WO2015009986A2 - Prétraitement par écoulement continu hydrothermique de biomasse lignocellulosique pour maximiser les rendements de sucre fermentescible et de lignine - Google Patents

Prétraitement par écoulement continu hydrothermique de biomasse lignocellulosique pour maximiser les rendements de sucre fermentescible et de lignine Download PDF

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WO2015009986A2
WO2015009986A2 PCT/US2014/047127 US2014047127W WO2015009986A2 WO 2015009986 A2 WO2015009986 A2 WO 2015009986A2 US 2014047127 W US2014047127 W US 2014047127W WO 2015009986 A2 WO2015009986 A2 WO 2015009986A2
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liquid
water
lignin
pretreatment
contacting
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WO2015009986A3 (fr
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Bin Yang
Lishi YAN
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Washington State University
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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
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0057—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Xylans, i.e. xylosaccharide, e.g. arabinoxylan, arabinofuronan, pentosans; (beta-1,3)(beta-1,4)-D-Xylans, e.g. rhodymenans; Hemicellulose; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
    • C08H6/00—Macromolecular compounds derived from lignin, e.g. tannins, humic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
    • C08H8/00—Macromolecular compounds derived from lignocellulosic materials
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L97/00—Compositions of lignin-containing materials
    • C08L97/005—Lignin
    • C—CHEMISTRY; METALLURGY
    • C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
    • C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G, C10K; LIQUEFIED PETROLEUM GAS; ADDING MATERIALS TO FUELS OR FIRES TO REDUCE SMOKE OR UNDESIRABLE DEPOSITS OR TO FACILITATE SOOT REMOVAL; FIRELIGHTERS
    • C10L1/00—Liquid carbonaceous fuels
    • C10L1/02—Liquid carbonaceous fuels essentially based on components consisting of carbon, hydrogen, and oxygen only
    • 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
    • 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
    • C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C1/00—Pretreatment of the finely-divided materials before digesting
    • D21C1/02—Pretreatment of the finely-divided materials before digesting with water or steam
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C1/00—Pretreatment of the finely-divided materials before digesting
    • D21C1/04—Pretreatment of the finely-divided materials before digesting with acid reacting compounds
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
    • D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
    • D21C5/02—Working-up waste paper
    • 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
    • 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
    • Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel
    • 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
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00—Technologies for solid waste management
    • Y02W30/50—Reuse, recycling or recovery technologies
    • Y02W30/64—Paper recycling

Definitions

  • the invention generally relates to improved methods of pretreating lignocellulosic biomass to maximize yields of fermentable sugars and lignin.
  • the invention provides improved water-only and dilute acid flowthrough pretreatment methods which are carried out at high temperature and which provide high yields of desired products, e.g.
  • Pretreatment is essential for achieving high yields of desirable products through overcoming the recalcitrance of lignocellulosic feedstocks, including: (1 ) hemicellulose, lignin and other compounds coating the surface of the cellulose microfibrils, and (2) the crystalline nature of the cellulose structure.
  • lignocellulosic feedstocks including: (1 ) hemicellulose, lignin and other compounds coating the surface of the cellulose microfibrils, and (2) the crystalline nature of the cellulose structure.
  • United States patent 8,765,428 teaches a flow-through reactor for biological conversion of lignocellulosic biomass.
  • the biological conversion e.g. with microbes, enzymes, etc.
  • the biological conversion follows any initial pretreatment with e.g. heat or acid, and is done at temperatures of e.g. 20 to 60°C and pH values of 4 to 8, in order to allow the
  • microorganisms and/or enzymes to function are microorganisms and/or enzymes to function.
  • What is needed is improved methods of treating lignocellulosic biomass that exhibit high levels of cellulose digestion, low chemical and water usage, and fewer safety and environment concerns, while providing high yields of desired products such as fermentable sugars and lignin.
  • novel methods are carried out in a flowthrough-style reactor at high temperatures with either water-only or dilute acid, and may be coupled with further processing of the resulting slurries using cellulases, either with or without the addition of bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the method comprises a step of contacting the lignocellulosic biomass with a stream of liquid, wherein the liquid is water or dilute acid; and wherein said step of contacting is carried out at a temperature in the range of from about 120 °C to about 290 °C; and wherein the liquid is continuously removed from the reaction vessel after contacting the biomass.
  • a flow rate of the liquid is in a range of from about 0.5 to about lOOml/min.
  • the liquid is water
  • the temperature range is from about 140 °C to about 290 °C
  • a pH ranges from about 4 to about 9.
  • the liquid is water, the temperature range is from about 220 °C to about 260 °C and the step of contacting is carried out for at least 2 minutes. In further aspects, the temperature of the reaction is about 240 °C. In another aspect, the liquid is water and the step of contacting is carried out with a severity of from about 5.5 to about 6.5. In other aspects, the liquid is dilute acid, the temperature range is about 140 °C to 290 °C and a pH ranges from about 2 to about 3. In another aspect, the liquid is dilute acid, the temperature range is from about 220 °C to about 260 °C and the step of contacting is carried out for at least 1 minute.
  • the method is carried out at a temperature of about 240 °C.
  • the liquid is dilute acid and the step of contacting is carried out with a severity of from about 4.0 to about 6.0.
  • the method may be carried out in a flowthrough reactor.
  • the method may further comprise a step of enzymatically hydrolyzing slurries produced in the contacting step by mixing the slurries with at least one hydrolytic enzyme, for example, at least one cellulase and/or at least one xylanase. Enzymatic hydrolysis is carried out with or without the addition of bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the invention also provides methods of making ethanol, the methods comprising the steps of i) pretreating lignocellulosic biomass by contacting the lignocellulosic biomass with a stream of liquid, wherein the liquid is water or dilute acid, the step of contacting is carried out at a temperature in the range of from 120 °C to 290 °C, and the liquid is continually removed from the reaction vessel; ii) enzymatically hydrolyzing slurries produced in the contacting step by mixing the slurries with at least one hydrolytic enzyme; and iii) microbial fermentation of glucose produced in the steps of pretreating and enzymatically hydrolyzing, wherein the step of fermenting produces ethanol.
  • a flow rate of the liquid is, for example, in the range of from about 0.5 to about lOOml/min.
  • the liquid is water
  • the temperature range is from about 140 °C to about 290 °C and the pH ranges from about 4 to about 9.
  • the liquid is water
  • the temperature range is from about 220 °C to about 260 °C and the step of contacting is carried out for at least 2 minutes.
  • the temperature is about 240 °C.
  • the liquid is water and the step of contacting is carried out with a severity of from about 5.5 to about 6.5.
  • the liquid is dilute acid
  • the temperature range is from about 140 °C to 290 °C and the pH range is from about 2 to about 3.
