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WO2013019822A1 - Elimination d'inhibiteurs de la fermentation microbienne d'hydrolysats cellulosiques ou d'autres compositions contenant un inhibiteur - Google Patents

Elimination d'inhibiteurs de la fermentation microbienne d'hydrolysats cellulosiques ou d'autres compositions contenant un inhibiteur Download PDF

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Publication number
WO2013019822A1
WO2013019822A1 PCT/US2012/049084 US2012049084W WO2013019822A1 WO 2013019822 A1 WO2013019822 A1 WO 2013019822A1 US 2012049084 W US2012049084 W US 2012049084W WO 2013019822 A1 WO2013019822 A1 WO 2013019822A1
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Prior art keywords
hydrolysate
acid
inhibitor
bioproduct
conditioned
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English (en)
Inventor
Jeffrey D. Keating
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COBALT TECHNOLOGIES Inc
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COBALT TECHNOLOGIES Inc
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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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • 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
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
    • 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 a method for removing or inactivating inhibitors of microbial growth and/or metabolite production from an inhibitor-containing composition such as a hydrolysate of cellulosic biomass, in particular by precipitation or inactivation with a combination of calcium hydroxide and potassium hydroxide.
  • a carbon source for such cultures is often provided by hydrolysis of lignocellulosic biomass materials. Soluble sugar molecules released by hydrolysis may be used to support microbial growth.
  • Hydrolysates of cellulosic materials often contain inhibitors of microbial growth and/or metabolite production, which reduces the amount of product produced in a culture that includes such a hydrolysate.
  • conditioning of hydrolysates to remove inhibitors prior to addition of the hydrolyzed material to microbial growth medium.
  • conditioning processes include vacuum or thermal evaporation, overliming, adsorption, enzymatic conditioning (e.g., peroxidase, laccase), chemical conversion (e.g., oxidation, reduction, sequestration), distillation, and ion exchange.
  • Methods for conditioning a composition that contains at least one inhibitor of microbial growth and/or bioproduct production and at least one compound suitable for use as a carbon source, such as a hydrolysate of a cellulosic material are provided.
  • Conditioned compositions, such as conditioned hydrolysates, prepared by the methods described herein are also provided, as well as methods for producing a bioproduct in a fermenting microorganism using the conditioned compositions described herein as a carbon source.
  • a method for removing at least one (i. e. , one or more) inhibitor of microbial growth and/or microbial bioproduct production (referred to as "an inhibitor" herein) from a composition that contains at least one inhibitor and at least one compound suitable for use as a carbon source in a microbial fermentation process, such as, for example, a hydrolysate of cellulosic biomass that contains inhibitor(s) and sugar molecule(s).
  • the method includes contacting the hydrolysate with calcium hydroxide and potassium hydroxide. At least a portion of at least one inhibitor forms a precipitate in the presence of the calcium hydroxide and potassium hydroxide. The precipitate is separated from the liquid composition, e.g., liquid cellulosic hydrolysate, thereby forming a conditioned composition.
  • At least a portion of one inhibitor or of each of a multiplicity (i. e. , two or more) of inhibitors forms a precipitate in the presence of the calcium hydroxide and potassium hydroxide.
  • substantially all of at least one inhibitor forms a precipitate.
  • substantially all of each of a multiplicity of inhibitors form precipitates.
  • a portion of at least one inhibitor and substantially all of at least one other inhibitor form precipitates.
  • the calcium hydroxide and potassium hydroxide may be added simultaneously or sequentially to the inhibitor-containing composition, e.g., cellulosic hydrolysate.
  • the calcium hydroxide and potassium hydroxide may be added sequentially to the composition in either order (calcium hydroxide first and potassium hydroxide second, or potassium hydroxide first and calcium hydroxide second).
  • the calcium hydroxide is added before the potassium hydroxide, and optionally the calcium hydroxide and potassium hydroxide are added to the composition in amounts such that the pH after potassium hydroxide addition is about 1 pH unit higher than the pH after calcium hydroxide addition.
  • calcium hydroxide is added in an amount sufficient to adjust the pH of the composition to about 10 to about 10.9, followed by addition of potassium hydroxide in an amount sufficient to adjust the pH of the composition to about 11.
  • the method is performed at a temperature of about 30°C to about 60°C.
  • the inhibitor-containing composition e.g., cellulosic hydrolysate, calcium hydroxide, and potassium hydroxide are incubated at a temperature of about 30°C to about
  • precipitated inhibitor(s) is(are) separated from the liquid composition, e.g., cellulosic hydrolysate, by filtration, centrifugation, pressing, decantation, and/or dissolved gas flotation.
  • the method may include reducing the pH of the conditioned composition, for example, neutralizing the conditioned composition, and removing further precipitate that forms, if any.
  • the pH may be reduced, e.g., neutralized or adjusted to close to neutral pH, with sulfuric acid or phosphoric acid, or a combination of sulfuric and phosphoric acids.
  • a method for inactivating ⁇ e.g. , reducing or eliminating inhibitory effect of) an inhibitor of microbial growth and/or microbial bioproduct production in a hydrolysate of cellulosic biomass including contacting the hydrolysate with calcium hydroxide and potassium hydroxide, wherein at least a portion of the inhibitor is inactivated in the presence of the calcium hydroxide and potassium hydroxide, thereby forming a conditioned hydrolysate.
  • the calcium hydroxide and potassium hydroxide may be added in amounts that are soluble in the hydrolysate and at which the inhibitor is inactivated.
  • substantially all of the inhibitor is inactivated.
  • a multiplicity of inhibitors are inactivated in the presence of the calcium hydroxide and potassium hydroxide.
  • substantially all of at least one of the inhibitors is inactivated.
  • the calcium hydroxide and potassium hydroxide are added sequentially. In one embodiment, the calcium hydroxide is added before the potassium hydroxide. In some embodiments, the calcium hydroxide and potassium hydroxide are added in amounts such that the pH after potassium hydroxide addition is about 1 pH unit higher than the pH after calcium hydroxide addition. In one embodiment, calcium hydroxide is added in an amount sufficient to adjust the pH of the hydrolysate to about 10 to about 10.9, followed by addition of potassium hydroxide in an amount sufficient to adjust the pH of the hydrolysate to about 11. [15] In one embodiment, the inhibitor-containing composition comprises or consists of a hydrolysate of cellulosic biomass.
  • the cellulosic biomass is a lignocellulosic biomass, for example, softwood or hardwood, or a combination thereof.
  • the lignocellulosic biomass contains beetle killed Lodgepole pine.
  • the lignocellulosic biomass contains mixed hardwoods, for example, mixed U.S. hardwoods, e.g., mixed Northern U.S. hardwoods.
  • the cellulosic biomass contains grass or straw, or a combination thereof.
  • grass include sugar cane, miscanthus, switchgrass, or a combination thereof.
  • straw include wheat straw, barley straw, rice straw, or a combination thereof.
  • the cellulosic biomass contains bagasse, cane trash, seaweed, algae, microalgae, agricultural waste or residue, hyacinth, sorghum, sugar beets, soybean residue, palm oil residue, or pulp mill liquor or effluent, or a combination thereof.
  • a hydrolysate is produced by acid hydrolysis of cellulosic biomass, for example, with nitric acid, formic acid, acetic acid, phosphoric acid, hydrochloric acid, sulfuric acid, or a combination thereof.
  • a hydrolysate is produced by hot water extraction of cellulosic biomass, optionally followed by addition of acid (for example, nitric acid, formic acid, acetic acid, phosphoric acid hydrochloric acid, or sulfuric acid, or a combination thereof) to further hydrolyze carbohydrate polymers.
  • the resulting material may be concentrated by thermal or other means prior to use as a fermentation feed material.
  • the inhibitor-containing composition comprises or consists of glycerol.
  • the composition comprises or consists of dairy effluent, for example, whey which contains lactose.
  • the composition comprises or consists of food waste, or a hydrolysate, e.g.. an acid hydrolysate, of food waste.
  • the inhibitor(s) is(are) selected from organic acids (e.g., acetic, formic, levulinic), aldehydes (e.g., furfural, 5-hydroxymethyl furfural, vanillin), lignins, lignin byproducts or derivatives, phenolic and/or phenylpropanoid compounds, inorganic salts (e.g., sulfates, phosphates, hydroxides), fatty acids, fatty alcohols, fats, waxes, polyesters (e.g., suberin), terpenoids, alkanes, wood extractives, Hibbert's ketones, and proteins, or a combination thereof.
  • organic acids e.g., acetic, formic, levulinic
  • aldehydes e.g., furfural, 5-hydroxymethyl furfural, vanillin
  • lignins lignin byproducts or derivatives
  • the inhibitor(s) include(s) one or more phenolic and/or phenypropanoid compound(s), including but not limited to, ferulic acid, syringaldehyde, syringic acid, vanillin, vanillic acid, 3,4- dihydroxybenzaldehyde, 3,5-dihydroxybenzaldehyde, catechol, sinapic acid, gallic acid, sinapyl alcohol, coniferyl alcohol, hydroquinone, 4-hydroxybenzaldehyde, and/or 4- hydroxybenzoic acid.
  • ferulic acid ferulic acid
  • syringaldehyde syringic acid
  • vanillin vanillic acid
  • 3,4- dihydroxybenzaldehyde 3,5-dihydroxybenzaldehyde
  • catechol sinapic acid
  • gallic acid sinapyl alcohol
  • coniferyl alcohol coniferyl alcohol
  • hydroquinone 4-hydroxybenzaldehyde
  • the inhibitor(s) include(s) one or more of sinapic acid, 3,4-dihydroxybenzaldehyde, coniferyl alcohol, hydroquinone, and catechol. In some embodiments, the inhibitor(s) are selected from sinapic acid, 3,4- dihydroxybenzaldehyde, coniferyl alcohol, hydroquinone, and catechol. In one
  • the inhibitor(s) include sinapic acid. In one embodiment, the inhibitor(s) include 3,4-dihydroxybenzaldehyde. In one embodiment, the inhibitor(s) include coniferyl alcohol. In one embodiment, the inhibitor(s) include hydroquinone. In one embodiment, the inhibitor(s) include catechol.
