JP2013544099A - Continuous feed biomass pretreatment method for packed bed reactors - Google Patents

Abstract

  Biomass pretreatment using anhydrous ammonia is a stationary reactor where ammonia can penetrate biomass particles or debris in the vapor state and when the biomass is continuously fed and moves through the reactor. Is effective. In order to achieve this condition, the water content of the entire system is kept below 40% by weight, based on the total weight in the system. This pretreated biomass product is effectively saccharified to produce fermentable sugar for biocatalytic production of the product.

Description

  This application claims the benefit of US Provisional Patent Application No. 61 / 416,484, filed Nov. 23, 2010, which is incorporated herein by reference in its entirety.

  The present invention relates to a method for treating biomass to obtain fermentable sugars. Specifically, the present invention provides a continuous supply pretreatment method of biomass in a packed bed reactor using anhydrous ammonia to produce a material that can be easily saccharified.

  Lignocellulosic raw materials and waste, such as crop residues, wood, forestry waste, paper sludge, and municipal and industrial waste are potentially large renewable raw materials for the production of chemicals, plastics, fuels, and feed I will provide a. Lignocellulosic raw materials and waste containing carbohydrate polymers, cellulose and hemicellulose, and lignin are generally treated by various chemical, mechanical, and enzymatic means to release mainly hexose and pentose sugars, It can then be fermented into a useful product.

  The pretreatment method is used to make lignocellulosic biomass carbohydrate polymers, ie polysaccharides, more easily susceptible to cellulolytic enzymes used for saccharification. Various pretreatment methods are known, including an ammonia pretreatment method for biomass. In general, ammonia has been used in an aqueous state during the treatment of Baymaos for saccharification.

  For example, US Pat. No. 7,932,063, owned by the owner of this application, discloses a method for pretreating biomass under conditions of high solids and low aqueous ammonia concentration. The concentration of ammonia used is the minimum concentration sufficient to keep the pH of the biomass-ammonia water mixture alkaline and is at most less than about 12% by weight based on the dry weight of the biomass. The dry mass of the biomass is an initial concentration of at least about 15% and up to about 80% of the mass of the biomass-ammonia water mixture.

  US Pat. No. 4,064,276 discloses the use of anhydrous ammonia for the treatment of straw and other plant materials to improve the nutritional value of the material. Straw having a dry matter content of at least 60% by weight is treated with 15-40 kg of anhydrous ammonia per ton of dry straw at ambient temperature and atmospheric pressure for at least 10 days.

  US Pat. No. 7,915,017 describes pretreatment of biomass with liquid or vaporous anhydrous ammonia and / or with a liquid or vaporous high concentration ammonia: water mixture to give a mixture of ammonia versus dry biomass. Disclosed is a process for obtaining a mixture having a ratio of about 0.2 to 1 to 1.2 to 1 and a water to dry biomass ratio of about 0.2 to 1.0 to 1.5 to 1. . These methods are used to increase the fraction of total ammonia in the liquid phase. The temperature is maintained at about 50 ° C. to 140 ° C. and the pressure is rapidly released by releasing ammonia from the tank to form the treated biomass.

  A packed bed flow-through reactor is used in the ammonia reuse percolation method for pretreatment of biomass (Yoon et al. (1995), Applied Biochemistry and Biotechnology 51/52: 5-19). In this method, ammonia water is continuously recirculated through the biomass bed.

  There remains a need for a method for efficiently pretreating biomass with ammonia and producing an easily saccharifiable material that reduces the cost of the reactor.

  The present invention provides a method for pretreating biomass with anhydrous ammonia in a continuous manner in a static bath to produce a material that can be easily saccharified.

Accordingly, the present invention provides a method for treating biomass to produce a pretreated biomass product, the method comprising:
a) providing biomass having a dry matter content of at least about 60%;
b) loading the biomass of (a) into a stationary pretreatment tank by continuously feeding it so that the loaded biomass in the tank travels through the tank;
c) At least about 4% (total mass in the tank) of the loaded biomass of (b) under conditions where the total moisture content in the tank remains below 40% by mass (measured based on the total mass in the tank). Contacting with the anhydrous ammonia of) and thereby allowing ammonia vapor to penetrate most of the biomass, and d) withdrawing the biomass from the pretreatment tank,
Biomass discharged from this tank is a pretreated biomass product.

It is a figure which shows the pre-processing system for supplying biomass continuously in a pre-processing tank.

  When treating biomass with ammonia, ammonia contact throughout the biomass is important for the effective preparation of biomass for saccharification. In general, mechanical agitation is used to mix ammonia and biomass to maximize contact. Under the conditions described herein, pretreatment is performed by continuously feeding biomass to the reactor and moving the biomass through the reactor without mechanical mixing, thereby providing ammonia. Eliminates the energy and maintenance costs associated with mixing reactors where is a reactant.

  The following definitions and abbreviations will be used for the interpretation of the claims and specification.

  As used herein, the terms “include” (“essentially” as “all”), “include”, “including” ”,“ has ”,“ having ")", "containing", "containing" ", or any other ending change thereof is intended to include non-exclusively included ones. For example, a composition, mixture, process, method, article, or device that includes a list of components is not necessarily limited to only those components, and components that are not explicitly listed, or such compositions An article, mixture, process, method, article, or other component specific to an apparatus can also be included. Further, unless stated to the contrary, “or” means inclusive or not, exclusive or not. For example, condition A or B is such that A is true (or exists) and B is false (or does not exist), A is false (or does not exist), and B is true (or Present) and both A and B are true (or present).