  • the liquid is dilute acid
  • the temperature range is from about 220 °C to about 260 °C and the step of contacting is carried out for at least 1 minute.
  • the temperature is about 240 °C.
  • the liquid is dilute acid and the step of contacting is carried out with a severity of from about 4.0 to about 6.0. The method may be carried out in a fiowthrough reactor.
  • the method may further comprise a step of enzymatically hydrolyzing a slurry produced in the contacting step by mixing the slurry with at least one hydrolytic enzyme, e.g. at least one cellulase and at least one xylanase. Enzymatic hydrolysis may be carried out with or without the addition ob bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the method may further comprise a step of separating the ethanol produced in the step of microbial fermentation.
  • the invention also provides methods of producing one or more C7 to CI 8 carbon biofuels.
  • the methods comprise the steps of: i) pretreating lignocellulosic biomass by contacting the lignocellulosic biomass with a mobile stream of liquid, wherein the liquid is water or dilute acid; the step of contacting is carried out at a temperature in the range of from 120 °C to 290 °C; and a flow rate of said liquid is in the range of from 0.5 to l OOml/min; ii) recovering lignin formed in the step of pretreating; and iii) reacting the lignin by catalytic chemical treatment in the presence of a 5% Ru/C and AI2O3 catalyst matrix, thereby producing the C7 to CI 8 carbon biofuel.
  • the biofuel may be jet fuel.
  • Figure 2A-C Effect of severity parameter (logR 0 ) on the removal of xylan, lignin and cellulose.
  • A Log Ro vs. pH: A water-only pretreatment; ⁇ 0.05% (w/w) H2SO4 pretreatment.
  • B Log R 0 vs. removal of xylan, lignin and cellulose with water-only pretreatment.
  • C Log R 0 vs. removal of xylan, lignin and cellulose with 0.05%o (w/w) H2SO4.
  • Figure 3A and B Effect of logRo on xylan recovery with A, water-only and B, 0.05% (w/w) H2SO4.
  • Figure 4A and B Effect of logRo on cellulose recovery by A, water-only and B, 0.05% (w/w) H 2 S0 4 pretreatment.
  • Figure 5A and B Effect of logR 0 on lignin recovery under A, water-only and B, 0.05% (w/w) H2SO4 conditions.
  • Figure 6A-F Soluble lignin-derived aromatic structures under both water-only and acid conditions.
  • A water only, 220 °C; B, water only, 240 °C; C, water only, 260 °C; D, water only, 280 °C; E, dilute acid, 200 °C; F, dilute acid, 240 °C. Vanillin and syringaldehyde were found as the predominant products.
  • FIG. 7A-D Comparison of results of water-only and dilute acid pretreatments at the indicated enzyme loadings.
  • a and C water-only; B and D, dilute acid.
  • Low enzyme loading 3 mg protein Ctec2 (2.8 FPU) with 0.6 mg protein Htec2/g glucan+xylan;
  • Medium enzyme loading 10 mg protein Ctec 2 (9.3 FPU) with 2mg Htec2/g glucan+xylan;
  • High enzyme loading 100 mg protein Ctec 2 (93 FPU) with 20mg Htec2/g glucan+xylan.
  • FIG. 8A and B Schematic representation of material balance of flowthrough pretreatment (Stage 1) and enzymatic hydrolysis (Stage 2).
  • Figure 9 Schematic representation of mass balance at the indicated stages of pretreatment.
  • a and B amounts of xylan, lignin and cellulose removed and product yields under the conditions indicated in C.
  • This disclosure describes methods for pretreating lignocellulosic biomass in order to convert or breakdown the biomass to products of interest.
  • the products of interest are typically "building blocks" or subunits of lignocellulosic such as sugars, lignin, etc.
  • the methods described herein overcome the chemical forces which hold together the small molecules which make up lignocellulose, releasing them so that they can be used for other purposes, e.g. sugars may be fermented by microorganisms to produce biofuels such as ethanol.
  • the initial step of the pretreatment method involves processing of the biomass at high temperature in a flowthrough reactor using either a water-only or a dilute acid liquid phase.
  • Pretreatment refers to treatment of a lignocellulosic material which results in production of at least one product of interest for further use.
  • the products of interest that are obtained by pretreatment are generally breakdown products (building blocks) of the lignocellulosic material.
  • Initial pretreatment or “Stage 1", as used herein, refers to high temperature treatment of lignocellulosic material in a flowthrough reactor using either water-only or dilute acid, to form a slurry.
  • Slurry refers to the mixture of products after initial pretreatment, which generally comprises some products of interest and some biomass that is still intact, or that is only partially broken down, e.g. hemicellulose, cellobiose, etc. Slurry is the combined mixture of hydrolysate (liquid) and pretreated solid residues (both intact and reacted).
  • Stage 2 pretreatment refers to a period of enzymatic hydrolysis of the slurry produced in Stage 1.
  • Cellulose is an organic compound with the formula (CgHioC ⁇ n, a polysaccharide having a linear chain of several hundred to over ten thousand ⁇ (1 ⁇ 4) linked D-glucose units.
  • Xylan refers to a group of hemicelluloses that are found in plant cell walls and some algae. Xylans are polysaccharides made from units of the pentose sugar xylose. Xylans are almost as ubiquitous as cellulose in plant cell walls and contain predominantly ⁇ -D-xylose units linked as in cellulose.
  • Plant "lignins” are polyphenolic substances derived from phenylalanine via dimerization of substituted cinnamic alcohols to a dibenzylbutane skeleton.
  • Severity Log Ro is defined as
  • a "flowthrough" reaction system includes a reaction vessel that is suitable for contacting lignocellulosic feedstock with a liquid by flowing a stream of liquid over, through, within, around, etc. particles of the feedstock (a solid phase).
  • the liquid phase flows in a substantially or net unidirectional manner and is continuously removed from the reactor after contacting the biomass feedstock.
  • the feedstock is generally particulate in nature, though this need not always be the case ("slivers", strips, sheets, etc of lignocellulosic material may also be used).
  • the effluent e.g.
  • liquid which has contacted the feedstock which contains one or more products of interest leached or dissolved from the feedstock during the flow, is generally collected continuously during the process, and optionally, may be recycled back through the feedstock one or more times.
  • This is in contrast to "batch" systems in which the liquid medium or hydrolysate is removed at the end of the reaction (when the reaction is complete or has proceeded to a desired point).
  • a key difference between flowthrough and batch reactors is that, in a batch reactor, the liquid is not removed until the end of pretreatment (e.g. by filtration, draining, pressing, centrifugation, etc.), while in a flowthrough system it is continuously removed from the reaction vessel, usually by an applied force, e.g.
  • the feedstock remains relatively (substantially) immobile (stationary, in a fixed position, etc.) while the mobile liquid phase flows (passes over and around, washes around, etc.) the particles of feedstock, flowing largely in a net unidirectional manner.
