WO2015142399A1 - Préparation de biomasse - Google Patents

Préparation de biomasse Download PDF

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Publication number
WO2015142399A1
WO2015142399A1 PCT/US2014/072278 US2014072278W WO2015142399A1 WO 2015142399 A1 WO2015142399 A1 WO 2015142399A1 US 2014072278 W US2014072278 W US 2014072278W WO 2015142399 A1 WO2015142399 A1 WO 2015142399A1
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Prior art keywords
acid
biomass
hydrolysate
hydrolysis
washed
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PCT/US2014/072278
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English (en)
Inventor
Yongming Zhu
David C. Walther
Xiadi GAO
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Cobalt Technologies, Inc.
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Publication of WO2015142399A1 publication Critical patent/WO2015142399A1/fr

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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
    • C12P7/16—Butanols
    • 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
    • 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 invention relates to reacting and/or removing compounds from biomass that may be deleterious to downstream pretreatment and/or fermentation processes by prewashing the biomass with acid hydrolysate.
  • Cel osic biomass provides a readily available source of sugar molecules, which may be used as a carbon source in microbial fermentations to produce bioproducts of interest.
  • Polymeric carbohydrate components of the biomass can be hydrolyzed to release soluble sugar molecules, hi acidic hydrolysis (e.g., dilute acid pretreatment) of biomass, acid represents a significant fraction of the cost of sugar production, so reducing the amount of acid required is very important to the process economics.
  • compounds such as ash in biomass can neutralize acid and reduce the effectiveness of the pretreatment. Tlie negative impact of these biomass components has been referred to as a neutralizing or buffering effect.
  • salts or dirt that are associated with tiie biomass can enter process streams and foul, contaminate, wear, and/or disable equipment such as hydrolysis or fermentation equipment, if not removed prior to pretreatment of the biomass or fermentation of hydrolysate prepared from the biomass.
  • a method for reacting and/or removing non-carbohydrate compounds from biomass including (a) washing a second biomass with a first acid hydrolysate that is produced by acidic hydrolysis of a first biomass, thereby producing an acid hydrolysate washed second biomass; and (b) separating the acid hydrolysate washed second biomass from at least a portion of the first acid hydrolysate that was used to wash the second biomass.
  • At least one compound (e.g., at least one non- carbohydrate compound) in the first biomass is reacted to produce another compound.
  • at least a portion of at least one non-carbohydrate compound is removed from the second biomass.
  • the first acid hydrolysate that is separated from the acid hydrolysate washed second biomass in (b) contains the at least one non-carbohydrate compound.
  • the hydrolysate of (b) is used as a primary substrate for fermentation.
  • the acid in the first acid hydrolysate used for washing the second biomass in (a) includes at least one acid selected from nitric acid, sulfiiric acid, sulfurous acid, SO2, hydrochloric acid, phosphoric acid, formic acid, and acetic acid, hi one embodiment, the acid is nitric acid.
  • non-carbohydrate compounds that are removed from the second biomass include inorganic salts, mineral oxides, and ' or organic acids, hi some embodiments, at least a portion of the non-carbohydrate compounds that are reacted and'or removed from the second biomass are capable of buffering and'or neutralizing acid, hi some embodiments, the reaction and or removal of the non-carbohydrate compounds in a method as described herein reduces or eliminates the buffering and/or neutralizing capacity of such compounds in the second biomass in a downstream pretreatment process such as acid (e.g., dilute acid) hydrolysis.
  • acid e.g., dilute acid
  • the reaction and/or removal of the non-carbohydrate compounds in a method as described herein improves downstream enzymatic hydrolysis and/or microbial fermentation performances.
  • removal of soluble (e.g., non-structural) sugar molecules reduces or prevents degradation of the sugar molecules from occurring in downstream prefreaiment processes, such as acid hydrolysis, avoiding or reducing formation of inhibitors of microbial fomentation and/or bioproduct production.
  • the method further includes: (c) contacting the acid
  • hydrolysate washed second biomass with acid and treating under conditions sufficient to depoly nerize at least one polymeric carbohydrate component of the second biomass, thereby producing: (i) a second acid hydrolysate that includes soluble sugar molecules; and (ii) residual solids.
  • less acid is required for said depolymerization from acid hydrolysate washed second biomass than from the same biomass that has not been prewashed as in (a), when tr eated with acid under identical conditions (e.g. , temperature, time, pH).
  • less base is required to raise the pH of the acid hydrolysate to a level thai is suitable for enzymatic hydrolysis and/or microbial
  • the acid used for production of acid hydrolysate in (c) includes at least one acid selected from nitric acid, sulfuric acid, siilfurous acid, SO 2 .
  • hydrochloric acid, phosphoric acid, formic acid, and acetic acid in one embodiment, the acid is nitric acid, hi some embodmieiits, the acid in the first acid hydrolysate used for washing second biomass in (a) and the acid used for production of second acid hydrolysate in (c) are the same acid, for example, selected from nitric acid, sulfuric acid, sitlfurous acid, SO2, hydrochloric acid, phosphoric acid, formic acid, and acetic acid, hi one embodiment, the acid is nitric acid, hi some embodiments, the acid in the first acid hydrolysate used for washing second biomass in (a) and the acid used for production of second acid hydrolysate hi (c) are different acids, for example, selected from nitric acid, sulfuric acid, sulfrirous acid, SO 2 . hydrochloric acid, phosphoric acid, formic acid, and acetic acid.
  • the method further includes: (d) separating the second acid hydrolysate produced i (c) from the residual solids produced in (c); and (e) using at least a portion of the second acid hydrolysate separated in (d) to wash a third biomass, as hi (a), in some embodiments, acid hydrolysis of acid hydrolysate washed biomass, separation of the resulting acid hydrolysate from residual solids, and use of the acid hydrolysate for washing of additional unhydrolyzed biomass (e.g., steps (c), (d), and (e)) are conducted in a continuous process.
  • the amount of acid in (c) that is required for about 60% to about.95% depolynierizatioii of the at least one polymeric carbohydrate component (e.g. , hemicelhilose) from the acid hydrolysate washed second biomass is reduced by about 20% to abou 60% in comparison with the same biomass that lias not been pretreaied as in (a).
  • die amount, of acid in (c) that is required for about 75% to about 85% depolynierizatioii of the at least one polymeric carbohydrate component e.g.,
  • the second biomass includes lignocelMosic biomass, for example, including but not limited to, rice straw, rice husks, wheat straw, barley straw, com stover, switchgrass, sugar cane bagasse, sugar cane straw or trash, palm empty fruit bunches, and/or Kenaf.
  • lignocelMosic biomass for example, including but not limited to, rice straw, rice husks, wheat straw, barley straw, com stover, switchgrass, sugar cane bagasse, sugar cane straw or trash, palm empty fruit bunches, and/or Kenaf.