  Also, the indefinite article “an” (“a”, “an”) preceding a component or component of the invention is non-limiting with respect to the number of those instances (ie, occurrences) of the component or component. Is intended. Thus, “an” (“a”, “an”) should be construed as including one or at least one, and a singular word of a component or component is clearly singular in number. The plural is also included unless it means.

  The term “invention” or “invention” as used herein is a non-limiting term, and is not intended to refer to any single embodiment of that particular invention. All possible embodiments described in the description and in the claims are included.

  As used herein, the term “about”, which modifies the amount used of a component or reactant of the invention, is a common measurement used in the real world, for example, to make concentrates or working solutions Through procedures and liquid handling procedures, through inadvertent errors in these procedures, and through the manufacture, sources, or differences in purity of ingredients used to make compositions or perform those methods, etc. Refers to a possible quantity change. The term “about” also encompasses amounts that differ due to different equilibrium conditions of the composition resulting from a particular initial mixture. The claims, whether modified or not by the term “about,” include equivalents of those amounts. In one embodiment, the term “about” means within 10% of the reported numerical value, preferably within 5% of the reported numerical value.

  The term “fermentable sugar” refers to oligosaccharides and monosaccharides that microorganisms can use as a carbon source during the fermentation process.

  The term “lignocellulosic” refers to a composition comprising both lignin and cellulose. The lignocellulosic material can also include hemicellulose.

  The term “cellulosic” refers to a composition comprising cellulose and additional ingredients including hemicellulose.

  The term “saccharification” refers to the production of fermentable sugars from polysaccharides.

  The term “pretreated biomass” means biomass that has been pretreated prior to saccharification.

  The term “butanol” refers to isobutanol, 1-butanol, 2-butanol, or combinations thereof.

  The term “lignocellulosic biomass” refers to any lignocellulosic material, including materials comprising cellulose, hemicellulose, lignin, starch, oligosaccharides, and / or monosaccharides. Biomass can also include additional components such as proteins and / or lipids. Biomass can be obtained from a single source or the biomass can include a mixture obtained from one or more sources. For example, the biomass can include a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Lignocellulosic biomass includes, but is not limited to, biofuel energy crops, agricultural residue, municipal waste, industrial waste, paper sludge, garden waste, wood, and forestry waste. Examples of biomass include, but are not limited to, corn cobs, crop residues such as corn hulls, corn stover, grass, wheat straw, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane Sorghum, sorghum plant material, soybean plant material, ingredients derived from cereal milling, trees, branches, roots, leaves, wood chips, sawdust, shrubs and shrubs, vegetables, fruits and flowers.

  The term “dry matter content” refers to the amount of material mass units present after removal of the liquid content of the subject material.

  The term “biomass hydrolyzate” refers to the product produced by saccharification of biomass. This biomass can also be pretreated or pre-processed prior to saccharification.

  The term “biomass hydrolyzate fermentation broth” is a broth containing a product resulting from the growth and production of a biocatalyst in a medium containing biomass hydrolyzate. The broth contains the components of biomass hydrolyzate that are not consumed by the biocatalyst, as well as the biocatalyst itself and the product made by the biocatalyst.

  The term “slurry” refers to a mixture of an insoluble material and a liquid. The slurry can also contain high levels of soluble solids. Examples of slurries include saccharification broth, fermentation broth, and distillation waste.

  The term “target product” refers to any product produced by a microbial production host cell during fermentation. These target products may be the result of a genetically engineered enzyme pathway in the host cell or may be produced by an endogenous pathway. Typical target products include, but are not limited to, acids, alcohols, alkanes, alkenes, aromatics, aldehydes, ketones, biopolymers, proteins, peptides, amino acids, vitamins, antibiotics, and pharmaceuticals. It is done.

  The term “static tank” refers to a tank that does not include a means for mixing the loading material. The packed bed reactor employs a stationary tank.

  The term “stationary pretreatment tank” refers to a stationary tank that can be used for pretreatment. This tank is constructed to withstand loading materials such as ammonia used in the method of the present invention.

  The term “vent” refers to allowing trapped gas to escape without the effects of an explosion.

  The term “gas” refers to both condensable low density phases below its critical temperature, for example both gas and steam, including water vapor.

  The term “total mass in the tank” refers to the total mass of the ingredients added to the tank.

  The term “majority” refers to more than 50%. The majority can refer to any integer greater than 50%.

Continuous pretreatment of biomass in a stationary tank In the method of the present invention, biomass is continuously fed into a stationary tank for pretreatment to produce a pretreated biomass product. One embodiment of the continuous feed stationary tank pretreatment method is shown in FIG. The method embodiment described below is illustrated in FIG. 1, which has the following numbering as shown in FIG.

  Biomass is fed to the first end or inlet (10) of the tank and travels through the tank (11) towards the second opposite end or outlet (12) of the tank. Biomass is supplied to the tank using a continuous supply device (13), such as an extruder, an industrial baler, or a concrete pump. Biomass enters a feed bin for loading into a continuous feed device, such as by gravity flow or by an energy input method such as by an auger driving the feed into the continuous feed device.

  Biomass continuously fed into the static pretreatment tank moves from the tank inlet to the tank outlet as a result of the force applied by the feeder. The vessel has an inlet-outlet shaft dimension that is larger than the width dimension. For example, the tub can be a cylinder or conduit (as in FIG. 1). A steam seal is used downstream of the biomass feed-in position to prevent pretreatment agents including ammonia and water vapor from escaping upstream from the inlet. This can be accomplished, for example, by providing sufficient swelling of the biomass between the biomass draw-in position and the ammonia and water vapor injection positions to block steam escape from the inlet. In one embodiment, two different biomass feedstocks, namely primarily switchgrass or corn cobs, and a continuous stack of pulverized corn kernels or starch zones that swell and plug when subjected to hot water are alternately stacked. Add to.