  • the inhibitor(s) includes formic acid.
  • formic acid is reduced to a level that is not toxic to a bioproduct producing microbial species, for example, a solventogenic microbial species, such as a Clostridium species.
  • growth of a microorganism and/or bioproduct production in the microorganism is higher in the presence of a composition from which formic acid concentration has been reduced or removed in a conditioning method as described herein in comparison to growth and/or bioproduct production in the presence of an identical composition which has not been conditioned according to a method as described herein.
  • the inhibitor(s) include(s) one or more phenolic and/or phenylpropanoid compound(s). In some embodiments, one or more phenolic and/or phenylpropanoid compound(s) is(are) reduced to a level that is not toxic to a bioproduct producing microbial species, for example, a solventogenic microbial species, such as a Clostridium species.
  • growth of a microorganism and/or bioproduct production in the microorganism is higher in the presence of a composition from which phenolic and/or phenylpropanoid compound(s) concentration has been reduced or removed in a conditioning method as described herein in comparison to growth and/or bioproduct production in the presence of an identical composition which has not been conditioned according to a method as described herein.
  • the phenolic and/or phenylpropanoid compound(s) include(s) one or more of sinapic acid, 3,4- dihydroxybenzaldehyde, coniferyl alcohol, hydroquinone, and catechol
  • the composition contains sugar molecules and no more than about 5% to about 10%, or about 10% to about 15%, are degraded in the conditioning methods described herein. In some embodiments, less than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% of the sugar molecules are degraded in the conditioning methods described herein.
  • a conditioned composition such as a conditioned hydrolysate, produced by any of the methods described herein, is provided.
  • a method for producing a bioproduct.
  • the method includes culturing a microorganism in a medium that contains a conditioned composition, such as a conditioned hydrolysate, produced by any of the methods described herein.
  • growth of and/or bioproduct production in the microorganism is greater in the medium that contains the conditioned composition than in an otherwise identical medium that does not contain the conditioned composition, i.e., a medium that is otherwise identical but contains the starting composition from which the conditioned composition was derived in place of the conditioned composition.
  • the bioproduct is produced in a greater amount, at a greater yield, and/or at a greater rate than in an otherwise identical medium that does not contain the conditioned composition.
  • the bioproduct is produced at a greater titer, yield, and/or productivity than in an otherwise identical medium that does not contain the conditioned composition.
  • the bioproduct is a solvent, for example, butanol, ethanol, acetone, isopropyl alcohol, or a combination thereof.
  • the bioproduct is a biofuel, for example, butanol, ethanol, acetone, or a combination thereof.
  • the bioproduct is a biochemical or biochemical intermediate, for example, formate, acetate, butyrate, propionate, succinate, methanol, propanol, hexanol, or a combination thereof.
  • Figure 1 shows the results of an experiment comparing two-stage overliming with calcium hydroxide plus sodium hydroxide or potassium hydroxide, in comparison overliming with calcium hydroxide alone.
  • Figure 2 shows lime input when calcium hydroxide is used in conjunction with potassium hydroxide, in comparison to calcium hydroxide alone.
  • Methods are provided for removal or inactivation of inhibitors of microbial growth and/or product production from compositions that contain such inhibitors and compounds that are suitable for use as a carbon source for microbial fermentation, such as hydrolysates of cellulosic material.
  • the methods provided herein include precipitation of inhibitors with a combination of calcium hydroxide and potassium hydroxide to produce a "conditioned composition" from which at least a portion of at least one inhibitor has been removed or inactivated.
  • Methods are also provided for using the conditioned compositions from which inhibitors have been removed or inactivated to support microbial growth for bioproduct production.
  • conditioning methods herein may be performed at relatively low temperature with minimal sugar loss, and precipitated inhibitors are easy to separate from the liquid composition, with the removal of the precipitates achievable with standard laboratory equipment.
  • Bioproduct refers to any substance of interest produced biologically, . e., via a metabolic pathway, by a microorganism, e.g., in a microbial fermentation process.
  • Bioproducts include, but are not limited to biofuels (e.g., butanol, acetone, ethanol), solvents, biomolecules (e.g., proteins (e.g., enzymes), polysaccharides), organic acids (e.g., formate, acetate, butyrate, propionate, succinate), alcohols (e.g., methanol, propanol, isopropanol, hexanol, 2-butanol, isobutanol), fatty acids, aldehydes, lipids, long chain organic molecules (for example, for use in surfactant production), vitamins, and sugar alcohols (e.g., xylitol).
  • Biofuel refers to fuel molecules (e.g., butanol, acetone, and/or ethanol) produced biologically by a microorganism, e.g., in a microbial fermentation process.
  • Biobutanol refers to butanol (i.e., «-butanol) produced biologically by a microorganism, e.g., in a microbial fermentation process.
  • “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, hydroxymethyl furfural, formic acid), and extractives (described infra).
  • Feestock refers to a substance that can serve as a source of sugar molecules to support microbial growth in a fermentation process.
  • the feedstock must be pretreated to release the sugar molecules.
  • the feedstock, which contains carbohydrate polymers is hydrolyzed to release 5 and/or 6 carbon containing carbohydrate molecules in monomeric and/or soluble oligomeric forms.
  • Deconstruction refers to mechanical, chemical, and/or biological degradation of biomass into to render individual components (e.g., cellulose, hemicellulose) more accessible to further pretreatment processes, for example, a process to release monomeric and oligomeric sugar molecules, such as acid hydrolysis.
  • Constanting refers to removal of inhibitors of microbial growth and/or bioproduct, e.g., biofuel, production from a hydrolysate produced by hydrolysis of a cellulosic feedstock, or rendering of inhibitors less inhibitory or noninhibitory to microbial growth and/or bioproduct production.
  • bioproduct e.g., biofuel
  • Tier refers to amount of a substance produced by a microorganism per unit volume in a microbial fermentation process.
  • biobutanol titer may be expressed as grams of butanol 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 that of the substance that would be produced if all of the feed substance were converted to product.
  • biobutanol yield may be expressed as % of biobutanol produced relative to a theoretical yield if 100% of the feed substance (for example, sugar) were converted to biobutanol.
  • Processivity refers to the amount of a substance produced by a microorganism per unit volume per unit time in a microbial fermentation process. For example, biobutanol productivity may be expressed as grams of butanol produced per liter of solution per hour.
  • Wild-type refers to a microorganism as it occurs in nature.
  • Biomass refers to cellulose- and/or starch-containing raw materials, including but not limited to wood chips, corn stover, rice, grasses, forages, perrie-grass, potatoes, tubers, roots, whole ground corn, grape pomace, cobs, grains, wheat, barley, rye, milo, brans, cereals, sugar-containing raw materials (e.g., molasses, fruit materials, sugar cane, or sugar beets), wood, and plant residues.
  • sugar-containing raw materials e.g., molasses, fruit materials, sugar cane, or sugar beets
  • Starch refers to any starch-containing materials.
  • the term refers to various plant-based materials, including but not limited to wheat, barley, potato, sweet potato, tapioca, corn, maize, cassava, milo, rye, and brans.
  • the term refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose, and amylopectin, with the formula (C 6 H 10 O5) x , wherein "x" can be any number.
  • ABE fermentation refers to production of acetone, butanol, and/or ethanol by a fermenting microorganism.
  • Advanced biofuels are high-energy liquid transportation fuels derived from low nutrient input/high per acre yield crops, agricultural or forestry waste, or other sustainable biomass feedstocks including algae.
  • “Lignocellulosic” biomass refers to plant biomass that contains cellulose, hemicelluloses, and lignin.
  • the carbohydrate polymers cellulose and hemicelluloses are tightly bound to lignin.
  • solvent refers to a liquid or gas produced by a microorganism that is capable of dissolving a solid or another liquid or gas.
  • solvents produced by microorganisms include «-butanol, acetone, ethanol, acetic acid, isopropanol, n-propanol, methanol, formic acid, 1,4-dioxane, tetrahydrofuran, acetonitrile, dimethylformamide, and dimethyl sulfoxide.
  • a "protic" solvent contains dissociable H + , for example a hydrogen atom bound to an oxygen atom as in a hydroxyl group or a nitrogen atom as in an amino group.
  • a protic solvent is capable of donating a proton (FT*). Conversely, an "aprotic" solvent cannot donate H + .
  • R-Butanol is also referred to as “butanol” herein.
  • “Lime” refers to Ca(OH) 2 (calcium hydroxide).
  • At least one inhibitor means one inhibitor substance or more than one different inhibitor substances.
  • a multiplicity of inhibitors means two or more inhibitor substances.
  • an “inhibitor” refers to a compound or substance which may reduce or eliminate microbial growth and/or reduce or eliminate production of one or more bioproduct(s) in a microbial growth medium, for example, one or more solvent(s), under conditions suitable for microbial growth and/or bioproduct production, such as microbial fermentation.
  • the carbon source contains sugar molecules, e.g., monosaccharides.
  • the carbon source contains glycerol.
  • "Conditioning" of the composition i.e., removal or inactivation of at least a portion of at least one inhibitor, improves its ability to support growth ⁇ e.g. , reproduction) of a microorganism and/or bioproduct production in a microbial fermentation.
  • conditioning improves the ability of the composition to support both growth of the microorganism and bioproduct production in a microbial fermentation.
  • conditioning improves the ability of the composition to support growth of the microorganism, but does not affect or substantially does not affect bioproduct production. In one embodiment, conditioning improves the ability of the composition to support bioproduct production, but does not affect or substantially does not affect growth of the microorganism.
