WO2012174459A2

WO2012174459A2 - Use of pressure to enhance ionic liquid pretreatment of biomass

Info

Publication number: WO2012174459A2
Application number: PCT/US2012/042790
Authority: WO
Inventors: Anthe George; Bradley M. Holmes; Blake Simmons
Original assignee: Sandia Corporation
Priority date: 2011-06-17
Filing date: 2012-06-15
Publication date: 2012-12-20
Also published as: WO2012174459A3

Abstract

The present invention provides novel and improved processes for pretreating a biomass using ionic liquids to increase the accessibility of the biomass to hydrolysis and subsequent reactions such as fermentation. The pretreatment processes of the present invention overcome the recalcitrance of the biomass and produce high sugar yields.

Description

USE OF PRESSURE TO ENHANCE IONIC LIQUID PRETREATMENT

OF BIOMASS

CROSS-REFERENCE TO RELATE APPLICATIONS

[0001] This application claims benefit of U.S. provisional application no. 61/498,436, filed June 17, 201 1, which is herein incorporated by reference for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] The invention was made with government support under Contract No. DE-AC02- 05CH1 1231 awarded by the U.S. Department of Energy. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] In recent years, tremendous efforts have been made to develop biofuels made from lignocellulosic biomass, which is derived from agricultural wastes, forest residues and dedicated energy crops. However, one of the greatest limitations facing the economic viability of this technology is the recalcitrant nature of the lignocellulosic biomass to enzymatic hydrolysis into its component sugars. This resistance to breakdown necessitates the use of pretreatment steps to enhance the accessibility to and hydrolysis of the carbohydrate components present in the lignocellulosic biomass. Most pretreatments processes are comprised of thermo-chemical processes that utilize combinations of high temperatures and pressures, or dilute acids or alkalis, to open up the structure of the biomass. Such processes necessitate the use of specialized equipment and high-energy inputs.

[0004] Ionic liquids (ILs) have come into prominence over recent years and have been used as innovative fluids for chemical processing. They are known as environmentally friendly solvents primarily due to their low volatility and their potential recyclability. Recently, the use of ILs for the pretreatment of biomass has been shown to be a promising technology, allowing for the solubilizing of crystalline cellulose and biomass under relatively mild conditions. Reconstitution of the biomass from the IL results in an amorphous product that significantly increases the rate of enzymatic hydrolysis to its component soluble sugars. For instance, the IL l-ethyl-3- methylimidazolium acetate [C₂Mim][OAc] has been found to be effective at the dissolution of biomass and the subsequent enhancement of enzymatic saccharification.

[0005] The ionic liquid pretreatment process can generally be described as the dissolution of biomass into the ionic liquid at an elevated temperature with stirring, followed by the addition of a precipitant (or, alternatively, an anti-solvent) that precipitates the biomass from solution. This precipitant or anti-solvent is typically either water or ethanol, or some other solvent with hydrogen bonding capacity. Once the biomass has been precipitated, solid/liquid separation and downstream enzymatic hydrolysis of the now amorphous biomass results in monosaccharides suitable for fermentation. Currently, the ionic liquid pretreatment process employs atmospheric pressure and temperatures ranging from about 120°C to 160°C. Recycling of ionic liquid is typically by distillation of all of the precipitating solvent.

[0006] Although pretreatment with ionic liquids has met with success, ionic liquids are expensive and the pretreatment process is both energy and time intensive. As such, what is needed in the art is a pretreatment process that employs less ionic liquid and that has reduced energy requirements, processing temperatures and processing times. The present invention provides a pretreatment process that fulfills these and other needs.

SUMMARY OF THE INVENTION

[0007] It has surprisingly been found that the use of pressure in the ionic liquid biomass pretreatment process increases glucose yields, while reducing energy requirements, processing temperatures and processing times. More particularly, it has surprisingly been found that: (1) the use of pressure increases glucose yield following hydrolysis of the pretreated biomass; (2) the use of pressure reduces temperature requirements, while increasing glucose yields; (3) the use of pressure enhances process kinetics, thereby reducing reaction times, while increasing glucose yields; (4) the use of pressure allows for the use of a co-solvent in the biomass pretreatment process, thereby reducing IL requirements and increasing loading; and (5) the use of pressure does not have an impact on the morphology or crystallinity of the biomass.

[0008] As such, in one aspect, the present invention provides a process for pretreating a biomass, the process comprising: contacting the biomass with an ionic liquid under pressure for a time sufficient to pretreat the biomass. Biomass suitable for use in the process of the present invention include, but are not limited to, a cellulose biomass, a hemicellulose biomass, a lignocellulose biomass and mixtures thereof. In a preferred embodiment, the biomass is a lignocellulose-containing biomass. Suitable lignocellulose-containing biomasses include, but are not limited to, corn stover, corn fiber, hard wood, softwood, cereal straw, switch grass, Miscanthus, rice hulls, municipal solid waste (MSW), industrial organic waste, office paper, or mixtures thereof.

