WO2006110891A2 - Treatment of biomass to obtain a target chemical - Google Patents

Treatment of biomass to obtain a target chemical Download PDF

Info

Publication number
WO2006110891A2
WO2006110891A2 PCT/US2006/014020 US2006014020W WO2006110891A2 WO 2006110891 A2 WO2006110891 A2 WO 2006110891A2 US 2006014020 W US2006014020 W US 2006014020W WO 2006110891 A2 WO2006110891 A2 WO 2006110891A2
Authority
WO
WIPO (PCT)
Prior art keywords
biomass
ammonia
reactor
saccharification
acid
Prior art date
Application number
PCT/US2006/014020
Other languages
French (fr)
Other versions
WO2006110891A3 (en
Inventor
James B. Dunson
Melvin Tucker
Richard Elander
Susan Hennessey
Original Assignee
E. I. Du Pont De Nemours And Company
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by E. I. Du Pont De Nemours And Company filed Critical E. I. Du Pont De Nemours And Company
Priority to BRPI0612944-7A priority Critical patent/BRPI0612944B1/en
Priority to EP06758339A priority patent/EP1869194A2/en
Priority to JP2008506725A priority patent/JP5149784B2/en
Priority to CN200680012124.5A priority patent/CN101160405B/en
Priority to CA 2604964 priority patent/CA2604964C/en
Publication of WO2006110891A2 publication Critical patent/WO2006110891A2/en
Publication of WO2006110891A3 publication Critical patent/WO2006110891A3/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M45/00Means for pre-treatment of biological substances
    • C12M45/02Means for pre-treatment of biological substances by mechanical forces; Stirring; Trituration; Comminuting
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/02Stirrer or mobile mixing elements
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/18Flow directing inserts
    • C12M27/20Baffles; Ribs; Ribbons; Auger vanes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M45/00Means for pre-treatment of biological substances
    • C12M45/09Means for pre-treatment of biological substances by enzymatic treatment
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/02Monosaccharides
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00Preparation of oxygen-containing organic compounds
    • C12P7/02Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06Ethanol, i.e. non-beverage
    • C12P7/08Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2201/00Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02EREDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00Technologies for the production of fuel of non-fossil origin
    • Y02E50/10Biofuels, e.g. bio-diesel
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02EREDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00Technologies for the production of fuel of non-fossil origin
    • Y02E50/30Fuel from waste, e.g. synthetic alcohol or diesel
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02WCLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00Technologies for wastewater treatment
    • Y02W10/40Valorisation of by-products of wastewater, sewage or sludge processing

Definitions

  • Methods for producing target chemicals using fermentable sugars derived from treating biomass are provided. Specifically, sugars obtained by pretreating biomass under conditions of high solids and low ammonia concentration, followed by saccharification, are used in fermentation by biocatalysts producing target chemicals.
  • Cellulosic and lignocellulosic feedstocks and wastes such as agricultural residues, wood, forestry wastes, sludge from paper manufacture, and municipal and industrial solid wastes, provide a potentially large renewable feedstock for the production of chemicals, plastics, fuels and feeds.
  • Cellulosic and lignocellulosic feedstocks and wastes composed of carbohydrate polymers comprising cellulose, hemicellulose, glucans and lignin are generally treated by a variety of chemical, mechanical and enzymatic means to release primarily hexose and pentose sugars, which can then be fermented to useful products.
  • Pretreatment methods are used to make the carbohydrate polymers of cellulosic and lignocellulosic materials more readily available to saccharification enzymes.
  • Standard pretreatment methods have historically utilized primarily strong acids at high temperatures; however due to high energy costs, high equipment costs, high pretreatment catalyst recovery costs and incompatibility with saccharification enzymes, alternative methods are being developed, such as enzymatic pretreatment, or the use of acid or base at milder temperatures where decreased hydrolysis of biomass carbohydrate polymers occurs during pretreatment, requiring improved enzyme systems to saccharify both cellulose and hemicellulose.
  • Teixeira, L., et al. (Appl. Biochem.and Biotech. (1999) 77-79:19-34) disclosed a series of biomass pretreatments using stoichiometric amounts of sodium hydroxide and ammonium hydroxide, with very low biomass concentration. The ratio of solution to biomass is 14:1.
  • Patent Application WO2004/081185 discusses methods for hydrolyzing lignocellulose, comprising contacting the lignocellulose with a chemical; the chemical may be a base, such as sodium carbonate or potassium hydroxide, at a pH of about 9 to about 14, under moderate conditions of temperature, pressure and pH.
  • a chemical such as sodium carbonate or potassium hydroxide
  • U.S. Patent Nos. 5,916,780 and 6,090,595 describe a pretreatment process wherein a specified ratio of arabinoxylan to total nonstarch polysaccharides (AX/NSP) is assessed and used to select the feedstock.
  • AX/NSP arabinoxylan to total nonstarch polysaccharides
  • a commercial process for the production of chemicals from a renewable resource biomass requires the hydrolysis of carbohydrates in lignocellulosic biomass to provide high yields of sugars at high concentrations, using low amounts of chemicals, to produce a source of fermentable sugars with low toxicity that are used by biocatalysts that produce value-added target chemicals.
  • the present invention provides methods for producing target chemicals that make use of biomass.
  • the process of the invention involves pretreatment of biomass, at relatively high concentration, with a low concentration of ammonia relative to the dry weight of biomass. Following pretreatment, the biomass is treated with a saccharification enzyme consortium to produce fermentable sugars. The sugars are then contacted with a biocatalyst that can ferment the sugars and produce a target chemical.
  • the method comprises: a) contacting biomass with an aqueous solution comprising ammonia, wherein the ammonia is present at a concentration at least sufficient to maintain alkaline pH of the biomass-aqueous ammonia mixture but wherein said ammonia is present at less than about 12 weight percent relative to dry weight of biomass, and further wherein the dry weight of biomass is at a high solids concentration of at least about 15 weight percent relative to the weight of the biomass-aqueous ammonia mixture; b) contacting the product of step (a) with a saccharification enzyme consortium under suitable conditions to produce fermentable sugars; and c) contacting the product of step b) with at least one biocatalyst able to ferment the sugars to produce the target chemical under suitable fermentation conditions.
  • FIG. 1 shows the growth of Zymomonas mobilis 8b (described in US Patent Application Publication 2003/0162271 A1 , examples IV, Vl and XII) in the presence or absence of acetamide and acetic acid.
  • the present invention provides methods for producing target chemicals that make use of biomass in the following manner: sugars are derived from biomass, which are then used as a carbon source for the growth of microorganisms that can make target chemicals as products of their metabolism.
  • the sugars are released from the biomass by pretreating the biomass, at relatively high concentration, with a relatively low concentration of ammonia relative to the dry weight of the biomass.
  • the ammonia-treated biomass is then digested with a saccharification enzyme consortium to produce fermentable sugars.
  • the sugars are used as a fermentation substrate for growth of a microorganism, or bioctalyst, that is able to produce a target chemical. Definitions:
  • transferable sugar refers to oligosaccharides and monosaccharides that can be used as a carbon source by a microorganism in a fermentation process.
  • lignocellulosic refers to a composition comprising both lignin and cellulose. Lignocellulosic material may also comprise hemicellulose. The term “cellulosic” refers to a composition comprising cellulose.
  • dry weight of biomass is meant the weight of the biomass having all or essentially all water removed. Dry weight is typically measured according to American Society for Testing and Materials (ASTM) Standard E1756-01 (Standard Test Method for Determination of Total Solids in Biomass) or Technical Association of the Pulp and Paper Industry, Inc. (TAPPI) Standard T-412 om-02 (Moisture in Pulp, Paper and Paperboard).
  • target chemical refers to a chemical produced by fermentation. Chemical is used in a broad sense and includes molecules such as proteins, including, for example, peptides, enzymes and antibodies.
  • a target chemical that is "derivable from biomass” is a target chemical produced by a process whereby biomass is hydrolyzed to release fermentable sugars, and the fermentable sugars are fermented using at least one biocatalyst to produce a desired target chemical.
  • plasticizer and “softening agent” refer to materials that cause a reduction in the cohesive intermolecular forces along or between polymer chains. Such materials may act, for example, to decrease crystallinity, or disrupt bonds between lignin and non-lignin carbohydrate fibers (e.g., cellulose or hemicellulose).
  • sacharification refers to the production of fermentable sugars from polysaccharides.
  • Suitable conditions to produce fermentable sugars refers to conditions such as pH, composition of medium, and temperature under which saccharification enzymes are active.
  • Suitable fermentation conditions refers to conditions that support the growth and target chemical production by a biocatalyst. Such conditions may include pH, nutrients and other medium components, temperature, atmosphere, and other factors.
  • biomass refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Biomass may also comprise additional components, such as protein and/or lipid. According to the present method, biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass could comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves.
  • Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste.
  • biomass include, but are not limited to, corn grain, corn cobs, crop residues such as com husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from processing of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers and animal manure.
  • biomass that is useful for the present method includes biomass that has a relatively high carbohydrate value, is relatively dense, and/or is relatively easy to collect, transport, store and/or handle.
  • biomass that is useful includes corn cobs, corn stover and sugar cane bagasse.
  • an "aqueous solution comprising ammonia” refers to the use of ammonia gas (NH 3 ), compounds comprising ammonium ions (NH 4 + ) such as ammonium hydroxide or ammonium sulfate, compounds that release ammonia upon degradation such as urea, and combinations thereof in an aqueous medium.
  • ammonia gas NH 3
  • compounds comprising ammonium ions NH 4 +
  • ammonium hydroxide or ammonium sulfate compounds that release ammonia upon degradation such as urea, and combinations thereof in an aqueous medium.
  • the concentration of ammonia used in the present method is minimally a concentration that is sufficient to maintain the pH of the biomass-aqueous ammonia mixture alkaline and maximally less than about 12 weight percent relative to dry weight of biomass.
  • This low concentration of ammonia is sufficient for pretreatment, and the low concentration may also be less than about 10 weight percent relative to dry weight o ⁇ Diomass.
  • alkaline is meant a pH of greater than 7.0.
  • Particularly suitable is a pH of the biomass-aqueous ammonia mixture that is greater than 8.
  • ammonia is present at less than about 10 weight percent relative to dry weight of biomass.
  • Particularly suitable is ammonia at less than about 6 weight percent relative to dry weight of biomass.
  • Ammonia as used in the present process provides advantages over other bases.
  • Ammonia partitions into a liquid phase and vapor phase. Gaseous ammonia can diffuse more easily through biomass than a liquid base, resulting in more efficacious pretreatment at lower concentrations.
  • Ammonia also is shown herein in Example 11 to compete with hydrolysis, via ammonolysis, of acetyl esters in biomass to form acetamide. Acetamide is less toxic than acetate to certain fermentation organisms, such as Zymomonas mobilis (as demonstrated herein in Example 12). Thus conversion of acetyl esters to acetamide rather than to acetic acid reduces the need to remove acetic acid.
  • ammonia also reduces the requirement to supplement growth medium used during fermentation with a nitrogen source.
  • ammonia is a low-cost material and thus provides an economical process.
  • Ammonia can also be recycled to the pretreatment reactor during pretreatment or following pretreatment, thus enabling a more economical process. For example, following pretreatment, as the temperature is decreased to that suitable for saccharification, ammonia gas may be released, optionally in the presence of a vacuum, and may be recycled. In a continuous process, ammonia may be continuously recycled.
  • the aqueous solution comprising ammonia may optionally comprise at least one additional base, such as sodium hydroxide, sodium carbonate, potassium hydroxide, potassium carbonate, calcium hydroxide and calcium carbonate.
  • the at least one additional base may be added in an amount that is combined with ammonium to form an amount of total base that is less than about 20 weight percent relative to dry weight of biomass.
  • the total second base plus ammonia is in an amount that is less than about 15 weight percent.
  • Additional base(s) may be utilized, for example, to neutralize acids in biomass, to provide metal ions for the saccharification enzymes, or to provide metal ions for the fermentation growth medium.
  • the dry weight of biomass is at an initial concentration of at least about 15% up to about 80% of the weight of the biomass-aqueous ammonia mixture. More suitably, the dry weight of biomass is at a concentration of from about 15% to about 60% of the weight of the biomass-aqueous ammonia mixture.
  • the percent of biomass in the biomass-aqueous ammonia mixture is kept high to minimize the need for concentration of sugars resulting from saccharification of the pretreated biomass, for use in fermentation.
  • the high biomass concentration also reduces the total volume of pretreatment material, making the process more economical.
  • the biomass may be used directly as obtained from the source, or energy may be applied to the biomass to reduce the size, increase the exposed surface area, and/or increase the availability of cellulose, hemicellulose, and/or oligosaccharides present in the biomass to ammonia and to saccharification enzymes used in the second step of the method.
  • Energy means useful for reducing the size, increasing the exposed surface area, and/or increasing the availability of cellulose, hemicellulose, and/or oligosaccharides present in the biomass to ammonia and to saccharification enzymes include, but are not limited to, milling, crushing, grinding, shredding, chopping, disc refining, ultrasound, and microwave. This application of energy may occur before or during pretreatment, before or during saccharification, or any combination thereof.
  • Pretreatment of biomass with ammonia solution is carried out in any suitable vessel.
  • the vessel is one that can withstand pressure, has a mechanism for heating, and has a mechanism for mixing the contents.
  • Commercially available vessels include, for example, the Zipperclave ® reactor (Autoclave Engineers, Erie, PA), the Jaygo reactor (Jaygo Manufacturing, Inc., Mahwah, NJ), and a steam gun reactor (described in General Methods; Autoclave Engineers, Erie, PA).
  • a steam gun reactor described in General Methods; Autoclave Engineers, Erie, PA
  • Much larger scale reactors with similar capabilities may be used.
  • the biomass and ammonia solution may be combined in one vessel, then transferred to another reactor.
  • biomass may be pretreated in one vessel, then further processed in another reactor such as a steam gun reactor (described in General Methods; Autoclave Engineers, Erie, PA).
  • vacuum Prior to contacting the biomass with an aqueous solution comprising ammonia, vacuum may be applied to the vessel containing the biomass. By evacuating air from the pores of the biomass, better penetration of the ammonia into the biomass may be achieved.
  • the time period for applying vacuum and the amount of negative pressure that is applied to the biomass will depend on the type of biomass and can be determined empirically so as to achieve optimal pretreatment of the biomass (as measured by the production of fermentable sugars following saccharification).
  • the contacting of the biomass with an aqueous solution comprising ammonia is carried out at a temperature of from about 4 0 C to about 200 °C. Initial contact of the biomass with ammonia at 4 0 C, allowing impregnation at this temperature, was found to increase the efficiency of saccharification over non-pretreated native biomass.
  • said contacting of the biomass is carried out at a temperature of from about 75 °C to about 150 °C.
  • said contacting of the biomass is carried out at a temperature of from greater than 90 0 C to about 150 °C.
  • the contacting of the biomass with an aqueous solution comprising ammonia is carried out for a period of time up to about 25 hrs.
  • the pretreatment process may be performed at a relatively high temperature for a relatively short period of time, for example at from about 100 0 C to about 150 °C for about 5 min to about 2 hr.
  • the pretreatment process may be performed at a lower temperature for a relatively long period of time, for example from about 75 °C to about 100 °C for about 2 hr to about 8 hr.
  • the pretreatment process may be performed at room temperature (approximately 22-26 °C) for an even longer period of time of about 24 hr. Other temperature and time combinations intermediate to these may also be used.
  • the temperature, time for pretreatment, ammonia concentration, concentration of one or more additional bases, biomass concentration, biomass type and biomass particle size are related; thus these variables may be adjusted as necessary to obtain an optimal product to be contacted with a saccharification enzyme consortium.
  • a plasticizer, softening agent, or combination thereof such as polyols (e.g., glycerol, ethylene glycol), esters of polyols (e.g., glycerol monoacetate), glycol ethers (e.g., diethylene glycol), acetamide, ethanol, and ethanolamines, may be added in the pretreatment process (i.e., step (a)).
  • a plasticizer may be added as a component of the aqueous ammonia solution, as a separate solution, or as a dry component.
  • the pretreatment reaction may be performed in any suitable vessel, such as a batch reactor or a continuous reactor.
  • a pressure vessel is required.
  • the suitable vessel may be equipped with a means, such as impellers, for agitating the biomass-aqueous ammonia mixture. Reactor design is discussed in Lin, K.-H., and Van Ness, H. C. (in Perry, R.H. and Chilton, C. H. (eds), Chemical Engineer's Handbook, 5 th Edition (1973) Chapter 4, McGraw-Hill, NY).
  • the pretreatment reaction may be carried out as a batch process, or as a continuous process.
  • a nitrogen source is required for growth of microorganisms during fermentation; thus the use of amm ⁇ a during pretfeatment provides a nitrogen source and reduces or eliminates the need to supplement the growth medium used during fermentation with a nitrogen source.
  • acids may be utilized to reduce pH.
  • the amount of acid used to achieve the desired pH may result in the formation of salts at concentrations that are inhibitory to saccharification enzymes or to microbial growth.
  • ammonia gas may be evacuated from the pretreatment reactor and recycled. Typically, at least a portion of the ammonia is removed, which reduces the pH but leaves some nitrogen that provides this nutrient for use in subsequent fermentation.
  • the biomass may be pretreated with an aqueous ammonia solution one time or more than one time.
  • a saccharification reaction can be performed one or more times. Both pretreatment and saccharification processes may be repeated if desired to obtain higher yields of sugars.
  • Saccharification Following pretreatment, the product comprises a mixture of ammonia, partially degraded biomass and fermentable sugars. Prior to further processing, ammonia may be removed from the pretreated biomass by applying a vacuum. Removing ammonia lowers the pH, and thus less neutralizing acid is used to obtain the desired pH for saccharification and fermentation. This results in a lower salt load in the pretreatment mixture. Typically some ammonia remains, which is desired to provide a nitrogen source for fermentation.
  • the pfefrea ⁇ ment mixture is then further hydrolyzed in the presence of a saccharification enzyme consortium to release oligosaccharides and/or monosaccharides in a hydrolyzate.
  • Saccharification enzymes and methods for biomass treatment are reviewed in Lynd, L. R., et al. (Microbiol. MoI. Biol. Rev. (2002) 66:506-577).
  • the entire pretreatment mixture comprising both soluble and insoluble fractions is utilized in the saccharification reaction.
  • the aqueous fraction comprising ammonia and solubilized sugars may be separated from insoluble particulates remaining in the mixture.
  • Methods for separating the soluble from the insoluble fractions include, but are not limited to, decantation and filtration.
  • the insoluble particulates may be recycled to the pretreatment reactor.
  • the insoluble particulates may optionally be washed with an aqueous solvent (e.g., water) to remove adsorbed sugars prior to being recycled to the pretreatment reactor.
  • the insoluble fraction may then be subjected to additional treatment with aqueous ammonia solution as described above for pretreatment, followed by saccharification with a saccharification enzyme consortium.
  • the soluble fraction may also be concentrated prior to saccharification using a suitable process, such as evaporation.
  • the pretreatment product Prior to saccharification, the pretreatment product may be treated to alter the pH, composition or temperature such that the enzymes of the saccharification enzyme consortium will be active, thus providing suitable conditions to produce fermentable sugars.
  • the pH may be altered through the addition of acids in solid or liquid form.
  • carbon dioxide (CO2) which may be recovered from fermentation, may be utilized to lower the pH.
  • CO 2 may be collected from a fermenter and fed, such as by bubbling, into the pretreatment product while monitoring the pH, until the desired pH is achieved.
  • the temperature may be brought to a temperature that is compatible with saccharification enzyme activity, as noted below. Any cofactors required for activity of enzymes used in saccharification may be added.
  • the saccharification enzyme consortium comprises one or more enzymes selected primarily, but not exclusively, from the group "glycosidases” which hydrolyze the ether linkages of di-, oligo-, and polysaccharides and are found in the enzyme classification EC 3.2.1.x (Enzyme Nomenclature 1992, Academic Press, San Diego, CA with Supplement 1 (1993), Supplement 2 (1994), Supplement s (1995, Supplement 4 (1997) and Supplement 5 [in Eur. J. Biochem. (1994) 223:1- 5, Eur. J. Biochem. (1995) 232:1-6, Eur. J. Biochem. (1996) 237:1-5, Eur. J. Biochem. (1997) 250:1-6, and Eur. J. Biochem.
  • Glycosidases useful in the present method can be categorized by the biomass component that they hydrolyze.
  • Glycosidases useful for the present method include cellulose-hydrolyzing glycosidases (for example, cellulases, endoglucanases, exoglucanases, cellobiohydrolases, ⁇ - glucosidases), hemicellulose-hydrolyzing glycosidases (for example, xylanases, endoxylanases, exoxylanases, ⁇ -xylosidases, arabinoxylanases, mannases, galactases, pectinases, glucuronidases), and starch-hydrolyzing glycosidases (for example, amylases, ⁇ -amylases, ⁇ -amylases, glucoamylases, ⁇ -
  • peptidases EC 3.4.x.y
  • lipases EC 3.1.1.x and 3.1.4.x
  • ligninases EC 1.11.1.x
  • feruloyl esterases EC 3.1.1.73
  • a "cellulase” from a microorganism may comprise a group of enzymes, all of which may contribute to the cellulose-degrading activity.
  • Commercial or non- commercial enzyme preparations, such as cellulase may comprise numerous enzymes depending on the purification scheme utilized to obtain the enzyme.
  • the saccharification enzyme consortium of the present method may comprise enzyme activity, such as "cellulase", However it is " recognized that this activity may be catalyzed by more than one enzyme.
  • Saccharification enzymes may be obtained commercially, such as Spezyme ® CP cellulase (Genencor International, Rochester, NY) and Multifect ® xylanase (Genencor).
  • saccharification enzymes may be produced biologically, including using recombinant microorganisms.
  • One skilled in the art would know how to determine the effective amount of enzymes to use in the consortium and adjust conditions for optimal enzyme activity.
  • One skilled in the art would also know how to optimize the classes of enzyme activities required within the consortium to obtain optimal saccharification of a given pretreatment product under the selected conditions.
  • the saccharification reaction is performed at or near the temperature and pH optima for the saccharification enzymes.
  • the temperature optimum used with the saccharification enzyme consortium in the present method ranges from about 15 °C to about 100 °C. In another embodiment, the temperature optimum ranges from about 20 °C to about 80 0 C.
  • the pH optimum can range from about 2 to about 11. In another embodiment, the pH optimum used with the saccharification enzyme consortium in the present method ranges from about 4 to about 10.
  • the saccharification can be performed for a time of about several minutes to about 120 hr, and preferably from about several minutes to about 48 hr.
  • the time for the reaction will depend on enzyme concentration and specific activity, as well as the substrate used and the environmental conditions, such as temperature and pH.
  • One skilled in the art can readily determine optimal conditions of temperature, pH and time to be used with a particular substrate and saccharification enzyme(s) consortium.
  • the saccharification can be performed batch-wise or as a continuous process.
  • the saccharification can also be performed in one step, or in a number of steps.
  • different enzymes required for saccharification may exhibit different pH or temperature optima.
  • a primary treatment can be performed with enzyme(s) at one temperature and pH, followed by secondary or tertiary (or more) treatments with different enzyme(s) at different temperatures and/or pH.
  • treatment with different enzymes in sequential steps may be at the same pH and/or temperature, or different pHs and temperatures, such as using hemicellulases stable and more active at higher pHs and temperatures followed by cellulases that are active at lower pHs and temperatures..
  • the degree of solubilization of sugars from biomass following saccharification can be monitored by measuring the release of monosaccharides and oligosaccharides.
  • Methods to measure monosaccharides and oligosaccharides are well known in the art.
  • the concentration of reducing sugars can be determined using the 1 ,3-dinitrosalicylic (DNS) acid assay (Miller, G. L., Anal. Chem. (1959) 31 :426-428).
  • sugars can be measured by HPLC using an appropriate column as described herein in the General Methods section. Fermentable sugars released from biomass can be used by suitable microorganisms to produce target chemicals.
  • the saccharification mixture may be concentrated by evaporation, for example, to increase the concentration of fermentable sugars.
  • liquid in the saccharification product may be separated from solids in a batch or continuous method.
  • the liquid or the entire saccharification product may be sterilized prior to fermentation.
  • the pH may be adjusted to that suitable for fermentation.
  • the saccharification mixture may be supplemented with additional nutrients required for microbial growth. Supplements may include, for example, yeast extract, specific amino acids, phosphate, nitrogen sources, salts, and trace elements.
  • Components required for production of a specific product made by a specific biocatalyst may also be included, such as an antibiotic to maintain a plasmid or a cofactor required in an enzyme catalyzed reaction.
  • additional sugars may be included to increase the total sugar concentration.
  • the saccharification mixture may be used as a component of a fermentation broth, for example, making up " between about 100% and about 10% of the final medium. Suitable fermentation conditions are achieved by adjusting these types of factors for the growth and target chemical production by a biocatalyst. Temperature and/or headspace gas may also be adjusted, depending on conditions useful for the fermentation microorganism(s). Fermentation may be aerobic or anaerobic.
  • Fermentation may occur subsequent to saccharification, or may occur concurrently with saccharification by simultaneous saccharification and fermentation (SSF).
  • SSF can keep the sugar levels produced by saccharification low, thereby reducing potential product inhibition of the saccharification enzymes, reducing sugar availability for contaminating microorganisms, and improving the conversion of pretreated biomass to monosaccharides and/or oligosaccharides.
  • Target chemicals that may be produced by fermentation include, for example, acids, alcohols, alkanes, alkenes, aromatics, 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, 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.
  • Biocatalysts may be microorganisms selected from bacteria, filamentous fungi and yeast. Biocatalysts may be wild type microorganisms or recombinant microorganisms, and include Escherichia, Zymomonas, Saccharomyces, Candida, Pichia, Streptomyces, Bacillus, Lactobacillus, and Clostridium.
  • biocatalysts may be selected from the group consisting of recombinant Escherichia coli, Zymomonas mobilis, Bacillus stearothermophilus, Saccharomyces cerevisi ⁇ , Clostridia thermocellum, Thermoanaerobacterium saccharolyticum, and Pichia stipitis.
  • biocatalysts used in fermentation to produce target chemicals have been described and others may be discovered, produced through mutation, or engineered through recombinant means. Any biocatalyst that uses fermentable sugars produced in the present method may be used to make the target chemical(s) that it is known to produce, by fermentation in the present method. Fermentation of carbohydrates to acetone, butanol, and ethanol
  • Lactic acid has been produced in fermentations by recombinant strains of E. coli (Zhou et al., (2003) Appl. Environ. Microbiol. 69:399- 407), natural strains of Bacillus (US20050250192), and Rhizopus oryzae (Tay and Yang (2002) Biotechnol. Bioeng. 80:1-12).
  • Recombinant strains of E. coli have been used as biocatalysts in fermentation to produce 1 ,3 propanediol (US 6013494, US 6514733), and adipic acid (Niu et al., (2002) Biotechnol. Prog. 18:201-211).
  • Acetic acid has been made by fermentation using recombinant Clostridia (Cheryan et al., (1997) Adv. Appl. Microbiol. 43:1-33), and newly identified yeast strains (Freer (2002) World J. Microbiol. Biotechnol. 18:271-275).
  • Production of succinic acid by recombinant E. coli and other bacteria is disclosed in US 6159738, and by mutant recombinant E. coli in Lin et al., (2005) Metab. Eng. 7:116-127).
  • Pyruvic acid has been produced by mutant Torulopsis glabrata yeast (Li et al., (2001 ) Appl. Microbiol. Technol. 55:680-685) and by mutant E.
  • E. coli (Yokota et al., (1994) Biosci. Biotech. Biochem. 58:2164-2167). Recombinant strains of E. coli have been used as biocatalysts for production of para-hydroxycinnamic acid (US20030170834) and quinic acid (US20060003429).
  • a mutant of Propionibacterium acidipropionici has been used in fermentation to produce propionic acid (Suwannakham and Yang (2005) Biotechnol. Bioeng. 91:325-337), and butyric acid has been made by Clostridium tyrobutyricum (Wu and Yang (2003) Biotechnol. Bioeng. 82:93-102).
  • Propionate and propanol have been made by fermentation from threonine by Clostridium sp. strain 17cr1 (Janssen (2004) Arch. Microbiol. 182:482-486).
  • a yeast-like Aureobasidium pullulans has been used to make gluconic acid (Anantassiadis et al., (2005) Biotechnol.
