Related Applications

This application claims priority to United States provisional application

61/359,331 filed on June 28, 2010, the entire contents of which are incorporated herein by reference.

Background

The resistance of cell wall components to degradation is a key source of strength and pathogen defense for plants. This resistance, commonly referred to as biomass recalcitrance, 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. However, before 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.

Summary

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. In addition, 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. According to certain aspects of the present invention, hydrating plant biomass and extracting the liquid produces a complex mixture of these polypeptides and other soluble chemicals. The inventors have discovered that this mixture, herein referred to as an "extract", has certain advantageous properties. For example, 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.

In one aspect of the invention, provided are methods comprising a step of incubating lignocellulolic biomass with an extract obtained from plant biomass. In some embodiments, such methods are used to increase yield from biomass conversion, e.g., increase conversion of glucans.

In one aspect of the invention, provided are 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.

In one aspect of the invention, provided are methods of starch hydrolysis comprising a step of incubating starch with an extract obtained from plant biomass.

In one aspect of the invention, provided are methods of bleaching cellulosic material comprising a step of incubating the cellulosic material with an extract obtained from plant biomass. In one aspect of the invention, provided are methods of reducing biomass recalcitrance of lignocellulosic biomass comprising a step of hydrating and pressing the lignocellulosic biomass before enzyme hydrolysis.

Brief Description of the Drawings

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 ACCELLERASE™ 1500 supplemented with corn extracts from the same source as the stover being hydrolyzed. Figure 2B depicts amounts of glucose in extracts themselves. In Figures 2A and 2B, "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 ACCELLERASE™ 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 ACCELLERASE™ 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 ACCELLERASE™ 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.

Figure 7 depicts glucose yields from hydrolyzing poplar biomass with

ACCELLERASE™ 1500 supplemented with poplar extract.

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.

Definitions

Throughout the specification, several terms are employed that are defined in the following paragraphs.

As used herein, 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.

As used herein, 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. In some embodiments of the invention, 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. In some embodiments of the invention, the extract is further purified in that certain substances, molecules, or combinations thereof are removed.

In certain embodiments of the invention, an "extract" is a preparation obtained from plant biomass. In some embodiments, such an extract is prepared by a process comprising hydrating the plant biomass in a liquid buffer. In some embodiments, an extract is prepared by a process comprising pressing the plant biomass. In some embodiments, an extract has one or more enzymatic activities. Non-limiting examples of such enzymatic activities of extracts of the invention are endoglucanase, exoglucanase, β- glucosidase, xylanase, β-xylosidase, ferulic acid esterase, pectinase, arabinase, arabinofuranosidase, acetylxylan esterase, galactanase, and alpha-rhamnosidase. In some embodiments, an extract contains ability to cleave one or more substrates selected from the group consisting of 4-methylumbelliferyl β-D-cellobioside (MUC), 4-nitrophenyl β- D-cellobioside (pNPC), 4-nitrophenyl β-D-lactopyranoside (pNPLac), 4-nitrophenyl β- D-glucopyranoside (pNPG), beechwood xylan, birchwood xylan, oat spelt xylan, azo- labeled wheat arabinoxylan, pectin, 4-methylumbelliferyl β-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), 4-methylumbelliferyl a-L-rhamnopyranoside (MU-Rh), and combinations thereof.

As will be clear from the context, the term "plant", as used herein, 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. For example, 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.

As used herein, the term "polypeptide", generally has its art-recognized meaning of a polymer of at least three amino acids. However, 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- β-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). For each such class, the present specification provides specific examples of known sequences of such polypeptides. Those of ordinary skill in the art will appreciate, however, that the term "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. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%>, 70%>, or 80%>, and further usually including at least one region of much higher identity, often greater than 90%) or even 95%, 96%, 97%, 98%>, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term "polypeptide" as used herein. 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.

