Process for producing fermentation product and lignin

Abstract

The present invention provides methods of producing one or more fermentation products and lignin. The method comprises the steps of pretreatment with sulfur dioxide and volatile alcohol, chemical recovery, whole pulp saccharification and fermentation. High monosaccharide yields are achieved, and alkoxylated and / or non-alkoxylated lignin fractions as well as lignosulphonate salts can be produced and separated if desired. Monosaccharides with or without lignin are fermented to produce one or more fermentation products, such as ethanol.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Patent Application No. 19 / 385,649, filed November 11, 2025, and U.S. Provisional Patent Application No. 63 / 773,846, filed March 18, 2025, the contents of which are incorporated herein by reference. Background Technology

[0003] Due to the inherent stubborn nature of lignocellulosic biomass, its economical and efficient conversion into monosaccharides, lignin, and fermentation products presents a challenge. A key step in this conversion process is pretreatment (or fractionation). Existing pretreatment / fractionation processes commonly suffer from problems including: high sugar degradation rates and high fermentation inhibitor formation rates; low cellulose enzymatic digestion rates; low monosaccharide and fermentation product yields; low lignin yield; poor lignin quality (e.g., condensation, derivatization); equipment blockage by sticky lignin precipitates; use of expensive / toxic chemicals; and low chemical recovery rates, among others.

[0004] Known pretreatment processes include hot water, steam flash explosion, dilute acid, deacetylation-mechanical refining (DMR) of NREL, sulfite / SPORL, sulfur dioxide, organic solvents (with or without acid / base catalysts), and SO2-organic solvent-water processes (e.g., the OR1 process described in U.S. Patent 11,118,017 (Iakovlev et al.), and... (Process).

[0005] Hot water (hydrothermal), steam flash explosion, and dilute acid pretreatment processes (150℃-220℃) are characterized by high sugar degradation rates, lignin condensation, low cellulase digestibility, formation of sticky lignin precipitates, unsuitability for softwood, and challenges in acid recovery under dilute acid conditions. For example, Perez et al. (2008) reported at least 47% loss of hemicellulose sugars in optimized hot water pretreatment of wheat straw. In another report, dilute sulfuric acid pretreatment of pine wood resulted in only 43% conversion of glucan to glucose when the enzyme dosage was approximately 22 filter paper units (FPU) / g dextran. The total recovery rate of monomeric sugars was only 248 kg per 'bone dry' (BD) metric ton of biomass feed (Zhu et al., 2010).

[0006] The deacetylation-mechanical refining (DMR) process of NREL (Chen et al. 2024, Chen et al. 2016) utilizes an alkaline solution to deacetylate hemicellulose, followed by mechanical refining. This treatment results in relatively low sugar yields even at high enzyme dosages, likely due to limited impact on cell wall structure, where most lignin and hemicellulose remain within cellulose, hindering enzyme accessibility. It has been reported that the total monosaccharide yield of carbohydrates in corn stalk feedstock is approximately 80% using 16 mg CTec3 protein / g cellulose (12 FPU / g cellulose) and 4 mg HTec3 protein / g cellulose; and approximately 74% using 8 mg CTec3-protein / g cellulose (6 FPU / g cellulose) and 2 mg HTec3-protein / g cellulose (Chen et al. 2016). Furthermore, alkali regeneration is difficult to achieve, increasing process costs.

[0007] Sulfite pulping and SPORL pretreatment processes (EP 2376642 (Sjoede) "LIGNOCELLULOSICBIOMASS CONVERSION BY SULFITE PRETREATMENT", Tian et al. 2011, Wang et al. 2012) utilize sulfur dioxide and sulfites, and occasionally sulfuric acid in the case of SPORL. Temperatures range from 125 to 180 °C, and pH values ​​are between 1 and 13, mostly in the acidic range. These processes have been reported to outperform dilute acid pretreatment (Tian et al. 2011, Wang et al. 2012) in terms of cellulase digestibility. However, the presence of bisulfite anions and acidity leads to sugar degradation. Most monosaccharides (>20-30%, Pfister and...) are degraded. (1977) Lost in the form of aldonic acid. The total recovery rate of monomeric sugars was reported to be approximately 520 kg per BD metric ton of Douglas Fir (Zhu et al., 2015). The recovery of the complex mixture of bisulfite, sulfite, thiosulfate, and sulfate is a nearly insurmountable challenge.

[0008] Sulfur dioxide-catalyzed pretreatment processes (Soderstrom et al. 2004, Stenberg et al. 1998, Galbe and Zacchi 2002, Ewanick et al. 2007, and van der Meulen: EP 2516660, US 8,834,633 (Van Der Meulen et al.), and US 9,528,129 (Van Der Meulen et al.)) typically utilize liquids with SO2 concentrations below 3% and temperatures of approximately 150-215°C. The pH of the pretreated liquid is typically 2-3, indicating a low residual sulfur dioxide concentration. The performance of low-SO2 pretreatment is similar to that of dilute acid pretreatment, as most of the SO2 is consumed to form strong lignin sulfonic acids. Therefore, lignin concentration and redeposition on the fibers are observed, and viscous precipitates are expected. The high residual lignin content of cellulose fibers results in below-optimal enzymatic digestibility of cellulose; at enzyme doses higher than or well above 20 FPU / g dextran, the conversion rate of dextran to glucose is only 60-70%. It has been reported that the total monomeric sugar yield per BD metric ton of biomass feed is only about 470-520 kg or less (Soderstrom et al., 2004; Stenberg et al., 1998; Galbe and Zacchi, 2002; Ewanick et al., 2007). High SO2 pretreatment leads to significantly higher sugar yields and efficient lignin solubilization, for example, as described in US Patent 11,306,113 (Iakovlev et al.). However, these processes convert most of the lignin into derivatives, namely lignin sulfonates, which have lower application potential compared to the non-sulfonated (natural) form of lignin.

[0009] Organic solvent processes (see US Patent No. 1,856,567 (Kleinert and Tayenthal), issued on May 3, 1932) involve treating lignocellulosic biomass, typically with or without a catalyst, at temperatures ranging from 160 to 210°C with varying proportions of organic solvents and water. The advantage of organic solvent processes is that the presence of the solvent promotes the dissolution of lignin, which is considered to be close to its natural form, although lignin is alkoxylated to some extent when alcohols are used as solvents. The most common organic solvents are alcohols, ketones, esters, and organic acids, while the most common catalyst is sulfuric acid. The combination of high temperature and acidity in organic solvent processes leads to significant sugar degradation, particularly in uncatalyzed organic solvent processes, where approximately 30% sugar loss has been observed (van Heiningen et al. 2018).

[0010] Another drawback of organic solvent processing is the limited removal of lignin, as well as the limited removal of hemicellulose from cellulose. Non-catalytic organic solvent processing of cork results in high residual lignin content in the fibers. For example, Aziz and Sarkanen (1989) reported that uncatalytically processed cork organic solvent pulp had a high residual lignin content (kappa number 80-100, i.e., approximately 13-20% residual lignin). Kleinert (1974) obtained cellulose pulp from spruce after 60 minutes of non-catalytic organic solvent pulping at 185°C, which had approximately 5% residual lignin and 8% residual hemicellulose at a liquid-to-solid ratio of 10, indicating that even at very high liquid-to-solid ratios, the removal efficiency of hemicellulose from cork is low. At lower liquid-to-solid ratios, lignin removal is considerably impaired.

[0011] The presence of catalysts improves the efficiency of organic solvent pretreatment, but producing pure cellulose pulp remains a challenge. For example, the residual lignin content of cellulose pulp from a mixture of spruce, pine, and Douglas fir wood residue from a timber mill, pretreated with sulfuric acid as a catalyst in 60% ethanol (185–198 °C, 30–60 min, pH 2.0–3.4; liquid-to-solid ratio 7–10 kg / kg), ranges from 6.4% to 27.4% (Pan et al. 2005). Residual organic solvent lignin, particularly in non-sulfonated forms, is known to reduce enzyme digestibility by increasing the adsorption of non-productive enzymes onto the lignin (Nakagame et al. 2010, del Rio et al. 2011).

[0012] For non-catalytic organic solvent processes using volatile alcohols, ketones, esters, or other volatile compounds as solvents, the solvent in the waste slurry can be easily recovered by distillation. However, without the addition of an acidic catalyst, alcohols chemically bound to lignin and sugars in organic solvent processes are difficult to recover, which may explain the high ethanol loss in ethanol-based Alcell processes.

[0013] In organic solvent processes using volatile solvents, solvent recovery is required, for example, through distillation. This can lead to lignin precipitation because organic solvent lignin is insoluble in water. Adding organic solvent lignin to cellulose reduces the yield of enzymatic hydrolysis. Therefore, to avoid lignin precipitation on cellulose, a cellulose washing step is used before the solvent recovery step, i.e., the separation of cellulose from dissolved substances. No literature mentions organic solvent pretreatment processes that do not include cellulose washing, with the entire pulp undergoing solvent recovery followed by enzymatic hydrolysis.

[0014] SO2-alcohol-water process (OR1 process, US Patent 11,118,017 (Iakovlev et al.), and The process (US Patents 8,038,842 and 8,268,125 to Retsina et al.) utilizes sulfur dioxide, alcohol, and water at a temperature of approximately 135-165°C. The OR1 process uses a sulfur dioxide concentration exceeding 9%, while the OR2 process uses approximately 3-8%. Both allow for efficient delignification and high sugar yields; however, The process favors lignin sulfonation to produce lignin sulfonic acid, but the lignin yield is lower compared to the OR1 process. Similar to organic solvent processes, the existing SO2-alcohol-water process requires numerous unit operations, including the separation and washing of cellulose pulp and dissolved substances, enzymatic hydrolysis of cellulose pulp, recovery of sulfur dioxide and ethanol, and heat treatment of dissolved substances to improve sugar yield.

[0015] Chemical recovery is a decisive factor in the economic feasibility of pretreatment processes. Non-volatile inorganic acids (dilute acids, SPORL), alkalis, and salts (sulfites, SPORL) cannot be effectively recovered, while sulfur dioxide and volatile organic solvents are relatively easy to recover, for example, by flash evaporation and / or steam stripping. The difficulty in recovering alkalis is a major drawback of most alkali-using processes.