  The pipe tank provides one or more skirts that allow the pipe diameter to be expanded beyond the diameter of the inlet end to provide sufficient residence time for pretreatment while at the same time reducing the overall tank length. It includes a portion (14) that has been expanded. An increase in diameter will reduce the overall frictional force compared to a longer constant diameter conduit. The increase in diameter also limits the back flow of the supplied biomass to the supply port.

  One or more turning sections (15) can be included in the bath to reduce the overall dimensions of the process. The tank is used to lift the biomass so that the pretreated biomass product exits at or near the withdrawal position of the downstream receiving tank (17) (which may be a saccharification device (18)). It can have a height (16) over all or part of its length. This avoids the need for buckets, augers, elevators, or other equipment to send the pretreated biomass product to a saccharification equipment or other receiving vessel.

  An inlet (19) is used at one or more locations along the length of the tank to supply pretreatment agents including anhydrous ammonia and water vapor. As indicated above, these inlets are at a distance downstream of the biomass feed and reducing the pretreatment agent from flowing out through the biomass feed. The pretreatment agent communicates with the biomass undergoing pretreatment and is fed into the tank at a rate sufficient to pretreat the loaded biomass as described below. Furthermore, heat is applied to the biomass by directly injecting water vapor into the tank or by heating the tank using a jacket that contains a heating medium such as water vapor or heated oil and surrounds the tank.

  The end portion (20) of the vessel may be non-insulated, or may be actively cooled, for example by using a reactor jacket, to allow the temperature to drop before the biomass exits the vessel. Sterile water may be injected directly into the process to accelerate cooling.

  The pretreated biomass product leaves the tank at the exit. The outlet can be an outlet valve such as a hinged plate (21) as used in a swing check valve. The discharge pressure can be adjusted by applying a controlled force against the flow.

  Excess reagent, such as ammonia, can be captured and reused in the pretreatment step. Ammonia and steam vapors can also be released from the pretreatment tank exhaust, but generally these vapors are discharged with the pretreated biomass and enter the receiving tank (17) or hopper. . In the receiving tank, the steam expands and evaporates to separate from the pretreated biomass, and is generally vented from the exhaust hole (22) and led to a pretreatment process for reuse.

  Alternatively, the added ammonia can be limited to the required stoichiometric amount of ammonia plus an acceptable ammonia residue to avoid the need to capture and reuse excess reagent.

Pretreatment Method Using Ammonia Vapor In the method of the present invention, continuously fed biomass loaded in a stationary tank is treated in a relatively dry system with anhydrous ammonia as it travels through the tank. If the system is relatively dry, ammonia can spread throughout the biomass in a vapor state that does not require mechanical mixing. An open interstitial space between biomass particles or debris through which vaporous ammonia passes is maintained by keeping the water content in the reactor low, so that the water present in the reactor is associated with the biomass, The gap is not filled. Therefore, the moisture content in the tank is not constant because the moisture is higher at the interface of biomass particles or debris and lower in the interstitial space between biomass particles or debris.

  The overall total moisture content in the tank is less than 40% by weight measured relative to the total mass in the tank including biomass, anhydrous ammonia, and any other components added to the tank, such as steam. To do. In the method of the present invention, without mechanical mixing, an effective pretreatment to produce a readily saccharifiable biomass product as determined by the yield of sugar produced in subsequent saccharification. It is shown herein that ammonia vapor can penetrate biomass. Samples taken at various distances from the ammonia loading point were able to produce high levels of sugar during saccharification. The sugars produced in the examples herein had xylose monomer in the range of 57-68% theoretical yield and glucose monomer in the range of 80-90% theoretical yield.

  The tank used in the pretreatment method of the present invention does not require a mixing mechanism and is a stationary tank. The tank can be any shape, such as a cylinder, and can be placed in a horizontal or vertical orientation, or it can include horizontal and vertical portions. The tank has one or more inlets / outlets for the introduction of biomass, ammonia, and water vapor. The tank can be constructed to operate from atmospheric pressure (0 gauge pressure) to 20 gauge pressure. The vessel can have means for raising its temperature directly, for example a heating jacket. The tank is of a material designed to withstand ammonia vapor and can be a packed bed reactor.

  The biomass loaded into the tank is typically lignocellulosic biomass and has a dry matter content of at least about 60%. The percentage of dry mass of the biomass can be about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more. For example, freshly harvested corn stover is typically about 70% dry mass, whereas biomass can be dried to a higher percentage of dry matter, for example by air drying. In addition, the biomass can be reduced in size before loading into the tank. In general, prior to loading the biomass into the tank, the size is reduced mechanically, for example, by chopping, cutting or grinding.

  The tank containing the biomass is loaded with anhydrous ammonia, and the biomass in the tank is brought into contact with the anhydrous ammonia by allowing the biomass to permeate the biomass. In the method of the present invention, most of the biomass in the tank comes into contact with anhydrous ammonia. In general, more than 50% of biomass is in contact with anhydrous ammonia. At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the biomass is in contact with anhydrous ammonia.

