WO2012009106A2 - Extraits de biomasse et utilisations de ceux-ci - Google Patents

Extraits de biomasse et utilisations de ceux-ci Download PDF

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Publication number
WO2012009106A2
WO2012009106A2 PCT/US2011/041109 US2011041109W WO2012009106A2 WO 2012009106 A2 WO2012009106 A2 WO 2012009106A2 US 2011041109 W US2011041109 W US 2011041109W WO 2012009106 A2 WO2012009106 A2 WO 2012009106A2
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WIPO (PCT)
Prior art keywords
plant
extract
biomass
enzyme
lignocellulosic biomass
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PCT/US2011/041109
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English (en)
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WO2012009106A3 (fr
Inventor
Kirk Pappan
Deisy Corridor
David Lee
Ramesh Nair
Bruce Ferguson
Michael Blaylock
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Edenspace Systems Corporation
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Publication of WO2012009106A2 publication Critical patent/WO2012009106A2/fr
Publication of WO2012009106A3 publication Critical patent/WO2012009106A3/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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
    • C08H8/00—Macromolecular compounds derived from lignocellulosic materials
    • C—CHEMISTRY; METALLURGY
    • 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
    • C13—SUGAR INDUSTRY
    • C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
    • C13K1/00—Glucose; Glucose-containing syrups
    • C13K1/02—Glucose; Glucose-containing syrups obtained by saccharification of cellulosic materials
    • 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

Definitions

  • the resistance of cell wall components to degradation is a key source of strength and pathogen defense for plants.
  • This resistance also represents a significant barrier in the conversion of lignocellulosic mass into simple sugars for production of biofuels and bio-based chemicals and for improvement of forage and silage digestibility.
  • Conversion of cellulose, for example, to fermentable sugars is accomplished by a series of enzymes known as cellulases.
  • cellulases can efficiently hydrolyze cellulose to simpler sugars, the surrounding matrix that includes hemicellulose, lignin, beta-glucans, homogalacturonans and rhamnogalacturonans typically should be partially or completely removed to expose the cellulose.
  • plants Numerous organisms have evolved the capability to hydrolyze plant biomass. Their methods of hydrolysis include the production of enzymes and chemicals that affect cell wall integrity, but these methods are not efficient to be directly used in large-scale industrial processes.
  • the present invention encompasses the recognition that many of these organisms are commonly associated with the plants they break down but they, and their active components, are destroyed by biomass pretreatment processes.
  • plants contain numerous polypeptides, such as cellulases, hemicellulases, peroxidases, and expansins, that act to loosen or break down cell walls and alter the presentation of structural carbohydrates in a manner that favors enzymatic hydrolysis. These polypeptides are generally inactivated by the high temperatures and extreme pH that characterize most biomass pretreatment methods.
  • the present invention encompasses the discovery that enabling the activities of such polypeptides before pretreatment and/or after pretreatment by shunting them around the pretreatment process confers significant benefits, including increased glucan conversion.
  • hydrating plant biomass and extracting the liquid produces a complex mixture of these polypeptides and other soluble chemicals.
  • This mixture herein referred to as an "extract”
  • both the process of hydration and extraction and the extract itself, separately or in combination substantially enhance biomass conversion, for example during enzymatic hydrolysis of dilute acid- pretreated plant material such as corn stover, corn fiber, poplar, sorghum, and switchgrass.
  • the inventors have shown that the extract exhibits moderate-to-high levels of activity with a range of natural and synthetic cell wall substrates and by itself leads to significant conversion of pretreated materials. Furthermore, the inventors have shown that when the extract is used to supplement commercial enzyme cocktails, such as those produced by Novozymes and Genencor, it can improve glucan conversion up to 50% and enables a 60% reduction in external enzyme usage.
  • methods comprising a step of incubating lignocellulolic biomass with an extract obtained from plant biomass.
  • such methods are used to increase yield from biomass conversion, e.g., increase conversion of glucans.
  • methods of increasing yield of fermentation product from a fermentative organism comprising a step growing a fermentative organism in a medium comprising an extract obtained from plant biomass.
  • provided are methods of starch hydrolysis comprising a step of incubating starch with an extract obtained from plant biomass.
  • provided are methods of bleaching cellulosic material comprising a step of incubating the cellulosic material with an extract obtained from plant biomass.
  • methods of reducing biomass recalcitrance of lignocellulosic biomass comprising a step of hydrating and pressing the lignocellulosic biomass before enzyme hydrolysis.
  • Figure 1 depicts a process flow diagram illustrating extraction of soluble components from biomass, pretreatment, and enzyme hydrolysis.