[0016] Enzymatic digestibility of cellulose is a key factor in economical process design. It is well known that cellulose is most efficiently digestible when its purity is high, i.e., when it contains only small amounts of residual hemicellulose and lignin. Solid, colloidal, or dissolved lignin is known to cause nonproductive enzyme adsorption, leading to low glucose yields. Lignin is also known to inhibit enzymatic hydrolysis (Lai et al. 2014, Pielhop et al. 2015, Huang et al. 2017). Therefore, processes for producing this lignin fraction (hot water, steam flash, dilute acid, organic solvents, SO2-alcohol-water, etc.) require separation of the cellulose pulp from the liquid (known as pulp washing) to avoid the inhibitory effect of the lignin component on enzymatic digestibility. This increases the capital and operating costs of the biorefinery.

[0017] It is well known that pre-treated fiber explosion (e.g., rapid pressure reduction leading to instantaneous evaporation of liquid within the fiber structure) increases the enzymatic digestibility of cellulose. This is likely due to the disruption of the fiber cell wall structure after flash explosion decompression, which increases the accessibility of enzymes to the cellulose surface.

[0018] Aside from the OR1 process, which involves the separation of cellulose pulp and dissolved substances, current pretreatment processes cannot economically and simultaneously yield fermentable monomeric sugars and non-condensed lignin in high yields without producing sticky lignin precipitates. High yield of fermentable monomeric sugars from softwood (gymnosperms) is defined as greater than approximately 75% of the available polysaccharides in the biomass, with an enzyme loading of approximately 3.3 FPU / odg biomass or less (“od” refers to oven-dried biomass). High yield of fermentable monomeric sugars from hardwood and herbaceous (non-woody) lignocellulosic biomass (angiosperms) is defined as greater than approximately 80%, based on the available polysaccharides in the biomass, with an enzyme loading of approximately 1.6 FPU / odg biomass or less. Total sugar-derived inhibitors (i.e., furfural, HMF, and levulinic acid) should be low, i.e., less than approximately 10 kg per BD metric ton of biomass. High lignin yield is defined as based on lignin content greater than 65% in the biomass (or, for softwood, greater than 180 BD kg lignin per BD metric ton of biomass). Furthermore, acidic pretreatment processes, such as those using sulfuric acid as a catalyst, typically produce sticky lignin precipitates that clog processing equipment, leading to increased downtime. Therefore, there is a need to improve processes to convert lignocellulosic biomass into non-condensation lignin fractions, lignocellulosic sugars, and fermentation products in high yields without producing sticky lignin precipitates that clog processing equipment. These processes should not require the separation of cellulose pulp and dissolved substances, and should allow for the hydrolysis and fermentation of the “whole pulp” after pretreatment, collectively reducing capital and operating costs. Summary of the Invention

[0019] This invention provides a method for converting lignocellulosic biomass into monosaccharides, lignin fractions, and one or more fermentation products, such that over 76-83% of the usable sugars are converted into monosaccharides from softwoods (e.g., pine, spruce, Douglas fir, larch, cedar, etc.), over 81-93% of the usable sugars are converted into monosaccharides from hardwoods (e.g., birch, beech, eucalyptus, poplar, maple, oak, etc.) and herbaceous biomass / agricultural residues / energy crops (e.g., corn stalks, corn cobs, wheat straw, barley straw, rice straw, rice husks, sugarcane stalks, energy sugarcane, sorghum stalks, miscanthus, switchgrass, giant reeds, elephant grass, kenaf, hemp residue, hollow fruit bundles, etc.), and lignin is converted into non-condensation reactive lignin (alkoxylated and / or non-alkoxylated forms) and optionally lignin sulfonates. The method involves pretreatment with a volatile alcohol solution containing sulfur dioxide, followed by chemical recovery, cellulose saccharification, and sugar fermentation. Non-condensed lignin and lignin sulfonate components can be separated after saccharification, fermentation, or after fermentation product separation. As described herein, the innovative combination of process steps significantly improves the yield of sugars and non-condensed lignin in lignocellulosic biomass and significantly simplifies downstream processing operations. The pulp washing step is eliminated compared to existing processes using organic solvents (organic solvents, SO2-alcohol-water, and other processes). These improvements enable a highly efficient and economical biorefining process.

[0020] In one aspect, the present invention includes a method for producing one or more fermentation products and lignin, comprising (a) contacting lignocellulosic biomass or cooked lignocellulosic biomass with a pretreatment liquid in a closed system under pressure and at elevated temperatures (e.g., from about 120°C to about 190°C); thereby releasing and / or dissolving lignin and hemicellulose in the lignocellulosic biomass into the pretreatment liquid, to obtain a mixture comprising lignin dissolved in the pretreatment liquid, hemicellulose dissolved and hydrolyzed into pretreatment liquid monosaccharides, and a solid portion comprising mainly cellulose from the lignocellulosic biomass, the mixture being referred to as pretreated material.

[0021] The method further includes one or more of the following steps: (b) recovering sulfur dioxide and alcohol to precipitate lignin, producing a first mixture comprising cellulose, lignin, and monosaccharides; (c) adjusting the pH of the first mixture (or a modified first mixture) to a level suitable for enzymatic hydrolysis; (d) contacting the pH-modified first mixture with cellulase, glucosidase, hemicellulase, glycosidase, or a combination thereof to provide a subsequent mixture comprising cellulose-derived glucose and hemicellulase-derived monosaccharides, as well as lignin (alkoxylated lignin and / or non-alkoxylated lignin); and (e) fermenting the subsequent mixture. Attached Figure Description

[0022] The following drawings are part of the specification and are used to further describe some embodiments or different aspects of the invention. In some instances, embodiments of the invention can be better understood by referring to the drawings and the detailed description provided herein. These descriptions and drawings may highlight certain specific embodiments or aspects of the invention. However, those skilled in the art will understand that embodiments or aspects of these parts can also be used in conjunction with other embodiments or aspects of the invention.

[0023] Figure 1 Example of a process flow diagram for producing one or more fermentation products and lignin fractions. In addition to producing one or more fermentation products, lignin fractions can be separated at different points in the process to provide commercial products.

[0024] Figure 2 Example of a process flow diagram for method 1, where the biomass feed is softwood (such as pine or spruce), the fermentation product is ethanol, and the product yield is based on 1 metric ton of dry biomass. Typical softwood biomass containing 650 kg of polysaccharides per 1 metric ton of dry biomass is used. This diagram corresponds to Example 1.1 (monosaccharide yield is 79% of the available polysaccharides in the biomass feedstock). The fermentation yield of 87% (theoretical percentage) is based on the yield obtained from separate fermentations of similar components. The yields of lignin and lignin sulfonates were determined in separate experiments.

[0025] Figure 3 Example of a flow chart for process method 2, where the biomass feed is softwood (such as pine or spruce), the fermentation product is ethanol, and the product yield is based on 1 metric ton of dry biomass. Typical softwood biomass containing 650 kg of polysaccharides per 1 metric ton of dry biomass is used. This figure corresponds to Example 1.7 (monosaccharide yield is 83% of the available polysaccharides in the biomass feedstock). The fermentation yield of 87% (theoretical percentage) is based on yields obtained from separate fermentations of similar components. The yields of lignin and lignin sulfonates were determined in separate experiments.

[0026] Figure 4 Example of a process flow diagram for method 1, where the biomass feed is corn stalks (agricultural residue), the fermentation product is ethanol, and the product yield is based on 1 metric ton of dry biomass. The corn stalks used in the experiment contained 555 kg of polysaccharides per 1 metric ton of dry biomass. This diagram corresponds to Example 2.9 (91% monosaccharide yield based on polysaccharides available in the biomass feedstock). The fermentation yield of 87% (theoretical percentage) is based on the results obtained in Example 4. The yields of lignin and lignin sulfonates are calculated based on the results obtained from pine alone.

[0027] Figure 5Example of a flow chart for process method 2, where the biomass feed is corn stalks (agricultural residue), the fermentation product is ethanol, and the product yield is based on 1 metric ton of dry biomass. The corn stalks used in the experiment contained 555 kg of polysaccharides per metric ton of dry biomass. This figure corresponds to Examples 2.3 and 2.4 (monosaccharide yields of available polysaccharides in the biomass feedstock were 85% and 93%, respectively). The fermentation yield of 87% (theoretical %) is based on the results obtained in Example 4. The yields of lignin and lignin sulfonates are yields calculated based on the results for pine alone.

[0028] Figure 6 The monosaccharide yield of corn straw biomass obtained by the method described in Example 2 was compared with the monosaccharide yield obtained by conventional hot water (hydrothermal) pretreatment (Example 3). Detailed Implementation

[0029] This invention generally relates to a pretreatment process for converting lignocellulosic biomass into various products, including but not limited to fermentable sugars and lignin fractions, wherein the pretreatment process is carried out in a closed system. As used herein, lignin fraction refers to one or more of lignin, alkoxylated lignin, and lignin sulfonates. Sugars are fermented into one or more fermentation products. Fermentation products may include, for example, ethanol, other alcohols, organic acids, biopolymers, microbial oils, microbial proteins, yeast biomass, etc.

[0030] Process Method 1:

[0031] In a first embodiment, referred to herein as Method 1, the present invention provides a method for producing one or more fermentation products and lignin from lignocellulosic biomass, comprising:

[0032] (a) Contacting lignocellulosic biomass or distilled lignocellulosic biomass with a pretreatment solution under elevated temperature and pressure;

[0033] The pretreatment solution contains sulfur dioxide, volatile alcohol, and water;

[0034] This releases and / or dissolves lignin and hemicellulose in lignocellulosic biomass into a pretreatment solution, resulting in a mixture containing lignin and hemicellulose dissolved in the pretreatment solution, and a solid portion mainly consisting of cellulose from lignocellulosic biomass. This mixture is referred to as the pretreated material.