  • the at least one polymeric component that is depolymerized from the acid hydrolysate washed second biomass in (c) includes heniicellulose. i some embodiments, the at least one polymeric component further includes cellulose,
  • the lignocellulosic biomass includes a high silica lignocellulosic biomass, for example, including but not limited to, rice straw, wheat straw, rice husks, and or corn stover, in some embodiments, at least a portion of the silica is removed by alkali extraction prior to step (a), and non-carbohydrate compounds that are removed include residual alkali.
  • acid in the first acid hydrolysate neutralizes a t least a portion of residual alkali in the sec ond biomass, and the first acid hydrolysate that is separated from acid hydrolysate washed second biomass contains neutralization product(s) of acid and residual alkali.
  • residual solids that are separated in (d) are subjected to a further hydrolysis process, for example, such as but not limited to, acid hydrolysis, enzymatic hydrolysis, or hydrolysis with a supercritical fluid, hi one embodiment, the residual solids are hydrolyzed with one or more enzyiiie ⁇ s) (for example, including at least csne ce ulase) to produce additional soluble sugar molecules in an enzymatic hydrolysate .
  • soluble sugar molecules in the enzymatic hydrolysate are fermented by a microorganism to produce a bioproduct of interest.
  • the second biomass contains non-stnictural sugar molecules (for example, but not limited to, sucrose), and at least a portion of re non-sinictural sugar molecules axe washed in to the first acid hydrolysate.
  • the first acid hydrolysate that is separated ' from acid hydrolysate washed second biomass may cont ain about 50% to about 95% of the non-structural sugar moieciiies thai were associated with the second biomass.
  • the acid hydrolysate washed second biomass retains a portion of the non-structural sugar molecules and the second acid hydrolysate produced in (c) includes soluble non-structural sugar molecules from the second biomass.
  • soluble sugar molecules in the first acid hydrolysate thai is separated in (b) (after washing of biomass) and or acid hydrolysate that is produced by acid hydrolysis of biomass that has been prewashed with acid hydrolysate as described herein are fermented by a microorganism to produce one or more bioproduct(s) of interest, for example, but not limited to. one or more solvent(s). organic acid(s). and or alcohol(s).
  • the acid hydrolysate that contains the soluble sugar molecules is conditioned to remove at least a portion of at least one substance that inhibits microbial growth and/or bioproduct production, prior to fermentation.
  • conditioning may include, but is not limited to, at least one process selected from evaporation, steam stripping, charcoal adsorption, electrodialysis, and reverse osmosis.
  • one or more solvents may be produced.
  • the solvent(s) may include ethaiiol, acetone, and/or butanol (e.g., n-butanol).
  • biomass is provided from which at least a portion of one or more non-carbohydrate eonipound(s) has been reacted and or removed, according to a process described herein. Hydrolysates that are prepared from such biomass are also provided.
  • Figure 1 schematically shows pretreatment of biomass to produce acid hydrolysate, and washing of fresh biomass with a stream of the acid hydrolysate to produce acid hydrolysate washed biomass
  • FIG. 21 j Figure 2 schematically shows prewashing of sugar cane bagasse with acid hydrolysate that is generated in a downstream acid hydrolysis operation and used for prewashing fresh bagasse.
  • Figure 3 schematically shows alkaline desilication and prewashing of rice stow with acid hydrolysate that is generated in a downstream acid hydrolysis operation and used for prewashing fresh rice straw.
  • FIG. 4 schematically depicts an embodiment of a biomass washing procedure, exemplified in Example 1.
  • FIG. 5 shows the co centration of free hydrogen ion in liquid phase of bagasse-water shinies (2.5 w ⁇ % solids) as a function of acid loading, as described hi Example 1.
  • the solid line represents pure aqueous solution (no solids).
  • Figure 6 schematically depicts an embodiment of a biomass washing procedure- exemplified in Example 2.
  • FIG. 8 schematically depicts an embodiment of a biomass sugar recovery procedure, exemplified in Example 3. This figure shows a flow diagram depicting an experiment that tested the effect of carry-over sugar on net sugar- production and compactability.
  • Methods and systems are provided herein for preparing biomass for pretreatmeiit (e.g., acid hydrolysis, enzymatic hydrolysis).
  • unwanted materials e.g. , non-carbohydrate components
  • compounds such as ash (e.g., mineral oxides), organic acids, extractives, and/or other soluble components, may be removed, and/or compounds such as salts (e.g. , calcium salts) and/or oxides may be reacted to form insoluble products in the biomass.
  • Compounds that are reacted and/or removed from biomass as described herein may otherwise affect the amount of one or more chemical(s) (e.g., acid(s)) required for
  • an acid hydrolysate is used to prewash biomass prior to downstream pretreatmeiit processes.
  • the acid hydrolysate may be a stream from downstream acid hydrolysis of biomass. For example, production of acid hydrolysate from prewashed bioniass and prewasfaiiig of further bioraass with a stream of the acid hydrolysate may be conducted m a continuous process.
  • Bioprodiict refers to any substance of interest produced biologically, i.e. , via a metabolic pathway, by a microorganism, e.g., in a microbial fermentation process.
  • Bioproducts include, but are not limited to fuel molecules (e.g. , n-butano , acetone, ethanoi, isobutanol, famesene, etc.) , solvents, biornolecules (e.g. , proteins (e.g., enzymes), polysaccharides), organic acids (e.g., formate, acetate, butyrate, propionate, succinate), alcohols (e.g. , methanol, ropane!
  • fuel molecules e.g. , n-butano , acetone, ethanoi, isobutanol, famesene, etc.
  • solvents e.g. , biornolecules (e.g. , proteins (e.g., enzymes), polysaccharides), organic acids (e.g., formate, acetate, butyrate, propionate, succinate), alcohols (e.g. , m
  • bioproducts may be used for catalysis, as a solvent, as a chemical intermediate, as a co-monomer, as a fuel (biofuel), or as a lubricant.
  • Byproduct refers to a substance that is produced and or purified and/or isolated during any of the processes described herein, which may have economic or environmental value, but that is not the primary process objective.
  • byproducts of the processes described herein include lignin compounds and derivatives, carbohydrates and carbohydrate degradation products (e.g. , furfural, hydroxyniethy! fiirrnral, formic acid), and extractives (described infra).
  • “Feedstock” refers to a substance that can serve as a source of sugar- molecules to support microbial growth in a fermentation process.
  • 'Reconstruction refers to mechanical, chemical, and/or biological degradation of bioniass to render individual components (e.g. , cellulose, hemic eilulose) more accessible to further pretreatment processes, for example, a process to release monomelic and oligomerie sugar molecules, such as acid hydrolysis.
  • “Conditioning” refers to removal of inhibitors of microbial gr owth and/or bioproduct production froin a hydrolysate produced by hydrolysis of a eeliulosic feedstock or adjustment of a physical parameter of the hydrolysate to render it more amenable to inclusion in a microbial culture medium, for example, adjustment of the pH to a pH that is suitable for growth of the microorganism when added to a microbial growth medium.