  • Corynebacterium, Brevibacterium, and Serratia For example, production of histidine using a strain resistant to a histidine analog is described in Japanese Patent Publication No. 8596/81 and using a recombinant strain is described Tn EP ⁇ 36359. Production of tryptophan using a strain resistant to a tryptophan analog is described in Japanese Patent Publication Nos. 4505/72 and 1937/76. Production of isoleucine using a strain resistant to an isoleucine analog is described in Japanese Patent Publication Nos. 38995/72, 6237/76, 32070/79. Production of phenylalanine using a strain resistant to a phenylalanine analog is described in Japanese Patent Publication No.
  • Phenylalanine was also produced by fermentation in Eschericia co// strains ATCC 31882, 31883, and 31884.
  • Example 9 The pretreatment and saccharification of biomass to fermentable sugars, followed by fermentation of the sugars to a target chemical is exemplified in Example 9 herein for the production of ethanol from pretreated corn cobs using Z mobilis as the biocatalyst for the fermentation of sugars to ethanol.
  • the present method may also be used for the production of 1 ,3-propanediol from biomass.
  • Biomass undergoes pretreatment and saccharification according to the present method; following (or during) saccharification, E. coli is used to produce 1 ,3- propanediol as described in Example 10 herein.
  • Target chemicals produced in fermentation by biocatalysts may be recovered using various methods known in the art.
  • Products may be separated from other fermentation components by centrifugation, filtration, micr ⁇ filtrat ⁇ n , " ancJ na ⁇ n ⁇ f iltration .
  • Products may be extracted by ion exchange, solvent extraction, or electrodialysis.
  • Flocculating agents may be used to aid in product separation.
  • bioproduced 1-butanol may be isolated from the fermentation medium using methods known in the art for ABE fermentations (see for example, Durre, Appl. Microbiol. Biotechnol. 49:639-648 (1998), Groot et al., Process. Biochem. 27:61-75 (1992), and references therein).
  • solids may be removed from the fermentation medium by centrifugation, filtration, decantation, or the like.
  • the 1-butanol may be isolated from the fermentation medium using methods such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, or pervaporation.
  • Purification of 1 ,3-propanediol from fermentation media may be accomplished, for example, by subjecting the reaction mixture to extraction with an organic solvent, distillation, and column chromatography (U.S. 5,356,812).
  • a particularly good organic solvent for this process is cyclphexane (U.S. 5,008,473).
  • Amino acids may be collected from fermentation medium by methods such as ion-exchange resin adsorption and/or crystallization.
  • Sulfuric acid, ammonium hydroxide, acetic acid, acetamide, yeast extract, 2-morpholinoethanesulfonic acid (MES), potassium phosphate, glucose, xylose, tryptone, sodium chloride and citric acid were obtained from Sigma-Aldrich (St. Louis, MO).
  • the 4-liter Zipperclave ® reactor (Autoclave Engineers, Erie, PA) is a batch pressure vessel equipped with a 2.5-liter Hastelloy ® pail for the biomass charge and an agitator to mix the biomass.
  • the reactor vessel is encircled by an electrical heater controlled at the desired pretreatment temperature. Direct steam injection is also used to rapidly bring the biomass up to pretreatment temperature. Steam pressure is adjusted and controlled to maintain the desired pretreatment temperature. Steam condensate formed by heating the Zipperclave ® reactor head plate, vessel and outside of the pail drain to a reservoir formed between the pail and the inner wall of the reactor to prevent excessive dilution of the pretreated slurry.
  • the Jaygo reactor is a 130-liter (approximately 51 cm diameter x 91 cm length), horizontal paddle type reactor (Jaygo Manufacturing, Inc., Mahwah, NJ) fabricated of Hastelloy ® C-22 alloy.
  • the reactor is equipped with a steam jacket capable of heating to approximately 177 0 C (862 kPa). Direct steam injection is also used to rapidly bring the biomass up to pretreatment temperature. Steam pressure is adjusted and controlled to maintain the desired pretreatment temperature. Numerous ports allow injection of other solvents and hot liquids.
  • the 4-liter steam gun reactor (Autoclave Engineers, Erie, PA) is a steam-jacketed reactor consisting of a length of 102 mm schedule 80
  • Hotfill ® nylon 0.21 m 3 bag supported within a heavy walled, jacketed, and cooled flash tank.
  • the disc refiner is a Sprout Waldron model 30.5 cm refiner (Andritz,
  • the inlet to the refiner was modified with six injection ports to allow the introduction of steam, hot water, or other sweep gases and liquids just ahead of the rotating refiner plate.
  • the refiner was equipped with plates (Durametal, Corp., Tulatin, OR) in either pattern D2A506 in Ni-Hard or pattern 18034-A in Ni-Hard.
  • PEHR Pretreatment and Enzymatic Hydrolysis Reactor
  • the 9L PEHReactor (constructed at NREL, Golden, CO; see co- pending US patent application CL3447) has an approximately 15 cm x 51 cm stainless steel reaction vessel with an injection lance for introduction of processing reactants.
  • the injection lance is connected using a rotary joint to a port in a cover on one end of the vessel, which has an additional port for vessel access.
  • Four baffles run the length of the vessel wall, and are attached perpendicularly to the wall.
  • the amount of cellulose in each starting biomass sample was determined using methods well known in the art, such as ASTM E1758-01 "Standard method for the determination of carbohydrates by HPLC".
  • Soluble sugars (glucose, cellobiose, xylose, galactose, arabinose and mannose), acetamide, lactic acid and acetic acid in saccharification liquor were measured by HPLC (Agilent Model 1100, Agilent
  • Injection volume 10 - 50 ⁇ L, dependent on concentration and detector limits
  • Mobile phase HPLC grade water, 0.2 ⁇ m filtered and degassed
  • Detector temperature as close to main column temperature as possible
  • Detector refractive index
  • Run time 35 minute data collection plus 15 minute post run (with possible adjustment for later eluting compounds)
  • Biorad Aminex HPX-87H (for carbohydrates, acetamide, lactic acid, acetic acid, and ethanol)
  • Detector temperature as close to column temperature as possible
  • Detector refractive index
  • Run time 25 - 75 minute data collection
  • concentrations in the sample were determined from standard curves for each of the compounds.
  • Condensate formed during preheating was removed by vacuum aspiration before pretreatment.
  • the Hastelloy ® pail was loaded with 0.635-cm (1/4- in.) milled stover (100 g, dry weight basis) and inserted into the pre- warmed reactor.
  • the reactor agitator was set to 20 rpm while a vacuum (approximately 85 kPa) was applied to the vessel interior and biomass charge.
  • Ammonium hydroxide solution of the necessary strength to give a dry weight of biomass concentration of 30 weight percent relative to the weight of the biomass-aqueous ammonia mixture, as well as the desired ammonia concentration listed in Table 1 was injected near the bottom of the vessel with a spray type nozzle.
  • Test samples had a final ammonia concentration of 12% relative to dry weight of biomass, while samples with a final ammonia concentration of 35% relative to dry weight of biomass were used as a comparison.
  • steam was introduced near the bottom of the reactor to fluidize and raise the temperature of the biomass charge to either 140 °C or 170 °C.
  • the reactor was depressurized through a vent condenser, and a vacuum (approximately 85 kPa) was applied for 3 minutes to lower the temperature and remove additional ammonia from the pretreated slurry prior to opening the reactor and recovering the pretreated biomass.
  • the sugar content of the resulting saccharification liquor was determined after 96 hr saccharification according to the sugar measurement protocol described in the General Methods. Sugar release after 96 hr is shown in Table 2. Controls for this experiment were 1 ) untreated corn stover, which yielded 23% of the theoretical yield of glucose (using 56 mg cellulase /g cellulose) and 2) steam (140 °C) pretreated corn stover, which yielded 40% of the theoretical yield of glucose (using 56 mg cellulase /g cellulose); xylose was not measured for the controls.
  • Table 1 Sugar release from pretreated corn stover with 96 hr saccharification
  • DWB dry weight of biomass.
  • the Jaygo reactor was charged with 0.635-cm milled stover (13 kg, dry weight basis).
  • a vacuum (67.7 kPa) was applied to the vessel, and dilute ammonium hydroxide solution was injected to give an ammonia concentration of 6.2 g ammonia/100 g dry weight of biomass and a dry weight of biomass concentration 30 weight percent relative to total weight of the biomass-aqueous ammonia mixture.
  • the vacuum was relieved, and steam was applied to the jacket to heat the stover to 100 0 C.
  • the soaked stover was held at temperature for 8 hr with constant mixing at 32 rpm, then allowed to cool overnight with continued mixing of the resulting slurry.
  • the vacuum was relieved and steam was applied to the jacket to heat the cobs while soaking to a temperature of 93 °C for the whole cob sample and 85 °C for fractured cob samples. Short periods of increased agitator speeds (up to 96 rpm) were applied in an effort to increase the heating rate.
  • the soaked cobs were held at temperature for 4 or 8 hr with constant mixing at 32 rpm then allowed to cool overnight with continued mixing.
  • pretreated biomass was removed from the reactor, the reactor was put under vacuum at 90 °C to strip ammonia out of the pretreated biomass.
  • the pH of the pretreated cob biomass was adjusted to 5.5 with solid citric acid.
  • About 10 kg of pretreated whole cob was saccharified in the Jaygo reactor at 50 °C.
  • About 1400 g of pretreated fractured cob was added to the PEHReactor, along with 22 ceramic attrition cylinders (3.2 cm diameter x 3.2 cm long; E. R. Advanced Ceramics, East furniture, OH), for saccharification.
  • Table 3 Sugar release from pretreated corn cobs using a high concentration of biomass (by dry weight) during saccharification.
  • DWB dry weight of biomass (percent is calculated relative to the total weight of the mixture)
  • the cooling water to the jacket was turned on.
  • the contents of the Jaygo reactor were cooled to between 33 0 C and 37 0 C; then CO 2 was used to pressurize the reactor to 138 kPa.
  • the pressurized CO 2 atmosphere was maintained for 30 min.
  • the final temperature of the reactor contents was between 27 0 C to 31 0 C.
  • the pH of the soaked/pretreated biomass was approximately 7.5.
  • Pretreated biomass was removed from the Jaygo reactor and transferred to the PEHReactor for saccharification at a final dry weight of biomass concentration at the beginning for saccharification of 30% relative to the total weight of the pretreated biomass-saccharification enzyme consortium mixture.
  • the pH was then adjusted to 5.5 with solid citric acid, and the material digested with 28 mg Spezyme CP ® /g cellulose and 28 mg Multifect Xylanase ® /g cellulose in untreated cob as described in Example 3.
  • the sugar content of the resulting saccharification liquor was determined according to the sugar measurement protocol in the General Methods. The sugar release after 96 hr digestion is shown in Table 4.
  • Whole cob was pretreated as described in Example 3 at a dry weight of biomass concentration of about 30% relative to the total weight of the biomass-aqueous ammonia mixture, 2 weight percent ammonia relative to dry weight of biomass, 100 °C, for 8 hr in the Jaygo reactor with 3 weight percent relative to dry weight of biomass of glycerol added to act as a plasticizer.
  • the pH of the resulting material was adjusted to 5 with solid citric acid.
  • Pretreated cob was then digested as described in Example 3. An enzyme mixture of 28 mg Spezyme CP ® /g cellulose in untreated stover plus 28 mg/g cellulose Multifect Xyianase ® in untreated cob was used. After 96 hr digestion, glucose concentration was 92.3 g/L and xylose concentration was 54.4 g/L.
  • Stover was pretreated in the manner described in Example 1 , with different samples having low ammonia (12%) or comparative ammonia (35%) concentrations, and temperature, time, and enzyme conditions as listed in Table 5.
  • Whole cob was pretreated as described in Example 3 with different samples having very low ammonia (3% or 6%), and other conditions as listed in Table 5.
  • the samples were passed through a Sprout Waldron disc refiner. The gap between the stationary plate and rotating plate was set at 0.254 mm (0.010 inch) and the feed auger speed at 7 rpm.
  • Refined material was saccharified as described in Example 2 and the sugar content of the resulting saccharification liquor was determined according to the sugar measurement protocol in the General Methods. Results of the saccharification at 96 hr are shown in Table 5. The results showed that with disc refining prior to saccharification, better digestibility was attained, or use of lower enzyme concentrations was effective.
  • Example 7 Steam Gun Treatment of Pretreated Biomass Stover was pretreated as described in Example 1 using conditions of 30% dry weight of biomass relative to total weight of biomass-aqueous ammonia mixture, 6 weight percent ammonia relative to DWB, 100 0 C, 8 hr, in the Jaygo reactor.
  • Cob was pretreated as described in Example 3 using conditions of 40% dry weight of biomass relative to total weight of biomass-aqueous ammonia mixture, 6 weight percent ammonia relative to DWB, 93 0 C, 8 hr, in the Jaygo reactor.
  • Samples of each pretreated biomass were separately loaded into a 4-liter steam gun reactor. Pretreated material was subjected to 170 °C for 5 min, or 140 0 C for 20 min before being released through a die. The resulting material was saccharified as described in Example 2. Results are given in Table 6 below. The results showed that steam gun treatment prior to saccharification improved release of glucose.
  • ammonia supplied fresh and from the recycle streams for each process are shown in Table 7.
  • the flash tanks operated in similar fashion so that ammonia recycle was similar. For both scenarios, more than half of the required ammonia was supplied through recycle, reducing the need for and cost of fresh ammonia.
  • Cob hydrolyzate was generated by pretreating whole cobs in the Jaygo reactor for 8 hr at 93 0 C with 6 weight percent ammonia relative to dry weight of biomass at a dry weight of biomass concentration of 40 weight percent relative to the total weight of the biomass-aqueous ammonia mixture, as described in Example 3. 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 sulfuric acid.
  • the pretreated biomass was saccharified in the Jaygo reactor at 30% dry weight of biomass relative to the total weight of the pretreated biomass-saccharification enzyme consortium mixture with 28 mg/g cellulose Spezyme ® cellulase and 28 mg/g cellulose Multifect ® xylanase for 168 hr at 50 °C and pH 5.
  • the resulting hydrolyzate was used for fermentation of Zymomonas mobilis 8b in Sixfors fermentors (INFORS AG, Switzerland).
  • Zymomonas mobilis 8b is a strain of Zymomonas mobilis that has been genetically engineered to give improved, over wild type, production of ethanol and is described in US Patent Application Publication 2003/0162271 A1 (Examples IV, Vl and XII).
  • the cob hydrolyzate comprised 78 g/L glucose, 51 g/L xylose, 6 g/L acetamide, and 7 g/L acetic acid.
  • the cob hydrolyzate was used at 40% and 80% strength, with the balance of the medium being concentrated aqueous medium consisting of yeast extract, and KH 2 PO 4 in quantities such that their concentrations in the final slurry were about 5 g/L, and 2 g/L respectively.
  • the pretreated biomass was enzymatically digested at 30% dry weight of biomass relative to the total weight of the pretreated biomass- saccharification enzyme consortium mixture with 224 mg/g cellulose Spezyme CP ® cellulase at 50 0 C and pH 5 to generate a high sugar concentration hydrolyzate for fermentation testing.
  • the resulting hydrolyzate comprised 88 g/L glucose, 52 g/L xylose, 9 g/L acetic acid and 15 g/L lactic acid.
  • Zymomonas mobilis 8b was fermented on either 40% or 80% (v/v) hydrolyzate slurry.
  • the remaining volume was made up of concentrated aqueous medium consisting of yeast extract, KH 2 PO 4 and MES buffer in quantities such that their concentrations in the final slurry would be about 10 g/L, 2 g/L and 0.1 M, respectively. Additionally, in the 40% hydrolyzate slurry, glucose and xylose were added in quantities sufficient to bring their concentrations to the same levels as in the 80% hydrolyzate slurry. Fermentation was done at 30 °C and pH 6 in 25 ml shake flasks with 20 ml working volume. Agitation was maintained at 150 rpm. Analysis was as for the cob hydrolyzate fermentation sample and results are given in Table 8.
  • Table 8 Sugar utilization and ethanol yields in fermentation on cob and stover hydrolyzates.
  • Hydrolyzate generated from pretreatment and saccharification of cob was fermented to produce 1 ,3-propanediol. Hydrolyzate was generated by pretreating cob pieces in the steam gun reactor. First cob biomass was loaded in the PEHReactor (described in General Methods), a vacuum applied, and dilute ammonium hydroxide solution was injected to give an ammonia concentration of 4 g ammonia/100 g dry weight biomass and a dry weight of biomass concentration of 30 g dry weight of biomass/100 g total biomass-aqueous ammonia mixture. The reactor vessel charged with ammonia and cob was rotated at 4°C for 30 min.
  • the contents were transferred to the steam gun reactor (described in General Methods), the temperature increased to 145°C, and the mixture was held at temperature for 20 minutes. Material from the steam gun was discharged into a flash tank, and vacuum was maintained on the flash tank to aid ammonia removal.
  • the pretreated biomass was saccharified at 30 g dry weight of biomass/100 g pretreated biomass- saccharification enzyme consortium mixture with 28.4 mg/g cellulose Spezyme CP ® cellulase and 10.1 mg active protein /g cellulose enzyme consortium consisting of ⁇ -glucosidase, xylanase, ⁇ -xylosidase and arabinofuranosidase for 72 hr at 50°C and pH 5.5.
  • strain RJ8n pBE93-k1 The construction of strain RJ8n pBE93-k1 is described in detail in PCT application WO/ 2004/018645 (Example 7), and it is a derivative of strain RJ8n, described in US 6358716.
  • the hydrolyzate was used at 10% with the balance being aqueous medium consisting of 7.5 g/L KH 2 PO 4 , 2.0 g/L citric acid*H 2 O, 4.0 ml/L 28% NH 4 OH, 3.0 g/L(NH 4 ) 2 SO 4 , 2.0 g/L
  • Results are shown in Table 9 below.
  • Products from glucose fermentation by the RJ8n pBE93-k1 strain of E. coli include glycerol (an intermediate metabolite) and 1,3-propanediol. Experiments were conducted in duplicate flasks and assayed at 24 hr. In this system, glucose in the hydrolysate was converted to both glycerol and 1 ,3-propanediol.
  • Table 9 Substrate utilization and product formation by fermentation with E. coli.
  • the acidified sample was autoclaved for 1 hr at 121 0 C; acetamide was quantitatively converted to acetic acid during this step. After autoclaving, the sample was allowed to cool. The sample was then passed through a 0.2 ⁇ m filter into a sample vial and analyzed according to the conditions listed below. Acetic acid and acetamide concentrations were determined from standard curves generated for each.
  • Detector temperature As close to column temperature as possible Detector: Refractive index
  • DWB dry weight of biomass. (percent is calculated relative to the total weight of the biomass-aqueous ammonia mixture)
  • Fermentation medium was composed of 10 g/L yeast extract, 2 g/L KH 2 PO 4 , 70 g/L glucose, 40 g/L xylose and 0.1 M MES buffer.
  • Z. mobilis 8b was grown in 25-mL baffled
  • the PEHReactor (described in General Methods), with no attrition media, was charged with 1.27 cm-milled bagasse (370 g, dry weight basis).
  • This sugar cane bagasse was NIST Reference Material RM8491 , from sugar cane clone H65-7052, originally obtained from the Hawaii Sugar Planters Association, Kunia substation, Oahu, HI. It was milled in a Wiley mill to pass through a 2 mm screen, with the fines (+74 mesh) removed.
  • the PEHReactor vessel was cooled to 4°C by rotation in contact with ice on the outer surface.
  • a vacuum was applied to the reactor vessel, and dilute ammonium hydroxide solution, that was pre-cooled in a cold room at 4 0 C and passed through tubing immersed in an ice-water bath, was injected to give an ammonia concentration of 4 g/100 g dry weight of biomass and a dry weight of biomass concentration of 45 g/100 g total biomass-aqueous ammonia mixture.
  • the reactor vessel charged with ammonia and bagasse was cooled to 4°C by applying ice to the surface of the rotating reactor vessel, and rotated at 4°C for 30 min. At this time the contents were transferred to the steam gun reactor that is described in General Methods.
  • the temperature was increased to 145 0 C and the mixture was held at temperature for 20 minutes.
  • the bagasse was discharged from the steam gun reactor through a 1-in circular die into a flash tank.
  • a sample of pretreated bagasse was subsequently saccharified in a shake flask and another sample (approximately 163 g dry weight) was saccharified in the PEHReactor.
  • the shake flask saccharification was carried out at 5% dry weight of biomass relative to the total weight of the pretreated biomass- saccharification enzyme consortium mixture, while the PEHReactor saccharification was carried out at 30% dry weight of biomass relative to the total weight of the pretreated biomass-saccharification enzyme consortium mixture.
  • the temperature was maintained at 50 °C.
  • Table 11 Yields following pretreatment and saccharification of bagasse.
  • the PEHReactor without attrition media, was charged with yellow poplar sawdust (596 g, dry weight basis; purchased from Sawmiller Inc., Haydenville, OH). A vacuum was applied to the reactor vessel, and dilute ammonium hydroxide solution was injected to give an ammonia concentration of 6 g/100 g dry weight of biomass and a dry weight of biomass concentration of 44 g/100 g total biomass-aqueous ammonia mixture.
  • the reactor vessel charged with ammonia and yellow poplar sawdust was brought to 4°C as described in Example 13, and rotated at 4°C for 30 min. At this time the contents were transferred to the steam gun reactor.
  • the temperature was increased to 145°C and the mixture was held at temperature for 20 minutes.
  • the yellow poplar sawdust was discharged from the steam gun reactor through a 1-in circular die into a flash tank. A sample of pretreated yellow poplar sawdust was subsequently saccharified as described in
  • Example 13 in a shake flask, and another sample was saccharified in the PEHReactor.
  • the shake flask saccharification was carried out at 5% dry weight of biomass relative to the total weight of the pretreated biomass- saccharification enzyme consortium mixture, while the PEHReactor saccharification (using ⁇ 279 g dry weight pretreated sawdust) was carried out at 30% dry weight of biomass relative to the total weight of the pretreated biomass-saccharification enzyme consortium mixture.
  • Unpretreated yellow poplar sawdust was also saccharified at 5% dry weight of biomass relative to the total weight of the pretreated biomass- saccharification enzyme consortium mixture in a shake flask.
  • Table 12 Yields following pretreatment and saccharification of yellow poplar sawdust.
  • Example 15 Ethanol Production by Yeast Fermentation on Hvdrolvzate from Very Low Ammonia-Pretreated and Saccharified Cob Biomass
  • Example 10 The same hydrolyzate used to produce 1 ,3-propanediol in Example 10 was also used to produce ethanol by yeast fermentation. This hydrolyzate was used as a source of fermentable sugar for conversion to ethanol by wild-type Saccharomyces cerevisiae in shake flasks. The hydrolyzate was used at 10% (v/v) strength, with the balance being aqueous medium consisting of 10 g/L yeast extract, and 20 g/L peptone. Yeast were cultured in 50 ml_ of medium in a 250 ml_ baffled flask. The cultures were incubated at 30 0 C with 250 rpm shaking for 24 hours. The amount of ethanol produced was measured by HPLC as described in Example 9, and results from duplicate flasks are listed in Table 13 below.
  • Table 13 Substrate utilization and product formation by fermentation with i yeast.
  • Example 10 The same hydrolyzate used to produce 1 ,3-propanediol in Example 10 was also used to produce lactic acid by fermentation with Lactobacillus brevis in shake flasks.
  • the hydrolyzate was used at 10% (v/v), with the balance being aqueous medium consisting of 5 g/L yeast extract, 10 g/L peptone, 2 g/L ammonium citrate, 5 g/L sodium acetate, 0.1 g/L MgSO 4 , 0.05 g/L MnSO 4 and 2 g/L K 2 HPO 4 and 1 g/L Tween.
  • the Lactobacillus was cultured in 50 mL broth in 250 mL baffled flasks. Duplicate cultures were incubated at 34 °C with 150 rpm shaking for 24 hours. The amount of lactic acid produced was measured by HPLC as described in Example 10 and is listed in Table 14. The two Flask 2 samples are duplicate assays of the same culture.
  • the appropriate amount of dilute ammonium hydroxide solution to give an ammonia concentration of 6 g ammonia/100 g dry weight of biomass and a solids concentration of 50 g dry weight of biomass/100 g total weight of biomass-ammonia mixture was then pumped into the reactor. Ethanol at 1 g/100 g dry weight of biomass was also added to the solution.
  • the ammonia solution was pumped through a heated loop in a water bath heated to -75 0 C, fabricated using a 2-gal Parr reactor.
  • the heated dilute ammonium hydroxide solution was injected via an injection lance into the reactor vessel and sprayed on the fractured cobs rotating and tumbling in the reactor.
  • the reactor was maintained at 75°C for 2 hr while turning at 19 rpm. At the end of that time, a vacuum (approximately 85 kPa) was applied to the reactor vessel for 30 minutes to remove ammonia and drop the temperature of the contents of the reactor to approximately 5O 0 C. Carbon dioxide was then injected into the reactor to relieve the vacuum and the reactor was pressurized to 103 kPa gauge pressure CO 2 and held at pressure for 30 min at 50 0 C.
  • a vacuum approximately 85 kPa
  • the reactor was depressurized, opened and attrition media were added.
  • the pH of the contents was adjusted to approximately 5.5 by injecting i M citric acid buffer at pH 4.8 using the injection lance, to increase the citric acid buffer strength to -75 mM, plus adding citric acid monohydrate. Not all of the ammonia was stripped off in the vacuum step nor neutralized with CO 2 .
  • the citric acid buffer was injected into the reactor following heating to 50 0 C and then the contents was allowed to equilibrate by incubating the reactor at 5O 0 C and 19 rpm for 1 hour. Injection of the citric acid buffer while rotating the reactor using the injection lance allowed for a more even spraying and distribution of the buffer on the pretreated cob particles.
  • the reactor was removed from the incubator, opened, and the pH of a sample determined. If the pH was above 5.5, then additional solid citric acid monohydrate was added and the reactor was incubated with mixing at 50 0 C for an additional hour. This process was repeated until the pH was approximately 5.5. Once the desired pH was reached, 12.9 mg/g cellulose Spezyme CP (Genencor) and 5 mg active protein /g cellulose enzyme consortium consisting of ⁇ - glucosidase, xylanase, ⁇ -xylosidase and arabinofuranosidase were loaded into the reactor. The reactor remained in the incubator at 50 0 C and 19 rpm for 72 hr. Following this pretreatment and saccharification, monomer glucose yield was 62.0% and monomer xylose yield was 31.0%. Total glucose yield was 75.2% and total xylose was 80.3%.
  • a vacuum (approximately 85 kPa) was applied to the reactor vessel before the start and the vessel was sealed off.
  • the rolling mechanism in the incubator was turned on and the rotation adjusted to 19 rpm.
  • the appropriate amount of dilute ammonium hydroxide solution to give an ammonia concentration of 6 g ammonia/100 g dry weight of biomass and a solids concentration of 50 g dry weight of biomass/100 g total weight of biomass-ammonia mixture was then pumped into the reactor.
  • the ammonia solution was pumped through a heated loop in a boiling water bath fabricated using a 2-gal Parr reactor.
  • the heated dilute ammonium hydroxide solution was injected via an injection lance into the reactor vessel and sprayed on the fractured cobs rotating and tumbling in the reactor.
  • the reactor was maintained at 95°C for 2 hr while turning at 19 rpm.
  • a vacuum approximately 85 kPa was applied to the reactor vessel for 30 minutes to remove ammonia and drop the temperature of the contents of the reactor to approximately 50 0 C.
  • Carbon dioxide was then injected into the reactor to relieve the vacuum and the reactor was pressurized to 103 kPa gauge pressure and held at pressure for 30 min at 50 0 C.
  • the reactor was depressurized, opened and the pH of the contents was adjusted to approximately 5.5 by injecting 1 M citric acid buffer, pH 4.8, into which citric acid monohydrate was added and dissolved.
  • the citric acid buffer was injected into the reactor following heating to 50 0 C and then the contents was allowed to equilibrate by incubating the reactor at 50°C and 19 rpm for 1 hour. Injection of the citric acid buffer while rotating the reactor using the injection lance allowed for a more even spraying and distribution of the buffer on the pretreated cob particles.
  • the reactor was removed from the incubator, opened, and the pH of a sample determined.
  • ammonium hydroxide solution to give an ammonia concentration of 3.2 g ammonia/10Og dry weight of biomass and NaOH to give a concentration of 1.9 g NaOH/100 g dry weight of biomass while maintaining a solids concentration of 30 g dry weight of biomass/100 g total weight of biomass-ammonia mixture was then pumped into the reactor.
  • the ammonia and additional base solution was pumped through a heated loop in a boiling water bath fabricated using a 2-gal Parr reactor.
  • the heated dilute ammonium hydroxide solution was injected via an injection lance into the reactor vessel and sprayed on the fractured cobs rotating and tumbling in the reactor. Following injection, the vacuum on the vessel was relieved to atmospheric pressure.
  • the reactor was maintained at 95°C 30 min, then the temperature was lowered to 85 0 C where it was maintained for 4 hr. At the end of that time, a vacuum (approximately 85 kPa) was applied to the reactor vessel for 30 minutes to remove ammonia and drop the temperature of the contents of the reactor to approximately 5O 0 C. Carbon dioxide was then injected into the reactor to relieve the vacuum and the reactor was pressurized to 103 kPa gauge pressure and held at pressure for 30 min at 50 0 C.
  • the reactor was depressurized, opened and the pH of the contents was adjusted to approximately 5.5 by injecting approximately 75 ml of 1 M citric acid buffer, pH 4.8, into which citric acid monohydrate was added and dissolved.
  • the citric acid buffer was injected into the reactor following heating to 50 0 C and the contents was then allowed to equilibrate by incubating the reactor at 50 0 C and 19 rpm for 1 hour. Injection of the citric acid buffer while rotating the reactor using the injection lance allowed for a more even spraying and distribution of the buffer on the pretreated cob particles.
  • the reactor was removed from the incubator, opened, and the pH of a sample determined.
  • the rolling mechanism in the incubator was turned on and rotation was adjusted to 19 rpm.
  • the appropriate amount of dilute ammonium hydroxide solution to give an ammonia concentration of 4 g ammonia/10Og dry weight of biomass and while maintaining a solids concentration of 30 g dry weight of biomass/total weight of biomass- ammonia mixture was then pumped into the reactor.
  • the dilute ammonium hydroxide solution was injected via an injection lance into the reacter vessel and sprayed on the fractured cobs rotating and tumbling in the reactor. Following injection, the vacuum on each vessel was relieved to atmospheric pressure.
  • the reactor was maintained at room temperature (22-26°C) for 24 hr.
  • a vacuum (approximately 81 kPa) was applied to the reaction vessel for 30 minutes to remove ammonia. Carbon dioxide was then injected into the reactor to relieve the vacuum and the reactor was pressurized to 103 kPa gauge pressure with CO 2 and held at pressure for 30 min at room temperature.
  • the reactor was depressurized, opened and the pH of the contents was adjusted to approximately 5.5 by adding citric acid monohydrate following heating to 5O 0 C, and then allowed to equilibrate by incubating the reactor at 50 0 C and 19 rpm.
  • the reactor was removed from the incubator, opened, and the pH of a sample determined. If the pH was above 5.5, then additional solid citric acid monohydrate was added and the reactor was incubated with mixing at 50 0 C. This process was repeated until the pH was approximately 5.5.