[0035] (b1) Recover sulfur dioxide and alcohol, thereby precipitating lignin and producing a first mixture containing cellulose, lignin and monosaccharides;

[0036] (c) Adjust the pH of the first mixture to a level suitable for enzymatic hydrolysis, such as 4 to 7, 4.5 to 6.5 or 5 to 6;

[0037] (d) Contact the pH-adjusted first mixture with cellulase, glucosidase, hemicellulase, glycosidase or a combination thereof to provide a second mixture comprising glucose derived from cellulose and monosaccharides derived from hemicellulase and lignin.

[0038] (d2) Optionally, remove lignin, alkoxylated lignin, and / or lignin sulfonate from the second mixture that provides the third mixture;

[0039] (e) Fermenting the second or third mixture, optionally combined with adjusting the pH of the second or first mixture to, for example, 3.5 to 7, 4.5 to 6.5 or about 5 to about 6, to produce a fermentation broth containing one or more fermentation products;

[0040] (e2) Optionally remove lignin, alkoxylated lignin and / or lignin sulfonate from the fermentation broth;

[0041] (f) Optionally, a mash column may be used to separate one or more fermentation products and to provide a lean mixture of fermentation products (e.g., lees); and

[0042] (f2) Optionally remove lignin, alkoxylated lignin and / or lignin sulfonate from the lean fermentation product mixture.

[0043] In some embodiments, the temperature increase in step (a) is from about 130°C to about 175°C.

[0044] In some implementations, step (a) is maintained at the elevated temperature for about 5 minutes to about 240 minutes.

[0045] In some implementations, the volatile alcohol in step (a) pretreatment is ethanol.

[0046] In some embodiments, the pretreatment liquid contains about 1% to about 50% by weight of sulfur dioxide, about 10% to about 80% by weight of volatile alcohols, and less than 89% by weight of water (including water from cooked lignocellulosic biomass).

[0047] In some embodiments, the pretreatment liquid contains about 5% to about 20% sulfur dioxide, about 20% to about 50% volatile alcohol and about 30% to about 75% water.

[0048] In some embodiments, the ratio of pretreatment liquid to lignocellulosic biomass is about 1 kg / kg to 12 kg / kg (including water from the cooked lignocellulosic biomass in the mass of the pretreatment liquid) based on the dry weight of the lignocellulosic biomass.

[0049] In some implementations, step (a) preprocessing is performed in a continuous mode. In other implementations, step (a) preprocessing is performed in a batch processing mode.

[0050] In some implementations, the recovery of free sulfur dioxide and alcohol in step (b1) utilizes one or more purge tanks, distillation columns, fractionation columns, and stripping columns. A series of steps may be employed.

[0051] In some embodiments, the first mixture in step (c) is maintained at an elevated temperature for about 5 minutes to about 15 hours before pH adjustment. In various embodiments, the elevated temperature is about 80°C to about 130°C.

[0052] In some embodiments, step (d) enzymatic hydrolysis is performed in a continuous mode. In other embodiments, step (d) enzymatic hydrolysis is performed in a batch mode. In some embodiments, step (d) enzymatic hydrolysis may be performed sequentially in two or more steps, for example, these steps may be liquefaction and saccharification. Some or all of the steps may be batch, feed-batch, or continuous.

[0053] In some embodiments, the high total solids content of step (d) enzymatic hydrolysis can be >9%, for example 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% or higher.

[0054] In some embodiments, step (d) enzymatic hydrolysis can be performed by adding the full amount of pretreated material and the full amount of enzyme at the start of enzymatic hydrolysis.

[0055] In some embodiments, step (d) enzymatic hydrolysis can be performed by adding the pretreated material in batches or steps to the enzymatic hydrolysis, or by adding the enzyme in batches, steps or all at once.

[0056] In some embodiments, step (d) enzymatic hydrolysis can be performed by adding the enzyme in batches or steps to the enzymatic hydrolysis, while adding the pretreated material in batches, steps, or in a single batch.

[0057] In all embodiments, there is no separation of cellulose from the remainder of the first mixture between step (b1) and step (d). In other words, both cellulose and precipitated lignin are present in the mixture undergoing enzymatic hydrolysis in step (d). The absence of a cellulose separation step reduces the equipment and energy required to operate the process.

[0058] In some embodiments, in step (d), the enzyme feed for enzymatic hydrolysis is less than 5 FPU, less than 4 FPU, less than 3.5 FPU, less than 3 FPU, less than 2.5 FPU, less than 2 FPU, or 0.2 to 5 FPU, 0.5 to 3.5 FPU, 0.5 to 3.3 FPU, 0.5 to 2.5 FPU, 0.5 to 2 FPU, or 0.8 to 1.6 FPU per odg of biomass.

[0059] In some embodiments, in step (d2), step (e2), or step (f2), lignin, alkoxylated lignin, and / or lignin sulfonate are partially or completely separated.

[0060] In some embodiments, before the enzymatic hydrolysis step (d), before the fermentation step (e), or before both steps (d) and (e), the pH of the first mixture after step (b), the second mixture after step (d), or both are adjusted to pH 7-11 and maintained at 20-100°C for 10-120 minutes, and then the pH is adjusted to less than 7.

[0061] In some embodiments, residual sulfur dioxide is removed from the first mixture after step (b), the second mixture after step (d), or both, by adding one or more aqueous solutions containing hydrogen peroxide and / or by adding air or steam.

[0062] In some embodiments, the lignocellulose biomass is hardwood, softwood, or herbaceous biomass. In some embodiments, the lignocellulose biomass is softwood. In some embodiments, the lignocellulose biomass is hardwood. In some embodiments, the lignocellulose biomass is herbaceous biomass. In other embodiments, the method can be carried out with a mixture of lignocellulose biomass, such as one or more combinations of softwood, hardwood, and herbaceous biomass. For example, two or more types of hardwood and / or softwood can be processed simultaneously using the same equipment and the same or similar process conditions.

[0063] Method 1 can be configured such that sugar and lignin dealkoxylation may not occur or may not be completed in step (b1), for example, due to low temperature and / or low retention time. The extent of the reaction can be adjusted by changing the conditions of step (b1). If desired, the dealkoxylation reaction can be carried out during the enzymatic hydrolysis step (d). In some embodiments, lignin dealkoxylation is not performed or only limited to produce alkoxylated lignin. The pretreatment conditions of process method 1 can be configured to minimize the production of alkoxylated sugars and / or alkoxylated lignin.

[0064] Process Method 2:

[0065] Process method 2 is similar to process method 1, but it has an additional step, step (b2), in which the dealkoxylation reaction is carried out by heat treatment. In some cases, such as when complete or near-complete decarboxylation of lignin and / or sugars is required, process method 2 is advantageous. Lignin dealkoxylation may be the preferred method for some downstream applications. Furthermore, or as an alternative, if maximizing sugar fermentation yield is important, then sugar decarboxylation in process method 2 may be preferable to the simplified method of process method 1. Lignin and sugar decarboxylation in process method 2 also provides higher pretreatment alcohol recovery rates.

[0066] In a second embodiment, referred to herein as process method 2, the present invention provides a method for producing one or more fermentation products and lignin from lignocellulosic biomass, comprising:

[0067] (a) Contacting lignocellulosic biomass or distilled lignocellulosic biomass with a pretreatment solution at an elevated temperature;

[0068] The pretreatment solution includes sulfur dioxide, volatile alcohols, and water;

[0069] This releases and / or dissolves lignin and hemicellulose in lignocellulosic biomass into a pretreatment solution, resulting in a mixture containing lignin and hemicellulose dissolved in the pretreatment solution, and a solid portion mainly consisting of cellulose from lignocellulosic biomass. This mixture is referred to as the pretreated material.

[0070] (b1) Recover sulfur dioxide and alcohol, thereby precipitating lignin and producing a first mixture containing cellulose, lignin and monosaccharides;

[0071] (b2) The first mixture is maintained at elevated temperature and desired pressure and mixing or recycling rate, whereby volatile alcohols and sulfur dioxide, optionally chemically or loosely bound to one or more sugars (e.g., in alkyl glycosides and α-hydroxysulfonic acids), lignin (e.g., in alkoxylated lignin) and other compounds (e.g., in esters and acetals), are released / separated and optionally recovered, and oligosaccharides and polysaccharides are optionally hydrolyzed to release monosaccharides and optionally decarboxylate lignin, providing a second mixture comprising cellulose, lignin and monosaccharides;

[0072] (c) Adjust the pH of the second mixture to a level suitable for enzymatic hydrolysis, such as 4 to 7, 4.5 to 6.5, or 5 to 6;

[0073] (d) Contact the pH-adjusted second mixture with cellulase, glucosidase, hemicellulase or a combination thereof to provide a third mixture comprising glucose derived from cellulose and monosaccharides derived from hemicellulase and lignin.

[0074] (d2) Optionally, lignin, alkoxylated lignin and / or lignin sulfonate are removed from the third mixture to provide a fourth mixture;

[0075] (e) Fermenting the third or fourth mixture, optionally combined with pH adjustment, for example to 3.5 to 7, 4.5 to 6.5 or about 5 to about 6, to produce a fermentation broth containing one or more fermentation products;

[0076] (e2) Optionally remove lignin, alkoxylated lignin and / or lignin sulfonate from the fermentation broth;

[0077] (f) Optionally, a mash column may be used to separate one or more fermentation products to provide a lean fermentation product mixture (e.g., lees); and

[0078] (f2) Optionally remove lignin, alkoxylated lignin and / or lignin sulfonate from the lean fermentation product mixture.

[0079] In some embodiments, the temperature rise in step (a) is from about 130°C to about 175°C.

[0080] In some implementations, step (a) is maintained at the elevated temperature for about 5 minutes to about 240 minutes.

[0081] In some implementations, the volatile alcohol in step (a) pretreatment is ethanol.

[0082] In some embodiments, the pretreatment liquid contains about 1% to about 50% by weight of sulfur dioxide, about 10% to about 80% by weight of volatile alcohols, and less than 89% by weight of water (including water from cooked lignocellulosic biomass).