  • DAP dilute acid pretreatment
  • EH enzyme hydrolysis.
  • Figure 2A depicts glucose yields from enzymatic hydrolysis of corn stover using ACCELLERASETM 1500 supplemented with corn extracts from the same source as the stover being hydrolyzed.
  • Figure 2B depicts amounts of glucose in extracts themselves.
  • “biomass source” indicates the source of extract.
  • Pioneer 3T55 and Edenspace HI II (ESC HI II) refer to different corn varieties.
  • Figure 3 depicts glucose yields from experiments in which corn stover was hydrolyzed with ACCELLERASETM 1500 supplemented with corn extracts of sources different than that of the corn stover being hydrolyzed.
  • Figure 4 depicts glucose yields from experiments in which corn pericarp was hydrolyzed with ACCELLERASETM 1500 supplemented with extract of various corn fractions.
  • Figure 5 depicts glucose yields from experiments in which corn stover was subject to a hydration and extraction process and the remaining solids were then hydrolyzed with ACCELLERASETM 1500 (without the addition of extract).
  • Figure 6 depicts glucose yields from hydrolyzing switchgrass biomass that is untreated, pressed before hydrolysis, or supplemented with switchgrass extract during hydrolysis.
  • FIG. 7 depicts glucose yields from hydrolyzing poplar biomass
  • Figure 8 depicts a summary of results from enzymatic activity assays. Corn extract was tested on a variety of substrates.
  • Figure 9 depicts a summary of results showing optimal pH and temperature ranges, as well as residual activity after a heating period, for enzymatic activities in corn extract.
  • Figure 10 shows results from temperature optimization experiments for hydrolysis of corn stover using corn extract.
  • Figure 11 shows results from pH optimization experiments for hydrolysis of corn stover using corn extract.
  • the terms “about” and “approximately,” in reference to a number, are used herein to include numbers that fall within a range of 20%, 10%>, 5%, or 1% in either direction (greater than or less than) the number unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value.
  • the term "extract,” when used as noun, refers to a preparation from a biological material (such as lignocellulolytic biomass) in which a substantial portion of solids such as proteins are in solution.
  • the extract is a crude extract, e.g., an extract that is prepared by disrupting cells such that proteins are solubilized and optionally removing debris, but not performing further purification steps.
  • the extract is further purified in that certain substances, molecules, or combinations thereof are removed.
  • an "extract” is a preparation obtained from plant biomass.
  • such an extract is prepared by a process comprising hydrating the plant biomass in a liquid buffer.
  • an extract is prepared by a process comprising pressing the plant biomass.
  • an extract has one or more enzymatic activities.
  • Non-limiting examples of such enzymatic activities of extracts of the invention are endoglucanase, exoglucanase, ⁇ - glucosidase, xylanase, ⁇ -xylosidase, ferulic acid esterase, pectinase, arabinase, arabinofuranosidase, acetylxylan esterase, galactanase, and alpha-rhamnosidase.
  • an extract contains ability to cleave one or more substrates selected from the group consisting of 4-methylumbelliferyl ⁇ -D-cellobioside (MUC), 4-nitrophenyl ⁇ - D-cellobioside (pNPC), 4-nitrophenyl ⁇ -D-lactopyranoside (pNPLac), 4-nitrophenyl ⁇ - D-glucopyranoside (pNPG), beechwood xylan, birchwood xylan, oat spelt xylan, azo- labeled wheat arabinoxylan, pectin, 4-methylumbelliferyl ⁇ -D-xylopyranoside (MUX), 4- methylumbelliferyl p-trimethylammoniocinnamate chloride (MUTMAC), arabinan, 4- methylumbelliferyl a-L-arabinofuranoside (MUARF), 4-methylumbelliferyl acetate (MUA), 4-methylumbelliferyl a-D-galactopyr
  • MUC
  • plant can refer to a whole plant, plant parts (e.g., cuttings, tubers, pollen), plant organs (e.g., leaves, stems, flowers, roots, fruits, branches, etc.), individual plant cells, groups of plant cells (e.g., cultured plant cells), protoplasts, plant extracts, seeds, and progeny thereof.
  • the class of plants which can be used in the methods of the present invention include both monocotyledonous and dicotyledonous plants, as well as certain lower plants such as algae.
  • the term includes plants of a variety of a ploidy levels, including polyploid, diploid and haploid. In certain embodiments of the invention, plants are green field plants.
  • suitable plants include, but are not limited to, corn, switchgrass, poplar, sorghum, miscanthus, sugarcane, pine, wheat, rice, soy, cotton, barley, turf grass, tobacco, bamboo, rape, sugar beet, sunflower, willow, and eucalyptus.