Abstract

Target chemicals were produced using biocatalysts that are able to ferment sugars derived from treated biomass. Sugars were obtained by pretreating biomass under conditions of high solids and low ammonia concentration, followed by saccharification.

Description

TREATMENT OF BIOMASS TO OBTAIN A TARGET CHEMICAL
This application claims the benefit of U.S. Provisional Application No. 60/670437, filed April 12, 2005.
STATEMENT OF GOVERNMENT RIGHTS
This invention was made with United States government support under Contract No. 04-03-CA-70224 awarded by the Department of Energy. The government has certain rights in this invention.
FIELD OF THE INVENTION
Methods for producing target chemicals using fermentable sugars derived from treating biomass are provided. Specifically, sugars obtained by pretreating biomass under conditions of high solids and low ammonia concentration, followed by saccharification, are used in fermentation by biocatalysts producing target chemicals.
BACKGROUND OF THE INVENTION
Cellulosic and lignocellulosic feedstocks and wastes, such as agricultural residues, wood, forestry wastes, sludge from paper manufacture, and municipal and industrial solid wastes, provide a potentially large renewable feedstock for the production of chemicals, plastics, fuels and feeds. Cellulosic and lignocellulosic feedstocks and wastes, composed of carbohydrate polymers comprising cellulose, hemicellulose, glucans and lignin are generally treated by a variety of chemical, mechanical and enzymatic means to release primarily hexose and pentose sugars, which can then be fermented to useful products. Pretreatment methods are used to make the carbohydrate polymers of cellulosic and lignocellulosic materials more readily available to saccharification enzymes. Standard pretreatment methods have historically utilized primarily strong acids at high temperatures; however due to high energy costs, high equipment costs, high pretreatment catalyst recovery costs and incompatibility with saccharification enzymes, alternative methods are being developed, such as enzymatic pretreatment, or the use of acid or base at milder temperatures where decreased hydrolysis of biomass carbohydrate polymers occurs during pretreatment, requiring improved enzyme systems to saccharify both cellulose and hemicellulose.
A number of pretreatment methods utilizing base have been proposed. Gould (Biotech, and Bioengr. (1984) 26:46-52) discloses a pretreatment method for lignocellulosic biomass using hydrogen peroxide (H2O2). The treatment is most efficient using H2O2 in an amount of at least 0.25 wt/wt with respect to substrate.
Teixeira, L., et al. (Appl. Biochem.and Biotech. (1999) 77-79:19-34) disclosed a series of biomass pretreatments using stoichiometric amounts of sodium hydroxide and ammonium hydroxide, with very low biomass concentration. The ratio of solution to biomass is 14:1.
Elshafei, A. et al. (Bioresource Tech. (1991) 35:73-80) examined the pretreatment of corn stover utilizing NaOH.
Kim, T. and Y. Lee (Bioresource Technology (2005) 96:2007-2013) report the use of high amounts of aqueous ammonia for the pretreatment of corn stover.
Patent Application WO2004/081185 discusses methods for hydrolyzing lignocellulose, comprising contacting the lignocellulose with a chemical; the chemical may be a base, such as sodium carbonate or potassium hydroxide, at a pH of about 9 to about 14, under moderate conditions of temperature, pressure and pH.
U.S. Patent Nos. 5,916,780 and 6,090,595, describe a pretreatment process wherein a specified ratio of arabinoxylan to total nonstarch polysaccharides (AX/NSP) is assessed and used to select the feedstock.
In order to be an economically competitive process, a commercial process for the production of chemicals from a renewable resource biomass requires the hydrolysis of carbohydrates in lignocellulosic biomass to provide high yields of sugars at high concentrations, using low amounts of chemicals, to produce a source of fermentable sugars with low toxicity that are used by biocatalysts that produce value-added target chemicals.
SUMMARY OF THE INVENTION
The present invention provides methods for producing target chemicals that make use of biomass. The process of the invention involves pretreatment of biomass, at relatively high concentration, with a low concentration of ammonia relative to the dry weight of biomass. Following pretreatment, the biomass is treated with a saccharification enzyme consortium to produce fermentable sugars. The sugars are then contacted with a biocatalyst that can ferment the sugars and produce a target chemical. In one embodiment of the invention, the method comprises: a) contacting biomass with an aqueous solution comprising ammonia, wherein the ammonia is present at a concentration at least sufficient to maintain alkaline pH of the biomass-aqueous ammonia mixture but wherein said ammonia is present at less than about 12 weight percent relative to dry weight of biomass, and further wherein the dry weight of biomass is at a high solids concentration of at least about 15 weight percent relative to the weight of the biomass-aqueous ammonia mixture; b) contacting the product of step (a) with a saccharification enzyme consortium under suitable conditions to produce fermentable sugars; and c) contacting the product of step b) with at least one biocatalyst able to ferment the sugars to produce the target chemical under suitable fermentation conditions.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the growth of Zymomonas mobilis 8b (described in US Patent Application Publication 2003/0162271 A1 , examples IV, Vl and XII) in the presence or absence of acetamide and acetic acid. DETAILED DESCRIPTION OF THE INVENTION
Applicants specifically incorporate the entire contents of all cited references in this disclosure. Further, when an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the invention be limited to the specific values recited when defining a range. The present invention provides methods for producing target chemicals that make use of biomass in the following manner: sugars are derived from biomass, which are then used as a carbon source for the growth of microorganisms that can make target chemicals as products of their metabolism. The sugars are released from the biomass by pretreating the biomass, at relatively high concentration, with a relatively low concentration of ammonia relative to the dry weight of the biomass. The ammonia-treated biomass is then digested with a saccharification enzyme consortium to produce fermentable sugars. The sugars are used as a fermentation substrate for growth of a microorganism, or bioctalyst, that is able to produce a target chemical. Definitions:
In this disclosure, a number of terms are used. The following definitions are provided:
The term "fermentable sugar" refers to oligosaccharides and monosaccharides that can be used as a carbon source by a microorganism in a fermentation process.
The term "lignocellulosic" refers to a composition comprising both lignin and cellulose. Lignocellulosic material may also comprise hemicellulose. The term "cellulosic" refers to a composition comprising cellulose.
By "dry weight" of biomass is meant the weight of the biomass having all or essentially all water removed. Dry weight is typically measured according to American Society for Testing and Materials (ASTM) Standard E1756-01 (Standard Test Method for Determination of Total Solids in Biomass) or 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. Chemical is used in a broad sense and includes molecules such as proteins, including, for example, peptides, enzymes and antibodies.
A target chemical that is "derivable from biomass" is a target chemical produced by a process whereby biomass is hydrolyzed to release fermentable sugars, and the fermentable sugars are fermented using at least one biocatalyst to produce a desired target chemical.
The terms "plasticizer" and "softening agent" refer to materials that cause a reduction in the cohesive intermolecular forces along or between polymer chains. Such materials may act, for example, to decrease crystallinity, or disrupt bonds between lignin and non-lignin carbohydrate fibers (e.g., cellulose or hemicellulose).
The term "saccharification" refers to the production of fermentable sugars from polysaccharides.
"Suitable conditions to produce fermentable sugars" refers to conditions such as pH, composition of medium, and temperature under which saccharification enzymes are active.
"Suitable fermentation conditions" refers to conditions that support the growth and target chemical production by a biocatalyst. Such conditions may include pH, nutrients and other medium components, temperature, atmosphere, and other factors.
The term "pretreated biomass" means biomass that has been subjected to pretreatment prior to saccharification. "Biomass" refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Biomass may also comprise additional components, such as protein and/or lipid. According to the present method, biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass could comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. Examples of biomass include, but are not limited to, corn grain, corn cobs, crop residues such as com husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from processing of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers and animal manure. In one embodiment, biomass that is useful for the present method includes biomass that has a relatively high carbohydrate value, is relatively dense, and/or is relatively easy to collect, transport, store and/or handle. In one embodiment of the invention, biomass that is useful includes corn cobs, corn stover and sugar cane bagasse.
For the purposes of this invention, an "aqueous solution comprising ammonia" refers to the use of ammonia gas (NH3), compounds comprising ammonium ions (NH4 +) such as ammonium hydroxide or ammonium sulfate, compounds that release ammonia upon degradation such as urea, and combinations thereof in an aqueous medium.
The concentration of ammonia used in the present method is minimally a concentration that is sufficient to maintain the pH of the biomass-aqueous ammonia mixture alkaline and maximally less than about 12 weight percent relative to dry weight of biomass. This low concentration of ammonia is sufficient for pretreatment, and the low concentration may also be less than about 10 weight percent relative to dry weight oτ Diomass. A very low concentration of 6 percent ammonia relative to dry weight of biomass, or less, also may be used for pretreatment. By alkaline is meant a pH of greater than 7.0. Particularly suitable is a pH of the biomass-aqueous ammonia mixture that is greater than 8. In one embodiment, ammonia is present at less than about 10 weight percent relative to dry weight of biomass. Particularly suitable is ammonia at less than about 6 weight percent relative to dry weight of biomass.
Ammonia as used in the present process provides advantages over other bases. Ammonia partitions into a liquid phase and vapor phase. Gaseous ammonia can diffuse more easily through biomass than a liquid base, resulting in more efficacious pretreatment at lower concentrations. Ammonia also is shown herein in Example 11 to compete with hydrolysis, via ammonolysis, of acetyl esters in biomass to form acetamide. Acetamide is less toxic than acetate to certain fermentation organisms, such as Zymomonas mobilis (as demonstrated herein in Example 12). Thus conversion of acetyl esters to acetamide rather than to acetic acid reduces the need to remove acetic acid. The use of ammonia also reduces the requirement to supplement growth medium used during fermentation with a nitrogen source. In addition, ammonia is a low-cost material and thus provides an economical process. Ammonia can also be recycled to the pretreatment reactor during pretreatment or following pretreatment, thus enabling a more economical process. For example, following pretreatment, as the temperature is decreased to that suitable for saccharification, ammonia gas may be released, optionally in the presence of a vacuum, and may be recycled. In a continuous process, ammonia may be continuously recycled.
According to the present method, the aqueous solution comprising ammonia may optionally comprise at least one additional base, such as sodium hydroxide, sodium carbonate, potassium hydroxide, potassium carbonate, calcium hydroxide and calcium carbonate. The at least one additional base may be added in an amount that is combined with ammonium to form an amount of total base that is less than about 20 weight percent relative to dry weight of biomass. Preferably the total second base plus ammonia is in an amount that is less than about 15 weight percent. Additional base(s) may be utilized, for example, to neutralize acids in biomass, to provide metal ions for the saccharification enzymes, or to provide metal ions for the fermentation growth medium.
In the present method, the dry weight of biomass is at an initial concentration of at least about 15% up to about 80% of the weight of the biomass-aqueous ammonia mixture. More suitably, the dry weight of biomass is at a concentration of from about 15% to about 60% of the weight of the biomass-aqueous ammonia mixture. The percent of biomass in the biomass-aqueous ammonia mixture is kept high to minimize the need for concentration of sugars resulting from saccharification of the pretreated biomass, for use in fermentation. The high biomass concentration also reduces the total volume of pretreatment material, making the process more economical.
The biomass may be used directly as obtained from the source, or energy may be applied to the biomass to reduce the size, increase the exposed surface area, and/or increase the availability of cellulose, hemicellulose, and/or oligosaccharides present in the biomass to ammonia and to saccharification enzymes used in the second step of the method. Energy means useful for reducing the size, increasing the exposed surface area, and/or increasing the availability of cellulose, hemicellulose, and/or oligosaccharides present in the biomass to ammonia and to saccharification enzymes include, but are not limited to, milling, crushing, grinding, shredding, chopping, disc refining, ultrasound, and microwave. This application of energy may occur before or during pretreatment, before or during saccharification, or any combination thereof.
Pretreatment of biomass with ammonia solution is carried out in any suitable vessel. Typically the vessel is one that can withstand pressure, has a mechanism for heating, and has a mechanism for mixing the contents. Commercially available vessels include, for example, the Zipperclave® reactor (Autoclave Engineers, Erie, PA), the Jaygo reactor (Jaygo Manufacturing, Inc., Mahwah, NJ), and a steam gun reactor (described in General Methods; Autoclave Engineers, Erie, PA). Much larger scale reactors with similar capabilities may be used. Alternatively, the biomass and ammonia solution may be combined in one vessel, then transferred to another reactor. Also biomass may be pretreated in one vessel, then further processed in another reactor such as a steam gun reactor (described in General Methods; Autoclave Engineers, Erie, PA).
Prior to contacting the biomass with an aqueous solution comprising ammonia, vacuum may be applied to the vessel containing the biomass. By evacuating air from the pores of the biomass, better penetration of the ammonia into the biomass may be achieved. The time period for applying vacuum and the amount of negative pressure that is applied to the biomass will depend on the type of biomass and can be determined empirically so as to achieve optimal pretreatment of the biomass (as measured by the production of fermentable sugars following saccharification).
The contacting of the biomass with an aqueous solution comprising ammonia is carried out at a temperature of from about 4 0C to about 200 °C. Initial contact of the biomass with ammonia at 4 0C, allowing impregnation at this temperature, was found to increase the efficiency of saccharification over non-pretreated native biomass. In another embodiment, said contacting of the biomass is carried out at a temperature of from about 75 °C to about 150 °C. In still another embodiment, said contacting of the biomass is carried out at a temperature of from greater than 90 0C to about 150 °C. The contacting of the biomass with an aqueous solution comprising ammonia is carried out for a period of time up to about 25 hrs. Longer periods of pretreatment are possible, however a shorter period of time may be preferable for practical, economic reasons. Typically a period of ammonia contact treatment is about 8 hours or less. Longer periods may provide the benefit of reducing the need for application of energy for breaking-up the biomass, therefore, a period of time up to about 25 hrs. may be preferable. In one embodiment, the pretreatment process may be performed at a relatively high temperature for a relatively short period of time, for example at from about 100 0C to about 150 °C for about 5 min to about 2 hr. In another embodiment, the pretreatment process may be performed at a lower temperature for a relatively long period of time, for example from about 75 °C to about 100 °C for about 2 hr to about 8 hr. In still another embodiment, the pretreatment process may be performed at room temperature (approximately 22-26 °C) for an even longer period of time of about 24 hr. Other temperature and time combinations intermediate to these may also be used.
For the pretreatment process, the temperature, time for pretreatment, ammonia concentration, concentration of one or more additional bases, biomass concentration, biomass type and biomass particle size are related; thus these variables may be adjusted as necessary to obtain an optimal product to be contacted with a saccharification enzyme consortium.
A plasticizer, softening agent, or combination thereof, such as polyols (e.g., glycerol, ethylene glycol), esters of polyols (e.g., glycerol monoacetate), glycol ethers (e.g., diethylene glycol), acetamide, ethanol, and ethanolamines, may be added in the pretreatment process (i.e., step (a)). A plasticizer may be added as a component of the aqueous ammonia solution, as a separate solution, or as a dry component.
The pretreatment reaction may be performed in any suitable vessel, such as a batch reactor or a continuous reactor. One skilled in the art will recognize that at higher temperatures (above 1000C), a pressure vessel is required. The suitable vessel may be equipped with a means, such as impellers, for agitating the biomass-aqueous ammonia mixture. Reactor design is discussed in Lin, K.-H., and Van Ness, H. C. (in Perry, R.H. and Chilton, C. H. (eds), Chemical Engineer's Handbook, 5th Edition (1973) Chapter 4, McGraw-Hill, NY). The pretreatment reaction may be carried out as a batch process, or as a continuous process.
It is well known to those skilled in the art that a nitrogen source is required for growth of microorganisms during fermentation; thus the use of ammόήϊa during pretfeatment provides a nitrogen source and reduces or eliminates the need to supplement the growth medium used during fermentation with a nitrogen source. If the pH of the pretreatment product exceeds that at which saccharification enzymes are active, or exceeds the range suitable for microbial growth in fermentation, acids may be utilized to reduce pH. The amount of acid used to achieve the desired pH may result in the formation of salts at concentrations that are inhibitory to saccharification enzymes or to microbial growth. In order to reduce the amount of acid required to achieve the desired pH and to reduce the raw material cost of ISIH3 in the present pretreatment process, ammonia gas may be evacuated from the pretreatment reactor and recycled. Typically, at least a portion of the ammonia is removed, which reduces the pH but leaves some nitrogen that provides this nutrient for use in subsequent fermentation. In order to obtain sufficient quantities of sugars from biomass, the biomass may be pretreated with an aqueous ammonia solution one time or more than one time. Likewise, a saccharification reaction can be performed one or more times. Both pretreatment and saccharification processes may be repeated if desired to obtain higher yields of sugars. To assess performance of the pretreatment and saccharification processes, separately or together, the theoretical yield of sugars derivable from the starting biomass can be determined and compared to measured yields. Saccharification: Following pretreatment, the product comprises a mixture of ammonia, partially degraded biomass and fermentable sugars. Prior to further processing, ammonia may be removed from the pretreated biomass by applying a vacuum. Removing ammonia lowers the pH, and thus less neutralizing acid is used to obtain the desired pH for saccharification and fermentation. This results in a lower salt load in the pretreatment mixture. Typically some ammonia remains, which is desired to provide a nitrogen source for fermentation. The pfefreaϊment mixture is then further hydrolyzed in the presence of a saccharification enzyme consortium to release oligosaccharides and/or monosaccharides in a hydrolyzate. Saccharification enzymes and methods for biomass treatment are reviewed in Lynd, L. R., et al. (Microbiol. MoI. Biol. Rev. (2002) 66:506-577). In one preferred embodiment, the entire pretreatment mixture comprising both soluble and insoluble fractions is utilized in the saccharification reaction.
In another embodiment, prior to saccharification, the aqueous fraction comprising ammonia and solubilized sugars may be separated from insoluble particulates remaining in the mixture. Methods for separating the soluble from the insoluble fractions include, but are not limited to, decantation and filtration. The insoluble particulates may be recycled to the pretreatment reactor. The insoluble particulates may optionally be washed with an aqueous solvent (e.g., water) to remove adsorbed sugars prior to being recycled to the pretreatment reactor. The insoluble fraction may then be subjected to additional treatment with aqueous ammonia solution as described above for pretreatment, followed by saccharification with a saccharification enzyme consortium. The soluble fraction may also be concentrated prior to saccharification using a suitable process, such as evaporation.
Prior to saccharification, the pretreatment product may be treated to alter the pH, composition or temperature such that the enzymes of the saccharification enzyme consortium will be active, thus providing suitable conditions to produce fermentable sugars. The pH may be altered through the addition of acids in solid or liquid form. Alternatively, carbon dioxide (CO2), which may be recovered from fermentation, may be utilized to lower the pH. For example, CO2 may be collected from a fermenter and fed, such as by bubbling, into the pretreatment product while monitoring the pH, until the desired pH is achieved. The temperature may be brought to a temperature that is compatible with saccharification enzyme activity, as noted below. Any cofactors required for activity of enzymes used in saccharification may be added. The saccharification enzyme consortium comprises one or more enzymes selected primarily, but not exclusively, from the group "glycosidases" which hydrolyze the ether linkages of di-, oligo-, and polysaccharides and are found in the enzyme classification EC 3.2.1.x (Enzyme Nomenclature 1992, Academic Press, San Diego, CA with Supplement 1 (1993), Supplement 2 (1994), Supplement s (1995, Supplement 4 (1997) and Supplement 5 [in Eur. J. Biochem. (1994) 223:1- 5, Eur. J. Biochem. (1995) 232:1-6, Eur. J. Biochem. (1996) 237:1-5, Eur. J. Biochem. (1997) 250:1-6, and Eur. J. Biochem. (1999) 264:610-650, respectively]) of the general group "hydrolases" (EC 3.). Glycosidases useful in the present method can be categorized by the biomass component that they hydrolyze. Glycosidases useful for the present method include cellulose-hydrolyzing glycosidases (for example, cellulases, endoglucanases, exoglucanases, cellobiohydrolases, β- glucosidases), hemicellulose-hydrolyzing glycosidases (for example, xylanases, endoxylanases, exoxylanases, β-xylosidases, arabinoxylanases, mannases, galactases, pectinases, glucuronidases), and starch-hydrolyzing glycosidases (for example, amylases, α-amylases, β-amylases, glucoamylases, α-glucosidases, isoamylases). In addition, it may be useful to add other activities to the saccharification enzyme consortium such as peptidases (EC 3.4.x.y), lipases (EC 3.1.1.x and 3.1.4.x), ligninases (EC 1.11.1.x), and feruloyl esterases (EC 3.1.1.73) to help release polysaccharides from other components of the biomass. It is well known in the art that microorganisms that produce polysaccharide- hydrolyzing enzymes often exhibit an activity, such as cellulose degradation, that is catalyzed by several enzymes or a group of enzymes having different substrate specificities. Thus, a "cellulase" from a microorganism may comprise a group of enzymes, all of which may contribute to the cellulose-degrading activity. Commercial or non- commercial enzyme preparations, such as cellulase, may comprise numerous enzymes depending on the purification scheme utilized to obtain the enzyme. Thus, the saccharification enzyme consortium of the present method may comprise enzyme activity, such as "cellulase", However it is "recognized that this activity may be catalyzed by more than one enzyme.
Saccharification enzymes may be obtained commercially, such as Spezyme® CP cellulase (Genencor International, Rochester, NY) and Multifect® xylanase (Genencor). In addition, saccharification enzymes may be produced biologically, including using recombinant microorganisms. One skilled in the art would know how to determine the effective amount of enzymes to use in the consortium and adjust conditions for optimal enzyme activity. One skilled in the art would also know how to optimize the classes of enzyme activities required within the consortium to obtain optimal saccharification of a given pretreatment product under the selected conditions.
Preferably the saccharification reaction is performed at or near the temperature and pH optima for the saccharification enzymes. The temperature optimum used with the saccharification enzyme consortium in the present method ranges from about 15 °C to about 100 °C. In another embodiment, the temperature optimum ranges from about 20 °C to about 80 0C. The pH optimum can range from about 2 to about 11. In another embodiment, the pH optimum used with the saccharification enzyme consortium in the present method ranges from about 4 to about 10.
The saccharification can be performed for a time of about several minutes to about 120 hr, and preferably from about several minutes to about 48 hr. The time for the reaction will depend on enzyme concentration and specific activity, as well as the substrate used and the environmental conditions, such as temperature and pH. One skilled in the art can readily determine optimal conditions of temperature, pH and time to be used with a particular substrate and saccharification enzyme(s) consortium.
The saccharification can be performed batch-wise or as a continuous process. The saccharification can also be performed in one step, or in a number of steps. For example, different enzymes required for saccharification may exhibit different pH or temperature optima. A primary treatment can be performed with enzyme(s) at one temperature and pH, followed by secondary or tertiary (or more) treatments with different enzyme(s) at different temperatures and/or pH. In addition, treatment with different enzymes in sequential steps may be at the same pH and/or temperature, or different pHs and temperatures, such as using hemicellulases stable and more active at higher pHs and temperatures followed by cellulases that are active at lower pHs and temperatures..
The degree of solubilization of sugars from biomass following saccharification can be monitored by measuring the release of monosaccharides and oligosaccharides. Methods to measure monosaccharides and oligosaccharides are well known in the art. For example, the concentration of reducing sugars can be determined using the 1 ,3-dinitrosalicylic (DNS) acid assay (Miller, G. L., Anal. Chem. (1959) 31 :426-428). Alternatively, sugars can be measured by HPLC using an appropriate column as described herein in the General Methods section. Fermentable sugars released from biomass can be used by suitable microorganisms to produce target chemicals. Following saccharification, but prior to fermentation, the saccharification mixture may be concentrated by evaporation, for example, to increase the concentration of fermentable sugars. Optionally, liquid in the saccharification product may be separated from solids in a batch or continuous method. Optionally, the liquid or the entire saccharification product may be sterilized prior to fermentation. Depending on the microorganism(s) used during fermentation and the pH used during saccharification, the pH may be adjusted to that suitable for fermentation. In addition, the saccharification mixture may be supplemented with additional nutrients required for microbial growth. Supplements may include, for example, yeast extract, specific amino acids, phosphate, nitrogen sources, salts, and trace elements. Components required for production of a specific product made by a specific biocatalyst may also be included, such as an antibiotic to maintain a plasmid or a cofactor required in an enzyme catalyzed reaction. Also additional sugars may be included to increase the total sugar concentration. The saccharification mixture may be used as a component of a fermentation broth, for example, making up" between about 100% and about 10% of the final medium. Suitable fermentation conditions are achieved by adjusting these types of factors for the growth and target chemical production by a biocatalyst. Temperature and/or headspace gas may also be adjusted, depending on conditions useful for the fermentation microorganism(s). Fermentation may be aerobic or anaerobic. Fermentation may occur subsequent to saccharification, or may occur concurrently with saccharification by simultaneous saccharification and fermentation (SSF). SSF can keep the sugar levels produced by saccharification low, thereby reducing potential product inhibition of the saccharification enzymes, reducing sugar availability for contaminating microorganisms, and improving the conversion of pretreated biomass to monosaccharides and/or oligosaccharides. Target chemicals that may be produced by fermentation include, for example, acids, alcohols, alkanes, alkenes, aromatics, 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, 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.
The fermentation of sugars to target chemicals may be carried out by one or more appropriate biocatalysts in single or multistep fermentations. Biocatalysts may be microorganisms selected from bacteria, filamentous fungi and yeast. Biocatalysts may be wild type microorganisms or recombinant microorganisms, and include Escherichia, Zymomonas, Saccharomyces, Candida, Pichia, Streptomyces, Bacillus, Lactobacillus, and Clostridium. In another embodiment, biocatalysts may be selected from the group consisting of recombinant Escherichia coli, Zymomonas mobilis, Bacillus stearothermophilus, Saccharomyces cerevisiθθ, Clostridia thermocellum, Thermoanaerobacterium saccharolyticum, and Pichia stipitis.
Many biocatalysts used in fermentation to produce target chemicals have been described and others may be discovered, produced through mutation, or engineered through recombinant means. Any biocatalyst that uses fermentable sugars produced in the present method may be used to make the target chemical(s) that it is known to produce, by fermentation in the present method. Fermentation of carbohydrates to acetone, butanol, and ethanol
(ABE fermentation) by solventogenic Clostridia is well known (Jones and Woods (1986) Microbiol. Rev. 50:484-524). A fermentation process for producing high levels of butanol, also producing acetone and ethanol, using a mutant strain of Clostridium acetobutylicum is described in US 5192673. The use of a mutant strain of Clostridium beijerinckii to produce high levels of butanol, also producing acetone and ethanol, is described in US 6358717. Genetically modified strains of E. coli have also been used as biocatalysts for ethanol production (Underwood et al., (2002) Appl. Environ. Microbiol.68:6263-6272). A genetically modified strain of Zymomonas mobilis that has improved production of ethanol is described in US 2003/0162271 A1.
Lactic acid has been produced in fermentations by recombinant strains of E. coli (Zhou et al., (2003) Appl. Environ. Microbiol. 69:399- 407), natural strains of Bacillus (US20050250192), and Rhizopus oryzae (Tay and Yang (2002) Biotechnol. Bioeng. 80:1-12). Recombinant strains of E. coli have been used as biocatalysts in fermentation to produce 1 ,3 propanediol (US 6013494, US 6514733), and adipic acid (Niu et al., (2002) Biotechnol. Prog. 18:201-211). Acetic acid has been made by fermentation using recombinant Clostridia (Cheryan et al., (1997) Adv. Appl. Microbiol. 43:1-33), and newly identified yeast strains (Freer (2002) World J. Microbiol. Biotechnol. 18:271-275). Production of succinic acid by recombinant E. coli and other bacteria is disclosed in US 6159738, and by mutant recombinant E. coli in Lin et al., (2005) Metab. Eng. 7:116-127). Pyruvic acid has been produced by mutant Torulopsis glabrata yeast (Li et al., (2001 ) Appl. Microbiol. Technol. 55:680-685) and by mutant E. coli (Yokota et al., (1994) Biosci. Biotech. Biochem. 58:2164-2167). Recombinant strains of E. coli have been used as biocatalysts for production of para-hydroxycinnamic acid (US20030170834) and quinic acid (US20060003429).
A mutant of Propionibacterium acidipropionici has been used in fermentation to produce propionic acid (Suwannakham and Yang (2005) Biotechnol. Bioeng. 91:325-337), and butyric acid has been made by Clostridium tyrobutyricum (Wu and Yang (2003) Biotechnol. Bioeng. 82:93-102). Propionate and propanol have been made by fermentation from threonine by Clostridium sp. strain 17cr1 (Janssen (2004) Arch. Microbiol. 182:482-486). A yeast-like Aureobasidium pullulans has been used to make gluconic acid (Anantassiadis et al., (2005) Biotechnol. Bioeng. 91 :494-501 ), by a mutant of Aspergillis niger (Singh et al., (2001 ) Indian J. Exp. Biol. 39:1136-43). 5-keto-D-gluconic acid was made by a mutant of Gluconobacter oxydans (Elfari et al., (2005) Appl Microbiol. Biotech. 66:668-674), itaconic acid was produced by mutants of Aspergillus terreus (Reddy and Singh (2002) Bioresour. Technol. 85:69 - 71), citric acid was produced by a mutant Aspergillus niger strain (Ikram- Ul-Haq et al., (2005) Bioresour. Technol. 96:645-648), and xylitol was produced by Candida guilliermondii FTI 20037 (Mussatto and Roberto
(2003) J. Appl. Microbiol. 95:331-337). 4-hydroxyvalerate-containing biopolyesters, also containing significant amounts of 3-hydroxybutyric acid 3-hydroxyvaleric acid, were produced by recombinant Pseudomonas putida and Ralstonia eutropha (Gorenflo et al., (2001 ) Biomacromolecules 2:45-57). L-2,3-butanediol was made by recombinant E. coli (Ui et al.,
(2004) Lett. Appl. Microbiol. 39:533-537).
Production of amino acids by fermentation has been accomplished using auxotrophic strains and amino acid analog-resistant strains of
Corynebacterium, Brevibacterium, and Serratia. For example, production of histidine using a strain resistant to a histidine analog is described in Japanese Patent Publication No. 8596/81 and using a recombinant strain is described Tn EP Υ36359. Production of tryptophan using a strain resistant to a tryptophan analog is described in Japanese Patent Publication Nos. 4505/72 and 1937/76. Production of isoleucine using a strain resistant to an isoleucine analog is described in Japanese Patent Publication Nos. 38995/72, 6237/76, 32070/79. Production of phenylalanine using a strain resistant to a phenylalanine analog is described in Japanese Patent Publication No. 10035/81. Production of tyrosine using a strain requiring phenylalanine for growth, resistant to tyrosine (Agr. Chem. Soc. Japan 50 (1) R79-R87 (1976), or a recombinant strain (EP263515, EP332234), and production of arginine using a strain resistant to an L-arginine analog (Agr. Biol. Chem. (1972) 36:1675-1684, Japanese Patent Publication Nos. 37235/79 and 150381/82) have been described. Phenylalanine was also produced by fermentation in Eschericia co// strains ATCC 31882, 31883, and 31884. Production of glutamic acid in a recombinant coryneform bacterium is described in US 6962805. Production of threonine by a mutant strain of E. coli is described in Okamoto and lkeda (2000) J. Biosci Bioeng. 89:87-79. Methionine was produced by a mutant strain of Corynebacterium lilium (Kumar et al, (2005) Bioresour. Technol. 96: 287-294). Useful peptides, enzymes, and other proteins have also been made by biocatalysts (for example, in US6861237, US6777207, US6228630). The pretreatment and saccharification of biomass to fermentable sugars, followed by fermentation of the sugars to a target chemical is exemplified in Example 9 herein for the production of ethanol from pretreated corn cobs using Z mobilis as the biocatalyst for the fermentation of sugars to ethanol. The present method may also be used for the production of 1 ,3-propanediol from biomass. Biomass undergoes pretreatment and saccharification according to the present method; following (or during) saccharification, E. coli is used to produce 1 ,3- propanediol as described in Example 10 herein.
Target chemicals produced in fermentation by biocatalysts may be recovered using various methods known in the art. Products may be separated from other fermentation components by centrifugation, filtration, micrόfiltratϊόn , "ancJ naϊnδf iltration . Products may be extracted by ion exchange, solvent extraction, or electrodialysis. Flocculating agents may be used to aid in product separation. As a specific example, bioproduced 1-butanol may be isolated from the fermentation medium using methods known in the art for ABE fermentations (see for example, Durre, Appl. Microbiol. Biotechnol. 49:639-648 (1998), Groot et al., Process. Biochem. 27:61-75 (1992), and references therein). For example, solids may be removed from the fermentation medium by centrifugation, filtration, decantation, or the like. Then, the 1-butanol may be isolated from the fermentation medium using methods such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, or pervaporation. Purification of 1 ,3-propanediol from fermentation media may be accomplished, for example, by subjecting the reaction mixture to extraction with an organic solvent, distillation, and column chromatography (U.S. 5,356,812). A particularly good organic solvent for this process is cyclphexane (U.S. 5,008,473). Amino acids may be collected from fermentation medium by methods such as ion-exchange resin adsorption and/or crystallization.
EXAMPLES GENERAL METHODS AND MATERIALS The following abbreviations are used:
"HPLC" is High Performance Liquid Chromatography, "C" is Centigrade, "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 millimolar, "cm" is centimeter, "g" is gram, "kg" is kilogram, "wt" is weight, "hr" is hour, "temp" or "T" is temperature, "theoret" is theoretical, "pretreat" is pretreatment, "DWB" is dry weight of biomass.
Sulfuric acid, ammonium hydroxide, acetic acid, acetamide, yeast extract, 2-morpholinoethanesulfonic acid (MES), potassium phosphate, glucose, xylose, tryptone, sodium chloride and citric acid were obtained from Sigma-Aldrich (St. Louis, MO). Pretreatment reactors
Zipperclave® reactor
The 4-liter Zipperclave® reactor (Autoclave Engineers, Erie, PA) is a batch pressure vessel equipped with a 2.5-liter Hastelloy® pail for the biomass charge and an agitator to mix the biomass. The reactor vessel is encircled by an electrical heater controlled at the desired pretreatment temperature. Direct steam injection is also used to rapidly bring the biomass up to pretreatment temperature. Steam pressure is adjusted and controlled to maintain the desired pretreatment temperature. Steam condensate formed by heating the Zipperclave® reactor head plate, vessel and outside of the pail drain to a reservoir formed between the pail and the inner wall of the reactor to prevent excessive dilution of the pretreated slurry.
Jaygo reactor The Jaygo reactor is a 130-liter (approximately 51 cm diameter x 91 cm length), horizontal paddle type reactor (Jaygo Manufacturing, Inc., Mahwah, NJ) fabricated of Hastelloy® C-22 alloy. The reactor is equipped with a steam jacket capable of heating to approximately 177 0C (862 kPa). Direct steam injection is also used to rapidly bring the biomass up to pretreatment temperature. Steam pressure is adjusted and controlled to maintain the desired pretreatment temperature. Numerous ports allow injection of other solvents and hot liquids.
Steam gun reactor batch digestion system
The 4-liter steam gun reactor (Autoclave Engineers, Erie, PA) is a steam-jacketed reactor consisting of a length of 102 mm schedule 80
Hastelloy® pipe closed by two ball valves. Additional electrical heaters are placed on all exposed, non-jacketed surfaces of the reactor and controlled to the pretreatment set point temperature. Direct steam injection is also used to rapidly bring the biomass up to pretreatment temperature. Steam pressure is adjusted and controlled to maintain the desired pretreatment temperature. The bottom of the reactor is necked down to 51 mm. All pretreated material exits through a replaceable die at the bottom of the reactor and is collected in a nylon (Hotfill®) 0.21 m3 bag supported within a heavy walled, jacketed, and cooled flash tank.
Disc refiner The disc refiner is a Sprout Waldron model 30.5 cm refiner (Andritz,
Inc., Muncy, PA) equipped with a 11 kW electric motor. The gap between the stationary and rotating plates is variable. The feed auger speed is also variable, from 0-88 rpm. The inlet to the refiner was modified with six injection ports to allow the introduction of steam, hot water, or other sweep gases and liquids just ahead of the rotating refiner plate. The refiner was equipped with plates (Durametal, Corp., Tulatin, OR) in either pattern D2A506 in Ni-Hard or pattern 18034-A in Ni-Hard.
Pretreatment and Enzymatic Hydrolysis Reactor (PEHR) The 9L PEHReactor (constructed at NREL, Golden, CO; see co- pending US patent application CL3447) has an approximately 15 cm x 51 cm stainless steel reaction vessel with an injection lance for introduction of processing reactants. The injection lance is connected using a rotary joint to a port in a cover on one end of the vessel, which has an additional port for vessel access. Four baffles run the length of the vessel wall, and are attached perpendicularly to the wall. The baffles and twenty-two ceramic attrition media cylinders of 3.2 cm X 3.2 cm (E. R. Advanced Ceramics, East Palestine, OH), free floating in the vessel, apply mechanical mixing of biomass and reactant as the vessel is rotated, promoting assimilation of reactant into the biomass. The PEHReactor is placed on a Bellco Cell- Production Roller Apparatus (Bellco Technology, Vineland, NJ) which provides a mechanism for rotation, and the reactor with roller apparatus is housed in a temperature controlled chamber which provides heat. Vacuum and pressure may be applied to the reaction vessel by attaching external sources to the lance-connected port in the cover. Analytical methods
Cellulose quantitation
The amount of cellulose in each starting biomass sample was determined using methods well known in the art, such as ASTM E1758-01 "Standard method for the determination of carbohydrates by HPLC".
Measurement of sugar, acetamide, lactic acid and acetic acid content
Soluble sugars (glucose, cellobiose, xylose, galactose, arabinose and mannose), acetamide, lactic acid and acetic acid in saccharification liquor were measured by HPLC (Agilent Model 1100, Agilent
Technologies, Palo Alto, CA) using Bio-Rad HPX-87P and Bio-Rad HPX- 87H columns (Bio-Rad Laboratories, Hercules, CA) with aft appropriate guard columns. The sample pH was measured and adjusted to 5-6 with sulfuric acid if necessary. The sample was then passed through a 0.2 μm syringe filter directly into an HPLC vial. The HPLC run conditions were as follows:
Biorad Aminex HPX-87P (for carbohydrates):
Injection volume: 10 - 50 μL, dependent on concentration and detector limits Mobile phase: HPLC grade water, 0.2 μm filtered and degassed
Flow rate: 0.6 mL / minute
Column temperature: 80 - 85 °C, guard column temperature <60°C
