forming conventional, substantially cylindrical, particulate, now commonly referred to as pellets (see Example 4).

In one embodiment, an integrated biomass pretreatment and densification process is provided. In certain embodiments, ammonia treatment, such as ammonia fiber expansion (AFEX (TM)) pretreatment or condensed AFEX (TM) pretreatment, is used in conjunction with a compression process to produce hydrolyzable microparticles in processes that do not require the added binder.

In one embodiment, the hydrolyzable particulate has a bulk density of chopped biomass (which is about 50 kg / m3) Hydrolyzable briquettes having a bulk density of at least ten (10) times. In one embodiment, the hydrolyzable particulate is a hydrolyzable pellet having a bulk density of about 550 kg / m3. The use of an integrated process as described herein eliminates the need for further pretreatment at the processing plant and further minimizes the distance the low density feed bale must be transported.

In one embodiment, the hydrolyzable microparticles may be combined with conventional carriers used in cereals for further processing such as further processing and / or hydrolysis and / or conversion (e.g., fermentation) to produce various bio- And handling infrastructure to centralized processing facilities.

In one embodiment, AFEX (TM) conditions are optimized according to the type of biomass being processed to improve the intrinsic binding properties of coarse biomass particles and improve the densification and hydrolysis efficiency after storage.

It is further contemplated that the downstream processing characteristics for briquettes will be better, or at least as good, as non-densified biomass in terms of conversion rate (e.g., foot efficiency), yield, and the like. Indeed, as mentioned herein, the hydrolytic improvement to the pellets is unexpectedly a result of a reduction in the water absorption capacity of the at least partially hydrolyzable microparticles.

Conventional knowledge suggests that poor water uptake will reduce the efficiency of enzyme hydrolysis. Rather, the hydrolyzable microparticles are capable of reducing the water absorption capacity of the hydrolyzable pellets and, even after the hydrolyzable pellets are completely disintegrated, free to move in the liquid and enzyme solution at high solids loading. In one embodiment, the hydrolyzable microparticles improve hydrolysis as a result of their ability to promote the mixing of materials, even at high solids loading.

In one embodiment, the hydrolysis occurs in a vertical stirred reactor having an impeller size to tank diameter ratio of 1: 4 to 1: 2. In one embodiment, hydrolysis occurs in a vertically agitated reactor having an impeller size to tank diameter ratio of about 1: 3, but various embodiments are not so limited. In one embodiment, downstream conversion, e. G., Fermentation, may also occur in such a reactor. Examples of reactors having an impeller having a ratio of impeller length to reactor diameter include, but are not limited to, marine impellers, pitched blade turbines, Rushton impellers, and the like. This is in contrast to conventional operations that do not involve a solid suspension requiring a more expensive special reactor throughout the hydrolysis and / or conversion step.

In one embodiment, hydrolysis with an enzyme is used. Any suitable enzyme capable of hydrolyzing the selected biomass may be used, including endoglucanase, cellobiohydrolases, xylanase, pectinase, ligninase, swarenin ( swollenin).

In one embodiment, AFEX (TM) -treated hydrolyzable microparticles are provided wherein no binder is added. In contrast to conventional binder-containing microparticles, the novel AFEX (TM) -treated hydrolyzable microparticles described herein are probably lignin on the outer surface of the hydrolyzable microparticles, which essentially acts as a type of coating, in some embodiments of hemicellulose Due to its presence, it has a substantially smooth non-flaky outer surface. As such, AFEX (TM) -treated hydrolyzable microparticles are not susceptible to flaking (loss of mass) as in conventional binder-containing microparticles without a coating and containing removable flakes on the outer surface thereof.

In some embodiments, the presence of lignin and / or hemicellulose is not only limited to the surface, but is also observed deep inside the fine pores of the hydrolyzable microparticles. Thus, the AFEX (TM) -treated hydrolyzable microparticles can be more advantageously added to co-ignition with burning / lignite than conventional binder-containing microparticles added with a binder that is chemically limited only to the surface of the binder- have.

AFEX (TM) -treated hydrolyzable microparticles are also less bendable than conventional non-pretreated microparticles and thus tend to be more linear. Surprisingly, the novel AFEX (TM) -treated hydrolyzable microparticles have a harder " feel " (and possibly less brittleness) against them as compared to the softer feel of conventional non-pretreated microparticles.

By the hardness test (e.g., Example 4), the AFEX (TM) -treated pellet was found to be stronger before the initial sudden destruction. In contrast, conventional pellets are essentially more " squeezable " or " crushed " than the novel AFEX (TM) -treated hydrolyzable pellets described herein while maintaining strength over a longer period of time ). In one embodiment, the AFEX (TM) -treated corn versus (CS) hydrolyzable pellets are at least 21% harder than non-pretreated CS hydrolyzable pellets and exhibit a variability of hardness of at least 20%. In one embodiment, the novel AFEX (TM) -treated hydrolyzable pellets exhibit less deformation than conventional non-pretreated CS hydrolyzable pellets (see, e.g., Table 7). Perhaps, AFEX (TM) -treated hydrolyzable pellets and AFEX (TM) -treated hydrolyzable briquettes, and other microparticles made from other types of biomass will show similar or better results.

Lignin is generally darker than other components in plant material, so the resulting material is significantly darker in appearance than a substance not substantially surrounded by lignin.

In one embodiment, the AFEX (TM) -treated CS pellets have a specific gravity of up to 1.16, which is comparable to a non-pretreated CS pellet, which may have a specific gravity of 0.87 or less, but various embodiments are not so limited. AFEX (TM) -treated hydrolyzable pellets appear to have less porosity and exhibit excellent hardness properties compared to conventional non-pretreated pellets, so that AFEX (TM) -treated hydrolyzable pellets may be more susceptible to degradation due to heat, Will exhibit improved short-term and long-term shelf-life, for example, fluidity, compressive strength, water solubility, absorption and overall shelf life, with reduced susceptibility.

In addition, AFEX (TM) -treated hydrolyzable microparticles are expected to have improved fluidity. Additional testing as mentioned in the Prediction Example will quantify the amount of improvement.

In one embodiment, some or all of the above-mentioned features are also present in hydrolyzable microparticles other than pellets (e.g., briquettes). In one embodiment, some or all of the above-mentioned characteristics are additionally or alternatively present in hydrolyzable microparticles that have been pretreated by methods other than AFEX (TM), for example, other ammonia treatments or other pretreatment methods described herein . See also Examples 6 to 11.

In one embodiment, one or more hydrolysable densified cellulosic biomass particles are hydrolyzed (by enzymatic hydrolysis) in a solid loading of greater than about 12% to about 35% (e.g., from about 18% to about 24% , ≪ / RTI > producing a convertible sugar-containing stream. In one embodiment, the conversion comprises fermentation of the sugar-containing stream to produce a biosynthetic product. In one embodiment, the biomass within the hydrolysable densified cellulose biomass particulates is corn vine, switchgrass, wood, precursor cordgrass, or a combination thereof.

In one embodiment, the hydrolysable densified cellulosic biomass particulate is a step of subjecting a sparse cellulosic fiber to a pretreatment (e.g., ammonia pretreatment), wherein at least a portion of the lignin contained in each fiber is present on the outer surface of each fiber To produce a quantity of spongy gum cellulosic biomass fiber; And densifying a quantity of coarse adhesive cellulosic biomass fibers to produce one or more hydrolyzable densified cellulose biomass particles, wherein the amount of cohesive biomass fiber is densified without the use of an added binder. In one embodiment, the pretreatment and densification steps form an integrated process. In one embodiment, the ammonia pretreatment is an ammonia fiber expansion (AFEX (TM)) treatment, for example, a gas AFEX (TM) treatment. In one embodiment, the method further comprises adding water and / or steam during the pretreatment step.

In the above method, the bio-product is a bio-fuel (for example, ethanol or butanol).

In one embodiment, there is provided a system comprising a hydrolysis facility for hydrolyzing at least one hydrolysable densified cellulose biomass particulate at a solid loading of greater than about 12% but no greater than about 35% to produce a convertible sugar-containing stream do. The hydrolysis facility may be part of a bio-product generation facility, for example, an ethanol production facility. In one embodiment, the biomass within the biomass particulates is corn.

