Amine and volatile alkaline aqueous solvents for biomass pretreatment

Abstract

The present invention provides for a method for increasing a sugar compound or saccharification yield from a biomass, the method comprising: (a) providing a first mixture comprising a solubilized biomass comprising an alkylamine or alkylammonium, or a distillable amine ionic liquid (DAIL), and (b) recovering, removing, or separating at least part of the alkylamine or alkylammonium, or DAIL, from the first mixture in order to separate the at least part of the alkylamine or alkylammonium, or DAIL, from the solubilized biomass or the first mixture; wherein optionally the alkylamine is a sterically unhindered, highly nucleophilic alkylamines.

Description

Attorney Docket: 2024-152-02 Lawrence Berkeley National LaboratoryAmine and Volatile Alkaline Aqueous Solvents for Biomass PretreatmentInventors: Joseph Palasz, Xueli Chen, Anagha Krishnamoorthy, Xihui Kang, Julius Choi, Venkataramana R. Pidatala, Alberto Rodriguez, Ning Sun, Hemant Choudhary, John M.Gladden, Blake A. SimmonsCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Ser. Nos. 63 / 709,270, filed October 18, 2024; 63 / 709,324, filed October 18, 2024; and, 63 / 733,942, filed December 13, 2024, which are hereby incorporated by reference.STATEMENT OF GOVERNMENTAL SUPPORT

[0002] The invention was made with government support under Contract Nos. DE-AC02- 05CH11231 awarded by the U.S. Department of Energy, and Grant No. AWD7196 (12023016) awarded by the U.S. Department of Defense. The government has certain rights in the invention.FIELD OF THE INVENTION

[0003] The present invention is in the field of pretreated biomass and recovering pretreatment solvent.BACKGROUND OF THE INVENTION

[0004] Biofuels and bioproducts derived from sustainable feedstocks are considered a potential solution to address the challenges associated with human population growth. For efficient biofuel production, the biochemical conversion of lignocellulosic biomass has been frequently discussed in terms of process optimization as well as the reaction mechanism of various thermochemical processing (e.g., pretreatment) and biochemical conversion (e.g., enzymatic hydrolysis and fermentation). Current challenges include the maximization of saccharification yields.SUMMARY OF THE INVENTION

[0005] The present invention provides for a method for increasing a sugar compound or saccharification yield from a biomass, the method comprising: (a) providing a first mixture comprising a solubilized biomass comprising an alkylamine or alkylammonium, or aAttorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory distillable amine ionic liquid (DAIL), and (b) recovering, removing, or separating at least part of the alkylamine or alkylammonium, or DAIL, from the first mixture in order to separate the at least part of the alkylamine or alkylammonium, or DAIL, from the solubilized biomass or the first mixture; wherein optionally the alkylamine is a sterically unhindered, highly nucleophilic alkylamines.

[0006] In some embodiments, the method comprises: (a) providing a first mixture comprising a solubilized biomass comprising an alkylamine or alkylammonium, or a distillable amine ionic liquid (DAIL) , and (b) recovering, removing, or separating at least part of the alkylamine or alkylammonium, or DAIL, from the first mixture in order to separate the at least part of the alkylamine or alkylammonium, or DAIL, from the solubilized biomass or the first mixture.

[0007] In some embodiments, the method comprises: (a) providing a first mixture comprising a solubilized biomass comprising an alkylamine or alkylammonium, and (b) recovering, removing, or separating at least part of the alkylamine or alkylammonium from the first mixture in order to separate the at least part of the alkylamine or alkylammonium from the solubilized biomass or the first mixture.

[0008] In some embodiments, the method comprises: (a) providing a first mixture comprising a solubilized biomass comprising a distillable amine ionic liquid (DAIL), and (b) recovering, removing, or separating at least part of the DAIL from the first mixture in order to separate the at least part of the DAIL from the solubilized biomass or the first mixture.

[0009] In some embodiments, the method comprises: (a) providing a first mixture comprising a solubilized biomass comprising an alkylamine, and (b) recovering, removing, or separating at least part of the alkylamine from the first mixture in order to separate the at least part of the alkylamine from the solubilized biomass or the first mixture; wherein the alkylamine is a sterically unhindered, highly nucleophilic alkylamines.

[0010] In some embodiments, the alkylamine, alkylammonium, or DAIL is a primary amine, a secondary amine, or a tertiary amine. In some embodiments, the alkylamine, alkylammonium, or DAIL is sterically unhindered and highly nucleophilic.

[0011] In some embodiments, the providing step (a) comprises introducing a pretreatment (PT) solvent comprising an alkylamine or alkylammonium to a biomass to solubilize theAttorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory biomass to form the first mixture. In some embodiments, the recovering, removing, or separating step (b) comprises recovering, removing, or separating at least part of the PT solvent from the solubilized biomass or the first mixture. In some embodiments, the recovering, removing, or separating step (b) comprises recovering, removing, or separating at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the PT solvent or alkylamine or alkylammonium.

[0012] In some embodiments, the providing step (a) comprises introducing a pretreatment (PT) solvent comprising the distillable amine ionic liquid (DAIL) to a biomass to solubilize the biomass to form the first mixture. In some embodiments, the recovering, removing, or separating step (b) comprises recovering, removing, or separating at least part of the PT solvent from the solubilized biomass or the first mixture. In some embodiments, the recovering, removing, or separating step (b) comprises recovering, removing, or separating at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the PT solvent or alkylamine or alkylammonium.

[0013] In some embodiments, the method further comprises: (c) introducing an enzyme and / or a microbe to the first mixture such that the enzyme and / or microbe produce a sugar from the solubilized biomass. In some embodiments, the method further comprises: (d) the sugar is separated from the first mixture.

[0014] In some embodiments, the recovering step (b) comprises distilling the at least part of the alkylamine or alkylammonium from the first mixture. In some embodiments, the method further comprises: (e) introducing at least part of the alkylamine or alkylammonium separated in the (b) recovering step to the first mixture in step (a). In some embodiments, the method further comprises: (f) introducing more biomass to the first mixture in step (a).

[0015] In some embodiments, the recovering step (b) comprises distilling the at least part of the DAIL from the first mixture. In some embodiments, the method further comprises: (e) introducing at least part of the DAIL separated in the (b) recovering step to the first mixture in step (a). In some embodiments, the method further comprises: (f) introducing more biomass to the first mixture in step (a).

[0016] In some embodiments, the DAIL comprises an alkanolammonium or alkylammonium cation, and an anion, such as a carboxylic acid. In some embodiments, the carboxylic acid is an alkanoic acid, such as a formate, acetate, or the like. In some embodiments, the alkanoicAttorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory acid has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. In some embodiments, the alkanolammonium is an ethanol ammonium. In some embodiments, the alkylammonium is a butylammonium. In some embodiments, the DAIL comprises ethanolammonium acetate and / or butyl ammonium acetate.

[0017] In some embodiments, the alkylamine has the following chemical structure:RiR2 — N — Rs (I). In some embodiments, the alkylamine is any alkylamine described herein wherein the nitrogen atom is protonated. In some embodiments, the alkylamine is an alkylammonium having the following chemical structure:RiR2— N — R3 (II);H

[0018] In some embodiments, Ri, R2, and R3 are each independently — H, or — (CH2)n — CH3, and n is 0, 1, 2, 3, or 4, and at least one of Ri, R2, and R3 comprises one carbon atom. In some embodiments, the alkylamine comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 14, or 15 carbon atoms. In some embodiments, the alkylamine comprises 1, 2, 3, 4, 5, or 6 carbon atoms. In some embodiments, the alkylamine is methylamine, ethylamine, propylamine, butylamine (w-butylamine), dimethylamine, diethylamine, dipropylamine, trimethylamine, triethylamine, tripropylamine, or the like. In some embodiments, the alkylammonium is methylammonium, ethylammonium, propylammonium, butylammonium (n- butylammonium), dimethylammonium, diethylammonium, dipropylammonium, trimethylammonium, triethylammonium, tripropylammonium, or the like. In some embodiments, the alkylammonium is a protonated form of any alkylamine depicted in Figure 1.5.

