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WO2010060050A2 - Hydrolyse enzymatique à haute consistance pour la production d’éthanol - Google Patents

Hydrolyse enzymatique à haute consistance pour la production d’éthanol Download PDF

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Publication number
WO2010060050A2
WO2010060050A2 PCT/US2009/065562 US2009065562W WO2010060050A2 WO 2010060050 A2 WO2010060050 A2 WO 2010060050A2 US 2009065562 W US2009065562 W US 2009065562W WO 2010060050 A2 WO2010060050 A2 WO 2010060050A2
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Prior art keywords
mixture
enzyme
hydrolysis
cellulase
alcohol
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WO2010060050A3 (fr
Inventor
Richard Phillips
Hasan Jameel
Hou-Min Chang
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North Carolina State University
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North Carolina State University
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Priority to US13/130,521 priority Critical patent/US20120036768A1/en
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Publication of WO2010060050A3 publication Critical patent/WO2010060050A3/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • Y—GENERAL 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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the presently disclosed subject matter relates to methods of converting lignocellulosic biomass to alcohol employing enzymatic hydrolysis reactions performed at high fiber consistency.
  • the use of high fiber consistency enzymatic hydrolysis reactions allows for the recovery of high sugar concentration mixtures that subsequently can provide high alcohol content solutions following fermentation.
  • Na 2 SO 4 sodium sulfate
  • Plant-derived lignocellulosic biomass represents a large, renewable source of potential starting materials for the production of a variety of chemicals, plastics, fuels and feeds.
  • lignocellulosic biomass feedstocks comprise cellulose, a polymer of glucose, which can be hydrolyzed to provide fermentable sugar for use in the production of ethanol.
  • Cellulose hydrolysis can be performed by acid or enzymatic hydrolysis
  • EH a family of enzymes can be used that works together to hydrolyze glycosidic bonds in polymeric lignocellulose molecules. Most EH is done at a lignocellulose fiber concentration (which can be referred to as a % K), between about 5-10%, to ensure proper contact between the enzymes and the fibers. At higher fiber concentrations, the cellulose can swell to provide very thick mixtures that are hard to handle (e.g., transfer from one reactor to another) and/or that make proper mixing of the enzymes and the fibers difficult, thus reducing hydrolysis efficiency. Unfortunately, the low concentration of fibers during hydrolysis results in solutions containing low concentrations of simple sugars, increasing the size of fermentation vessels that must be used during the ethanol production processes. Low sugar concentration also leads to lower alcohol concentration following fermentation, requiring larger distillation columns and higher energy input for purification of fermented mixtures.
  • a method of producing an alcohol from a lignocellulosic biomass comprising: providing lignocellulosic biomass; contacting the lignocellulosic biomass with a first enzyme composition for a first period of time to provide a first hydrolysis mixture; thickening the first hydrolysis mixture to form a second hydrolysis mixture; hydrolyzing the second hydrolysis mixture for a second period of time to provide a fermentable sugar mixture; and fermenting the fermentable sugar mixture to provide an alcohol.
  • the lignocellulosic biomass is selected from the group consisting of herbaceous material, agricultural residues, forestry residues, municipal solid wastes, waste paper, pulp and paper mill residues, or a combination thereof. In some embodiments, the lignocellulosic biomass is selected from the group consisting of corn stover, straw, bagasse, miscanthus, sorghum residue, switch grass, bamboo, water hyacinth, hardwood, hardwood chips, softwood chips, hardwood pulp, and softwood pulp.
  • the method further comprises pretreating the lignocellulosic biomass to increase enzymatic digestability.
  • the pretreating comprises one or more of the group consisting of removing or altering lignin, removing hemicellulose, decrystallizing cellulose, removing acetyl groups from hemicellulose, reducing the degree of polymerization of cellulose, increasing the pore volume of lignocellulose biomass, and increasing the surface area of lignocellulose.
  • the pretreating comprises one or more pretreatment technique selected from the group consisting of autohydrolysis, steam explosion, grinding, chopping, ball milling, compression mulling, radiation, flow-through liquid hot water treatment, dilute acid treatment, concentrated acid treatment, peracetic acid treatment, supercritial carbon dioxide treatment, alkali treatment, organic solvent treatment, cellulose solvent treatment, and treatment with an aerobic fungi.
  • the alkali treatment is selected from the group consisting of sodium hydroxide treatment, lime treatment, wet oxidation, ammonia treatment, and oxidative alkali treatment.
  • the alkali treatment is green liquor treatment.
  • contacting the lignocellulosic biomass with the first enzyme composition comprises mixing the lignocellulosic biomass with the first enzyme composition at a solids concentration of about 5%.
  • the first hydrolysis mixture comprises between about 5 filter paper units (FPU) and about 85 FPU of lignocellulose-hydrolyzing enzyme per gram of lignocellulosic biomass. In some embodiments, the first hydrolysis mixture comprises about 10 FPU of lignocellulose-hydrolyzing enzyme per gram of lignocellulosic biomass.
  • the first enzyme composition comprises cellulase. In some embodiments, the first enzyme composition further comprises xylanase and ⁇ -glucosidase.
  • the first period of time ranges from about 1 minute to about 20 minutes. In some embodiments, the first period of time ranges from about 5 minutes to about 10 minutes. In some embodiments, contacting the lignocellulosic biomass with the first enzyme composition is performed at a temperature of between about 4°C and about 70 0 C. In some embodiments, the contacting is performed at a temperature of about 38°C. In some embodiments, the contacting is performed at a pH of about 4.8. In some embodiments, the thickening step comprises increasing the fiber concentration of the first hydrolysis mixture to provide a second hydrolysis mixture having a solids concentration of between about 15% and about 30%. In some embodiments, the thickening step comprises filtering the first hydrolysis mixture to provide the second hydrolysis mixture and a filtrate. In some embodiments, the filtering is performed by vacuum filtering the first hydrolysis mixture using a filter press.
  • the filtrate comprises about 80% of the liquid from the first hydrolysis mixture. In some embodiments, the filtrate comprises water and unabsorbed lignocellulose-hydrolyzing enzyme, and wherein said filtrate is used to dilute lignocellulosic biomass, thereby recycling the lignocellulose-hydrolyzing enzyme.
  • the first enzyme composition comprises cellulase and wherein the filtrate comprises about 10% to about 20% of the cellulase from the first hydrolysis mixture.
  • the second period of time ranges between about 1 day and about 3 days.
  • hydrolyzing the second hydrolysis mixture comprises hydrolyzing the second hydrolysis mixture for a first portion of the second period of time, adding a second enzyme composition to the second hydrolysis mixture to increase the enzyme dosage in the second hydrolysis mixture, and continuing hydrolysis of the second hydrolysis mixture for a second portion of the second period of time to provide the fermentable sugar mixture.
  • the first portion of the second period of time ranges between about 0 hours and about 24 hours. In some embodiments, the first portion of the second period of time ranges between about 2 hours and about 3 hours.
  • the second enzyme composition comprises xylanase and ⁇ -glucosidase. In some embodiments, the second enzyme composition further comprises cellulase. In some embodiments, the first enzyme composition and the second enzyme composition each comprise cellulase, and the first enzyme composition comprises between about 25% and about 50% of the total cellulase dosage from the first and second enzyme compositions. In some embodiments, the first enzyme composition comprises about 50% of the total cellulase dosage.
  • the second portion of the first period of time ranges between about 24 hours and about 48 hours.
  • hydrolysis efficiency of cellulosic material originally present in the lignocellulosic biomass is about 70% or greater. In some embodiments, the hydrolysis efficiency is between about 78% and about 84%. In some embodiments, the fermentable sugar mixture comprises about 12% fermentable sugar by volume.