[0009] The biomass is contacted with an ionic liquid under pressure for a time sufficient to pretreat the biomass. Suitable ionic liquids for use in the pretreatment process of the present invention include, but are not limited to, l-alkyl-3-alkylimidazolium alkanate, 1-alky 1-3 - alkylimidazolium alkylsulfate, l-alkyl-3-alkylimidazolium methylsulfonate, l-alkyl-3- alkylimidazolium hydrogensulfate, l-alkyl-3- alkylimidazolium thiocyanate, and l-alkyl-3- alkylimidazolium halide, wherein an "alkyl" is an alkyl group comprising from 1 to 10 carbon atoms, and an "alkanate" is an alkanate comprising from 1 to 10 carbon atoms.

[0010] In some embodiments, the ionic liquid used in the pretreatment process includes, but is not limited to, one of the following: l-ethyl-3-methylimidazolium acetate (ΕΜΓΝ Acetate), l-ethyl-3-methylimidazolium chloride (ΕΜΓΝ CI), l-ethyl-3- methylimidazolium

hydrogensulfate (EMIM HOS0₃), l-ethyl-3 -methylimidazolium methylsulfate (EMIM MeOS0₃), l-ethyl-3 -methylimidazolium ethylsulfate (EMIM EtOS0₃), l-ethyl-3- methylimidazolium methanesulfonate (EMIM MeSC ), l-ethyl-3 - methylimidazolium tetrachloroaluminate (EMIM AIC1₄), l-ethyl-3 -methylimidazolium thiocyanate (EMIM SCN), l-butyl-3 -methylimidazolium acetate (BMIM Acetate), l-butyl-3- methylimidazolium chloride (BMIM CI), l-butyl-3 -methylimidazolium hydrogensulfate (BMIM HOSO₃), 1-butyl- 3 -methylimidazolium methanesulfonate (BMIM MeSC ), 1 -butyl- 3 -methylimidazolium methylsulfate (BMIM MeOS0₃), l-butyl-3 -methylimidazolium tetrachloroaluminate (BMIM AICI₄), l-butyl-3 -methylimidazolium thiocyanate (BMIM SCN), l-ethyl-2,3- dimethylimidazolium ethylsulfate (EDIM EtOSOs), Tris(2- hydroxyethyl)methylammonium methylsulfate (MTEOA MeOS0₃), 1 -methylimidazolium chloride (MIM CI), 1 - methylimidazolium hydrogensulfate (MIM HOSO₃), 1,2,4- trimethylpyrazolium

methylsulfate, tributylmethylammonium methylsulfate, choline acetate, choline salicylate, and mixtures thereof. In one embodiment, the ionic liquid used in the pretreatment process of the present invention is l-ethyl-3 -methylimidazolium acetate (ΕΜΓΝ Acetate or

[C₂mim][OAc]).

[0011] In the pretreatment process of the present invention, the biomass is contacted with an ionic liquid under pressure for a time sufficient to pretreat the biomass. This is in contrast to previously used ionic liquid biomass pretreatment processes that are carried out at atmospheric pressure. In one embodiment, the pressure (P_g) is about 1500 psi to about 4000 psi. In another embodiment, the pressure (P_g) is about 2000 psi to about 3500 psi. In one embodiment, the pressure (P_g) is about 2200 psi to 2700 psi and, in another embodiment, about 2500 psi. Typically, the contacting is at a temperature ranging from about 25°C to about 150°C. In one embodiment, the contacting is at a temperature ranging from about 115°C to about 130°C and, in another embodiment, at a temperature of about 120°C. In a further embodiment, the contacting is at a temperature ranging from about 75°C to about 85°C. In one embodiment, the contacting is at a temperature of about 80°C.

[0012] The biomass is contacted with the ionic liquid for a time sufficient to increase the accessibility to and hydrolysis of the carbohydrate components present in the biomass.

Typically, this results from the breaking of the lignin seal and disruption of the crystalline structure of cellulose. The contacting can comprising agitating or stirring (at, e.g., 200 to about 400 rpm) the biomass and the ionic liquid to ensure complete mixing of the two.

Typically, the biomass is contacted with the ionic liquid under pressure for a period of time ranging from about 0.5 to about 12 hours. In one embodiment, the biomass is contacted with the ionic liquid under pressure for a period of time ranging from about 0.5 to about 4 hours. In one embodiment, the biomass is contacted with the ionic liquid under pressure for about 3 hours.

[0013] One of the advantages of the pretreatment process of the present invention is that the use of pressure keeps liquids from boiling at higher temperatures, which allows for the use of co-solvents. Importantly, the use of co-solvents reduces the IL requirements, which helps increase the economic viability of this pretreatment process. In addition, the use of co- solvents reduces the viscosity of the biomass-IL-co-solvent mixture, thereby increasing loading. As such, in one embodiment, the biomass is contacted with the ionic liquid and a co-solvent for a time sufficient to pretreat the biomass. Suitable co-solvents include, but are not limited to water, ethanol and dimethyl sulfoxide (DMSO). In one embodiment, the co- solvent is water. Typically, the ionic liquid:co-solvent mixture is about 20%: 80% to about 80%:20%.