[0019] In some embodiments, the alkylammonium has the following chemical structure:RiR2— N — R3 (II);H

[0020] In some embodiments, Ri, R2, and R3 are each independently — H, or — (CH2)n —Attorney Docket: 2024-152-02 Lawrence Berkeley National LaboratoryCH3, and n is 0, 1, 2, 3, or 4, and at least one of Ri, R2, and R3 comprises one carbon atom. In some embodiments, the alkylammonium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 14, or 15 carbon atoms. In some embodiments, the alkylammonium comprises 1, 2, 3, 4, 5, or 6 carbon atoms. In some embodiments, the alkylammonium is methylammonium, ethylammonium, propyl ammonium, butylammonium (w-butylammonium), dimethylammonium, diethylammonium, dipropylammonium, trimethylammonium, triethylammonium, tripropylammonium, or the like. In some embodiments, the alkylammonium is a protonated form of any alkylamine depicted in Figure 1.5.

[0021] In some embodiments, the alkylamine or alkylammonium comprises one or more hydroxyl functional groups. In some embodiments, the alkylamine or alkylammonium comprises 1, 2, 3, 4, 5, or 6 hydroxyl functional groups. In some embodiments, the alkylamine is methanolamine (aminomethanol), ethanolamine (2-aminoethanol), dimethanolamine (DM0 A), diethanolamine (DEO A), trimethanolamine (TMOA), triethanolamine (TEO A), or the like.

[0022] In some embodiments, the alkanolammonium has the chemical structure (I) and comprising one or more hydroxyl functional groups. In some embodiments, the alkanolammonium comprises 1, 2, 3, 4, 5, or 6 hydroxyl functional groups. In some embodiments, the alkanolammonium is methanolammonium, ethanol ammonium, dimethanolammonium, diethanolammonium, trimethanolammonium, triethanolammonium, or the like.

[0023] In some embodiments, the providing step comprises adding a pretreatment (PT) solvent to a biomass. In some embodiments, the PT solvent comprises an ionic liquid, deep eutectic solution (DES) solvent, or any other solvent described in a reference incorporated by reference. In some embodiments, the PT solvent is a distillable protic ionic liquid (PIL) or any other PT solvent that can be recovered through distillation, such as an alkanolamine or alkanolammonium.

[0024] In some embodiments, the providing step comprises solubilizing, or deconstructing, or pretreating a biomass to obtain or release lignin from the biomass, prior to the (a) providing and (b) contacting steps. In some embodiments, the solubilizing, or deconstructing, or pretreating comprises contacting the biomass with an ionic liquid (IL) or a (DES).

[0025] In some embodiments, the water added or introduced is equal to about 50%, 60%,Attorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150% volume (or weight) of the volume (or weight) of PT solvent in the PT biomass, or a value within the range of any two preceding values. The glucose and / or xylose yield is about any of the values described herein.

[0026] In some embodiments, the method further comprises: (d) optionally introducing an enzyme to break down or depolymerize the cellulosic portion, including cellulose, hemicellulose, or a mixture thereof, of the solubilized, or deconstructed, or pretreated biomass, or biomass from which lignin has been obtained or released, into one or more sugar monomers, such as glucose, xylose, or a mixture thereof, and (e) introducing a microbe to the solubilized, or deconstructed, or pretreated biomass, or biomass from which lignin has been obtained or released, such that the microbe utilizes the cellulosic portion of the solubilized, or deconstructed, or pretreated biomass, or biomass from which lignin has been obtained or released, and / or one or more sugar monomers, as a carbon source to produces a biofuel or bioproduct (or chemical compound).

[0027] The present invention provides for 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. 2020 / 0216863, hereby incorporated by reference in its entirety.

[0028] In some embodiments, the method is a one-pot method, which does not require any solid-liquid separation step. In some embodiments, the one-pot method does not require adjustment of the pH level in the one-pot composition. In some embodiments, the one-pot method does not require any dilution, or addition of water or medium. In some embodiments, the growth of the microbe occurs in the same one-pot composition. In some embodiments, the IL, or mixture thereof, is renewable as it can be continuous in use.

[0029] In some embodiments, the method comprises a process in which water is added to a pretreated biomass slurry. In some embodiments, the biomass pretreatment involves a distillable protic ionic liquid or distillable solvent, such as alkanolamines. In some embodiments, the water is added to the pretreated slurry before the slurry is fed into a solvent recovery unit. The presence of water stabilizes the biomass components present and prevents them from undergoing unwanted chemical and physical changes during the solvent recovery process. The addition of water promotes higher sugar yields from a subsequent saccharification step that liberates glucose and xylose from the biomass. The addition ofAttorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory water is counterintuitive and is critical in terms of enabling a biomass deconstruction technology that enables both efficient solvent recovery and recycle and high yields of fermentable sugars.

[0030] In some embodiments, the PT solvent is any solvent, such as a distillable solvent, taught in PCT International Application No. PCT / US2021 / 18630 and U.S. Patent Application Publication No. 2023 / 0078811 (“Use of distillable volatile salt for the pretreatment of biomass”), and PCT International Application No. PCT / US2021 / 30023 and U.S. Patent Application Publication No. 2023 / 022921 (“Use of alkanolamines for lignin extraction in the pretreatment of biomass”), all of which are hereby incorporated by reference.

[0031] In some embodiments, the method comprises a process by which water is added to a pretreated slurry before the slurry is introduced into a solvent recovery unit. The solvents used for biomass pretreatment must be distillable, meaning they can be efficiently recovered using a wide range of recovery technologies such as thin film evaporators (vertical or horizontal), wiped film evaporators, flash tanks, rotary evaporators, distillation columns, vertical or horizontal evaporators, conical evaporators. These all involve elevated temperatures and vacuum. Without the addition of water then biomass components undergo unwanted physical and chemical transformations that have a negative impact on sugar yields obtained when enzymes are added to the pretreated slurry. The addition of water prevents these undesired transformations and enable significantly higher sugar yields. Pretreatment solvents are defined as distillable protic ionic liquids and any other pretreatment solvent that can be recovered through distillation, such as alkanolamines or alkanolammoniums.

[0032] In some embodiments, the alkylamine or alkanol ammonium is distillable. In some embodiments, the method lacks any washing step. In some embodiments, the biomass is a single or mixed biomass.

[0033] In some embodiments, pretreatment is carried out at about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% solid loading, or any percentage within a range of value between any two preceding values. In some embodiments, pretreatment is carried out at about 100 °C, 110 °C, 120 °C, 130 °C, 140 °C, 150 °C, 160 °C, 170 °C, or 180 °C, or any temperature with a range of value between any two preceding values. In some embodiments, pretreatment is carried out for about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0 hours, or any period of time within a range of value between any two preceding values.Attorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory

[0034] In some embodiments, the method results in a glucose yield of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or any percentage within a range of value between any two preceding values. In some embodiments, the method results in a xylose yield of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or any percentage within a range of value between any two preceding values.

[0035] This invention provides for distillable amine-based solvents for use in economic biomass pretreatment. This approach enables the development of a feedstock agnostic pretreatment technology which is more applicable than conventional approaches that generally require complex and expensive washing processes, due to its potential for efficient solvent recovery. These solvents exhibit effective pretreatment of single as well as mixed lignocellulosic feedstock samples and types, in small pressure tubes to large capacity reactors to process for sugar release at different scales. Therefore, the innovation opens avenues for a new solvent group to be considered for use within commercial biorefineries for biomass pretreatment.