Detector temperature: as close to main column temperature as possible Detector: refractive index
Run time: 35 minute data collection plus 15 minute post run (with possible adjustment for later eluting compounds)
Biorad Aminex HPX-87H (for carbohydrates, acetamide, lactic acid, acetic acid, and ethanol)
Injection volume: 5-10 μL, dependent on concentration and detector limits
Mobile phase: .01 N Sulfuric acid, 0.2 μm filtered and degassed Flow rate: 076 rn L / minute Column temperature: 55 °C
Detector temperature: as close to column temperature as possible Detector: refractive index Run time: 25 - 75 minute data collection
After the run, concentrations in the sample were determined from standard curves for each of the compounds.
Example 1
Stover Pretreatment at High Biomass Concentration, High Temperature and Comparison of Ammonia Concentrations The Zipperclave® reactor vessel and head plate were preheated to the target pretreatment temperature before introduction of the biomass charge by cycling steam into the reactor and venting several times.
Condensate formed during preheating was removed by vacuum aspiration before pretreatment. The Hastelloy® pail was loaded with 0.635-cm (1/4- in.) milled stover (100 g, dry weight basis) and inserted into the pre- warmed reactor. The reactor agitator was set to 20 rpm while a vacuum (approximately 85 kPa) was applied to the vessel interior and biomass charge. Ammonium hydroxide solution of the necessary strength to give a dry weight of biomass concentration of 30 weight percent relative to the weight of the biomass-aqueous ammonia mixture, as well as the desired ammonia concentration listed in Table 1 , was injected near the bottom of the vessel with a spray type nozzle. Test samples had a final ammonia concentration of 12% relative to dry weight of biomass, while samples with a final ammonia concentration of 35% relative to dry weight of biomass were used as a comparison. When the temperature of the biomass charge reached 50 0C, steam was introduced near the bottom of the reactor to fluidize and raise the temperature of the biomass charge to either 140 °C or 170 °C. At the end of pretreatment, the reactor was depressurized through a vent condenser, and a vacuum (approximately 85 kPa) was applied for 3 minutes to lower the temperature and remove additional ammonia from the pretreated slurry prior to opening the reactor and recovering the pretreated biomass.
Whole, unwashed pretreatment slurry containing 0.5 g of cellulose (based on initial feedstock composition) was added in a final volume of 50 ml_ to a 125-mL shake flask. Acetic acid (10-100 μl_) was added, to titrate the pH of the ammonia-pretreated biomass to 5.0 before enzyme addition because of the sensitivity of the enzymes to high pH environments. The pH was controlled at 5.0 during saccharification 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 the concentration listed for each sample in Table 1. The sugar content of the resulting saccharification liquor was determined after 96 hr saccharification according to the sugar measurement protocol described in the General Methods. Sugar release after 96 hr is shown in Table 2. Controls for this experiment were 1 ) untreated corn stover, which yielded 23% of the theoretical yield of glucose (using 56 mg cellulase /g cellulose) and 2) steam (140 °C) pretreated corn stover, which yielded 40% of the theoretical yield of glucose (using 56 mg cellulase /g cellulose); xylose was not measured for the controls.
Table 1: Sugar release from pretreated corn stover with 96 hr saccharification
DWB: dry weight of biomass.
Figure imgf000027_0001
Figure imgf000028_0001
These results indicate that a pretreatment using ammonia at 12% for 15 minutes at 1400C, followed by saccharification, releases more glucose and xylose than when pretreatment is using 35% ammonia for 5 minutes at 1400C. Thus, advantages of using lower ammonia can be incorporated by a small increase in pretreatment time.
Example 2 Stover Pretreatment at High Biomass Concentration, Low Temperature, and Very Low Ammonia
The Jaygo reactor was charged with 0.635-cm milled stover (13 kg, dry weight basis). A vacuum (67.7 kPa) was applied to the vessel, and dilute ammonium hydroxide solution was injected to give an ammonia concentration of 6.2 g ammonia/100 g dry weight of biomass and a dry weight of biomass concentration 30 weight percent relative to total weight of the biomass-aqueous ammonia mixture. The vacuum was relieved, and steam was applied to the jacket to heat the stover to 100 0C. The soaked stover was held at temperature for 8 hr with constant mixing at 32 rpm, then allowed to cool overnight with continued mixing of the resulting slurry.
Whole, unwashed pretreatment slurry containing 0.5 g of cellulose (based on initial feedstock composition) was added in a final volume of 50 mL to a 125-mL shake flask. Acetic acid (10-100 μL) was added, if necessary, to titrate the pH of the ammonia-pretreated biomass to 5.0 before enzyme addition because of the sensitivity of the enzymes to high pH environments. The pH was controlled at 5.0 during saccharification by the addition of 50 mM citrate buffer, and the temperature was maintained at 50 0C. Spezyme® CP cellulase (Genencor International, Rochester, NY) was added to 56 mg/g cellulose. The sugar content of the resulting saccharification liquor was determined after 96 hr saccharification according to the sugar measurement protocol described in the General Methods and is shown in Table 2. Tab"le"2. Sugar release from pretreated corn stover at 96 hr
Figure imgf000029_0001
The results indicate that these very low ammonia concentrations and low temperature pretreatment conditions, (for a period of 8 hr) are as effective as using 12% ammonia at 140 0C for 15 min.
Example 3
Cob Pretreatment at High Biomass Concentration, Low Temperature, and Very Low Ammonia Concentration Followed by High Biomass Concentration Saccharification
Whole or fractured corn cobs (approximately 13 kg, dry weight basis) were loaded into the Jaygo reactor. Cobs were fractured by passing through the disk refiner (General Methods) equipped with plates C-2975. Resulting fractured cobs were passed through a 1.27 cm screen. Any pieces retained were passed through the disk refiner again with a 0.5 cm smaller gap. A vacuum was applied to the reactor, and dilute ammonium hydroxide solution was injected to give the final desired ammonia concentration (2% or 6%) and concentration of dry biomass (30% or 40%), as given in Table 3. The vacuum was relieved and steam was applied to the jacket to heat the cobs while soaking to a temperature of 93 °C for the whole cob sample and 85 °C for fractured cob samples. Short periods of increased agitator speeds (up to 96 rpm) were applied in an effort to increase the heating rate. The soaked cobs were held at temperature for 4 or 8 hr with constant mixing at 32 rpm then allowed to cool overnight with continued mixing.
Before pretreated biomass was removed from the reactor, the reactor was put under vacuum at 90 °C to strip ammonia out of the pretreated biomass. Before saccharification, the pH of the pretreated cob biomass was adjusted to 5.5 with solid citric acid. About 10 kg of pretreated whole cob was saccharified in the Jaygo reactor at 50 °C. About 1400 g of pretreated fractured cob was added to the PEHReactor, along with 22 ceramic attrition cylinders (3.2 cm diameter x 3.2 cm long; E. R. Advanced Ceramics, East Palestine, OH), for saccharification. An enzyme mixture of 28 mg Spezyme CP®/g cellulose in untreated stover plus 28 mg/g cellulose Multifect Xylanase® was used for each saccharification reaction. The final dry weight of biomass concentration at the beginning of each saccharification was 30% relative to the total weight of the pretreated biomass-saccharification enzyme consortium mixture. The PEHReactor was rotated axially at 19 rpm while maintaining a temperature of 50 °C. The sugar content of the resulting saccharification liquor was determined according to the sugar measurement protocol in the General Methods. Sugar release after 96 hr is shown in Table 3.
Table 3: Sugar release from pretreated corn cobs using a high concentration of biomass (by dry weight) during saccharification.
DWB: dry weight of biomass (percent is calculated relative to the total weight of the mixture)
Figure imgf000030_0001
Example 4
Cob Pretreatment at High Biomass Concentration, High Temperature, and Very Low Ammonia Concentration Followed by High Biomass
Concentration Saccharification
Fractured corn cobs (13 kg, dry basis) were loaded into the Jaygo reactor. After pulling a vacuum on the reactor, ammonium hydroxide solution of the proper strength to give 2% ammonia and 30% dry weight of biomass concentration was pumped into the reactor with 32 rpm mixing at room temperature. The contents of the reactor were then heated to 95 0C using low-pressure jacket steam. Once the reactor reached 95 0C, direct steam injection was used to heat the contents of the reactor to 145 0C. When the reactor reached 145 0C, the reactor contents were held at that temperature for 20 minutes using jacket steam and some direct steam injection. After 20 minutes, a vacuum was pulled on the vent to the reactor and the shredder motor was turned on for 5 minutes. After 1 hr the cooling water to the jacket was turned on. The contents of the Jaygo reactor were cooled to between 33 0C and 37 0C; then CO2 was used to pressurize the reactor to 138 kPa. The pressurized CO2 atmosphere was maintained for 30 min. The final temperature of the reactor contents was between 27 0C to 31 0C. The pH of the soaked/pretreated biomass was approximately 7.5.
Pretreated biomass was removed from the Jaygo reactor and transferred to the PEHReactor for saccharification at a final dry weight of biomass concentration at the beginning for saccharification of 30% relative to the total weight of the pretreated biomass-saccharification enzyme consortium mixture. The pH was then adjusted to 5.5 with solid citric acid, and the material digested with 28 mg Spezyme CP®/g cellulose and 28 mg Multifect Xylanase®/g cellulose in untreated cob as described in Example 3. The sugar content of the resulting saccharification liquor was determined according to the sugar measurement protocol in the General Methods. The sugar release after 96 hr digestion is shown in Table 4.
Table 4. Sugar release from pretreated corn cobs using a high concentration of biomass (by dry weight) during saccharification.
Figure imgf000032_0001
Example 5
Pretreatmeπt with Addition of Plasticizer Whole cob was pretreated as described in Example 3 at a dry weight of biomass concentration of about 30% relative to the total weight of the biomass-aqueous ammonia mixture, 2 weight percent ammonia relative to dry weight of biomass, 100 °C, for 8 hr in the Jaygo reactor with 3 weight percent relative to dry weight of biomass of glycerol added to act as a plasticizer. After pretreatment, the pH of the resulting material was adjusted to 5 with solid citric acid. Pretreated cob was then digested as described in Example 3. An enzyme mixture of 28 mg Spezyme CP®/g cellulose in untreated stover plus 28 mg/g cellulose Multifect Xyianase® in untreated cob was used. After 96 hr digestion, glucose concentration was 92.3 g/L and xylose concentration was 54.4 g/L. Example 6
Disc Refining of Pretreated Biomass
Stover was pretreated in the manner described in Example 1 , with different samples having low ammonia (12%) or comparative ammonia (35%) concentrations, and temperature, time, and enzyme conditions as listed in Table 5. Whole cob was pretreated as described in Example 3 with different samples having very low ammonia (3% or 6%), and other conditions as listed in Table 5. Following pretreatment, the samples were passed through a Sprout Waldron disc refiner. The gap between the stationary plate and rotating plate was set at 0.254 mm (0.010 inch) and the feed auger speed at 7 rpm. Refined material was saccharified as described in Example 2 and the sugar content of the resulting saccharification liquor was determined according to the sugar measurement protocol in the General Methods. Results of the saccharification at 96 hr are shown in Table 5. The results showed that with disc refining prior to saccharification, better digestibility was attained, or use of lower enzyme concentrations was effective.
Table 5: Digestibility of pretreated material that was disc refined prior to saccharification
Figure imgf000033_0001
Example 7 Steam Gun Treatment of Pretreated Biomass Stover was pretreated as described in Example 1 using conditions of 30% dry weight of biomass relative to total weight of biomass-aqueous ammonia mixture, 6 weight percent ammonia relative to DWB, 100 0C, 8 hr, in the Jaygo reactor. Cob was pretreated as described in Example 3 using conditions of 40% dry weight of biomass relative to total weight of biomass-aqueous ammonia mixture, 6 weight percent ammonia relative to DWB, 93 0C, 8 hr, in the Jaygo reactor. Samples of each pretreated biomass were separately loaded into a 4-liter steam gun reactor. Pretreated material was subjected to 170 °C for 5 min, or 140 0C for 20 min before being released through a die. The resulting material was saccharified as described in Example 2. Results are given in Table 6 below. The results showed that steam gun treatment prior to saccharification improved release of glucose.
Figure imgf000034_0001
Example 8 Modeling of Pretreatmeπt with Ammonia Recycle
Advantages of ammonia recycle were examined with Aspen models (Aspen Technologies, Cambridge, MA, version 12.1 ) for two pretreatment schemes: low temperature (85 °C), long residence time (4 hr) and high temperature (130 °C), short residence time (20 min). In each model there was a series of three flash tanks operating at successively lower pressures after the pretreatment reactor to provide a means for ammonia recycle. As the feed stream entered into each tank, it split into vapor and liquid fractions due to the reduction in pressure. The vapor fraction was recycled to pretreatment, while the liquid fraction went on to the next step in the process. Assuming 2 weight percent ammonia relative to DWB and approximately 27% dry weight of biomass relative to the total weight of the biomass-aqueous ammonia mixture in pretreatment, ammonia supplied fresh and from the recycle streams for each process are shown in Table 7. In both models, the flash tanks operated in similar fashion so that ammonia recycle was similar. For both scenarios, more than half of the required ammonia was supplied through recycle, reducing the need for and cost of fresh ammonia.
Table 7: Ammonia recycle in pretreatment - Aspen model results.
Figure imgf000035_0001
Example 9
Ethanol Production from Low Ammonia-Pretreated and Saccharified Cob Biomass, and Comparison to High Ammonia-Pretreated and Saccharified
Stover
Cob hydrolyzate was generated by pretreating whole cobs in the Jaygo reactor for 8 hr at 93 0C with 6 weight percent ammonia relative to dry weight of biomass at a dry weight of biomass concentration of 40 weight percent relative to the total weight of the biomass-aqueous ammonia mixture, as described in Example 3. 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 sulfuric acid. The pretreated biomass was saccharified in the Jaygo reactor at 30% dry weight of biomass relative to the total weight of the pretreated biomass-saccharification enzyme consortium mixture with 28 mg/g cellulose Spezyme® cellulase and 28 mg/g cellulose Multifect® xylanase for 168 hr at 50 °C and pH 5. The resulting hydrolyzate was used for fermentation of Zymomonas mobilis 8b in Sixfors fermentors (INFORS AG, Switzerland). Zymomonas mobilis 8b is a strain of Zymomonas mobilis that has been genetically engineered to give improved, over wild type, production of ethanol and is described in US Patent Application Publication 2003/0162271 A1 (Examples IV, Vl and XII). The cob hydrolyzate comprised 78 g/L glucose, 51 g/L xylose, 6 g/L acetamide, and 7 g/L acetic acid. The cob hydrolyzate was used at 40% and 80% strength, with the balance of the medium being concentrated aqueous medium consisting of yeast extract, and KH2PO4 in quantities such that their concentrations in the final slurry were about 5 g/L, and 2 g/L respectively. Additionally, in the 40% hydrolyzate slurry, glucose and xylose were added in quantities sufficient to bring their concentrations to their same levels as in the 80% hydrolyzate slurry. The fermentation was carried out at 37 °C. Agitation in the fermentors was 100 rpm, and pH was maintained at 5.5 by addition of 2 N KOH. The results are shown in Table 8. Sugars and ethanol were analyzed as described in General Methods. For comparison, stover hydrolyzate was generated by pretreating stover with 35 weight percent ammonia relative to dry weight of biomass at a dry weight of biomass concentration of about 30 weight percent relative to the total weight of the biomass-aqueous ammonia mixture at 170 °C for 5 min in the Zipperclave® reactor, as described in Example 1. The pretreated biomass was enzymatically digested at 30% dry weight of biomass relative to the total weight of the pretreated biomass- saccharification enzyme consortium mixture with 224 mg/g cellulose Spezyme CP® cellulase at 50 0C and pH 5 to generate a high sugar concentration hydrolyzate for fermentation testing. The resulting hydrolyzate comprised 88 g/L glucose, 52 g/L xylose, 9 g/L acetic acid and 15 g/L lactic acid. For ethanol production, Zymomonas mobilis 8b was fermented on either 40% or 80% (v/v) hydrolyzate slurry. The remaining volume was made up of concentrated aqueous medium consisting of yeast extract, KH2PO4 and MES buffer in quantities such that their concentrations in the final slurry would be about 10 g/L, 2 g/L and 0.1 M, respectively. Additionally, in the 40% hydrolyzate slurry, glucose and xylose were added in quantities sufficient to bring their concentrations to the same levels as in the 80% hydrolyzate slurry. Fermentation was done at 30 °C and pH 6 in 25 ml shake flasks with 20 ml working volume. Agitation was maintained at 150 rpm. Analysis was as for the cob hydrolyzate fermentation sample and results are given in Table 8.
Table 8: Sugar utilization and ethanol yields in fermentation on cob and stover hydrolyzates.
Figure imgf000037_0001
These results showed that fermentation to produce ethanol from cob hydrolyzate pretreated with low ammonia was more efficient than from stover pretreated with high ammonia.
Example 10
1-3 Propanediol Production from Very Low Ammonia-Pretreated and
Saccharified Cob Biomass
Hydrolyzate generated from pretreatment and saccharification of cob was fermented to produce 1 ,3-propanediol. Hydrolyzate was generated by pretreating cob pieces in the steam gun reactor. First cob biomass was loaded in the PEHReactor (described in General Methods), a vacuum applied, and dilute ammonium hydroxide solution was injected to give an ammonia concentration of 4 g ammonia/100 g dry weight biomass and a dry weight of biomass concentration of 30 g dry weight of biomass/100 g total biomass-aqueous ammonia mixture. The reactor vessel charged with ammonia and cob was rotated at 4°C for 30 min. The contents were transferred to the steam gun reactor (described in General Methods), the temperature increased to 145°C, and the mixture was held at temperature for 20 minutes. Material from the steam gun was discharged into a flash tank, and vacuum was maintained on the flash tank to aid ammonia removal. After pH adjustment, the pretreated biomass was saccharified at 30 g dry weight of biomass/100 g pretreated biomass- saccharification enzyme consortium mixture with 28.4 mg/g cellulose Spezyme CP®cellulase and 10.1 mg active protein /g cellulose enzyme consortium consisting of β-glucosidase, xylanase, β-xylosidase and arabinofuranosidase for 72 hr at 50°C and pH 5.5. The resulting hydrolyzate was used as a source of fermentable sugar for conversion to 1 ,3-propanediol by recombinant E. coli strain RJ8n pBE93-k1. The construction of strain RJ8n pBE93-k1 is described in detail in PCT application WO/ 2004/018645 (Example 7), and it is a derivative of strain RJ8n, described in US 6358716. The hydrolyzate was used at 10% with the balance being aqueous medium consisting of 7.5 g/L KH2PO4, 2.0 g/L citric acid*H2O, 4.0 ml/L 28% NH4OH, 3.0 g/L(NH4)2SO4, 2.0 g/L
MgSO4*7H2O, 0.2 g/L CaCI2*2H2O, 0.33 g/L ferric ammonium citrate, 0.5 g/L yeast extract, 0.1 mg/L vitamin B12, 1.0 mg/L FeSO4*7H20, 1 mg/L ZnSO4*7H2O, 0.1 g/L CuSO4*5H2O, 1 mg/L CoCI2*6H2O, 0.3 mg/L MnSO4*7H2O, 0.1 g/L H3BO4, 0.10 g/L NaMoO4*2H2O, 10 mg/L NaCI with the final pH adjusted to 6.8. Cultures were started from frozen stocks (15% glycerol as cryoprotectant) in 50 mL of medium in a 250 mL baffled flask. The cultures were incubated at 34 0C and 300 rpm shaking for 24 hours. The amount of 1 ,3-propanediol produced was measured by HPLC under the following conditions: Column: Showdex SH1011
Sample volume: 20 μL
Mobile phase: 0.01 N H2SO4
Flow rate: 0.5 ml/min
Column temperature 50°C Detector: Waters 996 photodiode array
Detector temperature: 40°C
Run time: 40 min
Results are shown in Table 9 below. Products from glucose fermentation by the RJ8n pBE93-k1 strain of E. coli include glycerol (an intermediate metabolite) and 1,3-propanediol. Experiments were conducted in duplicate flasks and assayed at 24 hr. In this system, glucose in the hydrolysate was converted to both glycerol and 1 ,3-propanediol. Table 9: Substrate utilization and product formation by fermentation with E. coli.
Figure imgf000039_0001
Example 11
Formation of Acetamide During Pretreatment Samples derived from cobs pretreated according to the processes described in Example 3 and Example 4 were analyzed to determine the fate of the acetyl groups in the biomass. The pretreatment liquors (pretreatment mixture with insoluble solids removed) were assayed for acetic acid and acetamide content as follows. The pH of each sample was adjusted to about 3 with H2SO4 (72%). For measurement of acetamide, the sample was passed through a 0.2 μm filter and analyzed by HPLC according to the conditions listed below. For measurement of total acetate (includes acetate present as acetic acid and acetamide), the acidified sample was autoclaved for 1 hr at 121 0C; acetamide was quantitatively converted to acetic acid during this step. After autoclaving, the sample was allowed to cool. The sample was then passed through a 0.2 μm filter into a sample vial and analyzed according to the conditions listed below. Acetic acid and acetamide concentrations were determined from standard curves generated for each.
Mobile phase: 0.01 N H2SO4, 0.2 μm filtered and degassed Flow rate: 0.6 mL/min Column temperature: 55-65 0C
Detector temperature: As close to column temperature as possible Detector: Refractive index
Run time: 60 min
Column: Biorad Aminex HPX-87H column with corresponding guard column
Results for the 3 different pretreatment conditions assayed are shown in Table 10. In each case, all of the acetyl groups were solubilized to either acetic acid or acetamide.
10 Table 10. Conversion of acetyl groups in biomass to acetamide during pretreatment.
DWB, dry weight of biomass. (percent is calculated relative to the total weight of the biomass-aqueous ammonia mixture)
Figure imgf000040_0001
15 Using an ammonia concentration of 6%, nearly half of the acetyl groups were converted to acetamide, which is non-inhibitory for biocatalyst growth as shown in Example 12.
Example 12 Effect of Acetamide and Acetic acid on Zvmomonas Growth
20 To test the toxicity of acetamide and acetic acid, Z. mobilis strain 8b
(described in Example 9) was grown in fermentation medium at pH 6.0 with and without acetamide or acetic acid. Fermentation medium was composed of 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 was grown in 25-mL baffled
25 Erlenmeyer shake flasks rotating at 150 rpm at 30 0C in unsupplemented medium (control), medium supplemented with 6 g/L acetamide, or medium supplemented with 7.2 g/L acetic acid. As shown in Figure 1 , the presence of acetamide had no influence on the growth rate or final density of Z. mobilis, whereas the presence of acetic acid resulted in a reduced growth rate and lower cell yield (as measured by dry cell mass).
Example 13
Pretreatment of Bagasse at High Biomass Concentration, High Temperature, and Very Low Ammonia, and Saccharification at Low and High Concentration
The PEHReactor (described in General Methods), with no attrition media, was charged with 1.27 cm-milled bagasse (370 g, dry weight basis). This sugar cane bagasse was NIST Reference Material RM8491 , from sugar cane clone H65-7052, originally obtained from the Hawaii Sugar Planters Association, Kunia substation, Oahu, HI. It was milled in a Wiley mill to pass through a 2 mm screen, with the fines (+74 mesh) removed. The PEHReactor vessel was cooled to 4°C by rotation in contact with ice on the outer surface. A vacuum was applied to the reactor vessel, and dilute ammonium hydroxide solution, that was pre-cooled in a cold room at 40C and passed through tubing immersed in an ice-water bath, was injected to give an ammonia concentration of 4 g/100 g dry weight of biomass and a dry weight of biomass concentration of 45 g/100 g total biomass-aqueous ammonia mixture. The reactor vessel charged with ammonia and bagasse was cooled to 4°C by applying ice to the surface of the rotating reactor vessel, and rotated at 4°C for 30 min. At this time the contents were transferred to the steam gun reactor that is described in General Methods. Once the steam gun reactor was charged with the ammonia-bagasse mixture, the temperature was increased to 1450C and the mixture was held at temperature for 20 minutes. At the end of the pretreatment time, the bagasse was discharged from the steam gun reactor through a 1-in circular die into a flash tank. A sample of pretreated bagasse was subsequently saccharified in a shake flask and another sample (approximately 163 g dry weight) was saccharified in the PEHReactor. The shake flask saccharification was carried out at 5% dry weight of biomass relative to the total weight of the pretreated biomass- saccharification enzyme consortium mixture, while the PEHReactor saccharification was carried out at 30% dry weight of biomass relative to the total weight of the pretreated biomass-saccharification enzyme consortium mixture. The temperature was maintained at 50 °C.
For the PEHReactor saccharification, about 476 g (-163 g dry weight) pretreated biomass and 22 ceramic attrition cylinders were added to the reactor vessel. The pH was adjusted to 5.0-5.5 with solid citric acid. The reactor vessel was kept inside an incubator chamber controlled to 50 °C and rotated axially at 19 rpm. Unpretreated bagasse was also saccharified at 5% dry weight of biomass relative to the total weight of the pretreated biomass-saccharification enzyme consortium mixture in a shake flask. All saccharifications were done with 28.4 mg/g cellulose Spezyme CP® cellulase and 28.4 mg/g cellulose Multifect® xylanase at 5O0C and pH 5.5 for 96 hr. Yields given in Table 11 below are the release as percent of theoretical yield.
Table 11 : Yields following pretreatment and saccharification of bagasse.
Figure imgf000042_0001
ND: not determined The results demonstrate that pretreatment of bagasse with very low ammonia allows substantial sugar release as compared to the unpretreated control, and that saccharification at high dry biomass concentration in the PEHReactor is very effective in releasing sugars. Example 14
Pretreatment of Yellow Poplar Sawdust at High Biomass Concentration, High Temperature, and Very Low Ammonia, and Saccharification at Low and High Concentration
The PEHReactor, without attrition media, was charged with yellow poplar sawdust (596 g, dry weight basis; purchased from Sawmiller Inc., Haydenville, OH). A vacuum was applied to the reactor vessel, and dilute ammonium hydroxide solution was injected to give an ammonia concentration of 6 g/100 g dry weight of biomass and a dry weight of biomass concentration of 44 g/100 g total biomass-aqueous ammonia mixture. The reactor vessel charged with ammonia and yellow poplar sawdust was brought to 4°C as described in Example 13, and rotated at 4°C for 30 min. At this time the contents were transferred to the steam gun reactor. Once the steam gun reactor was charged with the ammonia- poplar mixture, the temperature was increased to 145°C and the mixture was held at temperature for 20 minutes. At the end of the pretreatment time, the yellow poplar sawdust was discharged from the steam gun reactor through a 1-in circular die into a flash tank. A sample of pretreated yellow poplar sawdust was subsequently saccharified as described in
Example 13 in a shake flask, and another sample was saccharified in the PEHReactor. The shake flask saccharification was carried out at 5% dry weight of biomass relative to the total weight of the pretreated biomass- saccharification enzyme consortium mixture, while the PEHReactor saccharification (using ~279 g dry weight pretreated sawdust) was carried out at 30% dry weight of biomass relative to the total weight of the pretreated biomass-saccharification enzyme consortium mixture. Unpretreated yellow poplar sawdust was also saccharified at 5% dry weight of biomass relative to the total weight of the pretreated biomass- saccharification enzyme consortium mixture in a shake flask. All saccharifications were done with 28.4 mg/g cellulose Spezyme CP® cellulase and 28.4 mg/g cellulose Multifect® xylanase at 50°C and pH 5.5 for 96 hr. Yields given in Table 12 below are the release or each sugar as a percentage of theoretical yield.
Table 12: Yields following pretreatment and saccharification of yellow poplar sawdust.
Figure imgf000044_0001
ND: not determined
The results demonstrate that pretreatment of yellow poplar sawdust with very low ammonia allows substantial sugar release as compared to the unpretreated control, and that saccharification at high dry weight of biomass in the PEHReactor is more effective in releasing sugars than the shake flask.
Example 15 Ethanol Production by Yeast Fermentation on Hvdrolvzate from Very Low Ammonia-Pretreated and Saccharified Cob Biomass
The same hydrolyzate used to produce 1 ,3-propanediol in Example 10 was also used to produce ethanol by yeast fermentation. This hydrolyzate was used as a source of fermentable sugar for conversion to ethanol by wild-type Saccharomyces cerevisiae in shake flasks. The hydrolyzate was used at 10% (v/v) strength, with the balance being aqueous medium consisting of 10 g/L yeast extract, and 20 g/L peptone. Yeast were cultured in 50 ml_ of medium in a 250 ml_ baffled flask. The cultures were incubated at 30 0C with 250 rpm shaking for 24 hours. The amount of ethanol produced was measured by HPLC as described in Example 9, and results from duplicate flasks are listed in Table 13 below.
Table 13: Substrate utilization and product formation by fermentation with i yeast.
Figure imgf000045_0001
Example 16 Lactic Acid Production by Lactobacillus Fermentation on Hydrolyzate from Very Low Ammonia-Pretreated and Saccharified Cob Biomass
The same hydrolyzate used to produce 1 ,3-propanediol in Example 10 was also used to produce lactic acid by fermentation with Lactobacillus brevis in shake flasks. The hydrolyzate was used at 10% (v/v), with the balance being aqueous medium consisting of 5 g/L yeast extract, 10 g/L peptone, 2 g/L ammonium citrate, 5 g/L sodium acetate, 0.1 g/L MgSO4, 0.05 g/L MnSO4 and 2 g/L K2HPO4 and 1 g/L Tween. The Lactobacillus was cultured in 50 mL broth in 250 mL baffled flasks. Duplicate cultures were incubated at 34 °C with 150 rpm shaking for 24 hours. The amount of lactic acid produced was measured by HPLC as described in Example 10 and is listed in Table 14. The two Flask 2 samples are duplicate assays of the same culture.
Table 14: Substrate utilization and product formation by fermentation with Lactobacillus brevis
Figure imgf000045_0002
Example 17 Cob Pretreatment at Higher Dry Biomass Concentration with Very Low
Ammonia Whole corn cobs were processed with a jaw crusher (2.2 kW motor) with a jaw spacing of approximately 0.95 cm, followed by a delumper (1.5 kW motor, Franklin Miller Inc., Livingston, NJ), followed by screening with a Sweco screen equipped with a 1.9 cm U.S. Standard screen. Approximately 805 g fractured cobs were loaded into the PEHReactor. Moisture content in the cobs was approximately 7%. The atmosphere in the reactor vessel was flushed 5 times with nitrogen prior to loading. The reactor, with no attrition media, was preheated to 75°C before the start of the experiment, without rotation. When the temperature within the reactor vessel stabilized at 75°C the rolling mechanism in the incubator was turned on and the rotation adjusted to 19 rpm. The appropriate amount of dilute ammonium hydroxide solution to give an ammonia concentration of 6 g ammonia/100 g dry weight of biomass and a solids concentration of 50 g dry weight of biomass/100 g total weight of biomass-ammonia mixture was then pumped into the reactor. Ethanol at 1 g/100 g dry weight of biomass was also added to the solution. The ammonia solution was pumped through a heated loop in a water bath heated to -750C, fabricated using a 2-gal Parr reactor. The heated dilute ammonium hydroxide solution was injected via an injection lance into the reactor vessel and sprayed on the fractured cobs rotating and tumbling in the reactor. The reactor was maintained at 75°C for 2 hr while turning at 19 rpm. At the end of that time, a vacuum (approximately 85 kPa) was applied to the reactor vessel for 30 minutes to remove ammonia and drop the temperature of the contents of the reactor to approximately 5O0C. Carbon dioxide was then injected into the reactor to relieve the vacuum and the reactor was pressurized to 103 kPa gauge pressure CO2 and held at pressure for 30 min at 500C.
Following this, the reactor was depressurized, opened and attrition media were added. The pH of the contents was adjusted to approximately 5.5 by injecting i M citric acid buffer at pH 4.8 using the injection lance, to increase the citric acid buffer strength to -75 mM, plus adding citric acid monohydrate. Not all of the ammonia was stripped off in the vacuum step nor neutralized with CO2. The citric acid buffer was injected into the reactor following heating to 500C and then the contents was allowed to equilibrate by incubating the reactor at 5O0C and 19 rpm for 1 hour. Injection of the citric acid buffer while rotating the reactor using the injection lance allowed for a more even spraying and distribution of the buffer on the pretreated cob particles. The reactor was removed from the incubator, opened, and the pH of a sample determined. If the pH was above 5.5, then additional solid citric acid monohydrate was added and the reactor was incubated with mixing at 500C for an additional hour. This process was repeated until the pH was approximately 5.5. Once the desired pH was reached, 12.9 mg/g cellulose Spezyme CP (Genencor) and 5 mg active protein /g cellulose enzyme consortium consisting of β- glucosidase, xylanase, β-xylosidase and arabinofuranosidase were loaded into the reactor. The reactor remained in the incubator at 500C and 19 rpm for 72 hr. Following this pretreatment and saccharification, monomer glucose yield was 62.0% and monomer xylose yield was 31.0%. Total glucose yield was 75.2% and total xylose was 80.3%.
Example 18
Cob Pretreatment at Higher Solids Concentration with Very Low Ammonia and Alternate Conditions Whole corn cobs were processed with a hammermill (10-inch hammer mill, Glen Mills Inc., Clifton, NH) to pass through a 1.27 cm screen. Approximately 805 g fractured cobs were loaded into the PEHReactor. Moisture content in the cobs was approximately 7%. Twenty-two ceramic attrition cylinders (3.2 cm diameter x 3.2 cm long; E. R. Advanced Ceramics, East Palestine, OH) were also added to the reactor. The reactor was preheated to 95°C before the start of the experiment, without rotation. A vacuum (approximately 85 kPa) was applied to the reactor vessel before the start and the vessel was sealed off. When the temperature within the reactor vessel stabilized at 95°C the rolling mechanism in the incubator was turned on and the rotation adjusted to 19 rpm. The appropriate amount of dilute ammonium hydroxide solution to give an ammonia concentration of 6 g ammonia/100 g dry weight of biomass and a solids concentration of 50 g dry weight of biomass/100 g total weight of biomass-ammonia mixture was then pumped into the reactor. The ammonia solution was pumped through a heated loop in a boiling water bath fabricated using a 2-gal Parr reactor. The heated dilute ammonium hydroxide solution was injected via an injection lance into the reactor vessel and sprayed on the fractured cobs rotating and tumbling in the reactor. The reactor was maintained at 95°C for 2 hr while turning at 19 rpm. At the end of that time, a vacuum (approximately 85 kPa) was applied to the reactor vessel for 30 minutes to remove ammonia and drop the temperature of the contents of the reactor to approximately 500C. Carbon dioxide was then injected into the reactor to relieve the vacuum and the reactor was pressurized to 103 kPa gauge pressure and held at pressure for 30 min at 500C.
Following this, the reactor was depressurized, opened and the pH of the contents was adjusted to approximately 5.5 by injecting 1 M citric acid buffer, pH 4.8, into which citric acid monohydrate was added and dissolved. The citric acid buffer was injected into the reactor following heating to 500C and then the contents was allowed to equilibrate by incubating the reactor at 50°C and 19 rpm for 1 hour. Injection of the citric acid buffer while rotating the reactor using the injection lance allowed for a more even spraying and distribution of the buffer on the pretreated cob particles. The reactor was removed from the incubator, opened, and the pH of a sample determined. If the pH was above 5.5, then additional solid citric acid monohydrate was added and the reactor was incubated with mixing at 5O0C for an additional hour. This process was repeated until the pH was approximately 5.5. Once the desired pH was reached, 12.9 mg/g cellulose Spezyme CP (Genencor) and 5 mg active protein/g cellulose enzyme consortium consisting of β-glucosidase, xylanase, β-xylosidase and arabinofuranosidase were loaded into the reactor. The reactor remained in the incubator at 500C and 19 rpm for 72 hr. Following this pretreatment and saccharification, monomer glucose yield was 50.7% and monomer xylose yield was 35.7%. Total glucose and xylose yields were 71.7% and 89.8%, respectively.
Example 19 Pretreatment of Cobs with Very Low Ammonia and Additional Base
Whole corn cobs were processed with a jaw crusher (2.2 kW motor) with a jaw spacing of approximately 0.95 cm, followed by a delumper (1.5 kW motor, Franklin Miller Inc.), followed by screening with a Sweco screen equipped with a 1.9 cm U.S. Standard screen. Approximately 460 g fractured cobs were loaded into the PEHReactor. Moisture content in the cobs was approximately 7%. The reactor was preheated to 95°C before the start of the experiment, without rotation. A vacuum (approximately 85 kPa) was applied to the reactor vessel before the start and the vessel was sealed off. When the temperature within the vessel re-stabilized at 950C the rolling mechanism in the incubator was turned on and the rotation was adjusted to 19 rpm. The appropriate amount of ammonium hydroxide solution to give an ammonia concentration of 3.2 g ammonia/10Og dry weight of biomass and NaOH to give a concentration of 1.9 g NaOH/100 g dry weight of biomass while maintaining a solids concentration of 30 g dry weight of biomass/100 g total weight of biomass-ammonia mixture was then pumped into the reactor. The ammonia and additional base solution was pumped through a heated loop in a boiling water bath fabricated using a 2-gal Parr reactor. The heated dilute ammonium hydroxide solution was injected via an injection lance into the reactor vessel and sprayed on the fractured cobs rotating and tumbling in the reactor. Following injection, the vacuum on the vessel was relieved to atmospheric pressure. The reactor was maintained at 95°C 30 min, then the temperature was lowered to 850C where it was maintained for 4 hr. At the end of that time, a vacuum (approximately 85 kPa) was applied to the reactor vessel for 30 minutes to remove ammonia and drop the temperature of the contents of the reactor to approximately 5O0C. Carbon dioxide was then injected into the reactor to relieve the vacuum and the reactor was pressurized to 103 kPa gauge pressure and held at pressure for 30 min at 500C.
Following this, the reactor was depressurized, opened and the pH of the contents was adjusted to approximately 5.5 by injecting approximately 75 ml of 1 M citric acid buffer, pH 4.8, into which citric acid monohydrate was added and dissolved. The citric acid buffer was injected into the reactor following heating to 500C and the contents was then allowed to equilibrate by incubating the reactor at 500C and 19 rpm for 1 hour. Injection of the citric acid buffer while rotating the reactor using the injection lance allowed for a more even spraying and distribution of the buffer on the pretreated cob particles. The reactor was removed from the incubator, opened, and the pH of a sample determined. If the pH was above 5.5, then additional solid citric acid monohydrate was added and the reactor was incubated with mixing at 500C for an additional hour. This process was repeated until the pH was approximately 5.5. Once the desired pH was reached, 28.4 mg/g cellulose Spezyme CP (Genencor) and 28.4 mg/g cellulose Multifect were loaded into the reactor. The reactor remained in the incubator at 500C and 19 rpm for 72 hr. Following this pretreatment and saccharification, monomer glucose yield was 56.1 % and monomer xylose yield was 39.5%. Total glucose and xylose yields were 82.8% and 84.2%, respectively. These values are the averages of 2 experiments. Example 20
Room Temperature and Very Low Ammonia Pretreatment Whole corn cobs were processed with a jaw crusher (2.2 kW motor) with a jaw spacing of approximately 3/8 inch, followed by a delumper (1.5 kW motor, Franklin Miller Inc.), followed by screening with a Sweco screen equipped with a 1.9 cm U.S. Standard screen. Approximately 460 g fractured cobs were loaded into the PEHReactor. Moisture content in the cobs was approximately 7%. Twenty-two ceramic attrition cylinders (3.2 cm diameter x 3.2 cm long; E. R. Advanced Ceramics, East Palestine, OH) were also added to the reactor. A vacuum (approximately 85 kPa) was applied to the reactor vessel before the start and the vessel was sealed off. When the temperature within the reactor re-stabilized at room temperature (22-26°C) the rolling mechanism in the incubator was turned on and rotation was adjusted to 19 rpm. The appropriate amount of dilute ammonium hydroxide solution to give an ammonia concentration of 4 g ammonia/10Og dry weight of biomass and while maintaining a solids concentration of 30 g dry weight of biomass/total weight of biomass- ammonia mixture was then pumped into the reactor. The dilute ammonium hydroxide solution was injected via an injection lance into the reacter vessel and sprayed on the fractured cobs rotating and tumbling in the reactor. Following injection, the vacuum on each vessel was relieved to atmospheric pressure. The reactor was maintained at room temperature (22-26°C) for 24 hr. At the end of that time, a vacuum (approximately 81 kPa) was applied to the reaction vessel for 30 minutes to remove ammonia. Carbon dioxide was then injected into the reactor to relieve the vacuum and the reactor was pressurized to 103 kPa gauge pressure with CO2 and held at pressure for 30 min at room temperature.
Following this, the reactor was depressurized, opened and the pH of the contents was adjusted to approximately 5.5 by adding citric acid monohydrate following heating to 5O0C, and then allowed to equilibrate by incubating the reactor at 500C and 19 rpm. The reactor was removed from the incubator, opened, and the pH of a sample determined. If the pH was above 5.5, then additional solid citric acid monohydrate was added and the reactor was incubated with mixing at 500C. This process was repeated until the pH was approximately 5.5. Once the desired pH was reached, 12.9 mg/g cellulose Spezyme CP (Genencor) and 5 mg active protein /g cellulose enzyme consortium consisting of β-glucosidase, xylanase, β- xylosidase and arabinofuranosidase were loaded into the reactor. The reactor remained in the incubator at 5O0C and 19 rpm for 72 hr. Following this pretreatment and saccharification, monomer glucose yield was 41.7% and the monomer xylose yield was 25.4%. Total glucose and xylose yields were 50.1% and 53.2%, respectively. These values were the averages of 2 experiments.

