Addition of water to pretreated biomass slurries to increase saccharification yields

Abstract

The present invention provides for a method for increasing saccharification yield comprising: (a) providing a pretreated biomass comprising a pretreatment (PT) solvent, (b) adding or introducing water to the pretreated biomass to form a water-added slurry, and (c) removing or separating the PT solvent from the water-added slurry.

Description

Addition of water to pretreated biomass slurries to increase saccharification yieldsInventors: Hemant Choudhary, Alberto Rodriguez, John M. Gladden, Blake A. SimmonsCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Ser. No. 63 / 553,034, filed February 13, 2024, which is incorporated by reference in its entirety.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. 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 saccharification yield comprising: (a) providing a pretreated biomass comprising a pretreatment (PT) solvent, (b) adding or introducing water to the pretreated biomass to form a water-added slurry, and (c) removing or separating the PT solvent from the water-added slurry.

[0006] 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, deepeutectic 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.

[0007] In some embodiments, the providing step (a) results in solubilizing or deconstructing the biomass such that lignin is released from the biomass. 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).

[0008] In some embodiments, the water added or introduced is equal to about 50%, 60%, 70%, 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. In some embodiments, the sugar release % of glucose is equal to or more than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or having a percentage with a range of any two preceding percentages. In some embodiments, the sugar release % of xylose is equal to or more than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or having a percentage with a range of any two preceding percentages.

[0009] In some embodiments, the removing or separating step (c) comprises maintaining a temperature of about 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, or 90 °C, or a temperature within a range of any two preceding temperatures, or between about 30-90 °C, or about 40-80 °C, while optionally continuously homogenizing or mixing the water-added slurry until at least about 75%, 100%, 125%, or 150% PT solvent and / or water (by weight) is removed from the water-added slurry.

[0010] In some embodiments, the biomass or pretreated biomass comprises a cellulosic portion. In some embodiments, the method further comprises: (d) 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, 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 cellulosicportion 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). In some embodiments, the sugar monomers comprise glucose, xylose, or a mixture thereof.

[0011] 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.

[0012] 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.

[0013] 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 of water in the method of the present invention increases the sugar yield from the method as compared to a method wherein there is no addition of water step.

[0014] The addition of 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. Addition of water typically results in the pretreatment inefficiencies of molecular and ionic solvents. Additionally, the presence of water also interferes with the recovery of pretreatment solvent by changing the pH of the system.

[0015] 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 ApplicationPublication 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.

[0016] 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.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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.

[0018] Figure 1 shows an embodiment of the present invention.DETAILED DESCRIPTION OF THE INVENTION

[0019] 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.

[0020] 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:

[0021] 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.

[0022] 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.

[0023] 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, 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.

[0024] Figure 1 shows a method of the present invention. The method comprises: (a) providing a pretreated biomass comprising a pretreatment (PT) solvent (10), (b) adding or introducing water to the pretreated biomass to form a water-added slurry (20), and (c) removing or separating the PT solvent from the water-added slurry (30).

[0025] In some embodiments, the pretreatment solvent comprises one or more of the following: ionic liquid, deep eutectic solvent (DES), and the like.IONIC LIQUID

[0026] 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 thebiomass 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.

[0027] 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 AICI4), l-ethyl-3- methylimidazolium thiocyanate (EMIM SCN), l-butyl-3-methylimidazolium acetate (BMIM Acetate), l-butyl-3-methylimidazolium chloride (BMIM Cl), l-butyl-3-methylimidazolium 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 methyl sulfate, tributylmethylammonium methyl sulfate, choline acetate, choline salicylate, and the like.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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 et 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.

[0032] In some embodiments, the protic ionic liquid (PIL) is disclosed by U.S. Patent Application Publication No. 2004 / 0097755, hereby incorporated by reference.

[0033] 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).

[0034] 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.

[0035] 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.

[0036] In some embodiments, the PILs are formed with 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. 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)

[0037] 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 ofquaternary 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.

[0038] 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.

[0039] 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.

[0040] 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 / or 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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

[0047] 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

[0048] 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 / 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).

[0049] Generally, although not necessarily, the microbe is a yeast or a bacterium. In some embodiments, the microbe is Rhodosporidium toruloides o Pseudomonas putida. In 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.

