Methods and compositions useful for biomass fractionation using Schiff base ionic liquids Inventors: Mohan Mood, Hemant Choudhary, Venkataramana R. Pidatala, Blake A. Simmons, Seema Singh, John M. Gladden

Abstract

The present invention provides a method of disintegrating biomass, the method comprising introducing a solvent comprising a Schiff base ionic liquid (SBIL) to biomass, such that the solvent solubilizes at least a portion of the biomass to form a solubilized biomass mixture.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 376,575, filed September 21, 2022, which is incorporated herein by reference.

[0002] Statement of government support This invention was made with government support under Contract No. DE-AC02-05CH11231 awarded by the U.S. Department of Energy. The government has certain rights in this invention.

[0003] The present invention relates to the field of biomass pretreatment. [Background technology]

[0004] Lignocellulosic biomass is a renewable feedstock that can be used to generate fuels and value-added chemicals. The conversion of lignocellulosic biomass is challenging because the structural arrangement of its constituent biopolymers (cellulose, hemicellulose, and lignin) prevents effective degradation of the biomass using water and many conventional organic solvents. Therefore, delignification requires an appropriate pretreatment solvent, which improves enzyme accessibility for converting biomass to fuels and platform chemicals. The discovery of alternative solvents, such as ionic liquids (ILs), has alleviated some of the challenges associated with biomass processing. To date, many of the ILs investigated for biomass pretreatment suffer from high viscosity, high cost, poor thermal stability, and high melting points. These factors necessitate the discovery of new ILs that can overcome these issues.

[0005] Despite extensive literature describing the associated environmental, health, and safety issues, fossil-derived organic solvents are still widely used in daily and industrial activities, especially in synthetic chemistry. 1 In this regard, ionic liquids (ILs; organic cation-containing salts with melting points below 100 °C) have emerged as promising alternatives to organic solvents. 2 Due to their ease of preparation, ILs can be designed to possess several advantages, such as chemical and thermal stability, solubility, and negligible vapor pressure. 3,4,5 For this reason, ILs have found applications in a variety of fields, including catalysis, 6,7 bioactive materials, 8,9 process development, 10 energy storage, 5,11 energy-dense materials, 12 biomedicine, 13 lubricants, and 14 other areas. 15 Although not all reported ILs are renewable, cost-effective, biodegradable, and nontoxic, the infinite combinations of cations and anions available facilitate the design and production of distinct ILs with unique physicochemical and desired properties to meet specific applications.

[0006] Among the several examples mentioned above, ILs have been found to be exceptional in dissolving biopolymers and facilitating their bioconversion into biofuels. 16,17,18,19,20,21 To accommodate the large volume of biofuels required, the application of IL technology to the sustainable processing of biomass requires a reliable and renewable source of ILs. Unfortunately, the majority of ILs produced today rely on rapidly depleting fossil fuels, limiting the exploration of the great potential offered by the billions of tons of virgin, available lignocellulosic biomass feedstocks, including agricultural, forest, and herbaceous residues. Therefore, the design of new renewable solvents, especially ILs, from renewable sources remains an open challenge for researchers in this field.

[0007] Lignin is an underutilized source of aromatics and is primarily utilized for generating heat and electricity through combustion in (bio)refineries and the pulp and paper industry. Lignin, primarily composed of phenylpropanoids, can be effectively oxidized to produce aldehydes such as vanillin and syringaldehyde. 22,23,24,25 These aldehydes undergo condensation reactions with amines to produce imines (also known as Schiff bases), which can be further protonated to produce ILs suitable for various applications. Previous efforts in our laboratory have focused on the reductive amination of biomass-derived aldehydes to synthesize renewable ILs. These ILs have been shown to be highly effective for lignocellulosic pretreatment, demonstrating the concept of a closed-loop biorefinery. 26 Summary of the Invention

[0008] The present invention provides a method of disintegrating biomass, the method comprising introducing a solvent comprising a Schiff base ionic liquid (SBIL) to biomass, such that the solvent solubilizes at least a portion of the biomass to form a solubilized biomass mixture.

[0009] In some embodiments, the method further includes separating the solubilized biomass from the SBIL; optionally, introducing enzymes and / or microorganisms to the solubilized biomass, where the enzymes and / or microorganisms produce sugars from the solubilized biomass; and (c) optionally, separating the sugars from the solubilized biomass.

[0010] In some embodiments, the method further comprises forming an SBIL prior to the introducing step, and optionally forming a solvent by providing the SBIL.

[0011] The present invention provides a composition comprising: (a) a solvent comprising a Schiff base ionic liquid (SBIL); and (b) biomass.

[0012] In some embodiments, the SBIL comprises a C=N moiety formed by condensation of an aldehyde or ketone with an amine, hi some embodiments, the SBIL is an azomethine.

[0013] In some embodiments, the solvent comprises an ionic liquid (IL) that is not a Schiff base, and / or a deep eutectic solvent (DES).

[0014] The present invention provides a method for improving biomass decomposition efficiency by utilizing lignin-derived Schiff base ionic liquids (SBILs). In some embodiments, the SBILs are organic salts containing iminium ions (formed by protonation of the resulting imine after reaction of an aldehyde or ketone with an amine). Depending on the specific acid or base used to produce the SBIL, physicochemical properties such as low melting points, low viscosity, and high thermal stability can be achieved, which are desirable features in ILs for efficient biomass pretreatment. The advantages of SBILs are that they enable cost-effective production of fermentable sugars and lignin removal, a major obstacle to commercially viable bioenergy production from waste biomass.

[0015] The present invention provides a solvent for biomass decomposition that involves the use of Schiff base ionic liquids. Schiff bases have been widely studied in biological and pharmaceutical research, and molecules with azomethine Schiff base skeletons are clinically approved drugs. Interestingly, SBIL has not been used for biomass pretreatment and processing. The present invention takes into account the inherent charge delocalization at the C=N moiety and provides for the use of SBIL as a solvent to improve biomass decomposition efficiency. Depending on the type of anion or cation supported by the Schiff base, several important properties related to its performance as an effective pretreatment solvent can be exploited.

[0016] Two different Schiff bases containing lignin-derived vanillin and ethylenediamine at different molar ratios (1:1 and 2:1) were synthesized. After the synthesis of the Schiff bases, three SBILs were prepared by mixing acetic acid with these two Schiff bases at molar ratios of 1:1 and 2:1. Biomass (sorghum) pretreatment experiments were conducted to test the effectiveness of the prepared SBILs as a function of sugar yield. Saccharification of SBIL-pretreated biomass (20 wt% biomass and 80 wt% IL loading) released up to 88% glucose and 76% xylose. Additional work will be conducted to optimize the pretreatment process in terms of SBIL screening, reaction conditions, lignin removal, sugar yield, and potential recycling. In some embodiments, the SBIL contains vanillin and / or ethylenediamine. In some embodiments, the vanillin and ethylenediamine have a molar ratio of about 1:1 to about 2:1. In some embodiments, the weight percent of biomass and the weight percent of IL (including SBIL) are about X% and Y% by weight, respectively, where X+Y equals 100. In some embodiments, X is 1, 5, 10, 15, 20, 25, 30, 40, or 50, or any two of the foregoing values; and Y is 99, 95, 90, 85, 80, 75, 70, 60, or 50, or any two of the foregoing values.

[0017] In some embodiments, the present invention is used to convert waste biomass (agricultural residues, wood / paper / pulping, grass, etc.) into biofuels and / or bioproducts. In some embodiments, the process helps achieve high concentrations of fermentable sugars while leaving residual lignin for valuable chemicals.

[0018] Advantages of the present invention may include one or more of the following: (1) SBILs can be developed from renewable and inexpensive resources such as lignin; (2) their biocompatibility and biodegradability make them highly compatible with downstream processes; (3) their versatility to accommodate a variety of biomass; (4) SBIL properties (e.g., low melting point, viscosity, better interaction) can be tailored; (5) lignin-derived ILs are easy to synthesize; no tedious derivatization is required; (6) unsaturated bonds (double bonds) are effective for lignin removal by π-staking and can reduce viscosity; and (7) the properties of SBILs may be different and unique compared to other common ionic liquids (1-ethyl-3-methylimidazolium acetate) and protic ionic liquids (ethanolamine acetate).