  • the individual particles may shift somewhat during processing, but they remain relatively immobile, compared to the liquid phase.
  • mixing may occur, e.g. with a static mixer that mixes the feedstock and liquid as the liquid passes through the vessel in an overall single direction from liquid inlet to liquid outlet.
  • the flow rate is an important parameter in flowthrough systems. Increasing the flow rate can increase the mass transfer of products into the bulk solution.
  • the solid biomass When lignocellulose undergoes a hydrolysis reaction, the solid biomass is surrounded by a diffusive boundary layer.
  • mass transfer of oligomers into the bulk may be reduced compared with systems having low solid to liquid ratios.
  • the boundary layer thickness is reduced when the flow is increased, accelerating movement of the oligomers from the pores in the biomass into the bulk liquid.
  • Different flow rates result in different mass transfer coefficients.
  • increases in flow rate generally enhance the solubility of oligomers since they are more continuously exposed to a stream of liquid.
  • the rate of dissolution of oligomers is proportional to the concentration gradient between oligomers at the surface of the solid and free water in the liquid.
  • the oligomer concentration of the liquid around the biomass is always close to zero due the short residence time. The gradient is thus enhanced compared to batch systems, leading to increased rates of dissolution.
  • a flowthrough reactor can provide more precise control of short residence times (e.g. the residence or removal time of the liquid) during the reaction process than can a batch reactor.
  • relatively precise residence times of less than about 10 minutes, e.g. about 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute or less
  • sugar decomposition byproducts such as furfural, 5-HMF and levulinic acid.
  • a residence time of less than 1 min e.g. 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10 or even 5 seconds
  • a residence time of less than 1 min cannot be obtained in batch reactors but can be achieved by increasing the flow rate of a flowthrough-type reactor.
  • any suitable reactor with a "flowthrough” capability may be used in the practice of the present invention, including those described in US 8,765,428, the complete contents of which is hereby incorporated by reference.
  • such reactors have at least one inlet through which biomass feedstock is introduced and at least one inlet through which liquid enters the reactor, which may be the same or different from the inlet for the feedstock; a vessel or containment area to contain the biomass while the liquid is in contact with the biomass, and at least one outlet via which the liquid continuously exits the reactor.
  • Spent (reacted) biomass may be removed, e.g. via an inlet or via the liquid outlet, or by another outlet.
  • the central container in which the biomass is placed for hydrolysis may be of any suitable shape, e.g. cylindrical, rectangular, etc.
  • a system comprising a flowthrough reactor may also comprise various monitoring devices to measure, e.g. temperature, pH, etc., as well as heaters, pumps, storage tanks, pipes and valves for transporting biomass and/or liquid before, during or after processing, etc.
  • the biomass to liquid ratio should be reduced or minimized as much as possible, and the total solids in the reactor should be increased or maximized as much as possible.
  • the reactor may be configured so that the biomass flows counter-current to the flow of the hot water such that the biomass to liquid ratio is reduced and the % total solids (TS%) in the reactor is greater than, for example, about 20-30% (e.g. about 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30%).
  • TS% total solids (TS%) in the reactor is greater than, for example, about 20-30% (e.g. about 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30%).
  • a single stage counter-current reactor is desirable because two-stage counter-current reactors suffer from problems associated with the loss of biomass ultrastructure in the first stage of pretreatment. Performing counter current pretreatments and extractions on "mud- like" biomass particles, such as those of the present disclosure, is difficult.
  • the reactors may thus employ counter-current or co-current continuous extraction equipment, such as a single or double screw conveyors or extrudors, vertical plate extractors, rotary extractors, centrifugal extractors, etc.
  • pressurized inclined screw extractors are modified to serve this purpose at the elevated temperatures and pressures needed for hot water pretreatments.
  • TS % in the hot water pretreatments is typical for flow- through systems (typically at 10-15%), but pressurized screw extruder-type of designs can be used for advantageously high TS% (up to 30%).
  • Low TS% in pretreatment not only adds extra amounts of water, thus increasing the size requirements of pretreatment and downstream equipment, but also increases the amount of energy required to remove the added water from a desired product. For example, the cost increases by 10% when the TS% is reduced from 30% to 15% (based on information from the National Renewable Energy Laboratory, NREL).
  • NREL National Renewable Energy Laboratory
  • hot water pretreatment offers at least three advantages: 1. minimal feedstock size reduction is required; 2. minimal hydrolyzate detoxification is required; and 3. there is no need for expensive, non-corroding alloys in the bioreactor (e.g. see Eggeman, T., Elander, R.T. 2005. Process and economic analysis of pretreatment technologies. Bioresource Technology, 96(18), 2019-2025).
  • Water-only pretreatment without any catalyst is an attractive way to treat biomass, since it does not require the use of toxic chemicals.
  • hydronium ions from the autoionization of water can catalyze the hydrolysis of ⁇ (1 -4) ether bonds between hemicellulose monomer units. Acidic side chains in hemicellulose are then liberated to further catalyze hydrolysis of hemicellulose.
  • water-only pretreatment is extremely effective under some conditions.
  • the conditions for water-only pretreatment are as follows: pH of about 5-9 (e.g. about 4..0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 or 9.0 or higher); temperature of about 240-290°C (e.g. about 200, 210, 220, 230, 240, 250, 260, 270, 280, or 290 °C or higher); a severity of about 4-7 (e.g.
  • reaction/processing time of about 2-120min (e.g. about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10, 1 15, or 120 minutes, or more).
  • desirable results are: 90%, e.g. about 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89 or 90% or more of the total mass is removed; at least about 98% e.g. about 90, 91 , 92, 93, 94, 95, 96, 97 or 98% or more of the total xylan is removed; at least about 95% e.g. about 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, or 95% of the total lignin is removed; and at least about 98% e.g.
  • the yield of glucose is generally at least about 50%, e.g. 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100%
  • the yield of xylose is generally at least about 85%, e.g. 86, 87, 88, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100%.
  • Optimal results and yields are generally obtained when the reactions are carried out at a temperature of about 220 to about 260 °C, e.g.
  • dilute acid we mean that the Stage 1 pretreatment step is carried out using an acidic solution.
  • dilute acid pretreatment is extremely effective under some conditions.
  • extremely low acid e.g. sulfuric acid
  • levels of 0.05— 0.1%) w/w
  • the decomposition of glucose at such a pH reached a minimum value.
  • the rate of glucose degradation can increase, and at higher pH, the decomposition increases due to other factors such as OH " induced reactions. This can be accomplished, for example, using 0.05% H 2 SO 4 (w/w).
  • other acids may also be used for this purpose, so long as the desired pH and other conditions are maintained.