  • “Titer' ' refers to amount of a substance produced fay a microorganism per unit volume ia a microbial fermentation process. For example, titer of butanol in a microbial iemientation may be expressed as grams ofbutanol produced per liter of solution.
  • Yield refers to amount of a product produced from a feed material (for example, sugar, relative to the total amount mat of the substance that would be produced if all of the feed substance were converted to product.
  • yield of butanol in a. microbial fermentation may be expressed as % of butanol produced relative to a theoretical yield if 100% of the feed substance (for example, soluble, e.g. , non-structural, sugar molecules) were converted to butanol.
  • Productivit ' refers to the amount of a substance produced by a microorganism per unit volume per unit time in a microbial fermentation process. For example, productivity of butanol in a microbial fermentation may be expressed as gr ams of butanol produced per liter of solution per hour.
  • “Sugar conversion” refers to grams of sugar consumed by a microorganism (e.g. , in a microbial fermentation process) per grams of sugar provided to the microorganism (e.g., grams of sugar- provided in a microbial growth medium).
  • Wild-type refers to a microorganism as it occurs in nature.
  • ABE fermentation ' refers to production of acetone, butanol, and/or ethanol by a fermenting macOorgamsm.
  • “LignoceHulosic” biomass refers to plant biomass that contains cellulose, hemicellulose, and ligiiin.
  • the carbohydrate polymers cellulose and hemicellulose are tightly bound to lignin.
  • y ains are macromolecular components of lignoeeMulosie biomass that contain phenolic propylbenzene skeletal units linked at various sites.
  • solvent refers to a liquid or gas that is capable of dissolving a solid or another liquid or gas.
  • a solvent may be produced as a bioproduct fay a microorganism as described herein.
  • Nonlimiting examples of solvents produced by microorganisms include ?:?-butanol, acetone, ethanol, acetic acid, isopropanol, w-propanol, methanol, formic acid, 1 ,4-dioxane,
  • ff-Butaaol is also referred to as "butanof ' herein.
  • Vinasse or “backset” or “stillage” refers to a fermentation broth from which one or rnore bioproduct has been removed.
  • fermentation broth of a microorganism that produces ethanol and from which ethanol has been removed is termed “ethanol vinasse.”
  • ethanol vinasse fermentation broth of a microorganism that produces bvrtanol and from which butanol has been removed
  • butanol vinasse fermentation broth of a microorganism that produces bvrtanol and from which butanol has been removed.
  • vinasse is the bottom fraction of distillation of a solvent-containing temientation medimii, and solvent and other volatile compounds are separated from the fermentation broth while the rest of the constituents (e.g., residual sugar, organic acids, glycerol, bioinass) are slightly concentrated in the vinasse.
  • Base or “alkali” are used interchangeably herein to refer to a molecule or compound that is characteristically basic (pH greater than 7) at room iemperaiiue.
  • Non- limiting examples of basic or alkaline compounds include oxides, carbonates, and hydroxides of alkali metals.
  • Acid refers herein to a molecule or compound that can donate a proton or that can accept an electron pair in reactions.
  • Hydrolysis refers to the chemical breakdown of a compound due to reaction with water.
  • hydrolysis of celhilosie bioinass refers to the breakdown of giycosidic bonds in sugar polymers that are contained within the bioinass.
  • a “hydrolysate” is the liquid product of a hydrolysis reaction, e.g., liquid product of bioinass hydrolysis containing soluble sugar molecules.
  • Pretreatineiit refers herein to use of a method (e.g., mechanical, thermal, chemical, and/or biological) to modify the characteristics of a biomass material.
  • pretreatment may modify biomass such that hydrolytic enzymes and/or microorganisms may access aad or hydroiyze or utilize carbohydrate molecules in the bioinass.
  • pretreatment operations include acid or enzymatic hydrolysis to release soluble sugar molecules as a liquid hydrolysate.
  • CSF or “combined severity factor” represents a combined effect of pretreatment temperature (°C), time (min), and pH (end of pretreatment ⁇ .
  • XMG refers to xylose, mannose, galactose.
  • unwanted non-carbohydrate compounds are reacted and/or removed from biomass feedstock prior to downstream processes such as dilute acid pretreatment and/or enzymatic hydrolysis, by prewashmg the bioinass with an acid h drolysaie of bioniass.
  • Unwanted non-carbohydrate compounds may include compounds that would be deleterious to one or more downstream proeess(es) if not removed, such as hydrolysis of the biomass (e.g., acid and/or enzymatic hydrolysis) and'or fom ntat on of a hydrolysate prepared from the biomass.
  • the acid hydrolysate that is used fo biomass feedstock prewashing is generated by acid hydrolysis of biomass.
  • the washed biomass solid material is separated from the liquid hydrolysate (separated from all, substantially all, or at least a portion of the liquid hydrolysate) that was used for prewashing, thereby producing ackl hydrolysate washed biomass from which at least a portion of at least one non-carbohydrate compound has been removed and/or reacted, hi embodiments in which at least one non-carbohydr ate compound is removed from the biomass in the prewashing process, the liquid hydrolysate that is separated from acid hydrolysate washed biomass solid includes at least one noil- carbohydrate compound from the biomass tliat was washed with the hydrolysate.
  • the method includes: reacting and/or removing non- carbohydrate compounds from biomass, including: (a) washing biomass with an acid hydrolysate that is produced by acidic hydrolysis of another portion of biomass (e.g. , acid hydrolysate fr om either the same or different bioniass as the biomass to be washed); and (b) separating the solid washed biomass fr om at least a portion of the liquid acid hydrolysate that was used for washing, thereby producing: (i) acid hydr olysate washed bioniass from which at least a portion of at least one non-carbohydrate compound has been reacted and/or removed; and (ii) acid hydrolysate, wherein in embodiments in which at least one
  • the acid hydrolysate contains at least one non- carbohydrate compound from the bioniass that was washed with the hydrolysate.
  • Compounds removed from biomass by prewashing with acid hydrolysate may include one or more compound(s) (e.g., non-carbohydrate compounds) that neutralize acid, compomid(s) that buffer acid, and/or comporaidf ' s) tliat would be deleterious to one or more downstream proeess(es), such as acid hydrolysis, enzymatic hydrolysis and/or microbial fermentation of a hydrolysate prepared from the biomass if not removed.
  • Nonliiniting examples of compounds (e.g., extractives) that may be removed in the prewashing procedur es described herein include ash (mineral oxides), inorganic salts (e.g..
  • the biomass are capable of buffering and or neutralizing acid.
  • the amount of acid thai is required for downstream acid hydrolysis of the solid washed biomass material may be reduced, and or the amount of base that is required to neutralize and or bring the pH of the hydrolysate produced from the washed biomass material to a suitable level for a downstream process such as fermentation may be reduced.
  • Compounds in the biomass that are reacted by prewashiiig with acid hydrolysate may include salts and/or oxides.