[0036] This invention provides for distillable ammonium-based solvents for use in economic biomass pretreatment. This approach enables the development of a feedstock agnostic pretreatment technology which is more applicable than conventional approaches that generally require complex and expensive washing processes, due to its potential for efficient solvent recovery. These solvents exhibit effective pretreatment of single as well as mixed lignocellulosic feedstock samples and types, in small pressure tubes to large capacity reactors to process for sugar release at different scales. Therefore, the innovation opens avenues for a new solvent group to be considered for use within commercial biorefineries for biomass pretreatment.

[0037] The present invention provides for a novel group of solvents for use in single and mixed feedstock agnostic biomass pretreatment. Suitable distillable amine-based or ammonium-based solvents, such as ethanolamine (EA), butylamine (BA) and triethylamine (TEA), are used for initial screening of the deconstruction of a wide range of lignocellulosic feedstocks that are representative of different environments such as agricultural (rice hulls, hay, sorghum), temperate (hardwood sawdust), tropical (palm oil fiber, coconut husks / fibers), and the like, into simple fermentable sugars.

[0038] In some embodiments, pretreatment is carried out at 15% solid loading at 140 °C for 3Attorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory hours. In some embodiments, solvent recovery comprises evaporation in a vacuum oven set at a temperature about 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, 90 °C, or 100 °C, 30 °C, 40 °C, or any temperature within a range of value between any two preceding values. In some embodiments, the temperature is set at about 80 °C, 40 °C, and 40 °C for EA, BA and TEA, respectively. In some embodiments, solvent recovery is followed by pH adjustment to about 4.0 pH, 4.5 pH, 5.0 pH, 5.5 pH, or 6.0 pH, or any pH within a range of value between any two preceding values. In some embodiments, solvent recovery and / or pH adjustment is followed by enzymatic hydrolysis using about 30 mg protein / g biomass with a suitable enzyme or mixture of enzymes, such as Cellic® CTec3:HTec3 (9:1) (Novozymes A / S, Bagsvserd, Denmark).

[0039] Preliminary results in pressure tubes reveal triethylamine to show best solvent removal capabilities with poor sugar release; however, butylamine showed excellent solvent removal with consistently higher sugar yields. Butylamine is chosen as the pretreatment solvent for the deconstruction of 22 different lignocellulosic feedstocks, followed by saccharification. The results reveal significantly higher sugar release among different biomass types, such as grasses and woody biomass, with over about 95% solvent removal. Following this, a mixed feedstock blend of agricultural residues consisting of sorghum, com stover, wheat straw and hay was pretreated with butylamine at bench-scale pressure tubes, 75 mL multi-Parr reactor and 1 L Parr reactor scales, with initial scale up revealing over about 90% glucose and over about 80% xylose yields with over about 99% solvent removal in the 1 L Parr scale. These results reveal the potential of amine-based solvents for single and mixed feedstock agnostic pretreatment for subsequent biofuel production.

[0040] A mixed feedstock blend of agricultural residues consisting of sorghum, corn stover, wheat straw and hay was pretreated with butylamine at bench-scale pressure tubes, 75 mL multi-Parr reactor and 1 L Parr reactor scales, with initial scale up revealing over about 90% glucose and over about 80% xylose yields with over about 99% solvent removal in the 1 L Parr scale. These results reveal the potential of amine-based solvents for single and mixed feedstock agnostic pretreatment for subsequent biofuel production.

[0041] The potential use of amine-based solvents would be as good pretreatment agents in refineries and industries that convert biomass waste such as agricultural residues, wood / paper / pulp, grasses into biofuels and / or sustainable aviation fuels and bioproducts.Attorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory

[0042] In some embodiments, the method provides for the use of amine-based solvents for single and mixed feedstock agnostic pretreatment offers several economic advantages. In some embodiments, the method has one or more of the following advantages: (1) The solvent is relatively easy to be removed by simple vacuum distillation, and also serve as cost- effective, multiuse solvents. (2) The method enables effective deconstruction of different types of biomasses such as sorghum, wheat straw, hardwood sawdust, poplar, or the like, irrespective of environment bias. (3) They serve as good pretreatment agents to breakdown single as well as mixed feedstock blends into simple sugars, a critical challenge of most other pretreatment technologies. (4) the method is affordable and scalable. (5) The method does not require any specialized process equipment. (6) The method has potential for scaling up from bench scale to larger scales.

[0043] The present invention also provides for a method for the pretreatment of lignocellulosic biomass substrates with an aqueous volatile base (AVB). In some embodiments, the methods allows for the efficient release of monosaccharides by cellulase and hemicellulose enzymes. In some embodiments, the PT solvent comprises one or more aqueous volatile base (AVB). In some embodiments, the AVB is an alkylamine that is in a gaseous state at room temperature (such as 25 °C). In some embodiments, the alkylamine has 1, 2, 3, 4, 5, or 6 carbon atoms. In some embodiments, the AVB has the chemical structure (I) having 1, 2, 3, 4, 5, or 6 carbon atoms. Suitable AVB include, but are not limited to, methylamine, ethylamine, propylamine, butylamine, dimethylamine, diethylamine, trimethylamine, and diisopropylamine.

[0044] In some embodiments, the AVB is aqueous, such as miscible amines, such as butyl amine, trimethyl amine, diisopropyl amine, propyl amine, pentyl amine, dimethyl amine, secbutyl amine, and isobutyl amine. Some amines which show poor performance in neat solution, however demonstrate improved performance in aqueous solution, which can be attributed to the interception of the amines nucleophilic activity with that of water.

[0045] As these volatile amines can be removed by distillation, this allows us to recover the active reagent in the pretreatment process via a distillation or vacuum distillation process, allowing for viable closed solvent loop processing designs where a quantity of solvent is continuously used to pretreat large quantities of biomass, with minimal solvent losses as a result of the pretreatment.Attorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory

[0046] Biomass pretreatment is an essential step in the production of biofuels and other products from non-food biomass sources like lignocellulose. The utilization of volatile bases (butylamine, trimethylamine, diisopropylamine, or the like) for pretreatment enables a closed-loop solvent recovery scheme which can minimize the process costs and environmental hazards associated with chemical biomass treatment steps. The pretreatment under these conditions has demonstrated excellent sugar releases (over about 90% glucose, over about 75% xylose yields for hybrid poplar with aqueous butylamine) and has proven effective even with low base concentration (about 6% by mass in water) and at modest temperatures (about 80 °C).

[0047] Biomass pretreatment is a necessary step for gaining access to the sugars contained in the lignocellulose structure. With an effective chemical or physical pretreatment process, high yields of monosaccharides can be extracted from arbitrary sources of lignocellulose, including potential bioenergy sources such as corn, sorghum, switchgrass, rice, miscanthus, and the like.

[0048] This extracted sugar can then be transformed through microbial fermentations into a variety of useful molecules which could be valuable feedstocks or fuel components. Alkaline chemical pretreatment has proven to be a widely versatile approach to treating biomass of many types, as it selectively unwraps the complex outer structure of lignocellulose fibers, dissolves lignin, and renders the internal polysaccharide core accessible for enzymatic digestion. Previously explored alkaline pretreatment approaches utilized aqueous bases such as sodium or ammonium hydroxide, or exposure to high pressure gas mixtures, such as supercritical liquid ammonia. The issues with those approaches for biomass pretreatment were in the high process costs associated with regenerating NaOH solution, or the ammonia gas.