  Anhydrous ammonia is added in an amount of at least about 4% based on the dry mass of biomass in the tank. The concentration range of anhydrous ammonia used is about 4% to about 20%. By using 6% ammonia, the yield of xylose is increased by saccharification of the resulting anhydrous ammonia permeation biomass as shown in Example 3 herein, compared to the effect of 3% anhydrous ammonia. did. The introduction of anhydrous ammonia raises the temperature of the biomass in the tank in which it is put.

  The reactor and / or biomass temperature may be raised before adding anhydrous ammonia. The temperature can be indirectly measured by any method including, for example, a method by applying heat to the vessel using a heating jacket or heating coil, or a method by introducing a hot gas or vapor, such as water vapor, into the reactor vessel. Can be raised manually or directly. Water vapor can be injected as superheated steam or dry steam to avoid the introduction of moisture in order to keep the total water content in the tank below 40% by mass of the total mass in the tank. The water in the tank may come primarily from biomass and added gas. When processing very dry biomass, such as biomass having a moisture content of less than about 15%, the steam need not be dry to maintain a low total moisture content of 40%. However, dry steam is used when the biomass has a higher moisture content, for example about 35%. The desired temperature is generally in the range of about 70 ° C to 190 ° C. Typically the temperature is in the range of about 90 ° C to 150 ° C.

  It is also possible to increase the pressure inside the tank. Steam injection can be used to increase the pressure. During pretreatment, the pressure is maintained from 0 gauge pressure to less than 20 gauge pressure. The residence time when the biomass moves through the pretreatment tank is about 10 minutes to 5 hours. This residence time can include the first section after biomass loading from when the biomass enters the tank until it meets an inlet for injecting pretreatment agents such as steam and ammonia. Typically, steam and ammonia are injected in the second section of the residence time to contact the biomass and pretreatment occurs. This second section can be from about 5 minutes to several hours. During this time, pressure and temperature are maintained in the vessel that gives the condition that ammonia continues to permeate through the interstitial spaces between biomass particles or debris in the vapor state. There may be a third segment of residence time as the biomass approaches the outlet where the temperature decreases as described above.

  At the end of the desired residence time, the pretreated biomass is discharged from the tank through the outlet as it is pushed out by the force applied by the feeder as described above. This pretreated biomass product is in a dry state. This can be loaded into the next tank by mechanical methods or by gravity.

  The associated pretreatment vapor can be reused as mentioned above. It is preferred to recycle ammonia in a commercially viable manner. Ammonia vapor can be recycled by methods known to those skilled in the art using an ammonia vapor handling system. For example, ammonia vapor can be condensed and reused as ammonia water. Alternatively, ammonia vapor can be reused primarily in the anhydrous state.

Lignocellulosic biomass The biomass used in the method of the present invention is lignocellulosic, which contains polysaccharides such as cellulose and hemicellulose, and lignin. Biomass polysaccharides can also be referred to as glucans and xylans. The types of biomass that can be used include, but are not limited to, biofuel energy crops, crop residues, municipal waste, industrial waste, paper sludge, garden waste, wood, and forestry waste. Examples of biomass include, but are not limited to, corn cob, corn peel, corn stover, grass, wheat straw, barley straw, oat straw, rapeseed straw, hay, rice straw, switchgrass, Japanese pampas grass, cord Ingredients obtained from glass, ragweed, waste paper, sugar cane pomace, sorghum pomace or straw, soy straw, cereal milling, trees, branches, roots, leaves, wood chips, sawdust, shrubs and shrubs, vegetables, fruits, Examples include flowers and animal manure. Biomass includes other grain residues, forest willows, other hardwoods, conifers, and sawdust and other waste products, used paper products after consumer use, corn fiber, beet pulp, pulp mill fines and defective products Fiber process residues, and other lignocellulosic materials present in sufficient amounts.

  Particularly useful biomass for the present invention includes biomass that has a relatively high carbohydrate content, is relatively dense, and / or is relatively easy to collect, transport, store, and / or manipulate.

  Lignocellulosic biomass can also be obtained from a single source, and the biomass can also include a mixture obtained from one or more sources. For example, the biomass can include a mixture of corn cobs and corn stover, or a mixture of stems or stems and leaves.

  The biomass obtained from the source can be used directly or it can be subjected to some pre-processing. For example, energy can be added to the biomass to reduce size or reduce moisture. The particle size reduction can be performed by using a method for producing a small and coarse material having a grain size exceeding 0.1 mm. Methods that can be used include mechanical methods such as knife milling, roughing, shredding, chopping, disc refining, and rough hammer milling. This type of particle size reduction can be done before or after treatment with anhydrous ammonia, but is generally before. Drying can be by any conventional means such as using a drying oven, rotary dryer, flash dryer, or superheated steam dryer. Furthermore, air drying may be sufficient to reach the desired biomass moisture content of less than about 40%. When used in the method of the present invention, the biomass desirably has a dry matter content of at least about 60, 65, 70, 75, 80, 85, 90, or 93% by weight.

Pretreated biomass product The pretreated biomass product obtained by the method of the present invention is used for saccharification to produce a saccharide for fermentation with a biocatalyst for producing a desired product.

Saccharification Typically, enzymatic saccharification degrades at least one saccharifying enzyme and often cellulose and hemicellulose to produce hydrolysates containing sugars including glucose, xylose, and arabinose. With the enzyme community. Saccharification enzymes are described in Lynd, L. et al. R. (Microbiol. Mol. Biol. Rev., 66: 506-577, 2002).