  • Genetically modified plants e.g., transgenic plants that have been obtained by transformation methods, are also suitable for use in the present invention.
  • polypeptide generally has its art-recognized meaning of a polymer of at least three amino acids.
  • the term is also used to refer to specific functional classes of polypeptides, such as, for example, lignocellulolytic enzyme polypeptides (including, for example, Acidothermus cellulolyticus El endo-1,4- ⁇ -glucanase polypeptide, Acidothermus cellulolyticus xylE polypeptide, Acidothermus cellulolyticus guxl polypeptide, Acidothermus cellulolyticus avilll polypeptide, Talaromyces emersonii cbhE polypeptide, and Pyrococcus furiosus faeE (ferulic acid esterase) polypeptide).
  • lignocellulolytic enzyme polypeptides including, for example, Acidothermus cellulolyticus El endo-1,4- ⁇ -glucanase polypeptide, Acidothermus cellulolyticus xylE polypeptide, Acid
  • polypeptide is intended to be sufficiently general as to encompass not only polypeptides having the complete sequence recited herein (or in a reference or database specifically mentioned herein), but also to encompass polypeptides that represent functional fragments (i.e., fragments retaining at least one activity) of such complete polypeptides.
  • polypeptides generally tolerate some substitution without destroying activity.
  • Other regions of similarity and/or identity can be determined by those of ordinary skill in the art by analysis of the sequences of various polypeptides presented herein.
  • source biomass refers to biomass that is used to obtain extracts used in accordance with the present invention.
  • source biomass refers to biomass obtained from the same source, e.g., the same plant variety, as that used to obtain extracts used in accordance with the present invention.
  • non-source biomass refers to biomass that is from a different source, e.g., a different plant species and/or variety, as that used to obtain extracts used in accordance with the present invention.
  • the present invention encompasses novel methods of using plant extracts for a variety of uses, including improving conversion of biomass.
  • novel methods of using plant extracts for a variety of uses including improving conversion of biomass.
  • Extracts used in accordance with the invention can be prepared from plant biomass from any of a variety of plants, plant parts, and/or plant organs. Extracts can be prepared from plant biomass from any plant part or combination of plant parts ⁇ e.g., cuttings, tubers, pollen) and/or from any plant organ or combination of plant organs (e.g., leaves, stems, culms, roots, seeds, grain, flowers, fruits, husks, hulls, straw, bark, stover, fiber, cobs, etc.), from individual plant cells, groups of plant cells (e.g., cultured plant cells), protoplasts, plant extracts, seeds, and progeny thereof.
  • plant part or combination of plant parts e.g., cuttings, tubers, pollen
  • plant organs e.g., leaves, stems, culms, roots, seeds, grain, flowers, fruits, husks, hulls, straw, bark, stover, fiber, cobs, etc.
  • Plants that can be used in the methods of the present invention include both monocotyledonous and dicotyledonous plants, as well as certain lower plants such as algae. Multicotyledenous plants may also be used.
  • Non-limiting examples of monocotyledonous plants suitable for use in the present invention include barley, bamboo, maize (corn), sorghum, switchgrass, miscanthus, sugarcane, wheat, rice, rye, turfgrass, oat, fescue, millet, and any grass species.
  • Non-limiting examples of dicotyledonous plants suitable for use in the present invention include oilseed rape, tobacco, tomato, sugar beet, potato, soybean, canola, sunflower, alfalfa, cotton, flax, pine, willow, eucalyptus, poplar, and any tree species. Hybrids of plant species may also be used.
  • Extracts can be prepared from any combination of plant biomass sources, e.g., biomass from more than one plant part and/or more than one plant species may be used to obtain extract.
  • Plants of a variety of a ploidy levels may be used. In certain embodiments of the invention, plants are green field plants.
  • plant biomass is obtained from a plant that comprises one or more soluble components that promote deconstruction of plant cell wells.
  • soluble components include polypeptides, metabolites, chemicals, and co-factors.
  • Polypeptides that promote deconstruction of plant cell walls include enzyme polypeptides, e.g., auto lytic enzyme polypeptides.
  • plant biomass may be obtained from a plant that comprises one or more lignocellulosic polypeptides (e.g., cellulases, hemicellulases, ligninases, or combinations thereof).
  • the plant may be genetically engineered to express one or more such lignocellulosic polypeptides.
  • any genetically modified plants e.g., transgenic plants that have been obtained by transformation methods, is also suitable for use in the present invention.
  • any genetically modified plants e.g., transgenic plants that have been obtained by transformation methods, is also suitable for use in the present invention.
