Treatment of biomass to obtain fermentable sugars

Cross-reference of related applications

This application claims the priority of US Provisional Patent Application No. 60/670437, filed April 12, 2005.

Statement on government rights The present invention was sponsored by the United States Government under Contract No. 04-03-CA-70224 awarded by the Department of Energy. Government has specific rights in the present invention.

A method for treating biomass is provided with the intention of obtaining fermentable sugars. Specifically, fermentable sugars are obtained by pretreating the biomass under conditions of high solid concentration and low ammonia concentration, followed by saccharification.

Cellulose and lignocellulosic feedstocks and wastes such as agricultural residues, wood waste, forestry waste, paper sludge, and urban and industrial solid waste are intended for the production of chemicals, plastics, fuels and feeds. To provide a potentially large renewable feedstock. Cellulosic and lignocellulose-based feedstocks and wastes, consisting of carbohydrate polymers containing cellulose, hemicellulose, glucan and lignin, are generally treated primarily by a variety of chemical, mechanical and enzymatic means. Hexose and pentose sugar are released and can then be fermented into useful products.

The pretreatment method produces carbohydrate polymers of cellulosic and lignocellulosic materials that are more readily available for saccharifying enzymes. Historically, standard pretreatment methods have mainly used strong acids at high temperatures, but due to high energy costs, high equipment costs, high pretreatment catalyst regeneration costs and incompatibility with saccharifying enzymes, enzymes Alternative methods such as the use of acids or bases at lower temperatures in the event of reduced hydrolysis of the biomass carbohydrate polymer during pretreatment with, or during pretreatment, are being developed for cellulose and hemicellulose. An improved enzyme system is needed to saccharify both.

Many pretreatment methods using bases have been proposed. Non-Patent Document 1 describes hydrogen peroxide (H).₂O₂) Is disclosed as a pretreatment method for lignocellulosic biomass. Processing is H₂O₂Is most efficient when used in an amount of at least 0.25 wt / wt with respect to the substrate.

Non-Patent Document 2 discloses a series of biomass pretreatments using stoichiometric amounts of sodium hydroxide and ammonium hydroxide with extremely low biomass concentrations. The solution ratio of biomass is 14: 1.

In Non-Patent Document 3, the pretreatment of corn stover using NaOH was examined.

Non-Patent Document 4 reports the use of large amounts of aqueous ammonia for the pretreatment of corn stovers.

In Patent Document 1 of the patent application, a step of contacting lignocellulose with a chemical substance which can be a base such as sodium carbonate or potassium hydroxide at a pH of about 9 to about 14 under appropriate conditions of temperature, pressure and pH is described. Methods for hydrolyzing lignocellulosic, which are contained, are being investigated.

Patent Document 2 and Patent Document 3 describe a pretreatment method, in which a specific ratio of arabinoxylan to the whole non-starch polysaccharide (AX / NSP) is evaluated and used for selecting a feedstock.

<patcit num="1"><text>International Publication No. 2004/081185 Pamphlet</text></patcit><patcit num="2"><text>U.S. Pat. No. 5,916,780</text></patcit><patcit num="3"><text>U.S. Pat. No. 6,090,595</text></patcit><nplcit num="1"><text>Gould (Biotech. And Bioengr. (1984) 26: 46-52)</text></nplcit><nplcit num="2"><text>Teixeira L. et al. (Appl. Biochem. And Biotech. (1999) 77-79: 19-34)</text></nplcit><nplcit num="3"><text>Elshafei A. et al. (Bioresource Tech. (1991) 35: 73-80)</text></nplcit><nplcit num="4"><text>Kim T. and Y. Lee (Bioresource Technology (2005) 96: 2007-2013)</text></nplcit>

<p> As an economically competitive method, commercial methods for producing fermentable sugars from renewable resource biomass use small amounts of chemicals to provide high concentrations and high yields of sugars. Carbohydrate hydrolysis in lignocellulosic biomass is required to produce a source of fermentable sugars that are less toxic to fermenting organisms that convert sugars into value-added chemicals and fuels.</p>

<p> The present invention provides a method for treating biomass intended to produce fermentable sugars. The method of the present invention comprises a pretreatment step in which a relatively high concentration of biomass is treated with a low concentration of ammonia relative to the dry weight of the biomass. After pretreatment, the biomass is treated with a saccharifying enzyme community to produce fermentable sugars. In one embodiment of the invention, the method a) The step of contacting biomass with an aqueous solution containing ammonia to form a biomass-aqueous ammonia mixture, wherein the ammonia is at least sufficient to maintain the alkaline pH of the biomass-aqueous ammonia mixture. Although present in concentration, the ammonia is present in less than about 12% by weight of the dry weight of the biomass, and the dry weight of the biomass is at least about 15% by weight of the weight of the biomass-aqueous ammonia mixture. In high solid concentration, process, b) The step of contacting the product of step (a) with the saccharifying enzyme community under appropriate conditions to produce fermentable sugar, Contains.</p><p> Biomass refers to any cellulosic or lignocellulosic material, such as bioenergy crops, agricultural residues, urban solid waste, industrial solid waste, factory waste, wood waste, forestry waste and combinations thereof. Aqueous solutions containing ammonia can be derived from ammonia gas, ammonium hydroxide, urea, and combinations thereof. According to the method of the present invention, an aqueous solution containing ammonia may contain at least one additional base. Further, according to the method of the present invention, a vacuum can be applied to the biomass prior to contacting the biomass with an aqueous solution containing ammonia. Ammonia may be removed prior to step (b), or ammonia may be returned to the pretreatment reactor for regeneration. Ammonia and biomass can be reacted at temperatures between about 4 ° C and about 200 ° C in the methods of the invention. Plasticizers, fabric softeners or combinations thereof can be used in the methods of the present invention. In addition, energy is applied before or during step (a) to reduce the size, increase the exposed surface area, and / or increase the accessibility of cellulose, hemicellulose and / or oligosaccharides present in the biomass. It may be applied to biomass before or during step (b), or in combination thereof.</p><p> The saccharifying enzyme community may consist of one or more glycosidases, wherein the glycosidases are selected from the group consisting of glycosidases that hydrolyze cellulose, glycosidases that hydrolyze hemicellulose, and glycosidases that hydrolyze starch. Can be done. Other enzymes in the saccharifying enzyme community may include peptidases, lipases, ligninases and ferroylesterases.</p>

Applicants specifically use the entire contents of all cited references in this disclosure. Further, if an amount, concentration, or other value or parameter is given as a range, preferred range, or a list of higher preferred and lower preferred values, this is with any higher range limit or preferred value. Any range formed from any lower range limit or any pair with a preferred value should be understood as specifically disclosing any range, whether or not the range is disclosed separately. Wherever a range of numbers is mentioned herein, the range is intended to include its endpoints and all integers and fractions within the range, unless otherwise specified. The scope of the invention is not intended to be limited to the particular values listed when defining the scope.

The present invention provides a method for treating biomass intended to produce fermentable sugar, wherein a relatively high concentration of biomass is treated with a relatively low concentration of ammonia relative to the dry weight of the biomass. Includes pretreatment steps to be performed. The ammonia-treated biomass is then digested by the saccharifying enzyme community to produce fermentable sugars.

Definition A number of terms are used in this disclosure. The following definitions are provided. "fermentation<u style="single">sex</u>The term "sugar" refers to oligosaccharides and monosaccharides that can be used as carbon sources by microorganisms in fermentation methods.

The term "lignocellulosic" refers to a composition comprising both lignin and cellulosic. The lignocellulose-based material may also contain hemicellulose.

The term "cellulosic" refers to a composition comprising cellulose.

"Dry weight" of biomass means the weight of biomass from which all or essentially all water has been removed. Dry weight is typically American Society for Testing and Materials (ASTM) standard E1756-01 (Standard Test Method for Determination of Total Solids in Biomass) or technical. Measured according to the Technical Association of the Pulp and Paper Industry, Inc. (TAPPI) standard T-412 om-02 (Moisture in Pulp, Paper and Paperboard) ..

The term "target chemical" refers to a chemical produced by fermentation. Chemicals are used in a broad sense and include molecules such as proteins, including, for example, peptides, enzymes and antibodies.

A target chemical that is "derivable from biomass" is that the biomass is hydrolyzed to release fermentable sugar, and the fermentable sugar is fermented with at least one biocatalyst to produce the desired target chemical. A target chemical produced by the method.

The terms "plasticizer" and "softener" refer to substances that induce reduction in the binding intermolecular force along or between polymer chains. Such substances can act, for example, to reduce crystallinity or to break the bond between lignin carbohydrate fibers and non-lignin carbohydrate fibers (eg, cellulose or hemicellulose).

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

The term "pretreated biomass" refers to biomass that has been pretreated prior to saccharification.

"Biomass" refers to any cellulosic or lignocellulosic material, including materials comprising cellulose and optionally further containing hemicellulose, lignin, starch, oligosaccharides and / or monosaccharides. Biomass may also contain additional components such as proteins and / or lipids. According to the present invention, the biomass can be derived from a single source, or the biomass can consist of a mixture derived from more than one source. That is, for example, biomass may contain a mixture of corn cobs and corn stovers, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, urban solid waste, industrial solid waste, paper sludge, factory waste, wood and forestry waste. Examples of biomass include crop residues such as corn grains, corn cobs, corn husks, corn stovers, grass, wheat, wheat straw, barley, barley straw, sorghum, rice straw, switchgrass, waste paper, sugar cane bibagus. , Corn, corn, ingredients obtained from grain milling, trees, branches, roots, leaves, wood chips, straw, shrubs and bushes, vegetables, fruits, flowers and animal manure. In one embodiment, the biomass useful for the present invention has a relatively high carbohydrate value, is relatively dense, and / or is relatively easy to recover, transport, store and / or process. including. In one embodiment of the invention, useful biomass includes corn cob, corn stover and sugar cane bagasse.

The "aqueous solution containing ammonia" for the purpose of the present invention is ammonia gas (NH) in an aqueous medium.₃), Ammonium ions such as ammonium hydroxide or ammonium sulfate (NH₄⁺), Compounds that release ammonia during decomposition, such as urea, and the use of combinations thereof.

The concentration of ammonia used in this method is at a minimum sufficient to maintain the alkaline pH of the biomass-aqueous ammonia mixture and at most less than about 12 weight percent of the dry weight of the biomass. .. This low concentration of ammonia is sufficient for pretreatment, and the low concentration can be less than about 10 weight percent of the dry weight of the biomass. Very low concentrations of ammonia, 6 percent or less of the dry weight of the biomass, can also be used in the pretreatment. Alkaline means a pH greater than 7.0. Particularly suitable are biomass-aqueous ammonia mixtures with a pH greater than 8. In one embodiment, ammonia is present in less than about 10 weight percent of the dry weight of the biomass. Particularly suitable is ammonia, which is less than about 6 weight percent of the dry weight of the biomass.

Ammonia used in this method offers advantages over other bases. Ammonia is divided into a liquid phase and a gas phase. Gaseous ammonia is more easily diffusible through biomass than liquid bases, resulting in lower concentrations and more effective pretreatment. Ammonia is also shown in Example 11 herein as forming acetamide by competing with hydrolysis of the acetyl ester in the biomass via ammonolesis. Acetamide (as shown in Example 12 herein) is Zymomonas. Less toxic than acetate to certain fermenting organisms such as mobilis). Therefore, the conversion of the acetyl ester to acetamide instead of acetic acid reduces the need to remove acetic acid. The use of ammonia also reduces the need to supplement the growth medium used during fermentation with a nitrogen source. In addition, the low cost of ammonia provides an economical method. Ammonia can also be regenerated into the pretreatment reactor during the pretreatment, or therefore a more economical method is realized after the pretreatment. For example, after pretreatment, when the temperature drops to a temperature suitable for saccharification, ammonia gas can optionally be released and regenerated in the presence of vacuum. In the continuous method, ammonia can be regenerated continuously.