Claims

CLAIMS What is claimed is:
1. A method for producing a target chemical derivable from biomass comprising: a) contacting biomass with an aqueous solution comprising ammonia, wherein the ammonia is present at a concentration at least sufficient to maintain alkaline pH of the biomass-aqueous ammonia mixture but wherein said ammonia is present at less than about 12 weight percent relative to dry weight of biomass, and further wherein the dry weight of biomass is at a high solids concentration of at least about 15 weight percent relative to the weight of the biomass-aqueous ammonia mixture; b) contacting the product of step (a) with a saccharification enzyme consortium under suitable conditions to produce fermentable sugars; and c) contacting the product of step (b) with at least one biocatalyst able to ferment the sugars to produce the target chemical under suitable fermentation conditions.
2. The method of Claim 1 wherein steps (b) and (c) are performed concurrently.
3. The target chemical of Claim 1 selected from the group consisting of acids, alcohols, alkanes, alkenes, aromatics, aldehydes, ketones, biopolymers, proteins, peptides, amino acids, vitamins, antibiotics, and pharmaceuticals.
4. The method of claim 1 , wherein the target chemical is selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol, propanediol, butanediol, glycerol, erythritol, xylitol, sorbitol, acetic acid, lactic acid, propionic acid, 3-hydroxypropionic acid, butyric acid, gluconic acid, itaconic acid, citric acid, succinic acid, levulinic acid, glutamic acid, aspartic acid, methionine, lysine, glycine, arginine, threonine, phenylalanine, tyrosine, methane, ethylene, acetone, and industrial enzymes.
5. The method of Claim 4, wherein the target chemical is lactic acid, propanediol, or ethanol.
6. The method of Claim 1 wherein said at least one biocatalyst is selected from the group consisting of bacteria, filamentous fungi and yeast.
7. The method of Claim 1 wherein said at least one biocatalyst is selected from the group consisting of wild type, mutant, or recombinant Escherichia, Zymomonas, Candida, Saccaromyces, Pichia, Streptomyces, Bacillus, Lactobacillus and Clostridium.
8. The method of Claim 1 wherein said at least one biocatalyst is selected from the group consisting of recombinant Escherichia coli, Zymomonas mobilis, Bacillus stearothermophilus, Saccharomyces cerevisiae, Clostridia thermocellum, Thermoanaerobacterium saccharolyticum, and Pichia stipitis.
9. The method of Claim 1 wherein the pH of the biomass-aqueous ammonia mixture is greater than 8.
10. The method of Claim 1 wherein vacuum is applied to the biomass prior to contacting the biomass with an aqueous solution comprising ammonia.
11. The method of Claim 1 wherein said dry weight of biomass is at a high solids concentration of from at least about 15% to about 80%.
12. The method of Claim 11 wherein said dry weight of biomass is at a high solids concentration of from at least about 15% to about 60%.
13. The method of Claim 1 wherein said ammonia is present at less than about 10 weight percent relative to dry weight of biomass.
14. The method of Claim 13 wherein said ammonia is present at about 6% or less weight percent relative to dry weight of biomass.
15. The method of Claim 1 wherein biomass is selected from the group consisting of bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, yard waste, wood and forestry waste.
16. The method of Claim 1 wherein biomass is selected from the group consisting of switchgrass, waste paper, sludge from paper manufacture, corn grain, corn cobs, corn husks, com stover, grasses, wheat, wheat straw, hay, barley, barley straw, rice straw, sugar cane bagasse, sorghum, soy, components obtained from processing of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers and animal manure.
17. The method of Claim 16 wherein biomass is selected from the group consisting of corn cobs, corn stover, corn husks, sugar cane bagasse, sawdust, switchgrass, wheat straw, hay, barley straw, rice straw, and grasses.
18. The method of Claim 17 wherein biomass is selected from the group consisting of com cobs, com stover, sawdust, and sugar cane bagasse.
19. The method of Claim 1 wherein ammonia selected from the group consisting of ammonia gas, ammonium hydroxide, urea, and combinations thereof.
20. The method of Claim 1 wherein (a) is carried out at a temperature of from about 4 0C to about 200 °C.
21. The method of Claim 20 wherein (a) is carried out at a temperature of from about 75 °C to about 150 °C.
22. The method of Claim 21 wherein (a) is carried out at a temperature of from greater than 90 °C to about 150 °C.
23. The method of Claim 1 wherein (a) is carried out for a period of time of up to about 25 hours.
24. The method of Claim 23 wherein (a) is carried out for a period of time of up to about 8 hours.
25. The method of Claim 1 or Claim 2 wherein at least a portion of the ammonia of (a) is removed prior to (b).
26. The method of Claim 25 wherein ammonia from (a) is recycled.
27. The method of Claim 1 wherein the contacting of (b) is at a dry weight of biomass concentration of at least about 15%.
28. The method of Claim 1 wherein (a), (b) or (a) and (b) are repeated at least one time.
29. The method of Claim 1 further comprising adding at least one plasticizer, softening agent or combination thereof in (a).
30. The method of Claim 29 wherein said at least one plasticizer, softening agent or combination thereof is selected from the group consisting of polyols, esters of polyols, glycol ethers, acetamide, ethanol, and ethanolamines.
31. The method of Claim 1 further comprising applying energy before or during (a), before or during (b), or a combination thereof.
32. The method of Claim 31 wherein said energy is selected from the group consisting of milling, crushing, grinding, shredding, chopping, disk refining, ultrasound and microwave.
33. The method of Claim 1 , wherein carbon dioxide from fermentation is used to adjust the pH of the pretreatment mixture prior to saccharification.
34. The method of Claim 1 wherein said saccharification enzyme consortium comprises at least one glycosidase.
35. The method of Claim 1 wherein said saccharification enzyme consortium comprises at least one enzyme selected from the group consisting of cellulose-hydrolyzing glycosidases, hemicellulose- hydrolyzing glycosidases, starch-hydrolyzing glycosidases, peptidases, lipases, ligninases and feruloyl esterases.
36. The method of Claim 1 wherein said saccharification enzyme consortium comprises at least one enzyme selected from the group consisting of cellulases, endoglucanases, exoglucanases, cellobiohydrolases, β-glucosidases, xylanases, endoxylanases, exoxylanases, β-xylosidases, arabinoxylanases, mannases, galactases, pectinases, glucuronidases, amylases, α-amylases, β-amylases, glucoamylases, α-glucosidases, isoamylases.
37. The method of Claim 1 wherein (b) is performed at a temperature of from about 15 °C to about 100 °C and at a pH of from about 2 to about 11.
PCT/US2006/014020 2005-04-12 2006-04-12 Treatment of biomass to obtain a target chemical WO2006110891A2 (en)

Priority Applications (5)

Application Number Priority Date Filing Date Title
BRPI0612944-7A BRPI0612944B1 (en) 2005-04-12 2006-04-12 METHOD OF PRODUCTION OF TARGET SUBSTANCE
EP06758339A EP1869194A2 (en) 2005-04-12 2006-04-12 Treatment of biomass to obtain a target chemical
JP2008506725A JP5149784B2 (en) 2005-04-12 2006-04-12 Biomass processing to obtain target chemicals
CN200680012124.5A CN101160405B (en) 2005-04-12 2006-04-12 Process for biomass treatment to obtain target chemical substance
CA 2604964 CA2604964C (en) 2005-04-12 2006-04-12 Treatment of biomass to obtain a target chemical

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US67043705P 2005-04-12 2005-04-12
US60/670,437 2005-04-12

Publications (2)

Publication Number Publication Date
WO2006110891A2 true WO2006110891A2 (en) 2006-10-19
WO2006110891A3 WO2006110891A3 (en) 2007-02-08

Family

ID=36782581

Family Applications (5)

Application Number Title Priority Date Filing Date
PCT/US2006/014147 WO2006110902A1 (en) 2005-04-12 2006-04-12 System and process for biomass treatment
PCT/US2006/014144 WO2006110899A2 (en) 2005-04-12 2006-04-12 Integration of alternative feedstreams in biomass treatment and utilization
PCT/US2006/014146 WO2006110901A2 (en) 2005-04-12 2006-04-12 Treatment of biomass to obtain fermentable sugars
PCT/US2006/014145 WO2006110900A2 (en) 2005-04-12 2006-04-12 Treatment of biomass to obtain ethanol
PCT/US2006/014020 WO2006110891A2 (en) 2005-04-12 2006-04-12 Treatment of biomass to obtain a target chemical

Family Applications Before (4)

Application Number Title Priority Date Filing Date
PCT/US2006/014147 WO2006110902A1 (en) 2005-04-12 2006-04-12 System and process for biomass treatment
PCT/US2006/014144 WO2006110899A2 (en) 2005-04-12 2006-04-12 Integration of alternative feedstreams in biomass treatment and utilization
PCT/US2006/014146 WO2006110901A2 (en) 2005-04-12 2006-04-12 Treatment of biomass to obtain fermentable sugars
PCT/US2006/014145 WO2006110900A2 (en) 2005-04-12 2006-04-12 Treatment of biomass to obtain ethanol

Country Status (7)

Country Link
US (3) US7998713B2 (en)
EP (5) EP1869201B1 (en)
JP (5) JP5804666B2 (en)
CN (5) CN101160405B (en)
BR (5) BRPI0612966B1 (en)
CA (5) CA2604964C (en)
WO (5) WO2006110902A1 (en)

Cited By (175)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008004853A1 (en) * 2006-07-03 2008-01-10 Hendriks Antonius Theodorus Wi Pre-treatment of biomass
JP2008154497A (en) * 2006-12-22 2008-07-10 Japan Science & Technology Agency Method for producing transformant and new mutant of yeast
JP2008161125A (en) * 2006-12-28 2008-07-17 Univ Of Tokyo Method for producing sugar, method for producing ethanol, method for producing lactic acid, cellulose for enzyme saccharification used for them and method for producing the same
WO2008134259A1 (en) 2007-04-24 2008-11-06 Novozymes North America, Inc. Detoxifying pre-treated lignocellulose-containing materials
WO2009030713A1 (en) 2007-09-03 2009-03-12 Novozymes A/S Detoxifying and recycling of washing solution used in pretreatment of lignocellulose-containing materials
JP2009112200A (en) * 2007-11-02 2009-05-28 Nippon Steel Engineering Co Ltd Method for producing ethanol
WO2009085935A2 (en) 2007-12-19 2009-07-09 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2010039812A2 (en) 2008-09-30 2010-04-08 Novozymes North America, Inc. Improvement of enzymatic hydrolysis of pre-treated lignocellulose-containing material with distillers dried grains
WO2010065830A1 (en) 2008-12-04 2010-06-10 Novozymes, Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
EP2204432A1 (en) 2006-10-26 2010-07-07 Xyleco, Inc. Processing biomass
WO2010078392A2 (en) 2008-12-31 2010-07-08 Novozymes North America, Inc. Processes of producing fermentation products
WO2010078391A2 (en) 2008-12-30 2010-07-08 Novozymes North America, Inc. Improvement of enzymatic hydrolysis of pretreated lignocellulose-containing material with dissolved air flotation sludge
WO2010080407A2 (en) 2008-12-19 2010-07-15 Novozymes, Inc. Methods for increasing hydrolysis of cellulosic material
WO2010080428A1 (en) 2008-12-19 2010-07-15 Xyleco, Inc. Processing biomass
WO2010080408A2 (en) 2008-12-19 2010-07-15 Novozymes, Inc. Methods for increasing enzymatic hydrolysis of cellulosic material in the presence of a peroxidase
WO2010080532A1 (en) 2008-12-19 2010-07-15 Novozymes, Inc. Methods for increasing hydrolysis of cellulosic material in the presence of cellobiose dehydrogenase
EP2207889A1 (en) * 2007-10-10 2010-07-21 Sunopta Bioprocess Inc. Treatment of lignocellutosic materials utilizing disc refining and enzymatic hydrolysis performed under vacuum
JP2010524473A (en) * 2007-04-19 2010-07-22 マスコマ コーポレイション Combined thermochemical pretreatment and refining of lignocellulose biomass
WO2010088387A1 (en) 2009-01-28 2010-08-05 Novozymes, Inc. Polypeptides having beta-glucosidase activity and polynucleotides encoding same
WO2010088463A2 (en) 2009-01-30 2010-08-05 Novozymes, Inc. Polypeptides having expansin activity and polynucleotides encoding same
WO2010108918A1 (en) 2009-03-24 2010-09-30 Novozymes A/S Polypeptides having acetyl xylan esterase activity and polynucleotides encoding same
WO2010138754A1 (en) 2009-05-29 2010-12-02 Novozymes, Inc. Methods for enhancing the degradation or conversion of cellulosic material
WO2010141325A1 (en) 2009-06-02 2010-12-09 Novozymes, Inc. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2011002832A1 (en) 2009-06-30 2011-01-06 Novozymes A/S Biomass hydrolysis process
WO2011005867A1 (en) 2009-07-07 2011-01-13 Novozymes, Inc. Polypeptides having cellulolytic enhancing activity activity and polynucleotides encoding same
WO2011008785A2 (en) 2009-07-17 2011-01-20 Novozymes A/S A method of analyzing cellulose decay in cellulosic material hydrolysis
WO2011035027A2 (en) 2009-09-17 2011-03-24 Novozymes, Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2011035029A1 (en) 2009-09-18 2011-03-24 Novozymes, Inc. Polypeptides having beta-glucosidase activity and polynucleotides encoding same
WO2011039319A1 (en) 2009-09-30 2011-04-07 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2011041405A1 (en) 2009-09-29 2011-04-07 Novozymes, Inc. Polypeptides having xylanase activity and polynucleotides encoding same
WO2011041504A1 (en) 2009-09-30 2011-04-07 Novozymes, Inc. Polypeptides derived from thermoascus crustaceus having cellulolytic enhancing activity and polynucleotides encoding same
WO2011041397A1 (en) 2009-09-29 2011-04-07 Novozymes, Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2011050037A1 (en) 2009-10-23 2011-04-28 Novozymes, Inc. Cellobiohydrolase variants and polynucleotides encoding same
WO2011057083A1 (en) 2009-11-06 2011-05-12 Novozymes, Inc. Polypeptides having xylanase activity and polynucleotides encoding same
WO2011057140A1 (en) 2009-11-06 2011-05-12 Novozymes, Inc. Compositions for saccharification of cellulosic material
WO2011057086A1 (en) 2009-11-06 2011-05-12 Novozymes, Inc. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2011059740A1 (en) 2009-10-29 2011-05-19 Novozymes, Inc. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2011080154A1 (en) 2009-12-21 2011-07-07 Novozymes A/S Biomass hydrolysis process
WO2011092136A1 (en) 2010-01-29 2011-08-04 Novozymes A/S Biogas production process with enzymatic pre-treatment
WO2011123450A1 (en) 2010-03-31 2011-10-06 Novozymes, Inc. Cellobiohydrolase variants and polynucleotides encoding same
WO2011126897A2 (en) 2010-03-30 2011-10-13 Novozymes A/S Methods for enhancing by-products from fermentation processes
WO2012003379A1 (en) 2010-06-30 2012-01-05 Novozymes A/S Polypeptides having beta-glucosidase activity and polynucleotides encoding same
WO2012006642A1 (en) 2010-07-07 2012-01-12 Novozymes North America, Inc. Fermentation process
WO2012012590A2 (en) 2010-07-23 2012-01-26 Novozymes A/S Processes for producing fermentation products
WO2012021401A1 (en) 2010-08-12 2012-02-16 Novozymes, Inc. Compositions comprising a polypeptide having cellulolytic enhancing activity and a bicyclic compound and uses thereof
WO2012030858A2 (en) 2010-08-30 2012-03-08 Novozymes A/S Polypeptides having hemicellulolytic activity and polynucleotides encoding same
WO2012030844A1 (en) 2010-08-30 2012-03-08 Novozymes A/S Polypeptides having endoglucanase activity and polynucleotides encoding same
WO2012030845A2 (en) 2010-08-30 2012-03-08 Novozymes A/S Polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same
WO2012030849A1 (en) 2010-08-30 2012-03-08 Novozymes A/S Polypeptides having xylanase activity and polynucleotides encoding same
WO2012030799A1 (en) 2010-08-30 2012-03-08 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2012030811A1 (en) 2010-08-30 2012-03-08 Novozymes A/S Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2012044915A2 (en) 2010-10-01 2012-04-05 Novozymes, Inc. Beta-glucosidase variants and polynucleotides encoding same
WO2012044835A1 (en) 2010-09-30 2012-04-05 Novozymes, Inc. Variants of polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2012044836A1 (en) 2010-09-30 2012-04-05 Novozymes, Inc. Variants of polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2012058293A1 (en) 2010-10-26 2012-05-03 Novozymes North America, Inc. Methods of saccharifying sugarcane trash
WO2012061432A1 (en) 2010-11-02 2012-05-10 Codexis, Inc. Compositions and methods for production of fermentable sugars
WO2012061382A1 (en) 2010-11-02 2012-05-10 Codexis, Inc. Improved fungal strains
WO2012061517A1 (en) 2010-11-02 2012-05-10 Novozymes, Inc. Methods of pretreating cellulosic material with a gh61 polypeptide
WO2012059053A1 (en) 2010-11-04 2012-05-10 Novozymes A/S Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2012062220A1 (en) 2010-11-12 2012-05-18 Novozymes A/S Polypeptides having endoglucanase activity and polynucleotides encoding same
WO2012068509A1 (en) 2010-11-18 2012-05-24 Novozymes, Inc. Chimeric polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
JP2012512658A (en) * 2008-12-19 2012-06-07 イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニー Organic solvent pretreatment of biomass to promote enzymatic saccharification
WO2012078656A1 (en) 2010-12-06 2012-06-14 Novozymes North America, Inc. Methods of hydrolyzing oligomers in hemicellulosic liquor
WO2012093041A1 (en) 2011-01-04 2012-07-12 Novozymes A/S Process for producing biogas from pectin and lignocellulose containing material
WO2012103322A1 (en) 2011-01-26 2012-08-02 Novozymes A/S Polypeptides having endoglucanase activity and polynucleotides encoding same
WO2012103293A1 (en) 2011-01-26 2012-08-02 Novozymes A/S Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2012101206A2 (en) 2011-01-26 2012-08-02 Novozymes A/S Novel glycoside hydrolases from thermophilic fungi
WO2012103288A1 (en) 2011-01-26 2012-08-02 Novozymes A/S Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2012103350A1 (en) 2011-01-26 2012-08-02 Novozymes A/S Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2012113340A1 (en) 2011-02-23 2012-08-30 Novozymes Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2012118767A1 (en) 2011-02-28 2012-09-07 Midori Renewables, Inc. Polymeric acid catalysts and uses thereof
WO2012122518A1 (en) 2011-03-09 2012-09-13 Novozymes A/S Methods of increasing the cellulolytic enhancing activity of a polypeptide
WO2012122477A1 (en) 2011-03-10 2012-09-13 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2012135659A2 (en) 2011-03-31 2012-10-04 Novozymes A/S Methods for enhancing the degradation or conversion of cellulosic material
WO2012130120A1 (en) 2011-03-25 2012-10-04 Novozymes A/S Method for degrading or converting cellulosic material
WO2012134626A2 (en) 2011-01-31 2012-10-04 Novozymes North America, Inc. Processes for enzymatic refining of pretreated cellulosic material for saccharification
WO2012135719A1 (en) 2011-03-31 2012-10-04 Novozymes, Inc. Cellulose binding domain variants and polynucleotides encoding same
WO2012149192A1 (en) 2011-04-28 2012-11-01 Novozymes, Inc. Polypeptides having endoglucanase activity and polynucleotides encoding same
WO2012149344A1 (en) 2011-04-29 2012-11-01 Novozymes, Inc. Methods for enhancing the degradation or conversion of cellulosic material
WO2012159007A1 (en) 2011-05-19 2012-11-22 Novozymes, Inc. Methods for enhancing the degradation of cellulosic material with chitin binding proteins
WO2012159009A1 (en) 2011-05-19 2012-11-22 Novozymes, Inc. Methods for enhancing the degradation of cellulosic material with chitin binding proteins
WO2013000945A1 (en) 2011-06-28 2013-01-03 Novozymes A/S Biogas from enzyme-treated bagasse
WO2013016115A1 (en) 2011-07-22 2013-01-31 Novozymes North America, Inc. Processes for pretreating cellulosic material and improving hydrolysis thereof
WO2013019780A2 (en) 2011-08-04 2013-02-07 Novozymes A/S Polypeptides having endoglucanase activity and polynucleotides encoding same
WO2013019827A2 (en) 2011-08-04 2013-02-07 Novozymes A/S Polypeptides having xylanase activity and polynucleotides encoding same
WO2013028927A1 (en) 2011-08-24 2013-02-28 Novozymes, Inc. Aspergillus fumigatus cellulolytic enzyme compositions and uses thereof
WO2013028928A1 (en) 2011-08-24 2013-02-28 Novozymes, Inc. Cellulolytic enzyme compositions and uses thereof
WO2013039776A1 (en) 2011-09-13 2013-03-21 Novozymes North America, Inc. Methods of hydrolyzing and fermenting cellulosic material
WO2013043981A1 (en) 2011-09-23 2013-03-28 Novozymes A/S Cellulolytic enzyme compositions and uses thereof
WO2013043910A1 (en) 2011-09-20 2013-03-28 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2013064075A1 (en) 2011-10-31 2013-05-10 Novozymes, Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2013074956A2 (en) 2011-11-18 2013-05-23 Novozymes, Inc. Polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same
WO2013075644A1 (en) 2011-11-22 2013-05-30 Novozymes, Inc. Polypeptides having beta-xylosidase activity and polynucleotides encoding same
WO2013079015A1 (en) 2011-12-01 2013-06-06 Novozymes, Inc. Polypeptides having beta-xylosidase activity and polynucleotides encoding same
WO2013083801A2 (en) 2011-12-09 2013-06-13 Novozymes A/S Biogas from substrates comprising animal manure and enzymes
WO2013087027A1 (en) 2011-12-16 2013-06-20 Novozymes, Inc. Polypeptides having laccase activity and polynucleotides encoding same
WO2013089889A2 (en) 2011-09-30 2013-06-20 Novozymes, Inc. Chimeric polypeptides having beta-glucosidase activity and polynucleotides encoding same
WO2013096369A1 (en) 2011-12-19 2013-06-27 Novozymes A/S Processes and compositions for increasing the digestibility of cellulosic materials
WO2013096652A1 (en) 2011-12-21 2013-06-27 Novozymes, Inc. Methods for determining the degradation of a biomass material
WO2013091547A1 (en) 2011-12-19 2013-06-27 Novozymes, Inc. Polypeptides having catalase activity and polynucleotides encoding same
WO2013096603A2 (en) 2011-12-20 2013-06-27 Novozymes, Inc. Cellobiohydrolase variants and polynucleotides encoding same
WO2013119302A2 (en) 2011-11-21 2013-08-15 Novozymes, Inc. Gh61 polypeptide variants and polynucleotides encoding same
WO2013148504A2 (en) 2012-03-26 2013-10-03 Novozymes North America, Inc. Methods of preconditioning cellulosic material
WO2013160247A2 (en) 2012-04-23 2013-10-31 Novozymes A/S Polypeptides having glucuronyl esterase activity and polynucleotides encoding same
WO2013163590A2 (en) 2012-04-27 2013-10-31 Novozymes, Inc. Gh61 polypeptide variants and polynucleotides encoding same
WO2013160248A2 (en) 2012-04-23 2013-10-31 Novozymes A/S Polypeptides having alpha-glucuronidase activity and polynucleotides encoding same
WO2014058896A1 (en) 2012-10-08 2014-04-17 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2014066141A2 (en) 2012-10-24 2014-05-01 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2014070844A1 (en) 2012-10-31 2014-05-08 Danisco Us Inc. Beta-glucosidase from neurospora crassa
WO2014070841A1 (en) 2012-10-31 2014-05-08 Danisco Us Inc. Compositions and methods of use
WO2014070837A1 (en) 2012-10-31 2014-05-08 Danisco Us Inc. Beta-glucosidase from magnaporthe grisea
WO2014092832A2 (en) 2012-09-19 2014-06-19 Novozymes, Inc. Methods for enhancing the degradation or conversion of cellulosic material
WO2014093835A1 (en) 2012-12-14 2014-06-19 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2014099798A1 (en) 2012-12-19 2014-06-26 Novozymes A/S Polypeptides having cellulolytic enhancinc activity and polynucleotides encoding same
WO2014138672A1 (en) 2013-03-08 2014-09-12 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
WO2014182990A1 (en) 2013-05-10 2014-11-13 Novozymes A/S Polypeptides having xylanase activity and polynucleotides encoding same
JP2014239694A (en) * 2005-09-30 2014-12-25 レネサイエンス・アクティーゼルスカブRenescience A/S Non-pressurized pretreatment, enzymatic hydrolysis and fermentation of waste fraction
WO2015007290A1 (en) 2013-07-16 2015-01-22 Advanced Substrate Technologies A/S Method for cycling biomasses between mushroom cultivation and anaerobic biogas fermentation, and for separating and drying a degassed biomass
WO2015035029A1 (en) 2013-09-04 2015-03-12 Novozymes A/S Processes for increasing enzymatic hydrolysis of cellulosic material
WO2015066492A1 (en) 2013-11-01 2015-05-07 Novozymes A/S Methods of saccharifying and fermenting a cellulosic material
WO2015081139A1 (en) 2013-11-26 2015-06-04 Novozymes A/S Enzyme compositions and uses thereof
WO2015084596A1 (en) 2013-12-04 2015-06-11 Danisco Us Inc. Compositions comprising a beta-glucosidase polypeptide and methods of use
WO2015105835A1 (en) 2014-01-07 2015-07-16 Novozymes A/S Process for degrading mannan-containing cellulosic materials
WO2015187935A1 (en) 2014-06-06 2015-12-10 Novozymes A/S Enzyme compositions and uses thereof
EP2963060A2 (en) 2008-04-30 2016-01-06 Xyleco, Inc. Processing biomass
WO2016004482A1 (en) * 2014-07-10 2016-01-14 Leaf Sciences Pty Ltd Methods for hydrolysing lignocellulosic material
WO2016004481A1 (en) * 2014-07-10 2016-01-14 Leaf Sciences Pty Ltd Methods for treating lignocellulosic material
US9238845B2 (en) 2012-08-24 2016-01-19 Midori Usa, Inc. Methods of producing sugars from biomass feedstocks
WO2016029107A1 (en) 2014-08-21 2016-02-25 Novozymes A/S Process for saccharifying cellulosic material under oxygen addition
WO2016037096A1 (en) 2014-09-05 2016-03-10 Novozymes A/S Carbohydrate binding module variants and polynucleotides encoding same
WO2016045569A1 (en) 2014-09-23 2016-03-31 Novozymes A/S Processes for producing ethanol and fermenting organisms
WO2016069541A1 (en) 2014-10-27 2016-05-06 Danisco Us Inc Compositions and methods related to beta-glucosidase
EP2403894B1 (en) 2009-03-03 2016-06-01 The Coca-Cola Company Bio-based polyethylene terephthalate packaging and method of making thereof
WO2016116113A1 (en) 2015-01-22 2016-07-28 Advanced Substrate Technologies A/S Methods for upgrading spent biomass material
WO2016138167A2 (en) 2015-02-24 2016-09-01 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
WO2016145363A1 (en) 2015-03-12 2016-09-15 Novozymes A/S Multi-stage enzymatic hydrolysis of lignocellulosic biomass employing an oxidoreductase with an aa9 polypeptide
WO2016145350A1 (en) 2015-03-12 2016-09-15 Novozymes A/S Multi-stage enzymatic hydrolysis of lignocellulosic biomass
WO2016145358A1 (en) 2015-03-12 2016-09-15 Novozymes A/S Enzymatic hydrolysis with hemicellulolytic enzymes
US9499783B2 (en) 2011-09-14 2016-11-22 Toray Industries, Inc. Production apparatus of sugar solution and production system of sugar solution
WO2016188459A1 (en) 2015-05-27 2016-12-01 Novozymes A/S Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2017019490A1 (en) 2015-07-24 2017-02-02 Novozymes Inc. Polypeptides having arabinofuranosidase activity and polynucleotides encoding same
WO2017019491A1 (en) 2015-07-24 2017-02-02 Novozymes Inc. Polypeptides having beta-xylosidase activity and polynucleotides encoding same
WO2017050242A1 (en) 2015-09-22 2017-03-30 Novozymes A/S Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2017070219A1 (en) 2015-10-20 2017-04-27 Novozymes A/S Lytic polysaccharide monooxygenase (lpmo) variants and polynucleotides encoding same
WO2017144670A1 (en) 2016-02-24 2017-08-31 Danmarks Tekniske Universitet Improved process for producing a fermentation product from a lignocellulose-containing material
WO2017151957A1 (en) 2016-03-02 2017-09-08 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
WO2017165760A1 (en) 2016-03-24 2017-09-28 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
WO2017205535A1 (en) 2016-05-27 2017-11-30 Novozymes, Inc. Polypeptides having endoglucanase activity and polynucleotides encoding same
WO2018026868A1 (en) 2016-08-01 2018-02-08 Novozymes, Inc. Polypeptides having endoglucanase activity and polynucleotides encoding same
WO2018050300A1 (en) 2016-09-13 2018-03-22 Institut National De La Recherche Agronomique (Inra) Polysaccharide-oxidizing composition and uses thereof
EP3305906A1 (en) 2008-04-30 2018-04-11 Xyleco, Inc. Processing biomass by electron beam irradiation
KR20180042416A (en) * 2009-08-06 2018-04-25 안니키 게엠베하 Process for the production of carbohydrate cleavage products from a lignocellulosic material
WO2018085370A1 (en) 2016-11-02 2018-05-11 Novozymes A/S Processes for reducing production of primeverose during enzymatic saccharification of lignocellulosic material
WO2018106792A1 (en) 2016-12-06 2018-06-14 Novozymes A/S Improved processes for production of ethanol from xylose-containing cellulosic substrates using engineered yeast strains
WO2018222990A1 (en) 2017-06-02 2018-12-06 Novozymes A/S Improved yeast for ethanol production
WO2018220116A1 (en) 2017-05-31 2018-12-06 Novozymes A/S Xylose fermenting yeast strains and processes thereof for ethanol production
WO2019025449A1 (en) 2017-08-02 2019-02-07 Institut National De La Recherche Agronomique (Inra) Methods of defibrillating cellulosic substrates and producing celluloses using a new family of fungal lytic polysaccharide monooxygenases (lpmo)
EP3461899A1 (en) 2008-04-30 2019-04-03 Xyleco, Inc. Processing biomass
WO2019148192A1 (en) 2018-01-29 2019-08-01 Novozymes A/S Microorganisms with improved nitrogen utilization for ethanol production
FR3083247A1 (en) 2018-07-02 2020-01-03 Institut National De La Recherche Agronomique (Inra) POLYPEPTIDES AND COMPOSITIONS WITH LYSTIC OXIDASE POLYSACCHARIDE ACTIVITY
WO2020023411A1 (en) 2018-07-25 2020-01-30 Novozymes A/S Enzyme-expressing yeast for ethanol production
US10590440B2 (en) 2014-08-22 2020-03-17 Cysbio Aps Process for producing a fermentation product from a lignocellulose-containing material
US10597595B2 (en) 2017-10-27 2020-03-24 Xyleco, Inc. Processing biomass
WO2020076697A1 (en) 2018-10-08 2020-04-16 Novozymes A/S Enzyme-expressing yeast for ethanol production
WO2020123463A1 (en) 2018-12-12 2020-06-18 Novozymes A/S Polypeptides having xylanase activity and polynucleotides encoding same
WO2021021458A1 (en) 2019-07-26 2021-02-04 Novozymes A/S Microorganisms with improved nitrogen transport for ethanol production
WO2021025872A1 (en) 2019-08-06 2021-02-11 Novozymes A/S Fusion proteins for improved enzyme expression
WO2021026201A1 (en) 2019-08-05 2021-02-11 Novozymes A/S Enzyme blends and processes for producing a high protein feed ingredient from a whole stillage byproduct
WO2021055395A1 (en) 2019-09-16 2021-03-25 Novozymes A/S Polypeptides having beta-glucanase activity and polynucleotides encoding same
WO2021119304A1 (en) 2019-12-10 2021-06-17 Novozymes A/S Microorganism for improved pentose fermentation
EP3848469A1 (en) 2013-02-21 2021-07-14 Novozymes A/S Methods of saccharifying and fermenting a cellulosic material
WO2022049250A1 (en) 2020-09-04 2022-03-10 Novozymes A/S Improved fermenting organism for ethanol production
WO2022090564A1 (en) 2020-11-02 2022-05-05 Novozymes A/S Glucoamylase variants and polynucleotides encoding same
WO2022261003A1 (en) 2021-06-07 2022-12-15 Novozymes A/S Engineered microorganism for improved ethanol fermentation
WO2023164436A1 (en) 2022-02-23 2023-08-31 Novozymes A/S Process for producing fermentation products and biogas from starch-containing materials