  This enzyme generally includes one or more types of glycosidases. Glycosidases hydrolyze the ether linkages of disaccharides, oligosaccharides, and polysaccharides. These glycosidases are classified according to the enzyme group EC 3.2.1x of the general group “hydrase” (EC 3) (Enzyme Nomenclature 1992, Academic Press, San Diego, CA, Addendum 1 (1993), Addendum 2 (1994), Addendum 3 (1995), Addendum 4 (1997), and Addendum 5 (Eur. J. Biochem., 223: 1-5, 1994, Eur. J. Biochem., 232: 1-6, 1995, respectively. Eur. J. Biochem., 237: 1-5, 1996, Eur. J. Biochem., 250: 1, 6, 1997, and Eur. J. Biochem., 264: 610-650, 1999. ))). Glycosidases useful in the methods of the present invention can be classified by the biomass components that they hydrolyze. Glycosidases useful in the methods of the invention include cellulose hydrolyzing glycosidases (eg, cellulase, endoglucanase, exoglucanase, cellobiohydrolase, β-glycosidase), hemicellulose hydrolyzing glycosidases (eg, xylanase, endoxylanase, exoxylanase, β-xylosidase, arabinoxylanase, mannase, galactase, pectinase, glucuronidase), and starch hydrolyzing glycosidase (eg, amylase, α-amylase, β-amylase, glucoamylase, α-glucosidase, isoamylase). In addition, peptidases (EC 3.4.x.y), lipases (EC 3.1.1.x and 3.1.4.x), ligninases (EC 1.11.1.x), and feruloyl esterases It may be useful to add other activities to the saccharifying enzyme consortium such as (EC 3.1.1.13) to promote the release of polysaccharides from other components of the biomass. It is well known in the art that microorganisms that produce polysaccharide hydrolases often exhibit activity catalyzed by several enzymes or groups of enzymes with different substrate specificities, such as cellulose degradation. Thus, microbial-derived “cellulases” can include a group of enzymes, all of which contribute to cellulolytic activity. Commercial or non-commercial enzyme preparations, such as cellulases, can contain a great variety of enzymes, depending on the purification scheme utilized to obtain the enzyme.

  Saccharification enzymes such as Spezyme® CP cellulase, Multifect® xylanase, Accelerase® 1500, and Accelerase® DUET (Danisco US Inc., Genencor International, Rochester, NY). Can be obtained on the market. Furthermore, the saccharifying enzyme can be unpurified or supplied as a single cell extract or whole cell preparation. Enzymes can also be produced using recombinant microorganisms that have been genetically engineered to express multiple types of saccharifying enzymes.

  The type of glycoside hydrolase (GH) is of the specific grade in the present invention, for example the families GH3, GH39, GH43, GH55, GH10 and GH11. These GHs are a group of enzymes that hydrolyze glycosidic bonds between two or more carbohydrates, or between carbohydrate and non-carbohydrate moieties. These families of GH are classified based on sequence similarity and are available in the Carbohydrate-Active enzyme (CAZy) database (Cantarel et al. (2009), Nucleic Acids Res. 37 (Database) published: D233-238). it can. These enzymes can act on multiple types of substrates, and are effective in the saccharification method of the present invention. Glycoside hydrolase family 3 (“GH3”) enzymes have several known activities: -Glucosidase (EC: 3.2.1.21), β-xylosidase (EC: 3.2.1.37), N -Acetyl-glucosaminidase (EC: 3.2.1.52), glucan β-1,3-glucosidase (EC: 3.2.1.58), cellodextrinase (EC: 3.2.1.74) ), Exo-1,3-1,4-glucanase (EC: 3.2.1), and β-galactosidase (EC: 3.2.1.23). Glycoside hydrolase family 39 (“GH39”) enzymes have α-L-iduronidase (EC: 3.2.1.76) or β-xylosidase (EC: 3.2.1.37) activity. Glycoside hydrolase family 43 (“GH43”) enzymes include L-α-arabinofuranosidase (EC: 3.2.1.55), xylosidase (EC: 3.2.1.37), endoarabinanase ( EC: 3.2.1.99), and galactan 1,3-galactosidase (EC: 3.2.1.145) activity. Glycoside hydrolase family 51 (“GH51”) enzymes have L-α-arabinofuranosidase (EC: 3.2.1.55) or endoglucanase (EC: 3.2.1.4) activity. Glycoside hydrolase family 10 (“GH10”) enzymes are more fully described in Schmidt et al. (1999, Biochemistry 38: 2403-2412) and Lo Leggio et al. (2001, FEBS Lett 509: 303-308). Have been described. The glycoside hydrolase family 11 (“GH11”) enzymes are more fully described in the paper by Hakuvainen et al. (1996, Biochemistry 35: 9617-24).

  These enzymes can be isolated from their natural host organisms or expressed in host organisms that have been genetically engineered for production. For example, a chimeric gene containing a promoter acting in a target expression host cell, the GH encoding sequence given above, and a termination signal can be expressed from a plasmid vector or standard known to those of skill in the art. Are integrated into the genome of the target expression host cell using standard methods. The coding sequence used can be codon optimized for the specific host used for expression. Commonly used expression host cells include bacteria such as Escherichia, Bacillus, Lactobacillus, Pseudomonas, and Streptomyces, and yeasts such as Saccharomyces, Schizosaccharomyces, Candida, Hansenula, Pichia, Pichia, Kluyveromyces, Kluyveromyces, Kluyveromyces For example, Acremonium, Aspergillus us), Aureobasidium, Bjerkanda, Seripoliopsis, Chrysoporum, Copurinus, Coriolus, Coriolus Corynascus, Chertomium, Cryptococcus, Filobasidium, Fusarium, Gibberella, Humicolap, Humikol (Mucor), Mycelioft ora), Mucor, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phanerochaete, Phanerochaete (Pyromyces), Pleurotus, Scytaldium, Schizophyllum, Sporotrichum, Talaromyces genus, Tararomys Radium (Tolypoclad) ium), Trametes, and Trichoderma.