  • genetically modified plants that may be suitable for use in accordance with the present invention, see e.g., see, e.g., International Patent Application Serial No. PCT/US09/48153 (published as WO 2009/155601).
  • Extracts may be used to enhance hydrolysis of lignocellulosic biomass that is from the same or different source of biomass.
  • the lignocellulosic biomass is from the same source as the plant biomass from which plant extract is obtained.
  • the lignocellulosic biomass is from the same species of plant as the plant biomass from which plant extract is obtained.
  • lignocellulosic biomass is from a different species of plant as the plant biomass from which plant extract is obtained.
  • plant biomass from which extract is obtained is freshly harvested.
  • plant biomass from which extract is obtained has undergone one or more procedures.
  • Non-limiting examples of such procedures include grinding, chopping, milling, ensilement, baling, binning, ginning, squeezing, pressing, composting, bagging, drying, tempering and combinations thereof.
  • the plant biomass is Dried Distillers Grains with Solubles (DDGS).
  • the plant biomass is a byproduct or waste product. For example, sawdust, cotton gin waste, and/or grass clippings may be used to obtain extract.
  • not pretreating or pretreating plant biomass under less harsh conditions may help preserve certain useful properties (e.g., enzymatic activities) of plant extracts.
  • plant biomass from which extract is obtained has not been pretreated under conditions to promote accessibility of celluloses within the plant biomass, or has been pretreated under conditions that are less harsh (e.g., less acid, less heat, wet milling etc.) than is typically used for pretreatment of lignocellulosic processing.
  • plant biomass from such extracts is used to supplement enzyme hydrolysis reactions on lignocellulosic biomass that has been pretreated.
  • an extract is existing plant steepage (also known as plant "steep water”).
  • plant steepage also known as plant "steep water”
  • Non-limiting examples of plant steepage suitable for use as an extract in accordance with the present invention include corn steep water, corn steep liquor, barley steep extract, rice steep extract, and wheat steep extract.
  • an extract is prepared by a process comprising hydrating the plant biomass in a liquid buffer.
  • a suitable liquid buffer is a sodium citrate solution.
  • a liquid buffer that has an acid pH is used.
  • the pH of the liquid buffer is between approximately 4.5 and approximately 5.5.
  • the pH of the liquid buffer is approximately 5.0.
  • plant biomass is hydrated for a period of up to about five days.
  • plant biomass is hydrated for a period of up to about three days (e.g., about 72 hours).
  • extract is prepared by a process comprising a step of separating solid from liquids, wherein the liquids so obtained are used (either without further processing or with one or more subsequent processing steps) as the extract.
  • techniques useful for separating solids from liquids include separation with a filter or filtering material (e.g., cheesecloth), centrifugation, allowing solids to settle to the bottom of a vessel or container, and combinations thereof.
  • an extract is prepared by a process comprising (a) subjecting the plant biomass to a procedure selected from the group consisting of rinsing, crushing, chopping, grinding, pureeing, pressing, extruding, milling, straining and combinations thereof; and (b) separating solids from liquid, thereby obtaining the liquid as an extract.
  • this process is performed at a pH between approximately 4.5 and approximately 5.5 (e.g., approximately 5.0).
  • this process is performed at a temperature between approximately 15 °C and approximately 50 °C (e.g., approximately 25 °C).
  • this process further comprises a period of hydration.
  • plant biomass can be incubated in a liquid buffer for hydration before the step of separating solids from liquids.
  • extracts are prepared by a process comprising a step of concentrating (e.g., to reduce volume of liquid extract), diluting (e.g., to increase the volume of liquid extract), or drying (e.g., to produce a solid extract).
  • extracts are prepared by a process comprising a step of removing soluble low molecular mass components from soluble high molecular mass components, thereby obtaining an extract that is enriched for enzyme activity.
  • components that are less than about 10 kDa in size are considered “low molecular mass components” and components that are about 10 kDa or more in size are considered “high molecular mass components”.
  • low molecular mass components for these purposes include, organic acids, furfurals, and HMF (5-Hydroxymethylfurfural).
  • a high molecular mass fraction may contain polypeptides such as enzyme polypeptides and therefore be enriched for enzymatic activity as compared to unseparated extract or to a low molecular mass fraction. Separation can be achieced, e.g., by any of various fractionation and filtration methods.
  • extracts are prepared by a process comprising a step of reducing the content of organic acids, e.g., of lactic acid.
  • an extract has one or more enzymatic activities.