[0014] Once the contacting step is complete, the biomass can be reconstituted. In one embodiment, the biomass is reconstituted using a precipitant (or an anti-solvent). In one embodiment, the precipitant is water, although ethanol can also be used as a precipitant. The ionic liquid is then extracted from the biomass and recycled using standard liquid-solid separation techniques. The solid biomass residue is then washed to ensure substantial removal of the ionic liquid, and the solid biomass is subjected to enzymatic hydrolysis to increase sugar yields. Following enzymatic hydrolysis, the sugars are fermented or can be used as a carbon source for a host cell to produce a biofuel or any other useful organic compound. Examples of such products include, but are not limited to, alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H₂ and CO₂); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B 12, teto-carotene); fatty acids and fatty acid derivatives (as described, e.g., in

PCT/US2008/068833); isoprenyl alkanoates (as described, e.g., PCT/US2008/068756, methyl butenol (as described, e.g., PCT/US2008/068831 ; fatty acid esters (as described, e.g., in PCT/US2010/033299), isoprenoid-based alternative diesel fuel (as described, e.g., in

PCT/US201 1/059784; a polyketide synthesized by a polyketide synthase, such as a diacid (see, e.g., PCT/US201 1/061900), biofuels (see, e.g., PCT/US2009/042132) and alpha-olefins (see, e.g., PCT/US2011/053787). Both the enzymatic hydrolysis and subsequent steps to produce a desired organic compound can be carried out using procedures known to and used by those of skill in the art.

[0015] In another embodiment, the present invention provides a process for solubilizing (dissolving) a polysaccharide in a biomass, the process comprising: contacting the biomass with an ionic liquid under pressure for a time sufficient to solubilize the polysaccharide in the biomass. In a preferred embodiment, the biomass is a cellulose biomass, a hemicellulose biomass, a lignocellulose biomass or a mixture thereof. In one embodiment, the process further comprises reconstituting the polysaccharide by contacting the polysaccharide and the ionic liquid with a precipitant (or an anti-solvent). In one embodiment, the precipitant is water, ethanol or a mixture thereof. In still another embodiment, the process further comprises hydrolyzing the polysaccharide using, for example, enzymatic hydrolysis to yield fermentable sugars. In yet another embodiment, the process further comprises fermenting the fermentable sugars resulting from the enzymatic hydrolysis.

[0016] As noted, the ionic liquid biomass pretreatment process of the present invention provides numerous advantages over the presently used pretreatment process, which is carried out at atmospheric pressure. The use of pressure facilitates pressure-induced phase transitions that mitigate the need for single phase addition of precipitants such as water and ethanol to recover the lignocellulosic biomass. Furthermore, the use of pressure opens up the pretreatment process to the use of supercritical conditions to enhance the selective fractionation and recovery of the biomass into its three major components: cellulose, hemicellulose and lignin, and, in addition, enhances downstream enzymatic hydrolysis. The use of pressure enables the use of cosolvents, such as water, DMSO and ethanol, which, in turn, reduces IL loading and facilitates precipitation post-pretreatment. For example, inclusion of -30% ethanol to a 5% solution of cellulose in l-ethyl-3-methylimidazolium acetate does not result in the precipitation of the biomass, whereas -40% ethanol does. The use of pressure allows a mixture of biomass, IL and ethanol to pass through a reactor without boiling off the ethanol. Subsequently, only 10% further ethanol needs to be added to precipitate the biomass, and only 10% needs to be removed before the IL can be recycled in the process.

[0017] As ionic liquids are incompressible, the pressurization of these systems is not energy intensive and lends itself to the use of a continuous process. In one embodiment, the process can consist of a traditional plug flow reactor, employing static mixing if the viscosity of the IL-biomass mixture is such that only laminar flow is achievable with the required flow rates. Following the pretreatment in the plug flow reactor, the reactor eluent, containing ionic liquid, biomass and cosolvent, passes into a vacuum flash separator to remove a portion of the precipitant from the mixture, thereby facilitating solid liquid separation with the overall goal being the efficient recycling of the ionic liquid.

[0018] Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figures 1, 2 and 3 illustrate the control pretreatment process and control parameters for pretreatment of various biomasses with the ionic liquid [C₂mim][OAc].

[0020] Figure 4 illustrates the glucose yield (% Glucose yield) for selected substrates as a function of pressure. [0021] Figure 5 illustrates the glucose yield (% Glucose yield) as a function of pressure and temperature.

[0022] Figure 6 illustrates the glucose yield (% Glucose yield) as a function of pressure and time.

[0023] Figure 7 illustrates the glucose yield (% Glucose yield) as a function of co-solvent (H₂0).

[0024] Figure 8 illustrates that pressure does not have an impact on the morphology or crystallinity of the biomass.