  • “Reacting” includes formation of an insoluble product in the biomass by reaction of a biomass component with one or more components) of the acid hydrolysate that is used for prewashiiig. such as, for example, the acid mat is used to produce the hydrolysate.
  • dilute sulfuric acid in an acid hydrolysate may react with a calcium salt or oxide in the biomass, fomiing calcium sulfate, which is insoluble and can form deposits within the biomass.
  • the amoimt of acid mat is required for acid hydrolysis of the biomass may be reduced, and'or the amount of base that is required to neutralize and or bring the pH of the hydrolysate to a suitable level for a downstream process such as fenneiitatioii maybe reduced.
  • the acid hydrolysate prewas ng is performed under conditions (e.g., temperature, pressure, pH, etc.) in which no or substantially o hydrolysis of me biomass occurs.
  • the pH of the acid hydrolysate that is used to prewash biomass is about 1 to about 2.
  • the process can cause an increase in pH to about 3 to about 4. depending on the washing conditions. No appreciable, eg., less than 10% of xylose yield, hydrolysis occurs within the washing step, owing to the lower reaction temperature, and relatively shorter contact time.
  • the acid hydrolysate that is used to prewash biomass is generated in a downstream process in which another portion of biomass is hydrolyzed by one or more acid.
  • the biomass that is hydrolyzed to provide acid hydrolysate for prewashiiig fresh biomass is generated either from prewashed biomass, as described herein, or fiom unwashed biomass.
  • the biomass that is hydrolyzed to provide acid hydrolysate for prewashing fresh biomass may be the same or different type of biomass as the biomass that is washed with the hydiOlysate.
  • acid hydiOlysate washed biomass is hydrolyzed with one or more acid(s) to produce an acid hydrolysate, at least a portion of which is then used to wash fresh biomass.
  • acid hydrolysis of biomass, washing of fresh biomass with the acid hydrolysate, and recycling of acid hydrolysate for the washing process are conducted in a continuous process. In other ernbcdiments, batch processes are employed.
  • Acid hydrolysate washed biomass may be contacted with, fresh acid and treated under conditions sufficient to depolymerize at least one polymeric carbohydrate component (e.g., hemicelHose, and optionally cellulose) from the biomass, thereby producing: (i) an acid hydrolysate that contains soluble sugar molecules; and (ii) residual solids.
  • at least one polymeric carbohydrate component e.g., hemicelHose, and optionally cellulose
  • residual solids Typically, less acid is required for the depoiymerization from acid hydrolysate washed biomass tha from the same biomass that has not been prewashed, when both the washed and unwashed biomass are treated with acid under identical conditions.
  • about 20% to about 50% less acid is required for depoiymerization of at least one polymeric component (e.g.. hemicelhilose. and optionally cellulose) from acid hydrolysate washed biomass.
  • at least one polymeric component e.g.. hemicelhilose. and optionally cellulose
  • less base is required to raise the pH of the acid hydrolysate to a level that is suitable for microbial fermentation ( .g., about 4.5 to about 7) than a hydrolysate that is produced from the same biomass that has not been prewashed, when both the washed and unwashed biomass are treated with acid under identical conditions.
  • about 20% to about 40% less base is required to adjust the pH of acid hydrolysate from acid hydrolysate washed biomass to a level that is suitable for microbial fermentation, hi some embodiments of the methods disclosed herein, reduction in acid required for hydrolysis of biomass or base required for neutralization of hydrolysate may reduce total dissoived solids, facilitating downstream wastewater treatment.
  • At least a portion of the acid hydrolysate produced by acid hydrolysis of a portion of biomass is used to prewash fr esh biomass.
  • about 65% to about 85% of hydrolysate that is produced from acid hydrolysis of a portion of biomass is used to prewash fresh biomass (e.g., another portion of biomass), although more or less acid hydrolysate may be used depending on various factors such as the amount of biomass hydrolyzed, the amount of biomass to be prewashed, the acid hydrolysis conditions used, or the volume ofhydrolysate produced.
  • Nonh iitmg examples of acid hydrolysate production and biomass prewash processes are shown hi Figure 2 for bagasse and Figure 3 for rice straw, and in Figures 4 and 6, which show acid hydrolysate production and biomass prewash processes exemplified in Examples 1 and 2, respectively.
  • the biomass to be prewashed is subjected to one or more upstream processes prior to washing with acid hydrolysate.
  • the biomass may be deconstructed prior to prewashing with acid hydroiysate.
  • decoiistraciioa may include mechanical disintegration in the presence of water aid under pressure, thereb producing liquid and/or vapor and disintegrated b omass solid.
  • mechanical disintegration may be performed at a pressure and residence time sufficient to shear- apart the biomass to render the carbohydrate polymers therein more accessible for acid-catalyzed depolymerizaiioii.
  • mechanical disintegration may include particle size reduction of the biomass.
  • one or more substance(s) may be removed from the biomass prior to washing with acid hydroiysate.
  • a high silica lignocel ulosic biomass such as, but not limited to, rice straw, wheat straw, rice husks, and or com stover, may be subjected to alkaline extraction of at least a portion of the silica prior to washing with acid hydroiysate.
  • a method for de-sili eating feedstock in which pre-puiping and low-consistency refining steps are used in conjunction with alkaline extraction, is described in U.S. Patent No. 7,364,640.
  • biomass may be washed in a single stage with acid hydroiysate, or may be washed in multiple stages with acid hydroiysate and optionally one or more other liqnid(s).
  • effective performance may be achieved in contactor systems in which the liquid (acid hydroiysate) and solids (biomass) are brought into intimate contact in a well-mixed environment and subsequently effectively separated into a liquid containing stream (with low suspended solids) and a relatively lower moistur e solids fraction.
  • biomass is washed in a coiitaetor/udixer system
  • the contactor/mixer is a tornado pulper.
  • the contactor/mixer is a multi-stage mixer (e.g., a mixer in which there is one unit operation with multiple parts), such as a multi-stage paddle mixer, hi other embodiments, existing biomass and liquid contacting devices may be modified to facilitate the mixing and separation of the biomass and acid hydroiysate.
  • One such embodiment includes the use of a sugarcane diffuser (e.g., currently used for the extraction of sugarcane juice from sugarcane fiber) and associated roller mill equipment for the treatment of biomass (e.g., bagasse) with acid hydroiysate.
  • a sugarcane roller mill or series of roller mills
  • associated equipment may be used.
  • Additional non-limiting examples include ribbon blending equipment, inclined screw mixers (e.g., a screw inside a cylinder where the cylinder is angled (so excess liquid flows)), roller drams (e.g., such as a sprayed bagasse washer (e.g., a rotating drum, where biomass is transferred axially while the druin rotates with water being sprayed onto the biomass and excess water drains down and can be recirculated) ⁇ , a sugar- beet diffiiser (e.g. , water is fed from top down and solids are conveyed 'upward'), or a conveyor belt with a sprayer.