  • Parameters for a dilute acid reaction include: the temperature of the reaction is at least about 120°C and is in the range of from about 120 to about 250°C (e.g. about 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 °C or higher); the severity logRo of the reaction is generally at least about 2 and is typically in the range of from about 2 to about 6 (e.g.
  • the flow rate is generally at least about 0.5ml/min and may be in the range of from about 0.5ml/min to about l OOml/min (e.g. about 0.1 , 0.5, 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 ml/min, or more); and the reaction time is at least about 2min and is in the range of from about 2min to about 120min (e.g. about 1 , 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10, 1 15, or 120 minutes, or more).
  • desirable results are: at least about 90%, e.g. about 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89 or 90% or more of the total mass is removed; at least about 98% e.g. about 90, 91 , 92, 93, 94, 95, 96, 97 or 98% or more of the total xylan is removed; at least about 95% e.g. about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% of the total lignin is removed; and at least about 98% e.g.
  • the yield of glucose is generally at least about 50%, e.g. 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100%
  • the yield of xylose is generally at least about 85%, e.g. 86, 87, 88, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100%.
  • Optimal results and yields are generally obtained when the reactions are carried out at a temperature of about 220 to about 260 °C, e.g.
  • the whole slurry from the reactor may be further treated to increase yields of desired products of interest, usually by
  • Some desired endproducts may be removed from the slurry prior to enzymatic hydrolysis (e.g. by centrifugation, filtering, etc.), or they may be left in the slurry mixture during enzymatic hydrolysis.
  • the enzymes include cellulase enzymes that are capable of hydrolyzing cellulose.
  • the enzyme mixture may include one or more enzymes including but not limited to: cellobiohydrolases (CBH), glucobiohydrolases (GBH), endoglucanases (EG), and ⁇ -glucosidases.
  • CBH cellobiohydrolases
  • GSH glucobiohydrolases
  • EG endoglucanases
  • ⁇ -glucosidases a cellulase mixture may include EG, CBH, and ⁇ -glucosidase enzymes.
  • the EG enzymes primarily hydrolyze cellulose polymers in the middle of the chain to expose individual cellulose chains.
  • CBH enzymes There are two types of CBH enzymes, CBHI and CBHII, which cleave the reducing and non-reducing ends of the cellulose chain, respectively, to produce cellobiose.
  • the conversion of cellobiose to glucose is typically carried out by a ⁇ -glucosidase, i.e. an enzyme that hydrolyzes the glucose dimer, cellobiose, to glucose, as defined by the Enzyme Commission (EC 3.2.1.21).
  • the ⁇ -glucosidase may come from any of various sources, and may be a Family 1 or Family 3 glycoside hydrolase, although other family members may also be used in the practice of this invention. It is also contemplated that the ⁇ -glucosidase enzyme may be modified to include a cellulose binding domain, thereby allowing this enzyme to bind to cellulose.
  • the enzymatic hydrolysis may also be carried out in the presence of one or more xylanase enzymes which degrade the linear polysaccharide P-l ,4-xylan into xylose, thus breaking down hemicellulose.
  • xylanase enzymes that may also be used for this purpose and include, for examples, xylanase 1 , 2 (Xynl and Xyn2) and ⁇ -xylosidase, which are typically present in cellulase mixtures.
  • cellulases which may be used in the practice of the invention include those obtained from filamentous fungi of the genera Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Theimoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma, and/or from bacteria of the genera Bacillus and Thermobifida
  • Suitable enzymes include those described in US patents 8,753,860; 8,759,040; 8,759,041 ; 8,759,023; 8,765,440 and US patent application 2012/0040410, the complete contents of each of which are hereby incorporated by reference in entirety.
  • the amount/activity of enzyme(s) is generally in the range of from about 0.5 FPU to about lOOFPU/mm, e.g. about 0.5, 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 FPU/mm.
  • Desirable yields after this stage are: xylose: at least about 90%, e.g. at least about 90, 91 , 92, 93, 94, 95, 96, 97, or 98%; glucose, at least about 90%, e.g.
  • lignin 85%, e.g. at least about 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95%.
  • Lignocellulosic biomass suitable for use as feedstock may be virgin biomass, waste biomass or energy crop biomass, or combinations of these.
  • Virgin biomass includes all naturally occurring terrestrial plants such as trees, bushes and grass.
  • Waste biomass refers to low value byproducts of various industrial sectors such as agricultural (e.g. corn stover, sugarcane bagasse, straw etc.), forestry (saw mill and paper mill discards), etc.
  • Energy crops are crops with high yield of lignocellulosic biomass produced to serve as a raw material for production of second generation biofuel, examples of which include but are not limited to switch grass (Panicum virgatum) and Elephant grass.
  • sources of lignocelluosic biomass that may be used as feedstock for pretreatment as described herein include but are not limited to wood, including both hardwood and softwood in forms such as trees, wood chips, "waste” wood, sawdust, wood pulp, etc.; various grasses, including switchgrass, elephant grass, grass clippings, bamboo, miscanthus, alfalfa, etc; agricultural crops (both purpose-grown and as waste byproducts) such as corn and/or cornstalks; corn cobs, corn stover, straw, bagasse, sorghum residue, cotton, jute, hemp, flax, sisal, abaca, straw, rice straw, barley straw, wheat straw, canola straw, oat straw, rice hulls, oat hulls, soybean stover, rice hulls, coconut hair, , etc., as well as others such as municipal waste solid waste, brush, tree limbs, forestry residues, agricultural residues, herbaceous material (e.g.
  • the feedstock is the poplar wood.
  • the biomass Prior to pretreatment as described herein, the biomass can be harvested and washed as necessary to remove any residual soil, dirt and the like; debarked if necessary; and/or reduced in size to a convenient size and certain quality that aids in moving the biomass or contacting the biomass with the liquids described herein in a flowthrough reactor.
  • the feedstock may be mechanically or physically processed so as to create as much accessible surface area as possible by shearing, grinding, mashing, chopping, pulverizing, milling (e.g., dry milling, wet milling, or vibratory ball milling), etc. or otherwise converting the feedstock to a suitable form, e.g. a particulate form.
  • milling e.g., dry milling, wet milling, or vibratory ball milling
  • Sugars produced by the methods described herein may be used for any purpose.
  • a particular use of interest is the production of biofuel, e.g. an alcohol such as ethanol. This is generally accomplished via fermentation by one or more fermentation microorganisms.
  • the sugars are generally substantially free of undissolved solids, such as lignin and other unhydrolyzed components using known techniques, including centrifugation, microfiltration, plate and frame filtration, crossflow filtration, pressure filtration, vacuum filtration and the like.
  • Any one of a number of known microorganisms may be used to convert sugar to ethanol or other alcohol fermentation products.