According to the method, the aqueous solution containing ammonia may optionally contain at least one additional base such as sodium hydroxide, sodium carbonate, potassium hydroxide, potassium carbonate, calcium hydroxide and calcium carbonate. .. The addition of at least one additional base in an amount bound to ammonium can result in the formation of an entire base in an amount less than about 20 weight percent of the dry weight of the biomass. Preferably, the entire second base, to which ammonia is added, is present in an amount of less than about 15 weight percent. The use of additional bases can, for example, neutralize the acid in the biomass, resulting in metal ions in the saccharifying enzyme or metal ions in the growth medium for fermentation.

In the method, the dry weight of biomass is at an initial concentration of at least about 15% to up to about 80% of the weight of the biomass-aqueous ammonia mixture. More preferably, the dry weight of the biomass is at a concentration of about 15% to about 60% of the weight of the biomass-aqueous ammonia mixture. The percentage of biomass in the biomass-aqueous ammonia mixture is kept high, minimizing the requirement for the concentration of sugars obtained from the saccharification of the pretreated biomass for use in fermentation. Higher biomass concentrations also reduce the total volume of pretreatment material, making the method more economical.

Biomass can be used directly from the source, or energy can be applied to the biomass to reduce its size, increase the exposed surface area, and / or in the second step of the method. The availability of cellulose, hemicellulose, and / or oligosaccharides present in the biomass for the ammonia and saccharifying enzymes used may be increased. Energy tools useful for reducing size, increasing exposed surface area, and / or increasing the availability of cellulose, hemicellulose, and / or oligosaccharides present in the biomass against ammonia and saccharifying enzymes are milling, sac. Includes, but is not limited to, lashing, crushing, crushing, chopping, disk refining, ultrasound, and biomass. This energy application can be made before or during pretreatment, before or during saccharification, or in any combination thereof.

Biomass pretreatment with ammonia solution is performed in any suitable container. Typically, the container is pressure resistant, has a mechanism for heating, and has a mechanism for mixing the contents. Commercially available containers include, for example, Zipperclave® reactors (Autoclave Engineers, Erie, NJ), Jaygo reactors (Jaygo). Manufacturing, Inc., Mahwah, NJ), and steam gun reactors ((described in "General Methods" Autoclave Engineers) Engineers), Erie, Pennsylvania). A much larger reactor with similar performance may be used. Alternatively, the biomass and ammonia solutions may be combined in one container and then transferred to another reactor. Biomass is also pretreated in one container and then another reactor such as a steam gun reactor (described in "General Methods"; Autoclave Engineers, Erie, Pennsylvania). May be further processed within.

A vacuum may be applied to the biomass-containing container prior to contact of the biomass with the aqueous solution containing ammonia. Better penetration of ammonia into the biomass can be achieved by expelling air from the pores of the biomass. The time to apply the vacuum and the amount of negative pressure applied to the biomass will depend on the type of biomass and will provide optimal pretreatment of the biomass (as measured by the production of fermentable sugar after saccharification). It can be empirically determined to do so.

Contact between the biomass and the aqueous solution containing ammonia is carried out at a temperature of about 4 ° C to about 200 ° C. Initial contact of biomass with ammonia at 4 ° C was found to allow injection at this temperature and increase saccharification efficiency over undenatured biomass. In another embodiment, the contact of biomass is carried out at a temperature of about 75 ° C to about 150 ° C. In yet another embodiment, the contact of biomass is carried out at temperatures above about 90 ° C and up to about 150 ° C.

Contact between the biomass and the aqueous solution containing ammonia takes up to about 25 hours. Longer pretreatments are possible, but shorter periods may be preferred for practical and economic reasons. Typically, the contact treatment period for ammonia is about 8 hours or less. A period of up to about 25 hours may be preferred, as longer periods may have the advantage of reducing the need to apply energy for biomass decomposition.

In one embodiment, the pretreatment method can be performed in a relatively short period of time at a relatively high temperature, eg, about 5 minutes to about 2 hours at about 100 ° C to about 150 ° C. In another embodiment, the pretreatment method can be carried out over a relatively long period of time at a lower temperature, eg, about 2 hours to about 8 hours, at about 75 ° C to about 100 ° C. In yet another embodiment, the pretreatment method can be carried out at room temperature (about 22-26 ° C) for a period of time longer than about 24 hours. Other intermediate temperature and time combinations for these may also be used.

The pretreatment method retains temperature, time for pretreatment, ammonia concentration, concentration of one or more additional bases, biomass concentration, biomass type and biomass particle size, thus requiring these variables. Depending on the adjustment, the optimum product to be contacted with the saccharifying enzyme community may be obtained.

Plasticizers, softeners, or combinations thereof, such as polyols (eg glycerol, ethylene glycol), polyol esters (eg glycerol monoacetate), glycol ethers (eg diethylene glycol), acetamides, ethanol, and ethanolamine, are pretreated. It can be added by method (ie step (a)). The plasticizer can be added as a component of an aqueous ammonia solution, a separation solution, or a dry component.

The pretreatment reaction can be carried out in any suitable vessel, such as a batch reactor or a continuous reactor. Those skilled in the art will understand that pressure vessels are needed at higher temperatures (above 100 ° C). Suitable containers may be equipped with means such as an impeller for stirring the biomass-aqueous ammonia mixture. For reactor design, see Lin KH (Lin, KH) and Van Ness, HC (Perry RH (Perry, RH) and Chilton CH (Chilton, CH) (eds.), Chemical Engineer's Handbook, No. It is considered in Edition 5 (1973), Chapter 4, McGraw-Hill, NY). The pretreatment reaction can be carried out as a batch method or a continuous method.

A nitrogen source is required for the growth of microorganisms during fermentation, and therefore the use of ammonia during pretreatment provides a nitrogen source and a growth medium used during fermentation with a nitrogen source. It is well known to those skilled in the art that the need for replenishment is reduced or eliminated. If the pH of the pretreated product is greater than the pH at the time of activation of the saccharifying enzyme or above the range suitable for microbial growth in fermentation, the use of acid can reduce the pH. The amount of acid used to obtain the desired pH can result in the formation of salts at concentrations that are inhibitory to the growth of saccharifying enzymes or microorganisms. Reduce the amount of acid required to obtain the desired pH and NH in this pretreatment method₃Ammonia gas can be discharged from the pretreatment reactor and regenerated in order to reduce the raw material cost. Typically, at least a portion of the ammonia is removed, which lowers the pH, but some nitrogen that provides this nutrient remains in subsequent fermentation use.

To obtain a sufficient amount of sugars from the biomass, the biomass can be pretreated with an aqueous ammonia solution one or more times. Similarly, the saccharification reaction may be carried out once or more times. If it is desirable to obtain higher yields of saccharides, both pretreatment and saccharification methods can be repeated. To evaluate the performance of the pretreatment and saccharification methods separately or in combination, the theoretical yields of saccharides derived from the initial biomass may be determined and compared to the measured yields.

Glycation After pretreatment, the product comprises a mixture of ammonia, partially decomposed biomass and fermentable sugar. Prior to further processing, ammonia can be removed from the pretreated biomass by applying a vacuum. Since the removal of ammonia lowers the pH, the use of less neutralizing acid gives the desired pH in saccharification and fermentation. This leads to a reduction in salt load in the pretreatment mixture. Typically, some ammonia remains, which is desirable to provide a nitrogen source in fermentation.

The pretreated mixture is then further hydrolyzed in the presence of the saccharifying enzyme community to release oligosaccharides and / or monosaccharides into the hydrolyzate. Methods for saccharifying enzymes and biomass treatment are reviewed in Lind LR (Lynd, LR) et al. (Microbiol.Mol.Biol.Rev. (2002) 66: 506-577). In one preferred embodiment, the entire pretreatment mixture, which comprises both soluble and insoluble fractions, is utilized in the saccharification reaction.

In another embodiment, prior to saccharification, the aqueous fraction containing ammonia and soluble sugars can be separated from the insoluble microparticles remaining in the mixture. Methods for separating solubles from the insoluble fraction include, but are not limited to, decantation and filtration. Insoluble microparticles can be regenerated into the pretreatment reactor. Adsorbed saccharides can be removed from the insoluble microparticles, optionally by washing with an aqueous solvent (eg, water) prior to being regenerated into the pretreatment reactor. The insoluble fraction can then be saccharified with a saccharifying enzyme community after further treatment with an aqueous ammonia solution as described above in the pretreatment. Soluble fractions can also be concentrated prior to saccharification using suitable methods such as evaporation.

Prior to saccharification, the pretreated product can be treated and the pH, composition or temperature can be changed so that the enzymes of the saccharifying enzyme community show activity. The pH can be changed through the addition of acid in solid or liquid form. Alternatively, carbon dioxide (CO)₂) Can be recovered from fermentation, and the pH can be lowered by use. For example, CO while monitoring the pH until the desired pH is obtained.₂Can be recovered from the fermenter and supplied to the pretreated product by bubbling or the like. As described below, the temperature can be induced to a temperature that is compatible with the activity of the saccharifying enzyme. Any cofactor necessary for the activity of the enzyme used in saccharification can be added.

The saccharifying enzyme community mainly comprises one or more enzymes selected (but not limited to) from the group "glycosidases" that hydrolyze the ether bonds of disaccharides, oligosaccharides, and polysaccharides. As an enzyme classification, in addition to EC3.2.1.x (Enzyme Nomenclature 1992, Academic Press, San Diego), California of the general group "hydrolase" (EC3.), Supplement 1 (1993) Year), Supplement 2 (1994), Supplement 3 (1995), Supplement 4 (1997) and Supplement 5 [Eur.J.Biochem. (1994) pp. 223: 1-5, Eur.J.Biochem. (1995) pp. 232: 1-6, Eur.J.Biochem. (1996) 237: It is found on pages 1-5, Eur.J.Biochem. (1997) 250: 1-6, and Eur.J.Biochem. (1999) 264: 610-650]). Glycosidases useful in this method may be classified according to the biomass components they hydrolyze. Glycosidases useful in this method are glycosidases that hydrolyze cellulose (eg, cellulase, endoglucanase, exoglucanase, cellobiohydrolase, β-glucosidase), glycosidases that hydrolyze hemicellulose (eg, xylanase, endoxylanase, exoxylanase). , Β-Xylanase, arabinoxylanase, mannase, galactase, pectinase, glucuronidase), and glycosidases that hydrolyze starch (eg, amylase, α-amylase, β-amylase, glucoamylase, α-glucosidase, isoamylase) .. In addition, peptidase (EC3.4.xy), lipase (EC3.1.1.x and 3.1.4.x), ligninase (EC1.11.1.x), and ferroylesterase (EC3.1.1.x). It may be useful to promote the release of polysaccharide biomass from other components by adding other activities to the saccharifying enzyme community such as 73). It is well known in the art that microorganisms that produce enzymes that hydrolyze polysaccharides often exhibit activities such as cellulolysis catalyzed by several enzymes or groups of enzymes with different substrate specificities. Thus, microbial-derived "cellulases" may include groups of enzymes, all of which can contribute to cellulolytic activity. Commercial or non-commercial enzyme preparations such as cellulases may contain a very large number of enzymes, depending on the purification scheme used to obtain the enzyme. Therefore, it is understood that the saccharifying enzyme community of the method may comprise an enzymatic activity such as "cellulase", which activity can be catalyzed by two or more enzymes.