  • substantially all of at least one inhibitor or substantially all of each of a multiplicity of inhibitors is removed or inactivated. In some embodiments, all of at least one inhibitor or all of each of a multiplicity of inhibitors is removed or inactivated. Different amounts of various inhibitors may be removed or inactivated in a method as described herein. For example, a portion of some inhibitor(s), substantially all of other inhibitor(s), and/or all of still other inhibitor(s) may be removed or inactivated.
  • the inhibitor(s) is(are) selected from organic acids (e.g., acetic, formic, levulinic), aldehydes (e.g., furfural, 5-hydroxymethyl furfural, vanillin), lignins, lignin byproducts or derivatives, phenolic and/or phenylpropanoid compounds, inorganic salts (e.g., sulfates, phosphates, hydroxides), fatty acids, fatty alcohols, fats, waxes, polyesters (e.g., suberin), terpenoids, alkanes, wood extractives, Hibbert's ketones, and proteins, or a combination thereof.
  • organic acids e.g., acetic, formic, levulinic
  • aldehydes e.g., furfural, 5-hydroxymethyl furfural, vanillin
  • lignins lignin byproducts or derivatives
  • the inhibitor(s) include(s) one or more phenolic and/or phenypropanoid compound(s), including but not limited to, ferulic acid, syringaldehyde, syringic acid, vanillin, vanillic acid, 3,4- dihydroxybenzaldehyde, 3,5-dihydroxybenzaldehyde, catechol, sinapic acid, gallic acid, sinapyl alcohol, coniferyl alcohol, hydroquinone, 4-hydroxybenzaldehyde, and/or 4- hydroxybenzoic acid.
  • ferulic acid ferulic acid
  • syringaldehyde syringic acid
  • vanillin vanillic acid
  • 3,4- dihydroxybenzaldehyde 3,5-dihydroxybenzaldehyde
  • catechol sinapic acid
  • gallic acid sinapyl alcohol
  • coniferyl alcohol coniferyl alcohol
  • hydroquinone 4-hydroxybenzaldehyde
  • the inhibitor(s) include(s) one or more of sinapic acid, 3,4-dihydroxybenzaldehyde, coniferyl alcohol, hydroquinone, and catechol. In some embodiments, the inhibitor(s) are selected from sinapic acid, 3,4- dihydroxybenzaldehyde, coniferyl alcohol, hydroquinone, and catechol. In one
  • the inhibitor(s) include sinapic acid. In one embodiment, the inhibitor(s) include 3,4-dihydroxybenzaldehyde. In one embodiment, the inhibitor(s) include coniferyl alcohol. In one embodiment, the inhibitor(s) include hydroquinone. In one embodiment, the inhibitor(s) include catechol.
  • the composition is conditioned by adding calcium hydroxide and potassium hydroxide for a period of time and at a temperature such that at least a portion of at least one inhibitor precipitates from the composition, and may then be separated and removed from the composition, thereby producing a "conditioned
  • the inhibitor-containing composition, calcium hydroxide, and potassium hydroxide are incubated at a temperature of about 30°C to about 60°C, or any of about 30°, 35°, 40°, 45°, 50°, 55°, or 60°C, for a period of time sufficient to precipitate at least a portion of at least one inhibitor, for example, about 1 minute to about 18 hours, about 1 minute to about 10 minutes, about 30 minutes to about 1 hour, about 1 hour to about 5 hours, about 5 hours to about 10 hours, or about 10 hours to about 18 hours.
  • the inhibitor may form a complex or undergo a chemical reaction with the calcium hydroxide and/or potassium hydroxide to form a precipitate that contains the inhibitor, or the formation of a precipitate in the hydrolysate may provide a condensation or nucleation site for the inhibitor.
  • the composition is conditioned by adding calcium hydroxide and potassium hydroxide at concentrations at which they are soluble in the hydrolysate and at which at least a portion of at least one inhibitor is rendered less inhibitory or not inhibitory ("inactivated").
  • calcium hydroxide and potassium hydroxide may be added simultaneously or sequentially.
  • the calcium hydroxide and potassium hydroxide may be added sequentially with calcium hydroxide added before potassium hydroxide.
  • calcium hydroxide and potassium hydroxide may optionally be added in amounts such that the pH after potassium hydroxide addition is about 0.1 to about 1 pH unit higher, or any of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 pH unit higher, than the pH after calcium hydroxide addition.
  • the pH difference between calcium and potassium hydroxide addition is about 1 pH unit.
  • calcium hydroxide is added in an amount sufficient to adjust the pH of the hydrolvsate to about 10 to about 10.9, or any of about 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, or 10.9, followed by addition of potassium hydroxide in an amount sufficient to adjust the pH of the hydrolysate to about 11.
  • the calcium hydroxide and potassium hydroxide may be added sequentially with potassium hydroxide added before calcium hydroxide.
  • the potassium hydroxide and calcium hydroxide may optionally be added in amounts such that the pH after calcium hydroxide addition is about 0.1 to about 1 pH unit higher, or any of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 pH unit higher, than the pH after potassium hydroxide addition.
  • the pH difference between potassium and calcium hydroxide addition is about 1 pH unit.
  • potassium hydroxide is added in an amount sufficient to adjust the pH of the hydrolysate to about 10 to about 10.9, or any of about 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, or 10.9, followed by addition of calcium hydroxide in an amount sufficient to adjust the pH of the hydrolysate to about 11.
  • the pH of the conditioned composition may optionally be lowered, for example, neutralized or adjusted to close to neutral pH, and further precipitate removed, if any.
  • the pH may be adjusted to a pH of about 7.2 to about 9, or any of about 7.2, 7.5, 7.8, 8, 8,2, 8.5, 8.8, or 9.
  • the pH adjustment may be effected, for example, with sulfuric acid or phosphoric acid, or a combination thereof.
  • At least a portion of at least one inhibitor ( . e. , at least a portion of one inhibitor or at least a portion of each of more than one inhibitor) of microbial growth and/or bioproduct production is precipitated from or inactivated in an inhibitor-containing composition by treatment with calcium hydroxide and potassium hydroxide in a method as described herein.
  • "a portion" of an inhibitor that forms a precipitate or is inactivated may be any of at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95%, but less than 100%.
  • at least about 50, 60, 70, 80, 90, 95, 98, or 99% of at least one inhibitor is removed or inactivated.
  • each of a multiplicity of inhibitors is removed or inactivated. In some embodiments, at least about 50, 60, 70, 80, 90, 95, 98, or 99% of each of a multiplicity of inhibitors is removed or inactivated. In some embodiments, substantially all of at least one inhibitor or substantially all of each of a multiplicity of inhibitors is removed or inactivated. In some embodiments, "substantially all" of an inhibitor may be about 99% to less than but close to 100%, or any of about 99, 99.5, 99.8, or 99.9%. Removal or inactivation of "all" of an inhibitor means removal of 100% of the inhibitor substance.
  • any of at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95%, or 100%) of at least one phenolic or phenylpropanoid compound is removed or inactivated.
  • any of at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95%, or 100%o of sinapic acid is removed or inactivated.
  • any of at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95%, or 100% of hydroquinone is removed or inactivated.
  • any of at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95%, or 100%) of coniferyl alcohol is removed or inactivated.
  • any of at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95%, or 100% of 3,4- dihyroxybnzaldehyde is removed or inactivated. In one embodiment, any of at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95%, or 100% of catechol is removed or inactivated.
  • Separation of the precipitate may be performed by any technique by which liquids and solids may be separated, including by not limited to, filtration, impingement, pressing, passive settling (e.g., lamella separator, settling tank), active settling (e.g., centrifugation, hydrocyclone), decantation, or dissolved gas flotation.
  • passive settling e.g., lamella separator, settling tank
  • active settling e.g., centrifugation, hydrocyclone
  • decantation e.g., decantation, or dissolved gas flotation.
  • hydrolyzed cellulosic feedstock e.g. , hydrolyzed lignocellulosic feedstock, which may be prepared as described infra
  • hydrolyzed lignocellulosic feedstock is conditioned to remove or inactivate inhibitors of microbial growth and/or bioproduct production, prior to addition of the hydrolyzed feedstock to a microbial growth medium.
  • inhibitors may include, but are not limited to, organic acids, furans, phenols, soluble lignocellulosic materials, extractives, and ketones.
  • Inhibitors present in wood hydrolysates may include, but are not limited to, 5-hydroxyy-methyl furfural (HMF), furfural, aliphatic acids, levulinic acid, acetic acid, formic acid, phenolic compounds, vanillin, dihydroconiferylalcohol, coniferyl aldehyde, vanillic acid, hydroquinone, catechol, acetoguaiacone, homovanillic acid, 4-hydroxy- benzoic acid, Hibbert's ketones , ammonium nitrate, />-coumaric acid, ferulic acid,
  • HMF 5-hydroxyy-methyl furfural
  • furfural furfural
  • aliphatic acids levulinic acid
  • acetic acid formic acid
  • phenolic compounds vanillin
  • dihydroconiferylalcohol coniferyl aldehyde
  • vanillic acid hydroquinone
  • catechol catechol
  • acetoguaiacone homo
  • the inhibitor(s) include(s) one or more phenolic and/or phenypropanoid compound(s), including but not limited to, ferulic acid, syringaldehyde, syringic acid, vanillin, vanillic acid, 3,4-dihydroxybenzaldehyde, 3,5-dihydroxybenzaldehyde, catechol, sinapic acid, gallic acid, sinapyl alcohol, coniferyl alcohol, hydroquinone, 4- hydroxybenzaldehyde, and/or 4-hydroxybenzoic acid.