  • yeasts or bacteria any one of a number of known microorganisms (for example, yeasts or bacteria) may be used to convert sugar to ethanol or other alcohol fermentation products.
  • microorganisms convert sugars, including, but not limited to glucose, mannose and galactose present in sugar solution to a fermentation product.
  • microorganisms may further be a yeast or a filamentous fungus of a genus Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera,
  • the fermentation may be performed with recombinant yeast engineered to ferment both hexose and pentose sugars to ethanol.
  • Recombinant yeasts that can ferment one or both of the pentose sugars xylose and arabinose to ethanol are described in U.S. Pat. Nos.
  • Xylose utilization can be mediated by the xylose reductase/xylitol dehydrogenase pathway (for example, WO9742307 Al 19971 1 13 and W09513362 Al 19950518) or the xylose isomerase pathway (for example, WO200702881 1 or WO2009109631).
  • the fermentation organism may also produce fatty alcohols, for example, as described in WO 2008/1 19082 and PCT US07/011 ,923 which disclosure is herein incorporated by reference.
  • the fermentation may be performed by yeast capable of fermenting predominantly C6 sugars for example by using commercially available strains such as Thermosacc and Superstart.
  • strains of Escherichia coli which transform glucose into biofuel gasoline that does not need to be blended may be used, as may strains capable of producing, from glucose, short-chain alkanes, free fatty acids, fatty esters and fatty alcohols through the fatty acyl (acyl carrier protein (ACP)) to fatty acid to fatty acyl-CoA pathway in vivo.
  • ACP acyl carrier protein
  • the fermentation is performed at or near the temperature and pH optima of the fermentation microorganism.
  • the temperature may be from about 25°C to about 55°C.
  • the pH of a typical fermentation employing microorganisms is between about 3 and about 6.
  • the dose of the fermentation microorganism will depend on other factors, such as the activity of the fermentation microorganism, the desired fermentation time, the volume of the reactor and other parameters. It will be appreciated that these parameters may be adjusted as desired by one of skill in the art to achieve optimal fermentation conditions.
  • the resulting fluids can be separated into products of interest by any known method or combination of methods. For example, distillation may be used to separate ethanol and other alcohols from the majority of water and residual solids, and vapor-phase molecular sieves and centrifugation may also be employed.
  • Alcohols are one type of desired end-product.
  • the alcohols produced can be a monohydroxy alcohol, e.g., ethanol, or a polyhydroxy alcohol, e.g., ethylene glycol or glycerin.
  • Examples of alcohols that can be produced include methanol, ethanol, propanol, isopropanol, butanol, e.g., n-, sec- or t-butanol, ethylene glycol, propylene glycol, 1 ,4-butane diol, glycerin or mixtures of these alcohols.
  • the alcohols have multiple uses, e.g. ethanol can be used as biofuel, or in the manufacture of varnishes and perfume.
  • Methanol can be used as a solvent
  • butanol can be used in plasticizers, resins, lacquers, and brake fluids, etc.
  • the alcohol is bioethanol, which also has multiple uses, e.g. it can be purified to food grade alcohol, used as a transportation fuel, e.g. blended with gasoline to produce "gasohol," which can be used as combustible fuel in a wide variety of applications, including automobile engines.
  • Other endproducts include various organic acids such as monocarboxylic acids or a polycarboxylic acids. Examples of organic acids include formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic, palmitic acid, stearic acid, oxalic acid, malonic acid, succinic acid, glutaric acid, oleic acid, linoleic acid, glycolic acid, lactic acid,
  • Lignin residue also has value e.g. as a high/medium energy fuel (e.g. to power the equipment required to carry out the methods described herein), as a dispersant in high performance cement applications, in water treatment formulations and textile dyes, as an additive in specialty oil field applications and agricultural chemicals, as raw material for several chemicals, such as vanillin, DMSO, ethanol, xylitol sugar, and humic acid, as an environmentally sustainable dust suppression agent for roads, for injection molding and to produce expanded polyurethane foam, etc.
  • the lignin is used to produce high quality C7 to C I 8 carbon fuels (e.g. suitable for use as jet fuel) by
  • HDO hydrodeoxygenation
  • Figure 12 illustrates the product distribution and selectivity of HDO ligninsubjected to catalytic chemical treatment in the presence of a 5% Ru/C and A1 2 0 3 catalyst matrix.
  • Figure 13 shows the product distribution and selectivity of several HDO model compounds produced when lignin undergoes catalytic chemical treatment in the presence of a 5% Ru/C and ⁇ 1 2 0 3 catalyst matrix.
  • Residual sugars may also be used to as fuel (e.g. alone or with lignin residue) to provide energy for carrying out the methods of the invention.
  • EXAMPLE 1 Enhancement of Total Sugar and Lignin Yields through Dissolution of Poplar Wood by Hot Water and Dilute Acid Flowthrough Pretreatment
  • Pretreatment is a vital but expensive step in biomass biofuel production. Overall, most past efforts have been directed at maximizing sugar yields from hemicellulose and cellulose through trials with different chemicals, operating conditions, and equipment configurations. Flowthrough pretreatment provides a promising platform for dissolution of lignocellulosic biomass to generate high yields of fermentable sugars and lignin for biofuel production.
  • poplar wood was pretreated in a flowthrough system at elevated temperatures (e.g. 200-280°C) under varied conditions (0-30 min, 3 ⁇ 4S0 4 0-0.05% (w/w), and flow rates of 10-62.5 mL/min) to investigate the effects on yields of total mass, lignin and sugars (mono and oligomer), as well as the subsequent enzymatic hydrolysis of pretreated whole slurries.
  • elevated temperatures e.g. 200-280°C
  • flow rates 10-62.5 mL/min
  • 5-HMF 5 -hydroxymethyl furfural
  • BSA Bovine serum albumin
  • FPU Filter paper unit
  • DP degree of polymerization.
  • Poplar wood provided by Forest concepts contains 48.8% cellulose
  • Poplar wood material was ground with Hammermill (Hammer 1067- A- 1 , Buffalo, NY) at 4500 rpm with a 1.59 mm screen. The particles were collected and passed between Sieve 20 and Sieve 40 meshes to obtain particles within a size range of 0.425— 0.850 mm for experiments and analysis. The materials were sealed in heavy-duty zipped bags and stored at -20°C in a laboratory freezer.
  • the flowthrough reactor was 1.3 cm i.d.x 15.2 cm length with an internal volume of 20.2 mL. It was constructed of 316 stainless-steel parts using VCR fittings, including one VCR male union (1.3 cm), two gasket filters (average pore size 5 ⁇ ), two VCR glands (1.3 cm x l .3 cm), two VCR nuts, and two VCR reducing fittings (1.3 cm ⁇ 0.3cm). All reactor parts were obtained from Swagelok Co., Richland, WA. A preheating coil (0.6 cm o.d.xO. l cm wall, stainless steel) was connected with the reactor system and the cooling coil (0.3 cm o.d.xO. l cm wall).