DETAILED DESCRIPTION OF THE INVENTION

I. OVERVIEW

[0025] Biomass, such as lignocellulose-containing biomass, can be hydrolyzed to glucose and other sugars. The sugars can be used for producing fermentation products, such as ethanol, that are useful as fuel, or for generating other organic compounds for which the sugar can serve as a carbon source. Typically, the production of fermentations products from lignocellulose-containing biomass includes pretreating, hydrolyzing and fermenting the lignocellulose-containing biomass. [0026] The pretreatment process is carried out because the structure of lignocellulose is not directly accessible to enzymatic hydrolysis. As such, the lignocellulose is pretreated in order to break the lignin seal and disrupt the crystalline structure of cellulose. This may cause solubilization and saccharification of the hemicellulose fraction. The cellulose fraction is then hydro lyzed enzymatically, e.g., by cellulolytic enzymes, which degrade the carbohydrate polymers into monosccharides. These sugars may then be converted into desired

fermentation products by a fermenting organism, which products may optionally be recovered, e.g., by distillation; or may be employed in other processes to generate organic compounds.

[0027] The present invention provides a ionic liquid biomass pretreatment process that has significant advantages over the presently used ionic liquid biomass pretreatment processes, which is carried out at atmospheric pressure. In the biomass pretreatment process of the present invention, the biomass is contacted with an ionic liquid under pressure for a time sufficient to pretreat the biomass (e.g., to break the lignin seal and disrupt the crystalline structure of cellulose). It has surprisingly been found that the use of pressure in the ionic liquid biomass pretreatment process increases glucose yields, while reducing energy requirements, processing temperatures and processing times. More particularly, it has surprisingly been found that the use of pressure in the ionic liquid biomass pretreatment process provides numerous advantages in that the use of pressure increases glucose yield following hydrolysis of the pretreated biomass; reduces temperature requirements, while increasing glucose yields; enhances process kinetics, thereby reducing reaction times, while increasing glucose yields; allows for the use of a co-solvent in the biomass pretreatment process, thereby reducing IL requirements and increasing loading; and provides no impact on the morphology or crystallinity of the biomass.

II. BIOMASS [0028] Biomass suitable for use in the process of the present invention include, but are not limited to, a cellulose biomass, a hemicellulose biomass, a lignocellulose biomass and mixtures thereof. In a preferred embodiment, the biomass is a lignocellulose biomass.

Lignocellulose-containing biomass primarily consists of cellulose, hemicellulose, and lignin. Woody biomass, for instance, is about 45-50% cellulose, 20-25% hemicellulose and 20-25% lignin. Herbaceous materials have lower cellulose, lower lignin and higher hemicellulose contents. Cellulose biomass, hemicellulose biomass and lignocellulose biomass are generally referred to herein as "biomass." [0029] Cellulose is a linear beta l->4 linked polymer of glucose. It is the principal component of all higher plant cell walls. In nature, cellulose exists in crystalline and amorphous states. The thermodynamic stability of the beta l->4 linkage and the capacity of cellulose to form internal hydrogen bonds gives it great structural strength. Cellulose is degraded to glucose through hydro lytic cleavage of the glycosidic bond. [0030] Hemicellulose is a term used to refer to a wide variety of heteropolysaccharides found in association with cellulose and lignin in both woody and herbaceous plant species. The sugar composition varies with the plant species, but in angiosperms, the principal hemicellulosic sugar is xylose. Like cellulose, xylose occurs in the beta l->4 linked backbone of the polymer. In gymnosperms, the principal component sugar is mannose. Arabinose is found as a side branch in some hemicelluloses.

[0031] Lignin is a phenylpropane polymer. Unlike cellulose and hemicellulose, lignin cannot be depolymerized by hydrolysis. Cleavage of the principal bonds in lignin require oxidation.

[0032] In a preferred embodiment, the biomass is a lignocellulose-containing material (or, alternatively, lignocellulose biomass). In some embodiments, the lignocellulose-containing material contains at least 30 wt.-%, or at least 50 wt.-%, or at least 70 wt.-%, or at least 90 wt.-% lignocellulose. It will be understood by those of skill that the lignocellulose- containing material can also comprise other constituents, such as proteinaceous material, starchy material, and sugars, such as fermentable sugars and/or un- fermentable sugars. [0033] Lignocellulose biomass is generally found, for example, in the stems, leaves, hulls, husks, and cobs of plants or leaves, branches, and wood of trees. Lignocellulose biomass can also be, but is not limited to, herbaceous material, agricultural residues, forestry residues, municipal solid wastes, waste paper, and pulp and paper mill residues. It is to be understood that lignocellulose biomass may be in the form of plant cell wall material containing lignin, cellulose and hemicellulose in a mixed matrix.

[0034] In some embodiments, the lignocellulose biomass includes, but is not limited to, corn stover, corn fiber, hardwood, such as poplar and birch, softwood, cereal straw, such as, wheat straw, switch grass, Miscanthus, rice hulls, or mixtures thereof. Other examples include corn fiber, rice straw, wheat bran, pine wood, wood chips, poplar, bagasse, paper and pulp processing waste.