  • fermenting comprises fermenting the fermentable sugar mixture using a microorganism to provide an alcohol mixture and distilling the alcohol mixture to provide the alcohol.
  • the microorganism is yeast.
  • the alcohol mixture comprises about 6% alcohol by volume.
  • the method further comprises dehydrating the alcohol.
  • the alcohol is ethanol.
  • the presently disclosed subject matter provides a composition comprising an alcohol prepared according to a method comprising: providing lignocellulosic biomass; contacting the lignocellulosic biomass with a first enzyme composition for a first period of time to provide a first hydrolysis mixture; thickening the first hydrolysis mixture to from a second hydrolysis mixture; hydrolyzing the second hydrolysis mixture for a second period of time to provide a fermentable sugar mixture; and fermenting the fermentable sugar mixture to provide the alcohol.
  • the alcohol is ethanol. In some embodiments, the composition comprises about 95% or greater ethanol by volume. In some embodiments, composition is a fuel mixture comprising ethanol and gasoline.
  • Figure 1A is a block diagram showing a method for preparing a sugar mixture from lignocellulosic biomass according to an embodiment of the presently disclosed subject matter.
  • Figure 1 B is a block diagram showing a method for preparing alcohol from lignocellulosic biomass according to an embodiment of the presently disclosed subject matter.
  • Figure 2A is a graph showing the effect of fiber consistency (%K) on the enzymatic hydrolysis (EH) of wood chips following pretreatment with green liquor (GL) having total titratable alkali of 12%.
  • the data shown by diamonds represents EH performed at 5K.
  • the data shown by squares represents EH performed at 7.5K.
  • the data shown by triangles represents EH performed at 10% K.
  • Enzymatic hydrolysis results are provided as % biomass conversion based on total sugars (glucose, xylose and mannose) in the hydrolysis mixture after EH.
  • Figure 2B is a graph showing the effect of fiber consistency (%K) on the enzymatic hydrolysis (EH) of wood following pretreatment with green liquor (GL) having total titratable alkali of 16%.
  • the data shown by diamonds represents EH performed at 5% K.
  • the data shown by squares represents EH performed at 7.5 %K.
  • the data shown by triangles represents EH performed at 10 %K.
  • Enzymatic hydrolysis results are provided as % biomass conversion based on total sugars (glucose, xylose and mannose) in the hydrolysis mixture after EH.
  • Figure 3 is a graph showing the influence of temperature on cellulase adsorption to wood pulp. Data is shown for cellulase adsorption at 4, 23, 38, and 50 0 C. Wood pulp was incubated with cellulase at 5 percent consistency
  • the amount of cellulase in the filtrate was determined and used to calculate the percentage of the cellulase adsorption (i.e., the % of the cellulase dosage remaining in the pulp mixture).
  • Figure 4 is a graph showing the influence of cellulase dosage on cellulase adsorption to wood pulp. Data is shown for cellulase dosages ranging from 5 filter paper units (FPU)/gram (gm) wood fiber to 40 FPU/gm. Wood pulp was incubated with cellulase at 5 percent consistency (%K) at the indicated dosage for ten minutes and then thickened to 20% K. The amount of cellulase in the filtrate was determined and used to calculate the percentage of the cellulase adsorption (i.e., the % of the cellulase dosage remaining in the pulp mixture).
  • FPU filter paper units
  • gm grams
  • Wood pulp was incubated with cellulase at 5 percent consistency (%K) at the indicated dosage for ten minutes and then thickened to 20% K. The amount of cellulase in the filtrate was determined and used to calculate the percentage of the cellulase adsorption (i.e., the % of the cellula
  • Figure 5 is a graph showing the influence of lignin content on cellulase adsorption to wood pulp. Data is shown for wood pulp having from 0% lignin content to 28% lignin content. Wood pulp was incubated with cellulase at 5 percent consistency (%K) for ten minutes and then thickened to 20% K. The amount of cellulase in the filtrate was determined and used to calculate the percentage of the cellulase adsorption (i.e., the % of the cellulase dosage remaining in the pulp mixture).
  • %K percent consistency
  • Figure 6 is a bar graph showing the effects of different green liquor (GL) pretreatments on enzyme adsoption to the pretreated wood pulp.
  • GL-12 represents GL pretreatment at 12% total titratable alkali (TTA).
  • GL-16 represents GL pretreatment at 16% TTA.
  • the pretreated wood pulp was incubated with cellulase at 5 percent consistency (%K) for ten minutes and then thickened to 20% K. The amount of cellulase in the filtrate was determined and used to calculate the percentage of the cellulase adsorption (i.e., the % of the cellulase dosage remaining in the pulp mixture).
  • Figure 7 is a graph showing the dependence of sugar recovery efficiency based on enzyme dosage after 48 hours of enzymatic hydrolysis (EH). Total enzyme dosage varied between 5 filter paper units (FPU) and 40 FPU/gram of wood pulp.
  • the shaded diamonds show data for sugar recovery after enzymatic hydrolysis at 20% fiber consistency (%K) when an enzyme composition including cellulase (c), xylanase (x) and ⁇ -glucosidase (b) are all added at the same time (cxb).
  • the shaded squares show data relating to sugar recovery after EH carried out at 20% K when cellulase is added first, followed by the addition of xylanase and ⁇ -glucosidase (c+xb).
  • the lightly shaded triangles show data relating to EH carried out at 20% K where cellulase is added 24 hours prior to addition of xylanase and ⁇ -glucosidase (c+24h+xb).
  • the open squares show the sugar recovery of EH carried out at 5% K.
  • Figure 8 is a graph showing the effects on enzymatic hydrolysis efficiency of adding xylanase and ⁇ -glucosidase (i.e., xb) at different times (i.e., between 0 and 8 hours) following thickening. Data is shown for hydrolysis mixtures where hydrolysis continues for a further 24 hours (diamonds) or a further 48 hours (squares) following addition of the xylanase and ⁇ -glucosidase.
  • xb ⁇ -glucosidase
  • Figure 9A is a graph showing the percentage (%) of overall enzymatic hydrolysis (EH) in bleached hardwood (HW) pulp (0% lignin content) at different enzyme dosages as determined based on total sugars produced during EH (10 minutes at 5% fiber consistency followed by 48 hours at 20% fiber consistency).
  • Figure 9B is a graph showing the percentage (%) of overall enzymatic hydrolysis (EH) in hardwood (HW) pulp with 2% lignin content at different enzyme dosages as determined based on total sugars produced during EH (10 minutes at 5% fiber consistency followed by 48 hours at 20% fiber consistency).
  • Figure 9C is a graph showing the percentage (%) of overall enzymatic hydrolysis (EH) in hardwood (HW) pulp with 10% lignin content at different enzyme dosages as determined based on total sugars produced during EH (10 minutes at 5% fiber consistency followed by 48 hours at 20% fiber consistency).
  • Figure 9D is a graph showing the percentage (%) of overall enzymatic hydrolysis (EH) in hardwood (HW) pulp with 28% lignin content at different enzyme dosages as determined based on total sugars produced during EH (10 minutes at 5% fiber consistency followed by 48 hours at 20% fiber consistency).
  • Figure 10 is a graph showing enzymatic hydrolysis (EH) efficiency using
  • FPU filter paper units
  • enzyme/gram (gm) wood pulp (28% lignin content) at either a 5% wood fiber concentration for 48 hours (stippled bars) or as described for Figure 9D (10 minutes at 5% fiber consistency followed by 48 hours at 20% fiber consistency, hatched bars).