Saccharifying enzymes include Spezyme® CP Cellulase (Genencor International, Rochester, NY) and Multifect® Xylanase (Genencor). It is commercially available. In addition, saccharifying enzymes can be biologically produced, including the use of recombinant microorganisms.

One of ordinary skill in the art will know how to determine the effective amount of enzyme for use in the community and to regulate the conditions for optimal enzyme activity. One of ordinary skill in the art will also know how to optimize the class of enzyme activity required in the community and obtain optimal saccharification of a given pretreated product under selected conditions.

Preferably, the saccharification reaction is carried out at or near the optimum temperature and pH of the saccharifying enzyme. The optimum temperature used for the saccharifying enzyme community in this method ranges from about 15 ° C to about 100 ° C. In another embodiment, the optimum temperature is in the range of about 20 ° C to about 80 ° C. The optimum pH value can be in the range of about 2 to about 11. In another embodiment, the optimum pH value used in the case of the saccharifying enzyme community in this method ranges from about 4 to about 10.

Saccharification may be carried out in a time of about several minutes to about 120 hours, and preferably about several minutes to about 48 hours. The time in the reaction will depend on the enzyme concentration and specific activity, as well as the substrate used and environmental conditions such as temperature and pH. One of ordinary skill in the art can easily determine the optimum temperature, pH and time conditions to be used in the case of a particular substrate and saccharifying enzyme community.

The saccharification may be carried out in batch or continuous manner. Further, saccharification may be carried out in one step or many steps. For example, the different enzymes required for saccharification may exhibit different optimum pH or temperature. After performing the primary treatment with an enzyme at a certain temperature and pH, a secondary or tertiary (or further) treatment may be performed with a different enzyme at a different temperature and / or pH. In addition, treatment with different enzymes in sequential steps involves hemicellulase that is stable and more active at the same pH and / or temperature, or at higher pH and temperature, followed by cellulase that is active at lower pH and temperature. Can be done at different pH and temperature, such as the use of.

The degree of solubilization of sugars from the saccharified biomass may be monitored by measuring the release of monosaccharides and oligosaccharides. Methods for measuring monosaccharides and oligosaccharides are well known in the art. For example, the concentration of reducing sugars may be measured using the 1,3-dinitrosalicylic (DNS) acid assay (Miller, GL, Anal. Chem. (1959), pp. 31: 426-428). .. Alternatively, sugars may be measured by HPLC using a suitable column, as described in the "General Methods" section herein.

Target chemicals may be produced by the appropriate microbial use of fermentable sugars released from the biomass. The concentration of fermentable sugar can be increased by concentrating the saccharified mixture, for example by evaporation, after saccharification but prior to fermentation. Optionally, the liquid in the saccharified product can be separated from the solid in a batch or continuous manner. In some cases, the entire liquid or saccharified product can be sterilized prior to fermentation. The pH depends on the microorganisms used during fermentation and the pH used during saccharification and can be adjusted to a pH suitable for fermentation. In addition, the saccharified mixture can be supplemented with additional nutrients needed for microbial growth. Supplements can include, for example, yeast extracts, certain amino acids, phosphates, nitrogen sources, salts, and trace elements. The components required for the production of a particular product produced by a particular biocatalyst, such as antibiotics, are also included, allowing the maintenance of the plasmid or cofactor required for the enzyme-catalyzed reaction. The inclusion of additional sugars can increase the total sugar concentration. The saccharified mixture is used as a component of the fermentation broth and can constitute, for example, about 100% to about 10% of the final medium.

Temperature and / or headspace gas can also be adjusted according to conditions useful to the fermenting microorganisms. Fermentation can be aerobic or anaerobic. Fermentation can follow saccharification or can occur at the same time as saccharification by co-saccharification and fermentation (SSF). SSF keeps the sugar levels brought about by saccharification low, which reduces the inhibition of possible saccharifying enzyme products, reduces the availability of sugars in contaminating microorganisms, and is a monosaccharide of pretreated biomass. Conversion to sugars and / or oligosaccharides may be improved.

Target chemicals that can be produced by fermentation include, for example, acids, alcohols, alkanes, alkenes, aromatic compounds, aldehydes, ketones, biopolymers, proteins, peptides, amino acids, vitamins, antibiotics, and pharmaceuticals. Alcohols include, but are not limited to, methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol, propanediol, butanediol, glycerol, erythritol, xylitol, and sorbitol. Acids include acetic acid, lactic acid, propionic acid, 3-hydroxypropionic acid, butyric acid, gluconic acid, itaconic acid, citric acid, succinic acid and levulinic acid. Amino acids include glutamic acid, aspartic acid, methionine, lysine, glycine, arginine, threonine, phenylalanine and tyrosine. Additional target chemicals include methane, ethylene, acetone and industrial enzymes.

Fermentation of sugars to target chemicals can be carried out in single or multi-step fermentations with one or more suitable biocatalysts. The biocatalyst can be a microorganism selected from bacteria, filamentous fungi and yeast. Biocatalysts can be wild or recombinant microorganisms, such as Escherichia, Zymomonas, Saccharomyces, Candida, Pichia, Streptomyces, Bacillus. Includes (Bacillus), Lactobacillus, and Clostridium. In another embodiment, the biocatalysts are recombinant Escherichia coli, Zymomonas mobilis, Bacillus stearothermophilus, Saccharomyces cerevisiae, Clostridia thermoselm ( Clostridia It can be selected from the group consisting of thermocellum), Thermoanaerobacterium saccharolyticum, and Pichia stipitis.

Numerous biocatalysts used in fermentation to produce target chemicals have been described, others can be found, produced by mutation, or designed by recombinant means. .. By using an arbitrary biocatalyst using fermentable sugar produced by this method, it is possible to produce a target chemical substance known to be produced by fermentation in this method.

Fermentation of carbohydrates to acetone, butanol and ethanol (ABE fermentation) by solventogenic Clostridia is well known (Jones and Woods (1986) Microbiol. Rev. 50: Pp. 484-524). A fermentation method using a mutant strain of Clostridium acetobutylicum to produce high levels of butanol, as well as acetone and ethanol, is described in US Pat. No. 5,192,673. Clostridium for producing high levels of butanol, as well as acetone and ethanol The use of mutants of beijerinckii) is described in US Pat. No. 6,358,717. Genetically modified strains of E. coli are also used as biocatalysts for ethanol production (Underwood et al. (2002) Appl.Environ.Microbiol.68: 6263-6272). .. A recombinant strain of Zymomonas mobilis that improves ethanol production is described in US Patent Application Publication No. 2003/0162271A1.

Lactic acid is a recombinant strain of E. coli (Zhou et al., (2003) Appl.Environ.Microbiol.69: 399-407), a natural strain of Bacillus (US patent application published). No. 2005/0250192), and produced in fermentation by Rhizopus oryzae (Tay and Yang (2002) Biotechnol. Bioeng. 80: 1-12). Recombinant strains of E. coli include 1,3 propanediol (US Pat. No. 6,013,494, US Pat. No. 6,514,733) and adipic acid (Niu et al., (2002) Biotechnol. It is used as a biocatalyst in fermentation to produce Prog. 18: 201-2-111). Acetic acid is a recombinant Clostridia (Cheryan et al., (1997) Adv. Appl. Microbiol. 43: 1-33) and a newly identified yeast strain (Freer, 2002). ) World It is made by fermentation using J. Microbiol. Biotechnol. 18: 271-275). For the production of succinic acid by recombinant E. coli (E. coli) and other bacteria in US Pat. No. 6,159,738, and for mutant E. coli (E. coli), Lin et al., (2005) Metab. Eng. 7: 116-127. Pyruvic acid is found in Torulopsis glabrata mutant yeast (Li et al., (2001) Appl. Microbiol.Technol. 55: 680-685) and mutant E. coli (Yokota). Et al. (1994) Biosci.Biotech.Biochem.58: 2164-2167). Recombinant strains of E. coli produce para-hydroxycinnamic acid (US Patent Application Publication No. 2003/0170834) and quinic acid (US Patent Application Publication No. 2006/0003429). It is used as a biocatalyst for the purpose.

Mutants of Propionibacterium acidipropionici have been used in fermentation to produce propionic acid (Suwannakham and Yang (2005) Biotechnol. Bioeng. .91: 325-337), butyric acid is made by Clostridium tyrobutyricum (Wu and Yang (2003) Biotechnol.Bioeng. 82: 93-102). Propionates and propanols are made by fermentation from threonine with Clostridium sp. Strain 17cr1 (Janssen, (2004) Arch. Microbiol. 182: 482-486). Aspergillus using yeast-like black yeast (Aureobasidium pullulans) Gluconic acid is produced by a mutant of niger (Singh et al. (2001) Indian J. Exp. Biol. 39: 1136-43) (Anantassiadis et al., (2005) ) Biotechnol. Bioeng. 91: 494-501). 5-keto-D-gluconic acid is made by a variant of Gluconobacter oxydans (Elfari et al., (2005) Appl Microbiol. Biotech. 66: 668-674), itaconic acid Acid is produced by a variant of Aspergillus terreus (Reddy and Singh (2002) Bioresour.Technol. 85: 69-71), and citric acid is Aspergillus. Produced by a niger mutant (Ikram-Ul-Haq et al. (2005) Bioresour.Technol. 96: 645-648), and xylitol is Candida guilliermondii. Produced by FTI 20037 (Mussatto and Roberto (2003) J. Appl. Microbiol. 95: 331-337). Biopolyesters containing 4-hydroxyvalerate also contain large amounts of 3-hydroxybutyric acid, 3-hydroxyvaleric acid, and are recombinants of Pseudomonas putida and Ralstonia eutropha. Produced by (Gorenflo et al., (2001) Biomacromolecules 2: 45-57). L-2,3-butanediol was produced by recombinant E. coli (Ui et al. (2004) Lett. Appl. Microbiol. 39: 533-537).

Fermentative amino acid production is carried out using auxotrophic strains and amino acid analogs-resistant strains of Corynebacterium, Brevibacterium and Serratia. For example, the production of histidine using a strain resistant to a histidine analog is described in Japanese Patent Application Publication No. 8596/81, and the production using a recombinant strain is described in European Patent No. 136359. .. The production of tryptophan using strains resistant to tryptophan analogs is described in Japanese Patent Application Publication No. 4505/72 and Japanese Patent Application Publication No. 1937/76. The production of isoleucine using strains resistant to isoleucine analogs is described in Japanese Patent Application Publication No. 38995/72, Japanese Patent Application Publication No. 6237/76, and Japanese Patent Application Publication No. 32070/79. Has been done. The production of phenylalanine using a strain that is resistant to phenylalanine analogs is described in Japanese Patent Application Publication No. 10035/81. A strain that requires phenylalanine for growth and is resistant to tyrosine (Agr. Chem. Soc. Japan) 50 (1) R79-R87 (1976)) or recombinant strains (European Patent No. 263515, EPO 332234) to produce tyrosine and tolerate L-arginine analogs Production of arginine using strains (Agr. Biol. Chem. (1972), pp. 36: 1675-1684, Japanese Patent Application Publication No. 37235/79 and Japanese Patent Application Publication No. 150381/82) is described. ing. Phenylalanine was also produced by fermentation within Eschericia coli strains ATCC 31882, 31883, and 31884. The production of glutamic acid in recombinant coryneform bacteria is described in US Pat. No. 6,962,805. The production of threonine by mutant strains of E. coli is described in Okamoto and Ikeda (2000) J. Biosci Bioeng. 89: 87-79. Methionine is Corynebacterium Produced by a mutant strain of lilium) (Kumar et al. (2005) Bioresour.Technol. 96: 287-294).