III. PRETREATMENT [0035] The present invention provides a process for pretreating a biomass, the process comprising: contacting the biomass with an ionic liquid under pressure for a time sufficient to pretreat the biomass. In this pretreatment process, the biomass, such as a lignocellulose biomass, is contacted, under pressure, with an ionic liquid for a time sufficient to pretreat the biomass. This is in contrast to previously used ionic liquid biomass pretreatment processes that are carried out at atmospheric pressure. In one embodiment, the pressure (P_g) is about 1500 psi to about 4000 psi. In another embodiment, the pressure (P_g) is about 2000 psi to about 3500 psi. In yet another embodiment, the pressure (P_g) is about 2200 psi to 2700 psi. In one embodiment, the pressure (P_g) is about 2500 psi. Typically, the contacting is at a temperature ranging from about 25°C to about 150°C. In one embodiment, the contacting is at a temperature ranging from about 1 15°C to about 130°C. In another embodiment, the contacting is at a temperature of about 120°C. In a further embodiment, the contacting is at a temperature ranging from about 75°C to about 85°C. In one embodiment, the contacting is at a temperature of about 80°C.

[0036] The biomass is contacted with the ionic liquid for a time sufficient to increase the accessibility to and hydrolysis of the carbohydrate components present in the biomass. The contacting can comprising agitating or stirring (at, e.g., 200 to about 400 rpm) the biomass and the ionic liquid to ensure complete mixing of the two. Typically, the biomass is contacted with the ionic liquid under pressure for a period of time ranging from about 0.5 to about 12 hours. In one embodiment, the biomass is contacted with the ionic liquid under pressure for a period of time ranging from about 0.5 to about 4 hours. In one embodiment, the biomass is contacted with the ionic liquid under pressure for about 3 hours.

[0037] Ionic liquids (ILs) are salts that are liquids rather than crystals at room temperatures. It will be readily apparent to those of skill that numerous ILs can be used in the pretreatment process of the present invention. In some embodiments of the invention, the IL is suitable for pretreatment of the biomass and for the hydrolysis of cellulose by thermostable cellulase. Suitable ILs are taught in ChemFiles (2006) 6(9) (which are commercially available from Sigma- Aldrich; Milwaukee, WI). Such suitable ILs include, but are not limited to, 1- alkyl- 3-alkylimidazolium alkanate, l-alkyl-3-alkylimidazolium alkylsulfate, l-alkyl-3- alkylimidazolium methylsulfonate, l-alkyl-3-alkylimidazolium hydrogensulfate, l-alkyl-3- alkylimidazolium thiocyanate, and l-alkyl-3-alkylimidazolium halide, wherein an "alkyl" is an alkyl group comprising from 1 to 10 carbon atoms, and an "alkanate" is an alkanate comprising from 1 to 10 carbon atoms. In some embodiments, the "alkyl" is an alkyl group comprising from 1 to 4 carbon atoms. In some embodiments, the "alkyl" is a methyl group, ethyl group or butyl group. In some embodiments, the "alkanate" is an alkanate comprising from 1 to 4 carbon atoms. In some embodiments, the "alkanate" is an acetate. In some embodiments, the halide is chloride.

[0038] In some embodiments, the IL includes, but is not limited to, l-ethyl-3- methylimidazolium acetate (ΕΜΓΝ Acetate), l-ethyl-3 -methylimidazolium chloride (ΕΜΓΝ CI), l-ethyl-3- methylimidazolium hydrogensulfate (EMIM HOS0₃), l-ethyl-3- methylimidazolium methylsulfate (EMIM MeOS0₃), l-ethyl-3 -methylimidazolium ethylsulfate (EMIM EtOS0₃), l-ethyl-3 -methylimidazolium methanesulfonate (EMIM MeS0₃), l-ethyl-3- methylimidazolium tetrachloroaluminate (EMIM AICI4), l-ethyl-3- methylimidazolium thiocyanate (EMIM SCN), l-butyl-3 -methylimidazolium acetate (BMIM Acetate), l-butyl-3- methylimidazolium chloride (BMIM CI), l-butyl-3 -methylimidazolium hydrogensulfate (BMIM HOSO₃), l-butyl-3 -methylimidazolium methanesulfonate (BMIM MeS0₃), 1-butyl- 3 -methylimidazolium methylsulfate (BMIM MeOS0₃), l-butyl-3- methylimidazolium tetrachloroaluminate (BMIM AICI₄), l-butyl-3 -methylimidazolium thiocyanate (BMIM SCN), l-ethyl-2,3-dimethylimidazolium ethylsulfate (EDIM EtOS0₃), Tris(2- hydroxyethyl)methylammonium methylsulfate (MTEOA MeOS0₃), 1 - methylimidazolium chloride (MIM CI), 1 -methylimidazolium hydrogensulfate (MIM HOS0₃), 1,2,4- trimethylpyrazolium methylsulfate, tributylmethylammonium methylsulfate, choline acetate, choline salicylate, and the like. The ionic liquid can comprises one or a mixture of the compounds. Additional ILs suitable for use in the present invention are taught in U.S. Patent No. 6,177,575, which is herein incorporated by reference. It will be appreciated by those of skill in the art that others ILs that will be useful in the process of the present invention are currently being developed or will be developed in the future, and the present invention contemplates their future use.