  • enzymatic activities of extracts of the invention are endoglucanase, exoglucanase, ⁇ -glucosidase, xylanase, ⁇ -xylosidase, ferulic acid esterase, pectinase, arabinase, arabinofuranosidase, acetylxylan esterase, galactanase, and alpha-rhamnosidase.
  • an extract contains ability to act on (e.g., cleave or otherwise alter) one or more substrates selected from the group consisting of 4- methylumbelliferyl ⁇ -D-cellobioside (MUC), 4-nitrophenyl ⁇ -D-cellobioside (pNPC), 4- nitrophenyl ⁇ -D-lactopyranoside (pNPLac), 4-nitrophenyl ⁇ -D-glucopyranoside (pNPG), beechwood xylan, birchwood xylan, oat spelt xylan, azo-labeled wheat arabinoxylan, pectin, 4-methylumbelliferyl ⁇ -D-xylopyranoside (MUX), 4-methylumbelliferyl p- trimethylammoniocinnamate chloride (MUTMAC), arabinan, 4-methylumbelliferyl a-L- arabinofuranoside (MUARF), 4-methylumbelliferyl acetate (MUA), 4- methyl
  • provided are methods comprising a step of incubating lignocellulosic biomass with an extract obtained from plant biomass.
  • provided methods are used to enhance conversion of lignocellulosic biomass, e.g., to increase production of sugars and/or alcohols from lignocellulosic biomass.
  • provided methods allow decreased enzyme loading of enzymes or enzyme cocktails that are typically added to a hydrolysate mixture during plant processing.
  • lignocellulosic biomass that can be processed in accordance with methods of the present invention can be obtained from any of a variety of plant parts, plants, and/or plant cells.
  • lignocellulosic biomass has been pretreated under under conditions to promote accessibility of celluloses within the lignocellulosic biomass.
  • lignocellulosic biomass has not been pretreated, or has been pretreated under conditions that are less harsh ⁇ e.g., less acid, less heat, wet milling, etc.) than is typically used for pretreatment of lignocellulosic processing.
  • lignocelluloisic biomass has been tempered to engage endogenous lignocellulosic enzymes favorably for hydrolysis. See, e.g., International Patent Application No. PCT/US 10/24505, the entire contents of which are herein incorporated by reference. Enzyme hydrolysis
  • lignocellulosic biomass is incubated with extract as part of a process converting the biomass, e.g., to sugars and/or alcohols ⁇ e.g., for industrial processing of ethanol, butanol, and/or methanol).
  • lignocellulosic biomass is incubated with an enzyme or enzyme cocktail in addition to being incubated with the extract during the enzyme hydrolysis reaction.
  • enzymes and enzyme cocktails are commercially available and useful for enzyme hydrolysis reactions.
  • suitable enzyme cocktails that may be used in conjunction with plant extracts in accordance with the invention include ACCELLERASETM, CELLICTM, CTec2, CELLICTM HTec2, and combinations thereof.
  • extract is mixed with or added to an enzyme or enzyme cocktail during enzymatic hydrolysis to convert glucans lignocellulosic biomass. In some embodiments, extract is mixed with or added to an enzyme or enzyme cocktail prior to enzymatic hydrolysis to convert glucans in the lignocellulosic biomass.
  • the amount of enzyme or enzyme cocktail that is added to an enzyme hydrolysate mixture or enzyme hydrolysis reacton is less in the presence of plant extract than is needed to hydro lyze an equivalent amount ⁇ e.g., in terms of yield) as compared to a hydrolysate mixture or enzyme hydrolysis reaction in which no plant extract is present.
  • plant extracts may be used for any of a variety of different uses.
  • plant extracts may be used as a nutrient source for fermentative organisms ⁇ e.g., yeast and other microbes).
  • methods of increasing yield of fermentation product from a fermentative organism comprising a step growing a fermentative organism in a medium comprising an extract obtained from plant biomass.
  • Non-limiting examples of fermentation products whose yields can be increased by provided methods include alcohols (such as ethanol, butanol, and methanol), other biofuels, and other renewable chemicals.
  • plant extracts are used as a nutrient source for fermentative organisms that produce products having commerical value, e.g., enzyme polypeptides enzyme cocktails, and/or supplemental or accessory enzyme polypeptides. Provided methods are compatible with commercial fermentation processes.
  • extracts may be used as a nutrient source for other organisms, e.g., humans and/or livestock (e.g., poultry).
  • livestock e.g., poultry
  • Extracts may also be used to enhance (e.g., increase yield from) starch hydrolysis.
  • Provided are methods comprising a step of incubating starch with an extract obtained from plant biomass.
  • starch sources compatible with provided mehtods include grain from maize, sorghum, rice, oat, barley, potato, or wheat.