  Those skilled in the art will know how to determine the effective amount of each enzyme to use in the community and how to adjust the conditions for optimal enzyme activity. Those skilled in the art will also know how to optimize the type of enzyme activity required in the community to obtain optimal saccharification of a given pretreatment product under selected conditions. is there. Examples of saccharification are described in US Pat. No. 7,932,063.

  Saccharification can be performed for about a few minutes to about 200 hours, generally about 24 hours to about 72 hours. The duration of the reaction will depend on the concentration and specific activity of the enzyme, as well as the substrate used and environmental conditions such as temperature and pH. One skilled in the art can readily determine the optimum temperature, pH, and time conditions to be used with a particular substrate and saccharifying enzyme consortium.

  Saccharification can be performed in a single batch, fed-batch, or as a continuous process. Saccharification can also be performed in one step or in multiple steps. For example, different enzymes required for saccharification may exhibit optimal conditions of different pH or temperature. Primary treatment with enzyme (or group of enzymes) at a certain temperature and pH followed by secondary or tertiary (or more) treatment with different enzyme (or group of enzymes) at different temperature and / or pH It can be performed. Furthermore, treatment with different enzymes in sequential steps may be the same pH and / or temperature, or use hemicellulase that is stable and more active at higher pH and temperature, followed by lower pH and temperature. Different pHs and temperatures may be used, such as using cellulases that are active in

  Prior to fermentation, the saccharified mixture can be concentrated, for example, by evaporation to increase the concentration of those fermentable sugars. Optionally, the liquid in the saccharification product can be separated from the solids in a batch or continuous process. Optionally, the liquid or the entire saccharified product can be sterilized prior to fermentation. Depending on the biocatalyst used during fermentation and the pH used during saccharification, the pH can be adjusted to be suitable for fermentation.

  Biomass hydrolyzate containing fermentable sugars is generally included in the medium as a proportion of the fermentation medium and forms all or part of the carbon source for biocatalyst growth and product production. The hydrolyzate in the fermentation medium is generally about 40% to 90% of the fermentation medium. Examples of hydrolysates used as 40% or 80% of the fermentation medium are shown in Example 9 of US Pat. No. 7,932,063. Depending on the fermentable sugar concentration in the hydrolyzate, additional sugar can be added to the medium. For example, if hydrolyzate containing about 80 g / L glucose and about 50 g / L xylose is included in 40% of the fermentation medium, additional glucose and xylose can be added to achieve the desired final sugar concentration. In addition to hydrolysates, the fermentation medium is a well-known element for those skilled in the art, other nutrients, salts, and elements that its specific biocatalysts used in the production of the product require for growth and production. And can be contained. These nutritional supplements can include, for example, yeast extract, specific amino acids, phosphates, nitrogen sources, salts, and trace elements. Components necessary for the production of specific products made by specific biocatalysts can also include, for example, antibiotics for maintaining plasmids, or cofactors required in enzyme-catalyzed reactions.

  As an alternative to preparing hydrolyzate, adding it to the fermentation medium, and then performing the fermentation, a biomass hydrolyzate fermentation broth can also be produced using the simultaneous saccharification and fermentation (SSF) method. In this method, sugars are produced from biomass as they are metabolized by the production biocatalyst.

Fermentation of biocatalyst and target product The fermentable sugar in the fermentation medium is metabolized by an appropriate biocatalyst to produce the target product. The sugar is brought into contact with the biocatalyst during the fermentation process. The biocatalyst then grows under conditions that produce the desired product made by the biocatalyst. The temperature and / or head space gas can be tailored to the fermentation depending on the conditions useful for the particular biocatalyst in use. Fermentation may be aerobic or anaerobic. These and other conditions, including temperature and pH, are tailored to the particular biocatalyst used.

  Examples of target products produced by the biocatalyst include 1,3-propanediol, butanol (isobutanol, 2-butanol, and 1-butanol), and ethanol. A recombinant microorganism that produces 1,3-propanediol is disclosed in US Pat. No. 7,504,250. The production of butanol by genetically modified yeast is disclosed, for example, in US Pat. No. 7,851,188. Genetically modified strains of E. coli have also been used as biocatalysts for ethanol production (Underwood et al. (2002), Appl. Environ. Microbiol. 68: 6263-6272). Ethanol is produced by genetically modified Zymomonas in lignocellulosic biomass hydrolyzate fermentation medium (US Pat. No. 7,932,063). A genetically modified strain of Zymomonas mobilis that can be used to improve ethanol production is disclosed in US Pat. No. 7,223,575 and US Pat. No. 7,998,722. Have been described.

  The invention will be further clarified in the following examples. While these examples illustrate preferred embodiments of the present invention, it should be understood that they are given for illustrative purposes only. From the foregoing discussion and these examples, those skilled in the art can ascertain the essential features of the present invention and depart from the spirit and scope of the present invention in order to adapt the present invention to various applications and conditions. Various variations and modifications can be made without it.