  • inclined screw mixers e.g., a screw inside a cylinder where the cylinder is angled (so excess liquid flows)
  • roller drams e.g., such as a sprayed bagasse washer (e.g., a rotating drum, where biomass is transferred axially while the druin rotates with water being sprayed onto the biomass
  • Other devices may be used, for example, any industrial device in which solids and liquid are contacted and then solids and liquids are separated.
  • Batch systems in which solids and liquid are added to a tank, and the liquid is drained may also be used (such as. for example, batch systems that have been used in the pulp and paper- industry).
  • materials, configurations, and/or operating parameters may be altered and/or adapted for use in the methods described herein, and are not limited to materials, configurations, and/or operating conditions of previously known systems.
  • biomass may be washed with water and or vinasse and/or process condensate (e.g., from an evaporator), e.g., in multiple unit operations in series, prior to washing with acid hydrolysate, optionally in a contactor/mixer system such as, for example, a tornado pulper or a multi-stage paddle mixer.
  • a contactor/mixer system such as, for example, a tornado pulper or a multi-stage paddle mixer.
  • biomass is conveyed into a multi-stage contactor/mixer system such as a paddle washer, where it is contacted with water, then vinasse, and then acid hydrolysate.
  • Different sequences and/or liquids may be used prior to the acid hydrolysate wash i other embodiments.
  • the washed biomass is conveyed to a pretreamient reactor, where it is liydrolyzed with acid, and then at least a portion of the liquid acid hydrolysate is used to wash another portion of unwashed biomass.
  • a biomass feedstock is a substance that provides the ba.se material from winch sugar molecules are generated.
  • Feedstock used in the methods described herein contains celhilose and kemicelhilose.
  • T e material may contain cellulose and iiemicellulose with or without lignin.
  • the feedstock is lignocellulosic biomass, which contains Iiemicellulose, cellulose, and lignin.
  • Cellulose which is a ⁇ -ghrcan built up of D-glucose units linked by ⁇ (1 ,4)- glycosidic bonds, is the main structural component of plant cell walls and typically constitutes about 35-60% by weight (%w/w) of lignocellulosic materials.
  • Hemicellulose refers to non-ceilittosie polysaccharides associated with cellulose in plant tissues.
  • Henikellulose frequently constitutes about 20-35% w w of iigtiocellutosic materials, and the majority of hemieelluloses consist of polymers based on pentose (five- carbon) sugar units, such, as D-xyiose and D-arabinose units, hexose (six-carbon) sugar units, such as D-ghieose and D-mannose units, and uronic acids such as D-glucuranic acid, f 70J Lignin, which is a complex, cross-linked polymer based on variously substituted p- hydroxyphenylpropaiie units, typically constitutes about 10-30% w/w of lignocellulosic materials.
  • the biomass feedstock is bagasse (e.g. , sugarcane or sorghum bagasse), cane trash (e.g., straw), rice straw, rice husks, empty fruit bunches, wheat straw, barley straw, com stover, switchgrass, palm biomass (e.g. , chips, empty fruit bunches, fronds). Kenaf, energy cane, wood chips and/or pulp, municipal solid waste, rapeseed, mustard, canofa straw, beet pulp, cassava pulp, or energy cane.
  • the feedstock contains grass, for example, sugar cane, miscanihus, and'Or switchgrass, and or straw, for example, wheat straw, barley straw, and or rice straw.
  • an amount of feedstock that is used in a method disclosed herein is calculated as dry weight of biomass.
  • the feedstock is an agricultural residue.
  • bagasse may be used as the feedstock. Bagasse is the residual fiber generated as part of the sugar extraction process from sugarcane or sorghum, for example, in a sugar mill or biorefiiiery. Bagasse contains hemicellulose, cellulose, lignin, and some residual sugars. In some embodiments, bagasse may contain residual sucrose that was not removed during sugar cane processing.
  • Residual sucrose may be extracted along with hemicellulose sugar s in a method disclosed herein and during acid hydrolysis, the sucrose may be hydrolyzed to glucose and fructose, which will be included in the soluble sugar molecules in the hydrolysate, in addition to sugar molecules extracted from hemicellulose and cellulose carbohydrate polymers. In some embodiments of the methods disclosed herein, at least a portion of residual sucrose may be removed into the acid hydrolysate that is used for prewashing the biomass.
  • a portion of the residual sucrose is removed into the acid hydrolysate that is used for prewashing the biomass and a portio of the residual sucrose remains with the solid biomass material, and at least a portion of the sucrose that is retained with the solid biomass material is hydrolyzed to glucose and fructose in a downstream hydrolysis process.
  • a high silica bgnocellulosic bioniass is used as die feedstock. Nominating examples of high silica bioniass ..include rice slraw, wheat straw, rice husks, sugar cane bagasse and straw, and com stover. In .some embodiments, silica is removed, for example, via alkaline extraction, prior to prewashnig with acid hydroiysate as described herein.
  • the feedstock is a hgnocellulosic material in the form of wood chips, sawdust, saw mill residue, or a combination thereof
  • the hgnoeeilulosie material is from a feedstock source that has been subjected to some form of disease in the growth and/or harvest production period.
  • the feedstock source is mountain pine beetle infested pine.
  • the feedstock source is sudden oak death syndrome infested oak, e.g. , coastal live oak, tanoak, etc.
  • the feedstock source is Dutch elm disease infested elm. In other
  • the feedstock source is lignoceilulosic material that has been damaged by drought or fire.
  • Lignoceilulosic bioniass may be derived from a fibrous biological material such as wood or fibrous plants.
  • suitable types of wood include, but are not limited to, spruce, pine, hemlock, fir, birch, aspen, maple, poplar, alder, salix, cottoiiwood, rubber tree, marantii, eucalyptus, sugi, and aease.
  • suitable fibrous plants include, but are not limited to, corn stover and fiber, flax, hemp, cannabis, sisal, hemp, bagasse, straw, cereal straws, reed, bamboo, mischantus, kenaf, canary reed, Phalaris aiiindinacea, and glasses.
  • lignoceilulosic materials may be used such as herbaceous material,
  • plant residues include, but are not limited to, stems, leaves, hulls, husks, cobs, branches, bagasse, cane trash, fronds, wood chips, wood pulp, wood pulp, and sawdust.
  • Aquatic plants such as kelp, algae, lily, and hyacinth, which contain proportionately hi gher levels of hemicellulose, can also be used.
  • lignoceilulosic materials may be byproducts of other bioniass industries such as soybean meal from soy oil extraction, rapeseed meal from rapeseed oil processing, empty fruit bunches from palm oil processing, or palm kernel meal from palm kernel oil extraction.
  • Biomass feedstock is pretreated with an acid hydrolysis process, to generate acid hydrolysate that is used for prewashing add ional biomass and/or to provide soluble sugar molecules for fermentation and bioproduct production by a microorganism.