[0019] In some embodiments, the method further includes ensiling the biomass prior to the introducing step to produce an ensiled biomass containing one or more organic acids, the ensiled biomass being the biomass of the introducing step. In some embodiments, the ensiled biomass contains about 10%, 20%, 30%, or 40% or more by weight of one or more organic acids. In some embodiments, the one or more organic acids include an alkanoic acid. In some embodiments, the alkanoic acid is lactic acid, acetic acid, butyric acid, or propionic acid, or a mixture thereof. In some embodiments, the ensiling step produces one or more toxic compounds in the ensiled biomass, and the microorganisms are resistant to the one or more toxic compounds. In some embodiments, the one or more toxic compounds are organic acids, such as linear or branched alkanoic acids (e.g., acetic acid, lactic acid, or formic acid) or aromatic organic acids (e.g., benzoic acid, vanillic acid, etc.). In some embodiments, the organic acid has about 2 to 10 carbon atoms.

[0020] In some embodiments, the method further comprises one or more steps taught in U.S. Provisional Patent Application Publication No. 63 / 016,877, filed April 28, 2021, and U.S. Patent Application Publication No. 17 / 242,256, filed April 27, 2020, both of which are incorporated by reference in their entireties.

[0021] In some embodiments, the method further includes (b) introducing an enzyme and / or a microorganism to the solubilized biomass mixture, wherein the enzyme and / or the microorganism produces sugars from the solubilized biomass mixture. In some embodiments, the method further includes (c) separating sugars from the solubilized biomass mixture.

[0022] The present invention provides compositions and methods described herein. In some embodiments, the compositions and methods further comprise steps, features, and / or elements described in U.S. Patent Application Publication No. 16 / 737,724, which is incorporated herein by reference in its entirety.

[0023] In some embodiments, the SBIL, IL and / or DES are biocompatible.

[0024] The present invention provides compositions and methods described herein.

[0025] In some embodiments, the compositions and methods further comprise steps, features, and / or elements described in US Patent Application Publication No. 16 / 737,724, which is incorporated herein by reference in its entirety. [Brief explanation of the drawings]

[0026] The foregoing and other aspects will be readily apparent to those skilled in the art from the following description of exemplary embodiments when read in conjunction with the accompanying drawings. [Figure 1-01] FT-IR profiles of vanillin (black), ethylenediamine (gray), and as-synthesized Schiff bases 1 (red) and 2 (blue). [Figure 1-02] FT-IR profiles of vanillin (black), ethylenediamine (gray), and as-synthesized Schiff bases 1 (red) and 2 (blue). [Figure 2-01] FT-IR profiles of acetic acid (dark grey), as-synthesized Schiff base 1 (red) or 2 (blue), 1:1 acetate IL 1A or 2A (olive), and 1:2 acetate IL 1B or 2B (cyan). [Figure 2-02] FT-IR profiles of acetic acid (dark grey), as-synthesized Schiff base 1 (red) or 2 (blue), 1:1 acetate IL 1A or 2A (olive), and 1:2 acetate IL 1B or 2B (cyan). [Figure 2-03] FT-IR profiles of acetic acid (dark grey), as-synthesized Schiff base 1 (red) or 2 (blue), 1:1 acetate IL 1A or 2A (olive), and 1:2 acetate IL 1B or 2B (cyan). [Figure 2-04] FT-IR profiles of acetic acid (dark grey), as-synthesized Schiff base 1 (red) or 2 (blue), 1:1 acetate IL 1A or 2A (olive), and 1:2 acetate IL 1B or 2B (cyan). [Figure 3-01] Differential scanning calorimetry and thermogravimetric analysis plots of Schiff bases 1 (red) and 2 (blue) and their respective ILs; 1:1 Schiff base:acetate (olive; 1A or 2A) and 1:2 Schiff base:acetate (cyan; 1B or 2B). [Figure 3-02] Differential scanning calorimetry and thermogravimetric analysis plots of Schiff bases 1 (red) and 2 (blue) and their respective ILs; 1:1 Schiff base:acetate (olive; 1A or 2A) and 1:2 Schiff base:acetate (cyan; 1B or 2B). [Figure 4] Activity coefficients of cellulose (dark grey) and lignin (grey) in IL 1A, 1B, and 2B. [Figure 5] (Left) Powder X-ray diffraction and (right) thermogravimetric analysis of untreated (black) and IL 2B pretreated (gray) sorghum biomass. [Figure 6-01] HSQC NMR profiles of untreated (left) and pretreated (right) sorghum. [Figure 6-02] HSQC NMR profiles of untreated (left) and pretreated (right) sorghum. [Figure 7] Synthetic scheme of Schiff bases and related ILs. DETAILED DESCRIPTION OF THE INVENTION

[0027] Before describing the present invention in detail, it is to be understood that unless otherwise indicated, this invention is not limited to particular sequences, expression vectors, enzymes, host microorganisms, or processes, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0028] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:

[0029] As used herein, the term "optional" or "optionally" means that the subsequently described feature or structure may or may not be present, or the subsequently described event or circumstance may or may not occur, and that the description includes instances where the particular feature or structure is present as well as instances where the feature or structure is not present, or instances where the event or circumstance occurs as well as instances where it does not occur.

[0030] The term "about," when applied to a value, refers to values ​​up to 10% greater than the stated value, and including values ​​up to 10% less than the stated value.

[0031] Where a range of values ​​is presented, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may each independently be included or excluded in the range, and each range where either limit is included in the smaller range, neither is included, or both limits are included in the smaller range is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0032] In some embodiments, the introducing step is performed in a vessel and homogenized. In some embodiments, the loading is a solids loading and is controlled at about 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40%, or within a range of any two of the foregoing values. In some embodiments, the biomass and solvent are heated to, for example, 100°C, 110°C, 120°C, 130°C, 140°C, 150°C, 160°C, 170°C, 180°C, 190°C, 200°C, 212°C, or within a range of any two of the foregoing values, for a time period such as, for example, about 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours, or within a range of any two of the foregoing values. In some embodiments, after pretreatment, the mixture is cooled for at least about 30 minutes, e.g., at room temperature or about 25°C, and / or then washed at least about 1, 2, 3, 4, or 5 times with water, e.g., deionized water. In some embodiments, the resulting solid is recovered, e.g., by separating the solid portion from the liquid portion.

[0033] In some embodiments, the biomass is lignocellulosic biomass. In some embodiments, the vessel is made of an inert material, such as stainless steel or glass, that does not react with or interfere with the reactions in the pretreatment mixture.

[0034] In some embodiments, the method further comprises heating the mixture, optionally also comprising the enzyme and / or microorganism, to a temperature equal to, about equal to, or near an optimal temperature for enzymatic activity of the enzyme and / or growth of the microorganism. In some embodiments, the enzyme is a genetically engineered host cell capable of converting cellulose in the biomass into sugars. In some embodiments, multiple enzymes are present. In some embodiments, the microorganism is a genetically engineered host cell capable of converting sugars produced from the biomass into biofuels, bioproducts, and / or chemical compounds. In some embodiments, multiple microorganisms are present. In some embodiments, the method produces sugars and lignin from the biomass. The sugars are used for growth by the microorganism.

[0035] In some embodiments, solubilization is complete, nearly complete (e.g., at least about 70, 80, or 90%), or partial (e.g., at least about 10, 20, 30, 40, 50, or 60%). In some embodiments, the mixture is a slurry.

[0036] ionic liquids Ionic liquids (ILs) are salts that are liquid rather than crystalline at room temperature. It will be readily apparent to those skilled in the art that numerous ILs can be used in the present invention. In some embodiments of the present invention, ILs are suitable for biomass pretreatment and cellulose hydrolysis with thermostable cellulases. Suitable ILs are taught in ChemFiles (2006) 6(9) (commercially available from Sigma-Aldrich, Milwaukee, WI). Suitable ILs include, but are not limited to, 1-alkyl-3-alkylimidazolium alkanates, 1-alkyl-3-alkylimidazolium alkyl sulfates, 1-alkyl-3-alkylimidazolium methyl sulfonates, 1-alkyl-3-alkylimidazolium hydrogen sulfates, 1-alkyl-3-alkylimidazolium thiocyanates, and 1-alkyl-3-alkylimidazolium halides, where "alkyl" is an alkyl group containing 1 to 10 carbon atoms and "alkanate" is an alkanate containing 1 to 10 carbon atoms. In some embodiments, "alkyl" is an alkyl group containing 1 to 4 carbon atoms. In some embodiments, "alkyl" is a methyl group, an ethyl group, or a butyl group. In some embodiments, "alkanato" is an alkanato containing 1 to 4 carbon atoms. In some embodiments, "alkanato" is acetate. In some embodiments, halide is chloride.