  • phenolic and/or phenypropanoid compound(s) including but not limited to, ferulic acid, syringaldehyde, syringic acid, vanillin, vanillic acid, 3,4-dihydroxybenzaldehyde, 3,5-dihydroxybenzaldehyde, catechol, sinapic acid, gallic acid, sinapyl alcohol, coniferyl alcohol, hydroquinone,
  • the inhibitor(s) include(s) one or more of sinapic acid, 3,4-dihydroxybenzaldehyde, coniferyl alcohol, hydroquinone, and catechol. In some embodiments, the inhibitor(s) are selected from sinapic acid, 3,4-dihydroxybenzaldehyde, coniferyl alcohol, hydroquinone, and catechol. In one embodiment, the inhibitor(s) include sinapic acid. In one embodiment, the inhibitor(s) include 3,4-dihydroxybenzaldehyde. In one embodiment, the inhibitor(s) include coniferyl alcohol. In one embodiment, the inhibitor(s) include hydroquinone. In one embodiment, the inhibitor(s) include catechol.
  • the hydrolysate is conditioned by adding calcium hydroxide and potassium hydroxide as described herein, wherein inhibitor(s) present in the hydrolysate form a precipitate.
  • the precipitate is separated from the liquid hydrolysate, thereby producing a conditioned hydrolysate in which the inhibitor(s) are depleted or absent.
  • hydrolysate produced in the first stage and optionally in subsequent stage(s) may be conditioned as described herein.
  • the calcium hydroxide and potassium hydroxide are added to the hydrolysate in amounts sufficient to form a separable precipitate with a substantial amount of one or more inhibitor compound(s) present in the hydrolysate.
  • any of the conditioning methods described herein with calcium and potassium hydroxide addition may be combined with one or more additional method(s) for removing one or more inhibitor(s), including but not limited to, chelation of metal ions, ion exchange, and/or treatment with activated carbon.
  • a calcium sequestrant ⁇ e.g. , EDTA) or cation exchange may be used to remove excess calcium, which may be a potential inhibitor, e.g., when present in excess.
  • a multivalent hydroxide such as a divalent hydroxide, e.g., magnesium hydroxide, may be used in addition to or instead of calcium hydroxide.
  • a conditioned composition from which at least a portion of at least one inhibitor of microbial growth and/or bioproduct production has been removed or inactivated by a method as described herein, is provided.
  • composition may be added to a growth medium or may be used as a growth medium for fermentation of a microorganism, for example, for production of one or more bioproduct(s).
  • the removed inhibitor(s) is(are) selected from organic acids (e.g., acetic, formic, levulinic), aldehydes (e.g., furfural, 5-hydroxymethyl furfural, vanillin), lignins, lignin byproducts or derivatives, phenolic and/or phenylpropanoid compounds, inorganic salts (e.g., sulfates, phosphates, hydroxides), fatty acids, fatty alcohols, fats, waxes, polyesters (e.g., suberin), terpenoids, alkanes, wood extractives, Hibbert's ketones, and proteins, or a combination thereof.
  • organic acids e.g., acetic, formic, levulinic
  • aldehydes e.g., furfural, 5-hydroxymethyl furfural, vanillin
  • lignins lignin byproducts or derivatives
  • the inhibitor(s) include(s) one or more phenolic and/or phenypropanoid compound(s), including but not limited to, ferulic acid, syringaldehyde, syringic acid, vanillin, vanillic acid, 3,4- dihydroxybenzaldehyde, 3,5-dihydroxybenzaldehyde, catechol, sinapic acid, gallic acid, sinapyl alcohol, coniferyl alcohol, hydroquinone, 4-hydroxybenzaldehyde, and/or 4- hydroxybenzoic acid.
  • ferulic acid ferulic acid
  • syringaldehyde syringic acid
  • vanillin vanillic acid
  • 3,4- dihydroxybenzaldehyde 3,5-dihydroxybenzaldehyde
  • catechol sinapic acid
  • gallic acid sinapyl alcohol
  • coniferyl alcohol coniferyl alcohol
  • hydroquinone 4-hydroxybenzaldehyde
  • the inhibitor(s) include(s) one or more of sinapic acid, 3,4-dihydroxybenzaldehyde, coniferyl alcohol, hydroquinone, and catechol. In some embodiments, the inhibitor(s) are selected from sinapic acid, 3,4- dihydroxybenzaldehyde, coniferyl alcohol, hydroquinone, and catechol. In one
  • the inhibitor(s) include sinapic acid. In one embodiment, the inhibitor(s) include 3,4-dihydroxybenzaldehyde. In one embodiment, the inhibitor(s) include coniferyl alcohol. In one embodiment, the inhibitor(s) include hydroquinone. In one embodiment, the inhibitor(s) include catechol. [73] In some embodiments, conditioned cellulosic hydrolysates, e.g., conditioned lignocellulosic hydrolysates, are provided, prepared by any of the methods described herein.
  • Precipitation of at least a portion of at least one inhibitor of microbial growth and/or product production with calcium hydroxide and potassium hydroxide, and removal of the precipitate, from a hydrolysate of a cellulosic material provides a conditioned hydrolysate.
  • calcium hydroxide and potassium hydroxide may be added at soluble concentrations that inactive at least a portion of at least one inhibitor of microbial growth and/or product production, to provide a conditioned hydrolysate.
  • Such a conditioned hydrolysate, which contains soluble carbohydrate molecules may be used as a carbon source in a microbial fermentation to produce one or more bioproduct(s) of interest.
  • a conditioned hydrolysate may be prepared by hydrolysis of any of the cellulosic biomass materials described herein, followed by conditioning with calcium hydroxide and potassium hydroxide, for example, under conditions as described herein.
  • the cellulosic biomass material is a lignocellulosic biomass material.
  • a feedstock is a substance that provides the base material from which sugar molecules are generated for inclusion in a microbial growth medium, to support the growth of the microorganism.
  • Feedstock used in the methods described herein may be cellulosic biomass, for example, lignocellulosic biomass.
  • Cellulose which is a ⁇ -glucan 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-cellulosic polysaccharides associated with cellulose in plant tissues. Hemicellulose frequently constitutes about 20-35% w/w of lignocellulosic materials, and the majority of hemicelluloses consist of polymers based on pentose (five- carbon) sugar units, such as D-xylose and D-arabinose units, hexose (six-carbon) sugar units, such as D-glucose and D-mannose units, and uronic acids such as D-glucuronic acid.
  • pentose five- carbon
  • sugar units such as D-xylose and D-arabinose units
  • hexose (six-carbon) sugar units such as D-glucose and D-mannose units
  • uronic acids such as D-glucuronic acid.
  • Lignin which is a complex, cross-linked polymer based on variously substituted >-hydroxyphenylpropane units, typically constitutes about 10-30% w/w of lignocellulosic materials.
  • the feedstock is woody biomass.
  • the feedstock is softwood, for example, pine, e.g., Lodgepole or Loblolly pine.
  • the feedstock contains mountain pine beetle infested pine, for example, dying ("red stage") or dead ("grey" stage).
  • the feedstock is hardwood, for example, maple, birch, or ash.
  • the feedstock is mixed hardwood and softwood.
  • the feedstock is mixed hardwood.
  • the woody biomass is in the form of wood chips, sawdust, saw mill residue, wood fines, or a combination thereof.
  • the feedstock is obtained as a process stream from a biomass processing facility, for example, a pulp mill.
  • the process stream may include reject pulp, wood knots or shives, pulp screening room rejects (e.g., essentially cellulose in water), prehydrolysis extraction stream, and/or black liquor.
  • Lignocellulose contains a mixture of carbohydrate polymers and non- carbohydrate compounds.
  • the carbohydrate polymers contain cellulose and hemicellulose, and the non-carbohydrate portion contains lignin.
  • the non-carbohydrate portion may also contain ash, extractives, and/or other components.
  • the specific amounts of cellulose, hemicelluloses, and lignin depends on the source of the biomass. For example, municipal solid waste may contain primarily cellulose, and extract streams from a paper and pulp plant may contain primarily hemicelluloses.
  • the remaining composition of lignocellulose may also contain other compounds such as proteins.
  • the feedstock is a lignocellulosic material in the form of wood chips, sawdust, saw mill residue, or a combination thereof.
  • the lignocellulosic material (e.g., wood chips sawdust, saw mill residue, or a combination thereof) 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.
  • the feedstock source is lignocellulosic material that has been damaged by drought or fire.
  • Lignocellulosic biomass 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, cottonwood, rubber tree, marantii, eucalyptus, sugi, and acase.
  • 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 arundinacea, and grasses.
  • lignocellulosic materials may be used such as herbaceous material, agricultural crop or plant residue, forestry residue, municipal solid waste, pulp or paper mill residue, waste paper, recycling paper, or construction debris.
  • suitable plant residues include, but are not limited to, stems, leaves, hulls, husks, cobs, branches, bagasse, wood chips, wood pulp, wood pulp, and sawdust.
  • suitable waste paper include, but are not limited to, discarded paper of any type (e.g., photocopy paper, computer printer paper, notebook paper, notepad paper, typewriter paper), newspaper, magazines, cardboard, and paper-based packaging material. Materials with high mineral content may potentially require additional pH adjustment (e.g., additional amounts of chemicals for pH adjustment) for effective processing.
  • feedstocks that may be used in the methods herein include hemicellulose extract from wood, beet extract, beet molasses, sorghum syrup, barley hulls, potato processing waste, and/or brewers mash.
  • a feedstock mix containing about 40% logging residues, about 20% sustainable roundwood, about 20% woody energy crops, and about 20% herbaceous energy crops may be used. This blend can account for regional variation and provide significant flexibility in selecting locations for facilities and in procuring feedstock supply contracts.
  • the feedstock contains grass, for example, sugar cane, miscanthus, and/or switchgrass, and/or straw, for example, wheat straw, barley straw, and/or rice straw.
  • Feedstocks such as those described herein can be pretreated using a variety of methods and systems prior to bioconversion.