As used herein, the phrase "source biomass" refers to biomass that is used to obtain extracts used in accordance with the present invention. Alternatively or additionally, the phrase "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.

As used herein, the phrase "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.

Detailed Description of Certain Embodiments of the Invention

The present invention encompasses novel methods of using plant extracts for a variety of uses, including improving conversion of biomass. In certain embodiments,

I. Extracts

Sources

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.

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.

For a more expansive list of plant types and plant parts that can be used as a source of plant biomass for preparing extracts, see, e.g., International Patent Application Serial No. PCT/US09/48153 (published as WO 2009/155601), the entire contents of which are herein incorporated by reference.

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, including polyploid, diploid and haploid, may be used. In certain embodiments of the invention, plants are green field plants.

In some embodiments, plant biomass is obtained from a plant that comprises one or more soluble components that promote deconstruction of plant cell wells. Non- limiting examples of such 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. Alternatively or additionally, plant biomass may be obtained from a plant that comprises one or more lignocellulosic polypeptides (e.g., cellulases, hemicellulases, ligninases, or combinations thereof). For example, the plant may be genetically engineered to express one or more such lignocellulosic polypeptides.

In general, any genetically modified plants, e.g., transgenic plants that have been obtained by transformation methods, is also suitable for use in the present invention. For more information on 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. In some embodiments, the lignocellulosic biomass is from the same source as the plant biomass from which plant extract is obtained. In some embodiments, the lignocellulosic biomass is from the same species of plant as the plant biomass from which plant extract is obtained. In some embodiments, lignocellulosic biomass is from a different species of plant as the plant biomass from which plant extract is obtained.

In some embodiments, plant biomass from which extract is obtained is freshly harvested.

In certain embodiments, 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. In some embodiments, the plant biomass is Dried Distillers Grains with Solubles (DDGS). In some embodiments, 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.

Without wishing to be bound by any particular theory, not pretreating or pretreating plant biomass under less harsh conditions may help preserve certain useful properties (e.g., enzymatic activities) of plant extracts. In some embodiments, 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. In some such embodiments, plant biomass from such extracts is used to supplement enzyme hydrolysis reactions on lignocellulosic biomass that has been pretreated.

In some embodiments, an extract is existing 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.

Preparation

Any of a variety of methods may be used to obtain extracts from plant biomass. Such methods include those exemplified in the Examples.

In some embodiments, an extract is prepared by a process comprising hydrating the plant biomass in a liquid buffer. A non-limiting example of a suitable liquid buffer is a sodium citrate solution. In some embodiments, a liquid buffer that has an acid pH is used. In some embodiments, the pH of the liquid buffer is between approximately 4.5 and approximately 5.5. In some embodiments, the pH of the liquid buffer is approximately 5.0. In some embodiments, plant biomass is hydrated for a period of up to about five days. In some embodiments, plant biomass is hydrated for a period of up to about three days (e.g., about 72 hours).

In some embodiments, 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. Non- limiting examples of 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.

For example, in some embodiments, 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. In some embodiments, this process is performed at a pH between approximately 4.5 and approximately 5.5 (e.g., approximately 5.0). In some embodiments, this process is performed at a temperature between approximately 15 °C and approximately 50 °C (e.g., approximately 25 °C). In some embodiments, this process further comprises a period of hydration. For example, plant biomass can be incubated in a liquid buffer for hydration before the step of separating solids from liquids.

In some embodiments, 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).

In some embodiments, 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. In some embodiments, 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". Non-limiting examples of "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.

In some embodiments, extracts are prepared by a process comprising a step of reducing the content of organic acids, e.g., of lactic acid.