  • Figure 1 1 is a graph showing enzymatic hydrolysis (EH) efficiency using
  • FPU filter paper units
  • enzyme/gram (gm) wood pulp (28% lignin content) at either a 5% wood fiber concentration for 48 hours (stippled bars) or as described for Figure 9D (10 minutes at 5% fiber consistency followed by 48 hours at 20% fiber consistency, hatched bars).
  • Figure 12 is a graph showing enzymatic hydrolysis (EH) efficiency using
  • FIG. 10 is a graph showing enzymatic hydrolysis (EH) efficiency using
  • Figure 14 is a graph showing enzymatic hydrolysis (EH) efficiency using 20 filter paper units (FPU) enzyme/gram (gm) wood pulp at either 5% fiber concentration (stippled bars), 20% fiber concentration with all enzymes added at once (hatched bars), 20% fiber concentration with the enzyme dosage added in two portions, where the first portion is 25% of the total enzyme dosage (darkly shaded solid bars), or 20% fiber concentration with the enzyme dosage added in two equal portions (medium shaded solid bars).
  • FPU filter paper units
  • gm filter paper units
  • Figure 15 is a graph showing enzymatic hydrolysis (EH) efficiency using 40 filter paper units (FPU) enzyme/gram (gm) wood pulp at either 5% fiber concentration (stippled bars), 20% fiber concentration with all enzymes added at once (hatched bars), 20% fiber concentration with the enzyme dosage added in two portions, where the first portion is 12.5% of the total enzyme dosage (darkly shaded solid bars), 20% fiber concentration with the enzyme dosage added in two portions, wherein the first portion is 25% of the total dosage (medium shaded solid bars), or 20% fiber concentration with the enzyme dosage added in two equal portions (unshaded open bars).
  • FPU filter paper units
  • an enzyme can refer to a plurality (i.e., two or more) enzymes.
  • the term "about" modifying any amount can refer to the variation in that amount encountered in real world conditions of producing sugars and ethanol, e.g., in the lab, pilot plant, or production facility.
  • the amounts can vary by about 5%, 1 %, or 0.5%.
  • all numbers expressing quantities of percentage (%), temperature, time, pH, distance, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.
  • saccharide refers to a carbohydrate monomer, oligomer or larger polymer.
  • the monomer units can include trioses, tetroses, pentoses, hexoses, heptoses, nonoses, and mixtures thereof.
  • each cyclized monomer unit is based on a compound having a chemical structure wherein n + m is 4 or 5.
  • saccharides can include monosaccharides including, but not limited to, aldohexoses, aldopentoses, ketohexoses, and ketopentoses such as arabinose, lyxose, ribose, xylose, hbulose, xylulose, allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, and tagatose, and to hetero- and homopolymers thereof. Saccharides can also include disacchahdes including, but not limited to sucrose, maltose, lactose, trehalose, and cellobiose, as well as hetero- and homopolymers thereof.
  • oligosaccharide refers to polysaccharides having a degree of polymerization of between about 2 and about 10.
  • the terms "fermentable sugar” and “sugar” can be used interchangeably and refer to oligosaccharides, monosaccharides and mixtures thereof that can be used as a carbon source in a fermentation process. Fermentable monosaccharides include arabinose, glyceraldehyde, dihydroxyacetone, erythrose, ribose, hbulose, xylose, glucose, galactose, mannose, fucose, fructose, sedoheptulose, neuraminic acid, or mixtures of these.
  • Fermentable disacchahdes include sucrose, lactose, maltose, gentiobiose, or mixtures thereof.
  • lignocellulosic refers to a composition comprising both lignin and cellulose.
  • lignocellulosic material can comprise hemicellulose, a polysaccharide which can comprise saccharide monomers other than glucose.
  • lignocellulosic materials comprise between about 38-50% cellulose, 15-30% lignin, and 23-32% hemicellulose.
  • Lignocellulosic biomass include a variety of plants and plant materials, such as, but not limited to, papermaking sludge; wood, and wood-related materials, e.g., saw dust, or particle board, leaves, or trees, such as poplar trees; grasses, such as switchgrass and sudangrass; grass clippings; rice hulls; bagasse (e.g., sugar cane bagasse), jute; hemp; flax; bamboo; sisal; abaca; hays; straws; corn cobs; corn stover; whole plant corn, and coconut hair.
  • papermaking sludge wood, and wood-related materials, e.g., saw dust, or particle board, leaves, or trees, such as poplar trees
  • grasses such as switchgrass and sudangrass
  • grass clippings rice hulls
  • bagasse e.g., sugar cane bagasse
  • bagasse e.g., sugar cane bagasse
  • lignocellulosic biomass is selected from the group including, but not limited to, herbaceous material, agricultural residues, forestry residues, municipal solid wastes, waste paper, pulp and paper mill residues, or a combination thereof. In some embodiments, lignocellulosic biomass is selected from the group including, but not limited to, corn stover, straw, bagasse, miscanthus, sorghum residue, switch grass, bamboo, water hyacinth, hardwood, hardwood, softwood, wood chips, and wood pulp.
  • Lignin is a polyphenol ⁇ material comprised of phenyl propane units linked by ether and carbon-carbon bonds. Lignins can be highly branched and can also be crosslinked. Lignins can have significant structural variation that depends, at least in part, on the plant source involved.
  • glucan refers to a polysaccharide comprising glucose monomers linked by glycosidic bonds.
  • cellulose refers to a polysaccharide of ⁇ -glucose (i.e., ⁇ -1 ,4- glucan) comprising ⁇ -(1-4) glycosidic bonds.
  • cellulosic refers to a composition comprising cellulose.
  • hemicellulose can refer polysaccharides comprising mainly sugars or combinations of sugars other than glucose (e.g., xylose).
  • xylan polymerized xylose
  • mannan polymerized mannose
  • Hemicellulose can be highly branched.
  • Hemicellulose can be chemically bonded to lignin and can further be randomly acetylated, which can reduce enzymatic hydrolysis of the glycosidic bonds in hemicellulose.
  • glycosidic bond and “glycosidic linkage” refer to a linkage between the hemiacetal group of one saccharide unit and the hydroxyl group of another saccharide unit.
  • biomass refers to a fuel that is derived from biomass, i.e., a living or recently living biological organism, such as a plant or an animal waste.
  • Biofuels include, but are not limited to, biodisel, biohydrogen, biogas, biomass- derived dimethylfuran (DMF), and the like.
  • biomass-derived alcohols e.g., bioalcohol
  • biomass-derived fuels such as ethanol, methanol, propanol, or butanol
  • biofuel can also be used to refer to fuel mixtures comprising biomass-derived fuels, such as alcohol/gasoline mixtures (i.e., gasohols).
  • Gasohols can comprise any desired percentage of biomass-derived alcohol (i.e., about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% biomass-derived alcohol).
  • biomass-derived alcohol i.e., about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% biomass-derived alcohol.
  • one useful biofuel-based mixture is E85, which comprises 85% ethanol and 15% gasoline.
  • pretreating generally refers to a chemical, microbial, or mechanical method of treating biomass to make it more amendable to enzymatic hydrolysis and/or microbial fermentation.
  • pretreating can relate to removing or altering lignin, removing hemicellulose, decrystallizing cellulose, removing acetyl groups (e.g., through chemical or enzymatic hydrolysis of the acetyl ester), reducing the degree of polymerization of cellulose (i.e., hydrolysis of glycosidic bonds), expanding the structure of the lignocellulosic material to increase pore volume and internal surface area.