In one embodiment, the system is a pretreatment facility in which a quantity of spongy cellulose biomass fibers is pretreated, wherein at least a portion of the lignin contained in each fiber is transferred to the outer surface of each fiber to produce a quantity of spongy gelling cellulosic biomass fiber A preprocessing facility for generating a preprocessing facility; And a densification facility for densifying a quantity of spongy gummed cellulosic biomass fibers to produce one or more hydrolyzable densified biomass particulates wherein the densification facility is densified without the use of a binder to which a quantity of viscous biomass fiber is added . In one embodiment, the pretreatment and densification facilities are co-located.

The resultant hydrolyzable microparticles can be used in the production of animal feeds, as a whole collection of other biochemicals using chemical catalysts or chemical conversion (e.g. fermentation), in other biochemical applications, in electric production applications (for example in combustion in boilers, For example, as a component in biofuels, solids such as fiberboards and extruded fibrous construction materials, including those for aerosol-burning stoves, and the like.

In the various AFEX (TM) processes described herein, ammonia pretreatment dissolves a given amount of lignin, and additionally a significant amount of lignin is brought from the interior of the plant material to the outer surface or outer edge of the fiber. As a result, the substance is more easily digested by the animal. In one embodiment, a combination of pretreated hydrolyzable microparticles, such as, for example, AFEXTM-treated briquettes or pellets as described herein with suitable additives and fillers as known in the art yields novel animal feeds .

In one embodiment, novel feedstocks in the power plant are provided by combining preprocessed hydrolyzable microparticles, such as AFEXTM-treated briquettes or pellets and coal.

Low bulk density The implementation plan for the acquisition, handling, transportation and storage of feedstock is a significant challenge to the developing bio-economy. Assuming a yield of 70 gal / ton, baled biomass at a density of 120 kg / m3 would require a 10-fold volume of material for ethanol of a given volume compared to corn grains. This lower bulk density will prevent the truck from reaching its maximum weight capacity and will increase the number of trucks required for additional feedstock supply.

As the biotech economy for alternative biotechnology evolves, individual producers will need the flexibility to sell their biomass to the bioenergy market for economic reasons. For example, using a regional biomass processing center (RBPC) (for example, within 5 to 10 miles of area), rounded veils can be transported using existing infrastructure and equipment in the transportation industry. Since the RBPC will be properly scaled, the transport distance to the rounded bail can be minimized. Moreover, the presence of multiple distributed RBPCs can minimize the need for long-term storage of rounded bale. Storage for shorter periods can use bale wrap and other current methods to minimize costs. Using the new integrated pretreatment (e.g., an AFEX (TM) - pretreatment) / densification system described herein, the hydrolyzable microparticles can be more efficiently transported to a central processing location.

Various embodiments will be further described with reference to the following examples, which are provided to further illustrate various embodiments. However, it should be understood that many changes and modifications may be made while remaining within the scope of the various embodiments.

Example 1

Corn corns (CS) from a hybrid maize plant ( Zea mays L. ) grown at the Michigan State University (MSU) Agronomy Center Field (typically stems without corn (cob) And leaves) were obtained in October 2007 and stored at room temperature in individual five (5) kg bags stored in a 30-gal waste basket. In October 2005, Switchgrass (SG), originating from Panicum virgatum L. , an "Alamo" lowland seed grown at Thelen Field in Farm Lane, MSU, And stored in a Ziploc® brand plastic bag sealed in a freezer at (4).

CS and SG, respectively, are similar to those described in the above-mentioned U. S. Patent Nos. 888, 176, 663 and 590, but subjected to AFEX (TM) treatment with certain modifications. Specifically, gaseous ammonia was used instead of applying liquid ammonia to the biomass and causing the ammonia and biomass to react as in conventional AFEX ™ treatment. By allowing the hot ammonia gas to condense directly in the cooler biomass, the ammonia and biomass are well mixed.

Gaseous AFEX ™ pretreatment was performed at the Biomass Conversion Research Laboratory of Michigan State University (East Lansing, Michigan, USA). Unless otherwise noted, standard laboratory equipment available in commonly available laboratories was used. AFEX (TM) pretreatment was carried out in an approved ventilation hood with a protective glass chassis minimum surface speed of 75 feet per minute.

A Parr Instruments Model 4524 bench top reactor (hereinafter " 4254 reactor ") was used for this test. The reaction chamber was first placed in a heating mantle of the 4254 reactor. A J-type T-bonding temperature probe is first introduced into a Farber Model 4843 Modular (thermal) regulator (see below) by placing a J-type T-bonding temperature probe against the inner wall of the reaction chamber Quot; 4843 controller "), and the other end was connected to the reaction chamber. The reaction chamber was then covered with a custom-made circular stainless steel sheet metal piece having a relief of about 12.7 cm (about five (5) inches) diameter cut for the temperature probe. The regulator turned on at low temperature (using the red heater switch), and the J-type temperature (blue) regulator showed room temperature measurements of about 25 ° C ± 5 ° C.

Simply connect the (yellow) K-type thermocouple (red display) and the (green) Omega brand CX105 pressure connector (located at Stamford, Conn.) (Green display) from the regulator to the 4254 reactor cover probe . The red display showed room temperature readings of about 25 ° C ± 5 ° C. The green display showed one (1) atm gauge pressure reading of -0.34 to about 0.34 atm (about -5 to about 5 psig). The 4254 reactor was then preheated to room temperature + 20 ° C by securing the yellow and green connectors and the 4254 reactor cover and turning on the blue pre-heating temperature. Observing the blue display for about five (5) minutes ensured that the blue temperature was increased at a rate of about three (3) ° C / min.

The water content of each of the biomass samples was determined using a Sartorius MA35 moisture analyzer (Göttingen, Germany). The initial moisture measurements for the samples were typically between 5 and 10%. The weight of each sample added to the 4254 reactor was 150 g dry weight, i.e., " dry biomass ". The amount of biomass was then weighed to 150 g dry biomass (as provided by total moisture calculation). For example, for a biomass sample containing an o (5)% water content, the following calculation will be made: Water of x (g) in biomass = (150 g (dry biomass) / - 150 g (dry biomass)). The solution to "x" was solved, resulting in 7.9 g of water being present in the biomass. Thus, in this example, the addition of 150 g dry weight of biomass will include weighing 157.9 g of biomass sample at 5% moisture content and adding it.

Calculation was then performed to determine the amount of deionized water to be added to each sample. For the corn stand, the desired water content was 37.5%. For the switchgrass, the desired water content was 45%. These values were chosen because these values represent the optimal individual biomass water for maximum glucose and xylose yields from hydrolysis by enzymes after AFEX ™.

Thus, for corn versus samples where 7.9 g of water are already present but require a 37.5% water content, the following calculation will be made: Water of x (g) to be added to the biomass = (150 g (Water already present in the biomass). By solving for "x", it was found that 82.1 g of water had to be added. In this example, 150 g The total weight of the corn versus sample of dry weight will be 82.1 + g + 7.9 g + 150 g = 240 g. The water will be added to the water bottle (g) until the total weight (dry biomass The biomass was evenly covered with water by stirring the biomass.

Pressure swag at each end, manufactured by Swagelok Co. (Swagelok Co.) located in Chicago, Illinois, USA, and an empty 500 ml ammonia cylinder (Parker 500 ml rotating 316 stainless steel pressure vessel (hereinafter " Parker cylinder ")) was weighed. The total weight of ammonia required for the cylinder and AFEX (TM) pretreatment was determined by adding arm (8) g to the required ammonia weight, since the approximate residual ammonia remaining in the cylinder after completion of this step was determined to be 8 g .

By opening the inlet valve on the ammonia tank and then opening the inlet valve on the parker cylinder, the Parker cylinder is purged with air gas (TM) brand manufactured by Airgas, Inc. (Radnor, PA) A stock ammonia tank (siphon tube was present) was attached. The parker cylinder was allowed to cool until it was cooled and no further charging noise from the cylinder could be heard (the elapsed time was about one day). The outlet valve on the ammonia tank was opened about 1/4. After several attempts, it was determined that it took about 20 seconds to add 158 grams of ammonia to the Parker cylinder. Then, starting with the outflow valve of the parker cylinder, finally closing all the valves to the outflow valve on the ammonia tank. The Parker cylinder was weighed to confirm that the total weight was equal to the expected weight. If the weight was too large, some ammonia was released under the hood. If this is not sufficient, the above steps are repeated.