[0083] In some embodiments, the pretreatment liquid comprises about 5% to about 20% by weight of sulfur dioxide, about 20% to about 50% by weight of volatile alcohol, and about 30% to about 75% by weight of water.

[0084] In some embodiments, the ratio of pretreatment liquid to lignocellulosic biomass is about 1 kg / kg to about 12 kg / kg based on the dry weight of the lignocellulosic biomass (including water from the cooked lignocellulosic biomass in the mass of the pretreatment liquid).

[0085] In some implementations, step (a) preprocessing is performed in a continuous mode. In other implementations, step (a) preprocessing is performed in a batch processing mode.

[0086] In some embodiments, the recovery of free sulfur dioxide and alcohol in step (b1) utilizes one or more purge tanks, distillation columns, fractionation columns, and stripping columns. A series of steps may be used.

[0087] In some embodiments, retention of the pretreated material in step (b2) is performed in a tank or series of tanks equipped with a heated recirculation line and optionally one or more distillation columns, fractionation columns, and stripping columns. Depending on the chemical substance to be removed, the columns may consist of a disk, packing material, or a combination of both. Transfer of the pretreated material to the tank can be achieved by differential pressure (purging), pumping, or other methods.

[0088] In some embodiments, step (b2) is carried out in a tank or series of tanks for a desired time, such as 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 hours, at a desired temperature, such as 50, 60, 70, 75, 80, 85, 90, 95, 97.5, 100, 102.5, 105, 107.5, 110, 112.5, 115, 117.5, 120, 122.5 or 125°C, and at the obtained or desired pressure, whereby alcohols and optional chemically or loosely bound sulfur dioxide to sugars, lignin and other compounds are released and optionally recovered, while one or more of alkoxylated lignins and alkyl glycosides (alkoxylated sugars) are optionally dealkoxylated, and oligosaccharides are optionally hydrolyzed into monosaccharides. The amount of alcohol released can be controlled by temperature and reaction time.

[0089] In some embodiments, in step (b2), free alcohol, optional sulfur dioxide, and optional pretreatment byproducts, including but not limited to acetic acid, formic acid, methanol, alkyl acetate, and furfural, are partially or completely removed from the pretreated material and concentrated in the desired manner.

[0090] In some embodiments, step (d) enzymatic hydrolysis is performed in a continuous mode. In other embodiments, step (d) enzymatic hydrolysis is performed in a batch mode.

[0091] In some implementations, step (d) enzymatic hydrolysis can be performed sequentially in two or more steps, such as liquefaction and saccharification. Some or all of these steps can be performed in batch or continuous mode.

[0092] In some embodiments, the high total solids content of step (d) enzymatic hydrolysis can be >9%, for example 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% or higher.

[0093] In some embodiments, step (d) enzymatic hydrolysis can be carried out by adding the full amount of pretreated material and the full amount of enzyme at the start of enzymatic hydrolysis.

[0094] In some embodiments, step (d) enzymatic hydrolysis can be performed by adding the pretreated material in batches or steps during enzymatic hydrolysis, while adding the enzyme in batches, steps, or all at once.

[0095] In some embodiments, step (d) enzymatic hydrolysis can be performed by adding the enzyme in batches or steps during enzymatic hydrolysis, while adding the pretreated material in batches, steps, or in a single batch.

[0096] In all embodiments, between steps (b1) and (d), or between steps (b2) and (d), there is no separation of cellulose from the remainder of the first or second mixture. In other words, both cellulose and precipitated lignin are present in the mixture where enzymatic hydrolysis occurs in step (d). The absence of a cellulose separation step reduces the equipment and energy required to operate this process.

[0097] In some embodiments, in step (d), the enzyme charge for the enzymatic hydrolysis process is less than 5 FPU, less than 4 FPU, less than 3.5 FPU, less than 3 FPU, less than 2.5 FPU, less than 2 FPU, or 0.2 to 5 FPU, 0.5 to 3.5 FPU, 0.5 to 3.3 FPU, 0.5 to 2.5 FPU, 0.5 to 2 FPU, or 0.8 to 1.6 FPU per odg of biomass.

[0098] In some embodiments, in step (d2), step (e2), or step (f2), lignin, alkoxylated lignin, and / or lignin sulfonate are partially or completely separated.

[0099] In some embodiments, the alkoxylated lignin is partially or completely dealkoxylated in step (b1) and / or step (b2), and the resulting lignin material is partially or completely separated in step (d2), step (e2), or step (f2).

[0100] In some embodiments, before the enzymatic hydrolysis step (d), before the fermentation step (e), or before both steps (b) and (e), the pH of the second mixture after step (b2), the third mixture after step (d), or both are adjusted to 7-11 and maintained at 20-100°C for 10-120 minutes, and then the pH is adjusted to less than 7.

[0101] In some embodiments, residual sulfur dioxide is removed from the second mixture after step (b2), the first mixture after step (d), or both, by adding one or more aqueous solutions containing hydrogen peroxide and / or by adding air or steam.

[0102] In some embodiments, the lignocellulosic biomass is hardwood, softwood, or herbaceous biomass. In some embodiments, the lignocellulosic biomass is softwood. In some embodiments, the lignocellulosic biomass is hardwood. In some embodiments, the lignocellulosic biomass is herbaceous biomass. In other embodiments, the process can use a mixture of lignocellulosic biomass, such as one or more combinations of softwood, hardwood, and herbaceous biomass. For example, two or more types of hardwood and / or softwood can be processed simultaneously using the same equipment and the same or similar process conditions.

[0103] Process Method 1 and Process Method 2:

[0104] In some embodiments, the volatile alcohol is methanol, ethanol, 1-propanol, 2-propanol, 2-methyl-2-propanol, or a mixture thereof. In a preferred embodiment, the volatile alcohol is ethanol.

[0105] In some embodiments, one or more volatile hydrocarbons (e.g., one or more pentanes, hexanes, cyclopentanes, and cyclohexanes), ethers (e.g., one or more diethyl ethers and methyl tert-butyl ethers (MTBE)) or mixtures thereof may be used instead of volatile alcohols or in addition to volatile alcohols.

[0106] In some embodiments, sulfur dioxide is recovered in step (b1) (process 1) or in step (b1) and optionally step (b2) (process 2), including:

[0107] (i) Releasing gaseous vapors from a pretreated material at elevated temperatures and / or low pressures, wherein the gaseous vapors released from the pretreated material include sulfur dioxide and one or more water vapors and alcohol vapors;

[0108] (ii) Concentrate at least a portion of the gas vapor to provide sulfur dioxide gas and a liquid containing at least one of water and alcohol;

[0109] (iii) Optionally, sulfur dioxide gas is purified by removing trace amounts of water vapor and volatile organic compounds;

[0110] (iv) To provide liquid sulfur dioxide by liquefying sulfur dioxide gas through pressure changes and / or temperature reductions; and optionally to store the liquid sulfur dioxide; and

[0111] (v) Optionally, liquid sulfur dioxide is introduced into the container of step (a) or another pretreatment container by pumping.

[0112] In some embodiments, ethanol is used as a volatile alcohol, obtained in step (ii), concentrated, and used in one or more desired ways, which are listed as optional: 1) recycling back to the pretreatment; and / or 2) mixing with the fermentation broth and recovering it together with the ethanol produced in the fermentation process.

[0113] In some embodiments, one or more of water, phosphoric acid, hydrochloric acid, sulfuric acid, nitric acid, acetic acid, formic acid, oxalic acid and process concentrate are added to step (b1) (process method 1) or step (b1) and / or step (b2) (process method 2).

[0114] In some embodiments, step (b1) of method 1 or 2 and step (b2) of method 2 are performed by retaining and recirculating the pretreated material for a period of time in a tank equipped with a heated recirculation line and a distillation column, fractionation column, or stripping column, thereby removing volatile compounds (e.g., volatile alcohols, sulfur dioxide, and pretreatment byproducts) from the pretreated material by flash evaporation; thereby removing volatile compounds from the pretreated material and concentrating and recovering volatile compounds. Volatile pretreatment byproducts may include, but are not limited to, acetic acid, formic acid, methanol, ethyl acetate, and furfural.

[0115] In some implementations, ethanol produced during fermentation is separated by distillation.

[0116] In some embodiments, after the removal of volatile alcohols (typically ethanol), alkoxylated and / or non-alkoxylated lignin fractions are separated, for example by centrifugation or by using a filter press, or both.

[0117] In some implementations, lignin sulfonates are produced, and if desired, can be separated at various points in the process, such as after enzymatic hydrolysis, after fermentation, or after the removal of volatile alcohols (typically ethanol). Lignosulfonates can be separated, for example, by membrane filtration.

[0118] In some embodiments, prior to step (a), the lignocellulosic biomass is contacted with steam, wherein the lignocellulosic biomass absorbs water from the steam and removes air and optional turpentine from the lignocellulosic biomass. The contact of the biomass with steam can take place in a first container, such as a cooking tank. The biomass can then be transferred to a separate container for pretreatment step (a).

[0119] In various implementations, distillation columns, fractionation columns, or stripping columns and concentrators are used to facilitate the separation of sulfur dioxide and volatile alcohol components.

[0120] In one specific embodiment of method 1, the present invention provides a method for producing one or more fermentation products and lignin, comprising:

[0121] (a) The cooked lignocellulosic biomass was contacted with the pretreatment liquid for 5 to 60 minutes at a temperature of about 145°C to about 175°C and under pressure.

[0122] The pretreatment liquid contains 3 to 30% by weight sulfur dioxide, 10 to 50% by weight ethanol and 20 to 87% by weight water (including water from cooked lignocellulosic biomass);

[0123] This releases and / or dissolves lignin and hemicellulose in lignocellulosic biomass into a pretreatment solution, resulting in a mixture comprising lignin, lignin sulfonic acid (LS), and hemicellulose dissolved in the pretreatment solution, as well as a cellulose-containing solid fraction from the lignocellulosic biomass. This mixture is referred to as the pretreated material.