[0037] In some embodiments, the IL is 1-ethyl-3-methylimidazolium acetate (EMIN acetate), 1-ethyl-3-methylimidazolium chloride (EMIN Cl), 1-ethyl-3-methylimidazolium hydrogen sulfate (EMIM HOSO), 1-ethyl-3-methylimidazolium methyl sulfate (EMIM MeOSO), 1-ethyl-3-methylimidazolium ethyl sulfate (EMIM EtOSO), 1-ethyl-3-methylimidazolium methanesulfonate (EMIM MeSO), 1-ethyl-3-methylimidazolium tetrachloroaluminate (EMIM AlCl), 1-ethyl-3-methylimidazolium thiocyanate (EMIM SCN), 1-butyl-3-methylimidazolium acetate (BMIM acetate), 1-butyl-3-methylimidazolium chloride (BMIM Cl), 1-butyl-3-methylimidazolium hydrogen sulfate (BMIM HOSO3), 1-butyl-3-methylimidazolium methanesulfonate (BMIM MeSO3), 1-butyl-3-methylimidazolium methyl sulfate (BMIM MeOSO3), 1-butyl-3-methylimidazolium tetrachloroaluminate (BMIM AlCl4), 1-butyl-3-methylimidazolium thiocyanate (BMIM SCN), 1-ethyl-2,3-dimethylimidazolium ethyl sulfate (EDIM EtOSO3), tris(2-hydroxyethyl)methylammonium methyl sulfate (MTEOA MeOSO3), 1-methylimidazolium chloride (MIM Cl), 1-methylimidazolium hydrogen sulfate (MIM HOSO3), 1,2,4-trimethylpyrazolium methyl sulfate, tributylmethylammonium methyl sulfate, choline acetate, choline salicylate, and the like.

[0038] In some embodiments, the ionic liquid is a chloride ionic liquid. In other embodiments, the ionic liquid is an imidazolium salt. In yet other embodiments, the ionic liquid is a 1-alkyl-3-imidazolium chloride, such as 1-ethyl-3-methylimidazolium chloride or 1-butyl-3-methylimidazolium chloride.

[0039] In some embodiments, the ionic liquids used in the present invention are pyridinium salts, pyridazinium salts, pyrimidinium salts, pyrazinium salts, imidazolium salts, pyrazolium salts, oxazolium salts, 1,2,3-triazolium salts, 1,2,4-triazolium salts, thiazolium salts, isoquinolium salts, quinolinium salts, isoquinolinium salts, piperidinium salts, and pyrrolidinium salts. Exemplary anions of ionic liquids include, but are not limited to, halogens (e.g., chloride, fluoride, bromide, and iodide), pseudohalogens (e.g., azide and isocyanate), alkyl carboxylates, sulfonates, acetates, and alkyl phosphates.

[0040] Additional ILs suitable for use in the present invention are described in U.S. Patent Nos. 6,177,575; 9,765,044; and 10,155,735; U.S. Patent Application Publication Nos. 2004 / 0097755 and 2010 / 0196967; and International Application Nos. PCT / US2015 / 058472, PCT / US2016 / 063694, PCT / US2017 / 067737, and PCT / US2017 / 036438 (all of which are incorporated by reference in their entirety). Those skilled in the art will recognize that other ILs useful in the methods of the present invention are currently being developed or will be developed in the future, and the present invention contemplates their future use. The ionic liquid may comprise one or a mixture of compounds.

[0041] In some embodiments, the IL is a protic ionic liquid (PIL). Suitable protic ionic liquids (PILs) include molten salts having a melting point below 100 °C with salts having a higher melting point, referred to as molten salts. Suitable PPILs are disclosed in Greaves et al., "Protic Ionic Liquids: Properties and Applications", Chem. Rev. 108(1):206 - 237(2008). PILs can be prepared by the neutralization reaction of specific Bronsted acids and Bronsted bases (generally, primary, secondary, or tertiary amines which are alkaline), and the basic characteristic of these types of ILs is that their cations have at least one available proton to form a hydrogen bond with anions. In some embodiments, the protic ionic liquid (PIL) is formed from a combination of an organic ammonium - based cation and an organic carboxylic acid - based anion. PILs are acid - base conjugate ILs that can be synthesized via the direct addition of their acid and base precursors. In some embodiments, the PIL is a hydroxyalkylammonium carboxylate. In some embodiments, the hydroxyalkylammonium contains a linear or branched C1, C2, C3, C4, C5, C6, C7, C8, C9, or C10 chain. In some embodiments, the carboxylate contains a linear or branched C1, C2, C3, C4, C5, C6, C7, C8, C9, or C10 chain. In some embodiments, the carboxylate is substituted with one or more hydroxyl groups. In some embodiments, the PIL is hydroxyethylammonium acetate.

[0042] In some embodiments, the protic ionic liquid (PIL) is disclosed by U.S. Patent Application Publication No. 2004 / 0097755, which is incorporated herein by reference.

[0043] Salts suitable for the present method include combinations of organic ammonium cations (such as ammonium, hydroxyalkylammonium, or dimethylalkylammonium) with organic carboxylate anions (such as acetate derivatives (C1-C8), lactic acid, glycolic acid, and DES such as ammonium acetate / lactic acid).

[0044] Suitable ILs, such as distillable ILs, are disclosed in Chen et al., "Distillable Ionic Liquids: Reversible Amide O Alkylation," Angewandte Comm. 52:13392-13396 (2013), King et al., "Distillable Acid-Base Conjugate Ionic Liquids for Cellulose Dissolution and Processing," Angewandte Comm. 50:6301-6305 (2011), and Vijayaraghavan et al., "CO2-based Alkyl Carbamate Ionic Liquids as Distillable Extraction Solvents," ACS Sustainable Chem. Engin. 2:31724-1728 (2014), all of which are incorporated herein by reference.

[0045] Suitable PILs, such as distillable PILs, are disclosed in Idris et al., "Distillable Protic Ionic Liquids for Keratin Dissolution and Recovery," ACS Sustainable Chem. Engineer. 2:1888-1894 (2014) and Sun et al., "One-pot integrated biofuel production using low-cost biocompatible protic ionic liquids," Green Chem. 19(13):3152-3163 (2017), all of which are incorporated herein by reference.

[0046] In some embodiments, PILs are formed by combining organic ammonium cations with organic carboxylate anions. PILs are acid-base conjugated ILs that can be synthesized via the direct addition of their acid and base precursors. Additionally, when sufficient energy is applied, they can dissociate back into neutral acid and base precursors, while PILs reform upon cooling. This offers a convenient method for recovering and recycling ILs after application. In some embodiments, PILs (such as hydroxyethylammonium acetate - [Eth][OAc]) are effective solvents for biomass pretreatment and are relatively inexpensive due to their ease of synthesis (Sun et al., Green Chem. 19(13):3152-3163 (2017)).

[0047] Deep eutectic solvents (DES) DES are systems formed from eutectic mixtures of Lewis or Brønsted acids and bases, which may contain various anionic and / or cationic species. DES can form eutectic points in two-component phase systems. DES are formed by complexing a quaternary ammonium salt (e.g., choline chloride) with a hydrogen bond donor (HBD), such as an amine, amide, alcohol, or carboxylic acid. The interaction between the HBD and the quaternary salt reduces the anion-cation electrostatic force, lowering the melting point of the mixture. DES share many characteristics of conventional ionic liquids (ILs) and are expected to have promising applications in fields such as biomass processing and electrochemistry. In some embodiments, DESs are any combination of a Lewis or Brønsted acid and a base. In some embodiments, the combination of a Lewis or Brønsted acid and a base used is distillable.

[0048] In some embodiments, DESs are prepared using an alcohol (such as glycerol or ethylene glycol), an amine (such as urea), and an acid (such as oxalic acid or lactic acid). The present invention can use renewable DESs with lignin-derived phenols as HBDs. Both phenolic monomers and phenolic mixtures readily form DESs when heated at 100°C in specific molar ratios with choline chloride. This class of DESs does not require multi-step synthesis. DESs are synthesized from lignin, a renewable source.

[0049] Both monomeric phenols and phenol mixtures can be used to prepare DES. DES can dissolve biomass or lignin, making it useful for biomass pretreatment. The use of DES derived from biomass can reduce the cost of biomass processing and provide a more environmentally friendly route for various industrially relevant processes.

[0050] The DES or mixture thereof is biocompatible, meaning that it does not reduce or significantly reduce the enzymatic activity of the enzyme and / or is non-toxic and / or does not reduce or significantly reduce the growth of microorganisms. A "significant" reduction is a reduction of 70, 80, 90, or 95% or less of the enzymatic activity of the enzyme and / or the growth (or doubling time) of the microorganisms in the absence of the DES or mixture thereof.