[0039] The ionic liquid (IL) is of a concentration of more than 0% and up to 100% of the composition or solution. In some embodiments, the biomass is mixed with ionic liquid, and the ionic liquid is of a concentration of more than 70% and up to 100% of the composition or solution, or of a concentration of more than 80% and up to 100%, or of a concentration of more than 90% and up to 100%. The upper range of the concentration of IL is equal to or less than 100%, equal to or less than 90%, equal to or less than 80%, equal to or less than 70%, equal to or less than 60%, or equal to or less than 55%. In other embodiments, the IL is of a concentration of more than 0% to less than 60% of the composition or solution. In some embodiments, the concentration of IL is equal to or more than 1%, equal to or more than 2%, equal to or more than 3%, equal to or more than 5%, equal to or more than 10%, equal to or more than 15%, or equal to or more than 20%.

[0040] In some embodiments of the invention, a co-solvent is used. Advantageously, the use of co-solvents reduces the IL requirements, which helps increase the economic viability of this pretreatment process. In addition, the use of co-solvents reduces the viscosity of the biomass-IL-co-solvent mixture, thereby increasing loading. As such, in one embodiment, the biomass is contacted with the ionic liquid and a co-solvent for a time sufficient to pretreat the biomass. Suitable co-solvents include, but are not limited to water, ethanol and dimethyl sulfoxide (DMSO). In one embodiment, the co-solvent is water. Typically, the ionic liquid:co-solvent mixture is about 20%: 80% to about 80%:20%.

[0041] It will be apparent to those of skill in the art that the solution containing the IL can further comprise NaCl, such as up to 10 mM of NaCl. In addition, the solution can further comprise a suitable buffer and other additives that are beneficial to the pretreatment process.

[0042] Once the contacting step is complete, the biomass can be reconstituted. In some embodiments, the biomass is reconstituted using a precipitant (or an anti-solvent). In one embodiment, the precipitant is water, although ethanol can also be used as a precipitant. The ionic liquid is then extracted from the biomass and recycled using standard liquid-solid separation techniques. The solid biomass residue is then washed to ensure substantial removal of the ionic liquid, and the solid biomass is subjected to enzymatic hydrolysis. The biomass and/or sugars from the biomass can then be used as desired, e.g., subjected to fermentation using procedures known to and used by those of skill in the art. IV. ENZYMATIC HYDROLYSIS/FERMENTATION

[0043] The pretreated biomass, e.g., the lignocellulose biomass, can be hydro lyzed enzymatically to break down, for example, hemicellulose and/or cellulose, into sugars.

Typically, the pretreated biomass is subjected to the action of one, or several or all enzyme activities selected from the group consisting of a cellulase, a protease, a pectate, a xylanase, a lyase, a ferulic acid esterase, and a mannanase. In one embodiment, the pretreated biomass is subjected to the action of one or more cellulases, such as a thermostable cellulase. Cellulases suitable for use in the present invention are commercially available from, for example, Genencor (USA) and Novozymes (Europe). For instance, Novozyme has a number of different enzymes and enzyme complexes that are specifically designed to be useful for the hydrolysis of lignocellulosic materials. Examples include, but are not limited to, the following: NS50013, which is a cellulose; NS50010, which is a teto-glucosidase; NS22086, which is a cellulose complex; NS22086, which is a xylanase; NS22118, which is β- glucosidase; NS221 19, which is an enzyme complex of carbohydrases, including arabinase, β-glucanase, cellulase, hemicellulase, pectinase, and xylanase; NS22002, which is a mixture of β-glucanase and xylanase; and NS22035, which is a glucoamylase. In addition, suitable thermostable cellulases are disclosed in PCT International Publication No. WO 2010/124266, the teachings of which are incorporated herein by reference. Other hydrolases suitable for hydro lyzing the pretreated biomass, i.e., the lignocellulosic material, will be known to those of skill in the art.

[0044] In certain embodiments, pretreatment of the biomass can be carried out

simultaneously with hydrolysis, such as simultaneously with addition of one or more hydrolyzing enzymes, and/or other enzyme activities, to release fermentable sugars, such as glucose and/or maltose. In these embodiments, the enzyme, such as a cellulase, is selected such that it can perform optimally in the presence of ionic liquids, which in such

embodiments are usually used in combination with a co-solvent, such as water. Successful development of IL tolerant cellulase provides potential candidates to be utilized for highly efficient deconstruction of biomass into monosaccharides in IL systems.

[0045] Following enzymatic hydrolysis, the sugars from the hydrolyzed biomass can be fermented using one or more fermenting organisms capable of fermenting fermentable sugars, such as glucose, xylose, mannose, and galactose, directly or indirectly into a desired fermentation product. The fermentation conditions depend on the desired fermentation product and can easily be determined by one of ordinary skill in the art.

[0046] In the case of ethanol fermentation with yeast, the fermentation is typically ongoing for between 5 and 120 hours, for example between 16 to 96 hours, or between 24 and 72 hours. In an embodiment, the fermentation is carried out at a temperature between 25 °C and 40 °C, such as between 29 °C and 35 °C, such as between 30 °C and 34 °C, such as around 32 °C. In some embodiments, the pH is from pH 3-7 or from 4-6.