[0049] By selecting alkylamines which can provide similar reactivities to NaOH and ammonia, the same effect of biomass pretreatment can be achieved. When sufficiently alkaline amines are selected, the alkylamine pretreatment performance can be maintained even in aqueous solution. When butylamine is used as the base for pretreatment of hybrid poplar, the degree of sugar release does not drop significantly (that is, below 85% glucose yield) until concentrations are as low as 6% by mass in water. The use of high percentages of water substantially improves both cost and safety parameters for any large scale iterations of this process.Attorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory

[0050] In some embodiments, the method has one or more of the following advantages: (1) removing the need for using a highly flammable organic solvent at 100% concentration, (2) replacing the majority of the solvent mixture with water which is a much more environmentally, and chemically benign solvent, (3) reducing the concentration of caustic alkaline base needed for pretreatment, and (4) diverting many chemical side reactions in the pretreatment towards hydrolysis reactions which generate potentially fewer inhibitory molecules, and thus benefitting downstream enzymatic hydrolysis and fermentation steps of biofuel and bioproduct processes.

[0051] This process has been demonstrated in the laboratory in 60 mL glass pressure vessels as well as in 75 mL multi-Parr reactors. The solvent removal has been demonstrated for all tests resulting in high removal (over about 95%) in all tests run. The process can be scaled under similar conditions to be run in 1 L parr reactors.

[0052] In some embodiments, the process has one or more advantages over previously explored process conditions with similar approaches. It would be likely that this approach would be the favorable way to treat bioenergy crops for the production of most biofuels and bioproducts from lignocellulose biomass streams. This process may help lower the cost of sugar production from lignocellulose biomass, which can either be purified and sold as a calorie stream, or further upgraded using microbial fermentation into a variety of high-value bioproducts.

[0053] The potential use of ammonium-based solvents would be as good pretreatment agents in refineries and industries that convert biomass waste such as agricultural residues, wood / paper / pulp, grasses into biofuels and / or sustainable aviation fuels and bioproducts.

[0054] This invention provides for a method using distillable ionic liquids in economic and sustainable biomass pretreatment. This approach enables the development of a feedstock agnostic pretreatment technology which is more applicable than conventional approaches that generally require complex and expensive washing processes, due to its potential for efficient solvent recovery. These solvents exhibit effective pretreatment of diverse lignocellulosic feedstock samples and types, in glass pressure tubes to process for sugar release. Therefore, the innovation opens avenues for a new solvent group to be considered for use within commercial biorefineries for biomass pretreatment.

[0055] In some embodiments, the method comprises using a novel group of solvents for useAttorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory in feedstock agnostic biomass pretreatment. Distillable ionic liquids, ethanolammonium acetate and butylammonium acetate are used for examining the deconstruction of a wide range of lignocellulosic feedstocks that are representative of different environments such as agricultural (rice hulls, hay, sorghum), temperate (hardwood sawdust), tropical (palm oil fiber, coconut husks / fibers), and the like, into simple fermentable sugars. In some embodiments, the pretreatment is carried out at 15% with a solid loading at about 140 °C for about 3 hours, and for solvent recovery, evaporation was performed in a vacuum oven set about 140 °C and 120 °C for ethanolammonium acetate and butylammonium acetate, respectively. In some embodiments, solvent recovery is, followed by pH adjustment to about 5.0, and subsequent enzymatic hydrolysis using about 30 mg protein / g biomass with Cellic® CTec3:HTec3 (9: 1) (Novozymes A / S, Bagsvserd, Denmark).

[0056] Preliminary results in glass pressure tubes show that both ethanol ammonium acetate and butylammonium acetate achieve superior sugar yields across various agricultural residues (hay, rice hulls, and sorghum), temperate (hardwood sawdust), tropical (palm oil fiber) feedstocks.

[0057] Over about 95% solvent removal is observed, except in the ethanolammonium acetate pretreatment of sorghum. Ethanolammonium acetate resulted in glucose yields exceeding about 85% for hay and palm oil fiber, and over about 70% for hardwood sawdust, with xylose yields above about 75%, 80%, and 60% respectively. Butylammonium acetate produced much higher sugar yields for sorghum, with glucose yields surpassing about 90% and xylose yields exceeding about 85%. These findings highlight the potential of distillable ionic liquids for feedstock agnostic pretreatment for the sustainable production of biofuels and bioproducts.

[0058] The potential use of distillable ionic liquids would be as good pretreatment agents in refineries and industries that convert biomass waste such as agricultural residues, wood / paper / pulp, grasses into biofuels and / or sustainable aviation fuels and bioproducts.

[0059] The present invention provides for using a distillable ionic liquid for feedstock agnostic pretreatment that offers several economic advantages; however, the key differences from the current state-of-art are described herein. In some embodiments, the method has one or more of the following advantages: (1) They are relatively easy to be removed by simple vacuum distillation, and also serve as cost-effective, multiuse solvents. (2) The solvents canAttorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory be recycled and reused. (3) They enable effective deconstruction of different types of biomasses such as sorghum, hardwood sawdust, palm oil fiber, etc. irrespective of environment bias. (4) The method is affordable and scalable. (5) The method does not require any specialized process equipment.BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The foregoing aspects and others will be readily appreciated by the skilled artisan from the following description of illustrative embodiments when read in conjunction with the accompanying drawings.

[0061] Figure 1.1. Initial screening with 8 different feedstocks and 3 amine-based solvents.

[0062] Figure 1.2. Screening of 22 different single feedstocks using butylamine as a pretreatment solvent.

[0063] Figure 1.3. Scaling up deconstruction to 1 L.

[0064] Figure 1.4. Initial scale-up of mixed agriculture residues improves performance.

[0065] Figure 1.5. Structure of suitable alkylamines.

[0066] Figure 2.1 shows a scheme for solvent recovery.

[0067] Figure 2.2 shows a scheme for the saccharification of a biomass. .

[0068] Figure 2.3 shows the sugar yield and solvent recovery using different amine solvents. Feedstock biomass tested are: 1. Palm oil fiber; 2. Coconut chips; 3. Hardwood sawdust-1; 4. Hardwood sawdust-3; 5. Hay; 6. Rice hulls; 7. 4-Crop mix pellets; 8. Sorghum.

[0069] Figure 2.4 shows the pretreatment properties of 22 feedstock biomass.

[0070] Figure 2.5 shows the correlation with the various pretreatment properties. * indicates a significant correlation between parameters (p < 0.05).

[0071] Figure 2.6 shows blending of feedstocks (A) and sugar yields and solvent removal for different feedstocks (B).

[0072] Figure 3.1. Examination with eight different feedstocks and two distillable ionicAttorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory liquids.

[0073] Figure 4.1. Solvents removal efficiency (A) and the sugar yields from untreated and pretreated corn. Stover with dimethylamine (40% aq. solution) and 1 -pentylamine (amylamine, neat) (B).

[0074] Figure 4.2. DMA (40% aq.) pretreatment of agriculture blend.

[0075] Figure 4.3. HSQC of DMA PT agriculture blend.DETAILED DESCRIPTION OF THE INVENTION

[0076] Before the invention is described 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 such may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting.

[0077] 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:

[0078] The terms "optional" or "optionally" as used herein mean that the subsequently described feature or structure may or may not be present, or that the subsequently described event or circumstance may or may not occur, and that the description includes instances where a particular feature or structure is present and instances where the feature or structure is absent, or instances where the event or circumstance occurs and instances where it does not.

[0079] The term “about” when applied to a value, describes a value that includes up to 10% more than the value described, and up to 10% less than the value described.

[0080] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value 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 independently be included or excluded in the range,Attorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory and each range where either, neither or both limits are included in the smaller ranges 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 invention.

[0081] In some embodiments, the pretreatment solvent comprises one or more of the following: ionic liquid, deep eutectic solvent (DES), and the like.IONIC LIQUID

[0082] 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 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, Wis.). Such suitable ILs include, but are not limited to, 1 -alkyl-3 - alkylimidazolium alkanate, 1 -alkyl-3 -alkylimidazolium alkylsulfate, l-alkyl-3- alkylimidazolium methyl sulfonate, 1 -alkyl-3 -alkylimidazolium hydrogensulfate, 1 -alkyl-3- alkylimidazolium thiocyanate, and 1 -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.