  • green liquor refers to an alkaline composition, such as that used in alkaline pulping during paper production, comprising sodium sulfide (Na 2 S) and sodium carbonate(Na 2 C03).
  • the green liquor can further comprise sodium sulfate (Na 2 SO 4 ).
  • total titratable alkali refers to the weight percentage of combined alkali species (e.g., Na 2 COs, Na 2 S, and NaOH) in a solution (e.g., a green liquor solution), expressed as Na 2 O.
  • sulfidity refers to the weight percentage of alkaline sulfur compounds in a solution (e.g., a green liquor solution) compared to the total titratable alkali.
  • delignification refers to the removal of some or all of the lignan present in a lignin-containing sample. Delignification can be performed via chemical, mechanical, or enzymatic processes or combinations thereof.
  • Oxygen delignification refers to a delignification process wherein biomass (e.g., green liquor pretreated biomass) is contacted with oxygen gas in a pressurized vessel at an elevated temperature in an alkaline environment. Oxygen delignification is used in the paper industry to treat paper pulp in part to reduce the consumption of bleaching chemicals.
  • refining refers to a mechanical process of treating lignocellulosic-containing solids in order to beat, bruise, cut, and/or fibrillate the fibers therein.
  • refining can be used to reduce lignocellulosic-containing solids in size as well as to providing material comprising bundles of cellulosic fibers, separate cellulosic fibers, fragments of cellulosic fibers, and combinations thereof.
  • enzyme refers to a protein that catalyzes the conversion of one molecule into another.
  • enzyme as used herein includes any enzyme that can catalyze the transformation of a biomass-dehved molecule to another biomass-derived molecule.
  • enzymes include those which can degrade or otherwise transform saccharide, cellulose, or lignocellulose molecules to provide fermentable sugars and/or alcohols.
  • an enzyme can be specifically selected based on the particular end product desired from the biomass.
  • the enzyme can also be selected to provide a desired property to a hydrolysis mixture.
  • an enzyme can be selected in order to produce a hydrolysis mixture of desired viscosity or pH.
  • lignocellulytic enzyme "lignocellulose-processing enzyme", and “lingocellulose-hydrolyzing enzyme” refer to enzymes that are involved in the disruption and/or degradation of lignocellulose.
  • the disruption of lignocellulose by lignocellulytic enzymes leads to the formation of substances including monosaccharides, disacchahdes, polysaccharides and phenols.
  • Lignocellulytic enzymes include, but are not limited to, cellulases, hemicellulases and ligninases.
  • lignocellulytic enzymes include sacchahfication enzymes, i.e., enzymes which hydrolyze (i.e., depolymerize) polysaccharides. Sacchahfication enzymes and their use in biomass treatments have been previously reviewed. See Lynd, L.R., et al., Microbiol. MoI. Biol. Rev., 66, 506-577 (2002).
  • cellulase when used generally can refer to enzymes involved in cellulose degradation.
  • Cellulase enzymes are classified on the basis of their mode of action.
  • Cellulases include cellobiohydrolases, endoglucanases, and ⁇ -D-glucosidases. Endoglucanases randomly attack the amorphous regions of cellulose substrates, yielding mainly higher oligomers.
  • Cellulobiohydrolases are exocellulases which hydrolyze crystalline cellulose and release cellobiose (glucose dimer). Both types of enzymes hydrolyze-1 ,4- glycosidic bonds. ⁇ -D-glucosidases or cellulobiase converts oligosaccharides and cellubiose to glucose.
  • cellulase is typically used more specifically to refer to the enzyme is cellulase (E. C. 3.2.1.4), also known as an endoglucanase, which catalyzes the hydrolysis of 1 ,4- ⁇ -D-glycosidic linkages.
  • the cellulase can be of microbial origin, such as derivable from a strain of a filamentous fungus (e.g., Aspergillus, Trichoderma, Humicola, Fusarium).
  • cellulase preparations which can be used include, but are not limited to, CELLUCLASTTM, CELLUZYMETM, CEREFLOTM, and ULTRAFLOTM (available from Novozymes A/S, Bagsvaerd, Denmark), SPEZYMETM CE and SPEZYMETM CP (available from Genencor International, Inc., Rochester, New York, United States of America) and ROHAMENT® CL (available from AB Enzymes GmbH, Darmstadt, Germany).
  • Hemicellulases are enzymes that are involved in hemicellulose degradation.
  • Hemicellulases include xylanases, arabinofuranosidases, acetyl xylan esterases, glucuronidases, mannanases, galactanases, and arabinases. Similar to cellulase enzymes, hemicellulases are classified on the basis of their mode of action: the endo-acting hemicellulases attack internal bonds within the polysaccharide chain; the exo-acting hemicellulases act progressively from either the reducing or non-reducing end of polysaccharide chains.
  • endo-acting hemicellulases include, but are not limited to, endoarabinanase, endoarabinogalactanase, endoglucanase, endomannanase, endoxylanase, and feraxan endoxylanase.
  • exo-acting hemicellulases include, but are not limited to, ⁇ -L-arabinosidase, ⁇ -L- arabinosidase, ⁇ -1 ,2-L-fucosidase, ⁇ -D-galactosidase, ⁇ -D-galactosidase, ⁇ -D- glucosidase, ⁇ -D-glucuronidase, ⁇ -D-mannosidase, ⁇ -D-xylosidase, exo- glucosidase, exo-cellobiohydrolase, exo-mannobiohydrolase, exo-mannanase, exo-xylanase, xylan ⁇ -glucuronidase, and conifehn ⁇ -glucosidase.
  • Ligninases are enzymes that are involved in the degradation of lignin. A variety of fungi and bacteria produce ligninases. Lignin-degrading enzymes include, but are not limited to, lignin peroxidases, manganese-dependent peroxidases, hybrid peroxidases (which exhibit combined properties of lignin peroxidases and manganese-dependent peroxidases), and laccases. Hydrogen peroxide, required as a co-substrate by the peroxidases, can be generated by glucose oxidase, aryl alcohol oxidase, and/or lignin peroxidase- activated glyoxal oxidase.
  • hydrolysis refers to the process of converting polysaccharides (e.g., cellulose) to fermentable sugars, e.g., through the hydrolysis of glycosidic bonds. This process can also be referred to as saccarification. Hydrolysis can be effected with enzymes or chemicals. Enzymes can be added to biomass directly (e.g., as a solid or liquid enzyme additive) or can be produced in situ by microbes (e.g., yeasts, fungi, bacteria, etc.). Hydrolysis products include, for example, fermentable sugars, such as glucose and other small (low molecular weight) oligosaccharides such as monosaccharides, disacchahdes, and thsacchahdes.
  • fermentable sugars such as glucose and other small (low molecular weight) oligosaccharides such as monosaccharides, disacchahdes, and thsacchahdes.
  • Hydrolysis products can also simply include lower molecular weight polysaccharides than those in the original cellulose or lignocellulose.
  • Suitable conditions for sacchahfication refer to various conditions including pH, temperature, biomass composition, and enzyme composition.
  • the term “filter paper unit” (or FPU) refers to the amount of enzyme required to liberate 2 mg of reducing sugar (e.g., glucose) from a 50 mg piece of Whatman No. 1 filter paper in 1 hour at 50 0 C at approximately pH 4.8.
  • “Fermentation” or “fermenting” can refer to the process of transforming an organic molecule into another molecule using a micro-organism.
  • “fermentation” can refer to transforming sugars or other molecules from biomass to produce alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid); ketones (e.g., acetone), amino acids (e.g., glutamic acid); gases (e.g., H 2 and CO 2 ), antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B12, beta-carotene); and/or hormones.