First, the Parker cylinder containing the ammonia is now surrounded by a BH Thermal branded Briskheat (Columbus, Ohio, USA) thermal tape, and a BH Thermal Brand Brisk Heat (Columbus, OH, USA) thermal tape conditioner Thereby heating the parker cylinder. The cylinder pressure was started at 0 to 125 psig (depending on the temperature of the cylinder internal ammonia as the cylinder internal ammonia cools during the charging phase). Parker cylinders were heated to 600 psig (40 bar), which can be adjusted from 400 psig (27 bar) for "colder" reaction (80 ° C) to 1000 psig (70 bar) for high temperature reaction (160 ° C) Do. The pressure was slowly increased at a rate of less than 0.034 atm / sec (5 (5) psig / sec) at all times.

The desired biomass was then added to the reaction chamber. (Black) temperature probe was removed from the reaction chamber and placed in a slot on the side of the heater mantle so that the outer surface temperature of the reaction chamber was measured. The (blue) display temperature was adjusted to +20 [deg.] C higher than the original preheating (using the arrow keys) to allow continuous 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 into the reaction chamber below the funnel. Once added, the (yellow) temperature probe tip was completely covered with biomass and observed to be about 2.54 cm (about one inch) from the ammonia inlet nozzle of the cover. The funnel was then removed, the cover was returned to the top of the 4254 reactor, and the bracket was tightened using bolts to seal it in place.

The Parker cylinder was then attached to the reaction chamber. A Welch Model 8803 vacuum pump (Niles, Ill., USA) was also attached to the reaction chamber. 4524 The vacuum valve on the reactor was opened, the vacuum turned on, and air was pumped from the 4254 reactor for one (1) minute. The vacuum valve was closed and the vacuum was turned off. (Yellow) temperature probe and (green) pressure connector were connected to the 4843 controller. The valve on the (only) ammonia cylinder was opened to guide it towards the reaction chamber.

The AFEX ™ reaction was initiated by opening the 4254 reactor valve connected to the Parker cylinder. When the pressure between the Parker ammonia cylinder and the reaction chamber became equal, the valve between the ammonia cylinder and the reaction chamber was closed (i.e., after about one (1) minute). In addition, the thermal tape on the parker cylinder was turned off. The 4843 reactor heater was left at a low temperature setting of 20 ° C above the original temperature used in the pre-heating. After about one (1) minute, the peak (red) display temperature and (green) pressure were recorded. (Red) If the display temperature does not reach more than 100 ° C within 1 minute, this means that the feedstock is not in contact with the temperature probe. Temperature and pressure were recorded approximately every five (5) minutes thereafter.

Initiated approximately 5 minutes prior to the inflation stage mentioned under vacuum, it was separated from the 4524 reaction chamber cover. The ammonia cylinder pipe was removed from the reaction chamber cover. The reaction chamber was turned to direct the 4524 pressure relief valve to the rear of the smoke vent hood. The vent hood sash was adjusted for maximum surface speed (recommended 75 feet per minute). Expansion phase: Ear protection was worn. The ammonia pressure in 4524 was released by quickly opening the pressure release valve.

The reaction chamber cover was removed. The biomass was recovered, placed in a tray, and left under a ventilation hood to volatilize the ammonia vapor. The AFEX (TM) biomass was allowed to dry overnight. The Parker cylinder was weighed to determine the grams of residual ammonia applied to the biomass and the weight recorded. Residual ammonia (approximately 8 g) was released from the parker cylinder inside the ventilation hood.

Example 2

Manufacture of materials and samples

(CS) obtained from the same source as described in Example 1 was used. Then, each (2) kg of each type of biomass was subjected to AFEX (TM) pretreatment according to the method described in Example 1. After pretreatment, the samples were densified using a briquetting machine (Federal Machine Co., d / b / a ComPAKco, LLC, Fargo, North Dakota) to produce AFEX ™ cornbars (AFEX ™ -CS) Briquettes and AFEX (TM) -SG briquettes.

Figure 1 shows an image of the four resulting products, including seven (7) g of AFEX (TM) -CS 102, 12 g of AFEX (TM) -SG 104, Briquettes and 23 g of AFEX (TM) -SG briquettes (108). AFEX (TM) -CS and AFEX (TM) -SG briquettes (106 and 108, respectively) had a substantially rectangular shape. Both briquettes 106 and 108 have a width of about 2.54 cm (about one inch), a depth of about 1.27 (0.5 inches), a length of about 10.16 to about 12.7 cm About five (5) inches). (The brewing length depends on the specific configuration use on the ComPAKco machine).

This image shows that only 7 to 12 grams of unbriquetted (i.e. spongy) biomass such as AFEX (TM) -CS (102) and AFEX (TM) -SG (104) For example, AFEX ™ -CS briquettes 106 and AFEX ™ -SG briquettes 108, as shown in FIG. In this example, the non-brittle biomass 102 and 104 occupies about 570 to about 980% more space than the brittle biomass 106 and 108.

Figure 2 includes images of binder-containing non-AFEX ™ -CS briquettes and AFEX ™ -CS briquettes according to various embodiments.

Test performed

Several additional samples were prepared in the manner described above and are described in Carr, RL Jr. 1965. Evaluating flow properties of solids . Chemical Engineering 72 (3): a preliminary physical tests such as the angle of repose ^(o) according to the method described in the 163-168] were given.

The thermal conductivity (W / m ^o C) was measured by Baghe - Khandan, M., S. Choi, and MR Okos. 1981, Improved line heat source thermal conductivity probe , J. of Food Science 46 (5): 1430-1432; KD2, Decagonal Devices, Pulau Peninsula, Decagon Devices).

The water activity was measured using a calibrated water activity meter (AW Sprint TH 500, Novasina, Tastrasse, Switzerland).

Bulk density (kg / m 3), true density (kg / m 3) and porosity are described in Sahin, S. and SG Sumnu. A multivolume pycnometer (Micromeritics Model 1305, Norcross, Ga.) As described in US Pat. Nos. 5,2005, Physical properties of foods , New York, NY: Springer Science Media, Respectively.

ASAE Standards . 51 ^st ed. 2004. S352.1: Moisture measurement - Grain and seeds , St. The water content was determined by the ASAE standard method S352.1 using an ISOTEMP laboratory scale (model number: 838F, Fisher Scientific, Pittsburgh, Pa.) As described in Joseph, Mich .: ASABE.

Color characteristics (L *, a *, b *) were measured using a spectrophotometer (LabScan XE, Hunter Associates Laboratory, Reston, Va.).

Using an Olympus SZH10 stereo microscope equipped with a DP digital camera, the original and sphericity were determined and the image of the particle was analyzed using Image Pro Plus software.

The water solubility index (%) and the water absorption index (-) are described by 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).

The results are shown in Table 1 below:

conclusion

AFEX ™ -CS briquettes (eg, 106) and AFEX ™ -SG briquettes (eg, 108) have relatively smooth surfaces and are well maintained during handling. The AFEX ™ briquettes of both corn and switchgrass have lower porosity, water absorption index, moisture activity and water content than non-briquetted AFEX ™ samples. These properties represent improved shelf-life for briquetted biomass. The lower porosity, higher bulk density and higher true density of the briquettes also indicate reduced shipping costs.

The briquettes exhibit other desired properties as shown in Table 1. [ In particular, the briquettes exhibit a high angle of repose. The angle of repose of the briquette is defined as the angle between the horizontal plane and the contact surface between the two briquettes when the upper briket is just sliding over the bottom briket. It is also known as the friction angle. Thus, the particles have an estimate of 45 degrees. Both the corn versus briquettes and switchgrass briquettes tested herein were greater than the anticipated angle of repose of 57.4 and 60.6, respectively, These values will probably be related to the substantially rectangular geometry of the briquettes.

Example 3

The purpose of this experiment was to compare the hydrolysis characteristics of AFEX ™ -CS briquettes with AFEX ™ -CS biomass (ie, non-briquetted).

Materials

(CS) obtained from the same source as described in Example 1 was used. AFEX (TM) pretreatment was carried out in CS in the same manner as described in Example 1. The briquettes were prepared according to the method described in Example 2.

To the samples tested, 100 ml of a solution of 100 ml of water was added at 25 [deg.] C for 5 minutes before hydrolysis to produce 1.7 g of AFEX (TM) -CS biomass, 1.6 g of AFEX-CS briquettes, 2.2 g of AFEX (TM) -CS immersed in positive deionized water is included.

step

After placing in a 500 ml beaker, enzymatic hydrolysis was performed in each sample according to standard laboratory protocols with one (1)% solids loading. See, for example, Shishir PS Chundawat , Balan Venkatesh, Bruce E. Dale, 2005, Effect of particle size based separation of milled corn stover on AFEX ™ pretreatment and enzymatic digestibility, Biotechnology and Bioengineering, Vol. 96, Issue 2, pp 219-231.