Families Citing this family (378)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7537826B2 (en) * 1999-06-22 2009-05-26 Xyleco, Inc. Cellulosic and lignocellulosic materials and compositions and composites made therefrom
BRPI0516665B8 (en) 2004-11-29 2022-08-09 Elsam Eng A/S PROCESSES FOR LIQUEFATION OF BIOMASSES CONTAINING POLYSACCHARIDE
US20150328347A1 (en) 2005-03-24 2015-11-19 Xyleco, Inc. Fibrous materials and composites
US7708214B2 (en) * 2005-08-24 2010-05-04 Xyleco, Inc. Fibrous materials and composites
US7781191B2 (en) * 2005-04-12 2010-08-24 E. I. Du Pont De Nemours And Company Treatment of biomass to obtain a target chemical
EP1869201B1 (en) * 2005-04-12 2017-12-27 E. I. du Pont de Nemours and Company Integration of alternative feedstreams in biomass treatment and utilization
US20070029252A1 (en) * 2005-04-12 2007-02-08 Dunson James B Jr System and process for biomass treatment
US8945899B2 (en) 2007-12-20 2015-02-03 Butamax Advanced Biofuels Llc Ketol-acid reductoisomerase using NADH
US20080274526A1 (en) 2007-05-02 2008-11-06 Bramucci Michael G Method for the production of isobutanol
AU2007210012B2 (en) * 2006-01-27 2012-04-05 University Of Massachussetts Systems and methods for producing biofuels and related materials
US8968515B2 (en) 2006-05-01 2015-03-03 Board Of Trustees Of Michigan State University Methods for pretreating biomass
US9206446B2 (en) 2006-05-01 2015-12-08 Board Of Trustees Of Michigan State University Extraction of solubles from plant biomass for use as microbial growth stimulant and methods related thereto
BRPI0722418B1 (en) * 2006-05-01 2023-10-10 Dartmouth College Process for treating lignocellulosic biomass with ammonia and water vapors
US8962298B2 (en) 2006-05-02 2015-02-24 Butamax Advanced Biofuels Llc Recombinant host cell comprising a diol dehydratase
US7527941B1 (en) * 2006-05-24 2009-05-05 Clear Water Technologies, Inc. Process for producing ethyl alcohol from cellulosic materials
JP5190858B2 (en) * 2006-07-12 2013-04-24 独立行政法人農業・食品産業技術総合研究機構 Production method of low molecular weight carbohydrates from materials containing polysaccharides
US8323923B1 (en) 2006-10-13 2012-12-04 Sweetwater Energy, Inc. Method and system for producing ethanol
WO2008048513A2 (en) * 2006-10-13 2008-04-24 Rowan University Ethanol resistant and furfural resistant strains of e. coli fbr5 for production of ethanol from cellulosic biomass
US9499635B2 (en) 2006-10-13 2016-11-22 Sweetwater Energy, Inc. Integrated wood processing and sugar production
AU2013202821B2 (en) * 2006-10-26 2015-02-12 Xyleco, Inc. Processing biomass
US20080131947A1 (en) * 2006-12-01 2008-06-05 Cellencor, Inc. Treatment of Cellulosic Material for Ethanol Production
CA2674534A1 (en) * 2007-01-23 2008-07-31 Basf Se Method for producing glucose by enzymatic hydrolysis of cellulose that is obtained from material containing ligno-cellulose using an ionic liquid that comprises a polyatomic anion
WO2008095033A2 (en) * 2007-01-30 2008-08-07 Verenium Corporation Enzymes for the treatment of lignocellulosics, nucleic acids encoding them and methods for making and using them
JP4984999B2 (en) * 2007-03-16 2012-07-25 独立行政法人産業技術総合研究所 Method for producing sugar
WO2008127629A1 (en) * 2007-04-14 2008-10-23 Gibbs Energy Llc Consortial growth of microorganisms for fuel feedstocks
NZ579780A (en) 2007-04-18 2012-07-27 Butamax Advanced Biofuels Llc Fermentive production of isobutanol using highly active ketol-acid reductoisomerase enzymes
US20080277082A1 (en) * 2007-05-07 2008-11-13 Andritz Inc. High pressure compressor and steam explosion pulping method
JP5385549B2 (en) * 2007-05-14 2014-01-08 大成建設株式会社 Preservation method of plant biomass
US20090011474A1 (en) * 2007-06-20 2009-01-08 Board Of Trustees Of Michigan State University Process for producing sugars from cellulosic biomass
US7819976B2 (en) 2007-08-22 2010-10-26 E. I. Du Pont De Nemours And Company Biomass treatment method
US8445236B2 (en) * 2007-08-22 2013-05-21 Alliance For Sustainable Energy Llc Biomass pretreatment
US7807419B2 (en) * 2007-08-22 2010-10-05 E. I. Du Pont De Nemours And Company Process for concentrated biomass saccharification
US20090053771A1 (en) * 2007-08-22 2009-02-26 Board Of Trustees Of Michigan State University Process for making fuels and chemicals from AFEX-treated whole grain or whole plants
WO2009040791A1 (en) * 2007-09-24 2009-04-02 Asher Vitner Ltd. A process for the recovery of a fermentation product
US8367378B2 (en) * 2007-10-03 2013-02-05 Board Of Trustees Of Michigan State University Process for producing sugars and ethanol using corn stillage
RU2010116359A (en) * 2007-10-09 2011-12-10 Маскома Канада Инк. (Ca) TWO-STAGE METHOD OF ENZYMATIC HYDROLYSIS FOR PROCESSING LIGNO CELLULAR MATERIALS
BRPI0816621A2 (en) * 2007-10-12 2016-08-30 Archer Daniels Midland Co "Method for increasing the amount of glucose and other sugars and peptides released from a fiber starting material and method for obtaining a solid hydrolyzed fiber for bio-oil production"
CN101848920A (en) * 2007-10-17 2010-09-29 新日铁化学株式会社 Production methods for solubilized lignin, saccharide raw material and monosaccharide raw material, and solubilized lignin
JP2011515066A (en) * 2007-10-30 2011-05-19 イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニー Zymomonas with improved ethanol production capacity in medium containing high concentrations of sugars and acetates
JP2011515067A (en) * 2007-10-30 2011-05-19 イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニー Method for producing ethanol from a medium containing xylose using a recombinant zymomonas strain with reduced expression of himA
JP5088951B2 (en) * 2007-11-22 2012-12-05 新日鐵化学株式会社 Method for producing water-soluble saccharide raw material from beer-added koji
JP2009125050A (en) * 2007-11-28 2009-06-11 Jfe Engineering Corp Pretreatment method for enzymatic hydrolysis of herbaceous biomass, ethanol production method using herbaceous biomass as raw material and ethanol production method using palm hollow bunch
EP2237679A4 (en) * 2007-12-14 2014-01-01 Abengoa Bioenergy New Technologies Inc Improved quality and value of co-products of the ethanol production industry
EP2238257B1 (en) * 2007-12-24 2017-08-23 Reliance Life Sciences Pvt., Ltd. Process for production of high yield of biobutanol
US8641910B2 (en) * 2008-02-05 2014-02-04 Syngenta Participations Ag Systems and processes for producing biofuels from biomass
CN101981199A (en) * 2008-02-27 2011-02-23 奎特罗斯公司 Methods for the conversion of plant materials into fuels and chemicals by sequential action of two microorganisms
US8058039B2 (en) * 2008-03-13 2011-11-15 North American Bioproducts Corporation Use of erythromycin as a selective antimicrobial agent in the production of alcohols
US20090238920A1 (en) * 2008-03-21 2009-09-24 Lewis Ted C Process for making high grade protein product
JP5690713B2 (en) * 2008-03-21 2015-03-25 ダニスコ・ユーエス・インク Hemicellulase enhanced composition for promoting hydrolysis of biomass
WO2009122728A1 (en) * 2008-03-31 2009-10-08 超音波醸造所有限会社 Biomass alcohol manufacturing method
PT2274433E (en) * 2008-04-03 2012-08-16 Cellulose Sciences International Inc Highly disordered cellulose
US20090286294A1 (en) * 2008-04-04 2009-11-19 University Of Massachusetts Methods and Compositions for Improving the Production of Fuels in Microorganisms
CN101250567B (en) * 2008-04-16 2011-06-15 中国石油化工股份有限公司 Method for preparing monosaccharide by steam blasting lignocellulosic materials and synchronous hydrolysis with soluble oligosaccharide
US20090269817A1 (en) * 2008-04-29 2009-10-29 Icm, Inc. Pretreatment of grain slurry with alpha-amylase and a hemicellulase blend prior to liquefaction
US7931784B2 (en) * 2008-04-30 2011-04-26 Xyleco, Inc. Processing biomass and petroleum containing materials
US8252566B2 (en) * 2008-05-20 2012-08-28 Jj Florida Properties Llc Ethanol production from citrus waste through limonene reduction
MX2010012736A (en) * 2008-05-20 2011-07-20 Jj Florida Properties Llc Removal of fermentation inhibiting compounds from citrus waste using solvent extraction and production of ethanol.
BRPI0915017B1 (en) 2008-06-09 2018-12-26 Lanzatech New Zealand Ltd a method for producing 2,3-butanediol by microbial fermentation of a gaseous substrate comprising co
US20100105114A1 (en) * 2008-06-11 2010-04-29 University Of Massachusetts Methods and Compositions for Regulating Sporulation
BRPI0915749A2 (en) * 2008-07-08 2018-07-10 Opx Biotechnologies Inc methods, compositions and systems for biosynthetic production of 1,4-butanediol
US20110177573A1 (en) * 2008-08-01 2011-07-21 Sarah All Microbial Treatment of Lignocellulosic Biomass
CN106978455A (en) * 2008-08-11 2017-07-25 帝斯曼知识产权资产管理有限公司 The degraded of ligno-cellulosic materials
US20110165639A1 (en) * 2008-08-15 2011-07-07 Brijen Biotech, Llc Refinery process to produce biofuels and bioenergy products from home and municipal solid waste
US8563282B2 (en) * 2008-08-27 2013-10-22 Edeniq, Inc. Materials and methods for converting biomass to biofuel
US20100068777A1 (en) * 2008-09-17 2010-03-18 Omnilytics, Incorporated Methods for producing biofuels and compositions for use in biofuel production methods
EP2337856A1 (en) 2008-09-29 2011-06-29 Butamax Advanced Biofuels Llc INCREASED HETEROLOGOUS Fe-S ENZYME ACTIIVTY IN YEAST
EP2331683A1 (en) * 2008-09-29 2011-06-15 Butamax Advanced Biofuels Llc Enhanced dihydroxy-acid dehydratase activity in lactic acid bacteria
EP2337851B1 (en) 2008-09-29 2014-08-13 Butamax Advanced Biofuels Llc IDENTIFICATION AND USE OF BACTERIAL Ý2Fe-2S¨DIHYDROXY-ACID DEHYDRATASES
US8455224B2 (en) * 2008-09-29 2013-06-04 Butamax(Tm) Advanced Biofuels Llc Enhanced pyruvate to 2,3-butanediol conversion in lactic acid bacteria
US8465964B2 (en) 2008-11-13 2013-06-18 Butamax (TM) Advanced Biofules LLC Increased production of isobutanol in yeast with reduced mitochondrial amino acid biosynthesis
US8828694B2 (en) * 2008-11-13 2014-09-09 Butamax Advanced Biofuels Llc Production of isobutanol in yeast mitochondria
US8715969B2 (en) * 2008-11-20 2014-05-06 E I Du Pont De Nemours And Company Delignification of biomass with sequential base treatment
US8524474B2 (en) * 2008-11-20 2013-09-03 E I Du Pont De Nemours And Company Process for producing a concentrated sugar solution by enzymatic saccharification of polysaccharide enriched biomass
CA2739704C (en) * 2008-11-20 2015-08-18 E.I. Du Pont De Nemours And Company Process for producing a sugar solution by combined chemical and enzymatic saccharification of polysaccharide enriched biomass
US8652823B2 (en) 2008-12-03 2014-02-18 Butamax(Tm) Advanced Biofuels Llc Strain for butanol production with increased membrane unsaturated trans fatty acids
US20100143995A1 (en) * 2008-12-04 2010-06-10 E. I. Du Pont De Nemours And Company Process for Fermentive Preparation of Alcohols and Recovery of Product
US20100143993A1 (en) * 2008-12-04 2010-06-10 E.I. Du Pont De Nemours And Company Process for fermentive preparationfor alcolhols and recovery of product
US20100143992A1 (en) * 2008-12-04 2010-06-10 E. I Du Pont De Nemours And Company Process for Fermentive Preparation of Alcohols and Recovery of Product
US20100143999A1 (en) * 2008-12-04 2010-06-10 E.I. Du Pont De Nemours And Company Process for fermentive preparation of alcohols and recovery of product
US20100140166A1 (en) * 2008-12-04 2010-06-10 E.I. Du Pont De Nemours And Company Process for fermentive preparation of alcohols and recovery of product
US20100143994A1 (en) * 2008-12-04 2010-06-10 E. I. Du Pont De Memours And Company Process for fermentive preparation of alcohols and recovery of product
ES2764124T3 (en) 2008-12-09 2020-06-02 Toray Industries Procedure to produce liquid containing sugar
CA2781862C (en) * 2008-12-09 2018-02-13 Sweetwater Energy, Inc. Ensiling biomass for biofuels production and multiple phase apparatus for hydrolyzation of ensiled biomass
WO2010080489A1 (en) 2008-12-19 2010-07-15 E. I. Du Pont De Nemours And Company Ozone treatment of biomass to enhance enzymatic saccharification
WO2010075213A2 (en) * 2008-12-22 2010-07-01 Mascoma Corporation Production of ethanol from lignocellulosic biomass
US8247208B2 (en) 2008-12-22 2012-08-21 Alliance For Sustainable Energy Llc Zymomonas with improved xylose utilization in stress conditions
WO2010075529A2 (en) * 2008-12-23 2010-07-01 Mascoma Corporation Heterologous biomass degrading enzyme expression in thermoanaerobacterium saccharolyticum
JP2010161997A (en) * 2009-01-19 2010-07-29 Tech Corporation:Kk Method for producing saccharified liquid by using seed of assai palm and method for producing ethanol by using the saccharified liquid
JP5381119B2 (en) * 2009-01-23 2014-01-08 王子ホールディングス株式会社 Method for producing saccharides from bark raw material
EP2393932A2 (en) * 2009-02-03 2011-12-14 Hercules Incorporated Process for treating biomass to derivatize polysaccharides contained therein to increase their accessibility to hydrolysis and subsequent fermentation
US8614085B2 (en) * 2009-02-27 2013-12-24 Butamax(Tm) Advanced Biofuels Llc Yeast with increased butanol tolerance involving a multidrug efflux pump gene
CA2754910A1 (en) * 2009-03-09 2010-09-16 Qteros, Inc. Production of fermentive end products from clostridium sp.
US20100086981A1 (en) * 2009-06-29 2010-04-08 Qteros, Inc. Compositions and methods for improved saccharification of biomass
WO2010113949A1 (en) * 2009-03-30 2010-10-07 三井造船株式会社 Method for producing cereal-origin saccharified solution
US20160369304A9 (en) * 2009-05-18 2016-12-22 Poet Research, Inc. System for treatment of biomass to facilitate the production of ethanol
US8636402B2 (en) 2009-05-20 2014-01-28 Xyleco, Inc. Processing biomass
AU2010249679B2 (en) 2009-05-20 2015-02-05 Xyleco, Inc. Processing biomass
EP2432887B1 (en) 2009-05-21 2016-03-09 Board Of Trustees Of Michigan State University Methods for pretreating biomass
EP2438183A2 (en) * 2009-05-26 2012-04-11 Lali, Arvind Mallinath Method for production of fermentable sugars from biomass
MX2011012841A (en) 2009-06-03 2012-01-27 Danisco Inc Cellulase variants with improved expression, activity and/or stability, and use thereof.
US8669397B2 (en) 2009-06-13 2014-03-11 Rennovia, Inc. Production of adipic acid and derivatives from carbohydrate-containing materials
BRPI1013087B1 (en) 2009-06-13 2018-05-15 Rennovia, Inc. PROCESS FOR PREPARING A GLUTARIC ACID PRODUCT
EP2440515B1 (en) 2009-06-13 2018-08-15 Archer-Daniels-Midland Company Production of adipic acid and derivatives from carbohydrate-containing materials
JP2011037719A (en) * 2009-08-06 2011-02-24 Nippon Rensui Co Ltd Purification method of bioethanol and purification apparatus of bioethanol
US8945245B2 (en) 2009-08-24 2015-02-03 The Michigan Biotechnology Institute Methods of hydrolyzing pretreated densified biomass particulates and systems related thereto
US10457810B2 (en) 2009-08-24 2019-10-29 Board Of Trustees Of Michigan State University Densified biomass products containing pretreated biomass fibers
ES2529510T3 (en) 2009-08-24 2015-02-20 Board Of Trustees Of Michigan State University Densified and pretreated biomass products and their manufacturing and use procedures
RU2012116081A (en) 2009-09-23 2013-10-27 ДАНИСКО ЮЭс ИНК. NEW GLYCOSYL HYDROLASE ENZYMES AND THEIR APPLICATIONS
AU2010300642B2 (en) 2009-09-29 2015-05-14 Butamax(Tm) Advanced Biofuels Llc Fermentive production of isobutanol using highly effective ketol-acid reductoisomerase enzymes
EP2483400A1 (en) 2009-09-29 2012-08-08 Butamax(tm) Advanced Biofuels LLC Improved flux to acetolactate-derived products in lactic acid bacteria
WO2011041426A1 (en) * 2009-09-29 2011-04-07 Butamax(Tm) Advanced Biofuels Llc Improved yeast production host cells
US20110195505A1 (en) * 2009-10-08 2011-08-11 Butamax(Tm) Advanced Biofuels Llc Bacterial strains for butanol production
EP2488655A2 (en) * 2009-10-12 2012-08-22 E. I. du Pont de Nemours and Company Ammonia pretreatment of biomass for improved inhibitor profile
US20110250645A1 (en) * 2009-10-12 2011-10-13 E.I. Du Pont De Nemours And Company Methods to improve monomeric sugar release from lignocellulosic biomass following alkaline pretreatment
ES2671798T3 (en) * 2009-10-14 2018-06-08 Xyleco, Inc. Edible waste production from ethanol production
CA2777130A1 (en) * 2009-11-02 2011-05-05 Hercules Incorporated Process for treating biomass to increase accessibility of polysaccharides contained therein to hydrolysis and subsequent fermentation, and polysaccharides with increased accessibility
US20110125118A1 (en) * 2009-11-20 2011-05-26 Opx Biotechnologies, Inc. Production of an Organic Acid and/or Related Chemicals
DK2599861T3 (en) 2009-11-20 2017-11-06 Danisco Us Inc BETA-GLUCOSIDASE VARIETIES WITH IMPROVED PROPERTIES
BRPI0904538B1 (en) 2009-11-30 2018-03-27 Ctc - Centro De Tecnologia Canavieira S.A. VEGETABLE BIOMASS TREATMENT PROCESS
EP2333151A1 (en) * 2009-12-11 2011-06-15 Nederlandse Organisatie voor toegepast -natuurwetenschappelijk onderzoek TNO Novel method for processing lignocellulose containing material
WO2011081658A2 (en) * 2009-12-15 2011-07-07 Qteros, Inc. Methods and compositions for producing chemical products from c. phytofermentants
US8906204B2 (en) 2009-12-21 2014-12-09 Butamax Advanced Biofuels Llc Methods for alcohol recovery and concentration of stillage by-products
US8628623B2 (en) * 2009-12-21 2014-01-14 Andritz Technology And Asset Management Gmbh Method and process for dry discharge in a pressurized pretreatment reactor
JP2011130680A (en) * 2009-12-22 2011-07-07 Ajinomoto Co Inc Method for producing sugar solution from residue of sugarcane-squeezed juice
US8647850B2 (en) 2009-12-23 2014-02-11 E I Du Pont De Nemours And Company Process for simultaneous saccharification and fermentation for production of ethanol
AU2010333801B2 (en) * 2009-12-23 2015-12-17 Danisco Us Inc. Methods for improving the efficiency of simultaneous saccharification and fermentation reactions
CN102762722B (en) 2009-12-29 2015-05-20 布特马斯先进生物燃料有限责任公司 Alcohol dehydrogenases (adh) useful for fermentive production of lower alkyl alcohols
CN102741401A (en) 2009-12-29 2012-10-17 布特马斯先进生物燃料有限责任公司 Expression of hexose kinase in recombinant host cells
WO2011092711A1 (en) * 2010-01-27 2011-08-04 Council Of Scientific & Industrial Research A one pot and single step hydrolytic process for the conversion of lignocellulose into value added chemicals
US8574406B2 (en) 2010-02-09 2013-11-05 Butamax Advanced Biofuels Llc Process to remove product alcohol from a fermentation by vaporization under vacuum
CA2787947A1 (en) * 2010-02-09 2011-08-18 Syngenta Participations Ag Systems and processes for producing biofuels from biomass
JP5950830B2 (en) 2010-02-17 2016-07-13 ビュータマックス・アドバンスド・バイオフューエルズ・エルエルシー Improvement of activity of Fe-S cluster requirement protein
US9034627B2 (en) 2010-03-01 2015-05-19 Toray Industries, Inc. Method for producing glucosidase, enzyme composition, and method for hydrolyzing biomass
US8669393B2 (en) 2010-03-05 2014-03-11 Rennovia, Inc. Adipic acid compositions
CA2791668A1 (en) 2010-03-10 2011-09-15 Toray Industries, Inc. Method for producing pure sugar solution, and method for producing chemical product
CN102791873A (en) 2010-03-15 2012-11-21 东丽株式会社 Manufacturing method for sugar solution and device for same
ES2689865T3 (en) 2010-03-15 2018-11-16 Toray Industries, Inc. Procedure for the production of a solution containing sugar
GB2478791A (en) * 2010-03-19 2011-09-21 Qteros Inc Ethanol production by genetically-modified bacteria
CN102939388B (en) * 2010-04-19 2016-08-03 密歇根州立大学董事会 Lignocellulose biomass and extract and preparation method thereof can be digested
US8906235B2 (en) 2010-04-28 2014-12-09 E I Du Pont De Nemours And Company Process for liquid/solid separation of lignocellulosic biomass hydrolysate fermentation broth
US8721794B2 (en) 2010-04-28 2014-05-13 E I Du Pont De Nemours And Company Production of high solids syrup from lignocellulosic biomass hydrolysate fermentation broth
CN101824439A (en) * 2010-04-28 2010-09-08 江苏绿丰生物药业有限公司 Method for fermentation preparation of L-lactic acid after microwave-alkali coupling pretreatment of distilled grain
JP5587028B2 (en) * 2010-05-12 2014-09-10 本田技研工業株式会社 Lignocellulosic biomass saccharification pretreatment equipment
CN101824339B (en) * 2010-05-12 2014-05-07 哈尔滨理工大学 Ethanol/water mixed solvent preprocessing biomass and method for preparing liquid fuel
US9701969B2 (en) 2010-06-03 2017-07-11 Danisco Us Inc. Filamentous fungal host strains and DNA constructs, and methods of use thereof
EP2579730A2 (en) * 2010-06-08 2013-04-17 Kenneth Hillel Peter Harris Methods for making animal feed from lignocellulosic biomass
CN103097437A (en) 2010-06-10 2013-05-08 阿文德·马立纳什·拉里 Process for fractionation of biomass
US9770705B2 (en) 2010-06-11 2017-09-26 Rennovia Inc. Oxidation catalysts
US8389243B2 (en) * 2010-06-16 2013-03-05 Catchlight Energy Llc Methods of spraying saccharification enzymes and fermentation organisms onto lignocellulosic biomass for hydrolysis and fermentation processes
US8871488B2 (en) 2010-06-18 2014-10-28 Butamax Advanced Biofuels Llc Recombinant host cells comprising phosphoketolases
US9012190B2 (en) 2011-06-15 2015-04-21 Butamax Advanced Biofuels Llc Use of thiamine and nicotine adenine dinucleotide for butanol production
BR112012032166A2 (en) 2010-06-18 2015-11-24 Butamax Tm Advanced Biofuels “COMPOSITION, METHOD FOR PRODUCING AN EXTRACTOR AND METHODS FOR PRODUCING AN ALCOHOL PRODUCT”
BR112012032172A2 (en) 2010-06-18 2015-11-24 Butamax Tm Advanced Biofuels method to produce an alcohol and composition
US9040263B2 (en) 2010-07-28 2015-05-26 Butamax Advanced Biofuels Llc Production of alcohol esters and in situ product removal during alcohol fermentation
JP2012005382A (en) * 2010-06-23 2012-01-12 Equos Research Co Ltd Biomass hydrolyzing device
EP2586871A1 (en) 2010-06-24 2013-05-01 Toray Industries, Inc. Process for production of aqueous refined sugar solution
EP3401410B1 (en) 2010-06-26 2020-12-30 Virdia, Inc. Methods for production of sugar mixtures
IL206678A0 (en) 2010-06-28 2010-12-30 Hcl Cleantech Ltd A method for the production of fermentable sugars
JP2012010638A (en) * 2010-06-30 2012-01-19 Honda Motor Co Ltd Process for producing saccharified solution of lignocellulosic biomass
US20130230624A1 (en) * 2010-07-01 2013-09-05 Commonwealth Scientific And Industrial Research Organisation Treatment of plant biomass
JP5713591B2 (en) * 2010-07-05 2015-05-07 本田技研工業株式会社 Saccharification pretreatment equipment for lignocellulosic biomass
JP5779323B2 (en) * 2010-07-05 2015-09-16 本田技研工業株式会社 Pre-saccharification method for lignocellulosic biomass
JP5759458B2 (en) 2010-07-05 2015-08-05 本田技研工業株式会社 Saccharification pretreatment equipment for lignocellulosic biomass
JP2012019730A (en) 2010-07-14 2012-02-02 Honda Motor Co Ltd Lignocellulosic biomass saccharification pre-treatment device
US8651403B2 (en) 2010-07-21 2014-02-18 E I Du Pont De Nemours And Company Anhydrous ammonia treatment for improved milling of biomass
IL207329A0 (en) 2010-08-01 2010-12-30 Robert Jansen A method for refining a recycle extractant and for processing a lignocellulosic material and for the production of a carbohydrate composition
WO2012024698A1 (en) 2010-08-20 2012-02-23 Codexis, Inc. Use of glycoside hydrolase 61 family proteins in processing of cellulose
IL207945A0 (en) 2010-09-02 2010-12-30 Robert Jansen Method for the production of carbohydrates
CA2807930A1 (en) 2010-09-02 2012-03-08 Butamax (Tm) Advanced Buofuels Llc Process to remove product alcohol from a fermentation by vaporization under vacuum
BR112013006004A2 (en) 2010-09-14 2016-06-07 Cellulose Sciences International Inc nano-disaggregated cellulose
BR112013008347A2 (en) 2010-10-06 2016-06-14 Bp Corp North America Inc cbh variant polypeptides i
US10334870B2 (en) 2010-10-07 2019-07-02 Tropicana Products, Inc. Processing of whole fruits and vegetables, processing of side-stream ingredients of fruits and vegetables, and use of the processed fruits and vegetables in beverage and food products
CA2806595A1 (en) * 2010-10-15 2012-04-19 Andritz Technology And Asset Management Gmbh High solids enzyme reactor or mixer and method
US8444810B2 (en) 2010-11-21 2013-05-21 Aicardo Roa-Espinosa Apparatus and process for treatment of fibers
US20120125548A1 (en) 2010-11-23 2012-05-24 E. I. Du Pont De Nemours And Company Continuously fed biomass pretreatment process for a packed bed reactor
US20120125551A1 (en) * 2010-11-23 2012-05-24 E. I. Du Pont De Nemours And Company Biomass pretreatment process for a packed bed reactor
PT106039A (en) 2010-12-09 2012-10-26 Hcl Cleantech Ltd PROCESSES AND SYSTEMS FOR PROCESSING LENHOCELLULOSIC MATERIALS AND RELATED COMPOSITIONS
US8476048B2 (en) 2010-12-17 2013-07-02 E I Du Pont De Nemours And Company Xylose utilizing zymomonas mobilis with improved ethanol production in biomass hydrolysate medium
US20130040340A1 (en) 2011-02-07 2013-02-14 E. I. Du Pont De Nemours And Company Production of alcohol esters in situ using alcohols and fatty acids produced by microorganisms
CN103328640A (en) * 2011-02-14 2013-09-25 希乐克公司 Processing paper stock
JP5867911B2 (en) * 2011-03-02 2016-02-24 日立造船株式会社 Method for producing ethanol from waste
CA2828505A1 (en) 2011-03-03 2012-09-07 Toray Industries, Inc. Method for producing sugar solution
RU2013146341A (en) 2011-03-17 2015-04-27 ДАНИСКО ЮЭс ИНК CELLULASE COMPOSITIONS AND WAYS OF THEIR APPLICATION FOR THE IMPROVED TRANSFORMATION OF LIGNO CELLULAR BIOMASS IN FERMENTABLE SUGAR
JP6290626B2 (en) 2011-03-17 2018-03-14 ダニスコ・ユーエス・インク Method for reducing viscosity in a saccharification process
AU2012229042B2 (en) 2011-03-17 2017-03-02 Danisco Us Inc. Glycosyl hydrolase enzymes and uses thereof for biomass hydrolysis
CN103562398A (en) 2011-03-22 2014-02-05 Opx生物工艺学公司 Microbial production of chemical products and related compositions, methods and systems
US8765425B2 (en) 2011-03-23 2014-07-01 Butamax Advanced Biofuels Llc In situ expression of lipase for enzymatic production of alcohol esters during fermentation
US8759044B2 (en) 2011-03-23 2014-06-24 Butamax Advanced Biofuels Llc In situ expression of lipase for enzymatic production of alcohol esters during fermentation
EP2689014B1 (en) 2011-03-24 2019-04-24 Butamax (TM) Advanced Biofuels LLC Host cells and methods for production of isobutanol
BR112013024898B1 (en) 2011-03-29 2019-10-22 Toray Industries methods for producing a sugar liquid and method for producing a chemical
US10179924B2 (en) 2011-03-29 2019-01-15 Toray Industries, Inc. Method for producing sugar solution
EP2694594A4 (en) 2011-04-07 2015-11-11 Virdia Ltd Lignocellulose conversion processes and products
US8765426B2 (en) 2011-04-07 2014-07-01 E I Du Pont De Nemours And Company Pantothenic acid biosynthesis in zymomonas
US9290728B2 (en) 2011-04-18 2016-03-22 Poet Research, Inc Systems and methods for stillage fractionation
JP2014516581A (en) 2011-06-17 2014-07-17 ビュータマックス・アドバンスド・バイオフューエルズ・エルエルシー Co-products from the biofuel manufacturing process and methods for manufacturing the same
US20130035515A1 (en) 2011-06-17 2013-02-07 Butamax(Tm) Advanced Biofuels Llc Lignocellulosic hydrolysates as feedstocks for isobutanol fermentation
US8329455B2 (en) 2011-07-08 2012-12-11 Aikan North America, Inc. Systems and methods for digestion of solid waste
BR112014001943A2 (en) 2011-07-28 2018-08-07 Butamax Advanced Biofuels Llc method of converting alpha-ketoisovalerate to isobutyraldehyde and method of isobutanol production
RU2597199C2 (en) 2011-07-29 2016-09-10 Торэй Индастриз, Инк. Method for sugar solution production
EP2737942B1 (en) 2011-07-29 2020-01-01 Toray Industries, Inc. Filter aid and method of manufacturing thereof
CN102391065B (en) * 2011-08-09 2014-02-05 山东省鲁洲食品集团有限公司 Method for producing dihydric alcohol and low molecular polyalcohol by taking corn husk as raw material
WO2013028701A1 (en) 2011-08-22 2013-02-28 Codexis, Inc. Gh61 glycoside hydrolase protein variants and cofactors that enhance gh61 activity
CN102321646B (en) * 2011-09-08 2013-06-05 深圳大学 Beta-endoglucanase PEGase-2 gene, its expressed products and application
EP2760997A4 (en) 2011-09-30 2015-02-11 Codexis Inc Fungal proteases
EP2764098A2 (en) 2011-10-06 2014-08-13 BP Corporation North America Inc. Variant cbh i polypeptides with reduced product inhibition
WO2013055785A1 (en) 2011-10-10 2013-04-18 Virdia Ltd Sugar compositions
WO2013067028A1 (en) 2011-10-31 2013-05-10 Bp Corporation North America Inc. Use of mammalian promoters in filamentous fungi
WO2013067026A1 (en) 2011-10-31 2013-05-10 Bp Corporation North America Inc. Use of plant promoters in filamentous fungi
US20140004571A1 (en) 2011-12-02 2014-01-02 Bp Corporation North America Inc. Compositions and methods for biomass liquefaction
AU2012347687A1 (en) 2011-12-09 2014-05-29 Butamax Advanced Biofuels Llc Process to remove product alcohols from fermentation broth by multistep flash evaporation
US9611462B2 (en) 2011-12-20 2017-04-04 Codexis, Inc. Endoglucanase 1B (EG1B) variants
US9447436B2 (en) 2011-12-20 2016-09-20 Codexis, Inc. Production of saturated fatty alcohols from engineered microorganisms
WO2013102084A2 (en) 2011-12-30 2013-07-04 Butamax (Tm) Advanced Biofuels Llc Fermentative production of alcohols
EP2797611B1 (en) * 2011-12-30 2017-09-06 SloIron, Inc. Methods for isolation, use and analysis of ferritin
KR20140113997A (en) 2011-12-30 2014-09-25 부타맥스 어드밴스드 바이오퓨얼스 엘엘씨 Genetic switches for butanol production
US8765430B2 (en) 2012-02-10 2014-07-01 Sweetwater Energy, Inc. Enhancing fermentation of starch- and sugar-based feedstocks
CN104254613A (en) 2012-02-13 2014-12-31 Bp北美公司 Methods for detoxifying a lignocellulosic hydrolysate
CN104254601A (en) 2012-02-13 2014-12-31 Bp北美公司 Methods for detoxifying a lignocellulosic hydrolysate
US10202660B2 (en) 2012-03-02 2019-02-12 Board Of Trustees Of Michigan State University Methods for increasing sugar yield with size-adjusted lignocellulosic biomass particles
US9751781B2 (en) 2012-03-20 2017-09-05 The Research Foundation For The State University Of New York Method to separate lignin-rich solid phase from acidic biomass suspension at an acidic pH
US9689004B2 (en) 2012-03-23 2017-06-27 Butamax Advanced Biofuels Llc Acetate supplemention of medium for butanologens
US8563277B1 (en) 2012-04-13 2013-10-22 Sweetwater Energy, Inc. Methods and systems for saccharification of biomass
JP6004321B2 (en) * 2012-04-18 2016-10-05 日立造船株式会社 Methods for controlling the growth of miscellaneous bacteria in ethanol fermentation of moss
WO2013159055A1 (en) 2012-04-20 2013-10-24 Codexis, Inc. Production of fatty alcohols from engineered microorganisms
JP2015517303A (en) 2012-05-04 2015-06-22 ビュータマックス・アドバンスド・バイオフューエルズ・エルエルシー Method and system for the production and recovery of alcohol
US9169467B2 (en) 2012-05-11 2015-10-27 Butamax Advanced Biofuels Llc Ketol-acid reductoisomerase enzymes and methods of use
US8715464B2 (en) * 2012-05-21 2014-05-06 Pure Pulp Products, Inc. Soy stalk and wheat straw pulp fiber mixtures
JP2013243954A (en) * 2012-05-24 2013-12-09 Kao Corp Xylanase and method for producing sugar therewith
JP2013243953A (en) * 2012-05-24 2013-12-09 Kao Corp Xylanase and method for producing sugar therewith
FR2991691B1 (en) 2012-06-08 2016-01-22 Toulouse Inst Nat Polytech PROCESS FOR ENZYMATIC TREATMENT OF SOLID LIGNOCELLULOSIC MATERIAL
CN102720083A (en) * 2012-06-11 2012-10-10 中国科学院过程工程研究所 Method for pretreating biomass by ball milling coupled with microwave
WO2013188305A2 (en) 2012-06-11 2013-12-19 Codexis, Inc. Fungal beta-xylosidase variants
CA3095401C (en) * 2012-06-12 2023-01-31 Renescience A/S Methods and compositions for biomethane production
UA116630C2 (en) * 2012-07-03 2018-04-25 Ксілеко, Інк. METHOD OF CONVERTING SUGAR TO FURFURYL ALCOHOL
WO2014015278A1 (en) 2012-07-20 2014-01-23 Codexis, Inc. Production of fatty alcohols from engineered microorganisms
US20140024064A1 (en) 2012-07-23 2014-01-23 Butamax(Tm) Advanced Biofuels Llc Processes and systems for the production of fermentative alcohols
JP5981263B2 (en) * 2012-08-09 2016-08-31 本田技研工業株式会社 Method for producing saccharification solution
BR112015002940A2 (en) 2012-08-10 2018-04-24 Opx Biotechnologies Inc microorganisms and methods for the production of fatty acids and products derived from fatty acids.
WO2014031831A1 (en) 2012-08-22 2014-02-27 Butamax Advanced Biofuels Llc Production of fermentation products
KR20150054832A (en) 2012-09-12 2015-05-20 부타맥스 어드밴스드 바이오퓨얼스 엘엘씨 Processes and systems for the production of fermentation products
CA2885427A1 (en) 2012-09-21 2014-03-27 Butamax Advanced Biofuels Llc A method for producing butanol using two-phase extractive fermentation and recyclable extractant compositions
US9512408B2 (en) 2012-09-26 2016-12-06 Butamax Advanced Biofuels Llc Polypeptides with ketol-acid reductoisomerase activity
AU2013323396B2 (en) 2012-09-28 2017-04-20 Butamax Advanced Biofuels Llc Production of fermentation products
US9273330B2 (en) 2012-10-03 2016-03-01 Butamax Advanced Biofuels Llc Butanol tolerance in microorganisms
NZ747168A (en) 2012-10-10 2019-12-20 Xyleco Inc Treating biomass
NZ706069A (en) 2012-10-10 2018-11-30 Xyleco Inc Processing biomass
AU2013329024A1 (en) 2012-10-11 2015-03-12 Butamax Advanced Biofuels Llc Processes and systems for the production of fermentation products
DE102012020166A1 (en) 2012-10-13 2014-04-30 Green Sugar Gmbh Produktinnovationen Aus Biomasse Process for the hydrolysis of pelletable biomasses by means of hydrohalic acids
US9434929B2 (en) 2012-10-26 2016-09-06 Roal Oy Esterases useful in the treatment of cellulosic and lignocellulosic material
AT513562A1 (en) 2012-11-14 2014-05-15 Annikki Gmbh Process for obtaining sugar derivatives
WO2014081848A1 (en) 2012-11-20 2014-05-30 Butamax Advanced Biofuels Llc Butanol purification
BR112015012986A2 (en) 2012-12-07 2017-09-12 Danisco Us Inc compositions and methods of use
CA2892054A1 (en) 2012-12-07 2014-06-12 Ling Hua Compositions and methods of use
CA2890849A1 (en) 2012-12-12 2014-06-19 Danisco Us Inc. Variants of cellobiohydrolases
CA2893053A1 (en) 2012-12-12 2014-06-19 Danisco Us Inc. Variants of cellobiohydrolases
CN104870644A (en) 2012-12-12 2015-08-26 丹尼斯科美国公司 Variants of cellobiohydrolases
EP2931890A1 (en) 2012-12-12 2015-10-21 Danisco US Inc. Variants of cellobiohydrolases
MX2015007233A (en) 2012-12-12 2015-10-29 Danisco Us Inc Variants of cellobiohydrolases.
BR112015013933A2 (en) 2012-12-14 2017-07-11 Bp Corp North America Inc Sequential fermentation of hydrolyzate and solids from a dilute acid hydrolysis of biomass to produce fermentation products
US9512447B2 (en) 2012-12-14 2016-12-06 Codexis, Inc. Modified native beta-ketoacyl-ACP synthases and engineered microorganisms
US8846357B2 (en) 2012-12-20 2014-09-30 E I Du Pont De Nemours And Company Stabilized chlorine dioxide for contamination control in Zymomonas fermentation
FR3000106B1 (en) * 2012-12-20 2015-01-30 Ifp Energies Now PROCESS FOR PRODUCING OLIGOSACCHARIDES FROM LIGNOCELLULOSIC BIOMASS
WO2014106107A2 (en) 2012-12-28 2014-07-03 Butamax (Tm) Advanced Biofuels Llc Dhad variants for butanol production
WO2014105840A1 (en) 2012-12-31 2014-07-03 Butamax Advanced Biofuels Llc Fermentative production of alcohols
JP6307789B2 (en) 2013-01-07 2018-04-11 東レ株式会社 Sugar solution manufacturing apparatus and sugar solution manufacturing method
WO2014120087A1 (en) 2013-01-29 2014-08-07 Singapore Technologies Dynamics Pte Ltd Method for modular design, fabrication and assembly of integrated biocolumn systems with multiple downstream outputs
CN103114099B (en) * 2013-02-07 2014-06-11 广西大学 Beta-glucosaccharase gene for coding glycosyl hydrolase family 1 and application thereof
JP6153341B2 (en) * 2013-02-15 2017-06-28 本田技研工業株式会社 Method for producing saccharification solution
DK3578054T3 (en) 2013-02-15 2021-05-31 Pepsico Inc MANUFACTURE AND INCORPORATION OF BEVERAGE BY-PRODUCTS TO IMPROVE NUTRITIONAL VALUE AND SENSORIC PROPERTIES
US20160002131A1 (en) 2013-02-21 2016-01-07 Butamax Advanced Biofuels Llc Vapor recompression
NZ706072A (en) 2013-03-08 2018-12-21 Xyleco Inc Equipment protecting enclosures
US20140273105A1 (en) * 2013-03-12 2014-09-18 E I Du Pont De Nemours And Company Gradient pretreatment of lignocellulosic biomass
WO2014159309A1 (en) 2013-03-12 2014-10-02 Butamax Advanced Biofuels Llc Processes and systems for the production of alcohols
WO2014160050A1 (en) 2013-03-14 2014-10-02 Butamax Advanced Biofuels Llc Glycerol 3- phosphate dehydrogenase for butanol production
US9809867B2 (en) 2013-03-15 2017-11-07 Sweetwater Energy, Inc. Carbon purification of concentrated sugar streams derived from pretreated biomass
WO2014151645A1 (en) 2013-03-15 2014-09-25 Butamax Advanced Biofuels Llc Process for maximizing biomass growth and butanol yield by feedback control
US9309548B2 (en) * 2013-03-15 2016-04-12 Valicor, Inc Process and method for improving the enzymatic hydrolysis of lignocellulosic biomass by addition of hydrothermally treated stillage
WO2014145768A2 (en) 2013-03-15 2014-09-18 Bp Corporation North America Inc. Use of non-fungal 5' utrs in filamentous fungi
US9771602B2 (en) 2013-03-15 2017-09-26 Butamax Advanced Biofuels Llc Competitive growth and/or production advantage for butanologen microorganism
US9580705B2 (en) 2013-03-15 2017-02-28 Butamax Advanced Biofuels Llc DHAD variants and methods of screening
US20150119601A1 (en) 2013-03-15 2015-04-30 Opx Biotechnologies, Inc. Monofunctional mcr + 3-hp dehydrogenase
CN105229156A (en) 2013-03-15 2016-01-06 布特马斯先进生物燃料有限责任公司 For utilizing the method extracting fermentative production butanols
WO2014144643A1 (en) 2013-03-15 2014-09-18 Butamax Advanced Biofuels Llc Method for producing butanol using extractive fermentation
US9850512B2 (en) 2013-03-15 2017-12-26 The Research Foundation For The State University Of New York Hydrolysis of cellulosic fines in primary clarified sludge of paper mills and the addition of a surfactant to increase the yield
CN103255659B (en) * 2013-04-26 2016-09-28 安徽安生生物化工科技有限责任公司 The method that ammonia associating diluted alkaline normal pressure processes lignocellulose-like biomass
WO2014186652A1 (en) * 2013-05-17 2014-11-20 Xyleco, Inc. Processing biomass
WO2014190342A1 (en) * 2013-05-24 2014-11-27 Valicor Inc. Process and method for improving fermentation by the addition of hydrothermally treated stillage
CN105452472B (en) 2013-06-12 2020-03-13 雷内科学有限公司 Method for treating Municipal Solid Waste (MSW) by microbial hydrolysis and fermentation
WO2015002913A1 (en) 2013-07-03 2015-01-08 Butamax Advanced Biofuels Llc Partial adaptation for butanol production
KR101536132B1 (en) * 2013-07-12 2015-07-13 한국화학연구원 Method for producing microbial inhibitor-free fermentable sugar solutions from lignocellulosic biomass
KR101447534B1 (en) 2013-08-23 2014-10-08 한국화학연구원 Method for producing fermentable sugar solution containing less toxic acetate from lignocellulosic biomass
CN105452478B (en) 2013-07-09 2019-10-08 东丽株式会社 The manufacturing method of liquid glucose
US11408013B2 (en) 2013-07-19 2022-08-09 Cargill, Incorporated Microorganisms and methods for the production of fatty acids and fatty acid derived products
BR112016001026A2 (en) 2013-07-19 2017-10-24 Cargill Inc genetically modified organism
US10167460B2 (en) 2013-07-29 2019-01-01 Danisco Us Inc Variant enzymes
US20150052805A1 (en) 2013-08-20 2015-02-26 Michael L Catto Oxygen-Deficient Thermally Produced Processed Biochar from Beneficiated Organic-Carbon-Containing Feedstock
US9593447B2 (en) 2013-08-20 2017-03-14 Biomass Energy Enhancements, Llc System and method using a reaction chamber to beneficiate organic-carbon-containing feedstock for downstream processes
WO2015031561A1 (en) 2013-08-30 2015-03-05 Bp Corporation North America Inc. Catalytic conversion of alcohols
US20160157437A9 (en) * 2013-09-15 2016-06-09 Freddie Hebert Lemna Based Protein Concentrate
US20150087041A1 (en) 2013-09-26 2015-03-26 E I Du Pont De Nemours And Company Production of ethanol with reduced contaminants in a cellulosic biomass based process
US20150087040A1 (en) 2013-09-26 2015-03-26 E I Du Pont De Nemours And Company Production of ethanol and recycle water in a cellulosic fermentation process
US20150101751A1 (en) 2013-10-10 2015-04-16 E I Du Pont De Nemours And Company Lignocellulosic biomass fermentation process syrup binder and adhesive
US20150101377A1 (en) 2013-10-10 2015-04-16 E I Du Pont De Nemours And Company Lignocellulosic biomass fermentation process co-product ash for land applications
US9725363B2 (en) 2013-10-10 2017-08-08 E I Du Pont De Nemours And Company Lignocellulosic biomass fermentation process co-product fuel for cement kiln
US20150147786A1 (en) * 2013-11-24 2015-05-28 E I Du Pont De Nemours And Company High force and high stress destructuring for starch biomass processing
FI127582B (en) * 2014-01-10 2018-09-14 Ab Bln Woods Ltd Method for extracting lignin
US9194012B2 (en) * 2014-02-02 2015-11-24 Edward Brian HAMRICK Methods and systems for producing sugars from carbohydrate-rich substrates
KR102136842B1 (en) * 2014-02-07 2020-07-24 한국과학기술원 Methods for Pretreating Lignocellulosic Biomass
US9951363B2 (en) 2014-03-14 2018-04-24 The Research Foundation for the State University of New York College of Environmental Science and Forestry Enzymatic hydrolysis of old corrugated cardboard (OCC) fines from recycled linerboard mill waste rejects
CN105001894A (en) * 2014-04-18 2015-10-28 栖霞市海怡污水处理材料厂 Modifier for converting biomass into crude oil, production method and application process thereof
US9701918B2 (en) 2014-06-16 2017-07-11 Biomass Energy Enhancements, Llc System of using a reaction chamber to beneficiate organic-carbon-containing feedstock for downstream processes
US9796940B2 (en) 2014-06-16 2017-10-24 Biomass Energy Enhancements, Llc Processed biomass pellets from organic-carbon-containing feedstock
US9683738B2 (en) 2014-06-16 2017-06-20 Biomass Energy Enhancements, Llc System for co-firing coal and beneficiated organic-carbon-containing feedstock in a coal combustion apparatus
US10024533B2 (en) 2014-06-16 2018-07-17 Ctp Biotechnology Llc System and process for combusting cleaned coal and beneficiated organic-carbon-containing feedstock
US9702548B2 (en) 2014-06-16 2017-07-11 Biomass Energy Enhancements, Llc System for co-firing cleaned coal and beneficiated organic-carbon-containing feedstock in a coal combustion apparatus
US10018355B2 (en) 2014-06-16 2018-07-10 CTP Biotechnology, LLC System and process for combusting coal and beneficiated organic-carbon-containing feedstock
WO2016007350A1 (en) 2014-07-09 2016-01-14 Danisco Us Inc. Preconditioning of lignocellulosic biomass
WO2016025425A1 (en) 2014-08-11 2016-02-18 Butamax Advanced Biofuels Llc Yeast preparations and methods of making the same
EP2993228B1 (en) 2014-09-02 2019-10-09 Cargill, Incorporated Production of fatty acid esters
MX2017003594A (en) 2014-09-19 2017-05-12 Xyleco Inc Saccharides and saccharide compositions and mixtures.
EP3201332A1 (en) 2014-09-30 2017-08-09 Danisco US Inc. Compositions comprising beta-mannanase and methods of use
WO2016054168A1 (en) 2014-09-30 2016-04-07 Danisco Us Inc Compositions comprising beta mannanase and methods of use
WO2016054205A1 (en) 2014-09-30 2016-04-07 Danisco Us Inc Compositions comprising beta mannanase and methods of use
US20170233707A1 (en) 2014-09-30 2017-08-17 Danisco Us Inc Compositions comprising beta-mannanase and methods of use
WO2016054185A1 (en) 2014-09-30 2016-04-07 Danisco Us Inc Compositions comprising beta-mannanase and methods of use
HRP20221024T1 (en) 2014-12-09 2022-11-11 Sweetwater Energy, Inc. Rapid pretreatment
WO2016100837A1 (en) 2014-12-18 2016-06-23 Danisco Us Inc Engineered multifunctional enzymes and methods of use
EP3234118A1 (en) 2014-12-18 2017-10-25 Danisco US Inc. Engineered multifunctional enzymes and methods of use
CN107108543A (en) 2015-01-07 2017-08-29 威尔迪亚公司 Extraction and the method for conversion hemicellulose sugar
EP3045234A1 (en) * 2015-01-16 2016-07-20 Clariant International Ltd. Process for the decomposition of biomass
CN104673846B (en) * 2015-01-19 2019-02-22 天津市天人世纪科技有限公司 A method of lactic acid is produced using agriculture and forestry organic waste material
US20160262389A1 (en) 2015-03-12 2016-09-15 E I Du Pont De Nemours And Company Co-products of lignocellulosic biomass process for landscape application
EA036683B1 (en) 2015-04-10 2020-12-08 Комет Биорефайнинг Инк. Methods and compositions for the treatment of cellulosic biomass and products produced thereby
CN104774877B (en) * 2015-04-10 2017-12-29 山东龙力生物科技股份有限公司 A kind of method of lignocellulose biomass co-producing ethanol, acetone and butanol
CN108291245A (en) 2015-07-07 2018-07-17 丹尼斯科美国公司 Use high concentration sugar mixture inducible gene expression
US9422663B1 (en) 2015-11-05 2016-08-23 Aicardo Roa-Espinosa Apparatus and process for treatment of biocomponents
CN105463031A (en) * 2015-11-11 2016-04-06 首都师范大学 Method for cooperatively producing ethyl alcohol and methane through energy grass
CN108350226B (en) 2015-11-24 2021-12-31 因比肯公司 Asphalt composition comprising lignin
CN109070092A (en) 2015-11-25 2018-12-21 富林特希尔斯资源有限公司 Corn and thus the method and system of ethyl alcohol is prepared for milling
US11718863B2 (en) 2015-11-25 2023-08-08 Poet Grain (Octane), Llc Processes for recovering products from a slurry
WO2017091766A1 (en) 2015-11-25 2017-06-01 Flint Hills Resources, Lp Processes for recovering products from a corn fermentation mash
US10059966B2 (en) 2015-11-25 2018-08-28 Flint Hills Resources, Lp Processes for recovering products from a corn fermentation mash
EP3397772A1 (en) 2015-12-28 2018-11-07 Danisco US Inc. Methods for the production of fermentation products from feedstock
MY187470A (en) 2016-02-17 2021-09-23 Toray Industries Method for producing sugar alcohol
US10759727B2 (en) 2016-02-19 2020-09-01 Intercontinental Great Brands Llc Processes to create multiple value streams from biomass sources
BR112018070645A2 (en) 2016-04-08 2019-02-05 Du Pont recombinant yeast cells, methods for producing a yeast cell and method for producing a target compound
CN106086083B (en) * 2016-06-08 2019-10-15 辽东学院 A method of cultivation chicken manure environmental pollution is administered using fermentation brewage process
WO2017218325A1 (en) 2016-06-15 2017-12-21 Codexis, Inc. Engineered beta-glucosidases and glucosylation methods
WO2018053058A1 (en) 2016-09-14 2018-03-22 Danisco Us Inc. Lignocellulosic biomass fermentation-based processes
CN106496019B (en) * 2016-10-13 2019-03-01 江南大学 A method of extracting α-ketoglutaric acid and pyruvic acid simultaneously from microbial fermentation solution or enzymatic conversion liquid
WO2018106656A1 (en) 2016-12-06 2018-06-14 Danisco Us Inc Truncated lpmo enzymes and use thereof
CN106755282A (en) * 2016-12-14 2017-05-31 曹书华 A kind of method that prediction Alcalase alkali proteases microwave digests grass carp albumen process
US20190330577A1 (en) 2016-12-21 2019-10-31 Dupont Nutrition Biosciences Aps Methods of using thermostable serine proteases
JP2020506702A (en) 2017-02-02 2020-03-05 カーギル インコーポレイテッド Genetically modified cells producing C6-C10 fatty acid derivatives
CA3053773A1 (en) 2017-02-16 2018-08-23 Sweetwater Energy, Inc. High pressure zone formation for pretreatment
US10730958B2 (en) 2017-03-08 2020-08-04 Board Of Trustees Of Michigan State University Pretreatment of densified biomass using liquid ammonia and systems and products related thereto
WO2018169780A1 (en) 2017-03-15 2018-09-20 Dupont Nutrition Biosciences Aps Methods of using an archaeal serine protease
CN107034241B (en) * 2017-05-17 2020-10-23 华中农业大学 Pretreatment process for saccharification and utilization of bagasse
US11440999B2 (en) 2017-07-07 2022-09-13 Board Of Trustees Of Michigan State University De-esterification of biomass prior to ammonia pretreatment
CN107501357A (en) * 2017-08-01 2017-12-22 华南理工大学 A kind of cellulose fermentable sugars and preparation method and application
WO2019074828A1 (en) 2017-10-09 2019-04-18 Danisco Us Inc Cellobiose dehydrogenase variants and methods of use thereof
CN107604012B (en) * 2017-10-25 2021-09-10 武汉凯迪工程技术研究总院有限公司 Biomass raw material pretreatment method based on high-frequency oscillation electromagnetic field
CN107904271A (en) * 2017-12-08 2018-04-13 齐颖 A kind of method of microwave reinforced soda lime preprocessing lignocellulose
CN108315359B (en) * 2018-03-23 2021-07-27 安玉民 Method for preparing alcohol and preparing feed by using potato straws
JP7094622B2 (en) * 2018-03-29 2022-07-04 株式会社ディスコ Circular whetstone
CN108315358A (en) * 2018-04-18 2018-07-24 黑龙江新天地能源开发有限公司 It is a kind of to prepare biogas method using stalk
WO2019209245A1 (en) 2018-04-23 2019-10-31 Dupont Nutrition Biosciences Aps Increasing activity of 2' fucosyllactose transporters endogenous to microbial cells
US11913046B2 (en) 2018-04-23 2024-02-27 Inbiose N.V. Increasing export of 2'fucosyllactose from microbial cells through the expression of a heterologous nucleic acid
CA3099535C (en) 2018-05-10 2024-01-16 Comet Biorefining Inc. Compositions comprising glucose and hemicellulose and their use
CN108374025A (en) * 2018-06-02 2018-08-07 山东省同泰维润食品科技有限公司 A kind of propionic acid preparation process
CN108823257A (en) * 2018-06-20 2018-11-16 齐齐哈尔龙江阜丰生物科技有限公司 The preparation process of Threonine Fermentation culture medium
US11193177B2 (en) 2018-06-28 2021-12-07 Indian Oil Corporation Limited Process for recovering higher sugar from biomass
CN109486969A (en) * 2018-10-26 2019-03-19 贵州茅台酒股份有限公司 A kind of method that directed screening generates normal propyl alcohol bacterial strain
CN111321173B (en) * 2018-12-14 2022-05-31 南京百斯杰生物工程有限公司 Application of mannase in alcohol fermentation
CN110655260B (en) * 2019-10-20 2021-11-12 广东新泰隆环保集团有限公司 Zero-emission treatment method and device for organic wastewater
US11692000B2 (en) 2019-12-22 2023-07-04 Apalta Patents OÜ Methods of making specialized lignin and lignin products from biomass
US11730172B2 (en) 2020-07-15 2023-08-22 Poet Research, Inc. Methods and systems for concentrating a solids stream recovered from a process stream in a biorefinery
CN115627277A (en) * 2022-11-18 2023-01-20 中国科学院过程工程研究所 Method for performing solid state fermentation on 2, 3-butanediol by high-solid-state enzymolysis of straws