  The meanings of the abbreviations used are as follows: “S” is seconds, “min” means minutes, “h” or “hr” means hours, “μL” means microliters, “mL” means milliliters, “ “L” means liter, “m” means meter, “nm” means nanometer, “mm” means millimeter, “cm” means centimeter, “μm” means micrometer "M" means millimole, "M" means mole, "mmol" means millimole, "μmole" means micromole, "g" means gram , “Μg” means microgram, “mg” means milligram, “kg” is kilogram, “rpm” means revolutions per minute, “C” is Celsius, “ppm” means parts per million and “psi” Is the pounds per square inch.

General Methods Saccharification Accelerase® 1500 (A1500) was obtained from Danisco US S. Inc. , Genencor, International (Rochester, NY).

Packed bed reactor A packed bed pretreatment reactor consisting of a vertically oriented stainless steel filter housing pressure tank and having a removable lid was used. The pressure vessel contains a stainless steel removable cylindrical basket with an open top, a solid wall, and a perforated base. The basket allowed the biomass to be statically introduced into the packed bed and allowed the passage of top-filled or bottom-filled liquids, vapors and / or gases throughout the interstitial volume between the biomass particles.

  The filter housing had an inner diameter of 8 inches (20.3 cm) and a chamber length of 29 inches (73.7 cm). The cylindrical basket had a diameter of 6.5 inches (16.5 cm), a depth of 29 inches (73.7 cm), and a volume of about 15.8 liters.

  A flexible copper tube surrounds approximately 30% of the outer surface area of the filter housing. This copper tube used to heat the outside of the bath was connected to a steam source of 164.7 psia (1135.6 kilopascals).

To suit the packed bed reactor,
Exhausting air to create a complete or incomplete vacuum,
Adding saturated steam from a boiler operating at 84.7 psia (584 kilopascals);
Adding anhydrous ammonia,
・ Exhaust process steam after reaction,
• After the reaction, a tube joint was made that allowed air to be added to break the vacuum.

Example 1
Pre-treatment of packed bed of biomass in saccharification preparation 4.07 kg corn cob is shredded and classified to a particle size greater than 1/4 inch (0.64 cm) and less than 3/8 inch (0.375 cm) A COB fraction with The corn cobs were placed in a basket and then placed in the pretreatment reactor described in the general method. The corn cobs had a moisture content of 8.6% and a bulk density of 227.5 g / L. The pre-reacted packed bed was approximately 29 inches (73.7 cm) deep.

  Air was evacuated from the reactor and an incomplete vacuum of 0.1 bar (10 kilopascals) was achieved. 8% by weight of anhydrous ammonia, based on the weight of the absolutely dry biomass, was added by loading 297.6 g of ammonia over a period of about 60 minutes. Saturated steam at a pressure of 84.7 psia (584 kilopascals) was then added to bring the reactor temperature to about 70 ° C. At this point, the reactor temperature was allowed to rise from 70 ° C. to 90 ° C. during a 40 minute hold time. The pressure in the reactor was approximately 85 psia (584 kilopascals) throughout this hold time. After this holding time, the pressurized gas and vapor were released and trapped in a cooled external uptake tank. When the reactor reached atmospheric pressure, the reactor was evacuated to about 0.1 bar (10 kilopascals) using a vacuum pump and held at that pressure for 5 minutes. The reactor was then opened and the vacuum was released to atmospheric pressure. After the process reached atmospheric pressure, the pretreatment reactor was opened.

  The pretreated biomass was divided into four fractions from the top to the bottom of the packed bed, ie the first quarter fraction (top), the second quarter fraction, the third as in Table 1. Removed from the quarter fraction, the fourth quarter fraction (bottom). These fractions were analyzed for moisture content and residual ammonia and the results are shown in Table 2. Residual ammonia was determined by extracting a known amount of pretreated solid in water for 1 hour and then determining the equivalent of acid to reach pH 5.3 using titration with 0.1N HCl. The equivalent weight was then normalized to the amount of dry matter in the extraction system. Further thereafter, equal-mass fractions were combined and then analyzed to determine the mixing cup composition of the pretreated biomass.

Table 1. Samples recovered from four separate layers in a 29 inch (73.7 cm) deep reactor bed

*DMは乾物 Table 2. Analysis of pretreated materials

* DM is dry matter

  Samples of each pretreated biomass fraction were saccharified enzymatically to determine the yield of glucan and xylan monosaccharides and oligosaccharides.

Saccharification Procedure 0.56 g of each fraction was weighed into individual 20 mL scintillation vials. An appropriate amount of 50 mM (pH 5) acetate buffer was added to obtain 18.6% solids per vial. The pH was adjusted to 5 by adding 1N H ₂ SO ₄ . This acetate buffer contained 0.005% sodium azide to inhibit bacterial growth in the raw material during incubation.

  Enzymatic saccharification can be achieved using Accelerase® combined with a reaction mixture of hemicellulases (Xyn3, Fv3A, Fv51A, and Fv43D) at either glucan + xylan 7.2 mg / g or glucan + xylan 21.7 mg / g 1500 was used. Enzyme was added to each vial, followed by a 1.1 / 2 inch (1.3 cm) steel ball to achieve sufficient attrition of the raw material during the incubation.

  The vial was covered tightly and left in a rotary shaker at 180 rpm on a rotary shaker for 72 hours for saccharification at about 48 ° C.

After incubation, the sample was diluted with water and then filtered and analyzed by HPLC. These samples were subjected to HPLC analysis using an HPX-87H column (BioRad) effluent at 60 ° C. using 0.01N H ₂ SO ₄ as the mobile phase at a flow rate of 0.6 mL / min.