[0047] Subsequent to fermentation, the fermentation product may optionally be separated from the fermentation medium in any suitable way. For instance, the medium may be distilled to extract the fermentation product or the fermentation product may be extracted from the fermentation medium by micro or membrane filtration techniques. Alternatively, the fermentation product may be recovered by stripping. Such recovery processes are well known in the art. The dry solids remaining after recovery comprising among other compounds lignin may be used in a boiler for steam and power production. [0048] The present invention may be used for producing any fermentation product or other product for which sugars obtained from hydrolysis can serve as a carbon source. Examples of products include, but are not limited to, alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H₂ and CO₂); antibiotics (e.g., penicillin and tetracycline); vitamins (e.g., riboflavin, B 12, beta-carotene), fatty acids and fatty acid derivatives (as described, e.g., in PCT/US2008/068833); isoprenyl alkanoates (as described, e.g., PCT/US2008/068756, methyl butenol (as described, e.g.,

PCT/US2008/068831; fatty acid esters (as described, e.g., in PCT/US2010/033299), isoprenoid-based alternative diesel fuel (as described, e.g., in PCT/US201 1/059784; a polyketide synthesized by a polyketide synthase, such as a diacid (see, e.g.,

PCT/US201 1/061900), biofuels (see, e.g., PCT/US2009/042132) and alpha-olefins (see, e.g., PCT/US2011/053787).

[0049] In one embodiment the fermentation product is an alcohol, such as ethanol. The fermentation product, e.g., ethanol, obtained according to the invention, may be used as fuel alcohol/ethanol. However, in the case of ethanol, it may also be used as potable ethanol.

[0050] The present invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results.

V. Examples

[0051] The following experiments were carried out to study the effects of the use of pressure on various process parameters in the biomass pretreatment process using ionic liquids. The process parameters studied were glucose yields, reaction temperatures, reaction times and process kinetics. [0052] As illustrated in Figures 1 to 3, the control pretreatment process was carried out as follows: biomass samples were mixed with EMIM Acetate [C₂mim][OAc] to form a biomass/IL mixture, and incubated at 120°C at ambient pressure in a nitrogen environment for 3 hours, while stirring at 300 rpm. The ionic liquid was extracted from the biomass by the addition of water, centrifuged and the supernatant was removed. The solid biomass residue was washed three times with 1L water, washed one time with 1L ethanol, and then washed one more time with 1L water. Once washed, the solid biomass was subjected to enzymatic hydrolysis for 24 hours using enzymes that are commercially available from Novozyme.

[0053] The experiments were carried out using the control pretreatment process set forth above, except that in order to assess the effect of pressure on the biomass pretreatment process, the reaction parameters were varied as set forth in Tables 1 and 2, below.

Table 1

[0054] In carrying out the biomass pretreatment process using the process parameters set forth in Tables 1 and 2, it was found that (1) the use of pressure in the biomass pretreatment process increases glucose yield for various biomass compositions, with the most pronounced effect seen with eucalyptus (see, Figure 4); (2) the use of pressure reduces temperature requirements, with an increase in glucose yield seen at each of the three temperatures studied (see, Figure 5); (3) the use of pressure enhances process kinetics, with an increase in glucose yields seen at each of the three time periods studied (see, Figure 6); (4) the use of pressure allows for the use of a co-solvent in the biomass pretreatment process, thereby reducing IL requirements and increasing loading (see, Figure 7); and (5) the use of pressure does not have an impact on morphology or crystallinity (see, Figure 8). It is noted that all efficiencies reported are reported with respect to glucose yield after 24 hours hydrolysis with Novozymes enzymes (NS50013 (cellulase) and S50010 (teto-glucosidase)) using standard hydrolysis procedures. [0055] The invention has been described by way of illustration, and not by limitation. It is to be understood that the particular embodiments depicted in the figures and the terminology which has been used has been intended in a nature of words of description rather then of limitation. It is to be further understood that any combination of the ingredients/therapeutic agents described in the foregoing paragraphs are deemed to be encompassed by the appended claims. It is to be further understood that all specific embodiments of the injection device are deemed to be encompassed by the appended claims. Many modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that the obvious modifications are deemed to be encompass within the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A process for pretreating a biomass, said process comprising: contacting said biomass with an ionic liquid under pressure for a time sufficient to pretreat said biomass.

2. The process in accordance with claim 1, wherein said biomass is a cellulose biomass, a hemicellulose biomass, a lignocellulose biomass or a mixture thereof is a lignocellulose-containing biomass.

3. The process in accordance with claim2, wherein said biomass is a lignocellulose biomass.

4. The process in accordance with claim 3, wherein said lignocellulose biomass is derived from corn stover, corn fiber, hard wood, softwood, cereal straw, switch grass, Miscanthus, rice hulls, municipal solid waste (MSW), industrial organic waste, office paper, or mixtures thereof.

5. The process in accordance with claim 1, wherein the ionic liquid is a member selected from the group consisting of is l-alkyl-3-alkylimidazolium alkanate, l-alkyl-3- alkylimidazolium alkylsulfate, l-alkyl-3-alkylimidazolium methylsulfonate, l-alkyl-3- alkylimidazolium hydrogensulfate, l-alkyl-3- alkylimidazolium thiocyanate, and l-alkyl-3- alkylimidazolium halide, wherein an "alkyl" is an alkyl group comprising from 1 to 10 carbon atoms, and an "alkanate" is an alkanate comprising from 1 to 10 carbon atoms.