Useful peptides, enzymes, and other proteins are also made by biocatalysts (eg, US Pat. No. 6,861,237, US Pat. No. 6,777,207, US Pat. No. 6,228,630).

Pretreatment and saccharification of biomass to fermentable sugars and subsequent fermentation of saccharides to target chemicals are pretreated using Z. mobilis as a biocatalyst in the fermentation of saccharides to ethanol. Illustrated in Example 9 herein for the production of ethanol from fermented corn stalks. The method of the present invention may also be utilized in the production of 1,3-propanediol from biomass. Biomass undergoes pretreatment and saccharification according to the present invention, i.e. 1,3-propane by using E. coli after (or during) saccharification as described in Example 10 herein. Diol is produced.

Target chemicals produced by biocatalytic fermentation can be recovered using a variety of methods known in the art. The product can be separated from other fermented components by centrifugation, filtration, microfiltration, and nanofiltration. The product can be extracted by ion exchange, solvent extraction, or electrodialysis. The use of flocculants facilitates product separation. As a specific example, bioproduced 1-butanol can be isolated from the fermentation medium using methods known in the art for ABE fermentation (eg, Durre, Appl. Microbiol. Biotechnol. 49: See pages 639-648 (1998), Groot et al., Process.Biochem. 27: 61-75 (1992), and references in them). For example, the solid can be removed from the fermentation medium by centrifugation, filtration, decantation or the like. 1-Butanol can then be isolated from the fermentation medium using methods such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, or dialysis evaporation. Purification of 1,3 propanediol from the fermentation medium can be performed, for example, by subjecting the reaction mixture to extraction with an organic solvent, distillation, and column chromatography (US Pat. No. 5,356,812). A particularly good organic solvent for this method is cyclohexane (US Pat. No. 5,008,473). Amino acids can be recovered from the fermentation medium by methods such as ion exchange resin adsorption and / or crystallization. The present invention can be summarized as follows. 1.a) The process of contacting biomass with an aqueous solution containing ammonia to form a biomass-aqueous ammonia mixture to produce a pretreated biomass product, where ammonia is a biomass-aqueous ammonia mixture. Although present at least in a concentration sufficient to maintain the alkaline pH of the biomass, the ammonia is present in less than about 12% by weight of the dry weight of the biomass, and the dry weight of the biomass is the weight of the biomass-aqueous ammonia mixture. The process, which is at a high solid concentration of at least about 15% by weight. b) The step of contacting the product of step (a) with the saccharifying enzyme community under appropriate conditions to produce fermentable sugar, A method of treating biomass for the production of fermentable sugars, comprising: 2. The method according to 1 above, wherein the pH of the biomass-aqueous ammonia mixture is greater than 8. 3. The method according to 1 above, wherein a vacuum is applied to the biomass prior to contact between the biomass and the aqueous solution containing ammonia. 4. The method according to 1 above, wherein the dry weight of the biomass is at a high solid concentration of at least about 15% to about 80%. 5. The method according to 4 above, wherein the dry weight of the biomass is at a high solid concentration of at least about 15% to about 60%. 6. The method according to 1 above, wherein the ammonia is present in less than about 10 weight percent of the dry weight of the biomass. 7. The method according to 6 above, wherein the ammonia is present in a weight percent of about 6% or less relative to the dry weight of the biomass. 8. The method according to 1 above, wherein the biomass is selected from the group consisting of bioenergy crops, agricultural residues, urban solid waste, industrial solid waste, factory waste, wood and forestry waste. 9. Biomass is switch glass, waste paper, paper sludge, corn grain, corn cob, corn husk, corn stover, grass, wheat, wheat straw, magusa, barley, barley straw, rice straw, sugar cane The method according to 1. .. 10. Biomass is selected from the group consisting of corn heads, corn stovers, corn husks, sugar cane bagasse, shavings, switchgrass, wheat straw, magusa, barley straw, rice straw, and grass. The method described. 11. The method according to 10 above, wherein the biomass is selected from the group consisting of corn cobs, corn stovers, sawdust, and sugar cane bagasse. 12. The method according to 1 above, wherein ammonia is selected from the group consisting of ammonia gas, ammonium hydroxide, urea, and combinations thereof. 13. The method according to 1 above, wherein the aqueous solution containing ammonia further contains at least one additional base. 14. The method according to 13 above, wherein the at least one base is selected from the group consisting of sodium hydroxide, sodium carbonate, potassium hydroxide, potassium carbonate, calcium hydroxide and calcium carbonate. 15. The method according to 1 above, wherein (a) is carried out at a temperature of about 4 ° C to about 200 ° C. 16. The method according to 15 above, wherein (a) is carried out at a temperature of about 75 ° C to about 150 ° C. 17. The method according to 16 above, wherein (a) is carried out at a temperature above 90 ° C and below about 150 ° C. 18. The method according to 1 above, wherein (a) is carried out for a period of up to about 25 hours. 19. The method according to 18 above, wherein (a) is carried out for a period of up to about 8 hours. 20. The method according to 1 or 2 above, wherein at least a part of the ammonia in (a) is removed prior to (b). 21. The method according to 1 above, wherein the ammonia from (a) is regenerated. 22. The method according to 1 above, wherein the contacting step of (b) is carried out with a dry weight of at least about 15% biomass concentration. 23. The method according to 1 above, wherein (a), (b) or (a) and (b) are repeated at least once. 24. The method according to 1 above, further comprising the step of adding at least one plasticizer, fabric softener or combination thereof in (a). 25. The method of 24 above, wherein the at least one plasticizer, fabric softener or combination thereof is selected from the group consisting of polyols, polyol esters, glycol ethers, acetamides, ethanol, and ethanolamines. 26. The method according to 1 above, further comprising the step of applying energy before or between (a), before or between (b), or a combination thereof. 27. The method of 26 above, wherein the energy is selected from the group consisting of milling, crushing, grinding, crushing, chopping, discriminating, ultrasonic and microwave. 28. The method according to 1 above, wherein the pH of the mixture in the pretreatment is adjusted using carbon dioxide from fermentation prior to saccharification. 29. The method of 1 above, wherein the saccharifying enzyme community comprises at least one glycosidase. 30. At least one enzyme selected from the group consisting of glycosidases that hydrolyze cellulose, glycosidases that hydrolyze hemicellulose, glycosidases that hydrolyze starch, peptidases, lipases, ligninases, and ferroyl esterases. The method according to 1 above, which comprises. 31. The saccharifying enzyme community is cellulase, endoglucanase, exoglucanase, cellobiohydrolase, β-glucosidase, xylanase, endoxylanase, exoxylanase, β-xylosidase, arabinoxylanase, mannase, galactase, pectinase, glucuronidase, amylase, The method according to 1 above, which comprises at least one enzyme selected from the group consisting of α-amylase, β-amylase, glucoamylase, α-glucosidase, and isoamylase. 32. The method according to 1 above, wherein (b) is carried out at a temperature of about 15 ° C to about 100 ° C and a pH of about 2 to about 11.

General methods and materials The following abbreviations are used. "HPLC" is high-performance liquid chromatography, "C" is in degrees Celsius, "kPa" is kilopascal, "m" is meter, "mm" is millimeter, "kW" is kilowatt, "μm" is micrometer, "μL" Is microliter, "mL" is milliliter, "L" is liter, "min" is minute, "mM" is mmol, "cm" is centimeter, "g" is gram, "kg" is kilogram, "wt" Is the weight, "hr" is the time, "temp" or "T" is the temperature, "theoret" is the theory, "pretreat" is the pretreatment, and "DWB" is the dry weight of the biomass.

Sulfuric acid, ammonium hydroxide, acetic acid, acetamide, yeast extract, 2-morpholinoetan sulfonic acid (MES), potassium phosphate, glucose, xylose, trypton, sodium chloride and citric acid, Sigma-Aldrich Obtained from (St. Louis, Missouri).

Pretreatment reactor Zipperclave® Reactor The 4-liter Zipperclave® reactor (Autoclave Engineers, Erie, Pennsylvania) is a 2.5-liter Hastelloy® vessel for biomass loading. It is a batch type pressure vessel provided with a shape vessel and a stirrer for mixing biomass. The reaction vessel is surrounded by an electric heater controlled at the desired pretreatment temperature. Direct steam injection is also used to quickly induce the biomass to the maximum pretreatment temperature. The vapor pressure is regulated and controlled to maintain the desired pretreatment temperature. The vapor condensate heats the outside of the Zipperclave® reactor head plate, vessel and tubular vessel drain leading to the reservoir formed between the reactor tubular vessel and the inner wall. To prevent excessive dilution of the pretreated slurry.

Jaigo reactor The Jaygo reactor is a 130-liter (approximately 51 cm in diameter x 91 cm in length) horizontal paddle reactor made of Hastelloy® C-22 alloy (Jaygo Manufacturing, Inc.), Mahwah, NJ). The reactor is equipped with a steam jacket that can be heated to approximately 177 ° C (862 kPa). Direct steam injection is also used to quickly induce the biomass to the maximum pretreatment temperature. The vapor pressure is regulated and controlled to maintain the desired pretreatment temperature. A large number of ports allow injection of other solvents and hot liquids.

Batch digestion system for steam gun reactor The 4-liter steam gun reactor (Autoclave Engineers, Erie, Pennsylvania) is a 102 mm long planned 80 Hastelloy (registered trademark) closed by two ball valves. ) A reactor with a steam jacket consisting of pipes. An additional electric heater is provided on the entire exposed surface without the reactor jacket and is controlled to the pretreatment constant temperature. Direct steam injection is also used to rapidly guide the biomass to the highest pretreatment temperature. The vapor pressure is regulated and controlled to maintain the desired pretreatment temperature. Neck down the bottom of the reactor to 51 mm. 0.21 m of all pretreated material drained through a replaceable die at the bottom of the reactor and supported inside a thick jacketed cooling flush tank³Collected in a nylon (Hotfill®) bag.

Disc refiner The disc refiner is a 30.5 cm refiner (Andritz, Inc., Muncy, PA) of the Sprout Waldron model equipped with an 11 kW electric motor. The gap between the fixed plate and the rotating plate is variable. The speed of the feed auger is also variable from 0 to 88 rpm. Improvements to the refiner inlet with six injection ports allow the introduction of steam, hot water, or other sweep gas and liquid just in front of the rotating refiner plate. The refiner was equipped with plates (Durametal, Corp., Tualatin, Oregon) with Ni-Hard pattern D2A506 or Ni-Hard pattern 18034-A.