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994003646A1 (en) * 1992-08-06 1994-02-17 The Texas A&M University System Methods of biomass pretreatment

Family Cites Families (72)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR656385A (en) * 1927-06-25 1929-05-07 Commercial Alcohol Company Ltd Glucose manufacturing process
JPS474505Y1 (en) 1967-12-25 1972-02-17
JPS4738995Y1 (en) 1969-10-07 1972-11-25
JPS516237A (en) 1974-07-05 1976-01-19 Tokyo Shibaura Electric Co TOFUHOSHIKI
JPS5710912B2 (en) 1974-08-08 1982-03-01
US4136207A (en) * 1977-01-24 1979-01-23 Stake Technology Ltd. Method of treating lignocellulose materials to produce ruminant feed
US4186658A (en) 1977-01-24 1980-02-05 Stake Technology Ltd. Apparatus for conveying particulate material
CH609092A5 (en) * 1977-04-01 1979-02-15 Battelle Memorial Institute
JPS5432070A (en) 1977-08-15 1979-03-09 Nec Corp Etching process method for semiconductor element
JPS5437235A (en) 1977-08-31 1979-03-19 Toshiba Corp Enclosed switchboard
US5366558A (en) * 1979-03-23 1994-11-22 Brink David L Method of treating biomass material
US4356144A (en) 1979-06-25 1982-10-26 General Atomic Company Closure hold-down system for a reactor vessel
JPS5610035A (en) 1979-06-30 1981-02-02 Tokyo Shibaura Electric Co Method of supplying current to electric device
US4461648A (en) 1980-07-11 1984-07-24 Patrick Foody Method for increasing the accessibility of cellulose in lignocellulosic materials, particularly hardwoods agricultural residues and the like
US4356196A (en) * 1980-10-20 1982-10-26 Hultquist Joe H Process for treating alfalfa and other cellulosic agricultural crops
US5037663A (en) 1981-10-14 1991-08-06 Colorado State University Research Foundation Process for increasing the reactivity of cellulose-containing materials
JPS5898093A (en) * 1981-12-08 1983-06-10 Chisso Corp Pretreatment of cellulosic material for saccharification
JPS5816872B2 (en) 1982-02-12 1983-04-02 協和醗酵工業株式会社 Corynebacterium glutamicum mutant strain
EP0332234B1 (en) 1983-02-17 1993-07-28 Kyowa Hakko Kogyo Co., Ltd. Process for preparing l-tyrosine
US4463648A (en) * 1983-05-02 1984-08-07 Fender C Leo Angled humbucking pick-up for an electrical musical instrument of the stringed type
JPS623776A (en) 1985-06-29 1987-01-09 Zojirushi Vacuum Bottle Co Thawing of frozen food and apparatus for controlling same
US5366553A (en) * 1985-11-07 1994-11-22 Burford Corporation Sequence controller
EP0236515A1 (en) 1986-03-05 1987-09-16 Kabushiki Kaisha Bandai Assembly set for confectionery model
DE3632397A1 (en) 1986-09-24 1988-03-31 Ruhrchemie Ag METHOD FOR PURIFYING PROPANDIOL-1,3
JPS6394985A (en) 1986-10-09 1988-04-26 Kyowa Hakko Kogyo Co Ltd Production of l-tyrosine
JPS643581A (en) 1987-06-26 1989-01-09 Nec Corp Ssr target detector
JPS6423776A (en) 1987-07-16 1989-01-26 Mitsubishi Electric Corp High tension pulse generator
JPH0193776A (en) 1987-10-06 1989-04-12 Canon Inc Image forming device
US4859283A (en) 1988-04-15 1989-08-22 E. I. Du Pont De Nemours And Company Magnesium ions in a process for alkaline peroxide treatment of nonwoody lignocellulosic substrates
JPH03207079A (en) 1990-01-10 1991-09-10 Mitsubishi Electric Corp Dynamic ram
US5192673A (en) * 1990-04-30 1993-03-09 Michigan Biotechnology Institute Mutant strain of C. acetobutylicum and process for making butanol
JP3074701B2 (en) 1990-06-15 2000-08-07 松下電器産業株式会社 Flow control valve
DE69123041T2 (en) 1990-08-10 1997-04-03 Daicel Chem MANUFACTURING PROCESS FOR OPTICALLY ACTIVE 3-PHENYL-1,3-PROPANDIOL
US5231017A (en) * 1991-05-17 1993-07-27 Solvay Enzymes, Inc. Process for producing ethanol
EP0553462B1 (en) 1992-01-28 1996-03-06 Voith Sulzer Papiermaschinen GmbH Support connection between two rolls
WO1994021785A1 (en) 1993-03-10 1994-09-29 Novo Nordisk A/S Enzymes with xylanase activity from aspergillus aculeatus
JPH0859681A (en) 1994-08-22 1996-03-05 Fujisawa Pharmaceut Co Ltd Cyclic-gmp-pde inhibiting substance, fr901,526
US5705369A (en) 1994-12-27 1998-01-06 Midwest Research Institute Prehydrolysis of lignocellulose
JP3207079B2 (en) 1995-06-23 2001-09-10 京セラ株式会社 Portable communication device
BR9600672A (en) * 1996-03-08 1997-12-30 Dedini S A Administracao E Par Acid hydrolysis process of lignocellulosic material and hydrolysis reactor
US5747320A (en) * 1996-08-02 1998-05-05 The United States Of America, As Represented By The Secretary Of Agriculture Glucose and cellobiose tolerant β-glucosidase from Candida peltata
ATE471984T1 (en) 1996-11-13 2010-07-15 Du Pont PRODUCTION PROCESS OF GLYCERIN BY RECOMBINANT ORGANISMS
ATE452979T1 (en) * 1996-11-13 2010-01-15 Du Pont PRODUCTION PROCESS OF 1,3-PROPANEDIOL BY RECOMBINANT ORGANISMS
JP3899572B2 (en) 1997-01-17 2007-03-28 カシオ計算機株式会社 E-mail display method and e-mail display device
WO1998051813A1 (en) 1997-05-14 1998-11-19 The Board Of Trustees Of The University Of Illinois A METHOD OF PRODUCING BUTANOL USING A MUTANT STRAIN OF $i(CLOSTRIDIUM BEIJERINCKII)
US5916780A (en) * 1997-06-09 1999-06-29 Iogen Corporation Pretreatment process for conversion of cellulose to fuel ethanol
US6159738A (en) * 1998-04-28 2000-12-12 University Of Chicago Method for construction of bacterial strains with increased succinic acid production
US6176176B1 (en) * 1998-04-30 2001-01-23 Board Of Trustees Operating Michigan State University Apparatus for treating cellulosic materials
HU224975B1 (en) * 1998-09-25 2006-04-28 Ajinomoto Kk Process for producing l-amino acids by fermentation and amino acid-producing bacterium strains
US6600077B1 (en) * 1999-01-29 2003-07-29 Board Of Trustees Operating Michigan State University Biocatalytic synthesis of quinic acid and conversion to hydroquinone
NZ514253A (en) * 1999-03-11 2003-06-30 Zeachem Inc Process for producing ethanol
WO2000072973A1 (en) * 1999-06-01 2000-12-07 Elan Pharma International Ltd. Small-scale mill and method thereof
BR9902607B1 (en) 1999-06-23 2010-08-24 biomass pre-hydrolysis apparatus and process.
US6254914B1 (en) 1999-07-02 2001-07-03 The Board Of Trustees Of The University Of Illinois Process for recovery of corn coarse fiber (pericarp)
US6521748B2 (en) * 1999-08-06 2003-02-18 E. I. Du Pont De Nemours And Company Polynucleotide encoding a mutant Rhodotorula glutinis tyrosine ammonia lyase polypeptide
JP4716634B2 (en) * 1999-08-18 2011-07-06 イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニー Biological production of 1,3-propanediol with high titer
KR100325975B1 (en) * 1999-11-26 2002-02-27 손재익 Methods and equipments of ammonia recycled percolation for biomass treatment
US6777207B2 (en) 1999-12-29 2004-08-17 Novo Nordisk A/S Method for making insulin precursors and insulin precursor analogues having improved fermentation yield in yeast
US7223575B2 (en) * 2000-05-01 2007-05-29 Midwest Research Institute Zymomonas pentose-sugar fermenting strains and uses thereof
US6861237B2 (en) * 2000-11-30 2005-03-01 Novo Nordisk A/S Production of heterologous polypeptides in yeast
US7272953B2 (en) * 2002-01-08 2007-09-25 Masterson James A Method and apparatus for separating and neutralizing ammonia
CA2477196C (en) * 2002-02-22 2012-02-21 Gibson W. Gervais Process of treating lignocellulosic material to produce bio-ethanol
AU2003208215A1 (en) 2002-03-15 2003-09-29 Iogen Energy Corporation Method for glucose production using endoglucanase core protein for improved recovery and reuse of enzyme
WO2004018645A2 (en) 2002-08-23 2004-03-04 E.I. Du Pont De Nemours And Company Utilization of starch products for biological production by fermentation
US20040231060A1 (en) 2003-03-07 2004-11-25 Athenix Corporation Methods to enhance the activity of lignocellulose-degrading enzymes
US7098009B2 (en) * 2004-03-04 2006-08-29 University Of Florida Research Foundation, Inc. Production of chemicals from lignocellulose, biomass or sugars
US20070029252A1 (en) 2005-04-12 2007-02-08 Dunson James B Jr System and process for biomass treatment
US7781191B2 (en) * 2005-04-12 2010-08-24 E. I. Du Pont De Nemours And Company Treatment of biomass to obtain a target chemical
EP1869201B1 (en) 2005-04-12 2017-12-27 E. I. du Pont de Nemours and Company Integration of alternative feedstreams in biomass treatment and utilization
US9297028B2 (en) 2005-09-29 2016-03-29 Butamax Advanced Biofuels Llc Fermentive production of four carbon alcohols
CA2622026C (en) 2005-10-26 2018-06-05 E.I. Du Pont De Nemours And Company Fermentive production of four carbon alcohols
US8962298B2 (en) 2006-05-02 2015-02-24 Butamax Advanced Biofuels Llc Recombinant host cell comprising a diol dehydratase

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994003646A1 (en) * 1992-08-06 1994-02-17 The Texas A&M University System Methods of biomass pretreatment

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
BEN-GHEDALIA D ET AL: "THE EFFECT OF CHEMICAL PRETREATMENTS AND SUBSEQUENT ENZYMATIC TREATMENTS ON THE ORGANIC MATTER DIGESTIBILITY IN VITRO OF WHEAT STRAW" NUTRITION REPORTS INTERNATIONAL, vol. 19, no. 4, April 1979 (1979-04), pages 499-505, XP008067476 *
CAO N ET AL: "PRODUCTION OF 2,3-BUTANEDIOL FROM PRETREATED CORN COB BY KLEBSIELLA OXYTOCA IN THE PRESENCE OF FUNGAL CELLULASE" APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, CLIFTON, NJ, US, vol. 63-65, 1997, pages 129-139, XP008067479 ISSN: 0273-2289 *
CAO N J ET AL: "Ethanol production from corn cob pretreated by the ammonia steeping process using genetically engineered yeast" BIOTECHNOLOGY LETTERS, vol. 18, no. 9, 1996, pages 1013-1018, XP008067477 ISSN: 0141-5492 *
IYER P V ET AL: "AMMONIA RECYCLED PERCOLATION PROCESS FOR PRETREATMENT OF HERBACEOUS BIOMASS" APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, CLIFTON, NJ, US, vol. 57/58, 1996, pages 121-132, XP008067478 ISSN: 0273-2289 *
JOBLIN K N ET AL: "Fermentation of barley straw by anaerobic rumen bacteria and fungi in axenic culture and in co-culture with methanogens." LETTERS IN APPLIED MICROBIOLOGY, vol. 9, no. 5, 1989, page 195, XP008067611 *
KURAKAKE M ET AL: "PRETREATMENT WITH AMMONIA WATER FOR ENZYMATIC HYDROLYSIS OF CORN HUSK, BAGASSE, AND SWITCHGRASS" APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, CLIFTON, NJ, US, vol. 90, no. 3, March 2001 (2001-03), pages 251-259, XP008067510 ISSN: 0273-2289 *
MOSIER NATHAN ET AL: "Features of promising technologies for pretreatment of lignocellulosic biomass" BIORESOURCE TECHNOLOGY, vol. 96, no. 6, April 2005 (2005-04), pages 673-686, XP002395515 ISSN: 0960-8524 *
See also references of EP1869194A2 *
TAYLOR F ET AL: "CORN-MILLING PRETREATMENT WITH ANHYDROUS AMMONIA" APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, CLIFTON, NJ, US, vol. 104, no. 2, February 2003 (2003-02), pages 141-148, XP008067530 ISSN: 0273-2289 *
WAISS A C ET AL: "IMPROVING DIGESTIBILITY OF STRAWS FOR RUMINANT FEED BY AQUEOUS AMMONIA" JOURNAL OF ANIMAL SCIENCE, NEW YORK, NY, US, vol. 35, no. 1, 1972, pages 109-112, XP008067636 ISSN: 0021-8812 *