[0051] In some embodiments, the DES or mixture thereof comprises a quaternary ammonium salt and / or glycerol. In some embodiments, the DES or mixture thereof comprises a quaternary ammonium salt and / or glycerol. In some embodiments, the quaternary ammonium salt and / or glycerol have a molar ratio of about 1:1 to about 1:3. In some embodiments, the quaternary ammonium salt and / or glycerol have a molar ratio of about 1:1.5 to about 1:2.5. In some embodiments, the quaternary ammonium salt and / or glycerol have a molar ratio of about 1:1.8 or 1:1.9 to about 1:2.1 or 1:2.2. In some embodiments, the quaternary ammonium salt and / or glycerol have a molar ratio of about 1:2. In some embodiments, the quaternary ammonium salt is a choline halide, such as choline chloride.

[0052] In some embodiments, a DES is distillable if the DES can be recovered in a yield of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% or more by vacuum distillation at a temperature of about 100°C, 110°C, 120°C, 130°C, 140°C, 150°C, or 160°C, or any temperature between any two of the foregoing temperatures.

[0053] In some embodiments, the DES may be one taught in WO 2018 / 204424 (Seema Singh et al.), which is incorporated by reference in its entirety.

[0054] In some embodiments, the method further comprises heating the one-pot composition, optionally also comprising the enzyme and / or microorganism, to a temperature equal to, about equal to, or near the optimum temperature for the enzymatic activity of the enzyme and / or growth of the microorganism. In some embodiments, the enzyme is a genetically engineered host cell capable of converting cellulose in the biomass into sugars. In some embodiments, multiple enzymes are present. In some embodiments, the microorganism is a genetically engineered host cell capable of converting sugars produced from the biomass into biofuels and / or chemical compounds. In some embodiments, multiple microorganisms are present. In some embodiments, the introducing step produces sugars and lignin from the biomass. The lignin can be further processed to produce DES. The sugars are used for growth by the microorganism.

[0055] In some embodiments, solubilization is complete, nearly complete (e.g., at least about 70, 80, or 90%), or partial (e.g., at least about 10, 20, 30, 40, 50, or 60%). In some embodiments, the one-pot composition is a slurry. When the processes described herein are continuous, the one-pot composition is at a steady state.

[0056] In some embodiments, the introducing step comprises heating the mixture, raising the temperature of the solution to a value within the range of about 75°C to about 125°C. In some embodiments, the heating step comprises raising the temperature of the solution to a value within the range of about 80°C to about 120°C. In some embodiments, the heating step comprises raising the temperature of the solution to a value within the range of about 90°C to about 110°C. In some embodiments, the heating step comprises raising the temperature of the solution to about 100°C.

[0057] enzyme In some embodiments, the enzyme is a cellulase. In some embodiments, the enzyme is thermophilic or hyperthermophilic. In some embodiments, the enzyme is any enzyme taught in U.S. Patent Nos. 9,322,042; 9,376,728; 9,624,482; 9,725,749; 9,803,182; and 9,862,982; and International Application Nos. PCT / US2015 / 000320, PCT / US2016 / 063198, PCT / US2017 / 036438, PCT / US2010 / 032320, and PCT / US2012 / 036007 (all of which are incorporated by reference in their entireties).

[0058] microorganisms In some embodiments, the microorganism is any prokaryotic or eukaryotic cell having any genetic modification, including those described in U.S. Patent Nos. 7,985,567; 8,420,833; 8,852,902; 9,109,175; 9,200,298; 9,334,514; 9,376,691; 9,382,553; 9,631,210; 9,951,345; and 10,167,488; and International Application Nos. PCT / US14 / 48293, PCT / US2018 / 049609, PCT / US2017 / 036168, PCT / U Nos. S2018 / 029668, PCT / US2008 / 068833, PCT / US2008 / 068756, PCT / US2008 / 068831, PCT / US2009 / 042132, PCT / US2010 / 033299, PCT / US2011 / 053787, PCT / US2011 / 058660, PCT / US2011 / 059784, PCT / US2011 / 061900, PCT / US2012 / 031025, and PCT / US2013 / 074214, all of which are incorporated by reference in their entireties.

[0059] Typically, but not necessarily, the microorganism is a yeast or bacterium. In some embodiments, the microorganism is Rhodosporidium toruloides or Pseudomonas putida. In some embodiments, the microorganism is a Gram-negative bacterium. In some embodiments, the microorganism is a microorganism of the phylum Proteobactera. In some embodiments, the microorganism is a microorganism of the class Gammaproteobacteria. In some embodiments, the microorganism is a microorganism of the order Enterobacteriales. In some embodiments, the microorganism is a microorganism of the family Enterobacteriaceae. Examples of suitable bacteria include, but are not limited to, species classified in the taxonomic groups Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, and Paracoccus. Suitable eukaryotic microorganisms include, but are not limited to, fungal cells. Suitable fungal cells are yeast cells, such as yeast cells of the genus Saccharomyces.

[0060] Yeast suitable for the present invention include, but are not limited to, Yarrowia, Candida, Bebaromyces, Saccharomyces, Schizosaccharomyces, and Pichia cells. In some embodiments, the yeast is Saccharomyces cerevisiae. In some embodiments, the yeast is a Candida species, including, but not limited to, C. tropicalis, C. maltosa, C. apicola, C. paratropicalis, C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. lipolytica, C. panapsilosis, and C. zeylenoides. In some embodiments, the yeast is Candida tropicalis. In some embodiments, the yeast is a non-oleaginous yeast. In some embodiments, the non-oleaginous yeast is a Saccharomyces species. In some embodiments, the Saccharomyces species is Saccharomyces cerevisiae. In some embodiments, the yeast is an oleaginous yeast. In some embodiments, the oleaginous yeast is a Rhodosporidium species. In some embodiments, the Rhodosporidium species is Rhodosporidium toruloides.

[0061] In some embodiments, the microorganism is a bacterium. Bacterial host cells suitable for the present invention include, but are not limited to, Escherichia, Corynebacterium, Pseudomonas, Streptomyces, and Bacillus. In some embodiments, the Escherichia cell is E. coli, E. albertii, E. fergusonii, E. hermanii, E. marmotae, or E. vulneris. In some embodiments, the Corynebacterium cell is Corynebacterium glutamicum, Corynebacterium kroppenstedtii, Corynebacterium alimapuense, Corynebacterium amycolatum, Corynebacterium diphtheriae, Corynebacterium efficiens, Corynebacterium jeikeium, Corynebacterium macginleyi, Corynebacterium matrcoti, Corynebacterium matruchotii, Corynebacterium minutissimum, Corynebacterium renale, Corynebacterium striatum, Corynebacterium ulcerans, Corynebacterium urealyticum, or Corynebacterium uropygiale.In some embodiments, the Pseudomonas cell is P. putida, P. aeruginosa, P. chlororaphis, P. fluorescens, P. pertucinogena, P. stutzeri, P. syringae, P. cremocolorata, P. entomophila, P. fulva, P. monteilii, P. mosselii, P. oryzihabitans, P. parafluva, or P. plecoglossicida. In some embodiments, the Streptomyces cell is S. coelicolor, S. lividans, S. venezuelae, S. amphotericinicus, S. avermitilis, S. albus, or S. scabies. In some embodiments, the Bacillus cell is B. subtilis, B. megaterium, B. licheniformis, B. anthracis, B. amyloliquefaciens, or B. pumilus.

[0062] biofuels In some embodiments, the biofuel produced is one or more of the biofuels described in U.S. Patent Nos. 7,985,567; 8,420,833; 8,852,902; 9,109,175; 9,200,298; 9,334,514; 9,376,691; 9,382,553; 9,631,210; 9,951,345; and 10,167,488; and International Application Nos. PCT / US14 / 48293, PCT / US2018 / 049609, PCT / US2017 / 036168, PCT / US2018 / 029668, PCT / US2008 / 036168, PCT / US201 ... and PCT / US2011 / 059784, PCT / US2011 / 061900, PCT / US2012 / 031025, and PCT / US2013 / 074214, all of which are incorporated by reference in their entireties.

[0063] biomass Biomass can be derived from one or more sources, such as softwood sources, hardwood sources, grass sources, and / or agricultural sources, or mixtures thereof.