  • Acids that may be used for hydrolysis include, but are not limited to, nitric acid, formic acid, acetic acid, phosphoric acid, hydrochloric acid, sulfuric acid, sulfurous acid, , or any combination thereof.
  • Any acid concentration may be used that is suitable for depolyrnerization of sugar molecules from at least one polymeric component and thai will produce soluble sugar molecules that will support a microbial fermentation process.
  • an acid ⁇ e.g. , nitric acid
  • nitric acid may be used for hydrolysis at a concentration of about 0.1% (w/w) to about 8.5% (w/w), for example, any of about 0.2% to about 1.5%, about 1.5% to about 3.0%, about 3.0% to about 4.5%, about 5.0% to about 6.5%, or about 6.5% to about 8.5%, or any of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%. 0.9%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, or 8.5%.
  • hernicelmlose and optionally some cellulose may be depolymeiized from the biomass material, and the hydrolysate contains soluble sugar molecules from hemicellulose and optionally some sugar molecules from cellulose.
  • Liquid hydrolysate that includes soluble sugar molecules ⁇ e.g. , from
  • depolyrnerization of hemicellulose ma y optionally be separated from residual solids prior to inclusion in a fermentation medium.
  • Residual solids remaining after acid hydrolysis of biomass as described herein may be subjected to one or more downstream processes.
  • the residual solids may be further hydrolyzed to release additional soluble sugar molecules (e.g., from depolyrnerization of cellulose) and/or may be used as a fuel source, e.g. , as fuel for a boiler and/or for electricity generation.
  • residual solids may be hydrolyzed in one or more process, including, but nest limited to acid hydrolysis, enzymatic hydrolysis, or supercritical fluid hydrolysis, hi embodiments in which residual solids are used as a fuel somce, removal of ash by prewashing biomass material with acid hydrolysate (de-ashing) may improve the burning characteristics of the residual solids.
  • further hydrolysis of residual solids may be conducted with one or more enzyme that is capable of depolymerizing cellulose ⁇ e.g. , hydrolysis of 1.,4-beta-D-glycosidic linkages in cellulose.
  • enzymes capable of depolymerizing cellulose ⁇ e.g. , hydrolysis of 1.,4-beta-D-glycosidic linkages in cellulose.
  • enzymatic hydrolysis may be perfomied with one or more cellulase enzyme(s).
  • NonHrnitmg examples of cellulase enzymes include endoeelliilases, exoeeilulases, eellobiases, oxidative cellulases, and cellulose phosphoiylases,
  • hydrolysis of residual solids may be conducted with acid, e.g. , under conditions suitable for depolynierizaiion of cellulose.
  • acid e.g., nitric acid
  • hydrolysis is perfomied with an acid, e.g., nitric acid, at a concentration of about 0.05% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 1%, about l% to about 4%, about 1.3% to about 3.5%, or about 1.3% (w/w of dry feedstock) at a temperatee of about 190° to about 230°C, and at the saturation pressure for steam at the reactor temperatee.
  • an acid e.g., nitric acid
  • solids may optionally be separated from liquids to produce a hydrolysate and residual solids hi a screw press, belt filter press, roller press, centr ifuge, settling tank, vacuum filter, sieve screen, or rotary dram dryer.
  • the hydrolysate from the second hydrolysis e.g. , hydrolysate containing depolymerized cellulose from residual solids
  • the hydrolysate from the first hydrolysis e.g. , hydrolysate containing depolymerized hemicel tlose from biomass
  • the hydrolysate from the second hydrolysis e.g. , hydrolysate containing depolymerized cellulose from residual solids
  • the hydrolysate from the first hydrolysis e.g.
  • hydrolysate containing depolymerized heniiceliulose f om biomass are separately fed to separate bioreactors for production of one or more bioproduct (s) of interest.
  • the separate bioreactors may contain the same or different macOorganisms and may produce the same or differeni bioproduct(s).
  • the first hydrolysate is fed to a bioreactor that contains a microorganism that is optimized for growth in the presence of this hydrolysate (e.g., hydrolysate containing C5 and C6 sugar molecules), and the second hydrolysate is fed to a bioreactor than contains a microorganism that is
  • this second hydrolysate e.g., hydrolysate containing C6 sugar' molecules.
  • the solid residue is prepared for use hi other downstream processes, such as burning for fuel or papemiakmg.
  • Solid residue mat has been prepared from
  • IS de-ashed biomass prepared as described herein by prewasliin biomass with acid hydrolysate, and consequent removal of at least a portion of ash into the hydrolysate that is used for prewasliing, may have advantageous characteristics as a fuel.
  • acid hydrolysate washed biomass from which, ash lias been removed e.g., biomass that has been washed with acid hydrolysaie as described herein
  • hyekolysis e.g., biomass that has been washed with acid hydrolysaie as described herein
  • die residual solids remaining after hydrolysis have higher heat value and'or less formation of ash in a conibustoi. or toiler than the same biomass that has not been acid hydrolysate washed, when both the washed and unwashed biomass are hydrolyzed under identical conditions.
  • Hie acid hydrolysate washed biomass may be processed in one or more downstream operations.
  • acid hydrolysate washed biomass may be subjected to acid
  • hydrolysis e.g., hydrolysis by acetic acid released frora biomass
  • enzymatic hydrolysis or hydrolysis with a supercritical fluid.
  • a stream of acid hydrolysate thus produced ma be used to wash f!irther biomass .
  • the acid hydrolysate that contains one or more non- carbohydrate compounds from the biomass may be used, for example, for microbial fermentation to produce one or more bioproduct(s) of interest, and'or may be reused to wash further unwashed biomass.
  • hydrolysaie is "conditioned" to remove inhibitors of microbial growth and/or bioproduct production and ' or to adjust one or more parameters of the hydrolysate to render it more suitable for addition to a microbial growth medium, for example, adjustment of pH and ' or temperature to a physiologically acceptable level for growth of a microorganism when added to microbial growth medium.
  • acid hydrolysate that has been used for prewashing biomass. as described herein is conditioned after the prewashing and before inclusion in a microbial fermentation medium.
  • conditioning includes evaporation, steam stripping, charcoal adsorption, ion exchange resin treatment., eieetrodiaiysis, and'or reverse osmosis.
  • a biomass hydrolysate is rendered fermentable, ie. suitable for microbial fermentation, after raising the pH to a physiologically acceptable level for growth of a particular microbial culture, for example, from the pH of the hydrolysate after acid hydrolysis (e.g. , about pH 1.7) to about pH 6 to pH 7, or about pH 5 to about pH 7 (e.g., about 6.7).
  • no further conditioning processes are required, other than the pH adjustment, for the hydrolysate to support microbial growth and/or bioproduct production (i.e., treatment of the hydrolysate to remove microbial growth and/or fermentation inhibitors is not required).
  • raising t e pH may result in deprotonation of certain organic acid inhibitor compounds, rendering them less inhibitory.