6. The process in accordance with claim 5, wherein the ionic liquid is a member selected from the group consisting of l-ethyl-3-methylimidazolium acetate (EMIN Acetate), l-ethyl-3-methylimidazolium chloride (EMIN CI), l-ethyl-3- methylimidazolium hydrogensulfate (EMIM HOS0₃), l-ethyl-3 -methylimidazolium methylsulfate (EMIM

MeOS03), l-ethyl-3 -methylimidazolium ethylsulfate (EMIM EtOS0₃), l-ethyl-3- methylimidazolium methanesulfonate (EMIM MeS0₃), l-ethyl-3- methylimidazolium

tetrachloroaluminate (EMIM AICI4), l-ethyl-3 -methylimidazolium thiocyanate (EMIM SCN), 1- buty 1-3 -methylimidazolium acetate (BMIM Acetate), l-butyl-3- methylimidazolium chloride (BMIM CI), l-butyl-3-methylimidazolium hydrogensulfate (BMIM HOS0₃), l-butyl-3- methylimidazolium methanesulfonate (BMIM MeS0₃), 1 -butyl- 3 -methylimidazolium methylsulfate (BMIM MeOS0₃), l-butyl-3 -methylimidazolium tetrachloroaluminate (BMIM AICI4), l-butyl-3 -methylimidazolium thiocyanate (BMIM SCN), l-ethyl-2,3- dimethylimidazolium ethylsulfate (EDIM EtOS0₃), Tris(2- hydroxyethyl)methylammonium methylsulfate (MTEOA MeOS0₃), 1 -methylimidazolium chloride (MIM CI), 1 - methylimidazolium hydrogensulfate (MIM HOS0₃), 1,2,4- trimethylpyrazolium methylsulfate, tributylmethylammonium methylsulfate, choline acetate, choline salicylate, and mixtures thereof.

7. The process in accordance with claim 6, wherein the ionic liquid is 1- ethyl-3 -methylimidazolium acetate (EMIN Acetate)

8. The process in accordance with claim 1, wherein the biomass is contacted with the ionic liquid and a co-solvent.

9. The process in accordance with claim 8, wherein said co-solvent is a member selected from the group consisting of water, ethanol, dimethyl sulfoxide (DMSO), acetone and mixtures thereof.

10. The process in accordance with claim 9, wherein said co-solvent is water.

11. The process in accordance with claim 9, wherein said co-solvent is ethanol.

12. The process in accordance with claim 8, wherein said ionic liquidxo- solvent mixture is 20%:80% to about 80%:20%.

13. The process in accordance with claim 1, wherein the pressure (P_g) is about 1500 psi to about 4000 psi.

14. The process in accordance with claim 1, wherein the pressure (P_g) is about 2000 psi to about 3500 psi.

15. The process in accordance with claim 1, wherein the pressure (P_g) is about 2500 psi.

16. The process in accordance with claim 1, wherein the contacting is at a temperature ranging from about 25°C to about 150°C.

17. The process in accordance with claim 1, wherein the contacting is at a temperature of about 120°C.

18. The process in accordance with claim 1, wherein the contacting is at a temperature of about 80°C.

19. The process in accordance with claim 1, further comprising reconstituting said biomass following the contacting step.

20. The process in accordance with claim 19, wherein reconstituting said comprises adding a precipitant to said biomass to form a precipitated biomass.

21. The process in accordance with claim 20, further comprising washing said precipitated biomass.

22. The process in accordance with claim 1, wherein said precipitant is water.

23. The process in accordance with claim 1, wherein the contacting is for a period of time ranging from 0.5 to 12 hours.

24. The process in accordance with claim 1, wherein the contacting is for a period of time ranging from 0.5 to 3 hours.

25. The process in accordance with claim 1, wherein contacting comprises stirring said biomass and said ionic liquid.

26. A process for pretreating a polysaccharide in a biomass, said process comprising: contacting said biomass with an ionic liquid under pressure for a time sufficient to pretreat said polypolysaccharide in said biomass.

27. The process in accordance with claim 26, wherein the biomass is a cellulose biomass, a hemicellulose biomass, a lignocellulose biomass or a mixture thereof.

28. The process in accordance with claim 26, further comprising reconstituting said polysaccharide by contacting said polysaccharide and said ionic liquid with a precipitant, thereby reconstituting said polysaccharide.

29. The process in accordance with claim 16, wherein said precipitant is a member selected from the group consisting of water, ethanol, and mixtures thereof.

30. The process in accordance with claim 29, wherein said precipitant is water.

31. The process in accordance with claim 29, further comprising hydrolyzing said polysaccharide.

32. The process in accordance with claim 29, wherein said hydrolyzing comprises enzymatic hydrolysis.

33. The process in accordance with claim 29, wherein said hydrolyzing produces glucose.

34. The process in accordance with claim 29, wherein said lignocellulose- containing biomass is derived from corn stover, corn fiber, hard wood, softwood, cereal straw, switch grass, Miscanthus, rice hulls, municipal solid waste (MSW), industrial organic waste, office paper, or mixtures thereof.