[0083] In some embodiments, the IL includes, but is not limited to, l-ethyl-3- methylimidazolium acetate (EMIN Acetate), l-ethyl-3-methylimidazolium chloride (EMIN Cl), l-ethyl-3-methylimidazolium hydrogensulfate (EMIM HOSO3), l-ethyl-3- methylimidazolium methylsulfate (EMIM MeOSCh), l-ethyl-3-methylimidazolium ethylsulfate (EMIM EtOSCh), l-ethyl-3-methylimidazolium methanesulfonate (EMIM MeSCh), l-ethyl-3-methylimidazolium tetrachloroaluminate (EMIM A1CL), l-ethyl-3- methylimidazolium thiocyanate (EMIM SCN), l-butyl-3-methylimidazolium acetate (BMIM Acetate), l-butyl-3-methylimidazolium chloride (BMIM Cl), l-butyl-3-methylimidazoliumAttorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory hydrogensulfate (BMIM HOSO3), l-butyl-3-methylimidazolium methanesulfonate (BMIM MeSCh), l-butyl-3-methylimidazolium methylsulfate (BMIM MeOSCh), l-butyl-3- methylimidazolium tetrachloroaluminate (BMIM A1C14), l-butyl-3-methylimidazolium thiocyanate (BMIM SCN), l-ethyl-2,3-dimethylimidazolium ethylsulfate (EDIM EtOSCh), Tris(2-hydroxyethyl)methylammonium methylsulfate (MTEOA MeOSCh), 1- methylimidazolium chloride (MIM Cl), 1-methylimidazolium hydrogensulfate (MIM HOSO3), 1,2,4-trimethylpyrazolium methylsulfate, tributylmethylammonium methyl sulfate, choline acetate, choline salicylate, and the like.

[0084] In some embodiments, the ionic liquid is a chloride ionic liquid. In other embodiments, the ionic liquid is an imidazolium salt. In still other embodiments, the ionic liquid is a 1 -alkyl-3 -imidazolium chloride, such as l-ethyl-3-methylimidazolium chloride or l-butyl-3-methylimidazolium chloride.

[0085] In some embodiments, the ionic liquids used in the invention are pyridinium salts, pyridazinium salts, pyrimidium 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 the ionic liquid include, but are not limited to halogens (e.g., chloride, fluoride, bromide and iodide), pseudohalogens (e.g., azide and isocyanate), alkyl carboxylate, sulfonate, acetate and alkyl phosphate.

[0086] 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, PCT International Patent Application Nos.PCT / US2015 / 058472, PCT / US2016 / 063694, PCT / US2017 / 067737, and PCT / US2017 / 036438 (all of which are incorporated in their entireties 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. The ionic liquid can comprise one or a mixture of the compounds.

[0087] In some embodiments, the IL is a protic ionic liquid (PIL). Suitable protic ionic liquids (PILs) include fused salts with a melting point less than 100°C with salts that have higher melting points referred to as molten salts. Suitable PPILs are disclosed in Greaves etAttorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory al. “Protic Ionic Liquids: Properties and Applications” Chem. Rev. 108(l):206-237 (2008). PILs can be prepared by the neutralization reaction of certain Bronsted acids and Bronsted bases (generally from primary, secondary or tertiary amines, which are alkaline) and the fundamental feature of these kinds of ILs is that their cations have at least one available proton to form hydrogen bond with anions. In some embodiments, the protic ionic liquids (PILs) are formed from the combination of organic ammonium-based cations and organic carboxylic acid-based anions. 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 comprises a straight or branched Cl, C2, C3, C4, C5, C6, C7, C8, C9, or CIO chain. In some embodiments, the carboxylate comprises a straight or branched Cl, C2, C3, C4, C5, C6, C7, C8, C9, or CIO chain. In some embodiments, the carboxylate is substituted with one or more hydroxyl groups. In some embodiments, the PIL is a hydroxyethylammonium acetate.

[0088] In some embodiments, the protic ionic liquid (PIL) is disclosed by U.S. Patent Application Publication No. 2004 / 0097755, hereby incorporated by reference.

[0089] Suitable salts for the method include combinations of organic ammonium-based cations (such as ammonium, hydroxyalkylammonium, or dimethylalkylammonium) with organic carboxylic acid-based anions (such as acetic acid derivatives (C1-C8), lactic acid, glycolic acid, and DESs such as ammonium acetate / lactic acid).

[0090] Suitable IL, such as distillable IL, 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 hereby incorporated by reference.

[0091] Suitable PIL, such as distillable PIL, are disclosed in Idris et al. “Distillable Protic Ionic Liquids for Keratin Dissolution and Recovery”, ACS Sustainable Chem. Engin. 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 hereby incorporated by reference.

[0092] In some embodiments, the PILs are formed with the combination of organicAttorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory ammonium-based cations and organic carboxylic acid-based anions. PILs are acid-base conjugate ILs that can be synthesized via the direct addition of their acid and base precursors. Additionally, when sufficient energy is employed, they can dissociate back into their neutral acid and base precursors, while the PILs are re-formed upon cooling. This presents a suitable way to recover and recycle the ILs after their application. In some embodiments, the PIL (such as hydroxyethylammonium acetate - [Eth][OAc]) is an effective solvent for biomass pretreatment and is also relatively cheap due to its ease of synthesis (Sun et al., Green Chem. 19(13):3152-3163 (2017)).DEEP EUTECTIC SOLVENT (DES)

[0093] DESs are systems formed from a eutectic mixture of Lewis or Bronsted acids and bases which can contain a variety of anionic and / or cationic species. DESs can form a eutectic point in a two-component phase system. DESs are formed by complexation of quaternary ammonium salts (such as, choline chloride) with hydrogen bond donors (HBD) such as amines, amides, alcohols, or carboxylic acids. The interaction of the HBD with the quaternary salt reduces the anion-cation electrostatic force, thus decreasing the melting point of the mixture. DESs share many features of conventional ionic liquid (IL), and promising applications would be in biomass processing, electrochemistry, and the like. In some embodiments, the DES is any combination of Lewis or Bronsted acid and base. In some embodiments, the Lewis or Bronsted acid and base combination used is distillable.

[0094] In some embodiments, DES is prepared using an alcohol (such as glycerol or ethylene glycol), amines (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 phenol mixture readily form DES upon heating at 100 °C with specific molar ratio with choline chloride. This class of DES does not require a multistep synthesis. The DES is synthesized from lignin which is a renewable source.

[0095] Both monomeric phenols and phenol mixture can be used to prepare DES. DES is capable of dissolving biomass or lignin, and can be utilized in biomass pretreatment and other applications. Using DES produced from biomass could lower the cost of biomass processing and enable greener routes for a variety of industrially relevant processes.

[0096] The DES, or mixture thereof, is bio-compatible: meaning the DES, or mixture thereof, does not reduce or does not significantly reduce the enzymatic activity of the enzyme, and / orAttorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory is not toxic, and / or does not reduce or significantly reduce, the growth of the microbe. A “significant” reduction is a reduction to 70, 80, 90, or 95% or less of the enzyme’s enzymatic activity and / or the microbe’s growth (or doubling time), if the DES, or mixture thereof, was not present.

[0097] 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 choline chloride.

[0098] In some embodiments, the DES is distillable if the DES can be recovered at least equal to or more than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% yield by distilling over vacuum at a temperature at about 100 °C, 110 °C, 120 °C, 130 °C, 140 °C, 150 °C, or 160 °C, or any temperature between any two of the preceding temperatures.

[0099] In some embodiments, the DES can be one taught in WO 2018 / 204424 (Seema Singh et al.), which is hereby incorporated in its entirety by reference.