[0064] Coniferous raw materials include Araucaria (Pinaceae) (e.g., Araucaria cunninghamii, Araucana angustifolia, Araucana araucana); Cedar (Pinaceae) (e.g., Juniperus virginiana, Thuja plicata, Thuja occidentalis, Chamaecyparis thyoides, Callitropsis nootkatensis); Cypress (Cypress) (e.g., Chamaecyparis cypress, Cupressus taxodium, Cupressus arizonica, Bald cypress, Taxodium distichum, Chamaecyparis obtusa, Chamaecyparis lawsoniana, Cupressus semperviren; Rocky Mountain Douglas fir; European yew; Fir (e.g., Abies balsamea, Abies alba, Abies procera, Abies amabilis); Hemlock (e.g., Tsuga canadensis, Tsuga mertensiana, Tsuga heterophylla); Kauri (kauri); Kaya (kaya); Larch (e.g., Larix arvensis) decidua), larch (Larix kaempferi), tamarack (Larix laricina), western larch (Larix occidentalis));Family Pinaceae: Genus Pinus (e.g., Pinus nigra, Pinus banksiana, Pinus contorta, Pinus radiata, Pinus ponderosa, Pinus resinosa, Pinus sylvestris, Pinus strobus, Pinus monticola, Pinus lambertiana, Pinus taeda, Pinus palustris, Pinus rigida, Pinus serrata, Pinus sylvestris, Pinus serrata ... echinata); Redwood; Rimu; Spruce (e.g., Picea abies, Picea mariana, Picea rubens, Picea sitchensis, Picea glauca); Sugi; and combinations / hybrids thereof, but are not limited thereto.

[0065] For example, coniferous raw materials that can be used herein include Cedrus (Pinaceae), Abies (Pinaceae), Pine (Pinaceae), Picea (Pinaceae), and combinations thereof. The coniferous raw materials of the present invention can be selected from loblolly pine (Pinus taeda), Radiata pine, jack pine, Picea (Picea) (e.g., white, interior, black), Douglas fir, Pinus sylvestris, Norway spruce, and combinations / hybrids thereof. The coniferous raw materials of the present invention can be selected from Pine (Pinaceae), Picea (e.g., Radiata pine, Pinus taeda), and combinations / hybrids thereof.

[0066] Hardwood raw materials include Acacia; Afzelia; Synsepalum duloificum; Albizia; Alder (e.g., Alnus glutinosa, Alnus rubra); Applewood; Arbutus; Ash (e.g., F. nigra, F. quadrangulata, F. excelsior, F. pennsylvanica), and others. Aspen (e.g., P. grandidentata, P. tremula, P. tremuloides); Australian Red Cedar (Toona ciliata); Ayna (Distemonanthus benthamianus); Balsa (Ochroma pyramidae) pyramidale); Basswood (e.g., T. americana, T. heterophylla); Beech (e.g., F. sylvatica, F. grandifolia); Birch (e.g., Betula populifolia, B. nigra, B. papyrifera, B. lenta, B. alleghaniensis / B. lutea, B. pendula, B. pubescens); Blackbean; Blackwood; Bocote;Boxelder; Boxwood; Brazilwood; Bubinga; Buckeye (e.g., Aesculus hippocastanum, Aesculus glabra, Aesculus flava / Aesculus octandra); Butternut; Catalpa; Cherry (e.g., Prunus serotina, Prunus pennsylvanica, Prunus avium) avium); Crabwood; Chestnut; Coachwood; Cocobolo; Corkwood; Cottonwood (e.g., Populus balsamifera, Populus deltoides, Populus sargentii, Populus heterophylla); Cucumbertree; Dogwood (e.g., Cornus florida, Cornus nuttallii); Ebony (e.g., Diospyros kurzii, Diospyros melanida, Diospyros crassiflora) crassiflora); Elms (e.g., Ulmus americana, Ulmus procera, Ulmus thomasii, Ulmus rubra, Ulmus glabra); Eucalyptus; Greenheart; Grenadilla;Gum (e.g., Nyssa sylvatica, Eucalyptus globulus, Liquidambar styraciflua, Nyssa aquatica); Hickory (e.g., Carya alba, Carya glabra, Carya ovata, Carya laciniosa); Hornbeam; Hophornbeam; Ipe; Iroko; Ironwood (e.g., Bangkirai, Carolina Hornbeam, Casuarina equisetifolia, Choricbangarpia subargentea, Copaifera spp., Eusideroxylon zwageri, Guajacum officinale, Guajacum sanctum, Hopea odorata, Ipe, Krugiodendron ferreum, Lyonothamnus lyonii (L. floribundus), Mesua ferrea, Olive (Olea spp.), Olneya tesota, Ostrya virginiana virginiana, Parrotia persica, Tabebuia serratifolia; Jacaranda; Jotoba; Lacewood; Laurel; Limba; Lignum vitae;Locust (e.g., Robinia pseudocacia, Gleditsia triacanthos); Mahogany; Maple (e.g., Acer saccharum, Acer nigrum, Acer negundo, Acer rubrum, Acer saccharinum, Acer pseudoplatanus); Meranti; Mpingo; Oak (e.g., Quercus macrocarpa, Quercus alba, Quercus stellata, Quercus bicolor, Quercus virginiana) virginiana, Quercus michauxii, Quercus prinus, Quercus muhlenbergii, Quercus chrysolepis, Quercus lyrata, Quercus robur, Quercus petraea, Quercus rubra, Quercus velutina, Quercus laurifolia, Quercus falcata, Quercus nigra, Quercus phellos, Quercus texana texana); Obeche; Okoume; Oregon Myrtle; California Bay Laurel; Pear;Poplar (e.g., P. balsamifera, P. nigra, Hybrid Poplar (Populus x canadensis)); Ramin; Red cedar; Rosewood; Sal; Sandalwood; Sassafras; Satinwood; Silky Oak; Silver Wattle; Snakewood; Sourwood; Spanish cedar; American sycamore; Teak; Walnut (e.g., Juglans nigra, Juglans serrata) regia); Willow (e.g., Salix nigra, Salix alba); Yellow poplar (Liriodendron tulipifera); Bamboo; Palmwood; and combinations / hybrids thereof, but are not limited to these.

[0067] For example, the hardwood raw materials of the present invention may be selected from acacia, aspen, beech, eucalyptus, maple, birch, gum, oak, poplar, and combinations / hybrids thereof. Hardwood raw materials for the present invention may be selected from the Populus genus (e.g., Populus tremuloides), Eucalyptus genus (e.g., Eucalyptus globulus), Acacia genus (e.g., Acacia dealbata), and combinations thereof.

[0068] Grass feedstocks include, but are not limited to, C4 or C3 grasses such as switchgrass, Indiangrass, big bluestem, little bluestem, Canada wildrye, Virginia wildrye, and goldenrod wildflower, among other species known in the art.

[0069] Agricultural feedstocks include, but are not limited to, agricultural by-products such as husks, stover, leaves, etc. Such agricultural by-products may be derived from crops intended for human consumption, animal consumption, or other non-consumer purposes. Such crops may include corn, wheat, sorghum, rice, soybeans, hay, potatoes, cotton, or sugarcane. Feedstocks may be derived from the harvest of intercropped, mixed, continuous, relay, or other crops.

[0070] In some embodiments, the biomass is ensiled biomass. In some embodiments, biomass is ensiled by placing the biomass in a sealed container or chamber, such as a silo, or by piling it into a heap covered with an air barrier, such as a plastic film. Ensiled biomass, known as silage, undergoes a bacterial fermentation process to produce volatile fatty acids. In some embodiments, ensiling involves adding an ensiling agent, such as sugar, lactic acid, or an inculant. In some embodiments, the ensiled biomass contains one or more toxic compounds. In some embodiments, when the ensiled biomass contains one or more toxic compounds, the microorganisms are resistant to the one or more toxic compounds.

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[0072] Example 1 Renewable Schiff base ionic liquids for lignocellulosic biomass pretreatment Growing interest in sustainable chemical and energy sources from renewable and reliable sources has spurred the design and synthesis of renewable ILs as alternatives to fossil-derived Schiff base (iminium) ionic liquids (ILs). In this study, we report the synthesis of three unique iminium-acetate ILs from lignin-derived aldehydes, aiming for a sustainable "future" lignocellulosic biorefinery. The synthesized ILs contained either imine-only or amine and imine groups in their structures. The imine-only IL achieved a sugar release rate of over 89%, demonstrating better pretreatment efficacy. Various analytical and computational tools were employed to understand the pretreatment efficacy of these ILs. This initial study, demonstrating the ease of synthesis of these renewable ILs, opens the door to a new class of "Schiff base ILs" and paves the way for further testing, potentially with task-specific design.

[0073] Herein, we report the first attempt, to our knowledge, to protonate imines formed by the direct condensation of lignin-derived vanillin with amines such as ethylenediamine (EDA). The applicability of these iminium ILs in the treatment of lignocellulosic biomass was also investigated. Iminium salts are a special class of organic compounds that can be visualized as □-amino carbocations with electrophilic properties that can potentially form pseudobases in the presence of water. Such chemical functionality may help elucidate some unknown interactions / chemistry when considering lignocellulosic biomass. While several examples of various iminium salts with various uses exist in the literature, it must be emphasized that the application of renewable iminium ILs in biomass treatment remains unexplored to date.