15 Enzyme of FPU (Filter Paper Unit), specifically, Spezyme ® CP (a subsidiary of Genencor® Danisco, Inc., Rochester, NY). Samples were incubated at 50 ° C in New Brunswick incubator Innova 44 (Edison, NJ) while shaking at 150 RPM in an incubator. 6 hours, 24 hours and 72 hours incubation time, and samples were taken.

result

Visual inspection of the obtained hydrolyzate revealed that each of the three samples was completely dissolved immediately upon addition of water (Fig. 3B). Thus, it is evident that all three samples are hydrolyzed to substantially the same extent within substantially the same amount of time.

Approximately two (2) ml of the sample taken from the incubator was filtered and analyzed by Shimadzu high pressure liquid chromatography (HPLC) Run through Model LC-2010HT w / ELSD-LT to determine glucan and xylan conversion.

Figures 3A-3E are images of three biomass samples taken at various times, including AFEX-CS, AFEX-CS pellets and immersed AFEX-CS pellets. Figures 4A and 4B are graphs of relative hydrolysis showing the glucan conversion of the samples shown in Figures 3A-3E. As can be observed, the glucan conversion remains substantially the same throughout each sample.

Table 2 shows the percentage of glucan converted to glucose at various times in each of the samples.

Table 3 shows the percentage of total glucose produced between samplings.

Table 4 shows the percentage of total xylan converted to xyros in each sample before hydrolysis and the total xylan.

Table 5 shows the percentage of total xyloses produced between samplings.

conclusion

By virtually instantaneous hydrolysis (e.g., wetting and dispersing) in AFEX ™ -CS briquettes, it is demonstrated that briquetting of corn-to-biomass does not affect hydrolysis. Perhaps other AFEX ™ briquettes made from other biomass materials will behave in a similar manner. Indeed, as shown in Figure 3b, in most briquettes most biomass is converted to sugar in six (6) hours, which is better than non-briquetted AFEX (TM) CS biomass samples. Both briquettes (AFEX ™ -CS briquettes and immersed AFEX ™ -CS briquettes) are hydrolyzed to approximately the same extent as non-briquetted samples. This determination was made by observing that there was no solid remaining after 72 hours (Figure 3e). Because the three samples had virtually the same conversion, the test was completed in 72 hours. These results are confirmed in Figs. 4A and 4B.

Example 4

This test was performed to determine the relative hardness between the AFEX ™ -CS pellet and the non-AFEX ™ -CS pellet, ie, the pellet not exposed to the pretreatment.

Materials

CS obtained from the same source as described in Example 1 was used for this test. Some CSs were subjected to AFEX (TM) pretreatment as described in Example 1. The AFEX (TM) treated biomass prior to pelletization, including that which does not include the added binder and does not include artificial drying (any evaporation occurring outdoors at room temperature is considered negligible during the course of the test procedure) No further processing was performed.

The remainder was subjected to a different (non-AFEX (TM)) procedure, which involved adding approximately five (5) to ten (10) grams of water per 100 grams of CS so that the moisture content of the biomass before pelleting was 15% .

Lodgepole pine biomass from the Driftmier Engineering Laboratory at the University of Georgia, Atlanta, Georgia, was also subjected to a similar non-AFEX ™ procedure and the biomass water content was 15 %, It was placed in a dryer until it had a water content of 12 to 15%.

Ten rows of AFEX (TM) -CS pellets and ten (10) non-AFEX (TM) -CS pellets were placed in a Yankee Pellet Machine Model 400 (Yankee Pellet Mill )) And a centrifugal die mill to produce pellets which are now considered as industry standards. Ten (10) non-AFX ™ pine pellets were pelletized using a California Pellet Machine, Model CL (CPM, Cropsville, IN).

The pellets produced in both of these devices have a substantially cylindrical shape and are about six (6) mm in diameter. The length can vary as desired, but is generally more uniform than the device used in Example 2 above. The pellet for the purpose of the test was about one (1) inch.

step

The pellets were tested for hardness using a 12T Carver Laboratory Hydraulic Press / Hardness tester equipped with a 400 PSI gauge (Carver, Wabash, IN). Specifically, this test measured the amount of force required to break each pellet beyond its yield strength. The determination of "yield strength" was made through trained observation and "touch". Specifically, pressure was applied to each pellet until the tester observed and felt that the pellets "gave". A number of pellets were tested and the average hardness, i.e., the pressure required to yield the pellets (Table 6) and mean strain (Table 7) were determined.

result

The relative hardness results are shown in Table 6 below:

A measurement of the final diameter of each pellet after each pellet was " crushed " was also performed. These measurements are shown in Table 7. (Note that the data is randomized compared to Table 6).

The average yield point of untreated, binder-added corn versus pellets was 98 psi + 25 psi. The average yield point of corn-to-pellets without AFEX ™ binder was 119 psi + 20 psi and the average yield point of non-AFEX ™ binder-added pine pellets was 98 psi + 23 psi.

All cylindrical pellets had a starting diameter of 6.00 mm. The average yield strain of untreated, binder - added corn versus pellets was 1.06 mm + 0.36 mm. The average yield strain of corn to pellet without AFEX ™ binder was 0.95 mm + 0.24 mm, and the mean yield strain of pine pellets with non-AFX ™ binder was 1.06 mm + 0.23 mm.

conclusion

AFEX ™ pellets show greater durability compared to non-AFX ™ pellets. AFEX ™ pellet quality is also more consistent than non-AFX ™ pellets. As such, it is expected that any provided AFEX ™ pellets will be less likely to be deformed or broken (non-cylindrical shape) compared to non-AFX ™ pellets.

Example 5

This test was performed to determine the bulk density of AFEX ™ -CS pellets relative to non-AFEX ™ -CS pellets.

AFEX ™ -CS pellet and non-AFX ™ CS (about six (6) mm in diameter and about one (1) inch in length) produced according to the method described in Example 4 were added to a 500 mL beaker, And weighed.

The non-AFX ™ -CS pellets had a bulk density of about 36 lb / ft ³ (553 g / l) while the AFEX ™ -CS pellets had a bulk density of about 38 lb / ft ³ (578 g / l) .

As shown by this preliminary test, the AFEX (TM) -CS pellets showed a higher bulk density than the non-AFX (TM) CS pellets. This is probably due to their smooth non-flaky outer surface (which is also expected to improve their fluidity) compared to the coarse flaky outer surface of non-AFX ™ pellets. A much larger difference in bulk density is expected to be demonstrated by tests conducted on a larger scale. Thus, the edge effect caused by the small size of the container is an important factor in this preliminary test.

It is also possible that pellets longer than one (1) inch pellets are crushed to each other so that higher masses can be produced at higher densities. Alternatively, shorter pellets can be packed better. Additional testing (including testing in larger containers) may be performed to optimize the pellet size for a given application, thereby optimizing the overall bulk density.

Example 6

In these tests, various properties of untreated corn versus briquettes were compared to AFEX ™ -treated corn versus briquettes.

Materials

(CS) obtained from the same source as described in Example 1 was used. AFEX (TM) pretreatment was carried out in CS in the same manner as described in Example 1. The briquettes were prepared according to the method described in Example 2.

step

Following the standard procedure, the results shown in Tables 8 and 9 were obtained. Specifically, total moisture: ASTM E871; Ash Content: ASTM D1102; Sulfur content: ATSM D4239; Gross Caloric Value at Constant Volume: ASTM E711; Chlorine content: ASTM D6721; Bulk density: ASTM E873; Particles (particles smaller than 0.32 cm (0.125 in)): Twin Peaks Test CH-P-06; Durability Index: Kansas State Method; 3.8 in. (1.5 in.) Of sample: 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 endurance index referred to herein is known as the " Kansas State Method ".

result

The results are shown in Tables 8 and 9 below:

conclusion

As shown by the results in Tables 8 and 9, the AFEX ™ briquet has an increased total calorie content, ie, the AFEX ™ briquet is about 4.8% more dampened by the presence of less moisture in the AFEX ™ briquettes compared to the untreated briquettes Burns efficiently. Specifically, the caloric increase of AFEX ™ in non-AFX ™ was calculated as 7388 Btu / lb - 7048 Btu / lb = 340 Btu / lb (or 748 Btu / kg) (340 Btu / lb) / (7048 Btu / lb) * 100% = 4.8%. In addition, the bulk density was increased by an average of seven (7)%, and compared to an untreated corn briquette bag having approximately the same weight, the AFEX ™ briquette bag weighed approximately 3.5 lb (1.6 kg) Diameter < / RTI > broken pieces) of about < RTI ID = 0.0 > 65% < / RTI >

In addition, the "durability index" between AFEX ™ and non-AFEX ™ briquettes was substantially the same in these tests, but the durability test method was a simple tumbling experiment ("the Kansas State Method") in comparison to the destructive test described in the above example. As such, insufficient energy is provided to cause the required separation to properly distinguish between briquettes. Nonetheless, the high durability index shows that AFEX ™ briquettes are suitable for use in the briquette industry.