  • Preparation of the feedstock can include chemical or physical modification of the feedstock.
  • the feedstock can be shredded, sliced, chipped, chopped, heated, burned, dried, separated, extracted, hydrolyzed, and/or degraded. These modifications can be performed by biological, non-biological, chemical, or non-chemical processes.
  • processes may be used to break down cellulose and/or hemicellulose into sugar molecules that may be more easily processed by a microorganism.
  • Processes that may be used include acid hydrolysis, enzymatic hydrolysis, gasification, pyrolysis, and cellulose degradation by a microorganism.
  • the feedstock such as lignocellulosic feedstock, for example, wood chips, sawdust, and/or sawdust residue
  • Deconstruction may include, but is not limited to, presteaming to swell and loosen material, mechanical grinding, mechanical explosion (e.g., steam or other chemical treatment followed by rapid decompression), vacuum treatment, acid-feedstock contact (diffusion of acid into feedstock), or a combination thereof.
  • deconstruction renders cellulose and/or hemicellulose in the feedstock more accessible for hydrolysis.
  • the feedstock such as lignocellulosic feedstock, for example, wood chips, sawdust, and/or sawdust residue
  • the feedstock is pretreated to remove extractives.
  • Extractives are material that is extracted from the feedstock by a process such as
  • Extractives include terpenes, resin acids, fatty acids, sterols, steryl esters, phenolic compounds, and triglycerides. Extractives may include, but are not limited to, -coumaric acid, ferulic acid, 4-hydroxybenzoic acid, vanillic acid, syringaldehyde, vanillin, furfural, hydroxymethylfurfural, and glucuronic acid. Extractives may be removed for other uses, such as production of sterols, or burned to provide energy for a bioproduct, e.g., biofuel, production process as described herein.
  • a bioproduct e.g., biofuel, production process as described herein.
  • extractives are removed prior to or in conjunction with deconstruction of the feedstock.
  • a feedstock contains sugar molecules in an oligomeric form, e.g., a polymeric form, and must be hydrolyzed to extract and release soluble monomeric and/or multimeric sugar molecules, which are converted to bioproduct, e.g., biofuel, in a microbial fermentation as described herein.
  • the sugar molecules are present in the feedstock in cellulose and optionally also in hemicellulose.
  • the feedstock is lignocellulosic biomass and the sugar molecules are present in the feedstock in cellulose and hemicellulose.
  • the feedstock is pretreated with an acid hydrolysis process.
  • Acids that may be used for hydrolysis include, but are not limited to, nitric acid, formic acid, acetic acid, phosphoric acid, hydrochloric acid, and sulfuric acid, or a combination thereof.
  • acid hydrolysis is performed in a single stage.
  • acid hydrolysis is performed in two or more stages, under different conditions in each stage to hydrolyze different components of the feedstock in each stage.
  • the second stage hydrolysis is performed at a higher temperature than the first stage hydrolysis. Acid hydrolysis performed in multiple stages may serve to limit the impact of kinetically controlled carbohydrate degradation mechanisms.
  • An acid hydrolysis system may be designed to submerge and flood the feedstock with the acid solution in the hydrolysis reactor, e.g., in a vertical section of the hydrolysis reactor, to insure even acid impregnation. Even heat distribution may be obtained by using both direct steam injection and a jacketed vessel in conjunction with a mechanical screw auger.
  • Variable speed drives may be used with temperature sensing instrumentation to control reactor residence time and temperature allowing reactor severity to be adjusted online.
  • Alternative reactor configurations with functionally similar properties may also be utilized. For example, a horizontal digestor configuration may be used. In this type of reactor, the material is only partially submerged.
  • the feedstock material in order to reach higher soluble sugar concentrations, is not completely submerged in the acid containing solution, thereby producing a hydrolysate that contains an increased sugar concentration (i.e., less dilution water added at the outset).
  • a multiple-stage dilute nitric acid hydrolysis process is used.
  • a two-stage dilute nitric acid process is used for hydrolysis of lignocellulosic feedstock.
  • conditions in the first stage are chosen to achieve hydrolysis of about 70% to about 90% of the hemicellulose in the feedstock and conditions in the second stage are chosen to achieve hydrolysis of about 40% to about 70% of the cellulose in the feedstock.
  • the first stage mainly targets the hydrolysis of the hemicellulose, yielding a mannose and/or xylose rich hydrolysate, whereas the second stage uses the solids remaining from the first stage and targets the cellulose, yielding a glucose rich hydrolysate.
  • first stage hydrolysate liquors contain a mix of 5-carbon and 6-carbon sugars, e.g., extracted primarily from hemicellulose and non-recalcitrant cellulose biomass components
  • second stage hydrolysate contains primarily 6-carbon sugars, e.g., extracted from cellulose fibers, in both cases as soluble monomeric and/or multimeric forms.
  • 6-carbon monosaccharides may include, but are not limited to, glucose, mannose, and galactose.
  • 6-carbon disaccharides may include, but are not limited to, cellobiose, mannobiose, glucomannose, and galactomannose.
  • 5-carbon monosaccharides may include, but are not limited to xylose and arabinose.
  • 5-carbon disaccharides and other multimeric forms may include, but are not limited to, xylobiose, xylotriose, and
  • the first stage hydrolysate contains about 60% to about 75% 5-carbon sugar by weight and about 25% to about 40% 6-carbon sugar by weight
  • the second stage hydrolysate contains about 80% to about 95% 6-carbon sugar by weight
  • the first stage hydrolysate contains about 20% to about 30% 5-carbon sugar by weight and about 70% to about 80% 6-carbon sugar by weight
  • the second stage hydrolysate contains about 90% to about 100% 6-carbon sugar by weight, wherein the second stage is performed at a higher temperature than the first stage.
  • a first stage hydrolysis module may be coupled to a second stage hydrolysis module, with solid residue separated from liquid hydrolysate generated in the first stage hydrolysis serving as substrate for the second hydrolysis process.
  • the residual solids may be rinsed/washed in order to increase the separation and recovery yield of soluble sugars separated from the biomass.
  • hydrolysis is performed at a nitric acid concentration of about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 4%, about 1.3% to about 3.5%), or about 1.3% (w/w of dry feedstock) for both hydrolysis stages, at a temperature of about 170° to about 175°C in the first stage and a temperature of about 210° to about 230°C in the second stage, and at the saturation pressure for steam at the reactor temperature for each hydrolysis stage.
  • the liquid (acid) to solid (feedstock) ratio for hydrolysis is about 10: 1 to about 5: 1 or about 7.5:1 to about 5:1.
  • the ratio of liquid to solid may be about 5 : 1 to about 3:1 or about 3.5 : 1 to about 3:1.
  • the ratio of liquid to solid may be about 4:1 to about 0.5:1.
  • a solvent-assisted hydrolysis may be performed.
  • a solvent may be added to solvate components of biomass.
  • a combination of glycerol and acid e.g., nitric acid
  • a hot water extraction process is used for hydrolysis.
  • biomass may be treated with hot water to solubilized oligomeric sugars, and then the sugars are hydrolyzed, for example, with acid.
  • liquids and solids may be separated prior to acid hydrolysis of the liquid fraction.
  • Methods are provided for producing a bioproduct.
  • the methods include culturing a microorganism in a medium that contains a conditioned composition, e.g., a conditioned hydrolysate, prepared according to any of the methods described herein.
  • the conditioned composition provides a carbon source, for example, soluble sugar molecules or glycerol.
  • microbial growth and/or bioproduct titer, yield, and/or productivity is increased when a conditioned composition, for example, conditioned hydrolyzed feedstock, as described herein is used in a microbial fermentation process, in comparison to identical hydrolyzed feedstock which has not been subjected to the conditioning process.
  • the bioproduct is a solvent, such as, for example, a polar aprotic or protic solvent.
  • the solvent is «-butanol, acetic acid, isopropanol, «-propanol, ethanol, methanol, formic acid, 1,4-dioxane, tetrahydrofuran, acetone, acetonitrile, dimethylformamide, or dimethyl sulfoxide, or a combination thereof.
  • the bioproduct is a biofuel, for example, butanol, ethanol, or acetone, or a combination thereof.
  • the bioproduct is a biochemical or biochemical intermediate, for example, formate, acetate, butyrate, propionate, succinate, methanol, propanol, or hexanol, or a combination thereof.
  • the methods for bioproduct production herein include fermentation of a bioproduct- producing microorganism in a bioreactor in a growth medium that contains a conditioned composition, for example, a conditioned hydrolysate, prepared as described herein.
  • a conditioned composition for example, a conditioned hydrolysate
  • the bioproduct production includes fermentation of a bioproduct-producing 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 stirred tank (suspended cell) bioreactor.
  • the microbial cells form a biofilm on the support and/or between support particles in the growth medium.
  • the bioproduct production process includes continuous fermentation of a microorganism (continuous addition of conditioned hydrolyzed feedstock and withdrawal of product stream). Continuous fermentation minimizes the unproductive portions of the fermentation 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 bioproduct-producing microorganism.
  • an immobilized bioproduct-producing Clostridium strain is fermented anaerobically in a continuous process as described herein.
  • One or more bioreactors may be used in the bioproduct production systems and processes described herein. When multiple bioreactors are used they can be arranged in series and/or in parallel. The advantages of multiple bioreactors over one large bioreactor include lower fabrication and installation costs, ease of scale-up production, and greater production flexibility. For example individual bioreactors may be taken off-line for maintenance, cleaning, sterilization, and the like without appreciably impacting the production schedule. In embodiments in which multiple bioreactors are used, the bioreactors may be run under the same or different conditions.
  • a conditioned composition such as conditioned hydrolyzed feedstock
  • medium containing a conditioned composition such as conditioned hydrolyzed feedstock
  • effluent from the bioreactors is removed.
  • the effluent may be combined from multiple bioreactors for recovery of the bioproduct, or the effluent from each bioreactor may be collected separately and used for recovery of the bioproduct.