[0100] In some embodiments, the method further comprises heating the one-pot composition, optionally also comprising the enzyme and / or microbe, to a temperature that is equal to, about, or near the optimum temperature for the enzymatic activity of the enzyme and / or growth of the microbe. In some embodiments, the enzyme is a genetically modified host cell capable of converting the cellulose in the biomass into a sugar. In some embodiments, there is a plurality of enzymes. In some embodiments, the microbe is a genetically modified host cell capable of converting a sugar produced from the biomass into a biofuel and / or chemical compound. In some embodiments, there is a plurality of microbes. In some embodiments, the introducing step(s) produce a sugar and a lignin from the biomass. The lignin can further be processed to produce a DES. The sugar is used for growth by the microbe.Attorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory

[0101] In some embodiments, the solubilizing is full, near full (such as at least about 70, 80, or 90%), or partial (such as at least about 10, 20, 30, 40, 50, or 60%). In some embodiments, the one-pot composition is a slurry. When the steps described herein are continuous, the one-pot composition is in a steady state.

[0102] In some embodiments, the introducing step comprises heating the mixture comprises increasing the temperature of the solution to a value within a range of about 75 °C to about 125 °C. In some embodiments, the heating step comprises increasing the temperature of the solution to a value within a range of about 80 °C to about 120 °C. In some embodiments, the heating step comprises increasing the temperature of the solution to a value within a range of about 90 °C to about 110 °C. In some embodiments, the heating step comprises increasing the temperature of the solution to about 100 °C.ENZYME

[0103] 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 PCT International Patent Application Nos. PCT / US2015 / 000320, PCT / US2016 / 063198, PCT / US2017 / 036438, PCT / US2010 / 032320, and PCT / US2012 / 036007 (all of which are incorporated in their entireties by reference).MICROBE

[0104] In some embodiments, the microbe is any prokaryotic or eukaryotic cell, with any genetic modifications, taught 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 PCT International Patent Application Nos. PCT / US14 / 48293, PCT / US2018 / 049609, PCT / US2017 / 036168, PCT / US2018 / 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 in their entireties by reference).

[0105] Generally, although not necessarily, the microbe is a yeast or a bacterium. In some embodiments, the microbe is Rhodosporidium toruloides o Pseudomonas putida. InAttorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory some embodiments, the microbe is a Gram-negative bacterium. In some embodiments, the microbe is of the phylum Proteobactera. In some embodiments, the microbe is of the class Gammaproteobacteria. In some embodiments, the microbe is of the order Enterobacteriales. In some embodiments, the microbe is of the family Enterobacteriaceae. Examples of suitable bacteria include, without limitation, those species assigned to the Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, and Paracoccus taxonomical classes. Suitable eukaryotic microbes include, but are not limited to, fungal cells. Suitable fungal cells are yeast cells, such as yeast cells of the Saccharomyces genus.

[0106] Yeasts suitable for the invention include, but are not limited to, Yarrowia, Candida, Bebaromyces, Saccharomyces, Schizosaccharomyces and Pichia cells. In some embodiments, the yeast is Saccharomyces cerevisae. In some embodiments, the yeast is a species of Candida, 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.

[0107] In some embodiments the microbe is a bacterium. Bacterial host cells suitable for the invention include, but are not limited to, Escherichia, Corynebacterium, Pseudomonas, Streptomyces, and Bacillus. In some embodiments, the Escherichia cell is an 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 matruchotii, Corynebacterium minutissimum, Corynebacterium renale, Corynebacterium striatum, Corynebacterium ulcerans, Corynebacterium urealyticum, or Corynebacterium uropygiale. In some embodiments, the Pseudomonas cell is a putida, P. aeruginosa, P. chlororaphis, P. fluorescens, P. pertucinogena, P. stutzeri, P. syringae, P. cremoricolorata, P. entomophila, P. fulva, P.Attorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory monteilii, P. mosselii, P. oryzihabitans, P. parafluva, or P. plecoglossicida. In some embodiments, the Streptomyces cell is a S. coelicolor, S. lividans, S. venezuelae, S. ambofaciens, S. avermitilis, S. albus. or S. scabies. In some embodiments, the Bacillus cell is a B. subtilis, B. megalerium. B. Ucheniformis. B. anlhracis. B. amyloliquefaciens, or B. pumilus.BIOFUEL

[0108] In some embodiments, the biofuel produced is ethanol, or any other organic molecule, described produced in a cell taught 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 PCT International Patent Application Nos. PCT / US 14 / 48293, PCT / US2018 / 049609, PCT / US2017 / 036168, PCT / US2018 / 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 in their entireties by reference).BIOMASS

[0109] The biomass can be obtained from one or more feedstock, such as softwood feedstock, hardwood feedstock, grass feedstock, and / or agricultural feedstock, or a mixture thereof.

[0110] Softwood feedstocks include, but are not limited to, Araucaria (e.g. A. cunninghamii, A. angustifolia, A. araucana); softwood Cedar (e.g. Juniperus virginiana, Thuja plicata, Thuja occidentalis, Chamaecyparis thyoides Callitropsis nootkatensis); Cypress (e.g. Chamaecyparis, Cupressus Taxodium, Cupressus arizonica, 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; Kaya; Larch (e.g. Larix decidua, Larix kaempferi, Larix laricina, Larix occidentalis); Pine (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 echinata); Redwood; Rimu; Spruce (e.g. Picea abies, Picea mariana, Picea rubens, Picea sitchensis, Picea glauca); Sugi; and combinations / hybridsAttorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory thereof.

[0111] For example, softwood feedstocks which may be used herein include cedar; fir; pine; spruce; and combinations thereof. The softwood feedstocks for the present invention may be selected from loblolly pine (Pinus taeda), radiata pine, jack pine, spruce (e.g., white, interior, black), Douglas fir, Pinus silvestris, Picea abies, and combinations / hybrids thereof. The softwood feedstocks for the present invention may be selected from pine (e.g. Pinus radiata, Pinus taeda); spruce; and combinations / hybrids thereof.

[0112] Hardwood feedstocks include, but are not limited to, 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 lanceolata, F. latifolia, F. profunda, F. americana ); Aspen (e.g. P. grandidentata, P. tremula, P. tremuloides ); Australian Red Cedar ( Toona ciliata ); Ayna ( Distemonanthus benthamianus ); Balsa ( Ochroma 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; Bubing a; Buckeye (e.g. Aesculus hippocastanum, Aesculus glabra, Aesculus flava / Aesculus octandra ); Butternut; Catalpa; Cherny (e.g. Prunus serotina, Prunus pennsylvanica, Prunus 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 ); Elm (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, Carpinus caroliniana, Casuarina equisetifolia, Choricbangarpia subargentea, Copaifera spp., Eusideroxylon zwageri, Guajacum officinale, Guajacum sanctum, Hopea odorata, Ipe, Krugiodendronferreum, Lyonothamnus lyonii ( L. floribundus ), Mesua ferrea, Olea spp., Olneya tesota, Ostrya virginiana, Parrotia persica, Tabebuia serratifolia ); Jacaranda; Jotoba; Lacewood; Laurel; Limba; Lignum vitae; Locust (e.g. Robinia pseudacacia, Gleditsia triacanthos ); Mahogany; Maple (e.g. Acer saccharum, Acer nigrum, Acer negundo, Acer rubrum, Acer saccharinum, Acer pseudoplatanus ); Meranti; Mpingo; Oak (e.g. QuercusAttorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory macrocarpa, Quercus alba, Quercus stellata, Quercus bicolor, Quercus 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 ); 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 regia); Willow (e.g. Salix nigra, Salix alba ); Yellow poplar ( Liriodendron tulipifera ); Bamboo; Palmwood; and combinations / hybrids thereof.

[0113] For example, hardwood feedstocks for the present invention may be selected from Acacia, Aspen, Beech, Eucalyptus, Maple, Birch, Gum, Oak, Poplar, and combinations / hybrids thereof. The hardwood feedstocks for the present invention may be selected from Populus spp. (e.g. Populus tremuloides), Eucalyptus spp. (e.g. Eucalyptus globulus), Acacia spp. (e.g. Acacia dealbata), and combinations thereof.