  • Extracts may also be used to bleach cellulosic material.
  • a non-limiting example of cellulosic material that may be used in accordance with the invention is wood pulp.
  • plant extracts are applied directly to green or ensiling biomass to improve silage quality or pretreatment efficiency.
  • lignocellulosic biomass comprising a step of hydrating and pressing the lignocellulosic biomass before enzyme hydrolysis.
  • the inventors have discovered that the mechanical process of hydrating and/or processing lignocellulosic biomass results in increased conversion to glucose. Such results were obtained even without using the plant extract that would be obtained by the hydration and pressing processes.
  • Example 1 Production and use of plant extracts to improve glucan conversion of corn biomass
  • the present Example illustrates the using corn biomass extracts improves glucan conversion of the same source of corn biomass.
  • Figure 1 shows a flow diagram that depicts a process for dilute acid pretreatment, extraction of soluble components from biomass, and enzymatic hydrolysis with and without supplementing plant extracts.
  • pressed biomass used to produce extract is also pretreated and hydrolyzed according to the DAP Pretreatment process shown.
  • corn stover material (leaves and stems) from senesced and dried corn plants were milled to pass a 20 mesh screen. A 1 g subsample of the milled material was then incubated with 10 mL of a 0.1 M sodium citrate buffer solution (pH 5.0) for 10 minutes at room temperature (25°C). After incubation, the resulting liquid extract was separated from the solid material by filtering through cheesecloth. Glucose concentration of an aliquot of the plant extract was quantified using ion chromatography.
  • Extract was then used to supplement enzymatic hydrolysis as described below.
  • Example 2 Production and use of plant extracts to improve glucan conversion of non- source biomass
  • extracts were prepared and used as described in Example 1, except that hydrolysis was performed on non-source biomass ⁇ i.e., biomass that is from a different source than the extract). Increased glucan conversion was also observed when extract is applied to non-source biomass.
  • Corn stover was pretreated with dilute sulfuric acid (1% H 2 S0 4 , 190°C for 15 minutes).
  • Extracts were prepared from a variety of biomass samples from corn plants other than the plants from which the corn stover that was pretreated were obtained. Dried biomass was harvested from outdoor field plots, and green biomass was collected from greenhouse-grown corn and a from field plot. Extracts from these biomass samples were added to the pretreated corn stover in hydrolysis reactions with 0.2 mL Accellerase/g glucan. For comparison purposes, a set of pretreated corn stover samples were hydrolyzed in 0.5 mL Accellerase/g glucan without extract. Enzymatic hydrolysis was carried out for all samples in this Example for 72 hours at 50 °C. Levels of glucose released from hydrolysis reactions were quantified using a commercial glucose oxidase kit (Sigma- Aldrich).
  • Example 3 Use of plant extracts to improve glucan conversion of corn grain pericarp
  • extract was tested on corn grain pericarp, the fibrous portion of corn grain, to determine if extract can provide processing benefits to non- stover biomass during hydrolysis.
  • Pericarp is also a ready source of biomass that could be used in existing corn grain ethanol plants.
  • Cobs and stover were dried in a forced air dryer and then milled to 1 mm particle size.
  • Corn grain pericarp tissue was obtained by a dry fractionation process and subjected to milling to 1 mm particle size.
  • Enzyme hydrolysis mixtures were prepared in the absence and presence of plant extracts and included ACCELLERASETM 1500 (Genencor) at a final concentration of either 0.2 or 0.5 mL/g biomass. Enzyme hydrolysis mixtures were added to dry pericarp (1 mm particle size) biomass and hydrolysis reactions were incubated at 50 °C for 72 hours. After incubation, samples were boiled for 5 minutes, centrifuged, and glucose in the supernatant was measured using a glucose oxidase-based assay kit from Sigma. Results
  • ACCELLERASETM 1500 with plant extracts increased the amount of glucose released from pericarp fiber.
  • the combination of a low concentration of ACCELLERASETM 1500 with any of the plant extracts tested led to a greater liberation of glucose than obtained with a high concentration (0.5 mL/g biomass) of ACCELLERASETM 1500 alone ( Figure 4).
  • Example 4 Increased glucan conversion by hydration and extraction
  • the present Example demonstrates that the mechanical process of hydration and extraction alone on biomass leads to increased glucan conversion of that biomass. Thus, increased glucan conversion can be obsereved even without adding extract to the biomass at a later time.