Example 7

These tests were performed to determine the water absorption capacity of pelleted AFEX (TM) treated corn versus non-pelletized AFEX (TM) treated corn versus non-pelleted.

Acquired a conventional multi-pass, low cob corn stover and made a veil on October 23, 2011 at Iowa State University (ISU). The cornstarch was supplied from a field located at the GPS coordinates of north (42.21, west -93.74). After harvesting the corn, the corn stands were lined up using a Hiniker 5600 series side discharge windrowing stalk chopper and were ground using a Massey Ferguson MF2170XD large square baler I made a veil. The bale was stored under a tarpaulin and then milled to approximately one inch granule size using Vermeer BG 480 mill. Then, the corn bale was dried at a water content of less than 5%.

The cornice was also obtained from a combination of multiple sources and the main source of supply was the National Renewable Energy Laboratory, as provided by a farm in Reale, Colorado in 2002 as a chopped corn stand. The cornstarch was dried and then milled to approximately 5 mm particle size in a Wiley Mill (Thomas Scientific, Suedesboro, NJ) prior to use.

AFEX (TM) pretreatment was carried out in two corn vaults samples by packing each with a density of 100 g dry matter per liter in a vertical pressure vessel (hereinafter " vessel ") with an inner diameter of 10 cm and a height of 90 cm. Water content was adjusted by adding distilled water to increase the water content to about 25%. Saturated steam was introduced into the top of the vessel at a mass flow rate of 10-15 psig and 1 gram per second and the resulting bed of corn was heated by venting the bottom for approximately 10 minutes. The final moisture content of the maize bar was about 40%.

The compressed anhydrous ammonia vapor was introduced upward while sealing the bottom of the vessel. The maximum pressure during this ammonia treatment step reached 200 psig. Ammonia was added until a ratio of 1: 1 ammonia: dry corn was achieved. The temperature of the cornice was initially about 80 to about 100 캜 and gradually decreased to about 30 to about 50 캜.

After a residence time of approximately 30 minutes, the pressure was released from the vessel by allowing the vapor to flow out through the bottom. The residual ammonia was then removed from the cornbodies by venting from the bottom while introducing the steam to the top of the vessel at a mass flow rate of 1 gram per second. After approximately 20 minutes, the steam flow was stopped and the corn bar was removed from the vessel. The AFEX (TM) -treated corn bones were then dried in a 50 캜 convection oven (Blue M Electric Company Class A Batch Oven, Blue Island, Ill.).

The pelletization was carried out using PM610 flat die pellet mill (hereinafter " pellet mill ") from Buskirk Engineering (Ocyan, IN USA). A die having a circular hole of 0.25 inches in diameter was used. Tap water was added to the AFEX (TM) treated cornbars and mixed manually until the desired water content was obtained. Three samples of corn weighing from about 3 to about 5 kilograms were manually added to the pellet mill at a rate sufficient to hold the maize mat on the die. The roller then pressed the cornbars through the die to create pellets. The pellets were collected and dried in a blue M convection oven.

Sample Nos. 1 and 2 consisted of corn fed from Colorado, ground to a particle size of 5 mm, and pelleted into 12% moisture and 50% moisture, respectively. Sample Nos. 3 and 4 were 1-inch corn bands obtained from ISU, each pelleted with 20% moisture and not pelleted.

The sample was added to distilled water in a total volume of 250 g in a 500 ml triangular baffle-type flask and placed in a 50 ° C shaking flask incubator overnight to absorb the water and destroy the pellet shape. The water content for pelleted biomass and spongy biomass was determined using OHaus (Parsippanny, NJ) MB25 moisture analyzer. For the pelletized samples (Nos. 1 to 3), a dry weight of corn-to-sample of 37.5 g was added to each flask, while for Sample No. 4 a corn weight of 25 g dry weight was added. Distilled water was added to each flask to increase the total weight to 250 g. After soaking overnight, the sample was removed and filtered through a Whatman # 1 cellulose filter through vacuum filtration.

Once all the liquid was drained, the vacuum was turned off. Then, the volume of the liquid was measured. The water absorption capacity was measured as the difference between the final volume of the recovered liquid and the total volume of water added. These measurements allowed the calculation of free water (as a percentage of the total weight of the components) present at 15% solids in the initial hydrolysis step, assuming complete mixing.

These results show that at various moisture contents the pelletized corn bran can be added to the water with a 15% solids loading, allowing the water to remain at about 18 to about 26% of its total mass as a liquid. The amount of free water is significantly increased in the pellets produced using the 1 inch particle size corn band (Sample No. 3) compared to the pellets generated in the 5 mm particle size. This may be due to increased compression of the larger grain size corn bushing through the die, which reduces the capillary volume in the corn band thereby reducing water absorption capacity. This amount of free water can ensure that the solids remain in suspension, which will enable homogeneous mixing for downstream processes, e.g., hydrolysis.

Example 8

These tests were performed to determine the bulk density and shelf life of the pelletized AFEX (TM) -treated corn bones, and the effect of mixing on the initial hydrolysis rate.

Storability and Bulk Density

The cornice was fed in the manner described in Example 7, AFEX-treated, and densified. In addition to the pellets previously described, the pellets were also made with a water content of 25% and 35% from AFEX (TM) -treated corn, obtained from the United States of America, and crushed through a 5 mm screen.

After pelleting, about 10 g of pellets were placed in a sealed plastic bag and observed over a period of one month. Also, pellets dried at moisture contents of less than 15% were sealed in plastic containers, which were also observed over the course of one month. If no visible fungal growth occurred, the sample was considered to have a sufficient shelf life. The remaining pellets were dried in the 50 캜 convection oven described in Example 7 until a water content of less than 15% was obtained.

Bulk density was measured by placing dried pellets in a 1000 mL beaker. The beaker was weighed slightly using a scale (O Haus GT 4000) with a sensitivity of 0.01 g, ensuring a uniform settling of the pellets, with slight shaking. The bulk density of the pellets was calculated as (fill weight - beaker weight) * (1 - water content) / 1 liter.

50% moisture content, and pellets placed in plastic bags began to show signs of fungal growth after 24 hours. Within 7 days, the pellet was completely covered with white fungi. 35% moisture content, and the pellets placed in plastic bags began to exhibit fungal growth within 3 days. Within 7 days, the pellet was completely covered with white fungi. Relatively, the pellets produced at moisture contents of 12%, 20% and 25% did not appear to cause any fungal growth for at least one month. Likewise, if the pellets are dried at a water content of less than 20%, all samples do not appear to have fungal growth for at least one month.

The bulk density of the pellets with the coarse untreated cornstarch and the coarse AFEXTM treated cornstarch as controls is shown in FIG. As shown in Figure 8, the bulk density of the pellets was increased from 50 g / l to untreated corn versus almost 600 g / l for the pelleted material at 12% moisture content. A significant reduction in bulk density was observed in the pelleted corn bands at higher moisture contents, but the bulk density was higher in the coarse AFEX (TM) -treated corn barrel with a bulk density of about 80 kg / m < 3 > It was much bigger than that.

AFEX (TM) -treated corn versus pellets can be produced with any moisture content of 12 to 50% based on the total weight with respect to bulk density, and can be produced with particle sizes ranging from 2 mm to 25 mm (1 inch) Kg / m < 3 >. It is possible that the pellets can be produced at much higher and / or lower moisture contents. However, drier pellets provide greater bulk density and longer shelf life.

Effect of mixing on hydrolysis rate

Inch corn band obtained from ISU was used. In addition, the same corn bran was obtained and treated with AFEXTM, but not pelleted.

In Sample Nos. 1, 2 and 3, the enzymatic hydrolysis was carried out in 18% solids loading. Hydrolysis was carried out in a 2.8 L baffled Erlenmeyer flask. To each flask was added 500 mL of 0.1 M sodium citrate / citrate buffer (Sigma Aldrich, St. Louis, Mo.), pH 4.5. Novozymes CTec2 cellulosic enzyme and Novozymes HTec2 hemicellulosic enzyme were added to each flask at a protein level of 1260 mg and 540 mg (7 mg and 3 mg per g of corn versus g, respectively). Distilled water was added to bring the total weight of the solution to 1000 g, which weighed 180 g of dry weight of corn.