Enzyme activities and substrates

In some embodiments, an extract has one or more enzymatic activities. Non- limiting examples of such enzymatic activities of extracts of the invention are endoglucanase, exoglucanase, β-glucosidase, xylanase, β-xylosidase, ferulic acid esterase, pectinase, arabinase, arabinofuranosidase, acetylxylan esterase, galactanase, and alpha-rhamnosidase. In some embodiments, 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 β-D-cellobioside (MUC), 4-nitrophenyl β-D-cellobioside (pNPC), 4- nitrophenyl β-D-lactopyranoside (pNPLac), 4-nitrophenyl β-D-glucopyranoside (pNPG), beechwood xylan, birchwood xylan, oat spelt xylan, azo-labeled wheat arabinoxylan, pectin, 4-methylumbelliferyl β-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), 4-methylumbelliferyl a-L- rhamnopyranoside (MU-Rh), and combinations thereof.

II. Methods of using extracts

In certain embodiments, provided are methods comprising a step of incubating lignocellulosic biomass with an extract obtained from plant biomass. In some embodiments, provided methods are used to enhance conversion of lignocellulosic biomass, e.g., to increase production of sugars and/or alcohols from lignocellulosic biomass. In some embodiments, provided methods allow decreased enzyme loading of enzymes or enzyme cocktails that are typically added to a hydrolysate mixture during plant processing.

Lignocellulosic biomass

As with plant biomass discussed above, 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.

In some embodiments, lignocellulosic biomass has been pretreated under under conditions to promote accessibility of celluloses within the lignocellulosic biomass. In some embodiments, 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. In some embodiments, 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

In certain embodiments, 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). In some embodiments, lignocellulosic biomass is incubated with an enzyme or enzyme cocktail in addition to being incubated with the extract during the enzyme hydrolysis reaction. A number of enzymes and enzyme cocktails are commercially available and useful for enzyme hydrolysis reactions. Non-limiting examples of suitable enzyme cocktails that may be used in conjunction with plant extracts in accordance with the invention include ACCELLERASE™, CELLIC™, CTec2, CELLIC™ HTec2, and combinations thereof. In some embodiments, 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.

In some embodiments, 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.

III. Other Uses

In accordance with various aspects of the present invention, plant extracts may be used for any of a variety of different uses.

For example, plant extracts may be used as a nutrient source for fermentative organisms {e.g., yeast and other microbes). In some embodiments, provided are 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. In some embodiments, 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.

Alternatively or additionally, extracts may be used as a nutrient source for other organisms, e.g., humans and/or 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. Non-limiting examples of 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. Provided are methods comprising a step of incubating the cellulosic material with an extract obtained from plant biomass. A non-limiting example of cellulosic material that may be used in accordance with the invention is wood pulp.

In some embodiments, plant extracts are applied directly to green or ensiling biomass to improve silage quality or pretreatment efficiency.

IV. Methods of reducing biomass recalcitrance

In one aspect of the invention, provided are methods of reducing biomass recalcitrance of lignocellulosic biomass, comprising a step of hydrating and pressing the lignocellulosic biomass before enzyme hydrolysis. As described in greater detail in Example 4, 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.

Examples

The following examples describe some of the preferred modes of making and practicing the present invention. However, it should be understood that these examples are for illustrative purposes only and are not meant to limit the scope of the invention. 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.

A. Preparation of plant extracts

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. In some embodiments, pressed biomass used to produce extract is also pretreated and hydrolyzed according to the DAP Pretreatment process shown.

In the present Example, 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.

B. Supplementation of enzyme hydrolysis reactions with plant biomass extract

Materials and Methods

For enzyme hydrolysis reactions supplemented with plant extract, a 5 mL aliquot of the extract solution was added to 2 g (wet weight) of dilute acid-pretreated stover with ACELLERASE™ 1500 (Genencor) at a concentration of 0.5 mL/g glucan or 0.2 mL/g glucan. (Dilute acid pretreatment was carried out using conditions of 0.5% H ₂ SO ₄ , 190°C for 10 minutes).

For enzyme hydrolysis reactions performed without plant extract, 5 mL of 0.1 M sodium citrate (pH 5.0) 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) stover from the same source material with ACELLERASE 1500 (Genencor) at a concentration of 0.5 mL/g glucan or 0.2 mL/g glucan.