[0124] (b1) Recover sulfur dioxide and ethanol, thereby precipitating lignin and producing a first mixture containing cellulose, lignin, lignin sulfonic acid (LS) and monosaccharides;

[0125] (c) Adjust the pH of the second mixture to between approximately 4.8 and approximately 5.8 for enzymatic hydrolysis;

[0126] (d) At a temperature of about 50°C to about 60°C, a pH-adjusted second mixture is contacted with a combination of cellulase, glycosidase, and hemicellulase to provide a third mixture comprising glucose derived from cellulose and monosaccharides derived from hemicellulase, as well as lignin and lignin sulfonate (salts of lignin sulfonic acid); and

[0127] (e) Fermenting the third mixture to produce one or more fermentation products, optionally combined with adjusting the pH of the third mixture. The preferred fermentation product is ethanol. However, this process can provide other fermentation products, including a group of fermentation products that are the main or only product other than ethanol.

[0128] In one specific embodiment of process method 2, the present invention provides a method for producing one or more fermentation products and lignin, comprising:

[0129] (a) The cooked lignocellulosic biomass was contacted with the pretreatment liquid for 5 to 60 minutes at a temperature of about 145°C to about 175°C and under pressure.

[0130] The pretreatment solution contains 3 to 30% by weight sulfur dioxide, 30 to 60% by weight ethanol and 10 to 67% by weight water (including water from cooked lignocellulosic biomass);

[0131] This releases and / or dissolves lignin and hemicellulose in lignocellulosic biomass into a pretreatment solution, resulting in a mixture comprising lignin, lignin sulfonic acid (LS), and hemicellulose dissolved in the pretreatment solution, as well as a cellulose-containing solid fraction from the lignocellulosic biomass. This mixture is referred to as the pretreated material.

[0132] (b1) Recover sulfur dioxide and ethanol, thereby precipitating lignin and producing a first mixture containing cellulose, lignin, lignin sulfonic acid (LS) and monosaccharides;

[0133] (b2) The first mixture is maintained at a temperature of about 90-130°C, a desired pressure and a mixing rate or recycling rate for 0.1 to 8 hours, so that ethanol and sulfur dioxide, optionally chemically or loosely bound to sugars, lignin and other compounds, are released / separated and optionally recovered, and oligosaccharides and polysaccharides are optionally hydrolyzed to release monosaccharides, providing a second mixture, wherein the second mixture contains a combination of furfural, hydroxymethylfurfural and levulinic acid in less than 10 kg per BD metric ton of wood;

[0134] (c) Adjust the pH of the second mixture to approximately 5 to approximately 6 for enzymatic hydrolysis;

[0135] (d) At a temperature of about 50°C to about 60°C, a pH-adjusted second mixture is contacted with a combination of cellulase, glycosidase, and hemicellulase to provide a third mixture comprising glucose derived from cellulose and monosaccharides derived from hemicellulase, as well as lignin and lignin sulfonate (salts of lignin sulfonic acid); and

[0136] (e) Fermenting the third mixture, optionally combined with adjusting the pH of the third mixture, to produce one or more fermentation products. The preferred fermentation product is ethanol. However, the process may provide other fermentation products, including a group of fermentation products that are the main or only product other than ethanol.

[0137] In some embodiments of this process, the ethanol obtained in steps (b1) and optionally (b2) is concentrated and recycled back to the pretreatment process; and / or at least partially mixed with the fermentation broth and recovered along with the ethanol produced during fermentation. In various embodiments, in steps (b1) and / or (b2), one or more pretreatment byproducts, including but not limited to acetic acid, formic acid, methanol, ethyl acetate, and furfural, are partially or completely removed from the pretreated material and concentrated in the desired manner.

[0138] As will be readily understood by those skilled in the art, in process methods 1 and 2, the pH of the composition to be enzymatically hydrolyzed or fermented is typically adjusted to an appropriate pH level for hydrolysis or fermentation, for example, to the pH described herein for the hydrolysis or fermentation step, in order to provide suitable conditions for hydrolysis or fermentation.

[0139] In some embodiments, the lignocellulosic biomass is derived from cork. Obtaining high yields of saccharified products from cork lignocellulosic biomass (e.g., pine, spruce, Douglas fir, larch, cedar, etc.) is more difficult than from hardwood and herbaceous lignocellulosic biomass. The methods described herein can economically provide high yields of 1) cellulose-derived fermentable glucose and 2) hemicellulose-derived fermentable monomeric sugars from all types of biomass (including cork-derived lignocellulosic biomass) simultaneously without producing sticky lignin precipitates, wherein the total yield of cork is based on approximately 76-83% or higher of polysaccharides available in the biomass and an enzyme loading of 3.3 FPU / odg biomass or lower. This yield is equivalent to or greater than 540-600 kg of monosaccharides per BD metric ton of cork biomass, containing 650 kg of polysaccharides (dehydrated) per BD metric ton. The total yield from hardwoods (e.g., poplar, birch, beech, eucalyptus, aspen, maple, oak), agricultural waste (e.g., corn stalks, wheat stalks, sugarcane stalks, empty fruit bundles, etc.) and energy crops (e.g., energy sugarcane) is based on approximately 81-85% or higher of available polysaccharides in the biomass and an enzyme loading of 0.8 FPU / odg biomass or less, or approximately 83-93% or higher of available polysaccharides in the biomass and an enzyme loading of 1.6 FPU / odg biomass or less. This yield is equivalent to or greater than 505-580 BD kg monosaccharides per BD metric ton of corn stalk biomass, containing 555 kg of polysaccharides (dehydrated) per BD metric ton.

[0140] The cellulose produced in this process exhibits high enzymatic digestibility, with dextran conversion to glucose exceeding 80%, 85%, 90%, or 95%, while the enzyme loading is low, for example, approximately 3.3 FPU per odg of biomass or less for softwood, or approximately 1.6 FPU per odg of biomass, or approximately 0.8 FPU per odk of biomass or less for hardwood and herbaceous biomass. The corresponding values ​​for hemicellulose conversion to monosaccharides exceed 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Alkyl glycosides, if present, are optionally converted to monosaccharides by enzymes (step (d) in process 1) or optionally by retaining the pulp (first mixture) at elevated temperatures (step (b2) in process 2). The sugars readily ferment without the need for detoxification, likely due to low sugar and lignin degradation during the process.

[0141] Surprisingly, despite the presence of relatively large amounts of water-insoluble lignin (in alkoxylated and / or non-alkoxylated forms), enzymatic hydrolysis proceeded smoothly with high glucose yields even under low enzyme loadings (e.g., 0.8 FPU or less per odg of biomass, or 1.6 FPU or less per odg of biomass). Equally surprising is that, after pretreatment, the undetoxified whole pulp can be enzymatically hydrolyzed under low enzyme loadings to obtain high yields of monosaccharides (e.g., 83% or higher yield for softwood and 93% or higher yield for herbaceous biomass, based on availability in the biomass). Therefore, this innovation eliminates the need for pulp washing.

[0142] In a preferred embodiment, the present invention provides a method for producing ethanol and lignin from softwood lignocellulosic biomass. The softwood lignocellulosic biomass may be contacted with a pretreatment liquid at about 150°C (±5°C) and pressure to provide a mixture comprising lignin and hemicellulose dissolved in the pretreatment liquid, and a solid fraction primarily comprising cellulose. The pretreatment liquid may contain about 7-15% sulfur dioxide, about 42-46.5% ethanol, and about 43-46.5% water. In a preferred embodiment, the pretreatment liquid contains 5-9% sulfur dioxide, preferably 6-8% sulfur dioxide. In another preferred embodiment, the pretreatment liquid contains 13-17% sulfur dioxide, preferably 14-16% sulfur dioxide. The pH of the first mixture comprising cellulose, lignin, and monosaccharides may be adjusted to a suitable level for enzymatic hydrolysis, for example, a pH of about 5 to about 6, preferably about 5.5. Contacting the first mixture with one or more enzymes provides a second mixture comprising glucose, other monosaccharides, and lignin, wherein the total monomeric sugar content in the second mixture is 79% or higher, based on polysaccharides available in cork lignocellulosic biomass. This process can then be completed as described above for process method 1.

[0143] In a preferred embodiment, the present invention provides a method for producing ethanol and lignin from softwood lignocellulosic biomass, comprising (a) contacting the softwood lignocellulosic biomass with a pretreatment liquid at about 150°C (±5°C) and pressure; wherein the pretreatment liquid comprises about 7-15% sulfur dioxide, about 42-46.5% ethanol, and about 43-46.5% water. In a preferred embodiment, the pretreatment liquid comprises 5-9% sulfur dioxide, preferably 6-8% sulfur dioxide. In another preferred embodiment, the pretreatment liquid comprises 13-17% sulfur dioxide, preferably 14-16% sulfur dioxide. In step (b1), sulfur dioxide and ethanol are preferably recovered, which results in lignin precipitation, producing a first mixture comprising cellulose, lignin, and monosaccharides. In step (b2), the first mixture is preferably maintained at about 99°C to about 120°C and at appropriate pressure and mixing or recycling rates, such that ethanol and sulfur dioxide, optionally chemically or loosely bound to one or more sugars (e.g., in ethyl glycosides and α-hydroxysulfonic acids), lignin (e.g., in ethoxylated lignin), and other compounds (e.g., in esters and acetals), are released / separated and optionally recovered, and oligosaccharides and polysaccharides are optionally hydrolyzed to release monosaccharides and optionally deethoxylate lignin, providing a second mixture. In step (c), the pH of the second mixture is adjusted to a level suitable for enzymatic hydrolysis, preferably a pH of about 5 to about 6. In step (d), the pH-adjusted second mixture is contacted with cellulase, glucosidase, hemicellulase, or a combination thereof to provide a third mixture comprising glucose derived from cellulose and monosaccharides derived from hemicellulosic acid, and lignin; wherein the total monosaccharide content in the second mixture, based on polysaccharides available in softwood lignocellulosic biomass, is 80% or higher. Then, the process can be completed as described above for process method 2.

[0144] Equally surprising is that some biomass species, including pine, which is unsuitable for acid sulfite pulping, can be used in the process described herein.