In Sample No. 4, hydrolysis by enzymes was performed with 24% solids loading. Hydrolysis was carried out in a 125 ml baffled Erlenmeyer flask. To each flask was added 25 mL of 0.1 M sodium citrate / citrate buffer (Sigma Aldrich, St. Louis, Mo.), pH 4.5. Novozymes CTec2 cellulase enzyme and Novozymes HTec2 hemicellulose enzyme were added to each flask at a protein level of 84 mg and 36 mg (7 mg and 3 mg per g of corn versus g, respectively). Distilled water was added to bring the total weight of the solution to 50 g, excluding the weight of dry weight of 12 g corn bran.

In Sample No. 1, the non-pelletized AFEX (TM) -treated corn bred was added in a fed-batch manner and half of the material (90 g dry weight) was added at the beginning of the hydrolysis and half (90 g dry weight) was added after 3 h. In Sample No. 2, all non-pelletized AFEX (TM) -treated corn bones were immediately added (180 g dry weight). In Sample No. 3, all pelleted AFEX (TM) -treated corn bones were immediately added (180 g dry weight). In Sample No. 4, pelletized AFEX (TM) -treated corn bran was added in a fed-batch fashion, and half (6 g dry weight) was added at the beginning of the hydrolysis and half (6 g dry weight) was added after 3 h. After the first addition of the biomass, the flask was placed in a shaking flask incubator at 50 DEG C and rotated at 200 RPM. Samples were visually inspected hourly and manually vortexed to determine the fluidity of the liquid medium and the ability to suspend the biomass particles.

One milliliter sample was obtained 6 hours and 24 hours after enzyme addition and analyzed for sugar production via HPLC. Using the Aminex HPX 87P column from Biorad (Hercules, CA), the individual sugars were separated at a flow rate of 0.6 ml / min and the column was heated to 85 ° C. The sugar was quantified using a Waters 2414 refractive index detector (Milford, Mass., USA).

Exemplary hydrolysis that may be performed according to various embodiments described herein, for example, a visual representation of the hydrolysis performed in this embodiment is shown in Figures 7a-7h. Hydrolysis of the hydrolyzable densified microparticles 706 (e.g., Sample No. 3) is illustrated in Figures 7A-7D. Hydrolysis begins at time 0 as shown in Figure 7a and a plurality of hydrolyzable densified microparticles 706 are disposed in the container 702 with a predetermined amount of liquid, e.g., water, having a water level 704A . The suspension 708A containing the particles 709 is formed within 0.5 hours as shown in Fig. 7B, and the hydrolyzable densified fine particles 706 are not visible on the water level 704A. The particles are retained in the suspension throughout the first 6 hours of hydrolysis and past it, as shown in Figures 7c and 7d. Optionally, additional hydrolyzable densified microparticles 706 may optionally be added at the 3 hour time point to further increase the solid loading as shown in Figure 7C (e.g., Sample No. 4).

In contrast, as shown in Figures 7e-7h, during the conventional hydrolysis of spongy biomass fibers (e. G., Sample No. 2), spongy biomass fibers and liquids, such as water, The coarse wet biomass fibers 710 as shown in Fig. 7e were formed, and even at 0.5 hour time point as shown in Fig. 7f, no mixing occurred. As shown in FIG. 7G, the water level 704B is visible at the beginning until the 3-hour time point. In a similar amount of material, this water level 704B is lower than the water level 704a shown in FIGS. 7A-7D, i.e., the case where the hydrolyzable densified fine particles 706 are used as a substrate.

7G, the suspension 708B containing the particles 709 is in the presence of unmixed coarse wet biomass fibers 710 present both above and below the water level 704B, Lt; / RTI > However, as shown in Figure 7h, at 6 hours, the coarse wet biomass fibers 710 are sufficiently hydrolyzed such that all the solids 710 are now converted to particles 709, which, similar to Figure 7d, While suspended in suspension 708B, the sugar concentration in suspension 708B is lower.

As these schematic diagrams show, hydrolysis occurs more rapidly initially using hydrolyzable densified microparticles 706, as well as that additional hydrolyzable densified microparticles 706 may optionally be present in a relatively short period of time, Can be added after approximately a half or less of the hydrolysis cycle, i.e., allowing for a higher solids loading so that the resulting suspension 708A of FIG. 7D has a higher sugar concentration than the sugar concentration of the suspension 708B of FIG. 7h .

Table 11 shows a visual observation of dissolution of the biomass during the first 6 hours after addition of the enzyme to sample Nos. 1, 2 and 3.

These results show that the use of densified corn bones significantly improves the initial hydrolysis step. Glucose released in the first 6 hours was 31% higher than sparse biomass without fed-batch addition and 11% higher than sparse biomass using fed-batch addition. Improved hydrolysis performance lasted for 24 hours. In addition, the pellet hydrolyzate was kept at a low apparent viscosity and was easily mixed for the first 6 hours, suggesting that the standard impeller could keep the biomass in suspension. Since the biomass can be easily retained in the suspension, the solid loading can be easily increased. In Sample No. 4, the biomass was retained in suspension and was readily mixed for the first 6 hours despite increased solid loading. A glucose concentration of 71 g / l was obtained after 24 hours and increased by 30% compared to the pellet at 18% solid loading.

Relatively, the cost-effective hydrolysis was not easily mixed in the first hour of hydrolysis as well as the first hour after the second addition of the enzyme. Coarse biomass without fed-batch addition remained unmixable for up to 5 hours.

Example 9

This test was performed to determine if 18% solids loading hydrolysis using AFEX ™ -treated corn versus pellets could be performed in a vertically agitated tank reactor with an impeller size to tank diameter ratio of 1: 3.

The cornstalks were AFEX (TM) -treated and pelleted in the manner described for Sample No. 1 in Example 7. A glass 6-liter Microferm reactor (New Brunswick Scientific, Enfield, Conn., USA) equipped with a 6-blade rushton impeller and a 3-blade marine impeller was used Respectively. For an impeller size to tank diameter ratio of 0.35 or about 1.3, the impeller diameter was about 7.5 cm and the tank inner diameter was about 21.5 cm. Four uniformly spaced vertical baffles were also present in the reactor. Distilled water and enzyme were added to a total weight of 4.60 kg. The enzyme used was 7,000 mg of Novozymes CTec2 and 3,000 mg of HTec2. Approximately 1 kg of dry weight pellet was added to the solution. The temperature was maintained at 50 < 0 > C and the pH was adjusted to 5 manually using 4M NaOH (Sigma Aldrich, St. Louis, Mo.). The impeller was rotated at 400 rpm. Visual observations were recorded throughout the first 30 minutes of hydrolysis and 20 ml samples were obtained at 1, 4 and 6 hours after the addition of the pellets. These samples were quantified for sugar analysis according to the previous examples.

After hydrolysis for 48 hours, the hydrolyzate broth was centrifuged to remove biomass particles. The supernatant was then fermented using Zymomonas mobilis AX101 as the fermentation organism. The pH was adjusted to 6 and the temperature was reduced to 30 占 폚. Zymomonas mobilis was grown in yeast extract and added to the hydrolyzate at an initial OD of 1 at 600 nm. A corn steep liquor of 1% (v / v) loading and 2 g / l of potassium phosphate were added as nutrients. Samples were taken 24 hours after inoculation to assess ethanol production and glucose utilization. Samples were analyzed for ethanol production and sugar consumption via HPLC as described in Example 8. For ethanol generation, a Biorad Aminex 87H column was used instead of Aminex 87P.

When agitation was initiated, the corn-to-pellet was immediately suspended and rapidly disrupted with individual particulates within 10 minutes. As the pellets disintegrated, a layer of cornice was deposited along the surface of the container. This layer appeared to be thin and was not permanent as the sections continued to break and re-enter the suspension. Within 20 minutes, all cornstalks were suspended and suspended during hydrolysis for a 48 hour period. Glucose concentrations were 21.9 g / l, 34.2 g / l and 44.1 g / l after 1, 4 and 6 hours, consistent with performance in shake flasks.