  • Corn stover material (leaves and stems) from senesced and dried corn plants were milled to pass a 20 mesh screen. A 1 g subsample of the milled material was then incubated with 10 mL of 50 mM sodium citrate buffer, pH 5.0, for 10 minutes at room temperature (25 °C). After incubation, the resulting extract solution was separated from solids by filtering through cheesecloth. 2 g (wet weight) of solids were dilute acid- pretreated (using conditions of 0.5% H 2 SO 4 , 190 °C for 10 minutes) and hydrolyzed.
  • ACCELLERASETM 1500 (Genencor) was added to the biomass slurry at a loading rate of 0.2 mL/g glucan with 100 L of 2% sodium azide to inhibit microbial growth. Reactions were brought to a final volume of 10 mL with distilled water. Slurries were then incubated for 72 hours at a temperature of 50 °C. After incubation, soluble glucose in the solution was measured colorimetrically using a commercial glucose oxidase kit (Sigma- Aldrich) or quantified by ion chromatography. Measured glucose levels were normalized to the starting quantity of biomass.
  • Example 5 Use of plant extracts to improve glucan conversion in switchgrass
  • the present Example demonstrates that extracting soluble components from plant biomass can improve glucan conversion in plants other than corn.
  • Wild type switchgrass (variety FA4) was grown in a greenhouse alongside a variety of Alamo switchgrass amenable to transformation (ESC Alamo) and biomass was harvested from mature plants. The biomass was milled to pass a 20 mesh screen. Three- gram samples of milled material was incubated with 30 mL of 50 mM sodium citrate buffer, pH 5.0 for 10 minutes at room temperature (25 °C). 100 L of 2% sodium azide was added to inhibit microbial growth. After incubation, the extract was separated from the solid by filtering through cheesecloth. Glucose concentrations in plant extracts were quantified using ion chromatography.
  • Each of the hydrolysis reactions in this Example contained 2 g of dilute acid- pretreated biomass, 0.2 mL of ACCELLERASETM 1500 (Genencor)/g glucan, andlOO
  • Example 6 Use of plant extracts to improve glucan conversion in poplar
  • the present Example demonstrates that use of plant extracts also improves glucan conversion in poplar.
  • Poplar biomass material (leaves and stems) from wild type poplar was dried and milled to pass a 20 mesh screen. A 1 g subsample of the milled material was then incubated with 10 mL 0.05 M sodium citrate buffer (pH 5.0) for 10 min at room temperature (25 °C). After incubation, the resulting extract solution was separated from the solid by filtering through cheesecloth. A 5 mL aliquot of the extract solution was added to 2 g (wet weight) of dilute acid-pretreated (using conditions of 0.5%> H 2 SO 4 , 190°C for 10 minutes) poplar with ACCELLERASETM 1500 (Genencor) at a rate of 0.2 ml/g glucan. For comparison, hydrolysis reactions were also carried out using
  • Figure 7 depicts results from this experiment. Addition of plant extracts during hydrolysis resulted in a greater than 30% increase in glucose production compared to that achieved by using an enzyme (ACCELLERASETM 1500) loading rate of 0.2 mL/g without extract. Addition of extract produces only slightly less glucose than is produced at the higher enzyme loading rate of 0.5 mL/g.
  • Corn stover was dried in a forced air dryer and then milled to 1 mm particle size. Milled stover source biomass material was weighed and extraction buffer (50 mM sodium acetate, pH 5.0) was added until a 10%> total solids concentration was achieved. Biomass was mixed with extraction buffer until it was thoroughly hydrated and was then strained through four layers of cheesecloth. Additional liquid was obtained by hand- wringing the cheesecloth. Total soluble protein concentration in the plant extract was determined using a Bradford protein assay reagent according to the manufacturer's instructions (Biorad) and a protein standard curve for the Bradford assay was prepared using bovine serum albumin purchased from Biorad.
  • extraction buffer 50 mM sodium acetate, pH 5.0
  • corn extracts The presence and characteristics of multiple enzyme classes in corn extracts was determined by activity assay using a range of natural, semi-pure, and synthetic substrates indicative of cellulases, xylanases, pectinases, hemicellulases, and cell wall accessory enzymes.