  • a series bioreactor arrangement medium containing a conditioned composition, such as conditioned hydrolyzed feedstock, is fed into the first bioreactor in the series, the effluent from the first bioreactor is fed into a second downstream bioreactor, and the effluent from each bioreactor in the series is fed into the next subsequent bioreactor in the series.
  • the effluent from the final bioreactor in the series is collected and may be used for recovery of the bioproduct.
  • Each bioreactor in a multiple bioreactor arrangement can have the same species, strain, or mix of species or strains of microorganisms or a different species, strain, or mix of species or strains of microorganisms compared to other bioreactors in the series.
  • feedstock is hydrolyzed in a multi-stage process as described herein, conditioned as described herein, and conditioned hydrolysate derived from hydrolysate generated in each stage is fed to separate bioreactors.
  • the bioreactors to which the different conditioned compositions are fed may contain the same or different microbial species or strains.
  • the bioreactors to which the different compositions are fed contain different microbial species or strains that have each been optimized for growth on the particular composition, e.g., hydrolysate, being fed to that bioreactor.
  • different sets of multiple bioreactors in series are fed hydrolysate from different stages of hydrolysis of a cellulosic feedstock.
  • Immobilized cell bioreactors allow higher concentrations of productive cell mass to accumulate and therefore, the bioreactors can be run at high dilution rates, resulting in a significant improvement in volumetric productivity relative to cultures of suspended cells. Since a high density, steady state culture can be maintained through continuous culturing, with the attendant removal of product-containing fermentation broth, smaller capacity bioreactors can be used. Bioreactors for the continuous fermentation of C. acetobutylicum are known in the art. (See, e.g., U.S. Patent Nos. 4,424,275, and 4,568,643.)
  • support material may be added to the reactor through bottom, top, or side loading to replenish support material that becomes degraded or lost from the bioreactor.
  • Fermentation media for the production of bioproduct contain a conditioned composition, e.g., a conditioned hydrolyzed feedstock, as described herein, as a source of fermentable carbohydrate molecules.
  • fermentation media contain suitable nitrogen source(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 the desired bioproduct.
  • nitrogen source(s) e.g., 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 the desired bioproduct.
  • salts and/or vitamin B12 or precursors thereof are included in the fermentation media.
  • hydrolyzed feedstock may contain some or all of the nutrients required for growth, minimizing or obviating the need for additional supplemental material.
  • the nitrogen source may be any suitable nitrogen source, including but not limited to, ammonium salts, yeast extract, corn steep liquor (CSL), and other protein sources including, but not limited to, denatured proteins recovered from distillation of fermentation broth or extracts derived from the residual separated microbial cell mass recovered after fermentation.
  • Phosphorus may be present in the medium in the form of phosphate salts, such as sodium, potassium, or ammonium phosphates.
  • Sulfur may be present in the medium in the form of sulfate salts, such as sodium or ammonium sulfates.
  • Additional salts include, but are not limited to, magnesium sulfate, manganese sulfate, iron sulfate, magnesium chloride, calcium chloride, manganese chloride, ferric chloride, ferrous chloride, zinc chloride, cupric chloride, cobalt chloride, and sodium molybdate.
  • the growth medium may also contain vitamins such as thiamine hydrochloride, biotin, and para-aminobenzoic acid (PABA).
  • the growth medium may also contain one or more buffering agent(s) (e.g., MES), one or more reducing agent(s) (e.g., cysteine HCl), and/or sodium lactate, which may serve as a carbon source and pH buffer.
  • buffering agent(s) e.g., MES
  • reducing agent(s) e.g., cysteine HCl
  • sodium lactate sodium lactate
  • the systems and processes described herein for bioproduct production include one or more microorganism(s) that is (are) capable of producing one or more bioproduct(s) of interest.
  • the microorganism(s) that is (are) capable of producing one or more bioproduct(s) of interest.
  • the microorganisms that is (are) capable of producing one or more bioproduct(s) of interest.
  • microorganisms may be the same or different microbial species and/or different strains of the same species.
  • the microorganisms are bacteria or fungi.
  • microorganisms may be a single species or a mixed culture of strains from the same species. In some embodiments, the microorganisms are a mixed culture of different species. In some embodiments, the microorganisms are an environmental isolate or strain derived therefrom. [124] In some embodiments of the processes and systems described herein for bioproduct production, different species or strains, or different combinations of two or more species or strains, are used in different bioreactors with different conditioned hydrolyzed feedstocks as a carbohydrate source.
  • a fungal microorganism such as a yeast.
  • yeasts include, but are not limited to, Saccharomyces cerevisiae, S. bayanus, S.
  • anaerobic or aerotolerant fungi include, but are not limited to, the genera Neocallimastix, Caecomyces, Piromyces and other rumen derived anaerobic fungi.
  • a bacterial microorganism is used, including Gram-negative and Gram-positive bacteria.
  • Gram-positive bacteria include bacteria found in the genera of Staphylococcus, Streptococcus, Bacillus, Mycobacterium, Enter ococcus, Lactobacillus, Leuconostoc, Pediococcus, and Propionibacterium.
  • Non-limiting examples of specific species include Enterococcus faecium and Enterococcus gallinarium.
  • Gram-negative bacteria include bacteria found in the genera Pseudomonas, Zymomonas, Spirochaeta, Methylosinus, Pantoea, Acetobacter, Gluconobacter, Escherichia and Erwinia.
  • the bacteria are Clostridium species, including but not limited to, Clostridium saccharobutylicum, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium puniceum, and environmental isolates of Clostridium.
  • Clostridium contemplated for use in this invention can be selected from C. aurantibutyricum, C. butyricum, C. cellulolyticum, C.
  • phytofermentans C. saccharolyticum, C. saccharoperbutylacetonicum, C. tetanomorphum, C. thermobutyricum, C. thermocellum, C. puniceum, C. thermosaccharolyticum, and C. paster ianum.
  • bacteria contemplated for use in the processes and systems herein include Corynebacteria, such as C. diphtheriae, Pneumococci, such as Diplococcus pneumoniae, Streptococci, such as S. pyogenes and S. salivarus, Staphylococci, such as S. aureus and S. albus, Myoviridae, Siphoviridae, Aerobic Spore-forming Bacilli, Bacilli, such as B.
  • Erysipelothrix rhusiopathiae Streptobacillus monilformis, Donvania granulomatis, Bartonella bacilliformis, Rickettsiae, e.g., Rickettsia prowazekii, Rickettsia mooseri, Rickettsia rickettsiae, and Rickettsia conori.
  • Other suitable bacteria may include
  • Escherichia coli Zymomonas mobilis, Erwinia chrysanthemi, and Klebsiella planticola.
  • the microorganisms comprise the genera Clostridium, Enterococcus, Klebsiella, Lactobacillus, or Bacillus. In some embodiments, the
  • microorganisms comprise Clostridium acetobutylicum, Clostridium beijerinckii,
  • Clostridium puniceum Clostridium saccharobutylicum, Enterococcus faecium
  • Clostridium aurantibutyricum Clostridium aurantibutyricum
  • Clostridium aurantibutyricum Clostridium tetanomorphum
  • Clostridium thermosaccharolyticum Clostridium thermosaccharolyticum.
  • the microorganisms are obligate anaerobes.
  • obligate anaerobes include Butyrivibrio fibrosolvens and Clostridium species.
  • microorganisms are microaerotolerant and are capable of surviving in the presence of small concentrations of oxygen.
  • microaerobic conditions include, but are not limited, to fermentation conditions produced by sparging a liquid media with a gas of at least about 0.01% to at least 5% or more 0 2 (e.g., 0.01%, 0.05%, 0.10%, 0.50%, 0.60%, 0.70%, 0.80%, 1.00%, 1.20%, 1.50%, 1.75%, 2.0%, 3%), 4%, 5% or more 0 2 ).
  • the microaerobic conditions include, but are not limited to, culture conditions with at least about 0.05ppm dissolved 0 2 or more (e.g., 0.05, 0.075, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0, 2.0, 3.0, 4.0, 5.0, 8.0, 10.0, ppm or more).
  • culture conditions with at least about 0.05ppm dissolved 0 2 or more (e.g., 0.05, 0.075, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0, 2.0, 3.0, 4.0, 5.0, 8.0, 10.0, ppm or more).
  • parent strains can be isolated from environmental samples such as wastewater sludge from wastewater treatment facilities including municipal facilities and those at chemical or petrochemical plants. The latter are especially attractive as the isolated microorganisms can be expected to have evolved over the course of numerous generations in the presence of high product concentrations and thereby have already attained a level of desired product tolerance that may be further improved upon.
  • Parent strains may also be isolated from locations of natural degradation of naturally occurring feedstocks and compounds (e.g., a woodpile, a saw yard, under fallen trees, landfills). Such isolates may be advantageous since the isolated microorganisms may have evolved over time in the presence of the feedstock and thereby have already attained some level of conversion and tolerance to these materials that may be further improved upon.
  • feedstocks and compounds e.g., a woodpile, a saw yard, under fallen trees, landfills.
  • Isolates including microbial consortiums can be collected from numerous environmental niches including soil, rivers, lakes, sediments, estuaries, marshes, industrial facilities, etc.
  • the microbial consortiums are strict anaerobes.
  • the microbial consortiums are obligate anaerobes.
  • the microbial consortiums are facultative anaerobes.
  • the microbial consortiums do not contain species of Enterococcus or Lactobacillus.
  • a selective growth inhibitor for undesired species or genera can be used to prevent or suppress the growth of these undesired microorganisms.
  • the culture conditions may be anaerobic, microaerotolerant, or aerobic. Aerobic conditions are those that contain oxygen dissolved in the media such that an aerobic culture would not be able to discern a difference in oxygen transfer with the additional dissolved oxygen, and microaerotolerant conditions are those where some dissolved oxygen is present at a level below that found in air or air saturated solutions and frequently below the detection limit of standard dissolved oxygen probes, e.g., less than 1 ppm.