[0114] Grass feedstocks include, but are not limited to, C4 or C3 grasses, e.g. Switchgrass, Indiangrass, Big Bluestem, Little Bluestem, Canada Wildrye, Virginia Wildrye, and Goldenrod wildflowers, etc, amongst other species known in the art.

[0115] Agricultural feedstocks include, but are not limited to, agricultural byproducts such as husks, stovers, foliage, and the like. Such agricultural byproducts can be derived from crops for human consumption, animal consumption, or other non-consumption purposes. Such crops can be corps such as corn, wheat, sorghum, rice, soybeans, hay, potatoes, cotton, or sugarcane. The feedstock can arise from the harvesting of crops from the following practices: intercropping, mixed intercropping, row cropping, relay cropping, and the like.

[0116] In some embodiments, the biomass is an ensiled biomass. In some embodiment, the biomass is ensiled by placing the biomass in an enclosed container or room, such as a silo, or by piling it in a heap covered by an airproof layer, such as a plastic film. The biomass undergoing the ensiling, known as the silage, goes through a bacterial fermentation process resulting in production of volatile fatty acids. In some embodiment, the ensiling comprises adding ensiling agents such as sugars, lactic acid or inoculants. In someAttorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory embodiments, the ensiled biomass comprises one or more toxic compounds. In some embodiments, when ensiled biomass comprises one or more toxic compounds, the microbe is resistant to the one or more toxic compounds.Example 1Amine-based solvents for single and mixed feedstock agnostic pretreatmentFEEDSTOCKS SELECTION

[0117] We have selected a wide range of feedstocks that are representative of different environments. Temperate - walnut, almond hulls, almond stems, pine, poplar, hardwood sawdust, pongamia. Tropical - energycane, eucalyptus, bamboo, rice hulls, coconut husks / fibers, pineapple, mango, or banana waste streams. Agricultural - com stover, wheat straw, hay, sorghum, hay, oatmeal.

[0118] The results of the experiment are shown in Figures 1.1-1.4. Figure 1.1. Initial screening with 8 different feedstocks and 3 amine-based solvents. Figure 1.2. Screening of 22 different single feedstocks using butylamine as a pretreatment solvent. Figure 1.3. Scaling up deconstruction to 1 L. Figure 1.4. Initial scale-up of mixed agriculture residues improves performance.CONCLUSION

[0119] Preliminary screening of three solvents and eight feedstocks revealed triethylamine to be the best at solvent recovery but with poor yields, identified butylamine as the best solvent for sugar yields and solvent recovery. Examined efficiency of butylamine in pretreatment of 22 single feedstocks and 1 feedstock blend agriculture mix (equal parts of sorghum, corn stover, wheat straw and hay), showed good sugar yields across different feedstock types. Successfully performed initial scale-up of ag mix in 1 L Parr reactor, achieving >90% yields of glucose, >80% of xylose, and >99% solvent recoveryExample 2Screening Distillable Amine-Based Solvents for Liberating Bioavailable Intermediates from Lignocellulosic FeedstocksAttorney Docket: 2024-152-02 Lawrence Berkeley National LaboratoryINTRODUCTIONExploring the potential of advanced distillable solvents as biomass pretreatment agents is critical for biorefmeries owing to their efficiency and recycling capabilities. Here, we employ distillable amine-based solvents for pretreating a wide range of lignocellulosic feedstocks, aiming to facilitate the industrial release of fermentable sugars through enzymatic hydrolysis. Diverse solvents, including ethanolamine, ethanolamine acetate, butylamine, butylamine acetate, and triethylamine, were tested for the pretreatment of 22 different biomasses, ranging from agricultural residues to woody and tropical biomass. Fig. 2.1 shows a scheme for solvent recovery. Fig. 2.2 shows a scheme for the saccharification of a biomass. .FEEDSTOCKS

[0120] Eight feedstocks, including palm oil fiber, coconut chips, hardwood sawdust, sorghum, hay, rice hulls, 4-crop mix pellets, were initially selected to screen the performance of different solvents. After determining the suitable solvent, more feedstocks representative of various sources, covering temperate, tropical and agricultural feedstocks, were examined to evaluate the performance of the selected solvent.SOLVENTS

[0121] Ethanolamine, ethanolamine acetate, butylamine, butylamine acetate, and triethylamine.EXPERIMENTALScreening Solvents for Deconstructing Diverse Feedstocks

[0122] Different solvents for pretreatment followed by enzymatic hydrolysis. Butylamine Pretreatment for A Variety of Feedstocks. Fig. 2.3 shows the sugar yield and solvent recovery using different amine solvents.

[0123] (A) Enzymatic hydrolysis efficiency of various feedstocks pretreated with butylamine. Effective for most feedstocks, especially grasses and hardwood. Solvent removal exceeds 95% for most feedstocks. (B) Chemical composition of various feedstocks. 22 feedstocks were analyzed for glucan, xylan, acid soluble lignin, and Klason lignin. NREL’s protocol was used for the analysis. (C) Lignin type of various feedstocks. Py-GC / MS.Attorney Docket: 2024-152-02 Lawrence Berkeley National LaboratoryQuantitatively and quantitatively analyzed the p-hydroxyphenyl (H) unit, guaiacyl (G) units, and syringyl (S) unit for 22 feedstocks prior to pretreatment. Matrix of Pearson correlation coefficients among sugar yield and feedstock composition properties. Note: H, G, or S relative percentage refers to the percentage of each value compared the total sum of H, G, and S. Fig. 2.4 shows the pretreatment properties of 22 feedstock biomass. Fig. 2.5 shows the correlation with the various pretreatment properties. * indicates a significant correlation between parameters (p < 0.05).Feedstock Mix and Scale Up

[0124] Initial scale-up of mixed agriculture residues improves performance. Liberated >90% of glucose from mixed ag feedstocks at 1 L scale. Removed >99% of butylamine after pretreatment. Fig. 2.6 shows blending of feedstocks (A) and sugar yields and solvent removal for different feedstocks (B).SUMMARY

[0125] Pretreated lignocellulosic feedstocks with distillable amine-based solvents, followed by solvent recovery and enzymatic hydrolysis for sugar release. Screened five solvents and eight feedstocks, identifying butylamine as the best solvent for sugar yields and solvent recovery. Examined butylamine pretreatment of 22 feedstocks, along with analysis of the relationship between sugar yield and feedstock composition properties. Successfully initial scale-up of ag mix, achieving >90% yields of glucose, >80% of xylose, and >99% solvent recovery.

[0126] References:(1) Ntakirutimana, Samuel, et al. “Amine-based pretreatments for lignocellulose fractionation and lignin valorization: a review.” Green Chemistry 24.14 (2022): 5460-5478.(2) Achinivu, Ezinne C., et al. "Alkanolamines as dual functional solvents for biomass deconstruction and bioenergy production." Green Chemistry 23.21 (2021): 8611-8631.(3) Achinivu, Ezinne C., et al. "Biomass pretreatment with distillable ionic liquids for an effective recycling and recovery approach." Chemical Engineering Journal 479 (2024): 147824.Attorney Docket: 2024-152-02 Lawrence Berkeley National LaboratoryExample 3Distillable ionic liquids for feedstock agnostic pretreatmentFEEDSTOCKS SELECTION

[0127] We have selected a wide range of feedstocks that are representative of different environments. Temperate -hardwood sawdust, pongamia. Tropical - palm oil fiber, coconut husks / ftbers. Agricultural - rice hulls, hay, sorghum.