  • fermentation includes alcohol fermentation. Fermentation also includes anaerobic fermentations.
  • Fermenting can be accomplished by any organism suitable for use in a desired fermentation step, including, but not limited to, bacteria, fungi, archaea, and protists.
  • Suitable fermenting organisms include those that can convert mono-, di-, and thsacchahdes, especially glucose and maltose, or any other biomass-derived molecule, directly or indirectly to the desired fermentation product (e.g., ethanol, butanol, etc.).
  • Suitable fermenting organisms also include those which can convert non-sugar molecules to desired fermentation products.
  • the fermenting is effected by a fungal organism (e.g., yeast or filamentous fungi).
  • the yeast can include strains from a Pichia or Saccharomyces species.
  • the yeast can be Saccharomyces cerevisiae.
  • the fermenting is effected by bacteria.
  • the bacteria can be Clostridium acetobutylicum (e.g., when butanol is the desired fermentation product) or Corynebacterium glutamicum (e.g., when monosodium glutamate (MSG) is the desired fermentation product).
  • the micro-organism e.g. yeast or bacteria
  • the organism can be yeast or other organism having or modified to be active in the presence of high concentrations of alcohol.
  • fermentation and grammatical variations thereof refer to the conversion of a fermentable sugar to an alcohol (e.g., methanol, ethanol, propanol, butanol, etc.).
  • alcohol e.g., methanol, ethanol, propanol, butanol, etc.
  • the particular product of a given alcohol fermentation can be determined by the biocatalyst used in the fermentation and/or the substrate of fermentation (i.e., the type of fermentable sugar being converted).
  • fermenting can comprise contacting a mixture including biomass-derived sugars with an alcohol-producing biocatalyst, such as a yeast or another alcohol-producing microbe.
  • an alcohol-producing biocatalyst such as a yeast or another alcohol-producing microbe.
  • fermenting involves simultaneous saccharification (e.g., hydrolysis) and fermentation (SSF).
  • SSF fermentation
  • the amount of fermentation biocatalyst employed can be selected to effectively produce a desired amount of ethanol in a suitable time and/or upon the sugar content of a given fermentation mixture.
  • the use of alcohol-producing biocatalyst can increase the rate of saccharification by reducing the concentration of sugars, which can inhibit saccharification biocatalysts.
  • Suitable conditions for alcohol fermentation can refer to conditions that support the production of ethanol or another alcohol by a biocatalyst. Such conditions can include pH, nutrients, temperature, atmosphere, and other factors.
  • Dehydrating refers to removing the residual water left in ethanol following distillation.
  • the residual water is generally about 5% by volume.
  • Dehydration can be performed using molecular sieves.
  • the presently disclosed subject matter provides methods of hydrolyzing lignocellulosic materials using enzymes (i.e., lignocellulose-degrading enzymes) at high fiber consistency, as well as methods of preparing alcohols from lignocellulosic biomass that involve enzymatic hydrolysis performed at high fiber consistency.
  • the methods involve mixing lignocellulose- hydrolyzing enzymes with cellulose fibers at a low concentration, thickening the mixture to increase the fiber content, and then hydrolyzing the fibers for a period of time at the increased fiber consistency.
  • the amount of water in the hydrolysis mixture can be reduced, thereby increasing the subsequent concentration of fermentable sugars that are available for fermentation.
  • the size of the equipment needed during fermentation of the sugars and recovery of the alcohol produced during fermentation is reduced. Therefore, while the overall conversion of biomass to fermentable sugars using high consistency enzymatic hydrolysis can result in lower levels of enzymatic hydrolysis of the biomass, the use of high consistency enzymatic hydrolysis can cut down on capital costs for the overall biomass-to- alcohol process.
  • additional enzymes are added after the thickening step, after the actions of the first portion of enzymes have reduced cellulose fibers in size somewhat to decrease the viscosity of the hydrolysis mixture and make the homogeneous mixing of the additional enzymes easier.
  • the first portion of the enzymes includes cellulase, which can absorb well to the biomass and disrupt the crystalline structure of the cellulose in the fibers, exposing individual fibers for additional enzymatic action.
  • the presently disclosed subject matter further relates to methods of recycling lignocellulose-hydrolyzing enzymes. As enzyme costs, particularly for cellulase, can be quite high, re-cycling the enzymes can provide significant savings.
  • the presently disclosed subject matter provides a method of producing an alcohol from a lignocellulosic biomass, wherein the method can comprise: providing lignocellulosic biomass; contacting the lignocellulosic biomass with a first enzyme composition for a first period of time to provide a first hydrolysis mixture; thickening the first hydrolysis mixture to from a second hydrolysis mixture; hydrolyzing the second hydrolysis mixture for a second period of time to provide a fermentable sugar mixture; and fermenting the fermentable sugar mixture to provide an alcohol.
  • the lignocellulosic biomass (such as the as- harvested biomass) is pretreated to increase enzymatic digestability prior to enzymatic hydrolysis by lignocellulose-degrading enzymes and/or to make handling of the biomass easier.
  • Pretreatments can be mechanical, chemical, or biochemical processes or combinations thereof.
  • the pretreating can involve removing or altering lignin, removing hemicellulose, decrystallizing cellulose, removing acetyl groups from hemicellulose, reducing the degree of polymerization of cellulose, increasing the pore volume of lignocellulose biomass, increasing the surface area of lignocellulose, or any combination thereof.
  • the pretreatment can comprise one or more technique known in the art of biomass-to-alcohol conversion, including, but not limited to, autohydrolysis, steam explosion, grinding, chopping, ball milling, compression mulling, radiation, flow-through liquid hot water treatment, dilute acid treatment, concentrated acid treatment, peracetic acid treatment, supercritial carbon dioxide treatment, alkali treatment, organic solvent treatment, cellulose solvent treatment, and treatment with an aerobic fungi.
  • the alkali treatment can include sodium hydroxide treatment, lime treatment, wet oxidation, ammonia treatment, and oxidative alkali treatment.
  • the alkali treatment comprises green liquor (GL) treatment, as described in the co-pending PCT International Patent Application titled “Production of Ethanol from Lignocellulosic Biomass Using Green Liquor Pretreatment” (based on U.S. Provisional Patent Application Serial No. 61/1 16,934).
  • GL green liquor
  • Green liquor treatment can involve treatment of biomass with an alkaline composition comprising sodium sulfide and sodium carbonate at a temperature of between about 100 0 C to about 220 0 C (e.g., about 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, or 220°C) for between about 0.25 and about 4 hours (e.g., about 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, or 4.0 hours) in a carbon steel pressure vessel.
  • an alkaline composition comprising sodium sulfide and sodium carbonate at a temperature of between about 100 0 C to about 220 0 C (e.g., about 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, or 220°C) for between about 0.25 and about 4 hours (e.g., about 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5,
  • the charge of total titratable alkali provided by the green liquor can be between about 4% and about 25% or between about 12% and about 20% (e.g., 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%).
  • the sulfidity of the green liquor can be between about 5% and about 50% (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50%). In some cases the sulfidity is about 25%.
  • the pretreatment can comprise a washing step, to remove any solubilized lignin, unfermentable solubilized cellulose products or any chemicals used in a pretreatment step.
  • green liquor pretreated biomass can be further pretreated via oxygen delignification (e.g., treatment with oxygen gas in a pressurized vessel at a temperature of between about 60 0 C and about 150 0 C for between about 10 minutes to about 4 hours), refining to reduce the size of the solid materials and/or separate pulp fibers (e.g., with refining equipment, such as a disk refiner, a PFI mill, or any other refiner, such as those typically used to refine paper pulp in the paper industry) or a combination thereof.