  • the following substrates were examined: 4-methylumbelliferyl ⁇ -D- cellobioside (MUC), 4-nitrophenyl ⁇ -D-cellobioside (pNPC), 4-nitrophenyl ⁇ -D- lactopyranoside (pNPLac), 4-nitrophenyl ⁇ -D-glucopyranoside (pNPG), beechwood xylan, birchwood xylan, oat spelt xylan, azo-labeled wheat arabinoxylan, pectin, 4- methylumbelliferyl ⁇ -D-xylopyranoside (MUX), 4-methylumbelliferyl p- trimethylammoniocinnamate chloride (MUTMAC), arabinan, 4-methylumbelliferyl a-L- arabinofuranoside (MUARF), 4-methylumbelliferyl acetate (MUA), 4- methylumbelliferyl a-D-galactopyranoside (MU-Gal), and 4-methylumbelliferyl a
  • Corn extract had relatively less activity toward MUC, pNPC, pNPLac, pNPG, xylans, and pectins than ACCELLERASETM, indicating that commercial enzyme blends contain high levels of cellulases, xylanases, and pectinases (Figure 8). Corn extract had relatively greater levels of activity toward MUA, MUX, MUTMAC, arabinan, MUARF, MU-Gal, and MU-Rh, indicating that it contains high levels of accessory enzymes that increase the efficiency of hemicellulases, pectinases, and cellulases.
  • Example 8 Temperature and pH optima for enzyme activities in corn extract
  • Extract from corn stover was prepared as described in Example 7.
  • Stock buffer solutions ranging in pH from 3.5 to 7.0 were made using 0.5 M sodium citrate and the final pH was adjusted using a pH meter and IN HCl or IN NaOH.
  • Stock buffer solutions ranging in pH from 7.5 to 8.5 were made in a similar fashion using Tris as the buffer.
  • thermostability of enzymatic activities in corn extract was investigated by heating plant extracts in the absence of substrate for 2 hours and for 24 hours at temperatures ranging from 20 °C to 80 °C. After incubation without substrate, plant extracts were mixed with substrates and residual activity at pH 5.0 and 50 °C was determined. Separate pH, temperature, and thermostability characterization experiments were conducted in parallel using ACCELLERASETM 1500 (Genencor) as an enzyme source to serve as a reference for the enzymes in corn extract. Relative activities of corn extract and of ACCELLERASETM was scored after activities of both samples were normalized to level of total source protein in the respective samples.
  • enzymes present in corn extracts had optimal activities in the moderately acidic range from pH 4.5-6.5 and temperature optima at or close to 50 °C (Figure 9).
  • Corn stover was dried in a forced air dryer and then milled to 1 mm particle size. Milled stover source biomass material was weighed and extraction buffer (50 mM sodium acetate, pH 5.0) was added until a 10% total solids concentration was achieved. Biomass was mixed with extraction buffer until it was thoroughly hydrated and was then strained through four layers of cheesecloth. Additional liquid was obtained by hand- wringing the cheesecloth.
  • extraction buffer 50 mM sodium acetate, pH 5.0
  • ACCELLERASETM 1500 was then added to the biomass slurry at a loading rate of 0.2 mL/g glucan.
  • pretreated biomass samples were hydrolyzed with either 0.2 mL/g or 0.5 mL/g ACCELLERASETM without corn extract.
  • 100 L of 2% sodium azide was added to each reaction in this Example to inhibit microbial growth. Reactions were brought to a final volume of 10 mL with distilled water.
  • Five separate slurries were then incubated for 72 hours in triplicate at various temperatures ranging from 37 °C to 60 °C. After incubation, soluble glucose in the solution was measured colorimetrically using a commercial glucose oxidase kit (Sigma- Aldrich).
  • Figure 11 shows results from pH optimization experiments. The maximum level of glucose production was observed when the slurry was incubated at pH 5.5, which is compatible with most commercial enzymes.

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Abstract

L'invention concerne des procédés d'utilisation d'extraits de biomasse végétale pour améliorer la transformation de glucane, accroître les rendements de fermentation, réduire la récalcitrance de la biomasse, améliorer la qualité d'ensilage, accroître l'hydrolyse d'amidon et/ou blanchir la matière cellulosique.
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CN103314673A (zh) * 2013-06-04 2013-09-25 新疆生产建设兵团第十三师农业科学研究所 棉花种子加工的方法
CN104527117A (zh) * 2014-12-18 2015-04-22 大名县荣凯生物质能壁炉有限公司 玉米秸秆压片机

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US20090270609A1 (en) * 2006-05-30 2009-10-29 Danisco A/S Separation process
US20080160514A1 (en) * 2006-12-27 2008-07-03 Weyerhaeuser Co. Enzymatic treatment of pulp
US20090093027A1 (en) * 2007-10-03 2009-04-09 Board Of Trustees Of Michigan State University Process for producing sugars and ethanol using corn stillage
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103314673A (zh) * 2013-06-04 2013-09-25 新疆生产建设兵团第十三师农业科学研究所 棉花种子加工的方法
CN104527117A (zh) * 2014-12-18 2015-04-22 大名县荣凯生物质能壁炉有限公司 玉米秸秆压片机

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