Pretreatment and Enzyme Hydrolase Reactor (PEHR) 9 L PEH Reactor (NREL, Golden, Colorado; co-pending US Patent Application CL3447) is approximately 15 cm x 51 cm with an infusion lance for the introduction of the treatment reactant. Has a stainless steel reaction vessel. The infusion lance is connected to a port in the cover on one end of the container using a rotary joint, which has an additional port as a means of access to the container. Four baffles are mounted perpendicular to the wall over the entire length of the vessel wall. The baffle floating in the vessel and 22 3.2 cm x 3.2 cm ceramic friction medium cylinders (ER Advanced Ceramics, East Palestine, Ohio) are biomass as the vessel rotates. A mechanical mixture of and the reactants is applied, thereby facilitating the absorption of the reactants into the biomass. Bellco Cell-Production, which provides a mechanism for rotation of PEHReactor. The reactor, which will be installed on the Roller MFP (Bellco Technology, Vineland, NJ) and equipped with a roller device, will be housed in a temperature control chamber that supplies heat. Vacuum and pressure can be applied to the reaction vessel by attaching an external source to the port connected to the lance in the cover.

Analytical method Quantification of cellulose The amount of cellulose in each biomass starting sample is measured using methods well known in the art such as ASTM E1758-01 "Standard method for the determination of carbohydrates by HPLC". Was measured.

Measurement of sugar, acetamide, lactic acid and acetic acid contents Soluble sugars (glucose, cellobiose, xylose, galactose, arabinose and mannose), acetamides, lactic acid and acetic acid in the saccharified solution, Bio-Rad HPX-87P and Bio-Rad with appropriate guard columns. -Rad Using HPX-87H columns (Bio-Rad Laboratories, Hercules, California), HPLC (Agilent Model 1100, Agilent Technologies, Palo Alto) Measured by Alto), California). The pH of the sample was measured and adjusted to 5-6 with sulfuric acid as needed. The sample was then passed directly through a 0.2 μm syringe filter into the HPLC vial. The operating conditions of HPLC were as follows. Biorad Aminex HPX-87P (for carbohydrates): Injection volume: 10-50 μL, depending on concentration and detector limits Mobile phase: HPLC grade water, filtered and degassed to 0.2 μm Flow velocity: 0.6 mL / min Column temperature: 80 ~ 85 ° C, guard column temperature &lt;60 ° C Detector temperature: as close to the main column temperature as possible Detector: Refractometer Run time: 35 minutes of data collection plus 15 minutes after run (make possible adjustments for later eluted compounds)

Biorad Aminex HPX-87H (for carbohydrates, acetamides, lactic acid, acetic acid, and ethanol) Injection volume: 5-10 μL, depending on concentration and detector limits Mobile phase: 0.01N sulfuric acid, filtered to 0.2 μm and degassed Flow velocity: 0.6 mL / min Column temperature: 55 ° C Detector temperature: as close to the column temperature as possible Detector: Refractometer Execution time: 25-75 minutes of data collection

After execution, the concentration in the sample was determined from the standard curve for each compound.

Example 1 Comparison of high biomass concentration, high temperature stober pretreatment and ammonia concentration Prior to the introduction of the biomass load, the Zipperclave® reaction vessel and headplate were preheated to the desired pretreatment temperature by circulating steam through the reactor and ventilating several times. Prior to pretreatment, the condensate formed during preheating was removed by vacuum suction. A Hastelloy® tubular vessel was loaded with a 0.635 cm (1/4 inch) crushed stover (100 g, based on dry weight) and placed in a preheated reactor. A vacuum (about 85 kPa) was applied inside the vessel and to the loaded biomass, while the reactor stirrer was set to 20 rpm. Biomass-Ammonia hydroxide of the strength required to provide the dry weight of biomass at a concentration of 30% by weight relative to the weight of the aqueous ammonia mixture as well as the desired ammonia concentration listed in Table 1 in a container with a spray nozzle. Injected near the bottom. A sample with a final ammonia concentration of 12% relative to the dry weight of the biomass while the test sample had a final ammonia concentration of 35% relative to the dry weight of the biomass was used for comparison. When the temperature of the biomass load reached 50 ° C, steam was introduced near the bottom of the reactor to fluidize and raise the temperature of the biomass load to 140 ° C or 170 ° C. At the end of the pretreatment, prior to opening the reactor and recovering the pretreated biomass, the reactor is depressurized through a vent capacitor and a vacuum (approximately 85 kPa) is applied for 3 minutes to reduce the temperature and add additional ammonia. It was removed from the pretreated slurry.

The entire unwashed pretreated slurry containing 0.5 g of cellulose (based on the initial feed raw material composition) was added to a 125 mL shaking flask in a final volume of 50 mL. Since the enzyme is sensitive to high pH environments, it was titrated with the addition of acetic acid (10-100 μL) prior to the addition of the enzyme to bring the pH of the biomass pretreated with ammonia to 5.0. During saccharification, the pH was adjusted to 5.0 by the addition of 50 mM citrate buffer and the temperature was maintained at 50 ° C. Spezyme® CP Cellulase (Genencor) International), Rochester, NY) were added to the concentrations listed in each sample in Table 1. After 96 hours of saccharification, the sugar content of the produced saccharified solution was measured according to the sugar measurement protocol described in "General Methods". Table 2 shows the release of sugar after 96 hours. The controls in this experiment were 1) an untreated corn stover with a theoretical yield of 23% glucose (using 56 mg cellulase / g cellulose), and 2) a theoretical yield of 40% glucose (56 mg cellulase). The corn stover was pretreated with steam (140 ° C) to give (using / g cellulose) and xylose was not measured in the control.

<tables num="1"><img file="JP5118626B2_D0001.tif" /></tables>

These results show that the pretreatment with 12% ammonia at 140 ° C for 15 minutes, followed by saccharification, results in more glucose and xylose than when 35% ammonia was used at 140 ° C for 5 minutes in the pretreatment. Indicates that is released. Therefore, by slightly increasing the pretreatment time, the advantage of using less ammonia can be enjoyed.

Example 2 Stover pretreatment with high biomass concentration, low temperature, and very small amounts of ammonia The Jaigo reactor was loaded with a 0.635 cm crushed stover (13 kg, based on dry weight). By applying a vacuum (67.7 kPa) to the vessel and injecting a diluted ammonium hydroxide solution, the ammonia concentration of 6.2 g of ammonia / 100 g of dry weight of ammonia and 30 weight percent of the total weight of the biomass-aqueous ammonia mixture. The dry weight of the ammonia at the concentration was obtained. The vacuum was released and steam was applied to the jacket to heat the stober to 100 ° C. The temperature of the soaked stover was maintained at 32 rpm for 8 hours with constant mixing, and then the produced slurry was cooled overnight with continuous mixing.

The entire unwashed pretreated slurry containing 0.5 g of cellulose (based on the initial feed raw material composition) was added to a 125 mL shaking flask in a final volume of 50 mL. Since the enzyme is sensitive to high pH environments, the pH of the biomass pretreated with ammonia was adjusted to 5.0 by titrating with the addition of acetic acid (10-100 μL), if necessary, before adding the enzyme. During saccharification, the pH was adjusted to 5.0 by the addition of 50 mM citrate buffer and the temperature was maintained at 50 ° C. Spezyme® CP cellulase (Genencor International, Rochester, NY) was added to 56 mg / g cellulose. Table 2 shows the sugar content of the saccharified solution produced after 96 hours of saccharification measured according to the sugar measurement protocol described in "General Method".

<tables num="2"><img file="JP5118626B2_D0002.tif" /></tables>

The results show that these extremely low ammonia concentrations and low temperature pretreatment conditions (8 hour period) are as effective as using 12% ammonia at 140 ° C for 15 minutes.

Example 3 Pretreatment of maize cobs at high biomass concentrations, low temperatures, and extremely low ammonia concentrations followed by saccharification at high biomass concentrations Whole-grain or ground corn cobs (approximately 13 kg, based on dry weight) were loaded onto the Jaigo reactor. The corn cob was ground by passing it through a disc refiner equipped with plate C-2975 (general method). The produced ground corn cobs were passed through a 1.27 cm screen. Any retained section was again passed through the disc refiner with a smaller gap of 0.5 cm. By applying a vacuum to the reactor and injecting a diluted ammonium hydroxide solution, the desired final ammonia concentration (2% or 6%) and dry biomass concentration (30% or 40%) can be obtained as shown in Table 3. Obtained. Steam was applied to the jacket to heat the maize cob while releasing the vacuum and immersing the whole maize cob sample at 93 ° C and the ground maize cob sample at 85 ° C. The stirrer speed was increased for a short time (up to 96 rpm) for the purpose of increasing the heating speed. The temperature of the soaked corn cob was maintained at 32 rpm for 4 or 8 hours with constant mixing, followed by continuous mixing and cooling overnight.

Prior to removing the pretreated biomass from the reactor, the reactor was placed under vacuum at 90 ° C to evaporate the ammonia from the pretreated biomass to the outside. Prior to saccharification, the pH of the pretreated maize cob biomass was adjusted to 5.5 with solid citric acid. Approximately 10 kg of pretreated whole grain cobs were saccharified in a 50 ° C Jaigo reactor. For saccharification, approximately 1400 g of pre-treated ground corn stalks are sewn into 22 ceramic friction cylinders (3.2 cm in diameter x 3.2 cm in length; ER Advanced Ceramics, East Palestine, Added to PEH Reactor with (Ohio). 28 mg / g of cellulose, Multifect An enzyme mixture of 28 mg Spezyme CP® / g cellulose in an untreated stover supplemented with Xylanase® was used in each saccharification reaction. The final concentration of dry weight of biomass at the start of each saccharification was 30% of the total weight of the pretreated biomass-saccharifying enzyme community mixture. The PEH Reactor was axially rotated at 19 rpm while maintaining a temperature of 50 ° C. The sugar content of the produced saccharified solution was measured according to the sugar measurement protocol in the "general method". Table 3 shows the release of sugar after 96 hours.

<tables num="3"><img file="JP5118626B2_D0003.tif" /></tables>

Example 4 Pretreatment of maize cobs at high biomass concentrations, high temperatures, and extremely low ammonia concentrations followed by saccharification at high biomass concentrations Crushed corn cobs (13 kg, dry) were loaded into the Jaigo reactor. After evacuating the reactor, a solution of ammonium hydroxide of suitable strength to give a concentration of 2% ammonia and 30% dry weight% of biomass was pumped into the reactor with mixing at room temperature, 32 rpm. The contents of the reactor were then heated to 95 ° C. using low pressure jacket steam. Once the reactor reached 95 ° C, the contents of the reactor were heated to 145 ° C using direct steam injection. When the reactor reached 145 ° C, the contents of the reactor were held at that temperature for 20 minutes using jacket steam and partial direct steam injection. Twenty minutes later, a vacuum was evacuated over the vent leading to the reactor and the shredder motor was run for 5 minutes. After 1 hour, cooling water was applied to the jacket. Cool the contents of the Jaigo reactor to 33 ° C-37 ° C, then CO₂Was pressurized to 138 kPa. Pressurized CO₂The atmosphere was maintained for 30 minutes. The final temperature of the reactor contents was 27 ° C to 31 ° C. The pH of the soaked / pretreated biomass was about 7.5.

Pretreated biomass is removed from the Jaygo reactor and saccharified at a final concentration of 30% dry weight of biomass relative to the total weight of the pretreated biomass-saccharifying enzyme community mixture at the start of saccharification. Moved to PEH Reactor for. The pH was then adjusted to 5.5 with solid citric acid and the material was adjusted to 28 mg Spezyme CP® / g cellulose and 28 mg Spezyme CP® / g cellulose in untreated corn cobs as described in Example 3. Digested with 28 mg of Multifect Xylanase® / g cellulose. The sugar content of the produced saccharified solution was measured according to the sugar measurement protocol in the "general method". Table 4 shows the release of sugar 96 hours after digestion.