  • oxygen delignification e.g., treatment with oxygen gas in a pressurized vessel at a temperature of between about 60 0 C and about 150 0 C for between about 10 minutes to about 4 hours
  • refining e.g., with refining equipment, such as a disk refiner, a P
  • the pretreatment (e.g., the green liquor pretreatment) further comprises the use of one or more additives to increase the yield of carbohydrate (e.g., cellulose and hemicellulose) during the pretreatment.
  • additives can include, but are not limited to, anthraquinone and sodium polysulfides.
  • biomass such as hardwood or softwood chips
  • diluted e.g., with water
  • 5-10% fiber consistency e.g., about 5, 6, 7, 8, 9 or 10% fiber consistency
  • the fiber concentration is diluted to about 5%.
  • a first enzyme composition comprising lignocellulose-hydrolyzing enzymes can be added and the mixture stirred for a period of time (e.g., between about 1 and 20 minutes). In some embodiments, the mixture can be stirred for about 5, 10, or 15 minutes.
  • the amount of enzyme added can vary depending upon the type of biomass and/or pretreatment used.
  • the first enzyme composition can comprise cellulase, either alone, or in combination with other lignocellulase- hydrolyzing enzymes (e.g, xylanase and ⁇ -glucosidase).
  • the cellulase can react quickly with the cellulose fibers, for example, to open physical pores for subsequent enzyme action.
  • the mixing and stirring can be done at any suitable temperature or pH to facilitate adsorption of the enzymes or enzymatic hydrolysis by the enzymes.
  • the temperature is between about 4°C and about 70 0 C.
  • the temperature is between about 4°C and about 50 0 C (e.g., about 4, 10, 15, 20, 25, 30, 35, 38, 40, 42, 45, or 50 0 C).
  • the temperature is about 38°C.
  • the temperature is about 50 0 C.
  • the pH can be optimized based on the type of enzymes being used.
  • the pH can be between about 4 and about 5 (e.g., about 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, and 5.0). In some embodiments, the pH is about 4.8.
  • the pH can be adjusted using pH-adjusting chemicals (e.g., acids, bases, buffers), so long as the pH-adjusting chemicals do not adversely affect the functioning of the enzymes.
  • Figure 3 shows the effect of temperature on cellulase adsorption to the biomass. While the effects of temperature are not large, in the embodiments implemented for Figure 3, the adsorption was observed to be best at about 38°C.
  • Figures 4 and 5 show the effect of cellulase dosage and lignin content on enzyme adsorption. While enzyme dosage has minimal effects on adsorption, higher lignin content can reduce enzyme adsorption.
  • Figure 6 shows the effect of two different green liquor pretreatments on enzyme adsorption.
  • GL-12 refers to green liquor pretreatment using an alkaline solution having a TTA of 12%.
  • GL-16 refers to green liquor pretreatment using an alkaline solution having a TTA of 16%. As seen in Figure 6, the effect of TTA on enzyme adsorption is minimal.
  • the mixture after allowing the mixing and adsorption of the enzymes from the first enzyme composition, can be thickened to between about 15% and about 30% K (e.g., about 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% K).
  • the mixture is thickened to about 20% K.
  • the thickening can be done by any suitable technique. In some embodiments, the thickening can be done by either gravity or vacuum filtration, for example, using a filter press.
  • the filtrate can comprise about 80% of the total volume from the first hydrolysis mixture. See Figure 1 A, which relates to an example where 4 liters (L) of an original 20 L 5% K hydrolysis mixture is left after filtration and carried on into further hydrolysis, while the filtrate comprises the remaining 16 L, some of which can optionally be feed back into the dilution/mix tank to dilute new incoming batches of biomass, which can comprise about 9 L of material straight from any pretreatment processing.
  • the filtrate can also comprise some cellulose fibers and non-adsorbed enzymes, which, if desired can be fed back into the system in the filtrate to dilute biomass, thereby reusing the unabsorbed enzyme.
  • cellulase adsorption can be reasonably high (e.g., between about 80 and 90%)
  • other enzymes such as xylanase
  • it can be cost effective to add cellulase prior to the thickening step, and to add other enzymes later, as part of a second enzyme composition, though this is not necessarily required.
  • Reference to particular volumes in Figure 1A is for purposes of illustration only, and not limitation.
  • the filtrate reuse described herein relates to methods wherein the main enzymatic hydrolysis of the fibers is performed at fiber consistencies above 10% K.
  • Reusing the filtrate can increase overall conversion of cellulose to sugar from 63-65% to up to 70% and above.
  • Enzymatic hydrolysis of the thickened mixture can proceed for any suitable time and temperature to provide sufficient fermentable sugars. In some embodiments, hydrolysis can proceed for between about 2 hours and about 3 days (e.g., about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, or 72 hours). In some embodiments, enzymatic hydrolysis in the thickened mixture is carried out at about 50 0 C.
  • a second enzyme composition (e.g., the "additional enzymes" in Figure 1A) is added at some time during the hydrolysis.
  • the addition of a second enzyme composition can be referred to as split enzyme dosing.
  • the second enzyme composition is added between about 0 hours (i.e., immediately after thickening) and about 24 hours following thickening (i.e., at 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 hours following thickening).
  • the second enzyme composition is added (e.g., in a "Mix" step as shown in Figure 1A) between about 2 hours and about 3 hours following thickening.
  • the second enzyme composition can comprise xylanase (i.e., x in Figure 1A), ⁇ -glucosidase (i.e., b in Figure 1A), combinations of x and b, and/or other lignocellulosic-hydrolyzing enzymes. All the enzyme or enzymes of the second enzyme composition can be added together, or portions of the second enzyme composition (e.g., an aliquot of the total second enzyme composition or each different type of enzyme) can be added sequentially over a period of time (e.g., a few minutes or hours apart).
  • xylanase i.e., x in Figure 1A
  • ⁇ -glucosidase i.e., b in Figure 1A
  • combinations of x and b lignocellulosic-hydrolyzing enzymes. All the enzyme or enzymes of the second enzyme composition can be added together, or portions of the second enzyme composition (e.g., an aliquot of the total second enzyme
  • the second enzyme composition comprises at least some additional cellulase (i.e., cellulase in addition to that remaining from the first enzyme composition).
  • the first enzyme composition can comprise between about 25% and about 50% of the total cellulase dose, while the second enzyme composition can comprise the remainder of the cellulase dose (e.g, about 50%, about 55%, about 60%, about 65%, about 70% or about 75%).
  • split enzyme dosing can refer to adding a portion of cellulase prior to thickening and a portion of cellulase after thickening.
  • the second enzyme composition can comprise some additional water or filtrate from the thickening step.
  • the diluent can include a sugar solution prepared from the enzymatic hydrolysis of another batch of biomass.
  • the second enzyme composition of additional enzymes has a volume of about 1 L.
  • Figure 7 shows the effect of total enzyme dosage and both single enzyme composition dosing and split enzyme dosing on sugar recovery efficiency in exemplary, non-limiting embodiments of the presently disclosed subject matter, also described in the Examples hereinbelow.
  • the period of time can range between about 2 hours and about 72 hours. In some embodiments, the period of time can range between about 24 and about 48 hours (e.g., about 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 hours).
  • Figure 8 shows the effects of adding a second enzyme composition comprising xylanase and ⁇ -glucosidase at between 2 and 8 hours following thickening and allowing the hydrolysis to continue for a further 24 or 48 hours in exemplary, non-limiting embodiments of the presently disclosed subject matter, also described in the Examples hereinbelow.