Glucose and xylose reverse transcripts were 51.6 g / l and 24.3 g / l at the beginning of fermentation. After 24 hours, the glucose was completely consumed and xylose was partially consumed, resulting in a final concentration of 13.1 g / l. This partial consumption is typical for the fermentation of AFEX (TM) -treated corn bands using such microorganisms, see for example Lau MW et al., Biotechnology for Biofuels 3:11 (2010). The final ethanol concentration was 32.3 g / l.

As demonstrated, hydrolysis and fermentation by the enzyme can be carried out at a level as high as 18% solids loading, and an excess of final ethanol concentration of 30 g / l is still achieved. The impeller size to tank diameter ratio of about 1: 3 is sufficient to maintain solids in the suspension and allow for even mixing. Perhaps much higher solid loading can be used, but additional testing will be performed to confirm this hypothesis.

Example 10

In this test, the pellets produced at different moisture contents were hydrolyzed with a high solids loading to determine their effect on the resulting glucose yield.

The cornice was obtained from a number of sources, but mainly from Colorado, USA, as described in Example 7. These corn bushes were ground to a 5 mm particle size as described in Example 7, treated with AFEX ™, and pelletized. The pellets were produced with 12% water content, 25% water content, 35% water content and 50% moisture content. Enzymatic hydrolysis was carried out with a total weight of 100 g in a 250 ml Erlenmeyer flask with 18% solids loading. 18 grams (dry weight) of pellets were added to each flask and water was added in an amount to cause a total weight of 100 g for all ingredients added.

Tetracycline and cycloheximide were added at final concentrations of 20 mg / L and 15 mg / L, respectively, to control fungal contamination. The pH was adjusted as described in Example 8 using a citrate buffer. Novozymes CTec2 and HTec2 enzymes were added at 7 mg and 3 mg protein loading per gram of pellet, respectively. After addition of the enzyme, the flask was sealed and placed in a shaking flask incubator set at 50 ° C and 200 rpm. 1 ml samples were obtained at 1, 6, 24, 48 and 72 hours after the addition of the enzyme and analyzed for sugar content as described in Example 9. The results are shown in Fig. (Note that it moves 0.5 hours to the left to clarify the line for 50% moisture).

As shown in Figure 6, a glucose concentration of greater than 60 g / l was obtained for all AFEX (TM) treated corn versus pellets within 48 hours. This concentration is sufficient for efficient fermentation into ethanol or other value-added products. In addition, the pellets are hydrolyzed at a rapid rate, resulting in more than 50% of the total sugar being produced within the first 6 hours. Pellets produced with a higher water content tend to have a higher sugar yield than pellets produced with a lower water content. However, the pellets produced at 50% water content did not release more glucose visibly than the pellets produced at 35% water content.

As demonstrated, AFEXTM-treated biomass can be pelletized over a wide range of water content and is still available as a feedstock for fermentable sugar production. Depending on the consumer's capital and desires, it may be possible to customize the water content to provide an appropriate combination of storage stability versus concentration for any number of applications.

Example 11 (Prediction)

Samples of biomass, such as switchgrass and pre-larg cordglas, will be harvested at various ages, and corn stalks will be collected after harvesting the grains. Biomass composition will be determined at acquisition, during storage at rounded bale, after initial AFEX ™ treatment and densification, and after storage of densified pellets. AFEX ™ pretreatment will be statistically optimized for hydrolysis and binding properties based on time, temperature, biomass moisture and parameters of ammonia to biomass ratio. Materials for densification will be prepared using AFEX ™ conditions that provide at least 90% conversion of glucan and 80% conversion of xylan.

The densification will be performed using any suitable method including the method used in Examples 2, 3 or 8.

The resulting pellets will be subjected to various environmental conditions to simulate long-term storage and then evaluated for flowability, compressive strength, and the like. The downstream processing characteristics will be evaluated using a set of standard hydrolysis and fermentation conditions, including individual hydrolysis and fermentation (SHF) versus 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 month), between stored pellets and non-densified biomass.

Example 12 (Prediction)

PREFRIEL COGURSH's AFEX ™ pretreatment will be statistically optimized for time, temperature, biomass moisture and ammonia to biomass ratio. A fairly wide range of AFEX (TM) pretreatment conditions provide similar hydrolysis results, giving the present inventors the assurance that there is also a set of pretreatment conditions to improve the binding properties. AFEX (TM) pretreatment conditions providing at least 90% glucose conversion and 80% xylan conversion are identified and used to produce materials for densification. We will characterize these pretreatment materials for surface properties using a variety of methods developed by our labs (ESCA, Prussian blue stain, SEM), and their properties include pellet density and durability, Will be relevant.

Example 13 (Prediction)

In order to optimize the operating conditions for converting the pretreatment biomass to densified pellets, operational parameters will be investigated. These variables include AFEX ™ preprocessing conditions, moisture content, particle size, die temperature vs. bond strength, compressibility versus yield quality, energy use, surface chemistry and deformation present, compression ratio and density obtained, do. The consumption and wear of mechanical components will also be evaluated.

Example 14 (Prediction)

The pretreatment of the biomass using any known AFEX ™ procedure, or according to the procedure of Example 1, or any other suitable modification of the AFEX ™ procedure, may be carried out using any of the methods described in Examples 2 and 3 Will be densified using the appropriate method of.

The densified biomass is then subjected to various environmental conditions including temperature (25-40 ° C), relative humidity (60-90%), consolidation stress (0-120 ° C) and storage time will be. After storage, the physical characteristics will be evaluated as described below:

The fluidity can be evaluated by a simple test in which multiple AFEX ™ -pellets are placed in a container, for example, a bed of a truck, and tilted about 45 degrees. Comparisons with conventional pellets can be made by noting the time it takes for the pellets to flow out of the container.

In addition, the fluidity will be evaluated using Carr Indices. ASTM D6393. 1999, Standard test method for bulk solids characterization by Carr indices , ASTM Standards , W. Conshohocken. PA]. Fluidity is fully defined as the ability of a substance to flow unexpectedly under the given environmental conditions. Fluidity measurements are often made by the Karl index by calculating the total liquidity index and the total immersion index. Carr, RL Jr. < / RTI > 1965, Evaluating flow properties of solids , Chemical Engineering 72 (3): 163-168.

A higher value for total fluidity index and a lower value for total immersion index will provide an ideal material with little or no flow problems. Another way to quantify fluidity is by measuring the Jenike Shear Stress properties. Jenike, AW 1964, Storage and flow of Bulletin No. < RTI ID = 0.0 > 123 , Utah Engineering station, Bulletin of University of Utah. We will also use the Zenick method to determine particle cohesion, yield locus, internal friction angle, yield strength and flow function and particle size distribution. ASTM D6128. 2000, S Tandard Test Method for Shear Testing of Bulk Solids Using the Jenike Shear Cell , ASTM Standards , W. Conshohocken. PA and ASAE S19.3. 2003, Method of determining and expressing fineness of feed materials by sieving , ASAE Standards . St Joseph, MI: ASABE].

In addition, we will assess the levels of glucan, xylan, galactan, arabinan, mannan, lignin, ash and fiber to determine their impact on storage and flow behavior. In addition, some other physical properties (i.e., particle size, particle shape, thermal properties, moisture properties, and color) will be measured as poor indicators of fluidity. Selig, M, et al., 2008, Enzymatic saccharification of lignocellulosic biomass , Technical report NREL / TP-510-42629 ; Determination of ash in biomass , Technical report NREL / TP-510-42622 ], which is incorporated by reference herein in its entirety ; 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 .

It will establish widespread rheological properties that affect the ability of biomass to be treated before and after densification. Such properties include, but are not limited to, bulk density, true density, compressibility, relaxation, springback, permeability, uniaxial yield strength and friction quality. These properties are a function of feedstock particle size and distribution, shape factor, moisture conditions and consolidation pressure and time. Commercial rheological testers are typically designed for use in small grains and fine powders; As a result, it can not accommodate particulates having a diameter greater than 1/4 inch; The present inventors will develop a novel measurement system for characterizing larger feedstock particles. The system can be scaled for various material sizes and includes compression and shear cells that can be integrated with a commercial load frame and can be operated over a variety of consolidation pressures.