[0074] Results and Discussion The reaction of amines with aldehydes (or ketones) to form carbinolamines and imines was reported by Hugo Schiff in 1864 as a new series of organic bases. 30 Since then, these imines, also known as Schiff bases, have been tested in a broader context, including catalysts and bioactive molecules. In this study, given its ease of synthesis, we used renewable lignin-derived vanillin as the aldehyde precursor and ethylenediamine (EDA) as the amine source. To obtain vanillin-based Schiff bases, an aqueous solution of vanillin was slowly added to a cold aqueous solution of EDA to dissipate the immediate heat generation. The presence of two amines in EDA afforded two unique Schiff bases, 1 and 2, by simply adding 1 and 2 equivalents of vanillin, respectively, as shown in Figure 7. The products were obtained in quantitative crude yield as high-melting yellow solids (mp > 230 °C) after filtration and air-drying.

[0075] Infrared (IR) spectra of the resulting products demonstrated the formation of imine bonds in 1 and 2 compared to vanillin and EDA (Figure 1). The primary amine NH stretching was weakened in 1 due to the absence of a primary amine in the molecule, but completely disappeared in 2. Furthermore, the aromatic aldehyde (C=O) stretching of vanillin was observed at 1667 cm. -1 So, in 1 it's 1641cm -1 , 2 is 1619cm -1 The red shift in the C=N stretching is characteristic of the conjugated C=N stretching. 1 H and 13 C NMR analysis suggested that the desired product had been formed (see Materials and Methods).

[0076] To prepare the ILs, the resulting Schiff bases 1 and 2 were then treated with acetic acid in a 1:1 and 1:2 ratio to protonate the imine N atoms in these bases, yielding four unique ILs, 1A, 1B, 2A, and 2B (see Figure 7). The synthetic protocol mirrors the acid-base reaction generally employed for the synthesis of protic ILs: a base (in this case, an imine) is mixed with an acid (acetic acid in this study) to expect proton transfer from the acid to the base (the degree of proton transfer depends on the physicochemical properties of the reagents). 31,32 It is important to note that acetic acid must be added slowly to a cold, stirred solution of the Schiff base. An increase in temperature or a high concentration of acid (H+ ions) leads to hydrolysis of the C=N bond of the Schiff base, producing water-soluble vanillin. To understand the proton transfer, FT-IR spectra of these ILs were recorded (Figure 2). Deprotonation of the acid results in a red shift from the carboxylic acid C=O stretch to the carboxylate C=O stretch. -1 Carboxylate CO stretching at 2600-2700 cm was observed in all four IL formulations. -1 Peaks around 2640 cm have been attributed in the literature to strong H-bonding features. 33 For example, intermolecular H-bonds in concentrated acids occur at approximately 2640 cm. -1This feature was also observed in 1 (primary amine) but not in 2 (no primary amine). Partial protonation of the amine / imine in 1 led to weaker intensity in 1A, whereas H-bonding appears negligible in 1B. Similarly, the absence of free amines in 2, 2A, and 2B obscures signals attributable to strong intermolecular H-bonding in these molecules. We believe that the partial protonation of 1 leads to a dynamic equilibrium in which the positive charge oscillates between the ammonium cation (1A') and the iminium cation (1A), as shown in Figure 7. Because amines are known to be more basic than imines, the equilibrium would be expected to favor protonation of the amine over the imine; however, we currently have no crystallographic or spectroscopic evidence to support this hypothesis.

[0077] Differential scanning calorimetry (DSC) profiles of these ILs revealed preferential proton occupancy sites in the 1:1 and 1:2 acetic acid derivatives of 1 and 2 (Figure 3). The high melting point characteristic of imines was also observed in the 1:1 acetic acid derivatives of these imines in the case of 2A. This indicates that 1:1 salts were not formed; instead, both nitrogen atoms in the imine appear to be fully protonated in the presence of acetic acid. Upon addition of one equivalent of acetic acid, only 50% of the imine was fully protonated, while the remainder remained neutral. This observation is consistent with the FT-IR data, in which the 1:1 salts exhibited characteristic peaks from both the imine and the 1:2 acetic acid derivative (see Figure 2). We hypothesize that no or full protonation does not apply to 1 because both the amine moiety (higher basicity) and the imine moiety coexist in the system. Additionally, thermogravimetric analysis (TGA) curves of the Schiff base and ILs complement the DSC curves. For example, the onset of decomposition of IL 2B was observed at approximately 125°C in both DSC and 131 TGA, compared to over 200°C for the corresponding Schiff base 2.

[0078] To maximize the chemical potential of lignocellulosic biomass, it is essential to fractionate the constituent biopolymers (cellulose, hemicellulose, and lignin) that are strongly retained in a complex and recalcitrant matrix through pretreatment processes. As previously mentioned, IL-based pretreatment technologies remain attractive for sustainable biorefineries due to the outstanding ability of ILs to dissolve, fractionate, and further transform biopolymers. 26,34,35,36,37 IL-based pretreatment technologies have been reported to be most effective when operated at temperatures between 90 and 160 °C for 3 h to obtain high sugar yields from a given biomass. 16,38,39,40,41 Next, we tested the performance of these ILs for the pretreatment of sorghum biomass under specified conditions. 20 wt.% sorghum biomass was mixed with 80 wt.% ILs and heated at 140 °C for 3 h. It is noteworthy that all synthesized ILs were solid at room temperature but were expected to melt at temperatures much lower than the pretreatment temperature (Figure 3). The pretreatment (PT) effectiveness of these ILs, including the solid recovery and sugar release rates, is shown in Table 1. Considering the amount of dry biomass recovered after pretreatment with the ILs and subsequent washing (see Materials and Methods for details), referred to herein as solid recovery, all ILs showed very high solid recovery rates, ranging from 83 to 87%. The general trend of higher solid recovery at lower pretreatment temperatures is consistent regardless of the biomass or IL used for pretreatment. 36,37,38,39,40,41 Snapshots of biomass mixed with ILs before and after pretreatment, included in Table 1, are in good agreement with the observed DSC trends. That is, IL 2A (a mixture of 2 and IL 2B) did not completely melt, whereas all other ILs melted under the pretreatment conditions. Visual observation of the results of 1A, 1B, 2A, and 2B showed that the pre-PT samples were light and the post-PT samples were all very dark, with 1B being the lightest colored of the "post-PT" samples.

[0079] [Table 1]

[0080] To understand the impact of pretreatment as a function of IL, the carbohydrate (glucan and xylan) and lignin contents of untreated and IL-pretreated sorghum were determined. Interestingly, the carbohydrate and lignin contents were found to be similar to those of untreated biomass. For example, the glucan, xylan, and lignin contents of IL-pretreated solids ranged from 26.1 to 27.8%, 14.9 to 15.6%, and 19.1 to 21.7%, respectively, whereas the glucan content of untreated sorghum biomass was 26.3%, the xylan content was 15.1%, and the lignin content was 19.2%. No significant reduction in biopolymers was observed with pretreatment using these ILs, even after accounting for solids recovery. Typically, the actual biopolymer removal rates of carbohydrate and lignin components, calculated as follows, achieved a reduction of less than 10% in all cases. Removal rate (%) = [100 - {(solid recovery rate (%)) * (Composition of pretreated biomass / Composition of untreated biomass)

[0081] To understand these results, we performed Conductor-Like Screening model for Real Solvent (COSMO-RS) calculations to understand the viability of IL-biopolymer interactions. In line with previous studies, we calculated the logarithmic activity coefficient (ln(γ)) to predict biopolymer dissolution in the ILs under investigation. 42,43 We also examined the intermolecular interactions between cellulose / lignin and the ILs (Figure 4). Because the compositions of the synthesized ILs differ, predictions could misinterpret experimental observations. IL 2A was not considered in these calculations. Generally, a lower ln(γ) (i.e., more negative) indicates a stronger interaction between the solute (cellulose or lignin) and the solvent (IL). Based on these predictions, none of the ILs under investigation tended to exhibit significant interactions with cellulose or lignin. IL 1B, in particular, had a positive ln(γ), indicating negligible interactions with both cellulose and lignin. Notably, all of these ILs (1A and 2B) had a higher affinity for cellulose than for lignin, which explains the negligible removal of biopolymers from biomass after pretreatment with these ILs (186).