  • Figures 9A-9D and 10-15 provide additional data regarding enzymatic hydrolysis efficiency in mixtures thickened to 20% K, in mixtures thickened to 20%K as compared to mixtures hydrolyzed at 5% K, and of various split enzyme dosing schedules according to exemplary, non-limiting embodiments of the presently disclosed subject matter, also described in the Examples hereinbelow.
  • enzymatic efficiency at high consistency is lower than at the typical 5% K used; however, capital saving costs can outweigh this yield loss.
  • enzymatic efficiency at higher consistency can be about the same as that for 5% K hydrolysis reactions.
  • the hydrolysis efficiency of the method can be about 60%, 65%, 70%, 75%, 80% or higher. In some embodiments, the hydrolysis efficiency can be about 84%.
  • the fermentable sugar mixture resulting from hydrolysis can comprise about 6% or more fermentable sugar by volume, instead of the 3-4% usually found following hydrolysis of 5% K mixtures.
  • the fermentable sugar mixture can comprise between about 10% and about 15% fermentable sugar by volume (e.g., about 10%, 1 1 %, 12%, 13%, 14%, or 15%).
  • the fermentable sugar mixture comprises about 12% fermentable sugar by volume.
  • the mixture is filtered to remove lignin, which can be burned as a fuel, and a sugar solution, which can be fermented with a suitable microorganism (e.g., yeast or another alcohol-producing microbe) as described herein to provide an alcohol.
  • a suitable microorganism e.g., yeast or another alcohol-producing microbe
  • the presently disclosed subject matter can provide a method of producing sugar from a lignocellulosic biomass, wherein the method comprises: providing lignocellulosic biomass, contacting the lignocellulosic biomass with a first enzyme composition for a first period of time to provide a first hydrolysis mixture, thickening the first hydrolysis mixture to form a second hydrolysis mixture; and hydrolyzing the second hydrolysis mixture for a second period of time to provide a sugar mixture.
  • the alcohol provided by the fermenting can be ethanol.
  • the alcohol mixture formed during fermentation can include between about 5% and about 7.5% alcohol (e.g., about 5%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, or about 7.5% alcohol).
  • Distillation and additional dehydration of the alcohol mixture e.g., using molecular sieves or another suitable hygroscopic material
  • the ethanol can be denatured, if desired, through the addition of a suitable additive (e.g., methanol, isopropyl alcohol, acetone, methyl ethyl ketone, or methyl isobutyl ketone).
  • a suitable additive e.g., methanol, isopropyl alcohol, acetone, methyl ethyl ketone, or methyl isobutyl ketone.
  • the alcohol can be used directly as a fuel or for another purpose, or can be mixed with another component to provide a fuel mixture.
  • the ethanol produced by one of the presently disclosed methods can be mixed with gasoline to provide a gasohol.
  • the presently disclosed subject matter provides a method of producing a biofuel.
  • Figure 1 B shows a scheme for the production of ethanol from biomass according to an exemplary embodiment of the presently disclosed subject matter.
  • biomass e.g., wood chips
  • green liquor or another pretreatment
  • the pretreated biomass can undergo an optional mechanical refining step (e.g., using a disc refiner or other mechanical refiner known in the art of paper manufacturing).
  • the mechanical refining can reduce the size of the pretreated chips and/or the size of the wood fibers or fiber bundles. Refining can also separate fiber bundles.
  • the pretreated biomass can comprise single fibers or mainly single fibers.
  • the pretreated biomass can be washed (e.g., with water).
  • the washing can remove the "black liquor” resulting from the green liquor pretreatment.
  • the black liquor can comprise alkaline chemicals, solubilized lignin, and solubilized (but unfermentable) cellulose-derived molecules.
  • the washed biomass can then be fed into a first enzyme reactor (i.e., enzyme reactor #1 , which can correspond to the dilution/mix tank of Figure 1A).
  • lignocellulase-hydrolyzing enzyme e.g., fresh cellulase
  • pulp coming from the pulp washing step can have a fiber consistency of about 14%.
  • liquid to dilute the biomass in the first enzyme reactor can include filtrate from the wash press (i.e., filtrate resulting from thickening the biomass mixture from the first enzyme reactor following adsorption of the enzymes).
  • Dilution liquid can also include other liquid, such as fresh water (i.e., water newly introduced into the biomass-to-ethanol process).
  • Biomass can reside in the first enzyme reactor from between about 1 and about 20 minutes, for example, prior to thickening in the wash press.
  • the thickened biomass mixture can be introduced to a second enzyme reactor (i.e., enzyme reactor #2 of Figure 1 B) and allowed to hydrolyze for a period of time (typically about 1 to 3 days).
  • the second enzyme reactor can be the same physical vessel as the first enzyme reactor, or can be a different vessel.
  • additional enzymes can be added as described hereinabove. Mixing of additional enzymes can be assisted as necessary by diluting the thickened mixture with, for example, reserved filtrate from the wash press, portions of filtered sugar solutions from prior hydrolysis mixtures, or fresh water.
  • any diluent used at this step will be no more than about 10% or 5% or less of the volume of the thickened mixture coming from the wash press.
  • the contents of the second enzyme reactor can be allowed to hydrolyze without further mixing (e.g, stirring or other agitation), if desired.
  • the hydrolyzed mixture can be filtered.
  • the hydrolyzed mixture can be introduced into a lignin filter to remove remaining solids, which can contain lignin.
  • the lignin filtercake can be added into a mix tank with the black liquor from the pulp washing step and burned to provide energy.
  • the energy can be used, for example, to heat an enzyme reactor, during the distillation process, and/or during another step of the biomass-to-ethanol process.
  • the energy from the lignin burning can also be used to fuel any external (i.e., non-biomass-to-ethanol) process.
  • the sugar-containing filtrate from the lignin filter can optionally be filtered through a fiber precoat filter, as shown in Figure 1 B.
  • the fiber precoat filter can be coated with some of the biomass (e.g., up to about 20%) from the pulp washing step.
  • the pulp coated in the filter can adsorb remaining lignocellulase-hydrolyzing enzymes present in the sugar- containing filtrate.
  • the pulp coating the fiber precoat filter can then be reintroduced into the process by being added as part of the pulp fed into the first enzyme reactor during a subsequent biomass conversion to reuse the readsorbed enzymes.
  • the sugar-containing filtrate can be fermented, for example in a conventional ethanol plant, to provide ethanol.
  • Enzymatic hydrolysis sugar yields and wood and/or pulp polysaccharide and lignin contents can be measured by any suitable method, as would be readily understood by one of ordinary skill in the art upon review of the instant disclosure. Such measurements can be performed, for instance, according to analytical procedures available from the National Renewable Energy Laboratory (NREL, Golden, Colorado, United States of America) and/or the Technical Association of the Pulp and Paper Industry (TAPPI, Norcross, Georgia, United States of America), among others.
  • polysaccharide content in a wood or pulp sample can be measured by sulfuric acid hydrolysis of given amount of a pulp or wood sample, followed by analysis of the resulting sugars, to calculate the amount of corresponding polysaccharide originally present in the wood or pulp.
  • enzymatic hydrolysis efficiencies can be calculated based on solids weight loss.
  • enzymatic hydrolysis efficiencies can be calculated by comparing sugar yield to pulp or wood polysaccharide content.