  • the cultures can be agitated or left undisturbed.
  • the pH of the media changes over time as the microorganisms grow in number, consume feedstock and excrete organic acids.
  • the pH of the media can be modulated by the addition of buffering compounds to the initial fermentation media in the bioreactor or by the active addition of acid or base to the growing culture to keep the pH in a desired range. Growth of the culture may be monitored by measuring the optical density, typically at a wavelength of 600 nm, or by other methods known in the art.
  • Clostridium fermentations are generally conducted under anaerobic conditions.
  • ABE fermentations by C. acetobutylicum are typically conducted under anaerobic conditions at a temperature in the range of about 25° C to about 40° C.
  • suspension cultures did not use agitators, but relied on evolved or sparged gas to mix the contents of the bioreactors. Cultures, however, can be agitated to ensure more uniform mixing of the contents of the bioreactor.
  • a bioreactor may be run without agitation in a fixed bed (plug flow) or fluidized/expanded bed (well-mixed) mode.
  • Thermophilic bacterial fermentations can reach temperatures in the range of about 50° C to about 80°C. In some embodiments, the temperature range is about 55° to about 70° C. In some embodiments, the temperature range is about 60°C to about 65° C.
  • Clostridium species such as C. thermocellum or C. thermohydrosulfuricum may be grown at about 60°C to about 65°C.
  • the pH of the Clostridium growth medium can be modulated by the addition of buffering compounds to the initial fermentation media in the bioreactor or by the active addition of acid or base to the growing culture to keep the pH in a desired range. For example, a pH in the range of about 3.5 to about 7.5, or about 5 to about 7, may be maintained in the medium for growth of Clostridium.
  • microorganisms are grown immobilized on a solid or semisolid support for production of one or more bioproduct(s) of interest.
  • Immobilization of the microorganism, from spores or vegetative cells can be by any known method.
  • entrapment or inclusion in the support is achieved by polymerizing or solidifying a spore or vegetative cell containing solution.
  • Useful polymerizable or solidifiable solutions include, but are not limited to, alginate, ⁇ - carrageenan, chitosan, polyacrylamide, polyacrylamide-hydrazide, agarose, polypropylene, polyethylene glycol, dimethyl acrylate, polystyrene divinyl benzene, polyvinyl benzene, polyvinyl alcohol, epoxy carrier, cellulose, cellulose acetate, photocrosslinkable resin, prepolymers, urethane, and gelatin.
  • the microorganisms are incubated in growth medium with a support.
  • a support include, but are not limited to, bone char, cork, clay, resin, sand, porous alumina beads, porous brick, porous silica, celite (diatomaceous earth),
  • microorganisms may adhere to the support and form an aggregate, e.g., a biofilm.
  • the microorganism is covalently coupled to a support using chemical agents like glutaraldehyde, o-dianisidine (U.S. Pat.
  • immobilized spores such as those of Clostridium, e.g., C. acetobutylicum, are activated by thermal shock and then incubated under appropriate conditions in a growth medium whereby vegetative growth ensues. These cells remain enclosed in or on the solid support. After the microorganisms reach a suitable density and physiological state, culture conditions can be changed for bioproduct production. If the immobilized cells lose or exhibit reduced bioproduct production ability, they can be reactivated by first allowing the cells to sporulate before repeating the thermal shock and culture sequence.
  • Vegetative cells can be immobilized in different phases of their growth.
  • cells can be immobilized after they enter the desired culture phase in order to maximize production of the desired products, where in the case of C.
  • acetobutylicum it is the organic acids acetic acid and butyric acid in the acidogenic phase and the solvents acetone, butanol and ethanol in the solventogenic phase.
  • biphasic cells can be immobilized in the acidogenic phase and then adapted for solvent production.
  • microorganisms to be immobilized in a bioreactor are introduced by way of a cell suspension. Generally, these microorganisms are dispersed in the media as single cells or small aggregates of cells. In other embodiments, the
  • microorganisms are introduced into the bioreactor through the use of suspended particles that are colonized by the microorganisms. These suspended particles can be absorbed onto the solid support and frequently are of sufficiently small size that they can enter and become immobilized in the pore structures of the solid support. Typically, regardless of the suspended particle size, microorganisms can be transferred by contact with the solid support. A biofilm on the introduced particles can transfer to and colonize these new surfaces. In some embodiments, the desired characteristics of the microorganisms can only be maintained by culturing on a solid support, thereby necessitating the use of small colonized particle suspensions for seeding a solid support in a bioreactor. Support for immobilized microbial growth
  • a bioproduct producing microorganism is grown in an immobilized form on a solid or semi-solid support material in a bioreactor as described herein.
  • the support contains a porous material.
  • suitable support materials include bone char, synthetic polymers, natural polymers, inorganic materials, and organic materials.
  • Natural polymers include organic materials such as cellulose, lignocellulose, hemicellulose, and starch.
  • Organic materials include feedstock such as plant residue and paper.
  • Composites of two or more materials may also be used such as mixtures of synthetic polymer with natural plant polymer.
  • Examples of semi-solid media include alginate, /c-carrageenan and chitosan, polyacrylamide, polyacrylamide-hydrazide, agarose, polypropylene, polyethylene glycol, dimethyl acrylate, polystyrene divinyl benzene, polyvinyl benzene, polyvinyl alcohol, epoxy carrier, cellulose, cellulose acetate, photocrosslinkable resin, prepolymers, urethane, and gelatin.
  • Examples of solid support include cork, clay, resin, sand, porous alumina beads, porous brick, porous silica, celite, wood chips or activated charcoal.
  • Suitable inorganic solid support materials include inorganic materials with available surface hydroxy or oxide groups. Such materials can be classified in terms of chemical composition as siliceous or nonsiliceous metal oxides.
  • Siliceous supports include, inter alia, glass, colloidal silica, wollastonite, cordierite, dried silica gel, bentonite, and the like.
  • Representative nonsiliceous metal oxides include alumina, hydroxy apatite, and nickel oxide.
  • the support material is selected from bone char,
  • the support material is bone char.
  • Useful support material has a high surface area to volume ratio such that a large amount of active, productive cells can accumulate in the bioreactor.
  • Useful supports may contain one or more macrostructured components containing one or more useful support material(s) that promotes good fluidmechanical properties, for example, a wire mesh/gauze packing material used for traditional distillation tower packing.
  • the support material includes a surface area of at least about
  • the support material comprises a bulk density of at least about 0.15 g/cm . In some embodiments, the support material comprises a ball-pan hardness number of at least about 60. In some embodiments, the support material comprises a yield strength of at least about 20 MPa.
  • the particle size for the support material will vary depending upon bioreactor configuration and operation parameters.
  • the support material is sized by sieving.
  • the particles are classified by the sieve number of the mesh that they can pass through.
  • the particles are sieved with a mesh that has a U.S. Sieve Number of 31 ⁇ 2, 4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, or 70.
  • the particles are sieved at least twice, first using a mesh with larger openings followed by a mesh with smaller openings to yield particles within a defined particle size distribution range.
  • the particles are at least about 100 ⁇ , 200 ⁇ , 300 ⁇ , 400 ⁇ , 500 ⁇ , 600 ⁇ , 700 ⁇ , 800 ⁇ , 900 ⁇ , 1000 ⁇ , 1,100 ⁇ , 1,200 ⁇ , 1,300 ⁇ , 1,400 ⁇ , 1,500 ⁇ , 1,600 ⁇ , 1,700 ⁇ , 1,800 ⁇ , 1,900 ⁇ , 2,000 ⁇ , 3,000 ⁇ , 4,000 ⁇ , 5,000 ⁇ , 6,000 ⁇ , 7,000 ⁇ , 8000 ⁇ , 9,000 ⁇ , 10,000 ⁇ , 12,500 ⁇ , 15,000 ⁇ , 17,500 ⁇ , 20,000 ⁇ , 22,500 ⁇ , or 25,000 ⁇ in diameter.
  • the particles are less than about 100 ⁇ , 200 ⁇ , 300 ⁇ , 400 ⁇ , 500 ⁇ , 600 ⁇ , 700 ⁇ , 800 ⁇ , 900 ⁇ , 1000 ⁇ , 1,100 ⁇ , 1,200 ⁇ , 1,300 ⁇ , 1,400 ⁇ , 1,500 ⁇ , 1,600 ⁇ , 1,700 ⁇ , 1,800 ⁇ , 1,900 ⁇ , 2,000 ⁇ in diameter.
  • At least about 80%, 85%, 90%, 95%, or 100% of the particle have diameters that are in the range of about 100-400 ⁇ , 100-600 ⁇ , 100- 800 ⁇ , 200-500 ⁇ , 200-800 ⁇ , 200-1000 ⁇ , 400-800 ⁇ , 400-1000 ⁇ , 500-1000 ⁇ , 600-1,200 ⁇ , 800-1,400, ⁇ 1,000-1,500, ⁇ 1,000-2000 ⁇ , 2,000-4,000 ⁇ , 4,000-6,000 ⁇ , 5,000-12,000 ⁇ , 3,000-15,000 ⁇ , or 6,000-25,000 ⁇ .
  • the particle diameters are the equivalent diameters, a parameter that takes into account the irregular shapes of the individual particles.
  • the semi-solid or solid support material should have a high surface area. This can be achieved through the use of small sized particles, particles with high porosity, or a combination thereof.