  Total sugar values were obtained by acid hydrolysis by addition of sulfuric acid, autoclaving, filtration, and analysis by HPLC. The oligomer value was determined by subtracting the monomer from the total sugar concentration for each sample. The results are shown in Table 3.

Table 3. Sugar produced from hydrolysis of each of the four fractions of treated biomass

Example 2
Pretreatment of packed bed of biomass at high temperature and short time in preparation for saccharification As in Example 1, 3.74 kg of ground corn cobs were placed in the reactor basket described in the general method. This basket was placed in a pretreatment reactor. The corn cobs had a moisture content of 8.6% and a bulk density of 227.5 g / L. The pre-reacted packed bed was approximately 29 inches (73.7 cm) deep.

  150 psig (1034.2 kilopascals) of steam was applied to the copper coil of the preheater. When the preheater lid reached 145 ° C, the air was evacuated until an incomplete vacuum of 0.6 psia was achieved. Steam was injected directly into the process, reaching a pressure of 17 psia (4.14 kilopascals). The preheater was then vented by opening the bottom valve and releasing the condensate formed in the system. To ensure that all condensate was purged from the system, steam was injected into the top of the preheater until dry steam was observed exiting the open bottom valve. At this point, the temperature in the reactor was about 100 ° C. The flow of water vapor was stopped and then the drain valve was closed. Due to heat absorption into the biomass, the reactor temperature dropped to 80 ° C., creating an incomplete vacuum of 8 psia. At this point, 190.3 g of anhydrous ammonia was added to the process to achieve an ammonia load of 6% by weight, based on the dry matter loaded in the reactor. After the ammonia addition, enough steam was added to bring the process to a reaction temperature of 150 ° C. and a pressure of 70 psia (482.6 kPa). After 30 minutes, pressurized gas and vapor were released and captured in a cooled external intake vessel. When the reactor reaches atmospheric pressure, the reactor is then evacuated to about 0.1 bar (10 kilopascals) using a vacuum pump to further reduce residual ammonia from the biomass and held at that pressure for 5 minutes. It was. The reactor was then opened and the vacuum was released to atmospheric pressure. After the process reached atmospheric pressure, the pretreatment reactor was opened.

  As in Table 4, the pretreated biomass was removed from each fraction that was fractionated into four from the top to the bottom of the packed bed. These fractions were analyzed for moisture content and residual ammonia and the results are shown in Table 5. Further thereafter, equal-mass fractions were combined and then analyzed to determine the mixing cup composition of the pretreated biomass.

Table 4. Samples recovered from four separate layers of reactor bed in a 30-inch deep reactor bed

*DM は乾物 Table 5. Analysis of pretreated materials

* DM is dry matter

  Samples of each pretreated biomass fraction were saccharified with enzymes and the yields of glucan and xylan monosaccharides and oligosaccharides were determined as described in Example 1.

Table 6. Sugar produced from hydrolysis of each of the four fractions of treated biomass

Claims (15)

A method of processing biomass to produce pretreated biomass,
a) providing biomass having a dry matter content of at least about 60%;
b) loading the biomass of (a) into a stationary pretreatment tank by continuously feeding, whereby the loaded biomass in the tank moves through the tank;
c) Under the condition that the total water content in the tank remains below 40% by mass (measured with respect to the total mass in the tank), the loaded biomass of (b) is treated with the pretreatment tank. While moving through, it is contacted with at least about 4% anhydrous ammonia (measured with respect to the dry mass of the biomass in the vessel), thereby penetrating the majority of the biomass with ammonia vapor. And d) discharging the biomass from the pretreatment tank,
The method wherein the biomass discharged from the tank is a pretreated biomass product.   The method of claim 1, wherein after the discharging step of (d), the steam is separated from the pretreated biomass product.   The method of claim 2, wherein the vapor comprises ammonia vapor, which is recovered and reused in a pretreatment tank or ammonia vapor handling system.   The method of claim 1, wherein after step (d), the pretreated biomass product is recovered.   The method of claim 1, wherein after step (c), the loaded biomass proceeds through the pretreatment tank with a residence time of about 10 minutes to about 5 hours.   2. The method of claim 1, wherein anhydrous ammonia is contacted with the biomass for less than about 5 hours.   The method of claim 1, wherein the temperature of the biomass in the tank is at least about 70 ° C.   The method of claim 1, wherein the temperature of the vessel is increased to at least about 70 ° C. prior to step (b).   The method of claim 8, wherein the temperature before step (b) is from about 70 ° C to about 190 ° C.   9. The method of claim 8, wherein the temperature is increased directly by injecting hot gas or indirectly by applying heat to the bath.   The method of claim 1, wherein the concentration of anhydrous ammonia in step (c) is less than about 20% (measured relative to the dry mass of biomass in the tank).   The method of claim 1, wherein the pressure in the bath is about 0 to 20 gauge pressure over steps (b)-(d).   The method according to claim 1, wherein the biomass in (a) is biomass that has been mechanically reduced in size.   The method of claim 1, wherein the biomass is a cellulosic biomass comprising cellulose, hemicellulose, and lignin.   Biomass, corn cob, corn peel, corn stover, grass, wheat straw, barley straw, oat straw, rapeseed straw, hay, rice straw, switchgrass, Japanese pampas grass, cordgrass, scallop, waste paper, sugarcane squeezed Ingredients from dregs, sorghum dregs or straw, soy straw, cereal milling, trees, branches, roots, leaves, wood chips, sawdust, shrubs and shrubs, vegetables, fruits, flowers, and animal manure 15. A method according to claim 14, wherein the method is selected.

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