[0082] The effectiveness of these IL pretreatments was also measured in terms of carbohydrate digestibility using commercially available enzymes, as described in the Materials and Methods section. Enzymatic hydrolysis to release monomeric sugars from pretreated biomass was carried out at 50°C for 72 hours with a protein loading of 10 mg per g of biomass. High sugar release rates were observed for ILs 1A, 1B, and 2B, resulting in glucose yields of 69–87% and xylose yields of 63–76% (Table 1). As expected based on the characterization data, low sugar release rates were observed for IL 2A. It is noteworthy that, under similar conditions, enzymatic saccharification of pretreated solids from a mixture of 20 wt% sorghum biomass and 80 wt% water at 140°C for 3 hours achieved glucose yields of 38% and xylose yields of 32%.

[0083] To further understand the pretreatment mechanism of the Schiff base ILs, we characterized the solids pretreated with these ILs, especially the solids after pretreatment with IL 2B, which showed the highest sugar release rate (Table 1). As suggested by the COSMO-RS predictions, these ILs exhibited higher interactions with cellulose (although only minimal removal from the biomass). To understand the interaction between cellulose and the ILs, we recorded powder X-ray diffraction patterns of untreated and IL 2B-pretreated sorghum (Figure 5, top right). We expected that lower cellulose crystallinity would increase enzyme accessibility, but no such reduction in crystallinity was observed in the pretreated solids. The crystallinity of the pretreated solid residue (23.9%) was similar to that of untreated sorghum (25.5%). Additionally, TGA of untreated and pretreated biomass showed similar profiles, except for the removal of extractives, particularly free sugars, aromatics, and soluble proteins, which are expected to be removed during washing of the pretreated biomass (Figure 5). Typically, most previous studies have considered delignification or a decrease in cellulose crystallinity (breaking of intermolecular hydrogen bonds) to explain the mechanism of pretreatment. 38,39,40 On the other hand, several other studies, including dilute acid pretreatment, have reported efficient sugar release rates without the significant decrease in crystallinity or delignification observed in this case. 44,45,46 Also, the increase in accessible area obtained using Simons staining and thermoporosimetry techniques was thought to explain the sugar release rate after pretreatment. 47 However, neither of these approaches explained the observed pretreatment effectiveness of these ILs in this study.

[0084] Finally, we examined the HSQC NMR of the lignin-rich residue obtained after saccharification of the pretreated biomass to examine changes in structural features after pretreatment (Figure 6). By comparing it with the lignin in the untreated biomass, we were able to examine the observed effectiveness of the Schiff base ILs for the pretreatment of lignocellulosic biomass. For example, signals corresponding to protons on hydroxyl-bearing carbons (see the Aα, Aβ, Aγ, A′γ, and Bγ structures in Figure 6) disappeared or weakened after pretreatment with IL 2B. 48,49,50 This is likely due to the chemical interaction of the Schiff base ILs with lignin, which abstracts protons, stripping the lignin from the recalcitrant biopolymer matrix (cellulose, xylose, and lignin), rendering active centers on the lignin to yield condensed lignin. The aromatic regions of the pretreated lignin in the HSQC NMR (Figure 6) supported the formation of condensed lignin. These results clearly demonstrate that the chemical interaction of Schiff base ILs with lignocellulosic biomass leads to high sugar yields. We propose that the Schiff base ILs investigated in this study act primarily by interacting with the lignin component of lignocellulosic biomass rather than with the cellulose, which remains crystalline after pretreatment. However, the observation of the resulting condensed lignin indicates that the lignin-carbohydrate bonds in lignocellulosic biomass are disrupted, increasing its accessibility to enzymes, as previously reported for acid pretreatment. To fully exploit the specific applications of Schiff base ILs for lignocellulosic biomass utilization for renewable fuels and products, detailed systematic studies are needed to gain a better understanding of the mechanisms of these ILs in lignocellulosic biomass processing.

[0085] Materials and Methods material All materials were used as supplied unless otherwise noted. Deionized water with a resistivity of 18 MΩ·cm at 25 °C was obtained from Purelab Flex (ELGA, Woodridge, IL). Choline hydroxide (45% in methanol), acetic acid (greater than 99.7%), sodium hydroxide pellets (greater than 97%), methanol, sodium azide, and sulfuric acid (98%) were obtained from Sigma-Aldrich (St. Louis, MO). Ethanol (200 proof) was purchased from Decon Labs, Inc. (King of Prussia, PA). Sulfuric acid (72%) was obtained from RICCA Chemical Company (Arlington, TX). JT Baker, Inc. (Phillipsburg, NJ) supplied hydrochloric acid and sodium citrate dihydrate, and citric acid monohydrate (greater than 99.99%) was obtained from Merck (Kenilworth, NJ).

[0086] Analytical standard grade glucose and xylose were also obtained from Sigma-Aldrich (St. Louis, MO) and used for calibration.

[0087] Sorghum (Sorghum bicolor, a gift from Idaho National Labs, Idaho Falls, USA) was dried in an oven at 40°C for 24 hours. It was then knife-milled using a 2 mm screen (Thomas-Wiley Model 4, Swedesboro, NJ). The resulting biomass was then placed in leak-proof bags and stored in a cool, dry place (4°C room during use).

[0088] Commercially available cellulase (Cellic® CTec3) and hemicellulase (Cellic® HTec3) mixtures were provided by Novozymes, North America (Franklinton, NC).

[0089] Synthesis of Schiff bases and related ionic liquids Synthesis of Schiff Bases. Ethylenediamine was weighed and suspended in water in an oven-dried round-bottom flask (RBF) containing a Teflon-coated magnetic stir bar. The flask was placed over a cold water bath (5°C) and an addition funnel was attached to the RBF. The aqueous solution of vanillin was transferred to the addition funnel and added dropwise to the stirring cold solution of ethylenediamine in water. The mixture was then stirred for an additional hour. After filtration and drying, the product was obtained as a yellow solid. Two different ratios of ethylenediamine to vanillin, namely 1:1 and 1:2, were used to obtain two different Schiff bases. The purity and identity of the synthesized molecules / ILs were determined and established by NMR, IR, and thermal analysis.

[0090] Ethylenediamine-vanillin (1:1), 1: 1 H NMR (800 MHz, DMSO-d6) δ 8.56, 7.30, 7.16, 6.87, 4.01, 3.84, 2.89. 13 C NMR(201 MHz,DMSO-d6)δ 162.7,151.7,149.9,133.4,122.7,118.2,113.4,58.1,53.1,40.3.

[0091] Ethylenediamine-vanillin (1:2), 2: 1 H NMR (800 MHz, DMSO-d6) δ 8.61,7.35,7.06,6.84,4.87,3.82. 13 C NMR(201 MHz,DMSO-d6)δ 162.4,150.9,149.8,132.8,122.1,118.7,113.5,60.9,55.4.

[0092] Synthesis of IL. A known amount of Schiff base was suspended in water in an oven-dried round-bottom flask (RBF) containing a Teflon-coated magnetic stir bar. The flask was placed on an ice bath, and an addition funnel was attached to the RBF. Acetic acid (Schiff base to acetic acid, 1:1 and 1:2) was transferred to the addition funnel and added dropwise to the stirring, cold suspension of base. The mixture was then stirred for an additional hour. The product was obtained after filtration and drying.

[0093] Biomass Pretreatment All pretreatment reactions were performed in duplicate. A 2 mm sorghum sample was mixed with IL at a 1:4 ratio (w / w) to give a biomass loading of 20 wt% in a 15 mL capped glass pressure tube and pretreated in an oil bath heated to 140 °C for 3 h. After pretreatment, the sample was removed from the oil bath and allowed to cool. 10 mL of DI water-ethanol (1:1 v / v) was slowly added to the biomass-IL slurry and mixed thoroughly. The mixture was transferred to a 50 mL Falcon tube and centrifuged at high speed (4000 rpm) to separate the solids and remove residual IL. The ethanol-water washed solids were freeze-dried to obtain dry pretreated biomass for further analysis.

[0094] Enzymatic saccharification All enzymatic saccharifications were performed in duplicate. Enzymatic saccharification of pretreated and untreated biomass was carried out at 50°C in a rotary incubator (Enviro-Genie, Scientific Industries, Inc.) using commercially available enzymes Cellic® CTec3 and HTec3 (9:1 v / v) from Novozymes. All reactions were carried out in 15 mL centrifuge tubes at a biomass loading of 5 wt%. To prevent microbial contamination, the pH of the mixture was adjusted to 5 with 50 mM sodium citrate buffer supplemented with 0.02% sodium azide. The total reaction volume contained a total protein content of 10 mg per g of biomass. The amount of released sugars was analyzed using an Agilent HPLC 1260 infinity system (Santa Clara, California, United States) equipped with a Bio-Rad Aminex HPX-87H column and a Refractive Index detector. Aqueous sulfuric acid (4 mM) was used as the eluent (0.6 mL min-1, column temperature 60 °C).