Cited By (249)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014239694A (en) * 2005-09-30 2014-12-25 レネサイエンス・アクティーゼルスカブRenescience A/S Non-pressurized pretreatment, enzymatic hydrolysis and fermentation of waste fraction
WO2008004853A1 (en) * 2006-07-03 2008-01-10 Hendriks Antonius Theodorus Wi Pre-treatment of biomass
EP2415808A2 (en) 2006-10-26 2012-02-08 Xyleco, Inc. Method of making organic acids from biomass
US10287730B2 (en) 2006-10-26 2019-05-14 Xyleco, Inc. Processing biomass
EP2415805A2 (en) 2006-10-26 2012-02-08 Xyleco, Inc. Method of producing a product from biomass
EP2415814A2 (en) 2006-10-26 2012-02-08 Xyleco, Inc. Method of preparation of alcohols, organic acids, sugars, hydrocarbons, and mixtures thereof from biomass
EP2415807A2 (en) 2006-10-26 2012-02-08 Xyleco, Inc. Method of making butanol from biomass
EP2415818A2 (en) 2006-10-26 2012-02-08 Xyleco, Inc. Method of preparation of alcohols, organic acids, proteins, sugars, hydrocarbons, and mixtures thereof from biomass
EP2415820A2 (en) 2006-10-26 2012-02-08 Xyleco, Inc. Method of changing the molecular structure of biomass
EP2204432A1 (en) 2006-10-26 2010-07-07 Xyleco, Inc. Processing biomass
EP2415810A2 (en) 2006-10-26 2012-02-08 Xyleco, Inc. Method for making sugars from biomass
EP2415806A2 (en) 2006-10-26 2012-02-08 Xyleco, Inc. Method of use of an intermediate fermentation product as an animal feed
EP2415819A2 (en) 2006-10-26 2012-02-08 Xyleco, Inc. Porcess of making sugars from biomass
US10704196B2 (en) 2006-10-26 2020-07-07 Xyleco, Inc. Processing biomass
EP2415809A2 (en) 2006-10-26 2012-02-08 Xyleco, Inc. Method of producing fuel from biomass
EP2415815A2 (en) 2006-10-26 2012-02-08 Xyleco, Inc. Method for making proteins from biomass
EP2415817A2 (en) 2006-10-26 2012-02-08 Xyleco, Inc. Method of making a sugar or a combustible fuel from biomass
EP2415811A2 (en) 2006-10-26 2012-02-08 Xyleco, Inc. Method of making an irradiated wood product
EP2415812A2 (en) 2006-10-26 2012-02-08 Xyleco, Inc. Method of making a combustible fuel from biomass
EP2415813A2 (en) 2006-10-26 2012-02-08 Xyleco, Inc. Method for generating energy
JP2008154497A (en) * 2006-12-22 2008-07-10 Japan Science & Technology Agency Method for producing transformant and new mutant of yeast
JP2008161125A (en) * 2006-12-28 2008-07-17 Univ Of Tokyo Method for producing sugar, method for producing ethanol, method for producing lactic acid, cellulose for enzyme saccharification used for them and method for producing the same
JP2010524473A (en) * 2007-04-19 2010-07-22 マスコマ コーポレイション Combined thermochemical pretreatment and refining of lignocellulose biomass
WO2008134259A1 (en) 2007-04-24 2008-11-06 Novozymes North America, Inc. Detoxifying pre-treated lignocellulose-containing materials
EP3219804A1 (en) 2007-04-24 2017-09-20 Novozymes North America, Inc. Detoxifying pre-treated lignocellulose-containing materials
WO2009030713A1 (en) 2007-09-03 2009-03-12 Novozymes A/S Detoxifying and recycling of washing solution used in pretreatment of lignocellulose-containing materials
EP2207889A4 (en) * 2007-10-10 2014-04-02 Sunopta Bioprocess Inc Treatment of lignocellutosic materials utilizing disc refining and enzymatic hydrolysis performed under vacuum
EP2207889A1 (en) * 2007-10-10 2010-07-21 Sunopta Bioprocess Inc. Treatment of lignocellutosic materials utilizing disc refining and enzymatic hydrolysis performed under vacuum
JP2009112200A (en) * 2007-11-02 2009-05-28 Nippon Steel Engineering Co Ltd Method for producing ethanol
EP2653539A1 (en) 2007-12-19 2013-10-23 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2009085935A2 (en) 2007-12-19 2009-07-09 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
EP3480217A1 (en) 2008-04-30 2019-05-08 Xyleco, Inc. Processing biomass
EP2963060A2 (en) 2008-04-30 2016-01-06 Xyleco, Inc. Processing biomass
EP3305906A1 (en) 2008-04-30 2018-04-11 Xyleco, Inc. Processing biomass by electron beam irradiation
EP3546587A1 (en) 2008-04-30 2019-10-02 Xyleco, Inc. Processing biomass
EP3461899A1 (en) 2008-04-30 2019-04-03 Xyleco, Inc. Processing biomass
WO2010039812A2 (en) 2008-09-30 2010-04-08 Novozymes North America, Inc. Improvement of enzymatic hydrolysis of pre-treated lignocellulose-containing material with distillers dried grains
WO2010065830A1 (en) 2008-12-04 2010-06-10 Novozymes, Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
EP3141609A1 (en) 2008-12-19 2017-03-15 Novozymes, Inc. Methods for increasing hydrolysis of cellulosic material in the presence of cellobiose dehydrogenase
WO2010080407A2 (en) 2008-12-19 2010-07-15 Novozymes, Inc. Methods for increasing hydrolysis of cellulosic material
WO2010080408A2 (en) 2008-12-19 2010-07-15 Novozymes, Inc. Methods for increasing enzymatic hydrolysis of cellulosic material in the presence of a peroxidase
WO2010080428A1 (en) 2008-12-19 2010-07-15 Xyleco, Inc. Processing biomass
WO2010080532A1 (en) 2008-12-19 2010-07-15 Novozymes, Inc. Methods for increasing hydrolysis of cellulosic material in the presence of cellobiose dehydrogenase
JP2012512658A (en) * 2008-12-19 2012-06-07 イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニー Organic solvent pretreatment of biomass to promote enzymatic saccharification
WO2010078391A2 (en) 2008-12-30 2010-07-08 Novozymes North America, Inc. Improvement of enzymatic hydrolysis of pretreated lignocellulose-containing material with dissolved air flotation sludge
WO2010078392A2 (en) 2008-12-31 2010-07-08 Novozymes North America, Inc. Processes of producing fermentation products
WO2010088387A1 (en) 2009-01-28 2010-08-05 Novozymes, Inc. Polypeptides having beta-glucosidase activity and polynucleotides encoding same
WO2010088463A2 (en) 2009-01-30 2010-08-05 Novozymes, Inc. Polypeptides having expansin activity and polynucleotides encoding same
EP2403894B1 (en) 2009-03-03 2016-06-01 The Coca-Cola Company Bio-based polyethylene terephthalate packaging and method of making thereof
WO2010108918A1 (en) 2009-03-24 2010-09-30 Novozymes A/S Polypeptides having acetyl xylan esterase activity and polynucleotides encoding same
WO2010138754A1 (en) 2009-05-29 2010-12-02 Novozymes, Inc. Methods for enhancing the degradation or conversion of cellulosic material
WO2010141325A1 (en) 2009-06-02 2010-12-09 Novozymes, Inc. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2011002832A1 (en) 2009-06-30 2011-01-06 Novozymes A/S Biomass hydrolysis process
WO2011005867A1 (en) 2009-07-07 2011-01-13 Novozymes, Inc. Polypeptides having cellulolytic enhancing activity activity and polynucleotides encoding same
WO2011008785A2 (en) 2009-07-17 2011-01-20 Novozymes A/S A method of analyzing cellulose decay in cellulosic material hydrolysis
KR101898849B1 (en) * 2009-08-06 2018-09-13 안니키 게엠베하 Process for the production of carbohydrate cleavage products from a lignocellulosic material
US9970038B2 (en) 2009-08-06 2018-05-15 Annikki Gmbh Process for the production of carbohydrate cleavage products from a lingnocellulosic material
KR20180042416A (en) * 2009-08-06 2018-04-25 안니키 게엠베하 Process for the production of carbohydrate cleavage products from a lignocellulosic material
EP3805348A2 (en) 2009-09-17 2021-04-14 Novozymes, Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
EP3269804A1 (en) 2009-09-17 2018-01-17 Novozymes, Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2011035027A2 (en) 2009-09-17 2011-03-24 Novozymes, Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2011035029A1 (en) 2009-09-18 2011-03-24 Novozymes, Inc. Polypeptides having beta-glucosidase activity and polynucleotides encoding same
WO2011041405A1 (en) 2009-09-29 2011-04-07 Novozymes, Inc. Polypeptides having xylanase activity and polynucleotides encoding same
WO2011041397A1 (en) 2009-09-29 2011-04-07 Novozymes, Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2011039319A1 (en) 2009-09-30 2011-04-07 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
EP2977382A2 (en) 2009-09-30 2016-01-27 Novozymes Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2011041504A1 (en) 2009-09-30 2011-04-07 Novozymes, Inc. Polypeptides derived from thermoascus crustaceus having cellulolytic enhancing activity and polynucleotides encoding same
WO2011050037A1 (en) 2009-10-23 2011-04-28 Novozymes, Inc. Cellobiohydrolase variants and polynucleotides encoding same
WO2011059740A1 (en) 2009-10-29 2011-05-19 Novozymes, Inc. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2011057140A1 (en) 2009-11-06 2011-05-12 Novozymes, Inc. Compositions for saccharification of cellulosic material
WO2011057083A1 (en) 2009-11-06 2011-05-12 Novozymes, Inc. Polypeptides having xylanase activity and polynucleotides encoding same
EP3222716A1 (en) 2009-11-06 2017-09-27 Novozymes, Inc. Composition for saccharification of cellulosic material
WO2011057086A1 (en) 2009-11-06 2011-05-12 Novozymes, Inc. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
EP3550016A1 (en) 2009-11-06 2019-10-09 Novozymes, Inc. Composition for saccharification of cellulosic material
WO2011080154A1 (en) 2009-12-21 2011-07-07 Novozymes A/S Biomass hydrolysis process
WO2011092136A1 (en) 2010-01-29 2011-08-04 Novozymes A/S Biogas production process with enzymatic pre-treatment
EP3070171A1 (en) 2010-03-30 2016-09-21 Novozymes A/S Processes of producing a fermentation product
WO2011126897A2 (en) 2010-03-30 2011-10-13 Novozymes A/S Methods for enhancing by-products from fermentation processes
WO2011123450A1 (en) 2010-03-31 2011-10-06 Novozymes, Inc. Cellobiohydrolase variants and polynucleotides encoding same
WO2012003379A1 (en) 2010-06-30 2012-01-05 Novozymes A/S Polypeptides having beta-glucosidase activity and polynucleotides encoding same
WO2012006642A1 (en) 2010-07-07 2012-01-12 Novozymes North America, Inc. Fermentation process
WO2012012590A2 (en) 2010-07-23 2012-01-26 Novozymes A/S Processes for producing fermentation products
WO2012021396A1 (en) 2010-08-12 2012-02-16 Novozymes, Inc. Compositions comprising a polypeptide having cellulolytic enhancing activity and an organic compound and uses thereof
WO2012021401A1 (en) 2010-08-12 2012-02-16 Novozymes, Inc. Compositions comprising a polypeptide having cellulolytic enhancing activity and a bicyclic compound and uses thereof
WO2012021399A1 (en) 2010-08-12 2012-02-16 Novozymes, Inc. Compositions comprising a polypeptide having cellulolytic enhancing activity and a nitrogen-containing compound and uses thereof
WO2012021395A1 (en) 2010-08-12 2012-02-16 Novozymes, Inc. Compositions comprising a polypeptide having cellulolytic enhancing activity and a sulfur-containing compound and uses thereof
WO2012021410A1 (en) 2010-08-12 2012-02-16 Novozymes, Inc. Compositions comprising a polypeptide having cellulolytic enhancing activity and a liquor and uses thereof
WO2012021408A1 (en) 2010-08-12 2012-02-16 Novozymes, Inc. Compositions comprising a polypeptide having cellulolytic enhancing activity and a dioxy compound and uses thereof
WO2012021394A1 (en) 2010-08-12 2012-02-16 Novozymes, Inc. Compositions comprising a polypeptide having cellulolytic enhancing activity and a quinone compound and uses thereof
WO2012021400A1 (en) 2010-08-12 2012-02-16 Novozymes, Inc. Compositions comprising a polypeptide having cellulolytic enhancing activity and a heterocyclic compound and uses thereof
WO2012030845A2 (en) 2010-08-30 2012-03-08 Novozymes A/S Polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same
WO2012030799A1 (en) 2010-08-30 2012-03-08 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
EP3470514A1 (en) 2010-08-30 2019-04-17 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2012030844A1 (en) 2010-08-30 2012-03-08 Novozymes A/S Polypeptides having endoglucanase activity and polynucleotides encoding same
WO2012030849A1 (en) 2010-08-30 2012-03-08 Novozymes A/S Polypeptides having xylanase activity and polynucleotides encoding same
EP2735611A2 (en) 2010-08-30 2014-05-28 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2012030858A2 (en) 2010-08-30 2012-03-08 Novozymes A/S Polypeptides having hemicellulolytic activity and polynucleotides encoding same
WO2012030811A1 (en) 2010-08-30 2012-03-08 Novozymes A/S Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2012044836A1 (en) 2010-09-30 2012-04-05 Novozymes, Inc. Variants of polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2012044835A1 (en) 2010-09-30 2012-04-05 Novozymes, Inc. Variants of polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
EP3023492A1 (en) 2010-10-01 2016-05-25 Novozymes, Inc. Beta-glucosidase variants and polynucleotides encoding same
WO2012044915A2 (en) 2010-10-01 2012-04-05 Novozymes, Inc. Beta-glucosidase variants and polynucleotides encoding same
WO2012058293A1 (en) 2010-10-26 2012-05-03 Novozymes North America, Inc. Methods of saccharifying sugarcane trash
WO2012061517A1 (en) 2010-11-02 2012-05-10 Novozymes, Inc. Methods of pretreating cellulosic material with a gh61 polypeptide
US8309328B1 (en) 2010-11-02 2012-11-13 Codexis, Inc. Compositions and methods for production of fermentable sugars
US9528098B2 (en) 2010-11-02 2016-12-27 Codexis, Inc. Fungal strains
US9068235B2 (en) 2010-11-02 2015-06-30 Codexis, Inc. Fungal strains
WO2012061382A1 (en) 2010-11-02 2012-05-10 Codexis, Inc. Improved fungal strains
WO2012061432A1 (en) 2010-11-02 2012-05-10 Codexis, Inc. Compositions and methods for production of fermentable sugars
US8236551B2 (en) 2010-11-02 2012-08-07 Codexis, Inc. Compositions and methods for production of fermentable sugars
WO2012059053A1 (en) 2010-11-04 2012-05-10 Novozymes A/S Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2012062220A1 (en) 2010-11-12 2012-05-18 Novozymes A/S Polypeptides having endoglucanase activity and polynucleotides encoding same
WO2012068509A1 (en) 2010-11-18 2012-05-24 Novozymes, Inc. Chimeric polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2012078656A1 (en) 2010-12-06 2012-06-14 Novozymes North America, Inc. Methods of hydrolyzing oligomers in hemicellulosic liquor
WO2012093041A1 (en) 2011-01-04 2012-07-12 Novozymes A/S Process for producing biogas from pectin and lignocellulose containing material
WO2012103293A1 (en) 2011-01-26 2012-08-02 Novozymes A/S Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2012103300A2 (en) 2011-01-26 2012-08-02 Novozymes A/S Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
EP3235903A1 (en) 2011-01-26 2017-10-25 Novozymes A/S Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2012103288A1 (en) 2011-01-26 2012-08-02 Novozymes A/S Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2012101206A2 (en) 2011-01-26 2012-08-02 Novozymes A/S Novel glycoside hydrolases from thermophilic fungi
WO2012103350A1 (en) 2011-01-26 2012-08-02 Novozymes A/S Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2012103322A1 (en) 2011-01-26 2012-08-02 Novozymes A/S Polypeptides having endoglucanase activity and polynucleotides encoding same
WO2012134626A2 (en) 2011-01-31 2012-10-04 Novozymes North America, Inc. Processes for enzymatic refining of pretreated cellulosic material for saccharification
WO2012113340A1 (en) 2011-02-23 2012-08-30 Novozymes Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2012118767A1 (en) 2011-02-28 2012-09-07 Midori Renewables, Inc. Polymeric acid catalysts and uses thereof
US9079171B2 (en) 2011-02-28 2015-07-14 Midori Usa, Inc. Polymeric acid catalysts and uses thereof
US8476388B2 (en) 2011-02-28 2013-07-02 Midori Renewables, Inc. Polymeric acid catalysts and uses thereof
EP3459975A1 (en) 2011-02-28 2019-03-27 Cadena Bio, Inc. Polymeric acid catalysts and uses thereof
US10131721B2 (en) 2011-02-28 2018-11-20 Cadena Bio, Inc. Polymeric acid catalysts and uses thereof
US9205418B2 (en) 2011-02-28 2015-12-08 Midori Usa, Inc. Polymeric acid catalysts and uses thereof
US10787527B2 (en) 2011-02-28 2020-09-29 Cadena Bio, Inc. Polymeric acid catalysts and uses thereof
WO2012122518A1 (en) 2011-03-09 2012-09-13 Novozymes A/S Methods of increasing the cellulolytic enhancing activity of a polypeptide
EP3339442A1 (en) 2011-03-09 2018-06-27 Novozymes A/S Methods of increasing the cellulolytic enhancing activity of a polypeptide
WO2012122477A1 (en) 2011-03-10 2012-09-13 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2012130120A1 (en) 2011-03-25 2012-10-04 Novozymes A/S Method for degrading or converting cellulosic material
EP3333258A2 (en) 2011-03-25 2018-06-13 Novozymes A/S Method for degrading or converting cellulosic material
WO2012135659A2 (en) 2011-03-31 2012-10-04 Novozymes A/S Methods for enhancing the degradation or conversion of cellulosic material
WO2012135719A1 (en) 2011-03-31 2012-10-04 Novozymes, Inc. Cellulose binding domain variants and polynucleotides encoding same
WO2012149192A1 (en) 2011-04-28 2012-11-01 Novozymes, Inc. Polypeptides having endoglucanase activity and polynucleotides encoding same
WO2012149344A1 (en) 2011-04-29 2012-11-01 Novozymes, Inc. Methods for enhancing the degradation or conversion of cellulosic material
WO2012159009A1 (en) 2011-05-19 2012-11-22 Novozymes, Inc. Methods for enhancing the degradation of cellulosic material with chitin binding proteins
WO2012159007A1 (en) 2011-05-19 2012-11-22 Novozymes, Inc. Methods for enhancing the degradation of cellulosic material with chitin binding proteins
WO2013000945A1 (en) 2011-06-28 2013-01-03 Novozymes A/S Biogas from enzyme-treated bagasse
WO2013016115A1 (en) 2011-07-22 2013-01-31 Novozymes North America, Inc. Processes for pretreating cellulosic material and improving hydrolysis thereof
WO2013019827A2 (en) 2011-08-04 2013-02-07 Novozymes A/S Polypeptides having xylanase activity and polynucleotides encoding same
EP3091073A2 (en) 2011-08-04 2016-11-09 Novozymes Inc. Polypeptides having xylanase activity and polynucleotides encoding same
EP3382016A1 (en) 2011-08-04 2018-10-03 Novozymes, Inc. Polypeptides having xylanase activity and polynucleotides encoding same
WO2013019780A2 (en) 2011-08-04 2013-02-07 Novozymes A/S Polypeptides having endoglucanase activity and polynucleotides encoding same
WO2013028927A1 (en) 2011-08-24 2013-02-28 Novozymes, Inc. Aspergillus fumigatus cellulolytic enzyme compositions and uses thereof
WO2013028928A1 (en) 2011-08-24 2013-02-28 Novozymes, Inc. Cellulolytic enzyme compositions and uses thereof
WO2013039776A1 (en) 2011-09-13 2013-03-21 Novozymes North America, Inc. Methods of hydrolyzing and fermenting cellulosic material
US9499783B2 (en) 2011-09-14 2016-11-22 Toray Industries, Inc. Production apparatus of sugar solution and production system of sugar solution
WO2013043910A1 (en) 2011-09-20 2013-03-28 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2013043981A1 (en) 2011-09-23 2013-03-28 Novozymes A/S Cellulolytic enzyme compositions and uses thereof
WO2013089889A2 (en) 2011-09-30 2013-06-20 Novozymes, Inc. Chimeric polypeptides having beta-glucosidase activity and polynucleotides encoding same
WO2013064075A1 (en) 2011-10-31 2013-05-10 Novozymes, Inc. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
EP3382017A1 (en) 2011-11-18 2018-10-03 Novozymes A/S Polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same
WO2013074956A2 (en) 2011-11-18 2013-05-23 Novozymes, Inc. Polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same
EP3409769A1 (en) 2011-11-18 2018-12-05 Novozymes A/S Polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same
EP3219794A1 (en) 2011-11-21 2017-09-20 Novozymes A/S Gh61 polypeptide variants and polynucleotides encoding same
EP3597736A1 (en) 2011-11-21 2020-01-22 Novozymes A/S Gh61 polypeptide variants and polynucleotides encoding same
WO2013119302A2 (en) 2011-11-21 2013-08-15 Novozymes, Inc. Gh61 polypeptide variants and polynucleotides encoding same
WO2013075644A1 (en) 2011-11-22 2013-05-30 Novozymes, Inc. Polypeptides having beta-xylosidase activity and polynucleotides encoding same
EP3342860A1 (en) 2011-11-22 2018-07-04 Novozymes, Inc. Polypeptides having beta-xylosidase activity and polynucleotides encoding same
WO2013079015A1 (en) 2011-12-01 2013-06-06 Novozymes, Inc. Polypeptides having beta-xylosidase activity and polynucleotides encoding same
WO2013083801A2 (en) 2011-12-09 2013-06-13 Novozymes A/S Biogas from substrates comprising animal manure and enzymes
EP3272862A1 (en) 2011-12-16 2018-01-24 Novozymes, Inc. Polypeptides having laccase activity and polynucleotides encoding same
WO2013087027A1 (en) 2011-12-16 2013-06-20 Novozymes, Inc. Polypeptides having laccase activity and polynucleotides encoding same
WO2013091547A1 (en) 2011-12-19 2013-06-27 Novozymes, Inc. Polypeptides having catalase activity and polynucleotides encoding same
WO2013096369A1 (en) 2011-12-19 2013-06-27 Novozymes A/S Processes and compositions for increasing the digestibility of cellulosic materials
WO2013096603A2 (en) 2011-12-20 2013-06-27 Novozymes, Inc. Cellobiohydrolase variants and polynucleotides encoding same
WO2013096652A1 (en) 2011-12-21 2013-06-27 Novozymes, Inc. Methods for determining the degradation of a biomass material
WO2013148504A2 (en) 2012-03-26 2013-10-03 Novozymes North America, Inc. Methods of preconditioning cellulosic material
WO2013160248A2 (en) 2012-04-23 2013-10-31 Novozymes A/S Polypeptides having alpha-glucuronidase activity and polynucleotides encoding same
WO2013160247A2 (en) 2012-04-23 2013-10-31 Novozymes A/S Polypeptides having glucuronyl esterase activity and polynucleotides encoding same
WO2013163590A2 (en) 2012-04-27 2013-10-31 Novozymes, Inc. Gh61 polypeptide variants and polynucleotides encoding same
EP3279320A2 (en) 2012-04-27 2018-02-07 Novozymes A/S Gh61 polypeptide variants and polynucleotides encoding same
US9238845B2 (en) 2012-08-24 2016-01-19 Midori Usa, Inc. Methods of producing sugars from biomass feedstocks
WO2014092832A2 (en) 2012-09-19 2014-06-19 Novozymes, Inc. Methods for enhancing the degradation or conversion of cellulosic material
WO2014058896A1 (en) 2012-10-08 2014-04-17 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
EP3586610A1 (en) 2012-10-08 2020-01-01 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2014066141A2 (en) 2012-10-24 2014-05-01 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2014070844A1 (en) 2012-10-31 2014-05-08 Danisco Us Inc. Beta-glucosidase from neurospora crassa
WO2014070841A1 (en) 2012-10-31 2014-05-08 Danisco Us Inc. Compositions and methods of use
WO2014070837A1 (en) 2012-10-31 2014-05-08 Danisco Us Inc. Beta-glucosidase from magnaporthe grisea
WO2014093835A1 (en) 2012-12-14 2014-06-19 Novozymes A/S Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same
WO2014099798A1 (en) 2012-12-19 2014-06-26 Novozymes A/S Polypeptides having cellulolytic enhancinc activity and polynucleotides encoding same
EP3848469A1 (en) 2013-02-21 2021-07-14 Novozymes A/S Methods of saccharifying and fermenting a cellulosic material
WO2014138672A1 (en) 2013-03-08 2014-09-12 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
WO2014182990A1 (en) 2013-05-10 2014-11-13 Novozymes A/S Polypeptides having xylanase activity and polynucleotides encoding same
WO2015007290A1 (en) 2013-07-16 2015-01-22 Advanced Substrate Technologies A/S Method for cycling biomasses between mushroom cultivation and anaerobic biogas fermentation, and for separating and drying a degassed biomass
US10196654B2 (en) 2013-07-16 2019-02-05 Advanced Substrate Technologies A/S Method for cycling biomasses between mushroom cultivation and anaerobic biogas fermentation, and for separating and drying a degassed biomass
WO2015035029A1 (en) 2013-09-04 2015-03-12 Novozymes A/S Processes for increasing enzymatic hydrolysis of cellulosic material
WO2015066492A1 (en) 2013-11-01 2015-05-07 Novozymes A/S Methods of saccharifying and fermenting a cellulosic material
WO2015081139A1 (en) 2013-11-26 2015-06-04 Novozymes A/S Enzyme compositions and uses thereof
WO2015084596A1 (en) 2013-12-04 2015-06-11 Danisco Us Inc. Compositions comprising a beta-glucosidase polypeptide and methods of use
EP3511418A1 (en) 2014-01-07 2019-07-17 Novozymes A/S Process for degrading mannan-containing cellulosic materials
WO2015105835A1 (en) 2014-01-07 2015-07-16 Novozymes A/S Process for degrading mannan-containing cellulosic materials
WO2015187935A1 (en) 2014-06-06 2015-12-10 Novozymes A/S Enzyme compositions and uses thereof
AU2015286229B2 (en) * 2014-07-10 2018-11-08 Leaf Sciences Pty Ltd Methods for treating lignocellulosic material
US11332768B2 (en) * 2014-07-10 2022-05-17 Leaf Sciences Pty Ltd Methods for hydrolysing lignocellulosic material
US20170211108A1 (en) * 2014-07-10 2017-07-27 Leaf Sciences Pty Ltd Methods for Hydrolysing Lignocellulosic Material
WO2016004481A1 (en) * 2014-07-10 2016-01-14 Leaf Sciences Pty Ltd Methods for treating lignocellulosic material
AU2015286230B2 (en) * 2014-07-10 2018-11-08 Leaf Sciences Pty Ltd Methods for hydrolysing lignocellulosic material
WO2016004482A1 (en) * 2014-07-10 2016-01-14 Leaf Sciences Pty Ltd Methods for hydrolysing lignocellulosic material
WO2016029107A1 (en) 2014-08-21 2016-02-25 Novozymes A/S Process for saccharifying cellulosic material under oxygen addition
US10590440B2 (en) 2014-08-22 2020-03-17 Cysbio Aps Process for producing a fermentation product from a lignocellulose-containing material
EP3594335A1 (en) 2014-09-05 2020-01-15 Novozymes A/S Carbohydrate binding module variants and polynucleotides encoding same
WO2016037096A1 (en) 2014-09-05 2016-03-10 Novozymes A/S Carbohydrate binding module variants and polynucleotides encoding same
WO2016045569A1 (en) 2014-09-23 2016-03-31 Novozymes A/S Processes for producing ethanol and fermenting organisms
WO2016069541A1 (en) 2014-10-27 2016-05-06 Danisco Us Inc Compositions and methods related to beta-glucosidase
WO2016116113A1 (en) 2015-01-22 2016-07-28 Advanced Substrate Technologies A/S Methods for upgrading spent biomass material
EP3739045A2 (en) 2015-02-24 2020-11-18 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
WO2016138167A2 (en) 2015-02-24 2016-09-01 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
WO2016145363A1 (en) 2015-03-12 2016-09-15 Novozymes A/S Multi-stage enzymatic hydrolysis of lignocellulosic biomass employing an oxidoreductase with an aa9 polypeptide
WO2016145350A1 (en) 2015-03-12 2016-09-15 Novozymes A/S Multi-stage enzymatic hydrolysis of lignocellulosic biomass
WO2016145358A1 (en) 2015-03-12 2016-09-15 Novozymes A/S Enzymatic hydrolysis with hemicellulolytic enzymes
WO2016188459A1 (en) 2015-05-27 2016-12-01 Novozymes A/S Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2017019491A1 (en) 2015-07-24 2017-02-02 Novozymes Inc. Polypeptides having beta-xylosidase activity and polynucleotides encoding same
WO2017019490A1 (en) 2015-07-24 2017-02-02 Novozymes Inc. Polypeptides having arabinofuranosidase activity and polynucleotides encoding same
WO2017050242A1 (en) 2015-09-22 2017-03-30 Novozymes A/S Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
WO2017070219A1 (en) 2015-10-20 2017-04-27 Novozymes A/S Lytic polysaccharide monooxygenase (lpmo) variants and polynucleotides encoding same
WO2017144670A1 (en) 2016-02-24 2017-08-31 Danmarks Tekniske Universitet Improved process for producing a fermentation product from a lignocellulose-containing material
WO2017151957A1 (en) 2016-03-02 2017-09-08 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
WO2017165760A1 (en) 2016-03-24 2017-09-28 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
WO2017205535A1 (en) 2016-05-27 2017-11-30 Novozymes, Inc. Polypeptides having endoglucanase activity and polynucleotides encoding same
WO2018026868A1 (en) 2016-08-01 2018-02-08 Novozymes, Inc. Polypeptides having endoglucanase activity and polynucleotides encoding same
WO2018050300A1 (en) 2016-09-13 2018-03-22 Institut National De La Recherche Agronomique (Inra) Polysaccharide-oxidizing composition and uses thereof
WO2018085370A1 (en) 2016-11-02 2018-05-11 Novozymes A/S Processes for reducing production of primeverose during enzymatic saccharification of lignocellulosic material
WO2018106792A1 (en) 2016-12-06 2018-06-14 Novozymes A/S Improved processes for production of ethanol from xylose-containing cellulosic substrates using engineered yeast strains
WO2018220116A1 (en) 2017-05-31 2018-12-06 Novozymes A/S Xylose fermenting yeast strains and processes thereof for ethanol production
WO2018222990A1 (en) 2017-06-02 2018-12-06 Novozymes A/S Improved yeast for ethanol production
WO2019025449A1 (en) 2017-08-02 2019-02-07 Institut National De La Recherche Agronomique (Inra) Methods of defibrillating cellulosic substrates and producing celluloses using a new family of fungal lytic polysaccharide monooxygenases (lpmo)
US10597595B2 (en) 2017-10-27 2020-03-24 Xyleco, Inc. Processing biomass
WO2019148192A1 (en) 2018-01-29 2019-08-01 Novozymes A/S Microorganisms with improved nitrogen utilization for ethanol production
WO2020007880A2 (en) 2018-07-02 2020-01-09 Institut National De La Recherche Agronomique (Inra) Polypeptides and compositions with lytic polysaccharide oxidase activity
FR3083247A1 (en) 2018-07-02 2020-01-03 Institut National De La Recherche Agronomique (Inra) POLYPEPTIDES AND COMPOSITIONS WITH LYSTIC OXIDASE POLYSACCHARIDE ACTIVITY
WO2020023411A1 (en) 2018-07-25 2020-01-30 Novozymes A/S Enzyme-expressing yeast for ethanol production
WO2020076697A1 (en) 2018-10-08 2020-04-16 Novozymes A/S Enzyme-expressing yeast for ethanol production
WO2020123463A1 (en) 2018-12-12 2020-06-18 Novozymes A/S Polypeptides having xylanase activity and polynucleotides encoding same
WO2021021458A1 (en) 2019-07-26 2021-02-04 Novozymes A/S Microorganisms with improved nitrogen transport for ethanol production
WO2021026201A1 (en) 2019-08-05 2021-02-11 Novozymes A/S Enzyme blends and processes for producing a high protein feed ingredient from a whole stillage byproduct
WO2021025872A1 (en) 2019-08-06 2021-02-11 Novozymes A/S Fusion proteins for improved enzyme expression
WO2021055395A1 (en) 2019-09-16 2021-03-25 Novozymes A/S Polypeptides having beta-glucanase activity and polynucleotides encoding same
WO2021119304A1 (en) 2019-12-10 2021-06-17 Novozymes A/S Microorganism for improved pentose fermentation
WO2022049250A1 (en) 2020-09-04 2022-03-10 Novozymes A/S Improved fermenting organism for ethanol production
WO2022090564A1 (en) 2020-11-02 2022-05-05 Novozymes A/S Glucoamylase variants and polynucleotides encoding same
WO2022261003A1 (en) 2021-06-07 2022-12-15 Novozymes A/S Engineered microorganism for improved ethanol fermentation
WO2023164436A1 (en) 2022-02-23 2023-08-31 Novozymes A/S Process for producing fermentation products and biogas from starch-containing materials

Also Published As

Publication number Publication date
WO2006110899A3 (en) 2007-03-29
CA2603128C (en) 2014-04-08
JP2008535523A (en) 2008-09-04
JP5149785B2 (en) 2013-02-20
WO2006110901A2 (en) 2006-10-19
BRPI0612966B1 (en) 2017-12-05
CA2603774A1 (en) 2006-10-19
CA2604100A1 (en) 2006-10-19
CN101155925B (en) 2012-05-09
JP2008535664A (en) 2008-09-04
EP1885840A1 (en) 2008-02-13
WO2006110902A1 (en) 2006-10-19
EP1869201A2 (en) 2007-12-26
US7998713B2 (en) 2011-08-16
JP2008537886A (en) 2008-10-02
US20070037259A1 (en) 2007-02-15
US20070031953A1 (en) 2007-02-08
JP2008535524A (en) 2008-09-04
CN101160409B (en) 2013-04-24
WO2006110899A2 (en) 2006-10-19
US7910338B2 (en) 2011-03-22
EP1869201B1 (en) 2017-12-27
CN101155925A (en) 2008-04-02
CN101155928B (en) 2013-04-24
CN101155928A (en) 2008-04-02
BRPI0612966A2 (en) 2010-12-14
CN101160405A (en) 2008-04-09
JP5118626B2 (en) 2013-01-16
WO2006110900A2 (en) 2006-10-19
BRPI0612939A2 (en) 2010-12-07
BRPI0612944A2 (en) 2010-12-07
CN101160409A (en) 2008-04-09
BRPI0612937B1 (en) 2015-07-07
JP5149784B2 (en) 2013-02-20
CA2604100C (en) 2013-04-02
BRPI0612944B1 (en) 2017-11-28
EP1885840B1 (en) 2012-02-29
BRPI0612207A2 (en) 2010-10-26
JP2008537682A (en) 2008-09-25
US20070031918A1 (en) 2007-02-08
WO2006110901A3 (en) 2007-03-29
EP1869202B1 (en) 2018-02-14
CA2603128A1 (en) 2006-10-19
CA2603774C (en) 2015-11-17
EP1869194A2 (en) 2007-12-26
WO2006110891A3 (en) 2007-02-08
BRPI0612939B1 (en) 2018-05-02
CA2604964A1 (en) 2006-10-19
CA2604961A1 (en) 2006-10-19
CN101160405B (en) 2014-01-01
EP1869197A2 (en) 2007-12-26
EP1869202A2 (en) 2007-12-26
JP4991700B2 (en) 2012-08-01
US7932063B2 (en) 2011-04-26
BRPI0612937A2 (en) 2010-12-07
WO2006110900A3 (en) 2007-03-29
CN101160388A (en) 2008-04-09
JP5804666B2 (en) 2015-11-04
CA2604964C (en) 2014-12-16
CN101160388B (en) 2013-05-01

Similar Documents

Publication Publication Date Title
US7781191B2 (en) Treatment of biomass to obtain a target chemical
US7932063B2 (en) Treatment of biomass to obtain fermentable sugars
US8445236B2 (en) Biomass pretreatment
JP2010536557A (en) Biomass processing equipment
EP2190883A2 (en) Biomass treatment method

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 200680012124.5

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application
REEP Request for entry into the european phase

Ref document number: 2006758339

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2006758339

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2604964

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2008506725

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 7956/DELNP/2007

Country of ref document: IN

NENP Non-entry into the national phase

Ref country code: RU

ENP Entry into the national phase

Ref document number: PI0612944

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20071011