<tables num="4"><img file="JP5118626B2_D0004.tif" /></tables>

Example 5 Pretreatment with the addition of plasticizer Whole corn cobs are placed in a Jaygo reactor with 3% by weight glycerol to the dry weight of biomass added with the intention of acting as a plasticizer, as described in Example 3. Pretreated at 100 ° C. for 8 hours with a concentration of about 30% dry weight of biomass relative to the total weight of the biomass-aqueous ammonia mixture and 2% by weight of ammonia relative to dry weight of biomass. After pretreatment, the pH of the produced material was adjusted to 5 with solid citric acid. The pretreated corn cobs were then digested as described in Example 3. Enzyme with 28 mg Spezyme CP® / g cellulose in untreated corn and 28 mg / g cellulose in untreated corn cob, Multifect Xylanase® The mixture was used. 96 hours after digestion, the glucose concentration was 92.3 g / L and the xylose concentration was 54.4 g / L.

Example 6 Discrimination of pretreated biomass As described in Example 1, the stover was previously subjected to different samples, temperatures, times, and enzymatic conditions with low ammonia concentration (12%) or comparative ammonia concentration (35%), as listed in Table 5. Processed. Whole-grain maize cobs were pretreated under different samples with very small amounts of ammonia (3% or 6%) and other conditions listed in Table 5, as described in Example 3. After pretreatment, the sample was passed through a Sprout Waldron disc refiner. The gap between the stationary plate and the rotating plate was set to 0.254 mm (0.010 inch) and the feed auger speed was set to 7 rpm. The purified material was saccharified as described in Example 2, and the sugar content of the produced saccharified solution was measured according to the sugar measurement protocol in the "general method". Table 5 shows the results of saccharification after 96 hours. The results showed that better digestibility or the use of lower enzyme concentrations was effective for discriminating prior to saccharification.

<tables num="5"><img file="JP5118626B2_D0005.tif" /></tables>

Example 7 Steam gun treatment of pretreated biomass As described in Example 1, in a Jaigo reactor, 30 dry weight percent biomass relative to the total weight of the biomass-aqueous ammonia mixture, 6 weight percent ammonia relative to DWB, 100. Pretreatment was performed using the condition of 8 hours at ° C. Corn stalks, as described in Example 3, in a Jaygo reactor, 40 dry weight% biomass relative to the total weight of the biomass-aqueous ammonia mixture, 6 weight% ammonia relative to DWB. , Pretreated at 93 ° C for 8 hours. Each pretreated biomass sample was loaded separately into a 4 liter steam gun reactor. The pretreated material was subjected to 170 ° C for 5 minutes or 140 ° C for 20 minutes before being released through the die. The produced material was saccharified as described in Example 2. The results are shown in Table 6 below. The results showed that steam cancer treatment prior to saccharification improved glucose release.

<tables num="6"><img file="JP5118626B2_D0006.tif" /></tables>

Example 8 Modeling of pretreatment with ammonia regeneration Aspen model (Aspen) on the benefits of ammonia regeneration in two pretreatment schemes: low temperature (85 ° C), long residence time (4 hours) and high temperature (130 ° C), short residence time (20 minutes) Technologies (Aspen Technologies), Cambridge, Massachusetts, version 12.1). Each model provided a means for ammonia regeneration by providing a series of three flush tanks operating at low pressure sequentially after the pretreatment reactor. As the feed stream entered each tank, it separated into gas and liquid fractions due to the drop in pressure. The gas fraction was regenerated to pretreatment, while the liquid fraction proceeded to the next step in the method. Assuming 2 weight percent ammonia to DWB and about 27 dry weight% biomass to the total weight of the biomass-aqueous ammonia mixture in the pretreatment, from the newly supplied ammonia and regeneration stream in each method Table 7 shows the ammonia. The flash tanks in both models were operated similarly so that ammonia regeneration was similar. In both scenarios, supplying more than half of the required ammonia through regeneration reduced the need and cost for new ammonia.

<tables num="7"><img file="JP5118626B2_D0007.tif" /></tables>

Example 9 Production of ethanol from biomass in corn cobs pretreated and saccharified with a small amount of ammonia, and comparison with a stover pretreated and saccharified with a large amount of ammonia. Whole grain corn cobs were added in a Jaygo reactor, 6% by weight to dry weight of biomass, 40 to the total weight of the biomass-aqueous ammonia mixture, as described in Example 3. A hydrolyzate of corn stalks was produced by pretreatment at 93 ° C. for 8 hours under the dry weight of biomass at a concentration of weight percent. After pretreatment, ammonia was removed by heating the reactor to 90 ° C. under vacuum. The pH of the pretreated biomass was then adjusted to 5 with sulfur acid. Pretreated biomass in a Jaygo reactor using 28 mg / g cellulose, Spezyme® cellulase and 28 mg / g cellulose, Multifect® xylanase. The dry weight of the biomass, which is 30% of the total weight of the pretreated biomass-saccharifying enzyme community mixture, was saccharified at 50 ° C. and pH 5 for 168 hours. The hydrolyzate produced is put into a Sixfors fermenter (INFORS). Used in the fermentation of Zymomonas mobilis 8b in AG), Switzerland). Zymomonas mobilis 8b produces a product of ethanol that has been genetically engineered to no longer be wild and is described in US Patent Application Publication No. 2003/0162271A1. mobilis) strain (Examples IV, VI and XII). The corn cob hydrolyzate consisted of 78 g / L glucose, 51 g / L xylose, 6 g / L acetamide, and 7 g / L acetic acid. Hydrolyzate of corn cob, yeast extract and KH in final slurry₂PO₄Was used at 40% and 80% intensities in equilibrium with concentrated aqueous media consisting of them in amounts such that the concentrations of were about 5 g / L and 2 g / L, respectively. In addition, glucose and xylose were added into the 40% hydrolyzate slurry in sufficient amounts to bring their concentrations to the same concentration as in the 80% hydrolyzate slurry. Fermentation was carried out at 37 ° C. The agitation in the fermenter was 100 rpm and the pH was maintained at 5.5 by the addition of 2N KOH. The results are shown in Table 8. Sugars and ethanol were analyzed as described in "General Methods".

For comparison, as described in Example 1, in a Zipperclave® reactor, the stover is placed at a concentration of approximately 30% by weight based on the total weight of the biomass-aqueous ammonia mixture. The Stover hydrolyzate was produced by pretreatment at 170 ° C. for 5 minutes under 35 weight percent ammonia to dry weight of the biomass. In the fermentation test, the pretreated biomass was subjected to 224 mg / g of cellulose at 50 ° C and pH 5, using Spezyme CP® cellulase, and the total weight of the pretreated biomass-saccharifying enzyme community mixture. It was enzymatically digested with 30% dry weight of biomass to produce a hydrolyzate with a high sugar concentration. The hydrolyzate produced contained 88 g / L glucose, 52 g / L xylose, 9 g / L acetic acid and 15 g / L lactic acid. In the production of ethanol, Zymomonas mobilis 8b was fermented on a 40% or 80% (v / v) hydrolyzate slurry. Residual volume is yeast extract, KH₂PO₄It consisted of a concentrated aqueous medium consisting of and MES buffer, and quantitatively, their concentrations in the final slurry were about 10 g / L, 2 g / L and 0.1 M, respectively. In addition, glucose and xylose were added to the 40% hydrolyzate slurry in sufficient amounts to bring them to the same concentration as in the 80% hydrolyzate slurry. Fermentation was carried out at 30 ° C. and pH 6 in a 25 ml shaking flask with an effective volume of 20 ml. Stirring was maintained at 150 rpm. Table 8 shows the results of analysis of the fermented sample of the hydrolyzate of maize cob.

<tables num="8"><img file="JP5118626B2_D0008.tif" /></tables>

These results indicate that fermentation to produce ethanol from corn cob hydrolyzate pretreated with a small amount of ammonia is more efficient than from a stover pretreated with a large amount of ammonia. It was.

Example 10 1 from corn cob biomass pretreated and saccharified with very small amounts of ammonia , 3-Propanediol production The hydrolyzate produced from the pretreatment and saccharification of maize cobs was fermented to produce 1,3-propanediol. The hydrolyzate was produced by pretreating corn cob pieces in a steam gun reactor. Ammonia concentration of 4 g of ammonia / 100 g of dry weight of biomass by loading the first corn cob biomass into the PEH Reactor (described in "General Methods"), applying a vacuum and injecting a diluted ammonium hydroxide solution. And a dry weight of biomass 30 g / dry weight of biomass at a concentration of 100 g overall of the biomass-aqueous ammonia mixture was obtained. The reaction vessel loaded with ammonia and corn cob was rotated at 4 ° C for 30 minutes. The contents were transferred to a steam gun reactor (described in "General Methods"), the temperature was raised to 145 ° C and the temperature of the mixture was maintained for 20 minutes. The material obtained from the steam gun was discharged into the flash tank and the vacuum was maintained on the flash tank to promote the removal of ammonia. After pH adjustment, pretreated biomass was subjected to 28.4 mg / g of cellulose, Spezyme CP® cellulase and 10.1 mg of active protein / g of β-glucosidase, xylanase, β-xylosidase and arabinofurano. Using a cellulosic enzyme community consisting of cedase, the dry weight of biomass was 30 g / 100 g of the pretreated biomass-saccharifying enzyme community mixture was saccharified at 50 ° C and pH 5.5 for 72 hours. Fermentation to convert the produced hydrolyzate to 1,3-propanediol by recombinant E. coli strain RJ8n pBE93-k1<u style="single">sex</u>Used as a sugar source. The preparation of the strain RJ8n pBE93-k1 is described in detail in WO 2004/018645 of the PCT application (Example 7), which is a derivative of the RJ8n strain described in US Pat. No. 6,358,716. It is a stock. Hydrolyzate with final pH adjusted to 6.8 or 7.5 g / L KH₂PO₄, 2.0g / L citric acid^*H₂O, 4.0 ml / L 28% NH₄OH, 3.0g / L (NH₄)₂SO₄, 2.0g / L DDL₄^*7H₂O, 0.2g / L CaCl₂^*2H₂O, 0.33 g / L ammonium ferric citrate, 0.5 g / L yeast extract, 0.1 mg / L vitamin B12, 1.0 mg / L FeSO₄^*7H₂O, 1 mg / L ZnSO₄^*7H₂O, 0.1g / L CuSO₄^*5H₂O, 1 mg / L CoCl₂^*6H₂O, 0.3 mg / L MnSO₄^*7H₂O, 0.1g / L H₃BO₄, 0.10g / L NaMoO₄^*2H₂It was used at 10% in equilibrium with an aqueous medium consisting of O and 10 mg / L NaCl. Cultures were initiated from frozen stock (15% glycerol as antifreeze) in 50 mL medium in a 250 mL baffled flask. Cultures were incubated for 24 hours under shaking at 34 ° C and 300 rpm. The amount of 1,3-propanediol produced was measured by HPLC under the following conditions. Column: Showdex SH1011 Sample volume: 20 μL Mobile phase: 0.01NH₂SO₄ Flow velocity: 0.5 ml / min Column temperature: 50 ° C Detector: Waters 996 photodiode array Detector temperature: 40 ° C Execution time: 40 minutes

The results are shown in Table 9 below. The product obtained from glucose fermentation with the RJ8n pBE93-k1 strain of E. coli contains glycerol (intermediate metabolite) and 1,3-propanediol. Experiments were performed in two flasks and assayed for 24 hours. In this system, glucose in the hydrolyzate was converted to both glycerol and 1,3-propanediol.