[0095] Composition analysis All compositional analysis experiments were performed in duplicate. Compositional analysis of biomass before and after pretreatment was performed using NREL's two-step acid hydrolysis protocols (LAPs) LAP-002 and LAP-005.51. 51 Briefly, 200 mg of biomass and 2 mL of 72% sulfuric acid (H2SO4) were incubated at 30 °C for 1 h with shaking at 200 rpm. The solution was diluted to 4% H2SO4 with 56 mL of DI water and autoclaved at 121 °C for 1 h. The reaction was stopped by cooling the flask, and then filtered using a medium-porosity filtering crucible to remove solids. The filtrate was analyzed spectrophotometrically for acid-soluble lignin or ASL (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA) using absorbance at 240 nm. Additionally, glucose and xylose concentrations were determined from the filtrate using HPLC (as previously described). The amounts of glucan and xylan were calculated by multiplying the glucose and xylose contents by anhydrous correction factors of 162 / 180 and 132 / 150, respectively. Finally, the acid-insoluble lignin was determined gravimetrically from the solid after heating at 105°C overnight (weight of acid-insoluble lignin and ash) and then at 575°C for at least 6 h (weight of ash).

[0096] Powder X-ray diffraction Powder X-ray diffraction (PXRD) data were collected using a Rigaku MiniFlex 6G sixth-generation benchtop X-ray diffractometer equipped with a 600W sealed source Cu tube and a HyPix-400MF Hybrid Pixel Array 0D / 1D / 2D detector. Data collection and analysis were performed using SmartLab Studio II.

[0097] The crystallinity index (CI) was determined from the crystalline and amorphous peak areas of the measured diffraction patterns using the following equation as previously reported: 52 %CI=[(I 002 -I am ) / I 002 ] * 100 where I002 is the intensity of the crystal plane (002) and I amis the minimum between the (002) and (101) peaks, and is about 18°.

[0098] More about COSMO-RS COSMO-RS calculations were used to predict the dissolution and / or interaction of cellulose and lignin in Schiff-base ILs. To perform these calculations, initial structures of cellulose, lignin, and ILs were drawn in the Avogadro freeware software. 53 The structures of all investigated molecules were optimized using the Gaussian09 package with B3LYP (Becke three-parameter hybrid function combined with Lee-Yang-Parr correlation) theory and the 6-311+G(d,p) basis set. 54,55 After the geometry optimization step, COSMO files were further generated using the BVP86 / TZVP / DGA1 level of theory and basis set. 56 The same level of theory, i.e., BVP86 with the "scrf=COSMORS" keyword, was used to calculate ideal screening charges on the molecular surfaces. The generated COSMO file was then used as input in the COSMOtherm (version 19.0.1, COSMO-logic, Leverkusen, Germany) package using the BP_TZVP_19 parameterization. In the COSMORS calculations, the lignin molar fraction was set to 0.2, while the solvent molar fraction was set to 0.8, mimicking the experimental pretreatment setup with a biomass-to-IL loading ratio of 1:4 (w / w). The activity coefficient of component i is related to the chemical potential μi and is given by:

number

[0099] In the formula, μi 0 is the chemical potential of the pure component i, R is the real gas constant, and T is the absolute temperature. Details of the COSMO-RS calculation are given in the COSMOtherm user manual. 60

[0100] FT-IR analysis The identity of the Schiff bases and related ILs was established using FT-IR spectroscopy using a Bruker VERTEX 70 / 80 system (Billerica, MA). Data were analyzed using OPUS (version 8.2, build 8, 2, 28 (20190310)) software.

[0101] thermal analysis Thermal behavior was determined under nitrogen (50 mL / min) using a Mettler Toledo Stare TGA / DSC1 unit (Mettler Toledo, Leicester, UK). 3–10 mg samples were placed in alumina crucibles (70 μL) and heated from room temperature to 800 °C at a heating rate of 10 °C / min to obtain thermal decomposition profiles. Similarly, Schiff bases and related ILs were sealed in Hermetic Al pans and heated from room temperature to 250 °C at a heating rate of 10 °C / min to obtain thermal transition profiles. Data were analyzed using STARe Evaluation software.

[0102] HSQC NMR The untreated and pretreated biomass obtained after enzymatic saccharification were mixed in a mixer mill (Qiagen MM300 Mixer, Retsch) using stainless steel balls with a diameter of 2 mm for 30 s. -1 The samples were milled at a mixing frequency of 100 Hz for 15 minutes. The milled material was dispersed in DMSO-d6 and allowed to stand overnight to extract the lignin. 2D heteronuclear single quantum coherence (HSQC) spectra were collected on a Bruker Avance I 800 MHz spectrometer equipped with a TXI probe at 310 K. A standard Bruker pulse sequence (hsqcetgpsisp2.2) was used with the following parameters typical for plant cell wall samples: HSQC spectra were obtained using F2 ( 1 H) dimension from 11 to -1 ppm, collected with 1024 data points for an acquisition time of 53 ms, and F1( 13256 data points were collected for a 3.5 ms acquisition time from 165 to -10 ppm in the F and C dimensions. A total of 256 scans were recorded for each t point with a 1 s pulse delay. The central DMSO solvent peak was used as the reference for chemical shift calibration of all samples (δC 39.5 ppm, δH 2.5 ppm). All HSQC spectra were processed using typical 90° sine-squared apodization in both the F and F dimensions, and contours were integrated with MestreNOVA software (v.14). Peaks were assigned according to published data.

[0103] conclusion In summary, we developed lignin-based renewable Schiff base ILs and investigated their application in the pretreatment of lignocellulosic biomass. It was noted that imines are preferred to be fully protonated rather than in a dynamic equilibrium where protons are transferred from one iminium center to the other. Furthermore, the fully protonated iminium IL 2B most efficiently released glucose (approximately 87%) and xylose (approximately 76%), while experimental data (no significant removal of biopolymer after water washing) and simulation data indicated negligible interaction with biopolymers. Interestingly, HSQC NMR spectra suggested changes in lignin structure after pretreatment with IL 2B, suggesting an interaction between the IL and biopolymers. We emphasize that this study represents an example of many lignin-derived aldehyde and amine combinations that can be designed and applied for various applications, including lignocellulosic biomass pretreatment, with overall reduced environmental and economic impacts. Furthermore, rigorous techno-economic and life cycle modeling is essential to understand the overall impact and optimal applications of new classes of Schiff base ILs.

[0104] While the present invention has been described in conjunction with preferred specific embodiments thereof, it is to be understood that the foregoing description is intended to illustrate, but not limit, the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0105] All patents, patent applications, and publications mentioned herein are incorporated by reference in their entirety.

[0106] While the present invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various modifications may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

Claims

1. 1. A method for disintegrating biomass, comprising: (a) introducing a solvent comprising a Schiff base ionic liquid (SBIL) to biomass, wherein the solvent solubilizes at least a portion of the biomass to form a solubilized biomass mixture.

2. 10. The method of claim 1, further comprising: (b) separating the solubilized biomass from the SBIL; (c) optionally introducing enzymes and / or microorganisms to the solubilized biomass, such that the enzymes and / or microorganisms produce sugars from the solubilized biomass; and (d) optionally separating the sugars from the solubilized biomass.

3. 10. The method of claim 1, further comprising forming the SBIL prior to the introducing step, and optionally forming the solvent by providing the SBIL.

4. 10. The method of claim 1, further comprising ensiling the biomass prior to the introducing step (a).

5. 10. The method of claim 1, wherein the SBIL is derived from or obtained from lignin.

6. A composition comprising: (a) a solvent comprising a Schiff base ionic liquid (SBIL); and (b) biomass.

7. 7. The composition of claim 6, wherein the SBIL comprises a C=N moiety formed by condensation of an aldehyde or ketone with an amine.

8. The composition of claim 7 , wherein the SBIL is an azomethine.

9. 7. The composition of claim 6, wherein the solvent further comprises an ionic liquid (IL) that is not a Schiff base, and / or a deep eutectic solvent (DES).

10. 7. The composition of claim 6, wherein the SBIL comprises vanillin and ethylenediamine.

11. 11. The composition of claim 10, wherein the vanillin and the ethylenediamine have a molar ratio of from about 1:1 to about 2:

1.

12. 7. The composition of claim 6, wherein the biomass comprises biomass from sorghum.

13. 7. The composition of claim 6, comprising 20% ​​by weight biomass and 80% by weight solvent.

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