  • a high-pressure pump (Acuflow Series III Pumps, Fisher) with a flow rate range of 0 to 100 mL/min, a pressure gauge (pressure range 0 to 1500 psi; Cole-Parmer Instrument Co., IL), and a back-pressure regulator (Valve and Fitting Co., WA) were used to control flow through the system.
  • 0.5g of biomass substrate was loaded into the reactor.
  • Distilled water or 0.05% (w/w) sulfuric acid was pumped through the reactor to purge air and then used to pressurize the reactor to a set pressure of 225-1245 psi.
  • the reactors were heated to the target temperature (200-280°C) in a 4-kW fluidized sand bath (model SBL-2D, Omega engineering, Inc., CT).
  • a thermal monitor combined with a 0.3cm stainless steel thermocouple (Omega Engineering Co., Stamford, CT) was connected to the outlet of the flow reactor to precisely control the reaction temperature.
  • Glucose, xylose, furfural, and 5-HMF in hydrolyzates of pretreatment and enzymatic hydrolysis were analyzed using a Waters HPLC system (model 2695) equipped with a 410 refractive detector and a Waters 2695 autosampler using Waters Empower Build 1 154 software (Waters Co., Milford, MA).
  • Bio-Rad Aminex HPX-87H column Bio-Rad Laboratories, Hercules, CA was operated at a temperature or 65°C. Yields of glucose, xylose, furfural, and 5-HMF were calculated as follows [12].
  • Furfural% F Xn x 100% (4)
  • Wo n and Wx represent the initial weight of glucan and xylan, respectively.
  • WG, Wx, WS.HMF and Wp ur represent the weight of glucose, xylose, 5-HMF and furfural, respectively.
  • the unit of W consistently refers to g/lOOg dw raw biomass.
  • Pretreatment hydrolysate flowing out of the flowthrough system was collected and then filtered through a 0.45 ⁇ polypropylene membrane filter (VWR, Radnor, PA). The filtrate was autoclaved in 4% (w/w) sulfuric acid for 1 h at 121°C to break down glucose oligomers and xylooligomers into their monomeric sugars based on standard NREL LAPs [13]. Yields of soluble glucose oligomers and xylooligomers were then calculated as below [ 14]:
  • Glucose oligomers% — 2- x 100%
  • WTG and WJX represent the total glucose and total xylose after autoclaving of the filtrate
  • WG and Wx represent glucose and xylose in the pretreatment filtrate before autoclaving
  • WOG and Wox represent the original glucan (as glucose) and original xylan (as xylose); and the unit of W consistently refers to g/lOOg dw raw biomass.
  • Pretreatment hydrolyzate (without filtration, not including solid residue in the reactor) was presoaked with 1 % (w/w) BSA at pH 4.8 and then followed by enzymatic hydrolysis at 50 °C for 168 hours with a high enzyme loading (100 mg protein Ctec 2 (93 FPU) with 20mg Htec2/g glucan + xylan) for maximum glucan conversion.
  • the final glucose concentration after enzymatic hydrolysis was used to determine the total glucan recovery in the pretreatment hydrolyzate.
  • the total glucan recovery by pretreatment was calculated as follows:
  • WEG is the total glucose after enzymatic hydrolysis
  • WOG is the original glucan as glucose.
  • the unit W consistently refers to g/lOOg dw raw biomass.
  • the structure characterization of soluble lignin was determined by GC-MS analysis.
  • the pretreated samples were filtered and extracted with dichloromethane [17] then analyzed with an Agilent gas chromatography mass spectrometer (GC, Agilent 7890A; MS, Agilent 5975C) equipped with a DB-5MS column (30m ⁇ 320 ⁇ ⁇ 0.25 ⁇ ).
  • the oven temperature was programmed from 45°C to 250°C at ramping rate of 5°C /min. Both the initial and final temperature was held for 5 minutes.
  • the flow rate of carrier gas (helium) was 1.3 ml/min.
  • Novozymes Cellic® CTec2 (220mg protein/mL, preserve 200 mg glucose/mL, 205 FPU/mL) and Novozymes Cellic® HTec2 (230mg protein/mL, preserve 180 mg xylose/mL) were generously provided by Dr. Melvin Tucker from NERL for all hydrolysis experiments.
  • the filter paper activity of CTec2 was determined according to the standard filter paper assay [18].
  • Liquid samples were taken at 4, 24, 48, 72, 96 and 120 hours and measured directly by HPLC for monomeric sugars.
  • BSA treatment was conducted for parts of experiments. Prior to enzyme addition to start hydrolysis, the whole pretreated slurries were presoaked with 1% (w/w) BSA 10 mg/L sodium azide for 24 hours [2] .
  • W G i and W X are the glucose and xylose released in the pretreatment; WG2 and ⁇ 2 are the glucose and xylose released in enzymatic hydrolysis; WTG and WTX are the total potential glucose and xylose released after enzymatic hydrolysis of whole pretreated slurries (including solid residue) with the high enzyme loading (100 mg protein Ctec 2 (93 FPU) with 20mg Htec2/g glucan+xylan) in 168hrs.
  • the unit of W consistently refers to g/lOOg dw raw biomass.
  • a severity parameter logRo which is widely applied in hot water and dilute acid pretreatment [13, 19, 20] was used to unify the data obtained at different combinations of temperature and reaction time, which includes the preheating time.
  • the severity Log RQ is defined as [21].
  • T reaction time in minutes (including the preheating time); T is the hydrolysis temperature in °C. 100°C is the reference temperature.
  • logRo is the function of temperature and time as described in Equation 10, its value was calculated based on Equation 10 using the measured value of the target reaction temperature from the thermal monitor and the reaction time.
  • Elevating the target temperature or adding acid increased the removal of both xylan and lignin. Overall, up to 100% of xylan, 49% cellulose and 87%o lignin were removed into the hydrolyzate through the preheating processes under tested conditions. Most of the dissolved xylan and cellulose during these preheating processes was in the form of oligomers with small amount of xylose and glucose, and negligible degradation compounds.
  • 62.5mL/min could improve lignin removal by 5-15% for water-only treatments, and around 5% for dilute acid treatments, respectively.
  • cellulose Unlike xylan and lignin, cellulose consists of cellulose ⁇ and la which are both held together via a network of hydrogen hydrophobic interactions, causing deconstruction of the crystals challenging.
  • cellulose removal rapidly increased to 40% at lOmL/min and 50% with 62.5mL/min flow rate when severity logRo reached 4.8 at 240°C.
  • Cellulose ⁇ underwent a transition into an amorphous structure when the temperature increased to around 220-230°C.