The data will be analyzed to determine conditions that result in improved (or optimized) fluidity using normal statistical methods, such as general linear models, regression, response surface analysis, multivariate analysis, and other suitable techniques. [Myers, HR 1986, Classical and modern regression applications , 2 ^nd edition. Duxbury publications, CA. USA. Draper, NR] and Smith, H. 1998, Applied Regression Analysis , New York, NY: John Wiley and Sons,

Example 15 (Prediction)

We will evaluate at least three types of biomass, cornstalk, switchgrass and pre-larg cordglas. For each of these feedstocks, raw raw biomass, AFEX (TM) pretreatment biomass and AFEX (TM) pretreatment and densified biomass (before and after storage) samples will be collected. Therefore, we will evaluate 3 x 4 = 12 total biomass sample types. Individual hydrolysis and fermentation (SHF) will be assessed. For glycation, the flask will be incubated in an orbital shaker at 50 < 0 > C and 250 rpm for 48 hours. The sample will be removed at 0, 2, 4, 6, 8, 18, 24, 30, 36 and 48 hours. Then, the flask was cooled to 30 DEG C, and saccharomyces cerevisiae (1 g), which had a pentose-effective potency, grown in a medium containing (2) g / l of glucose and 2 ml of culture for 12-18 hours of the recombinant strain of Saccharomyces cerevisiae were inoculated. The flask will be incubated in an orbital shaker at 30 < 0 > C and 150 rpm for an additional 96 hours. The sample will be removed at 0, 3, 6, 9, 18, 24, 36, 48, 60, 72, 84 and 96 hours during fermentation.

In addition, simultaneous saccharification and fermentation (SSF) will be performed to evaluate the conversion. The main difference will be that the flask is dosed with the enzyme, immediately inoculated with yeast as mentioned above, and then incubated at 30 DEG C for 144 hours. The sample will be removed at 0, 2, 4, 6, 8, 18, 24, 36, 48, 60, 72, 96, 120 and 144 hours. Enzyme and biomass loading and other conditions will be the same as those listed above.

New densified biomass products and methods for making and using the same are described herein. In one embodiment, conventional pretreatment is used to produce tacky biomass, which is surprisingly easily convertible to solid hydrolyzable microparticles, without the use of added binders. In addition, the hydrolyzable microparticles are surprisingly dense and exhibit excellent hardness properties compared to conventional densified microparticles containing at least binder (s) produced and / or added using at least one additive (s).

In one embodiment, hydrolyzable microparticles are provided comprising more than one biomass material (e.g., cornice, grass and / or wood, etc.). In this way, there is provided a hydrolysable solid biomass product article having relatively uniform properties that can be more easily adapted to the biomass processing industry. Such properties may include, but are not limited to, BTU content, sugar content, and the like.

Any suitable type of densification process can be used to produce products of various sizes and shapes. In one embodiment, the densification process apparatus uses a gear engagement system to compress the biomass through tapering channels between adjacent gear teeth to form dense hydrolyzable microparticles. In one embodiment, the system operates at lower temperature, pressure, and energy requirements than conventional processes.

In one embodiment, the pretreatment hydrolyzable particulates are better "retained", ie, more resistant to physical forces during transport, handling and / or storage, as compared to particulates that are not pretreated. In one embodiment, the resulting product has increased fluidity compared to conventional biomass solids, which enables automated loading and unloading of transport vehicles and storage systems and transport through processing facilities.

All publications, patents, and patent documents are incorporated herein by reference in their entirety, each as if each individually incorporated by reference. In the event of inconsistency, the present disclosure, including any definitions, will control.

Although specific embodiments have been illustrated and described herein, it will be appreciated by those skilled in the art that any of the procedures that are calculated to achieve the same purpose may be substituted for the specific embodiments shown. For example, although the process has been discussed using certain types of plant biomass, any type of plant biomass or other type of biomass or biofuel may be used, for example, agricultural biofuels. This application is intended to cover any adaptations or variations of the subject matter of the present invention. Accordingly, it is intended that the embodiments of the invention be apparently limited only by the claims and their equivalents.

Claims (23)

A process for producing a sugar-bearing stream comprising hydrolyzing at least one hydrolysable densified biomass particulate in an agitated reactor with an enzyme to produce a sugar-containing stream,
Wherein said stirred reactor is a reactor having an impeller disposed therein and having a diameter ratio of impeller to reactor of 1: 4 to 3: 4, or a capacity of 6 liters or more, said hydrolysis being in the range of 12% to 24% And solid loading of the densified biomass particles in the reactor,
The densified biomass particles
a) pretreating plant biomass fibers containing lignin and / or hemicellulose with ammonia or sodium hydroxide to move at least a portion of the lignin and / or hemicellulose in the biomass fiber to the outer surface, producing tacky plant biomass fibers; And
b) densifying the pretreated viscous plant biomass fibers to produce at least one hydrolysable densified biomass particulate, wherein the densified plant biomass fibers are densified without the use of an additional binder. Lt; RTI ID = 0.0 > of: < / RTI > The process according to claim 1, wherein the capacity of the reactor is at least 500,000 liters. The method of claim 1 wherein the impeller to reactor diameter ratio is from 1: 2 to 3: 4 or from 1: 4 to 1: 2. The method according to claim 1,
Wherein the solid loading ranges from 12% to 20%, or from 18% to 24%. The method of claim 1, wherein the biomass fibers are corn stover fibers, switchgrass fibers, wood fibers, prairie cord grass fibers, or combinations thereof. The method of claim 1, wherein steps a) and b) are performed as a single process at a single location. The method of claim 1, wherein the ammonia pretreatment is ammonia fiber expansion (AFEX ™) pre-treatment or gaseous AFEX ™ pretreatment. 8. The method of claim 7, wherein the ammonia pretreatment is performed in a pretreatment vessel, and the method further comprises removing residual ammonia remaining in the pretreatment vessel after the pretreatment by introducing steam into the pretreatment vessel. The method of claim 1, wherein the method further comprises adding water and / or steam to the plant biomass fibers prior to and / or during pretreatment. The method according to claim 1, wherein the convertible sugar-containing stream is fermented to produce a bioproduct. 11. The method of claim 10, wherein the bio-product is biofuel. The method of claim 1, wherein the sugar-containing stream contains greater than 50% of available sugar in the densified biomass particulate. 2. The method of claim 1, wherein hydrolyzing the enzyme comprises adding the densified biomass particles to the stirred reactor at two or more stages separated by hydrolysis activity. 11. The process of claim 10 wherein the sugar in the convertible sugar-containing stream is produced from the densified biomass particles at a rapid rate as compared to the sugar-bearing stream prepared using the non-densified biomass. 15. The method according to any one of claims 1 to 14,
Wherein the pretreatment is performed in a vertical reactor. 1) In a pretreatment vessel, plant biomass fibers containing lignin and / or hemicellulose are pretreated with ammonia or sodium hydroxide to provide at least a portion of lignin and / or hemicellulose in the biomass fiber to the outer surface of the fiber To produce pretreated adhesive plant biomass fibers;
2) removing residual ammonia remaining in the pretreatment vessel by introducing steam into the pretreatment vessel;
3) densifying the pretreated viscous plant biomass fiber to produce at least one hydrolyzable densified biomass particulate, wherein the biomass fiber is densified without the use of an additional binder; And
4) hydrolyzing one or more hydrolysable densified biomass microparticles in an agitated reactor with an enzyme to produce a convertible sugar-containing stream,
Wherein the stirring type reactor is a reactor having an impeller disposed therein and a ratio of the diameter of the impeller to the reactor being 1: 4 to 3: 4, or a capacity of 6 liters or more,
The steps 1), 3) and 4) are performed as an integrated process at a single location,
Wherein the hydrolysis comprises a solid loading of the densified biomass particles in the reactor in the range of 12% to 24%. 17. The method of claim 16, wherein the method further comprises adding water and / or steam to the plant biomass fibers prior to and / or during pretreatment. 17. The process of claim 16, wherein the capacity of the reactor is at least 500,000 liters. Providing a hydrolyzable densified biomass particulate or a plurality of hydrolyzable densified biomass particulates, wherein the hydrolyzable densified biomass particulate comprises a plurality of plant biomass fibers containing lignin and / or hemicellulose on the outer surface of the fibers, Characterized in that the hydrolyzable densified biomass particles are in the form of compressed particulates and are free of added binders; And
Comprising the step of hydrolyzing hydrolyzable densified biomass particles or a plurality of hydrolyzed densified biomass particles in an agitated reactor into an enzyme to produce a convertible sugar-containing stream, ,
Wherein said stirred reactor is a reactor having an impeller disposed therein and having a diameter ratio of impeller to reactor of 1: 4 to 3: 4, or a capacity of 6 liters or more, said hydrolysis being in the range of 12% to 24% Characterized in that it comprises the solid loading of the densified biomass particles in the reactor. 20. The method of claim 19, wherein the impeller to reactor diameter ratio is at least 1: 2. delete delete delete

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