[0128] The results of the experiment are shown in Figure 3.1. Figure 3.1. Examination with eight different feedstocks and two distillable ionic liquids.CONCLUSION

[0129] Both ethanolammonium acetate and butylammonium acetate achieve superior sugar yields across diverse feedstocks, with over 95% solvent for most. The efficiency of these solvents vary depending on the feedstock. This highlights the important of selecting the appropriate solvent for optimal results.Example 4Unhindered volatile amine reagents for highly efficient biomass pretreatment

[0130] We describe a novel group of solvents for biomass pretreatment, such as the agricultural residue, corn stover. Distillable amine solvents including dimethylamine (40% aq. solution) and 1 -pentylamine (amylamine, neat) were used for evaluating the deconstruction of a typical agricultural by-product - com stover - that account for the largest part of the agricultural waste in the United States into simple fermentable sugars.Pretreatment was carried out at 20% solids loading at 80 °C for 3 hours, and for solvent recovery, simple evaporation was performed in a vacuum oven set at 80 °C. After solvent recovery, the pH was adjusted to 5.0, and subsequent enzymatic hydrolysis was carried out using 20 mg protein / g biomass with Cellic® CTec3:HTec3 (9: 1) at 5% solid loading at 50 °C for 72 hours.

[0131] Preliminary results at small scales show that both dimethylamine (40% aq. solution) and 1 -pentylamine (amylamine, neat) achieve high sugar yields from corn stover (Figure 4.1). As shown in Fig. 4.1 (A), within detection limits complete (100%) solventAttorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory removal was obtained for both solvents. 1 -pentylamine (amylamine, neat) resulted in glucose yield exceeding 90% with xylose yield above 70% (Fig. 4.1 (B)), respectively.Dimethylamine (40% aq. solution) produced even higher sugar yields with preliminary glucose yield surpassing 99% and xylose yield exceeding 85% (Fig. 4.1 (B)). These findings highlight the promising potential of these two amine solvents for feedstock pretreatment for the sustainable production of biofuels and bioproducts. Other similarly nucleophilic amines (e.g. ethylamine and methylamine) are being actively investigated as similarly promising candidates.

[0132] Two reactors ran on 3 g scale with 15% mass loading agriculture blend. The PT conditions are 80 °C for 3 hours, and stirred at 200 rpm. Drying is at 80 °C overnight in a house vacuum. Solvent + Moisture Removal. RCT A: 97.4%, RCT B: 98.1%. pH adjusted to 5+-0.25 with 5% H2SO4 before adding citrate buffer, NaNs and enzymes (30 mg / g BM).Average pH after drying is 6.60 pH. Similar lignin and hemicellulose solubilization when compared to butylamine pretreatments. Clear dimethylamine residuals still functionalize biomass. Figures 4.2 and 4.3 show the results.

[0133] It is to be understood that, while the invention has been described in conjunction with the preferred specific embodiments thereof, the foregoing description is intended to illustrate and 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.

[0134] All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entireties.

[0135] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes 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 claims appended hereto.

Claims

Attorney Docket: 2024-152-02 Lawrence Berkeley National LaboratoryWhat is claimed is:

1. A method for increasing a sugar compound or saccharification yield from a biomass, the method comprising: (a) providing a first mixture comprising a solubilized biomass comprising an alkylamine or alkylammonium, or a distillable amine ionic liquid (DAIL), and (b) recovering, removing, or separating at least part of the alkylamine or alkylammonium, or DAIL, from the first mixture in order to separate the at least part of the alkylamine or alkylammonium, or DAIL, from the solubilized biomass or the first mixture.

2. The method of claim 1, wherein the alkylamine, alkylammonium, or DAIL is a primary amine.

3. The method of claim 1, wherein the alkylamine, alkylammonium, or DAIL is a secondary amine.

4. The method of claim 1, wherein the alkylamine, alkylammonium, or DAIL is a tertiary amine.

5. The method of claim 1, wherein the alkylamine, alkylammonium, or DAIL is sterically unhindered and highly nucleophilic.

6. The method of claim 1, wherein the method comprises: (a) providing a first mixture comprising a solubilized biomass comprising an alkylamine or alkylammonium, and (b) recovering, removing, or separating at least part of the alkylamine or alkylammonium from the first mixture in order to separate the at least part of the alkylamine or alkylammonium from the solubilized biomass or the first mixture.

7. The method of claim 6, wherein the first mixture comprises an alkylamine having the following chemical structure:RiR2— N— R3 (I); wherein Ri, R2, and R3 are each independently — H, or — (CH2)n— CH3, and n is 0, 1, 2, 3, or 4, and at least one of Ri, R2, and R3 comprises one carbon atom8. The method of claim 7, wherein the alkylamine comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 14, or 15 carbon atoms.Attorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory9. The method of claim 8, wherein the alkylamine comprises 1, 2, 3, 4, 5, or 6 carbon atoms.

10. The method of claim 9, wherein the alkylamine is methylamine, ethylamine, propylamine, butylamine (w-butylamine), dimethylamine, diethylamine, dipropylamine, trimethylamine, triethylamine, or tripropylamine.

11. The method of claim 6, wherein the first mixture comprises an alkylammonium having the following chemical structure:RiR2— N — R3 (II);H wherein Ri, R2, and R3 are each independently — H, or — (CH2)n— CH3, and n is 0, 1, 2, 3, or 4, and at least one of Ri, R2, and R3 comprises one carbon atom.

12. The method of claim 11, wherein the alkylammonium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 14, or 15 carbon atoms.

13. The method of claim 12, wherein the alkylammonium comprises 1, 2, 3, 4, 5, or 6 carbon atoms.

14. The method of claim 13, wherein the alkylammonium is methylammonium, ethylammonium, propyl ammonium, butylammonium (w-butylammonium), dimethylammonium, diethylammonium, dipropylammonium, trimethylammonium, triethylammonium, or tripropylammonium15. The method of claim 1, wherein the method comprises: (a) providing a first mixture comprising a solubilized biomass comprising a distillable amine ionic liquid (DAIL), and (b) recovering, removing, or separating at least part of the DAIL from the first mixture in order to separate the at least part of the DAIL from the solubilized biomass or the first mixture.

16. The method of claim 15, wherein the DAIL comprises an alkanolammonium or alkylammonium cation, and an anion.

17. The method of claim 16, wherein the anion is a carboxylic acid.

18. The method of claim 17, wherein the carboxylic acid has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms.

19. The method of claim 16, wherein the alkanolammonium is an ethanolammonium.Attorney Docket: 2024-152-02 Lawrence Berkeley National Laboratory20. The method of claim 16, wherein the alkylammonium is a butylammonium.

21. The method of claim 15, wherein the providing step (a) comprises introducing a pretreatment (PT) solvent comprising the distillable amine ionic liquid (DAIL) to a biomass to solubilize the biomass to form the first mixture.

22. The method of claim 15, wherein the recovering, removing, or separating step (b) comprises recovering, removing, or separating at least part of the PT solvent from the solubilized biomass or the first mixture.

23. The method of claim 22, further comprising: (c) introducing an enzyme and / or a microbe to the first mixture such that the enzyme and / or microbe produce a sugar from the solubilized biomass.

24. The method of claim 23, further comprising: (d) the sugar is separated from the first mixture.

25. The method of claim 15, wherein the recovering step (b) comprises distilling the at least part of the DAIL from the first mixture.

26. The method of claim 25, further comprising: (e) introducing at least part of the DAIL separated in the (b) recovering step to the first mixture in step (a).

27. The method of claim 26, further comprising: (f) introducing more biomass to the first mixture in step (a).

28. The method of claim 15, wherein the DAIL comprises an alkanolammonium or alkylammonium cation, and a carboxylic acid.

29. The method of claim 1, wherein the method comprising: (a) providing a first mixture comprising a solubilized biomass comprising an alkylamine, and (b) recovering, removing, or separating at least part of the alkylamine from the first mixture in order to separate the at least part of the alkylamine from the solubilized biomass or the first mixture; wherein the alkylamine is a sterically unhindered, highly nucleophilic alkylamines.

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