100 L of 2% sodium azide was added to each enzymatic hydro lvysis reaction in this Example to prevent microbial growth. Reactions were brought to a final volume of 10 mL with distilled water. Slurries were then incubated for 72 h at a temperature of 50 °C. After incubation, soluble glucose was measured colorimetrically using either a a commercial glucose oxidase kit (Sigma-Aldrich) or quantified by ion chromatography. Measured glucose levels were normalized to the starting quantity of biomass.

Results

As depicted in Figure 2A, increased glucose yields were obtained by adding extract before enzymatic hydrolysis. Extracts contain small concentrations of glucose (Figure 2B) that are insufficient to account for the increased glucose yield shown in Figure 2A, indicating that other components of the extract are primarily responsible for increased glucose yields observed after hydrolysis.

Example 2: Production and use of plant extracts to improve glucan conversion of non- source biomass

In the present Example, 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.

Materials and Methods

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).

Results

In all cases in this Example, higher levels of glucose were obtained from biomass supplemented with extract than were obtained from biomass treated with 0.5 mL

ACCELLERASE™/g glucan (but with no extract) (Figure 3). These results illustrate that extract does not need to come from the same biomass that it is added to for hydrolysis to increase glucan conversion.

Example 3 : Use of plant extracts to improve glucan conversion of corn grain pericarp In the present Example, 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.

Materials and Methods

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.

Milled cob, stover, and pericarp 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 then was strained through four layers of cheesecloth. Additional liquid was obtained by hand- wringing the cheesecloth. Enzyme hydrolysis mixtures were prepared in the absence and presence of plant extracts and included ACCELLERASE™ 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

Supplementation of a low concentration (0.2 mL/g biomass) of

ACCELLERASE™ 1500 with plant extracts increased the amount of glucose released from pericarp fiber. The combination of a low concentration of ACCELLERASE™ 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 ACCELLERASE™ 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.

Materials and Methods

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. ACCELLERASE™ 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.

Results

Depending on the source of the biomass, pressed biomass yielded between 10- 40% more soluble glucose than biomass samples that did not undergo a hydration and extraction process (Figure 5). 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.

Materials and Methods

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.

Solid fractions were collected and subject to dilute acid pretreatment (using conditions of 0.5% H ₂ SO ₄ , 190 °C for 10 minutes) in parallel with dried ESC Alamo and FA4 biomass that was not hydrated and pressed. Three sets of hydrolysis reactions were set up using the two types of biomass (FA4 and ESC Alamo):

1) "Untreated" reactions used the pretreated biomass that did not undergo

hydration and pressing with 5 mL of 0.1 M pH 4.8 sodium citrate buffer.

2) "Pressed" reactions used the pretreated biomass that did undergo hydration and pressing with 5 ml of 0.1 M pH 4.8 sodium citrate buffer.

3) "+Extract" reactions used the pretreated pressed biomass with 5 mL of extract from the biomass.

Each of the hydrolysis reactions in this Example contained 2 g of dilute acid- pretreated biomass, 0.2 mL of ACCELLERASE™ 1500 (Genencor)/g glucan, andlOO

L of 2% sodium azide. 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). Measured glucose levels were normalized to the starting quantity of biomass. Results

Increased glucan conversion was observed in samples in which switchgrass extract was added to pretreated switchgrass biomass (Figure 6). The "+Extract" reactions produced up to 50% higher levels of glucose than the "Pressed" reactions in both FA4 and ESC Alamo samples.

These results suggest that the active components in corn extracts that allow increased glucan conversion are also found in switchgrass extracts. As with the experiments described in Example 4, simply hydrating and pressing switchgrass biomass prior to dilute acid pretreatment also resulted in increased glucan conversion. The "Pressed" reactions showed greater level of glucan conversion than the "Untreated" reactions.