  • Figure 3A represents xylose and xylooligomer yields from poplar wood by water-only flowthrough pretreatment. Results showed that xylooligomers were predominant recovered xylan in filtered pretreatment hydrolyzate at all tested severities. Higher than 75% xylooligomers yield were observed while xylose yields were found less than 25%.
  • Xylooligomers yield decreased slightly as logRo increased while the corresponding xylose yield increased. It indicated that the increased severity could shift the distribution of generated sugars to monomers. Conversely, increasing flow rate from 10 mL/min to
  • biomass-derived monomeric sugars can be further dehydrated into furans (furfural and 5-HMF) which in turn can degrade into organic acids, such as levulinic acid, resulting in reduced fermentable sugar yield.
  • furans furans
  • 5-HMF organic acids
  • Table 2 As shown in Table 2, at a flow rate of 10 mL/min, 3.1% 5-HMF yield was observed at 240°C after 10 min with water-only, whereas elevating flow rate up to 25mL/min resulted in negligible 5-HMF yield. Even when the temperature was raised to 270°C, 5-HMF yield remained negligible with flow rate of 25 mL/min. 0.7% furfural was formed under 250°C at 10 min when employing a flow rate of 10 mL/min.
  • Lignin is believed to depolymerize and micellarize under acidic conditions via both homolytic and acidolytic cleavage into low molecular weight lignin globules.
  • acidic water passes through the material, especially at high flow rates, highly reactive nucleophilic carbonium ion intermediates are formed within the lignin structure, and can react further leading to the cleavage of predominant ⁇ - ⁇ -4 bonds.
  • This brings about efficient depolymerization of lignin, which can then be quickly and continuously swept out of the reactor to limit simultaneous repolymerization reactions and re-precipitation of the depolmerized lignin at ambient temperature.
  • dilute acid resulted in a high glucose yield of 52.7% at 4hrs and 73.3% at 120hrs with even the lowest enzyme loading. With the medium enzyme loading, about a 93% glucose yield was reached within 120hr. At high enzyme loading, the glucose yield was found >90% without BSA within 4hrs.
  • the enzymatic xylose yield of pretreated whole slurries reached 94.1% and 96.8% for water-only and dilute acid, respectively, within 24hrs at high enzyme loading (Table 4A and B).
  • the medium enzyme loading resulted in 92.2% and 89.2% of enzymatic xylose yield for water-only and dilute acid pretreated whole slurries in 72hrs, respectively.
  • Similar enzymatic xylose yields were found at lower enzyme loadings for water-only and dilute acid pretreated whole slurries.
  • the enzyme loading employed during Stage 2 for water-only and dilute acid pretreated slurries were high and medium, respectively, both of which led to > 90% enzymatic glucose yield and >95% enzymatic xylose yield from corresponding samples.
  • the results showed that, on the basis of 100 g poplar wood, more than half the cellulose and nearly all the xylan was converted to soluble sugars at Stage 1 for the water-only operation: 4.0 g xylose plus 14.7 g xylooligomers, and 7.5 g glucose plus 19.7 g glucose oligomers were obtained.
  • A 270 °C, water-only, 25mL/min, lOmin
  • B 240 °C, 0.05% (w/w) H 2 S0 4 , 25mL/min, 8min
  • the water consumption for water-only (i.e. 270°C, lOmin, 25 mL/min) and dilute acid (i.e. 240°C, 0.05% (w/w) H2SO4, 8 min, 25mL/min) operations was approximately 250 mL and 200 mL, respectively, which led to around 0.2-0.25% (w/w) overall solid to liquid ratio with a solid loading of 0.5 g.
  • bHigh enzyme loading 100 mg protein Ctec 2 (93 FPU) with 20mg Htec2/g glucan+xylan; Medium enzyme loading: 10 mg protein Ctec 2 (9.3 FPU) with 2mg Htec2/g glucan+xylan.
  • Glu glucose, Xyl— xylose, XOS— xylooligomers, GOS— soluble glucose oligomers;
  • L-GOS insoluble glucan derivatives
  • SL soluble lignin
  • ISL insoluble lignin
  • Poplar wood was pretreated through water-only and dilute acid flowthrough approaches at temperatures in the range of 200-280°C and this treatment resulted in more than 98% removal of solids. Temperature was considered as the most significant factor for cellulose degradation. Cellulose removal significantly increased as the temperature reached 240°C for water-only and 220°C for dilute acid. Up to 100% xylan and 90% cellulose were hydro lyzed with negligible furfural and 5-HMF formation during pretreatment. Dilute acid pretreatment also resulted in higher yields of recovered xylan and cellulose as monomelic sugars in the hydro lyzate, compared to water-only pretreatment.
  • Insoluble lignin accounted for the majority of the original lignin (-90%) while a small amount (-15%) became soluble in the pretreated whole slurries. A larger fraction of recovered lignin was soluble with water-only pretreatment. Increasing severity enhanced total mass removal, xylan removal, lignin removal, and cellulose removal, and adding dilute sulfuric acid significantly accelerated all of the above. Dissolution of almost all biomass in the hydroyzate was obtained at logRo around 6.0 without acid added while a faster rate was achieved with dilute acid (logRo around 5.0).
  • the pretreated whole slurries under selected dilute acid conditions (240°C, 0.05% (w/w) H2SO4, 25mL/min, 8min) resulted in a much higher soluble glucose plus glucose oligomers yield (-90%) at stage 1 than did the water-only operation (270°C, 25mL/min, lOmin), and required only 10 FPU/g glucan+xylan enzyme to achieve >90% glucose yield and >95% xylose yield.
  • BSA testing showed that the limited inhibitory compounds in the pretreated slurries had an insignificant impact on the performance of enzymes on pretreated whole slurries, especially after dilute acid pretreatment.
  • the insoluble lignin was recovered from hydrolyzate with low molecular weight ( ⁇ 1800 Dalton, with a low molecular weight peak at 320 Dalton and a high MW peak at 1500 Dalton).
  • Catalytic techniques have been developed to convert such technical lignin into C7 to C9 range hydrocarbons through a novel hydrodeoxygenation process [International patent application PCT/ US2013/38927].
  • Poplar was used as substrate. It contains 42% cellulose, 21% xylan and 23% lignin. The compositions of poplar wood were determined based on Laboratory Analytical
  • the flowthrough reactor used in this work was 1.3 cm i.d.x l 5.2 length with an internal volume of 14.3 mL. These units were constructed of 316 stainless-steel parts using VCR (Swagelock Corp.) fittings, including one VCR male union (1.3 cm), two gasket filters (316 stainless-steel, average pore size 5 ⁇ ), two VCR glands (1.3 cm ⁇ 1.3 cm), two VCR nuts, and two VCR reducing fittings (1.3 cmx0.3cm). All reactor parts were obtained from the Maine Valve and Fitting Co. A 0.3cm stainless-steel thermocouple (Omega Engineering Co., Stamford, CT) was installed at the outlet of the reactor to monitor temperature.