[0050] 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.

[0051] 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 P. putida, P. aeruginosa, P. chlororaphis, P. fluorescens, P. pertucinogena, P. stutzeri, P. syringae, P. cremoricolorata, P. entomophila, P. fulva, P. 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. megaterium, B. licheniformis, B. anthracis, B. amyloliquefaciens, or B. pumilus.BIOFUEL

[0052] 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 theirentireties by reference).BIOMASS

[0053] 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.

[0054] 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 / hybrids thereof.

[0055] 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.

[0056] 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; Chemy (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. Quercus 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*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.

[0057] 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.

[0058] 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.

[0059] 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 com, 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.

[0060] 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 some 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 1Addition of water to pretreated biomass slurries to promote high saccharification yieldsEXPERIMENTAL METHODSConventional Biomass Pretreatment (no solvent recovery)

[0061] All pretreatment reactions were conducted in duplicate. 4: 1 w / w mixture of almond / walnut was mixed with pretreatment solvent in a 15:85 ratio (w / w) to afford a biomass loading of 15wt% in a IL Parr reactor and pretreated for 3 h at 140 °C. After pretreatment, samples were removed from the oil bath and allowed to cool. Pretreated slurry was washed with DI water to wash off the pretreatment solvent using centrifugation at high speed (4000 rpm). The water washed solid was freeze-dried to obtain dried pretreated biomass for further analysis.Biomass Pretreatment (solvent recovery)

[0062] All pretreatment reactions were conducted in duplicate. 4: 1 w / w mixture of almond / walnut was mixed with pretreatment solvent in a 15:85 ratio (w / w) to afford a biomass loading of 15wt% in a IL Parr reactor and pretreated for 3 h at 140 °C. After pretreatment, pretreatment solvent was removed under reduced pressure. The temperature was maintained between 40-80 °C while continuously homogenizing the slurry and thorough mixing until >98% solvent (by weight) was removed from the slurry. In order to compare results with conventional biomass pretreatment, the dried-solvent removed-pretreated slurry was washed with DI water to wash off the pretreatment solvent using centrifugation at high speed (4000 rpm). The water washed solid was freeze-dried to obtain dried pretreated biomass for further analysis.Biomass Pretreatment (solvent recovery after water addition)

[0063] All pretreatment reactions were conducted in duplicate. 4: 1 w / w mixture of almond / walnut was mixed with pretreatment solvent in a 15:85 ratio (w / w) to afford a biomass loading of 15wt% in a IL Parr reactor and pretreated for 3 h at 140 °C. After pretreatment, DI water was added to the pretreatment slurry (equal amount as the pretreatment solvent) followed by the removal of pretreatment solvent under reduced pressure. The temperature was maintained between 40-80 °C while continuously homogenizing the slurry and thorough mixing until >125% solvent (by weight) was removed from the slurry. In order to compare results with conventional biomass pretreatment, the dried-solvent removed-pretreated slurry was washed with DI water to wash off the pretreatment solvent using centrifugation at high speed (4000 rpm). The water washed solid was freeze-dried to obtain dried pretreated biomass for further analysis.Enzymatic Saccharification

[0064] All enzymatic saccharifications were conducted in duplicate. Enzymatic saccharification of pretreated and untreated biomass was carried out using commercially available enzymes, Cellic® Ctec3 and Htec3 (9: 1 v / v) from Novozymes, at 50 °C in a rotary incubator (Enviro-Genie, Scientific Industries, Inc.). All reactions were performed at 5wt% biomass loading in a 15 mL centrifuge tube. The pH of the mixture was adjusted to 5 with 50 mM sodium citrate buffer supplemented with 0.02% sodium azide to prevent microbial contamination. The total reaction volume included a total protein content of 20 mg per gbiomass. The amount of sugars released was analyzed on 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. An aqueous solution of sulfuric acid (4 mM) was used as the eluent (0.6 mL min column temperature 60 °C).Compositional Analysis - Glucan and Xylan