[0145] Furthermore, contrary to existing techniques, the pretreatment surprisingly does not require the explosion of fiber structures to achieve high monosaccharide yields. Therefore, in some embodiments, the methods described herein exclude the explosion of fiber structures after pretreatment.

[0146] In a preferred embodiment, the present invention provides a method for producing one or more fermentation products and lignin from herbaceous biomass / agricultural residues (e.g., corn stalks), comprising:

[0147] (a) Herbaceous lignocellulosic biomass or distilled herbaceous lignocellulosic biomass can be brought into contact with the pretreatment solution at approximately 140-150°C and pressure;

[0148] The pretreatment solution contains approximately 3-7% sulfur dioxide, approximately 23-24% ethanol, and approximately 69-74% water;

[0149] This releases and / or dissolves lignin and hemicellulose in lignocellulosic biomass into a pretreatment solution, resulting in a mixture comprising lignin and hemicellulose dissolved in the pretreatment solution and a solid portion consisting mainly of cellulose from lignocellulosic biomass, referred to as the pretreated material.

[0150] (b1) Recover sulfur dioxide and alcohol, thereby precipitating lignin and producing a first mixture containing cellulose, lignin and monosaccharides;

[0151] (c) Adjust the pH of the first mixture to an appropriate level for enzymatic hydrolysis, for example, to about pH 5 to about pH 6;

[0152] (d) Contact the pH-adjusted first mixture with cellulase, glucosidase, hemicellulase, glycosidase or a combination thereof to provide a second mixture comprising glucose derived from cellulose and monosaccharides derived from hemicellulase and lignin.

[0153] The monomeric sugar content in the second mixture, based on the polysaccharides available in herbaceous lignocellulosic biomass, is 86% or higher. This process can then be completed as described above for process method 1.

[0154] In another preferred embodiment, the present invention provides a method for producing one or more fermentation products and lignin from herbaceous lignocellulosic biomass / agricultural residues (e.g., corn stalks), comprising:

[0155] (a) Contact herbaceous lignocellulosic biomass or distilled herbaceous lignocellulosic biomass with a pretreatment solution at approximately 150°C and pressure;

[0156] The pretreatment solution contains approximately 5% sulfur dioxide, approximately 47.5% ethanol, and approximately 47% water;

[0157] This releases and / or dissolves lignin and hemicellulose in lignocellulosic biomass into a pretreatment solution, resulting in a mixture containing lignin and hemicellulose dissolved in the pretreatment solution, as well as a solid portion mainly consisting of cellulose from lignocellulosic biomass. This mixture is referred to as the pretreated material.

[0158] (b1) Recover sulfur dioxide and ethanol, thereby precipitating lignin and producing a first mixture containing cellulose, lignin and monosaccharides;

[0159] (b2) The first mixture is maintained in a temperature range of about 99°C to about 120°C, and the required pressure and mixing or recycling rate are maintained, so that ethanol and sulfur dioxide, which are optionally chemically or loosely bound with one or more sugars (e.g., in ethyl glycosides and α-hydroxysulfonic acid), lignin (e.g., in ethoxylated lignin) and other compounds (e.g., in esters and acetals), are released / separated and optionally recovered, and oligosaccharides and polysaccharides are optionally hydrolyzed to release monosaccharides and optionally deethoxylated lignin, to provide the second mixture;

[0160] (c) Adjust the pH of the second mixture to a level suitable for enzymatic hydrolysis, for example, to about pH 5 to about pH 6;

[0161] (d) Contact the pH-adjusted second mixture with cellulase, glucosidase, hemicellulase or a combination thereof to provide a third mixture comprising glucose derived from cellulose and monosaccharides derived from hemicellulase and lignin.

[0162] The monomeric sugar content in the second mixture, based on the polysaccharides available in herbaceous lignocellulosic biomass, is 85% or higher. This process can then be completed as described above for process method 2.

[0163] Furthermore, hardwood biomass (angiosperms) is easier to pretreat than softwood biomass (gymnosperms), and its pretreating efficiency is generally between that of softwood and herbaceous biomass. Therefore, the methods described herein can be carried out on hardwood biomass to provide monosaccharide yields equal to or greater than those of the softwood (gymnosperms) described herein under similar enzyme loading, and wherein the monosaccharide yield can be close to the yields of the herbaceous (non-woody) biomass (angiosperms) (e.g., corn stalks) described herein under similar enzyme loading.

[0164] Figure 1-5 A flowchart of the process described in this article is shown.

[0165] Figure 1 Process methods 1 and 2 are described. Step (a) pretreatment, steps (b1) and (b2) chemical recovery, step (c) pH adjustment before enzymatic hydrolysis, step (d) enzymatic hydrolysis, optional step (d2) separation of lignin, alkoxylated lignin and / or lignin sulfonate before fermentation, step (e) fermentation, optional step (e2) separation of lignin, alkoxylated lignin and / or lignin sulfonate after fermentation, step (f) separation of fermentation products, and optional step (f2) separation of lignin, alkoxylated lignin and / or lignin sulfonate after fermentation product separation.

[0166] Lignin, alkoxylated lignin, or a mixture thereof can be obtained by adjusting the pretreatment conditions and the conditions of step (b2) in process method 2 through processes 1 and 2. One or more lignins, alkoxylated lignins, mixtures thereof, and lignin sulfonates can be separated at desired process points, such as before fermentation (optional step (d2)), after fermentation (optional step (e2)), or after separation of fermentation products (optional step (f2)).

[0167] Figure 2 A flowchart example is provided when using cork biomass as raw material and using process method 1.

[0168] Figure 3 A flowchart example is provided when using cork biomass as raw material and using process method 2.

[0169] Figure 4 A flowchart example is provided when using corn stalk biomass as raw material and using process method 1.

[0170] Figure 5 A flowchart example is provided when using corn stalk biomass as raw material and using process method 2.

[0171] Figure 6 A comparison is provided of the monosaccharide yield obtained by the process described in Example 2 with the monosaccharide yield obtained by conventional hot water (hydrothermal) pretreatment of corn stalks (Example 3). Notably, the hot water process resulted in a lower sugar yield compared to the process described herein, under all test parameters. Even at much higher enzyme loadings, the hot water process did not achieve the yields of the process described herein. A comparison of the enzymatic hydrolysis monosaccharide yield of the process described herein with that of conventional hot water pretreatment at an enzyme loading of 1.6 FPU / odg biomass shows that the enzymatic hydrolysis monosaccharide yield increased from an average of 54% to an average of 90%, a significant increase of approximately 67%. Furthermore, at an enzyme loading of 6.6 FPU / odg biomass, the average monosaccharide yield of the conventional hot water process was 71%, while the average monosaccharide yield of the process described herein was 83% at an enzyme loading of only 0.8 FPU / odg biomass, representing a 17% increase in monosaccharide yield when the enzyme loading was reduced by 8-fold.

[0172] As described herein, one or more lignins, alkoxylated lignins, and lignin sulfonates can be removed from process method 1 or process method 2 at different points in the process. See Figure 1-5 However, the removal of lignin, alkoxylated lignin, and lignin sulfonate is optional and not required for high monosaccharide yields during enzymatic hydrolysis or for high fermentation product yields.

[0173] definition:

[0174] The following definitions are provided to provide a clear and consistent understanding of the specification and claims. The terms used herein have the following meanings. Other terms and phrases used in this specification have their common meanings as understood by those skilled in the art. Such common meanings can be obtained by referring to technical dictionaries, such as Hawley's Condensed Chemical Dictionary 14. th Edition, by RJ Lewis, John Wiley & Sons, New York, NY, 2001.

[0175] The terms "one embodiment" and "an embodiment" used in this specification indicate that the described embodiment may include specific aspects, features, structures, portions, or characteristics, but not every embodiment must include these specific aspects, features, structures, portions, or characteristics. Furthermore, these phrases may, but do not necessarily, apply to the same embodiments mentioned in other parts of this specification. Moreover, when a specific aspect, feature, structure, portion, or characteristic is described in conjunction with a certain embodiment, those skilled in the art should be able to understand that these specific aspects, features, structures, portions, or characteristics are applied to or associated with other embodiments, whether or not explicitly stated.

[0176] The singular articles "a," "an," and "the" include plural meanings unless the context clearly distinguishes them. Thus, for example, "compound" can refer to a plurality of such compounds, such that compound X includes a plurality of compounds X. It is further noteworthy that the claims may be drafted to exclude any optional elements. Therefore, this statement is intended as a precondition for the use of exclusionary terms such as "solely," "only," etc., associated with any element described herein and / or as a limitation on the elements stated in the claims or as a "negative" restriction.

[0177] The term "and / or" refers to any one, any combination, or all of the related items. The phrases "one or more" and "at least one" are readily understood by those skilled in the art, especially in context. For example, these phrases can refer to one, two, three, four, five, six, ten, one hundred, or any upper limit approximately 10, 100, or 1000 times higher than the lower limit already listed.

[0178] Those skilled in the art will understand that all figures, including those representing amounts of components, properties such as molecular weight, reaction conditions, etc., are approximate values ​​and are understood to be modifiable in all cases by the term "about". These values ​​may vary depending on the desired properties sought by those skilled in the art using the teachings described herein. It is also understood that these values ​​themselves contain variability inherent in the standard deviations found in their respective test measurements. When the values ​​are expressed as approximate, it is understood, by using the antecedent "about", that specific values ​​without the modifier "about" also constitute another aspect.

[0179] The term "about" indicates that a particular value can vary by ±5%, ±10%, ±20%, or ±25%. For example, a percentage of "about 50" can vary from 45% to 55% in some embodiments. For integer ranges, the term "about" can include each end of the range being one or two integers smaller or larger than the listed integer. Unless otherwise stated herein, the term "about" is used to include numerical values ​​that are close to the listed range and are equivalent to the function of the ingredients, combinations, or embodiments. The term "about" can also be used to adjust the endpoints of the ranges mentioned above in this paragraph.

[0180] The term "sticky lignin" refers to concentrated lignin that precipitates from aqueous solutions and binds to the surface of processing equipment, which can lead to decreased equipment performance by clogging and / or blocking equipment valves and pipes. The precipitated sticky lignin hardens over time, eventually requiring operation to be stopped to remove it from the equipment.