  • conditioning processes are included for removal of inhibitors from the hydrolysate.
  • Inhibitors of microbial growth and/or bi ⁇ product production may include, but are not limited to. organic acids, furans, phenols, soluble lignocellulosic materials, extractives, and ketones.
  • Inhibitors present in bydrolysates may include, but are not limited to, 5-hydroxyy-niethyl ftirfural (HMF), furfural, aliphatic acids, levulinic acid, acetic acid, foiinic acid, phenolic compounds, vamllin, dmyckoconiferylalcohoi, coniferyl aldehyde, vanillic acid, hydioquiiioiie, catechol, acetognaiacone, hornovanillic acid, 4- hydroxy-beiizoic acid, Hibbert's ketones , ammonium nitrate and/or other salts, -coumaric acid, ferulic acid, vanillic acid, syringaldehyde. sinapyl alcohol, and glucuronic acid.
  • HMF 5-hydroxyy-niethyl ftirfural
  • furfural aliphatic acids
  • levulinic acid
  • microbial growth and or bioproduct titer, yield, and/or productivity is increased when conditioned hydrolyzed feedstock is used, in comparison to identical hydrolyzed feedstock winch has not been subjected to the conditioning process.
  • a microorganism that is tolerant to inhibitors in hydrolyzed feedstock is used, or the rnicroorganism used for bioproduct production develops increased tolerance to inhibitors over time, e.g. , by repeated passaging, rendering the conditioning step unnecessary or uneconomical.
  • Methods are provided for producing one or more bioproduct(s) of interest in a microbial fermentation.
  • the methods include cultuihig a microorganism that produces the bioproduct of interest in a medium that contains soluble sugar molecules produced from biomass (e.g. , in an acid and/or enzymatic hydrolysate of biomass) to support microbial growth for production of one or more bioproduct(s) of interest.
  • biomass e.g. , in an acid and/or enzymatic hydrolysate of biomass
  • fermentable sugar molecules that are generated from biomass that has been piewashed with acid bydrolysate, as described herein, are included in the consumeriitation medium for
  • the bioproduct is a biofuel, for example, butanol, acetone, and/or ethaool.
  • the bioproduct is solvent (e.g. , a polar protic or aprotic solvent), biomoiecule, organic acid, alcohol, fatty acid, aldehyde, lipid, long chain organic molecule, vitamin, or sugar alcohol.
  • the bioproduct is a solvent or organic acid.
  • the methods for bioproduct production herein include fermentation with a bioprodiict-producing microorganism in a bioreactor in a growth medium that contains liquid sugar-containing extract from biomass, such as a bydrolysate or conditioned iiydrolysate of biomass.
  • a liquid sugar containing extract such as cane juice and/or molasses is included in the growth medium.
  • the bioproduct production includes fermentation with a bioproduct-prochicing microorganism in an immobilized cell bioreactor (i.e.. a bioreactor containing cells that are immobilized on a support, e.g., a solid support),
  • an immobilized cell bioreactor provides higher productivity due to the accumulation of increased productive cell mass within the bioreactor compared with a stiixed tank (suspended cell) bioreactor.
  • the microbial cells form a biofiim on the support and/or between support particles in the growth medium.
  • microorganisms may be grown in a non-immobilized system, such as an agitated
  • fermentation reactor e.g., designed to provide adequate conditions for fermentation, including but not limited to mixing of components, gas removal, temperature control, and or the ability to add and/or remove material from the reactor.
  • fermentation operational moieties exist, including but not limited to batch, fed-batch, and continuous in single or multiple reactor configurations.
  • Exemplar reactor types include but are not limited to agitated tanks, e.g., where agitatio is effected by a mechanical impeller, the addition and withdrawal of material, the addition of gas, and/or the recirculation of fermentation gas; com and/or cane ethanol fermentation tanks: pharmaceutical fermentation vessels; vacuum fermentation systems; air-lift type reactors; fmidized bed reactors; anaerobic digesters; and activated sludge reactors, hi some embodiments, an extractive fermentation process is used (e.g. gas stripping, liquid extraction, vacuum fermentation, extraction by absorption and/or adsorption by a solid material such as a polymeric material).
  • an extractive fermentation process is used (e.g. gas stripping, liquid extraction, vacuum fermentation, extraction by absorption and/or adsorption by a solid material such as a polymeric material).
  • the bioproduct production process herein includes continuous fermentation of a inicroorganism (eontinuoiis addition of conditioned hydrolyzed feedstock and withdrawal of product stream). Continuous femientation minimizes the unproductive portions of the femientatio cycle, such as lag, growth, and turnaround time, thereby reducing capital cost, and reduces the number of inoculation events, thus minimizing operational costs and risk associated with human and process error.
  • Fermentation may be aerobic or anaerobic, depending on the requirements of the bioproduet-proditcing microorganism.
  • fermentation media in addition to an appropriate carbon source, fermentation media must contain suitable nitrogen souree(s). mineral salts, cofactors, buffers, and other components suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for the production of die desired bioproduct. i some embodiments, salts and/or vitamin B12 or precursors thereof are included in the fermentation media.
  • hydrolyzed biomass e.g. , bagasse and or cane straw
  • microorganism that is capable of producing one or more bioproduct(s) of interest is used in the fermentation methods described herein.
  • the microorganisms may be the same or different microbial species and'or different strains of the same species.
  • the microorganisms are bacteria or fungi. In some embodiments, the microorganisms are a single species, hi some embodiments, the
  • mia'oorganisms are a mixed culture of strains from the same species.
  • the microorganisms are a mixed culture of different species. In some embodiments, the microorganisms are an environmental isolate or strain derived therefrom.
  • Biomass Sugarcane bagasse was reduced in size to less than one centimeter by knife miiling. 1110] Neutralization (buffering) effect; A 100 g bagasse/deionized water mixture was prepared from 2.5 dry grams of bagasse. Three drops of I KCl was added to the bagasse/water mixture by small diameter pipette to increase the conductivity for pH measurement, and the pH of the ' bagasse/water mixture was measured with a portable pH meter. Two hundred micro liters of 2 wt% HNO3 was added in increments to the
  • Acid hydrolysis (pretreatment) reactor A 1-inch diameter x 8-inch length tubular stainless steel reactor with flange sealing on each end was used, heated in a sand bath, f 112] Acid hydrolysis and washing of bagasse with acid hydrolysate: This process is shown schematically in Fig. 4. Bagasse (about 10 g dry bagasse) and acid were well mixed in a beaker before loading into the reactor. The pH before pretreatment was 1.22. Bagasse was hydrolyzed with 0.028 g nitric acid/g unwashed dry solids at 20 wr% solids
  • Cycle #1 started with washing (de-ashing) fresh bagasse with the hydrolysate (liquid 1) produced Cycle #0.
  • Ten dry grams of bagasse were mixed with liquid 1 at room temperature for 15 minutes.
  • Cheesecloth and manual pressing was used to separate liquids from solids.