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.

Materials and Methods

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 ACCELLERASE™ 1500 (Genencor) at a rate of 0.2 ml/g glucan. For comparison, hydrolysis reactions were also carried out using

ACCELLERASE™ 1500 at a rate of 0.2 and 0.5 mL/g glucan without added extract. 100 L of 2% sodium azide was added for microbial inhibition. Reactions were brought to a final volume of 10 mL with distilled water. Slurries were then incubated for 72 h at a temperature of 50 °C. After incubation, soluble glucose in the solution was measured colorimetrically using the glucose oxidase method. Results

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 (ACCELLERASE™ 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.

These results suggest that plant extracts can provide processing benefits in a wide range of plant biomasses, as poplar trees are not closely related phylogenetically to corn or switchgrass.

Example 7: Enzymatic activities in corn extract

In the present Example, enzyme activities present in corn extract were

characterized. Enzyme activities on a variety of substrates were examined.

Materials and Methods

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.

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 β-D- cellobioside (MUC), 4-nitrophenyl β-D-cellobioside (pNPC), 4-nitrophenyl β-D- lactopyranoside (pNPLac), 4-nitrophenyl β-D-glucopyranoside (pNPG), beechwood xylan, birchwood xylan, oat spelt xylan, azo-labeled wheat arabinoxylan, pectin, 4- methylumbelliferyl β-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-L- rhamnopyranoside (MU-Rh).

Results

Corn extract had relatively less activity toward MUC, pNPC, pNPLac, pNPG, xylans, and pectins than ACCELLERASE™, 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

In the present Example, enzymatic activities in corn extract were tested at a variety of temperature and pH conditions to determine optimal conditions for using corn extract in enzyme hydrolysis and other applications.

A. Enzymatic activities across temperature and pH ranges

Materials and Methods

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.

Effect of pH on enzyme activity was investigated by measuring enzyme activities present in corn extracts at pHs over the range from 3.5-8.5 at 50 °C using a range of substrates.

Effect of temperature on enzyme activity was investigated by measuring enzyme activities present in corn extracts at temperatures ranging from 20 °C to 80 °C at pH 5.0 using a range of substrates. In some substrate profiling experiments, 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 ACCELLERASE™ 1500 (Genencor) as an enzyme source to serve as a reference for the enzymes in corn extract. Relative activities of corn extract and of ACCELLERASE™ was scored after activities of both samples were normalized to level of total source protein in the respective samples.

Results

In general, 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).

B. Optimal temperature and pH for enzymatic activities in corn extract

Materials and Methods

1. Preparation of corn extract

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.

2. Enzyme hydrolysis reactions

A 5 mL aliquot of each extract solution was added to 2 g of dilute acid-pretreated (using conditions of 0.5%> H ₂ S0 ₄ , 190 °C for 15 minutes) corn stover.

i. Temperature optimization

ACCELLERASE™ 1500 (Genencor) was then added to the biomass slurry at a loading rate of 0.2 mL/g glucan. As controls, pretreated biomass samples were hydrolyzed with either 0.2 mL/g or 0.5 mL/g ACCELLERASE™ 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).

ii. pH optimization

Additional hydrolysis reactions were performed with pretreated corn stover and extract. In these reactions, the temperature remained constant at 50 °C but the pH of the hydrolysis reactions ranged from 4.5 to 7.0. After 96 hours of incubation, soluble glucose in the solution was measured colorimetrically using a commercial glucose oxidase kit (Sigma- Aldrich).

Results

Results from temperature optimization experiments are shown in Figure 10. The maximum level of glucose production was observed when the slurry was incubated at temperatures between 45°C and 55°C, which is the optimal temperature for

ACCELLERASE™ activity.

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.

Other Embodiments

Other embodiments of the invention will be apparent to those skilled in the art from a consideration of the specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope of the invention being indicated by the following claims.