[0181] Those skilled in the art will understand that, for any purpose, especially for the purpose of providing a textual description, any range provided herein also includes all possible subranges and combinations of such subranges, as well as individual values, especially integer values, that constitute the range. Thus, it is understood that each unit between two specific units is also disclosed. For example, if 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed individually and as part of the range. The range (e.g., weight percentage or carbon groups) includes each specific value, integer, decimal, or characteristic within that range. Any listed range can be readily identified as sufficiently descriptive and decomposed into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily decomposed into a lower third, middle third, and upper third, etc. Those skilled in the art will also understand that all terms such as “at least,” “greater than,” “less than,” “more than,” “or above,” etc., include the cited numbers, and these terms refer to ranges that can subsequently be decomposed into subranges as described above. In the same manner, all ratios described herein also include all subratios falling within a wide ratio. Therefore, the specific values ​​for groups, substituents, and ranges are for illustrative purposes only; they do not exclude other limiting values ​​for groups and substituents or other values ​​within the defined ranges. It will be further understood that each endpoint of a range is meaningful relative to and independent of the other endpoint.

[0182] Those skilled in the art will also readily understand that when the units are combined in a conventional manner, for example, within a Markush group, the invention includes not only the entirety of the listed unit combinations, but also each individual unit of the group and any possible subgroups of the basic group. Furthermore, for all purposes, the invention includes not only the basic group, but also groups of the basic group without one or more units. Therefore, the invention may include any one or more units that explicitly exclude the cited groups. Consequently, constraints may be attached to any disclosed scope or implementation where any one or more units, types, or implementations may be excluded from that scope or implementation, for example, in cases of explicit negative limitation.

[0183] The following embodiments are provided to further describe the invention and should not be construed as limiting its scope. Those skilled in the art will readily recognize that the embodiments demonstrate that the invention can be implemented in many other ways. It should be understood that many variations and modifications are possible within the scope of the invention.

[0184] Example

[0185] Example 1: Pretreatment and enzymatic hydrolysis of Scots pine

[0186] Scots pine sawdust (biomass) containing 7% moisture (on a receiving basis), 37.8% dextran (on a drying basis), 5.4% xylan, 2.6% galactan, 1.7% arabinogalactan, 10.8% mannan, 1.2% acetyl, 23.4% lignin, 6.1% acetone extract, and 0.6% ash was prepared as a liquid for pretreatment, yielding a moisture-adjusting composition of 7-15% sulfur dioxide, 42-46.5% ethanol, and 43-46.5% water (by weight), added at a ratio of approximately 4 parts liquid to 1 part dry biomass (i.e., a liquid-to-solid ratio of 4 kg / kg). Deionized water was used. The biomass and liquid were mixed and pretreated under pressure at a final temperature of 150°C, reached approximately 10 minutes after the start of pretreatment. The heat applied to the pretreatment liquid and biomass causes an increase in pressure within the closed system of the small reactor. The time to final temperature is 60 minutes; the cooling time is approximately 2 minutes (by immersing the reactor in ice water).

[0187] Unreacted sulfur dioxide is slowly removed from the system via exhaust gas, without the use of fiber explosion. Pulp and liquid are not separated. The material is transferred to a rotary evaporator and evaporated at approximately 75°C for about 25 minutes, with the pressure gradually reduced from atmospheric pressure to approximately 150 Torr to provide a final total solids content of approximately 23-26%. To further remove ethanol, water is added and the evaporation cycle is repeated.

[0188] In various experiments, materials were heat-treated using rotary evaporation flasks or microreactors immersed in oil baths under various conditions (total solids content of approximately 20-27%, temperature of 99°C or 120°C, duration of 1-8 hours).

[0189] In all experiments, the material was transferred to shake flasks and diluted with deionized water. The pH of the mixture was adjusted to 4 by adding 30% calcium hydroxide slurry, and then adjusted to 5.3-5.5 with 15% ammonium hydroxide solution. The material was then subjected to enzymatic hydrolysis for 96 hours. A commercially available enzyme mixture containing cellulase, hemicellulase, and glucosidase was used. In all experiments, the total solids content in the enzymatic hydrolysis process was approximately 12-13%, the pH was maintained at 4.8-5.5 using 15% ammonium hydroxide solution, and the temperature was maintained at approximately 53°C. The shaking rate was 200 RPM.

[0190] Tables 1 and 2 show surprisingly high sugar yields at a low enzyme load of 3.3 FPU / odg biomass. Despite the presence of a significant amount of water-insoluble lignin fraction that typically inhibits enzymatic hydrolysis, the overall glucose yield was 81–91% (based on glucans available in the biomass). The total yield of hemicellulose sugars reached 68–74% (based on hemicellulose available in the biomass), which may be explained by the fact that the enzyme mix used was not optimized for softwood hemicellulose (i.e., glucomannan). At an enzyme dose of 3.3 FPU / odg biomass, the total monosaccharide yield of pine was 76–83% (based on polysaccharides available in the biomass). Further increases in yield can be expected through optimization of the enzyme mix or other means.

[0191] Component analysis to measure the content of sugar degradation products—furfural, hydroxymethylfurfural, and levulinic acid—revealed that their total content was less than 5 kg (<0.5%) per BD metric ton of biomass, a surprisingly low percentage given the relatively high pretreatment temperature (150°C).

[0192] Table 1: Sugar yield of Example 1 (7% sulfur dioxide).

[0193]

[0194] Table 2: Sugar yield of Example 1 (15% sulfur dioxide).

[0195]

[0196] Example 2: Pretreatment and enzymatic hydrolysis of corn stalks

[0197] Corn stalks (biomass) containing 8% moisture (on a receiving basis), 33.7% dextran (on a drying basis), 18.2% xylan, 0.0% galactan, 3.2% arabinogalactan, 0.4% mannan, 2.5% acetyl, 17.4% lignin, 1.8% acetone extract, and 5.0% ash were pretreated in a rotating microreactor immersed in hot oil. Liquids for pretreatment were prepared, yielding the moisture-adjusting compositions provided in Tables 3 and 4. Approximately 5.5 parts liquid were added per part dry biomass (i.e., a liquid-to-solid ratio of 5.5 kg / kg). Laboratory Type II water was used. The biomass and liquid were mixed and pretreated at a final temperature of 140–155 °C, reached approximately 10 minutes after the start of pretreatment. The time at the final temperature was 60–120 minutes; the cooling time was approximately 2 minutes (by immersing the reactor in ice water).

[0198] Unreacted sulfur dioxide is slowly removed from the system by venting, without the use of fiber explosion. Pulp and liquid are not separated. The material is transferred to a rotary evaporator flask and evaporated at approximately 75°C, with the pressure gradually reduced from atmospheric pressure to approximately 150 Torr to provide a final total solids content of approximately 14–17%. Laboratory type II water is added to bring the total solids content to approximately 12%, followed by a second evaporation under the same conditions. Laboratory type II water is added again to bring the total solids content to approximately 12%, followed by a third evaporation under the same conditions.

[0199] In some experiments, materials were heat-treated using microreactors immersed in an oil bath under various conditions (total solids content of approximately 11-14%).

[0200] In all experiments, the material was transferred to shake flasks and diluted with laboratory type II water. The pH of the mixture was adjusted to 4 by adding 30% calcium hydroxide slurry, and then adjusted to 5.4-5.5 with 15% ammonium hydroxide solution. As will be readily recognized by those skilled in the art, other alkali metal or alkaline earth metal hydroxides (e.g., solutions of KOH and NaOH or slurries of Mg(OH)₂) or oxides (e.g., slurries of MgO and CaO) (one or more) can be used instead of calcium hydroxide or ammonium hydroxide to achieve the desired pH level. The material was then subjected to enzymatic hydrolysis for 96 hours. A commercially available enzyme mixture containing cellulase, hemicellulase, and glucosidase was used. The total solids content in the enzymatic hydrolysis process was about 9-10%, the pH was maintained at 4.8-5.5 using 15% ammonium hydroxide solution, and the temperature was maintained at about 53°C. The shaking rate was 200 RPM.

[0201] Table 3-6 shows the surprisingly high sugar yields and surprisingly low degradation products at low enzyme loadings of 0.8 and 1.6 FPU / odg biomass. Despite the presence of a significant amount of water-insoluble lignin fraction that typically inhibits enzymatic hydrolysis, the overall glucose yield was 84–97% (based on glucans available in the biomass). Total hemicellulose sugar yields reached 76–95% (based on hemicellulose available in the biomass). At enzyme doses of 0.8 and 1.6 FPU / odg biomass, the total monosaccharide yields from corn stalks were 81–85% and 83–93% (based on polysaccharides available in the biomass), respectively.

[0202] Table 3: Sugar yield and degradation products in Example 2 (5% sulfur dioxide, 47.5% ethanol)

[0203]

[0204] Analysis of the composition to measure the content of sugar degradation products revealed that the sugar degradation products were less than about 8 kg per BD metric ton of original biomass (see Table 4), which was unexpectedly low based on the relatively high pretreatment temperature (150°C).

[0205] Table 4: Sugar yield and degradation products in Example 2 (5% sulfur dioxide, 24% ethanol)

[0206]

[0207] Table 5: Sugar yield and degradation products in Example 2 (3% sulfur dioxide, 24% ethanol)

[0208]

[0209]

[0210] Table 6: Sugar yield and degradation products in Example 2 (7% sulfur dioxide, 23% ethanol)

[0211]

[0212] Based on experimental and literature data, using other biomass such as softwood, hardwood, or corn stalks will provide similar or higher monosaccharide yields.

[0213] Example 3: Conventional hot water (hydrothermal) pretreatment and enzymatic hydrolysis of corn stalks (comparative example)

[0214] For comparison, the corn stalk batch used in Example 2 was pretreated with conventional hot water (hydrothermal) followed by enzymatic hydrolysis. The amount of water added was approximately 5.5 parts water per 1 part dry biomass (i.e., a liquid-to-solid ratio of 5.5 kg / kg). The biomass and water were mixed and pretreated at a final temperature of 150–180°C, reached approximately 10 minutes after the start of pretreatment. The time at the final temperature was 20–180 minutes; the cooling time was approximately 2 minutes (by immersing the reactor in ice water).