  • Stainless-steel tubing (316) was used as a preheating coil (0.6 cm o.d. ⁇ 0.1 cm wall) and to connect the reactor with other system components as well the cooling coil (0.3 cm o.d. 0.1 cm wall).
  • the preheating coil was long enough to allow the incoming water to reach the desired temperature before it entered the reactor, as measured experimentally.
  • a high pressure pump (Acuflow Series ⁇ Pumps, Fisher) with a flow rate range of 0 to 100 mL/min, a pressure gauge (pressure range 0 to 1500 psi; Cole-Parmer Instrument Co., Vernon Hills, IL), and a back-pressure regulator (Maine Valve and Fitting Co.) were used to control flow through the system.
  • Liquid samples were centrifuged at 10,000 rpm/min for lh, the supernatants were analyzed using a Waters HPLC system (model 2695) equipped with a 410 refractive detector and a Waters 2695 autosampler using Waters Empower Build 1 154 software (Waters Co., Milford, MA).
  • the Bio-Rad Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA) were operated under 65° C for separation of compounds for quantification.
  • the mobile phase was 0.005M H2SO4 with a flow rate at 0.6 ml/min.
  • the supernatant liquid hydrolysate samples were also autoclaved in 4% (w/w) sulfuric acid for 1 h at 121 °C to breakdown glucose oligomers and xylooligomers into their monomeric sugars as described (Sluiter et al., 2008).
  • Sugar standards containing known sugar concentrations were also autoclaved for the same time and at the same acid
  • Glucose oligomers(g) Total glucose(g) in the hydrolysate corrected for degradation - Monomers(g) in the hydrolysate liquid before autoclaving.
  • Xylooligomers(g) Total xylose(g) in the hydrolysate corrected for degradation - Monomers(g) in the hydrolysate liquid before autoclaving.
  • NREL LAP "Determination of Structural Carbohydrates and Lignin in Biomass”
  • Solid residues were analyzed through hydrolysis with 72% (w/w) H 2 SO 4 at 30 °C for 2 h, followed by dilution into 4% (w/w)of the reaction mixture and then a second hydrolysis at 121 °C for 1 h.
  • the hydrolysate was neutralized and the sugar content was analyzed using a Waters HPLC system (model 2695) as described above (for Analytical methods).
  • the pretreatment temperature for hydrolysis of lignocellulosic biomass is on the range of 120-220 °C. Few researchers reported temperature above 220 °C
  • Hemicellulose and lignin can be effectively removed in such temperature range.
  • cellulose cannot be effectively degraded at such temperatures, likely due to extensive hydrogen bonding.
  • the intra- and inter-molecular hydrogen bonds formed in cellulose, and the hydrogen bonds formed between cellulose and the water molecules provide a physical barrier for protons penetrating into the cellulose to interact with glycoside oxygen, which results in cleavage of C-O-C bond. It has been reported that hydrogen bonds begin to break down at temperatures above 200 °C, and that the temperature for effective degradation of cellulose is around 260 °C in hot water pretreatment.
  • An optimal temperature in the range of 220-300 °C is found at which effective hydrolysis of cellulose, hemicellulose and lignin occurs with fewer byproducts (such as furfural, 5-HMF and levulinic acid).
  • the cellulosic biomass is hydrolyzed in a flowthrough reactor with water-only and extremely dilute sulfuric acid (0.05%— 0.1% w/w) at a temperatures in the range of 240-300 °C and pressure range of 485psi— 1245 psi (the corresponding saturated vapor pressure of the temperature), and at flow rates ranging from 10-50mL/min for 0-10 min.
  • the effects of temperature, acid concentration and flow rate on sugar recovery are determined for both xylan and cellulose.
  • Optimal conditions to achieve 90% yields of sugar monomers and oligomers from both xylan and cellulose are determined.
  • Ehrman T Determination of acid-soluble lignin in biomass. Golden, CO: National Renewable Energy Laboratory Analytical Procedure; 1996.
  • numeric ranges present herein are intended to encompass intervening numeric values to at least one decimal point, e.g. a range of 1-2 represents the values 1.0, 1 ,1 , 1 ,2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 2.0.

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Abstract

L'invention concerne des procédés à écoulement continu hydrothermique d'acide dilué et d'eau uniquement pour prétraiter de la biomasse lignocellulosique. Les procédés à écoulement continu sont effectués à haute température et sont généralement suivis par une hydrolyse enzymatique de la boue obtenue pour libérer d'autres produits de dégradation de cellulose. Ces procédés permettent d'obtenir des rendements élevés de produits souhaités, par exemple des sucres fermentescibles et de la lignine, lesquels peuvent être traités par la suite, par exemple, pour produire des biocarburants.
PCT/US2014/047127 2013-07-19 2014-07-18 Prétraitement par écoulement continu hydrothermique de biomasse lignocellulosique pour maximiser les rendements de sucre fermentescible et de lignine WO2015009986A2 (fr)

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* Cited by examiner, † Cited by third party
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US20160251690A1 (en) * 2013-10-07 2016-09-01 Showa Denko K.K. Method for treating cellulose-containing biomass
WO2020033633A1 (fr) * 2018-08-08 2020-02-13 Casad Robert C Jr Procédés et dispositifs de traitement de biomasse lignocellulosique à récupération de lignine purifiée et fractions de cire purifiées

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US20110300586A1 (en) * 2008-12-19 2011-12-08 Chaogang Liu Two-Stage Process for Biomass Pretreatment
WO2010102060A2 (fr) * 2009-03-03 2010-09-10 Poet Research, Inc. Système de prétraitement d'une biomasse pour la production d'éthanol
BRPI1009093A2 (pt) * 2009-08-24 2016-03-01 Abengoa Bioenergy New Technologies Inc "método para pré-tratamento do estoque de alimentação de biomassa celulósica que compreende celulose, hemicelulose, e lignina".
IT1399078B1 (it) * 2010-03-24 2013-04-05 Eni Spa Procedimento per la conversione della lignina a idrocarburi liquidi
EP2471940A1 (fr) * 2010-12-31 2012-07-04 Süd-Chemie AG Hydrolyse efficace de la lignocellulose avec production d'enzymes intégrée

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160251690A1 (en) * 2013-10-07 2016-09-01 Showa Denko K.K. Method for treating cellulose-containing biomass
WO2020033633A1 (fr) * 2018-08-08 2020-02-13 Casad Robert C Jr Procédés et dispositifs de traitement de biomasse lignocellulosique à récupération de lignine purifiée et fractions de cire purifiées

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