  • 400 .grams of water (water 3) was used to wash the de-ashed ba gasse, producing liquid 2.
  • Fig. 5 shows the concentration of See hydrogen ion in bagasse-acid mixtures as a function of acid loading.
  • unwashed bagasse (“Fresh bagasse 1" in Fig, 4) was used, approximately 5.5 mg of nitric acid was .neutralized by the ash in one gram of bagasse (the offset in pH response in Fig. 5).
  • the hydrolysate de-ashed bagasse was vised, adding nitric acid to the mixture led to an immediate and nearly linear increase of free hydr ogen ion concentration. No offset in pH response was observed, indicating little neutralizing capacity.
  • Knife milled rice straw ( ⁇ 3mm; 85% dry matter content) and nitric acid solution (0.035 g nitric aeid/g rice straw) were well mixed i a 1000 mL beaker .
  • the mixture was loaded into four 1 inch diameter by 8 inch length stainless steel tubular reactors with flanges sealing at each end. Each reactor was loaded with 50 grams of rice straw/acid mixture at a solids concentration of 20% (g/g).
  • a fhiidized sand bath (SBL-2D, Teehne, Princeton, NJ) was used as the heating source for the pretreatnient reactors.
  • the sand bath was preheated to 2°C higher tha the desired pretreatnient temperature.
  • the sand bath temperature controller was set to the desired pretreatnient temperature.
  • the reactors were then submerged in the sand bath. Due to the cold reactor bodies, the sand bath temperature dropped by about 5°C in 2 minutes, but stabilized to the set level in about another 3-5 minutes. It took approximately 5 minutes for the reactor centers to reach 5°C from the desired point. (The cold reactors were placed in the hot sand bath. The reactor outer wall was heated up first, and it took time for the heat to transfer from the reactor outer wall to the center of the biomass bed.
  • Cycle #1 started with washing rice straw (Fresh rice straw 1) with acid hydrolysate from Cycle #0 (C5 liquid 0).
  • the feedstock rice straw was mixed with C5 liquid 0 at a ratio of 3.59 g liquid dry rice straw at room temperature for 10 m i.
  • the acid hydrolysate washed rice straw was then washed with 50 niL deionized water/g dry rice straw to remove soluble sugars and acid that were carried by the hydrolysate.
  • the acid hydrolysate washed rice str aw 1 was mixed with fresh acid solution as well as some C5 liquid 0 at a ratio of 0.228 g C5 liquid g dry rice straw. (It was estimated that 6.36% of the hydrolysate from the previous pretreatment was earned over to the next pretreatment due to incomplete washing of the soluble sugars from the solids, which is equivalent to 0.228g hydiolysate g rice straw.)
  • the mixture was loaded into two reactors for pretreatment using the same procedure as described above for Cycle #0. After pretreatment, the two reactors were unloaded and s iny 1 was collected.
  • Buffering capacity measurement To measure the buffering capacity. 2.5 g (dry matter) of rice straw was mixed with 97.5 g deionized water to make 2.5% solids mixture. Three drops of 3M KI was added to the mixture to increase the conductivity for pH
  • Dilute nitric acid solution (5 wt% was added to about 100 mL of the mixture in increments of 200 ⁇ or 500 ⁇ .
  • the mixtur e was mixed for 30 seconds and then pH was measured and recorded at room temperature. Free hydrogen ion values were calculated from pH.
  • Fig. 7 shows the concentration of free hydr ogen ion concentration in the liquid phase of rice straw slurry (2.5 wi.% solids) as a function of acid loading.
  • the acid hydroiysate washed rice straw in this figure was feedstock that was mixed with acid hydroiysate (C5 liquid 0) and then thoroughly washed with deionized water, as described above.
  • the intercepts of the dashed lines with the x-axis were read as buffering capacities, which are shown in Table 2.
  • C6 ghteose
  • C5 x lose oiaiinose/ ' galactose (XMG) + arabmose
  • Total gittceset- XMG ⁇ arabinose
  • Knife milled rice straw ( ⁇ 3mm; 85% w/w dry matter content) and deionized water were mixed at 10.5% total solids loading and incubated at 55° C, 1 5 rpm for 1 hour to extract soluble sugars. Liquid was the ' separated from residual solids through cheese cloth. Solids were further pressed to a dry matter content of approximately 50% w/w and more liquid was collected. All collected liquid was combined and filter ed though 0.7 um filter paper to remove insoluble solids. The resulting liquid is termed "soluble sugar solution" in following experiment.
  • the temperature in the center of the reactor reached 5°C from the set level within 5 mm.
  • the pretreatment conditions in this experiment were 145° C. 35 mm (not including the 5 min ramp up time).
  • Cycle #1 For 10% carry-over sugar, in Cycle #1, depicted schematically in Fig. 8, deashed rice straw was mixed with fresh acid solution as shown in Table 5, C5 Liquid 0 at ratio of 0.36g liquid'g deashed rice straw, and soluble sugar- solution at a ratio of 0.9 g liquid ' g deashed rice straw. Mixing was performed by hand in a beaker. The mixture was then loaded into a reactor for pretreatment using the same procedure as described above for Cycle #0. After pretreatment, the reactor was unloaded and "Slurry 1" was collected. Thirty grams of Slimy 1 was saved for enzymatic hydrolysis, and the remaining Slimy 1 was pressed to collect hydroiysate ("C5 Liquid I"). The C5 Liquid 1 hydroiysate was filtered through ⁇ . ⁇ filter paper to remove insoluble solids. This completed Cycle #1. Cycles #2 and #3 were performed in the same mariner as Cycle #1 , and as shown schematically in Fig, 8.
  • a hutariol-producmg Clostridium strain was used in a femienia ioii test.
  • 15mL seed media 60 g 1 sugar from molasses, and other growth nutrients
  • Tlie medium was de-oxygenated fo at least 24hr.
  • Tlie solution was swirled gently and allowed to incubate hi the anaerobic chamber overnight When culture was at appropriate growth conditions, the seed culture was used for inoculation.
  • Enzymatic hydrolysate prepared as described above, was subjected to solid-liquid separation by eentrifiigatioii. Liquid was then filtered through 0.2 ⁇ filter hi a laminar hood for sterilization. Hydrolysate growth media was prepared under laminar hood to contain 55 g L total sugar, with 0 g L from hydrolysate and 5 g/L from molasses, and other .growth nutrients.

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Abstract

L'invention concerne des procédés et des systèmes de préparation d'une biomasse pour des opérations de prétraitement en aval afin de libérer des molécules de sucre. La biomasse est prélavée avec un hydrolysat acide avant le prétraitement. Cela permet de réduire considérablement la quantité de produits chimiques qui sont nécessaires pour traiter de façon efficace une biomasse pour des utilisations en aval.
PCT/US2014/072278 2014-03-18 2014-12-23 Préparation de biomasse WO2015142399A1 (fr)

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