[0215] The pulp and liquid were not separated. The material was transferred to a shake flask and diluted with laboratory type II water. The pH of the mixture was adjusted to 4 by adding 30% calcium hydroxide slurry, and then adjusted to 5.4-5.5 with 15% ammonium hydroxide solution. The material was then subjected to enzymatic hydrolysis for 96 hours. A commercially available enzyme mixture containing cellulase, hemicellulase, and glucosidase was used. The total solids content in the enzymatic hydrolysis process was approximately 9-10%, and the pH was maintained at 4.8-5.5 using 15% ammonium hydroxide solution, with the temperature maintained at approximately 53°C. The shaking rate was 200 RPM.

[0216] The results of conventional hot water (hydrothermal) pretreatment of corn stalks are shown in Table 7. Monosaccharide yields were very similar at the four temperatures studied. There was little correlation between increasing pretreatment time and obtaining higher monosaccharide yields. At enzyme loadings of 1.7 and 3.3 FPU per odg of biomass, monosaccharide yields ranged from 46% to 68%, which is quite low.

[0217] Table 7: Sugar yield in Comparative Example 3 (Conventional hot water pretreatment)

[0218]

[0219] Figure 6 A comparison is shown between the conventional hot water (hydrothermal) pretreatment of corn stalks (this example; Example 3) and the process described in Example 2. The process described in Example 2 was observed to achieve a monosaccharide yield of 85% at 0.8 FPU per odg of corn stalks (Experiment No. 2.3), and over 90-93% at 1.6 FPU per odg of corn stalks (Experiments Nos. 2.4 to 2.9, 2.11, 2.13, and 2.15). In contrast, the hot water process achieved a monosaccharide yield of approximately 46-62% at 1.7 FPU per odg of corn stalks and approximately 56-68% at 3.3 FPU per odg of corn stalks. Therefore, the process described in Example 2 allows for at least 50% higher monosaccharide yields than achievable with conventional hot water processes at a reasonable enzyme load of 1.6-1.7 FPU per odg of biomass.

[0220] The process of Example 2 allows for a further reduction in enzyme loading to 0.8 FPU per odg of biomass with a very high monosaccharide yield (85%); wherein the yield is higher than that achievable even with a very high loading of 6.6 FPU per odg of biomass in a hot water process (60-77% yield).

[0221] Example 4: Fermentation of the enzyme hydrolysate obtained in Example 2

[0222] The enzymatic hydrolysate obtained in Example 2 was fermented into ethanol in two replicates using a commercially available genetically modified (GMO) yeast strain capable of utilizing glucose, mannose, galactose, xylose, and arabinose in shake flasks at pH 5.0–5.5 (200 mL volume, shaking speed 200 RPM). Suitable examples of yeast cells and fermentation techniques are described in U.S. Patent Publication 2019 / 0106464 (Oeser et al.). The fermentation process can optionally use non-GMO yeast or bacterial strains, GMO yeast or strains, or a combination of non-GMO and GMO strains. Fermentation was carried out at 32°C. The fermentation time was less than 24 hours, at which point almost all the sugar was consumed, and the ethanol yield was 83–87% of the theoretical value, i.e., 0.42–0.44 g ethanol / g sugar.

[0223] Based on experimental and literature data, conducting fermentation processes on a larger scale than that described in this embodiment can increase fermentation yield by approximately 3%, and up to approximately 10%. For example, compared to the 0.2-liter scale used in this embodiment, using a bioreactor with a working volume of 1-50 liters can provide an additional 3-8% yield, while an industrial fermenter with a working volume of at least 100 cubic meters can provide an additional 5-10% yield.

[0224] All publications, authorized patents, and patent applications are incorporated herein by reference as if they were individually incorporated herein by reference. No limitation inconsistent with the disclosure herein should be introduced by these disclosures. The description of the invention references various specific and preferred embodiments and technical solutions. It should be understood that many variations and modifications may be adopted, all of which should still fall within the spirit and scope of the invention.

[0225] The above-described embodiments and examples are merely illustrative and do not limit the scope of the invention. Modifications and alterations may be made using techniques common in the art, without departing from the broad scope defined by the following claims.

Claims

1. A method for producing one or more fermentation products and lignin, comprising: (a) Contacting lignocellulosic biomass or distilled lignocellulosic biomass with a pretreatment solution under elevated temperature and pressure; The pretreatment solution includes sulfur dioxide, volatile alcohols, and water; The lignin and hemicellulose in the lignocellulosic biomass are released and / or dissolved in the pretreatment liquid to obtain a mixture containing lignin, hemicellulose and a solid fraction mainly composed of cellulose dissolved in the pretreatment liquid. The mixture is referred to as the pretreated material. (b1) Recover sulfur dioxide and alcohol, thereby precipitating lignin and producing a first mixture containing cellulose, lignin and monosaccharides; (b2) Optionally, the first mixture is maintained at a desired temperature and desired pressure and mixing or recycling rate, thereby releasing and optionally recovering chemically bound volatile alcohols, providing a second mixture comprising cellulose, lignin and monosaccharides; (c) Adjust the pH of the first mixture or the second mixture to an appropriate level for enzymatic hydrolysis; (d) Contacting the pH-adjusted first or second mixture with cellulase, glucosidase, hemicellulase, or a combination thereof to provide a third mixture comprising cellulose-derived glucose and hemicellulase-derived monosaccharides, and lignin; and (e) Ferment the third mixture; To produce one or more fermentation products.

2. The method according to claim 1, wherein step (d) further comprises removing one or more of lignin, alkoxylated lignin, and lignin sulfonate from the third mixture.

3. The method according to claim 1 or 2, wherein the volatile alcohol in the pretreatment liquid in step (a) comprises methanol, ethanol, 1-propanol, 2-propanol, 2-methyl-2-propanol or a mixture thereof; and optionally, in addition to the volatile alcohol, the pretreatment liquid further comprises one or more volatile hydrocarbons, wherein the volatile hydrocarbons are selected from alkanes such as pentane, hexane, cyclopentane and cyclohexane; ethers such as diethyl ether and MTBE; and mixtures thereof.

4. The method according to any one of claims 1-3, wherein in step (b1) and / or step (b2): Sulfur dioxide that is chemically or loosely bound to sugars, lignin, or other compounds is also released and optionally recovered; and / or Oligosaccharides are hydrolyzed, thereby releasing monosaccharides; and / or Lignin decarboxylation, sugar decarboxylation, or both lignin and sugar decarboxylation.

5. The method according to any one of claims 1-4, wherein the recovery of sulfur dioxide in step (b1) or steps (b1) and (b2) comprises: (i) Releasing gaseous vapors from the pretreated material at elevated temperature and / or low pressure, wherein the gaseous vapors released from the pretreated material comprise sulfur dioxide and one or more water vapors and alcohol vapors; the gaseous vapors containing sulfur dioxide are optionally purified by removing trace amounts of water vapor and volatile organic compounds; (ii) Concentrate at least a portion of the gas vapor to provide a sulfur dioxide gas and liquid containing at least one of water and alcohol; (iii) liquefying sulfur dioxide gas by pressure change and / or temperature reduction, thereby providing liquid sulfur dioxide; and optionally storing the liquid sulfur dioxide; and (iv) Optionally, the liquid sulfur dioxide is introduced into the container of step (a) or another pretreatment container by pumping.

6. The method of claim 5, wherein ethanol is used as a volatile alcohol, obtained in step (ii) of claim 5, concentrated and recycled back to the pretreatment and / or at least partially mixed with the fermentation broth, and recovered together with the ethanol produced during fermentation.

7. The method according to any one of claims 1-6, wherein the preprocessing in step (a) is performed in continuous mode or batch mode.

8. The method according to any one of claims 1-7, wherein step (b1), step (b2), or both are performed using one or more of a purge tank, a stripping column, a distillation column, and a fractionation column, and / or wherein step (b1), step (b2), or both are performed in one or more containers, the containers being tanks equipped with heated recirculation lines and distillation columns, fractionation columns, or stripping columns, wherein the columns consist of column disks, packing, or combinations thereof, and the pretreated material is transferred into the tank by differential pressure (purge) or pumping.

9. The method according to any one of claims 1-8, wherein between step (b1) and step (d), cellulose is not separated from the remainder of the first mixture or the second mixture, such that both cellulose and precipitated lignin are present in the first mixture or the second mixture in step (d).

10. The method according to any one of claims 1-9, wherein the enzyme loading in step (d) is less than 5 FPU, less than 4 FPU, less than 3.5 FPU, less than 3 FPU, less than 2.5 FPU, less than 2 FPU, or 0.2 to 5 FPU, 0.5 to 3.5 FPU, 0.5 to 3.3 FPU, 0.5 to 2.5 FPU, 0.5 to 2 FPU, or 0.8 to 1.6 FPU per odg of biomass.

11. The method according to any one of claims 1-9, wherein the enzymatic hydrolysis in step (d) is carried out in a continuous or batch mode and / or the pretreated material in the enzymatic hydrolysis in step (d) is added in a single, batch, or stepwise manner, and the enzyme is added in a single, batch, or stepwise manner.

12. The method according to any one of claims 1-11, wherein the enzymatic hydrolysis in step (d) is carried out with a total solids content of more than 9%.

13. The method according to any one of claims 1-12, wherein the lignin is partially or completely separated after the enzymatic hydrolysis step (d) or after the fermentation step (e); or wherein the alkoxylated lignin is partially or completely dealkoxylated in step (b1) and / or (b2), and the resulting lignin material is partially or completely separated after the enzymatic hydrolysis step (d) or after the fermentation step (e).

14. The method according to any one of claims 1-13, wherein the fermentation product is ethanol.

15. The method according to any one of claims 1-14, wherein prior to step (a), the lignocellulosic biomass is contacted with steam, wherein the lignocellulosic biomass absorbs water from the steam, thereby removing air from the lignocellulosic biomass.

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