[0065] All compositional analysis experiments were conducted in duplicate. Compositional analysis of biomass before and after pretreatment was performed using NREL two-step acid hydrolysis protocols (LAP) LAP-002 and LAP-005 (A. Sluiter, National Renewable Energy Laboratory (NREL) Analytical Procedures, 2004). Briefly, 200 mg of biomass and 2 mL of 72% sulfuric acid (H2SO4) were incubated at 30 °C while shaking at 200 rpm for 1 h. The solution was diluted to 4% H2SO4 with 56 mL of DI water and autoclaved at 121 °C for 1 h. The reaction was quenched by cooling down the flasks before removing the solids by filtration. Glucose and xylose concentrations were determined from the filtrate using HPLC (as described previously). The amount of glucan and xylan was calculated from the glucose and xylose content multiplied by the anhydro correction factors of 162 / 180 and 132 / 150, respectively. The compositional profiles for the untreated and pretreated biomass are included in Table 1.RESULTS AND DISCUSSION

[0066] The pretreatment efficacy was measured in terms of sugar release and is tabulated in Table 1. The untreated biomass afforded 32.2% glucose and 39.8% xylose yields due to high recalcitrance of the biomass material. Solvent-based pretreatment of the biomass is an effective methodology to enhance sugar release from the biomass for further bioconversion. Conventionally, pretreatment solvents are removed from the pretreated slurry by water washing before addition of enzyme cocktails to prevent any enzyme activity inhibition. When the pretreatment solvent was removed by water washing, sugar release was boosted to 79.4% glucose and 85.3% xylose compared to untreated biomass. Both glucose and xylose yields dropped to 49.6% and 50.7% when solvent was removed under reduced pressure while being exposed to heat after pretreatment step. This could be due to following reasons, a) decomposition of the biomass (in dry state) due to heat, and b) formation of products (attached to biomass) that inhibit enzyme activity among others. To overcome this issue, water was added to the pretreated slurry before removal of solvent under reduced pressure.The addition of water boosted back glucose (81.6%) and xylose (66.1%) yields.

[0067] Table 1. Compositional analysis and sugar release profiles of biomass samples

[0068] 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.

[0069] All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entireties.

[0070] 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

What is claimed is:

1. A method for increasing saccharification yield comprising: (a) providing a pretreated biomass comprising a pretreatment (PT) solvent, (b) adding or introducing water to the pretreated biomass to form a water-added slurry, and (c) removing or separating the PT solvent from the water-added slurry.

2. The method of claim 1, wherein the providing step comprises adding a pretreatment (PT) solvent to a biomass.

3. The method of claim 1, wherein the PT solvent comprises an ionic liquid or deep eutectic solution (DES) solvent.

4. The method of claim 3, wherein the PT solvent is a distillable protic ionic liquid (PIL).

5. The method of claim 1, wherein the PT solvent is an alkanolamine.

6. The method of claim 1, the providing step (a) results in solubilizing or deconstructing the biomass such that lignin is released from the biomass.

7. The method of claim 1, wherein the adding or introducing step (b) comprises adding or introducing water equal to equal to or more than about 50% volume (or weight) of the volume (or weight) of PT solvent in the pretreated biomass.

8. The method of claim 1, wherein the removing or separating step (c) comprises maintaining a temperature of about 30 °C to 90 °C, while optionally continuously homogenizing or mixing the water-added slurry until at least about 75% to 150% of the PT solvent and / or water (by weight) is removed from the water-added slurry.

9. The method of claim 1, wherein the pretreated biomass comprises a cellulosic portion, and the method further comprises (d) introducing an enzyme to the pretreated biomass and / or water-added slurry to break down or depolymerize the cellulosic portion into one or more sugar monomers.

10. The method of claim 9, wherein the sugar monomers comprise glucose, xylose, or a mixture thereof.

11. The method of claim 10, wherein the glucose obtained is equal to or more than about 60% of the total glucose contained in the pretreated biomass.

12. The method of claim 10, wherein the xylose obtained is equal to or more than about 60% of the total xylose contained in the pretreated biomass.

13. The method of claim 9, wherein the (e) introducing a microbe to the pretreated biomass and / or water-added slurry, wherein the microbe utilizes the cellulosic portion or one or more sugar monomers as a carbon source to produce a biofuel or bioproduct.

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