  • the surface area of the particles is at least about 0.003 m 2 /g, 0.01 m 2 /g, 0.02 m 2 /g, 0,05 m 2 /g, 0.1 m 2 /g, 0.5 m 2 /g, 1 m 2 /g, 5 m 2 /g, 10 m 2 /g, 25 m 2 /g, 50 m 2 /g, 75 m 2 /g, 100 m 2 /g, 125 m 2 /g, 150 m 2 /g, 175 m 2 /g, 200 m 2 /g, 225 m 2 /g, 250 m 2 /g, 275 m 2 /g, 300 m 2 /g, 325 m 2 /g, 350 m 2
  • the bulk density should be sufficiently high so that the smallest particles settle out of the fluid stream in the column expansion zone and/or particle disengagement zone and are thereby retained in the bioreactor.
  • the bulk density of the support is at least about 0.1 g/cm , 0.2 g/cm , 0.3g/cm , 0.4 g/cm3, 0.5 g/cm , 0.6 g/cm , 0.7 g/cm , 0.8 g/cm , 0.9 g/cm , 1.0 g/cm , 1.1 g/cm , 1.2 g/cm , or 1.3 g/cm .
  • the support material should have sufficient hardness to resist abrasion and thereby avoid appreciable dust formation when the support particles touch or collide with each other.
  • the support has a ball-pan hardness number of at least about 20, 40, 60, 80, 100, 120, 140, 160 or 200.
  • the support material should also have sufficient tensile strength to resist shattering due to internal stresses, which may be caused by the growth of biofilms inside support material pores.
  • the support has a yield strength of at least about 20 MPa, 40 MPa, 60 MPa, 80 MPa, 100 MPa, 120 MPa, 140 MPa, 160 MPa, 180 MPa, 200 MPa, 300 MPa, or 400 MPa.
  • the support material should also have the ability to resist being crushed by the accumulated weight of material above it.
  • Crush strength is another measurement of the mechanical strength of the support and is typically a function of the composition, shape, size, and porosity of the material (increase in port volume may negatively impact particle strength). In some embodiments, the crush strength is at least about 8 kg.
  • the support material is chosen to support growth of the fermenting bioproduct producing microorganism as a biofilm.
  • the biofilm may grow on exterior surfaces of support particles, in the fluid space between support particles, and/or on surfaces in the interior of pores of the support material.
  • a continuous process for bioproduct production is provided.
  • a conditioned composition such as a conditioned carbohydrate-containing feedstock containing soluble sugar molecules, e.g., a conditioned hydrolyzed cellulosic feedstock, is continuously fed to one or more bioreactors for microbial production of the bioproduct, the bioproduct is continuously produced by immobilized microorganism(s) in the one or more bioreactors, and bioproduct-containing effluent, i.e., fermentation broth, is continuously withdrawn from the one or more reactors, for the duration of fermentation.
  • bioproduct-containing effluent i.e., fermentation broth
  • feedstock is continuously hydrolyzed to release soluble sugar molecules, and continuously conditioned prior to introduction of the conditioned hydrolyzed feedstock into the bioreactor(s).
  • the conditioning process may operate continuously downstream from a feedstock hydrolysis process, and upstream from the bioreactor(s), and conditioned hydrolyzed feedstock may be continuously fed to the bioreactor for the duration of fermentation.
  • the feedstock is lignocellulosic feedstock, and is hydrolyzed with nitric acid to release soluble sugar molecules from cellulose and hemicellulose, as described supra.
  • the continuous process may also include downstream continuous concentration and/or purification processes for recovery of the bioproduct, wherein continuously withdrawn effluent is continuously processed in one or more concentration and/or purification processes to produce a bioproduct.
  • the process may also include deconstruction of the feedstock and/or removal of extractives from the feedstock, as described herein.
  • Deconstruction and/or removal of extractives may be continuous or may occur prior to or periodically throughout the continuous process.
  • the process operates continuously for at least about 50, 100, 200, 300, 400, 600, 800, 1000, 1350, 1600, 2000, 2500, 3000, 4000, 5000, 6000, 7000, 8000, or 8400 hours.
  • a "continuous" process as described herein may include periodic or intermittent partial or complete shutdowns of one or more parts of the bioproduct production system for processes such as maintenance, repair, regeneration of resin, etc.
  • Continuous fermentation with constant feed of feedstock and withdrawal of product-containing microbial broth, can minimize the unproductive portions of a fermentation cycle, such as lag, growth, and turnaround time, thereby reducing the capital cost, and can reduce the number of inoculation events, thus minimizing operational costs and risk associated with human and process error.
  • the continuous methods and systems described herein can utilize one or more, e.g., one, two, or three or more, bioreactors. When multiple (two or more) bioreactors are used, they may be arranged in parallel, series, or a combination thereof.
  • the bioreactors can grow the same or different strains of microorganism(s). The strains can be different based on the type of sugar they metabolize to maximize bioproduct production.
  • a first bioreactor or multiple bioreactors arranged in parallel, series, or a combination thereof can grow a strain that has been selected to metabolize C5 sugars and a second bioreactor or multiple bioreactors arranged in parallel, series, or a combination thereof can grow another strain that has been selected to metabolize C5 and C6 sugars.
  • the bioreactors may be coupled to upstream feedstock hydrolysis and conditioning units, and may also be coupled to a downstream recovery/separation unit.
  • an acid hydrolysis unit may be coupled to an in fluid communication with a downstream conditioning unit, for conditioning with a metal salt as described herein, which is coupled to a downstream bioreactor, whereby conditioned hydrolysate, which has been separated from metal salt complexed inhibitor(s) is fed to the downstream bioreactor for production of bioproduct(s) of interest.
  • Hydrolysis, conditioning, product production, and product recovery may proceed continuously.
  • a first bioreactor or multiple bioreactors arranged in parallel, series, or a combination thereof with a strain that metabolizes C5 sugars can be coupled to an upstream first stage hydrolysis module of a nitric acid hydrolysis unit for hydrolysis of lignocellulosic feedstock and an upstream conditioning unit downstream from the hydrolysis unit and upstream from the bioreactor(s).
  • a second bioreactor or multiple bioreactors arranged in parallel, series, or a combination thereof with a strain that metabolizes C5 and C6 sugars can be coupled to an upstream second stage hydrolysis module of a nitric acid hydrolysis unit for hydrolysis of a lignocellulosic feedstock and an upstream conditioning unit downstream from the hydrolysis unit and upstream from the bioreactor(s).
  • the same bioreactor or multiple bioreactors arranged in parallel, series, or a combination thereof may be used for conversion of both C5 and C6 sugars to bioproduct.
  • both first and second stage conditioned nitric acid hydrolysates of a lignocellulosic feedstock may be added either separately or as a combined mixture to the bioreactor(s).
  • BBY Balanced butanol yield
  • BY Balanced solvent yield
  • HPLC HPLC - Sugars, solvent products, organic acids, and sugar degradation products were identified and quantified.
  • t (tinitiai) refers to the time point at the start of the conditioning process
  • tf (tfmai ) refers to the time point at the end of the conditioning process
  • to refers to the time point at beginning of fermentation.
  • t 96 refers to the time point after 96 hours of fermentation.
  • hydrolysates were subject to electrochemistry.
  • a hydrolysate of sugarcane bagasse was prepared by nitric acid hydrolysis. The resulting hydrolysate was treated with a combination of Ca(OH) 2 to pH 10, followed by KOH to pH 11, as described in Example 1.
  • the mixture was transferred to an appropriately sized plastic syringe, and the precipitate was removed by filtration using the filter discs.
  • the phases were equilibrated for 15 min by gentle end-over-end inversion on a rotating wheel (30 rpm) at 25.0 ⁇ 1°C.
  • the biphasic mixture was centrifuged at 3400 ⁇ g rpm for 3 min to ensure complete phase disengagement, and the MTBE phase was transferred to one or more glass test tubes, depending on the particular apparatus used for solvent evaporation.
  • test tube(s) containing the combined MTBE extracts were placed into the Turbo Vap and the volume of MTBE was reduced to 1-2 ml under a stream of nitrogen at 55°C.
  • the aqueous mixture was transferred to a 5 -ml volumetric flask, a drop of concentrated sulfuric acid was added, and water was added to dilute to volume.
  • a 1-ml aliquot of the aqueous solution from step 10 above was transferred to a labeled 2-ml autosampler vial, 25 ml of internal standard mixture was added, and the vial was capped and placed in the autosampler tray of HPLC-UV.
  • composition of samples is widely variable, and concentrations of target analytes typically span several orders of magnitude in a single sample. Accordingly, 10-fold and 100- fold dilutions of the solution from step 10 were also prepared.
  • a nitric acid hydro lysate of sugarcane bagasse was conditioned with a combination of Ca(OH) 2 to pH 10, followed by KOH to pH 11 , as described in Example 1.
  • butanol titer was 1.90 times greater in media that contained conditioned hydrolysate at a sugar concentration of 54.0 g/1 than in media that contained unconditioned hydrolysate at a sugar concentration of 53.2 g 1.
  • butanol titer was 4.21 times greater in media that contained 54.0 g/1 than in media that contained unconditioned hydrolysate at a sugar concentration of 53.2 g/1.

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Abstract

L'invention concerne des procédés de conditionnement d'un hydrolysat de biomasse cellulosique afin d'éliminer ou d'inactiver des inhibiteurs de la croissance microbienne et/ou de production de métabolite. Les procédés comprennent la précipitation ou l'inactivation des inhibiteurs par de l'hydroxyde de calcium et de l'hydroxyde de potassium.
PCT/US2012/049084 2011-08-01 2012-07-31 Elimination d'inhibiteurs de la fermentation microbienne d'hydrolysats cellulosiques ou d'autres compositions contenant un inhibiteur Ceased WO2013019822A1 (fr)

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US10759727B2 (en) 2016-02-19 2020-09-01 Intercontinental Great Brands Llc Processes to create multiple value streams from biomass sources
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EP4574984A1 (fr) * 2023-12-21 2025-06-25 Tirlán Limited Procédé de production d'acides organiques ou de sels de ceux-ci

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