<tables num="9"><img file="JP5118626B2_D0009.tif" /></tables>

Example 11 Formation of acetamide during pretreatment Samples derived from pretreated maize cobs were analyzed according to the methods described in Example 3 and Example 4 to determine the whereabouts of acetyl groups in the biomass. The content of acetic acid and acetamide in the pretreatment solution (the pretreatment mixture from which the insoluble solid was removed) was assayed as follows. Set the pH of each sample to H₂SO₄Adjusted to about 3 using (72%). In the measurement of acetamide, the sample was passed through a 0.2 μm filter and analyzed by HPLC according to the conditions listed below. In the measurement of whole acetate (including acetic acid and acetate present as acetic acid), the acidified sample was autoclaved at 121 ° C for 1 hour and acetamide was quantitatively converted to acetic acid during this step. After autoclaving, the sample was cooled. The sample was then passed through a 0.2 μm filter into a sample vial for analysis according to the conditions listed below. The concentrations of acetic acid and acetamide were determined from the standard curves produced in each.

Mobile phase: 0.01NH₂SO₄, 0.2 μm filtration and degassing Flow velocity: 0.6 mL / min Column temperature: 55 ~ 65 ° C Detector temperature: as close to the column temperature as possible Detector: Refractometer Execution time: 60 minutes Columns: Biorad HPX-87H columns and corresponding guard columns

The results under the three different pretreatment conditions assayed are shown in Table 10. In each case, all of the acetyl groups were solubilized in acetic acid or acetamide.

<tables num="10"><img file="JP5118626B2_D0010.tif" /></tables>

As shown in Example 12, using a 6% ammonia concentration, nearly half of the acetyl groups were converted to acetamide, which is non-inhibitory to biocatalytic growth.

Example 12 Effects of acetamide and acetic acid on the growth of Zymomonas To test the toxicity of acetamide and acetic acid, Z. mobilis strain 8b (described in Example 9) with and without acetamide or acetic acid was grown in a fermentation medium at pH 6.0. It was. Fermentation medium, 10 g / L yeast extract, 2 g / L KH₂PO₄, 70 g / L glucose, 40 g / L xylose and 0.1 M MES buffer. Z.mobilis 8b in unsupplemented medium (control), 6 g / L acetamide-supplemented medium, or 7.2 g / L acetic acid-supplemented medium at 30 ° C., 150 rpm. It was grown in a 25 mL Erlenmeyer shaking flask with a rotating baffle. As shown in FIG. 1, the presence of acetamide had no effect on the growth rate or final density of Z. mobilis, whereas the presence of acetic acid reduced the growth rate and the cell yield. It led to a decrease (measured by dry cell mass).

Example 13 Pretreatment of bagasse with high biomass concentration, high temperature, and very small amounts of ammonia, and saccharification at low and high concentrations A PEH Reactor (described in the "General Method"), which had no friction medium, was loaded with bagasse (370 g, based on dry weight) crushed to 1.27 cm. This sugar cane bagasse was originally from the Hawaii Sugar Planters Association. It was NIST reference material RM8491 from sugarcane clone H65-7052 obtained from Association, Kunia bureau, Oahu, Hawaii. It was ground in a Wiley mill and passed through a 2 mm screen to remove fine powder (+74 mesh). The PEHReactor vessel was cooled to 4 ° C by rotating on the outer surface in contact with ice. By applying vacuum to the reaction vessel, pre-cooling at 4 ° C in a cold chamber and injecting a diluted ammonium hydroxide solution that has passed through a tubing immersed in an ice water tank, 4 g / biomass dry weight 100 g of ammonia A dry weight of biomass was obtained at a concentration of 45 g / biomass-a total concentration of 100 g of aqueous ammonia mixture. The reaction vessel loaded with ammonia and bagasse was applied to the surface of the rotating reaction vessel with ice, cooled to 4 ° C. and rotated at 4 ° C. for 30 minutes. At this point, the contents were transferred to the steam gun reactor described in "General Methods". Once the steam gun reactor was loaded with the ammonia-bagasse mixture, the temperature was raised to 145 ° C and the temperature of the mixture was maintained for 20 minutes. At the end of the pretreatment time, bagasse was discharged from the steam gun reactor through a 1-inch cyclic die into the flush tank. The pretreated bagasse sample was then saccharified in a shaking flask and another sample (dry weight of about 163 g) was saccharified in PEH Reactor. Saccharification in a shaking flask is performed with 5 dry weight% of biomass relative to the total weight of the pretreated biomass-saccharifying enzyme community mixture, while saccharification with PEH Reactor is performed with the pretreated biomass-saccharifying enzyme community mixture. It was carried out with 30 dry weight% of biomass based on the total weight of. The temperature was maintained at 50 ° C.

For saccharification with PEH Reactor, about 476 g (about 163 g dry weight) of pretreated biomass and 22 ceramic friction cylinders were added to the reaction vessel. The pH was adjusted to 5.0-5.5 with solid citric acid. The reaction vessel was held in an incubator chamber controlled at 50 ° C., and the shaft was rotated at 19 rpm. The untreated bagasse was also saccharified in a shaking flask with 5 dry weight% of biomass relative to the total weight of the pretreated biomass-saccharifying enzyme community mixture. All saccharifications at 50 ° C and pH 5.5 with 28.4 mg / g of cellulose, Spezyme CP® cellulase and 28.4 mg / g of cellulose, Multifect® xylanase. It took 96 hours. The yields shown in Table 11 below are published as percentages of theoretical yields.

<tables num="11"><img file="JP5118626B2_D0011.tif" /></tables>

The results show that bagasse pretreatment with a very small amount of ammonia allows for the release of large amounts of sugar when compared to untreated controls and saccharification at high dry biomass concentrations within the PEH Reactor. It is shown to be extremely effective when releasing saccharides.

Example 14 Pretreatment of yellow-poplar sawdust with high biomass concentration, high temperature, and very small amounts of ammonia, and saccharification at low and high concentrations Yello-Poplar Sawdust (596g, based on dry weight; Sawmiller) on PEH Reactor without friction medium Inc.), Heydenville, purchased from Ohio). A vacuum was applied to the reaction vessel and a diluted ammonium hydroxide solution was injected to obtain a dry weight of biomass at a concentration of 100 g dry weight of 6 g / biomass and 100 g of 44 g / total biomass-aqueous ammonia mixture. The reaction vessel loaded with ammonia and yellow-poplar sawdust as described in Example 13 was set to 4 ° C and rotated at 4 ° C for 30 minutes. At this point, the contents were transferred to the steam gun reactor. Once the steam gun reactor was loaded with the ammonia-poplar mixture, the temperature was raised to 145 ° C and the temperature of the mixture was maintained for 20 minutes. At the end of the pretreatment time, yellow-poplar sawdust was discharged from the steam gun reactor through a 1-inch cyclic die into the flash tank. Then, as described in Example 13, a sample of yellow-poplar sawdust pretreated in a shaking flask was saccharified, and another sample was saccharified in PEH Reactor. Saccharification in a shaking flask is performed with 5 dry weight% of biomass relative to the total weight of the pretreated biomass-saccharifying enzyme community mixture (using pretreated waste with a dry weight of approximately 279 g). Saccharification with PEH Reactor was performed at 30% dry weight of biomass relative to the total weight of the pretreated biomass-saccharifying enzyme community mixture. The untreated yellow-poplar sawdust was also saccharified with 5 dry weight% of biomass based on the total weight of the pretreated biomass-saccharifying enzyme community mixture in the shaking flask. All saccharifications at 50 ° C and pH 5.5 with 28.4 mg / g of cellulose, Spezyme CP® cellulase and 28.4 mg / g of cellulose, Multifect® xylanase. It took 96 hours. The yields shown in Table 12 below are published for each sugar as a percentage of the theoretical yield.

<tables num="12"><img file="JP5118626B2_D0012.tif" /></tables>

The results show that pretreatment of yellow-poplar sawdust with very small amounts of ammonia allows for the release of large amounts of sugar when compared to untreated controls, and the dry weight of biomass in the PEH Reactor is high. It is shown that saccharification of time is more effective in releasing saccharides than in the case of shaking flasks.

Example 15 Production of ethanol by yeast fermentation on the hydrolyzate obtained from saccharified corn cob biomass pretreated with a very small amount of ammonia The same hydrolyzate used to produce 1,3-propanediol in Example 10 was further used to produce ethanol by yeast fermentation. Fermentation to convert this hydrolyzate to ethanol in a shaking flask by wild-type Saccharomyces cerevisiae<u style="single">sex</u>Used as a sugar source. The hydrolyzate was used at an intensity of 10% (v / v) in equilibrium with an aqueous medium consisting of 10 g / L yeast extract and 20 g / L peptone. Yeast was cultured in 50 mL of medium in a 250 mL baffled flask. Cultures were incubated at 30 ° C. for 24 hours under shaking at 250 rpm. The amount of ethanol produced is HP as described in Example 9. The results from the two flasks measured by LC are listed in Table 13 below.

<tables num="13"><img file="JP5118626B2_D0013.tif" /></tables>

Example 16 Production of lactic acid by Lactobacillus fermentation on hydrolyzate obtained from saccharified corn panicle biomass pretreated with a very small amount of ammonia Lactic acid is produced in a shaking flask by fermentation with Lactobacillus brevis, further using the same hydrolyzate used to produce 1,3-propanediol in Example 10. did. Hydrolyzate, 5 g / L yeast extract, 10 g / L peptone, 2 g / L ammonium citrate, 5 g / L sodium acetate, 0.1 g / L DDL₄, 0.05g / L MnSO₄And 2g / L K₂HPO₄It was used at 10% (v / v) in equilibrium with an aqueous medium consisting of 1 g / L Tween. Lactobacillus was cultured in 50 mL culture medium in a 250 mL baffled flask. The two cultures were incubated at 34 ° C for 24 hours under shaking at 150 rpm. Table 14 shows the amount of lactic acid produced measured by HPLC as described in Example 10. Two samples in two flasks are two assays in the same culture.

<tables num="14"><img file="JP5118626B2_D0014.tif" /></tables>

Example 17 Pretreatment of corn cobs with very small amounts of ammonia at higher dry biomass concentrations Whole corn cobs, jaw crusher (2.2kW motor) with a jaw spacing of about 0.95cm, then delumper (1.5kW motor, Franklin Miller) Inc.), Livingston, NJ) and then screened on a Sweco screen with a 1.9 cm US standard screen. Approximately 805 g of ground corn cob was loaded onto the PEH Reactor. The water content in the corn cob was about 7%. Prior to loading, the atmosphere in the reaction vessel was flushed with nitrogen 5 times. Prior to the start of the experiment, the reactor, which had no friction medium, was preheated to 75 ° C without rotation. When the temperature in the reaction vessel stabilized at 75 ° C, the rotation mechanism in the incubator was activated to adjust the rotation to 19 rpm. An appropriate amount of diluted ammonium hydroxide solution was then pumped into the reactor to obtain an ammonia concentration of 6 g of ammonia / 100 g of dry weight of biomass and a total dry weight of 50 g of biomass / 100 g of biomass-ammonia mixture as a solid concentration. .. Ethanol with a dry weight of 1 g / biomass of 100 g was also added to the solution. Ammonia solution was pumped through a heating loop into a water tank heated to about 75 ° C, created using a 2 gal Parr reactor. A heated diluted ammonium hydroxide solution was injected into the reaction vessel via an infusion lance and sprayed onto crushed corn cobs that rolled and rolled in the reactor. The reactor was maintained at 75 ° C. for 2 hours while rotating at 19 rpm. At the end of that time, a vacuum (about 85 kPa) was applied to the reaction vessel for 30 minutes to remove ammonia and reduce the temperature of the reactor contents to about 50 ° C. Then carbon dioxide is injected into the reactor to release the vacuum and the reactor is CO.₂The pressure was increased until the gauge pressure of was 103 kPa, and the pressure was maintained at 50 ° C. for 30 minutes.