  • Hardwood chips were pretreated with green liquor (12% or 16% TTA) at 160 0 C as briefly described hereinabove, refined in a disc refiner and washed. The pretreated chips were then diluted with water to a fiber consistency of 5%, 7.5% or 10%. 20 FPUs/gram wood was added and the mixtures allowed to hydrolyze for up to 48 hours. At 6, 12, 24, or 48 hours, the mixtures were analyzed for monomeric sugar content (glucose, xylose, and mannose). The amount of sugars produced was compared to the amount of sugar theoretically present based on analysis of the original wood polysaccharide content to determine the percentage of total sugar yield.
  • Results are shown in Figure 2A for the chips pretreated with green liquor at 12% TTA (GL-12) and in Figure 2B for the chips pretreated with green liquor at 16% TTA (GL-16).
  • increasing the fiber consistency can reduce the amount of sugar produced during enzymatic hydrolysis. Without being bound to any one theory, it is believed that this decrease can be due, at least in part, to inefficient mixing of the pulp and enzyme at the higher consistencies.
  • enzymes can be premixed with biomass at a lower consistency and the premixed material can be filtered to produce a thickened biomass mixture.
  • the premixed material can be filtered to produce a thickened biomass mixture.
  • the adsorption characteristics of enzymes to biomass fibers can help to determine which enzymes to use during a premix step.
  • a variety of cellulase/biomass mixtures were prepared to study the affects of temperature, enzyme dosage, and lignin content on cellulase adsorption.
  • Bleached softwood or hardwood pulp was mixed with water and cellulase to provide mixtures having a fiber consistency of 5%.
  • the mixtures were incubated for 10 minutes and vacuum filtered (using a filter press) to increase the fiber consistency to 20%.
  • the filtrate was then analyzed for free (i.e., non-adsorped) cellulase.
  • Cellulase adsorption was calculated as 100% - (amount of enzyme in the filtrate/amount of enzyme added to the pulp).
  • Mixtures were also prepared using hardwood pulps with 2%, 10%, or 28% lignin and using green liquor pretreated pulps. Enzyme dosages used included 5, 10, 20, and 40 FPU. Incubation temperatures included 4, 23, 28, and 50 0 C.
  • TTA did not appear to greatly affect enzyme adsorption. See Figure 6.
  • Xylanase adsorption to bleached hardwood pulp (0% lignin) was studied in a similar manner, using xylanase dosages of between 1 and 10 FPU. In contrast to cellulase, xylanase does not appear to become adsorbed to the pulp. The amount of xylanase that was found in the filtrate was proportional to the amount of hydrolysis mixture liquid that was in the filtrate.
  • Pulp mixtures that had been incubated for 10 minutes at 5% K with cellulase alone were thickened to 20% K using vacuum filtration.
  • Xylanase and ⁇ -glucosidase was added to the thickened mixture immediately and the mixture allowed to hydrolyze for 48 hours prior to sugar analysis.
  • the thickened mixture was hydrolyzed for 24 hours and then the additional enzymes were added and then the mixture was hydrolyzed for another 24 hours.
  • FIG. 7 shows the sugar recovery efficiency results from the various samples. Enzymatic hydrolysis at 20% K was somewhat less efficient than at
  • Example 3 bleached hardwood pulp was placed in a dilution/mixing tank and diluted to 5% K with water. Cellulase (20 FPU/gram pulp) was added and the resulting mixture incubated for 10 minutes. The mixture was thickened using vacuum filtration to increase the fiber consistency to 20% K. Hydrolysis was then allowed to proceed for 15 minutes to 10 hours prior to addition of xylanase and ⁇ -glucosidase. Following the adding of the hemicellulose- degrading enzymes, hydrolysis was continued for a further 24 or 48 hours.
  • Figure 8 shows the effect of varying the addition time of the hemicellulose-degrading enzymes. Waiting to add the additional enzymes for 2-3 hours appeared to provide the highest enzymatic efficiency, particularly over shorter total hydrolysis times.
  • FIG. 13 shows how splitting the cellulase from a 10 FPU/gm pulp dose into two equal parts affected enzymatic hydrolysis. In addition to showing the results from a control sample where all of the cellulase was added prior to thickening, results from a sample hydrolyzed for 48 hours at 5% K are shown.
  • Figure 14 shows how splitting the cellulase charge so that only one fourth or one half of the total cellulase from a 20 FPU/gram pulp dose was added prior to thickening affected hydrolysis. Results are also provided from a sample where all the cellulase was added prior to thickening and from a sample where the mixture was not thickened.
  • Figure 15 shows how splitting the cellulase charge so that only one eighth, one fourth, or one half of the cellulase from a 40 FPU/gram pulp dose was added prior to thickening affected hydrolysis. Results are also provided for a sample where all of the cellulase was added prior to thickening to 20% K and a sample where hydrolysis was performed at 5% K.
  • the highest hydrolysis was observed when 50% of the cellulase charge was added to the pulp at low consistency and the remaining cellulase and other enzymes were added after thickening. Without being bound to any one theory, it appears that the initial cellulase charge can be well mixed with the pulp at low consistency and then serves to reduce viscosity in the thickened mixture so that the remaining enzymes can be easily mixed to contact and hydrolyze the remaining poly- and oligosaccharides.
  • Hardwood pulp was hydrolyzed at 5% K (20 L total volume) using 20 FPU/gm pulp enzymes (cellulase, xylanase and ⁇ -glucosidase) for 48 hours. Baseline sugar conversion was determined to be about 63-65%. The pulp was then thickened to a consistency of 20% K via vacuum filtration. The filtrate (15 L) was used as a part of the diluent for a new batch of pulp, which was diluted to 5% K overall and to which was also added a new 20 FPU /gram dose of enzymes. The new batch was allowed to hydrolyze for 48 hours. Sugar conversion of the new batch was determined to be about 70%. The new batch was then thickened to a consistency of 20 % K via vacuum filtration.
  • 20 FPU/gm pulp enzymes cellulase, xylanase and ⁇ -glucosidase
  • the filtrate (15 L) was used as part of the diluent for a second new batch of pulp.
  • the second new batch of pulp was diluted to 5% K, mixed with a new 20 FPU/gram dose of enyzmes, and hydrolyzed for 48 hours.
  • Sugar conversion of the second new batch of pulp was analyzed and determined to be about 72%. Accordingly, it appears that recycling of the filtrate can be employed for increasing sugar concentration, leading to slightly higher overall pulp-to-sugar conversion.

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Abstract

La présente invention concerne des procédés permettant de transformer des matières lignocellulosiques en alcool qui reposent sur l’augmentation de la consistance fibreuse de mélanges d'hydrolyse enzymatique. Plus particulièrement, les procédés consistent à mettre une biomasse lignocellulosique en contact avec une composition enzymatique sur une certaine durée, puis épaissir le mélange et hydrolyser le mélange épaissi. L'épaississement peut être obtenu par filtration, en réutilisant éventuellement le filtrat et/ou toutes les enzymes qui y sont contenues dans le procédé de transformation de la lignocellulose pour augmenter son efficacité. L'hydrolyse du mélange épaissi permet d'obtenir un mélange de sucres fermentables avec une concentration en sucres supérieure à celle des mêmes mélanges obtenus à partir de mélanges biomasse/enzymes moins concentrés. L’invention concerne également des compositions contenant de l’alcool préparées par les procédés ci-décrits.
PCT/US2009/065562 2008-11-21 2009-11-23 Hydrolyse enzymatique à haute consistance pour la production d’éthanol Ceased WO2010060050A2 (fr)

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WO2014028368A1 (fr) * 2012-08-14 2014-02-20 Arisdyne Systems, Inc. Procédé pour augmenter le rendement en alcool à partir de grains
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CN108315058A (zh) * 2018-03-08 2018-07-24 利胜强 新型清洁乙醇汽油及其制备方法
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