After this, the reactor was depressurized, opened and a friction medium was added. The pH of the contents is adjusted to about 5.5 by injecting 1M citrate buffer with pH 4.8 using an infusion lance to increase the strength of the citrate buffer to about 75 ml and citrate monohydrate. Was added. Removed in vacuum or CO₂It was not all of the ammonia that was neutralized with. The contents were equilibrated by heating the citrate buffer to 50 ° C and then injecting it into the reactor and then incubating the reactor at 50 ° C and 19 rpm for 1 hour. Injecting the citrate buffer using an infusion lance while rotating the reactor allowed for more uniform spraying and distribution of the buffer onto the pretreated corn cob particles. The reactor was removed from the incubator, opened and the pH of the sample was measured. If the pH was greater than 5.5, additional solid citrate monohydrate was added and the reactor was incubated at 50 ° C. for an additional hour with mixing. This method was repeated until the pH reached about 5.5. Once the desired pH is reached, 12.9 mg / g of cellulose, Spezyme CP (Genencor) and 5 mg of active protein / g from β-glucosidase, xylanase, β-xylosidase and arabinofuranosidase. The cellulosic enzyme community was loaded into the reactor. The reactor was maintained in the incubator at 50 ° C and 19 rpm for 72 hours. After this pretreatment and saccharification, the yield of monomeric glucose was 62.0% and the yield of monomeric xylose was 31.0%. The total glucose yield was 75.2% and total xylose was 80.3%.

Example 18 Pretreatment and alternative conditions with very small amounts of ammonia at higher solid concentrations of corn cob Whole-grain corn cobs were treated with a hammer mill (10-inch hammer mill, Glen Mills Inc., Clifton, New Hampshire) and passed through a 1.27 cm screen. Approximately 805 g of ground corn cob was loaded onto the PEH Reactor. The water content in the corn cob was about 7%. 22 ceramic friction cylinders (3.2 cm in diameter x 3.2 cm in length; ER Advanced Ceramics, East Palacetine (East) Palestine), Ohio) was also added to the reactor. Prior to the start of the experiment, the reactor was preheated to 95 ° C without rotation. Vacuum (approximately 85 kPa) was applied to the reaction vessel prior to initiation and the vessel was sealed. When the temperature in the reaction vessel stabilized at 95 ° C, the rotation mechanism in the incubator was activated and the rotation was adjusted to 19 rpm. An appropriate amount of diluted ammonium hydroxide solution was then pumped into the reactor to obtain an ammonia concentration of 6 g of ammonia / 100 g of dry weight of biomass and a total dry weight of 50 g of biomass / 100 g of biomass-ammonia mixture as a solid concentration. .. Ammonia solution was pumped through a heating loop into a boiling water tank made using a 2 gal Parr reactor. A heated diluted ammonium hydroxide solution was injected into the reaction vessel via an infusion lance and sprayed onto crushed corn cobs that rolled and rolled in the reactor. The reactor was maintained at 95 ° C. for 2 hours while rotating at 19 rpm. At the end of that time, a vacuum (about 85 kPa) was applied to the reaction vessel for 30 minutes to remove ammonia and reduce the temperature of the reactor contents to about 50 ° C. Carbon dioxide was then injected into the reactor to release the vacuum, the reactor was pressurized to a gauge pressure of 103 kPa and held at 50 ° C. for 30 minutes.

After this, the reactor was depressurized and opened, the pH of the contents was adjusted to about 5.5 by injecting a 1 M citrate buffer of pH 4.8, into which citrate monohydrate was added and Dissolved. The contents were equilibrated by heating the citrate buffer to 50 ° C and then injecting it into the reactor and then incubating the reactor at 50 ° C and 19 rpm for 1 hour. Injecting the citrate buffer using an infusion lance while rotating the reactor allowed for more uniform spraying and distribution of the buffer onto the pretreated corn cob particles. The reactor was removed from the incubator, opened and the pH of the sample was measured. If the pH was greater than 5.5, additional solid citrate monohydrate was added and the reactor was incubated at 50 ° C. for an additional hour with mixing. This method was repeated until the pH reached about 5.5. Once the desired pH is reached, 12.9 mg / g of cellulose, Spezyme CP (Genencor) and 5 mg of active protein / g from β-glucosidase, xylanase, β-xylosidase and arabinofuranosidase. The cellulosic enzyme community was loaded into the reactor. The reactor was maintained in the incubator at 50 ° C and 19 rpm for 72 hours. After this pretreatment and saccharification, the yield of monomeric glucose was 50.7% and the yield of monomeric xylose was 35.7%. Yields for total glucose and total xylose were 71.7% and 89.8%, respectively.

Example 19 Pretreatment of corn cobs with very small amounts of ammonia and additional bases A jaw crusher (2.2kW motor) with a jaw spacing of about 0.95cm on the whole corn cob, followed by a delamper (1.5kW motor, Franklin Miller). Inc.)) and then screened on a Sweco screen with a 1.9 cm US standard screen. Approximately 460 g of ground corn cob was loaded onto PEH Reactor. The water content in the corn cob was about 7%. Prior to the start of the experiment, the reactor was preheated to 95 ° C without rotation. Vacuum (approximately 85 kPa) was applied to the reaction vessel prior to initiation and the vessel was sealed. When the temperature inside the vessel was re-stabilized at 95 ° C, the rotation mechanism inside the incubator was activated and the rotation was adjusted to 19 rpm. Then, while maintaining the dry weight of biomass 30 g / total weight of the biomass-ammonia mixture 100 g as the solid concentration, the dry weight of ammonia 3.2 g / ammonia and NaOH to obtain a concentration of NaOH 1.9 g / dry weight of biomass 100 g. An appropriate amount of ammonium hydroxide solution was pumped into the reactor to obtain 100 g of ammonia concentration. A solution of ammonia and additional base was pumped through a heating loop into a boiling water tank made using a 2 gal Parr reactor. A heated diluted ammonium hydroxide solution was injected into the reaction vessel via an infusion lance and sprayed onto crushed corn cobs that rolled and rolled in the reactor. After injection, the vacuum in the container was released to atmospheric pressure. The reactor was maintained at 95 ° C for 30 minutes, then the temperature was reduced to 85 ° C, where it was maintained for 4 hours. At the end of that time, a vacuum (about 85 kPa) was applied to the reaction vessel for 30 minutes to remove ammonia and reduce the temperature of the reactor contents to about 50 ° C. Carbon dioxide was then injected into the reactor to release the vacuum, the reactor was pressurized to a gauge pressure of 103 kPa and held at 50 ° C. for 30 minutes.

After this, the reactor was depressurized and opened, and the pH of the contents was adjusted to about 5.5 by injecting about 75 ml of 1 M citrate buffer with pH 4.8, in which citrate monohydrate was added. Added and dissolved. The contents were equilibrated by heating the citrate buffer to 50 ° C and then injecting it into the reactor and then incubating the reactor at 50 ° C and 19 rpm for 1 hour. Reactor by the fact that by using the injection lance is rotated to inject citrate buffer Ri, buffer was possible more uniform spray and distribution to the pretreated corn cob on particles. The reactor was removed from the incubator, opened and the pH of the sample was measured. If the pH was greater than 5.5, additional solid citrate monohydrate was added and the reactor was incubated at 50 ° C. for an additional hour with mixing. This method was repeated until the pH reached about 5.5. Once the desired pH was reached, the reactor was loaded with 28.4 mg / g of cellulose, Spezyme CP (Genencor) and 28.4 mg / g of cellulose Multifect. The reactor was maintained in the incubator at 50 ° C and 19 rpm for 72 hours. After this pretreatment and saccharification, the yield of monomeric glucose was 56.1% and the yield of monomeric xylose was 39.5%. Yields for total glucose and total xylose were 82.8% and 84.2%, respectively. These values are the average of the two experiments.

Example 20 Pretreatment at room temperature and with very small amounts of ammonia Whole-grain corn cobs are processed with a jaw crusher (2.2 kW motor) with a jaw spacing of approximately 0.95 cm, followed by a delamper (1.5 kW motor, Franklin Miller Inc.), and then 1.9 cm in the United States. Screening was performed on a Sweco screen equipped with a standard screen. Approximately 460 g of ground corn cob was loaded onto the PEH Reactor. The water content in the corn cob was about 7%. 22 ceramic friction cylinders (3.2 cm in diameter x 3.2 cm in length; ER Advanced Ceramics, East Palacetine (East) Palestine), Ohio) was also added to the reactor. Prior to the start, a vacuum (approximately 85 kPa) was applied to the reaction vessel and the vessel was sealed. When the temperature in the reactor was re-stabilized at room temperature (22-26 ° C), the rotation mechanism in the incubator was activated to adjust the rotation to 19 rpm. Then, while maintaining the dry weight of biomass 30 g / total weight of the biomass-ammonia mixture as the solid concentration, an appropriate amount of diluted ammonium hydroxide solution to obtain an ammonia concentration of 4 g ammonia / 100 g dry weight of ammonia was placed in the reactor. Pumped. Diluted ammonium hydroxide solution was injected into the reaction vessel via an infusion lance and sprayed onto crushed corn cobs that rolled and rolled in the reactor. After injection, the vacuum in each container was released to atmospheric pressure. The reactor was maintained at room temperature (22-26 ° C) for 24 hours. At the end of that time, vacuum (about 81 kPa) was applied to the reaction vessel for 30 minutes to remove ammonia. Then carbon dioxide is injected into the reactor to release the vacuum and the reactor is CO.₂The pressure was increased to 103 kPa gauge pressure using, and the pressure was maintained at room temperature for 30 minutes.

After this, the reactor is depressurized and opened, the pH of the contents is adjusted to about 5.5 by heating to 50 ° C and then adding citric acid monohydrate, then the reactor is adjusted to 50 ° C. And equilibrated by incubating at 19 rpm. The reactor was removed from the incubator, opened and the pH of the sample was measured. If the pH was greater than 5.5, additional solid citrate monohydrate was added and the reactor was incubated at 50 ° C. with mixing. This method was repeated until the pH reached about 5.5. Once the desired pH is reached, it consists of 12.9 mg / g of cellulose, Spezyme CP (Genencor) and 5 mg of active protein / g of β-glucosidase, xylanase, β-xylosidase and arabinofuranosidase. The cellulosic enzyme community was loaded into the reactor. The reactor was maintained in the incubator at 50 ° C and 19 rpm for 72 hours. After this pretreatment and saccharification, the yield of monomeric glucose was 41.7% and the yield of monomeric xylose was 25.4%. Yields for total glucose and total xylose were 50.1% and 53.2%, respectively. These values are the average of the two experiments.

<figref num="1">It shows the growth of Zymomonas mobilis 8b (described in US Patent Application Publication No. 2003/0162271A1, Examples IV, VI and XII) in the presence or absence of acetamide and acetic acid.</figref>